X-Message-Number: 4474
From: Mike Darwin <>
Date: 31 May 95 00:03:14 EDT
Subject: SCI.CRYONICS BPI Tech Brief 16: Canine Brain Cryopreservation (2/2)
HUMAN CRYOPRESERVATION
PROTOCOL ON THE ULTRASTRUCTURE OF THE CANINE BRAIN
by Michael Darwin, Sandra Russell, Larry Wood, and Candy Wood
(continued from previous message)
III EFFECTS OF CLOSED CHEST CARDIOPULMONARY SUPPORT
At the start of Thumper support MAP was between 25mmHg and 30
mmHg and increased to between 35mmHg to 45mmHg with the
administration of initial bolus of high dose (0.2 mg/kg)
epinephrine. End-tidal CO2 at this time was 1-2% and cardiac
output was 0.5 to 0.7 liters per minute (LPM). After 30
minutes of CCCS, MAP had declined to 30mmHg to 35mmHg with a
corresponding decrease in responsiveness to each bolus of
epinephrine. End-tidal CO2 declined to 1% to 0.5% and CO
declined to 0.3 to 0.5 LPM. Esophageal temperature at the
end of CCCS and immediately prior to the start of bypass had
declined to 21 to 28 C depending on the mass of the animal
and the amount of subcutaneous fat covering the animal
(subcutaneous fat served as a good insulator and greatly
slowed cooling, somewhat independent of total body mass).
IV. EFFECTS OF GLYCEROLIZATION
Blood washout was rapid and complete in all the animals. MAP
rose sharply as glycerol concentration increased, probably as
a result of the increasing viscosity of the perfusate as is
shown in Figure 7.
Within approximately 5 minutes of the beginning of the
cryoprotective ramp, bilateral ocular flaccidity was noted.
As the perfusion proceeded, ocular flaccidity progressed
until the eyes had lost approximately 30% to 50% of their
volume. Gross examination of the eyes revealed that initial
water loss was primarily from the aqueous humor, with more
significant losses from the posterior chamber of the eyes
apparently not occurring until later in the course of
perfusion. Within 15 minutes of the start of glycerolization
the corneal surface became dimpled and irregular and the eyes
had developed a concave appearance.
Dehydration was also apparent in the skin and skeletal
muscles and was evidenced by a marked decrease in limb girth,
profound muscular rigidity, cutaneous wrinkling, a "waxy-
leathery" texture and a mummified appearance of both cut
skin and skeletal muscle. Tissue water evaluations conducted
on ileum, kidney, liver, lung, and skeletal muscle confirmed
the gross observations. Preliminary observations suggest
that water loss was in the range of 30% to 40% in most
tissues as was previously observed both in previous animal
studies (5,6) and in humans undergoing cryopreservation using
a similar protocol. (13)
Examination of the cerebral hemispheres upon cranitomy
revealed an estimated 30% to 50% reduction in cerebral
volume, presumably as a result of osmotic dehydration
secondary to glycerolization. The cortices also had the
"waxy" amber appearance previously observed as characteristic
of glycerolized brains.
The gross appearance of the kidneys, spleen, mesenteric and
subcutaneous fat, pancreas, and reproductive organs (where
present) were unremarkable. The ileum and mesentery appeared
somewhat dehydrated, but did not exhibit the dense
mummified/waxy appearance that was characteristic of muscle,
skin, and brain.
Oxygen consumption (determined by measuring the
arterial/venous difference) throughout perfusion was fairly
constant to about 3M glycerol and then dropped off sharply as
6M glycerol concentration was approached (the high viscosity
of the perfusate above 6M made measurement by the Nova Stat 5
Profile blood gas-electrolyte system used in these
experiments impossible. Oxygen consumption versus glycerol
concentration is shown in Figure 8. Arterial and venous pH,
PO2, PCO2, and electrolytes are showon in Figures 9, 10, 11,
and 12 respectively.
IV. GROSS EFFECTS OF COOLING TO AND REWARMING FROM -90 C
The gross appearance of the animals' skin, thoracic and
abdominal viscera was surprisingly good (Figure 13). In
contrast to subtle post-thaw alterations in the appearance of
the tissues of cryopreserved animals in our previous
studies, the tissue colors were "normal"; i.e., normal for
organs and tissues subjected to TBW with MHP-2 ( a survivable
procedure). Particularly absent was the previously observed (14, 15)
altered texture of the tissues following thawing, with no pulpy
material coating gloves or instruments on sectioning. Also, in
contrast to prior post-cryopreservation evaluation of both
humans (14) and animals, the vasculature contained perfusate
in noticeable amounts after thawing and the "filling time"
required to achieve venous return was far shorter.
