Illustrations by Mike Darwin

This is a work of fiction  {or is it?}

We Froze the First Fly.

Great title.

I could have written it.

I should have written it.

I’m an insect endocrinologist.

This futon in the lab lounge is so hard and lumpy I’d’ rather crash on the floor. But it’s nearly as sticky-gray as the table cum journal holder, cum lamp stand at the end of it. I am waiting on some gel tracks to finish. I wearily sit up, grab the ratted copy of PNASty on the coffee-juice soaked table next to the fridge. It comes away from the faux wood-grain surface with a stickysssssss.  The journal opens, on cue to,  ”Conversion of the chill susceptible fruit fly larva (Drosophila melanogaster) to a freeze-tolerant organism.”

Did I mention I’m also a cryonicist?

My middle name is Drosophila.

Humiliation.

Embarrassment.

Feelings of worthlessness.

Should I call GOD (Grand Old (Mike) Darwin) when I get home? That’s a conversation I can’t have here, or at Starbucks across the street.

GOD knows everything, well, almost everything.

Yeah, I should call him.

He hates it when I call him that, so I guess I should call him Darwin here, or maybe just “him”, when it’s grammatically correct.

I started phoning him after I got turned onto the history of the interaction between scientists and cryonics by something Chris Hayworth wrote.

Then I was pulled into his blog.

This place (where I work) is close to one of the Great Libraries. Periodicals. Films. There’s maybe two places  you can go to find out about the history of cryonics and science as it happened: Mike Darwin, and the Library of Congress.  When I started, I didn’t know to start with Mike Darwin. I’d have saved a lot of time. But I think it would’ve warped my perspective .

Digging through the stacks of magazines and newspapers from the 1960s and the 1970s, ordering up 35 mm film, kinescopes, and videotapes that were the size of hard drives from 1980′s, was like opening old tombs. That stuff smells. It feels ancient. Dead. Gone.

Darwin is alive. Electric. Now. He ruins the past by making it present.

The gels are done.

I’m done.

Gone.

Home.

The bugdust can wait till tomorrow.

I get Darwin on the Droid and start pouring out my woes about the missed opportunity with frozen flies. He is only mildly moved. “It’s good work,” he says, “not so much because it’s great science, but because it shows people straining to do something, to try, to be clever. I know this will sound impossibly, prickishly arrogant, but this is work that could have been done, and should have been done by a kid in high school, or middle school as a Science Fair project 10, or even 15 years ago. No, no, not the DSC (differential scanning calorimetry), and all the sophisticated science, but the basic work of trying to successfully introduce cryoprotectants into flies, or other larger organisms, and then freeze them successfully. Planaria would be a great model for that!”

“Really?” I replied with some skepticism.

The image of a Justin Bieber, working studiously at my bench,  just didn’t crystallize in my mind?

“Hell yes!”

“In the 1970s, students, children,  were freezing mammals – reproducing Smith’s work – and Greg Fahy and I had both done experiments with invertebrates (and me with vertebrates) before the Science Fair banned such work. In fact, you can introduce 6% DMSO into gold fish. I never tried to see how much additional ice that lets them tolerate. Now, because all such biological “hacking ” is banned, no kid is going to try things like introducing combinations of molecules like perhaps a  membrane protecting sugar such as trehalose,  a protective amino acid such as proline,  and a small amount of a colligative agent, such as glycerol, DMSO or ethylene glycol into a common pest, like the California garden snail. Can’t be done. They’d send the poor bastard off  for a psych referral and counseling.  “Tsk, tsk, you maladjusted, mean little bugger,” they’d say.  ”Why, the next thing you know, you’ll be pulling the wings off song birds and sniffing your mates’ jockstraps in the locker room.”

“I had to admit, he had a point. ”

“So you’re saying I shouldn’t feel so bad that I didn’t do this  experiment 10 years ago?”

“No, I’m saying that as far as your likelihood of  brilliant scientific contributions to cryobiology goes, you’re fucked.  In my opinion, that window probably closed when you were a graduate student, and it certainly closed after you were a post doc. Any mark you make scientifically now in cryobiology/cryonics will be along the lines of what Donaldson did, and Donaldson was a fucking genius.”

“And I’m guessing you think I’m not?” I replied.

“Who do people always put words in my mouth, and then get royally pissed off at me? I’m glad you’re recording these calls, and I hope you not only save them, but that you actually listen to them some day. Because when you do, you’re going find that, to your considerable surprise,  after 20 or 30 years of telling people that “Mike Darwin called you a fucking moron,” in fact, what I really said  was nothing at all. Literally, nothing at all. Please, try and remember that.

People have this remarkable tendency to substitute their own dire adjectives at junctures like this when they are forced to confront the hard reality that they are not geniuses, or millionaires, or movies stars, or any other of those nearly impossible ideals and that, at least during  this life cycle, they are not going to be.  That is one of the most important reasons why we are tangled up in cryonics in the first place! Because, if you stop and think about it even a little, not even George Clooney, or Bill Gates, or Barac Obama, or anybody gets it all. They only get a teeny tiny bit of it: and then they die. Whitney Houston. Fantastic, angelic voice. Beautiful woman. Rich, rich rich! Miserable life. Dead. Great stuff, huh? ” Cryonics isn’t just about any of those things, it’s about all of those things, minus death, and infinitely more,  and that’s what makes its transcendent.  That’s why the prefix trans keeps popping up spontaneously in cryonics (and everywhere else in human culture).”

“So what do you think I should do?” I ask.

“If you mean what specifically, the answer is, ‘I don’t know.’ And that’s because you are not a PFC and I’m not a general. You’re not a grunt with an IQ of 90, under the authority of a nation-state, that I can order about at my pleasure. If I try to do that, you’ll turn on me like a cornered rat. In fact, odds are, you’ll do that no matter how I choose to interact with you. It’s just that the odds are a lot better that it will happen later, rather than sooner.

So I can’t give you orders. I can’t even really give you specific suggestions, because as soon as I do, you’ll start returning with all kinds of ‘well but’s', because again, it will rapidly degenerate into my planning your life. That won’t work.”

“So what does work?”

“The nature of an insurgency is that, in its early stages, it is self organizing.  Still, it must reach a critical mass. How it does that is still a mystery to me. I think it is part chance, part timing, part the presence of the right individual – the nucleating individual.”

“Do you think you’re that nucleator?”

“It doesn’t matter what I think.  At any one time there are a thousand, ten thousand, maybe a million guys who think they are the nucleators. I was in the UK at the baths and all the action had stopped. All the men had gathered around the telly  to watch this ghastly, absolutely ghastly woman with Asperger’s from Scotland sing.[i] There was no sex to be had anywhere; these men had paid good money to get laid and they’re watching this ghastly woman on TV! She sang. Objectively, her voice was good. Not great, not fantastic. Definitely the kind of voice that can make a meager living for you at the low end of the industry if you have a good personality and a great manager; clearly neither of which she had. Good singing voices are common. Great singing voices, truly great singing voices, are not. Now this, on the telly, commanding the attention of gay men in a city where you can hear the most magnificent voices in the world at St. Martin’s in the Field for fucking free (if you can read)!

As it turned out, she became a sensation, went onto fame in the U.S., sold millions of albums! It was mad, absolutely mad! And I assure you, it had nothing to do with her raw talent. She was one of millions and millions of would-be nucleating agents trying for that peculiar niche, and she was in exactly the right place at exactly the right time. Did she think she was going to be a multimillionaire hit recording star? It doesn’t really matter, because she is. It’s very much like the lottery if you are poor , disenfranchised, have no other options and desperately want to get hold of millions. Well it’s really your only chance, and if you don’t play, you can’t win.

I’d also hasten to add that you’d best be careful what you wish for and be damn sure you have the tools and the talent to handle it if you get it, because most people who  win the lottery are destroyed by it. And the results of winning for most insurgents and insurgencies are disastrous for them.”

“But back to me? Where do I fit in?”

“You say you’ve become ‘obsessed’ with the war between the cryobiologists and us. What have you learned?”

“That you single-handedly squashed those Cacks . Reading that history, the history that you wrote of the battle royale between the cryobiologists and the cryonicists,  between them and us, I mean, that was the catalyst. When I began looking at the source material, it didn’t compute. ”

“Why not?”

“They caved too quickly. It was all over as if they’d been hit in the taint with a sledge hammer. That didn’t make sense. Cacks don’t wage a 20 year war, invest their reputations and take the time to go on TV and talk to journalists, and then just stop. Not. Doesn’t happen.”

“So?”

“So I wanted to know what did happen. I know that you threatened to sue them. They’re herd animals.  But some of them are mavericks. And some of them are stupid, too. ”

“Like Dr. Arthur Rowe, who, in fact, is still alive, and recently, like a frozen Woolly Mammoth in some bad B-movie, has come back to life, eons later, and is making TV appearances again, trashing cryonics.”

“Yeah, like  Arthur Rowe.”

“There are colleagues of mine here who won’t talk to any journalist, but if someone from Wired or Scientific American comes sniffing around, they can’t help themselves. Greed and ego, ego and greed.”

“Exactly.”

“So, I wanted to know what happened and that’s when I started digging. I guess that’s when I began to understand your message on Chronosphere and to understand what the word insurgency meant. I think it’s Chris Hayworth who mentions that you threatened to sue the Society for Cryobiology.

When your name comes up in cryonics, everybody thinks they know you, and everyone has a story to tell about you. In a small group of people who’ve been involved for a while, I’m usually the only one that hasn’t got anything to say. Listening to that kind of talk is funny. I sit and think about the letters written to those scientists’ bosses. And to the bosses of those scientists’ bosses. About the phone calls, probably hundreds of phone calls made to university chancellors, blood bank officials, trustee members, university board members, grant committee remembers. About all the letters, hundreds and hundreds of letters on different letter heads, on no letter heads; letters written and mailed to the same types of people complaining about the unscientific, unethical, overreaching and improper behavior of their scientist employees.  Courteous letters and not so courteous letters.

And I have to wonder what kinds of letters some of those scientists, or their families, the ones who didn’t stop their unscientific and irrational attacks on cryonics, might have received?”

“I’m sure I wouldn’t know.”

“You know, a few of the secretaries and support staff who worked for some of the most outspoken scientific critics of cryonics are still around. They offer an interesting peek into that time. You ground those people down. In fact, you sacred the crap out of them.”

“I had help.”

“I’m sure you did. But it was you. It was your idea. It was your leadership. It was your insurgency, as you would put it.”

“Yes.”

“Melody Maxim?”

“What about her?”

“She was not merely annoying, she was becoming dangerously destructive. Not because of the true things she was saying. Had she spoke the truth – no matter how malignantly or viciously, no matter with what calls for regulation and policing, I would have remained silent. But she began to lie, to defame good men who were cryopreserved and who could not defend themselves; to threaten the lives of innocent people, and to try to destroy cryonics on the basis of fraud and force. Interestingly, the response of the cryonics organizations (and their members) twenty years after the cryobiologists’ attacks on cryonics organizations that were now orders of magnitude bigger in size and with assets larger still, was to revert to type. It was exactly the same as it had been before 1980. They simply argued with these creeps in their own forums, were picked off one by one, took it, watched the opposition grow dangerously and did nothing. And in the bargain, they fought with each other!

I was stunned. Frankly, I was more stunned than I am today, having just been informed that both my parents have  been dead for four months and that I was deliberately not informed about it. It shook me to core.  I realized, as I read over that traffic, that cryonics was in no way going to work. It wasn’t an opinion, or a guess, or a hunch, it was a simple fact. It was like turning on the TV on 9/11 and seeing those people falling from one of the Twin Towers. There could be absolutely no question in your mind that whilst those people were alive, they were absolutely certain to be dead within a (short) and quantifiable period of time.

You have to realize that I was not following any of that traffic in real-time. I was busy doing all kinds of other things. In fact, during that immediate time interval, I was in London,  soaking up art, music, food, culture and having more sex than any one person should ever have. It was only because of the persistence of this fellow with the handle of Finance Director (FD), who kept intruding into my life to tell me how I was being slandered by this Melody Maxim person, that I even began to read that pap.

And then it took awhile , a long while, to deal with the shock of that “cryonics 9/11.” At least credit me with a lot more sense than George W. Bush. My measured response was to write the “Failure Analysis Lectures” which have been, I must say, a spectacular failure.

But I also began Chronosphere, and I began efforts to squelch the attacks on cryonics. I believe those were successful. Of course, Alcor was also suing Larry Johnson, and I think that that was enormously useful in that it sent the clear message that lies, even if mixed with the truth, will be very costly. They can and will cost you your home, your job, your reputation.

Unfortunately, it is in the nature of the U.S. tort system, and of insurgencies, that they  have an inherent dark side. It’s in the nature of any force, of any weapon or technology that there is the capability for harm equal to or greater than that which is present for good. Insurgencies are more like projectile weapons, than, say,  bladed weapons, such as knives or swords. As such, they are more suited for warfare and they are mostly of use for killing and mayhem. This is also the difference between the National Guard and the Army, and between the Police and the Army, and it is why you never use the Army in place of the Police. Never. The problem with the Johnson victory is that while most of the book is lies, there is still a meta-truth to it. The “victory”, which was also a shallow one, is thus further diluted, because it was not a completely just one.

There is so little second guessing the fight against the Nazi/Axis ~70 years later because:

the Nazis were  kooks,

they behaved with abominable aggressiveness,

their European allies were kooks,

they behaved with disgusting barbarity,

they left the concentration camps to be filmed and photographed,

they were utterly and completely defeated and humiliated,

it was all beautifully documented.

What you witnessed in the ultimate response to Maxim was the rekindling of a mini-insurgency. I gave no orders. Before I came on the scene, Alcor was already prosecuting Johnson, albeit neglecting their flanks with Maxim and Arnold. However, that was not enough then and it is not enough now.

It’s not just about “enemies.” It ‘s about not making progress, about not doing science. It’s about not being excited, planning, thinking, innovating and being obsessed with, and in love with cryonics. The failure to defend ourselves; that’s a symptom of all those other things being absent. Only  the sick, the weak, the distracted or the demented fail to defend themselves.”

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

By Mike Darwin

I used to love science fiction. The trouble is that it simply became too believable to be any fun anymore. Space travel? Sure, everybody knows that’s possible, and thanks to Industrial Light & Magic, we all know what it will look like, too. Multiple universes? That’s almost received dogma in physics these days,[1] and it’s made it into Scientific American,[2] not once, but twice! You can turn on your TV and watch “Fringe” if you want to have an adventure in the universe next door. Yes, it has gotten really hard for a person to have a good time these days. So I was thinking, maybe I should sit down and write some uplifting cryonics science fiction. You know, the kind of really unbelievable stuff that get’s your pulse pounding and respiratory rate up – when you’re not holding your breath, that is! Because there sure isn’t much that’s very escapist or uplifting here on Chronosphere most of the time.

The question is, am I up to the task? The good ideas have already been well exploited by literary talents far greater than mine, and it’s hard to find inspiration for “the almost impossible” from the advancing front of science, these days. Or so I thought. Then I came across a remarkable new discovery, one made just this year, in fact. It turns out that there are two Alcors! That’s right, the “star” we’ve called Alcor for about 500 years years is really two stars.[3] One star is your run of the mill M-type star, and the other is a small, dim red dwarf star about one fourth the mass of Sol. There is thus an Alcor-A and an Alcor-B. Wow! That really got me thinking, what if there were an Alcor-A and an Alcor-B cryonics organization?

Of course, we know all about Alcor-A, because we can see it. It’s everywhere; on TV, on the Internet, everywhere we look for information about cryonics. But what if there were an Alcor-B, sort of an alternate, utterly fantastical and impossible Alcor? An Alcor that could say, take $20 million dollars over 20 years or so, and, and…what? Well, in order to find out the answer to that question, you have to read my story. Now let me warn you that I’m not much of a story teller. My story is one of those newfangled ones, which has no beginning, no middle and no end! It’s just a press release by an alternate Alcor, Alcor-B. Alcor-B may seem much like Alcor-A, but I assure you that any resemblance is purely coincidental. They are enough alike, however, that I was able to use pictures, illustrations and the like from Alcor-A, to tell my story, and for that I am very grateful.

Similarly, I do what most science fiction writers do. I build heavily on existing science. Even though my story is utterly incredible, indeed completely impossible (like time travel or faster than light travel), I need the real science to try to persuade my readers (you) to suspend your disbelief just long enough to read the story through, and hopefully to thoroughly enjoy it. My premise is simple. Just a few days from now on 06 August, 2011, in a universe much like ours, a cryonics organization called Alcor-B holds a press conference and makes the following announcement. Regrettably, due to constraints on data transmission between adjacent universes, all we are able to retrieve from that event is the media handout. And so, without further ado, here it is.

An Extensive Press Briefing from the Alcor-B Foundation on the Debut of its Novel Near Vitrification Cryopreservation Technology for Humans

Alcor-B Cryopreservation Research Foundation (ABCRF)

FOR IMMEDIATE RELEASE

06 August, 2006

ALCOR-B FOUNDATION DEVELOPS NEAR VITRIFICATION TECHNOLOGY (NVT)

NEAR PERFECT STRUCTURAL

PRESERVATION OF THE HUMAN BRAIN ACHIEVED

 Figure 1: The Alcors are the second, smaller and dimmer companion stars to the Mizars, the bright stars that comprise the crook in the handle of the Big Dipper constellation. In the Arab world of the 5^th Century CE, Mizar’s much less bright (and more difficult to see) companion stars, Alcor-A and Alcor-B, were used as tests for good vision. Only someone with the clearest and most acute vision could see the Alcor’s. Alcor-B was discovered early in 2011 using Project 1640m, which makes use of the Hale Telescope’s adaptive optics system. Project 1640 gives the Hale a view almost equal to what is possible in space with the Hubble telescope. The instrument also has the ability to block out the light of a star, allowing faint objects located next to a star to be seen. The Hale, armed with Project 1640, was pointed at Alcor earlier this year and found that it isn’t a single star. Alcor has a small stellar companion that hadn’t been seen before: Alcor-B, a small, dim, red dwarf star about one fourth the mass of our Sun. To see Alcor-B you must have the superior vision that only mastery of the most sophisticated technology allows. Alcor-B is thus a test for the clearest and most acute vision – vision capable of seeing things as they really are – not just as they appear to be.

Introduction

Over the past five years the Alcor-B Cryobiology Research Foundation (Alcor-B) has been working to develop a fundamentally new and profoundly improved human cryopreservation technology platform.  We have expended $3.5 million dollars on this effort and what we have developed is nothing less than a quantum advance in the quality of cryopreservation now available to Alcor-B patients who present for treatment under optimum conditions.

Figure 2: At left, above, is a light micrograph (400x) of the molecular layerof the rabbit cerebral cortex subjected to freezing to -79^oC in the absence of cryoprotection (straight freezing). The tissue is compressed between blocks of ice that have osmotically extracted the intracellular water. At right is the molecular layer of rabbit cerebral cortex tissue (10,000x) following thawing and fixation after straight freezing. The ultrastructure of the tissue resembles that of a tissue homogenate, rather than that of the molecular layer of the cerebral cortex.[4]

Previous cryopreservation techniques have relied on freezing, usually after the introduction of a cryoprotective chemical(s) to reduce the amount of ice being formed. While this technology reduces the amount of freezing injury that occurs when human patients are cryopreserved, there is still a great deal of serious disruption of the fine structure of the brain (ultrastructure) that will require very sophisticated molecular-level repair by a mature nanotechnology – a technology that is likely to be many decades, or even a century or more in the future. Alcor-B’s development of minimal ice, or Near-Vitrification Technology (NVT) all but eliminates damage from ice formation. In fact, in experimental animals subjected to Alcor-B  NVT, damaging ice formation is confined to a few small areas of the brain comprising less than 1% of total brain volume. Similarly, NVT, delivered under optimal conditions, results in only approximately 3-5% ice formation in entire body of dogs subjected to this procedure.

What is NVT?

Under normal conditions, when a living system is cooled, the water in it, which comprises about 60% of its mass, is converted into ice. This ice forms outside of cells, not inside of them, and in the process the cells become dehydrated and shrunken. This dehydration due to ice formation increases the concentration of the salts normally present in body fluids, which in turn causes chemical injury to the proteins and the lipids (fats) that make-up the cells’ structures (Figures 2 & 3). The addition of modest (10-20% w/v) or moderate (20-50%) amounts of cryoprotective agents which do not freeze, and which interact with cellular water to reduce the amount of that water which freezes during cooling, markedly reduces freezing damage.

Figure 3: A ribbon model of a protein depicting the kind of conformational changes typically seen in protein denaturation as a result of freezing injury.

Figure 4: Above, top, shows a false-color rendering of the normal configuration of membrane lipids and the membrane protein sodium-potassium-ATPase in a bacterial cell membrane. The membrane exhibits a smooth, lamellar character, and there are only a few aggregated and displaced particles of protein evident (yellow granules). In B, at right above, there is evidence of an alteration in membrane structure after the cell has been incubated at ~ -6^oC for 1 hour in the presence of 20% w/v dimethylsulfoxide (DMSO). The membrane has developed a pebbly appearance and there are many extruded granules of protein on the membrane surface. The lower illustration (above) is a computer rendering of various lipid phase transitions in a model system (Langmuir trough), some of which result in perforations of the normally lamellar membrane structure.

In fact, such “cryo-protection” almost completely eliminates the damage that normally occurs to the lipids and proteins that cells are comprised of. This kind of cryopreservation technique is what forms the basis for the successful freeze-preservation of sperm, blood, bone marrow and many other types of cells and tissues that are amorphous in structure – in other words, cells and tissues where the cells are not attached to each other and organized in a highly structured way.

Figure 5: Typical representation of how freezing proceeds in cells and tissues. Ice begins forming outside cells, forming crystals of pure water. The salts and other solids that were formerly dissolved in the crystallized water are forced into a progressively smaller volume of unfrozen solution. This increase in the concentration of solids dissolved in the extracellular fluid osmotically extracts water from the cells, causing them to shrink. At ~ -20^oC no further water can be converted into ice and the interior of the cells remains in an unfrozen state – a highly concentrated solution of cell proteins and salts, from both inside and outside the cells. With further cooling this electrolyte gel will be converted to a crystal free glass at ~ -100^oC

Unfortunately, most multi-cellular animals, including human beings, consist of highly organized and structurally complex aggregations of cells which have specific jobs to do – jobs which can only be carried out if those structures are intact and un-disrupted. Ice formation in tissues can disrupt those important inter-cellular connections, and while the cells themselves may survive freezing and cryopreservation, the function of the tissue or organ is compromised (Figure 5).

Vitrification: A New Preservation Technology

Over the past two decades a new technology of cryopreservation has emerged and is being perfected. This technology is called “vitrification” (from the Latin vitrum, glass + Latin -ficre, -fy)because it completely suppresses the formation of damaging ice crystals during the cryopreservation process, as can be seen in Figure 6, below.

