Declaration of a Leading Cryobiologist
This was excerpted from court records, and retyped, with some changes of format. Original document is on file in reference to Case No. 191277, Kent v. Coroner, Superior Court, County of Riverside, California, Appendix of Declarations, dated Feb. 1, 1988. The cryonics people won the case, and the coroner was enjoined against interfering with the cryonics operation.
For professional reasons, the identity of the author of this declaration has been withheld from this web site.
What then would be required for the brain to be restorable? First, the brain must be preserved well enough to repair, i.e. it must be possible today to preserve with some reasonable fidelity the basic biological components of the brains of humans shortly after these humans have clinically died. Second, repair technology must be available to carry out any repairs required.
The two indispensable premises of cryonics, then, are preservation and the development of advanced molecular scale (nanotechnological) biological repair devices. Both premises are fully open to scientific scrutiny and falsification by experiment or calculation and, in fact, both seem at present to withstand such scrutiny. The more detailed declaration testimony which follows documents only the results of scientific tests of the premise of preservation. I understand that K. Eric Drexler will address, in broad scope, the issue of biological repair. To return to the point: If both premises are valid then, assuming the procedure is done under reasonable conditions and non-scientific problems do not intervene, cryonics should work to at least some extent.
6. Cryobiology and Preservation. It can be stated quite firmly that cell bodies, cell membranes, synapses, mitochondria, general axon and dendroid patterns, metabolites such as neurotransmitters, chemical constituents such as proteins and nucleic acids, and general brain architecture are preserved reasonably well or excellently with current techniques. The brain can withstand severe mechanical distortion by ice without impairment of subsequent cognition, and a glycerol concentration of 5.15M can be shown to limit ice formation to quantities currently thought to be consistent with good function or recovery of the intact brain. Information is lacking about the ultrastructure of frozen thawed brains, but much can be inferred from the customary observation of a high level of functional recovery of frozen thawed brains, brain tissue or brain cells which depends on a high degree of both local and long range ultrastructural integrity. Absolute proof is lacking about the quality of preservation in each and every brain region, since not all brain regions have been examined by neurobiologists to date. However, in my experience, no clear differences in preservation quality from one brain region to another have ever been apparent to me while examining entire cross-sections of the frozen - thawed brain at many different levels.
A reasonable way of summarizing the world literature on the subject at present is to say that whenever either brain structure or brain function has been evaluated after freezing to low temperatures and thawing, robust preservation has almost always been demonstrable provided that some attention was paid to providing at least token cryoprotection, and in some cases good preservation has been documented in the complete absence of reasonable cryobiological technique. The implication of these findings is that structures and functions not examined to date will also respond in a favorable way to freezing and thawing.
7. A detailed review of relevant current cryobiological knowledge follows:
(A) General cryobiological background. Freezing is not a process of total destruction. It is well known that human embryos, sperm, skin, bone, red and white blood cells, bone marrow, and tissues such as parathyroid tissue survive deep freezing and thawing, and the same is true for systems of animal origin. In 1980 I published a table listing 3 dozen mammalian organized tissues and even a few mammalian organs which had been shown to survive cooling to low temperatures (1), and this list could now be expanded due to additional experiments on other systems. Such survival could not occur if the molecules comprising biological systems were generally altered by freezing and thawing, and, in general, freezing does not cause chemical changes or protein denaturation. (Frozen food might be an exception, but it is an irrelevant exception).
Contrary to popular imagination, cells never burst as a result of intracellular freezing. The expansion of water as it is converted to ice causes less than a 10% increase in volume, whereas cells can withstand far larger increases in volume, e.g., 50% to 100% increases. But the primary flaw in this concept is the idea that ice forms in cells at all under ordinary conditions of slow freezing: it does not. Indeed, ice forms between cells and water actually travels from the interior of the cell to the ice outside the cell, causing shrinkage rather than bursting of the cell.
