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

Adapted from Source Document: http://www.docstoc.com/docs/88919930/Cryostat-Preparation—Cryonics-Institute

PURPOSE: To detail the procedures used for set-up and final preparation of Almax fiberglass-composite resin long-term patient care cryostats. This standard operating procedure (SOP) (aka Best Practices) details the vendors, materials and techniques used to prepare the Almax cryostats for full operational status after receipt from the manufacturer.

1.0. Detail of configuration and a brief overview of the manufacturing procedure used to produce cryostats.

Almax cryostats are cylindrical, double walled vessels that employ perlite and low vacuum (1-12 torr) insulation to facilitate highly efficient long-term liquid nitrogen refrigeration of cryopatients. Each unit has an overall height of 327.7 cm, an external diameter of 182.9 cm, an internal diameter of 121.9 cm and a useable internal height of 218.4 cm. The static liquid nitrogen capacity of Almax cryostats is approximately 2550 liters with a static boil-off rate in the range of 10.5 to 12.5 liters per day. Adult, human, whole body patient capacity is between 4 and 6 patients, depending upon patient diameter and the method of packaging used.

1.2.  Engineering details are presented Figure 1.1-1.2.

Figure 1.1: Detailed engineering specifications for the Almax long-term patient care Cryostats.

The cryostats are fabricated from a fibreglass mat-modified vinyl ester (Hetron 922, Ashland Chemical Co.) composite. The basic procedure for fabrication consists of building up layers of glass mat saturated with a resin monomer that is reinforced with carboxyl-terminated butadiene-acrylonitrile copolymer. The resin is polymerized (cured) using methyl ethyl ketone peroxide (2-butanone peroxide, or MEK-peroxide), which initiates free-radical cross-linkage of the monomer. This technique avoids incorporation of the MEK peroxide catalyst into the finished polymer, rendering it more stable, more corrosion resistant and less chemically reactive. Five millilitres of MEK peroxide are used per pound of Hetron 922. The inner vessel (can) of the cryostat is an open- topped cylinder with a concave bottom made from of vinyl ester resin and glass mat with a wall thickness of ~13 mm. The outer cylinder (can) is comprised of the same material, has a wall thickness of ~15 mm and is connected to the inner can only by a glue bond where the two are joined at the opening of the inner can on the top of the cryostat.

The opening of each cryostat is closed with a snug-fitting insulating neck-plug with an external cover of 14 gauge grade #2, 304 stainless steel. The insulating neck-plug is made from 22 layers of 2.5 cm thick Owens-Corning high density extruded polystyrene insulating foam board (~121.9 cm in diameter by 55.9 cm thick.) which are sandwiched between the stainless steel cover and an inner cover of painted chip board or marine plywood using 4 threaded nylon rods to compress and secure the foam to the inner and outer covers of the cryostat lid. A section of 5.1 cm diameter PVC plastic pipe penetrates the neck-plug and external cover in the center allowing access to the inside of the cryostat for temperature and liquid level monitoring.

Figure 1.2: Detailed engineering specifications for the Almax long-term patient care cryostats.

1.3.  Cryostats are manufactured under contract with Almax Products, a company owned and operated by Bruce Alter, located in Bearsville, New York:

Almax Products                    Mailing address:  Almax Products

363 Coldbrook Road                                            P.O. Box 441

Bearsville, NY 12409                                           Bearsville, NY 12409

Phone: 845-679-4615  FAX: 845-679-8620   email: Almax441@aol.com

Almax subcontracts the work of building the cryostats shells to Polymil Products, (contacts Sam Yacuzzo and Tammy Shultz) of LeRoy, NY:

Polymil Products, Inc                 585-768-8170

51 North Street

Leroy, NY 14482

Purchase price for 1 cryostat, ordered in May 2009 was $23,000 US, half payable on issuance of the purchase order and half payable by 45 days after delivery.

Perlite insulation is for the units is obtained from:

Noble Perlite                             405-872-5660

312 W Chestnut

Noble, OK 73068-8545

On average, 70 thirty-pound bags of perlite are used by Almax in a preliminary filling of the annular space prior to shipment of the cryostat. An additional 14 bags of perlite are shipped with the unit and used to top-up the annular space after shipping; the perlite settles en route due to handling and movement of the cryostat. Cost per bag as of 16 May, 2009 was ~ $20 US, per bag, including wrapping and palletizing, in preparation for shipment.

Currently shipment is being arranged by Almax and charges for the last load of perlite were $__________ US.

The stainless steel cover for the cryostat is manufactured by:

Beck Industries, Inc.

24462 Sorrentino Court,

Clinton Township,MI, United States, 48035
(586) 790-4060 PHONE
(586) 790-4982 FAX
EMAIL: mbeck6@sbcglobal.net

Figure 1.3: Stainless steel cryostat covers manufactured by Beck Industries, Clinton Township, MI.

The covers are 127 cm in diameter x 7.6 cm deep with a 20.3 cm circular central access port cover. The cover has 1/8″ diameter holes at 116.8 cm bolt circle, 22.9 cm bolt pattern with 1/8″ screw holes and 7.6 cm sides which are skip welded around the 127 diameter of the cover. The covers are fabricated from 14 gauge, grade #2, 304 stainless steel.

Price is $860.00 US per cover. Charge for palletizing and shipping to Bearsville, NY is $200.00 US.

TOTAL PRICE $_________ US

Figure 1.4: Removal of cryostat from shipping vehicle/container.

1.4  Atmospheric air is withdrawn from the annular space of the cryostat in order to create a vacuum in two stages. The first stage employs a roughing pump which is capable of reducing pressure in the annulus to ~ 5 x 10^-2 torr, however it will only be necessary to achieve a stable vacuum of ~ 500 torr before switching to the polishing/ maintenance vacuum pump. The roughing pump used is  in an Alcatel ACP-15, 8.2 cubic ft/min with a peak pumping speed of 14 m³ /hr and a final vacuum capacity of 5 x 10^-2 torr. The ACP-15 employs Roots blower technology. Roots pumps are positive displacement machines using two synchronized rotors rotating in opposite directions. The rotors feature profiles usually shaped like the figure 8.During the rotation, molecules of gas are isolated between the lobes and the stator and then led to the exhaust side of the pump without variation of volume.

Figure 1.5: Alcatel ACP-15 roughing pump.

The ACP-15 features a frictionless pumping module that is optimized for operation without internal lubrication. Complete technical specifications, operation and servicing instructions for the ACP-15 are present as Appendix 1 to this SOP.

Figure 1.6: Welch 1376C-03, DUOSEAL®, two-stage, belt drive high vacuum pump.

Final ‘polishing’ evacuation of the cryostat annulus as well as maintenance of the vacuum, is achieved using a Welch 1376C-03,DUOSEAL®, two-stage, belt drive high vacuum pump. The Welch pump has a peak pumping capacity of 300 LPM (10.6 CFM) with a final achievable vacuum of 1 x10^-4 torr. The Welch pump motor is configured to operate on 220V, 50 Hz,1 PH and is supplied with Schuko plug which must be replaced with a ____________ plug prior to be being placed into service.

Complete technical specifications, operating and servicing instructions for the Welch Welch 1376C-03,DUOSEAL® pump are present as Appendix II to this SOP.

2.0.  Shipment and unloading of the cryostat.

2.1.  The cryostats is palletized and prepared for shipment via commercial freighter in a sea-land container. It is then shipped, either by semi-trailer, or by truck, within the sea-land container, on wooden skids (generally skids of very poor quality). Drag chains are placed around the skids and they are pulled to the end of the trailer. Then they are pulled out further with the forklift so that the rear end of the skid rests firmly on the trailer and the opposite end of the skid is then lowered to rest on a wooden support frame so that the pallet holding the cryostat can be can be picked-up from the side with the forklift, removed from the  truck and moved into the facility where the cryostat is placed on custom made steel frame castered trolley for additional preparation, prior to placement into service.

Figure 2.2: The forklift is repositioned at the side of the cryostat/pallet and the unit is removed from the vehicle and placed on the ground..

 Figure 2.3: The evacuation port cover plate used to hold perlite in place and prevent contamination of the perlite with moisture during shipping is unbolted and removed.

 Table 2.1: Equipment, Tools and Supplies Required to Remove Cryostats from Delivery Vehicle

2.2.  The cryostat is shipped from the manufacturer with a resin-composite cover plate and sealing gasket secured to the evacuation port opening of the unit with 12 bolts (Figure 1.3). This cover plate serves both to contain the perlite insulation material and keep it dry during shipment. Perlite is moderately hygroscopic and will absorb water from the atmosphere in high humidity environments. Once the cryostat is in the storage facility, the cover plate is unbolted and the cover plate and the neoprene rubber gasket that seals it to the evacuation port flange are removed and set aside. The evacuation port opening is then immediately covered with a heavy-duty, 3 mil plastic refuse bag that is tightly secured in place with a ratchet-type nylon tie-down strap. It is important to immediately and tightly cover this opening to prevent moisture from entering the annular space and contaminating the perlite, since this would make subsequent evacuation of the annulus difficult, or impossible.

  Figure 2.4:  A custom built trolley fabricated from powder coated welded steel tube stock and high quality 3″ diameter urethane casters is used to safely move the  cryostat around the facility in the horizontal position during post -manufacturing preparation. Wooden skids are used to protect the cryostat from damage by the steel frame of the trolley.

 2.3.  The cryostat is transported to the work-area at the facility by placing it on a custom built metal trolley. The unit is left on the trolley until all preparative work (prior to hoisting the unit into the upright position) is completed.

3.0 Topping up the cryostat with perlite.

3.1 Protective clothing consisting of a heavy-duty, hooded Tyvek work coverall, fabric reinforced vinyl gloves and a full face N-100 respirator are donned. Duct tape is used to secure the hood opening of the of Tyvek suit to the edges of the respirator, the sleeves of the Tyvek suit to the work gloves and the tops of the work boots to the leggings of the Tyvek coverall, as shown in Figure 3.1, below. It is important to achieve a seal at all joints in the protective clothing in order to prevent the highly irritating perlite dust from contaminating the worker’s skin.

3.2  The plastic bag covering the evacuation port is removed and perlite is poured from the bags into the evacuation port opening as shown in Figure 3.2. The perlite is spread out inside the annular space and packed tight with wooden spreading and tamping paddles that are made in-house, as shown in Figure 3.3, below. Considerable force is required to tamp the perlite solidly into place, and typically the full weight of the worker must be brought to bear on the tamping paddle.

Figure 3.1: Duct tape is used to secure and seal the respirator, gloves and boots to the protective Tyvek coverall in order to prevent perlite dust from coming into contact with the workers’ skin. An full-face N-100 respirator is to provide respiratory protection from the perlite dust. Note perlite spreading and tamping tools resting on the cryostat at the middle left of the photo.

 Figure 3.2: Perlite is poured from the 20 lb bags into the cryostat annular space with the workman standing atop the cryostat.

Figure 3.3: A spreading and tamping tool are fabricated from plywood and a 24 x 24 x 61 cm piece of lumber (which serves as the handle). The spreading tool has the handle offset to one side of the plywood plate, while the tamping tool has the handle secured to the center of the plate allowing for stability and even distribution of load when compressing the perlite. The handles are secured to the plywood plates using  1/4″  by 3″ wood screws reinforced with quick-set epoxy adhesive.