Figure 6: At bottom left a rabbit kidney that has been frozen following treatment with ~ 40% cryoprotectant agents. The kidney was submerged in solution that did not have enough cryoprotective agents present to allow it to vitrify. The kidney has a chalky, opaque appearance is due the presence of large amounts of ice in the tissue. At bottom right is a kidney that has been perfused and equilibrated with sufficient cryoprotectant to allow cooling to -140^oC with no ice formation.  Because this kidney has no ice crystals in it to refract light, it remains translucent and appears unfrozen – which is in fact the case – even though it has been converted to a solid, glassy state.^[43] At top; Even in the everyday world, the difference between ice and glass is clearly visible when the two are compared side by side.

This non-frozen glassy solid state is achieved by replacing ~60% of the water in the tissues of an organ, or a whole organism, with a combination of antifreeze molecules that completely prevent ice formation. The technique of vitrification as applied to whole organs is relatively new, and only recently has a whole mammalian organ, the rabbit kidney, been subjected to vitrification and recovered function. However, there are still many obstacles to be overcome before this technology can be routinely applied to organs to allow for the creation of organ banks, wherein a large reserve of organs and tissues can be stored indefinitely, for transplantation. And there are many additional obstacles to be overcome before whole organisms, such as human beings, can be cooled to very low temperatures for stable, indefinite storage.

Figure 7: Visual appearance of ice in a rabbit kidney that was cross-sectioned during rewarming. The kidney was perfused with a cryoprotective mixture called M22 at -22°C, cut in half, immersed in M22, vitrified at -135°C, and eventually re-warmed at ~1°C/min while being periodically photographed. Times (1:30 and 1:40) represent times in hours and minutes from the start of slow warming. The temperatures refer to ambient atmospheric temperatures near the kidney but not within the kidney itself. The upper panel shows the kidney at the point of maximum ice cross-sectional area, and the lower panel shows the kidney after complete ice melting. Both panels show the site of an inner medullary biopsy taken for differential scanning calorimetery in order to determine the actual concentration of cryoprotectants in the tissue with high precision. [http://cryoeuro.eu:8080/download/attachments/425990/FahyPhysicBiolAspectsRenalVitri2010.pdf?version=1&modificationDate=1285892563927]

The ability to cryopreserve people indefinitely would effectively allow for ‘medical time travel,’ whereby terminally ill patients with currently incurable illnesses could wait in suspended animation until medicine developed not just the cure for the particular disease that caused them to opt for cryopreservation, but also for aging and other degenerative diseases. Such a technology of fully reversible suspended animation would thus allow for a broad cross-section of the terminally ill population to have an opportunity to take advantage of indefinitely long lives in youthful good health, or in other words, what the neurosurgeon and medical commentator Dr. Sanjay Gupta, has termed “practical immortality.”

Near Vitrification Technology

Until suspended animation is developed it is necessary to cryopreserve today’s terminally ill patients with less than perfect techniques. These techniques inflict some injury, but we believe that this injury will be reversible in the future and we have excellent evidence right now that it is reversible, in principle. This is the theoretical underpinning of cryonics – the idea that if we cryopreserve today’s terminally ill patients with the best available techniques, it may well be possible to not only cure the lethal illness the patient is suffering from, but also to cure the damage incurred through the use of still imperfect cryopreservation techniques.

However, the damage inflicted by conventional freezing techniques is extensive, and it will take many decades, and perhaps even a century or two, before a mature “nanotechnology,” one capable of effecting repair at the molecular level, is likely to be developed. It would obviously be much better to be able to cryopreserve patients in such a way that the kind of damage being done could be reversed (and the patient restored to life) by biomedical technologies currently under development, and which may well become available within the next 30 to 60 years.

Alcor-B has been working relentlessly to develop such a cryopreservation platform and now, after 5 years and expenditure of $3.5 million dollars, we have succeeded. We call this cryopreservation modality Near Vitrification Technology (NVT). NVT is basically vitrification applied to the human body, or the human head in isolation, with the understanding that small islands of tissue will still be undergoing freezing, despite our best efforts to completely suppress ice formation. As you will see in the discussion that follows, the amount of ice that forms is surprisingly low; < 5% of the body as a whole and only ~1% of the brain.

It is important to point out that this ice formation is not global in nature, but rather is confined to a few tissues that are poorly circulated with blood under normal conditions, and are thus difficult to equilibrate with a sufficient amount of cryoprotectant to completely inhibit ice formation (Figure 7). It is also important to point out that even where some ice formation does take place and “freezing” occurs, it is freezing in the presence of very high concentrations of cryoprotectant drugs, and therefore the damage is much less than would be the case if freezing were to have occurred in the absence of cryoprotection.

Alcor B’s Laboratory Experience

To achieve NVT, Alcor-B is using technology similar to that developed by 21^st Century Medicine (21CM), a cryobiological research and development company located in Fontana, CA. We have used M-22 solution, a vitrification solution developed by 21CM primarily for kidney vitrification. The composition of M-22 is shown Figure 8, below. This complex mixture of antifreeze and actively ice-growth inhibiting (ice-blocking) cryoprotectants exhibits comparatively low toxicity, even at concentrations of ~60%. A unique feature of M-22 is the presence of two synthetic molecules that inhibit ice growth by binding to both the  a and c axes of ice. These molecules stabilize the solution against ice nucleation and propagation, allowing for use of the much slower cooling and rewarming rates needed for ice free cryopreservation of large tissues masses, such as humans organs or entire human beings.

Figure 8: Twenty First Century Medicine’s M-22 vitrification solution contains 5 penetrating colligative cryoprotective agents as well as 6% of non-penetrating polymers – two of which are highly active ice-blocking molecules; Supercool X-1000 and Supercool Z-1000. X-1000 contains 80% of the syndiotactic stereochemical form of polyvinyl alcohol and 20% vinyl acetate and Z-1000 is a linear polymer of polyglcerol with an average molecular weight of 750 Da. Both bind to the a and c axes of ice crystals, stabilizing solutions they are present in against ice formation during slow rates of cooling and rewarming.^[95

A fair summary of the current technological state of the art with respect to the vitrification of organs for the purpose of developing organ banks is that under ideal (laboratory) conditions it is likely now possible to place complex mammalian organs, such as the rabbit kidney, into indefinitely long suspended animation with little or no loss of viability, and no damage as a consequence of structural disruption due to ice formation. The use of radio frequency, or microwave illumination to speed rewarming, the use of warm gas (such as helium) to perfuse the organ’s circulation, or a combination of these modalities, may offer a workable solution to the problem of ice formation during rewarming. Perhaps most impressively, one mammalian kidney has survived vitrification and rewarming sufficiently intact to permit immediate support of the rabbit from which it was removed (as the sole kidney), until the animal was sacrificed for evaluation 29 days after the organ was re-implanted.

Figure 9: The first kidney to survive vitrification shortly before it was removed from the animal for evaluation after supporting its life as the sole kidney for 29 days.[5]

It is not possible to directly apply the 21CM vitrification technology to humans, or to the human brain, because of constraints on the rate at which the necessary cryoprotective drugs can be loaded into and unloaded from the brain. Kidneys can be perfused with M-22 to temperatures as low as -20^oC, whereas the brain can be perfused only to -3-4 ^oC. This means that the brain will be exposed to toxicity from the cryoprotectants in the vitrification solution, which cause injury very much like that shown in Figures 4 & 5 above – although much, much less extensively. Alcor-B research indicates that perhaps a total of 25 proteins undergo some kind of denaturation, and that fewer than 20% of the cells in an animal treated with NVT undergo alterations in membrane structure that would interfere with function upon reanimation.

Viability studies conducted by Alcor-B indicate that even with extended exposure to M-22 at -3^oC, there is recovery of ~40% of pre-preservation viability (as measured by Na++/K+ ratio). Very importantly, two dog brains out of a series of 17, subjected to NVT and stored for 7 and 11 days, respectively, at -130^oC, demonstrated brief recovery of electrical activity (EEG). Rabbit brain slices treated with M-22 in the laboratories of 21CM, have demonstrated complete recovery of viability, electrical activity and Long Term Potentiation (LTP). Importantly, LTP is the biochemical change in brain cells currently thought to encode memory. Brain slices in which LTP was induced, by simulating a learning experience with a weak electrical current, were able to ‘recall’ this event following cryopreservation and reanimation.

An additional complicating factor in achieving reversible (viable) vitrification of the mammalian brain has been the inability to continue cryoprotectant perfusion at the same subzero temperatures (-20^oC) that have proven essential for recovery of rabbit kidneys following loading and unloading with M-22. As can be seen in Figure 10, below, perfusion of the terminal concentration of M-22 is not possible below ~ -3-4^oC. Exposure to ~8.2M M-22 at such a relatively high temperature, for the final ~60 min of perfusion required to load the brain with the CPA mixture, results in major loss of viability, but does not visibly affect brain ultrastructure, as imaged using Transmission Electron Microscopy (TEM).

Figure 10: Cryoprotection and cooling protocol used to achieve structural vitrification of the rabbit brain at 21^st Century Medicine, Inc., CPA loading commences at a temperature of ~+4^oC and continues at that temperature for ~ 60 minutes while the M-22 concentration is gradually increased to ~4 M. The temperature is then reduced to ~ -3^oC while the CPA concentration is increased to ~ 8M. The total time required to achieve full equilibration of the brain with M-22 is ~ 180 minutes, after which the organ is immediately transferred to an air-blast cooler for very rapid cooling to ~ -135^oC. [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

The ultrastructure of dog brains cryopreserved using the 21CM vitrification technique shows no evidence of gross ice formation, as can be seen in the photo at the top of Figure 11, below. Indeed, there is no evidence of ice formation in any of the tissues of the head and neck with simple visual inspection.

Figure 11: Dog brains subjected to NVT under optimum laboratory conditions show no visual evidence of ice formation. However, when false-color, polarized light imaging is used, areas of minimal ice formation can be seen. Measurements of the ice content in the brain regions seen to contain ice (as above) using differential scanning calorimetery typically show ice formation in the range of 4-12% of the tissues volume. A=brain, B=space from cryoprotectant-induced brain dehydration, C=spinal cord, D=soft palate.

However, if the exposed tissues of the brain and head are subjected to false-color, polarized light imaging, the areas where ice has formed become visible as green, highlighted areas. As can be seen in Figure 11, above, small amounts of ice have formed in several brain areas, as well as in the muscle of the neck, and in the frontal sinus. The rest of the brain appears ice free, fully vitrified, and thus spared any mechanical disruption of the tissues.

Figure 12: Sections of the experimental animals were cut at deep subzero temperatures using a specially modified Bright Instruments 8000 (BI-8000) sledge microtome with electro-linear drive (left, above). The modified BI-8000 can section specimens up to 250mm long, and is liquid nitrogen cooled to maintain stable temperatures during sectioning, and to avoid artifact-crystallization due to inadvertent re-warming. The BI-8000 employs a fully automated cutting sequence and an electro-linear drive for high cutting forces. Cut sections were then removed from the BI-8000 and photographed for subsequent analysis of the effects of the NVT procedure on the tissues, including polarimetric evaluation of ice formation (e.g., when and where it occurred).

In order to resolve the question of whether or not ice is forming at the cellular and intra-cellular level, Alcor-B has conducted extensive Transmission Electron Microscopy (TEM) studies of animals’ brains subjected to NVT. Exposure to the vitrifying cryoprotectants results in extensive (but fully reversible) dehydration of the tissues (Figure 11) and this makes interpreting the TEM pictures more difficult. Aside from increased density of the ground substance (which is the molecular fabric of the brain) due to dehydration, the brain cells (neurons), their long processes (axons) and their connections (synapses) are intact.  As is evident in Figures 13-15, the architecture of NVT treated brains is essentially normal (aside from the cryoprotectant-induced dehydration). Cell membranes are crisp and intact, as are the intra-cellular membranes, including the synaptic vesicles that contain neurotransmitting chemicals.

Figure 13: TEM of rabbit cerebral cortex gray matter (~ 15,000x) subjected to vitrification, rewarming and perfusion fixation using M-22 and the perfusion protocol shown in Figure 10, above. The extensive dehydration induced by cryoprotective loading makes it difficult to visualize the finer elements of the ultrastructure such as vesicles and microtubules. The overall appearance of tissue in terms of the larger structural elements and their relationship to each other is apparently normal. [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

Figure 14: High magnification TEM (~ 40,000x) of vitrified rabbit brain tissue discloses the presence of difficult to visualize fine structures – in this case a synapse (S) with synaptic vesicles visible as dark densities in the synaptic bouton and a small myleinated (M) axon containing condensed axoplasm (A). Importantly, the topographical and structural relation of the synapse to the surrounding structures appears intact. [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

Figure 15: TEM of rabbit cerebral cortex white matter (15,000x) subjected to vitrification, rewarming and perfusion fixation using M-22 and the perfusion protocol shown in Figure 10, above. There is severe dehydration of the axoplasm and separation between some of the layers of myelin. There is no evidence of ice formation, and all structural changes appear to be a consequence of CPA-induced dehydration. These changes are reversible with controlled removal of CPA and return of the tissue to incubating medium (see Figure 17, below). [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

Figure 16: Mosaic of TEM’s demonstrating continuity of a long axon (red arrows) in a rabbit brain subjected to vitrification, rewarming and perfusion fixation using M-22 and the perfusion protocol shown in Figure 10, above. The tear in the tissue (green arrow) is believed to be a processing artifact. Two capillaries visible near the middle and top of the mosaic (blue arrows). [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

As can be seen quite dramatically above, in Figure 16, the axons are intact over long distances within the NVT treated brains. The areas of open space seen in Figure 15 are not due to ice formation, but rather appear to be tears in the brain tissue resulting from the cryoprotectant-induced dehydration. This is a worrisome problem that Alcor-B is actively working to solve. However, it should be emphasized that such damage does not destroy any information about the structure of the brain tissue. Thus, it should be possible to restore the tissue to its pre-cryopreservation state. It is also the case that micro-tears of this nature occur in concussion and other kinds of closed head trauma and that they are survivable, albeit it often with some cognitive impairment, with even the very limited repair abilities the brain naturally possesses.

Figure 17: Hippocampal CA4 cells following recovery from vitrification using a fully reversible, viability conserving technique. Following rewarming and unloading of the CPA the tissue was incubated in artificial cerebrospinal fluid at 35^oC for >60 min before being fixed in low-osmolality Karnovsky’s and examined by TEM.[6]

Most reassuringly, when brains subjected to NVT are cleared of cryoprotectant and reperfused with a life-supporting physiological solution, the dehydration induced changes in the fine structure of the brain observed in the NVT- state disappear, as can be seen in Figure 17, above.

Both freezing and vitrification have the potential to disrupt the structures that encode LTM in ways that would leave them non-infer able. Vitrification may do this by the expedient of altering membrane structure irreversibly by dehydration, or by changing the molecular structure of the membranes (or membrane components) by directly perturbing their structure. Vitrification solution is not water, and water is critical to the structure of many of the molecules inside cells. Indeed, a good part of the science behind designing tolerable vitrification solutions is to make them behave as much like water as possible – while at the same time behaving as good, or good enough, glass forming agents when cooled.On a purely structural basis it would seem that vitrification, applied under ideal (laboratory) conditions, is preserving the structures that encode memory and personality. To the extent that structural vitrification (as opposed to fully reversible, viable vitrification) perturbs or damages the biochemistry associated with LTP, there are grounds for concern. However, it seems unlikely that such injury would render the biochemistry of the brain non-infer-able, and therefore nonviable.

Real World Considerations

While laboratory investigations conducted under ideally controlled conditions provide considerable reassurance that existing cryopreservation techniques can conserve the essential structural and biochemical elements that comprise personal identity, such techniques are rarely available to human cryonics patients. Due to medico-legal and logistical constraints, most patients presenting for cryopreservation suffer extensive peri- and post-cardiac arrest global ischemia. When cryoprotection is delivered under these conditions in the laboratory setting, the results are very discouraging.

Figure 18: Feline cerebral cortex frozen, thawed and fixed in the presence of 4 M glycerol after 30 minutes of normothermic ischemia, followed by 24 hours of cold ischemia at ~2-4^oC. There was severe disruption of the tissue fine structure by ice (A,B), in addition to changes associated with ischemia such as mitochondrial swelling and blebbing of the endothelial cells (A). [TEMs by the author.][7]

In 1983, studies were undertaken by Alcor-B to determine the effects of 30 minutes of normothermic ischemia followed by 24 hours of cold ischemia at ~ 2-4^oC.^[101] Healthy adult cats were anesthetized; heparinized and cardiac arrest was induced. The animals were allowed to remain undisturbed on the operating table for 30 minutes, and then were packed completely in water ice, where they were allowed to remain for 24 hours. They were then perfused to 4 M glycerol using a linear increase in glycerol concentration during the ~ 60 minute perfusion interval. Following cryoprotective perfusion, the animals were cooled to dry ice temperature at ~ 3^oC/hour, and to liquid nitrogen temperature at ~4^oC/hour. Due to the unexpected presence of fracturing in the brain and other viscera, fixative reperfusion following thawing was not possible, and brain tissue samples for TEM were fixed by immersion.

As can be seen in Figure 18, above, there was extensive freezing damage superimposed over ischemic injury to the tissue. The mitochondria were swollen and often showed little internal structure. Neuronal plasma membranes were impossible to identify and the neuropil was macerated by what appeared to be ice artifacts. Large peri-capillary ice holes were almost uniformly present and large islands of tissue had the appearance of a tissue homogenate, as was observed in animals subjected to straight freezing. We thus wish to emphasize that for cryonics patients to obtain the maximum benefit from NVT, they must present for care immediately upon cardiac arrest, so that procedures can be undertaken to minimize the effects of lack of blood circulation on the brain and other vital organs (Figure 19).

The Alcor-B Cryopreservation Procedure

Figure 19: Ideally, immediately following the pronouncement of medico-legal death, circulation and respiration are restored by mechanical means while the patient is rapidly cooled. Medications to protect the brain against damage from lack of blood flow (ischemic injury) are also administered at this time. A new technique for cooling employing chilled liquid perfluorocarbon cycled in and out of the patient’s lungs allows for even faster cooling of the brain (~0.5 ^oC/min).[8]

Initial Stabilization & Cooling

As shown in Figure 19, above, care of Alcor-B cryonics patients commences the instant that medico-legal death is pronounced. Circulation and respiration are temporarily restored by mechanical means, initially employing chest compressions and mechanical ventilation. Cooling is also initiated at this time; both externally, and by the infusion of cold fluids into the abdominal cavity as well as chilled perfluorochemical into the lungs. Cooling using these technique can approach that achievable with cardiopulmonary bypass (1.0^oC/min) for the first 10 minutes of closed-chest cardiopulmonary support.

Figure 20: The Alcor-B extracorporeal membrane oxygenation, or ECMO cart, being used to provide circulation, gas exchange and cooling to a cryonics patient who has experienced medico-legal death in his home, under the care of home hospice.

As soon as possible, usually with 45-90 minutes of the onset of cardiac arrest, circulation and gas exchange are taken over by the use of a blood pump and a membrane oxygenator (extracorporeal membrane oxygenation, or ECMO). ECMO allows any deficits in artificial circulation or ventilation due to organ failure (such as fluid accumulation or tumor in the lungs) to be side-stepped, and it also allows for far more rapid cooling, typically in the range of 1^oC/min all the way down to a few degrees above freezing. The Alcor-B ECMO cart is shown in Figure 20, above.

Cryoprotective Perfusion

Figure 21: At left above are the process control refractometrs (inside perspex fronted cabinet) which monitor the concentration of cryoprotectants going into (arterial) and coming out of (venous) the patient. The data stream from these refractometers feeds into the cryoprotective perfusion and cooling control computer. Next to the refractometers are the cryoprotectant addition pump and the recirculating perfusate withdrawal pump. The third pump is for ‘cardiotomy suction’ to recover perfusate leaking into the chest wound and return it to the circuit. At right above is the recirculating and mixing reservoir (yellow top) sitting atop a magnetic stirring table. A magnetically driven stir bar mixes the concentrated cryoprotectant solution with the much more dilute perfusate being recirculated through the patient.

Once the patient has been stabilized and temporarily protected against further ischemic injury, he is transported to Alcor-B’s facilities for cryoprotective treatment and cooling to -150^oC for long term care. The introduction of cryoprotectants is carried out using a sophisticated, computer controlled perfusion system developed in-house by Alcor-B. The key elements of the cryoprotectant introduction circuit are shown in Figure 21, above, and in schematic form in Figure 22, below. Two low capacity pumps deliver cryoprotectant to the perfusate being recirculated through the patient, and withdraw fluid from the circuit – fluid containing water from the patient’s tissues – which is discarded.

Figure 22: A schematic diagram of the extracorporeal circuit used to replace ~60% of the water in a cryonics patient’s body with vitrification (cryoprotective) drugs.

The process of cryoprotective perfusion requires very careful control over not just the pressure and the flow rate of the cryoprotective solution through the patient’s circulatory system, but also of the temperature. The toxicity of the cryoprotective drugs is a function of both their concentration and the temperature at which they are introduced. The lower the temperature; the lower the toxicity of the cryoprotective agents. That is why, in order for the rabbit kidney to recover from cryoprotectant loading to a vitrifiable concentration of cryoprotectant, the final phase of introduction must be carried out at -20^oC. If this is done, 100% of kidneys treated with a vitrifiable amount of M-22 cryoprotectant will recover completely, and support the animal as the sole kidney after re-implantation.

Figure 23: At left, above, is the controlled temperature enclosure for cryoprotective perfusion of the patient and cooling to -150^oC. The contoured aluminum module on which the patient rests is the bottom half of the protective pod that will enclose the patient during long-term cryogenic storage. Once perfusion and deep cooling are complete, the pre-cooled upper half of the patient pod is attached and the patient is transferred to long-term storage. At right, above, is close-up view of the liquid nitrogen (LN2) vapor circulating fans and the LN2 dispensing manifold. A solenoid, under computer control, open and closes the valve to the LN2 reservoir to maintain the temperature at the desired point.

However, as previously noted, it is not yet possible to introduce M-22 into mammalian brains at such a low temperature. Nevertheless, it is still very important to control the temperature precisely and to keep it as low as possible during cryoprotectant loading. The current procedure for perfusing cryonics patients with M22 is to wash out the blood with a specially designed carrier solution called B1, and then continue perfusion with B1 at ~+3.5^oC. Over a period of ~90 minutes, a concentrated form of M22 (125%) is gradually added to effect a linear increase in concentration in the perfusion circuit in order to allow the patient’s cells adequate time to equilibrate, and thus avoid injury from too much cellular dehydration.[9]

When 50% of target concentration of M-22 is reached, there is a pause in the addition of M-22 to the circuit, again in order to allow time for cryoprotectant to equilibrate more completely, and also to allow for the patient’s temperature to be reduced to -3^oC. When the venous and arterial concentrations of M-22 are roughly equal, the concentration of M-22 is rapidly increased to 100% of the target concentration. This is done in order to minimize the toxic effects of the cryoprotectants that would occur if they were introduced at these (high) concentrations at temperatures at or above 0^oC. At this point, the arterial concentration is held between 100% and 105% as long needed, but not exceeding 5 hours, until the concentration of M-22 in the venous perfusate reaches 100% of the target concentration.[9]

Figure 24: Once the patient is connected to the cryoprotective perfusion system and flushed with B1 carrier solution, the control of all parameters of cryoprotective perfusion is assumed by the computer. Arterial and venous pressures, perfusate flow rate, cryoprotectant concentration increase and all temperatures are under computer control. This is necessary because the rapid changes to these parameters that are required to minimize perfusion time and reduce toxicity by keeping the patient’s temperature just above the freezing point of the cryoprotectant-water mixture in his tissues cannot be managed by humans – we’re too slow – and too easily distracted. 

