X-Message-Number: 0035
Subject: The Cryobiological Case For Cryonics
THE CRYOBIOLOGICAL CASE FOR CRYONICS
Contents
Introduction
A. Premises and their scientific evaluation
B. Short introductory summary of general conclusions
C. Detailed review of relevant current cryobiological
knowledge
1. General cryobiological background
2. Living adult animal brains
3. Living adult human and animal brain tissue
4. Living fetal human and animal brain tissue
5. Living human and animal isolated brain cells
6. Post-mortem human and animal brains
7. Post-mortem human spinal cord and outflowing nerves
Summary
List of references cited
* * * * * * * * * * * * * * * *
* * * *
Introduction
Any casual newspaper reader will have decided quite confidently by now that
cryonics has no chance whatever of success, due to the systematic
misinformation contained in all media coverage of this subject to date.
Not only has the scientific evidence supportive supportive of cryonics not
been presented, but the unchallenged, supposedly scientific criticisms of
cryonics presented in the media have been as harsh as they have been vapid
and without merit. In reality, it seems that no supposedly scientific
criticism of cryonics has ever addressed the real issues involved or ever
been based on a grasp of them. The purpose of this discussion is to
provide a summary of the extensive cryobiological evidence which
exists to support cryonicists' premise that existing freezing techniques
preserve the molecular basis of human memory and personality and thus offer
a reasonable chance of allowing future restoration of cryonics patients to
life.
Why has this evidence not been presented previously? The reasons are
largely political. Also it should be appreciated that even a neutral
position with respect to the emotionally charged subject of cryonics is
hazardous for a cryobiologist because of hardened opposition on the part
of many key scientists who control job availability and grant support.
This opposition is generally based on a gut reaction and/or philosophical
objections that do not invite further consideration. Unfortunately, almost
no one ever seriously asks whether anything as seemingly outrageous as
cryonics could have any compelling scientific foundation, despite the fact
that it does. The problem is that the relevant scientific facts are far
from obvious or readily available, and that no well- established scientist
has ever dared or even been able to enunciate them.
The result has been the suppression of discussion, the creation of
anxiety, the propagation of gross misinformation among the general public,
and the censorship of valid scientific observations: in short, the
antithesis of what science is supposed to be all about. It is time to
consider the scientific facts and to show that what is really outrageous
is not cryonics but the notion that there is no scientific basis for cryonics
or that cryonics cannot possibly work.
A. Premises and their scientific evaluation
What are the cryobiological issues? Another way of asking this question is:
what is the minimum cryobiological requirement for "success" with the
cryonics endeavor? Since the one indispensable goal of cryonics is
restoration of the brain, we can limit our attention to the cryobiological
requirements for the achievement of this goal. Questions concerning
maintenance of the brain after restoration are not cryobiological and can
therefore be neglected here.
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 reasonable brain
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,
as the experimental evidence which is presented in this paper as well as
the work of others on the problems of biological repair (see K. Eric
Drexler's book, Engines of Creation, and his technical papers) should
show. If both premises are valid (assuming cryonic suspension is done
under reasonable conditions and nonscientific problems do not intervene),
then in principle cryonics should work to at least some extent.
As noted above, this article is about the cryobiological basis of cryonics
rather than the cell repair aspect. But because the cryobiological premise
of cryonics loses significance without the nanotechnological premise of
cryonics, it is necessary to comment at least briefly on
nanotechnology in order to clarify the relevance of the evidence to be
presented about cryobiology. There appear to be no significant flaws in K.
Eric Drexler's concepts of molecular scale cell repair devices, and this
judgment is supported by the absence of even a single significant and
coherent objection to his concepts. The concepts involved are powerful
enough to make it easy to imagine the technology not only for repairing
the fine structure of the brain but also the technology for transplanting
a brain into a new body. It seems not only possible but inevitable that
such technologies will be developed, and a person waiting in liquid nitrogen
should remain changeless for centuries if need be while such developments
proceed.
B. Summary of general conclusions
It can be stated quite firmly that cell bodies, cell membranes,
synapses, mitochondria, general axon and dendrite 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 less than 4M -- a concentration achieved in
current cryonics procedures -- can be shown to limit ice formation to
quantities currently thought to be consistent with good functional 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 the experience
of those who have histologically examined entire cross-sections through
the frozen-thawed brain at many different levels, no clear differences in
preservation quality from one brain region to another have ever been
apparent.