Cell death during slow freezing may be related to changes in the cell membrane produced by cell shrinkage or to toxicity of cryoprotectants as they are progressively concentrated as a consequence of the formation of pure ice in initially dilute solutions. Both of these putative causes of death are relatively mild on the molecular level and are certainly not irreversible in principle. But whatever the cause of death, cells examined in the frozen state appear to be structurally intact even when they are known to be nonviable upon thawing (with very few exceptions on the part of nonmammalian systems not relevant to the brain). This is true both for single plant and animal cells and for cells that comprise animal tissue. Hence, lack of functional recovery after thawing is not proof of lack of structural preservation in the frozen state before thawing, and it is the latter that is relevant to cryonics.
A truism of cryobiology is that different types of cells require different protocols of cryoprotectant treatment, cooling and warming rate, and cryoprotectant washout in order to exhibit maximal survival. Some kinds of cells are particularly difficult to freeze without killing them. All of these differences can be minimized greatly by using high concentrations of cryoprotectant, provided such concentrations are tolerated. Nevertheless, other than a few generalizations such as those described above, it is impossible to extrapolate from one biological system to another in terms of predicting the details of its cryobiological behavior.
For this reason, if we wish to understand what happens to the brain when it is frozen, we cannot argue on the basis of results obtained with kidneys or plant cells or embryos or granulocytes, but must, instead, focus specifically on the brain. Herein lies one of the largest errors cryobiologists and other scientists have made in dismissing the prospects for cryonics: making sweeping negative statements without knowing anything about the cryobiology of the brain (or, for that matter, the primacy of the brain, or the concepts of nanotechnology).
In order to examine the scientific evidence bearing on the only indispensable cryobiological premise of cryonics, then, the balance of this testimony will be devoted to an extensive review of the contents of a large number of scientific papers othe freezing of brains, brain tissue, and/or brain cells. As extensive as the following remarks are, it should be understood that they are not exhaustive. No attempt has been made to obtain the complete scientific literature describing the state of brains after freezing in ways which are relevant to the issue of cryonics. The review simply reflects all relevant information already in my files at the same time this deposition was requested.
(B) Living Adult Animal Brains. Dr. Robert J. White, the Chairman of the Dept. of Neurology at Case Western Reserve University's School of Medicine, has favorably discussed the prospects for the eventual successful cryopreservation of human brains (2,3,4). (Dr. White is also an expert on cephalic transplantation and hypothermic brain preservation and has published several scientific papers on these subjects.) However, it is clearly impossible to experiment with entire living human brains, so the closest we can come to evaluating the degree of total brain preservation achieved in best-case cryonics procedures is to review the results of freezing the brains of animals.
The earliest observations of this sort were made by Lovelock and Smith (5,6) in 1956. These investigators froze golden hamsters to colonic temperatures between -0.5 C and -10 C and quantitated the amount of ice formed in the brain, allowing them to determine how much ice formed in the brains of animals which made full neurological recoveries. They determined that at least 60% of the water in the brain could be converted into ice without damaging the ability of the hamsters to regain normal behavior after thawing. Considerably more ice was consistent with restoration of breathing, a complex neural function. The exact quantity of ice consistent with full neurological recovery could not be clearly determined, however, because of death due to intestinal, pulmonary, and renal bleeding, and was probably significantly in excess of 60% of the initial water volume. Tolerance of so much ice by the brain shows that this organ is considerably more tolerant of freezing than is the kidney.
The prospects for successfully avoiding damage due to the formation of ice at much lower temperatures can be assessed to a first approximation based on this finding of Lovelock and Smith. The quantity of glycerol required in theory to prevent mechanical injury from ice (Cgr) an be calculated from the equation (derivable from reference 7) cgr = 9.3 - .093Vt, where Vt is the percentage of the liquid volume of the brain which can be converted into ice without causing injury. Assuming Vt = 60%, Cgr is 3.72M. In the specific case of Mrs. Kent, 4.15M glycerol was perfused, more than required according to this calculation.
The work of Lovelock and Smith was followed up by Suda and his associates (8,9,10), who made a number of critical observations on frozen glycerolized cat brains. Their first publication, in 1966, demonstrated that cat brains gradually perfused with 15% v/v glycerol at 10 C and frozen very slowly for storage for 45-203 days at the very unfavorable temperature of -20 C regained normal histology, vigorous unit activity in the cerebral cortex, hypothalamus, and cerebellar cortex, and strong if somewhat slowed EEG activity (8) after very slow thawing.