Control over the temperature of the patient, and the perfusate flowing through his circulatory system, is achieved by the use of a sophisticated, computerized temperature controlled enclosure, in addition to the computerized perfusion system already described. This enclosure was developed by Alcor-B, and is shown in Figure 23, above. The refrigerated enclosure is cooled by liquid nitrogen vapor and it can maintain the patient’s temperature at any desired point between +15^oC and -150^oC. This means that when cryoprotective perfusion is completed, the patient can be cooled, in place within the enclosure he was perfused in, all the way to his long-term storage temperature of -150^oC. The entire perfusion and cooling to storage procedure are completely automated and continuously monitored by Alcor-B’s highly trained cryo-biomedical staff (Figure 24).

Long-Term Cryogenic Care

Figure 25: Long-term cryogenic storage is carried out using a newly developed technology known as intermediate temperature storage (ITS). ITS holds the patient at a temperature of ~ -150^oC, which is a sufficiently low temperature to stop all biochemical activity, and yet not so cold that it could cause fracturing in the patient’s tissues.

Once the patient has been cooled to storage the temperature, the other half of the protective aluminum storage pod, which has been pre-cooled to -180 ^oC, is placed atop the bottom half of the pod that the patient has rested upon during cryoprotective perfusion and cooling. With the two halves of the pod secured, the patient is them removed to long-term storage in one of Alcor-B’s unique, Intermediate Temperature Storage (ITS) units (Figure 25). There the patient can be held at -150^oC for centuries, if need be, to await rescue by more sophisticated medical technology. And in the meantime, Alcor-B will be there to care for him, and to assist in developing both the social and the technological infrastructure required to restore him to life, health and youth

Validating NVT in Cryonics Patients

Figure 26: Quality control and validation of our procedures is critically important to Alcor-B. For that reason we treat each human case as an experiment; an undertaking to be carefully documented and to be learned from. At left above is an Alcor-B patient following cooling to -150^oC. The chalky white areas present on the skin are areas where ice formation has occurred. The rest of the patient’s skin appears somewhat translucent and is not frozen, but rather is vitrified – converted into a glassy state. At right is a section of spinal cord taken from an Alcor-B neuropatient (i.e., a head-only patient). This section of cord is completely free of ice and demonstrated normal ultrastructure for tissue subjected to NVT, as can be seen in Figure 28, below.

Alcor-B is not content to rely solely on animal experiments conducted under ideal laboratory conditions; we have carried out careful examinations of patients undergoing NVT both during cooling, and after 8 months of storage, in order to evaluate the amount of superficial freezing that is occurring. As can be seen at left in Figure 26, above, there is always some ice formation in the skin of NVT patients, which is typically associated with peri-cardiac arrest trauma, or with injury to the skin secondary to the placement of temperature probes and the creation of “monitoring widows” in the skull. This ice formation is not of concern, and the amount of ice formed in the skin is within survivable limits for that tissue.

Figure 27: At left above is a dog treated with NVT under ideal conditions of 5 minutes of cardiac arrest at normal body temperature followed by 45 minutes and closed-chest CPS and then extracorporeal cooling to 10 ^oC. Cryoprotective perfusion was then carried out followed by cooling to -150^oC and solidification. There is no visible ice and the animal presents the appearance of being uniformly equilibrated with cryoprotective solution. At right, above, is a dog which experienced cardiac arrest followed by cooling with ice bags. Blood washout and cryoprotective perfusion were not initiated until 18 hours after the start of both cardiac arrest and external cooling. This animal underwent freezing and the distribution of cryoprotective agents was very inhomogeneous in the brain and in the skeletal muscle and other tissues.

We have conducted many experiments simulating the non-ideal conditions to which cryonics patients are sometimes subjected and we have also taken non-vital samples from human patients in our care that have been treated under a variety of conditions, ranging from optimum, to highly undesirable (Figure 27). In patients treated under ideal conditions, with only a few minutes of ischemia between the time of cardiac arrest (and the pronouncement of legal death) and the start of the procedure, the spinal cord is uniformly vitrified, as is evident in at right in Figure 26, above. TEM examination of tissue taken from patients treated under such conditions is indistinguishable in appearance from that observed in experimental animals (dogs) subjected NVT (Figure 28). Examination of the surface of the brain through the two observation windows (burr holes) made in the skull at the start of the procedure also show no evidence of ice formation when a patient is treated with NVT under ideal conditions.

Figure 28: Above are gray (left) and white (right) matter from the spinal cord of an Alcor-B neuropatient who underwent NVT under optimum conditions. The gray matter (left) shows two normal appearing capillaries and dehydrated, but otherwise normal fine structure. The white matter (right) shows more dehydration from cryoprotection. The interiors of the axons (axoplasm) are shrunken and the myelin sheaths that surround the axons have a rumpled and somewhat unraveled appearance.  Alcor-B is currently conducting research to try to overcome these problems. Despite these admittedly undesirable alterations, the overall structure of the spinal cord appears beautifully preserved.

Summary

Figure 29: Patients treated with NVT & ITS may be recoverable before this century’s end using biologically derived organogenesis and tissue repair technologies. This offers considerable risk reduction and improved odds that cryopreservation will be successful.

With the advent of NVT and ITS technology, Alcor-B is now able to cryopreserve patients in a state of near viability, with little structural injury. It is conceivable that patients so treated may be recoverable before this century’s end, if the pace of biomedical advance continues at the rate that it has over the past five decades. The ability to recover cryonics patients within the limits of normal corporate and human undertakings (i.e., 60 to 90 years) offers a tremendous reduction in the degree of risk to which the patients are subjected. For example, very few business entities of any kind survive long-term. Even non-profit organizations (NPOs), such as Alcor-B, have a ~95% failure rate by the 30 year mark, and of those that survive to 30 years, only ~1 % will survive to 100 years.

Figure 30: Using the “Cryonics Calculator” developed by Brook Norton (http://www.cryonicscalculator.com/), and assuming a very conservative risk of organizational failure of 30% for the first two decades of cryopreservation, 75% for the second 20 year interval, 10% for the third 20 year interval, 3% for the fourth 20 year interval and 2% for last 20 year interval the probability of being recovered from cryopreservation is only 17%. [This assumes that you are currently 50 years old and will be cryopreserved at age 90 and that you have a 5% risk of autopsy, or other catastrophic destruction of your remains prior to cryopreservation.]

 Using a very simple model of the impact of institutional failure on the chances of recovery from cryopreservation, and (approximately) applying the historical NPO failure rate, the chances that a person will be recovered from cryopreservation over a 100 year period of storage are only 8%. This outcome does not consider other risks, such as government proscription of cryonics, or existential risks, such as fire, flood, earthquake, pandemic disease, etc. Very importantly, it also does not take into account the probability that existing cryopreservation procedures may not be sufficiently advanced to allow for recovery of today’s patients (the default assigned autopsy risk is 5%, which is also quite low). Given such a high probability of failure solely from lack of institutional continuity, it should be clear why so many people, especially those who are knowledgeable and world-wise, fail to find cryonics sufficiently attractive to commit to it personally.

Figure 31: Alcor-B’s timeline to achieving fully reversible suspended animation for both the human brain and the intact human.

This is one of the principal reasons that Alcor-B is working so hard to improve the quality of cryopreservation, and to ultimately achieve fully reversible suspended animation. While the current odds of cryonics working are anything but good, we strongly believe that we can change that situation. With our commitment to research to achieve suspended animation, and our deep commitment to achieve truly long-term institutional stability, we believe cryonics will become an increasingly attractive choice. We invite you to join us in our effort to break the limiting chains of time and open a future for all of humanity that is as potentially limitless in time, as it is boundless in space.

References

1.            Deutsch D: The Fabric of Reality: The Science of Parallel Universes and Its Implications New York: Penguin; 1998.

2.            Tegmark M: Paralell Universes: http://space.mit.edu/home/tegmark/PDF/multiverse_sciam.pdf. Scientific American 2003(May, 2003):41-51.

3.            http://www.sciencedaily.com/releases/2009/12/091210092005.htm. Science Daily 2009.

4.            Darwin M, Russell, S, Wakfer, P, Wood, L, Wood, C.: Effect of a human cryopreservation protocol on the ultrastructure of the canine brain. (Originally published by BioPreservation, Inc, as BPI Tech Brief 16 on CryoNet and SciCryonics, May 31, 1995), http://wwwalcororg/Library/html/braincryopreservation2html and http://wwwalcororg/Library/html/braincryopreservation1html 1995.

5.            Fahy G, Wowk, B, Pagotan, R, et al.: Physical and biological aspects of renal vitrification. Organogenesis 2009, 5(3):167-175.

6.            Pichugin Y, Fahy, GM, Morin, R.: Cryopreservation of rat hippocampal slices by vitrification. Cryobiology 2006, 52(2):228-240.

7.            Darwin M, Leaf, JD.: Cryoprotective perfusion and freezing of the ischemic and nonischemic cat: http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1389, http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1390, http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1391, http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1392  See also: Federowicz,  MG. and Leaf JD. Cryonics. issue 30, p.14,1983. 1983.

8.            Darwin M, Russell, S, Rasch, C, O’Farrell, J, Harris, S.: A novel method of rapidly inducing or treating hypothermia or hyperpyrexia, by means of ‘mixed-mode’ (gas and liquid) ventilation using perfluorochemicals. In: In: Society of Critical Care Medicine 28th Educational and Scientific Symposium. vol. 27. San Francisco: Critical Care Medicine; 1999: A81.

9.            de Wolf A: Vitrification agents in cryonics: http://www.depressedmetabolism.com/2008/07/08/vitrification-agents-in-cryonics-m22/. 2008.

 Selected Bibliography

1. Fahy GM, Wowk B, Wu J, Phan J, Rasch C, Chang A, Zendejas E. Cryopreservation of organs by vitrification: perspectives and recent advances. Cryobiology, 2004 Apr;48(2):157-78.
2. Fahy GM, Wowk B, Pagotan R, Chang A, Phan J, Thomson B, Phan L. Physical and biological aspects of renal vitrification. Organogenesis. 2009 Jul-Sep;5(3):167-75.
3. Fahy, G.M. “A personal view of the Alcor research fund-raiser.” Cryonics, Volume 12, December 1991, Issue 137, pp. 13-14.
4. Fahy GM, Wowk B, Wu J, Paynter S. Improved vitrification solutions based on the predictability of vitrification solution toxicity. Cryobiology, 2004 Feb;48(1):22-35.
5. Wowk B, Leitl E, Rasch CM, Mesbah-Karimi N, Harris SB, Fahy GM. Vitrification enhancement by synthetic ice blocking agents. Cryobiology. 2000 May;40(3):228-36.
6. Wowk B. Anomalous high activity of a subfraction of polyvinyl alcohol ice blocker. Cryobiology. 2005 Jun;50(3):325-31.
7. Gomez-Angelats M, Cidlowski JA. Cell volume control and signal transduction in apoptosis. Toxicologic Pathology. 2002 Sep-Oct;30(5):541-51.
8. Schliess F, Haussinger D. The cellular hydration state: a critical determinant for cell death and survival. Biological Chemistry. 2002 Mar-Apr;383(3-4):577-83.
9. de Wolf, A. de Wolf, C. Advances in Cryonics Protocols, 1990-2006: http://www.alcor.org/Library/html/protocoladvances.html

Figure 1:

Understanding the Environment

The history of Alcor during the period 1983 and 1991 can be subdivided into two eras. The first was 1983 to 1987 and the second was from 1987 to 1991. The first era was the time period during which the most remarkable and enduring technical and administrative accomplishments occurred, despite the fact that there was very little cash flowing into or otherwise available to either Cryovita or Alcor at this time, and that there were virtually no paid staff. There were many reasons for this, and I feel certain that I will not succeed in identifying them all here.

Certainly one material factor was our ability to focus our efforts on research, technical matters, and core administrative development without distractions, and in a quiet, peaceful environment. Alcor neither sought nor had a significant media presence during these years and in fact, both Jerry Leaf and I were actively hostile to media coverage of the activities of Cryovita and Alcor.  The staff was small, cohesion and sense of shared mission was present, and the character and quality of activist recruits was uniformly high. Whilst money was scarce, there were neither distractions nor conflicts that interfered with its focused application.

In the decades that have passed since those days, I have had the opportunity to observe, first-hand, the workings of many enterprises of many kinds, the world over. My experience in this respect was much more limited before and during 1991. Most of the companies that I did interact with were at least “sanely” operated in that it was uncommon to observe blatantly self destructive or even criminal behavior. Many regulations were flouted, but usually not in what I would describe as in suicidal or careless ways. My experience in this regard may be an artifact of the kinds of businesses I was dealing with, and in their location.

Since that time, I have observed acts of corporate careless, negligence and outright stupidity that have had a profound effect on my attitude towards regulation. It would be easy to attribute these bad behaviors to exploitation on the part of owners or managers. However, I have all too often observed these people not only operate their facilities in blatantly dangerous ways, but to do so while exposing themselves to the same (or greater) risks of injury or death than they do to their employees, or customers. As but one example, I have repeatedly observed facilities here in the US with locked fire exists, disabled sprinkler systems, no working fire extinguishers, heavy burdens of flammable materials, makeshift and highly dangerous wiring, poor lighting, no lighted or marked fire exits and which also permitted smoking on the premises. The most shocking thing about this was that the owners worked in this environment alongside their employees and customers, and thus shared the same risk!  I have seen pharmaceutical and food handling facilities operate under conditions of not just grossly deficient worker safety, but also of disgustingly poor or absent procedures for maintaining basic cleanliness and sanitary products. I would also note that this phenomenon has recently been observed (repeatedly) amongst the giants of the pharmaceutical industry; the disgraceful behavior of McNeil Pharmaceuticals (Tylenol) /Johnson & Johnson are but one of many examples.[1, 2]

These experiences have altered my attitude towards to government regulation. First, the anger and outrage these experience have provoked in me have created a strong desire to see to it that such callous disregard for human life do not go uncorrected. Second, I have observed that much of the most basic regulation and code enforcement are carried out with at least a modicum of common sense and flexibility and that, despite its many shortcomings, basic government regulation at the community level is effective at both reducing the number of scofflaws, and educating businesses in what they need to do to have a reasonably safe, or at least not overtly dangerous, workplace. In short, I have become to believe that an unacceptably large cross-section of the business community is (mix and match as appropriate): ignorant, expedient, lacking in common sense, in denial, careless, negligent, and in some cases criminally indifferent to even the most fundamental elements of workplace safety; pretty much in that order. Behave badly, and someone is likely to come mind your business for you.

Figure 2: The first human cryonics case done at Cryovita Laboratories on 14 July, 1978 prompted a visit from the Fullerton police and the Orange County Coroner. Only photos were taken and footprints left behind. No harm was done to cryonics or to its patients in the process…

I’ve engaged in the above digression because I believe that one very significant reason for the fast pace of progress from 1983-1997 was that we were left unmolested. The fire department came and saw our non-permitted construction, but also saw that it was safe and sound and met UBC. They noted that we had extension cords running many places, including on the floor; but they also noted that where they trod upon, they were covered with vinyl protectors and that they conducted power not to space heaters or motors, but to low wattage analytical equipment, or to briefly and intermittently used devices like a sternal saw or a video recorder. It was a different time and place; an era where regulation was flexibly interpreted to accommodate both common sense and people who were acutely aware of their infrastructure shortcomings and were working to mitigate, or improve them.

When Cryovita received its first patient for perfusion and freezing the coroner and the police were called by a nosy neighbor. They came, they photographed, they pondered the law, and they left. Years later, in the dead of night, we were unloading a dual patient dewar from a Ryder rental truck (the HiCube, the same style used by Timothy Mc Veigh to blow up the Murrah Building in Oklahoma City) transported at ~45 degree angle from Emeryville, in Northern California. The dewar’s castered cradle barely fit onto the sloping lift gate and it was heavily belayed to the truck with ropes to prevent a mishap. As the dewar was moved from the box of the truck and positioned on the lift gate, the jostling liquid nitrogen inside began to aggressively boil. It was a warm, humid night in North Orange County and clouds of steamy vapor began to issue from the top of the dewar. On routine patrol, a Fullerton Police Department cruiser pulled up and stopped in the driveway ahead of us. The two uniformed officers got out and said, “Wow! that is really neat! Do you mind if we watch?” Of course we didn’t mind. They sat and watched the 15 minute unloading in respectful silence, seemingly aware of the difficulty and risk attending the operation. When we were done, they thanked us, asked a few questions and left. I can still see them in my memory driving away in the midnight moonlight.

Because we had little media interface or attention during that time, and because we were free to focus exclusively on the things we deemed both important and possible to do, we were free to progress as fast as our capital and our abilities would allow. The move from Fullerton to Riverside in 1987 seemed rich with promise. The new facility offered more space, improved credibility, and increased cash flow – the last as a result of reducing the expense of rent, insurance and the increased member support due to the “mobilizing effect” of the insurance crisis. However, the move to Riverside signaled the end of invisibility and of “flying below the radar.” When the Dora Kent incident began to unfold in the closing days of that same year,[3] the era of tranquil progress was over, in part because of the media and government assaults that followed, but also to a significant degree because of the way we ultimately responded to them.

Figure 3: In 1990 Dr. Thomas Donaldson, a long-time Alcor member and an important thinker and activist in cryonics sued the Attorney General (AG) of the state of California for the right to be cryopreserved while still legally alive. Dr. Donaldson had been diagnosed with a Grade II astrocytoma in 1988, a usually lethal malignant brain tumor. Dr. Donaldson responded well to a course of radiotherapy, but ultimately succumbed to the cancer and was presumably cryopreserved in 2006. The suit against the AG was unsuccessful.[4]

There can be no question that Alcor had to respond, and respond exactly as it did, to both the Dora Kent and DHS cases. To have done otherwise would have destroyed Alcor and very likely some or all of its patients.[5] The successful outcome of these, and associated legal cases, resulted both in membership growth and in lasting high public visibility for Alcor. With those changes came an alteration in asset allocation and in priorities. Alcor became focused on growth over both research and improved technical and biomedical performance. In addition to its intrinsic justice, the litigation launched against the California Attorney General by Alcor and Thomas Donaldson to allow “pre-mortem” cryopreservation was seen as promotional tool, and as a potential practical bonanza, should Donaldson have prevailed and cryopreservation prior to legal death become permissible in California. With the sudden and unexpected cryopreservation of Jerry Leaf in July of 1991,[6] the die was cast, and Alcor became almost exclusively focused on matters other than research or charitable activity, inside or outside the cryonics community.

Figure 4: Spreadsheet of some of Alcor’s financial parameters from fiscal year ending 1984 through the end of fiscal year 2007. The total revenues column in highlighted in purple and the expenditures for research column is highlighted in red. Expemditures for research as a fraction of total revenues declined dramatically after the Dora Kent crisis in 1988. In the ensuing 19 year expenditure for research remained a tiny fraction of Alcor’s total revenues, and with the exception of three years, failed to even approach in absolute dollar amounts the yearly disbursements for research in the years prior to 1988.

This shift in priorities was by no means subjective, or a matter of opinion. It is reflected in Alcor’s financial reports, as can be seen in Figure 4, above. The reality, in terms of the impact on research productivity, was actually much grimmer in terms of value returned for the few research dollars expended after 1988, because of two factors which do not appear on any balance sheet generated to date; the absence of donated labor and the end of the era of free parenteral products, drugs and medical consumables recovered from the medical/drug supplier waste-stream.

Figure 5: Alcor’s all-volunteer research team provided an otherwise unaffordable asset in the form of thousands of hours of contributed labor in support of Alcor’s various research undertakings.

Another development which increased costs and decreased the buying power of research dollars was the effective end of “pound seizure” in the closing years of the 1980s in Southern California. Pound seizure is the process whereby animals slated for destruction in municipal impound facilities, primarily dogs and cats, were sold, for a small fee, to federally licensed animal research facilities.

This practice was effectively eliminated by animal rights activists, although a few cities and counties still have pound seizure laws on the books.[7] The end of pound seizure raised the cost of research dogs from ~$60.00 in the early 1980s to ~ $600 by the mid-1990s. Animal rights activism also dramatically raised the cost of non-rodent animal research by requiring costly infrastructure and specialized training and certification of personnel.[8] Thus, even the very small amounts expended on research as compared to the 1980s, both relatively and absolutely, had greatly reduced buying power.

The Scope of the Progress

What follows is a listing of what was achieved by Alcor from ~ 1982 to 1990 for an estimated total of $1,772,081 in 2010 dollars.  

Basic & Applied Cryonics Research

Beginning in 1982, Alcor continued the research to establish the degree to which ultrastructure was being preserved using existing human cryopreservation techniques begun by IABS in the late 1970s. The work at IABS employed rabbits, however it was decided to use cats as the experimental animals in the Alcor work because of the previous work done on brain cryopreservation by Isamu Suda and because it was anticipated that this work would progress to the evaluation of brain viability using electrophysiology – a model that was, at that time, largely confined to the cat. This research disclosed a number of previously unknown and unexpected findings, including the presence of macroscopic fractures in the animals as a result of cooling to below the glass transition point (Tg) of the water-cryoprotectant solution present in the tissues, as well as much more serious ultrastructural disruption due to freezing damage, than was previously expected.  This work constituted the first comprehensive evaluation of human cryopreservation techniques and was also the first to examine the effects of ischemia on cryopreservation injury.

Darwin, M. And Leaf, JD. Cryoprotective perfusion and freezing of the ischemic and nonischemic cat: http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1389 –  http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1390 — http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1391 — http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1392

See also: Federowicz,  MG. and Leaf JD. Cryonics. issue 30, p.14, January, 1983.   http://www.alcor.org/cryonics/cryonics8301.txt __________________________________________

Leaf and Darwin believed that cryonics procedures should be validated in-house to the extent that it was technologically possible to do so. Building on the pioneering work of Gerald Klerbanoff and his associates at Lackland Air Force base,[9-13]  Alcor and Cryovita undertook to apply the technology being developed for extended hypothermic solid organ preservation (for transplant) to whole animals. In the early 1980s, the use of “intracellular perfusates,” principally Collins’ Solution, [14-18] and what was later to become University of Wisconsin (UW) solution,[19, 20] was allowing for 12-24 hour cold storage of human kidneys, livers and pancreases. An experimental solution which had demonstrated even better results at the Red Cross Blood Research Laboratory, Renal Preservative Solution-2 (RPS-2) was adapted for use on dogs. This solution, Mannitol-HEPES Perfusate-1 (MHP-1), allowed for the consistent recovery of dogs from 4 hours of perfusion at ~5^○C without neurological deficit and with uneventful survival of the animals into old age. The record of 4 hours for asanguineous ultraprofound hypothermic perfusion remains unbroken today.