A reasonable way of summarizing the world literature on this subject at
present is to say that wherever either brain structure or brain function
has been evaluated after freezing to low temperatures and thawing,
robust preservation has almost always been demonstrable provided at
least some minimal attention was paid to providing 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.
C. Detailed review of relevant current cryobiological knowledge
1. 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 a table was
published listing three 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. In general, freezing does not cause chemical changes or protein
denaturation.
Contrary to popular imagination, cells do not 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- 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. Instead, ice forms
between cells, and water actually travels from the interior of the cell to the
ice outside the cell, causing shrinkage rather than expansion 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 rates, and
cryoprotectant washout in order to exhibit maximal survival. All of these
differences can be minimized greatly by using high concentrations of
cryoprotectant, provided such concentrations can be tolerated.
Nevertheless, other than a few generalizations such as those described above,
it is difficult 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 can't 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
article will be devoted to an extensive review of the contents of a large
number of scientific papers on the 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. This review
simply reflects all relevant information currently at hand.
2. 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 -1*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. However, the exact quantity
of ice (above 60%) consistent with full neurological recovery could
not be clearly determined, because of death due to intestinal, pulmonary,
and renal bleeding. Nevertheless, tolerance of at least 60% 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) can 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.
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 (individual
cell) 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, 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 capability. In short, these brains
appeared to be basically viable based both on function and on structure.
"Pial oozing" was noted (though not described adequately) after about an
hour of blood reperfusion, but this defect seems minor.
Their second publication, in 1974 (9), went considerably farther. 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,
the time spent near -20*C is measured in 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*C 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 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 3.72 M
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*C 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 brains of cryonic suspension patients perfused with more than 3.72M
glycerol.
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 reasonable prospect, but these studies do not
describe the histological effects of freezing brains to the low
temperatures required for truly long-term preservation. This information was
provided by Fahy and colleagues (12-14a). They 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 (-79*C). In fact, there was no difference in structure
between brains which had been perfused with glycerol only and brains which
had been perfused, frozen, and thawed. Although Fahy et al. 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 Fahy et al. observed brain shrinkage if the perfusion
temperature was held constant below room temperature (14a). But Suda and his
colleagues also observed the same degree of brain shrinkage (10), yet this
did not prevent apparent survival of their frozen cat brains.
One report (14b) has appeared which briefly documented the ultrastructural
effects of a now-obsolete cryonics procedure on the brain. A single dog was
perfused directly with 15% DMSO for 55 minutes at 10-17*C. The head was
then cooled at 0.1*C/min to -14*C and then cooled at 0.5*C/min to lower
temperatures. The brain was estimated to have reached - 79*C after 3 hours,
after which it was shipped cross-country for thawing, fixation, and
examination by light and electron microscopy. Histochemical staining of
undefined nature showed evidence for appreciable enzymatic activity and
cellular retention of histochemical reaction product, i.e., intact cell
membranes. Ultrastructure, as documented in a single electron micrograph,
revealed intact cell bodies, an intact double nuclear membrane, intact
myelin sheaths around small myelinated fibers, recognizable
organelles (mitochondria and endoplasmic reticulum), and recognizable
synapses. Extensive damage was also apparent, but it was not clear
whether this was due to freezing and thawing, perfusion with DMSO in one
step as opposed to gradual addition, or abrupt dilution of DMSO upon
fixation. No details were provided as to DMSO washout and fixation
procedures. Significantly, the concentration of DMSO employed was not
sufficient to prevent mechanical damage according to "the Smith criterion"
mentioned earlier. The presumption would be that current cryonics
procedures, employing the preferred cryoprotectant glycerol in higher
concentrations, better preserve ultrastructure. Nevertheless, it is not
obvious from the published micrograph that the original brain structure could
not be inferred.
3. 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 frozen 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 4*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 6*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
rats and humans. Incorporation of glucose-derived carbon into acetylcholine
was 89-100% of control incorporation for rats and 85% of control for humans.
Incorporation of glucose-derived carbon into CO2 was 86-100% of control for
rats, 78% of control for humans.
Haan and Bowen noted that their tissue prisms are mostly synapses, so their
results imply that synapses of both rats and humans 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. Once 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 sole
cryoprotectant was 0.32 M sucrose (Far from an optimal regimen!). After
thawing, synaptosomes were prepared from the tissue samples and tested for
functional recovery. Here is a summary of the results:
Percent
recovery*
Measurement human rat
---------------------------------------------------------------------
number of synaptosomes recovered not done 80
number of mitochondria recovered 133** 67
increase in number of unidentifiable
(damaged) structures 29 24
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 (aspartate, good good
glutamate, GABA)
stimulated transmitter release (amount, good good
selectivity, and drug modulation
---------------------------------------------------------------------
* recovery compared to unfrozen control samples.
