X-Message-Number: 1392
Date: 03 Dec 92 06:53:15 EST
From: Paul Wakfer <>
Subject: CRYONICS: Freezing Damage (Darwin) Part 4
FIGP Brain
The FIGP brains presented an "exploded" appearance at the
ultrastructural level. Virtually every structure appeared swollen and
there were large amounts of interstitial space. A uniform but not
universal alteration was massive swelling and unraveling of the
myelin. Typically there was about a 5-fold increase in the thickness
of the myelin sheath, with a corresponding decrease in electron
density. Often the individual sheets or "turns" of myelin could be
easily discerned, with separating spaces between each layer. The
presence or absence of intact axons within this disrupted myelin was
highly variable; in some regions the axons appeared well preserved,
with neurofibrils and microtubules clearly visible, while in others
apparently nothing but debris remained.
Mitochondria were uniformly swollen and presented varying degrees
of internal structure ranging from easily identifiable cristae to a
fine-grained amorphous appearance. In contrast to FGP brains there
was virtually no dehydration in evidence in the FIGP brains and
intracellular structures and small processes such as neurites, where
intact, were easily identified. The nuclei appeared more like those
present in the control and did not show the peculiar gaps or cavities
present in the FGP group.
Small cavities and large gaps peppered the tissue as in the FGP
cerebral cortex. These cavities contained considerably more debris
than those in the FGP brains and the debris were less structured and
frequently appeared flocculent and/or granular in nature. Cell
membranes were frequently disrupted and masses of free cytosol were
common. Synapses, synaptic vesicles and what appeared to be
occasional synaptic debris were noted with a frequency comparable to
that of the control.
VI. SUMMARY AND DISCUSSION
Glycerolization
Cryoprotective perfusion of non-ischemically injured animals
resulted in profound dehydration. This dehydration was particularly
pronounced (in terms of visual appearance) in the brain, eyes,
skeletal muscle, and skin. While it can be argued that removal of
interstitial and intracellular water may be useful in minimizing
mechanical injury during subsequent freezing since less water means
less ice, it can also be argued that glycerol is failing to adequately
penetrate cells and thus is providing less than optimum
cryoprotection. Certainly the profound dehydration documented in
these animals (and similarly noted in human patients) is indicative of
a failure of cellular equilibration of glycerol, particularly in the
brain and skeletal muscle, and in and of itself is probably a
significant source of osmotic injury.
In an unpublished pilot study we tried to determine if better
glycerol equilibration could be facilitated by carrying out
cryoprotective perfusion at 18*C. Both the gross effects of
dehydration and the measured water losses from tissues (including in
the brain, which was determined to be 28% in the single experiment
conducted) indicated that glycerolizing at higher temperatures is not
the solution to this problem. Clinically it has been known for many
years that infusion of significant amounts of glycerol at normal body
temperatures, as in the case of inadvertent transfusion of frozen-
thawed red cells without deglycerolization, results in rapid death
from cerebral dehydration (14). Indeed, glycerol has been used as an
osmotic agent to control cerebral edema in the traumatized brain (15).
Thus, glycerol would seem to be a poor choice of cryoprotectant, at
least in terms of its cellular permeability, for the brain. Clearly,
a cryoprotective agent(s) capable of better equilibration with the
intracellular space of the brain is needed.
In the ischemic animals, the gross effects of dehydration were
less obvious or were not seen due to the occurrence of interstitial
edema. However, cellular dehydration might not have occurred in these
animals, perhaps as a result of increased cell membrane permeability
due to ischemic changes such as phospolipase (16) or free radical (17)
mediated degradation of cellular and organelle membranes. Certainly
the intracellular organelles and axons did not have the dense,
collapsed, dehydrated appearance of these structures in the
nonischemic animals.
This noticeable change in cellular glycerol permeability, the
loss of capillary integrity as evidenced by the development of serious
interstitial edema in the brain and virtually all other body organs
with the exception of the liver (which apparently failed to perfuse
significantly), the patchy nature of perfusion due to clotting, and
the failure to reach target glycerol concentration as a result of all
of these effects is indicative of the profound deleterious impact of
ischemia and of the importance of minimizing ischemic time and
inhibiting mechanisms of ischemic pathology in human suspension
patients if adequate distribution and terminal concentration of
cryoprotectant is to be achieved.
