X-Message-Number: 1391
Date: 03 Dec 92 06:55:56 EST
From: Paul Wakfer <>
Subject: CRYONICS: Freezing Damage (Darwin) Part 3


     The  most striking change noted upon thawing of the  animals  was 
the presence of multiple fractures in all organ systems.  As had  been 
previously noted in human cryonic suspension patients, fracturing  was 
most pronounced in delicate, high flow organs which are poorly  fiber-
reinforced.   An exception to this was the large arteries such as  the 
aorta, which were heavily fractured.

     Fractures  were most serious in the brain, spleen, pancreas,  and 
kidney.   In these organs fractures would often completely  divide  or 
sever  the  organ  into one or more discrete  pieces.   Tougher,  more 
fiber-reinforced tissues such as myocardium, skeletal muscle, and skin 
were less affected by fracturing; there were fewer fractures and  they 
were smaller and less frequently penetrated the full thickness of  the 

     In both FGP and FIGP animals the brain was particularly  affected 
by  fracturing  and  it  was not uncommon to  find  fractures  in  the 
cerebral hemispheres penetrating through to the ventricles as seen  in 
Figure  15, or to find most of both cerebral hemispheres and the  mid-
brain  completely  severed from the cerebellum by a  fracture  (Figure 
16).  Similarly, the cerebellum was uniformly severed from the medulla 
at the foramen magnum as were the olfactory lobes, which were  usually 
retained  within  the olfactory fossa with severing  fractures  having 
occurred at about the level of the transverse ridge.  The spinal  cord 
was  invariably transversely fractured at intervals of 5 mm to  15  mm 
over  its  entire  length.  Bisecting CNS fractures  were  most  often 
observed  to  occur  transversely  rather  than  longitudinally.    In 
general,  roughly  cylindrical structures such as  arteries,  cerebral 
hemispheres, spinal cord, lungs, and so on are completely severed only 
by transverse fractures.  Longitudinal fractures tend to be shorter in 
length and shallower in depth, although there were numerous exceptions 
to this generalization.

     In  ischemic animals the kidney was usually grossly fractured  in 
one  or  two locations (Figure 17).  By  contrast,  the  well-perfused 
kidneys of the nonischemic FGP group exhibited multiple fractures,  as 
can  be  seen in Figure 18.  A similar pattern was observed  in  other 
organ  systems  as well; the nonischemic animals  experienced  greater 
fracturing injury than the ischemic animals, presumably as a result of 
the   higher   terminal  glycerol  concentrations  achieved   in   the 
nonischemic group.

     Cannulae  and attached stopcocks where they were externalized  on 
the  animals  were  also frequently  fractured.   In  particular,  the 
polyethylene pressure-monitoring catheters were usually fractured into 
many  small  pieces.   The  extensive  fracture  damage  occurring  in 
cannulae,  stopcocks, and catheters was almost certainly a  result  of 
handling  the animals after cooling to deep subzero  temperatures,  as 
this  kind of fracturing was not observed in these items upon  cooling 
to  liquid nitrogen temperature (even at moderate rates).  It is  also 
possible that repeated transfer of the animals after cooling to liquid 
nitrogen  temperature may have contributed to fracturing  of  tissues, 
although the occurrence of fractures in organs and bulk quantities  of 
water-cryoprotectant  solutions  in the absence of  handling  is  well 
documented in the literature (12, 13).

     There were subtle post-thaw alterations in the appearance of  the 
tissues of all three groups of animals.  There was little if any fluid 
present  in the vasculature and yet the tissues exhibited  oozing  and 
"drip"  (similar to that observed in the muscle of frozen-thawed  meat 
and  seafood)  when cut.  This was most pronounced  in  the  straight-
frozen  animal.  The tissues (especially in the ischemic  group)  also 
had  a somewhat pulpy texture on handling as contrasted with  that  of 
unfrozen,  glycerolized  tissues  (i.e.,  those  handled  during  pre-
freezing  sampling for water content).  This was most in  evidence  by 
the accumulation during the course of dissection of small particles of 
what appeared to be tissue substance with a starchy appearance and  an 
oily  texture on gloves and instruments .  This phenomenon  was  never 
observed  when handling fresh tissue or glycerolized tissue  prior  to 
freezing and thawing.

     There were marked differences in the color of the tissues between 
the three groups of animals as well.  This was most pronounced in  the 
straight-frozen  control  where the color of almost  every  organ  and 
tissue examined had undergone change.  Typically the color of  tissues 
in  the  straight-frozen animal was darker, and white  or  translucent 
tissues such as the brain or mesentery were discolored with hemoglobin 
released from lysed red cells.