** suboptimal technique
As Hardy et al. stated, it is apparent that both human and rat brain
tissue frozen to -70*C with almost no cryoprotection has synapses
"closely comparable to [those from]. . . fresh tissue".
As if this were not demonstration enough, Walder (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.
4. Living fetal human and animal brain tissue
In 1986, Groscurth et al. reported the successful freezing of human fetal
brain tissue (19). 1x2x2 mm brain fragments from a 9-14 week abortus were
treated with 10% DMSO and 20% fetal calf serum and placed into a -30*C
environment for 3 hours or overnight, then stored at -80*C for several
weeks, then finally transferred to liquid nitrogen. After storage for
3-12 months, the samples were "thawed at room temperature", trypsinized, and
seeded on glass cover slips for 2-4 weeks of tissue culture at 37*C. The
brain cells were found to be alive and to grow in culture: "Twenty-four
hours after trypsinization the cells formed clusters of variable size....
During further cultivation numerous fiber bundles were found to grow
from the margin of the clusters. Single fibers showed varicosities as
well as growth cones at the terminal projection. Bipolar spindle-shaped
cells with a smooth surface were regularly apposed along the bundles."
The first reports of attempts to freeze fetal animal brain tissue seem to be
those of Houle and Das in 1980 (20-22). These attempts were fully
successful, the frozen-thawed transplanted cerebral cortex being
indistinguishable from non-frozen brain tissue transplants in every
way. Das et al. have more recently described their technique in finer
detail (23). Briefly, they use 10% DMSO, a cooling rate of 1*C/min,
storage at -90*C, and rapid thawing. Survival was best if the tissue was
not dissociated or minced before freezing.
Although a variety of conditions allowed for 100% success rates for 16 and
17-day neocortex, brainstem tissue from 16-day fetuses showed at best a 50%
survival rate, and Das et al. suggested that these more differentiated
cells, which have a low transplant survival rate even in the absence of
freezing and thawing, might be more damaged by freezing and thawing.
On the other hand, it should be kept in mind that, as should be clear from
the earlier discussion of cryoprotectant concentrations necessary for
protection at low temperatures, 10% DMSO is a rather low concentration of
a possibly suboptimal cryoprotectant (Suda indicated that glycerol was
superior to DMSO for brain), and better survival might well have been
obtained using the more gentle freezing/thawing conditions employed in
cryonics procedures.
Jensen and colleagues (24) reported their work on freezing fetal hippocampal
tissue in 1984, again using 10% DMSO, a cooling rate of 1*C/min, storage in
liquid nitrogen, and rapid thawing. Treatment with DMSO at 4*C was for 2
hr, with rapid washout at room temperature (not necessarily an innocuous
approach; unfortunately, no DMSO controls were done). Although 21% of the
cryopreserved hippocampi showed ideal structural preservation after
development in oculo, in general there was some structural alteration
compared to nonfrozen control hippocampal transplants. It was felt that
this may have been due to the extra manipulations of the cryopreserved
tissue (controls were not washed in DMSO solutions, etc.). Only half
of the cryopreserved transplants at most were found to be present after
20-68 days in oculo, survival rate being dependent upon fetal age. It was
felt that this once again may have been due to loosening of the hippocampal
structure by the experimental manipulations.
This tended to be confirmed by transplants into the brain rather than into
the eye: the brain provides more confinement to transplanted
hippocampi, helping to prevent disintegration of the grafts, and, in
fact, 100% of hippocampi transplanted to the brain survived. (It should
be obvious that the hippocampus of a frozen intact brain will of course
receive support from all surrounding structures and will thus be more
analogous to the intracerebral transplants noted by Jensen et al. than to
the intraocular transplants, in addition to being spared from disruptive
manipulations in vitro.)
Frozen-thawed hippocampi grown in oculo were smaller than control grafts, and
frozen- thawed hippocampi transplanted either to the eye or to the brain
showed a loss of dentate granule cells (a 35% loss was seen in oculo). In
several other ways, this complex brain structure important for encoding
and decoding memories appeared to be unaffected by freezing and
thawing. Moreover, freezing in 10% DMSO, as noted above, might not be an
ideal procedure. It should be noted that Fahy et al. were not impressed by
any loss of dentate cells in whole adult rabbit brains after freezing and
thawing (12-14a).
