By Mike Darwin

Introduction

In order to understand the significance of the results of the Cryonics Intelligence Test and the discussion of priorities in cryonics research that is to follow (and in particular the relationship of such research to the Alcor Life Extension Foundation and the cryonics community at large), it will first be necessary to provide a substantial amount of background and context.

The first part of this context is to understand the statistics of Chronosphere. Even a decade and a half ago, the data I am about to present and discuss would have been virtually impossible to obtain. Small-scale paper, and even Internet publications, were mostly black boxes in terms of feedback. Knowing how many people viewed a given article, looked at your publication (no matter how cursorily) or took the time to download specific materials was essentially impossible. Letters to the Editor and the total number of paid and gratis subscribers (e.g., basic circulation), as well as advertisers, if any, were all the data available – plus, perhaps some demographic data on subscribers, such as what part of the country and what part of the world they resided in.

Today, with powerful statistical engines, it is possible to obtain in real time a large body of data that was heretofore not only inaccessible, it was unimaginable that it would ever be available, let alone be available at virtually no cost and with almost no effort. Having said this, it still not possible to capture the core demographic data that would provide the most useful information about the scope and depth of Chronosphere’s impact; namely the detailed demographic characteristics of the individuals accessing the site, the individual articles, the identities of those individuals, which articles they actually read, and finally, what impact those articles have on their world view, or subsequent actions. To a very limited extent it is possible to track the effect that articles and ideas in Chronosphere have on others by using Google search tools to monitor the mention of discrete articles or ideas that have appeared uniquely on Chronosphere in the blogosphere and on the Internet in general. However, this is still far from satisfactory, and such data is necessarily anecdotal, rather than comprehensive.

A Preliminary Look at the Numbers

The graph below shows the total number of hits, by month, that  Chronosphere has received since its inception through, 23 May, 2012, at 1307. Since the start of Chronosphere, there have been 101,929 unique visitors to the site. During 2012 the average number of unique viewers, excluding individuals who subscribe via Google Reader Subscriber Service (RSS), is approximately 300 per day. The number of RSS subscribers has increased from ~ 80 as of October 2011 to 101 as of 23 May, 2012. The average number of new posts to Chronosphere has been 2.1 per week since its inception in February of 2011. The table below shows the statistics for the top 10 articles being accessed as of 22 and 23 May, 2012. There is substantial variability on a day to day basis as to which articles achieve “top ten” status. The following table shows the ranking of all articles that have appeared, from the first one, which was posted on 06 February 2011, through 23 May, 2012. These data show the number of unique hits these articles received, independent of RSS subscribers and of individuals who may have read the article, copied or downloaded it from the “Home Page.”

To understand what this means in practical terms, the article Robert C. W. Ettinger, First Life Cycle: 1918 to 2011, shows 2,762 discrete hits.

However, any examination of the aggregate number of hits for the two week period following Ettinger’s cryopreservation (boxed in red in the top graph, above), when his obituary, and a related article on media obituaries were the articles featured on the Homepage, show that the number of hits to Chronosphere increased from ~ 1,000 per day to ~ 3,000 per day. Thus, a more realistic number for views that article received is probably in the range of ~ 4,000, total.  Therefore, the total “viewership” for any given article will be some total of the number of discrete hits the article receives, plus some fraction of the number of Homepage hits it received when it was the featured (Homepage) article on Chronosphere.

 Making Sense of it All

Missing from all these data is the critically important “time on page” number. This metric helps to distinguish between “accidental,” or very casual viewers, and those who have a serious interest in the content of the article. Unfortunately, all efforts to date to add this capability (a function of Google Analytics) have proved unsuccessful. Nevertheless, the absolute number of hits a given article receives after it ceases to be the Homepage feature is very likely representative of its popularity and readership.

My personal (completely arbitrary) rule of thumb has been to assume that ~ 10% of the hits to lengthily and highly technical posts  represent serious readers and that ~25% of hits for shorter, topical posts are actually read and seriously considered.  What the “real” number of serious readers is for any given article is, of course, unknown. I have set my numbers so low primarily because of the nearly complete lack of commentary or embedded URL actuations most of the articles generate.

As a case in point, the extensive four-part series of articles Last Aid as First Aid for Cryonicists generated a total of only 5 comments, most of which were not of a practical nature consonant with the subject matter of the articles, which dealt specifically with how to prepare for a cryonics emergency. Some of the suggestions in the articles should have proved controversial (based on past experience in discussing them in the cryonics community) and yet, there were no dissenting comments, nor any alternative or additional suggestions offered, either on Chronosphere, or in the publications, blogs, or list-serves of the various cryonics organizations (or those serving the cryonics community as a whole, such as New Cryonet). This is in striking contrast to the author’s past experience with printed periodicals serving the cryonics community and having a comparable, or  smaller number of readers  (e.g., 200-300).

Some of the likely reasons for these differences between print and epublications are:

a)      Subscribers paid for paper publications and thus were more heavily invested in recovering the value expended.

b)      Because of the time, effort and money required to gain access to paper publications, the readership was highly filtered compared to epublications.

c)       Prior to the Internet era, the total volume of information being disseminated about cryonics was very small and the available technology (e.g., the printing press) further compressed and limited dissemination of that information to a very few venues.

d)      Cryonics itself was far smaller and the overlap between “activists” and “customers” was more nearly complete.

e)      Cryonics publications prior to the Internet were necessarily more diverse in content than is Chronosphere due to the need to cater to a broader audience.

f)       The content in Chronosphere leans heavily towards the technical and historical and is lengthily; all of which are likely to discourage the casual reader. In other words it is, by definition, a niche publication.

g)      Chronosphere and its author are frequently critical of how cryonics is currently practiced as well as of  the major (extant) cryonics organizations, and sometimes  specific individuals who are, or who have been active in cryonics.

h)      Chronosphere does not (yet) offer a blog roll nor high profile links to other organizations, sites, or publications (paper or electronic). This, coupled with the hostility generated by g) above, has resulted in a near complete lack of on-line and paper publication referrals to Chronosphere.

With these considerations in mind, let’s again take a look at how Chronosphere  has performed from its inception, thru 24 May, 2012, but this time in greater detail with attention to daily and weekly numbers:

But what do these metrics really mean? Is Chronosphere doing what it is supposed top do: raise awareness and change fundamental thinking about the way cryonics and interventive gerontological research is being pursued,  as well as attracting other, like minded contributors to the site? The number of RSS feeds, the number of unique viewers and even the number of comments aren’t necessarily very useful metrics (certainly not in isolation) to determine if the effort being expended on Chronosphere is worth the return. Probably the best indicators are the combination of:

a)      Number of comments,

b)      Number of RTs,

c)       Number of downloads of white-papers, pingbacks, and “critical” URL’s accessed from the site.

d) Number of people who contribute articles to Chronosphere.

Of course, context is everything, or almost everything in this case, because Chronosphere is catering to what is, both relatively and absolutely, a miniscule community of people. To put these numbers in context, the graph below shows the traffic on the Wikipedia “cryonics” page.

In the past 90 days there have been ~78,000 visitors or ~25,000 visitors per month, as compared with ~8,400 visitors to Chronosphere over the same time period.

There are perhaps something on the order of 2,000 living cryonicists[i] in the world, the majority of them in the English speaking/reading world. Of these, optimistically, perhaps 15% are technically/scientifically/philosophically oriented “activists” with an interest in the mechanics of cryonics, as opposed to people who have chosen cryonics as a service or product “as is,” and are content to accept it without further improvement as a result of their own efforts. That would yield a number of ~ 300 people within the cryonics community who are sufficiently interested to read a publication like Chronosphere.

Even using these far more restrictive criteria, it is hard to know just how well or poorly Chronosphere is doing. Consider the cryonics self-help series of articles, Last Aid as First Aid for Cryonicists:

The overall performance of this series of articles is pretty dismal. However, interestingly, Part 4 in the series received the most hits, roughly three times the total that each of the preceding three parts received. This might be explained on the basis that the fourth part of the article contained the bulk of the practical suggestions for how to deal with an emergency (such as the equipment and supplies needed for cooling).

Articles that are likely to be of interest primarily to cryonicists, such as A Brief Pictorial History of Extracorporeal Technology in Cryonics show a viewership that is broadly similar to that seen for this five part  series of article (below).

By monitoring the search engine terms (and their frequency) being used, it is possible to get some idea of how many people are accessing these articles for reasons unrelated to cryonics, such as for information on extracorporeal medicine, specific devices mentioned in the articles and for illustrations of equipment or procedures (again, unrelated to cryonics, per se). Roughly a third of all hits fall into this category of what could be fairly called “extraneous viewers.” Again, the number of likely seriously interested viewers is probably quite small, being somewhere in the rage of 50 to 100.

The intermittent spiky nature of the number of hits over time is most likely the result of referrals; one person sees an article of interest, passes the URL to others and there is a brief burst of activity until that pocket of interested people is exhausted.

Similarly, technical posts which have direct relevance to medicine or biomedical research are clearly attracting viewers who are not accessing them because of any interest in cryonics. Indeed, it can reasonably be presumed they are accessing them in spite of their cryonics orientation and content, as can be seen from the data for the articles The Pathophysiology of Ischemic Injury: Impact on the Human Cryopreservation Patient, I Know this is Going to be Shocking: A Review of Wearable Continuous Monitoring Systems to Detect and Treat Sudden Cardiac Arrest in Cryonicists, Does Personal Identity Survive Cryopreservation?, Achieving Truly Universal Health Care and Induction of Hypothermia in the Cryonics Patient: Theory and Technique.

It is possible that articles that deal solely with technical issues related to cryonics, but which do not explicitly mention it, such as Liquid Assisted Pulmonary Cooling in Cardiopulmonary Cerebral Resuscitation may provide some insight into how many of the visitors accessing the explicitly cryonics-oriented technical articles are doing so for reasons completely unrelated to any interest in cryonics:

If If this line of reasoning is indeed valid, then the number of explicitly cryonics-interested viewers is again probably somewhere in the range of 50 to 150 people.

This number is also consonant with the number of viewers that access a post which is almost exclusively of interest to cryonicists, such as the series of articles, Much Less Than Half a Chance,  on using medical imaging to reduce the number of sudden and unexpected deanimations (cardiac arrests) amongst cryonicists:

and Your Picture Won’t Be Hanging Here?:

More problematic to interpret are articles which deal with technical matters of a nature, interest in which one would expect would be largely or completely confined to cryonicists, such as the (so far) three-part series, The Effects of Cryopreservation on the Cat, which has generated sustained and (relatively) substantial interest, with Parts 2 & 3, wherein the results of the study are presented, having received a total number of views of ~ 1,800:

This is comparable to the degree of interest shown in most “data/conclusion-dense” part of the series of articles on brain degeneration in aging, Going, Going, Gone…:

However, it bears pointing out again that the more specifically cryonics oriented an article is, the smaller its readership will typically be, as was the case in the Cryonics: Failure Analysis Lectures, 1 & 2:

and the article Freezing People is Easy:

Below, I’ve presented the statistics on a range of other types of articles without comment, in large measure because it is hard to know how to interpret the data:

And finally, we come to Take the Cryonics Intelligence Test which was designed as a seminal experiment to probe both the readership of Chronosphere and the cryonics community at large. Leaving out of consideration the number of people who may have read this article during its tenure on the Homepage, 193 people accessed it as of 24, May, 2012:

and only 82 people were interested enough to view the results of the test:

Even more interesting (and telling) is metric for the number of people who downloaded the Resource Materials for the test from Yousendit, a mere 22 souls: of whom two bothered to actually take the test.

These numbers seem dismal to me, all the more so when, in the next few installments here, the issues involved (dealing with the principal subject matter in the Cryonics Intelligence Test and the Cryonics Intelligence Test Responses) are explicated and put into context and their importance (hopefully) made apparent to even the least technically inclined readers of Chronosphere.

Finally, it would be most useful to see similar performance metrics from other cryonics and life extension related blogs and websites. It is virtually impossible to evaluate the performance of this effort without any benchmarks to compare it to.

Footnote

On 06 May, 2012 responses were solicited to what was termed The Cryonics Intelligence Test which was posted here on Chronosphere (see: http://wp.me/p1sGcr-vV). Two people responded to this public request to “take the test” and provide input on possible solutions to the problems posed by the resource material that accompanied the test. The test consisted of the resource materials and the following  instructions:

Dear ______,

If you can figure out the scientific take home message for cryonics in what is to follow, you will have demonstrated extraordinary insight into “thinking in a cryonics-medical context.”

You will also have the tool to be able to understand why I believe that cryonics must, on a purely scientific-medical basis, be pursued in a fundamentally different way, both biomedically and socially.

The Test: The test resource materials are available for download at ___________, you will find a number of full text peer reviewed scientific papers. In addition, you will be sent several cryopatient case Hxs. Together, these resources contain data which should give a reasonably intelligent person with a properly prepared mind a fundamentally new insight into a major, indeed overwhelming flaw in how cryonics has been, and currently is practiced.

Your task is to:

a) identify the problem(s)

b) identify one or more possible solutions

You have 5 days to complete this task. Your response should be in the form of a succinct statement of the problem, and an itemization, and if you like, a discussion of possible solutions.

Thanks for your patience and cooperation.

Mike Darwin

Purposes

The reasons for  this exercise were as follows (in no particular order):

To answer the question posed to me by Alcor CEO on what was the most important research to be undertaking in cryonics at this time.

To determine if a representative cross section of people not actively employed in cryonics, or working in cryonics-related research, would independently reach the same or same similar conclusions about a heretofore not understood or appreciated major problem in cryonics and propose the same possible solutions (or novel ones) to said problem.

To evaluate the caliber of the intellects (who chose to participate) who read Chronosphere.

To attempt to determine the number of Chronosphere readers who were willing to accept the challenge of  exposing their judgment and intellectual performance to scrutiny, either by myself, publicly, or both.

To determine the approximate number of people who took the time and exerted the effort to at least peruse the article and download the Test Resource Materials.

To attempt to get a preliminary idea of the nature of the readers of Chronosphere and their interest in highly technical topics of serious relevance to cryonics.

To gauge the impact and reaction of both the leadership of the cryonics community, and the cryonics community itself, to the revelations that result from this exercise and the commentary that is to shortly follow it.

To solicit novel solutions to the central problem posed in the exercise.

To inform the community at large, both the cryonics community and the public, of this serious problem in the way human cryoprerservation is currently being pursued (e.g., informed consent).

Participants

Two people (Alexander McLin and Gerald Monroe ) responded to the public request on Chronosphere to take the test. Prior to publicly soliciting responses, fifteen individuals of diverse backgrounds in cryonics were privately asked to take the test. Of these, eleven agreed to do so and of those eleven, ten completed the test. Of the ten privately solicited respondents, three agreed to allow publication of their answers; two with the use of their names. One individual, a young academic pursuing advanced graduate degrees, asked for and was granted anonymity, due to the likelihood that open involvement in cryonics could prejudice his academic career.

Since it is not possible for the responses of those who chose not to allow publication to be evaluated here, I will not make any comment on them beyond noting that they exist and that they, along with those of the respondents who did allow publication, were material in making the decision to pursue an open solicitation here for additional respondents.

At this time, the answers of the respondents are being presented absent any biographical/background information, so as not to bias the reader as he reads and considers each response. At a later date, I will edit this post to add a brief (few sentences) background description on each of the participants in order to provide demographic data on the participants as a group (e.g., how many were biomedically sophisticated, laypersons, long-time cryonicists, novices, etc.).[1]

 Responses

Responses are presented in alphabetical order (by name of the respondent). The only editing that has been done is to to correct typographical errors.

Alexander McLin

After studying the test materials, I have come to the following conclusions about how cryonics is currently practiced today and the problem with its current standards of practice. The problem is that cryonics isn’t effectively managing ischemia, nor it doesn’t seem to be incorporating medical findings about how the brain is affected by hypotension, hypoventilation, and hyperventilation.

Moreover, research in determining a method to predict onset of cardiac death after life-saving treatments is withdrawn indicate that this is difficult to do so, this in conjunction with other papers, show that the brain damage begins almost as soon as a patient’s circulatory system begins to fail. This is problematic from the cryonics point of view, because long before cardiac death is declared, the brain may have already suffered irreversible ischemia damage preventing optimal cryonics suspension.

The research materials furthermore show that hyperventilation when administrated for whatever reason actually makes things worse and that hypoventilation is preferred. With this in mind, do cryonics providers incorporate that finding when administrating oxygen to patients as part of the stabilization protocol?

To summarize, the conclusions I arrived at are that current cryonics providers are failing to manage ischemia, failing to research ways to predict the degree of severity of ischemia, failing to engage in proactive activities to minimize ischemia pre- and post-deanimation, and not incorporating medical findings in improving brain survivability in presence of hypotension and hypoventilation. In addition, there appear to be a lack of an attempt to maintain extensive database of patient medical history, collection of body fluids for pre and post-deanimation, and pre- and post-suspension which is essential for research intended to improve cryonics practices.

Here I will discuss solutions I have come up to address some of the conclusions I have arrived at. The biggest problem is the issue of ischemia and how likely it is to occur once oxygen is interrupted and also how sensitive the brain is to reperfusion injury. I would review the existing protocols to ensure whether they’re adequately taking the reperfusion injury into account, whether medicines need to be updated(add or remove medicines) with respect to the latest medical findings. It should be verified via meaningful actual research whether the cool-down equipment is really minimizing ischemia.

Finally, how can cryonics address the crucial issue of the existing medical-legal atmosphere that require patients to be declared dead according either to the cardiac or brain death definitions. Both which ensure that the brain will suffer ischemia damage before suspension occurs. How can cryonicists safely arrange for optimal cryonic suspension free of problematic legal implications? This suggests a need to engage in policy lobbying and pushing for legislation aimed towards changing the legal situation for the betterment of cryonics. To put it so bluntly, it appears that voluntary euthanasia is a cryonicist’s best friend, as distressing and stressful it may sound.

Lastly, cryonics providers need to establish a medical database and engage in much more data collection than they are doing at present. Some of the patient histories show recurrent problems with their collection equipment, do they need to be upgraded or replaced? Research in minimizing or preventing ischemia should be undertaken to determine how to optimize brain preservation prior to beginning suspension.

Mark Plus

Many cryonicists in hospice conditions currently deanimate and are pronounced after agonal periods similar to shock which result in prolonged hypoperfusion and hypoxia of the brain. These lead to significant compromises of the brain’s vasculature (e.g., the brain’s ability to self-regulate its blood flow to certain regions like the hypothalamus when the arterial pressure drops below 40 mm Hg) and interfere with cardiopulmonary support, washout and especially perfusion with cryoprotectants, not to mention the havoc they must cause to the brain’s fine structure.

Also, the trend towards harvesting organs from patients who are pronounced cardiac-dead after as little as two minutes of asystole is probably not a good thing for cryonicists, if the laws change to make it harder to opt out of such donations which will have the effect of ensuring thorough brain death.

My suggestions:

Use people with professional training in shock medicine and anesthesiology to perform the cardiopulmonary support after pronouncement. Monitor the level of brain perfusion with the proprietary bispectral index technology (which I had to look up and I’d like to read more about) to determine if brain hypoperfusion happens. Hypoventilate the patients.

Premedicate cryonicists before pronouncement with drugs like piracetam, arginine vasopressin and NO inhibitors, mentioned in the papers you sent me. You also wrote that Jerome White had attempted to premedicate himself with over the counter supplements until a few weeks before his suspension.

Cryonicists with terminal illnesses should consider moving to places where the laws allow assisted euthanasia so that they can go into arrest and undergo the suspension procedure well before their agonal decline.

Cryonics organizations need to gather a lot more data when they perform suspensions based on the current state of the medical art. The S-100B assay should be used along with other assays to measure brain injuries. These assays plus the bispectral index data can provide badly needed feedback on the effectiveness of brain perfusion procedures.

If the patient can’t deanimate at the time of his choosing, use some of the medical models developed by the DCD researchers to better estimate the patient’s time of cardiac death during standby.

I hope my answers and recommendations are not too off the mark, and I suspect I’ve misunderstand or failed to notice some key points. You gave me a lot of unfamiliar material to absorb in a short amount of time. After a few more weeks of study, I could probably understand it better. Some kind of primer would also help. A few years ago I speculated that based on actuarial considerations, the ideal candidate for cryosuspension would have to be a healthy ten year old who could walk into the lab and lie down on the table. That leaves the rest of us somewhere away from optimal candidacy for cryosuspension. But then, what can we do about it?

And I do plan to study this further, so thank you very much for the scientific background information, and feel free to send me additional papers.

Other observations:

I notice the contrast between the thorough reports you’ve written for the suspensions you’ve performed versus the ones written by Alcor’s “pod people,” which apparently includes Aaron Drake. Several things seem to go wrong with about every suspension Alcor has done lately, including basic preparations like not having the tray of all the necessary surgical tools ready for Dr. Nancy or the surgeon. I knew in a vague way that things had gotten bad, but you’ve given me some idea of how bad.

The scientific literature started to report the effects of shock and hypoperfusion decades ago, but you wouldn’t know that from the “official” cryonics propaganda. It seems like the cryonics movement should have incorporated this knowledge from the very beginning, but then physicians, surgeons and neuroscientists have mostly avoided cryonics and deprived us of their expertise. Dr. Ravin Jain, a neurologist, sits on Alcor’s board, and he should know this stuff, but I don’t get the impression that he’s done anything to incorporate his knowledge into Alcor’s suspension procedures. The neglect gives cryonics a reputation for “scienciness” and pseudoscience which it doesn’t necessarily have to have.

Gerald Monroe

a. The current techniques practiced for all the cryonics cases most likely result in long periods of ischemic hypoperfusion to the brain. Instruments now exist to detect this, combining the bispectral index with near infrared spectroscopy, and apparently even when top notch experts support cardiac surgeries on children, the hypoperfusion is common.

The ischemia and the hypoperfusion are very, very bad. Of course, so is the freezing. And the storage in liquid nitrogen where dissolved oxygen can reach the tissues and oxidize them. And the shoestring budget (compared to even a single hospital) the cryonics organizations have to do everything on.

b. It doesn’t sound like these problems are insoluble if there were real resources (compared to those spent to delay death from cancer by a few months, for instance) dedicated to the problem. Tomorrow, if cryonics had the resources of a single major metropolitan hospital, it could actually solve these problems in a systemic way.

There have to be experiments done on animals, where many different techniques* are attempted and evaluated. Evaluations should be done by preparing synapses of slices of the subject’s brain following the freezing. Also, rewarming and function tests (of slices), once the state of the art reaches the point that this is practical.

The human patients have to be part of this evaluation. If no one looks, the mistakes made will never be corrected. Somehow very small pieces need to be removed as samples from the human patients, following each cryonics procedure, small portions mostly taken from sections of the patient’s brain not thought to contain unique personality information.

And so on. Real improvements don’t come easily or cheaply – they come incrementally, with great effort, and honest evaluation of the results of each change. The last element is probably the most important of all.

The history of medicine is littered with many, many examples where something becomes common practice without honest testing of the results. Pretty much universally it fails.

With all that said, for those of us right here, alive in an era where cryonics does not have the resources it deserves, it is simply Pascal’s wager. No matter how dim the odds are, some chance of a form of survival is better than none. Information is probably duplicated inside the human brain many times over, and all of the decay processes that work against cryonics are things that happen according to predictable laws of physics. In a future world where a brain could be scanned at the molecular level, there is probably at least some recoverable memory and personality data for even the worst cryonics case.

For some, the prospective of saving even an incomplete fragment of yourself is better than the guaranteed destruction by rotting in the ground or burning in an incinerator.

Why it is like it is : the cryonics organizations don’t have any money. There’s probably a hundred new things that could be tried, and most of them are not better than what is being done now. Every dollar spent now is a buck less that could go to protecting the existing patients over many more decades.

Moreover, without any way to evaluate the current baseline : how effective is cryonics actually preserving the patients, right now? Making changes blindly is stupid. In the history of medicine, time and time again, it has been found that when a simple and dumb medical technique is compared honestly to a more expensive and advanced technique, almost universally the difference is minimal to none. A few examples : diuretics work as well as the far more expensive and specific beta blockers, film X-rays provide basically the same therapeutic improvement as the vastly more expensive CTs and MRIs, physical therapy works about as often as spine surgery, etc.

This is why in countries with socialized medicine, with outdated equipment and techniques and long wait lists, the patients live almost as long. (and the population lives years longer due to better lifestyles)

* A few ideas that might or might not work :
1. More rapid cooling by exposing the brain to coolant with burr holes and connecting pumps directly to cerebral perfusion
2. Drugs to prevent the cerebral arterioles from closing when exposed to cold perfusate.
3. Calcium blockers to prevent apoptotic pathways from triggering
4. Oscillating magnets like the Japanese claim work for transplanting teeth
5. Skipping cryonics entirely and plastinating the brain

Jordan Sparks, DMD

Well, I’ve read all the papers. I’ve attached the notes I made. I know you said I could skim them a little more quickly, but I was having trouble understanding and remembering. I needed to use a more aggressive approach this time. I did the references to help me get organized, and if I had to do that again I would do it without listing out all the names. Anyway, this is where I’m at.

I have a tentative answer which I may refine later. I’m continuing to think about it. You only gave me one cryopatient case Hx. I notice that it’s rich with hematology and chemistry data. Repeated samples were taken and charted over time. Both the TBW circuit and the cryoprotective perfusion circuit are well documented. Pressures and flow rates are nicely charted. Also, glycerol, blood gas, and pH were monitored during cryoprotective perfusion. The lab samples, in particular, are notable because that is not the current practice of Alcor or CI. It would take me some time to look back through case reports to see when was the last time this was done.

a) Cryonics providers are currently disregarding complexity associated with the biochemical milieu. I’m not quite sure how to state it, but all of the 22 papers treated their problems as a complex interplay of the mechanical issues as well as the biochemistry. Reading current Alcor and CI reports, on the other hand, there is a total disregard for the role of biochemistry.

That’s my first stab at it. I wish I could state it better, and I might try to rewrite it. I might wait for feedback from you before I go much further in case I’ve missed your point.

1.  Fast recovery from shock used vasopressor combined with hypertonic saline starch.  Slow recovery used fluid resuscitation.  Propofol and Hb concentrations were comparable in both groups.  The fast recovery resulted in better cerebral perfusion and a higher BIS that was likely due to the better perfusion.  CPP =MAP−ICP.

2.  Three resuscitation protocols: 1=FR (fluid resuscitation), 2=NA/HS (noradrenaline/ hypertonic starch), and 3=AVP/ HS (arginine vasopressin/HS).  The AVP/HS group had faster and higher increase in MAP and CCP as well as better survival.  Also, ICP was lower.

3.  After significant hypervolemia, cerebral circulation decompensation occured.  There were significant regional variations in cerebral blood flow.  The redistribution favored the areas related to cardiovascular control.

4.  Patients in shock can have normal physiological, hematological, fluid, and electrolyte balance but still die due to metabolic abnormalities.

5.  In spite of mechanisms for preferential shunting of blood to the brain, low MAP will result in poor perfusion.  This results in inadequate oxygenation as well as inadequate lactate washout.  Decreased perfusion leads to ischemic damage.

6.  Hemorrhagic hypotension was induced in dogs which was still above the lower limit of cerebral autoregulation.  This resulted in an increased turnover of free fatty acids in the CSF.

7.  Moderate reduction of MAP in anesthetized cats resulted in no significant EEG changes.  Below 40 mm Hg, cortical rhythms slowed and then stopped.  Cell damage was only found below 40 mm Hg.

8.  Baboons were pretreated with Phenoxybenzamine (PBZ) before hypovolemic shock, and it prevented the fall in cerebral blood flow.  EEG does not normally return after reinfusion.

9.  Bispectral index (BIS) dropped to 0 during cerebral hypoperfusion.

10.  For donation after cardiac death (DCD) kidneys, prolonged severe hypotension was a good predictor of subsequent organ function.  Donor age also correlated with worse outcome.

11.  Dogs anesthetized and hypovolemic shock induced for 2 hours.  NMR used to monitor phosphate metabolism.  Upon fluid resuscitation, phosphate pools quickly returned to near baseline values, but intracellular acidosis persisted.

12.  Hemorrhagic shock combined with increased ICP is particularly damaging.  Increased ICP leads to cerebral ischemia which causes release of thromboxane A2 (TxA2), a potent vasoconstrictor and hypertenstive agent.  The increase in TxA2 persists for at least two hours after reperfusion and results in further cerebral hypoperfusion.  Pretreatment with COX inhibitor ibuprofen decreases TxA2 levels and improves total cerebral blood flow after global cerebral ischemia.

13.  Brain is vulnerable during hypotension and shock, especially long-lasting shock.  Patchy areas of ischemia developed through sludge formation and persisted even after hyperperfusion, indicating the role of local factors.  Phenoxybenzamine pretreatment significantly reduced rCBF changes during shock.

14.  DCD livers result in inferior graft survival compared to donation after brain death (DBD).  A DCD risk index was developed.  The lowest risk is with donor age <= 45 years,  warm ischemia time (DWIT) <= 15 minutes, and cold ischemia time (CIT) <= 10 hours.

15.  CNS activity was measured during hemorrhagic shock under light central anesthesia.  After reinfusion, if neurons failed to recover electrical activity, this was an early indication of eventual irreversibility.  There is a relationship between irreversibility and cumulative oxygen debt and excess lactate.

16.  Rats were subjected to hypoxia and hypotension followed by resuscitation.  Rather than the no reflow that the authors were expecting, they observed hyperemia in some areas for at least two hours.  They concluded that therapy aimed at increasing cerebral blood flow and oxygenation would be insufficient.

17.  Guidelines for controlled DCD are given.  DBD is superior.

18.  DCD score system is described.  Kidneys may benefit from therapeutic interventions before transplantation.

19.  Average values for basal respiratory functions in adolescents and adults.

20.  Severe hypotension causes brain damage.  Microvascular damage results in hemorrhage upon reinfusion.

21.  Prolonged agonal time did not influence kidney transplantation outcome when other variables were closely considered instead.  For example, elderly donors were not included.

22.  During hypovolemic shock, electrical activity and ICP was minimally altered.  The authors interpret this as a lessening of the role of the brain in the genesis and perpetuation of irreversible shock.

References

1: Cavus E, Meybohm P, Doerges V, Hoecker J, Betz M, Hanss R, Steinfath M, Bein B.  Effects of cerebral hypoperfusion on bispectral index: A randomized, controlled animal experiment during haemorrhagic shock.  Resuscitation.  2010;81:1183-1189.

2: Cavus E, Meybohm P, Doerges V, Hugo HH, Steinfath M, Nordstroem J, Scholz J, Bein B.  Cerebral effects of three resuscitation protocols in uncontrolled haemorrhagic shock: a randomized controlled experimental study.  Resuscitation.  2009;80:567-572.

3: Chen RY, Fan FC, Schuessler GB, Simchon S, Kim S, Chien S.  Regional cerebral blood flow and oxygen consumption of the canine brain during hemorrhagic hypotension.  Stroke.  1984;15:343-350.

4: Cowley RA, Attar S, LaBrosse E, McLaughlin J, Scanlan E, Wheeler S, Hanashiro P, Grumberg I, Vitek V, Mansberger A, Firminger H.  Some significant biochemical parameters found in 300 shock patients.  J Trauma.  1960;9:926-938.

5: Feldman RA, Yashon D, Locke GE, Hunt WE.  Cerebral tissue lactate in experimental oligemic shock.  J Neurosurg.  1971;34:774-778.

6: Fritschka E, Ferguson JL, Spitzer JJ.  Increased free fatty acid turnover in CSF during hypotension in dogs.  Am J Physiol.  1979;236(6):H802-H807.

7: Gregory PC, McGeorge AP, Fitch W, Graham DI, MacKensie ET, Harper AM.  Effects of hemorrhagic hypotension on the cerebral circulation.  II.  Electricocortical function.  Stroke.  1979;10:719-723.

8: Hamar J, Kovach AGB, Reivich M, Nyary I, Durity F.  Effect of phenoxybenzamine on cerebral blood flow and metabolism in the baboon during hemorrhagic shock.  Stroke.  1979;10:401-407.

9: Hemmerling TM, Olivier JF, Basile F, Le N, Prieto I.  Bispectral index as an indicator of cerebral hypoperfusion during off-pump coronary artery bypass grafting.  Anesth Analg.  2005;100:354-6.

10: Ho KJ, Owens CD, Johnson SR, Khwaja K, Curry MP, Pavlakis M, Mandelbrot D, Pomposelli JJ, Shah SA, Saidi RF, Ko DSC, Malek S, Belcher J, Hull D, Tullius SG, Freeman RB, Pomfret EA, Whiting JF, Hanto DW, Karp SJ.  Donor postextubation hypotension and age correlate with outcome after donation after cardiac death transplantation.  Transplantation.  2008;85:1588-1594.

11: Horton JW, McDonald G.  Heart and brain nucleotide pools during hemorrhage and resuscitation.  Am J Physiol.  1990;259:H1781-H1788.

12: Kong DL, Prough DS, Whitley JM, Taylor C, Vines S, Deal DD, DeWitt DS.  Hemorrhage and intracranial hypertension in combination incresae cerebral production of thromboxane A2.  Critical Care Medicine.  1991;19:532-538.

13: Kovach A, Sandor P.  Cerebral blood flow and brain function during hypotension and shock.  Ann Rev Physiol.  1976;38:571-596.

14: Lee KW, Simplins CE, Montgomery RA, Locke JE, Segev DL, Maley WR.  Factors affecting graft survival after liver transplantation from donation after cardiac death donors.  Transplantation.  2006;82:1683-1688.

15: Peterson CG, Haugen FP.  Hemorrhagic shock and the nervous system.  1. Spinal cord reflex activity and brain stem reticular formation.  Annals Surgery.  1965;485-496.

16: Proctor HJ, Wood JJ, Palladino W, Woodley C.  Effects of hypoxia and hypotension on oxygen delivery in the brain.  J Trauma.  1979;19:682-685.

17: Reich DJ, Mulligan DC, Abt PL, Pruett TL, Abecassis MMI, D’Alessandro A, Pomfret EA, Freeman RB, Markmann JF, Hanto DW, Matas AJ, Roberts JP, Merion RM, Klintmalm GBG.  A J Transplant. 2009;9:2004-2011.

18: Plata-Munoz JJ, Vazques-Montes M, Friend PJ, Fuggle SV.  The deceased donor score system in kidney transplants from deceased donors after cardiac death.  European Society Organ Transplant.  2010;23:131-139.

19: Shock NW, Soley MH.  Average values for basal respiratory functions in adolescents and adults.  J Nutrition.  1939;143-153.

20: Tamura H, Witoszka MM, Hopkins RW, Simeone FA.  The nervous system in experimental hemorrhagic shock: morphology of the brain.  J Trauma.  1972;12:869-875.

21: van Heurn LWE.  Prolonged agonal time–not a contraindication for transplantation.  Nat Rev Nephrol.  2011;7:432-433.

22: Yashon D, Locke GE, Bingham WG, Wiederholt WC, Hunt WE.  Cerebral function during profound oligemic hypotension in the dog.  J Neurosurg.  1971;34:494-499.

“Synaptic”

As you wrote in 1994, the three sources of damage to cryopatients are 1) the underlying disease process, 2) shock and global and trickle flow ischemia secondary to dying and cardiac arrest, and 3) cryoprotectant toxicity and cryoinjury from freezing. This, as far as I can tell, has not changed. So, a flaw in how cryonics is practiced would have to mean that providers are not minimizing the damage from these processes as well as they could be. #1 is out as that is not the primary mission of cryo providers, although I agree with the arguments on your blog that they could add some value here too. #3 is also basically out, because gains over M22 seem unlikely to come in the near future, at least outside of 21CM.