Leaf, JD, Darwin, M, Hixon, H. A mannitol-based perfusate for reversible 5-hour asanguineous ultraprofound hypothermia in canines: http://www.alcor.org/Library/html/tbwcanine.html ­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­­

____________________________________________

By carrying out autopsies and conducting histological and ultrastructural studies on the bodies of human patients converted from whole body cryopreservation to neurocryopreservation, Alcor was the first to discover that the macroscopic fracturing of organs and tissues that were occurring in experimental animals were also occurring in human patients. This work demonstrated that existing perfusion techniques were not delivering an adequate amount of cryoprotectant to the tissues. It was also the first demonstration that at the histological level, 3M glycerol was effective in preserving tissue architecture in a state indistinguishable from that of seen in unfrozen humans.

Noble, C. Histological Examination of a temporarily cryopreserved human. Cryonics. # 52, November, 1984, pp. 13-32: http://www.alcor.org/cryonics/cryonics8411.txt

Federowicz, M., Hixon, H., and Leaf J. Post-mortem examination of three cryonic suspension patients. Cryonics.  5(9);16-28:1984: http://www.alcor.org/cryonics/cryonics8409.txt __________________________________________________

Alcor research demonstrated that hollow fiber dialyzers could be effectively used as oxygenators for extracorporeal support of animals, including adult ~25 kg dogs. This work was conducted 4 years before the first hollow fiber blood oxygenators entered clinical trials and five years before they entered routine clinical use.[21, 22]

Darwin, M. Report on the use of the Cordis-Dow hollow fiber dialyzer as a membrane oxygenator in profound hypothermia. Cryonics.  4(9);3-5:1983: http://www.alcor.org/cryonics/cryonics8309.txt

Leaf, JD, Federowicz, M, Hixon, H. Hemodialyzers as experimental hollow fiber oxygenators for biological research: a preliminary report. Cryonics. 5(5);10-19:1984: http://www.alcor.org/cryonics/cryonics8405.txt ___________________________________________________________

In 1983 Alcor developed a silicone heat exchange medium as a replacement for flammable alcohol, acetone and methanol, all of which has been previously used as heat exchange media for cooling cryonics patients. This non-toxic, low flammability liquid was later marketed by several firms, including Dow Chemical, the original supplier of the polydimethylsiloxane species used by Alcor as its low temperature heat exchange medium. The Dow product is currently sold under the brand name Syltherm.

Darwin, M, Hixon, H. Evaluation of heat exchange media for use in human cryonic suspensions. Cryonics.  5(7);17-36:1984: http://www.alcor.org/cryonics/cryonics8407.txt Retrieved 2010-08-31. _________________________________________________________

A wide range of technical and scientific advances resulted from Alcor treating each human case as what it was; an experimental procedure to be learned from by careful observation and documentation of data. Observations of the inadequacy of conventional closed chest mechanical cardiopulmonary support (CPS) led to the development and application of High Impulse CPS. Observations showing unacceptably slow rates of cooling with ice bags led to the development of the Portable Ice Bath and profound improvement in the efficiency with which hypothermia was induced. Alcor also pioneered pharmacotherapy to mitigate the ischemia-reperfusion injury cryonics patients necessarily suffer as a result of having to wait for the pronouncement of medico-legal death prior to the start of the procedure.

Leaf, JD, Federowicz, M, Hixon,H. Case report: two consecutive suspensions, a comparative study in experimental human suspended animation. Cryonics.  6(11):13-38;1985:  http://www.alcor.org/Library/html/casereport8511.html

Editorial Staff.  Meds prep: changes in suspension transport protocol. Cryonics. 10(7);15-6:1989: http://www.alcor.org/cryonics/cryonics8907.txt

Darwin, MG, Leaf, JD, Hixon, H. Case report: neuropreservation of Alcor patient A-1068. 1 of 2, Cryonics. 7(2);17-32, 1986: http://www.alcor.org/cryonics/cryonics8602.txt and Part 2 of 2: Cryonics. 7(3);15-29, 1986:   http://www.alcor.org/cryonics/cryonics8603.txt

Basic Research: Applied Biomedical Technology

Alcor applied research led to the development of a field-able platform for rapid post-cardiac arrest extracorporeal support of cryonics patients.

Leaf, J.D.,  Phases of cryonic suspension. Lecture given at the Lake Tahoe Life Extension Festival, Lake Tahoe, CA,May, 1986: http://www.lifepact.com/tahoe.htm  [68 minutes, 263 mb, .wmv, 320x240, 30 fps].

Legal & Administrative Advances

Throughout the 1980s Alcor led the way both the legal and administrative spheres of cryonics. Alcor developed the first informed consent documents for cryonics members/patients and the administrative paperwork developed by Steve Bridge (pictured below), Ron Buth and Mike Darwin has served as the model for cryonics organizations in the US and Europe. Staff, SUMS updated. Cryonics. 7(6)1986;2: http://www.alcor.org/cryonics/cryonics8606.txt

Darwin, M. The cost of cryonics. Cryonics, 11(8);15-36:1990: http://www.alcor.org/Library/html/CostOfCryonics.html 

Applied Research: Cryogenic Engineering & Patient Storage

Alcor pioneered high efficiency storage for cryonics patients with the use of the MVE A-2542 cryogenic dewar for storing neuropatients and the subsequent development of  the “bigfoot” dewar which can store four whole body patients and 4 neuropatients. Alcor was the first cryonics organization to offer seismic, ballistic and blast protection to its patients with the development of the “neurovault” in 1984.

Darwin, M. Cephalarium vault arrives. Cryonics. December, 1984, p. 1: http://www.alcor.org/cryonics/cryonics8412.txt

Editorial Staff, The cephalarium vault. Cryonics, Oct 1988, page 24:  http://www.alcor.org/cryonics/cryonics8410.txt

Editorial Staff: Bigfoot Arrives. Cryonics. 1990, 11(6):12: http://www.alcor.org/cryonics/cryonics9101.pdf

Charitable Cryonics Care Alcor engaged in considerable charity work during the 1980s taking over the care of three unfunded patients and assisting two other patients with inadequate funding into cryopreservation. This record of charitable cryogenic care of patients in need is unique to Alcor through to the present.

Editorial Staff, Making charity do good work. Cryonics, January, Issue #30, 1983: http://www.alcor.org/cryonics/cryonics8301.txt

Editorial Staff, Three patients converted to neuropreservation. Cryonics,  Issue #42, January,1984 p. 3: http://www.alcor.org/cryonics/cryonics8401.txt

Darwin, M. Dear Dr. Bedford (and those who will care for you after I do). Originally published in Cryonics (Alcor Life Extension Foundation), July, 1991.  Now at:  http://www.alcor.org/Library/html/BedfordLetter.htm

Scientific Education & Promotion of Cryonics

Throughout the 1980s and into the 1990s Alcor engaged in a vigorous program of public education and of the promotion of cryonics. Speaking engagements and outreach to the community were commonplace and an extensive package of scientific, technical and organizational information was mailed out to anyone who requested it.

Editorial Staff, Molecular engineering. Cryonics. Issue 45, April 1984 p. 5: http://www.alcor.org/cryonics/cryonics8404.txt

Drexler, KE, Molecular technology and cell repair machines, Part 1. Cryonics. 6(12)1985; 16-24: http://www.alcor.org/cryonics/cryonics8512.txt

Drexler, KE, Molecular technology and cell repair machines, Part 2. Cryonics. 7(1)1985; 19-28: 7(1)

Wowk, B, Darwin, M, Cryonics: Reaching for Tomorrow, Alcor Life Extension Foundation (February 1989), Riverside, CA, 1990: ISBN-101880209004: http://cryoeuro.eu:8080/download/attachments/425990/AlcorReachingForTomorrow1989.pdf

Alcor pioneered promotion of the idea of nanotechnology and was the first organization to promote nanotechnology as possible pathway to allowing the recovery of cryonics patients. Wowk, BW, The death of death in cryonics. Cryonics.  9(6);30-71:988:  http://www.alcor.org/cryonics/cryonics8806.txt

Alcor also pioneered paradigm shifting ideas in the promotion of cryonics, not the least of which was the excision of the words “death” and “dead” from discussions regarding the status of cryonics patients.

Teaching and Training in Cryonics Procedures

Darwin, MG. Transport Protocol for Cryonic suspension of Humans.  Alcor Life Extension Foundation, Fullerton, CA, 1986. http://www.alcor.org/Library/html/1990manual.html

Improved Emergency Response & Readiness

This period was also one of great growth in the technology for providing in-field support of patients experiencing cardiac arrest remote from Alcor’s facilities. Alcor conceived of an implemented the idea of remote standby and was the first cryonics organization to offer extended extracorporeal support during ultraprofound hypothermic transport of patients.

Editorial Staff. Alcor Coordinators: more progress. Cryonics. 6(12);2-4:1985: http://www.alcor.org/cryonics/cryonics8512.txt 54)

Editorial Staff. Alcor Coordinators: more progress. Cryonics. 6(12);2-4:1985: http://www.alcor.org/cryonics/cryonics8512.txt

Similarly, Alcor was the first cryonics organization to develop facilities for remote cryoprotective perfusion with the creation of the Alcor facility in South Florida.

Editorial Staff. Alcor Coordinators: training and equipment deployment. Cryonics. 7(1);2-4:1986: http://www.alcor.org/cryonics/cryonics8601.txt Retrieved 2010-08-31.

Darwin, M. A major advance in suspension patient support,. Cryonics. 10(8)1989:7-14. http://www.alcor.org/cryonics/cryonics8908.txt

Social & Legal Issues

Alcor’s aggressive defense of its patients and its litigation to establish the legality of cryonics in the state of California are without peer. The Dora Kent and Department of Health Services cases consumed hundreds of thousands of dollars in legal expenses and exacted a heavy human toll on the Alcor staff. End of Part 8 References

1.            Loftus P: Whistleblower’s Long Journey: http://online.wsj.com/article/SB10001424052702303443904575578713255698500.html. In: Wall Street Journal. 2010.

2.            National News Briefs; Schering-Plough Recalls Medication for Asthma:  http://www.nytimes.com/1999/12/03/us/national-news-briefs-schering-plough-recalls-medication-for-asthma.html. New York Times 1999.

3.            Darwin M: Multiple articles relating to the Dora Kent case: http://www.alcor.org/cryonics/cryonics8801.txt. Cryonics 1988, 9(1):1-35.

4.            Wikipedia: Thomas K. Donaldson: http://en.wikipedia.org/wiki/Thomas_K._Donaldson. In.; 2011.

5.            Mondragon C: A stunning legal victory for Alcor: http://www.alcor.org/Library/html/LegalVictory.html. Cryonics 1990, 11(11):3-7.

6.            Darwin M: Jerry Leaf enters cryonic suspension: http://www.alcor.org/cryonics/cryonics9109.txt. Cryonics 1991, 12(9):19-25.

7.            Hecht L (ed.): Pound seizure: when will it end :http://www.banpoundseizure.org/ps2.pdf. Park City: Citizens for Alternatives to Animal Labs.

8.            Hubel D: Animal rights movement  threatens progress of US medical research. The Scientist 1993, 7(22):11.

9.            Cline RE, Klebanoff G, Armstrong RG, Stanford W: Extracorporal circulation in hypothermia as used for total-body washout in stage IV hepatic coma. Ann Thorac Surg 1973, 16(1):44-51.

10.          Haff RC, Klebanoff G, Brown BG, Koreski WR: Asanguineous hypothermic perfusion as a means of total organism preservation. J Surg Res 1975, 19(1):13-19.

11.          Klebanoff G, Hollander D, Cosimi AB, Stanford W, Kemmerer WT: Asanguineous hypothermic total body perfusion (TBW) in the treatment of stage IV hepatic coma. J Surg Res 1972, 12(1):1-7.

12.          Klebanoff G, Langdon D, Wilen S, Tobias H: Total-body washout in hepatic coma. N Engl J Med 1973, 289(15):807.

13.          Klebanoff G, Phillips J: Temporary suspension of animation using total body perfusion and hypothermia: a preliminary report. Cryobiology 1969, 6(2):121-125.

14.          Carter JN, Collins GM, Halasz NA: Subzero nonfreezing kidney preservation. Transplant Proc 1981, 13(1 Pt 2):718-720.

15.          Collins GM, Bravo-Shugarman M, Novom S, Terasaki PI: Kidney preservation for transplantation. I. Twelve-hour storage in rabbits. Transplant Proc 1969, 1(3):801-807.

16.          Collins GM, Halasz NA: Forty-eight-hour ice storage of kidneys: importance of flush solution cation content. Surg Forum 1975, 26:337-338.

17.          Collins GM, Halasz NA: Forty-eight hour ice storage of kidneys: importance of cation content. Surgery 1976, 79(4):432-435.

18.          Hartley LC, Collins GM, Clunie GJ: Kidney preservation for transportation. Function of 29 human-cadaver kidneys preserved with an intracellular perfusate. N Engl J Med 1971, 285(19):1049-1052.

19.          Belzer FO, Glass NR, Sollinger HW, Hoffmann RM, Southard JH: A new perfusate for kidney preservation. Transplantation 1982, 33(3):322-323.

20.          Southard JH, Belzer FO: Control of canine kidney cortex slice volume and ion distribution at hypothermia by impermeable anions. Cryobiology 1980, 17(6):540-548.

21.          Haworth WS: The development of the modern oxygenator. Ann Thorac Surg 2003, 76(6):S2216-2219.

22.          Karlson KE, Massimino R, Singh AK, Cooper GN, Jr., Moran JM: Initial clinical experience with a more efficient hollow fiber oxygenator of unique design. J Cardiovasc Surg (Torino) 1987, 28(4):384-387.

Audrey U. Smith, circa 1960s

Audrey U. Smith (1915–1981), the mother of Cryobiology, was born in India on 21 May 1915. She was educated King’s College, London (first-class B.Sc., 1935); Bedford College for Women (first-class B.Sc. in physiology, 1936); registered for a Ph.D. degree at King’s College (1937); Vassar College (1937–1938); Marine Biological Institute at Woods Hole; King’s College (M.D., 1956). Professional experience: King’s College Hospital, house physician (1942), clinical pathologist (1943–1944); Epsom, public health laboratory, pathologist (1944–1945); Emergency Public Health Laboratory Service, Nottingham, pathologist (1945–1946); National Institute for Medical Research, then at Hampstead, researcher (1946–1970); Royal National Orthopaedic Hospital at Stanmore, staff (1970–1981).

Smith began her research in cryobiology in 1946 at Mill Hill in Britain’s Medical Research Council (MRC). Working with Sir Alan Parkes and Christopher Polge Smith attempted to develop a workable cryopreservation technique for animal semen. After many failed efforts Smith had success, however attempts to replicate the feat using the concentrated egg albumen that had previously conferred cryoprotection were unsuccessful. Smith noted that the particular bottle of albumen that had yielded success was not the one used in the subsequent, unsuccessful experiments and she undertook to analyze it. As she prepared to evaluate the contents of the bottle she accidentally dropped it in a laboratory sink where it shattered. As the bottle broke, a droplet of the liquid it contained traveled “in a long arc” from the sink where it landed on a laboratory hot plate. The result was a pungently acrid puff of smoke. Smith immediately recognized the odor of acrolein in the smoke; acrolein is a primary pyrolysis product of glycerol.

Hypothesizing that bottles in the chemical cupboard may have become mixed up – perhaps two loose labels had been affixed to the wrong bottles – Smith decided to try glycerol in the next round of experiments. This proved successful, and thus the first practical cryoprotectant molecule was discovered. Smith quickly extended her initial success with fowl semen to bull and human semen. This led to the development of the artificial insemination industry for dairy cattle, and the creation of human sperm banks, which were initially used as a resource primarily for married couples where the husband was infertile.

The impact of artificial insemination (AI) on milk production has been astonishing. Mostly as a result of AI, annual milk production per cow in the US has risen from 252.27 kg (13,555 lbs) in 1961 to 9,164.4 kg (20,204 lbs) in 2007, exclusive of the milk consumed by cow’s the calves. This is the primary reason why milk remains a highly affordable foodstuff and that it and its byproducts can be produced in vast surplus for export to the developing world as a major source of high quality protein.

Smith and her colleagues extended their work to tissue culture, and to a wide range of cells and tissues, including mammalian ovaries and embryos. In 1950, Smith published a paper documenting the successful cryopreservation of red blood cells. Smith expanded this technique into a workable method for cryobanking red blood cells for transfusion – a technique that is still in use to treat anemia from a variety of causes. Smith was also the first to achieve successful cryopreservation of lymphocytes and of bone marrow stem cells. The latter discovery is responsible for the existence of contemporary bone marrow banks and thus the feasibility of heterologous bone marrow transplantation in medicine.

In addition to her work on cryoprotection and the mechanics of cryoinjury, Smith, along with Sir James Lovelock and Alan Parkes, published an extraordinary series of papers documenting the ability of the golden hamster to withstand conversion of ~ 50% of their body water into ice and recover with no lasting harm. Smith and Lovelock extended this to work to rabbits and galagoes (a small primate) demonstrating acute success, but not long term survival of the animals.

In 1967 Smith was appointed head of the Division of Low Temperature Biology of the Clinical Research Centre. However when the Centre moved from Mill Hill to Harrow in 1970, Smith did not move with it due to her strong ethical objections to the application of cryopreservation to human embryos. Whilst Smith had worked closely with Robert Edwards and Patrick Steptoe, who were the first to achieve in vitro fertilization and cryopreservation of human embryos (with the birth of Louise Brown in 1978, in Oldham) she was virulently opposed to the cryopreservation of human embryos. When Smith headed the Division of Low Temperature Biology she successfully petitioned Sir Charles Harrington, then Director of the MRC, to ban all in vitro fertilization research on humans.

Smith strongly disapproved of cryonics and had such antipathy towards the father of cryonics, Robert Ettinger, that she would refer to him in conversation only as “that man.” Smith died of cancer in 1981 in Stanmore, which is located in Northwest London.

Selected Bibliography

A. U. Smith. Prevention of Haemolysis during Freezing and Thawing of Red Blood Cells.” Lancet 2 (1950):910–911.

A. U. Smith, J.E. Lovelock, A. S. Parkes. Resuscitation of hamsters after supercooling or partial crystallization at body temperature below 0 degrees C. Nature. 1954 Jun 12;173(4415):1136–1137.

C. Polge, A. Smith, and A. Parkes. Revival of spermatozoa after vitrification and dehydration. Nature (London), 164:666, 1949.

D. E. Pegg. The history and principles of cryopreservation. Semin Reprod Med, 20(1):5–13, 2002.

A. U. Smith. Biological Effects of Freezing and Supercooling, Williams and Wilkinns, London, 1961.

Introduction

Someone who wants to understand the critical technical, social, political or personal issues involved in cryonics may well turn to any of several FAQ’s (Frequently Asked Questions) sites hosted by the various cryonics organizations.^[1],[2],[3] As someone who was responsible for writing some of the answers to the technical questions used in these FAQs, I was interested to find upon revisiting them for the first time in many years that they contained little more scientific and technical information than was available more than a decade ago. Of greater concern was the realization that in some cases, the rapid and sustained advances in neuroscience and cryobiology over the past two decades offer the possibility for far more definitively bounding answers to questions such as, “under what conditions  is long term memory (LTM) and personality likely to survive (or not survive) cryopreservation, or be badly degraded?”

Beyond satisfying the intellectual curiosity of the public, these issues are a key component to informed consent for individuals considering cryopreservation for themselves, or for a family member, or other person for whom they may have the responsibility and authority to make such a decision. Furthermore, if it can be demonstrated that the biochemical and structural basis upon which memory and personality (personal identity) rest are degraded or destroyed by some cryopreservation techniques, while being preserved by others, then the issue of what treatment to choose within the sphere of human cryopreservation procedures becomes paramount.

Figure 1: At top above (A) is a typical transmission electron micrograph (TEM) of cerebral cortex tissue at 10,000 x magnification. The slice from which this image was created was ~50 nanometers (nm) thick and was cut from a minute block of brain tissue (B) in which the water was replaced with a polymer that was subsequently plasticized. Generation of useful 3-dimensional images usually requires ~100 scanned, high resolution photographs (C) for input into the tomographic program.

In the mid-1980s, neuroscientists studying the mechanics of how memory is encoded in the brain began to develop techniques precisely analogous to those used in medicine to create 3-dimensional images of tissue structure – but in this case, on the nanoscale as opposed to the macro-scale images produced from serial, uni-dimensional x-rays of tissue with Computerized Tomographic Scanning (CT-Scanning). Two techniques have been developed to allow such 3-dimensional nanoscale imaging of brain tissue: Electron Microscopic tomography^{[4, 5]} and Ultrathin Serial Section Transmission Electron Microscopy (USSTEM).^[6],[7] This latter technique consists of making ~ 100 serial sections of tissue (in this case of brain tissue), of 40 to 60 nm thickness and imaging them with conventional Transmission Electron Microscopy (TEM). The resultant images are captured on conventional high resolution photographic film[1], digitized using a standard flatbed scanner, and then subjected to computer processing using a standard PC running MS Windows to yield a 3-dimensional image which can be further manipulated, based on available software and the investigators’ objectives.^[8] The process of USSTEM is shown in Figure 1. The serial images obtained using TEM are aligned and stacked, at which point software is used to render a 3-dimensional representation of the imaged tissue (Figure 2 and Figure 19).^[9]

Figure 2: Sample volume reconstruction and analysis. Top, a section of the series (A) showing the sampling frame (blue) and identified synapses (red). The left and top edges of the frame are exclusion edges. A synapse that contacts an exclusion edge is marked with a yellow contour. Middle (B), the stack of serial sections for the aligned series is depicted in semi-transparent gray. After alignment, the sections form a volume with an irregular boundary due to the different transformations applied to each section. Inside this irregular volume, a rectangular prism or brick (purple) is defined as a reference volume for making density measurements. Finally, further processing (C) allows extraction of the desired reconstructed images from the sampled tissue block. In C, above, a set of 11 dendrite segments has been reconstructed from the serially TEM imaged tissue volume. The dendrites appear in the three-dimensional configuration that they have in the reconstructed volume. They are colored to help distinguish the individual segments and their synapse bearing spines. The red dendrite is a segment from an interneuron, as determined by the frequency and clustering of shaft synapses and the lack of mature-looking dendritic spines. [Fiala J.C., Harris K. M. (1999) Dendrite Structure. In Dendrites (eds. G. Stuart, N. Spruston, M. Häusser), Oxford University Press, in press.]^[9]

When consideration is given to the kinds of research cryonics organizations have funded over the past two decades,^{[10], [11]} it is astonishing that they have not conducted in-house, nor commissioned extramural studies of this kind on brain tissue subjected to the cryopreservation techniques they are currently using (under both ideal, and the actual clinical conditions in which they are being applied). This is especially the case because the volume reconstruction system needed to perform this kind of imaging is freely available on-line as a Windows (2000, 95, 98, and NT) application called Serial EM (sEM) Align, and is straightforward to use. The sEM Align program was developed with the funding of the Human Brain Project and is available online at http://synapses.bu.edu./.  [If cryonics organizations have retained existing tissue blocks from brain cryopreservation studies conducted in the past, these could easily be further sectioned, and the needed TEM photographs for tomographic reconstruction generated.]