These results are remarkable in a number of ways. First, it is clear that no other organ would be capable of the same degree of activity after such prolonged storage at such a high subfreezing temperature. Second, Suda et al made no attempt to supplement their perfusion fluid (diluted cat blood) with dextrose, which must have become depleted fairly rapidly thereby worsening the EEG results. Third, Suda and colleagues did not wash the glycerol from the brain carefully, and this may have caused injury during brain reperfusion. Fourth, the presence of EEG activity implies preservation of long-range neural connections and synaptic transmission, and unit activity indicates preservation of cell membrane integrity, energy metabolism, and sodium and potassium pumping capacity. In short, these brains appeared to be basically viable.based both on function and on structure. Although "pial oozing" was noted (but not described adequately) after about an hour of blood reperfusion, this defect seems minor. It may be noted that the cryoprotectant and the temperature of cryoprotectant perfusion were similar to those used for Mrs. Kent.
Their second publication, in 1974 (9), went considerably further. After 7.25 years of storage at -20 C, "well synchronized discharges of Purkinje cells were observed" (i.e., normal cerebellar unit activity) as well as "spontaneous electrical activity ... from the thalamic nuclei and cerebellar cortex", and short-lived EEG activity from the cerebral cortex. Another brain stored for 777 days showed cortical EEG activity for 5 hours after reperfusion. In both cases, EEG activity was of lower quality than EEG activity of fresh brains, but the existence of any activity at all after such extraordinary conditions is amazing. Cell loss after 7.25 years and hemorrhage after reperfusion of brains stored for 5-7 years is not surprising.
More important was a comparison of the frequency distribution of EEG activity in a fresh brain before perfusion and then after storage at -20 C for 5 days. The EEG pattern before freezing and after thawing was very nearly the same (9). It should be noted that in a typical cryonics operation, and in Mrs. Kent's case specifically, the time spent near -20 C is measured in minutes or hours rather than days or years and, based on the work of Suda et al., should not therefore involve appreciable deterioration of the brain.
It is noteworthy that in both reports of Suda's group, the brains were successfully reperfused with diluted cat blood after thawing. The quality of reperfusion was not documented in detail, but the autocorrelogram comparing the EEG of the 5-day cryopreserved brain to the EEG of the same brain before freezing could not have been as good as it was without relatively complete restoration of cerebral circulation. This is an important question not only with respect to viability and functional recovery but also with respect to the accessibility of the brain to nanotechnological repair devices which might be administered via the vascular system.
Also relevant were unpublished results mentioned in passing (9) on storage at -60 and -90 C and on the effectiveness of other cryoprotectants (dimethyl sulfoxide or polymers). Evidently, EEG activity could be obtained after freezing to -60 C and storage for weeks, but not after freezing to -90 C, and dimethyl sulfoxide was effective but not as effective as glycerol. This is confirmed in an unpublished manuscript sent to me by Suda (10), which reveals also that unit (single cell) activity can still be recorded in brains frozen to -90 C. This unpublished paper (written in Japanese) also shows that brain reperfusion was better after thawing when glycerol rather than DMSO was used.
These results can be evaluated with respect to the information obtained previously by Lovelock and Smith. For protection against mechanical injury at -90 C, as noted above, the results with hamsters suggest that 3.72M glycerol, or 27.2% glycerol by volume, might be required, whereas Suda and colleagues used only 15% glycerol by volume. It can be calculated (11) that at Suda's storage temperature of -20 C, 62% of the liquid content of the brain was converted into ice, while at -60 C, 77% of the liquid volume of the brain was converted to ice, a quantity which equals or exceeds the tolerable degree of distortion by ice in the hamster brain. Therefore, the finding by Suda and his colleagues of no injury at -20 C for 5 days but of injury after freezing to -60 and especially to -90 C is entirely consistent with predictions from the work of Lovelock and Smith and is also entirely consistent with an absence of any such mechanical injury in the brain of Dora Kent, which was perfused with enough glycerol to protect at any temperature.