Figure 3.5: Perlite is tamped into place in the annulus of the cryostat using the wooden tamping tool.

Figure 3.6: When the annular space is filled with packed perlite to the level of the bottom of the evacuation port no additional perlite is added and the top of the cryostat is brushed off with a household broom.

Figure 3.7: The evacuation port is again tightly covered with a plastic bag to prevent entry of water vapor into the annular space.

Figure 3.8: A jet of compressed air is used to clean the perlite dust off of the cryostat.

4.0.  Preparation of the evacuation port and evacuation valve assembly.

The first step in preparing the evacuation plumbing assembly is to sweat solder a 27 cm long x 3/4″ piece of copper onto a 3/4″ NPT Stainless Steel Ball Valve Full Port WOG1000 SS304 SUS304 0.75 .75 Female Ports.

Assemble the tools and supplies required for sweating the section of pipe into the valve. Prepare the copper pipe by sanding both ends using fine grit sand paper. Apply solder paste to the end to be sweated to the ball valve and insert the pipe into the 3/4″ copper T-connector. Don gloves and heat the copper pipe and connector with the torch for approximately 30 seconds. Apply solder by touching a J-shaped piece of solder to the joint 7 times; the solder will be drawn into the joint between the pipe and connector by capillary action. If the metal is not hot enough, reheat it with the torch as necessary. Allow the solder to cool and set-up for 60 seconds and then wipe the joint clean with a shop towel. Any remaining excess solder may be removed with a wire brush.

The threaded copper NPT to pipe slip fitting is then attached to the vacuum shut-off valve using Teflon thread sealing tape to insure a gas-tight seal.

Table 4.1 Tools and Supplies Required for Sweating Joints in Copper Pipe

Figure 4.1: A section of copper pipe is sweat-soldered into the female end of a brass NPT connector which is then screw threaded into a ball type shut off valve using Teflon pipe joint sealing tape.

The valve and pipe assembly are then attached to the evacuation port cover plate by drilling a hole just large enough to admit the copper pipe in the center of the 41.9 cm diameter cover plate. It is important that the hole be a tight fit to the valve and pipe assembly so that the pipe can be securely cemented into place without any possibility of leaks (there must be a gas-tight seal). The copper pipe is prepared for cementing into place by sanding with fine grit sand paper, after which it is degreased using acetone and a clean rag (or lint-less disposable shop towel). The end of the pipe to be attached to the evacuation port cover is then painted with Special Blend MFR-10 lb laminating resin (low volatile organ compound, mixed 100 to1 with methyl ethyl ketone (MEK) peroxide (supplied by Michigan Fiberglass Sales, St. Claire Shores, MI)  and the pipe is inserted into the previously drilled hole. Additional coats of laminating resin and glass mat, as needed, are used to secure the evacuation pipe in place, with care being taken to ensure that the pipe opening remains clean and unobstructed by resin. Each coat of applied resin is allowed to fully cure before the next coat is applied.

Figure 4.2: Top: the evacuation port cover plate with the stub of copper pipe to which the vacuum valve will be attached already in position. Bottom: schematic of the evacuation port, vacuum valve and T assembly housing the thermocouple vacuum gauge.

The back of evacuation port cover plate and the tip of the copper evacuation pipe assembly is then prepared for bonding to the flange of the evacuation port by being sanded with fine grit sandpaper. Once the plate has been “roughed-up” so that the adhesive epoxy will adhere, it is blown clean of particulates with a jet of compressed air, and then wiped with a clean rag dampened with acetone. Seven 6”x6” squares of cotton batting for filtration are painted with special blend MFR-10 lb laminating resin, low V.O.C. mixed  100/1 with MEK Peroxide (both from Michigan Fiber Glass Sales, St. Claire Shores) for hardening and adhesion.

Figure 4.3: Cotton batting filter pads are shown being cemented in place on back of evacuation port cover plate.

Figure 4.4.: The edges of the cotton bats are saturated with adhesive resin and smoothed onto the back of evacuation port cover plate.

The neoprene rubber gaskets that were between the evacuation port cover plates and the evacuation port flanges during shipment from Almax are used as templates for cutting the 3/4 ounce chopped strand FG-03438 fiberglass cloth rings.

Figure 4.5: The rubber sealing gaskets used to protect the annulus from the ingress of dirt and moisture during transport of the cryostat from the manufacturer are used as templates for cutting rings of fiberglass cloth which will act as the permanent sealing gasket.

Figure 4.6: It is important to wear respiratory protection whenever working with or around fiberglass. N-95 masks are suitable for such work, whereas a full-face N-100 respirator is required for work where perlite dust is being generated.

The fiberglass cloth rings are then applied to the cryostat evacuation plate flange using the same laminating resin that was used to adhere the cotton filter pads.

Figure 4.7: This illustrates proper preparation for cementing the fiberglass cloth rings to the evacuation port plate flange. Note the presence of a piece of protective (black) plastic to prevent damage or marring of the surface of the cryostat with the resin being used to cement the rings in place.

Figure 4.8: Household fiberglass building insulation (Owens-Corning) is used to plug the opening of the evacuation port. This prevents the perlite from migrating into the vacuum line, and it also serves as a coarse pre-filter for the larger particles of perlite dust, preventing them from entering the vacuum pumps.

Owens-Corning fiberglass “wool”  building insulating is packed against the perlite to prevent the perlite from plugging the filter.  The edges are then painted with laminating resin to facilitate adherence of the fiberglass cloth rings.

Figure 4.9: The edges of the evacuation port flange are carefully painted with resin to insure adhesion of the fiberglass cloth rings and to facilitate a thorough seal when the port cover is applied and clamped in place for final bonding to the flange.

 4.10: A small paint application roller is used to evenly apply (and assure saturation of) the fiberglass cloth rings to the flange.

A roller applicator is used to apply more laminating resin to the fiberglass cloth  rings. Three fiberglass “cloth” rings are applied in this manner to each cryostat. [The non-disposable parts of the roller may be cleaned up with acetone after use.] Once preparation of the fiberglass cloth rings is completed, the back surface of the evacuation port cover plate is painted with resin, taking care not to contaminate the cotton batting filters.

Figure 4.11: After the prep of the filter is completed and the final coat of adhesive has been applied, the back of the evacuation port cover plate is carefully and completely painted with adhesive resin taking care not to get resin on the cotton filter pads.

Figure 4.12: The evacuation port cover is then attached to the flange and held in place tightly with 4 equidistantly spaced C-clamps which are left in place until the resin has dried and fully hardened (~72 hours under normal working conditions).

The evacuation port cover with its integral filter (i.e., glued-on assembly of 3 cotton bats) is then clamped onto the flange opening and held in position for the adhesive resin to set up and cure.

5.0 Initial (rough) evacuation of the cryostat

 Initial evacuation of the cryostat is undertaken using the Alcatel ACP-15 roughing pump to a stable vacuum of ~ 500 torr. The Welch 1376C-03,DUOSEAL®, two-stage, belt drive high vacuum pump. must not be used for initial evacuation of the cryostat.  Failure to pre-evacuate the cryostat using a roughing pump will result in contamination of the oil in the two-stage pump with water and can damage the pump mechanism. Additionally, two-stage vacuum pumps are not designed to pump high density atmospheric gas – they are to be used only as “polishing” pumps to  harden and subsequently maintain the vacuum to ~ 1.0 torr.

Figure 5.1: Initial evacuation of the perlite filled annulus is accomplished using the Alcatel roughing pump. An inexpensive Bourdon tube vacuum  gauge (VG350-14CBM) is interposed in the vacuum line (mounted on a 3/4″ copper T-connector) to monitor the progress of the initial pump-out.

Figure 5.2: Once a vacuum of ~ 1.0 torr is achieved, the vacuum valve is closed, the roughing pump is removed, and the 2-stage vacuum pump is connected to the annulus. For this preliminary hardening of the vacuum a thermocouple vacuum gauge is used and is placed near the pump, for convenience.

 6.0 Preparing the base of the cryostat prior to erection upright.

A five foot diameter circle of 3/4″ plywood is used to seal and secure the bottom of the cryostat. The plywood circle has three 5″diameter holes cut in it, arranged as shown in Figure 6.1, to allow for 2-part  urethane foam resin to be poured into the space between the plywood circles and the bottom of the cryostat. Once the urethane resin foams, expands and sets, it serves to stabilize and reinforce the plywood so the bottom of the cryostat and ensure that  it is well supported and stable on the floor when the unit is finally filled with liquid nitrogen.

Figure 6.1:Circles of 3/4″ plywood are cut so as to fit into the opening of the base of the cryostat’s outer cylinder. Three 5″ diameter holes, spaced equidistant from each other are cut into the plywood to allow for filling of the space between the plywood discs and the bottom of the cryostat with urethane foam. The discs are placed with the holes at the top of cryostat base so that the urethane resin-activator mixture does not leak out onto the floor during loading into the base of the cryostat.

 Figure 6.2: The plywood disc is initially held in place with duct tape until it can be firmly anchored with steel tube stock or metal bars to prevent it from being displaced by the expanding urethane foam.

The plywood disc is initially secured to the bottom of the cryostat with duct tape and then clamped firmly into place using rigid steel tube stock or metal bars and heavy-duty C-clamps, as shown in Figures 6.2 and 6.6

The  space between the plywood disc and the bottom of the cryostat can now be filled with supporting, rigid, closed-cell urethane foam. The foam used for this is MF-1002 1.2 lb density urethane foam (from Michigan Fiberglass Sales). The foam is prepared from a two component kit consisting of  urethane resin (part-A) and activator (Part-B) which are mixed in equal parts using a wooden paint mixing-type stick in disposable 2-gallon paper pails. The resin, activator, paper pails and wooden mixing paddle are included with each MF-1002  kit.

Figure 6.3: The two (A&B) components of the urethane foam are mixed in disposable paper pails using a wooden mixing paddle (also disposable). The foaming reaction begins almost immediately and is well underway within a minute.

Figure 6.4: Foaming action of the combined resin and activator less than a minute after being combined and thoroughly mixed in the mixing-dispensing pail.

Once the components are mixed, the activated urethane resin will expand to ten times its starting volume and will subsequently harden into dense foam. The foaming action begins within 60 seconds of the start of mixing of the resin and activator, so it is necessary to quickly pour the mixture into the holes in the plywood. The activated urethane resin is poured into the headspace using disposable funnels made from lightweight aluminum sheet metal (~22 gauge). The resulting urethane foam requires approximately an hour to set and  is fully cured in 24 hours.

Figure 6.5: Lightweight flexible aluminum sheet metal is formed into half-cones which are taped in place to form funnels. These disposable funnels are then used to facilitate pouring the mixture into the 5″ holes cut into the plywood discs, starting with the lower holes and finishing up with the top holes.