Defining the Elements of Personal Identity – and it’s Destruction

What constitutes personal identity is a matter of considerable controversy and contention, and there may in fact be no single answer to the question that applies universally.[2] An excellent discussion of these issues, including a superb bibliography on the ‘problem’ of personal identity is available here: http://plato.stanford.edu/entries/identity-personal/.

Despite the uncertainty attending the definition of personal identity, it is possible to define and explore the biological and structural elements that comprise it. This is the case because only a limited number of biochemical and structural elements are candidates for encoding memory and personality, and it is now increasingly possible to image both this chemistry and structure.^{[12],[13],[14],[15],[16],[17],[18],[19, 20],[21]} Similarly, without being able to succinctly define personal identity, we nevertheless find ourselves in the position of being able to determine when it is irretrievably lost by using the criterion of “information-theoretic death” as applied to the physical structures which encode and instantiate memory and personality. Information-theoretic death is the destruction of the human brain (or any cognitive structure capable of constituting a person) and the information within it to such an extent that recovery of the original person is theoretically impossible by any physical means. The concept of information-theoretic death (ITD) arose in the 1990s in response to the problem that as medical technology advances, conditions previously considered to be death, such as cardiac arrest, become reversible and are no longer considered to be death.^[22] The criterion of ITD will be used to judge whether or not personal identity survives any given cryopreservation modality throughout this discussion.

“Information-theoretic death” is intended to mean death that is absolutely irreversible by any technology, as distinct from clinical death and legal death, which denote limitations to contextually-available medical care rather than the true theoretical limits of survival. In particular, the prospect of brain repair using molecular nanotechnology raises the possibility that medicine might someday be able to resuscitate patients even hours after the heart stops. The term “information-theoretic” is used in the sense of information theory.

Figure 3: DNA, the molecular starting recipe for constructing the individual.

The molecular foundation upon which personal identity is built is the genomes – the nuclear genome, which is comprised of genetic material from both parents – and the mitochondrial genome, which is inherited from the mother in the form of the mitochondria in the cytoplasm of the maternal oocyte. In particular, the instructions for constructing the individual that are present in the form of the nuclear DNA most powerfully contribute to the fundamental structural composition of the individual.

It is important to understand that DNA is a recipe, not a blueprint.^[23] Nuclear DNA does not specify where every cell, let alone every molecule in the human body will be placed, just as a recipe for a cake does not specify where each gas bubble in the cake will be, what their precise relationship will be to each other, or even how many bubbles there will be. The proof of this in humans is seen in the case of identical twins. Twins do not share the same fingerprints, the same retinal (or cerebral) blood vessel arrangements, and where one twin is homosexual, there is only a 38% chance that the sibling male twin will be gay, and a 30% chance of shared sexual orientation in the case of female twins.^{[24],[25],[26],[27]}

Additional determinants of brain and body structure occur during fetal development as a result of influence from the maternal biochemical environment; maternal circulating nutrient and hormone levels, harmful or beneficial maternal transmission of chemicals from the environment, and so on.

Similarly, there are biophysical influences from the environment during the growth and development of the child – and continuing through life, which may shape personal identity. Once maturation is reached, such influences are likely to be of less significance in shaping brain structure and biochemistry critical to memory and personality, although there are clearly exceptions to this rule.[3] During and after the completion of neurogenesis in the brain (~ 2 years of age)^[28] the primary determinants of memory and personality will be experiential, and will take the form of long-term memories encoded in the molecular structure of the brain. It is the complex interaction of these “recorded” experiences with the hardware encoding and processing them, that constitute personhood. The nuclear genome continues to be of importance in that it is responsible for maintaining the neuronal machinery that encompasses the person, and it is even critical to the elaboration of proteins needed to modify brain structure in order to create and maintain long-term memories.^[19]

Freezing Damage

In order to understand if personal identity survives cryopreservation, it is first necessary to understand the nature and extent of the damage that results from cryopreservation, via either freezing or vitrification, and under a range of clinically relevant conditions.

Figure 4: The water-sodium chloride-glycerol phase diagram above shows the enormous increase in salt concentration that cells are exposed to in the absence of cryoprotection (red line) and in the presence of increasing, but still modest concentrations of the cryoprotective agent (CPA) glycerol. Absent cryoprotection, there is a ~12 fold increase in the concentration of dissolved solids cells are exposed to by -20^oC; the point at which no more water can be converted into ice upon further cooling.^[29] The effect of this increase in the extracellular solute load on cell volume is shown in Figure 5, below.

A typical cartoon rendering of the effects of freezing at slow to moderate rates is shown in Figure 5, below. This scenario is accurate in that it correctly shows that under these conditions, ice forms first outside of cells, and the ice crystals consist of pure water.^[30] Ice begins forming outside of cells rather than inside of them for at least two reasons. First, the concentration of dissolved materials in the water inside cells is much higher than that present in the water outside of the cells (in the extracellular space). These concentrated materials, primarily salts, such as phosphates and potassium chloride, exert a small amount of antifreeze activity, depressing the freezing point of the intracellular milieu about a degree Celsius (C) below that of the extracellular fluid.^{[31, 32]}

The second factor in play is that for ice to begin forming at temperatures at or just below 0^oC, it is necessary for molecules known as nucleators to be present.^[33] Nucleators, or nidi as they are sometimes called, mostly consist of bacterial proteins and poorly characterized inorganic molecules, and serve as a template for ice growth to begin. This has the effect of raising the freezing point of water from -40^oC to 0^oC. It is believed that the intracellular space does not contain these nucleating agents.^[34]

Figure 5: Typical representation of how freezing proceeds in cells and tissues. Ice begins forming outside cells, forming crystals of pure water. The salts and other solids that were formerly dissolved in the crystallized water are forced into a progressively smaller volume of unfrozen solution. This increase in the concentration of solids dissolved in the extracellular fluid osmotically extracts water from the cells, causing them to shrink. At ~ -20^oC no further water can be converted into ice and the interior of the cells remains in an unfrozen state – a highly concentrated solution of cell proteins and salts, from both inside and outside the cells. With further cooling this electrolyte gel will be converted to a crystal free glass at ~ -100^oC

Once ice formation begins, as previously stated, it forms crystals of pure water, and this means that solids that were previously dissolved in the extracellular water being converted into ice are excluded from the ice crystals, and become dissolved in the progressively smaller volume of water that remains unfrozen. This has the effect of increasing the concentration of dissolved salts and proteins present outside of the cells, and as a result, water is removed from the cells by the increased extracellular osmotic pressure generated by the rising concentration of salts.^[35],[36] As the temperature is reduced, and progressively more ice forms, more and more water is osmotically extracted from the cells until an equilibrium state is reached, and all the ice that can form has done so.^[37] The result is what you see at right in Figure 5, above: severely dehydrated, shrunken cells surrounded by extracellular ice.^[38] The interiors of such cells contain such highly concentrated salts and proteins that ice cannot form there. As cooling proceeds, eventually, at a temperature of ~ -100^oC, this very viscous electrolyte gel that now comprises the intracellular space transitions from a liquid state to a solid state – in this case to a non-crystalline solid, a glass.^[29]

Chemicals that provide cryoprotection are typically antifreeze compounds that are virtually identical in their action to that of ethylene glycol and propylene glycol, the chemicals used in automobile radiators, and to winterize plumbing in recreational vehicles to protect them against freezing damage.^{[36],[35],[39]} These antifreeze compounds work not only by decreasing the point at which a mixture of the agents and water will freeze, but also by reducing the amount of ice that forms when freezing does occur.^[40] They do this by two mechanisms: a) by the ‘bulk effect’ of taking up so much space in the solution that they physically interfere with the interaction of water molecules with each other, by providing a kinetic obstacle, and b) by so strongly hydrogen bonding to water that they prevent the water from continuing to be converted into ice, once a certain amount of ice has been formed. These mechanisms of action are termed “colligative cryoprotection.”^{[36],[35],[41]} Instead of the entire volume of solution freezing, only part of it does, whilst the rest becomes a thick, un-freezable liquid that, upon further cooling, solidifies into a crystal-free and molecularly immobile glass (see Figure 6).^[42] The more of the colligative cryoprotectant molecule(s) present in the solution (or in the cells or tissues), the less ice is formed (Figure 7).^[36],[35]

Figure 6: At left a rabbit kidney that has been frozen following treatment with ~ 40% cryoprotectant agents. The kidney was submerged in solution that did not have enough cryoprotective agents present to allow it to vitrify. The kidney has a chalky, opaque appearance due the presence of large amounts of ice in the tissue. At right is a kidney that has been perfused and equilibrated with sufficient cryoprotectant to allow cooling to -140^oC with no ice formation.  Because this kidney has no ice crystals in it to refract light, it remains translucent and appears unfrozen – which is in fact the case – even though it has been converted to a solid, glassy state.^[43]

Figure 7: The fraction of a solution that is converted to ice upon freezing is a function of the amount of colligative cryoprotectant agent (CPA) present, as shown at left, above: the higher the CPA concentration, the smaller the fraction of the solution that is converted to ice at any given temperature. By contrast, in cryopreservation by vitrification the concentration of cryoprotectants is sufficiently high to prevent any ice formation during cooling, regardless of how low the temperature of the solution is cooled to. [Graphic courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

If enough antifreeze chemicals are present in the solution, no ice forms, and the solution is said to be vitrified (converted into a glassy state) if it is cooled below its solidification point.^[44] Since crystallization does not accompany solidification of solutions when they vitrify, the point at which the solution transitions from a liquid to a solid state is called the Glass Transition Point, which is abbreviated T_g. The difference between vitrified and frozen tissue is readily apparent to the naked eye, as can clearly be seen in Figure 6, above.^[43]

Figure 8: A ribbon model depicting the kind of conformational changes typically seen in protein denaturation.

The obvious advantage that vitrification enjoys over freezing is the absence of ice formation. Because the molecules that comprise the solution both inside and outside the cells remain in an unorganized state that is virtually indistinguishable from its aqueous state, there is no mechanical injury to cells due to ice growth dehydrating and compressing them – and tearing, or otherwise degrading the connections between cells. The cells are also spared the many fold increase in intra- and extra-cellular ion (salt) concentration. This latter advantage is important, because one way in which cells are injured by freezing is at the molecular level. Many of the proteins that comprise the workhorses of cellular activity, including enzymes, DNA, RNA and the structural proteins critical to the cell maintaining its shape and membrane integrity, require the presence of water to maintain their functional structure.^[45]

The water normally suspending and surrounding these proteins provides “conformational support” and is essential to many proteins maintaining their functional shape. When too much water is removed from the medium suspending proteins, the proteins may become denatured. What this means is that that the string of amino acids that make up the backbone of the protein can become unstable, and lose its necessary folding configuration, as seen in Figure 8, above. Since a protein is made up of a “bead work” of amino acids forming a long chain, it is often possible for that chain to be folded, or configured, in different ways. Unfortunately, usually only one folding configuration allows the protein to function in the lock and key fashion it requires in order to function properly – or at all.

When very high concentrations of salts or cryoprotective chemicals replace the native cellular water, protein can become completely denatured (Figure 8), or partially denatured. Sometimes this is reversible, but most often it is not, and the only solution to protein denaturation is for the cell to synthesize a replacement protein. However, if all of a species of protein that is essential to cellular metabolism is rendered inactive by denaturation, then the cell does not have the opportunity to restart metabolism, and thus replace the damaged protein(s).

Figure 9: Cartoon of cell membrane structure.The plasma membrane is composed of a lipid bilayer made up primarily of phospholipids with cholesterol interdigitated in the interior of the phospholipid bilayer. A large variety of proteins, carbohydrates, and glycolipids are studded into, or decorate the membrane and serve as receptors, signal transducers, molecular transporters, pores, ion channels and pumps. At physiological temperatures the membrane lipids exist in a disordered, highly liquid state which is converted to a more organized gel state upon cooling to ambient temperature (~ 20^oC). Further cooling can result in profound phase change in the membranes lipids resulting poration of the membrane and/or the redistribution or exclusion of embeded proteins and other structures essential to normal cell function.^[46]

Of course, cells are not just made up of proteins; they are also composed of lipids (fats) and carbohydrates. The lipids, in combination with the proteins, constitute the primary structural elements of the cells: the intracellular organelle membranes and the plasma membrane that separates the intra- from the extra-cellular spaces. The cellular membranes are the walls, floors and tanks that make up the organized structure of the cell. However, far from being passive dividers or containers, the cellular membranes are incredibly complex molecular machines which are studded with proteins that perform a vast range of functions, from the mundane to the nearly miraculous (Figure 9).^[47],[48] The cell membrane selectively controls the passage of nutrients, ions, water and a nearly endless array of signaling molecules into and out of the cell. Embedded in the membrane are structural support elements, and complex protein and protein-lipid complexes that act as molecular gatekeepers and message senders and receivers, facilitating communication between the cell and its environment.

Vitrification Damage

Both freezing and vitrification can damage cell membranes and cell proteins in the same ways. The antifreeze chemicals that comprise vitrification solutions are not water, and the very property that makes them useful in tightly binding water and preventing it from forming ice, makes then unable to fully pinch hit for water biochemically.[4] As a consequence, replacement of too much of the cellular water content with cryoprotectants in order to secure vitrification upon cooling, can result in protein denaturation.^[49] Additionally, the colligative compounds used to achieve vitrification have the potential to solubilize or dissolve membrane components,^[41] and they may also destabilize the lipid bilayer that comprises the cell membranes – or further facilitate its disturbance during cooling.

As seen in Figure 9, the cell membrane is composed of a laminar bilayer of phospholipids and cholesterol, studded with proteins, glycolipids and carbohydrates which serve as regulatory portals, signal transducers, and transporters for wastes and for molecules essential  for metabolism and structural maintenance. This bilayer is normally lamellar – a smooth arrangement of phospholipids with the hydrophilic polar heads pointing out and the hydrophobic portion forming the core of the membrane bilayer. These phospholipids and cholesterol which comprise the membrane are mostly present in the liquid state at body temperature. However, these lipids, independent of the cellular water, undergo freezing, or phase change when cooled. Many cell membrane lipids freeze at or near room temperature, and almost all cellular lipid species are frozen at high subzero temperatures (i.e., above -20^oC).^[50],[51]

Figure 10: Above, top, shows a false-color rendering of the normal configuration of membrane lipids and the membrane protein sodium-potassium-ATPase in a bacterial cell membrane. The membrane exhibits a smooth, lamellar character, and there are only a few aggregated and displaced particles of protein evident (yellow granules). In B, at right above, there is evidence of an alteration in membrane structure after the cell has been incubated at ~ -6^oC for 1 hour in the presence of 20% w/v dimethylsulfoxide (Me₂SO₄). The membrane has developed a pebbly appearance and there are many extruded granules of protein on the membrane surface.

The lower illustration (above) is a computer rendering of  various lipid phase transitions in a model system (Langmuir trough), some of which result in perforations of the normally lamellar membrane structure.

Lipid phase change can result in a variety of alterations to membrane structure, and thus function.^{[52], [53]} Reorganized lipids in the membrane may open up holes or pores which allow the leakage of water and ions into or out of the cells.^[54] Cooling, independent of freezing, can also irreversibly alter the structure of glycolipids and lipid-protein complexes, rendering them inactive. Phase change in the membrane can also result in the precipitation, relocation, or extrusion of membrane proteins critical to cellular function, as seen in Figure 10.^[55] Figure 10A shows the normal configuration of a cell membrane after rapid cooling to a stable, deep subzero temperature. The smooth lamellar character of the membrane is conserved, and very few of the particles (yellow) of sodium-potassium-ATPase protein are seen adjacent to the embedded lipoprotein structure (pink) in the membrane. In Figure 10B it is evident that the membrane has lost its smooth lamellar character and has undergone phase change. There is extensive relocation of the sodium-potassium-ATPase from the interior domain of the membrane to the surface, as well as aggregation of the protein into visible particles.

TEMs of membranes subjected to cooling to below the phase transition point of the lipids comprising them show the presence of long cylinders, which resemble the inverted hexagonal phase formed by some lipid-water dispersions under conditions of extreme dehydration. In this phase, water is found in long narrow cylinders on a hexagonal array, wherein each cylinder is surrounded by the hydrophilic moiety of the lipids. This morphology creates pores in the membrane forming a semipermeable barrier. A variety of other topological alterations in membrane structure have also been reported as a result of cooling.^{[56],[46],[57]} Lateral phase separations may also occur in the fluid state, and under conditions of low hydration, whether from the removal of water from the cellular milieu by ice formation, or as a consequence of replacement of a large fraction of the cellular water with cryoprotectants, islands of protein free membrane are observed in TEMs. Further, phase separation of membrane components may occur, which result in one phase which is enriched in highly hydrating lipid species, and another which is enriched in weakly hydrating lipid species. The significance of this alteration is that the inverted hexagonal phases can most easily be formed by weakly hydrating lipids in the absence of protein – and the change from the lamellar to the hexagonal phase compromises the relative impermeability of the membrane to ions, and even to larger molecular species.^[58]

The longer a cell is held at temperatures intermediate to the freezing points of membrane lipids, and stable, very low subzero temperatures (i.e., below – ~50^oC), the more likely lipid phase change is to occur, and the more extensive such changes will likely be.^[59] The chemical composition of the medium inside and outside the cells may also act to either stabilize, or destabilize membrane lipids, with respect to phase change.^[60] So, while these changes can and do occur in the course of freezing, they are likely to be more extensive and more biologically significant in organs (or other large systems) subjected to vitrification, where there is necessarily prolonged exposure to high concentrations of cryoprotectants in the presence of high subzero temperatures, before the system is cooled to sufficiently to arrest these adverse alterations in membrane morphology.^[61],[62]

Figure 11: Peri-capillary tear in the parenchyma of the cerebral cortex as a result of hyperosmotic dehydration secondary to cryoprotective perfusion prior to vitrification. The space resulting from the dissection of the neuropil from the capillary basement membrane has been colored with a light purple tint.

There is also osmotic injury which occurs during brain cryopreservation employing currently available vitrification techniques. The permeability of cell membranes to most cryoprotectants is very slow when compared with water. As a consequence, these osmotically active CPAs extract water from the intracellular space more rapidly than they can equilibrate with it, given the constraints imposed by the toxicity of these agents: namely, that they must be introduced at low temperatures (sometimes well below 0^oC) and that the exposure time at these relatively high temperatures must not be too long. The relative impermeability of colligative cryoprotectants to brain tissue is not, as is commonly misunderstood, a function of the blood brain barrier (BBB). Most colligative CPAs freely cross the BBB because they are lipid soluble. Rather, it is due to the kinetics of water and CPA movement across the individual brain cell membranes. The multiply membrane wrapped myleinated axons that comprise the white matter pose a special diffusion barrier, in this respect. The cerebral dehydration that results from perfusion with high molarity CPA solutions causes occasional peri-capillary tears (Figure 11) as well as infrequent small tears within the brain parenchyma.

Fracturing

Figure 12: At left, above, is a flask of vitrification solution cooled to its solidification point of ~ -135^oC. The solution is unfrozen and solid – a glass. At right, is what happens when the solution is further chilled to ~ -160^oC; the solution has extensively fractured. The center insert shows a rabbit kidney equilibrated with 4M glycerol and then frozen to -196^oC. It has extensively fractured, as well. The degree of fracturing seen in the photos above is worst case. Since the time this phenomenon was discovered, considerable work has been done to reduce the number and severity of fractures. This has been accomplished by slowing the rate of cooling during and after T_g, as well as providing for a period “stress relaxation” by annealing, or holding at a temperature near T_g. [Solution in flasks images are courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

A type of injury common to cryopreservation by both freezing and vitrification is fracturing injury.^{[42, 63],[64],[65]}When cryoprotective solutions, or organs loaded with them are cooled to significantly below the glass transition point of the CPA-water solution, internal stresses begin to build up.^[66],[67] Since CPA-water glasses are inherently weak (and most tissues are little stronger), the result is cracking, or fracturing of the solution, or of the organ, as seen in Figure 12, above. Various protocols of controlled, ultra-slow cooling, and of annealing by holding the temperature steady for an interval at or near T_g, help to reduce the number and size of fractures, but no technique has yet been identified which eliminates them. Even storing just below T_g still causes some fracturing to occur.^{[42, 68]}

Biological versus Electromechanical Machines

It is probably no accident that a large fraction of the people recruited to cryonics since its inception have been engineers, mathematicians, computer scientists, programmers, and, in general, physical science types.[5] One likely reason for this is that there is a fundamental difference in the way biological ‘machines,’ and electromechanical machines are structured and operate. Almost all electromechanical systems may be fairly described as solid state – even those in existence before the era of “modern electronics” and the advent of the transistor. By solid state, what is meant here is that virtually all their components are, literally, solid at their normal operating temperatures.

Figure 13: Living cells are not electro-mechanical machines comprised of solid-state, rigid parts which fracture, or break into discrete, easily identifiable pieces of debris when mechanically damaged.

A consequence of this is that it is possible to take a 21^st Century mobile phone, or even a 17^th century mechanical clock, and hit them with a sledgehammer, and the end result would broadly be the same: a collection of solid pieces of varying sizes and shapes that just sit there – and of course, neither the phone nor the clock would be functional. However, all the pieces would continue to exist unchanged – and they would retain their individuality and unique identity (Figure 12). And such would be the case indefinitely, as long as they are protected from the elements (moisture, oxidation, corrosion) and are kept at a temperature below their melting or combustion points.

Figure 14: Even the simpler cells in the human body are surprisingly complex, as is the case with the red blood cells (RBCs), as seen above.  At left, (A) is an electron micrograph of RBCs with a 2 micron scale bar present – each RBC is ~ 8 microns in diameter. Red cells lack nuclei and the complex interior structure of other somatic cells; however their structure is still enormously complex. As can be seen in (B) at left, the cytoskeleton, the molecular framework that gives the RBC its unique biconcave shape and deformability, is comprised of an intricate web work of proteins. The membrane itself (C) is studded and interdigiated with a wide array of complex proteins arranged into pores, pumps, and signaling devices.^[48]

Biological systems are not solid state devices, and they operate in very different ways from such instruments. The core of biological systems is the membrane; and, as previously noted,  membranes in living systems are not just passive walls or “compartment makers.” They are the engines of chemistry and action in living systems (Figures 9, 14 and 16). They have enormous complexity and they derive a great deal of their unique ability to function as living systems from their liquidity and plasticity.^[48] A physical analogy in the everyday macro-world would be that of soap bubbles (Figure 15). Cell membranes are very much like soap bubbles and they behave in broadly similar ways when stressed. If a cell  is osmotically stressed by shrinking or swelling it too much, it does not behave like a glass sphere and shatter into discrete pieces, which can be collected and reassembled. Rather it buds and ‘blebs’ and behaves like what it is, a liquid. And if it is stressed enough, it comes apart into little droplets and into smaller ‘cells;’ blebs and vesicles that have formed from the original membrane (Figure 17).