Jensen's group followed up this work with more extensive work on many
different subregions of the fetal rat brain, i.e., the neocortex,
habenula, septum and basal forebrain, cerebellum, and retina (25). All
of these regions showed good survival and preservation of normal
structural organization after transplantation into an adult recipient's
cerebral cortex, despite wide, uncontrolled variations in cooling protocol
from run to run. The only exception was the cerebellum: only 2 of 7 grafts
were found at the time of sacrifice, although they were structurally
normal. The numbers involved are too small for adequate statistical
analysis, and no control cerebellar grafts were performed to determine if
this rate of takes is normal for this tissue. All in all, then, this paper
tends to confirm the impression from other studies that tissue from many
quite different brain areas survives freezing and thawing quite well.
5. Living human and animal isolated brain cells
Silani et al. (26) dissociated human fetal cerebral cortex into cells and
froze the cells at 1*C/min in 7% DMSO plus 20% fetal calf serum. After
more than 12 months in liquid nitrogen, the cells were thawed rapidly.
Immediately after thawing, the cell recovery was 96.5+/-2.1%, showing
that brain cells are not physically destroyed by freezing even under
rather severe conditions. After 72 hours of culture, 53% of the total cell
population was alive, but only 24% of the neurons were alive. The surviving
neurons were, however, morphologically and functionally normal, as were
astrocytes. Silani et al. considered their yield of human neurons to be
high. These results show unequivocally that human brain cells can survive
freezing and thawing and imply that, as was the experience of Hardy et al.
(17) and Das et al. (23) (and as is suggested by the experience of Jensen et
al. (24)), it is best to use undissociated tissues (analogous to the intact
brain in cryonics procedures) rather than dissociated cells to obtain
optimal results.
Kim et al. (27) isolated living oligodendrocytes and astrocytes from the white
matter of brains of human cadavers aged 62, 86, and 93 years after 5,
14, and 6 hours of clinical death, respectively. These cells were
cultured for 2-28 days, then scraped from their substratum, exposed
abruptly to 10% DMSO, frozen to -70*C at an unknown and uncontrolled,
exponentially decreasing rate, immersed in liquid nitrogen for 1-3 weeks,
thawed rapidly, and abruptly diluted to 1% DMSO, further washed, and
recultured. The excellent morphology of the cultured cells after thawing
and the robust presence of membrane markers was not different from what
existed before freezing. 70%, 60%, and 55% survival was obtained after 2,
7, and 28 days of culture before freezing, respectively.
Kim et al. (27) also reported informally the following. "Recently, we have
frozen various types of neural tissue cultures and found that the recovery of
frozen neurons and glial cells was excellent. The neural cultures tested
were: (a) dissociated chick embryo spinal cord and dorsal root ganglia; (b)
dissociated newborn mouse cerebellum and dorsal root ganglia; (c)
dissociated adult mouse dorsal root ganglia, and; (d) dissociated or
explant fetal human brain cultures."
Kawamoto and Barrett (28) froze rat fetus striatal (including overlying
cortical) and spinal cord cells by dissociating these tissues in 5-10%
DMSO and placing them into uninsulated boxes in a -90*C freezer and
leaving them there for up to 88 days. They were then thawed rapidly and
exposed immediately to DMSO-free solution, a procedure these scientists
found to be damaging. Nevertheless, they observed "neuronal survival rates
comparable to those of brain tissues plated immediately after dissection".
Preliminary results indicated similar survival of neuroglia frozen in the
same way. Survival was roughly independent of DMSO concentration above
5%. Increased sensitivity of the cells to mechanical forces was observed
after thawing or after simple cold storage, but this was reduced by using
cryoprotectant carrier solutions low in sodium. Beautiful morphology was
seen after thawing, and vigorous regrowth of cellular processes occurred after
thawing, to yield mature cultures indistinguishable from controls.
Surprisingly, dissociated cells survived freezing and thawing better than
cells embedded in undissociated tissue.
Scott and Lew (29) gradually exposed undisturbed cultured adult mouse
dorsal root ganglion cells to 10% DMSO, placed them in a -15*C environment
for 30 min, then placed them in liquid nitrogen vapor. Thawing took 5
min, after which the DMSO was removed gradually. Other cultured neurons
were dissociated and frozen and thawed similarly as a cell suspension.