     The FGP and FIGP groups did not experience the profound post-thaw 
changes  in tissue color experienced by the straight-frozen  controls, 
although  the  livers and kidneys of the FIGP  animals  appeared  very 
dark, even when contrasted with their pre-perfusion color as  observed 
in those animals laparotomized for tissue water evaluation.


     Histology was evaluated in two animals each from the FIG and FIGP 
groups, and in one control animal.  Only brain histology was evaluated 
in the straight-frozen control animal.


     The  histological appearance of the liver in all three groups  of 
animals  was  one  of  profound injury.  Even in  the  FGP  group  the 
cellular integrity of the liver appeared grossly disrupted.  In  liver 
tissue  prepared using Yajima stain the sinusoids and spaces of  Disse 
were  filled  with  flocculent debris and it was  often  difficult  or 
impossible  to  discern cell membranes.   The  collagenous  supporting 
structures of the bile  canaliculi were in evidence and the nuclei  of 
the hepatocytes appeared to have survived with few alterations evident 
at the light level, although occasional pyknotic nuclei were noted  in 
the FIGP group.  Indeed, the nuclei often appeared to be floating in a 
sea of amorphous material.  Not surprisingly, the density of  staining 
of  the cytoplasmic material was noticeably reduced over that  of  the 
fixative-perfused control.  Few intact capillaries were noted.

     FGP  liver  tissue prepared with PAS stain  exhibited  a  similar 
degree  of disruption.  However, quite remarkably, the borders of  the 
hepatocytes  were defined by a clear margin between  glycogen  granule 
containing  cytoplasm  and non-glycogen containing membrane  or  other 
material  (membrane debris?) which failed to stain with  Yajima  stain 
due to gross disruption or altered chemistry.


     PAS  stain  was used to prepare the control, FGP and  FIGP  renal 
tissue for light microscopy.  The histological appearance of FGP renal 
tissue was surprisingly good.  The glomeruli and and tubules  appeared 
grossly  intact  and stain uptake was normal.  However,  a  number  of 
alterations  from  the appearance of the control were  apparent.   The 
capillary tuft of the glomeruli appeared swollen and the normal  space 
between the capillary tuft and Bowman's capsule was absent.  There was 
also marked interstitial edema, and marked cellular edema as evidenced 
by the obliteration of the tubule lumen by cellular edema.  

     By contrast, the renal cortex of the FIGP animals, when  compared 
to  either  the control or the FGP group, showed a  profound  loss  of 
detail, absent intercellular space, and altered staining.  The  tissue 
appeared frankly necrotic, with numerous pyknotic nuclei and  numerous 
large  vacuoles  which peppered the cells.   One  striking  difference 
between FGP and FIGP renal cortex was that the capillaries, which were 
largely  obliterated in the FGP animals, were consistently  spared  in 
the FIGP animals.  Indeed, the only extracellular space in evidence in 
this  preparation was the narrowed lumen of the  capillaries,  grossly 
reduced in size apparently as a consequence of cellular edema.

     Both ischemic and nonischemic sections showed occasional evidence 
of  fracturing, with fractures crossing and severing tubule cells  and 

Cardiac Muscle

     Yajima  stain  was  used to prepare the  Control,  FGP  and  FIGP 
cardiac  tissue for light microscopy.  The histological appearance  of 
FGP  cardiac muscle was grossly normal with one exception;  there  was 
increased  interstitial  space, probably  indicative  of  interstitial 
edema.    The  banding  pattern  was  normal  and  the   nuclei   were 
unremarkable.   Similarly,  FIGP cardiac  tissue  appeared  relatively 
normal  histologically.   The principal alterations from  control  and 
from  the  FGP  group  were  the  noticeable  presence  of   increased 
interstitial  space  and a more "ragged" or rough  appearance  of  the 
myofibrils where they are silhouetted against interstitial space.

     Most surprising was the general absence of thaw-rigor in the  FGP 
group and only the occasional presence of rigor in the FIGP group.  No 
microscopic evidence of fracturing was noted in either the FGP or  the 
FIGP groups.


