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Path: spies!sgiblab!zaphod.mps.ohio-state.edu!pacific.mps.ohio-state.edu!linac!att!cbnewse!cbnewsd!att-out!cbnewsl!kqb
From: kqb@cbnewsl.cb.att.com (kevin.q.brown)
Subject: Freezing Damage - Part 1
Organization: AT&T Bell Laboratories
Date: Fri, 4 Dec 1992 17:55:23 GMT
Message-ID: <1992Dec4.175523.9470@cbnewsl.cb.att.com>
References: <merkle.722467022@manarken> <1992Dec1.204713.17920@cbnewsl.cb.att.com>
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------------------------ Forwarded Message ------------------------
> Date: 03 Dec 92 06:49:03 EST
> From: Paul Wakfer <70023.3041@CompuServe.COM> <- Mike Darwin
> Message-Subject: CRYONICS: Freezing Damage (Darwin) Part 1
Note: This posting is from Mike Darwin
There have been several requests for information about the kind
of damage done during cryonic suspension. In particular, there have
been requests for detailed, objective studies. As a result of this
interest I have decided to post a research paper which is now in the
(hopefully) final stages of preparation for publication.
However, there is a serious shortcoming to this posting, namely
that of necessity there can be no accompanying light or electron
micrographs. In this case this is a serious handicap, although for
many readers the micrographs would mean little. In any event, it is
to be hoped that this paper can be prepared for more formal
publication within 6 to 12 months. Anyone who wishes to assist me in
this capacity should feel free to do so (the EM's and light
micrographs need to be captioned and laid out -- a formidable task).
Many caveats about the validity of this work are contained in its
closing paragraphs. However, I would like to add the following: The
presence of pericapillary ice holes has been verified by freeze
substitution work done by Dr. Gregory M. Fahy of the Red Cross Organ
preservation lab. Similarly Dr. Fahy's freeze-substitution work has
documented the presence of massive ice crystals which comprise about
60% of the tissue volume. I gather that Dr. Fahy feels somewhat more
optimistic about preservation of neuronal connectivity than I do, but
one thing we are both agreed on: our work clearly demonstrates serious
histogical disruption with tears or fractures in the brain tissue
appearing at approximately 3 to 5 micron intervals.
I think it is also fair to say that anyone, layman or
neurophysiologist who looks at either the pictures in the study by
Darwin, et al., or the pictures generated by Fahy of freeze-
substituted brains (showing massive histological disruption by ice)
will be given pause for thought about the workability of cryonics.
I have great admiration for Dr. Merkle and his work. But I would
also point out that in the theoretical domain where there is a will
there is almost always a way. Alas, the real world is somewhat
harsher. We all want and need to believe very desperately that
cryoinjury can and will be reversed. However, there is no direct
evidence of this. The kind of damage my colleagues and I observed in
the study below is carefully "qualified," to make it good science.
However, in this preamble I am a bit freer and I can say that I
believe that the damage is at least as bad as we saw -- it would be
hard to imagine it being any worse short of calling it a tissue
homogenate -- something you get when you run a brain through a
blender. It is my gut feel that post-thaw artifactual stirring was
not the main reason things look bad. I think things look bad because
they *are* bad.
Does that mean patients frozen with today's techniques will not
be revived? I do not know. Does that mean we should *not* continue
to freeze people? No. What it does mean is that we need to do some
serious work to improve the situation. The kind of damage we observed
and are observing is a consequence of ice formation. At a minimum we
can do a great deal to reduce or eliminate ice formation. A major,
comprehensive research proposal is under development at this time and
should be ready for submission to the cryonics community by late
Spring or early Summer.
In the meantime my colleagues and I at Biopreservation and
Cryovita are working hard to make further improvements on hypothermic
brain presevation which will allow us to conduct the necessary
cryopreservation work with greater ease.