The work with hamsters and with cat brains demonstrates that extensive freezing of the brain at high temperatures is compatible with its full functional recovery and that at least partial functional recovery from low temperatures is a iasonable prospect, but these studies do not describe the histological effects of freezing brains to the low temperatures -quired for truly long term preservation. This information was provided by myself and my colleagues (12-14). We reported that with either 3M or 6M glycerol, excellent histological preservation of the cerebral cortex and the hippocampus was observed after slow freezing to dry ice temperature. In fact, there was no difference in structure between brains which had been perfused with glycerol only or brains which had been perfused, frozen, and thawed. Although we did not report it formally, this finding was also true in every other region of the brain examined, such as the cerebellum and the area of the ventral brain containing giant neurons and well-organized axonal bundles. It is of interest that we observed brain shrinkage if the perfusion temperature was held constant below room temperature (14). But Suda and his colleagues observed the same degree of brain shrinkage (10), yet this did not prevent apparent survival of their frozen cat brain. Again, Mrs. Kent's brain was perfused with a concentration in the 3-6M range studied by us and found to be protective despite shrinkage.
(C) Living Adult Human and Animal Brain Tissue. In 1981, Haan and Bowen (15) reported that they had collected sections of cerebral cortex from living human patients (during brain operations requiring removal of cortex to allow access to deep tumors) and froze them using 10% v/v dimethyl sulfoxide (DMSO) as the cryoprotectant. The DMSO was added and removed essentially in one step each, with some agitation of tissue samples to promote equilibration in the short times allowed for equilibration at 40 C. Freezing was accomplished by a two-step method in which the tissue was placed at -30 C for 15 min (5 min required to reach -30 C, for a cooling rate of about 60 C/min, and 10 min of equilibration at -30 C) and then transferred directly to liquid nitrogen. Thawing was rapid. For comparison, rat brain tissue was obtained by decapitating rats and removing their brains (probably involving a warm ischemic insult of 5-10 min), and this rat brain tissue was equilibrated with dimethyl sulfoxide and frozen in the same way.
The results? Norepinephrine uptake was 94-95% of control uptake for both rat and man. Incorporation of glucose-derived carbon into acetylcholine was 89-100% of control incorporation for rat and 85% of control for human. Incorporation of glucose-derived carbon into C02 was 86-100% of control for rat, 78% of control for man.
Haan and Bowen noted that their tissue prisms are mostly synapses, so their results imply that synapses of both rat and man survive freezing by their technique. This agrees with inferences noted above that synapses survive in whole brains frozen with completely different techniques. Although not strictly brain tissue, the superior cervical ganglion, considered part of the central nervous system, also demonstrated 100% recovery of synaptic function after freezing to dry ice temperature in 15% glycerol according to Pascoe's report in 1957 (16). It was noteworthy that Pascoe's ganglia also showed 100% recovery of action potential amplitude and conduction velocity after thawing from dry ice temperature (16).
In 1983, Hardy et al. (17) confirmed the extreme survivability of synapses in human brain tissue beyond any doubt. Or, again, normal living adult human cerebral cortex was removed during operations on deep brain structures and compared to viable rat forebrains in terms of freeze-thaw recovery. The best results were obtained by freezing 1-5 gram pieces of human brain (or 1 gram rat forebrains), as opposed to freezing homogenates. The cooling rate to -70 C was slow but was not measured or controlled; the thawing rate was fast but not measured or controlled; the cryoprotectant was 0.32M sucrose (which would be expected to provide almost no cryoprotection!). After thawing, synaptosomes; were prepared from the tissue samples and tested for functional recovery. Here is a summary of the results:
Measurement Human Rat
number of synaptosomes recovered not done 80
amount of protein recovered 91 70
oxygen uptake/100 mg of protein 78 59
stimulation of oxygen uptake by veratrine 86 86
potassium accumulated/100 mg protein 86 70
loss of potassium stimulated by veratrine 39 85
retention of neurotransmitters good good
stimulated transmitter release (amount, selectivity
and drug modulation) good good
*recovery compared to unfrozen control samples.
As Hardy et al. stated, it is apparent that both human and rat brain tissue frozen to -70 C with almost no cryoprotection had synapses "closely comparable to (those from) ... fresh tissue".
As if this were not demonstration enough, Waler(18) has shown that not even cryosurgery destroys synapses. He applied a -60 C cryoprobe to the brain of cats for 5 min and examined the resulting lesions in the electron microscope. Not only were well preserved synapses found, but also cell bodies, organelles, and neuronal processes could be identified, despite considerable damage to the organization of the neuropil and to astrocyte cell membranes.