Figure 15: The soap bubble is a tolerable working analogy to the cell membrane – both the plasma membrane that encases the cells – and the intracellular membranes that bound the cellular organelles, such as the nuclei and the mitochondria, exhibit the same liquid based, space enclosing behavior.

Before disintegration or blebbing of the cell membrane occurs in response to stress (e.g., osmotic, temperature reduction, dehydration, chemical destabilization), the structure of the membrane can undergo reorganization in many ways, and the proteins embedded in the membrane may be rearranged as well. This is true not just for the plasma membrane that encloses the cell, but also for the membranes that comprise the cellular organelles. In fact, it is just such a rearrangement of mitochondrial membrane structure that underlies some of the damage that occurs in ischemia.^[69] [6] Freezing causes enormous biophysical stress to the plasma membrane, and to organelle membranes, and some of the response to this stress is to radically alter membrane structure.

Figure 16: Far from being simple, passive, devising walls, cell membranes are complex biological machines consisting of structural elements, pumps, channels, and gated pores. In turn, these various functional units are comprised of complex proteins that translate the energy generated by the cell, and stored in ATP, into the work required to regulate ion homeostasis, transport nutrients and pump out wastes.

In the worst case, the cell membranes disappear as the structures they were, and reappear as new structures; droplets of membrane material, brand new micro-cells, and so on.^[70] And they shed structures that were embedded, or enclosed in them; thus the explanation for the debris fields seen in frozen-thawed tissues that are inadequately cryoprotected.

Figure 17: At left (A), the shadow of a hepatocyte is superimposed over the debris field (B) remaining after the cell was exposed to a hypertonic medium yielding approximately the same osmotic stress as would be experienced during freezing to -20^oC (i.e., increase in incubating medium osmolality from ~310 mOsm to 3,720 mOsm). The cell has partially lyzed and has formed numerous large blebs (B) containing cytoplasmic material, as well as smaller plasma membrane vesicles devoid of interior structure. The intracellular organelles are largely unrecognizable with the exception of a few swollen mitochondria (M) and the cell nucleus (N). At right (C-D) hypertonicity-induced plasma membrane blebs and vesicles on the surface of a frog oocyte. The blebs and vesicles vary in size and were induced by exposure to a solution of approximately 3x normal tonicity (800 mOsm) – a fourth of the hyperosmotic stress imposed as a result of freezing to -20^oC.^[71]

Because biological systems are not solid state devices, if they are damaged badly enough, they do not remain as discrete, broken pieces waiting to have their pre-injury, functioning structure inferred from the debris. And it is important to understand that some of the “re-morphing” that goes on during the cryopreservation process occurs as a consequence of the enormous osmotic and mechanical stresses imposed by ice formation – not as consequence of thawing (see Figure 17, above). Further, as previously noted, there are significant changes to membrane structure, such crystallization of the lipids that occur solely as a result of cooling, and completely independent of freezing. To return to the mobile phone analogy, it would be as if the phone were made up of liquids and gels encased in soap bubble membranes, and the device was crushed. Crushing is thus a much better analogy to use when describing cryoinjury to biological systems, than is breaking or shattering.

Imagine a soap bubble with an exquisitely detailed picture embedded into its surface. A picture made up of millions of tiny pixels comprised of colored micro-particles. If you burst the bubble, some of the bubble wall material will return to a simpler, all-fluid state, and some of it may reform into new bubbles. But in any event, the picture is gone, and what’s more, it is not obvious that the image can be inferred from the puddle of particles in liquid, and the new bubbles that result. In fact, given our current understanding of physical law, inference of the image that was on the surface of the bubble before it burst is not possible.

Figure 18: Simple schematic of a neuron and its axon, dendrites and synapses. The dendritic arbors that grow out of the neuronal soma and from the axon generate hundreds of thousands of synapses (up to 1 million per neuron) that serve as the signal switching mechanisms allowing inter-cellular communication and encoding LTM.

Figure 19: At left above, the cytoskeleton of a typical cerebral cortex neuron from the hippocampus. A representative dendrite has been circled in red and is shown in greater detail in Figure 20, below. This image was created using Rotary shadow electron microscopy of a cultured, wild-type, hippocampal neuron. The plasma membrane has been removed, allowing a detailed view of the underlying cytoskeleton. At right (above) is an artist’s cutaway rendering of the axon, showing the cytoskeleton, and microtubules that carry skeletal proteins, and other nutrients from the cell soma, to the dendrites and synapses. [Stern, S, Debre, E,  Stritt, C, Berger, J, Posern, G, Knöll, B. A nuclear actin function regulates neuronal motility by serum response factor-dependent gene transcription. The Journal of Neuroscience, April 8, 2009, 29(14):4512-451: http://www.jneurosci.org/cgi/content/short/29/14/4512]

A typical cerebral cortex neuron in a mammal may have as many as one million synaptic interconnections with its neighboring neurons, yet each one is distinct, and will have different configurations of membrane proteins and structure at any instant in time. These synapses can cover the entire body of the neuron, including the dendrites, where they are observed as “synaptic spines,” using various kinds of electron microscopy. Electron microscopic examination with 3-dimensional reconstruction of a microscopic block of brain tissue demonstrates that these synapses are present at very high density in relation to each other^{.[72],[73],[74]} In Figure 20 the incredible density and exquisitely complex synaptic connections of three dendritic spines branching from three different axons is shown. Each point of color (excluding green) is a functional point of synaptic contact. The topology and apposition of these structures must be conserved during cryopreservation in order to preserve LTM.

Figure 20: A: A 10 µ segment of pyramidal cell dendrite from stratum radiatum (CA1) with thin, stubby, and mushroom-shaped spines. Spine synapses colored in red, stem (or shaft) synapses colored in blue. The dendrite was made transparent in the lower image to enable visualization of all synapses^.[75] B: Graphic reconstruction (i.e. manually shaded serial contour tracings) of dendritic spines arising from three dendrites (D1, D2, D3) participating in synaptic glomerule in thalamic ventrobasal nucleus. Some of spines are branched (asterisk). Multiple macular synapses of different afferent origin are marked in blue, red and orange. Extensive reticular adherent zones free of synaptic vesicles are marked in green. (Rat, thalamic ventrobasal nucleus.) (Adapted from Spacek and Lieberman 1974)^[76]

Figure 21: At left above is a three dimensional block of brain hippocampal tissue tomographically reconstructed from hundreds of slices of tissue 0.25 micron thick, using the same mathematical algorithms used in computerized tomography (CT) scanning employed in medical imaging. In the image above, a single dendrite has been isolated from the tissue block to show the number and configuration of its synapses. There are approximately 100 billion neurons in a typical 1.4 kg human brain, with an aggregate average of 0.15 quadrillion synapses.^{[77, 78]} At right is a representation of a neuron having undergone synaptic remodeling in response to the encoding of long-term memory; with new synaptic connections highlighted in yellow.

It is critically important, especially for the engineers, information technology, and computer scientists who are reading this to understand that the brain is not a computer, but rather, it is a massive, 3-dimensional hard-wired circuit. It does not use programming, addresses, or coding; and in engineering terms it most closely resembles pre-digital computer integrated circuits. Such circuits were constructed and wired for a discrete purpose and they did not function as multiple use processors. Similarly, the neuronal circuitry in the brain shows no evidence of using the biophysical equivalent of “addresses,” such as are used in digital computers. Each neuron has a single output axon which cannot select a receiver. Before addresses can work, they have to be agreed upon between the sender and the receiver, and such a function is problematic to have been generated as a result of biological evolution via natural selection.[7]

And of course, without addresses, there can be no ordered set of bits that carries information; and consequently no bytes, and no record. Coding, as is used in computers, is thus not possible: no location can be given to send or retrieve information from, and information cannot be moved from neuron to neuron. Another consequence of the inability of the brain to process information using programming, addresses and coding, is that information cannot be put side by side and compared. This means that information must be encoded and retrieved at the same location. A consequence of these insights is that, from an information processing standpoint, the brain is most properly viewed as a massive 3-dimensional homogenous memory array, with each memory location having its own processor, and every node and connection a site-dependent, dedicated purpose. Acute losses of circuit elements (neurons) are thus of far greater significance than are the loss of processors in a digital computer because they represent loss of the memory information they contain.

Molecular and Ultrastructural Basis of Long Term Memory

Of concern in the setting of cryonics is the nature of long term memory (LTM) and its likely response to the biophysical changes that may be imposed as a result of freezing and vitrification under different conditions. Several mechanisms are currently understood to be in play in the formation of LTM. The earliest of these is long-term potentiation (LTP), which is an enduring strengthening of signal transmission between two neurons that results from stimulating them synchronously^.[79] LTP is but one of many mechanisms that facilitate the ability of chemical synapses in the brain to change their strength, and thus exhibit what is known as synaptic plasticity.^[80] LTP appears to be the first step by which memories are encoded in the brain and it operates by modification of synaptic strength in response to artificial stimulation (simulated inter-neuronal signaling) via the delivery of carefully modulated electrical pulses, or naturally, as a result of multi-pass filtered and integrated somatosensory signaling, generated as a result of life experience.^{[81],[82],[83]}

Figure 22: Artist’s rendering of some of the structural elements underlying long term potentiation (LTP): neuronal plasma membrane (NMP), dendrite (D), bouton (B), mitochondria (M), chemical synapse (CS), neurotransmitter vesicles (V), dendritic spine (S), cytoskeleton (C) and golgi apparatus (G). ). A necessary ‘defect’ to this rendering is that it shows far more extracellular space and much lower density of synapses than are actually present in the mammalian brain. A single axonal arbor is capable of sprouting tens of thousands of synapses.

LTP has been the focus of a decade’s long investigation because it has in common with LTM rapid induction in response to experience or artificial stimulation, rapid induction of the synthesis of new proteins, the property of being associative in nature, and both LTP and LTM can last for many months in vivo in the free ranging animal.^[79],[84] It has been posited that LTP may be the mechanism for encoding both broad classes of learning and memory: ranging  from the most basic conditioned-responses present in unicellular organisms, to procedural learning, such as mastering a musical instrument, or learning how to drive a car, through the high-level cognitive memory involved in intellectual tasks such as understanding a scientific theory, comprehending complex social relationships, and recognizing individual faces and facial expressions.^[79]

At the level of individual neurons, LTP enhances synaptic transmission by lowering the threshold for two neurons, one presynaptic and the other postsynaptic, to communicate with one another across a synapse. The precise molecular mechanics of how this alteration in synaptic sensitivity to signaling occurs are still not fully understood. Under laboratory conditions (usually employing brain slices in vitro) LTP occurs primarily as a result of alterations to the biochemistry and structure of the postsynaptic cell’s sensitivity to signals received from the presynaptic cell.^[82] These signals occur secondary to the electrochemical stimulation of the presynaptic neuron, which results in the release of neurotransmitter chemicals (i.e., dopamine, serotonin, norephenephrine, etc.,) which activate neurotransmitter receptors present on the surface of the postsynaptic cell.

Figure: 23: Simplified schematic of the expression of LTP: An increase in calcium within the dendritic spine binds to calmodulin (CaM) to activate CaM Kinase II, which undergoes autophosphorylation, thus maintaining its activity after calcium returns to basal levels. CaMKII phosphorylates AMPA receptors (AMPARs) already present in the synaptic plasma membrane, thus increasing their single-channel conductance. CaMKII is also postulated to influence the sub-synaptic localization of AMPA receptors, such that more AMPA receptors are delivered to the synaptic plasma membrane. The localization of these “reserve” AMPA receptors is unclear, and thus they are shown in three different possible locations. Before the triggering of LTP, some synapses may be functionally silent in that they contain no AMPA receptors in the synaptic plasma membrane. Nevertheless, the same expression mechanisms would apply.^[85]

This activation of postsynaptic receptors results in a complex biochemical cascade inside the  postsynaptic neuron which results in an increase of the postsynaptic cell’s sensitivity to neurotransmitter, in large measure by increasing the activity of existing neurotransmitter receptors (Figure 23), and by increasing the number of receptors on the postsynaptic cell surface.^[82] In the minutes, hours or days following the initial induction of LTP there is gene activation in the neuronal nucleus resulting in the synthesis and incorporation of new proteins into the neuronal membrane, the creation of new synapses,^[84] alterations to the structure of the neuronal cell membranes, and changes in the number and/or distribution of neurotransmitter containing vesicles in the synapses. There is also remodeling of the pattern and of the character of synaptic connections between neurons. LTP may also involve the participation of the glial cells – supportive cells which surround neurons and which secrete neurotrophic factors responsible for maintaining neuronal health and viability.^[86] There is also emerging evidence that changes in neuronal membrane lipid structure may be important to LTP; for instance it has recently been demonstrated that lipids can interact directly with glutamate transporters.^[87]

The creation of indefinitely durable LTM is presumably an even more complex process, and involves not just the addition, subtraction, or modification of one type of synapse, but of many. In fact, there are at least 161different synapse morphologies, a few of which are shown in Figure 24 and 25, below.^[18] These synapse morphologies and distributions undergo both transient and long-lasting changes during LTP and LTM.^[88],[89]

Figure 24: Most synapses cover a small area and have a compact, roughly convex shape, such as numbers 51, 59, and 81, above. These are referred to as macular synapses. Larger synapses are often exhibit ‘holes’ in the middle. These holes are regions of cell membrane devoid of the specializations characteristic of the synapse, e.g. postsynaptic density, synaptic cleft, presynaptic active zone, etc. Synapses with holes, such as numbers 45, 46, 86, 90, 94, 96, and 100, are referred to as perforated synapses. Of the 161 synapses so far classified in the neuropil, 148 are macular, while the remaining 13 are perforated. The difference between macular and perforated synapses can be seen in electron micrographs in which the postsynaptic densities have been stained (Figure 25).^[89]

Figure 25: Synapses in Hippocampal Area CA1 of the Rat: Scale: 1 micron. Most synapses in stratum radiatum (>90%) occur on dendritic spines. As shown in Figure 3, spines come in a variety of shapes. A thin spine (T) has a small head with a macular postsynaptic density. The length of the spine neck is much greater than its diameter. The mushroom spine (M) has a large head, typically greater than 0.6 microns in diameter. An elaboration of the endoplasmic reticulum, called a spine apparatus (sa) is often visible within the neck of a mushroom spine. Mushroom spines also tend to have perforated (perf) postsynaptic densities on the spine head. The stubby spine (S) does not have a constricted neck, and its overall length is roughly equal to its diameter. The stubby spine illustrated above possesses a macular postsynaptic density. Occasionally synapses occur directly on the shaft of a dendrite (shaft) without the participation of a dendritic spine. Symmetric (inhibitory) synapses in stratum radiatum tend to be shaft synapses. All of the symmetric synapses of the neuropil shown in Figure 23 are shaft synapses. [Sorra KE, Harris KM (1993) Occurrence and three-dimensional structure of multiple synapses between individual radiatum axons and their target pyramidal cells in hippocampal area CA1. J. Neurosci. 13:3736-3748. (5,414K PDF)]

Another feature observed in LTP is that while each dendritic spine in the hippocampus typically receives only one excitatory synapse on its head, sometimes these synaptic heads are segmented into multiple active zones. These “segmented synapses” have evoked much speculation regarding their possible role in synaptic plasticity.^[88] Recent reports show that segmented synapses increase transiently after LTP induction in the hippocampus, then return to control levels within an hour.^[84],[90] The complex morphology and multi-axonal interface of a typical segmented synapse of the hippocampus is shown in Figure 26, below. While not common, if segmented synapses are indeed lasting, and material to LTM, then the number of effective connections in the brain will have to be revised substantially upwards.

Figure 26: Reconstruction of ‘same-dendrite, multiple synapse boutons’ (sdMSBs) and related structures in a hippocampal brain slice. (a) The sdMSB makes a synapse with the head of one spine (x) on this section. Three of the axons (4,6,7) are visible between the spine head and the dendrite (Dend). (b) Three-dimensional reconstruction of the dendrite (gray), the sdMSB axon, and all seven axons (1–7) passing through the gap between the spines (x,y). Four of the axons (2,4,5,6) are cross-sectioned to avoid obscuring the other axons. Scale bar, 0.75 μm. [Fiala JC, Allwardt B, Harris KM. Dendritic spines do not split during hippocampal LTP or maturation. Nat Neurosci. 2002 Apr;5(4):297-8. PubMed PMID: 11896399.]

Understanding the Biophysical Consequences of Cryoinjury

As the foregoing discussion should make clear, the encoding of LTM likely relies upon a multiplicity of structural and biochemical changes, including the critical spatial relationship and apposition between dendrites, dendritic spines, neurons, and possibly even neuronal and glial cell membranes. It is within the context of this current understanding of the neurobiology of LTM that the effects of cryopreservation will now be considered.

Straight Freezing

Straight freezing is the cryopreservation of cells, tissues or organisms in the absence of added cryoprotection (in the form of colligative, or other cryophylactic molecules). Since most cells and organisms lack endogenous cryoprotection, the result is the conversion of almost all of the available water in the system into ice.

Figure 27: At left, above, is a light micrograph (400x) of the molecular layer[8] of the rabbit cerebral cortex subjected to freezing to -79^oC in the absence of cryoprotection (straight freezing). The tissue is compressed between blocks of ice that have osmotically extracted the intracellular water. At right is the molecular layer of rabbit cerebral cortex tissue (10,000x) following thawing and fixation after straight freezing. The ultrastructure of the tissue resembles that of a tissue homogenate, rather than that of the molecular layer of the cerebral cortex.^[91]

Undoubtedly the compressive forces are enormous under these conditions. What happens to the multiple species of brain cell membranes and their connections and embedded structures when they are dehydrated, biochemically destabilized, and then osmotically and mechanically compressed as a result of ice formation in straight freezing? Obviously, there will be shape changes in discrete membrane structures, but beyond that, to what extent will membranes merge, reorganize into novel structures, or otherwise become physically transformed in ways that would render inference of their pre-frozen state impossible – all of this occurring without thawing, and as a direct consequence of freezing? Definitive answers to these questions are not known, in part because the intense dehydration and compression of the tissue makes visualization of its ultrastructure virtually impossible in the frozen state.

In cryonics patients treated with straight freezing, these delicate and easily re-morphed structures will be crushed together and it might be impossible to tell, even with a complete 3-dimensional molecular level understanding of the remaining structure, what the original configuration was.  It may be that receptors, membrane proteins, and other uniquely configured membrane structures, like the micro-particles comprising the hypothetical image on a soap bubble, will be scattered in debris fields and intermingled with each other.

The character of the changes observed in thawed-fixed, straight frozen brain tissue, as seen in Figure 27, above, suggest that irreversible structural degradation has occurred during the freezing process (and is undoubtedly amplified during thawing). The tissue shows no evidence of plasma cell membranes, and most intracellular structures are no longer identifiable; with the exception of the nucleus and mitochondria – both of which show major morphological abnormalities. Compare the TEM of the molecular layer of the straight frozen—thawed rabbit cerebral cortex at right in Figure 27, above, with that of the molecular layer of the cerebral cortex of a control animal in Figure 28, below. These data provide little grounds for optimism about the conservation of the fine structures of the brain believed to be responsible for encoding LTM.

Figure 28: Control (perfusion fixed) TEM of the molecular layer of the rabbit cerebral cortex (10,000x ). [TEM by author]

Low to Moderate Molarity Cryoprotected Freezing

To the extent that colligative cryoprotectant replaces water in the brain, the effects seen in Figure 27 are attenuated. In the case of freezing in the presence of ~ 4 M glycerol, the fine structure of the tissue before, during, and after freezing can be directly imaged. The sequence of events and the ultrastructural sequelae of cryopreservation under those conditions are shown in Figure 29, A-C, below.

A: At left above is a rendering based upon the rotary shadow electron micrograph of a hippocampal neuron shown Figure 19. The neuron and the extracellular space have been equilibrated with 3.7 M glycerol, and freezing is beginning to take place as the neuron is progressively cooled to -79^oC. The ice freezes out as pure water pushing an advancing front of hyperosmolar solution (purple) in front of it. At right is the condition of the tissue after glycerolization, but prior to the onset of freezing. [Artistic rendering and TEM by the author.]

B: At top left is an artistic rendering of the neuron and the surrounding extracellular medium in the frozen state at -79^oC. The neuronal cell body, as well as the axon and dendrites are dehydrated and compressed between masses of ice. The interior of the neuron, as well as small islands in the interstices between ice masses, contains highly concentrated, vitreous glycerol-water-salt solution (purple). At right(and below) is a TEM of rabbit hippocampus (21,000x ) that was frozen in the presence of 3.7 M glycerol and fixed in the frozen state with osmium tetroxide using a technique known as freeze substitution.[92] [PMC free article] The neuropil is compressed between masses of ice and several tears in the tissue are evident (red arrows). [Artistic rendering by the author; TEM courtesy of Brian Wowk, Ph.D.]

C: At left is an artistic rendering of the nature and extent of worst-case cryoinjury to a hippocampal neuron following freezing and thawing in the presence of 3.7 M glycerol. The axon has been transected by ice, the cell membrane has been osmotically stressed to the point of lysis, and there are debris surrounding the cell, in the form of membrane and cytoplasmic contents, as well as detached dendrites and synapses. Some of the plasma membrane has reformed into blebs and vesicles. At right is a TEM (9,000x) from the hippocampus of rabbit cerebral cortex that has been frozen to -79^oC, rewarmed, perfused with fixative and prepared for TEM. The nucleus of a large neuron is visible in the center of the upper third of the micrograph, however the plasma membrane appears fragmented and there are ice-induced cavities with debris present in a band that spans the lower, middle third of the image. [Artistic rendering and TEM by the author.]

Figure 29: A-C, above: Impact of cryopreservation (-79^oC) and thawing on tissue from the CA-1 area of the hippocampus in the presence of 4 M glycerol.

 Figure 30: Appearance of feline cerebral cortex gray matter (medial temporal lobe) following freezing, thawing and fixation in the presence of 4 M glycerol (9000x). There is enhanced density of the ground substance (tissue texture) as a result of dehydration, and numerous small perforations of the neuropil that appear to be a result of ice formation. Overall, the fine structure appears well conserved and would seem to be inferable. [TEM by the author.]