Also in order is a word about Jerry Leaf, who entered suspension
over a year ago. This work was completed in the mid 1980's and this
paper was completed in draft form circa 1988. Jerry read and
commented on the draft shortly after it was written. Most, but not
all of his suggested changes were incorporated. The final two
paragraphs of summary were written after his suspension. I feel
comfortable that Jerry would want this paper published, warts and all.
The work that underpins it took a great deal of his time and effort.
Indeed, while none of us knew it at the time, this work comprised a
major block of Jerry's cryonics-science productive life. In drawing
the few conclusion I draw, I have striven to be as objective as Jerry
would have been. I have also submitted this work for review by a
prominent cryonicist-cryobiologist whom I know Jerry respected
greatly. I have incorporated all of this cryobiologists substantive
revisions.
Finally, to Jerry: may you someday have the pleasure of reading
these words and proving us both wrong about the prospects for
recovery. Jerry, I miss you more than words can tell.
THE EFFECTS OF CRYOPRESERVATION ON THE CAT
by Michael Darwin, Jerry Leaf, Hugh L. Hixon
I. Introduction
II. Materials and Methods
III. Effects of Glycerolization
IV. Gross Effects of Cooling to and Rewarming From -196*C
V. Effects of Cryopreservation on Histology of Selected Tissues
VI. Effects of Cryopreservation on Ultrastructure of Selected Tissues
VII. Summary and Discussion
I. INTRODUCTION
The immediate goal of cryonic suspension is to use current
cryobiological techniques to preserve the brain structures which
encode personal identity adequately enough to allow for resuscitation
or reconstruction of the individual should molecular nanotechnology be
realized (1,2). Aside from two previous isolated efforts (3,4) there
has been virtually no systematic effort to examine the fidelity of
histological, ultrastructural, or even gross structural preservation
of the brain following cryopreservation in either an animal or human
model. While there is a substantial amount of indirect and
fragmentary evidence in the cryobiological literature documenting
varying degrees of structural preservation in a wide range of
mammalian tissues (5,6,7), there is little data of direct relevance to
cryonics. In particular, the focus of contemporary cryobiology has
been on developing cryopreservation techniques for currently
transplantable organs, and this has necessarily excluded extensive
cryobiological investigation of the brain, the organ of critical
importance to human identity and mentation.
The principal objective of this pilot study was to survey the
effects of glycerolization, freezing to liquid nitrogen temperature,
and rewarming on the physiology, gross structure, histology, and
ultrastructure of both the ischemic and non-ischemic adult cats using
a preparation protocol similar to the one then in use on human cryonic
suspension patients. The non-ischemic group was given the designation
Feline Glycerol Perfusion (FGP) and the ischemic group was referred to
as Feline Ischemic Glycerol Perfusion (FIGP).
The work described in this paper was carried out over a 19-month
period from January, 1982 through July, 1983. The perfusate employed
in this study was one which was being used in human cryonic suspension
operations at that time, the composition of which is given in Table I.
The principal cryoprotectant was glycerol.
II. MATERIALS AND METHODS
Preperfusion Procedures
Nine adult cats weighing between 3.4 and 6.0 kg were used in this
study. The animals were divided evenly into a non-ischemic and a 24-
hour mixed warm/cold ischemic group. All animals received humane care
in compliance with the "Principles of Laboratory Animal Care"
formulated by the National Society for Medical Research and the "Guide
for the Care and Use of Laboratory Animals" prepared by the National
Institutes of Health (NIH Publication No. 80-23, revised 1978).
Anesthesia in both groups was secured by the intraperitoneal
administration of 40 mg/kg of sodium pentobarbital. The animals were
then intubated and placed on a pressure-cycled respirator. The EKG
was monitored throughout the procedure until cardiac arrest occurred.
Rectal and esophageal temperatures were continuously monitored during
perfusion using YSI type 401 thermistor probes.