Peerhaps most striking was the excellent reperfusion of
virtually every organ system in the animals (Figures 13, 14,
15) with the exception of the spleen (Figure 16), which
failed to perfuse almost completely. Distribution of carbon
was uniform, occurred rapidly and evenly after the start of
perfusion, and venous return was excellent. In fact, MAP
dropped steadily during the first 5-10 minutes of reperfusion
from 140 mmHg to 80mmHg to 90mm Hg, before beginning to rise,
presumably as fixation took place rendering the capillaries
both rigid and freely permeable to colloid. Fixative flow
rates were in the range of 800cc/min to 1.2 LPM.
In two of the animals an area of obvious failed perfusion
occurred (Figure 17) in the dependent part of the stomach as
evidenced by the normal whitish pink appearance of an island
of tissue as contrasted with the uniform black of the
reperfused areas. Upon opening the stomach it was discovered
that stomach fluid/contents were partially frozen over the
area of failed reperfusion.
The logical explanation for this is that dilution of cryoprotectant
concentration in the stomach wall underlaying the stomach contents,
by diffusion of water from the stomach contents during the long
time-course of cooling reduced the tissue glycerol concentration
to a low enough level to compromise vascular integrity.
Presumably such dilution would have resulted in more ice
formation in the affected tissue and thus greater cryoinjury
with subsequent compromise of the capillary bed.
The chamber of the left ventricle which is sequestered behind
the aortic valve was uniformly found to contain large ice
crystals in a slushy mass (Figure 18) with associated failed
perfusion of the endocardium (again, presumably as a result
of dilution of cryoprotectant to below the threshold required
to provide capillary protection). This left ventricular ice
was observed to have a strong pink cast and many red cell
ghosts were observed when the ice was melted and examined
under the light microscope.
Perhaps most importantly, there was no evidence of cracking
or fracturing, even though these animals were cooled to near
Tg for glycerol water solutions and rewarmed by transfer from
-90 C to a 0 C liquid bath creating a large surface to core
thermal differential. In order to explore the fragility and
ductility of animals loaded with 7.4M glycerol and cooled to
-90 one animal was loaded with 30 kilos of dry ice placed
accross the thorax and abdomen with the animal suspended
(head and hindquarters) on two blocks of styrofoam (without
supports between). This static loading was maintained for 48
hours at -90 C with no evidence of sagging, flexion or
cracking at either the gross, histological, or
ultrastructural levels.
Particularly striking was uniform fixative perfusion of the
brain. (Figures 19, 20, 21) An advantage of carbon particle
marker over dye is that it is possible to demonstrate not
only filling of large vessels, but of perfusion of the
capillaries as well, as evidenced by uniform darkening of the
tissue to black or charcoal gray. A drawback of dyes is that
they rapidly diffuse out of vessels into areas of failed
perfusion. Solid particles of carbon (1-2 micons in
diameter) cannot do this and thus remain where they are
deposited during perfusion (14).
IV. EFFECTS OF CRYOPRESERVATION ON BRAIN ULTRASTRUCTURE
In sharp contrast to all of the previously cited studies,
the high degree of ultrastructural preservation observed in
this series of animals is unprecedented. In order to better
characterize both the degree of preservation and the degree
of injury, the discussion of these two facets of the results
will be handled in seperate sections, beginning with an
overview of the injury/alterations in brain tissue
ultrastructure which were observed.
Injury and Alterations of Ultrastructure
There are basically four classes of lesions or alterations in
appearance of ultrastructure observed in these animals: The
first are changes seen in both glycerolized-fixed (but not
frozen) animals and those observed in animals which were
subjected to glycerolization, freezing, thawing and fixation.
In both groups of animals there are characteristic changes in
the density of the cytoplasm and ground substance that we
associate with dehydration; there are packs of "stacked"
ribosomes occupying large fractions of the cytoplasm (Figure
22), small mitochondria with dense cristae (Figure 23), and
shrunken nucleoli. (Figure 24) The density of the ground
substance appears enhanced in both groups, and some non-
neuronal cells (possibly astrocytes) appear to have lost
plasma membrane integrity and appear as naked nuclei
surrounded by vesicular debris (Figure 24).
