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.
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