Following placement of temperature probes, an IV was established
in the medial foreleg vein and a drip of Lactated Ringer's was begun
to maintain the patency of the IV and support circulating volume
during surgergy. Premedication (prior to perfusion) consisted of the
IV administration of 1 mg/kg of metubine iodide to inhibit shivering
during external and extracorporeal cooling and 420 IU/kg sodium
heparin as an anticoagulent. Two 0.77 mm I.D. Argyle Medicut 15"
Sentinel line catheters with Pharmaseal K-69 stopcocks attached to the
luer fittings of the catheters were placed in the right femoral artery
and vein. The catheters were connected to Gould Model P23Db pressure
transducers and arterial and venous pressures were monitored
throughout the course of perfusion.
Surgical Protocol
Following placement of the monitoring catheters, the animals were
transferred to a tub of crushed ice and positioned for surgery. The
chest was shaved and a median sternotomy was performed. The aortic
root was cleared of fat and a purse-string suture was placed, through
which a 14-gauge angiocath was introduced. The angiocath, which
served as the arterial perfusion cannula, was snared in place,
connected to the extracorporeal circuit and cleared of air. The
pericardium was opened and tented to expose the right atrium. A
purse-string suture was placed in the apex of the right atrium and a
USCI type 1967 16 fr. venous cannula was introduced and snared in
place. Backties were used on both the arterial and venous cannulae to
secure them and prevent accidental dislodgment during the course of
perfusion. Placement of cannulae is shown in Figure 2.
Extracorporeal Circuit
The extracorporeal circuit (Figure 3) was of composed of 1/4" and
3/8" medical grade polyvinyl chloride tubing. The circuit consisted
of two sections: a recirculating loop to which the animal was
connected and a glycerol addition system. The recirculating system
consisted of a 10 liter polyethylene reservoir positioned atop a
magnetic stirrer, an arterial (recirculating) roller pump, an Erika
HPF-200 hemodialyzer which was used as a hollow fiber oxygenator (8)
(or alternatively, a Sci-Med Kolobow membrane oxygenator), a Travenol
Miniprime pediatric heat exchanger, and a 40-micron Pall LP 1440
pediatric blood filter. The recirculating reservoir was continuously
stirred with a 2" teflon-coated magnetic stir bar driven by a Corning
PC 353 magnetic stirrer. Temperature was continuously monitored in
the arterial line approximately six inches from the arterial cannula
using a Sarns in-line thermistor temperature probe and YSI 42SL remote
sensing thermometer. Glycerol concentrate was continuously added to
the the recirculating system using a Drake-Willock hemodialysis pump.
Storage and Reuse of the Extracorporeal Circuit
After use the circuit was flushed extensively with filtered tap
and distilled water, and then flushed and filled with 3% formaldehyde
in distilled water to prevent bacterial overgrowth. Prior to use the
circuit was again thoroughly flushed with filtered tap water, and then
with filtered distilled water (including both blood and gas sides of
the hollow fiber dialyzer; Kolobow oxygenators were not re-used). At
the end of the distilled water flush, a test for the presence of
residual formaldehyde was performed using Schiff's Reagent. Prior to
loading of the perfusate, the circuit was rinsed with 10 liters of
clinical grade normal saline to remove any particulates and prevent
osmotic dilution of the base perfusate.
Pall filters and arterial cannula were not re-used. The circuit
was replaced after a maximum of three uses.
Preparation of Control Animals
Fixative Perfused
Two control animals were prepared as per the above. However, the
animals were subjected to fixation after induction of anesthesia and
placement of cannulae. Fixation was achieved by first perfusing the
animals with 500 cc of bicarbonate-buffered Lactated Ringer's
containing 50 g/l hydroxyethyl starch (HES) with an average molecular
weight of 400,000 to 500,000 supplied by McGaw Pharmaceuticals of
Irvine, Ca (pH adjusted to 7.4) to displace blood and facilitate good
distribution of fixative, followed immediately by perfusion of 1 liter
of modified Karnovsky's fixative (Composition given in Table I).
Buffered Ringers-HES perfusate and Karnovsky's solution were filtered
through 0.2 micron filters and delivered with the same extracorporeal
circuit described above.