Figure 31: Appearance of feline cerebral cortex gray matter (medial temporal lobe) following freezing, thawing and fixation in the presence of 4 M glycerol (15,000x). There is enhanced density of the ground substance as a result of dehydration, but very little evidence of injury as a result of ice formation. Overall, the fine structure appears well conserved and would seem to be inferable. [TEM by the author.]

High Molarity Cryoprotection Freezing

Figure 32: Artistic (left) rendering of a hippocampal neuron subjected to freezing to -79^oC after equilibration with 7.5 M glycerol. Cell volume is conserved and ice formation is reduced to ~ 30% of the starting aqueous volume. At right is cerebral cortex tissue from the medial temporal lobe of a dog cryopreserved with 7.5 M glycerol, thawed, reperfused with fixative, and examined with transmission electron microscopy (15,000x). There is excellent conservation of the fine structure of the neuropil, and a notable absence of ice crystal artifacts, or debris. The capillaries (C) are intact and demonstrate uninterrupted adhesion of the endothelial cells to the basement membrane. The increased density of ground substance (tissue texture) and the presence of small spaces between ultrastructural elements are due to dehydration from the cryoprotectant agent (glycerol) and are reversible when the agent is removed. [Artistic rendering and TEM by the author.]

As the tissue cryoprotectant concentration approaches a vitrifiable amount (with concurrent suppression of ice formation) freezing damage is dramatically reduced. Not only is the volume of the system that is converted to ice greatly decreased, there is also a reduction in ice crystal size as result of the high cryoprotective agent (CPA) concentration. In the case of a CPA like glycerol, where the viscosity of the glycerol-water solution increases sharply, both as a function of increasing glycerol concentration and decreasing temperature, there is also a marked reduction in consolidation of smaller ice crystals into larger ones (recrystallization). The intracellular milieu becomes a glassy (vitreous) solid; in the case of systems cryoprotected with glycerol the glass transition point occurs at ~ -100^oC.^[93],

As can be seen in Figure 32 (above) and Figures 33 and 34 (below) the result of high molarity (glycerol) cryoprotection during freezing is dramatically improved conservation of tissue ultrastructure across anatomical locations and morphological levels. Synapses are intact with neurotransmitter vesicles exhibiting normal density and distribution. Cell membranes appear crisp and continuous, and there is no sign of blebbing or budding of vesicles from neuron, or other cell membranes.^[91],[94]

Figure 33: A synapse in gray matter from the hippocampus at 40,200x magnification. The presynaptic junction contains small packets of neurotransmitter (A) visible as granules. Note the overall crisp appearance of both the synaptic membranes and adjacent structures of the neuropil. This degree of preservation at the synaptic level was uniformly observed in all samples examined. [TEM by the author.^[91]]

Figure 34: White matter from the corpus collosum at 6700x magnification. Note the excellent preservation of the capillary (A) and its endothelial cell plasma membranes. The nucleus (B) shows typical loss or reorganization of nucleoplasm; this is seen more frequently in frozen-thawed brains than in brains just perfused with glycerol and fixed without freezing. Several axons (C) exhibit typical shrinkage of axoplasm and alteration in myelin structure. The increase in free space between axons and other structures is the result of glycerol-induced dehydration. [TEM by the author.^[91]]

Vitrification

Laboratory Experience

The only structural studies of vitrified mammalian brains which are known to exist are those conducted by, or under the auspices of 21^st Century Medicine (21CM), a cryobiological research and development company located in Fontana, CA. The most definitive data so far disclosed employed M-22, a vitrification solution developed by 21CM, primarily for renal vitrification.^[95]The composition of M-22 is shown Figure 35, below. This complex mixture of colligative and actively ice inhibiting cryoprotectants exhibits comparatively low toxicity, even at concentrations of ~60%. A unique feature of M-22 is the presence of two synthetic molecules that inhibit ice growth by binding to both the  a and c axes of ice.^{[96],[97],[98]} These molecules stabilize the solution against ice nucleation and propagation, allowing for use of the much slower cooling and rewarming rates needed for ice free cryopreservation of large tissues masses, such as humans organs.

Figure 35: Twenty First Century Medicine’s M-22 vitrification solution contains 5 penetrating colligative cryoprotective agents as well as 6% of non-penetrating polymers – two of which are highly active ice-blocking molecules; Supercool X-1000 and Supercool Z-1000. X-1000 contains 80% of the syndiotactic stereochemical form of polyvinyl alcohol and 20% vinyl acetate and Z-1000 is a linear polymer of polyglcerol with an average molecular weight of 750 Da. Both bind to the a and c axes of ice crystals, stabilizing solutions they are present in against ice formation during slow rates of cooling and rewarming.^[95][Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

As a result of very recent advances in vitrification technology, it is now possible to vitrify entire mammalian organs.^[99] However, two significant remaining problems are to inhibit ice growth  during rewarming (“devitrification”) in some tissues that do not equilibrate well with the cryoprotectant chemicals, and to overcome the problem of fracturing, which occurs during cooling to or below T_g.

Ice formation occurs during rewarming as a result of the generation of ultramicroscopic ice nuclei during cooling, which cannot grow or propagate, because there is too little energy in the system and too little time for ice to grow as a result of steady and continued cooling to T_g. While devitrification is a significant hurdle to be overcome, it is a technological, rather than a theoretical one. Additionally, virtually all research on reversible vitrification of organs has been conducted on the kidney, and this presents a unique challenge, because the interior of the organ, the renal medulla, is very poorly circulated (Figure 36). It is thus difficult to load a sufficient concentration of cryoprotectants into this poorly vascularized tissue to completely avoid ice formation^.[99]

Figure 36: Visual appearance of ice in a rabbit kidney that was cross-sectioned during rewarming. The kidney was perfused with a cryoprotective mixture called M22 at -22°C, cut in half, immersed in M22, vitrified at -135°C, and eventually re-warmed at ~1°C/min while being periodically photographed. Times (1:30 and 1:40) represent times in hours and minutes fom the start of slow warming. The temperatures refer to ambient atmospheric temperatures near the kidney but not within the kidney itself. The upper panel shows the kidney at the point of maximum ice cross-sectional area, and the lower panel shows the kidney after complete ice melting. Both panels show the site of an inner medullary biopsy taken for differential scanning calorimetery in order to determine the actual concentration of cryoprotectants in the tissue with high precision. [http://cryoeuro.eu:8080/download/attachments/425990/FahyPhysicBiolAspectsRenalVitri2010.pdf?version=1&modificationDate=1285892563927]

A fair summary of the current technological state of the art is that under ideal (laboratory) conditions, it is likely now possible to place complex mammalian organs, such as the rabbit kidney, into indefinitely long suspended animation with little or no loss of viability, and no damage as a consequence of structural disruption due to ice formation. The use of radio frequency, or microwave illumination to speed rewarming, the use of warm gas (such as helium) to perfuse the organ’s circulation, or a combination of these modalities, may offer a workable solution to the problem of ice formation during rewarming. Perhaps most impressively, one mammalian kidney has survived vitrification and rewarming sufficiently intact to permit immediate support of the rabbit from which it was removed (as the sole kidney), until the animal was sacrificed for evaluation 29 days after the organ was re-implanted.^[99]

Figure 37: The first kidney to survive vitrification shortly before it was removed from the animal for evaluation after supporting its life as the sole kidney for 29 days. [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc, http://www.21cm.com/]

An additional complicating factor in achieving reversible (viable) vitrification of the mammalian brain has been the inability to continue cryoprotectant perfusion at the same subzero temperatures (-20^oC) that have proven essential for recovery of rabbit kidneys following loading and unloading with M-22. As can be seen in Figure 38, below, perfusion of the terminal concentration of M-22 is not possible below ~ -3-4^oC. Exposure to ~8.2M M-22 at such a relatively high temperature, for the final ~60 min of perfusion required to load the brain with the CPA mixture results in major loss of viability, but does not visibly affect brain ultrastructure, as imaged using TEM.

Figure 38: Cryoprotection and cooling protocol used to achieve structural vitrification of the rabbit brain at 21^st Century Medicine, Inc., CPA loading commences at a temperature of ~+4^oC and continues at that temperature for ~ 60 minutes while the M-22 concentration is gradually increased to ~4 M. The temperature is then reduced to ~ -3^oC while the CPA concentration is increased to ~ 8M. The total time required to achieve full equilibration of the brain with M-22 is ~ 180 minutes, after which the organ is immediately transferred to an air-blast cooler for very rapid cooling to ~ -135^oC. [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

The ultrastructure of brains cryopreserved using the 21CM vitrification technique shows no evidence of ice formation. As previously noted, there is extensive dehydration of all imaged areas of the brain, with associated increase in ground substance density which makes assessment of very fine structures difficult. Reassuringly, it is possible to not only viably recover brain slices from this state, but to demonstrate the persistence of pre-vitrification induction of LTP in such vitrified and recovered brain slices.

Figure 39: TEM of rabbit cerebral cortex gray matter (~ 15,000x) subjected to vitrification, rewarming and perfusion fixation using M-22 and the perfusion protocol shown in Figure 38, above. The extensive dehydration induced by cryoprotective loading makes it difficult to visualize the finer elements of the ultrastructure such as vesicles and microtubules. The overall appearance of tissue in terms of the larger structural elements and their relationship to each other is apparently normal. [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

Figure 40: High magnification TEM (~ 40,000x) of vitrified rabbit brain tissue discloses the presence of difficult to visualize fine structures – in this case a synapse (S) with synaptic vesicles visible as dark densities in the synaptic bouton and a small myleinated (M) axon containing condensed axoplasm (A). Importantly, the topographical and structural relation of the synapse to the surrounding structures appears intact. [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

Figure 41: TEM of rabbit cerebral cortex white matter (15,000x) subjected to vitrification, rewarming and perfusion fixation using M-22 and the perfusion protocol shown in Figure 38, above. There is severe dehydration of the axoplasm and separation between some of the layers of myelin. There is no evidence of ice formation, and all structural changes appear to be a consequence of CPA-induced dehydration. These changes are reversible with controlled removal of CPA and return of the tissue to incubating medium (see Figure 42, below). [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

Figure 42: Mosaic of TEM’s demonstrating continuity of a long axon (red arrows) in a rabbit brain subjected to vitrification, rewarming and perfusion fixation using M-22 and the perfusion protocol shown in Figure 39, above. The tear in the tissue (green arrow) is believed to be a processing artifact. Two capillaries visible near the middle and top of the mosaic (blue arrows). [Image is courtesy of Brian Wowk, Ph.D., of 21^st Century Medicine, Inc., http://www.21cm.com/]

Figure 43: Hippocampal CA4 cells following recovery from vitrification using a fully reversible, viability conserving technique. Following rewarming and unloading of the CPA the tissue was incubated in artificial cerebrospinal fluid at 35^oC for >60 min before being fixed in low-osmolality Karnovsky’s and examined by TEM.^[100]

Both freezing and vitrification have the potential to disrupt the structures that encode LTM in ways that would leave them uninferrable. Vitrification may do this by the expedient of altering membrane structure irreversibly by dehydration, or by changing the molecular structure of the membranes (or membrane components) by directly perturbing their structure. Vitrification solution is not water, and water is critical to the structure of many of the molecules inside cells. Indeed, a good part of the science behind designing tolerable vitrification solutions is to make them behave as much like water as possible – while at the same time behaving as good, or good enough, glass forming agents when cooled.^[49] On a purely structural basis it would seem that vitrification, applied under ideal (laboratory) conditions, is preserving the structures that encode memory and personality. To the extent that structural vitrification (as opposed to fully reversible, viable vitrification) perturbs or damages the biochemistry associated with LTP, there are grounds for concern. However, it seems unlikely that such injury would render the biochemistry of the brain uninferable, and therefore nonviable by the information-theoretic criterion for death.^[22]

Real World Considerations

While laboratory investigations conducted under ideally controlled conditions provide considerable reassurance that existing cryopreservation techniques can conserve the essential structural and biochemical elements that comprise personal identity, such techniques are rarely available to human cryonics patients. Due to medico-legal and logistical constraints, most patients presenting for cryopreservation suffer extensive peri- and post-cardiac arrest global ischemia. When cryoprotection is delivered under these conditions in the laboratory setting, the results are very discouraging.

Figure 44: Feline cerebral cortex frozen, thawed and fixed in the presence of 4 M glycerol after 30 minutes of normothermic ischemia, followed by 24 hours of cold ischemia at ~2-4^oC. There was severe disruption of the tissue fine structure by ice (A,B), in addition to changes associated with ischemia such as mitochondrial swelling and blebbing of the endothelial cells (A). [TEMs by the author.^[101]

In 1983 studies were undertaken to determine the effects of 30 minutes of normothermic ischemia followed by 24 hours of cold ischemia at ~ 2-4^oC.^[101] Healthy adult cats were anesthetized; heparinized and cardiac arrest was induced. The animals were allowed to remain undisturbed on the operating table for 30 minutes, and then were packed completely in water ice, where they were allowed to remain for 24 hours. They were then perfused to 4 M glycerol using a linear increase in glycerol concentration during the ~ 60 minute perfusion interval. Following cryoprotective perfusion, the animals were cooled to dry ice temperature at ~ 3^oC/hour, and to liquid nitrogen temperature at ~4^oC/hour. Due to the unexpected presence of fracturing in the brain and other viscera, fixative reperfusion following thawing was not possible, and brain tissue samples for TEM were fixed by immersion.

As can be seen in Figure 44, above, there was extensive freezing damage superimposed over ischemic injury to the tissue. The mitochondria were swollen and often showed little internal structure. Neuronal plasma membranes were impossible to identify and the neuropil was macerated by what appeared to be ice artifacts. Large peri-capillary ice holes were almost uniformly present and large islands of tissue had the appearance of a tissue homogenate, as was observed in animals subjected to straight freezing.

The Problem of Ischemia and Ischemia-Reperfusion Injury

Hypoperfusion Following Reperfusion

Figure 45: The no reflow phenomenon as documented by Ames, et al., in the rabbit brain in 1968. The control brain on the left was perfused with a colloidal carbon solution while the animal was still alive and under anesthesia. At right, the brain of an animal reperfused with carbon solution after 15 minutes of global normothermic cerebral ischemia – an interval of ischemia all too common in human cryopreservation patients. The pale white areas are zones of no-reflow or failed reperfusion, and account for 93% of the surface area in this cortical slice, despite use of hypertensive reperfusion at 120 mm Hg.^[104]

In cryonics patients, a significant contributor to reperfusion injury is hypoperfusion after the artificial restoration of circulation using closed chest cardiopulmonary support (CPS). Not only is CPS very inefficient at restoring adequate perfusion, blood flow to the brain, in particular, is impeded as a consequence of the cerebral “no-reflow” (CNR) phenomenon.^[102] The no-reflow phenomenon was first described by Ames and Fisher in 1968,^[103],[104] and was observed in the brains of animals subjected to reperfusion after experimentally induced global cerebral ischemia (GCI) from cardiac arrest lasting for periods of 5 minutes or longer. Interestingly, no-reflow does not usually develop in the heart, or other organs and tissues, until they have experienced periods of ≥ 30 min of normothermic global ischemia. Ames, et al., originally proposed three mechanisms as the cause of CNR; 1) glial and endothelial cell edema causing a reduction of the capillary lumen diameter to below the critical threshold for the passage of RBCs, 2) hyperviscosity of micro-vessel blood due to concentration of plasma proteins and formed blood elements as water is relocated from the microvascular space to intracellular space under the influence of the Gibbs-Donan Equilibrium in ischemia, and  3) alterations in the neurovascular endothelial surface as a result of bleb formation due to endothelial cell edema (See Figure 46).

Figure 46: The no reflow phenomenon, first demonstrated by Ames and Fisher in 1968, constitutes a major barrier to successful cerebral resuscitation. Beginning at ~5 minutes after the start of normothermic global cerebral ischemia (GCI) a variety of biomechanical changes can be observed at the microscopic and especially at the ultramicroscopic level. Due to the failure of ion pumping cellular edema becomes pronounced at this point, resulting in swelling of both the brain parenchymal and capillary endothelial cells. This edema decreases capillary diameter to considerably less than that of the red blood cells (RBCs), leading to RBC plugging of the vessels. The cellular response to edema is to shed small vesicles of cell membrane material, apparently in an attempt to regulate cell volume and prevent hypervolemic cell lysis. These vesicles or “blebs” may interfere with the flow of the formed elements of the blood, and when they occur between the endothelium and the sarcolemma of the pre-capillary sphincters, they may also act to decrease capillary diameter resulting in reduced or absent blood flow.

Additionally, there are rheological changes in the blood itself. As water translocates from the blood in the capillary lumen, plasma proteins and formed blood elements become concentrated and hyper-viscous, and consequently resistant to flow. The surfaces of the RBCs are prevented from sticking to each other due the existence of a charge barrier, the zeta potential, which is pH sensitive. Under conditions of acidosis, as pertain in ischemia, the zeta potential collapses and the RBCs become sticky and aggregate into masses which cannot pass through the micro-vessels. The RBCs themselves also undergo changes, most notably becoming rigid as a result of intracellular ATP depletion .This reduces RBC deformability, making their passage through brain capillaries difficult or impossible.

Finally, there is growing evidence that prolonged period of GCI cause activation of immune/inflammatory cascade resulting in neutrophil adhesion and activation, platelet aggregation and activation of the clotting cascade, the latter resulting in the possible formation of micro-thrombi in the small vessels, particularly in the brain venules.

Since the initial work by Ames, et al., it is has become apparent that the pathophysiology of CNR is considerably more complex. Other factors that have been identified as causative are changes in the character of the endothelial surface during ischemia, which make it stickier due to increases Nuclear factor kB (NFkB) and Tumor Necrosis Factor-α  (TNF-α), resulting in elevated levels of a wide range of other pro-adhesion and pro-inflammatory chemokines.^[105],[106] Additionally, the generation of free radicals, in particular peroxynitrite and the hydroxyl radical, degrade the extracellular matrix and the capillary basal lamina, resulting in increased permeability of the BBB with consequent development of interstitial cerebral edema^.[107] Both free radical and leukocyte activity can also degrade the endothelial cell tight junctions to the point that frank extravasation of formed blood elements occurs, along with activation of the clotting cascade, resulting in the formation of fibrin lactoids and frank clots in the capillary lumen^.[108] Free radical and leukocyte activation can also result in complete destruction of cells in the neurovascular endothelium, resulting in patches of exposed basement membrane, which in turn result in micro-thrombus formation. Blebs, debris from lyzed RBCs and endothelial cells, aggregated platelets, and precipitated macromolecules can also accumulate to form capillary occluding masses of debris (Figure 46).

The diameter of the average RBC is ~7.7μ, about 1μ larger than the diameter of the average brain capillary. In order for RBCs to pass through capillaries it is necessary for them to deform (and in so doing place the maximum amount of surface area in contact with the vascular endothelium to facilitate gas exchange). RBC deformability is critically dependent upon RBC intracellular adenosine triphosphate (ATP) concentration being adequate. With periods of ischemia of ~ 7 minutes, RBCs become depleted of ATP and become rigid, making passage through brain capillaries more difficult (higher arterial pressure required) or impossible.^[109]

Figure 47: The zeta potential is the degree of negative charge on the surface of the RBC; the potential difference between the negative charges on the RBC and the cations in the fluid portion of the blood which constitutes of zone of charge the cells that prevents them from adhering to each other (above left). Changes in the cation content, or reduction of the pH of the plasma can collapse the zone of repulsive charge around the RBCs,  allowing them to agglomerate and stick together in clumps.

The surface of RBC (and most cell) membranes is intrinsically sticky, and what keeps them from adhering to each other and aggregating into a solid mass is a complex interaction between sialic acid groups (sialoglycoprotein) on the RBC membrane, which give the cell a negative charge, and positive ions in plasma that are attracted to the negatively charged RBC membrane.^[110] This zone of oppositely charged ions surrounding the RBC surface is called the “fixed layer.” Outside the fixed layer, there are varying compositions of ions of opposite polarities, forming a cloud-like zone. This area is called the “diffuse double layer,” and the whole of the diffuse double zone is electrically neutral. Thus, the net positive charge surrounding the RBCs keeps them apart due to electrostatic repulsion. The electrostatic potential of this zone, measured at the plane of hydrodynamic slippage outside the surface of the cell, is called the zeta potential.^[111] Zeta potential is considered to be the electric potential of this inner area, including this conceptual “sliding surface” (Figure 48). As this electric potential approaches zero, particles tend to aggregate. Put more succinctly, the zeta potential is the degree of negative charge on the surface of the RBC, the potential difference between the negative charges on the RBC and the cations in the fluid portion of the blood.

Figure 48: Graphic illustration of the zeta potential surrounding the RBC. The zeta potential consists of 3 layers of charge; the negative charge of the RBC membrane, a boundary layer of positively charged cations that travel with the cell as it moves through the blood plasma, and an outer layer of positive charge that is more dynamic composed of varying compositions of ions of opposite polarities, forming a cloud-like area that exists at the boundary of shear, or the “slipping phase” at the plane of hydrodynamic slippage outside the RBC. These last two layers are known as the “diffusible double layer.”

A critical determinant of the zeta potential is pH. The zeta potential of RBCs is optimized around a pH of 7.4. Under conditions of acidosis, and in particular where the pH drops below 6.6, as is the case in ischemia, the zeta potential of the RBC collapses and the intrinsic stickiness of the membrane surface is unmasked.^[112] RBCs in contact with each other will tend to stick, and in the absence of flow (which acts as a dispersing agent) and under the influence of gravity, RBCs begin to aggregate and sediment out of the plasma.^[113] Where RBCs are in contact with capillary endothelium, they will tend to stick, and the force required to dislodge them will be far larger than under physiological conditions. The aggregation of RBCs under acidic conditions was, at one time, used as an alternative method to centrifugation for removing RBCs from solution during washout of cryoprotectant following freezing, using a technique known as cytoagglomeration.^[114]

Even in the absence of ischemia, dynamic variations in flow, pH and plasma chemistry can increase the amount of time RBCs in flowing blood spend in contact with each other. The longer the contact times between RBCs, the greater the viscosity of the blood. Increased blood viscosity, and the presence of loosely bound clumps or aggregates of RBCs, result in a blood sludging; a condition of greatly slowed and irregular flow in the micro-vessels (arterioles, capillaries and venules).^[115]

Figure 49: Aggregation or agglomeration of RBCs into irregular clumps is the most common pattern of RBC adhesion seen in both RCIRI and GCIRI. The impact on flow is devastating resulting in either severe blood sludging or complete arrest of microcirculatory flow.

Within 60 seconds of the start of  ischemia, pH in the cerebral micro-vessels quickly drops to ~6.8, and then declines to ~6.2 after only 5 minutes or more of GCI.^[116]Sedimentation of RBCs, WBCs and platelets becomes pronounced after 6 minutes and is difficult to reverse due to the adhesion of the cells to each other.^[117] The acidemia of prolonged ischemia may also adversely affect the configuration of some plasma proteins causing them to act as bridging molecules between the RBCs thus further increasing agglomeration.