That leaves #2. A number of the papers you sent me study animal models of hemorrhagic shock, and the results are not pretty for preservation of cellular structure. For example, the amount of necrotic cells in Ozkan et al’s paper is pretty high–up to 50% necrotic in the temporal lobe, after just 3 hours. The natural question is: if a cell undergoes necrosis, has it irretrievably lost the information coded in its cellular state? The answer is unclear. On one hand, it may be possible to reverse engineer the process of cell degradation from the surviving clues and thus recover the position of crucial membrane receptors and/or neurites. On the other hand, if the degradation process is random enough, that may not be the case. Probably it depends on the specifics — “cell necrosis” is a broad class.

A number of the other papers look at the acceptability of donors who died of cardiac death. It seems that kidneys can last up to 4 hr’s of warm ischemia with similar function post-transplant, while lungs following can hardly withstand 15 mins of warm ischemia time and still offer good function post-transplant. Meanwhile, it is practically common knowledge that the organ which is least able to survive following ischemic time is the brain. Finally, there is regional susceptibility variation within the brain, and there are reasons to think that regions like CA1 that may be especially important for identity (i.e., memory) are especially vulnerable to ischemia.

To me, this emphasized how quick the interventions must be and how essential it is to maximize the time period during which oxygen perfusion in the brain is high. There’s no reason why neurons have to be able to withstand lack of oxygen for long before randomly decaying — evolution has little reason to select for it. It is a bias of operating on human timescales to think that not much can happen within five minutes, but molecular timescales unfold much faster.

You also sent a few papers that evaluated measures to query brain activity via EEG. You seem to have a particular interest in one EEG-derived algorithm called the Bispectral Index, which in a few fascinating cases actually went to zero in the absence of cerebral blood flow during surgery. These are interesting in part because they could potentially be used to monitor CBF in cryo patients.

Which brings me to the major problem that we see in many of the case reports you sent me. That is, we have good reason to believe that all of them had already experienced a very low brain oxygen perfusion prior to clinical death. The signs of this are many, and include the hyperventilation of A2435 and A2361, the terrible peripheral perfusion of A1556, the hypotension and fluid loss of A1614, ACS9577′s poor perfusion and very low coma scale score, and the long periods of apnea and low blood pressure of A2420. One of the papers that you sent me called the period after removal from life support and cardiac death the “agonal phase”, and this phrase has been aptly used in cryonics to describe the period during which a patient is known to be eminently terminal but has not yet reached cardiac death.

One key question is whether these patients are ever in fact technically brain dead, meaning no neural activity at all, as measured by EEG or CT. If they are, then it is possible that clinical death could be pronounced and preservation techniques could be started much sooner. When I first thought of this, I was hopeful that I had discovered your “problem.” But on further contemplation I’m not so sure, in part because it seems like people would have thought of this. So, I am going with the more obvious, and indeed in some senses more troubling, problem that many or most cryonics patients experience torrents of brain damage during their agonal period.

What to do about this?

1) Somehow establish, in the US, legal recognition of the rights of cryo patients to initiate procedures to preserve brain-encoded identity when the patient is diagnosed by independent physicians to be terminal, in a similar way that organ transplants are.

2) Use a workaround by going to a country like Switzerland that already allows assisted suicide in such cases, perform the cryopreservation there, and then ship the patients back on dry ice to the US.

3) #2, except establish a new storage facility in the foreign country.

4) Develop, drawing off of the “normal” biomedical literature, substantially improved methods for preserving brain oxygen perfusion in agonal cryonics patients, and implement these on a routine basis.

One of the interesting things about this problem is that it is not specific to cryopreservation but would also apply to plastination, and may even be more pronounced there. So this is one area where progress, if any is made on either front, would certainly be synergistic.

A meta thought of mine about this assignment is that I didn’t like the assumption that I would be able to diagnose problems and suggest solutions so quickly to a problem that many people have spent lots of time thinking about. I doubt that what I have written above is at all novel.

Still, I did find it to be a very worthwhile exercise to learn about some details of cryopreservation and its associated medical concepts, and for that, I thank you for offering it to me.

——————————————————————————————————

I want to extend a sincere thank you to all who participated in this exercise, and especially to Alexander McLin, Mark Plus, Gerald Monroe, Jordan Sparks, DMD, and “Synaptic” for publicly participating. It takes an enormous amount of courage to undertake such an exercise on the Internet, where it both is and will remain open to public scrutiny, more or less indefinitely. Congratulations gentlemen, you have my unreserved admiration for your courage and for your willingness to take a personal risk in pursuit of the truth. — MD

Footnote

When you give the answer to a question over and over again and it is not understood, perhaps not even perceived, and the question gets asked repeatedly, you know you’ve got a communication problem. I suppose the classic example is a friend, a family member or colleague who keep asking the same question repeatedly, but either can’t hear, or don’t want to hear the answer.

It’s a frustrating situation, because it raises another question that often has no answer; “How do I parse my answer or give the information in a way that will be understood?” The cliché answer to that question, and one my mother frequently gave is, “That’s something they’re just going to have to figure out for themselves; you can lead a horse to water, but you can’t make him drink.”

Over the past six months or so, I’ve been doing an experiment. I confess that I’m surprised that the first part of that experiment has worked as well as it has. What the experiment consisted of was asking a cross section of people in cryonics to whom I have personal access (correspondents, queries for information, old cryonics friends…) to take something I call “The Cryonics Intelligence Test.” My expectation was that few, if any, would participate. I was thus gratified when 10 people out of 12 agreed to take the test. Of those, 9 completed it. The results were fascinating – at least to me – and they convinced me that, as a prelude to discharging another obligation I have relating to Chronosphere, that I should offer the test to all and sundry who are willing to take it.

You needn’t be concerned about  your “performance”; this is an instance where anonymity on Chronosphere is permitted. If you like, you can submit your answers using a pseudononymous name and email address. If someone out there knows how to format the test to Survey Monkey, or some similar anonymous data gathering engine, please contact me and I’ll work with you to set it up (contact me at m2darwin@aol.com).

The test itself consists to of two parts: a simple introductory letter with the two test questions and a file of resource materials which must be evaluated in order to answer the two questions. The answers will necessarily be essay style and expositive.

You can submit your answers to either the Comments section of this post (here on Chronosphere), or to me directly at m2darwin@aol.com. Obviously, if you submit to the Comments section, your answers will be published. If you submit to me, they will be held in confidence, unless permission is granted from you, in writing, to post them. Privately submitted answers, and the fact that the individual participated in the Test will not be circulated, either privately or publicly, without the prior written consent of the participant, although statistical data obtained as a result will be used at my discretion.

I will be commenting on the issues raised by the answers to the test extensively in the near future.

The test is below, and should you choose to take it, I offer both my thanks and good luck.

  Cryonics Intelligence Test

Dear ______,

If you can figure out the scientific take home message for cryonics in what is to follow, you will have demonstrated extraordinary insight into “thinking in a cryonics-medical context.”

You will also have the tool to be able to understand why I believe that cryonics must, on a purely scientific-medical basis, be pursued in a fundamentally different way, both biomedically and socially.

The Test: The test resource materials are available for download at http://www.yousendit.com/download/M3BsQndOR0ZsMHhjR05Vag , you will find a number of full text peer reviewed scientific papers. In addition, you will be sent several cryopatient case Hxs. Together, these resources contain data which should give a reasonably intelligent person with a properly prepared mind a fundamentally new insight into a major, indeed overwhelming flaw in how cryonics has been, and currently is practiced.

Your task is to:

a) identify the problem(s)

b) identify one or more possible solutions

You have 5 days to complete this task. Your response should be in the form of a succinct statement of the problem, and an itemization, and if you like, a discussion of possible solutions.

Thanks for your patience and cooperation.

Mike Darwin

Screening for the Risk of Deanimation

The term “screening” is used in medicine to describe routine examinations or diagnostic procedures of a defined group of individuals to identify diseases or risk factors for same at an early stage. Screening is usually categorized as a  “preventive medical examination” or a  “checkup,” and its aim is to increase the life expectancy of those examined  by reducing the incidence or severity of life threatening disease and enhancing the quality of life. The most accurate examination methods possible should be used to identify as many diseases as possible still in their non-symptomatic phase, so that early treatment or change in life style can be initiated.

It is critically important to understand that the purpose of a “deanimation screening scan” (DSS) is not primarily to interfere with the course of disease or to extend the duration of life during this life cycle. Rather, it is to predict or to warn of impending  deanimation with increased accuracy and precision. Any contemporary medical or health benefits are thus incidental. Indeed, it is precisely when DSSing is used to determine or influence current medical interventions that it becomes dangerous. Knowing when you are likely to deanimate with greater precision, for sole purpose of improving your cryopreservation, carries little if any risk of iatrogenesis beyond that which would be present if you found out you were dying at a later time, or didn’t find out and suddenly collapsed in cardiac arrest from a heart attack, or suffered a massive stroke. It is only when the course of treatment is altered by obtaining the data, or looking at it (see “The Black Box of the Baseline,” below) that DSSing becomes either a practical or an ethical conundrum.

The first problem we confront in a screening test for deanimation risk is that we are moving in completely uncharted waters. We have no benchmarks or baselines on which to structure our screening program, save for a modest number of pilot programs that have been undertaken to evaluate full body scanning as a primary tool for the detection of cancer and atherosclerosis in the general population, or in selected subpopulations. For now, these will have to serve as the basis for our protocols, as well as the important cautionary lessons learned from other screening programs.

For reasons of safety, (see Radiation & Risk, below) Magnetic Resonance Imaging (MRI) is preferred over Computerized Tomography (CT), because no ionizing radiation is employed in making the image. MRI has some important limitations at this time, most notably only a few centers have devices that image the coronary vessels with sufficient precision  to allow risk  assessment for coronary artery disease (CAD).  Similarly, screening for Alzheimer’s Disease (AD),(beta amyloid deposits) also requires CT-PET scanning and the associated exposure to ionizing radiation.  So, for the present, CT is the only way to screen for CAD and AD. For this reason, and for those who for economic reasons may need to use CT imaging, it is worthwhile to briefly discuss the much hyped “risks” of radiation from whole body CT scans and this is done in some detail below.

Figure 25: Typical finding in an elderly woman who under prophylactic full body MRI scanning during a clinical trial in Germany to determine if full body scanning would reduce morbidity and mortality from cardiovascular disease and cancer. (Gohde, et al.)

A specimen imaging protocol is presented as Appendix 1 and is taken from the study by Gohde, et al., “Prevention without radiation – a strategy for comprehensive early detection using magnetic resonance imaging,” which was itself a pilot study in the use of MRI as a screening tool for cancer and cardiovascular disease.

The Mechanics

Currently, there is only one way to get a  DSS and that is to do it yourself.  There are several reasons, which will be discussed directly, why that is not a good idea, or certainly not the ideal way  to pursue DSSing. There are a number of reasons for this, starting with the potential for harm. Primum non nocere is the first dictum in medicine: first do no harm. Information is the most powerful force in the universe and information concerning you own health and welfare is especially important. It is also information that you cannot be objective about. It just isn’t possible. It is for this reason that no good physician treats himself or his immediate family in life or death matters as the sole or usually even the primary caregiver. In fact, speaking from experience as a person knowledgeable in medicine, I have found that wise counsel and advice I can (and do) easily give to others  is strangely absent from my own ears when I am the patient.

This lack of objectivity is more than a nuisance, it can be truly dangerous; and here I will have recourse to an actual example. The first four people to undergo DSSing have done so over the past 11 months. These were all individuals who were over 60 and who had not had consistent (or recent) “physicals.” All were counseled about the dangers of VOMIT and about the negative psychological impact of potentially finding out “something was wrong.” All four individuals had significant anomalies on their scans – two of which were life threatening and these were (or are) being medically managed.

In the other two cases, the scans revealed anomalies that might merit further medical evaluation in testing, and in both cases, the decision was wisely made not to pursue those tests. Why? That’s a complicated question, and I’ll answer it by explaining the circumstances of one of these people:

Mr. Ling is an 82 year old man who is in excellent health. He is physically active, mentally sharp and still working part time in his profession of many years.  He underwent a DSS five months ago. The findings were, overall, very good. His coronary calcium score was roughly a third lower than expected for his age, he had no signs of neoplasms, or of peripheral or central atherosclerosis, and the only abnormal cardiovascular finding was evidence of mitral valve regurgitation, which was deemed not serious and not likely to progress rapidly. However, a number of nodules were found in his right lung, along with some enlarged lymph nodes. The radiologist who reviewed the scan suggested a possible biopsy, with or without “bronchoalveolar lavage” (BAL).

While Mr. Ling is in good health, he is an 82 year old man and BAL requires sedation with propofol or a similar drug, and carries with it the risk of significant complications.  As to a CT-guided needle biopsy of the lung masses or the lymph nodes, this is this discussion that took place between Mr. Ling and the radiologist who interpreted his scan: “OK, let’s consider what this could be? I’m not sick – never felt better, so it’s not TB or something infectious? And if it’s cancer, well, what kind of treatment options would I have at my age for lung cancer with lymph node involvement?”

Those were great questions, and as it turned out, the radiologist was only playing it safe – he doesn’t want to get sued if Mr. Ling finds out he has cancer and a lawyer says to  a jury, “The doctor who imaged him said, ‘You’re in you 80s, I see this kind of thing all the time. Don’t worry about it.”  The radiologist ended by noting, “Since you are planning on following up in a year with another scan, we’ll see if anything has changed then.” And Mr. Ling is fortunate to have sufficient financial means that if he wants to pop in for a scan two months later, he can do that, too.

The problem is, most people aren’t in Mr. Ling’s position, and many will be unable to reason their way past the information that they have “masses” or “lumps” in their lungs and “enlarged lymph nodes in their chests!” That kind of worry cannot only be expensive, it can be damaging to one’s health, and corrosive to one’s quality of life. The information from DSSing should be given in the proper context, in the proper way, by the proper people, with the proper knowledge.  Absent that, it can do real harm. And if the scan does reveal a grave or untreatable medical condition, then there is all the more reason for the person to have the necessary resources at hand to help him cope and plan.

Ideally, this program would be part of a comprehensive Member Survival Program (MSP) administered by the cryonics organization (CO) and there would be a staff person whose job it would be to maintain communications with members, encourage compliance with MSP protocols (including the preferred imaging protocol) and collect and manage the resulting data stream.

Under such a scheme, upon intake (approval of cryopreservation arrangements) all members would have (at their option) completed a comprehensive health history and demographic information questionnaire, most of which would be completed as part of their membership application. The data from this questionnaire, as well as any electronic medical records the member may choose to provide, would be entered into the CO’s comprehensive member data base. The availability of this data would then allow for downstream refinement of the “one size fits all” scan protocol being proposed here, by allowing for individual risk assessment for CVD and cancer. This would flag members at elevated risk of early onset of these diseases to consider commencing scanning surveillance at an earlier age.

The Schrödinger Scan: the Black Box of The Baseline

Unless otherwise indicated, the first (baseline) scan would be done at age 45 for men and age 50 for women. In order to completely avoid any deleterious negative psychological effects, as well any potentially harmful effects from VOMIT (as discussed above), the baseline scan remains blinded and unexamined for 1 year after it is made. This done by providing written instructions to the radiologist reviewing the scan to seal the report unless there are unequivocal findings of life threatening pathology.

At the end of the year long blind period, the scan is examined and any anomalies noted. If the member chooses, a repeat scan can be done to resolve any questions or concerns raised by the baseline imaging. For example, if what appears to be a suspicious mass or nodule was found, a rescan a year later will very likely disclose if it is a neoplasm e.g., it will have grown or spread). It may seem counter intuitive to not look at data which you have paid for, experienced inconvenience to get, and which “might” save your life, but that is the necessary price that must be paid for this intervention to be used safely.

The baseline scan must be regarded as the first part of something that will not “happen,” or be completed for another year – like a bulb that has been planted to bloom in the spring, or a bond that will not mature for another 12  months. The scan itself is only a part of the process: the necessary information to safely interpret it does not appear until the required interval of time has elapsed. After all, before this protocol was proposed, no one ever got scanned and they felt just fine about it (until they dropped over in cardiac arrest).  For those of a quantum bent, consider it an extended version of Schrödinger’s famous experiment, except instead of the cat in the box, it’s a CAT scan in the box.

Scan Intervals & Exceptions

If the baseline is “negative,” showing no evidence of evolving pathological processes that merit intervention or further monitoring, then it is being proposed that the next scan take place 5 years later. Similarly, with each subsequent negative “healthy” scan, the next scan would be 5 years hence until age 81, at which point scans would be done every 2 years until cryopreservation ensues.

Figure 26: Proposed algorithm for Deanimation Screening Scan intervals and actions.

These scan intervals are arbitrary and will no doubt need to be refined over time as experience is gained. Intuitively, it seems that there should be a relationship between scan intervals and increasing age, and it is possible to configure scan intervals based on things like increasing risk of SCA or terminal illness with age. However, until some real world experience is gained, a conservative approach which minimizes costs and maximizes the opportunity for benefit, seems best. There are lots of programmers, mathematicians and similarly qualified people in cryonics and if any are interested in working with me, I am interested in generating scan interval algorithms based on the rising risk of disease and death with age (if you are interested, contact me at m2darwin@aol.com)

Going it Alone?

If a decision is made to proceed with DSSing on an individual basis, there are a number of important things to keep in mind and to do:

* Do consider carefully the possible impact this decision will have on you and on your family. In fact, give some thought to discussing this with your spouse or significant other before moving ahead.

* Do select a good imaging center with competent and caring staff who can give you good counsel about the procedure and the results. Imaging centers that offer full body scans are often used to counseling patients: make sure the one you select is a good one. Talk with the staff about your concerns before you commit to being imaged.

* Do explain to the radiologist who will interpret your images that you are having a baseline scan done and you only want to know if there is unequivocal pathology present that requires immediate or urgent medical intervention. If you can’t get that assurance from him, ask for your results only in writing on the same disk on which your scan is written.

* Don’t look at your scan or the written report that accompanies it. If you have a reliable and willing CO, send a copy to them and ask them to send you the results a year from when they receive the media with the images and the report on it. Duplicate CDs are typically made and given upon request at no charge, or for a small fee at the time you are imaged, or when you come for your results. Bring your own media to save money!

* Do provide a copy of the disk with the scan on it to your medical surrogate and to anyone who is on you ICE (in case of emergency) contact list on your mobile phone. The reason for doing so is that, should you experience SCA during the blinded waiting period, the scan may still save you from autopsy if it documents the presence of CAD, or some other pathology that could have caused your sudden and unexpected deanimation.

* Don’t  rely on the DSS to keep you out of trouble, or to reassure that everything is OK, should you develop serious health concerns. Just because a scan shows no indication of pathology does not necessarily mean that there is none. If you have signs or symptoms that would have prompted medical attention absent scanning, act on them in the same way after scanning. Let your physician decide if the scan is significant in the context of any illness or concerns.

* Don’t forget that the scan intervals are 5 years and that is more than enough time for serious disease to develop. Indeed, the 5 year window is a long one, especially where cancer is concerned. A DSS is not a health promotion or a disease prevention program. It’s primary purpose is to let you know you are terminally ill, not to assist you in avoiding that eventuality.

* Do know that if you have atherosclerosis, “vasculopathy” and you want to monitor progression of the disease, your scan intervals will have to be much shorter than 5 years – probably 6 months to 1 year, depending upon the severity, your response to medical intervention, and so on.

Economies of Scale?

Medical imaging is a highly competitive, non-monolithic industry consisting of many operators, large and small, both independent and institutionally affiliated. Such market environments inevitably encourage the drive to survive, and thus typically offer the discriminating consumer the opportunity for real bargains. I made a number of calls to imaging centers around the US and discussed the possibility of group discounts and “scan plans” wherein members of an organization or group, even just a group of like minded individuals, could get deep discounts on scans. The majority of centers I spoke with were receptive to this idea, and several discussed specific numbers which were anywhere from 20% to 60% lower than their standard walk-in fee.

Thus, it should be possible for groups of cryonicists in a given geographical area to make arrangements with a local imaging center for scans. The same was also true when I inquired about group or institutional discounts for carotid and abdominal ultrasound screenings, with the difference being that in some cases, prices went from ~ $350 per screen to ~ $60 per screen, providing the group could be scheduled for the same time and place.

The Pre-Cryopreservation Baseline CT Scan

Figure 27: A hypothetical pre- and post-cryopreservation  CT cerebral angiogram. The post-perfusion image would be obtained by administering radiocontrast agent(s) into the perfusate immediately, or shortly before discontinuing cryoprotective perfusion, prior to deep cooling to storage temperature.

If it is at all possible, a final vital CT scan of the head (at least) should be done as close to the time of cryopreservation as possible. This scan should be done with contrast and with no concerns about clinical radiation dose limitations, since the member will be terminal. The objective of this scan is to document, in as much detail (highest resolution) possible, the morphology of the brain and its vasculature. The imaging technique used should be one that optimizes resolution of the cerebral angiogram. The reason for making these images is that they should allow for many important determinations about the quality of initial stabilization and cryoprotective perfusion and cryoprotectant distribution in the brain to be made, at leisure, during the period the patient is in storage.

If contrast agent(s) is injected into the perfusion circuit shortly, or immediately prior to the discontinuation of perfusion, it should be possible to obtain a post-vitrification angiogram, which in turn should allow for evaluation of cerebrovascular patency, as well as assist in determining the anatomical landmarks within the cryopreserved tissue. It should also be possible to add other kinds of tracers to the perfusate, which might allow for quantification of regional distribution of cryoprotectants, or of other molecular species of interest not only within the brain vasculature, but within the brain parenchyma, as well. Again, the presence of a baseline pre-cryopreservation scan will likely be of great importance in allowing accurate interpretation of post-cryopreservation images.

This scan must be a CT, as opposed to an MRI, since MRI scans are unobtainable in deep hypothermia, or in the solid state.

Radiation & Risk

When the mass media talk about the “risks” from radiation associated with CT scanning, the first question that should spring to mind is, “Risks to who?” Sensitivity to ionizing radiation varies based on the cell age and mitotic cycle, and what this means in practical terms is that the younger you are, the greater the risk radiation presents to you.  Children thus have a much higher relative risk when compared to adults due to their rapid cell division and cell differentiation rate.

Figure 28: The risk of developing cancer as a result of radiation exposure is strongly age dependent and decays dramatically as people age. By the time an individual is in his 60s, 70s or 80s, the risk of neoplastic disease from medical imaging becomes negligible. Adapted from ICRP Publication 60 (1990).
 

These data also demonstrate that you cannot simply use the average relative risk shown in Table 1 to estimate the increased incidence of cancer due to radiation exposure. In order to do this analysis correctly, you need take into consideration the age of all individuals in the irradiated group. For instance, a man of 80 has a life expectancy of about 8 years, versus 33 years for a man of 45. Thus the risk to individuals over the age of 70 is, for all practical purposes, essentially nil. Table 2 illustrates what the  American College of Radiology considers minimal to high radiation doses in “absolute” terms.

This is why there is an increase in the relative risk values for the “entire population”  if children are included in that evaluation. However, even a quick glance at Figure 28 (above), where the estimated lifetime risk that radiation will result in cancer (carcinogenesis) is presented relative to the person’s age, shows that children have a 10% – 15% lifetime risk from radiation exposure, while individuals over the age of 60 have minimal to no risk (due to the latency period for cancer and the person’s life expectancy).  The accepted latency period is, by the way ~ 10 years.

Table 1 shows the relative risk of developing cancer per sievert (Sv) unit of radiation exposure. Tables 3 and 4 provide some comparison benchmarks of radiation exposure both in relative terms (low, medium, high) and in terms of common, specific medical imaging procedures used in regional CT.

So, let’s put this information in the context of a cryonicist wishing to reduce his risk of unexpected deanimation. The protocol being proposed here assumes a baseline scan at age 45 for males (50 for females) which, if free of any indication of ongoing morbid processes, is to  be repeated in 5 years, at age 51. If than scan is negative, subsequent scans would be performed at intervals of 5 years (if negative) until age 81, at which time the scan interval would decrease to 2 years. If we assume a lifetime cancer risk of approximately 1 in 1000 and a total of 7 scans  until age 81, at which point any further risk from radiation exposure becomes irrelevant, we might expect to see an increase in the lifetime risk of cancer from approximate 33% to 34%.  Even if the number of scans were more than doubled to 20; one per two years during the interval between age 50 and age 80, the lifetime risk of cancer would increase at most to ~ 35%.[1] This of course, assumes that all DSSs are CT, as opposed to MRI.

These risk calculations are based on the linear no-threshold (LNT) model of radiation risk.  This model assumes that the carcinogenicity of radiation is proportional to dose, even down to the lowest levels.  No one really knows how carcinogenic low-dose radiation is, because the carcinogenicity of low doses is so small that it’s practically impossible to measure. The official position of the Health Physics Society is that quantitative estimates of risk for doses below 50 mSv per year (100 mSv lifetime) cannot be made.[2]

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As useful aside, if you are interested in the progress being made in medical imaging, I would highly recommend the blog Magnetic Resonance Imaging: To See and Be Amazed: http://limpeter-mriblog.blogspot.com/ The site contains many beautiful images and is a treasure trove of information on both the mainstream progress, and the esoterica of MRI

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End of Part 4

(ischemia) ~85% of the time!

By Mike Darwin

Removing a Central Objection to Cryonics

In case you missed it, what I just said in that slim paragraph at the end of the preceding part of this article has profound implication because it has the potential to remove what is unarguably one of  the largest and the most rational objections that there are to cryonics. That objection is that roughly two-thirds of those who have made cryonics arrangements will not be cryopreserved under good conditions, and that half of all those signed up will be cryopreserved under very adverse conditions, such as autopsy or long (greater than 12 hours) post cardiac arrest delay. The recent advances in non-invasive medical imaging I’m about to discuss here offer the opportunity to we cryonicists to make many, if not most such losses all but unnecessary.

Figure 17: False color CT 3-D reconstruction of a patient’s intracranial arterial vascular tree. The orange-red, cheery shaped anomaly behind the right eye is a large aneurysm. The brain and other intracranial soft tissues have been digitally subtracted to facilitate a complete and unobstructed view of the patient’s arterial vasculature.

The image that you see in Figure 17 is now a perfectly pedestrian medical image that can be obtained from a garden variety CT scanner available at most diagnostic imaging centers in mid-sized cities anywhere in the world. This particular image has the brain, the soft tissue and everything digitally subtracted from it but the patient’s arterial tree and skull. The cherry shaped protrusion on the right is an aneurysm which, if were to rupture, could cost the patient his life or leave him profoundly disabled.

Figure 18: Many brain aneurysms can be treated non-surgically by passing a very thin platinum wire within the aneurysm where the wire coils up to form a yarn-like ball inside the weakened, ballooned-out area of the vessel wall. A clot subsequently forms around the coil and the vessel eventually closes off the opening to what was once the aneurysm.

Fortunately, there is a procedure  called “coiling” (Figure 18) which allows most such aneurysms to be successfully treated. Sadly, very people with brain aneurysms know that they have one until it ruptures – by which time it is almost always too late treat it effectively.

Scan Your Troubles Away?

The question logically arises, “Why not look inside everyone’s head if we have the technology to do so? Wouldn’t that allow us to identify not only the people who have aneurysms they don’t know about, but also everyone who has a tumor, or a narrowed coronary or carotid artery, or a gallstone, or anything else wrong with them that they don’t know about? In fact, why not scan their whole bodies and see if anything is amiss? Wouldn’t that allow us to nip most slowly progressing degenerative diseases in the bud?”

The answer to that question is a qualified “Yes and no.” The first and most important qualification to consider is the very substantial difference between them and us. They are going to die and, hopefully, we are not. Once you are content to die, it doesn’t really make a great difference exactly how it happens and it certainly doesn’t make any difference what happens to you afterwards. They will pay exactly nothing to avoid laying around dead for x-hours, or to avoid being autopsied. We, on the other hand, will pay something. That is a huge divide, because, as it turns out, the first and greatest barrier to such universal screening using CT and/or MRI is its adverse cost to benefit ratio.

Figure 19:  The rapid advance of computing and the high demand for ever more sophisticated medical images has driven the cost of 3-D CT and MRI scanning down to ~ $200 for a head scan $800 for a whole body scan.  http://www.superiorbodyscan.com/?gclid=CP_d5Neyiq8CFWwGRQodsHQX-w

While there are many CT and MRI machines, they are kept adequately busy, or perhaps just a little less busy than some of their owners would like, imaging sick and the worried well or hypochondriacal people. If the entire population, or even some modest fraction of it were to suddenly present for imaging, the system would crash. CT and MRI machines are very expensive and while the cost of scans has dropped dramatically, they are still not free. On the macro-level, governments, insurance companies and economists are constantly struggling to determine which therapeutic and diagnostic interventions offer the best return for the money invested in them.

The Problems of Bite Back and VOMIT

Surprisingly, information obtained from diagnostic tests can sometimes not only fail to yield any benefit, in which the case the money spent on the test is wasted, they can also cause harm. A recent example of this, much in the news, is the Prostate Specific Antigen (PSA) test used as a screening tool for prostate cancer (Figure 20). (http://www.pbs.org/newshour/rundown/2011/10/psa-testing-controversy-reignites-over-screening-debate.html) The problem with the PSA test as a screening tool is that to be effective in that capacity it requires a fairly long baseline, a good deal of contextual information (the patient’s race, family history, medications, and so on) and it requires good clinical judgment as well as a ‘patient’ patient.

Figure 20: It was anticipated that the PSA test, used as a screening tool for prostate cancer, would significantly reduce both the morbidity and mortality from the disease. It has so far failed to do so.

A single high PSA reading, or even several, may mean nothing. Most often it is the trend, rather than the absolute number; this is particularly true for black men.  In short, it’s a test that takes a lot of time and thought to interpret and use well and as such is probably not well suited to mass screening where a “yes” or “no” answer is sought before proceeding to costly, invasive and possibly injurious further evaluation.  Yet another problem is that even when prostate cancer is found and treated, it turns out that very few lives are saved because most of those cancers are slow growing and in men who will die of something else before the cancer kills them. Thus, the cost to benefit ratio of the PSA is being questioned, not the least of which because it causes many men to suffer and even die from treatments from which they did not benefit!

This is very much where medicine is today with respect to the “medical imaging singularity.” While it is possible to “look inside” just about everybody, the cost to benefit ratio for the health care system and for the “man on the street” would not justify it. In fact, it would be a medical catastrophe.

To understand why this is so it is necessary to understand three things. The first and most important of these is something called VOMIT, which is a very serious form of bite back associated with our new found ability to see inside patients with increasing exactitude. VOMIT stands for Victim of Medical Imaging Technology and refers to patients who suffer unnecessary interventions for abnormalities observed by imaging or other investigational technology, but which were not found during surgery or subsequent invasive diagnostic interventions. (Hayward, 2003) Here, I will go further and extend the definition of VOMIT to include any diagnostic finding which result in a diagnostic or therapeutic intervention which is not cost effective or causes harm to the patient. That is a very important caveat and tall order to fill, as we shall soon see.

The second is the relatively straightforward one of the ratio of the dollar benefit of resources expended to dollar benefit returned in years of productive life saved as a result of the intervention. Even in cases where early diagnosis saves lives, such as in breast cancer screening, the economic returns are equivocal. It is also often the case that “early” diagnosis with existing imaging technology is still not early enough to cure the disease. As a result, the patient suffers a longer, more miserable course of treatment and the healthcare system is subjected to greater expense with no return.

The third is the problem of information overload and it is somewhat related to VOMIT. The truism that a picture is worth a thousand words is probably a vast understatement. A single 3-D medical image contains a vast wealth of information – information which has heretofore been unavailable to both the clinician and his patient.  This might seem like a good thing, and in the long run it will be, but for now, and for a long while to come the details of the landscapes being revealed will, to a great extent, be terra incognito.

The Danger of TMI

When advances in microelectronics allowed for 24-hour ECG monitoring in the 1970s,  it became possible for clinicians for the first time to see the beat by beat electrical activity of their patients’ hearts for up to a day at a time, or longer. Prior to that, they were limited by the enormous quantities of paper tracings that would be required and the need to confine the patient to the clinic or laboratory. Now, with the advent of the compact and mobile “Holter monitor,” it was possible to capture the patient’s ECG data continuously under ambulatory, real-world conditions (Figure 21). Physicians were awash in a veritable sea-tide of data!

Figure 21: The Model 445 Mini-Holter Recorder which was released in 1976 allowed clinicians for the first time to “see” their patients’ ECGs under real-world conditions and for prolonged periods of time.

The problem was , they assumed, quite understandably, that they knew what it all meant. After all, doctors had been looking at patients’ ECGs for decades in their offices, in hospitals, at bedsides in homes and in physiology laboratories. They knew how to read  an ECG! So, when they discovered that some of their patients had periodic bouts or “runs” of very worrisome arrhythmias, they did the prudent and rational thing – they treated them for these arrhythmias with medications. Unfortunately, the result was the opposite of that expected; a significant increase in morbidity and mortality in these patients, because it turns out that in a subpopulation of healthy people, those arrhythmias were benign and not indicative of any health problem.  Thus, misinterpretation of the “same” information they were confident in dealing with in small chunks, presented in bulk and in a different context, was one of the unforeseen and arguably unforeseeable bite back consequences of Holter monitoring technology. (Harrison, 1978)

The Last Heart Attack?

If you assemble and then read over the Alcor case summaries of the last 40 years it is impossible not to be shocked by the seemingly high incidence of sudden and unexpected cardiac arrests. Because my data set is incomplete for Alcor, I can’t be definitive, but the number seems to be somewhat higher than for the same subpopulation of people from the general population (white, middle class, etc). Until, that is, you consider that most cryonicists are male. So, as you read accounts of cryonicists in their 40s and 50s arresting while scuba diving, while taking a nap or watching television, in part what you are seeing is selection bias at work. The point is, no one ever died of “sudden heart disease” a “sudden aneurysm” or, for that matter “a sudden cancer.” These are degenerative disease that takes years to decades to develop. While still difficult to detect in their nascent stages, their terminal lesions are usually very visible many months and sometimes for even for many years before they end lives.

Figure 22: Coronary artery calcium scoring using computed tomography and carotid intima media thickness and plaque using B-mode ultrasonography offer the prospect of detecting almost all coronary artery disease before it reaches the stage where it can cause a heart attack or sudden cardiac arrest.

There has been a great deal of media attention lately to an initiative called SHAPE; The Society for Heart Attack Prevention and Eradication,  which aims to all but eliminate heart attacks by combining CT of the heart to obtain a “myocardial calcium score” (a powerful risk predictor of heart attack)(Figure 22) and carotid intima media thickness and plaque using B-mode ultrasonography as part of a three step program to eliminate heart disease. The next two steps in SHAPE’s plan are a “polypill” combination of blood pressure and anti-atherosclerosis drugs and finally, perhaps, a vaccine. A similar “Last Heart Attack in America” initiative focused on coronary scanning along with dietary interventions to reverse atherosclerosis has been the focus of a feature length documentary on CNN in which former US President Bill Clinton is prominently  featured as a spokesman and advocate. The common ground of these two initiatives is that almost no one dies of a heart attack without there being  glaring evidence present in their hearts years before the infarct occurs. It is only necessary to look for it!