(F) Postmortem Human and Animal Brains. Human brain banks are now in existence for investigators interested in understanding human brain biochemistry and pathology (30-33). Sections or subregions of postmortem human brains, frozen rapidly several hours after death, are sent to medical researchers who analyze these brains for neurotransmitters, proteins, enzyme activity, lipids, nucleic acids, and even histology. There would be no reason for such banks if no molecular or structural preservation were achieved by freezing.
Haberland et al. (34) isolated synaptosomes after freezing the nucleus accumbens of rats and 72 (plus or minus 5 years) year old humans. The humans were dead 15 +/- 5 hours before this brain structure was removed and frozen. Previous studies indicated that dopamine uptake by such synaptosomes could still achieve 55% of the values of fresh brains even 24 hours after death. In this study, the humans were not refrigerated until 3-5 hours after death. Freezing was done with varying concentrations up to 10% DMSO, 1.2 deg C/min to -25 C, and subsequent immersion in liquid nitrogen.
Experiments on rat nucleus accumbens (NA) removed 5-10 min after decapitation of the rat indicated that freezing to -25 C allowed about 100% of control dopamine uptake. When rat NA was frozen to -196 C, survival ranged from 96% of control using 0.07M DMSO to 99.7% of control using 0.7M DMSO. Human NA frozen to -196 C as described in the presence of 0.7M DMSO (5% v/v) yielded dopamine uptakes equaling 102.9+/-5.2% of unfrozen control uptakes.
Schwarcz (35) subjected rat brains to postmortem conditions comparable to those experienced generally by humans: 4 hours of storage in situ at room temperature followed by 24 hours of storage in situ at 4 C followed by brain isolation and freezing of brain regions by placement in a -80 C freezer for 5 days. Glutamate uptake by striatal synaptosomes prepared from striata frozen in this way amounted to 26% of control uptake by fresh tissue synaptosomes, an amazing degree of preservation. (Schwarcz noted, however, that glutamate uptake processes may be more resistant than serotoninergic, dopaminergic, and cholinergic uptake mechanisms.)
Brammer and Ray (36) confirmed that it is possible to isolate intact if not living oligodendroglia cells from bovine brain white matter frozen to -30 C without any cryoprotective agent more than 1 hour after the slaughter of the cow. (The original paper describing isolation of human oligodendroglia under similar circumstances is that of Igbal et al. (37), not in my files.) If the white matter was treated with polyvinyl pyrrolidone before freezing, cytoplasmic enzyme activities were not different from enzyme activities in unfrozen cells (without PVP, enzyme activities were one-half to one-fourth of control values, which is significant preservation of enzyme structure and function even under these highly adverse circumstances.) Although no data were shown concerning the effects of glycerol or DMSO, it was stated that these agents did not improve enzyme activity. Nevertheless, it should be recalled that Kim (27) isolated the same cells from postmortem human brains before freezing and found that pretreatment with 10% DMSO allowed them to survive freezing to liquid nitrogen temperature.
Morrison and Griffin (38) isolated undegraded messenger RNA from human brains after 4 or 16 hours of death, with or without freezing in liquid nitrogen. The mRNA was used to direct protein synthesis in vitro, which was then analyzed by 2-D O'Farrell gel electrophoresis. Normal protein populations were observed, causing them to conclude "that postmortem storage for 4 and 16 hours at room temperature had little effect on the spectrum of isolated mRNAs" and "the profile of proteins synthesized ... was not changed ... when the tissues were stored in liquid nitrogen."
Stahl and Swanson (39) looked at the fidelity of subcellular localization of 6 brain enzymes and total brain protein after guinea pig or postmortem human brain tissues were frozen to -70 C without cryoprotectant simply by being placed into a freezer. Their conclusion: "subcellular fractionation of brain material is possible even with postmortem tissues removed from the cranial cavity some hours after death. Two other groups have subsequently fractionated human postmortem brain and have come to a similar conclusion . . . Even after freezing and prolonged storage, human and guinea pig brains can be separated into biochemically distinguishable subcellular fractions . . . Frozen storage for several months did not strikingly modify the fractionation characteristics of freshly homogenized cerebral cortex."