Figure 50: The adhesion of RBCs to each other in a “stack of coins” configuration, known as rouleaux is the less prevalent, but still common pattern of RBC aggregation observed during and after both RCIRI and GCIRI. As is the case with irregular aggregation of RBCs rouleaux formation has a profound negative impact on perfusion.

When flow is re-established, higher pressures and higher flow rates will be needed to generate the shear required to re-suspend sedimented blood cells, and to disrupt agglomerations of cells in the microvasculature.^[118] With the passage of enough time (~30 min), agglomeration and sedimentation extend to the large vessels, where much larger masses of adherent cells will form, fall under the influence of gravity, and consolidate. This macro-scale hyper-viscous sludge will be very difficult to disrupt, and can be expected to compromise flow for prolonged periods of time following the start of reperfusion.

The work of Hossman, et al.,^[119] and Sterz, et al.,^[120] has demonstrated the critical importance of providing adequate circulatory support following global cerebral ischemia.  Loss of autonomic regulation, depressed myocardial function secondary to ischemic insult of the myocardium, and autonomic dysfunction all serve to depress MAP and cerebral perfusion following restoration of circulation. Both Hossman’s and Sterz’s work has demonstrated significant improvements in neurological outcome if circulation is immediately supported extracorporeally during reperfusion – an option which is not available to cryonics patients.

In-house research conducted by the author has demonstrated that the cerebral microcirculation remains profoundly compromised for 30-60 minutes following reperfusion, even when circulation is restored using cardiopulmonary bypass. Brain parencymal and endothelial cell swelling, as well as changes in the zeta potential of the red blood cells, may all be contributing to the extensive blood sludging and microvascular stasis observed after reperfusion following 10 minutes of global normothermic ischemia in the laboratory.

Figure 51: Canine cerebral cortical (gray matter) capillary plugged with a mass of red cells following extensive washing (5 L) with a colloid containing hyperosmotic solution, followed by fixation perfusion with Karnofsky’s fixative. [TEM by the Author.]

As the electron micrograph above demonstrates, even following attempts at reperfusion with a hyperosmotic physiological solution at a MAP of 90 mmHg, there are many instances of capillaries plugged with aggregated red cells, as seen above.

Figure 52: At left, baseline pial circulation in the dog brain prior to induction of cardiac arrest and the 15 minute ischemic insult. At right, flow is seen to be sluggish with obvious sludging in the larger vessels, and complete stasis observed in many of the smaller ones ~30 minutes after the onset of reperfusion. [Intravital microscopy by Jerry Leaf and the author.]

The microcirculation in the pia remains disturbed and sluggish even 30 minutes after reperfusion following 15 minutes of normothermic GCI as can be seen in Figure 51 which shows baseline flow in pial vessels and flow 30 minutes after ROSC at a MAP of 80 mmHg.

Consequences of Ischemia-Induced Derangement of the Microcirculation for the Human Cryopreservation Patient

In the case of human cryopreservation patients, it is apparent that the warm and cold ischemia suffered by many has effects on the brain (and somatic tissues) that combine features of GCIRI, RCIRI and Multisystem Organ Failure (MSOF, or the post-resuscitation syndrome). Injury associated with the post-resuscitation syndrome does not develop under normal clinical conditions until 12 to 24 hours after the ischemic insult. Leukocyte activation is “slow” (hours) and compromise to the BBB is typically delayed until 12 to 24 hours after reperfusion.^[121]

Figure 53: In the terminally ill patient suffering from progressive degenerative disease, the immune-inflammatory cascade is already activated. In cancer patients, NFkB and TNF-α will often be expressed and are responsible for the cachexia of the wasting syndrome. In some cancers (lung, prostate, colon), elements of the clotting cascade may be activated, and the patient will be in a hypercoagulable state weeks or months prior to cardiac arrest.

Activation of the Immune-Inflammatory Cascade

In the human cryopreservation patient a number of factors appear to be responsible for the acceleration of the inflammatory and tissue destroying components of reperfusion injury, causing them to manifest during the period of initial stabilization following cardiac arrest and the pronouncement of legal death (and especially by the time cryoprotective perfusion is initiated if there has been a prolonged period of cold ischemia; i.e., transport packed in ice absent perfusion and gas exchange). The first of these is the terminal illness, and any satellite pathologies that preceded, or were a consequence of it (Figure 53). As an example, a patient experiencing terminal decline from metastatic adenocarcinoma will inevitably experience up-regulation of a host of pro-inflammatory cytokines.^[122],[123] With some cancers there will be chronic, low level activation of the clotting cascade as a direct result of the tumor’s biochemistry, and it should be noted that 50% of all cancer patients show evidence of deep vein thrombosis, or other kinds of intravascular clotting at autopsy.^[124],[125]

Such patients will almost invariably experience ongoing localized ischemia as tumor compresses and compromises blood flow to healthy tissues; and there will also be ongoing necrosis in the center of the poorly vascularized tumor masses with attendant release of pro-inflammatory tissue breakdown products. Chronic and acute pain also up-regulate the immune-inflammatory cascade. Many medications used to treat the disease or its complications will adversely affect blood rheology leading to blood sludging and additional incomplete regional ischemia. Most neoplasms provoke enormous and sustained release of TNF-α which will have adverse effects on capillary integrity and blood coagulation. Finally, chemotherapy and radiation therapy both have dramatic pro-inflammatory systemic effects, as do the intercurrent infections that are so often complications of these treatments. Malnutrition with protein catabolism and cell death is yet another likely inflammation inducing factor.

Figure 54: A prominent feature of moderate to severe ischemic injury in the absence of prompt blood washout and extracorporeal support in the cryonics patient is red trapping in the tissues. At left are photos taken of the arterial pump raceway and arterial filter at the conclusion of cryoprotective perfusion (4.5M glycerol). The circuit (L) of the patient given prompt CPS, followed by blood washout and continuous ECMO support until the start of CPA perfusion, shows only trace amounts of RBCs and has an unreadable hematocrit. The circuit shown (R) was from a patient who experienced sudden cardiac arrest, was given heparin and a brief interval of CPS in the hospital ED, and was then transported packed in ice with CPA perfusion starting ~20 hours later.

As a result of these destructive processes, the terminally ill patient is not only primed to experience the destructive effects of ischemia on the vascular endothelium, they have, in fact, often begun to experience many of the pathophysiological mechanisms of ischemia-reperfusion injury weeks, or even months before cardiac arrest occurs.

With the immune–inflammatory cascade already significantly activated, the peri-arrest period of severe regional ischemia (as tumor mass critically encroaches on vital organ structure and function) coupled with what are often very long (6-48 hours) peri-arrest intervals of severe hypoxic and ischemic injury during the agonal period, there is full-scale activation of the immune-inflammatory cascade. The result of this is that the patient is primed for reperfusion injury, and in fact may be experiencing reperfusion-type injury on both a regional and a systemic basis prior to cardiac arrest.

Pre-Cardiac Arrest Regional and Global Cerebral Ischemia?

Many patients dying slowly will experience the clinical signs of failed cerebral perfusion (fixed, unresponsive pupils, absence of corneal and deep tendon reflexes) anywhere from 30 minutes, to an hour or more prior to cardiac arrest. This typically occurs during a period of profound bradycardia (20-30 bpm) accompanied by 3-4 agonal breaths per minute, or erratic and infrequent agonal breaths (1-2 minute)^[126] In some patients this condition may persist for many hours. In such cases there is clinical evidence at autopsy of the body (neuropatients) of systemic mixed regional and global ischemic injury, as indicated by the presence of clots in the femoral veins, pulmonary inflammation/edema, erosion of the gastric mucosa, and areas of focal necrosis in the liver, kidneys, and ileum, as determined by light microscopy.^[127],[126] Rarely, onset of rigor has been observed in the gastrocnemius, peroneus longus and peroneus brevis muscles of the lower limbs an hour or more prior to cardiac arrest^.[126]

Patients experiencing this kind of injury respond to CPA perfusion by developing severe peripheral and visceral edema, sometimes accompanied by transudation of large volumes of perfusate from the lungs (ET tube) and leakage of similarly large volumes of perfusate into the upper and lower gut, resulting in abdominal distension, and in some cases, compartment syndrome. Such patients invariably experience red cell trapping in the tissues which persists throughout cryoprotective perfusion.^{[128],[129],[130],[131],[132],[133]} This is especially remarkable given the large volume of solution passed through the patient’s vasculature (120 L), the hyperosmolality of the CPA solution, and the relative cellular impermeability of a number of the cryoprotectants (i.e., glycerol, ethylene glycol). Patients with very short warm ischemic times (<8 minutes) who receive prompt and effective CPS, followed by blood washout and/or extracorporeal support, do not exhibit red cell trapping, and do not typically develop CPA perfusion limiting cerebral edema.^{[128],[129],[130],[131],[132],[133]}

Figure 55: Patients with minimal peri-arrest ischemic injury who are given prompt and effective CPS followed by blood washout and either immediate CPA perfusion, or extended (~8-10 hours) ECMO support respond to CPA perfusion with massive cerebral dehydration (above right) and relatively uniform CPA perfusion of the skin. The patient shown below has a few infarcted areas evident on the bridge of the nose, probably secondary to pressure from ice bags, and is already evidencing cerebral cortical retraction from the dura, even during the first 30 minutes of CPA perfusion (7.5 M glycerol).

Cerebral Edema

Figure 56: The burr hole of a human cryopreservation patient who experienced a brief period of CPS with heparinization, followed by packing in ice, and transport to the CPA perfusion facility. One hour into CPA perfusion, the brain abuts the burr hole opening, and a steady stream of aggregated RBCs can be seen exiting the burr hole in the perfusate leaking from the torn bridging veins between the dura and pia matters. The bridging veins tore as a consequence of the brief interval of cerebral dehydration which accompanied the initiation of CPA perfusion.

Compromise of the BBB, as indicated by the development of cerebral edema, may range from moderate to severe, and is often the reason for discontinuation of CPA perfusion.^{239, 240} In patients who are stabilized immediately post-arrest under good conditions, and who respond well to CPS (adequate EtCO₂, SpO₂, MAP) the BBB remains intact as evidenced by massive (~50%) shrinkage of the brain during CPA perfusion.^[134],[135] Patients who have experienced high quality transport in the presence of minimal peri-arrest injury, but who are subsequently transported by air without continuous asanguineous cardiopulmonary bypass support, also suffer damage to the BBB, as evidenced by failure of brain to shrink (and/or remain shrunken) during CPA perfusion. In such patients there is often initial shrinkage of the brain in response to CPA loading, followed by return to isovolemia, or the development of a slight degree of cerebral edema by the end of CPA perfusion.^[136],[137]

Figure 57: The impact circulatory obstruction on cryoprotectant equilibration in a cryonics patient who underwent ~24 hours of cold ischemia is evident in the inhomogeneous glycerolization of the skin at the conclusion of CPA perfusion. False color imaging discloses a patchwork of well, poorly, and completely unglycerolized areas of the patient’s skin. This pattern of patchy, compromised, or failed perfusion is present in the brain, as well as in the skin.

Cryonics patients who experience significant ischemic insult, be it peri- or post-cardiac arrest, warm or cold, will, as a consequence, suffer from multifocal areas of cerebral infarction and severely reduced flow on both the micro- and the macro-level. This will result in failed or inadequate cryoprotection of the brain, with many micro- and macro-domains of tissue undergoing straight freezing, or freezing in the absence of adequate cryoprotection. Patients who have suffered sufficiently long periods of warm and/or cold ischemia will be un-perfusable, and will of necessity be straight frozen. Given our current understanding of the biophysical basis of LTM, and in particular of “declarative” or “biographical” memory,” ^{[138],[139],[140]}[9] it would appear that this critical element of personal identity is unlikely to survive cryopreservation under conditions of straight freezing, or in circumstances where the level of cryoprotection is very low, or very inhomogeneous, with substantial areas of the brain being subjected to neuronal membrane re-morphing, and severe topographical and spatial distortions of the neuropil as a result of ice formation and the attendant biochemical, osmotic, and mechanical stresses.

Summary

The prospects for the conservation of personal identity via cryopreservation under optimum conditions, particularly with the use of no, or very low ice forming methods, such as high molarity cryoprotective freezing or vitrification, seem excellent. TEM studies in animals demonstrate good preservation of both the gross and ultrafine structure of the neuropil, as well as of the white matter of the cerebral cortex. Synaptic connectivity and attachment to dendrites and dendritic spines appears undisturbed by these preservation methods, and where ice formation does cause injury, the structure appears to be displaced, rather than crushed or re-morphed – and as such, should allow inference of its undamaged, pre-cryopreservation state. The demonstration of the persistence of LTP following vitrification of mammalian hippocampal brain slices provides considerable grounds for optimism that the biochemical basis for encoding memory is also being conserved; at least in the case of vitrification under ideal conditions.

The criticality of avoiding both warm and cold ischemia cannot be overemphasized. The consequences of un-cryoprotected, or inadequately cryoprotected freezing are dire. There is nearly uniform loss of neuronal membrane structure, maceration of the neuropil, and obvious re-morphing of cell membrane components. These kinds of changes, especially if they are occurring during freezing, as well as during thawing, are not compatible with the survival of the patient using the information-theoretic criterion for determining death. Cryonicists may wish to rethink the way they communicate the viability of cryonics to the public – as well as how they conduct their personal lives – if they realistically hope to benefit from cryopreservation as a potentially reversible method of medical time travel.

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97.       Wowk B: Anomalous high activity of a subfraction of polyvinyl alcohol ice blocker. Cryobiology 2005, 50(3):325-331.

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99.       Fahy G, Wowk, B, Pagotan, R, et al.: Physical and biological aspects of renal vitrification. Organogenesis 2009, 5(3):167-175.

100.     Pichugin Y, Fahy, GM, Morin, R.: Cryopreservation of rat hippocampal slices by vitrification. Cryobiology 2006, 52(2):228-240.

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Selected Bibliography of Full Text PDFs

Brain Ultrastructure and LTP:

Harris KM (1980) Relationships between dendrite and spine neck diameters in freeze-fractured rat hippocampal formation. Biol. Bull. 159:470-471. (646K PDF)

Harris KM, Landis DM (1986) Membrane structure at synaptic junctions in area CA1 of the rat hippocampus. Neurosci. 19:857-872. (3,253K PDF)

Harris KM, Stevens JK (1989) Dendritic spines of CA1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. J. Neurosci. 9:2982-2997. (7,143K PDF)

Chicurel ME, Harris KM (1992) Three-dimensional analysis of the structure and composition of CA3 branched dendritic spines and theirsynaptic relationships with mossy fiber boutons in the rat hippocampus.J. Comp. Neurol. 325:169-182. (6,353K PDF)

Sorra KE, Harris KM (1993) Occurrence and three-dimensional structure of multiple synapses between individual radiatum axons and theirtarget pyramidal cells in hippocampal area CA1. J. Neurosci. 13:3736-3748. (5,414K PDF)

Harris KM, Sultan P (1995) Variation in number, location, and size of synaptic vesicles provides an anatomical basis for the non-uniformprobability of release at hippocampal CA1 synapses. J.Neuropharmacology34:1387-1395.   (851K PDF)

Spacek J, Harris KM (1997) Three-dimensional organization of smooth endoplasmic reticulum in hippocampal CA1 dendrites and dendriticspines of the immature and mature rat. J. Neurosci. 17:190-203.  (2,560K PDF)

Spacek J, Harris KM (1998) Three-dimensional organization of cell adhesion junctions at synapses and dendritic spines in area CA1of the rat hippocampus. J. Comp. Neurol. 393:58-68.  (947K PDF)

Shepherd GMG, Harris KM (1998) Three-dimensional structure and composition of CA3–>CA1 axons in rat hippocampal slices: implicationsfor presynaptic connectivity and compartmentalization. J. Neurosci. 18:8300-8310. (964K PDF)

Sorra KE, Fiala JC, Harris KM (1998) Critical assessment of the involvement of perforations, spinules, and spine branching in hippocampalsynapse formation. J. Comp. Neurol. 398:225-240.  (1,643K PDF)

Ventura R, Harris KM (1999) Three-dimensional relationships between hippocampal synapses and astrocytes. J. Neuroscience 19(16):6897-6906. (888K PDF)

Cooney JR, Hurlburt JL, Selig DK, Harris KM, Fiala JC (2002) Endosomal compartments serve multiple hippocampal dendritic spines from a widespread rather than a local store of recycling membrane. J. Neurosci 22:2215-2224. (763K PDF)

Harris KM, Cruce WLR, Greenough WT, Teyler TJ (1980) A Golgi impregnation technique for thin brain slices maintained in vitro. J. Neurosci. Methods.2:363-371. (3,633K PDF)

Sorra KE, Harris KM (1998) Stability in synapse number andsize at 2 hr after long-term potentiation in hippocampal area CA1. J. Neurosci.18:658-671. (1,407K PDF)

Kirov SA, Sorra KE, Harris KM (1999) Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats. J Neurosci. 19(8):2876-2886.  (1,426K PDF)

Ventura R, Harris KM (1999) Three-dimensional relationships between hippocampal synapses and astrocytes. J. Neurosci. 19(16):6897-6906. (888K PDF)

Sorra KE, Harris KM (1998) Stability in synapse number and size at 2 hr after long-term potentiation in hippocampal area CA1. J. Neurosci.18:658-671. (1,407K PDF)

Sorra KE, Fiala JC, Harris KM (1998) Critical assessment of the involvement of perforations, spinules, and spine branching in hippocampalsynapse formation. J. Comp. Neurol. 398:225-240 (1,643K PDF)

Kirov SA, Sorra KE, Harris KM (1999) Slices have more synapses than perfusion-fixed hippocampus from both young and mature rats. J Neuroscience 19(8):2876-2886.  (1,426K PDF)

Kirov SA, Harris KM (1999) Dendrites are more spiny on mature hippocampal neurons when synapses are inactivated. Nature Neuroscience 2(10):878-883. (643K PDF)

Cooney JR, Hurlburt JL, Selig DK, Harris KM, Fiala JC (2002) Endosomal compartments serve multiple hippocampal dendritic spines from a widespread rather than a local store of recycling membrane.  J. Neurosci. 22:2215-2224. (763K PDF)

Fiala JC, Allwardt B, Harris KM (2002) Dendritic spines do not split during hippocampus LTP or maturation. Nat. Neurosci. 5:297-298. (311K PDF)

Ostroff LE, Fiala JC, Allwardt B, Harris KM (2002) Polyribosomes redistribute from dendritic shafts into spines with enlarged synapses during LTP in developing rat hippocampal slices. Neuron 35:535-545. (552K PDF)

This website is a superb resource for visual information as well as full text papers on the fine structure of the mammalian central nervous system:

http://synapses.clm.utexas.edu/lab/lab.stm

http://synapses.clm.utexas.edu/anatomy/chemical/synapse.stm

Vitrification:

Fahy, GM, Wowk, B, et al. Cryopreservation of organs by vitrification: perspectives and recent advances. Cryobiology 48 (2004) 157–178: http://www.21cm.com/pdfs/cryopreservation_advances.pdf

Fahy, GM, Wowk, B, Wu, J. Cryopreservation of Complex Systems: The Missing Link in the Regenerative Medicine Supply Chain. Rejuvenation Research. 2004, 9(2):279-91: http://www.21cm.com/articles/Missing_Link.pdf

Fahy G, Wowk, B, Pagotan, R, et al.: Physical and biological aspects of renal vitrification. Organogenesis 2009, 5(3):167-175: http://cryoeuro.eu:8080/download/attachments/425990/FahyPhysicBiolAspectsRenalVitri2010.pdf?version=1&modificationDate=1285892563927

Wowk B: Anomalous high activity of a subfraction of polyvinyl alcohol ice blocker. Cryobiology 2005, 50(3):325-331: http://www.21cm.com/pdfs/anomalous.pdf

Pichugin Y, Fahy, GM, Morin, R.: Cryopreservation of rat hippocampal slices by vitrification. Cryobiology 2006, 52(2):228-240: http://www.21cm.com/pdfs/hippo_published.pdf

Brain Cryopreservation by Freezing:

Darwin, M, Russell, S, Wakfer, P, Wood, L, Wood, C, Effect of a human cryopreservation protocol on the ultrastructure of the canine brain. (Originally published by BioPreservation, Inc., as BPI Tech Brief 16 on CryoNet and sci.cryonics, May 31, 1995): http://www.alcor.org/Library/html/braincryopreservation2.html and http://www.alcor.org/Library/html/braincryopreservation1.html.

Darwin M, Leaf, JD.: Cryoprotective perfusion and freezing of the ischemic and nonischemic cat: http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1389, http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1390, http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1391, http://www.cryonet.org/cgi-bin/dsp.cgi?msg=1392  See also: Federowicz,  MG. and Leaf JD. Cryonics. issue 30, p.14,1983.

Suda, I., Kito, K, Adachi, C. Viability of Long Term Frozen Cat Brain in Vitro. Nature. v. 212, Oct. 15, p. 167:1966: http://cryoeuro.eu:8080/download/attachments/425990/SudaNature1966.pdf.

Suda, I., Kito, K, Adachi, C. Bioelectric discharges of isolated cat brain after revival from years of frozen storage. Brain Res.  70;527-531:1974: http://www.ncbi.nlm.nih.gov/pubmed/5970120?dopt=Abstract.   Retrieved 2010-08-31

[1]The resolution of direct digital imaging technology using charge-coupled devices (CCDs) is still well below that pf the best fine grain photographic films – color or black and white.

[2] For instance, does a person remain the ‘same’ person after head injury that causes major and permanent changes in personality or in cognitive ability? Or, alternatively, does a person ‘survive’ brain injury that leaves his personality and cognitive capabilities intact, but which deprives the individual of some or all of his memories? If permanent amnesia does occur, will the loss of some memories prove identity critical, whilst the loss of others be merely inconvenient, but not represent a fundamental compromise to personal identity?

[3] Exposure to environmental toxins such as mercury or to psychoactive drugs may also permanently alter cognition and personality in adults.

[4] By interacting with water so strongly via hydrogen bonding CPAs may also make the remaining water less biological available to perform the solventing and stabilizing functions it normally provides.

[5] Biologists arguably ‘know better’ in that they presumably have a better grasp of the fluid nature of cell and cell component morphology.

[6] Ischemia is absent or inadequate blood flow to tissues as occurs in cardiac arrest, heart attack and stroke.

[7] Rather, such a configuration is a product of conscious design at the meta-level.

[8] The outer lamina of the cerebral cortex, containing the neuronal soma and dendrites of Purkinje cells, the axons of the granule cells, and the cell bodies, dendrites, and axons of basket cells.

[9] Declarative memory (sometimes referred to as explicit memory) is one of two types of long term human memory. It refers to memories which can be consciously recalled such as facts and events.[1]  Its counterpart is known as non-declarative or Procedural memory, which refers to unconscious memories such as skills (e.g. learning to ride a bicycle). Declarative memory can be divided into two categories: episodic memory which stores specific personal experiences and semantic memory which stores factual information.