There can be no question that as imaging technology evolves, and as medical acumen catches up with what is available, that such imaging will become a routine part of any checkup  for patients whose age and risk profile merit it (and eventually, if they live long enough, that means most patients). As it stands right now, if you are a middle aged man or woman with a significant risk profile for heart disease, and you have a heart attack, it’s my personal opinion you have ample grounds to sue your physician for negligence.  Right now, that’s just my opinion, so it doesn’t count for anything, but the point is that sooner or later this, or a better coronary imaging modality is going to become the standard of care and heart attacks will become a rare event – a thing of the past – a relic from a time when doctors couldn’t see inside of you.

Ultrasound Investigations

There are cheaper, simpler and completely risk free ways (in terms of radiation) to  find out whether you have atherosclerosis or not.  The most predictive of these for money is the carotid ultrasound (CUS) test.

Figure 23: The carotid ultrasound scan is  a simple, non-invasive diagnostic investigation that employs sound waves to create an image of the two large blood vessels in the neck that supply most of the blood to the brain. If there is a buildup of plaque or a thickening of the limning of these two arteries the person is at increased risk of stroke and there is a high probability that there is also systemic atherosclerosis present. If there is evidence of severe narrowing of one or both of the vessels, then it becomes urgent that medication and possibly surgery be used to correct the condition in order to avoid the likelihood of a crippling or lethal stroke.

This simple, non-invasive test takes just a few minutes and uses ultrasound waves to image the carotid arteries and the blood flowing through them (Figure 23). If there is thickening of the arterial wall, or plaque present, then it is a virtual certainty that the person has systemic atherosclerosis and warrants a more extensive workup. This test is often also “packaged”  with a quick “look-see” at the abdominal aorta also using ultrasound, to rule out the possibility of an abdominal aortic aneurysm – something that is more common in smokers once they reach middle age, and beyond.

If you shop around diligently, the cost a CUS can be as little as your transportation costs to the health fare or community center where it is being offered, often as a “loss leader” by health care providers or medical imaging companies seeking more remunerative business opportunities (if they find something amiss during the CUS).  The cost of such an evaluation can range from as little as $60, to as much as $380.

A CUS is ideal for people on a budget and for those under age 45 with no history of heart disease, cancer or other pathology or risk factors that might put them at increased risk of sudden cardiac arrest.

Why Full Body Scans?

Figure 24: The full body CT or MRI scan is often offered as “add-on” to the complete or the “executive’s” physical. Many imaging centers offer these scans without the need of the patient’s person physician prescribing the scan using their in-house radiologists to write the order for the test. http://www.prevenium.com/contact.asp

 Put simply, there is no substitute for seeing, or to put a new twist on an old adage: a picture is worth a thousand medical tests. While the origins of all of the degenerative diseases that kill us are at the molecular level, mostly we die as a consequence of the macro-level changes they inflict on our bodies, even if the coup de gras is rooted in the action of things like adhesion molecules and inflammatory pathways; as is the case with most heart attacks. It is the large, easily “seen” bulges of aneurysms, masses of plaque or tumor that kill, and these almost always take years to develop. What this means practically is that, with a few exceptions, aside from suicide, homicide and accident, virtually no one has to die – or to deanimate without plenty of advance warming. The implications for cryonics are as obvious as they are profound.

End of Part 3

Left Ventricle

Figure 43: The myofibrils of each cardiac muscle cell are branched and contain a single nucleus. The branches interlock with those of adjacent fibers by adherens junctions which act to prevent scission of the cardiomycytes during the high-shear, forceful contractions of the heart. The muscle is richly supplied with mitochondria which are largely confined to the spaces between the fibrils. The fibrils are covered with a membrane, the Sarcolemma, which is frequently invaginated to form the Transverse tubules. These invaginations of the plasma membrane or sarcolemma, are called transverse tubules and they reach deep into the myofibrils and  bring the action potential deep into the fibers. Specialized intercellular junctions, the Intercalated discs, facilitate rapid transmission of the electrical signals which initiate myocyte contraction. The myofibrils are formed by myosin and actin fibers aligned in a distinct pattern which is visible under light microscopy as the A-, H- and I- bands.

      Yajima stain was used to prepare the Control (Figure 44), FGP  and FIG cardiac tissue for light microscopy. The FGP cardiac muscle showed increased interstitial space, probably indicative of interstitial edema. In many areas the sarcolemma appeared to be separated from the cytoplasm of the myocyte and, occasionally, appeared to have disintegrated into debris in the interstitial spaces (Figure 45). The myofilaments appeared maximally relaxed with widened I-bands . The mitochondria were grossly swollen and contained numerous amorphous matrix densities. The sarcolemma was fragmented beneath an intact basement membrane and there was increased space between the capillary endothelium/basement membrane and intact areas of the sarcolemma of the cardiomyocytes. The cell nuclei  were unremarkable.

Figure 44: Control-1, Left  Ventricle, Yajima, 100x. Control cardiac muscle demonstrated crisp, well defined membranes and the normal density and pattern of myofibril structure. Capillary endothelium appeared intact and the capillary basement membrane was well anchored to adjacent myocytes and appeared intact.

Figure 45: FGP-1 Left Ventricle, Yajima, 100x. In the FGP animals the myocardium exhibited increased interstitial space (IIS) as well as the presence of debris in the IIS which appeared to be disrupted sarcolemma (yellow arrows). The capillary basement membrane was often observed to be separated from the sarcolemma of the adjacent myocytes and endothelial cell nuclei were sometimes observed devoid of plasma membranes or cytoplasm (red arrow).The occasional naked myocyte nucleus could also be observed (green arrow).

The same changes were also present in the FIG group with the added presence of a “ragged” or rough appearance of the myofibrils where they were silhouetted against interstitial space (Figure 46). There also appeared to be holes or spaces, possibly as a result of edema, in the fabric of the myofibrils that were not present in the myocardium of either the control, or the FGP animals.

Most surprising was the general absence of contraction band necrosis in the FIG group, possibly as a consequence of the protective effect of reasonably prompt post-cardiac arrest refrigeration. No microscopic evidence of fracturing, either gross or microscopic, was noted in the myocardium of either the FGP, or  the FIG groups.

Figure 46: FIG-2 Left Ventricle, Yajima, 100x. Separation and fragmentation of the sarcolemma were observed in the FIG myocardium to a greater extent than that seen the in myocardium from the FGP animals (yellow arrow). Additionally, the fibers of myofibrils had a more ragged appearance and consistently displayed open spaces in the bands which were not seen in the myocardium of either the Control or the FGP animals (red arrows).

 Figure 47: The myofibrils of both the FGP and FIG animals appeared maximally relaxed with a marked increase in the thickness of the I-band. Intact red blood cells (RBCs) were observed in the FIG animals and represent incomplete blood washout (red cell trapping) despite perfusion with large volumes of washout, cryoprotectant and fixative solution (~8-10 L) over a time course of ~140 minutes of perfusion.

Cerebral Cortex

 Figure 48: The cerebral cortex consists of six distinct layers, beginning with the first layer, the Molecular Layer (Stratum zonale), which consists of finely branched medullated and non-medullated nerve fibers. The molecular layer is largely devoid of neuronal cell bodies. Those neuronal cell bodies which are present are the cells of Cajal which possess irregular cell bodies and typically have four or five  dendrite that terminate within the molecular layer and a long nerve fiber process, or neuraxon, which runs parallel to the surface of the cortical convolutions.

 The second layer of the cortex consists of a layer of small Pyramidal cells with the apices of the pyramids being directed towards the surface of the cortex. The apex of the small Pyramidal cells terminates in a dendron, which reaches into the molecular layer, giving off several collateral horizontal branches. The final branches in the molecular layer take a direction parallel to the surface. Smaller dendrites arise from the lateral and basal surfaces of these cells, but do not extend far from the body of the cell. The neuronal axon (neuraxon) always arises from the base of the small Pyramidal cells and passes towards the central white matter, thus forming one of the nerve-fibers of the white matter. In its path, the neuraxon gives off a number of collaterals at right angles, which are distributed to the adjacent grey matter.

The third cortical layer consists of Pyramidal neurons which are characterized by the presence of cells of the same type as those of the preceding layer, but of a larger size. The nerve-fiber process becomes a medullated  fiber of the white matter.

 The fourth layer is comprised of  Polymorphous neurons which  are irregular in outline and give off several dendrites which branch into the surrounding grey matter. The neuraxons of the Polymorphous neurons give off a number of collaterals, and then become a nerve-fiber of the central white matter. Scattered through these three layers are the cells of Golgi, whose neuraxon divides immediately with the divisions terminating in the immediate vicinity of the Polymorphous neuron cell-bodies. Some cells are also found in which the neuraxon, instead of extending into the white matter of the brain, passes towards the surface of the cortex; these are called cells of Martinotti.

 The fifth cortical layer contains the largest pyramidal neurons which send outputs to the brain stem and spinal cord and comprise the the pyramidal tract. Layer 5 is particularly well-developed in the motor cortex.

 Layer 6 consists of pyramidal neurons and neurons with spindle-shaped cell bodies. Most cortical outputs leading to the thalamus originate in layer 6, whereas most outputs to other subcortical nuclei originate in layer 5.

The cortical blood supply is via the pia mater which overlies the cerebral hemispheres.

Bodian stain was used to prepare the control, FGP, and FIG brain tissue  samples for light microscopy. Three striking changes were apparent in FGP cerebral cortex histology: 1) marked  dehydration of both cells and cell nuclei, 2) the presence of  tears or cuts at intervals of 10 to 30 microns throughout the tissue on a variable basis (some areas were spared while others were heavily lesioned), and 3) the increased presence (over Control) of irregular, empty spaces in the neuropil as well as the occasional presence of large peri-capillary spaces (Figures 54,56, and 57). These  changes were fairly uniform throughout both the molecular layer and the second layer of the cerebral cortex. Changes in the white matter paralleled those in the cortex, with the notable exception that dehydration appeared to be more pronounced (Figure 55).

Other than the  above changes, both gray and white matter histology appeared remarkably intact, and only careful inspection could distinguish it  from control (Figures 52, 58, 59 and 60). The  neuropil appeared normal (aside from the aforementioned holes and tears) and many long  axons and collaterals could be observed traversing the field. Cell membranes appeared crisp, and apart from appearing dehydrated, neuronal architecture  appeared comparable to control. Similarly, staining was comparable to that observed in Control  cerebral  cortex. Cell-to-cell connections appeared largely intact.

The histological appearance of FIG brain differed from that  of FGP animals in that ischemic changes such as the presence of pyknotic and fractured nuclei were much in evidence and cavities and tears in the neuropil appeared somewhat more frequently. The white matter of the FIG animals presented a macerated appearance, in addition to exhibiting the rips or tears observed in the white matter of the FGP brains (Figure 61).

Both FGP and FIG brains  presented occasional  evidence of microscopic fractures.

Figure 49: Control-1, 1st (molecular) cell layer, cerebral cortex, Bodian, 40x. Cells of Cajal (N) and a dense weave of axons (A) are visible. The tissue is perforated by numerous capillaries (C) and a  small venue containing many red blood cells (RBCs).

Figure 50: Control-1, 2nd cell layer, cerebral cortex, Bodian, 40x, showing a pyramidal neuron (N, lower left) multiple capillaries (C) and the interwoven connections of dendrites that comprise the neuropil.

Figure 51: Control-1, white matter, cerebral cortex, Bodian, 40x. Myelinated axons (MA) appear both in cross section (yellow arrows) and laterally (green arrows). Unmyelinated axons are present inside the black circle. Glial cell nuclei (GN) are scattered throughout the tissue.

 Figure 52: FGP-1, Cerebral Cortex, 1st cell layer, Bodian, 40x. Two large capillaries (LC) are present, one with a red blood cell present (right). Neurons (N, cells of Cajal) are present in normal density and the neuropil appears intact. This section appears indistinguishable from that of the Control animal.

Figure 53: FGP-1, Cerebral Cortex, 2nd cell layer, Bodian, 40x. This area of FGP cerebral cortex shows injury typical of that seen in both FGP and FIG animals. There are a number of large tears in the neuropil (red arrows) approximately 10 to 30 microns across. A pyramidal neuron is present in the lower left of the micrograph and it appears somewhat dehydrated. There are a number of naked glial cell nuclei (yellow arrows), as well some nuclei with what appears to adherent cytoplasm visible at the margins of the tears in the neuropil.  

Figure 54: FGP-1, Cerebral Cortex, 2nd cell layer, Bodian, 40x. In this area of the 2nd layer of the cerebral cortex the neuropil presents a somewhat “moth eaten” appearance, with numerous tears and vacuoles in evidence (red arrows). One large tear appears to be a pericapillary ice hole (yellow arrow).

Figure 55: FGP-3, Cerebral Cortex, white matter, Bodian, 100x. There are numerous open spaces in the white matter that appear to be ice holes (red arrows). The density of the tissue appears markedly increased over that of the Control white matter, possibly as a result of glycerol-induced dehydration. This apparent dehydration is also evident in the increased density of the axoplasm seen in the myelinated axons (green arrows).

Figure 56: FIG-3, Cerebral Cortex, 1st cell layer, Bodian, 40x. Extraordinarily normal appearing Molecular layer of the FIG cerebral cortex. The neuropil appears intact with the exception of what appear to be scattered tears or ice holes (red arrows).

Figure 57: FIG-2, Cerebral Cortex, 1st cell layer, Bodian, 40x. Large tears are evident (red arrows) and naked glial cell nuclei and fragmented cytoplasm are apparent (nn). Several intact capillaries are in evidence (C) as well as what appears to be two capillaries that have been separated from the neuropil and appear largely surrounded by open (pericapillary) space (green arrows). A mass of debris appears to occupy some of the luminal space of what appears to have been a capillary (Cd).

Figure 58: FIG-2, Cerebral Cortex, 2nd cell layer, Bodian, 40x. Remarkably intact neuropil with several capillaries, including several capillaries sectioned oblique to the plane of the tissue (OC). A neuron (N) with what appears to be a crisp plasma membrane is present at the upper right of the micrograph.

Figure 59: FIG-2, Cerebral Cortex, 2nd cell layer, Bodian, 40x.Normal appearing cerebral cortex in an FIG animal. There are multiple intact neurons with normal appearing dendrites (D) and axons (A). An intact large capillary (LC) is present and appears free of red cells.

Figure 60: FIG-2, Cerebral Cortex, 2nd cell layer, neuropil, Bodian, 100x. Normal appearing layer 2 of the cerebral cortex with intact neurons (N), axons (A), and neuropil. A capillary (C)with intact endothelial cells and an endothelial cell nucleus (EN) is also visible (left, center).

Figure 61: FIG-2, Cerebral Cortex, white matter, Bodian, 40x. Severely injured white matter typical of that seen in FIG animals. The tissue presents a macerated appearance (black circles) with numerous rips and tears, possibly as a result of ice formation (red arrows). The capillaries (C) are separated from the tissue parenchyma (yellow arrow) and what appears to be a naked endothelial cell nuclei projected into the intraluminal space of one capillary (green arrow).

END OF PART 3

Histology was evaluated in two animals each from the FIG and FIGP groups, and in one control animal.  Only brain histology was evaluated in the straight-frozen control animal.

Liver

      The histological appearance of the liver in all three groups  of animals was  one  of  profound injury.  Even in the FGP group, the cellular integrity of the liver appeared grossly disrupted.  In liver tissue prepared using Yajima stain, the sinusoids and spaces of Disse were filled with flocculent debris, and it was often  difficult or impossible to  discern cell membranes (Figures 30-32). The collagenous  supporting structures of the bile canaliculi were in evidence and the nuclei of the hepatocytes appeared to have survived with few alterations evident at the light level, although frequent pyknotic nuclei were noted in the FIGP group (Figures 31 & 34).  Indeed, the nuclei often appeared to be floating in a sea of amorphous material (Figure 34).  Not surprisingly, the density of  staining of the cytoplasmic material was noticeably reduced over that  of  the fixative-perfused control. Few intact capillaries were noted.

FGP  liver  tissue prepared with PAS stain  exhibited  a  similar degree  of disruption (Figure 32).  However, quite remarkably, the borders of  the hepatocytes  were defined by a clear margin between  glycogen granule containing cytoplasm  and non-glycogen containing membrane or other material (membrane debris?) which failed to stain with Yajima due to gross physical disruption, or altered tissue chemistry (Figure 35).

Figure 28: Control-1 Liver, Yajima, 100x. Liver sections from the Control animal demonstrated normal morphology as can be seen in the image above.

Figure 29: Control-1 Liver, PAS, 100x. Liver sections were prepared with both Yajima and PAS stain in order to allow visualization of structures that neither stain discloses alone; in this case, most importantly, the presence or glycogen granules in the hepatocytes of the Control animal. Note the presence of normal intralobular architecture with crisp cell membranes in evidence, normal appearing sinusoid spaces, and residual sinusoidal red blood cells (RBCs) not washed out during fixative perfusion.

Figure 30: FGP-1 Liver, Yajima, 100x. The livers of FGP animals demonstrated extensive histological disruption. The sinusoids were all but obliterated and appeared filled with debris (ds) and the cytoplasm was extensively vacuolated (v). 

Figure 31: FIG-2 Liver, Yajima, 100x. As was the case with the FGP animals, the sinusoids were barely discernable and appeared filled with cellular debris (cd). In addition to extensive cytoplasmic (cv) and nuclear vacuolization (nv), pyknotic nuclei (pn) were also present. Cell membranes were difficult to discern and in many areas, frank cell lysis appears to have occurred with flocculent cellular debris (cd) appearing to fill the sinusoids.

Figure 32: FGP-1, Liver, PAS, 100x. The intensely red-stained granules present in the cytoplasm of the hepatocytes are glycogen deposits selectively stained by PAS. There is extensive cytoplasmic (cv) and nuclear vacuolization (nv) and the sinusoids appear filled with flocculent cellular debris (d). Indeed, it is only possible to discern the outlines of the original individual hepatocytes from the pattern of the intracellular glycogen granules disclosed by the PAS stain.

Figure 33: FIG-2, Liver, Yajima, 100x, well preserved area. While the bulk of the hepatic parenchyma exhibited the severe injury seen in Figures 30-32, there were frequently observed islands of comparatively well preserved tissue visible in both the FGP and FIGP sections suggesting that freezing injury is occurring non-homogenously.

Figure 34: FIG-2, Liver, PAS, 100x, necrotic area. There were patchy areas of frank necrosis visible in the livers of the FIGP animals that were not present in the livers of the FGP animals. This area, adjacent to  a central vein, shows extensive cell lysis with heavy vacuolization of the cytoplasm (v) and many pyknotic nuclei (pn) in evidence.

Figure 35: FIG-2, Liver, PAS, 100x. Note the presence of a few scattered glycogen granules (GG). Interestingly, in this comparatively well preserved area of FIGP liver it is possible to see some remaining deposits of glycogen that were not consumed during the long post-arrest ischemic interval. The absence of pyknotic nuclei and the relative absence of large intracellular vacuoles is also remarkable.

 Kidney

Figure 36: The functional unit of the kidney is the nephron, consisting of the glomerulus and the uriniferous tubule ( the renal corpuscle: a).The capillary tuft of the nephron, the glomerulus, is enclosed within a double cell layered structure; Bowman’s capsule. Bowman’s capsule and the capillary tuft it encloses comprise the glomerulus. Bowman’s capsule and the glomerular capillary tuft constitute the renal (or Malpighian) corpuscle (b).

PAS  stain  was used to prepare the control, FGP and  FIGP  renal tissue for light microscopy.  The histological appearance of FGP renal tissue was surprisingly good (Figures 329, 40 & 41).  The glomeruli and tubules  appeared grossly intact  and stain uptake was normal.  However,  a  number  of alterations  from  the appearance of the control were  apparent.  The capillary tuft of the glomeruli appeared swollen and the normal  space between the capillary tuft and Bowman’s capsule was absent.  There was also marked interstitial edema, and marked cellular edema as evidenced by the obliteration of the tubule lumen by cellular edema.

By contrast, the renal cortex of the FIGP animals, when  compared to  either  the control or the FGP group, showed a  profound  loss  of detail, absent intercellular space, and altered staining (Figures 40 & 42). The  tissue appeared frankly necrotic, with numerous pyknotic nuclei and  numerous large  vacuoles  which peppered the cells.  One  striking  difference between FGP and FIGP renal cortex was that the capillaries, which were largely  obliterated in the FGP animals, were consistently  spared  in the FIGP animals. Indeed, the only extracellular space in evidence in this  preparation was the narrowed lumen of the  capillaries,  grossly reduced in size apparently as a consequence of cellular edema.

Both ischemic and non-ischemic sections showed occasional evidence of  fracturing, with fractures crossing and severing tubule cells  and glomeruli (Figure 41).

Figure 37: Control, Renal Cortex, PAS 40x. Three glumeruli are present (G) adjacent to crisp, well defined proximal (P) and distal (D) convoluted tubules. The intertubular capillaries (C) show normal diameter with lumens free of red cells or debris. There is normal capsular space between Bowman’s capsule (BC, yellow arrows) and the glomerular capillary tuft and vascular pole (VP) are also normal in appearance.

Figure 38: Control-1, PAS 40x. Collecting ducts (CD), distal tubules (D) and a glomerulus  (G) are present in 6this micrograph of renal apical column. At right, a glomerulus is present with normal Bowman’s space (BS) and the macula densa (MD) in evidence.

Figure 39: FGP-1, Renal Cortex, PAS, 40x. The intertubular space (ITS) is great expanded and the tubule cells are heavily vacoulated (V) and lack definition. The intratubular space (IS) is no longer in evidence and the architecture of the glomerluar capillary tuft (GT) is radically altered and there is an absence of the normal architecture of Bowman’s space (yellow arrows). The intertubular capillaries appear to have been reduced to debris (D) visible in the intertubular spaces.

 Figure 40: FIG-2 Renal Cortex, PAS, 40x. There is massive cellular edema present with almost complete obliteration of Bowman’s space. The tubule (T) lumens are no longer visible and the tubule cells are extensively vacoulated with many pyknotic nuceli in evidence. Individual tubular cell membranes are impossible to resolve. The afferent glomerular arteriole (AA) appears intact (red arrow).

Figure 41: FIG-2 Renal Cortex, fracture present (arrows), PAS, 40x. Two renal tubules, possibly a proximal and distal convoluted tubule (T) are dissected by a fracture as is the macula densa (MD) of the glomerulus (G). Remarkably, there is still a small amount of intertubular space present in this micrograph.

Figure 42: FIG-2 Renal Cortex, PAS 40x. vacuolization (black arrows) and extensive vacuolization (blue arrows) accompanied by necrotic changes, such as the frequent presence of pyknotic nuclei (red arrows).

END OF PART 2

I.    Introduction                                  

II.   Materials and Methods

III   Effects of Glycerolization

IV.  Gross Effects of Cooling to and Rewarming From -196°C

I. INTRODUCTION

The  immediate  goal  of human cryopreservation  is  to  use  current cryobiological  techniques  to  preserve the  brain  structures  which encode personal identity adequately enough to allow for  resuscitation or reconstruction of the individual should molecular nanotechnology be realized (1,2).  Aside from two previous isolated efforts (3,4)  there has  been  virtually no systematic effort to examine the  fidelity  of histological,  ultrastructural, or even gross structural  preservation of  the brain following cryopreservation in either an animal or  human model.    While  there  is  a  substantial  amount  of  indirect   and fragmentary  evidence  in the  cryobiological  literature  documenting varying  degrees  of  structural  preservation  in  a  wide  range  of mammalian tissues (5,6,7), there is little data of direct relevance to cryonics.   In particular, the focus of contemporary  cryobiology  has been   on   developing  cryopreservation  techniques   for   currently transplantable  organs,  and this has necessarily  excluded  extensive cryobiological  investigation  of  the brain, the  organ  of  critical importance to human identity and mentation.

The  principal  objective of this pilot study was to  survey  the effects of glycerolization, freezing to liquid nitrogen  temperature, and  rewarming  on  the physiology, gross  structure,  histology,  and ultrastructure of both the ischemic and non-ischemic adult cats  using a preparation protocol similar to the one then in use on human cryopreservation patients.  The non-ischemic group was given the designation Feline Glycerol Perfusion (FGP) and the ischemic group was referred to as Feline Ischemic Glycerol Perfusion (FIGP).

The work described in this paper was carried out over a  19-month period from January, 1982 through July, 1983.  The perfusate  employed in this study was one which was being used in human cryopreservation operations at that time, the composition of which is given in Table I.

The principal cryoprotectant was glycerol.

II. MATERIALS AND METHODS

Pre-perfusion Procedures

Nine adult cats weighing between 3.4 and 6.0 kg were used in this study.  The animals were divided evenly into a non-ischemic and a  24-hour mixed warm/cold ischemic group.  All animals received humane care in  compliance  with  the  “Principles  of  Laboratory  Animal   Care” formulated by the National Society for Medical Research and the “Guide for  the Care and Use of Laboratory Animals” prepared by the  National Institutes  of  Health  (NIH Publication  No.  80-23,  revised  1978).  Anesthesia   in  both  groups  was  secured  by  the   intraperitoneal administration of 40 mg/kg of sodium pentobarbital.  The animals  were then  intubated and placed on a pressure-cycled ventilator.   The  EKG was monitored throughout the procedure until cardiac arrest  occurred. Rectal and esophageal temperatures were continuously monitored  during perfusion using YSI type 401 thermistor probes.

Following placement of temperature probes, an IV was  established in  the medial foreleg vein and a drip of Lactated Ringer’s was  begun to  maintain  the  patency of the IV and  support  circulating  volume during  surgery. Premedication (prior to perfusion) consisted of  the IV  administration of 1 mg/kg of metubine iodide to inhibit  shivering during  external  and  extracorporeal cooling  and  420  IU/kg  sodium heparin  as  an anticoagulant.  Two 0.77 mm I.D.  Argyle  Medicut  15″ Sentinel line catheters with Pharmaseal K-69 stopcocks attached to the luer fittings of the catheters were placed in the right femoral artery and vein.  The catheters were connected to Gould Model P23Db  pressure transducers   and  arterial  and  venous  pressures   were   monitored throughout the course of perfusion.

Surgical Protocol

Following placement of the monitoring catheters, the animals were transferred  to a tub of crushed ice and positioned for surgery.   The chest  was shaved and a median sternotomy was performed.   The  aortic root was cleared of fat and a purse-string suture was placed,  through which  a  14-gauge  Angiocath was introduced.   The  Angiocath,  which served  as  the  arterial  perfusion cannula,  was  snared  in  place, connected  to  the  extracorporeal circuit and cleared  of  air.   The pericardium  was  opened  and tented to expose the  right  atrium.   A purse-string  suture was placed in the apex of the right atrium and  a USCI  type  1967 16 fr. venous cannula was introduced  and  snared  in place.  Back-ties were used on both the arterial and venous cannulae to secure  them and prevent accidental dislodgment during the  course  of perfusion.  Placement of cannulae is shown in Figure 1.

Figure 1: Vascular access for extracorporeal perfusion was via median sternotomy. The arterial cannula consisted of a 14-gauge  Angiocath (AC) which was placed in the aortic root (AR) and secured in place with a purse string suture. A USCI  type  1967 16 fr. venous cannula (VC) was placed in the right atrium (RA) and snared in place using 0-silk ligature and a length of Red Robinson urinary catheter (snare). The chest wound was kept open using a Weitlander retractor. The left ventricle (LV) was not vented.

  Extracorporeal Circuit

Figure 2: Cryoprotective perfusion apparatus: RR = recirculating reservoir, PMC = arterial pressure monitor and controller, MBD = micro-bubble detector, US = ultrasonic sensor, ADC = arterial drip chamber, D/0 = dialyzer/oxygenator, RP = cryoprotective ramp pump, HEX = arterial heat exchanger, 40 MFH = 40 micron filter holder, PT = arterial pressure transducer, CR = glycerol concentrate reservoir, EKG = electrocardiograph, TT = thermistor thermometer, TS = thermistor switch box, IB = ice bath, EC = electrocautery, APD = arterial pressure display.

The extracorporeal circuit (Figures 2&3) was of composed of 1/4″ and 3/8″  medical grade polyvinyl chloride tubing.  The circuit  consisted of  two  sections:  a  recirculating loop  to  which  the  animal  was connected  and a glycerol addition system.  The  recirculating  system consisted  of  a  10 liter polyethylene reservoir  positioned  atop  a magnetic  stirrer, an arterial (recirculating) roller pump,  an  Erika HPF-200  hemodialyzer which was used as a hollow fiber oxygenator  (8) (or alternatively, a Sci-Med Kolobow membrane oxygenator), a  Travenol Miniprime  pediatric  heat  exchanger, and a 40-micron  Pall  LP  1440 pediatric blood filter.  The recirculating reservoir was  continuously stirred with a 2″ Teflon-coated magnetic stir bar driven by a  Corning PC  353 magnetic stirrer.  Temperature was continuously  monitored  in the  arterial line approximately 15.2 cm from the arterial  cannula using a Sarns in-line thermistor temperature probe and YSI 42SL remote sensing  thermometer.  Glycerol concentrate was continuously added  to the recirculating system using a Drake-Willock dual raceway hemodialysis pump, while venous perfusate was concurrently withdrawn from the circuit and discarded using a second raceway in the same pump head.

Figure 3: Schematic of cryoprotective perfusion circuit.

Storage and Reuse of the Extracorporeal Circuit

After  use the circuit was flushed extensively with filtered  tap and distilled water, and then flushed and filled with 3%  formaldehyde in distilled water to prevent bacterial overgrowth.  Prior to use  the circuit was again thoroughly flushed with filtered tap water, and then with  filtered distilled water (including both blood and gas sides  of the hollow fiber dialyzer; Kolobow oxygenators were not re-used).   At the  end  of  the distilled water flush, a test for  the  presence  of residual formaldehyde was performed using Schiff’s Reagent.  Prior  to loading  of  the perfusate, the circuit was rinsed with 10  liters  of clinical  grade normal saline to remove any particulates  and  prevent osmotic dilution of the base perfusate.

Pall filters and arterial cannula were not re-used.  The  circuit was replaced after a maximum of three uses.

Preparation of Control Animals

Fixative Perfusion

Two control animals were prepared as per the above.  However, the animals  were subjected to fixation after induction of anesthesia  and placement  of cannulae.  Fixation was achieved by first perfusing  the animals   with  500  mL  of  bicarbonate-buffered  Lactated   Ringer’s containing 50 g/l hydroxyethyl starch (HES) with an average  molecular weight  of  400,000 to 500,000 supplied by  McGaw  Pharmaceuticals  of Irvine, Ca (pH adjusted to 7.4) to displace blood and facilitate  good distribution of fixative, followed immediately by perfusion of 1 liter of  modified  Karnovsky’s  fixative (Composition given  in  Table  I).  Buffered Ringers-HES perfusate and Karnovsky’s solution were  filtered through 0.2 micron filters and delivered with the same  extracorporeal circuit described above.

Immediately   following  fixative  perfusion  the  animals   were dissected and 4-5 mm thick coronal sections of organs were cut, placed in  glass screw-cap bottles, and transported, as detailed  below,  for light or electron microscopy.

Straight Frozen Non-ischemic Control

One animal was subjected to straight freezing (i.e., not  treated with   cryoprotectant).    Following  induction  of   anesthesia   and intubation  the  animal  was supported on  a  ventilator  while  being externally  cooled  in  a  crushed  ice-water  bath.   When  the   EKG documented  profound bradycardia at 26°C, the animal was  disconnected from  the  ventilator,  placed  in a  plastic  bag,  submerged  in  an isopropanol  cooling bath at -10°C, and chilled to dry ice and  liquid nitrogen  temperature  per the same protocol used for  the  other  two experimental groups as described below.

Preparation of FGP Animals

Following  placement of cannulae, FGP animals were  subjected  to total  body  washout  (TBW) by open-circuit perfusion  of  500  mL  of glycerol-free  perfusate.  The extracorporeal circuit was then  closed and constant-rate addition of glycerol-containing perfusate was begun.

Cryoprotective  perfusion continued until the target concentration  of glycerol  was reached or the supply of glycerol-concentrate  perfusate was exhausted.

Preparation of FIGP Animals

In   the  FIGP  animals,  ventilator  support  was   discontinued following anesthesia and administration of Metubine.  The endotracheal tube was clamped and the ischemic episode was considered to have begun when cardiac arrest was documented by absent EKG.

After the start of the ischemic episode the animals were  allowed to  remain on the operating table at room temperature ( 22°C to  25°C) for  a  30  minute period to simulate  the  typical  interval  between pronouncement  of legal death in a clinical environment and the  start  of  external cooling at that time.  During the 30 minute  normothermic ischemic  interval the femoral cut-down was performed  and  monitoring lines were placed in the right femoral artery and vein as per the  FGP animals.  Prior to placement, the monitoring catheters were  irrigated with normal saline, and following placement the catheters were  filled with 1000 unit/mL of sodium heparin to guard against clot  obstruction of the catheter during the post-arrest ischemic period.

Figure 4: Typical cooling curve of FIGP animals to ~1°C following cardiac arrest.

After the 30 minute normothermic ischemic period the animals were placed  in  a  1-mil polyethylene bag,  transferred  to  an  insulated container  in  which  a bed of crushed ice had  been  laid  down,  and covered  over with ice.  A typical cooling curve for a FIGP animal  is presented in Figure 4. FIGP animals were stored on ice in this fashion for a period of 24 hours, after which time they were removed from  the container and prepared for perfusion using the surgical and  perfusion protocol described above.

Perfusate

 TABLE I

Perfusate components were reagent or USP grade and were dissolved in USP grade water for injection.  Perfusate was pre-filtered through a Whatman GFB glass filter (a necessary step to remove precipitate)  and then passed through a Pall 0.2 micron filter prior to loading into the extracorporeal circuit.

Perfusion

Perfusion  of both groups of animals was begun by carrying out  a total body washout (TBW) with the base perfusate in the absence of any cryoprotective agent.  In the FGP group washout was achieved within  2 –  3 minutes of the start of open circuit asanguineous perfusion at  a flow rate of 160 to 200 mL/min and an average perfusion pressure of 40 mm Hg.   TBW  in  the  FGP  group  was  considered  complete  when  the hematocrit  was  unreadable and the venous effluent was  clear.   This typically was achieved after perfusion of 500 mL of perfusate.

Complete blood washout in the FIGP group was virtually impossible to  achieve (see “Results” below).  A decision was made prior  to  the start  of  this  study (based on  previous  clinical  experience  with ischemic human cryopreservation patients) not to allow the  arterial pressure  to  exceed  60  mm Hg for any  significant  period  of  time.  Consequently, peak flow rates obtained during both total body  washout and subsequent glycerol perfusion in the FIGP group were in the  range of 50-60 mL/min at a mean arterial pressure of 50 mm Hg.

Due to the presence of massive intravascular clotting in the FIGP animals  it  was necessary to delay placement of the  atrial  (venous) cannula (lest the drainage holes become plugged with clots) until  the large  clots present in the right heart and the superior and  inferior vena  cava  had been expressed through the atriotomy.  The  chest  was kept  relatively  clear of fluid/clots by active suction  during  this interval.   Removal  of  large clots and reasonable  clearing  of  the effluent  was usually achieved in the FIGP group after 15  minutes  of open  circuit asanguineous perfusion, following which the circuit  was closed and the introduction of glycerol was begun.

Figure 5: pH of non-ischemic Δ•▪*(FGP) and ischemic ●●● ᴑ  (FIGP) cats during cryoprotective perfusion. The FIGP animals were, as expected, profoundly acidotic with the initial arterial pH being between 6.5 and 6.6.