Figure 6.6:Once the urethane foam has filled the headspace and has stopped exhausting from the filling holes, the holes are covered with squares of plywood which are screwed into place. The plywood disc should then be primed and painted with a waterproof oil-based, or two-part epoxy concrete floor paint, to prevent subsequent water damage due to efflorescence from the concrete slab, or insect (termite) infestation.

Four to six 2-gallon pails of the activated resin mixture is typically enough for each cryostat. [ The density of the foam may be altered by changing the ratio of resin and activator: more part-B than part-A results in a larger final volume of foam with less density.]

The cryostat is now ready for movement to the patient storage area of the facility for erection to a vertical position, fire-retardant coating, final hardware outfitting, painting and placement into service.

_______________________________________________________

By  Mike Darwin

SPECIMEN PROPOSED INTERNATIONAL PURCHASE CONTRACT AS OF 2009

TERMS AND CONDITIONS OF CONTRACT FOR PURCHASE OF ALMAX LIQUID NITROGEN CRYOPATIENT STORAGE CRYOSTAT

These Conditions may only be varied with the written agreement of the Purchaser.. No terms or conditions put forward at any time by the Supplier (Almax)  shall form any part of the Contract unless specifically agreed in writing by the Purchaser.

1. DEFINITIONS

In these Conditions:

“Purchaser” means the Purchaser, a limited liability company located at OOO “Purchaser_______________________________________________, hereinafter referred to as ‘Purchaser.’

“Supplier” means Almax Products, 363 Coldbrook Road, P.O. Box 441, Bearsville, NY, United States of America, 12409, Phone: 845-679-4615, FAX: 845-679-8620   email: Almax441@aol.com hereinafter referred to as ‘Almax.’

“Goods” means any goods as are to be supplied to Purchaser by Almax Products (or by any of the Supplier’s subcontractors) pursuant to or in connection with this Contract, as detailed in the Purchase Order attached to this contract and in Section 2.4, below.

“Contract” means the Contract between Purchaser and the Almax consisting of the Purchase Order, these conditions and any other documents (or parts thereof) specified in the Purchase Order and in A.

“Purchase Order” means the document setting out Purchaser’ requirements for the Contract.

2. GOODS

2.1       The Goods shall be to the reasonable satisfaction of Purchaser and shall conform in all respects with any particulars specified in the Contract and in any variations thereto.

2.2       The Goods shall conform in all respects with the requirements of any statutes, orders, regulations or bye-laws from time to time in force.

2.3       The Goods shall be fit and sufficient for the purpose for which such Goods are ordinarily used and for any particular purpose by Almax in the supply of the Goods and the execution of the Contract.

2.4       Specifically, Almax agrees to provide the following goods and services:

2.4.1   A double walled, cylindrical, composite vinyl ester resin fiberglass, perlite and vacuum (10^-3 mm Hg) insulated cryogenic liquid nitrogen biological specimen storage container (cryostat) based on the engineering drawings provided by Almax Products and attached to this Contract as Exhibit A. The inner vessel diameter is 1220 mm, and the inner vessel height is 2440 mm (tolerance ± 2 mm). The outer vessel diameter is 1830 mm, and the outer vessel height 2740 mm (without stand). With the stand the overall height of the cryostat is 3200 mm. The empty weight with the stand attached is 1814 kg. The inner cylinder wall thickness is a minimum of 12.7 thick. The approximate working volume for liquid nitrogen of the cryostat is 2142 liters.

All drawings are included in the price. Almax will send detailed drawings, blueprints and photos as requested, upon signing the contract.

Materials of construction for the cryostat are as follows:

Outer cylinder or shell: H-992  MEKP/COBALT STRUCT

Inner cylinder or shell:  H-992 MEKP/COBALT STRUCT

Heads:  H-992 MEKP/COBALT STRUCT

Flanges: H-992 MEKP/COBALT STRUCT

Nozzle necks: H-992  MEKP/COBALT STRUCT

External nuts/bolts: CS

CS Gaskets: 11 mm Neoprene rubber

Corrosion Barrier: 1-ply “C” backed W 2-ply chopped strand fiberglass laminate

Exterior: Five (5) coats of FireFree FF88 tumescent fire protective coating as supplied by FIREFREE Coatings, Inc., 580 Irwin Street, Suite 1, San Rafael, CA 94901, Phone: (888) 990-3388, USA and applied per the manufacturer’s specifications and instructions attached as Exhibit B to this Contract.

Design Pressure: (4′) + 15 PSIG, (6′) – 15 PSIG

Design standards: ASTM-D3299

2.4.2   A stand for the cryostat is provided equipped with 4 casters capable of easily rolling over finished concrete floors with the unit fully loaded with liquid nitrogen at  a gross weight of 2,430 kg including the cryostat stand, neck-plug and cover.

2.4.3   Cryostat will be loaded with perlite prior to shipment. Additional perlite will be furnished for “top off” as per 2.4.4, below. Price of cryostat inclusive of above: $25,000 with $3,000 discount on a second cryostat if ordered with 90 days of the receipt of the unit specified in this Contract.

2.4.4   Fourteen (14) bags Grefco Minerals HP-500 grade perlite as supplied Noble Perlite, 312 W Chestnut, Noble, OK 73068-8545 USA, phone:405-872-5660.@ $ 30 a bag (30 pound bag) plus a $15.00 pallet charge, price: $435.00

2.4.5   One (1) each resin kit to include: 2 ea: 10″ wide x 50 yards rolls of 1.5 oz FRP mat and 1 each 5-gallon drum of 411-400 resin, price: $545.00

2.4.6   Annular space vacuum burst disc to be provided by Purchaser or Purchaser’ designated vendor FOB to Almax.( Rupture disc set pressure: 15 psi rupture temp: ambient (-20 to +45 deg C) normal operating pressure: 2.5 x 10-5 torr (high vacuum) on one side, ambient pressure (1 atmosphere) Almax installation charge: $175.00

2.4.7   One (1) each extra 41.9 cm diameter evacuation port/filter cover plate to be supplied by Almax, price $245.00

2.4.8   One (1) each 41.9 cm diameter evacuation port/filter cover plate fully outfitted with 7-ply cotton filter and 3/4″ copper pipe and fittings, including Mueller brand 3/4″ ball brass ball valve (Home Depot part #06P115) sealed and assembled per the procedure detailed in Exhibit C, attached to this Contract, price included in base cryostat price.

2.4.9   Five (5) each: steel clevises for lifting cryostat capable of bearing a weight of at least 1,000 kg each, price: $148.50.

2.5.0   One (1) each R-06413-30 Tygon® vacuum tubing, 3/8″ID x 7/8″OD, 10 ft/pack, price $115.00

2.5.1   One (1) each 10 ft length Fischer Scientific red rubber vacuum tubing 9.5mm ID 22.3mm OD, 3/8″ ID 7/8″ OD., price: $69.90

2.5.2   Almax agrees to work with the subcontractor selected for the cryostat cover, Beck Industries of 24454 Sorrentino Court, Clinton Township,MI, 48035, USA, Phone number (586)790-4060, to ensure that the stainless steel cover fabricated by Beck Industries fits the cryostat supplied by Almax. In the event the cover does not fit due to incorrect specification supplied to Beck Industries by Almax, then Almax shall be fully liable for the replacement cost of said cover.

3. PRICE

3.1       The price of the Goods shall be as stated in the Contract and no increase will be accepted by Purchaser unless agreed by them in writing before the execution of the Contract.

3.2       Unless otherwise agreed in writing by Purchaser, Almax shall render a separate invoice in respect of each consignment delivered under the Contract. Payment shall be due 30 days after receipt of the Goods or the correct invoice therefore, whichever is the later.

3.3       Taxes, where applicable, shall be shown separately on all invoices as a strictly net extra charge.

3.4       The cost of palletizing and preparing the cryostat for shipment and for shipping the container is to be paid by Almax. Shall employ a licensed and bonded forwarder to handle the entire shipping procedure to include arranging the pick-up and delivery of Goods, filing and completing all required paperwork, and clearing of  the Goods through customs.

3.5       The total price is $27,733.34

3.6       The price shall be paid as follows:

•           1/3rd deposit upon initiation of this Contract and issuance of the Purchase Order

•           1/3rd upon completion of unit/system and or photo or inspection at factory

•           Final 1/3rd prior to ship and confirming positive test results done by Purchaser at its facility in Moscow, Russian federation

•           Prices are FOB shipping point.

•           All payments are in US dollars.

4. DELIVERY

4.1       The Goods shall be delivered to Purchaser, _________________________. Any access to premises and any labor and equipment that may be provided by Purchaser in connection with delivery shall be provided without acceptance by the Purchaser of any liability whatsoever and Almax shall indemnify Purchaser in respect of any actions, suits, claims, demands, losses, charges, costs and expenses which the Purchaser may suffer or incur as a result of or in connection with any damage or injury (whether fatal or otherwise) occurring in the course of delivery or installation to the extent that any such damage or injury is attributable to any act or omission of the Supplier or any of his subcontractors.

4.2       Where any access to the premises is necessary in connection with delivery or installation, the Supplier and his sub contractors shall at all times comply with the reasonable requirements of the Purchaser’ staff.

4.3       The time of delivery shall be of the essence and failure to deliver within eighty (80) days shall enable Purchaser (at its option) to release itself from any obligation to accept and pay for the Goods and/or to cancel all or part of the Contract therefore, in either case without prejudice to its other rights and remedies.

5. PROPERTY AND RISK

5.1       Property and risk in the Goods shall without prejudice to any of the rights or remedies of the Purchaser (including Purchaser’ rights and remedies under condition 7 hereof) pass to Almax at the time of delivery.

5.2       The property in the Goods shall pass to Purchaser upon payment for the Goods unless delivery of the Goods is made prior to payment, when it shall pass to Purchaser once the Goods have been delivered.

5.3       Any Goods for which the Supplier has received payment but which have not been delivered will, for the avoidance of doubt, remain the exclusive property of Purchaser and may be removed at any time by Purchaser or its representatives from wherever they are stored.

6. DAMAGE IN TRANSIT

6.1       On dispatch of any consignment of the Goods Almax shall send to Purchaser at the address for delivery of the Goods an advice note specifying the means of transport, the place and the date of dispatch, the number of packages and their weight and volume. Almax  shall free of charge and as quickly as possible either repair or replace (as the Purchaser  shall elect) such of the Goods as may either be damaged in transit or having been placed in transit fail to be delivered to Purchaser provided that: (a) in the case of damage to such goods in transit the purchaser shall within 30 days of delivery give notice to Purchaser that the Goods have been damaged, (b) in the case of non delivery Purchaser shall (provided that Almax has been advised of the dispatch of the Goods) within 10 days of the notified date of delivery give notice to the Supplier that the Goods have not been delivered.

7. INSPECTION, REJECTION AND GUARANTEE

7.1       Almax Products guarantees and warrants that the cryostat will maintain a vacuum of 10^-3 mm Hg between inner and outer containers with no more than 24 hours of pumping (using a standard laboratory vacuum pump with a minimum of 20 LPM of free air displacement and capable of delivering an ultimate vacuum of 1 x10 ^-4) per 30 day period. Almax further warrants that the cryostat (inner and outer containers and joint  at the neck-tube) will retain their structural integrity without leaking or cracking at a pressure difference of one atmosphere while storing a full load of liquid nitrogen (at least 2142 liters) and that the cryostat will conform to the description and drawings attached hereto as exhibit

7.2       Almax shall permit Purchaser or his authorized representatives to make any inspections or tests they may reasonably require and Almax shall afford all reasonable facilities and assistance free of charge at his premises. No failure to make complaint at the time of such inspection or tests and no approval given during or after such tests or inspections shall constitute a waiver by Purchaser’ of any rights or remedies in respect of the Goods.