There are also alterations in nuclear density in both groups
suggestive of either loss or redistribution of nuclear
material. The nuclear membranes appear crisp and intact in
both groups, so it is difficult to draw conclusions from
this. In both frozen and nonfrozen glycerolized gray and
white matter there is a modest increase in the inter-cellular
space (Figure 25, 26) as compared to the unglycerolized
control perfused with a beating heart (Figure 27). These
increases in inter-cellular space are probably also as a
result of dehydration secondary to glycerolization.
Finally, at least five other changes both groups have in
common when compared to the beating-heart fixed control are partial
unraveling of the myelin,(Figure 28, 29) shrinkage of the axoplasm
within the myelin, dehydration of the mitochondria and nucleoli,
the presence of occassional debris strewn "tears" in the tissue
(Figure 30, 31), and increased difficulty in discerning plasma
membranes. These tears are very uncommon in the glycerolized
non-frozen controls and more common in the frozen-thawed controls;
although they still occur infrequently in the frozen-thawed
group as well.
Further, the etiology of these tears appears different
between the two groups; in the frozen thawed groups the
fissures or tears are relatively neat edged, the spaces
contain minimal debris and the edges appear complementary,
like two halves of a torn piece of paper. Perhaps the
degree of "match" between the sides of these fissures could
be best characterized by the degree of "match" observed in
orbital photographs of continents experiencing millions of
years of continental drift; that the patterns are related is
obvious, but the match is not precise.
The fissures observed in the glycerolized non-frozen tissue
(both grey and white matter) appear less clean and more
debris strewn. The etiology of these tears remains more of
a mystery.
Lesions observed exclusively or more extensively in the
frozen-thawed brains are as follows:
a) Areas at high magnification (40,000 x) where the myelin
appears to have lost its lamellar structure and presents an
amorphous or disintegrated appearance, as if a coarse
charcoal line-drawing of tightly concentric rings had been
smeared or smudged (Figure 31).
b) Loss or alteration of nucleoplasm which is evident at both
low maginification (6700x) and higher magnifications
(40,000x). This change is not uniformly observed in all
nuclei, but is very common (Figure 32).
c) Pericapillary holes or spaces (Figure 33) occasionally
strewn with vesicles or debris (Figures 34, 35) are still
present; these have been observed in prevous work with cats
and rabbits and their location and appearance correlate well
with the observed presence of ice in freeze-substituted grey
and white matter (Figures 36, 37). However, it should be
noted that these "ice holes" occur with far less frequency in
the 7.4M glycerolized brains than has been observed in brains
cryopreserved with 3M glycerol, (or lower concentrations)
(Figures 38,39).
Preservation of Ultrastructure
The most striking difference between this work and previous
brain cryopreservation studies is the overall recognizability,
inferrability and even "normality" which is present in the micrographs.
(Figures 40, 41, 42) Examination of neuropil, individual synapses and
axons at magnifications from 40,000x to 80,000x reveal excellent
preservation of fine structure (Figures 43, 44, 45). Synapse
morphology is normal in appearance and synaptic vesicles, membrane
structure and general appearance are almost indistinguishable from
unglycerolized, nonfrozen control, (Figure 46) and are virtually
indistinguishable from glycerolized-fixed non-frozen controls
(Figure 47). The relationship of the neurons to each other and of
fine processes such as dendritic spines seems very well preserved
with exception of the occasional 5-10 micron tears or fissures.
Capillary integrity is excellent with intact endothelial cell
membranes, clearly visible intra-endothelial cell
ultrastructure and intact basement membranes. Capillary
lumens are either clear or show occassional dark black
particles of carbon (Figure 48). Very rarely, small
vesicles or bits of membrane material well under 0.2 micon
in diameter can be observed in the lumen of the capillary
adjacent to an endothelial cell (Figure 49). Blood washout
appears to be complete as there are no red cells or other
formed elements of the blood present in the capillaries in
any micrograph.
Intracellular organelles while somewhat dehydrated in
appearance are readily identifiable; the endoplasmic
reticulum, mitochondria, golgi apparatus, lysosomes and the
fine structure of the axoplasm are all well preserved.
Mitochondria are rarely swollen, show (dehydrated,
compressed) cristae, and are absent of calcium crystals.
Similarly, the polyribosomes appear normal in architecture
and are nondissociated.