The  arterial pO₂ of animals in both the FGP and FIGP groups  was kept  between  600  mm Hg and 760 mm Hg throughout  TBW  and  subsequent glycerol  perfusion.  Arterial pH in the FGP animals was  between  7.1 and  7.7  and was largely a function of the degree of  diligence  with which  addition of buffer was pursued.  Arterial pH in the FIGP  group was 6.5 to 7.3.  Two of the FIGP animals were not subjected to  active buffering during perfusion and as a consequence recovery of pH to more normal  values  from the acidosis of ischemia (starting  pH  for  FIGP animals was typically 6.5 to 6.6) was not as pronounced (Figure 5).

Figure 6: Calculated versus actual increase in arterial and venous glycerol concentration in the FGP animals. Arrow indicates actual time of termination of perfusion.

Introduction  of glycerol was by constant rate addition  of  base perfusate  formulation  made up with 6M glycerol  to  a  recirculating reservoir  containing 3 liters of glycerol-free base  perfusate.   The target  terminal tissue glycerol concentration was 3M and  the  target time  course for introduction was 2 hours.  The volume of 6M  glycerol concentrate  required  to  reach  a  terminal  concentration  in   the recirculating   system  (and  thus  presumably  in  the  animal)   was calculated as follows:

Vp

Mc = ——— Mp

Vc + Vp

where

Mc = Molarity of glycerol in animal and circuit.

Mp = Molarity of glycerol concentrate.

Vc = Volume of circuit and exchangeable volume of animal.*

Vp = Volume of perfusate added.

* Assumes an exchangeable water volume of 60% of the pre-perfusion  weight of the animal.

Glycerolization  of  the FGP animals was carried out at  10°C  to 12°C.   Initial  perfusion  of FIGP animals was at  4°C  to  5°C  with warming  (facilitated  by  TBW with warmer perfusate  and  removal  of surface  ice packs) to 10°-12°C for cryoprotectant introduction.   The lower  TBW  temperature of the FIGP animals was a consequence  of  the animals  having  been refrigerated on ice for the 24  hours  preceding perfusion.

Following  termination  of the cryoprotective ramp,  the  animals were  removed  from bypass, the aortic cannula was left  in  place  to facilitate  prompt reperfusion upon rewarming, and the venous  cannula was removed and the right atrium closed.  The chest wound was  loosely closed using surgical staples.

Concurrent with closure of the chest wound, a burr hole craniotomy 3  to  5  mm in diameter was made in the right parietal  bone  of  all animals  using a high speed Dremel “hobby” drill.  The purpose of  the burr hole  was  to  allow for  post-perfusion  evaluation  of  cerebralvolume, assess the degree of blood washout in the ischemic animals and facilitate  rapid expansion of the burr hole on re-warming to allow  for the visual evaluation of post-thaw reperfusion (using dye).

The  rectal  thermistor probe used to  monitor  core  temperature during  perfusion was replaced by a copper/constantan thermocouple  at the  conclusion  of perfusion for monitoring of the  core  temperature during cooling to -79°C and -196°C.

Cooling to -79°C

Figure 7: Representative cooling curve (esophageal and rectal temperatures) of FGP and FIGP animals from ~ 10°C to ~ -79°C. The ragged curve with sharp temperature excursions and rebounds is an artifact of the manual control of temperature descent via the addition of chunks of dry ice.

Cooling  to -79°C was carried out by placing the  animals  within two 1 mil polyethylene bags and submerging them in an isopropanol bath which  had  been  pre-cooled to -10°C.   Bath  temperature  was  slowly reduced  to  -79°C  by the periodic addition of dry  ice.   A  typical cooling curve obtained in this fashion is shown in Figure 7.   Cooling was at a rate of approximately 4°C per hour.

Cooling to and Storage at -196°C

Figure 8: Animals were cooled to -196°C by immersion in liquid nitrogen (LN₂) vapor in a Linde LR-40 cryogenic dewar. When a core temperature of ~-180 to -185°C was reached, the animals were immersed in LN₂.

Following cooling to -79°C, the plastic bags used to protect  the animals  from  alcohol were removed, the animals  were  placed  inside nylon  bags with draw-string closures and were then positioned atop  a 6″ high aluminum platform in an MVE TA-60 cryogenic dewar to which 2″-3″ of liquid nitrogen had been added.  Over a period of  approximately 15  hours  the liquid nitrogen level was gradually  raised  until  the animal  was  submerged.  A typical cooling curve  to  liquid  nitrogen temperature  for animals in this study is shown in Figure 8.   Cooling rates to liquid nitrogen temperature were approximately 0.178°C per  hour.  After  cool-down  animals  were maintained in liquid  nitrogen  for  a period  of  6-8  months until being removed  and  re-warmed  for  gross structural, histological, and ultrastructural evaluation.

Re-warming

Figure 9: Rewarming of all animals was accomplished by removing the animals from LN2 and placing them in a pre-cooled box insulated with 15.2 cm of polyurethane (isocyothianate) foam to which 1.5 L of LN2 (~2 cm on the bottom of the box)  of LN2 had been added. When the core temperature of the animals reached -20°C the animals were transferred to a mechanical refrigerator at 3.4°C.

The  animals  in  both groups were re-warmed to -2°C  to  -3°C  by removing them from liquid nitrogen and placing them in a pre-cooled box insulated on all sides with a 10.2 cm thickness of Styrofoam and containing a small quantity of liquid nitrogen.  The animals were then allowed to re-warm to approximately -20°C, at which time they were transferred  to a  mechanical  refrigerator at a temperature of 8°C.   When  the  core temperature  of the animals had reached -2°C to -3°C the animals  were removed to a bed of crushed ice for dissection, examination and tissue collection  for  light and electron microscopy.  A  typical  re-warming curve is presented in Figure 9.

Modification of Protocol Due To Tissue Fracturing

After the completion of the first phase of this study  (perfusion and  cooling  to  liquid nitrogen temperature)  the  authors  had  the opportunity  to evaluate the gross and histological condition  of  the remains  of three human cryopreservation patients who  were  removed from  cryogenic  storage  and  converted  to  neuropreservation  (thus allowing  for post-arrest dissection of the body, excluding the  head) (10).  The results of this study confirmed previous, preliminary, data indicative of gross fracturing of organs and tissues in animals cooled to  and  re-warmed from -196°C.  These findings led us to  abandon  our plans  to  reperfuse  the  animals  in  this  study  with  oxygenated, substrate-containing  perfusate  (to have been  followed  by  fixative perfusion  for histological and ultrastructural evaluation) which  was to be have been undertaken in an attempt to assess post-thaw viability by  evaluation  of post-thaw oxygen consumption, glucose  uptake,  and tissue-specific enzyme release.

Re-warming  and  examination  of the first  animal  in  the  study confirmed  the presence of gross fractures in all organ systems.   The scope  and severity of these fractures resulted in disruption  of  the circulatory system, thus precluding any attempt at reperfusion as  was originally planned.

Preparation of Tissue Samples For Microscopy

Fixation

 TABLE II.

Samples of four organs were collected for subsequent histological and  ultrastructural  examination:  brain, heart,  liver  and  kidney.  Dissection  to  obtain  the tissue samples was begun as  soon  as  the animals  were  transferred to crushed ice.  The brain  was  the  first  organ  removed  for sampling.  The burr hole created at  the  start  of perfusion  was  rapidly extended to a full craniotomy  using  rongeurs (Figure  14).   The  brain was then removed en bloc to  a  shallow  pan containing  iced,  modified Karnovsky’s fixative  containing  25%  w/v glycerol  (see  Table  II  for composition)  sufficient  to  cover  it.  Slicing of the brain into 5 mm thick sections was carried out with the brain  submerged  in fixative in this manner.  At  the  conclusion  of slicing  a 1 mm section of tissue was excised from the  visual  cortex and  fixed  in a separate container for electron  microscopy.   During final  sample  preparation for electron microscopy care was  taken  to avoid  the  cut  edges  of the tissue block  in  preparing  the  Epon embedded sections.

Figure 10: The sagitally sectioned (5 mm thickness) brains of the animals were placed in a  perforated basket immersed in Karnofsky’s fixative. This assembly was placed atop a magnetic stirring table and the fixative was gently  stirred with a magnetic stirring bar.

      The  sliced  brain  was  then placed in  350  ml  of  Karnovsky’s containing  25%w/v glycerol in a special stirring apparatus  which  is illustrated  in Figure 10.  This  fixation/de-glycerolization  apparatus consisted of two plastic containers nested inside of each other atop a magnetic stirrer.  The inner container was perforated with numerous  3 mm holes and acted to protect the brain slices from the stir bar which continuously  circulated the fixative over the slices.   The  stirring reduced  the likelihood of delayed or poor fixation due to overlap  of slices  or stable zones of tissue water stratification.   (The  latter was a very real possibility owing to the high viscosity of the  25%w/v glycerol-containing Karnovsky’s.)

De-glycerolization of Samples

Figure 11: Following fixation, the tissues slices of all organs evaluated by microscopy were serially de-glycerolized using the scheme shown above. When all of the glycerol was unloaded from the tissues they were shipped in modified Karnovsky’s to outside laboratories for histological and electron microscopic imaging.

          To avoid osmotic shock all tissue samples were initially immersed in Karnovsky’s containing 25%w/v glycerol at room temperature and were subsequently  de-glycerolized  prior  to  staining  and  embedding   by stepwise    incubation    in   Karnovsky’s    containing   decreasing concentrations  of  glycerol  (see  Figure  11  for the de-glycerolization protocol).

Figure 12: Fixation and de-glycerolization set up employed to prepare tissues for subsequent microscopic examination. Karnofsky’s fixative (A) was added to the tissue slice fixation apparatus (B) and the tissue slices were then subjected to serial immersion in fixative bathing media containing progressively lower concentrations of glycerol (C) (see Figure 11).

      To  prepare  tissue sections from heart, liver,  and  kidney  for microscopy,  the  organs  were  first removed  en  bloc  to  a  beaker containing an amount of ice-cold fixative containing 25% w/v  glycerol sufficient  to cover the organ.  The organ was then removed to a  room temperature  work  surface at where 0.5 mm sections were made  with  a Stadie-Riggs microtome.  The microtome and blade were pre-wetted  with fixative,  and cut sections were irrigated from the microtome  chamber into  a beaker containing 200 ml of room-temperature fixative using  a plastic  squeeze-type  laboratory  rinse  bottle  containing  fixative solution.   Sections  were  deglycerolized using  the  same  procedure previously detailed for the other slices.

Osmication and Further Processing

At  the  conclusion  of de-glycerolization of  the  specimens  all tissues  were  separated into two groups; tissues to be  evaluated  by light microscopy, and those to be examined with transmission  electron microscopy.   Tissues for light microscopy were shipped  in  glycerol-free  modified  Karnovsky’s solution to American  Histolabs,  Inc.  in Rockville,  MD  for  paraffin  embedding,  sectioning,  mounting,  and staining.

Tissues   for  electron  microscopy  were  transported   to   the facilities  of the University of California at San Diego in  glycerol-free  Karnovsky’s at 1° to 2°C for osmication, Epon embedding, and  EM preparation of micrographs by Dr. Paul Farnsworth.

Due  to  concerns  about the osmication and  preparation  of  the material processed for electron microscopy by Farnsworth, tissues from the  same  animals  were also submitted  for  electron  microscopy  to Electronucleonics of Silver Spring, Maryland.

III. EFFECTS OF GLYCEROLIZATION

 Perfusion of FGP Animals

Blood  washout  was  rapid and complete in the  FGP  animals  and vascular  resistance  decreased  markedly  following  blood   washout.  Vascular  resistance increased steadily as the glycerol  concentration increased,  probably  as a result of the increasing viscosity  of  the perfusate.

Within   approximately  5  minutes  of  the  beginning   of   the cryoprotective ramp, bilateral ocular flaccidity was noted in the  FGP animals.   As  the perfusion proceeded, ocular  flaccidity  progressed until  the  eyes had lost approximately 30% to 50%  of  their  volume.

Gross  examination  of the eyes revealed that initial water  loss  was primarily  from the aqueous humor, with more significant  losses  from the posterior chamber of the eyes apparently not occurring until later in  the  course  of  perfusion.  Within 15 minutes  of  the  start  of glycerolization  the corneal surface became dimpled and irregular  and the eyes had developed a “caved-in” appearance.

Dehydration  was also apparent in the skin and  skeletal  muscles and  was  evidenced  by  a marked decrease  in  limb  girth,  profound muscular  rigidity,  cutaneous  wrinkling (Figure 11),  and  a  “waxy-leathery” appearance and texture to both cut skin and skeletal muscle.

Tissue water evaluations conducted on ileum, kidney, liver, lung,  and skeletal  muscle  confirmed  and  extended  the  gross   observations.

Figure 13: Cutaneous dehydration following glycerol perfusion is evidenced by washboard wrinkling of the thoraco-abdominal skin (CD). The ruffled appearance of the fur on the right foreleg (RF) is also an artifact of cutaneous dehydration. The sternotomy wound, venous cannula and the Weitlaner retractor (R) and the retractor blade (RB) holding open the chest wound are visible at the upper left of the photo.

Preliminary  observation suggest that water loss was in the  range  of 30%  to 40% in most tissues. As can be seen in Table III,  total  body water  losses  attributable  to dehydration, while  typically  not  as profound, were still in the range of 18% to 34%.  The gross appearance of  the heart suggested a similar degree of dehydration, as  evidenced by modest shrinkage and the development of a “pebbly” surface  texture and a somewhat translucent or “waxy” appearance.

TABLE III.

Figure 14: Cerebrocortical dehydration as a result of 4M glycerol perfusion. The cortical surface (CS) is retracted ~5-8 mm below the margin of the cranial bone (CB).

Examination  of  the cerebral hemispheres through the  burr  hole (Figure  14) and of the brain in the brain brainpan (Figure 19) revealed an estimated 30% to 50% reduction  in  cerebral volume,  presumably  as a result of osmotic dehydration  secondary  to glycerolization.   The cortices also had the “waxy”  amber  appearance previously observed as characteristic of glycerolized brains.

The  gross  appearance  of the kidneys,  spleen,  mesenteric  and subcutaneous  fat, pancreas, and reproductive organs  (where  present) were   unremarkable.   The  ileum  and  mesentery  appeared   somewhat dehydrated,  but  did  not  exhibit  the  waxy  appearance  that   was characteristic of muscle, skin, and brain.

Figure 15: Oxygen consumption was not apparently affected by glycerolization as can be seen in the data above from the perfusions of FGP-5 and FGP-5.

Oxygen  consumption (determined by measuring the  arterial/venous difference)  throughout  perfusion  was fairly constant  and  did  not appear to be significantly impacted by glycerolization, as can be seen Figure 12.

Perfusion of FIGP Animals

As previously noted, the ischemic animals had far lower flow rates at  the  same  perfusion  pressure as  FGP  animals  and  demonstrated incomplete  blood  washout.   Intravascular  clotting  was  serious  a barrier  to  adequate perfusion.   Post-thaw  dissection  demonstrated multiple  infarcted areas in virtually all organ systems; areas  where blood  washout  and  glycerolization were incomplete  or  absent.   In contrast  to  the even color and texture changes observed in  the  FGP animals,  the  skin of the FIGP animals  developed  multiple,  patchy, non-perfused   areas  which  were  clearly  outlined  by   surrounding, dehydrated, amber-colored glycerolized areas.

External  and internal examination of the brain and  spinal  cord revealed  surprisingly  good  blood washout  of  the  central  nervous system.  While grossly visible infarcted areas were noted, these  were relatively  few  and  were generally no larger than 2 mm to  3  mm  in diameter.   With few exceptions, the pial vessels were free  of  blood and appeared empty of gross emboli.  One striking difference which was consistently  observed  in  FIGP  animals  was  a  far  less  profound reduction  in brain volume during glycerolization (Figure  17).   This may  have  been due to a number of factors: lower flow  rates,  higher perfusion  pressures,  and the increased  capillary  permeability  and perhaps increased cellular permeability to glycerol.

Figure 16: The eye of an FGP animal following cryopreservation. The cornea has  become concave due to the glycerol-induced osmotic evacuation of the aqueous humor. The vitreous humor is completely obscured by the lens which has become white and opaque as a result of the precipitation of the crystallin proteins in the lens.

Whereas   edema   was   virtually   never   a   problem    during glycerolization  of  FGP  animals, edema was  universal  in  the  FIGP animals  after as little as 30 minutes of perfusion.  In  the  central nervous  system this edema was evidenced by a “rebound”  from  initial cerebral  shrinkage  to  frank  cerebral  edema,  with  the  cortices, restrained by the dura, often abutting or slightly projecting into the burr hole.   Marked  edema of the nictating membranes,  the  lung,  the intestines,  and  the  pancreas  was also a  uniform  finding  at  the conclusion  of cryoprotective perfusion.  The development of edema  in the central nervous system sometimes closely paralleled the  beginning of “rebound” of ocular volume and the development of ocular turgor and frank ocular edema.

Figure 17: The appearance of the brain of an FIGP animal following cryoprotective perfusion as seen through a craniotomy performed over the right temporal lobe. The cortical surface (CS) is retracted ~3-5 mm from the cranial bone (CB) and appears

In contrast to the relatively good blood washout observed in  the brain,  the  kidneys  of  FIGP animals had a  very  dark  and  mottled appearance.   While  some  areas (an estimated  20%  of  the  cortical surface) appeared to be blood-free, most of the organ remained  blood-filled throughout perfusion.  Smears of vascular fluid made from renal biopsies  which  were collected at the conclusion  of  perfusion  (for tissue  water determinations) revealed the presence of many  free  and irregularly clumped groups of crenated and normal-appearing red cells, further evidence of the incompleteness of blood washout.   Microscopic examination  of recirculating perfusate revealed some free, and a  few clumped  red  cells.   However, the concentration  was  low,  and  the perfusate  microhematocrit  was  unreadable  at  the  termination   of perfusion (i.e., less than 1%).

The  liver  of  FIGP  animals  appeared  uniformly   blood-filled throughout  perfusion,  and  did not exhibit even  the  partial  blood washout evidenced by the kidneys.  However, despite the absence of any grossly  apparent blood washout, tissue water evaluations in one  FIGP animal  were  indicative  of  osmotic dehydration  and  thus  of  some perfusion.

The mesenteric, pancreatic, splanchnic, and other small  abdominal vessels  were  largely free of blood by the conclusion  of  perfusion.  However,  blood-filled  vessels  were not  uncommon,  and  examination during   perfusion   of   mesenteric   vessels   performed   with   an ophthalmoscope  at 20x magnification revealed stasis in  many  smaller vessels, and irregularly shaped small clots or agglutinated masses  of red  cells in most of the mesenteric vessels.   Nevertheless,  despite the   presence  of  massive  intravascular  clotting,  perfusion   was possible, and significant amounts of tissue water appear to have  been exchanged for glycerol.

One  immediately  apparent difference between the  FGP  and  FIGP animals  was  the  accumulation in the lumen of  the  ileum  of  large amounts  of  perfusate  or perfusate  ultrafiltrate  by  the  ischemic animals.  Within approximately 10 minutes of the start of reperfusion, the  ileum  of the ischemic animals that had  been  laparotomized  was noticed  to  be  accumulating fluid.  By the  end  of  perfusion,  the stomach  and the small and large bowel had become massively  distended with  perfusate.   Figure  14 shows both FIGP and  FGP  ileum  at  the conclusion  of glycerol perfusion.  As can be clearly seen,  the  FIGP intestine  is markedly distended.  Gross examination of the  gut  wall was   indicative  of  tissue-wall  edema  as  well   as   intraluminal accumulation  of  fluid.  Often by the end of perfusion, the  gut  had become  so  edematous  and  distended  with  perfusate  that  it   was impossible  to completely close the laparotomy  incision.   Similarly, gross  examination of gastric mucosa revealed severe erosion with  the mucosa being very friable and frankly hemorrhagic.

Escape  of  perfusate/stomach contents from the  mouth  (purging) which occurs during perfusion in ischemically injured human suspension patients did not occur, perhaps due to greater post-arrest  competence of the gastroesophageal valve in the cat.

Oxygen  consumption  in  the two ischemic cats in  which  it  was measured  was dramatically impacted, being only 30% to 50% of  control and deteriorating throughout the course of perfusion (Figure 12).

IV. GROSS EFFECTS OF COOLING TO AND REWARMING FROM -196°C

The  most striking change noted upon thawing of the  animals  was the presence of multiple fractures in all organ systems.  As had  been previously noted in human cryopreservation patients, fracturing  was most pronounced in delicate, high flow organs which are poorly  fiber-reinforced.   An exception to this was the large arteries such as  the aorta, which were heavily fractured.

Fractures  were most serious in the brain, spleen, pancreas,  and kidney.   In these organs fractures would often completely  divide  or sever  the  organ  into one or more discrete  pieces.   Tougher,  more fiber-reinforced tissues such as myocardium, skeletal muscle, and skin were less affected by fracturing; there were fewer fractures and  they were smaller and less frequently penetrated the full thickness of  the organ.

Figure 18: All of the animals in the study exhibited fractures of the white matter that transected the brain between the cerebellum and the cerebral cortices. Similarly, the spinal cord was invariable severed by fractures in several locations and exhibited the appearance of a broken candle stick. The yellow box encloses a sampling area used to determine brain water content.

Figure 19: Deep fracture of the left occipital cortex. Note the absence oif fracturing in the adjacent skeletal muscle (M) observed in FGP-1. Note that the brain appears shrunken and retracted in the brainpan.

Figure 20: Appearance of the brain after removal from the brainpan. There is a massive fracture of thew right frontal=temporal cortex which penetrates the full thickness of the cerebral hemisphere to expose the right cerebral ventricle observed in FIGP-2. The cortex appears buff colored and gives the appearance of being incompletely washed out of blood.

Figure 21: Typical fracture sites in the brain (arrows and yellow shading). The olfactory cortices and the brainstem were invariably completely severed by fractures.

In both FGP and FIGP animals the brain was particularly  affected by  fracturing  (Figures 18, 19 & 22) and  it  was not uncommon to  find  fractures  in  the cerebral hemispheres penetrating through to the ventricles as seen  in Figure  20, or to find most of both cerebral hemispheres and the  mid-brain  completely  severed from the cerebellum by a  fracture  (Figure 18).  Similarly, the cerebellum was uniformly severed from the medulla at the foramen magnum as were the olfactory lobes, which were  usually retained  within  the olfactory fossa with severing  fractures  having occurred at about the level of the transverse ridge.  The spinal  cord was  invariably transversely fractured at intervals of 5 mm to  15  mm over  its  entire  length (Figure 21).  Bisecting CNS fractures  were  most  often observed  to  occur  transversely  rather  than  longitudinally.   In general,  roughly  cylindrical structures such as  arteries,  cerebral hemispheres, spinal cord, lungs, and so on are completely severed only by transverse fractures.  Longitudinal fractures tend to be shorter in length and shallower in depth, although there were numerous exceptions to this generalization.

Figure 22: Crisp olfactory lobe fracture which also partially penetrated the pia matter in FGG-4.

In  ischemic animals the kidney was usually grossly fractured  in one  or  two locations (Figure 25).  By  contrast,  the  well-perfused kidneys of the non-ischemic FGP group exhibited multiple fractures,  as can  be  seen in Figure 24.  A similar pattern was observed  in  other organ  systems  as well; the non-ischemic animals  experienced  greater fracturing injury than the ischemic animals, presumably as a result of the   higher   terminal  glycerol  concentrations  achieved   in   the non-ischemic group.

Figure 23: Appearance of a fractured kidney before removal of the renal capsule. The renal capsule has only one fracture, however when the capsule is removed, the extensive fracturing of the renal cortex and medulla become evident (Figure 24, below).

Figure 24: Fractured renal cortex from FGP-1 after removal of the renal capsule. The renal cortex is extensively fractured, the renal medulla slightly less so. Note the uniform, tan/light brown color of the cortex indicating complete blood washout and the absence of red cell trapping.

Cannulae  and attached stopcocks where they were externalized  on the  animals  were  also frequently  fractured.   In  particular,  the polyethylene pressure-monitoring catheters were usually fractured into many  small  pieces.   The  extensive  fracture  damage  occurring  in cannulae,  stopcocks, and catheters was almost certainly a  result  of handling  the animals after cooling to deep subzero  temperatures,  as this  kind of fracturing was not observed in these items upon  cooling to  liquid nitrogen temperature (even at moderate rates).  It is  also possible that repeated transfer of the animals after cooling to liquid nitrogen  temperature may have contributed to fracturing  of  tissues, although the occurrence of fractures in organs and bulk quantities  of water-cryoprotectant  solutions  in the absence of  handling  is  well documented in the literature (12, 13).

There were subtle post-thaw alterations in the appearance of  the tissues of all three groups of animals.  There was little if any fluid present  in the vasculature and yet the tissues exhibited  oozing  and “drip”  (similar to that observed in the muscle of frozen-thawed  meat and  seafood)  when cut.  This was most pronounced  in  the  straight-frozen  animal.  The tissues (especially in the ischemic  group)  also had  a somewhat pulpy texture on handling as contrasted with  that  of unfrozen,  glycerolized  tissues  (i.e.,  those  handled  during  pre-freezing  sampling for water content).  This was most in  evidence  by the accumulation during the course of dissection of small particles of what appeared to be tissue substance with a starchy appearance and  an oily  texture on gloves and instruments .  This phenomenon  was  never observed  when handling fresh tissue or glycerolized tissue  prior  to freezing and thawing.

There were marked differences in the color of the tissues between the three groups of animals as well.  This was most pronounced in  the straight-frozen  control  where the color of almost  every  organ  and tissue examined had undergone change.  Typically the color of  tissues in  the  straight-frozen animal was darker, and white  or  translucent tissues such as the brain or mesentery were discolored with hemoglobin released from lysed red cells.

Figure 25: The (ventral) dependent and dorsal (less dependent) surfaces of the right kidney from FIGP-1. There is extensive mottling evidencing incomplete blood washout despite perfusion with many liters of CPA solution. Fracturing is much less extensive than that observed in FGP animals not subjected to prolonged periods of post-arrest ischemia. Note the pink colored “drip” from the organ that is present on sectioning board.

Figure 26: Appearance of the kidney from FIGP-1 shown above on cross-section. The renal medulla appears congested and blood filled.

The FGP and FIGP groups did not experience the profound post-thaw changes  in tissue color experienced by the straight-frozen  controls, although  the  livers and kidneys of the FIGP  animals  appeared  very dark, even when contrasted with their pre-perfusion color as  observed in those animals laparotomized for tissue water evaluation.

END OF PART 1

Perfluorchemicals (PFCs)

Figure 3-1: Fluorine and carbon; the two building blocks of the remarkable molecules knows as the perflurochemicals (PFC)s.

Physical Chemistry and Synthesis

Perfluorchemicals (PFCs) are derived from hydrocarbons by replacing hydrogen atoms with fluorine atoms, typically using common organic hydrocarbons as substrates. This is accomplished by one of three methods; the oldest of which is via a highly exothermic vapor-phase reaction employing fluorine gas. An alternative method is the more stable cobalt trifluoride technique which was developed during the Manhattan Project in World war II (WWII) [290]. Electrochemical fluorination, developed by Simmons in1950 [291] has increasingly replaced the earlier techniques.

Ectrochemical fluorination yields more homogeneous products with less carbon-carbon bond cleavage and is better suited to smaller scale production of molecules for use in research and development applications. However, whether done by electrolysis or by using the pre-Simmons method of reaction with high-valent metal fluorides, both laboratory and industrial scale PFC manufacturing and synthesis have in the past resulted in impure and poorly characterized compounds (unless perfluorinated building blocks are employed as starters). This occurs because the large difference (~15 kcal per M^-1) in the carbon-hydrogen bond versus the carbon-fluorine bond energies (and the release of this energy during the synthetic process) results in undesired side reactions, isomerisations, and  polymerizations creating a mélange of compounds which are not fully characterized, let alone purified.[292]

PFC carbon chain length is variable, and these, in addition to the attached moieties, determine the individual properties of a given molecule. Liquid PFCs that are useful as gas and/or heat exchange media in the lungs all exploit the utterly unique properties of the carbon-fluorine (C-F) bond and the larger size of fluorine atoms compared to hydrogen atoms.  The C-F bond is the strongest covalent bond found in organic chemicals (average 485 kJ mol^-1, compared to ~413 kJ mol^-1 for a standard C-H bond)  [293] and this bond-strength is amplified as the number of fluorine atoms on each carbon atom increases.[293]

The electroattracting nature of the fluorine atoms further increases the strength of the C-C bonds and the larger size of fluorine atoms (estimated van der Waals radius of 147 versus 120 pm) [294] and their high electron density result in a compact electron shield that ensures effective protection of the molecule’s backbone. The dense electron shell of the fluorine atoms may also provide protection against nucleophilic attack. The PFCs thus have very high intra-molecular (covalent) bonding and very low intermolecular forces (van der Waals interactions).[295]

Physical Properties

These liquids are clear, colorless, odorless, non-conducting, nonflammable, and are both hydrophobic and lipophobic. Replacement of hydrogen atoms by fluorine atoms results in high thermal stability and chemical inertness. PFCs do not undergo decomposition (except at temperatures above ~350ºC) and they are not metabolized or acted upon by any enzymatic system. They are ~1.8 times as dense as water; and are capable of dissolving large amounts of physiologically necessary gases (45 to 55 ml O₂/dl and16 to 210 ml CO₂/dl).[296]  O₂ dissolution in PFCs is via O₂ occupying intermolecular sites in the liquid, unlike the pH and concentration dependent porphyrin binding in hemoglobin which is responsible for the sigmoidal oxyhemoglobin dissociation curve.[297]   O₂ solubility in PFCs is a linear function of the pO₂ (per Henry’s law) and the structure of the particular PFC.[298]  Solubility of O₂ and other gases in PFCs is a function of the NMR T1 relaxation constant which determines the intermolecular cavity size and the presence and character of large channels within the liquid. Linear aliphatic structures more easily allow the formation of such channels while planar structures result in more closely interlocked layers which accommodate less gas. While the solubility of O₂ is greater in aliphatic than in aromatic PFCs this is not the case with CO₂. O₂ solubility increases with the degree of fluorination and the O₂-dissolving capacity of aliphatic PFCs having 9-11 carbons is in the range of 40 to 60 mole fractions of O₂, or roughly twice that of aromatic molecules.

The O₂ content of perfluocarbons at 1 atmosphere (atm) of 100% O₂ is approximately 20 times that of water, twice that of blood, and 1.5 times that of an equal volume of gaseous O₂. If the PFCs are compared to blood in terms of O₂ carrying capacity under normal atmospheric conditions it is immediately obvious that PFCs carry only a fraction of the O₂ that hemoglobin does. However, it is not simply the O₂ dissolving capacity of PFCs that is important, but also their O₂ delivery capability. The O₂ diffusion rate from the PFC-filled alveoli to the alveolar capillaries is quite high when saturated with O₂ at 760 torr [299] and due to their 50% greater O₂ carrying capacity than O₂ delivered as a gas at an FiO₂ of 100%, the delivered O₂ to the blood is 25% to 50% greater than is possible using 100% gaseous O₂ for ventilation. The use of such a high FiO₂ is sustainable with PFCs because, for reasons not yet understood, O₂ toxicity does not occur in their presence as a liquid in the lung under normobaric conditions.

A remarkable and essential feature of the PFCs in liquid assisted ventilation is that they are essentially insoluble in both water (7 to 11 ppm at STP) and alcohols, and are only sparingly soluble in some lipids.[299] The PFCs have a kinematic viscosity (ratio of viscosity to density) similar to that of water [300] and an extremely low surface tension (15 to 19 dyn/cm²) and dielectric constant; again secondary to weak intermolecular forces resulting from the exterior fluorine atoms.[301] The volatility of PFCs varies widely depending upon the molecular weight of the molecule, with vapor pressures ranging from ~1 to over 80 torr at 25ºC. Vapor pressure is the critical determinant of the half-life of elimination of a given PFC from the lungs following both intrapulmonary and intravenous administration. Vapor pressure also dictates the viscosity of a given PFC and thus PFC-gas and PFC-surface shear interactions.[302],[303]  Vapor pressure is also a critical determinant of toxicity as will be discussed under the heading Toxicology, below.

Commercially Available PFCs

Historically, the requirements of a PFC for use as a gas exchange medium in liquid ventilation are excellent solubility for respiratory and putative therapeutic gases such CO, NO, and H₂S, kinematic viscosity in the range of ~ 0.50 to 2.2, low to moderate vapor pressure (1 to 15 torr) with a cutoff of 20 torr, a high spreading coefficient and a very low surface tension.[303]   Additionally, a PFC that is to be used for intrapulmonary heat exchange must have a viscosity and freezing point appropriate to the application. These criteria, particularly with respect to vapor pressure and viscosity, may change in the future, depending upon the application, medical condition being treated, or large airway diameter of the patient.

No existing PFC combines all of these desirable properties, and certainly no single PFC can be tailored to have the widely varying physical properties required for a particular pathology or patient. In addition, from a physical chemistry standpoint, no single PFC is likely to combine all these properties, even in the sphere of providing assisted ventilation in ARDS; and recent research has begun to focus not only on the creation of novel of PFCs for liquid assisted ventilation applications, but also on investigating mixtures of different PFCs to provide an optimum ventilating medium which can be formulated to meet the needs of a given application.[304]

Unfortunately, de novo flurochemical synthesis involves the use of extremely toxic and hazardous materials such as fluorine gas, hydrogen fluoride, silver difluoride, or the halogen fluorides, and yields a poor ratio of desired end product to undesired (and costly to dispose of) side-products and waste.[293] Even flurochemical synthesis using per- and poly-fluroinated reagents, the ‘building block’ approach is costly, has low selectivity for many compounds and requires formidable expertise [305], although this is changing.[306]

Once the synthesis is completed, numerous and costly purification steps such as lengthily refluxing, spinning band distillation and reparative vapor phase chromatography must be undertaken, adding greatly to the cost, and making well characterized synthesis and purification of quantities sufficient for use in liquid assisted ventilation or blood substitutes a full-time effort and the province of expert chemists and dedicated facilities.[293]  Historically, this has severely limited the development and commercial production of high purity, completely chemically characterized novel PFCs.

As a result, most of the initial work in liquid ventilation (including that done for liquid assisted pulmonary cooling (LAPC)) was carried out using commercially available PFCs such as perflurodecalin, Rimar-101™ (Miteni Corporation, Milan, Italy), or the Fluorinert™ liquids (3M Company, St. Paul, MN). Table 3, below, shows some of the physical properties of a number of commercially available PFCs used for leak testing, heat exchange and for cleaning applications in the electronics industry marked by 3M Corporation as the Fluorinert™  ‘liquids,’ as well as those of Rimar-101 and Perflubron™ (Alliance Pharmaceuticals, San Diego, CA).

A serious disadvantage to Fluorinert™ PFCs and all other industrial grade PFCs (as well as most reagent grade materials available from laboratory chemical suppliers) is that they are not chemically defined in terms of chain length or even precise chemical composition. As examples, FC-75 has been shown to have as many as 6 peaks when evaluated by gas chromatography and F-tripropylamine (FTPA), one of the components in the first FDA approved blood substitute, Flusol-DA, contained only 27% of perfluorinated FTPA in addition to a number of other uncharacterized compounds.[307]  FC-43, a PFC used extensively as an experimental oxygen carrying blood substitute and for liquid assisted ventilation is chemically ~ 85% perfluoro-tri-n-butylamine (C₁₂F₂₇) with the unit structure shown in Figure 3-2. However, the perfluoro-tri-n-butylamine molecules may be present as polymers of varying lengths, and other related fluorinated molecular species are also present.