7.3       Purchaser may by written notice to Almax reject any of the Goods which fail to meet the requirements specified herein. Such notice shall be given within a reasonable time after delivery to Purchaser of Goods concerned. If Purchaser shall reject any of the Goods pursuant to this Condition, Purchaser shall be entitled (without prejudice to his other rights and remedies) either (a) to have the Goods concerned as quickly as possible either repaired by Almax or (as the Purchaser shall elect) replaced by Almax with Goods which comply in all respects with the requirements specified herein, or (b) to obtain a refund from Almax in respect of the Goods concerned with no charge, either in materials or labor, to Purchaser.

7.3       The guarantee period applicable to the cryostat shall be 3 years from putting into service or 3 years from delivery, whichever shall be the shorter (subject to any alternative guarantee arrangements agreed in writing between Purchaser and Almax). If Purchaser shall within such guarantee period, or within 30 days thereafter, give notice in writing to Almax of any defect in any of the Goods as may have arisen during such guarantee period under proper and normal use Almax shall (without prejudice to any other rights and remedies which Purchaser may have) as quickly as possible remedy such defects (whether by repair or replacement as the Purchaser may elect) without cost to Purchaser.

7.4       Prior to shipment of the cryostat Almax shall perform a successful vacuum confirmation and spark test and provide detailed results of these test to Purchaser.

7.5       Any Goods rejected or returned by Purchaser as described in paragraph 7.2 or 7.3 shall be returned to the Almax at Almax’s risk and expense.

8. LABELLING AND PACKAGING

8.1       The Goods shall be packed and marked in a proper manner and in accordance with the Purchaser’s instructions and any statutory requirements and any requirements of the carriers. In particular, the Goods shall be marked with the Purchase Order number, the net gross and tare weights, the name of the contents shall be clearly marked on each container and all containers of hazardous goods (and any documents relating thereto) shall bear prominent and adequate warnings. Almax shall indemnify Purchaser against all actions, suits, claims, demands, losses, charges, costs and expenses which Purchaser may suffer or incur as a result of, or in connection with, any breach of this Condition.

8.2       All packaging materials will be considered nonrefundable and will be destroyed unless Almax’s advice note states that such materials will be charged for unless returned. The Purchaser accepts no liability in respect of the non-arrival at the Supplier’s premises of empty packages returned by Purchaser unless Almax shall within 10 days of receiving notice from the Purchaser that the packages have been dispatched notify Purchaser of such non-arrival.

8.3       Almax agrees to accept for placement in the sea-land container transporting the Goods to Purchaser at ____________________________ such other accessory items and equipment as will reasonably fit in the container upon the mutual agreement of both parties at no additional charge to Purchaser.

9.0 CORRUPT GIFTS OR PAYMENTS

Almax shall not offer or give or agree to give, to any employee or representative of Purchaser any gift or consideration of any kind as an inducement or reward for doing or refraining from doing or having done or refrained from doing, any act in relation to the obtaining or execution of this or any other contract with Almax or showing or refraining from showing favor or disfavor to any person in relation to this or any such contract.

10. PATENTS AND INFORMATION

10.1    It shall be a condition of the Contract that the Goods are made up in accordance with designs furnished by Almax that none of the Goods will infringe any patent, trademark, registered design, copyright or other right in the nature of industrial property of any third party and Almax shall indemnify Purchaser against all actions, suits, claims, demands, losses, charges, costs and expenses which Purchaser may suffer or incur as a result of or in connection with any breach of this Condition.

10.2    All rights (including ownership and copyright) in any specifications, instructions, plans, drawings, patterns, models, designs or other materials (a) furnished to or made available to Almax Purchaser pursuant to the Contract, shall remain vested solely in Purchaser (b) prepared by or for Almax for use, or intended use, in relation to the performance of this Contract are hereby assigned to and shall be vested in the Purchaser solely and (without prejudice to condition 14.2). Almax shall not, and shall procure that his servants and agents shall not (except to the extent necessary for the implementation of the Contract) without the prior written consent of Purchaser, use or disclose any such specifications, instructions, plans, drawings, patterns, models, designs or other materials as aforesaid, or any other information (whether or not relevant to the Contract) which Purchaser may obtain pursuant to or by reason of this Contract, except information which is in the public domain, otherwise than by reason of a breach of this provision, and in particular (but without prejudice to the generality of the foregoing) Almax shall not refer to Purchaser or the Contract in any advertisement without Purchaser’ prior written agreement.

10.3    The provision of this Condition 10 shall apply during the continuance of this Contract and after its termination, howsoever arising.

11. HEALTH AND SAFETY

Almax represents and warrants to Purchaser that Purchaser has satisfied itself that all necessary tests and examinations have been made or will be made prior to delivery of the Goods to ensure that the Goods are designed and made so as to be safe and without risk to the health and safety of persons using the same, and that Almax has made available Purchaser adequate information about the use for which the Goods have been designed and which have been tested and about any Conditions necessary to ensure that when put to use the Goods will be safe and without risk to health. Almax shall indemnify Purchaser against all actions, suits, claims, demands, losses, charges, costs and expenses which Purchaser may suffer or incur as a result of or in connection with any breach of this Condition.

12. INDEMNITY AND INSURANCE

12.1    Without prejudice to any rights or remedies of Purchaser’ (including Purchaser’ rights and remedies under condition 7 hereof) Almax shall indemnify Purchaser, its agents and employees against all actions, suits, claims, demands, losses, charges, costs and expenses which Purchaser may suffer or incur as a result of or in connection with any damage to property or in respect of any injury (whether fatal or otherwise) to any person which may result directly or indirectly from any defect in the Goods or the negligent or wrongful act or omission of the Almax.

12.2    Purchaser shall have in force and shall require any sub-contractor of Almax to have in force; (a) employer’s liability insurance in accordance with any legal requirements for the time being in force, and (b) public liability insurance for such sum and range of cover as Almax deems to be appropriate but covering at least all matters which are the subject of indemnities or compensation obligations under these Conditions in the sum of not less than $1,000,000 for any one incident and unlimited in total, unless otherwise agreed by Almax in writing.

12.3    The policy or policies of insurance referred to in paragraph 12.2 shall be shown to Purchaser whenever it requests, together with satisfactory evidence of payment of premiums.

13. CONFIDENTIALITY

13.1    Almax’s shall take all reasonable steps to ensure that all persons engaged in any work in connection with this Contract have notice that the statutory provisions apply to them and will continue so to apply after the expiry or termination of this Contract.

13.2    Almax shall keep secret and not disclose and shall procure that his employees shall keep secret and do not disclose any information of a confidential nature obtained by him by reason of the Contract except information which is in the public domain otherwise than by reason of a breach of this Provision.

13.3    The provisions of paragraphs 14.1 and 14.2 shall apply during the continuance of this Contract and after its termination howsoever arising.

14. TERMINATION

14.1    Almax shall notify Purchaser in writing immediately upon the occurrence of any of the following events:

a) where Almax is an individual and if a petition is presented for Almax’s bankruptcy or the sequestration of its estate or a criminal bankruptcy order is made against Almax  or Almax is apparently insolvent or Almax  makes any conveyance or assignation for the benefit of creditors, or if an administrator is appointed to manage his affairs; or b) where Almax is not an individual but is a firm; or a number of persons acting together in any capacity, if any event in (a) or (c) of this Condition occurs in respect of any partner in the firm or any of those persons or a petition is presented for Almax to be wound up as an unincorporated company; or c) where the Almax is a company, if the company passes a resolution for a winding-up or dissolution (otherwise than for the purposes of and followed by an amalgamation or reconstruction) or the court makes an administration order or a winding-up order, or the company makes a composition or arrangement with its creditors, or an administrative receiver, receiver or manager is appointed by a creditor or by the court, or possession is taken of any of its property under the terms of a floating charge.

14.2    On the occurrence of any of the events described in paragraph 15.1, or if Almax shall have committed a material breach of this contract and (if such breach is capable of remedy) shall have failed to remedy such breach within 30 days of being required by Purchaser in writing to do so, or, where Almax is an individual, if he shall die or be adjudged incapable of managing his affairs by determination of a court of law, Purchaser shall be entitled to terminate this Contract by notice to Almax with immediate effect. Thereupon, without prejudice to another of its rights, Purchaser may itself complete the Services or have them completed by a third party using for that purpose (making a fair and proper allowance therefore in any payment subsequently made to Almax) all materials, plant and equipment on the Premises belonging to the Almax, and the Purchaser shall not be liable to make any further payment to Almax until the Services have been completed in accordance with the requirements of this Contract, and shall be entitled to deduct from any amount due to the Almax the costs thereof incurred by Purchaser (including the Purchaser’ own costs). If the total cost to the Purchaser exceeds the amount (if any) due to Almax, the difference shall be recoverable by the Purchaser from Almax.

14.3    In addition to his rights of termination under paragraph Purchaser shall be entitled to terminate this contract by giving to Almax  not less than 30 days’ notice to that effect. In the event of such termination Almax shall, if required to do so by Purchaser , prepare and submit to  Purchaser a report on the work done prior to the termination and making such recommendations as may be based on the work done prior to termination.

14.4    Termination under paragraphs 14.2 or 14.3 shall not prejudice or affect any right of action or remedy which shall have accrued or shall thereupon accrue to Purchaser and shall not affect the continued operation of Conditions 10 and 14.

15. RECOVERY OF SUMS DUE

Wherever under the Contract any sum of money is recoverable from or payable by Almax, that sum may be deducted from any sum then due, or which at any later time may become due, to the Supplier under this Contract or under any other agreement or contract with Purchaser

16. ASSIGNATION AND SUB CONTRACTING

16.1    Almax shall not assign or sub-contract any portion of the Contract without the prior written consent of Purchaser. Sub-contracting any part of the Contract shall not relieve Almax of any obligation or duty attributable to it under the Contract or these conditions.

16.2    Where Purchaser has consented to the placing of subcontracts, copies of each sub-contract shall be sent by the Supplier to the Purchaser immediately it is issued.

16.3    Where Almax enters a sub-contract with a supplier or contractor for the purpose of performing the Contract, Almax shall cause a term to be included in such sub-contract which requires payment to be made to the supplier or contractor within a specified period not exceeding 30 days from receipt of a valid invoice as defined by the sub-contract terms.

17. FORCE MAJEURE

17.1    For the purposes of this Contract the expression “force majeure” shall mean any cause affecting the performance by a party of its obligations arising from acts, events, omissions, happenings or non happenings beyond its reasonable control including (but without limiting the generality thereof) governmental regulations, fire, flood, or any disaster or an industrial dispute affecting a third party for which a substitute third party is not reasonably available. In the case of Almax, each cause will only be considered force majeure if it is not attributable to the willful act, neglect or failure to take reasonable precautions of Almax, its agents or employees.