     Bodian stain was used to prepare the control, FGP, and FIGP brain 
tissue  samples  for light microscopy.  Three  striking  changes  were 
apparent  in FGP cerebral cortex histology: 1) marked  dehydration  of 
both  cells  and  cell nuclei, 2) the presence of  tears  or  cuts  at 
intervals  of  10 to 30 microns throughout the tissue  on  a  variable 
basis (some areas were spared while others were heavily lesioned), and 
3) the increased presence (over control) of irregular, empty spaces in 
the neuropil as well as the occasional presence of large pericapillary 
spaces.   These  changes  were  fairly  uniform  throughout  both  the 
molecular layer and the second layer of the cerebral cortex.   Changes 
in  the white matter paralleled those in the cortex with  the  notable 
exception that dehydration appeared to be more pronounced.

     Other  than  the  above  changes,  both  gray  and  white  matter 
histology  appeared  remarkably intact, and  only  careful  inspection 
could  distinguish  it  from control.  The  neuropil  appeared  normal 
(aside  from the aforementioned holes and tears) and many  long  axons 
could be observed traversing the field.  Cell membranes appeared crisp 
and  apart from appearing dehydrated, neuronal  architecture  appeared 
comparable  to  control.  Similarly, staining was comparable  to  that 
observed   in  control  cerebral  cortex.   Cell-to-cell   connections 
appeared largely undisrupted.

     The  histological appearance of FIGP brain differed from that  of 
FGP animals in that ischemic changes such as the presence of  pyknotic 
and  fractured nuclei were much in evidence and cavities and tears  in 
the neuropil appeared somewhat more frequently.

     Both  FGP  and  FIGP  brains  presented  occasional  evidence  of 
microscopic fractures.


     Ultrastructure was evaluated in two animals from the FIG and FIGP 
groups,  and  in one control animal.  Only  brain  ultrastructure  was 
evaluated in one straight-frozen control animal.


     Hepatic  ultrastructure  was grossly disrupted, with  the  tissue 
presenting  more as a homogenate than as an organized  tissue.   While 
organelle membranes, particularly rough endoplasmic reticulum, nuclear 
membranes,  and  mitochondrial membranes were frequently  intact,  the 
presence  of intact cell membranes was the exception rather  than  the 
rule.  The  sinusoids, bile canaliculi, and capillaries,  where  these 
structures  were identifiable, were largely filled with  debris.   The 
character of this debris ranged from the relatively amorphous granular 
and  flocculent debris observed in the other organ systems of FGP  and 
FIGP  animals  to  relatively organized  fragments  of  cytosol,  free 
organelles  (naked nuclei and mitochondria being the  most  frequently 
observed),  as well as somewhat structured but unidentifiable  debris.  
In  areas  where discernible hepatocyte membranes  were  visible,  the 
intracellular  contents  appeared washed out and  depleted  of  ground 
substance.  Similarly, the spaces of Disse were hard to  identify  and 
where identifiable were both collapsed and filled with debris.

     In the FIGP animals, intact red cells were frequently in evidence 
as  well  as sinusoids full of what appeared to be  leukocytes  and/or 
leukocyte debris, indicating failed blood washout and probable  failed 
cryoprotective  perfusion as well.  Mitochondria, where  identifiable, 
rarely had much ground substance and presented only faint evidence  of 
cristae.   Nuclei in the livers of both FGP and FIGP animals  appeared 
reasonably  well  preserved  and  the  double  nuclear  membrane   was 
frequently (although not universally) intact.


     The ultrastructure of FGP renal tissue was intact to a surprising 
degree.   The  desmosomes, endoplasmic  reticulum,  and  intracellular 
organelles,  with the exception of the mitochondria  appeared  normal.  
Most  mitochondria demonstrated marked enlargement,  decreased  matrix 
density,  disruption of cristae and a few amorphous matrix  densities.  
The nuclei were largely free of margination and clumping of chromatin.  
The glomeruli appeared intact as did tubule and mesangial cells.   The 
architecture of the brush border and urinary space compared  favorably 
to  control  with little debris in evidence.  While there  was  little 
debris in the intercellular spaces, there was extensive debris in  the 
capillary spaces, where it was common to find the capillary completely 
obliterated  and  free  red cells present.   Intact  capillaries  were 
occasionally  observed  in FGP renal tissue.  However,  this  was  the 
exception rather than the rule.