Immediately following fixative perfusion the animals were
dissected and 4-5 mm thick coronal sections of organs were cut, placed
in glass screw-cap bottles, and transported, as detailed below, for
light or electron microscopy.
Straight Frozen Non-ischemic Control
One animal was subjected to straight freezing (i.e., not treated
with cryoprotectant). Following induction of anesthesia and
intubation the animal was supported on a respirator while being
externally cooled in a crushed ice-water bath. When the EKG
documented profound bradycardia at 26*C, the animal was disconnected
from the respirator, placed in a plastic bag, submerged in an
isopropanol cooling bath at -10*C, and chilled to dry ice and liquid
nitrogen temperature per the same protocol used for the other two
experimental groups as described below.
Preparation of FGP Animals
Following placement of cannulae, FGP animals were subjected to
total body washout (TBW) by open-circuit perfusion of 500 cc of
glycerol-free perfusate. The extracorporeal circuit was then closed
and constant-rate addition of glycerol-containing perfusate was begun.
Cryoprotective perfusion continued until the target concentration of
glycerol was reached or the supply of glycerol-concentrate perfusate
was exhausted.
Preparation of FIGP Animals
In the FIGP animals, respirator support was discontinued
following anasthesia and administration of Metubine. The endotracheal
tube was clamped and the ischemic episode was considered to have begun
when cardiac arrest was documented by absent EKG.
After the start of the ischemic episode the animals were allowed
to remain on the operating table at room temperature ( 22*C to 25*C)
for a 30 minute period to simulate the typical interval between
pronouncement of legal death in a clinical environment and the start
of external cooling at that time. During the 30 minute normothermic
ischemic interval the femoral cut-down was performed and monitoring
lines were placed in the right femoral artery and vein as per the FGP
animals. Prior to placement, the monitoring catheters were irrigated
with normal saline, and following placement the catheters were filled
with 1000 unit/cc of sodium heparin to guard against clot obstruction
of the catheter during the post-mortem ischemic period.
After the 30 minute normothermic ischemic period the animals were
placed in a 1-mil polyethylene bag, transferred to an insulated
container in which a bed of crushed ice had been laid down, and
covered over with ice. A typical cooling curve for a FIGP animal is
presented in Figure 1. FIGP animals were stored on ice in this fashion
for a period of 24 hours, after which time they were removed from the
container and prepared for perfusion using the surgical and perfusion
protocol described above.
Perfusate
The perfusate was an intracellular formulation which employed
sodium glycerophosphate as the impermeant species and hydroxyethyl
starch (HES)(av. MW 400,000 - 500,000) as the colloid. The
composition of the base perfusate is given in Table I. The pH of the
perfusate was adjusted to 7.6 with potassium hydroxide. A pH above
7.7, which would have been "appropriate" to the degree of hypothermia
experienced during cryoprotective perfusion (9), was not achievable
with this mixture owing to problems with complexing of magnesium and
calcium with the phosphate buffer, resulting in an insoluble
precipitate.
Perfusate components were reagent or USP grade and were dissolved
in USP grade water for injection. Perfusate was prefiltered through a
Whatman GFB glass filter (a necessary step to remove precipitate) and
then passed through a Pall 0.2 micron filter prior to loading into the
extracorporeal circuit.
Perfusion
Perfusion of both groups of animals was begun by carrying out a
total body washout (TBW) with the base perfusate in the absence of any
cryoprotective agent. In the FGP group washout was achieved within 2
- 3 minutes of the start of open circuit asanguineous perfusion at a
flow rate of 160 to 200 cc/min and an average perfusion pressure of 40
mmHg. TBW in the FGP group was considered complete when the
hematocrit was unreadable and the venous effluent was clear. This
typically was achieved after perfusion of 500 cc of perfusate.
Complete blood washout in the FIGP group was virtually impossible
to achieve (see "Results" below). A decision was made prior to the
start of this study (based on previous clinical experience with
ischemic human cryonic suspension patients) not to allow the arterial
pressure to exceed 60 mmHg for any significant period of time.