17.2    Neither party shall, in any circumstances, be liable to the other for any loss of any kind whatsoever including, but not limited to, any damages or abatement of charges whether directly or indirectly caused to or incurred by the other party by reason of any failure or delay in the performance of its obligations hereunder which is due to force majeure.

17.3    If either of the parties shall become aware of circumstances of force majeure which give rise to or which are likely to give rise to any such failure or delay on its part, it shall forthwith notify the other by the most expeditious method then available and shall inform the other of the period which it is estimated that such failure or delay shall continue.

17.4    It is expressly agreed that any failure by Almax to perform or any delay by Almax in performing its obligations under this Contract which results from any failure or delay in the performance of its obligations by any person, firm or company with which Almax shall have entered into any contract, supply arrangement or sub-contract or otherwise shall be regarded as a failure or delay due to force majeure only in the event that such person, firm or company shall itself be prevented from or delayed in complying with its obligations under such contract, supply arrangement, subcontract or otherwise as a result of circumstances or force majeure.

17.5    For the avoidance of doubt, it is hereby expressly declared that the only events which shall afford relief from liability for failure or delay shall be any event qualifying for force majeure hereunder

18. REFERENCES

Almax shall provide details of two reference bodies including names and telephone numbers of contacts, for whom similar work has been, or is currently, undertaken.

19. WAIVER

19.1    The failure of either party to insist upon strict performance of any provision of the Contract, or the failure of either party to exercise any right or remedy to which it is entitled under the Contract, shall not constitute a waiver thereof and shall not cause a diminution of the obligations established by the agreement.

19.2    A waiver of any default shall not constitute a waiver of any subsequent default.

19.3    No waiver of any of the provisions of the Contract shall be effective unless it is expressly stated to be a waiver and communicated to the other party in writing.

20. SEVERABILITY

If any provision of the Contract is held invalid, illegal or unenforceable for any reason by any court of competent jurisdiction, such provision shall be severed and the remainder of the provisions hereof shall continue in full force and effect as if the Contract had been executed with the invalid, illegal or unenforceable provision eliminated. In the event of a holding of invalidity so fundamental as to prevent the accomplishment of the purpose of the agreement, the Purchaser and Almax shall immediately commence good faith negotiations to remedy such invalidity.

21. NOTICES

Any notice given under or pursuant to the Contract may be sent by hand or by post or by registered post or by the recorded delivery service or transmitted by telex, telemessage, facsimile transmission or other means of telecommunication resulting in the receipt of a written communication in permanent form and if so sent or transmitted to the address of the party shown in the Purchase Order, or to such other address as the party may by notice to the other have substituted therefore, shall be deemed effectively given on the day when in the ordinary course of the means of transmission it would first be received by the addressee in normal business hours.

22. ARBITRATION

Any controversy or claim arising out of or relating to this Contract, or the breach thereof shall be settled by binding arbitration in accordance with the Commercial Arbitration Rules of the American Arbitration Association, and judgment upon the award entered by the arbitrator(s) may be entered and enforced by any court having jurisdiction thereof. Additionally, the parties intend that the arbitrators have power to issue any provisional relief appropriate to the circumstances, including but not limited to: temporary restraining orders, injunctions and attachments. The parties intend that this agreement to arbitrate be irrevocable and agree that either party is entitled to injunctive relief to quash litigation by the other part which breaches the agreement

21. HEADINGS

The headings to Conditions shall not affect their interpretation.

22. GOVERNING LAW

The Contract shall be governed by and construed in accordance with United States of America law and Almax hereby irrevocably submits to the jurisdiction of the US courts. The submission to such jurisdiction shall not (and shall not be construed so as to) limit the right of the Purchaser to take proceedings against Almax  in any other court of competent jurisdiction, nor shall the taking of proceedings in any one or more jurisdictions preclude the taking of proceedings in any other jurisdiction, whether concurrently or not.

IN WITNESS WHEREOF, the parties hereto have executed this

Agreement as of the date and year indicated below.

______Month _______Day ___________Year

Purchaser

By : _____________________________

XXXXX X. XXXXXXX,

Title: General Director, “Purchaser”

Date__________________________

Almax Products, Inc.

_________________________________

Bruce Alter

Title: President, Chief Executive Officer

Date____________________________

—————————————————————————————–

PURCHASE ORDER

Almax Products agrees to supply

At this time Price for one (1) complete unit, per contract to include:

double wall fiberglass liquid nitrogen storage system complete with required load of perlite, fill/load service fitting installed and 4 extra bags of perlite for “toping off” system…

Lifting lugs (3) placed per details

Stand:

PRICE $ 25,000 USD

2 plus units:

@ $ 22,000  Each  USD

***IF A SECOND UNIT IS PURCHASED WITHIN 3 MONTHS OF THE FIRST P.O. THEN DEDUCT $ 3,000 USD…

***AGAIN, THESE PRICES WILL CHANGE IF RESIN IS NOT AVAILABLE AT OLD PRICE..

_____________________________________________________________________________________

Options:

55 gallons Hetron 922 Resin** @ $ 595  USD

** Catalyst can not be shipped due to regulations.

This can be obtained via web site or local hardware or DIY store.

Extra Perlite: 14 bags Grefco Minerals HP-500 grade   @ $ 30 a bag ( 30 pound ) plus a $ 15 pallet charge

5-layers of Fire Flame 88 equal  Flame Control 20-20 A @ $ 1320  USD

1-Extra sealing Filter Plate Fiberglass @ $ 540 USD

1-Welch Model # 1376C-03  Vacuum pump wired for 220V, 50Hz 1 phase with Schuko Plug @ $ 4480 USD

5-clevises for lifting with a capacity 2,000 kg @ $ 29.70 each USD

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

THE CONSEQUENCES OF FAILURE

SLIDE 137

Alcor had achieved an exponential rate of membership growth by the time Jerry Leaf was cryopreserved. Since that time, there has been only modest growth of membership and in fact, in the years since 2007, membership growth has flattened.

SLIDE 138

The growth in the patient population has been similarly stunted with almost all increase being due to the cryopreservation of members, rather than at-need cases. The time when Alcor selected the highest quality at-need cases and delivered state-of-the-art care to those patients has now become a dim memory and, with one exception, the staff at Alcor has no experience with cases where immediate CPS, followed by prompt extracorporeal support, proceeded smoothly and without incident.

While it is easy to see the risks associated with at-need cases, particularly in the absence of careful vetting and strict adherence to predetermined (and protective) acceptance criteria, it is not so easy to see the even greater cost of foregoing them.

The quality of any complex procedure, medical or otherwise, is directly dependent upon the amount of experience staff have in doing it. Even highly trained and skilled personnel benefit from the experience gained by doing large numbers of cases. In fact, in medicine it has been a consistent finding that outcome in terms of morbidity and mortality in areas as diverse as open heart surgery, radiation oncology and HIV management improves steadily as a function of the number of procedures performed, or cases handled annually. The benefit of an increased case load is even more pronounced when the transition is made from a few cases per year to a few dozen per year, or more.

Absent a case load that keeps the cryopreservation team continuously busy, the only way to maintain even a semblance of competence is to carry out a program of animal research using a survival model that employs the same equipment, facilities and procedures that are employed in human cryopreservation cases. Absent this kind of day-in, day-out experience, it becomes impossible for staff to remember (or even know) where supplies are, how to calibrate, operate and troubleshoot equipment, and just as importantly, how to work together cohesively as a team.

SLIDE 139

The person leading that team and directing that research must be a competent and motivated „mountain climber‟ – otherwise the work will be a meaningless and gruesome exercise that achieves nothing but the demoralization of those participating in it.

SLIDE 139

Medical malpractice is a pretty common thing and as we have seen iatrogenic deaths are commonplace. That this is so, given the extensive training and mentoring physicians receive, should give us pause for thought. To become a General Practitioner in the UK or the US requires 12 years of postgraduate training. That is a huge commitment in terms of both time and money and it requires substantial motivation over and above the likely financial returns (in the UK or the US). This level of training and commitment act as a human filter – effectively removing many people who are not suited to the task of being physicians either as a result of „defects‟ in temperament or due to lack of intellect or skill.

However, this slide is misleading in that most of the real filtration has already taken place before a student enters medical school, or perhaps I should say more appropriately, is accepted to medical school. Roughly 95% of those who score well on the MCAT (Medical College Admission Test) or the UKCAT (UK Clinical Aptitude Test) and are subsequently admitted to medical school will finish it! Most of the separation of the wheat from the chaff takes place as a result of the MCAT/UKCAT scores and during the admissions process when the complete academic and behavioral profile of the candidate is evaluated.

SLIDE 140

What this means is that in practice only about 0.01% of the ~12% of graduating secondary school students who say they want to become physicians actually do so. Yet despite this high degree of selection and the extensive and costly training that follows, iatrogenesis is still a leading cause of death in both the UK and the US!

The implications of this for cryonics are pretty straightforward, although still hard to comprehend. In fact, most cryonicists simply refuse to believe what is on the previous slide and the 5 slides that follow.

SLIDE 141

All of these errors have occurred in the period of 1991 thru the present. Some, such as reversing the arterial and venous bypass lines or pouring sterile perfusate into a feces soiled container before perfusing it through a patient defy understanding even when it is accepted that they actually took place.

SLIDE 142

As we’ve just seen, as is the case with iatrogenic errors in medicine, mistakes happen even when practitioners are highly trained and carefully vetted. Without exception all of the well respected and highly qualified critical care physicians and surgeons whom I’ve known well have told me that in the course of their careers they made errors that cost patients their lives or resulted in serious and lasting morbidity. Indeed, I’ve made mistakes in caring for patients – the most serious of which involved errors in judgment that resulted in extra minutes of exposure to warm ischemia. In hindsight, both of these errors were easily avoidable by the simple expedient of insisting that reliable, trained cryonics organization personnel stay with the patient continuously after the start of Standby – regardless of how uncomfortable or problematic that might be for the family so long as our ability to provide Standby for the patient was not compromised.

SLIDE 143

The issue here is not that errors were made, but rather the underlying reasons, the frequency and the repetitiveness of the errors. Because of the enormous surface tension of water any air bubbles present in blood that are larger in diameter than the capillaries act as obstructions, or emboli. Thus, any air introduced into the arterial circulation of a patient receiving extracorporeal treatment will result in blockage or embolization of the arteries supplying the tissues with blood. Depending upon the amount of air and the area it embolizes, “pumping air” will result in either serious injury or death.

There is an old saying amongst perfusionists: “There are two types of perfusionists: those who have pumped air (into a patient‟s circulatory system), and those who will.” Particularly in the days before microbubble detectors with automatic interrupts to shut down flow and clamp the line supplying blood to the patient were developed and put into universal use, it was typically only a matter of time until any given perfusionist made a mistake that resulted in air being perfused into a patient. This might happen once in the course of a 20 year career during which time thousands of patients would have been perfused for an aggregate of tens of thousands of clinical hours.