Histology
The histological preservation achieved in brain, kidney, and
heart in both ischemic (excluding ischemia-associated alterations to
nuclei) and non-ischemic animals was surprisingly good considering the
magnitude of the insult. In the case of the FGP brains structural
preservation appeared excellent and almost indistinguishable from
control, with the exceptions of the presence of an increased number of
empty cavities and more light-lucent areas, and the presence of
obvious tears at 10 to 20 micron intervals in the neuropil.
Similarly, the histological preservation of the renal cortex was
surprisingly good in both the FGP and the FIGP animals. The glomeruli
were generally intact and this is surprising considering the body of
data from renal cryopreservation studies documenting destruction of
the glomerulus due to ice formation (18, 19). Perhaps the reason this
did not occur in our animals was the very slow rate at which cooling
was carried out (4*C/hour) as contrasted with the comparatively rapid
rate at which kidneys are cooled during cryopreservation experiments.
Such comparatively slow cooling rates may have allowed time for water
to migrate out of the glomerulus to other sites during freezing (20),
and/or the distortive and disruptive effects of ice formation may have
been minimized by the plasticity of these structures at the higher
temperatures at which most ice formation and growth occurs.
Histological preservation in cardiac tissue in both FGP and FIGP
animals was also remarkably good and it was often difficult to
distinguish ischemic from non-ischemic tissue without careful
observation.
Ultrastructure
The ultrastructural preservation of the brain was unexpectedly
poor in all three groups of animals: ischemic, non-ischemic and
straight-frozen. Not unexpectedly, the straight-frozen animal
presented the worst ultrastructural appearance. The ischemic animals
also suffered extensive ultrastructural disruption. This was somewhat
unexpected given the relatively good appearance of brain tissue at the
light level; in particular it appeared that membranes were crisp and
well preserved that cellular ground substance was of reasonably normal
density, and that the overall ground substance density of the
neuropil, as well as the preservation of long individual axon fibers
and cell-to-cell connections, were largely intact. Unfortunately, the
degree of ultrastructural injury observed was in sharp contrast to the
apparently good histological preservation. The profound loss of
ground substance, gross and widespread loss of membrane integrity,
presence of extensive debris, and the widespread destruction of the
myelin all underscore, yet again, the critical importance of
protection of suspension patients from cerebral ischemia.
While the degree of ultrastructural disruption was not as
profound in the brains of the FGP animals, it was far from acceptable.
The presence of frequent ice holes, tears in the neuropil, and the
cellular dehydration and fracturing observed are all indicative of
unacceptably poor preservation and point to the urgent need for
additional research to ameliorate or eliminate these problems.
Given the severity of the ultrastructural disruption observed in
the brains of all three groups of animals, it is certainly open to
question whether or not sufficient structure is being preserved to
allow for resuscitation of cryonic suspension patients treated with
similar techniques (and presumably injured comparably) with their
memories and personalities intact.
Freezing Versus Thawing
The especially poor perfusion of the liver in the ischemic
animals was unexpected. Additionally, the poor ultrastructural
preservation observed in the nonischemic animals is puzzling,
especially in light of the apparent good perfusion and amounts of
water loss (which were comparable to those experienced by the heart
and kidney during glycerolization).
The relatively good ultrastructure of the kidney and heart in the
FGP and to a lesser extent in the FIGP group stand in sharp contrast
to widespread disruption seen in the brain. The reason(s) for this
are not clear. However, a possible explanation might be the failure
of glycerol to penetrate brain cells and provide adequate
cryoprotection. It should be noted that the amount of water lost from
the brain during glycerolization, while not directly measured,
appeared by gross examination to be roughly comparable to that
observed in the heart and kidney, both of which were, by comparison,
much better preserved.
Some caveats regarding these results should be considered. First
of all, examination of the tissues was conducted following thawing.