The degree of polymerization as well as the physical properties of the species which comprise the ~15% balance of FC-84, are such that the average vapor pressure, boiling point, melting point, thermal conductivity and other physical properties of FC-84 are fairly uniform from lot-to-lot.[308],[184] However, two of the most critically important determinants of the utility of PFCs in liquid ventilation applications are their vapor pressure (and thus their viscosity) and their direct chemical toxicity. Vapor pressure is of great concern, because even if the average vapor pressure of the liquid is quite low (i.e., 1.3 torr at STP for FC-43) if even a small percentage of the species present have a far higher vapor pressure, then that fraction of the liquid can turn into a gas and create long-lasting and mechanically disruptive bubbles in lung tissue under conditions of baro- and/or volu-trauma; and will create ‘sponge rubber lung’ syndrome due to stable intra-alveolar gas bubble formation by vaporizing between surfactant and the alveolar epithelium. This phenomenon, known as hyper-inflated non-collapsible lungs (HNCL) occurs even under the relatively non-traumatic conditions of PLV if the vapor pressure is low enough; as is the case with F-alkylfuran in FC-75.[309] Similarly, the unspecified and often uncharacterized other perflurocompounds (or even incompletely fluorinated compounds) may be chemically toxic to cells.[310],[311],[292]

 Some Perfluorchemicals Used in Liquid Ventilation Research

 Table 3: Physical properties of some PFCs that have been used for liquid assisted ventilation: 3-M Fluorinert Liquids ™, Rimar-101, perflurodecalin and perflurooctylbromide (PFOB, Perflubron™). Perflubron™ is the first completely defined PFC intended for medical applications. Sources: [312],[292] ,[313].

Figure 3-2: Chemical structure of perfluoro-tri-n-butylamine (FC-43).

For these reasons a completely chemically defined molecule, perflurooctylbromide F₃(CF₂)₇Br, Perflubron,™ LiquiVent™), was developed by Alliance Pharmaceuticals of San Diego, CA  for use in clinical trials of liquid ventilation (Figure 25, below). Unfortunately, Perflubron™, (Figure 3-3) with a molecular weight (MW) of 498.97, has a freezing point of +6.0ºC which makes it unsuitable for use in inducing ultraprofound hypothermia (0-5^oC) where the temperature of the ventilating liquid must be in the range of 2ºC to 4ºC for optimum efficacy.

Toxicology

Figure 3-3: Perflurooctylbromide (LiquiVent™)

The high stability, chemical inertness, and nearly total insolubility in both water and lipids of PFCs used in liquid assisted ventilation preclude their metabolism and limit their interaction with biomolecules. Despite 40-plus years of use in biomedicine little published work has been done on the toxicology of these compounds. Based on data from the available literature their toxicity can be divided into two categories: biophysical/biomechanical and immunological. When administered intravenously or intraperitoneally as neat (pure) chemicals injury results from the biophysical interaction with the animal rather than from biochemical interactions. Because PFCs are not miscible in water they form vascular emboli in the same way that injecting intravascular oil or air would.

PFCs with higher vapor pressures can form vascular gas emboli even if emulsified [314] and can lethally distend closed body viscuses such as the peritoneum, or cause perfluorocarbon vapor pneumothoraces (‘perflurothorax’).

PFCs of intermediate vapor pressure may accumulate in the lungs and be converted to vapor which is retained for weeks or months in the alveoli or lung parenchyma resulting in what Clark, et al., termed hyperinflated non-collapsible lungs (HNCL). [309] This phenomenon is noted at necropsy after IV administration of emulsified perflurodecalin containing blood substitutes [315] and after LAPC with intermediate vapor pressure PFCs such as FC-75 or FC-77.[272]  Schutt, et al., call this ‘pulmonary alveolar gas trapping’ and they propose that the phenomenon occurs as a result of PFC liquid or vapor migration through the pulmonary surfactant-liquid bridges where it forms stable, long lasting, PFC vapor micro-bubbles. They propose that these intra-alveolar micro-bubbles are part of the ‘normal pulmonary elimination of perfluorocarbon vapor’ from the body.[316]  While HNCL or ‘pulmonary alveolar gas trapping’ may not be clinically evident, and does not perturb blood gases or interfere with gas exchange, it does interfere with normal respiratory mechanics and can cause ‘stiff lungs’ in dogs following LAPC using FC-75 or FC-77.[161],[272] Stiff lungs increase the work of breathing until the vapor dissipates enough to relieve the acute tension in the alveoli.[272]  As such, the author believes this phenomenon should properly be classified as an adverse biophysical effect, rather than an acceptable or normal mechanism of PFC elimination. It is interesting to note that in a chemical model of lung injury using inhaled kerosene even brief PLV with FC-77 increased mortality and resulted in extensive gas trapping.[317]

Depending upon the emulsion size intravascular PFCs may be phagocytized by PMNL’s and macrophages and be deposited in the reticuloendothelial system where they may cause enzyme induction or mild inflammation from their space occupying, mechanical effects distorting normal tissue architecture. [318]  These changes are typically reversible as the PFC is eliminated via the lungs and the hepatomegaly and splenomegaly dissipate (~ 3-weeks).

The immunological effects of PFCs vary with the molecule, method of preparation and particle size (if administered intravascularly). Some PFCs appear to be directly cytotoxic to PMNLs and macrophages.[319] However, as a class, the PFCs seem to interfere with PMNL and macrophage chemotaxis, activation and de-granulation without inducing apoptosis or necrosis, by mechanisms that are not understood.[320], [321],[322]  Augustin, et al., have observed that PFCs alter the cytoskeleton of hepatic macrophages in a dose dependent manner that varies with the compound. [323]  Inhibition of PMNL and macrophage chemotaxis and respiratory bursts gives the PFCs moderately potent anti-inflammatory effects and by the same token makes them immunosuppressive.  While this effect is immunomodulatory and probably beneficial in ARDS [324],[325],[326], it also has the potential to impair pulmonary and systemic immune surveillance and presumably increase the risk of infection and neoplasm. FC-43 has been used to delay neutrophil mediated xenograft rejection [327],[328] and Perflubron™ has been demonstrated to inhibit neutrophil activation in the rat heart after 2-hours of cold ischemia and whole blood reperfusion.[329]

A recently discovered novel and unexpected effect of at least one PFC, Perflubron™ [326], is direct inhibition of oxidative damage in both cultured pulmonary artery endothelial cells exposed to hydrogen peroxide and in linoleic acid micelles subjected to varying concentrations of the azo initiator 2,2’-diazo-bis-(2-amidinopropane) dihydrochloride . This result is unexpected because it has previously been presumed that the antioxidant activity of PFCs was secondary to their immunomodulating and immunosuppressive effects. The protective effect of Perflubron™ in a non-biological system raises many questions about its basic pharmacology, and possibly about the chemistry and environmental interactions of the PFCs as a class, should this effect prove replicable with similar compounds.

Environmental Impact and Future Availability

The PFCs may be justifiably described as the penultimate atmospheric (greenhouse) poison. The PFCs, like water vapor and methane, both absorb and emit long wave (infrared) radiation; effectively trapping heat from the sun and warming the terrestrial surface and atmosphere.

Unlike CO₂ and methane, PFCs are not subject to biological cycling, are unaffected by electrochemical reactions, and do not dissociate in aqueous media. They are essentially already fully oxidized and are unaffected by standard oxidizing agents such as permanganates, chromates, and the like. As previously noted, degradation via oxidation occurs only at very high temperatures. Because of their inertness, they are similarly resistant to degradation by reduction, except under extreme conditions, requiring reducing agents such as metallic sodium. This leaves photochemical decomposition, primarily via hydroxyl radical (^.OH) mediated degradation, as the only means of terrestrial disposition. Both Cicerone [330] and Yi Tang [331] have shown that the reaction of  ^.OH  with the C3 and CF4 moieties is negligible under ground-state conditions, and that the lifespan of the molecules, once they enter the atmosphere, is likely in excess of 10,000 years. The heavier, higher MW and lower vapor pressure PFCs which are ideal for liquid assisted ventilation can be expected to remain in the lower reaches of the troposphere indefinitely, and thus not reach the upper atmosphere where, however slowly, they might be photo-degraded. In any event, the PFCs are so resistant to photo-degradation that the Flourinerts, and related compounds that are used as chemically stable cooling agents in photochemical reactors, as carrier solvents for photo-decomposition of other organic molecules, and are likewise classed as ‘radiation durable compounds’ for use in the photolithography industry.[332]

At present, the PFCs constitute an insignificant contribution to greenhouse gas emissions. However, widespread medical use could change this, and in any event, 3M, DuPont and other manufactures of industrial quantities of PFCs are aggressively encouraging the use of alternative compounds which they manufacture, principally the hydrofluoroethers [333] and the perfluorinated alkyl vinyl ethers. In 1982 Riess and Le Blanc estimated that if PFC-based blood substitutes came into wide use the quantities required would be in the ‘multi-thousand-tons-per-year range’ [292] all of which would end up in the atmosphere. Widespread use of PFC-facilitated LAPC and PLV could easily require a similar amount of product.

Extensive medical use of PFCs would seem to mandate associated efforts at recovery and recycling to minimize environmental contamination. However, this is not easy to do even in hospital under controlled conditions. Recovery of PFCs used emergently for LAPC (i.e., in-field induction of hypothermia in stroke, myocardial infarction, cardiac arrest) and as the O₂ carrying molecules in blood substitutes would seem to preclude effective recovery. The indefinite lifespan of these compounds makes their use akin to radiation exposure wherein the effect is cumulatively damaging, and ultimately lethal to the biosphere (as it exists now) as a consequence of their greenhouse effects and indefinite atmospheric lifespan.

The PFCs used in liquid assisted ventilation do not seem likely to accumulate or concentrate in biota. However, they do have comparatively long dwell times in patients when used clinically, and the exposure of health care workers to these volatile compounds would seem unavoidable. In light of these facts and the recent discovery that PFCs have direct radical quenching effects (with possible important environmental ramifications), as well as immunosuppressive properties, it seems reasonable to question the future large scale production, and thus the biomedical availability of these molecules.

Section 4:

History of Liquid

Assisted Ventilation and Implications for LAPC

History of Liquid Ventilation

Figure 4-1: An ultra-deep sea diver breathing PFC liquid in the 1989 motion picture ‘The Abyss’ Directed by James Cameron. [Photo courtesy 20^th Century Fox and Lightstorm Entertainment.]

As was the case with the first great rationalization of surgery and wound management by Pare’ [352], the creation of scientific nursing by Nightingale [353], and the development of fluid resuscitation and the first effective medical management of shock by Cannon [354] and Blalock [355], the impetus for the development of liquid ventilation was also initially warfare. Interest in the use of liquid as a breathing medium in mammals originated in the early 1960s in response to the U.S. Navy’s need to develop rescue systems for submariners that would allow them to transiently breathe saline or some other aqueous liquid. Mortality among submariners in World War I (WWI) and WW II was greater than in any other branch of military service. In WWII 22% of U.S. and 75% German submariners were killed in action.[356] With the advent of nuclear submarines in 1951, and the use of submarines to carry and deliver nuclear weapons, prolonged and complex missions while continuously submerged created the need (still largely unmet) to carry out rescue of submariners, and recovery of nuclear weapons and other strategically critical materiel, from extreme depths.

Johannes Arnold Klystra, M.D. is the Dutch pulmonologist and clinical researcher responsible for developing saline lavage of the lung as a treatment for advanced cystic fibrosis in 1958.[357]  Klysta’s interests extended well beyond clinical innovation in the management of lung diseases, and in the late 1950s this maverick physician approached the Dutch Navy to explore possible ways to allow deep ocean recovery of submariners as well as the development of liquid breathing systems that would allow divers to be free from the constraints imposed by gas breathing under conditions of high pressures (Figure 4-1):

“Man has tried for centuries to invade the oceans, perhaps driven by a subconscious nostalgia for atavistic weightlessness in the vast hydrosphere that covers more than 70 per cent of the earth, but gas in his lungs, compressed by a layer of water above, confines his activities to the shallow. Nitrogen, for instance, produces a progressively severe intoxication at depths greater than100 feet and usually incapacitates a diver by ‘rapture of the deep’ at no more than300 feet. Moreover, relatively large amounts of carrier gas dissolve in blood and tissues to be released as bubbles whenever the diver returns to the surface too rapidly. These hazards are all due to the compressibility of gases. The properties of water, on the other hand, hardly change at all with pressure, and I have observed mice with fluid filled airspaces move around in no apparent distress at a simulated depth of 3000 feet. If man were able to breathe oxygenated water instead of an oxygenated carrier gas, exploration of the oceans would no longer be limited by gas toxicity and decompression sickness.”[358]

The first mammal to survive liquid breathing was a mongrel dog named ‘Snibby.’  [158] Snibby was shaved, bathed, anesthetized, intubated, and submerged in a tub of buffered salt solution in a large hyperbaric chamber at 5 atmospheres of pressure while O₂ was bubbled through the saline bath. The dog breathed the liquid, which was held at a temperature of 32ºC, for 24 minutes. As Klystra noted in his published account of the experiment: “Snibby’s recovery was uneventful and he was adopted by the officers and crew of H. M. Cerberus to serve as a mascot aboard this submarine rescue vessel of the Royal Netherlands Navy.”[359]

In 1962 Klystra documented survival of mice breathing a balanced, buffered salt solution under 8 atmospheres of pressure at 20ºC for 18-hours.[158] Throughout the 1960s Klystra and his associates probed the limits of liquid breathing using aqueous solutions and they were the first to document the problem of profound hypercarbia as a fundamental limitation in tidal liquid breathing.[157]  Klystra was also the first to demonstrate survival of mammals following extreme hyperbaria using spontaneous liquid breathing of a buffered salt solution.[357]  A further testimony to the highly creative and innovative nature of Klystra’s work was his use of the selectively liquid lavaged lung lobe as a possible replacement for the kidney; in other words, as a mass exchanger for nitrogenous wastes and as an osmotically driven ultrafilter for removal of excess water in the setting of renal failure.[360]

One of the most remarkable things about this pioneering work is that saline and other aqueous solutions denude the alveoli of surfactant, the stiff molecular cage that supports the 3-dimensional alveolar structure and keeps the acinar airways open to ventilation. Removal of surfactant is a primary cause of serious pulmonary injury and a major pathophysiological mechanism in both acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS). Through the present, saline lavage of the lungs remains a standard model for inducing pulmonary injury to simulate ALI and ARDS in experimental animals.[361],[362] The inadequate gas carrying capacity of aqueous solutions under normobaric conditions, and the inherently injurious nature of water-based ventilating media made clinical or undersea application of liquid breathing infeasible. Indeed, breathing of balanced salt solutions, even under hyperbaric conditions in the presence of 100% O₂ was only possible for extended periods of time if the animals were hypothermic. While O₂ delivery was adequate, CO₂ elimination was not, and hypercarbia and respiratory acidosis were lethal complications of liquid breathing under normothermic conditions.[363]

Figure 4-2: Chemical structure of polydimethyl(siloxane). (Image from Wikimedia Commons: http://en.wikipedia.org/wiki/Image:Silicone-3D-vdW.png.)

Shortly after the publication of Klystra’s pioneering work Leland C. Clark began looking for more suitable liquid breathing media. Cark’s initial efforts focused on using polyunsaturated vegetable oils such as corn and safflower oil. These oils proved injurious to lungs and Clark next evaluated the organosilanes octamethyltrisiloxane, dodecamethylpentasiloxane, decamethyltetrasiloxane, polydimethylcyclosiloxane, and polydimethylsiloxane. These compounds, commonly referred to as ‘silicone oils,’ were manufactured by the Dow Chemical Co. of Midland, MI in various chain lengths, and thus vapor pressures and physiochemical properties.[364]  The organosilanes are made from a Si-O backbone to which a variety of organic groups are attached to the silicon atoms via a Si-C bond. Polydimethylsiloxane is the most common of the commercially produced organosilanes and is a polymer with a backbone consisting of a repetition of the (CH₃)₂SiO unit (see Figure 4-2, above). The organosilanes proved less toxic than vegetable oils, and better able to dissolve O₂ and CO₂, but were still too toxic to be used for liquid ventilation.

The problem of an inert and non-injurious breathing medium capable of carrying enough dissolved O₂ to support life under normobaric conditions was finally solved by Leland C. Clark and Frank Gollin in 1966 with their report that a variety of fluorocarbons performed well as liquid breathing media without apparent injury to the lungs and with long-term survival of the animals.[163]

Figure 4-3: Dr.  Leland C. Clark, Jr., 1918–2005 (Photo courtesy of Richard Bindstadt – Blackstar)

The PFC identified by Clark and Gollin, a ~50/50 mixture of isomers of F-alkylfurans (FC75), was evaluated under conditions of spontaneous respiration with the animals submerged in the liquid.[309]  As proved the case with aqueous solutions, the PFCs delivered adequate amounts of O₂, but failed to allow for effective clearance of CO₂ resulting in lethal hypercarbia and acidosis. The vastly greater density and viscosity of PFCs relative to air or other gases limited the diffusion of CO₂ into the liquid. An added problem contributing to the hypercarbia observed in liquid ventilation was the vastly greater work of breathing (WOB) imposed by a liquid 1,000 times as dense as air and the increased time for exhalation in the absence of active (negative pressure) pumping of the PFC from the lungs.

Figure 4-4: Archetypical tidal liquid ventilation (TLV) system. PFC completely replaces gas in the lungs and is moved in and out of the lungs using mechanical (usually roller) pumps. After exhalation the PFC is passed through a filter, and then through an oxygenator-heat exchanger to be scrubbed of CO₂, oxygenated and warmed to body temperature before being re-infused into the lungs.

In 1970 Gordon D. Moskowitz and Thomas H. Shaffer (Figure 4-5) began work on a mechanical liquid ventilator (Figure 4-4) to overcome the problem of the increased inspiratory workload accompanying liquid breathing.[365]  During the next 5-years these investigators developed progressively more sophisticated demand-regulated liquid ventilators which also began to address the need for assisted exhalation. [366],[367],[368] During the 1980s development of a variety of tidal liquid ventilators was undertaken by Shaffer, et al., and applied to animals ranging from cats [300] to preterm and neonatal lambs [369], culminating in the first clinical application of tidal liquid ventilation (TLV) to human neonates in 1989.[370]

Figure 4-5:  Dr. Thomas H. Shaffer, MSE, PhD. (Photo Courtesy of Dr. Thomas Shaffer.)

Partial Liquid Ventilation (PLV)

From the beginning of liquid ventilation research with the work of Klystra in 1960, and continuing until the publication of the work of Furhman, et al. in 1991, only one kind of liquid ventilation existed; tidal or total liquid ventilation (TLV). As early as 1976 Shaffer, et al., had noticed that peak airway pressures were dramatically improved in pre-term lambs after they were returned to gas ventilation following 20 minutes of TLV.[368] The observation that lung mechanics and gas exchange remained transiently improved following TLV was extended by Shaffer, et al., in 1983 with the observations that pulmonary compliance and paO₂ were increased and paCO₂ was decreased during conventional gas ventilation following TLV to values lower than those that could be achieved during TLV.[300]

Bradley Furhman, an anesthesiologist and critical care physician at the University of Pittsburgh Medical School, noticed the enduring salutary effects of PLV following reinstitution of conventional gas ventilation in a model of infant respiratory distress syndrome (IRDS) using pre-term lambs, and he further noted that Shaffer, et al., had reported that these improvements in lung function did not occur following TLV in the healthy lung.[371]  Furhman hypothesized that these salutary effects of TLV might be due to possible surfactant-like and alveolar recruitment effects of the comparatively large amounts of PFC retained in the lungs following TLV, since it was well documented that even with aggressive efforts to remove PFC following TLV, a volume approximately equal to the animal’s functional residual capacity (FRC) of 30 ml/kg remained in the lungs until it was eventually eliminated by evaporation.[370]

Figure 4-6: When filled to Functional Residual Capacity (FRC) the PFC forms a meniscus in the endotracheal tube. As shown at left, FRC constitutes all the volume in the lungs and trachea at the end of exhalation. (Modified by the author from the original art at Wikimedia Commons: http://en.wikipedia.org/wiki/Image:3DScience_respiratory_labeled.jpg.)

Furhman, et al., tested this hypothesis by administering FC-77 (a perfluorinated butyl-tetrahydrofuran isomer mixture) via the endotracheal tube in a volume equal to the FRC of neonatal swine.[372]  As opposed to using TLV, conventional positive PPV was continued using the same parameters employed before the PFC was instilled into the lungs (Figure 4-6). This technique, christened partial liquid ventilation (PLV), proved as effective, or more effective, than TLV in decreasing airway resistance, as well as peak and mean airway pressures. PLV also seemingly abolished the need for PEEP in healthy lungs, while providing adequate gas exchange. This study not only established the effectiveness of PLV, it also posited (correctly) the mechanisms by which PLV was achieving restoration of gas exchange, increasing pulmonary compliance, and homogenizing ventilation in the lungs thus greatly reducing volutrauma (Figure 4-7, below).

Figure 4-7: Lungs from two rabbits subjected to a model of ischemia-reperfusion injury. The lung on the left (A) shows severe volutrauma to the upper lobe (‘baby lung’) with obvious consolidation in the ventral, dependent areas of the lung. The lung on the right (B) is from an animal treated with PLV and shows no evidence of injury and is homogenously ventilated with PFC and gas. Note that the letter A has been placed on an apical ‘baby lung.’

The investigators noted that due to its higher density than water the PFC rapidly flowed to the most dependent areas of the lungs and in so doing it filled collapsed alveoli and displaced serous transudate in flooded alveoli (Figure 4-9, below). With each gas breath, liquid from the large and medium caliber airways was admixed with ventilating O₂ under conditions of turbulent flow, thus oxygenating it and washing it of CO₂. During exhalation some, but not all of the PFC in the alveoli, flowed out into the larger airways where it was also admixed with both ventilating gas and already oxygenated PFC. During the next inspiration the alveoli were refilled with PFC that was oxygenated and cleansed of CO₂. Because PFC is retained in the lungs to FRC, and gas is present in the alveoli only as micro-bubbles, if at all, the alveoli never completely empty and thus are vastly more compliant to re-inflation.  As Furhman, et al., noted, the alveoli of the lungs appear to be ventilated almost exclusively with PFC throughout the ventilatory cycle with direct gas admixing occurring mostly in the larger airways (trachea, bronchi and bronchioles). Gas exchange between the blood and PFC most likely occurs due to direct PFC-alveolar membrane contact.

Figure 4-8: Lung volumes.

In addition to recruiting alveoli to liquid-mediated gas exchange, PLV also displaces alveolar transudate, mucus, and cellular debris by continuously lavaging the airways; macroscopic to microscopic.[373]  PLV is cytoprotective of Type II alveolar epithelial cells, decreases PMNL adhesion in pulmonary capillaries [326], reduces alveolar hemorrhage, and generally preserves alveolar ultrastructure in the setting of ALI [324] and ARDS.[326]

Also of great importance is that PLV is simple to implement; it does not require novel, complex tidal liquid ventilators with an oxygenator, heat exchanger, water trap and filters – all under complex computer control. Because there is no bulk movement of PFC over long distances of airways, the resistance to PFC flow is greatly reduced, effectively eliminating the constraint of only 5 to 7 breaths per minute in TLV.[374] Because the transit times and distances between the alveoli and the bronchioles are very small in PLV (where ventilation gas admixing and gas exchange is occurring) and because CO₂ is probably exchanged in micro-bubbles in the PFC which are far smaller than would be the case in a ‘sphere’ of PFC the diameter of the alveolus (~250µ), the problem of hypercarbia is also eliminated.[375]

Figure 4-9: A: Alveoli are flooded alveoli in ARDS or pulmonary edema. When PFC is, literally, poured down the endotracheal tube it flows under gravity (due to its ~1.8x density of water) to the most dependent areas of the lungs. B: In so doing it opens the collapsed alveoli and displaces edema fluid from them. (Modified from original art by Patrick J. Lynch, medical illustrator, and is from Wikimedia Commons.)

From 1991 to 2000, PLV was extensively investigated in a number of different animals employing a variety of models of lung injury; saline FTLV, oleic acid injury, smoke inhalation, prematurity, intestinal ischemia, lung transplantation injury, and pneumococcal pneumonia.[376], [377], [378],[379],[380],[381] These studies, with no notable exceptions, showed marked benefit for PLV in ALI and ARDS.

In 1993 Leach, et al., [164] carried out the first clinical trial of PLV in IRDS (Figure 4-10). This was followed by a number of clinical trials for ARDS in both children [382] and adults.[383]  These trials were sponsored by Alliance Pharmaceuticals, Inc. of San Diego, CA (Alliance) in an effort to obtain FDA approval for the use of Perflubron™ as the first gas exchange PFC in PLV.

In 2006, after 5 years of delay, the ‘definitive,’ Phase III, prospective, randomized clinical trial (RCT) of PLV in ARDS was published (LiquiVent™ study).[384]  For reasons that are only now being understood, this trial showed PLV (using Perflubron™ at a dose equal to FRC (30 ml/kg), and at a lower dose of 10 ml/kg, to yield a worse outcome than conventional PPV with increased overall mortality and increased days requiring mechanical ventilation: The 28-day mortality in the control group was 15%, versus 26.3% in the low-dose (p=0.06) and 19.1% in the high-dose (p = 0.39) PLV groups. There were more ventilator-free days in the control group (13.0 ± 9.3) compared with both the low-dose (7.4 ± 8.5; p=0.001) and high-dose (9.9 ± 9.1; p =0.043) groups. Most remarkably, there was a high incidence of barotrauma: 34% pneumothoraces in the phase II LiquiVent™ trial (20% in the control group) and 29% and 28% in the phase III LiquiVent™ trial (control, 9%). Pneumothoraces requiring the placement of chest tubes is an ominous complication of PPV in ALI and ARDS and is associated with increased mortality, duration of ventilator time and length of stay in the ICU. In view of the consistently diverse, positive and well conducted animal studies demonstrating unequivocal benefit, this result was surprising.

Figure 4-10: Initiation of PLV in a neonate with IRDS. The only novel piece of equipment used was a luer-loc one-valve interposed between the endotracheal tube and the 16 mm connector to the ventilator to facilitate intra-tracheal administration of Perflubron™ without having to disconnect the patient from the breathing circuit. (Photo courtesy of Alliance Pharmaceutical, Inc.)

The reasons for the failure of the Phase III LiquiVent ™ RCT are both complicated and subtle – and are still being debated today. [385] The likely reasons for the failure of the Phase III study have important implications, not only for the future of PLV in ALI and ARDS, but also for the optimum use of PLV and LAPC in emergency and critical care medicine. The reasons for PLV’s failure are rooted in difficulties and errors that have plagued translational research from animals to humans in many areas of medical research.[386]  What follows is a point-by-point evaluation of the possible causes of failure of the Phase III PLV trial as well as an analysis of the implications of these problems for LAPC.

 Unanticipated Effect of Lung Protective Ventilation Strategies

The Phase III trial was designed in 1997, begun in 1998, and completed in 2002. This was well before the first report of the efficacy of lung-protective ventilation in reducing mortality in ARDS and ALI was reported in 2000 [387] and 7 years before the first influential ARDSnet study was published.[388]  The criticality of minimizing barotrauma and volutrauma, even over maintaining gas exchange at optimum physiological levels, was thus not taken into consideration in the LiquiVent™ study design. This was especially significant because, due to subtle shortcomings in the design of most of the animal studies, it was not understood that PLV is a source of barotrauma and volutrauma; even when far less than full FRC-dosing is used under circumstances most like those encountered clinically (see ‘Failure to Establish a Dose-Response Curve,’ below).

While the LiquiVent™ study experimental group had a worse outcome than the control group, it should be noted that the absolute results were actually no better (or worse) than those reported in the previously cited ARDSnet studies validating lung protective ventilation (i.e., 15 to 26%).  This is especially worth noting because all 3 groups of the LiquiVent™ study patients were considerably sicker than the patients in the ARDSnet studies. The objective entry criteria for the LiquiVent™ study were an initial PaO₂/FiO₂ <200mmHg followed by a failed (PaO₂/FiO2 < 300 mmHg) response to a PEEP of ≥ 13 cm H₂O at a FiO₂ ≥ 0.5. By contrast, the ARDSnet patients only needed to have a PaO₂/FiO2 of < 300 mmHg with no requirements for PEEP or FiO₂ upon randomization. One possible reason for the superior outcome in the LiquiVent™ control group, compared to that seen in most other studies of ARDS at that time, is that Alliance selected only the very best centers of excellence in the management of ALI and ARDS to conduct the trials. By contrast, ARDSnet studies were also conducted on a contract basis by NIH at institutions that were more representative of the actual quality of care available at large metropolitan hospitals in the U.S. Alliance thus placed LiquiVent™, and consequently the entire field of PLV, on trial in a setting with the sickest patients receiving the best medical management for ARDS

Defective Translational Research Models

The animal models of ALI and ARDS used to evaluate PLV did not model the real-world course of lung injury in clinical illness. Researchers typically inflict an insult and then wait a uniform, and often unrealistically short time, for the injury to develop. By the time human patients in respiratory distress enter the ICU they have usually been ill for many hours, or even days, and as a consequence the degree of pulmonary compromise, and in particular, pulmonary edema, may be greater. Furthermore, animal models of lung injury are inherently more homogenous than is usually the case in human ARDS. Heterogeneity of injury is one of the hallmarks of both ALI and ARDS in humans, and heterogeneity means that normal or minimally injured areas of lung will be subjected to the same conditions as more severely injured areas (see discussion of varying requirements for PEEP depending upon the degree of injury individual alveoli under the heading, ‘The PFC Air Interface and Shear Effects in the Small Airways,’ below).

These observations have other important implications because loading to the theoretical FRC (30 ml/kg) in ill and often aged humans may not be possible, in the sense that the ‘normal’ or predicted volume of lung to be recruited may not be available. Alveoli can be filled with PFC only if there is enough room in the thorax to allow them to fill. If fluid in severely edematous lung parenchyma stubbornly resists relocation to the vascular compartment due to hypoalbuminemia and an interstitial pressure in excess of the hydrostatic force generated by the PFC, or if for other mechanical reasons there is not sufficient volume to accommodate a FRC-dose of PFC, then the result will be volutrauma and barotrauma to the less dependent lung. This possibility was noted by Cox, et al., as early as 1997 and was later raised in a study done by Lim et al., in 2000.[389]

Failure to Establish a  Dose-Response Curve

While as previously noted, a wide range of animal models, and models of injury were investigated with respect to PLV, there was little study to determine the optimum dose-response curve of either LiquiVent™ or other PFCs used in PLV. In hindsight this seems strange because in the experimental evaluation of any novel drug the first step is usually to establish a dose-response curve and thus to bound the ‘safe and effective dose.’ This was not done in PLV and those studies which documented injury from PLV in normal lungs at FRC dosing were arguably not given the attention they deserved.[390],[391]

Recently, the work of Dreyfuss and Ricard [392] has demonstrated that dosing to FRC in healthy rats actually causes alveolar capillary leak and induces lung injury. In a series of elegant studies they have examined the complex relationship between PFC dose, PEEP and Pplat. Their studies indicate that the probable ideal dose of PFC (or of Perflubron™ in this case) is ~ 3 ml/kg; 10% of FRC, and still only 30% of the 10 ml/kg dose used in the ‘low dose’ LiquiVent™ study.[393] Furthermore, these investigators have documented that FRC dosing with Perflubron™ causes gas trapping in the lungs, and that under these circumstances, paradoxically, PEEP is protective.[391]

 Gas Trapping and Selection of the Appropriate PFC

Perflubron’s™ comparatively low vapor pressure is similar to that of perflurodecalin and thus probably results in a lower spreading coefficient relative to most other PFCs used in the animal studies. This would likely result in slower dynamic flow during inspiration and in ‘plugging’ of medium caliber airways (with the previously noted effect of gas-trapping) due to high gas-fluid interfacial tension.[394],[395] These effects may contribute to barotrauma and volutrauma when Perflubron™ is administered to full FRC.[396],[397]

It now appears that if PLV is to have any chance at conventional clinical application in the West, clinical trials will have to start from scratch, quite possibly with a different PFC, or blend of PFCs, being used to achieve the pharmaco-physical properties required for the particular application – including possibly tailoring the medium to the size of the patient’s intermediate sized airways (which  are greatly different between neonates, children and adults) and to the particular application at hand. For instance, as Jeng, et al., (from Schaffer’s group) states:

“In terms of clinical application, the appropriate fluid for liquid assisted ventilation will depend on the clinical situation. For example, a more viscous fluid may be more appropriate for supporting the lung during extracorporeal membrane oxygenation during which the PFC application is aimed at preventing atelectasis and fluid flux across lung-at-rest conditions. In contrast, a less viscous fluid may be preferred for TLV during which tidal volumes of fluid are exchanged. For PLV, a fluid with low vapor pressure would reduce dosing requirements during the course of the treatment. Thus, the data presented herein further relate fluid physical properties with liquid ventilation applications.”[304]

The PFC Air Interface and Shear Effects in Small Airways

Figure 4-11: The shear- inducing effect of a single flooded alveolus on neighboring alveoli. Open alveoli (A) expand evenly in unison and experience no shear. After alveolar flooding and collapse (B) shear forces occur on the adjoining alveolar septa. PFC filled; partially filled and unfilled alveoli and other acinar structures will be subject to the same damaging shear forces as is the case when aqueous media are present in the acini.

Although the alveolar air-liquid interface is eliminated during PLV, giving PLV its PEEP and surfactant-like properties, a PFC-gas interface is created. The law of Laplace ( P = 2γ/r) describes the relationship between the pressure to stabilize an alveolus (P) and surface tension at the gas-PFC interface of an alveolus (γ) in relation to the radius of the alveolus r. [398]  In the normal lung, surfactant present at the gas-liquid interface lowers the air-liquid surface tension in lockstep with decreasing alveolar radius (to nearly 0 mN · m^-1 for low alveolar radii), thus keeping the ratio of γ/r of the alveolus constant and guaranteeing alveolar end expiratory stability at low pressures.[399],[400] By contrast, PFCs exhibit a constant gas-PFC surface tension for any alveolar radius. This means that at the end of exhalation, the alveolar radius will be quite small, while the surface tension remains unchanged, and therefore very high compared to when the alveolus is inflated. Thus, the alveoli will collapse unless they are supported by PEEP from the ventilating gas.[401]  The implication is that PFC-derived ‘liquid PEEP’ must always be balanced by precisely the right amount of ‘gas PEEP’ to prevent end-expiratory collapse of non-PFC-filled alveoli (Figure 4-11). This presents a formidable challenge in both the laboratory and the clinic.

Partial Liquid Ventilation and the Law of Laplace

Figure 4-12: In attempting to determine the extent to which PFC is likely to cause alveolar injury due to gas-liquid mediated shear forces, and variable distention of alveoli filled with PFC as opposed to gas, it is instructive to compare the dynamic behavior of PFC (red line) with surfactant (green) as well as with other liquids, such as pulmonary edema transudate (yellow) (which contains dissolved surfactant), and saline (blue line). While PFC exerts far less surface tension than saline, it still exerts a force at the air-PFC interface of ~20 dynes cm^-1, and, like saline, lacks the dynamic responsiveness to changes in surface area which are the unique property of surfactant and surfactant containing solutions.