SLIDE 144

It should also be understood that this aphorism includes incidents where introduction of air into the patient‟s circulatory system was arguably unavoidable. Here I‟d like to speak from personal experience. For about 8 years I was a hemodialysis technician both in the outpatient and acute care (ITU) setting. During that time I „pumped air‟ once. In this photo you see me doing hemodialysis in 1978 in Indianapolis, IN.

Microbubble detection equipment was available at that time, but not used at the institution where I worked. If you look at the schematic of the extracorporeal circuit used in dialysis you‟ll note that the leg of tubing connecting the patient‟s arm (artery) to the pump will be under negative pressure with respect to the atmosphere. In order for ~250 ml/min of blood to be withdrawn from the small caliber radial artery it is necessary to “suck” on the vessel. A consequence of this is that if there are any holes – even ones too tiny to see – in the tubing between the artery and the pump raceway air will enter. The dialyzer is inverted to serve as a bubble trap and there is yet another bubble trap before the blood is returned to the patient.

However, in the event the breach in the tubing is very small the resulting bubbles are microscopic and remain suspended in the blood even as it passes through the dialyzer and the bubble trap. Fortunately, in dialysis, we are returning blood to the venous circulation as opposed to the arterial circulation and that means that we have another safety feature – an air bubble filter in the form of the lungs. In the case I‟m discussing here there was a manufacturing defect in the arterial tubing set such that where the blood conducting tubing from the patient was joined to the pump raceway there was an incomplete seal. While the defect was invisible to the eye it was of sufficient size to allow the creation of a steady stream of microbubbles.

Approximately an hour into the treatment my patient began to complain of back pain and shortly thereafter shortness of breath (SOB). I rechecked the composition of the dialysate (blood washing solution) and checked the integrity of the circuit and found nothing amiss. However, as the back pain and SOB increased in severity I became extremely concerned. I realized that these were symptoms of micro-air embolism and I got a flashlight and carefully examined the tubing carrying blood back to the patient.

There was a barely visible fine whitish line at the top of some of the tubing. This was an accumulation of microbubbles that had risen to the top of the blood flowing through the tubing. The patient was immediately removed from the machine and recovered uneventfully and with no lasting harm.

Interestingly, it took the deaths of two patients from air embolism at that institution before ultrasonic air bubble detectors were purchased and added to the dialysis machines.

SLIDE 145

At left is the Travenol RSP dialysis machine that I began my career with and at right is a contemporary, highly automated hemodialysis machine. There are bubble traps on both the arterial and venous legs of the circuit and, of course, sophisticated ultrasonic microbubble detectors which will shut down the pumps and clamp the lines in the event air in the blood is detected. Additionally, these machines mix the dialysate in real time and ensure it is safe, calculate and implement water removal from the patient and otherwise carry out a myriad of tasks we never dreamed would be possible to „automate‟ in 1978.

Most of these advances came at the price of injury or death to patients who were treated with earlier generations of less sophisticated equipment. In 1978 universal chronic hemodialysis was only 6 years old in the US and I worked in one of the pioneering units making the treatment available to hundreds of patients who previously would have died. While some of the errors and shortcomings of that program were avoidable – many were not – they came as part of the price tag for implementing a then new and demanding technology on a scale previously undreamed of.

SLIDE 146

I understand errors and I understand their increased frequency and probable severity when implementing any complicated new technology. However, that is not the kind of failure I‟m talking about here in cryonics. The errors listed in these slides are not occasional but rather have become routine. Many are so base that they rise to the level of uncaring negligence.

Consider, for example, the case where a patient frozen to dry ice temperature was removed from dry ice storage and packed in water ice for air shipment to the cryonics facility because of airline restrictions on the amount of dry ice that could be used to refrigerate the patient in transit. Obviously, the patient thawed out before arriving at the cryonics facility and had to be refrozen. That means that tissue ultrastructure that was compressed and fragmented by initial straight freezing (but ostensibly locked in place by ice) would be returned to an aqueous and diffusible state – indeed a state characterized by intense fluid turbulence and “stirring” as concentrated pools of electrolyte diffused and re-equilibrated with the large masses of nearly pure water created by melting ice crystals!

When “average” cryonicists with no technical background or training are told that an “experienced” cryopreservation team leader took a patient out of dry ice and packed him in water ice they are uniformly appalled. Most cannot even understand how or why such a decision would be made by anyone, let alone a highly experienced cryonics caregiver. The same is true of many of the other errors just discussed.

But what is perhaps most shocking and seemingly inexplicable is the complete absence of any visible emotional reaction to these errors. When I discovered microbubbles in the venous return line of the patient I was dialyzing I had an immediate and strong reaction of fear and anxiety bordering on terror. Was the patient going to be all right? Had any permanent harm been done? Next came a wave of dread and worry that I had not delivered good care. Was there something I could have or should have done to prevent the injury to the patient? Could I have detected the problem sooner and acted to prevent some of the pain the patient experienced? With years of experience in medicine I’ve come to understand that this kind of emotional response is both normal and healthy. Strong feelings of discomfort in such situations are an essential part of not repeating the error. This empathetic and self critical emotional response to iatrogenic events seems to be completely absent in an increasing number of cryonics caregivers.

THE POD PEOPLE

SLIDE 147

As it turns out, I was not alone in having noticed this phenomenon. Aschwin de Wolf, then employed at Suspended Animation, Inc. in South Florida, was observing the same kind of behavior in a range of settings within the cryonics community. We both found it puzzling to the point of incomprehensibility that people who were delivering care to cryonics patients, in some cases medically trained professionals, could be so indifferent to errors that would, in a conventional medical setting, be career ending or at very least result in costly and traumatic litigation.

This phenomenon was most pronounced in non-cryonicist medical and technical professionals who had been hired to deliver care to cryonics patients. Superficially these individuals seemed to be competent and caring, but a closer examination revealed this to be anything but the case. This was especially surprising to me because I had hired and worked with non-cryonicist medical professionals in the past and had never encountered behavior even remotely like that which Aschwin first identified. In my correspondence with Aschwin I likened such individuals to the “Pod People” in the novel and films Invasion of the Body Snatchers.

SLIDE 148

While we speculated as to the possible motivation such people might have in becoming and remaining involved in delivering cryonics services (financial gain aside) we did not have to speculate as to what constituted a “Pod Person” in cryonics.

SLIDE 149

I want to credit Aschwin with first articulating most of these characteristics. He put into words things which I had observed myself, but had not fully understood and he identified a number of traits which I had not (at that time) observed myself. Since he was a cryonicist and he was in intimate contact with a culture of non-cryonicist “employee professionals” he was uniquely situated to observe and understand what was going on.

SLIDE 150

What he discovered was that people who are not cryonicists, and who are not selected and mentored to hold the values of people who are, behaved with uncaring indifference towards their patients. Not infrequently they actually held cryonicists in contempt considering them “chumps” or “fools” who are tilting at windmills while being consumed with an unnatural and cowardly fear of death.

It seems likely that these people are, in effect, recruited from and filtered out of the larger population of caring and empathetic health care providers and professionals. Absent a cohesive program of instruction and mentoring coupled with meaningful and results-driven day to day activity it would be difficult for anyone, cryonicist

or not, to remain engaged and committed to such a job. More to the point, few if any truly competent and caring persons (professional or otherwise) would accept and remain in a job where there was no “real” day-to-day work, no leadership, and no sense of mission or accomplishment. The kind of people who stay in such a position – especially given their active contempt for their employers and patients – are not psychologically healthy and are certainly lacking not only in compassion, but in work ethic.

Such “sterile” cryonics service operations led by people who lack vision, passion and commitment to cryonics themselves become highly efficient recruitment facilities for individuals who are, at best, borderline sociopaths.

SLIDE 151

In considering the history of cryonics it became all too apparent that the existence of Pod People was by no means a new phenomenon. As many people in cryonics over the years have observed, cryonics is a magnet for frauds and charlatans. Important extensions to that observation are that the majority of these individuals are also sociopaths and that they are routinely placed in positions of power by cryonicists and cryonics organizations.

This was true in 1966 when Robert Nelson arrived on scene and it has remained the case over the course of the subsequent four decades. The Olga Visser episode is only the most public of many, many other situations where deeply disturbed or frankly sociopathic individuals have been placed in positions of power and authority in cryonics, often within weeks or months of arriving on the scene!

Charles Platt chronicled the Visser saga very well:

http://www.cryocare.org/index.cgi?subdir=ccrpt10&url=visser.html, and I excerpt it only briefly here:

“On October 9th, 1995, readers of the sci.cryonics Usenet news group found themselves confronted with a strange report quoted from the South African Sunday Times. Supposedly, a 37-year-old cardiovascular perfusionist named Olga Visser had developed a new cryoprotectant that would enable human hearts to be frozen with virtually no damage, opening up exciting possibilities in the field of transplants, where organs usually have to be utilized within several hours after removal.

According to the Times Ms. Visser had started her cryoprotectant research two years previously when she helped to establish a heart-valve organ bank. Since valves can be cryopreserved using DMSO, she saw no reason why she shouldn’t be able to freeze whole hearts as well. Undeterred by her lack of knowledge of cryobiology, she consulted some experts, read some journals, and formulated her own cryoprotectant.

When she applied it to a pig heart, she reported “no damage” after the heart was rewarmed from liquid nitrogen. She described similar success with human heart tissue. Finally, “a rat heart was frozen, unfrozen, and then warmed by a special process–and started beating.

On September 8th an astonishing press release was issued jointly by Robert Ettinger, president of The Cryonics Institute (CI), and Steve Bridge, president of Alcor Foundation. Apparently Ettinger had been in discreet contact with Ms. Visser earlier in the year, had satisfied himself that her work was genuine, and then contacted Alcor.

The two groups formed an unprecedented secret alliance, contributing money to Ms. Visser’s research and ultimately flying her to Alcor’s facility in Scottsdale, Arizona. From August 30th through September 4th she demonstrated her experiment to Ettinger, Bridge, and several officers and directors of Alcor. She also gave CI and Alcor an exclusive license to use her present and future technology for cryonics applications.”

SLIDE 152

Ultimately, Visser was shown to be at best a misguided incompetent, and at worst a calculating con artist. When her „novel cryoprotectant‟ was put to an objective test at Alcor‟s facilities in February of 1997, it failed utterly to protect rat hearts against freezing. The net financial hit cryonics, including licensing fees paid to Visser, air fare, equipment purchases, and contributions to support her research was estimated by Alcor‟s then President Steve Bridge to be ~ $50K. Charles Platt sums it up aptly:

“Olga Visser’s brief passage through cryonics could still turn out to be a positive, salutary event if it reminds us to be more circumspect in the future. The next time a character out of a Heinlein novel turns up with a secret formula to fix our deepest fears, we may be a little less willing to pay cash for the recipe. We may even be a little more tolerant of the smart-asses who insist on reminding us that death is not an easy adversary, human biology is infernally delicate and difficult to preserve, and scientific rigor is a fundamental necessity, not a tiresome detail.”

