X-Message-Number: 1391
Date: 03 Dec 92 06:55:56 EST
From: Paul Wakfer <>
Subject: CRYONICS: Freezing Damage (Darwin) Part 3
IV. GROSS EFFECTS OF COOLING TO AND REWARMING FROM -196*C
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
organ.
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.
IV. EFFECTS OF CRYOPRESERVATION ON THE HISTOLOGY OF SELECTED TISSUES
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.
Liver
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.
Kidney
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
glomeruli.
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.
Brain
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.
V. EFFECTS OF CRYOPRESERVATION ON
THE ULTRASTRUCTURE OF SELECTED TISSUES
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.
Liver
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.
Kidney
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.
Heart
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
group.
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
tears.
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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