In addition to the problem of end-tidal alveolar collapse, high shear forces at the alveolar membrane as a result of insufficient PEEP during PLV may cause alveolar rupture and consequently gas/PFC leak and the development of pneumo- and/or perflurothorces.[402]  The complexity and difficulty of this problem becomes clearer when consideration is given to the fact that the amount of ventilator-applied ‘gas PEEP’ will be a function of the pathological condition of each alveolus (which will vary widely in the same lung, let alone from patient to patient). This is so because the presence of a thin film of PFC in the alveolus will only be beneficial in alveoli where the native surfactant has failed to maintain alveolar patency (diameter).

In those compromised alveoli that are unable to reduce surface tension to the gas-PFC interfacial tension, the level of ‘gas PEEP’ required to keep them open at the end of exhalation will be lower during PLV than the level of ‘gas PEEP’ during conventional mechanical ventilation. Conversely, in alveoli with functioning surfactant, (i.e., able to reduce their surface tension to below that of the gas-PFC interface) there will be an interaction between PFC and the normal alveolar membrane fluid, leading to levels of ‘gas PEEP’ with PLV higher than those required to keep the alveoli open with ‘gas PEEP’ required in conventional ventilation alone (Figure 4-12). In the clinical milieu this implies careful titration of PFC dose during initiation and maintenance of PLV and equally careful titration of PFC ‘removal’ (i.e. evaporation) or weaning from PFC during the transition from PLV to gas-only PPV.[401]

Even assuming that the means can be developed to determine and control these parameters, the seemingly intractable problem of inhomogeneous gas distribution during tidal gas ventilation in PLV remains.  Gas will go preferentially to the least PFC liquid loaded and least dependent airways and this will likely produce over-distension and volutrauma much as happens in the edematous consolidated lung.  At a minimum it will be necessary combine fluid PEEP with pressure-controlled ventilation in a way such that the pressure in any alveolus does not exceed the pressure of the ventilating gas.[381]  Failure to prevent shear or alveolar hyperinflation will result not only in direct mechanical injury to the alveolar membrane and pulmonary capillaries, [403] but also in both local and systemic injury from pro-inflammatory cytokines whose release by alveolar epithelial cells is triggered by even modest shear stress or over-distension.[404],[405]

An additional source of shear injury in PLV and thus presumably LAPC is only now beginning to emerge. Research on VLI has predominately focused on the role of high inflation pressures and large tidal volumes.[404],[406],[407] However, ventilation at low lung volumes and pressures results in a different type of lung injury, in which airway instability leads to repetitive collapse and reopening of the terminal airways.[408]  This type of injury is relevant to LAPC and PLV because the air-PFC interface behaves similarly to the cyclically re-inflated collapsed airway. During reopening of collapsed airways a finger of air moves through the airway generating stresses on airway walls and injuring the airway epithelium.[409],[410],[411],[412]  This does not occur during normal tidal ventilation because pulmonary surfactant stabilizes the airways and prevents their collapse during exhalation.

Figure 4-13: The power of surface tension is perhaps most easily illustrated by meniscus formation at tripartite air-cylinder-liquid interface. When water wets a small diameter tube the liquid surface inside the tube forms a concave meniscus, which is a virtually spherical surface having the same radius, r, as the inside of the tube. The tube experiences a downward force of magnitude 2πrdσ.The gas-liquid interface under the influence of the tidal forces of ventilation generates enormous shear stress on the respiratory epithelium of small caliber airways. The ‘pull’ exerted by PFC on a capillary wall is approximately 1/5^th that of saline, or ~ 0.4 πrdσ; more than enough to cause endothelial cell injury during tidal ventilation. A metal paperclip floating atop a glass of water illustrates the power of surface tension.

However, in ARDS, and where bulk liquid mixed with gas is present in the small airways (including PFC) surfactant cannot act to protect the airway epithelium against the stress field exerted on the walls of these airways as bubbles move to and fro through the liquid inside them. Bilek, et al., have investigated this phenomenon in vitro and have modelled it computationally as a semi-infinite bubble progressing through a compliantly collapsed airway, as well as a bubble progressing through a rigid tube occluded by fluid. [413]  In this process, the airway walls are separated in a peeling motion as the bubble traverses the airway. This effect has been indirectly documented by experimental observation in vivo. [414],[415] Airway reopening induces large and rapid changes in normal and shear stress along the airway walls. These spatial and temporal gradients of stress exert dynamic, large, and potentially damaging stresses on the airway epithelium that do not occur under one-phase steady-flow conditions.[411], [416],[415] These forces are shown schematically in Figure 4-14. The shear stresses induced by bubble progression along both collapsed and liquid filled airways cause direct trauma due to substrate stretch-induced injuries of pulmonary epithelial cells as a result of  stretching of the plasma membrane causing small tears.[417],[404] Additionally, mechanical stresses from the fluid flow may stretch the plasma membrane either directly, or as a consequence of cellular deformation.

For a low profile, predominately flat region of a cell, the non-uniformly distributed load may regionally deform the membrane. In addition, the normal-stress difference could induce transient internal flows within the cell that exert hydrodynamic stresses on the intracellular surface of the cell membrane, which might injure the membrane by the same mechanisms as extracellular stresses, and additionally be disruptive to the cytoskeleton. Bilek, et al., also describe the effects of irregular airway topology on forces generated at the air-liquid interface. They note that bulges of as little as 2 μ into the lumen of an airway result in greatly amplified shear stresses. Such bulges are commonplace in healthy airway epithelium as a result of the protrusion of epithelial cell nuclei into the airway lumen. The smoothness of the pulmonary epithelium is greatly compromised in ARDS and pulmonary edema and this may be expected to exacerbate topologically mediated shear injury to the small airways. It is interesting to note that Bilek, et al., found that the addition of surfactant to their model systems abolished shear injury in both reopening and bubble- traversing, saline occluded models; perhaps as a result of moderating liquid film thickness over the surface of the airway epithelium thereby decreasing the flow resistance and stress amplification. To what extent this may be applicable in the setting of PLV or LAPC using PFCs is unclear, since presumably surfactant is only effective by being dissolved in the bulk liquid present in the airways (i.e., in the Bilkek, et al., model, saline).

Figure 4-14: Hypothetical stresses imparted on the epithelial cells of an airway during reopening. A: a collapsed compliant airway is forced open by a finger of air moving from left to right. A dynamic wave of stresses is imparted on the airway tissues as the bubble progresses. Circles show the cycle of stresses that an airway epithelial cell might experience during reopening. The cell far downstream is nominally stressed. As the bubble approaches, the cell is pulled up and toward the bubble. As the bubble passes, the cell is pushed away from the bubble. After the bubble has passed, the cell is pushed outward.

B: A fluid ‘occlusion’ in a rigid narrow channel is cleared by the progression of a finger of air moving from left to right. A dynamic wave of stresses is imparted on the pulmonary epithelial cells lining the channel wall. The circles show the cycle of stresses that the cells might experience during reopening. Far downstream, the cell is pushed forward and slightly out. As the bubble approaches, a sudden rise in pressure and a peak in shear stress occurs, pushing the cell forward and outward with much greater force. After the bubble has passed, the cell is pushed outward. Pressure gradients generated in the presence of an air-PFC interface can also be expected to create normal stress imbalances on the cell membrane over the length of the cell. (Illustration and accompanying text reproduced from Bilek, AK, Dee, KC, Gaver, DP, III. Mechanisms of surface-tension-induced epithelial cell damage in a model of pulmonary airway reopening. J Appl Physiol 94:770-783; 2003.)

 These problems may only (if ever) be solved when applying PLV to ALI and ARDS by eliminating tidal gas ventilation and replacing it with high frequency oscillating ventilation (HFOV) which yields uniform and far lower mean airway and P_t pressures and abolishes peak pressures associated with inspiration [418]; although this modality did not prove superior to either PLV or HFOV alone in animal studies – albeit using high (FRC) doses of PFC.[419]

In the case of LAPC, the short duration of FTLV as compared to that required for the treatment of ARDS using PLV may not cause sufficient shear injury to be of clinical concern, particularly in the absence of extensive preexisting pulmonary pathology. In the event that shear injury does prove to be a problem, it may be possible to use HFOV in combination with a more or less continuous process of PFC introduction and removal at or near the level of the carina.  Although, the extent to which HFOV would be effective in rapidly exchanging liquid, as opposed to gas, between the large and small airways is unknown.

The Best as the Enemy of the Good

Finally, another possible factor in the discrepancy between the human and animal trials of PLV was the very close control over the availability of Perflubron™ to investigators exerted by Alliance. In part, this tightly controlled and highly selective distribution of Perflubron™ to only a small number of carefully vetted researchers was driven by the high intrinsic cost of the molecule, and by the cultural and regulatory milieu currently present in the West. Gone are the days when pharmaceutical companies freely distributed putative new drugs for studies more or less upon request. The staggering cost of regulatory approval for a new drug, coupled with often irresponsible ‘research’ aimed at inciting the media frenzy that occurs whenever the toxicity or carcinogenicity of any novel or synthetic molecule (the so-called ‘cyclamate-effect’) is newly demonstrated (regardless of the lack of soundness of the experimental design), has understandably made drug developers cautious about to whom they entrust the evaluation of potentially multibillion dollar compounds. An unfortunate effect of this abundance of caution is that novel drugs are often protected from robust evaluation under more widely varying conditions that more closely approximated those seen in the real world.

Implications for SCA and LAPC

To a much greater degree than was the case with the LiquiVent™ trials (and PLV studies in general), the study being used to initially determine the feasibility of  LAPC for SCA using a TLV-type approach conducted by the author and his colleagues [272] suffers from the same defects that plagued the Alliance PLV studies. This study was conducted on healthy dogs – not on animals that were undergoing CPR with the associated very high peak and mean airway pressures. As was the case in the LiquiVent™ studies, this work preceded the ARDSnet data demonstrating the importance of low tidal volume ventilation and lung protective strategies in general, including minimizing peak and plateau airway pressures. At the time this study was published the authors were, like the LiquiVent™ investigators, unaware of the adverse effects of loading with PFC to FRC – although we certainly observed volutrauma and barotrauma – and became very sensitive to the need for controlling peak and mean airway pressures, as well as to a nuanced shaping of the flow-pressure curve. Indeed, the problem of automating the ideal flow-pressure algorithm has reportedly remained elusive, and ventilation using the technique of LAPC we reported is still least traumatically performed by hand.[420]

Figure 4-15 (left):  The first human cryopatient, Eleanor Williams, undergoing LAPC on 02 March, 2002; several 1,500 ml FTLVs of PFC chilled to ~4ºC were administered by the author using gravity delivery from a flexible 2-liter peritoneal dialysis bag (blue arrow) and then suctioned out. This patient experienced massive hemoptysis immediately after the start of CPS and before LAPC was initiated (red arrow). The patient hemorrhaged ~1,500 ml of blood in less than 5 minutes: underscoring the catastrophic nature of pulmonary bleeds in the setting of friable lungs and CPR. (Photo courtesy of the Alcor Life Extension Foundation.)

The recent insights into the reasons for the failure of PLV Phase III clinical trials suggest that to the extent it is possible to reduce the volume of gas breaths and of the FTLVs required to facilitate heat exchange (i.e., delivery of PFC to and retrieval of PFC from the lungs on a cyclical basis) this will likely reduce the severity of lung injury resulting from LAPC. The measured intrapulmonary pressure in patients undergoing TLV in the Phase III LiquiVent™ study were ~60 cmH₂0 and this resulted in a high incidence of air leak. Intrathoracic pressure in CPR is typically in the same range; 45 to 55 mmHg or ~61 to 75 cmH₂0.[253] The combination of LAPC at FRC with CPR is unknown territory and should be approached with caution; and hopefully also approached with additional studies in animal models of extended duration CPR with long term follow up of the animals.

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393.    Ricard, J., Dreyfuss, D, Laissy, J. P., et al., Dose-response effect of perfluorocarbon administration on lung microvascular permeability in rats. Am J Respir Crit Care Med, 2003. 168: p. 1378-82.

394.    Tredici, S., Tredici, F, Brant, DO, Hirschl, RB, Bull, JL., Effect of viscosity on instilled perfluorocarbon distribution in rabbit lungs. J Biomech Eng, 2006. 128: p. 857-61.

395.    Zheng, Y., et al., Effects of inertia and gravity on liquid plug splitting at a bifurcation. J Biomech Eng, 2006. 128(5): p. 707-16.

396.    Hirschl, R., et al., Prospective, randomized, controlled pilot study of partial liquid ventilation in adult acute respiratory distress syndrome. Crit Care Med, 2002. 165: p. 781-87.

397.    Wiedemann, H., et al., A multicenter, randomized, feasibilitystudy of two doses of perflubron administered for partial liquid ventilation in patients with acute respiratory distress syndrome. Abstr. Am J Respir Crit Care Med, 1999. 159:A80.

398.    Griese, M., Griese M. Pulmonary surfactant in health and human lung diseases: state of the art. Eur Respir J, 1999. 13: p. 1455-76.

399.    Rosenzweig, J., Jensen, OE., Capillary-elastic instabilities of liquid-lined lung airways. J Biomech Eng, 2002. 124(6): p. 650-55.

400.    Kamm, R., Schroter, RC., Is airway closure caused by a liquid film instability? Respir Physiol, 1989. 75(2): p. 141-56.

401.    Salman, N., Fuhrman, BP, Steinhorn, DM, et al., Prolonged studies of perfluorocarbon-associated gas exchange and of the resumption of conventional mechanical ventilation. Crit Care Med, 1995. 23: p. 919-24.

402.    Kaisers, U., et al., Partial liquid ventilation with small volumes of FC 3280 increases survival time in experimental ARDS. Eur Respir J 1997. 10.

403.    West, J., Tsukimoto K, Mathieu-Costello, O, et al., Stress failure in pulmonary capillaries. J Appl Physiol, 1991. 70: p. 1731-42.

404.    Dos Santos, C., Slutsky, AS., Invited review: Mechanisms of ventilator-induced lung injury: a perspective. J Appl Physiol, 2000. 89: p. 1645-1655.

405.    Chu, E., Whitehead, T, Slutsky, AS., Effects of cyclic opening and closing at low- and high-volume ventilation on bronchoalveolar lavage cytokines. Crit Care Med, 2004. 32: p. 168-74.

406.    Dreyfuss, D., Saumon, G, Ventilator-induced lung injury: Lessons from experimental studies. Am J Respir Crit Care Med, 1998. 157: p. 294-323.

407.    Hughes, J., Rosenzweig, DY, Kivitz, PB., Site of airway closure in excised dog lungs: histological demonstration. J Appl Physiol, 1970. 29: p. 340-344.

408.    Ghadiali, S.N. and D.P. Gaver, Biomechanics of liquid-epithelium interactions in pulmonary airways. Respir Physiol Neurobiol, 2008. 163(1-3): p. 232-43.

409.    Argiras, E., Blakeley, CR, Dunnill, MS, Otremski, S, Sykes, MK., High PEEP decreases hyaline membrane formation in surfactant deficient lungs. Br J Anaesth 1987. 59: p. 1278-1285.

410.    Gaver, D., Halpern, D, III, Jensen, O, Grotberg, JB., The steady motion of a semi-infinite bubble through a flexible-walled channel. J Fluid Mech, 1996. 319: p. 25-65.

411.    Mead, J., Takishima, T, Leith, D., Stress distribution in lungs: a model of pulmonary elasticity. J Appl Physiol, 1970. 28: p. 596-608.

412.    Yap, D., Gaver, D., The influence of surfactant on two-phase flow in a flexible-walled channel under bulk equilibrium conditions. Phys Fluids, 1998. 10: p. 1846-1863.

413.    Bilek, A., Dee, KC, Gaver, DP, III., Mechanisms of surface-tension-induced epithelial cell damage in a model of pulmonary airway reopening. J Appl Physiol, 2003. 94: p. 770-783.

414.    Naureckas, E., Dawson, CA, Gerber, BS, Gaver, DP, Gerber, HL III, Linehan, JH, Solway, J, and Samsel, RW., Airway reopening pressure in isolated rat lungs. J Appl Physiol, 1994. 76: p. 1372-1377.

415.    Yap, D., Liebkemann, W, Solway, J, Gaver, D., Influences of parenchymal tethering on the reopening of closed pulmonary airways. J Appl Physiol, 1994. 76: p. 2095-2105.

416.    Jensen, O., Horsburgh, MK, Halpern, D, Gaver, DP, III., The steady propagation of a bubble in a flexible-walled channel: asymptotic and computational models. Phys Fluids 2002. 14: p. 443-457.

417.    Vlahakis, N., Hubmayr, RD., Invited review: plasma membrane stress failure in alveolar epithelial cells. J Appl Physiol, 2000. 89: p. 2490-2497.

418.    Doctor, A., et al., High-frequency oscillatory ventilation of the perfluorocarbon-filled lung: preliminary results in an animal model of acute lung injury. Crit Care Med, 1999. 27(11): p. 2500-7.

419.    Rotta, A.T., et al., Combining lung-protective strategies in experimental acute lung injury: The impact of high-frequency partial liquid ventilation. Pediatr Crit Care Med, 2006. 7(6): p. 562-70.

420.    Harris, S.B., New Breakthrough in Rapid Cooling of Cryopreservation Patients after Cardiac Arrest, presented at the Suspended Animation, Inc. Advances in Cryopreservation Conference. 2007: 19 May, Ft. Lauderdale.

Appendix B: Explanation of the Mechanics of Blood Flow during Closed Chest CPR: The Thoracic Pump Theory

The following text, with the accompany references, is reproduced from Weisfeldt, M., Chandra, N., Physiology of cardiopulmonary resuscitation. Ann Rev Med, 1981. 32: p. 43-42.

“At least three general mechanisms are now thought to contribute to the generation of the extrathoracic arterial-venous pressure gradient observed during conventional CPR in the dog. These factors are (a) various venous valving mechanisms are operating, (b) peripheral venous capacitance greater than arterial capacitance, and (c) arterial resistance to collapse greater than venous resistance.

 The most easily understood of these mechanisms is that of a valving mechanism. Veins at the thoracic inlet and other veins leading from the brain appear to have anatomic valves that prevent retrograde flow of blood during increases in intrathoracic pressures (9, 10, 16, 18). The valves along the extrathoracic veins, and the one at the thoracic inlet appear to be important.

 The second factor contributing to the generation of the peripheral arterial-venous pressure gradient is that venous capacitance is greater than arterial. Clearly, if the same amount of blood were to move from the intrathoracic arterial and from the intrathoracic venous systems into the extrathoracic arterial and venous systems, arterial pressure would rise more than venous pressure because of the differences in extrathoracic arterial and venous capacitance.

 The third contributor to the peripheral arterial-venous pressure gradient is the difference in arterial and venous resistance to collapse. Venous structures readily collapse when inside pressures fall below surrounding pressures by even a small amount. Recently we showed that the in vivo carotid artery at the thoracic inlet exhibits considerable intrinsic resistance to collapse.

 This resistance to collapse can be increased in the presence of vasoconstrictor agents (23). Resistance to collapse of the arterial vessels would allow blood flow to continue toward the brain despite surrounding intrathoracic pressures which exceed intravascular pressures. The veins would readily collapse at the exit to the high pressure region, i.e. at the thoracic inlet (1, 13).

 Blood returns from the periphery to the central circulation between compression cycles. Extrathoracic venous pressure rises (20) when blood flows from arteries to veins during compression. Between compressions intrathoracic pressure falls to near atmospheric, and an extrathoracic-to intrathoracic venous pressure gradient appears, which leads to flow into the chest. Right heart and pulmonary blood flow is also diastolic, at least in part (7). With conventional CPR, fight heart compression may be a component of the mechanism for pulmonary flow.”

References

1. Brecher, G. A. 1952. Mechanism of venous flow under different degrees of aspiration. Am. J. Physiol. 169-423.

7. Cohen, J. M., Alderson, P. O., Van AswegenA, ., Chandra,N ., Tsitlik, J.,

Weisfeldt,M .L . 1979.T imingo f intrathoracic blood flow during resuscitation

with high intrathoracic pressure. Circulation. 59 & 60:II-19.

10. Franklin,K . J. 1927.V alvesin veins: an historical survey. In Proc. R. Soc. ed. Sect. History Med., pp. 4-6.

13. Holt, J. P. 1941. The collapse factor in the measurement of venous pressure.

Am. J. Physiol. 134:292.

16. MacKenzie, J. 1894. The venous and liver pulses, and the arhythmie (sic) contraction of the cardiac cavities, d. Pathol. Bacteriol. 2:113.

18. Niemann, J. T., Garner, D., Rosborough, J., Criley, J. M. 1979. The mechanism of blood flow in closed chest cardiopulmonary resuscitation. Circulation 59 & 60:I1-74.

21. Weale,F . E., Rothwell-JacksonR, . L. 1962. The efficiency of cardiac  massage. Lancet 1:990-92.

23. Yin, F. C. P., CohenJ,. M., Tsitlik, J., Weisfeldt, M. L. 1979. Arterial resistance

to collapse: A determinant of peripheral flow resulting from high intrathoracic

pressure. Circulation 59 & 60:I1-196.

Reproduced from the  Annual Reviews www.annualreviews.org/aronline

Annu. Rev. Med. 1981.32:435-442. Downloaded from: arjournals.annualreviews.org by PALCI on 10/25/08.

Experimental Studies to Determine the Effectiveness of LAPC under Laboratory Conditions

Experimental Studies to Determine the Effectiveness of LAPC under Laboratory Conditions

 [This section is an edited version of an article authored by Steven B. Harris, Michael G. Darwin, Sandra R. Russell, Joan M. O’Farrell, Mike Fletcher and Brian Wowk entitled, Rapid (0.5°C/min) minimally invasive induction of hypothermia using ~4ºC perfluorochemical lung lavage in dogs, which first appeared in Resuscitation, 2001. 50: p. 189-204.)]

 1. Introduction

The potential utility of profound and ultra-profound hypothermia (0-5^oC ) to arrest deleterious neurological changes has long been understood in both biology and medicine.[168],[169],[170],[171],[172] In 1959 Benson, et al., reported good outcome using profound hypothermia (10-22^oC ) as a treatment following cardiac arrest. [173] This work was followed up by a number of clinicians [168],[174],[175],[176] who also reported favourable results. However, due to coagulopathy, arrhythmias, and the increased incidence of pneumonia and sepsis associated with such deep and prolonged cooling, post arrest hypothermia failed to gain acceptance and was abandoned. It was not until the work of Safar, et al., [160],[53],[82] that the utility of mild therapeutic hypothermia (MTH) (DT = −2 to −3°C) as an active treatment for the post-resuscitation syndrome was rigorously demonstrated, and subsequently validated by others. [177],[178],[55],[179-181]  As noted in Section 1, while CPB offers the most rapid core cooling possible, it is logistically unsupportable as currently practiced. Additionally, CPB carries the added risks of anticoagulation, further activation of the immune-inflammatory cascade, RBC aggregation, and the danger of gas embolism, as chilled, nitrogen-saturated blood is rapidly re-warmed as it perfuses warm tissues.[182]

As was also previously noted, less invasive modalities with the potential for in-field application, such as surface cooling and lavage of body viscuses with a balanced salt solution, are only effective in achieving cooling rates in the range of 0.10–0.15°C/min. The seemingly straightforward  experimental technique of ‘tidal liquid ventilation’ (TLV) with chilled, oxygenated PFC uses the ~20 m² surface area of the lungs for heat exchange, but thus far has been no more effective in inducing hypothermia than surface cooling with ice bags or chilled water blankets.[155] After preliminary experiments demonstrated the technical adequacy of LAPC at achieving heat exchange in range of 0.25 to 0.35°C/min [183] a comprehensive study was undertaken by 21^st Century Medicine Inc., (21CM) and Critical Care Research, Inc., (CCR), beginning in 1999, to define and validate this cooling modality in a canine model. The goals of this research were to, a) demonstrate the fundamental safety and efficacy of the technique, b) determine the optimum cycle and volume of liquid and gas fractional tidal liquid ventilations (FTLVs), and c ) attempt to determine safe airway pressures and define liquid and gas ventilation strategies that minimized or eliminated baro- and volutrauma.

This technique, developed at 21CM/CCR, was initially called ‘mixed-mode liquid ventilation cooling’ and was later renamed ‘gas-liquid ventilation’ (GLV). However, neither of these names adequately describes the technique, and this author (Darwin) has chosen to the use the term liquid assisted pulmonary cooling (LAPC) instead. In previous studies where large fractional tidal liquid volumes and shorter cycles of FTLV were used, the performance of LAPC deteriorated towards that seen when TLV-cooling (or warming) was used. In practice however, certain significant differences remained and understanding these differences proved essential to optimization of the technique.

In LAPC the critical elements of gas ventilation are retained allowing for flexibility in selecting ventilation parameters independently for heat and gas-exchange, allowing for liquid-mediated heat-exchange to be easily undertaken using existing ventilation systems[1]. The combination of gas and liquid FTLVs may also play a role in the surprisingly good thermal efficiency of LAPC as compared with TLV.

The following study explored the performance of LAPC using a prototype automated FTLV device, and discussed the basic mechanics and intrinsic limitations of heat-exchange using FTLV.

2. Materials and methods

These experiments were approved by 21st Century Medicine, Inc.’s Institutional Animal Care and Use Committee and were in compliance with the Animal Welfare Act and the National Research Council’s Guide for the Care and Use of Laboratory Animals. Fifteen mongrel dogs weighing 13.8–25.7 kg were used (Table 1). Dogs were pre-medicated with I.M. acepromazine (1.0 mg/kg) and atropine (0.02 mg/kg) prior to induction of general anesthesia using sodium pentobarbital (30 mg/kg I.V., with maintenance dosing). Anesthetized dogs were intubated with a reinforced 10.0 mm I.D. (Willy Rusch AG, Kernen, Germany) endotracheal tube (E.T.), and ventilated on room air using a Bennett MA1 or Siemens Servo 900 C ventilator. Ventilator parameters, unless otherwise noted, were 12 gas-breaths/min, gas tidal-volume of 15 ml/kg, I:E ratio of 1:3, and a maximal positive inspiratory pressure (PIP) limit of 26 cm H₂O (2.5 kPa). Gas pressures were measured at the E.T. adapter. Gas minute-volume (V_g) was adjusted to maintain PaCO₂ between 35 and 40 torr. Animals were maintained at ~37.5°C prior to LAPC, using a temperature-controlled water blanket. Rectal and bilateral tympanic temperatures (Ttym) were monitored continuously using a type-T thermocouple system (Cole-Parmer, Vernon Hills, IL) with a response time constant (to) of 5 s.

Combination pressure, blood sampling, and temperature-probe catheters were constructed from rigid polyethylene pressure-monitoring catheters, threaded centrally with 0.05 in. O.D. Teflon™-sheathed type-T thermocouples (to=0.3 s, Physitemp Instruments, Clifton, NJ). In order to reduce the risk of catheter-associated clot formation, I.V. sodium heparin was given to adjust activated clotting times to 300–500 s, prior to central line placement. Femoral vessels were isolated surgically, and arterial and venous catheters placed and advanced to a level above the renal vessels, as confirmed by X-ray. During surgery, bupivacaine (0.5%) was infiltrated into wounds to mitigate post-operative pain.

In one dog (Trial I-2), a femorally-placed pulmonary artery thermodilution catheter replaced the venous combination catheter. Blood and ventilator pressures were acquired through a Hewlett Packard 78532-B monitor/transducer system.

Immediately prior to LAPC, dogs were assessed for adequacy of general anesthesia, and then given pancuronium bromide (2 mg) to inhibit shivering and spontaneous breathing. FIO₂ was increased to 100% and external temperature control discontinued. To serve as a cannula for both delivery and removal of PFC liquid, a 19-Fr. flat-wire reinforced Bio-Medicus® venous catheter (Medtronic, Eden Prairie, MN) was introduced through the suction port of the E.T. adapter, and advanced ~45 cm to approximately the level of the carina (Figures 2-1 and 2-2) as confirmed by X-ray. This cannula was connected to the LAPC apparatus described below. LAPC was performed using the PFC liquid ‘FC-75’ (3M Corporation, St. Paul, MN), a perfluorinated butyl-tetrahydrofuran isomer mixture.[184]

Figure 2-1 (right): PFC delivery and withdrawal catheter threaded through the endotracheal tube with the tip positioned at the level of the carina.

A two reservoir circuit (Figure 2-3) was used to deliver and remove PFC from the lungs (FTLV) via the cannula, in cycle periods of 37 s (Trial I) or 16 s (Trial II). During timed PFC infusions (t_in=20 s for Trial I, or 10 s for Trial II), PFC was pumped through the cannula by a continuously-operating Travenol CPB roller-pump (Sarns, Ann Arbor, MI). A bypass loop, open during suction, allowed the roller-pump to divert (recirculate) PFC flow back into the storage reservoir whenever flow was not directed by line clamps V1–V3 into the animal. PFC was pumped continuously through an in-line 0.2 μ ‘pre-bypass’ filter (Pall PP 3802, Pall, East Hills, NY), a primary heat-exchanger (Torpedo-T, Sarns, Ann Arbor, MI), and a combination silicone membrane oxygenator/heat-exchanger (SciMed II-SM35, SciMed Life Systems, Minneapolis, MN). The oxygenator was supplied with 5–6.5 l/min O₂ (maximal device design rate), and the reservoir PFC was allowed to circulate and equilibrate with heat-exchangers and O₂, before LAPC was initiated. The circuit tubing was constructed of S-50 HL TYGON® 3/8 and 1/2 in. I.D. class VI tubing (Norton/Performance Plastics, Akron, OH) with the exception of a length of silicone tubing (Masterflex® 96410-73, Barrant Co., Barrington, IL) used in the roller-pump head in order to allow flexibility at low temperatures. PFC suction was driven by a vacuum pump (model 107CAB18B, Thomas Compressors, Sheboygan, WI), and suction reservoir negative pressure was limited to −35 torr by a vacuum relief valve. Figure 2-2: Detail of LAPC PFC introduction and removal catheter and ET Tube and gas ventilator configuration. A Biomedicus CPB venous return catheter was threaded through the suction port of a standard 16 mm respiratory ET tube swivel connector.

Figure 2-3: The LAPC system. The LAPC system was connected to a catheter inserted into the suction port of the E.T. adapter. PFC flows were directed by manual or mechanical clamps at V1–3. During the suction phase, FC from the lungs was removed into a sealed ‘suction’ reservoir, for later addition to the primary circuit (via adjustment of V4 and V5), while ‘infusion ready’ PFC was re-circulated through a bypass loop. Negative pressure was limited by a vacuum relief valve (VrV). Photo (right) A) Suction Reservoir, B) Storage Reservoir, C) Solenoid Valves (V1-V3), D) SciMed Oxygenator & Heat Exchanger, E)Sarns Roller Pump, F) PFC Suction/Delivery Catheter, G) Pump Controller, H) Heat Exchanger Return Line with weighted water diffuser (yellow), I) Thermocouple Probes.

Figure 2-4: Gas ventilator and respiratory monitoring equipment used in the LAPC experiments; a) Novametrix CO₂SMO respiratory function monitor and capnograph, b) Siemens Servo 900 C ventilator, c) Korr Medical, Inc., automated device used to perform rapid-cycle LAPC, d) LAPC apparatus.

  Concurrent FC-75 FTLV and gas ventilation (LAPC) was performed for 18 min in Trials I and II (n=12). This time was chosen, on the basis of preliminary work (data not shown), to achieve rapid systemic-cooling of greater than 5°C. For Trials I and II, the PFC recirculation rate within the LAPC device (=PFC infusion rate, ˙V _inf), was set at 50 ml/kg per min rate, V_FTLV) was set at 50 ml/kg per min.

Immediately after a timed FTLV, PFC was removed as rapidly as possible. Infusion of PFC for the next FTLV began immediately after suction was discontinued. In LAPC experiments, PFC was chilled to ~4°C prior to FTLV (Table 1), whereas in normothermic (control) dogs, isothermic PFC was delivered to the dog within ~2°C of tympanic temperature (T_tym). The PFC inflow and outflow temperature was measured continuously by a thermocouple inserted into the PFC path at the base of the delivery/removal cannula. Temperature data was collected throughout LAPC, and for 22 min after LAPC was completed. Arterial blood gas (ABG) samples were taken from the femoral arterial line before the start of LAPC, and every 2 min during LAPC. Following the post-LAPC equilibration period, monitoring devices were removed and incisions closed.

Table 1:

Table 2:

2.3. Trial I (manually-controlled LAPC)

Trial I was designed to investigate the variability in the response of individual animals to LAPC and to investigate the physiological effects of the LAPC technique with and without cooling (i.e., ~4ºC PFC vs. isothermic PFC FTLV). Either isothermic (near-body temperature) or ~4ºC PFC FTLV was administered using a manually-controlled system (V1–V5 in Fig. 1 represent CPB tubing-occluders in this Trial). One FTLV (period t_c ~37 s) was composed of a timed FTLV (t_inf = 20 s), followed by PFC suction (t_s ~17 s). Suction was stopped when PFC liquid return became sparse, or gas pressure in the ventilator circuit fell below −5 cm H2O (−0.5 kPa). Five dogs received ~4ºC FTLV (Trial I-1–5), while two controls received the same protocol using isothermic FTLV (Trial I-6 and 7).

 2.4. Trial II (machine-controlled LAPC)

Trial II assessed the utility of using an automated device (custom manufactured by Korr Medical, Inc., Salt Lake City, UT) to perform rapid-cycle LAPC. Computer-controlled solenoid clamp-valve occlusion of circuit lines at V1–V3 allowed smaller FTLV volumes (V_FTLV) and smaller t_c. While t_inf was decreased to 10 s in Trial II, V_FTLV remained constant, and the effective PFC FTLV rate (V_FTLV) remained in the range of V_FTLVfor Trial I. Table 1 gives relevant trial parameters. In Trial II, suction of PFC from the lungs began immediately after infusion, and was automatically stopped whenever a ventilator circuit pressure of −5 cm H₂O was reached (t_s ~6 s, giving t_c ~16 s). Three dogs received ~4ºC PFC (Trial II-1–3), while two controls (Trial II-4 and 5) received isothermic PFC.

 2.5. Animals A, B and C

 Selected data from three dogs in an earlier method development series was used. These dogs had been prepared as above, then manually given 1, 15 and 21 FTLVs, respectively with ~4ºC PFC, at much slower rates than in Trials I and II (Table 1). Data from these animals allowed independent measurements of FTLV volume heat-contents and temperatures, and thus the heat capacities and heat transfer efficiencies, by a more thorough thermal accounting method (Table 2, Appendix A).

2.6. Data collection and correction, statistical methods, graphical display and presentation

 Temperature and pressure data were collected using a PCI E series data acquisition board and LabVIEW™ software (National Instruments, Austin, TX). Graphical analysis and display of temperature data, and curve fitting, was done using the software package Origin™ (Microcal Software, Northhampton, MA). Statistical comparison of Trial group values was done using GraphPad Prism (GraphPad Software, San Diego, CA). Group means are reported ± standard

Figure 2-5 (above): Body temperature changes observed during LAPC (Method of Trial I). In this illustrative experiment from Trial I (I-4), FTLVs of ~4ºC FC-75 were infused (~20 s) and removed (~17 s) from the lungs. LAPC was performed for 18 min (hatched bar), then stopped to allow thermal equilibration (22 min). Arterial temperature (˜Tart), central venous temperature (˜T_ven), tympanic temperature (˜T_tym), and rectal temperature (˜T_rec) are shown. Inset: Enlarged view of temperature changes recorded during the first two cycles of PFC infusion (gray bar) and removal (yellow bar).

 deviation (SD) except as otherwise noted. For each animal, the T_tym from whichever probe cooled most rapidly, was used (right probe in 12/15 dogs). In order to facilitate comparison of cooling rates between sites in the same animal, temperatures at all probe sites were corrected to the baseline aortic temperature (T_art), as measured immediately prior to the start of LAPC. For ease of description, LAPC-cooling is presented in terms of thermal-deficit (‘cold’) moving from the lungs into successive body compartments. A compartmental analysis of thermal transfer in this model, and a glossary of notation and equations used, is given in Appendix A.