SLIDE 153

Why this happens is not much of a mystery when it is examined in the context of other disciplines that command power over and control of peoples‟ lives. Medicine is not more overrun with psychopathic quacks than it is only because there is a profession of medicine, and there are also vast bodies of regulation and law with serious penalties attached, that govern its practice. Cryonics lacks all of these safeguards. Imagine, if you will, what the situation would be if such psychopaths were empowered to fly airplanes, captain ships, or design large, heavy structures such as multi-story buildings, bridges and dams? Indeed, when such people do succeed in occupying these positions disaster is the inevitable result.

Absent these controls, both internal and external, cryonics will continue to fall prey to quacks, frauds and most dangerously, sociopaths seeking positions of perceived psychological power and control with the bonus of being increasingly well paid for indefensibly careless and sloppy work.

SLIDE 154

Remember my example of repetitive iatrogenesis associated with ascites? Just a few weeks after I gave the first version of this lecture in 2008 it happened yet again, this time to cryonics pioneer (and my personal mentor), Curtis Henderson. See :

http://www.suspendedinc.com/cases/Stabilization%20and%20Transport%20Case%20Report%20CI95.pdf

and:

http://cryonics.org/reports/CI95.html.

I am a deeply committed and seasoned veteran of cryonics and I am telling you, without hesitation, that what happened to Curtis had a devastating impact on me. Anyone with medical sophistication who reads those two case reports will most likely just walk away and dismiss cryonics as perhaps an interesting idea with some potential – but clearly not one whose time has not yet come.

THE DUNNING-KRUGER EFFECT

SLIDE 155

Finally, how do we explain the actions of people in cryonics who are sincere and committed cryonicists and yet who take on technical tasks that are beyond their knowledge and skill sets with terrible results? Much of what happened to Curtis Henderson, particularly with respect to the errors made which prevented him receiving effective cryoprotective perfusion, fall into this category.

I believe the explanation lies in something called the Dunning–Kruger Effect (DKE) The DKE was put forward in 1999 by Justin Kruger and David Dunning and it posits that unskilled people make poor decisions and reach

erroneous conclusions, but their incompetence denies them the meta-cognitive ability to realize their mistakes. Thus, the unskilled suffer from an illusion of superiority, rating their own abilities as above average and much higher than they actually are. This leads to the situation in which less competent people rate their own abilities higher than more competent people.

SLIDE 156

It also explains why actual competence may weaken self-confidence. Competent people often falsely assume that others have an equivalent understanding and degree of skill or competence. A very simple and pithy way to sum up the DKE was put to me by a Russian cryonicist in an elevator at Birkbeck College: “We are so ignorant that we do not even know what we need to know, or what we don‟t know that we don‟t know – and that is a very dangerous situation indeed.”

SLIDE 157

The moment at which I first truly understood the role of the DKE in causing technical mayhem in cryonics was actually documented by a journalist doing a story on the Cryonics UK (CUK) group at one of their meetings, held in Brighton, in the fall of 2009. I had met the new leader of the group the year before, and was more than a little surprised to hear him dismiss the Alcor ATP in-field cardiopulmonary bypass system as being “simple to operate and something any mortician would be capable of immediately mastering.” When I incredulously asked if this young man had ever actually seen the ATP, he replied that he had and that it was “just a box with tubes going in and out of it.” I wasn’t the only one who was surprised at this assessment: there was a professional perfusionist in the room from a prestigious UK hospital, and he also (to put it mildly) took considerable issue with this assertion.

SLIDE 158

A year later I was having much the same discussion with what constituted virtually all of the technical people in the CUK group. After much heated and futile discussion, I proposed that rather than argue about it, they simply get the equipment and simulate putting a patient on bypass starting from the time pronouncement had occurred. At this point, I think it best to let the newspaper account pick up the narrative:

“Tim put any doubts to the back of his mind. He’s raring to go. “There’s a patient on the table dying. Hurry up, Darwin says.”

But, of course, the patient is imaginary. Tim takes the lead, explaining the ins and outs of the tubing to his less experienced fellow travellers. Meanwhile Mike Darwin watches, arms crossed reprovingly, his concern for the patient growing by the second.

“Right, I started timing you three minutes ago,” he says.

A good few minutes later Tim and his not-so-crack team are still working out where the red and blue bits plug into. “The only thing that goes wrong is if you switch it on without all the bits plugged in. It doesn’t like it and it has been known to go bang,” he says cheerily.

Darwin can’t contain himself. “If I had that kit here, I’d be scared shitless. Shitless. There are some critical things wrong with the setup of that circuit.” He tells the team they have made so many mistakes the patient would have suffered irreversible brain damage by now. Darwin suggests technology has regressed since he was in his cryonic prime 20 years ago.

But the water is pumping through the system, and Sinclair’s team are fully focused on saving their imaginary patient. Whatever Darwin tells them, they believe they are ahead of their time, not behind it.”

I will add one thing that the reporter didn‟t because he had left the room to photograph some of the other CUK members before he lost his light to the setting sun. And that is that the venous blood reservoir bag in the circuit of the ATP exploded due to a misplaced clamp. The reporter apparently missed the timid request made to the meeting‟s hostess, Sylvia Sinclair, for a mop and towels to clean up the water that was all over the kitchen.

While it is true that cryonicists often have no choice but to undertake to provide and deliver care for themselves, it is equally true that they should not attempt to do so in ways that make the situation worse for the patient than had they taken a simpler approach that was, in fact, within their ability to master.

I had spent most of that day at the meeting trying to convince the CUK group that rather than the ATP, what they really needed was to use a simple, inherently „safe‟ open circuit system open circuit system equipped with a microbubble detector and auto-line clamp, to start cryoprotective perfusion as soon as was logistically feasible and to follow that with cooling of the patient to dry ice.

My lack of success in persuading obviously sincere and concerned cryonicists to undertake a course of action that was at once simpler, easier, much less costly, and vastly more likely to benefit the patient speaks to the power of the DKE and to the over-optimism and lack of realism that is endemic to cryonicists, the same over-optimism and lack of realism that makes them easy prey for con men and sociopaths.

End of Inherent Failure Mechanisms and Risks and of Lecture 2

(ischemia) ~85% of the time!

By Mike Darwin

Foreplay

It has taken me roughly 30 years to learn that having the technological capability to achieve some marvelous end is only a small part of the battle to actually achieving it.  This is profoundly true in the world of biology and medicine because, unlike as was the case with “free speech” and “private life,” there was no Martin Luther and no Thomas Paine to definitively divorce these areas of human endeavor from the grasp of the religious moralists, the secular ethicists, and the social busybodies of the earth. The life sciences have yet to have their Martin Luther’s 95 theses nailed to the doors of the places in which this culture’s moral tyrants currently reside. The separation of Church from private life which began with Luther, and of private life from state, which began with the Magna Carta and the US Declaration of Independence, could take us only so far.

Now, we are in an interesting place and time, because never before have potentially lifesaving technologies been being generated at such a phenomenal rate. And yet, they remain outside our grasp as surely and solidly as if there were an impenetrable Prespex wall between them and us. We can look, but we can’t touch.

Beyond our physical inability – or seeming physical inability – to access those lifesaving capabilities, we also pay a heavy price in a different way. Our vision and perspective becomes warped. We literally become unable to see how we might help ourselves, because we have been conditioned to be dis-empowered. We lose the ability to think outside the box and we begin endlessly replaying the failed or marginal strategies that the existing system does allow us to pursue.

However, a close look at our predicament will reveal that that Perspex wall works mostly for the masses – for them – and not for us. If we are careful and clever, we can reach through it and extract much of the technological benefit sitting there. We can do this, but they can’t. Once we understand that, it has the potential to change our perspective on everything in terms of our chances for survival, and for our chances of living productively and in comfort, while much of the rest of world may well pursue a very different path.

That’s what this article, and the ones that follow it, are about. This article is preparatory, it’s a kind of foreplay to prepare you for the powerful penetration of the ideas that are to follow.

Of  Singularities & Hams

Figure 1: Jamón ibérico de bellota is a gourmet ham made from black Iberian pigs fed only acorns during the months prior to their slaughter.

 The first few times it happened, I hardly noticed, and I can’t remember the specifics. But when it really began to annoy me I can  remember, quite clearly, perhaps because I was already in a foul mood and the surroundings were extraordinary. We had been taken out earlier in the day to see the pigs from which the jamón ibérico de bellota is made. The vile, dusty, slobbering and altogether horrid beasts are fed nothing but acorns so that their flesh is rendered especially succulent and flavorful after elaborate smoking and aging. They were moving about with indifferent belligerence, unaware that their kin were to  be on the supper menu late that afternoon. The visit to their quarters made me thankful I did not eat land vertebrates and reminded me uncomfortably of some of my compadres at the Hacienda; the several “Mr. Bigs” who had gathered to discuss the creation of a new cryonics enterprise.

As we sat down to dinner in the courtyard of the Hacienda that evening, I was seated at a table with several middle aged cryonicists and two older ones, (sadly, including myself). It wasn’t long before I was bombarded with the question I would soon find irritating, and eventually come to loathe: “Have you had genomics testing done?”

Figure 2: The courtyard of the Hacienda where my dinner companions assailed me over my lack of diligence in having my genotype analyzed to determine my disease risks.

“And why would I have that done, I asked?” My questioner, an enthusiastic thirty-something, leaned forward a bit and explained to me how rapidly the cost of sequencing DNA base pairs was dropping, and that it was now possible to tell all kinds of things about an individual’s risk for diseases by genotypic analysis.

“It costs only  $200 US; I just had mine done.”

Others began to chime in. Since it was an international crowd, the stories were fascinating and I was content to listen. Some had discovered they had Neanderthal lineage, others had discovered less exotic, but no less unexpected genetic heritage. Finally, the conversation returned to me, the apparent elder statesman and, presumably, the example setting cryonicist at the table: why hadn’t I had my genotype evaluated, and much more importantly, why didn’t I have any plans to do so?

“Look, ” I said, “I think genomics  technology is going to be incredibly valuable. I think its most immediate value is going to be in pharmacogenomics – in determining which drugs work for which individual people and which drugs don’t work, or are actually dangerous for given individuals. A bit later, this technology will likely have real prognostic value. But not now, and not for me. I’m in my early-50s. My relatives are already sick, dying or dead of illnesses that are genetically mediated. I know what my genetic risks are. In fact, from my family history alone, I’ve known what those risks are for roughly 20 years now. Both my parents are now in their 80s, and I have a very good idea of what they are going to die of. And if they don’t die of those things, well, it will be from an accident, an infection or something not likely to be readable in the tea leaves of my genome.

 Figure 3: The Hacienda on the arid Spanish countryside outside Madrid where we took our repast and discussed singularities, past, present and future.

Interestingly, my parents have had every single disease that has also killed their parents, their aunts and their uncles: cancer, hypertension, atherosclerosis, alcoholism, type II diabetes, and Alzheimer’s Disease (AD). I’m pretty sure that AD is going to claim my mother’s life, and I’d say it is probably down to atherosclerosis, and possibly cancer or emphysema, in the case of my father. With the help of modern medicine, my folks have so far dodged all of the other genetically mediated bullets that have been shot at them. So, I know my genetic risks  (and to those I’d add the risk of some peculiar autoimmune diseases in late life are present in my maternal bloodline).