This introduces the possibility of significant "stirring" of damaged
structure not only during thawing, but also during sectioning and
fixation, since re-perfusion with fixative was not possible owing to
disruption of the vasculature by fractures. This is potentially a
particularly troubling "artifact" because a major concern is the
presence of debris many microns from the likely source of origin (as
observed in the liver and brain). When and how this debris was
translocated from its point of origin, as well as its character (i.e.,
how unique are the fragments of debris; can their precise point of
origin and orientation be determined?) is of critical importance in
determining whether or not repair can be undertaken. If the extensive
ultrastructural and molecular-level stirring observed in these animals
occurred as a result of diffusion/stirring which took place during, or
even after thawing and/or during sectioning and fixation, then the
situation is considerably more hopeful than if the damage occurred
during the freezing process.
It will not be easy to determine how much of the observed
disruption is a result of freezing, and how much is a result of
thawing and/or post-thaw diffusion-driven processes. Depending upon
the degree to which the microvasculature is intact following freezing
and thawing it should be possible to eliminate pre-fixation sectioning
and handling of the tissue as a source of artifacts by the expedient
of not cooling to below the glass transition temperature of the the
water-cryoprotectant mixture, thus effectively avoiding fracturing and
allowing for fixative reperfusion upon thawing. However, evaluating
the degree to which freezing, as opposed to freezing followed by
thawing, results in the disruption of, and perhaps more importantly,
the translocation of cell structures would not be resolved by this
means.
Finally, it is especially important to point out that this was a
pilot study. During the evaluation of the light and electron
microscopy it became apparent that additional control groups were
needed to resolve many important questions left unanswered by this
work. In particular, post glycerolization/pre-freezing ultrastructure
and histology should have been evaluated to separate the effects of
glycerolization from the effects of subsequent cryopreservation.
Similarly, a group of post-thaw cryopreserved tissues should have been
deglycerolized prior to fixation in order to allow for evaluation
without the confounding effects of glycerol-induced dehydration.
Freeze substitution studies at both the light and EM levels would
also be useful in helping to relate the lesions observed (gaps, tears,
cavities and so on) to mechanical injury resulting from the presence
of ice.
Technical Issues
Some of our most serious caveats are technical in nature. Brain
slicing with a Stadie-Riggs microtome could have obliterated structure
in and of itself, particularly if the frozen-thawed brain is
structurally weaker than a control brain (as is to be expected) .
However, this criticism does not apply to the the electron microscopy
since tissue examined by EM was from the center of the slice, away
from the cut surface.
Summary
Evaluation of a cryopreservation protocol which is broadly
similar to that being used in human cryonic suspensions today
discloses poor ultrastructural preservation of the brain, the target
organ of the preservation process. The comparatively good
ultrastructural preservation of the heart and kidney indicate that
better results are possible and strongly suggest that the preservation
protocol currently in use is not optimal for the brain and results in
unacceptable levels of ultrastructural disruption. There is an urgent
need for additional research to address this problem.
The impact of prolonged ischemia on tissue histology,
ultrastructure, and perfusion was profound and underscores the need to
protect suspension patients from ischemia.
TABLE I.
Composition Of Modified Karnovsky's Solution
Component g/l
Paraformaldehyde 40
Glutaraldehyde 20
Sodium Chloride 0.2
Sodium Phosphate 1.42
Calcium Chloride 2.0 mM
pH adjusted to 7.4 with sodium hydroxide.
_________________________________________
TABLE II
Perfusate Composition
Component mM
Potassium Chloride 2.8
Dibasic Potassium Phosphate 5.9
Sodium Bicarbonate 10.0
Sodium Glycerophosphate 27.0
Magnesium Chloride 4.3
Dextrose 11.0
Mannitol 118.0
Hydroxyethyl Starch 50 g/l
TABLE III.
Total Water-Loss Associated With
Glycerolization Of The Cat
____________________________________________________
Animal Pre-Perfusion Post-Perfusion Kg./ % Lost As
# Weight Kg. Weight Water Dehydration
FGP-1 4.1 3.6 2.46 18
FGP-2 3.9 3.1 2.34 34
FGP-3 4.5 3.9 2.70 22
FGP-4 6.0 5.0 3.60 28
FIGP-1 3.4 3.0 2.04 18
FIGP-2 3.4 3.2 2.04 9
FIGP-3 4.32 3.57 2.59 29
References
In Preparation
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