 3. Results

 LAPC allowed FTLV of dogs during concurrent gas ventilation. Suction from the submerged catheter tip at the carina allowed collection of PFC even during forced gas inspiration. It was discovered that a long suction catheter was necessary to insure that adequate suction pressure could be used to withdraw PFC throughout the liquid removal sequence, without prolonged exposure of the gas filled portion of the airways to the negative pressure of the suction system/reservoir. Additional protection of the airways against excessive negative pressure during the relatively brief time after liquid no longer filled the suction line was provided by incorporating a negative pressure relief valve on the suction reservoir. Suction in this manner was efficient, although FTLV volume measurements showed that the lungs retained ~12 ml/kg PFC (approximately FRC) between FTLVs.

The PFC pump circulation/infusion rate (˙V _inf), measured volumetrically preceding and following LAPC, was stable to within 1% over the duration of LAPC, and was not significantly different between trials (P = 0.28). The V_FTLV, calculated as tinf  _inf/t_c, was 30.7±2.3 ml/kg per min (Trial I) and 36.4 ± 3.2 ml/kg per min (Trial II). The ˙V_FTLV was significantly (P = 0.023) larger in Trial II because machine-controlled suction made more efficient use of available non-infusion time, resulting in faster net PFC removal.

Figure 2-6: Thermal equilibration after LAPC. Mean Tart and T_ven values (Fig. 2) are shown for Trial I, dogs 1–5. To highlight equilibration changes, T_tart curve nadirs (n=5) were superimposed before calculation of means, and T_ven data (n = 4) for each dog was adjusted with its corresponding Tart curve. Incompatible T_ven data from a pulmonary artery thermodilution catheter in I-2 has been omitted. Inset: The sigmoidal mean (N = 5) T_art recovery during the first ~12 s after final LAPC. FTLV is approximated by linear fitting.

Figure 2-7: Body temperature changes during manual and mechanical LAPC (Trial I vs. Trial II). The relative rates of core body cooling in dogs undergoing 18 min (hatched region) of manual (Trial I, solid squares) or machine-driven (Trial II, open circles) ~4ºC LAPC, were assessed by comparing changes in group mean T_tym. Symbols represent the mean and SEM (n = 5 for manual, and 3 for machine groups).

 3.1. Thermal results of LAPC

3.1.1. Cooling time delay

 Figure 2-5 illustrates LAPC cooling in a representative dog (I-4) from Trial I. The T_art began to decrease 3–6 s after the start of each PFC FTLV. Since this delay included circulation delay from lungs to aorta, the transfer of thermal-deficit from newly-introduced PFC to pulmonary blood was very rapid. The venous temperature (T_ven) began to decrease 10.4 ± 6.9 s after T_art decline, representing the minimum systemic circulation time. Though exhibiting delay, damping, and broadening behavior (presumably due to peripheral heat-exchange and varying systemic blood-return path lengths), T_ven transients from FTLVs mirrored T_art transients. T_tym temperatures, presumably reflecting brain and viscera temperatures, were non-oscillatory. The T_tym did not begin to decrease until ~24 s after the start of LAPC. This decrease occurred in three phases: an initial phase lasting ~100 s, an exponential phase lasting for ~900 s, and a final linearly-decreasing phase lasting until the end of LAPC. Core cooling as measured by T_tym continued for about 120 s after the end of LAPC (Figure 2-5), then exhibited a marked rebound effect [185] with exponential dampening (t ~20 min, Figures 2-5–2-7). These phases of cooling and equilibration were consistent with a five-compartment thermal model, in which the three compartments representing animal tissues corresponded roughly with (1) the blood and vasculature; (2) the classical thermal core; and (3) the classical thermal periphery (Figure 2-8). Modelling equations and estimation of compartment sizes are given in the Appendix A.

3.1.2. Cooling rate

 Crude cooling rates were determined numerically from appropriate T vs. t graph segments. The mean cooling rate from LAPC initiation, or DT_tym/Dt, reached a maximum value in Trial I at −0.49±0.09°C/min (t=6.6 min). The differential cooling rate d (DT_tym)/ dt = dT_tym/dt reached a maximum (max) value of -0.59 ± 13°C/min at t ~100 s, near the end of the initial heat exchange development region. (This value is comparable to analytic d (DT_tym)/dt  (max) from (Eq. (1)) = DT_k/t_o  = −0.63°C/min). Corresponding cooling rates in Trial II were DT_tym / Dt (max) = −0.33 ± 0.02°C/min (at t = 7.3 min) and dT_tym / d_t (max) = −0.37 ± 0.06°C/min (at t = 100 s).

3.1.3. Mean cooling power

 The mean heat removal rate (cooling-power) P over the entire duration of LAPC, for each animal, was estimated from DT_e according to P = m C_m DT_e/t (total).

Here t (total) is the entire LAPC application time = ~1080 s. (Note: for this calculation, the more accurate Trial I mean C_m is used for all Trial II animals.) The mean cooling power of Trial I was 336 ± 60 watts, while that of Trial II (using the Trial I value of C_m) was 207 ± 49 watts (P = 0.02). Variation in animal size was the major source of intra-group variability.

Figure 2-8: Heat transfer among body compartments during LAPC. Heat transfer during LAPC in the dog may be modeled using 5 thermal compartments. Heat transfer between compartments (which is by blood circulation, except as noted) is shown in the box diagram as double-headed arrows. The pair of arrows connecting Compartments 2 and 3 represents the different processes of lung equilibration with (1) pulmonary artery flow; and (2) with the complete blood volume and selected viscera.

 3.2. Gas exchange

 ABG measurements demonstrated that infusion of ~4ºC PFC stabilized PaO₂ and PaCO₂ during LAPC. In contrast, LAPC using isothermic PFC failed to maintain baseline PaO₂ or PaCO₂ levels (Figure 2-9). In Trial II-4, hypercarbia during the first 13 min of isothermic LAPC was abolished by increasing the tidal volume from 15 to 25 ml/kg (final V_g = 375 ml/kg per min). In Trial II-5, V_g was pre-set to 375 ml/kg per min in an attempt to avoid hypercarbia, and no significant ABG changes were observed.

Figure 2-9: LAPC does not maintain normocarbia at isothermic temperatures without alteration of the gas ventilation parameters. Animals in Trials I and II underwent LAPC using either ~4ºC (˜) or isothermic (r£ ™¯) FC-75. Both arterial PaO₂ (Panel A) and PaCO₂ (Panel B) levels were affected by FTLV temperature. ~4ºC FTLV data from Trials I and II were very similar in magnitude, and therefore, have been combined (n=8). Isothermic LAPC is shown as four separate experiments (Trial I-6 and 7, and Trial II-4 and 5). Gas tidal volume was increased from 15 to 25 ml/kg in Trial II-4 at t=13 min, and at t=0 in Trial II-5, normalizing PaO₂ and PaCO₂ in both animals. Declining PaO₂ in Trial I-7 was due to inadvertent failure to pre-oxygenate PFC.

Figure 2-10: Effect of LAPC on VCO₂ and EtCO₂ during 20 minutes of ~4ºC FTLV. LAPC allows superior CO₂ removal due to gas ventilation because ^·V _PFC = no more than 30 mL/kg/min of liquid, allowing gas ventilation of at least 200 mL/kg/min, resulting in a maximum ^·VCO₂ removal rate of  >8 mL/kg/min or a minimum of 400% of basal metabolic rate.

3.3. Clinical observations and gross pathology

 With the exception of one dog, animals subjected to LAPC displayed mild tachypnea and increased expiratory sounds, but otherwise exhibited unremarkable recovery from anesthesia, including the ability to walk and drink. The exception was an eosinophilic animal (Trial II-1) which had normal oxygenation during LAPC, but developed severe hypoxemia shortly after LAPC.

Chest X-ray pre- and post-procedure showed no (comparatively) remarkable features. This dog was sacrificed at 9 h.

Necropsy revealed a mass of D. immitis (heart- worm) embolized into the pulmonary arterial circulation, possibly as a result of local chilling of the parasite mass due to LAPC (this animal had been heartworm seronegative). Necropsies performed on nine remaining Trial I and II animals sacrificed 24 h post-procedure revealed diffuse spongy, resilient hyperinflated non-collapsible lungs (HNCL) seen in animals exposed to a high-vapor pressure PFC at high PIP pressures.[186]  HNCL was most prominent in the anterior, least dependent areas of the lung lobes. This trapped intra-alveolar PFC was thought to be the cause of broncho-constriction and wheezing found in post-LAPC animals. There was also evidence gross, dependent-lung damage evidenced by pulmonary edema with consolidation in both isothermic and ~4^oC PFC-FTLV animals.

Other organ systems in this series were grossly normal. Two animals in Trial II (II-3 and II-4) were not sacrificed, and were held for long term evaluation. They were neurologically normal at 1 year post-LAPC.

3.4 Impact on Hemodynamics

 FTLV with both ~4ºC and isothermic PFC resulted in an almost immediate modest decrease central venous pressure (CVP) which persisted for the duration of the FTLV and recovered to pre-FTLV values at the conclusion of each FTLV cycle (Figure 2-11 – 2-13).

Figure 2-11: Impact of LAPC on HR.

In animals subjected to FTLV with ~4ºC PFC there was an immediate, transient reduction in heart rate (HR) and mean arterial blood pressure (MAP) and a corresponding increase CVP during the FTLV cycle. In animals undergoing ~4ºC FTLV these effects could be attributed to the acute, cyclical chilling of the coronary blood supply during each FTLV with chilled PFC. The temperature of the blood entering the coronary os was 10^o to 15^oC colder than systemic blood (as measured by pulmonary artery catheter), and this would be expected to have an immediate depressive effect on myocardial contractility due to the transient hypothermia the myocardium would experience as a consequence of perfusion with chilled blood. In fact, consonant with this interpretation, HR decreased steadily during ~4ºC FTLV, recovering progressively less after each FTLV cycle, as systemic hypothermia was induced (Figure 21).

Figure 2-12: Cyclical variation in CVP is response to FTLVs with isothermal PFC.

Figure 2-13: Effect of FTLV with ~4ºC PFC on MAP and CVP over the course of 6 minutes of LAPC.  MAP is transiently markedly depressed and CVP is concurrently increased in response to loading with ~4ºC PFC; this effect is reversed when the PFC load is suctioned from the lungs; although there is increasing depression of MAP in response to the induction of systemic hypothermia. Cardiac output (CO) was not measured in these studies and mathematical analysis of the MAP waveforms generated during isothermic FTLV were not done. Thus, the precise extent to which FTLV with isothermic PFC (i.e., without the thermal-metabolic effects of chilled blood on the myocardium as occurs in LAPC) impairs cardiac output or coronary perfusion pressure is unknown.

Interestingly, the cyclical increase and recovery of the CVP remained constant during both ~4ºC and isothermic FTLV. Initially, the effect of FTLV on CVP was thought to be due to compression of the thoracic vena cavae by the relatively dense PFC load in the lungs. It was hypothesized that this effect might be more pronounced in the dog due to the V-shape of the canine thorax with the cavae resting at the bottom of the thoracic ‘trough’ in a dependent position under the lungs. To test this hypothesis, isothermic FTLV was carried out with a dog in the prone position. Pronation had no effect on the transient, cyclical depression of HR and increase in the CVP associated with FTLV. It thus seems possible that loading of the lungs with dense PFC liquid results in increased pressure on the thoracic vasculature, in particular on the thoracic venous vasculature, in much the same way gas PEEP reduces thoracic venous capacitance and raises CVP; reducing right ventricular preload and right ventricular output (cardiac output). These effects would seem the most likely explanation for the reduction in MAP observed during maximal PFC loading during both ~4ºC and isothermic FTLV.

CO was not measured during these LAPC studies nor was mathematical analysis of the aortic pressure waveforms undertaken to determine with precision the degree to which FTLV depressed MAP. Crude analysis of MAP during isothermic FTLV suggests that cyclical PFC loading is responsible for ~15-20% reduction in MAP over baseline.  Mean CVP is increased ~35% over basal levels during FTLV. To what extent CO will be impacted as a result of FTLV with PFC during CPR will have to be determined experimentally (see discussion in 4.5.3. Overcoming Increased Intrathoracic Pressure and Preserving CO, below).

4. Discussion

 4.1. Apparent effect of temperature on gas exchange

 Isothermic LAPC in this model was surprisingly poor at removing CO₂, considering that the CO₂ carrying capacity in FC-75 decreases by only ~23% from 0 to 40°C (extrapolated from [184]). A useful observation was that pO₂ values decreased even in isothermic animals, indicating an influence on total ventilation and possibly also reflecting decreased CO as evidenced by the (average) decline in MAP and increase in systemic vascular resistance during LAPC.

Capnographic analysis of LAPC in Trials I and II (data not shown) indicated that isothermic LAPC had a much larger negative effect on pressure-limited total gas ventilation V_g, as compared to ~4ºC LAPC using the same technique and the same gas ventilator settings. Since LAPC at a ˙V_FTLVr of 30–36 ml/kg per min relies on gas ventilation V_g for ~50% of total alveolar ventilation, a differential loss of pressure-limited V_g with temperature appeared to be the basis of CO₂ retention in isothermic LAPC. The mechanism of the implied differential change in lung compliance may be related to the depressive effects of FTLV on CO, and presumably, on perfusion. Thus, gas ventilation adjustments similar to those in Trial II-4 and 5 may be required if LAPC is used as a re-warming technique, and it may additionally be necessary to abolish gas PEEP, or even apply continuous negative airway pressure [187],[188] to counteract the PEEP-like effects of PCF loading and to generally improve CO and coronary perfusion pressure (CPP) during CPR.[189]

4.2. Thermal transfer efficiency and kinetics

 The optimal LAPC cooling (or warming) protocol remains unknown. However, the finding that the thermal equilibration of non-dead space PFC and local pulmonary blood flow proceeds very rapidly (t_o <12 s) suggests that FTLV infusion times need to be no longer than this time scale. When PFC lung dwell times exceed this duration, the FTLV load is in place longer than is required to transfer the most labile part of its thermal potential to the pulmonary blood and parenchyma.

Figure 2-14: Relative insensitivity of PFC dwell time to heat exchange was demonstrated by progressively shortening the duration of FTLVs and increasing their frequency. Even at the maximum achievable rate of FTLV at ˙V_FTLV  rates of 30 ml/kg per min and ˙V_FTLV  rates of 50 ml/kg per min no deterioration in the efficiency of heat exchange was observed. This suggests that thermal equilibration at the level of the alveolus is practically instantaneous and that the shortest FTLV dwell time should be used for optimum heat exchange.

Since FTLV ventilation rates (˙V_FTLVr) in the present study are already at least a third of the maximal rates possible in TLV, it seems probable that PFC infusion rates and pressures, rather than heat transfer rates from PFC to lung, will be the fundamentally limiting factor to power transfer in LAPC. These observations suggest that, as least to ˙V_{FTLV r} rates of 30 ml/kg per min and ˙V_FTLV  rates of 50 ml/kg per min, the total cooling power (cooling rate) in LAPC will be greatest if no PFC dwell time is allowed, and all available time during the FTLV cycle is used to either introduce, or remove, PFC.

4.3. Question of diffusion dead space in LAPC

 Mammalian lungs depend on simple gas diffusion for CO₂ transport through the acinar airways during normal tidal ventilation. An intractable problem in experimental TLV has been that simple diffusion is not sufficient to similarly move CO₂ through liquid PFC at physiologic CO₂ partial pressure gradients. This limitation appears in TLV as a ‘CO₂ diffusion dead space’ which effectively lowers alveolar ventilation. In part due to such extra physiologic dead space, TLV of adult humans has been estimated to require liquid minute-volumes near 70 ml/kg per min.[190] This value is at the upper bound of realistically attainable liquid flow rates [191],[192] and leaves little leeway for treating hypercarbia, hypermetabolic states, or lung disease. Such difficulties are not a theoretical limitation in LAPC, however, since LAPC does not require high liquid flow rates for ventilation. In the most rapid-cooling LAPC protocol used in this trial, ˙V_FTLV was 31 ml/kg per min—a low baseline value which permitted the addition of 10 times this minute-volume of gas ventilation (see Fig. 4, Trial II-4, 5). Moreover, since normal gas minute volumes were required to maintain normocapnia in Trial I, there is as yet no evidence for any CO₂ diffusion limitations caused by intrapulmonary PFC in LAPC. Possible reasons for this are discussed below.

Thermal-diffusion limits in TLV have not been studied per se, but their presence is suggested by the results of Shaffer and co-workers.[155] In their cat TLV model using a ˙V _FTLV of 75 ml/kg per min, a decrease in PFC inspiration temperature from 20 to 10°C (increasing the thermal gradient by a factor of 1.6) increased the cooling rate from −0.13 to −0.15°C/min. This small rate change represented a significant loss of efficiency. By contrast, in the present LAPC study using PFC at 4°C, there was no evidence of a thermal-diffusion limit at rates up to 4 FTLVs/min. Notably, in Trial I, where 100% of the V_FTLVr, and 40% of the ˙V_FTLVr of the cat TLV model was used, cooling rates for LAPC were more than three times those reported for cats subjected to TLV at 4.5 liquid breaths/min at 10°C.[155]

The possible quantitative presence of a thermal diffusion limit for LAPC at 4 FTLVs/min may be evaluated using a modified version of the concept of gas-exchange dead space (V_D). The respiratory system of a dog undergoing LAPC heat-exchange may be considered, by analogy with gas exchange dead space (V_D), to also contain a ‘thermal exchange dead space’ (V_Dtherm). Each thermal FTLV volume ˙V_FTLV of PFC then also contains a V_Dtherm , which by definition does not participate in heat-exchange. Thus, cycle thermal transfer efficiency E_f may be expressed as (V_FTLVr − V_Dtherm)  / ˙V_FTLV, and any measured value of mean E_f may be expressed as an equivalent mean V_Dtherm = ˙V_FTLVr  (1− E_f). For Trial I (E_f = 0.6, Appendix A for calculation), mean V_Dtherm was then seen to be 7.5±1.6 ml/kg, and in Trial II (using Eq. (6) E_f  Value = 0.40), V_Dtherm was 5.3±0.8 ml/kg (P = 0.072). The absence of an increase in V_Dtherm in Trial II vs. Trial I indicated that the size of V_Dtherm in these LAPC protocols was non-dynamic at time-scales of one FTLV cycle, providing evidence against the presence of a ‘thermal diffusion dead space’ (analogous to a CO₂ diffusion dead space) at these FTLV rates. In absolute terms, it may be useful to compare calculated V_Dtherm in the LAPC dog model to the expected physiologic gas-exchange dead space, VDCA, which in healthy animals is close to the dog anatomic VD = ~6.5 ml/kg.[193]

In thermal diffusion, as in gas diffusion, diffusion physiologic dead space would be expected to significantly add to anatomical dead space. However, the sum of mechanical-VD in the LAPC circuit (~1.5 ml/kg) plus the anatomic VD for dogs is found to be more than the calculated V_Dtherm in either trial in this study, leaving little room for a large heat-diffusion contribution to V_Dtherm. For these reasons it is suggested that the loss of cooling power observed in Trial II was not due to heat diffusion limitations, but instead due to a loss of efficiency effect similar to that seen with low tidal volumes in ordinary gas ventilation. In these terms, low FTLV volumes in LAPC result in an increase in ‘thermal dead space ventilation’ at the expense of PFC flow involved in active heat-exchange, resulting in a larger ‘wasted’ FTLV V_Dtherm / ˙V_FTLV. VD in heat transfer (V_Dtherm) that is analogous to VD in gas-transfer; in as much as all dead space is ‘diffusion dead space’ at long-enough time-scales. However, some of the mechanisms for diffusion modification of V_Dtherm are unique. By contrast with gas molecules, heat diffuses rapidly through device tubing into the PFC in the LAPC circuit dead space, and heat also diffuses directly through the tracheal wall into the anatomic-VD. Thus, heat diffusion from dead space liquid at sufficiently slow FTLV rates might be expected to have a pronounced effect on E_f in LAPC, due to slow heat-diffusion reduction in V_Dtherm.

Some evidence for such a process was found, though at FTLV dwell times too long to be of interest for rapid cooling. At the relatively small tc of Trials I and II, the calculated V_Dtherm was found to be ~VDCA; but in animal B, with a much longer t_c of 7 min, the V_Dtherm was only 2.6 ml/kg. The limit of this process was reached in animal A, in which the V_Dtherm of a single retained ‘breath’ of highly-oxygenated PFC fell to nearly zero after 10 min. Disappearance of V_Dtherm by thermal equilibration, estimated from individual cycle E_f variations in animals B and C, was estimated to occur with a half-time of ~5 min (data not shown).

This process was slow enough to be neglected when the duration of FTLV intervals (t_c) was less than several minutes.

Thus, at the FTLV rates of Trials I and II, a full-sized V_Dtherm of ~6 ml/kg appeared, and accounted for significant loss of cooling power at low ˙V_FTLV (e.g. Trial II where V_FTLV was only 8.8 ml/kg). The characteristic size of V_Dtherm at all but the slowest FTLV rates (~1 FTLV per 5 min) implies that the only thermally-efficient solution for performing LAPC at faster rates is maintenance of [˙V_FTLVr  / VDCA] or [˙V_FTLVr / V_Dtherm] ratios >3, in order to avoid excessive ‘wasted’

V_Dtherm  ventilation. This requires a ˙V_FTLV of ~18 ml/kg in dogs. In humans, where the anatomic-VD is <3 ml/kg, less than half the value for dogs, both the V_Dtherm, and therefore, most-efficient ˙V_FTLV values, might also be expected to be correspondingly less. In any case, it is clear that rapid-cooling LAPC techniques cannot wait for the relatively slow thermal equilibration of PFC within the anatomical VD, since equilibration in the remaining non-VD parts of the lung is so rapid (i.e. less than Trial II t_c of 16 s).

4.3.2. Possible synergy of combined gas and liquid ventilation in assisting mass (CO₂) and heat transfer

 The absence of expected heat-diffusion and gas-diffusion limitations in LAPC suggests that some assistive process for both gas and heat transfer through PFC in the peripheral lung may occur in LAPC. The authors’ fluoroscopic observations (made with the non-brominated and relatively radiolucent FC-75) have been that each gas breath in PLV produces a flash of fine bubbles which spread uniformly throughout the lung. As compared to the more familiar behavior of water, the low surface tension of PFCs (15 dyne-cm for FC-75, about 1/5th that of water) lowers the energy barrier to producing small bubbles in forced gas/liquid flows. Such

bubbles moving within small airways may induce eddies and turbulence in laminar liquid flows at small scales, contributing significantly to heat and mass (CO2) transport though PFC liquid by means other than diffusion.

It is hypothesized that the lack of bubble-induced turbulence in TLV may account for the large diffusion-dead-space for heat and CO₂ which seems to be present in TLV at even low breathing rates – an effect which is apparently absent in both PLV and LAPC.

4.4. Potential development of clinical LAPC

 Rapid cooling of the CNS has now become a primary goal in the clinical management of the post resuscitation syndrome [49] and potentially in the acute management of spinal cord injury.[194],[195],[196] Based on their work, Safar, et al., have noted that clinical implementation of mild resuscitative hypothermia, which was highly effective in the dog model of SCA, will depend upon the development of truly rapid MTH.[197]  A recent editorial in Stroke [83] commented on the striking ability of the combination of  (33-35^oC ) [198] and pharmacological pre-treatment to ameliorate ischemic brain damage in the rat middle cerebral artery occlusion model, and  then addressed similar concerns: “A problem for use of this technique for acute stroke therapy is that the time required to induce hypothermia in patients is likely to be considerably longer than for rats. […]. To substantially increase the rate of hypothermia induction in humans, it will almost certainly be necessary to use some sort of invasive procedure, such as a heat-exchanger, to cool the circulation.”

The technique of LAPC may eliminate the need for such invasive measures. For example, in the cited trial [199], rats were cooled from 37 to 33°C (−4°C) over 40 min, using surface cooling with ice packs. By contrast, the present study demonstrates cooling of the canine body core and brain by −4°C in less than 10 min.

4.5. Challenges Ahead

 4.5.1. PFC Selection

Development of clinical LAPC awaits identification of suitable PFCs for various LAPC applications. For example, the pharmaceutical PFC perfluorooctylbromide (Perflubron™ Alliance Pharmaceuticals) would presumably not be suitable for rapid LAPC cooling due to its freezing point of +6°C, but might be useful for slower cooling or for LAPHE facilitated re-warming. Some industrial PFCs have pour-points low enough to make them potentially useful as LAPC rapid-cooling media; however, most of these agents also have unacceptably high vapor pressures at 37°C or are not chemically defined in terms of chain length or even precise chemical composition (see Section 3: Perfluorchemicals). PFCs with such high vapor pressures exacerbate barotrauma by causing HNCL.

High vapor pressure PFCs may also increase the danger of long lasting lung collapse as a result of PFC, secondary to pneumothoraces, entering the pleural space and vaporizing (‘perfluorothorax’). FC-75, (formerly FX-80) was the first PFC used in liquid ventilation [10], but its relatively high vapor pressure (31.5 torr) makes it an undesirable LAPC agent. Assuming that a PFC with the desired biophysical properties is identified and produced to medical standards, LAPC should be easily scalable to adult humans. For example, the viscosity of FC-75 is similar to water [11], and under standard suction a 19 Fr. An adult pulmonary toilet catheter will remove FC-75 at ~2 l/min. As in the system described, a LAPC system may interface with a conventional gas ventilator system via a simple liquid-carrying catheter which extends through the endotracheal tube adapter suction port.

 4.5.2. Coronary Perfusion during LAPC

The effect of LAPC on coronary perfusion in the setting of ROSC following cardiac arrest due to myocardial infarction (MI) or in the presence of coronary artery disease (CAD) is unknown but is a possible cause of concern. One possible adverse effect is the potential compromise of the coronary circulation in CAD due to perfusion of the heart with profoundly chilled blood (i.e., blood temperature  @10°C below systemic temperature) leaving the pulmonary circulation and entering the coronary os. One of the authors (Darwin) has observed the onset of severe, acute angina in two hemodialysis patients with stable angina who were inadvertently dialyzed using very cold dialysate (Q_B of 250 ml/min at a blood temp of ~10° to 15°C) which resolved only when the dialysate temperature was increased to 30°C or above.

It is well established that exercise in cold environments, with associated inhalation of cold air, can trigger angina and lead to cardiac arrest in patients with coronary artery disease.[200],[201]  There has been considerable debate as to whether the cause of angina in this setting is due to cold air inhalation per se, or to the effects of a cold environment. It has been suggested that exposure to a cold environment is the primary factor in inducing cold weather angina, presumably by increasing peripheral vascular resistance resulting in an increase in cardiac workload at any given level of exercise [202],[203] in the same way that the cold pressor test produces acutely increased afterload and thus increased left ventricular wall stress.[204],[205],[206],[207] Hattenhauer and Neill [208] studied 33 male patients with coronary artery disease,11 of whom gave a positive history of angina when exposed to cold winter air. Seventeen of these patients were subjected to inhalation of cold air at -20°C for 4 min, and this resulted in angina at rest in four of these patients.

Cold air inhalation also produced angina in four of the seven patients who were paced at a heart rate which was well below the threshold for angina at room temperature. Cold air inhalation did not significantly increase myocardial oxygen consumption, or alter coronary blood flow as determined by the xenon clearance method. In a separate arm of the Hattenhauer and Neill study, cold air inhalation for 90 s in 18 patients produced no detectable-constriction of coronary arteries visualized angiographically. The investigators concluded from these findings that cold air inhalation induced angina could not be explained by an increase in cardiac work and myocardial oxygen consumption.

This study also demonstrated that there was no evidence of large or intermediate coronary artery or coronary arteriole constriction in response to the inhalation of very cold air. As a consequence, the authors proposed that cold air inhalation induced angina might be the result of constriction of minute coronary collaterals, or to other vessels compromising blood flow to potentially ischemic regions of the diseased myocardium. The work of Lassvik and Aveskog [209], confirmed the effects of cold air inhalation in inducing angina and failed to demonstrate any decrease in workload at either the onset of angina or at maximal workload during inhalation of moderately cold air (-10°C) in a room at 20°C. Dodds, et al., [210] evaluated 12 male patients with stable angina inhaling cold air (-8.8°C) during exercise to investigate if the vasoconstrictor peptides endothelin-l (ET-1) and angiotensin-II (AT-lI) played a material role in the etiology of this phenomenon. They concluded that neither ET-1 nor ATII had any significant role in the pathophysiology of cold air inhalation induced angina.

In contrast to Hattenhauer and Neill, and Lassvik and Aveskog, these investigators documented decreased myocardial oxygen consumption during peak exercise in cold air inhalation and concluded that the cause of this was a centrally operating mechanism such as a reduction in coronary flow. These investigators noted that the response to cold air inhalation was biphasic, and posit that the initial response; earlier onset of angina during exercise while breathing cold air, was due to activation of cold receptors in the upper airways stimulating a systemic increase in peripheral vascular resistance; and thus the observed accompanying rise in blood pressure and increased cardiac workload (i.e., the cold pressor test response). The secondary response; reduction in total exercise time, which occurred at a significantly lower rate-pressure product compared with the same patients breathing ambient temperature air, was thought to be due to peripheral reflex responses. Thus, these investigators hypothesize that the early onset of angina during exercise is due to sympathetic stimulation from inhaled cold air, but as exercise continues, central mechanisms play an increasing role in the pathophysiology of cold air inhalation induced angina.

The implications of cold air induced angina for LAPC in the setting of coronary artery disease are troubling and certainly bear careful investigation in animal models of compromised myocardial circulation. Patients in all of these studies developed significant ST depression (³ 1 mm) concurrent with the onset of cold air inhalation induced angina, suggesting clinically significant myocardial ischemia. What is unclear is whether the concomitant profound reduction in myocardial temperature seen in LAPC (both transient and long-term) will be protective against any perturbation in myocardial perfusion induced as a result of the sympathetic or central effects of cold FTLV.

Such a protective effect is suggested by the well established finding that ‘cold’ (~34°C) hemodialysis (HD) is protective against both intra-dialytic hypotension and angina [211], [212],[213],[214], as a result of sympathetic stimulation from the return of ‘cool’ blood to the systemic circulation. [215],[216],[217] In addition to its protective effect on hemodynamics during aggressive ultrafiltration, cold HD also attenuates the hypoxemia leucopoenia, [218] and the production of Complement 5a induced by blood exposure to the dialyzer membrane.[218],[219] It should also be noted that catecholamine administration (mimicking profound sympathetic stimulation) in the form of epinephrine is still a mainstay treatment of ventricular fibrillation and aystole in SCA.[59, 220],[221]

Finally, it is essential to point out that mild and even moderate induced hypothermia not only do not interfere with defibrillation from cardiac arrest, but actually greatly facilitate conversion of VF to perfusing NSR.[222],[223],[224],[225],[226]

 4.5.3. Potential for Regional (Myocardial) Overcooling

Under the low flow conditions of CPR, the possibility exists that due to thermal compartmentalization myocardial temperature could be reduced to below the fibrillation threshold or to adversely affect contractility. In the laboratory and the clinical setting moderate (28-32 ^oC ) and deep (10-22 ^oC) hypothermia are known to induce both benign and malignant cardiac arrhythmias; [227],[228] and  ventricular fibrillation is the most common cause of death in accidental hypothermia. [229],[230] In addition to the arrythymogenic effect of deep hypothermia (J waves, prolonged PR, QRS, QT intervals, and atrial arrhythmias) there is evidence that defibrillation becomes increasingly problematic as myocardial temperature decreases below 30^oC.[231],[232],[233]

The temperature of blood leaving the pulmonary circulation under normal flow conditions (during spontaneous circulation) within 5 minutes of the start of LAPC can reach temperatures of 17^-20^oC (see Figure 2-14, FC Suction Reservoir Temperature; this temperature closely approximates the PA blood temperature during the FTLV cycle). This is benign under high flow conditions because heat is being rapidly and continuously transferred between body compartments (Figures 2-5 – 2-7). However, under low flow conditions, and especially in the presence of pharmacologically induced severe peripheral vasoconstriction, rapid, selective core over-cooling could occur.

Disproportionate cooling of the body core happens under normal conditions in humans given large volumes of intravenous fluid chilled to 4^oC, and this is the basis for cold IV fluid induced hypothermia for cardiac arrest.[234],[235],[236],[237] This surprisingly durable core cooling, well beyond that predicted on the basis of calculations for the whole body, occurs because the thermal distribution volume in humans given rapid  cold IV infusions turns out to be much lower than total body volume. The result is that chilled IV fluids are ~3 times more effective in inducing hypothermia than suggested by all-compartment equilibrium calculations.[234]  Several explanations have been offered for this apparent thermodynamic inconsistency; most plausibly that peripheral vasoconstriction and inherently slower kinetics of heat exchange between central and peripheral compartments act to keep core temperature below the all-compartment equilibrium for at least 60 min after the conclusion of the cold IV infusion [238],[239] which is long enough for external cooling to begin contributing to cooling and for endovascular cooling to be initiated under ideal conditions.

These findings provide additional reason for vigilance in avoiding excessive myocardial or core cooling during CPR and suggest that a surrogate for myocardial temperature be sought and that the use of warmer PFC FTLVs be explored; trading off rapidity of temperature systemic reduction against the danger of over-cooling the heart.

 4.5.4. Overcoming Increased Intrathoracic Pressure and Preserving CO

Following the development of active compression decompression CPR (ACD-CPR) by Cohen, et al., in 1992 [240] the critical importance of maintaining negative intrathoracic pressure during the decompression phase of the CPR duty cycle has become increasingly understood.[241],[242]  There is a rapidly growing body of both animal and clinical CPR research documenting improved survival and decreased neurological morbidity when the intrathoracic pressure is kept negative during the  decompression (release of chest compression) phase of CPR by the use of inspiratory impedance threshold devices and active ACD-CPR.[188],[243],[244],[241] Similarly, there is accumulating evidence that the increased intrathoracic pressure that results from excessive positive pressure ventilation (PPV) during CPR dramatically reduces CO and causes increased morbidity and mortality.[245],[246],[247],[248],[249],[250]

In 2004, Yannopoulos, et al., reported the development of a device which allows for the continuous application of negative intrathoracic pressure (Figure 2-15) by applying controlled suction to the airway.[187]  This device, called the intrathoracic pressure regulator (ITPR) allows PPV to be delivered as needed during ACD-CPR, while maintaining negative intrathoracic pressure at all times when PPV is not being administered. The device effectively transforms the thoracic cavity into a low negative pressure (vacuum) chamber; increasing venous return from the body and consequently increasing preload and cardiac output. The ITPR also markedly increases CPP while at the same time decreasing intracranial pressure (ICP). In CPR ICP is typically elevated from the basal value of 12-16 mm Hg to 22-30 mm Hg (as the result of pressure transmission by blood in non-valved veins and by transmission of intrathoracic pressure via the cerebrospinal fluid) further compromising already inadequate cerebral perfusion.[251] Reduction of ICP during CPR has been shown to improve both survival and neurological outcome in an animal model of CPR.[252]

Figure 2-15: Prototype ITPR (Advanced Circulatory Systems, Inc.) in position in a typical bag-vale resuscitator – ET tube set-up.