But by far my biggest risks, which would not yet (to my knowledge) show up on any genotypic test are Bipolar-2 Disorder and homosexuality, both of which have a devastating impact on longevity, dramatically increasing the risk of a broad range of pathologies, including cardiovascular disease, cancer, dementia, substance abuse, other mental illness, and all cause mortality. My point is that in most cases where genes influence destiny, you’re best clue is the evolved or evolving fate of your kin – unless you are an anonymous orphan, that is.”

Still, they wouldn’t give up. The implication was that I must have genomic testing. And, truth to tell, I had, and have, no objection to it. It’s not like I am opposed on religious grounds, as if it were fortune telling. “In fact, I think it’s a nifty conversation piece and personally interesting in the bargain. It’s just that I’d have a lot higher priority uses for my $200 in terms of the dramatic medical advantages it could buy me as a cryonicist, if I had $200 to spend on such things! It would make a wonderful Newton Day gift, the kind of thing you’d like, but would never buy for yourself.”

Now that, that statement really set them off! I had thrown gasoline on a fire. Didn’t I know that the exponential decrease in the cost of DNA sequencing constituted a Singularity in biomedicine, one that was, even as were sitting there that very moment, revolutionizing medicine? “Sure.” I said, “But  there are singularities happening all the time. The thing is, most singularities in medicine unfold over a period of decades, and very few individual patients gain benefit from them on the basis of special, unique, or insider knowledge.”

But, I had lost them. They were having none of it, and I wouldn’t be the least bit surprised if I’ve lost you as well. I was irritated and frustrated and I had already lost my temper badly earlier that day. So, I decided to bite my tongue and proceed in relative silence with the rest of the meal. But what I really wanted to say to those gentleman was that, “you wouldn’t know what to do if a medical singularity were to come right up here and bite you in the ass, because it already has!”

One of the (many) reasons the meeting had crumbled was the intransigence of one of the Mr. Bigs, who wanted cryonics with the stipulation that there be essentially no ischemic time. He had his approach to solving the problem which was, well, this meeting was some years ago, and I wonder if Mr. Big is still alive?

It was a strange situation. Mr. Big was clearly not a well man and he knew this to be the case. What I suggested was straightforward, involved nothing either exotic nor illegal and was something that I knew would work, based on the sorry experience of seeing it not work with men exactly like him. I tried to explain to Mr. Big that it was now possible to “simply” look inside of him, from top to bottom, and get a fairly accurate assessment of what his risks were for deanimating in the near future. Given his medical history, which he shared with me,  I also suggested that he have a condition treated which would, probably sooner rather than later, cost him his life, or leave him profoundly disabled. He was having none of that, either!

Instead, a few hours later, here we were seated together at dinner and Mr. Big was extolling the virtues of genomic testing as a way of avoiding premature cryopreservation-  to me.  A true, nearly unalloyed medical singularity had arrived for cryonicists, and for the previous two days they had snuffled and shuffled around each other with same indifferent belligerence of the hogs in the pen nearby who were awaiting their conversion to jamón and their journey away from the Hacienda in someone’s belly. It is at moments like this, which come with increasing frequency, that I sneak a quick look out of the corners of my eyes to see if I can catch a glimpse of some dimple or ripple in the fabric of reality that will clue me into the fact that my life has really been just a joke in very poor taste  – on me.

I’ve struggled mightily with how to effectively communicate the idea that for cryonicists, a singularity of truly incredible magnitude has arrived and that it is one which, in theory, should be available for use by us now. I’m reasonably sure I’ll fail in that task and that no matter how I might have framed the argument, or presented the evidence, the outcome will remain the same. And therein probably lies yet another powerful lesson about why Singularities, wherein everything is transformed in the blink of an eye, never really happen.

How ‘Fast’ are Most Medical Singularities?

Medicine, ironically  much more so than entertainment or warfare, is bound up with the sensitive issues of ethics and morality, which have historically complicated and often slowed the propagation of paradigm changing, or so called “singularity events” within its confines.  Vaccination, contraception, anesthesia, organ transplantation, mechanical life support, resuscitation medicine, in vitro fertilization and embryo and gamete cryopreservation have all been slowed or blocked altogether as a result of religious or ethical concerns. (1,2,3) Indeed, surf the net or turn on TV today and you will see hordes of angry people decrying vaccination, contraception, and arguing furiously over life support. Support for vaccination, ~212 years after Jenner, is even eroding in the nation that spawned it!

The idea that wound infections – sepsis – were caused by a contact-transmissible agent was definitely proved by 1848, in the form of the exhaustive statistical work documenting the effectiveness of antisepsis conducted by Semmelweis. By 1860, the theoretical grounding for the basis of that transmissible agent, germ theory, was in place. Scattered throughout Europe there were a few men who understood the new paradigm and could no doubt foresee many of its practical implications in medicine. These men must have been as frustrated as cryonicists in the middle of this last( 20th) century – men like Pasteur and Koch. If ever there was a singularity in medicine, this was it. And yet, what happened?

Figure 4: President (then General) Robert E. Lee of the Confederate States of America receiving his critical Magic Lantern briefing on the revolutionary, but heretofore unappreciated work of the Hungarian physician Dr. Ignaz Phillip Semmelweis, concerning the importance of antisepsis for the control of infections in battlefield and surgical wounds. The information proved of a vital strategic advantage in helping the Confederacy to successfully prosecute the war against Union forces. Lee is seen here in the sitting room of his home in Arlington, Virginia in this classic painting by John Elder.

Perhaps it might be more instructive if we ask ourselves what should have happened according to the Singulatarian, or even according to the “popular” model of how  powerful, beneficial ideas with virtually no downsides spread through the culture. For instance, one of the most popular “what if” questions in the realm of alternate history is, what if this or that had been different that would have altered the outcome of the United States Civil War?(4) Military historians all have their favorite “what ifs” in this regard, but mine, well mine wouldn’t be military at all, but would come down to a long, drawn out Magic Lantern (PowerPoint) presentation given to a very receptive General Robert E. Lee, on the eve of the Secession. The subject of that presentation would be the revolutionary findings of two maverick Europeans; Dr. Ignaz Philipp Semmelweis, and  Dr. Louis Pasteur, as they apply to battlefield medicine and the recovery and survival of injured troops in the conflict to come.  The Confederacy lost the war for many reasons, but in the end it came down to a lack of manpower and the disproportionately draining and depressing effect that combat related sepsis had on the South. [At least, that's my story and I'm sticking to it ;-).]

Lee would listen, his military surgeons would be briefed on the Confederacy’s “secret weapon” and the tide of history would be turned. Wild and playful imaginings? Yes, but they constitute a considerably more reasonable scenario for the rapid adoption of asepsis in the US (or even half of it!) than just about any other you are likely to come up with, because the reality of what happened is almost incomprehensibly tragic.

Figure 5: In his magnificent painting entitled The Gross Clinic, Thomas Eakins graphically captures the state of surgery in the US during the decades following the US Civil War. These grotesquely unsanitary conditions had by this time to a large extent become a thing of the past in surgical theaters through much of Europe.

Figure 6: Even 14 years later, when Eakins revisits the them of the operating theater in his painting The Agnew Clinic, full adoption of asespsis and antisepsis had not occurred in the US.

Semmelweis’ work had already been published and disseminated around Europe by 1848, and by 1861, the year the American Civil War was opening, Lister was reprising Semmelweis’ discovery of antisepsis in Scotland, not with chlorine, but with carbolic acid. The sad reality was that the Americans (North and South) were so pigheaded regarding germ theory and the value of asepsis and antisepsis to medicine, that it would not be until well into the 19th century before that particular singularity fully took hold of the United States.(5)

Indeed, Lister made an “evangelical” tour of US medical schools in 1876 to little avail.(6)  Whilst the Listerian revolution was well underway in Europe by then, the situation in the US was to remain, as it was so vividly portrayed by Thomas Eakins in his magnificent oil, The Gross Clinic, which was painted the year before Lister’s missionary visit to the germ loving heathens across the pond. Fourteen years later, when Eakins painted The Agnew Clinic, we can see the beginnings of asepsis just starting to take root in the form of basic cleanliness being imposed in theatre. Clearly, antisepsis/asepsis are an example of a technological singularity in medicine, albeit one that took onto a century to fully unfold!

The Problem of Bite Back

But beyond these arguably irrational roadblocks slowing the progress of technological singularities in medicine, there are two others: the very real problems of their rational management on both the macro and the individual (patient) scale.

Figure 5: Edward Tenner’s excellent book, Why Things Bite Back explores many examples and a number of reasons why technological advances often fail to reach their expected potential, and in fact, not infrequently turn out to be self limiting, or even self defeating.

Some of the technological singularities just listed, vaccination, for instance, can have very serious practical, economic and societal consequences. Rapid and widespread introduction of vaccination into equatorial Africa by Christian missionaries, absent the concurrent introduction of agricultural and other infrastructure, resulted in a population explosion and mass famine which has not abated to this day. Oral contraception has resulted in huge demographic and social changes occurring within a single human generation; a heretofore unprecedented event in the history of our species.

While medical advances are usually perceived as an unalloyed good for the patients who will benefit from them, this is rarely, if ever the case. The discovery of x-rays opened the interior of the human body to non-invasive examination, but it also exposed the patients so viewed to initially unsuspected exposure to damaging radiation – a problem that persists in radiologic medicine through the present. Beyond the problem of unforeseen or unknown dangers, there is also the problem of technological bite back, or what Edward Tenner has called the “revenge of unintended consequences.”(7) This is a major adverse effect of technological singularities, and one which often robs them of much of their anticipated bounty – not just for societies, but for individuals as well.

As I’ve just pointed out,  new medical technologies are sharply constrained in their utility at their start due to our inexperience with their bite back potential, and with the possibility of unknown  direct adverse affects of the technology  itself. However, every great once in awhile there are peculiar exceptions, and it just so happens that cryonicists are ideally positioned to enjoy just such an exception, starting now.

References

1. Fasouliotis, Sozos J, Schenker, Joseph G, TI, Cryopreservation of embryos: Medical, ethical, and legal issues. Journal of Assisted Reproduction and Genetics. 13:10 56-76;1996.

2. Simmons , RG, Fulton , J, Fulton, RF. The Prospective Organ Transplant Donor: Problems and Prospects of Medical Innovation. OMEGA–Journal of Death and Dying. 3:4;319-339:1972

3. Carrell. JL, The Speckled Monster: A Historical Tale of Battling the Smallpox Epidemic, Dutton, 2003, ISBN-10: 0525947361.

4. McKinlay, Kantor, If The South Had Won The Civil War, Forge Books, 2001, ISBN-10: 0312869495.

5. Murphy, FP, “Ignaz Philipp Semmelweis (1818–1865): An Annotated Bibliography,” Bulletin of the History of Medicine 20(1946), 653-707: 654f.

6. Herr, HWJ, Ignorance is bliss: the Listerian revolution and  the education of American surgeons. Urology;177:457-60,2007.

7. Tenner, EW, Why Things Bite Back: The Revenge of Unintended Consequences, Vintage, 1997, ISBN-10: 0679747567.

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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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.