The relative number of surviving neurons was not quantitated in this study,
although there was evidently considerable cell death (probably due to the
high cooling rate below -15*C, which would be expected to induce
intracellular freezing and cell death). Nevertheless, many neurons
survived and were capable of basically normal electrical activity as well
as regeneration of new nerve fibers.
6. Post-mortem 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 post-mortem 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 of 72 (plus or minus 5) 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 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*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 caused no
measurable reduction of dopamine uptake. When rat NA was frozen to -196*C,
survival ranged from 96% of control using 0.07 M DMSO to 99.7% of control
using 0.7 M DMSO. Human NA frozen to -196*C as described in the
presence of 0.7 M DMSO (5% v/v) yielded dopamine uptakes equaling
102.9+/-5.2% of unfrozen control uptakes.
Stahl and Swanson (35) looked at the fidelity of subcellular localization of 6
brain enzymes and total brain protein after guinea pig or post-mortem human
brain tissues were frozen to -70*C without a cryoprotectant simply by
being placed into a freezer. Their conclusion: "subcellular fractionation
of brain material is possible even with post-mortem tissues removed from the
cranial cavity some hours after death." Two other groups have subsequently
fractionated human post-mortem brain and have come to a similar conclusion:
"Our present study further shows that 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."
Schwarcz (36) subjected rat brains to post-mortem 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 (37) confirmed that it is possible to isolate intact, if not
living, oligodendroglial cells from bovine brain white matter after freezing
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 Iqbal et al. (38))
If the white matter was treated with polyvinyl pyrollidone (PVP) 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 demonstrates 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 post-mortem human brains before freezing and found that
pretreatment with 10% DMSO allowed them to survive freezing to liquid
nitrogen temperature.
Morrison and Griffin (39) 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 post-mortem
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."
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 ischemia,
after isolation from the dead animal and freezing (40-42). Hopefully, the
point is clear that brain structure and even some brain functions and
enzymatic activity survive freezing even when freezing is done after
hours of unprotected clinical death and even with minimal or no
cryoprotection.
7. Post-mortem human spinal cord and outflowing nerves
One report (43) is available documenting the effects of cryonics procedures
on the spinal cord, which is part of the central nervous system. A human
cryopreserved by now- obsolete cryonics procedures 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, to 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 will inevitably
work. But it is to say that cryonics may work and that it is a reasonable
undertaking.
List of references cited
General cryobiological background
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frozen in the presence of dimethyl sulfoxide., Cryobiology, 17, 371-388
(1980).
Living adult animal brains
2. White, R.J., Brain, In: Organ Preservation for Transplantation, A.M.
Karow, Jr., G.J.M. Abouna, and A.L. Humphries, Jr., Eds., Little, Brown, &
Company, Boston, 1974. pp. 395-407.
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1981. pp. 655-674.
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Biological Effects of Freezing and Supercooling, A.U. Smith, Ed.
Edward Arnold, London, 1961. pp. 304-368.
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cooling to and rewarming from body temperatures below 0*C. III.
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427-442 (1956).
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underlying the physical properties, biological actions, and utility
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brain in vitro, Nature (London), 212, 268-270 (1966).
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cat brain after revival from years of frozen storage, Brain Res, 70,
527-531 (1974).
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based on a talk given by Dr. Suda (President of Kobe University) in
Japan and reportedly being prepared for publication in English.
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of rat and rabbit brains, Cryo-Letters, 5, 33-46 (1984).
14a. Fahy, G.M., and A.M. Crane, Histological cryoprotection of rabbit
brain with 3M glycerol, Cryobiology, 21, 704 (1984).
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Living adult human and animal brain tissue
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243-246 (1981).
16. Pascoe, J.E., The survival of the rat's superior cervical ganglion
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17. Hardy, J.A., P.R. Dodd, A.E. Oakley, R.H. Perry, J.A. Edwardson, and
A.M. Kidd, Metabolically active synaptosomes can be prepared from
frozen rat and human brain, J Neurochem, 40, 608-614 (1983).
18. Walder, H.A.D., The effect of freezing and rewarming on feline brain
tissue: an electron microscope study In: The Frozen Cell, G.E.W.
Wolstenholme and M. O'Connor, Eds., J. & A. Churchill, London, 1970.
pp. 251-266.
Living fetal human and animal brain tissue
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Cryopreservation of human fetal organs, Anat Embryol, 174, 105-113 (1986).