 The ITPR has been shown to dramatically improve gas exchange, hemodynamics, blood flow, vital organ perfusion, and short-term survival rates during VF cardiac arrest in a porcine model of SCA and CPR [187] The ITPR is able to not only overcome the high intrathoracic pressures. associated with CPR (45 to 55 mmHg or ~61 to 75 cmH₂0 [253]) but to both create and sustain negative intrathoracic pressure (determined indirectly by measuring the ET tube pressure) continuously during prolonged periods of ITPR-CPR, even in the presence of induced hypovolemia.[189]   In hemorrhaged (hypovolemic) pigs, Yannopoulos, et al., were able to sustain CPP at >15 mm Hg (the accepted threshold for successful defibrillation in human SCA) and the isovolemic VF animals in the study maintained CPP at >25 mm Hg throughout the full 15 minutes of ITPR-CPR. In both groups, ETCO₂ was consistently maintained >25 mm Hg, and the 1-hour survival was 100%, as contrasted with 10% in control animals receiving AHA standard CPR (P = 0.0001).

By comparison, after 3 minutes of conventional CPR the control animals had a mean coronary perfusion pressure of <15 mm Hg and all had developed pseudo-respiratory alkalosis indicative of the V/Q mismatch of standard CPR.[254]  Blood gases in VF animals were strikingly preserved during ITPR-CPR; paO₂, which was 96±2 mm Hg at baseline, was   214±12.37 mm Hg after 10 min and 198±6.75 mm Hg after 15 min of ITPR-CPR. These findings would seem to suggest that ITPR-CPR may be reducing or eliminating the pulmonary edema that accompanies CPR and the high intrathoracic (and thus pulmonary arterial and venous pressures) generated during CPR.  ITPR is similarly effective at improving both hemodynamics and survival in a swine model of severe hypovolemic hypotension.[255]

The use of an ITPR during LAPC offers the prospect of reducing or abolishing the undesirable hemodynamic effects of FTLV with PFC. These effects, while not clinically significant in the healthy animal with spontaneous circulation undergoing LAPC (and the ability to dynamically respond to alterations in preload, CVP and SVR), may be unacceptable in the setting of cardiac arrest and CPR.

While it is clear that FTLV with PFC reduces MAP and elevates the mean CVP,[2] the extent to which these effects will reduce CO during CPR is unknown and will necessarily require further experimentation in animal models of SCA and CPR that closely approximates those experienced in humans under clinical conditions. The mechanics of CO and perfusion in CPR are radically different than those that pertain under conditions of spontaneous circulation.[256],[257],[258] In the healthy beating heart, modest increases in CVP (~5-15 mm Hg) result in increased cardiac output via the Frank-Starling mechanism (Starling’s Law of the Heart). Increased CVP increases left ventricular diastolic end pressure (LVEDP) increasing ventricular volume and stretching the ventricular myofibrils. Myofibrillary stretch results in sarcomere extension thereby increasing the affinity of troponin C for calcium, causing a greater number of cross-bridges to form within the myofibrils; this increases the contractile force of the heart increasing CO. This biochemical mechanism is not operational during cardiac arrest and CPR. Indeed, as outlined in the discussion below, little of the mechanics of perfusion under normal physiological conditions pertain in the setting of CPR.

4.5.5. Consideration of the Mechanics of Blood Flow in CPR and Implications for LAPC

During CPR in humans ventricular volume is not a primary determinant of CO, at least not in an appreciable fraction of patients undergoing CPR.[258] Despite the fact that it has been forty-eight years since the invention of closed chest CPR – the mechanics of blood flow during CPR in humans have not been definitively established – and a great deal of controversy surrounds the subject. Broadly, three mechanisms of antegrade blood flow have been proposed in (human) CPR:

• The Direct Cardiac Compression (Cardiac Pump) Theory posits that mechanical compression of the ventricles between the sternum and vertebral column creates a pressure gradient between the ventricle and the aorta (or pulmonary artery in the case of the right ventricle); as a consequence the mitral and tricuspid valves are closed, and blood is moved antegrade out of the ventricle. The ventricle then refills during the decompression phase of CPR and the process is repeated with each compression cycle.[259]  The cardiac pump theory was most definitively challenged with the publication of case data documenting the ineffectiveness of CPR in patients with flail chest. This could be reversed only be restoring elastic recoil to the chest by binding it; indicating that it was the generation of a net negative intrathoracic pressure upon recoil of the chest during the decompression phase of CPR that was essential for blood flow.[260]
• The Thoracic Pump Theory asserts that chest compression increases intrathoracic pressure forcing blood to flow from the thoracic to the extrathoracic circulation[3]. Retrograde flow from the right heart to the systemic veins is prevented by the venous valves; with the heart serving only as a passive conduit, and having no function as a pump.[261], [262] In the thoracic pump paradigm blood circulates because increased intrathoracic pressure is transmitted more or less equally to all of the intrathoracic vascular structures [256] and the atrioventricular valves remain open during the compression phase of CPR.[263]  However, these intravascular pressures are not equally transmitted to the extrathoracic arterial and venous beds, thus creating an extrathoracic arterial-venous pressure gradient resulting in antegrade blood flow during the period of high thoracic pressure. During the decompression phase of CPR, blood flows into the lungs as a result of the extrathoracic venous-to-intrapulmonary pressure gradient.  Angiographic [256] and echocardiographic studies in dogs documented static ventricular volumes during CPR [262], and case reports of human patients with cardiac tamponade recovering successfully after closed chest CPR both supported the thoracic pump mechanism as the explanation for antegrade flow in CPR.[264]
• In the Lung Pump Theory, as in the thoracic pump theory, the increased intrathoracic pressure during the compression phase of CPR is similarly transmitted equally to all intrathoracic vascular structures and there is insignificant regurgitation of blood from the pulmonary artery into the right ventricle and vena cavae until the pulmonary valve closes. [265] Thus, blood under pressure within the pulmonary vasculature will flow out of the lungs via the left side of the heart.[263],[266] During the decompression phase of the cycle the intrathoracic pressure drops below that present in the extrathoracic vasculature and blood flows into the thorax via the vena cavae and aorta. The pulmonary vascular volume is replenished by blood flowing from the right side of the heart through the open tricuspid and pulmonary valves. [266], [267] Retrograde aortic flow is prevented by competent closure of the aortic valve.[268],[265]

Transthoracic echocardiographic studies reported in the early 1980s during CPR in humans supported the thoracic pump theory.[268],[265] However,more recent studies employing transesophageal echocardiography (TEE) have concluded that because the mitral valve was observed to close during the compression phase, and open during the decompression phase, and the left and right ventricular volumes decreased during the compression phase, the  mechanism of antegrade flow during CPR was consistent with the direct cardiac compression theory.[269],[263]

Perhaps the most likely explanation of these seemingly incompatible findings is that all three mechanisms are responsible for antegrade flow, but not in every patient. In other words, the mechanics of forward flow may differ from patient to patient.  In fact, in the 1981 paper in which they proposed the thoracic pump theory of antegrade blood flow in CPR, Weisfeldt and Chandra stated, “It is not essential in the human to think about these mechanisms in an exclusive fashion. Direct cardiac compression is useful when possible; when it is not potent enough to maintain cerebral perfusion, manipulation of intrathoracic pressures would likely have a favorable additive effect on carotid blood flow.”[261]

In support of this view is the study by Ma, et al., which evaluated 17 patients undergoing CPR using TEE to measure both pulmonary and trans-mitral flow.[270]  Five of the 17 patients demonstrated closure of the mitral valve during the compression phase of CPR, with associated mitral regurgitation and forward aortic flow occurring which is consistent with the cardiac pump theory.

In the remaining 12 patients, the mitral valve remained open during both the compression and decompression phases of CPR with maximal antegrade mitral flow occurring during the compression phase of CPR. Eight of these 12 patients demonstrated antegrade mitral flow during the compression phase that was accompanied by antegrade pulmonary vein flow; consistent with the classic ‘thoracic pump’ mechanism. The last 4 patients in this study evidenced retrograde pulmonary vein flow concurrent with antegrade mitral flow during the compression phase; which is most consistent with lung pump theory as the primary cause of antegrade flow, at least in these patients.

5.0 Review of the Literature on LAPC

From the foregoing, it should be easy to understand the difficulty of establishing by experiment, let alone extrapolating on any theoretical basis, what the hemodynamic impact of FTLV or LAPC will be under clinical conditions in humans undergoing CPR. Similarly, the effect of LAPC on regional myocardial blood flow, myocardial irritability, coronary electrophysiology, and susceptibility to defibrillation will require further laboratory and, likely, experimental clinical investigation as well. Since publication of the 21CM/CCR LAPC research in 2001 there have been 4 subsequent studies to evaluate various aspects of the utility and safety of LAPC for application in CPR, or as a therapy in MI.

The first of these studies was published by Hong, et al., in 2002 and announced, “Our study is the first to demonstrate an induction of hypothermia by adopting PLV (no need of an extracorporeal circuit) and 0°C PFC.” [271] failing to cite the previously published work of either Darwin, et al., or Harris, et al. [272] These investigators attempted to validate the utility of LAPC using FTLV to achieve rapid reduction in core temperature and also studied the physiological impact of the procedure. The cooling rate achieved appears to have been ~0.11°C/min and this slow rate was almost certainly an artifact of the small volume of PFC used for each intrapulmonary exchange (~20 ml) and the small number of exchange (10 – 12 exchanges over 38 minutes). In fact, the cooling rate was faster in the animals in this study that were cooled externally by repeated application of ice water slush (0.17°C/min).

The most interesting result of this investigation were that MAP, mean PA pressure, HR, and CO, CBC and lactate of the LAPC treated animals were not significantly different than in the surface cooled animals. One variable that was altered in the LAPC group was an increase in pulmonary vascular resistance which appeared immediately after liquid loading and did not begin to return to baseline until halfway through the LAPC period. It is also remarkable that 1 animal in both the LAPC and the surface cooled groups (n = 7 for both groups) developed pulmonary hypertension and lethal pulmonary edema. The authors speculate that the observed pulmonary hypertension could have been a result of pulmonary venous constriction and/or increased pulmonary microvascular blood sludging due to the profound local cooling of lung vasculature resulting from instillation of 0°C PFC. They also raise the possibility that the observed elevation in PAP may have resulted from the mechanical effects of the PFC load; but offer no explanation for this.

Otherwise, hemodynamics were unaffected by LAPC. The paCO₂ became progressively elevated during the hypothermic interval in the LAPC group, perhaps due to inadequate PEEP or inappropriate parameters of mechanical ventilation in the face of evaporating PFC (which was not replenished during the course of the experiments).

The second study to be published was by Ko, et al., in 2002.[273] The purpose of this study was to evaluate the effect of PLV on pulmonary blood flow under low blood flow conditions designed to simulate those encountered during CPR in order to further validate the use of LAPC as an adjunct to cardiopulmonary cerebral resuscitation. Isolated perfused rat lungs were subjected to 20 min of PLV with room temperature Perflubron™ and segmental (i.e. pre-capillary, capillary, and post-capillary) hemodynamics were studied at a perfusate flow rate of 6 ml/min (~5% normal cardiac output [274]). Lungs received either gas ventilation or 5 or 10 ml/kg PLV. Segmental pressures and vascular resistances were determined, as was transcapillary fluid flux. PLV at both the 5 and 10 ml/kg Perflubron™ dose produced no detectable changes in pulmonary blood flow or in transcapillary fluid flux and the investigators concluded that, “These data support further investigation of this technique as an adjunct to cardiopulmonary resuscitation.”

Figure 2-16: Dramatic reduction in myocardial infarction size in rabbits rapidly cooled to ~34 by the use of TLV LAPC: infarct volume was 4.0 ± 5% in the LAPC groups vs. 37.7 ± 1.3% in the normothermic gas ventilated group (p = 0.001). Redrawn from Tissier, et al.[275]

Unfortunately, this study did not explore the effect of instilling cold PFC into the lungs, nor did it employ FTLV. Because it was an ex vivo study it was not possible to determine the effects of PFC loading, cold or isothermic, on the sympathetic response, coronary blood flows or other physiological parameters of concern in LAPC.

In 2007 a study by Tissier, et al., [275] used a rabbit model of evolving MI to evaluate the efficacy of TLV LAPC administered during the ischemic interval in achieving rapid core cooling to reduce infarct size and provide myocardial protection [276],[277],[278] even after substantial delay  [279] and when induced during reperfusion. [280],[281] It is well established that intra- and post-ischemic myocardial hypothermia dramatically reduces infarct size in animal models of MI and this observation has recently been extended to humans in the clinic.[282]

These investigators note that in the US the time to coronary revascularization following infarction is 120 min in 41.5% of patients admitted during off-hours and that 27.7% of patients presenting during normal business hours still averaged delays to revascularization in excess of 120 min.[283]  Rapid induction of hypothermia in these patients could be effective in providing substantial myocardial salvage during the delay between presentation and revascularization. This study demonstrated a remarkable reduction in infarct size in the LAPC treated group; 4.0 ± 5% vs. 37.7 ± 1.3% in the normothermic, gas ventilated animals (p = 0.001). The cooling rate achieved with TLV was 1.32°C/min; 6.6°C in 5 minutes. The effectiveness of TLV LAPC is not consistent with results obtained in larger animals in this author’s experience. It may be that the relatively short distances between the large and small airways and the very small diameter of even the largest airways in the rabbit as compared with the dog or human, may have improved the efficiency of heat exchange.

The most recent study by Staffey, et al., [284] is more on-point and provides considerable reassurance that cold PFC loading of the lungs during CPR and subsequent resuscitation is not only not deleterious, but in fact is markedly beneficial. In this study swine were subjected to 11 min of VF and treated either with a static intrapulmonary infusion of PFC chilled to -12 C to ~75% of total lung volume (40 ml/kg), static infusion of isothermic PFC (33^oC) under the same conditions, TLV with -15^oC PFC at 6-cycles per minute during 10.5 minutes of the arrest interval, and a control group that was arrested with no intervention during the ischemic interval. The animal were then given AHA standard CPR with defibrillation and advanced cardiac life support (ACLS). The specific objectives of this study were to determine what the effects LAPC; tidal and static, administered during the period of cardiac arrest would be on hemodynamics, CPP, gas exchange, defibrillation, and hemodynamic stability during a 1 hour period of evaluation post ROSC. The endpoint was ROSC for 1 hour without ionotropic support.

The global objective of the study was to determine if LAPC could be used to rapidly induce cardiopulmonary hypothermia during the arrest period as opposed to cerebral or systemic hypothermia. Work by Rhee, et al., and Boddicker et al., also using swine models, had previously demonstrated that systemic hypothermia induced before resuscitation from cardiac arrest resulted in more rapid and consistent defibrillation from VF as well as earlier and more stable ROSC. Since the purpose of this study was to cool only the thoracic viscera to determine the effect of local cardiopulmonary hypothermia on resuscitation; ‘targeted cardiopulmonary intra-arrest moderate hypothermia (28-32^oC),‘ LAPC was not continued beyond the period of cardiac arrest.

Both static and TLV LAPC succeeded in reducing the pulmonary artery temperature to the desired temperature of 34^oC by 6 and 10 min post arrest, respectively. Eighty two percent of the animals in both LAPC groups were resuscitated successfully and survived the 1 hour evaluation period without pharmacological support, as contrasted with only 27% of the controls animals (p = 0.03).  Interestingly, 73% of the isothermic  TLV swine achieved and maintained ROSC as opposed to 27% of the swine in the control group (p = 0.09), suggesting that the presence of PFC in the lungs during either the arrest or resuscitation interval may improve the odds of successful defibrillation.

Figure 2-17: Results of the study by Staffey, et al., to evaluate the effect of cold and isothermic PFC infusion and TLV on resuscitability of swine after 11 min of electrically induced VF cardiac arrest. PFC loading, and in particular cold PFC loading or TLV improved 1 hour stable hemodynamic survival by 73% versus 27% in controls. Infusion of isothermic PFC to near vital capacity also showed a trend towards increased survival. Redrawn from Staffey, et al.,[284].

Chamberlain et al., have recently proposed that dilation of the right ventricle (RV) that occurs after the first ~5 min of cardiac arrest results in compression of the left ventricle (LV) within the confines of the pericardium. The effect of this would be to reduce myocyte stretch and reducing the contractile strength of the left ventricle in response to defibrillation (see discussion of the Frank-Starling curve above). They posit that an arrested heart that has a reduced contractile state, and in which left ventricular volume is reduced by the right ventricle over-distended with venous blood will not be able to initiate effective contraction even if coordinated electrical activity is restored. Distension of the RV occurs post-arrest due to centrally mediated sympathetic contraction of the vasculature, as well as movement of blood into the venous circulation as arterial and venous pressure equilibrate; this is a commonplace finding at both necropsy and autopsy in normovolemic subjects.

Unloading the RV prior to defibrillation results in an immediate improvement in successful ROSC independent of any known metabolic that might accrue from ~10 to ~15 sec of coronary perfusion that might also result. Chamberlain, et al., believes that it is the decompression of the left ventricle and restoration of a more physiologic morphology that facilitates or even enables defibrillation under these conditions. Instillation of a large volume of dense PFC may antagonize distension of the RV and prevent LV volume loss and compression of the LV to below the threshold required to established effective contractile activity in response to defibrillation. PFC to vital capacity (VC) should effectively preclude this post-arrest pooling of blood in the thoracic caval and pulmonary vessels and may act to preserve left ventricular morphology during prolonged cardiac arrest.

Especially encouraging findings from the Staffey, et al., study were the absence of any noticeable adverse hemodynamic impact from either isothermic PFC loading or from cold PFC loading or cold TLV. MAP, CVP, CPP, pH and blood gases were not statistically different between the four groups of animals in the study.

These four studies of LAPC aimed at answering questions bearing on the feasibility of LAPC in MI, SCA and CPR are very encouraging. They indicate that LAPC is being seriously considered as a potential therapeutic application in humans. That this research is being undertaken in independent academic and medical research both in the US and elsewhere is especially heartening and would seem to indicate that the enormous therapeutic potential of LAPC-induced hypothermia has been successfully communicated.

6. Conclusions

LAPC is capable of inducing hypothermia in a fraction of the time that it takes to prepare a patient for cooling via CPB. In addition, automated LAPC need not have the spatial and technical restrictions of the hospital setting. Although relatively simple methods of continuous arterio-venous shunt heat-exchange that do not require a blood pump or carry the most of the risks attendant to CPB have been described which might be potentially applicable in the field [285], these techniques also have the drawback of requiring skilled personnel for cannulation of a major artery and vein. Intracaval heat exchange catheters can reduce core temperature,  but only at rates of ~1.46 ± 0.42°C/h [286]; far too slowly to achieve the maximum benefit from post-reperfusion hypothermia

Figure 2-18: Large negative excursions in airway pressure occurred during suctioning of PFC at the end of most FTLVs when this operation was carried out manually (A). This occurred because when the PFC suction catheter was no longer filled with PFC, evacuation of gas from the airways occurred very rapidly owing to the much lower viscosity of gas compared to PFC. It was impossible for the operator to anticipate when the last of the bulk liquid would be removed, or to react rapidly enough when this occurred. Computerized sensing and control eliminated this potential source of baro-injury (B).

By contrast, LAPC may be a candidate for a much wider range of emergency field-uses in civilian and military settings since the primary technical skill required to initiate LAPC in the field is endotracheal intubation; a skill possessed by paramedical personnel throughout Canada, the U.S. and Europe. LAPC has also potential as a very rapid treatment for heatstroke and malignant hyperthermia. While  not the subject of this report, LAPHE clearly also holds promise for core rewarming in severe hypothermia, although an absolute maximal PFC temperature of 42°C would in theory limit the re-warming rate to about one-third of that possible in cooling.

Computer control of both gas and FTLVs is effective at eliminating excessive positive and negative airway pressures during LAPC (Figure 2-16) and computer control can easily be extended to encompass cooling rate, depth, and duration and can also be extended to control gas ventilation, as necessary.

Whether used inside or outside hospital, successfully implemented LAPC might more generally serve as a neuroprotective bridge [287] in order to gain time for more technically sophisticated supportive or definitive treatment (e.g. neurovascular thrombolysis or interventional thrombectomy, emergency CPB, spinal cord decompression or definitive management of hemorrhagic shock in trauma or surgery).

Although some modalities of liquid ventilation have been clinically evaluated [288], the safety parameters of rapid and cold liquid delivery to the lungs remain to be determined. As noted in this study, LAPC can cause pulmonary injury. The mechanism of such damage suggest by the location of lesions indicates that both barotrauma (dependent lung) and volu-trauma (nondependent lung) are the primary, if not the sole factors. It has been observed that LAPC causes little permanent lung injury in long term survival animals. Similar pathology seen in lungs exposed to either isothermic or ~4ºC LAPC in the present study (data not shown) suggest that thermal/chilling-injury per se is not the major insult. Although more subtle biochemical and immune problems secondary to hypothermia itself are suggested by reports from some longer duration studies of MTH (pneumonia and sepsis), it is not clear that the short duration of treatment necessary to achieve the benefit of post-resuscitative MTH will pose such problems. It is hypothesized that the pulmonary injury observed in LAPC may be reduced with better control of LAPC pressure and volume limits, and by use of PFC liquids having more physiologically suitable properties.

A significant, unresolved concern is the potential negative impact of LAPC on myocardial perfusion and the danger of overcooling the heart during the low flow conditions of CPR with possible adverse effects on achieving ROSC and maintaining a stable rhythm following defibrillation. As already noted, these questions can only be resolved with further study.

Acknowledgements

 The authors thank Saul Kent and William Faloon for support, and Casey Brechtel for helpful discussions. Several of the authors have applied for LAPC device patents. This trial was funded by a grant from the Life Extension Foundation (Hollywood, FL).

Appendix A

 (The abbreviations contained in this appendix are also reproduced in the table of abbreviations and acronyms at the beginning of this document.)

 A.1. Abbreviations and notation

 In the text, volumes (V) are given in ml/kg, and flows (dV/dt =V’= ˙V ) in ml/kg per min. Since all V and ˙V are expressed in mass-specific (per kg animal) terms, derived quantities DQ and Cm are automatically mass-specific. C_m and C_vf are given in calories / (g or ml) per °K for easy comparison with water.

PFC = perfluorochemical; hydrogen-free organic molecule in which most of the peripheral atoms are fluorine.

TLV = tidal liquid ventilation is a modality in which liquid completely fills the lungs and ventilator.

PLV = partial liquid ventilation is a modality in which all gas exchange is via gas ventilation, with ~1/2 FRC of PFC liquid present in the lungs to recruit dependent lung in ALI or ARDS.

LAPC = liquid assisted pulmonary cooling is a heat-exchange modality in which ventilation occurs via both gas and liquid ventilation proceeding concurrently.

T_tym = tympanic temperature

T_art = arterial temperature

T_ven = central venous temperature

T_rec = rectal temperature

DT_enet DT_tymp = resulting from LAPC, after equilibration at t=40 min

T_FTLV  = FTLV cycle infusion time

t_s = FTLV cycle suction time

t_c = FTLV cycle period (=tinftc +ts)

V_FTLV = single-cycle PFC FTLV infusion volume=tinf V_ inf

 V_S = single-cycle PFC FTLV suction-return volume

V_D  = ventilatory dead space (any type)

V_DCA = expected gas ventilation VD=sum of circuit (mechanical) VD plus anatomic VD

V_DTherm = thermal or heat-exchange VD (ml/kg, in reference to liquid PFC infusion)

˙V _inf = PFC infusion rate (set to _50 ml/kg per min in Trials I and II)

˙V_FTLV = effective PFC FTLV rate=LAPC liquid FTLV minute-ventilation (ml/kg per min) = V_FTLV/ t_c

˙V_g = gas minute-ventilation (ml/kg per min) m animal mass Ch heat capacity

 C_T = total heat capacity of the animal (= mC_m)

m = mean mass-specific heat capacity of the animal ( = DQ_T / DT_e)

C_vf = volume-specific heat capacity of FC-75 (mean of 0 and 25°C values) = 0.45 cal/ml per °K

DQT = total heat removed during LAPC (kJ/kg animal) = S DQ_c

DQc = heat removed during one FTLV cycle?????

E_f = mean cycle heat transfer efficiency=mean of [DQ_c / (theoretic DQ_c (max)] for all cycles in a single experiment

n = number of FTLV cycles in LAPC experiment

S = sum entire quantity following, for all cycles i = 1 through n

 T_inf   = PFC infusion temperature

T_S = PFC suction removal temperature (time-averaged PFC suction flow temperature)

T_SM= PFC mixed suction return-volume temperature (temperature of mixed VS)

 A.2. Thermal kinetics

 During LAPC cooling and equilibration, the blood and tympanic temperature changes in the animals were modelled by a simple five compartment model (Figure 2-8). During the initial ~100 s of LAPC (value used as empiric time mark), full development of heat-exchange behavior is established between the lungs, blood volume, and the thermal core of the animal, as suggested by the characteristic half-times for equilibration of these systems (see below).

Modelling of cooling during LAPC:

After the initial ~100 s of cooling, the data for tympanic DT(t) = DT_tym during LAPC in Trial I and II were modelled by a single time-constant exponential decline.

Mean DTtym data for each trial from times t=100 to 1080 s were fit using (Eq. (1)).

DT (t) = T₁₀₀ + DT_k [1− exp (−t / t_o) ],

DT(t), total T_tym change from baseline T_tym at start of LAPC; t, =time in seconds after empiric time mark, 100 s after start of LAPC; T₁₀₀, observed DT at empiric time mark, 100 s after start of LAPC; DT_k, observed temperature-interval constant, specific to each LAPC method; to, observed natural-base time-constant, in sec (t_o = halftime / ln 2).

Best-fit values for Trial I data were: T₁₀₀ = −0.52 ± 0.02°C; DT_k = −11.2 ± 0.02°C; and to = 1064 ~3 s. Trial II values were T₁₀₀ = −0.24 ± 0.02°C; DT_k = −8.14 ± 0.02°C; and t_o = 1107 ± 5 s. The relatively long time-constant associated with this thermal phase, which was similar in the two trials, presumably reflects the long time-constant (~700 s, see below) associated with heat transfer from the thermal core of the animal to the thermal periphery; thus the exponential phase represents full development of heat exchange between the LAPC cooling device and the entire animal. The final linear segments of cooling occurring after this phase, measured at −0.29°C/min (Trial I) and −0.21°C/min(Trial II), represent the final relatively simple state which exists after heat exchange equilibrium between cooling device and animal has been fully established.

Cooling in blood and tympanic sites during LAPC, and thermal evolution in these sites during equilibration phase after LAPC was discontinued, was in accordance with a five-compartment thermal model (Figure 2-8). In this model, the tissues of the animal are divided into three thermal compartments, corresponding loosely with the vascular system, the thermal core, and the thermal periphery.

Modelling of equilibration after LAPC:

Perfusion-driven convection is the major heat transfer mechanism in very rapid systemic cooling processes. This fact allowed T_art and T_ven changes to be used to quantify some features of heat transfer between body thermal compartments during the equilibration period after LAPC. The mean T_art curve in Trial I increased nearly linearly (R² = 0.9976) for 12 s after the end of LAPC, rising at a rate of 7.9°C/min. After this initial 12 s, T_art departed from linearity (Figure 2-6 inset), and was modelled by the sum of three exponential terms with respective time constants (t₀) of 12 ± 0.4, 102 ± 2 and 701 ± 8 s. These t₀ times differed to a large enough extent that their respective influences could be considered to be controlling over discrete time periods of about twice their value. Thus, the four equilibration phases seen after the end of LAPC lasted approximately 12, 24, 200, and 1400 s (23 min), respectively and represented 34, 14, 25, and 27% of the 5.1°C rise in T_art during equilibration after LAPC.

These data may be interpreted as follows: during each phase of the equilibration process, one or more thermal compartments in the animal equilibrated with the next-most closely-connected compartment (Figure 2-8). Afterwards, the newly captured compartment(s), as part of a larger unit bound together by blood mediated convection, equilibrated with the next-most closely connected compartment, and so on. The 12 s linear first equilibration phase (Figure 2-7, inset) most likely represents development of heat transfer from the lungs to local pulmonary blood flow. This phase was not associated with blood recirculation since it was seen as a rise in T_art but not T_ven. The second equilibration phase (duration ~24 s) was characterized by an increase in dT_ven / dt to the value of d_Tart  /dt, indicating that the lungs, blood-volume, and certain other well-perfused viscera, such as the kidneys, were now evolving into a single thermal system. Since the observed to for this phase was 12 s, less than the animal’s mean circulation time (= cardiac output / blood volume ~30 s), this process appeared to be driven by blood circulation via the most rapid paths (e.g. renal circulation). Such short paths for circulatory heat transfer were evident in the relatively small lag times (10.4 ± 6.9 s) noted between T_art and T_ven changes in these animals.

During the first two equilibration processes, the pulmonary circulation added thermal potential to the blood-volume more rapidly than it could be removed by the systemic circulation. By the end of the second equilibration phase, however, lung-to-blood heat transfer no longer dominated, and the gap between T_art and T_ven was set by the magnitude of heat transfer from the circulating blood volume to the tissues that comprise the ‘thermal core’ of the animals. In this third equilibration phase (duration ~200 s), the viscera and blood-volume, as a unit, equilibrated with the remainder of the ‘well-perfused’ tissues of the body (thermal core, comprising about 70% of the animal’s heat capacity). Heat capacities for thermal compartments are calculated below. The t_o for this process is seen most directly in the ~2 min. delay between maximal DT_ven and maximal DT_tym (Figure 2-7).

Finally, heat transfer within well-perfused tissues fell to a new minimum, and the T_art to T_ven gap decreased to a value set by the fourth equilibration phase (duration ~23 min) during which the well-perfused tissues equilibrated, as a unit, with a succession of the more poorly-perfused compartments, e.g. gut contents, fat, and other tissues comprising the thermal ‘periphery’ [12]. These processes could be consolidated into a single exponential term. Due to the long time-scale, heat transfer during phase four was probably partly conductive. Estimates of basal metabolism in the anesthetized, non-shivering dog (@90 J/kg per minute) indicate that as much as 0.6°C of warming per 20 min in this model may be due to metabolism.

A.3. Thermal accounting

 Heat transfer efficiency:

Although machine-LAPC allowed 2.3 times the FTLV frequency of the manual method, and resulted in a larger ˙V_FTLV by a factor of 1.2, the cooling magnitudes and rates for machine-LAPC significantly (P < 0.001) fell short of those obtained with manual-LAPC (Fig. 3). The strategy of increasing FTLV frequency (1/t_c) and decreasing ˙V_FTLV, in order to arrive at approximately the same FTLV rate (˙V_FTLV), therefore, significantly decreased the fraction of thermal potential which was transferred from each FTLV (= heat transfer efficiency, E_f). Unexpectedly, when the E_f for each of the 8 LAPC cooled animals of Trials I and II (Table 2) was calculated using (Eq. (2)), the value did not differ (P = 0.46) between trials; nor did total heat removed per kg (DQT), as calculated using (Eq. (3)), differ (P=0.14) between Trials. Both of these quantitative methods were therefore inaccurate for some dogs. Calculation of whole-animal mass-specific heat capacities C_m (= DQT / DT_e) suggested that the Trial II values of ~DQT and E_f principally were inaccurate, since the mean C_m value of 0.70 ± 0.1 cal/g per °K for Trial I was consistent with the C_m reported in the literature for mice and humans [289], whereas C_m values calculated for Trial II using (Eq. (2)) were unrealistic, being greater than the C_m of water.

E_f  (method 1) = 1/nS (T_S−T_inf) / (T_ven−T_inf),     (2)

DQ_T (method 1) = ˙V_FTLV C_vf ST_S−T_inf.                     (3)

To independently check the accuracy of Trial I values, we computed DQT and C_m for three dogs from an earlier study (Tables 1 and 2: dogs A, B, and C) that had been given ~4ºC PFC with FTLV times sufficiently long to allow the volumes and mixed-temperatures of suction-return liquid to be measured for each FTLV.

This allowed computation of DQ_T and E_f by a more detailed method (Method 2, Eqs. (4) and (5)), which used the extra thermal data (not available for Trials I and II) to more directly estimate FTLV heat transfer.

DQT (method 2)

= C_vf S V_FTLV (T_ven−T_inf) − V_S (T_ven−T_SM),                              (4)

When this was done, the mean Cm for dogs A, B, and C was found to be 0.68 ± 0.06 cal/g per °K, consistent with the Cm in Trial I (P=0.80). Method 1 (Eqs. (2) and (3)) required the assumptions that PFC suction volume equalled infusion volume, that suction flows remained constant, and that thermal hysteresis was negligible. These assumptions apparently held true at the larger ˙V_FTLV and t_c values of Trial I, but not for the smaller values of Trial II.

With this information, a new E_f for Trial II was estimated using method 3 (Eq. (6)), which employed an estimate for the heat required for the observed DT_e, vs. the total PFC thermal-deficit theoretically available.

This estimate required a presumed value of C_m. However, if the mean Trial II Cm was assumed to be the same as that of Trial I, then the true Trial II Ef could be calculated (Eq. (6)) to be 0.40 ± 0.06.

This value agreed with the rough estimation that since Trial II achieved only 73% of the DTe of Trial I, despite using 1.19 times more total PFC (Table 1), the

E_f in Trial II was expected to be about 73%/1.19 = 61% that of Trial I.

Thermal compartment size:

Heat removal for each cycle (DQ_c) in Trial I was calculated from the individual terms of Eq. (3), and individual-cycle mean cooling power calculated as DQ_c/t_c. The latter parameter was useful since thermal compartments in the dog are relatively isolated at short time scales (Figure 2-8), and thus the ratio of cooling-power to cooling rate (Eq. (7)) at a given probe site was expected to give the heat capacity (C_h) of the system of thermal compartments that were in equilibrium with each other, and with the site, at the time of the measurement:

C_h (Comp N), total heat capacity of thermal compartments N = 2 + 3, or N = 2 + 3 + 4.

Both t_c and dT/dt values were chosen at a time t, of interest when compartment system N has not yet equilibrated with slower half-time compartment(s). Thus, in

Trial I, near the end of FTLV cycle c1 (t = 30 s), the FTLV thermal-deficit had equilibrated within thermal Compartments 2 + 3 (PFC/viscera/blood-volume), but had not yet significantly reached Compartments 4 or 5.

If the cooling rate of T_ven at t = 30 s (−1.9 ± 0.8°C/min) was then taken as the cooling rate of the system of PFC/viscera/blood-volume, the C_h for this compartment system could be estimated from (Eq. (7)) as 20 ± 9% of C_T, the total heat capacity of the animal (C_T = m C_m). Subtracting the C_h contribution of lung PFC (=m ˙V_FTLV C_vf) allowed estimation of the remaining tissue C_h for Compartment 3 as ~19 ± 9% of C_T.

Similarly, the Ch of Compartments 2, 3, and 4 together, was estimated at t = ~140 s (cycle 4) as 71 ± 17% of C_T, corresponding to the classical whole-body ‘thermal core.’ The Compartment 5 C_h was then calculated to be the remainder (100−71%) = 29 ± 17% of C_T, corresponding to the classical ‘thermal periphery.’