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.  



     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.


     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 


     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 

     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. 


     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.


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.


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


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


In Preparation

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