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and its successful transplantation in the rat brain, Anat Rec, 196, 81A
(1980).
21. Houle, J.D., and G.D. Das, Freezing of embryonic neural tissue and its
transplantation in the rat brain, Brain Res, 192, 570-574 (1980).
22. Houle, J.D., and G.D. Das, Freezing and transplantation of brain tissue
in rats, Experientia, 36, 1114-1115 (1980).
23. Das, G.D., J.D. Houle, J. Brasko, and K.G. Das, Freezing of neural
tissues and their transplantation in the brain of rats: technical
details and histological observations, J Neurosci Methods, 8, 1-15 (1983).
24. Jensen, S., T. Sorensen, A.G. Moller, and J. Zimmer, Intraocular grafts
of fresh and freeze-stored rat hippocampal tissue: a comparison of
survivability and histological and connective organization, J Comp Neurol,
227, 558-568 (1984).
25. Jensen, S., T. Sorensen, and J. Zimmer, Cryopreservation of fetal rat
brain tissue later used for intracerebral transplantation, Cryobiology,
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Living human and animal isolated brain cells
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cell cryopreservation, (abstract from unidentified literature source)
27. Kim, S.U., G. Moretto, B. Ruff, and D.H. Shin, Culture and
cryopreservation of adult human oligodendrocytes and astrocytes,
Acta Neuropathol (Berlin), 64, 172-175 (1984).
28. Kawamoto, J.C., and J.N. Barrett, Cryopreservation of primary neurons
for tissue culture, Brain Res, 384, 84-93 (1986).
29. Scott, B., and L. Lew, Neurons in cell culture survive freezing, Exp
Cell Res, 162, 566-573 (1986).
Post-mortem human and animal brains
30. Itabashi, H.H., W.W. Tourtellotte, B. Baral, and M. Dang, A freezing
method for the preservation of nervous tissue for concomitant molecular
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31. Tourtellotte, W.W., R.C. Cohenour, J. Raj, A. Morgan, R. Warwick, J.
Sweeder, et al, The NINCDS/NIMH human neurospecimen bank,
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32. Bird, E.D., Brain tissue banks, Trends in Neurosci, 1(5), I-II (1978).
33. Tourtellotte, W.W., H.H. Itabashi, I. Rosario, and K. Berman, National
neurological research bank: A collection of cryopreserved human
neurological specimens for neuroscientists, Ann Neurol, 14, 154 (1983).
34. Haberland, N., L. Hetey, H.A. Hackensellner, and G. Matthes,
Characterization of the synaptosomal dopamine uptake from rat and
human brain tissue after low temperature preservation, Cryo-Letters,
6, 319-328 (1985).
35. Stahl, W.L., and P.D. Swanson, Effects of freezing and storage on
subcellular fractionation of guinea pig and human brain, Neurobiology,
5, 393-400 (1975).
36. Schwarcz, R., Effects of tissue storage and freezing on brain glutamate
uptake, Life Sci, 28, 1147-1154 (1981).
37. Brammer, M.J., and P. Ray, Preservation of oligodendroglial cytoplasm
in cryopreservative-pretreated frozen white matter, J Neurochem, 38,
1493-1497 (1982).
38. Iqbal, K., et al., Oligodendroglia from human autopsied brain. Bulk
isolation and some chemical properties, J Neurochem, 28, 707-716 (1977).
39. Morrison, M.R., and W.S.T. Griffin, The isolation and in vitro
translation of undegraded messenger RNAs from human post-mortem brain,
Anal. Biochem, 113, 318-324 (1981).
40. Tower, D.B., S.S Goldman, and O.M. Young, Oxygen consumption by frozen
and thawed cerebrocortical slices from warm-adapted or hibernating
hamsters: the protective effects of hibernation, J Neurochem, 27,
285-287 (1976).
41. Tower, D.B., and O.M. Young, The activities of butyrylcholinesterase
and carbonic anhydrase, the rate of anaerobic glycolysis, and the
question of a constant density of glial cells in cerebral cortices of
various mammalian species from mouse to whale, J Neurochem, 20, 269-278
(1973).
42. Tower, D.B., and O.M. Young, Interspecies correlations of cerebral
cortical oxygen consumption, acetylcholinesterase 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).
Spinal cord and spinal nerves
43. Anonymous, Histological study of a temporarily cryopreserved human,
Cryonics, #52, 13-32 (Nov, 1984).
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