SUMMARY
We believe this study demonstrates, for the first time,
preservation of brain ultrastructure in sufficient detail to
provide, in a qualifed fashion, an evidentiary basis for
reconstruction of cryopreserved humans using the information-
theoretic criterion (15). Without a full understanding of how
memory, personality and identity are encoded in the human
brain it is not possible to state with certainty that these
functions are being preserved, even with the comparatively
good ultrastructural preservation reported here, and this
remains the major "qualifier" on the optimism expressed
above.
While there is much ultrastrucural and histological
preservation in evidence in the micrographs obtained in this
series, there is also evidence of considerable damage.
Particularly disturbing are the continued presence of large
(5 to 15 micron diamater in cross section) tears of unknown
"depth" in both the grey and white matter. Dehydration of
structures and the presence of what appear to be free nuclei
and lysed glial cells are also disturbing.
Another important caveat to consider in the context of the
comparatively positive results demonstrated in this study is
the relatively benign pre-mortem (i.e., pre cardiac arrest)
and post cardiac arrest insult that these animals were
exposed to. Complete noromthermic ischemia was brief and at
the margin of contemporary clinical reversibility. The post
arrest Thumper support (even with the use of high dose
epinephrine) was grossly inadequate as indicated by low CO,
EtCO2 aMAP and SaO2. This period of trickle-flow due to the
failure of CCCS to deliver adequate CO was brief compared to
the typical clinical cryonics patients' course. At a
minimum, this study confirms the poverty of circulatory
support provided by closed chest cardiopulmonary
resuscitation and it can be reliably presumed that it was
only the unrealistic brevity of this period of inadequate
circulation and ventilation which prevented even more
ischemic injury from occurring. Clearly, more effective
means of circulatory support are needed to bridge the gap
between pronouncement (cardiac arrest) and vascular access
and the beginning of extracorporeal circulatory support.
Thus, while this study demonstrates substantial preservation
of brain ultrastructure and histology, it also points out
that much remains to done before either reversible brain
cryopreservation can be achieved or there can be a high
degree of confidence that the structures responsible for
memory and personality remain sufficiently intact to allow
recovery of cryopreserved patients on a reasonable time scale
(50 to 150 years).
TABLE I.
Composition Of Trump's Storage Fixative
Component g/l
Paraformaldehyde 40 g
50% Glutaraldehyde 20 ml
Sodium Hydroxide 2 7g
Dibasic Sodium Phosphate .H2O 11.6 g
Distilled Water 980 ml
pH adjusted to 7.4 with sodium hydroxide.
_________________________________________
TABLE II
Perfusate Composition
FORMULA FOR MHP-2 BASE
PERFUSATE
Component Molar Concentration Grams/Liter Grams/20 Liters mM
Mannitol 170.0 (MW 182.20) 30.97 619.40
Adenine HCl 0.94 (MW 180.6) 0.17 3.4
D-Ribose 0.94 (MW 150.2) 0.14 2.82
Sodium Bicarbonate 10.00 (MW 84.0) 0.84 16.8
Potassium Chloride 28.3 (MW 74.56) 2.11 42.2
Calcium Chloride
10% (w/v) soln. 1 (MW 111) 0.28 ml 5.6 ml
Magnesium Chloride
20% (w/v) soln. 1 (MW 95.2) 1.0 ml 20.0 ml
Sodium HEPES 15 (MW 260.3) 3.90 78.0
Glutathione (free acid) 3 (MW 307.3) 0.92 18.44
Hydroxyethyl Starch ---- 50.00 1,000.00
Glucose 5 (MW 180.2) 1.80 36.0
Heparin ---- 1,000 IU 20,000 IU
Insulin (Humulin U-100) 40 IU 800 IU
----------------------------------------------------------------------
Adjust pH to 8.0 with potassium hydroxide. pH must be
measured after glutathione is added because the glutathione
is supplied in the free acid form and it will substantially
decrease perfusate pH.
Filter through 0.2 micron Pall prebypass filter (Do not use
other 0.2 micron filters!)
References are available upon request by e-mail
Figures will be sent to cooperating colleagues as a matter of course.
Those wishing to see copies of the figures prior to publication may
request copies and an estimate of the cost (color photocopying).
Copies will be supplied at the discretion of the senior author at cost
(i.e., copy cost plus postage and handling). Interested individuals
may inquire for an estimate.
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