     By  contrast,  the  capillaries in the  FIGP  animals  were  more 
consistently  intact.   The  narrow lumens of  these  relatively  well 
preserved  capillaries  constituted virtually the  only  extracellular 
space  visible.   Also remarkable, given the poor  appearance  of  the 
tissue  at the light level, was the presence of a considerable  amount 
of renal ultrastructure.  The microvilli, glomeruli, and the mesangial 
cells  were all present and reasonably intact.  However,  the  urinary 
space  and  capillary  lumens  were  filled  with  flocculent  debris.  
Ultrastructural  changes in cell organelles were more pronounced  with 
the  nuclei  exhibiting  clumping of  the  chromatin,  the  consistent 
presence of megamitochondria exhibiting loss of integrity of membranes 
and many amorphous matrix densities.


     Cardiac   ultrastructure  in  both  FGP  and  FIGP  animals   was 
reasonably  well  preserved.  The sarcoplasmic  reticulum,  transverse 
tubules,  intercalated  discs,  and banding  of  the  myofibrils  were 
comparable to that of control.  A notable abnormality in both the  FGP 
and FIGP myocardium was the presence of severe interstitial edema,  as 
evidenced by greatly increased interstitial spaces littered with  both 
granular  and  flocculent  debris.   There did  not  appear  to  be  a 
significant  difference  in the quantity, character,  or  location  of 
debris  between  the FGP and FIGP animals.  No significant  amount  of 
fibrolysis was noted in either the FGP or the FIGP groups.

     Also notable was the presence of megamitochondria, with decreased 
matrix  density  and disruption of cristae.   Occasional  mitochondria 
with  normal density were observed in FGP animals.  However, this  was 
virtually never the case in the FIGP animals.  Myocardial  capillaries 
were  grossly  intact,  with  only the  infrequent  presence  of  what 
appeared  to be very small areas of focal injury involving  separation 
of the endothelial cell membrane from basement membrane.  Small  areas 
of  rigor evidenced by the presence of severe contraction  bands  were 
sometimes  present  in the FIGP group but were not noted  in  the  FGP 

FGP Brain

     At  the  outset it should be noted that evaluation  of  the  fine 
ultrastructure of FGP cerebral cortex is complicated by the degree  of 
apparent  dehydration  of intracellular  structures  present.   Ground 
substance  was  markedly increased over control and most,  though  not 
all,  axons  appeared shrunken, electron-dense, and  surrounded  by  a 
periaxonal space.  Intraorganelle structures were frequently difficult 
to  identify  as  a  result  of  dehydration,  with  many   structures 
presenting an electron dense but amorphous interior.  

     These  effects notwithstanding, the overall architecture  of  the 
tissue  could be discerned. Intact neuronal, glial, and vascular  cell 
membranes  were  uniformly present.  Such interstitial  space  as  was 
present  consisted  of  periaxonal shrinkage  spaces  and  irregularly 
shaped  cavities,  apparently artifacts of ice  formation,  of  widely 
varying  size, often containing small quantities of organized  debris, 
which  peppered  the  tissues at intervals of 5 to  10  microns.   The 
largest  of these cavities appeared to be 20 to 30 microns across  and 
presented the appearance of tears or rips, with the two opposing sides 
of the gap presenting a rough match.  Smaller cavities 1-3 microns  in 
diameter  were  more frequently present than  these  relatively  large 

     The  capillaries  appeared  reasonably intact,  with  the  lumens 
containing  no or modest amounts of relatively well organized  debris.  
However,  the  capillaries  were frequently  surrounded  by  cavities.  
These  cavities varied in size from a few microns to 10 to 15  microns 
in diameter with the cavity separating the capillary from  surrounding 
brain  cells usually circumscribing from one-third to one-half of  the 
capillary perimeter.

     Axons usually appeared intact, but shrunken.  However, it  should 
be  noted  that  some spaces characteristic of  axons  and  containing 
myelin debris (but no axon) were also present in most sections of  FGP 
brain  examined.  Myelinated tracts were often difficult  to  evaluate 
due  to the degree of dehydration.  However, unraveled  and  disrupted 
myelin  was  commonplace, often surrounding  an  intact-looking  axon.  
Synapses  were  present  in numbers comparable to  that  seen  in  the 
control and were especially well preserved, presenting grossly  normal 
architecture, including clear pre- and post-synaptic densities and the 
presence of synaptic vesicles.

     The  nuclei  were  highly condensed (presumably  an  artifact  of 
dehydration)  and  sometimes contained unusual gaps  or  spaces  which 
might  have occurred as a result of dehydration from  glycerolization, 
the formation of intranuclear ice, or ultramicroscopic fractures as  a 
result  of  differential  contraction during cooling  below  Tg.   The 
mitochondria appeared dense and amorphous.

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