Many similar reports exist in the literature. Tower et al. showed preservation of oxygen consumption and enzyme activities in brains of many species, including whales subject to many hours of warm ischernia, after isolation from the dead animal and freezing (40-42). Hopefully, the point is clear that brain structure and enzymatic activity and even some brain functions survive freezing even when freezing is done after hours of unprotected clinical death and even with minimal or no cryoprotection.
Postmortem Human Spinal Cord and Outflowing Nerves. One report (43) is available to my knowledge documenting the effects of cryonics procedures on the spinal cord, which is a part of the central nervous system. A human cryopreserved by now-obsolete cryonics procedures and was decapitated while frozen, the body thawed, and the spinal cord and spinal nerves examined histologically after aldehyde fixation and osmication. The basic finding was that myelin sheaths were intact and shrunken axoplasm could be seen within the myelin sheaths, conceivably indicating intact axolemmas. Large neuronal cell bodies were observed which appeared intact and normal in shape. In general, the histological preservation was impressive. Apparently intact blood vessels were observed within the spinal cord. (Other, non-neuronal tissues were also examined and were found to be surprisingly intact, with the exception of the liver and a lesser extent the kidney.)
Summary. The scientific literature allows no conclusion other than that brain structure and even many brain functions are likely to be reasonably well preserved by freezing in the presence of cryoprotective agents, especially glycerol in high concentrations. Thus, cryonics' premise of preservation would seem to be well supported by existing cryobiological knowledge. This is not to say that cryonics may work. Therefore, the implications of autopsy in the present case should be considered serious and different from the implications of autopsy in the usual case.
Attached hereto following the curriculum vitae is a list of the references cited. The numbers appearing next to the cited references in the body of the text of this declaration are those appearing to the left hand side of the noted articles in the attached list.
If called upon to testify in a court of law I could and would competently testify to each and every fact set forth hereinabove.
I declare under penalty of perjury that all of the foregoing is true and correct and that this declaration was executed at Rockville, Maryland, on January 26, 1988.
[Following in the original document is a list of 43 references cited, the author's curriculum vitae, and a list of 30 publications and 22 abstracts by the author. To save space, we list just a few of the references.]
A. List of references cited
General cryobiological background:
5. Smith, A.U. "Revival of mammals from body temperatures below zero." In: BIOLOGICAL EFFECTS OF FREEZING AND SUPERCOOLING (A.U. Smith, editor). Edward Arnold, London, 1961. pp. 304-368.
9. Suda, I., Kito, K., and Adachi, C. "Bioelectric discharges of isolated cat brain after revival from years of frozenstorage." BRAIN RES. 70:527-531, 1974.
Living adult human and animal brain tissue:
15. Haan, E.A., and Bowen, D.M. "Protection of neocortical tissue prisms from freeze-thaw injury by dimethylsulphoxide." J. NEUROCHEM. 37:243-246, 1981.
16. Pascoe, J.E. "The survival of the rat's superior cervical ganglion after cooling to -760C." PROC. ROY. SOC. (London) B, 147:510-519, 1957.
17. Hardy, J.A., Dodd, P.R., Oakley, A.E., Perry, R.H., Edwardson, J.A., and Kidd, A.M. "Metabolically active synaptosomes can be prepared from frozen rat and human brain." J. NEUROCHEM. 40:608-614. 1983.
35. Schwarcz, R. "Effects of tissue storage and freezing on brain glutamate uptake." LIFE SCI. 28:1147-1154, 1981.
39. Stahl, W.L., and Swanson, P.D. "Effects of freezing and storage on subcellular fractionation of guinea pig and
human brain." NEUROBIOLOGY 5:393-400, 1975.
42. Tower, D.B., and Young, O.M. "Interspecies correlations of cerebral cortical oxygen consumption, acety1cholinesterase activity and chloride content: studies on the brains of the fin whale (Balaenoptera physalus) and the sperm whale (Physeter catodon)." J. NEUROCHEM. 20:253-267, 1973.
End of Excerpted Declaration
Cryoletter is an important endorsement by recognised professionals for cryonics and could be printed and used when dealing with naysayers, particularly other professionals. It is a product of the Immortality Institute.