Consequently, peak flow rates obtained during both total body washout
and subsequent glycerol perfusion in the FIGP group were in the range
of 50-60 cc/min at a mean arterial pressure of 50 mmHg.
Due to the presence of massive intravascular clotting in the FIGP
animals it was necessary to delay placement of the atrial (venous)
cannula (lest the drainage holes become plugged with clots) until the
large clots present in the right heart and the superior and inferior
vena cava had been expressed through the atriotomy. The chest was
kept relatively clear of fluid/clots by active suction during this
interval. Removal of large clots and reasonable clearing of the
effluent was usually achieved in the FIGP group after 15 minutes of
open circuit asanguineous perfusion, following which the circuit was
closed and the introduction of glycerol was begun.
The arterial pO2 of animals in both the FGP and FIGP groups was
kept between 600 mmHg and 760 mmHg throughout TBW and subsequent
glycerol perfusion. Arterial pH in the FGP animals was between 7.1
and 7.7 and was largely a function of the degree of diligence with
which addition of buffer was pursued. Arterial pH in the FIGP group
was 6.5 to 7.3. Two of the FIGP animals were not subjected to active
buffering during perfusion and as a consequence recovery of pH to more
normal values from the acidosis of ischemia (starting pH for FIGP
animals was typically 6.5 to 6.6) was not as pronounced.
Introduction of glycerol was by constant rate addition of base
perfusate formulation made up with 6M glycerol to a recirculating
reservoir containing 3 liters of glycerol-free base perfusate. The
target terminal tissue glycerol concentration was 3M and the target
time course for introduction was 2 hours. The volume of 6M glycerol
concentrate required to reach a terminal concentration in the
recirculating system (and thus presumably in the animal) was
calculated as follows:
Vp
Mc = --------- Mp
Vc + Vp
where
Mc = Molarity of glycerol in animal and circuit.
Mp = Molarity of glycerol concentrate.
Vc = Volume of circuit and exchangeable volume of animal.*
Vp = Volume of perfusate added.
* Assumes an exchangeable water volume of 60% of the preperfusion
weight of the animal.
Glycerolization of the FGP animals was carried out at 10*C to
12*C. Initial perfusion of FIGP animals was at 4*C to 5*C with
warming (facilitated by TBW with warmer perfusate and removal of
surface ice packs) to 10*-12*C for cryoprotectant introduction. The
lower TBW temperature of the FIGP animals was a consequence of the
animals having been refrigerated on ice for the 24 hours preceding
perfusion.
Following termination of the cryoprotective ramp, the animals
were removed from bypass, the aortic cannula was left in place to
facilitate prompt reperfusion upon rewarming, and the venous cannula
was removed and the right atrium closed. The chest wound was loosely
closed using surgical staples.
Concurrent with closure of the chest wound, a burrhole craniotomy
3 to 5 mm in diameter was made in the right parietal bone of all
animals using a high speed Dremel "hobby" drill. The purpose of the
burrhole was to allow for post-perfusion evaluation of cerebral
volume, assess the degree of blood washout in the ischemic animals and
facilitate rapid expansion of the burrhole on rewarming to allow for
the visual evaluation of post-thaw reperfusion (using dye).
The rectal thermistor probe used to monitor core temperature
during perfusion was replaced by a copper/constantan thermocouple at
the conclusion of perfusion for monitoring of the core temperature
during cooling to -79*C and -196*C.
Cooling to -79*C
Cooling to -79*C was carried out by placing the animals within
two 1 mil polyethylene bags and submerging them in an isopropanol bath
which had been precooled to -10*C. Bath temperature was slowly
reduced to -79*C by the periodic addition of dry ice. A typical
cooling curve obtained in this fashion is shown in Figure 5. Cooling
was at a rate of approximately 4*C per hour.
Cooling to and Storage at -196*C
Following cooling to -79*C, the plastic bags used to protect the
animals from alcohol were removed, the animals were placed inside
nylon bags with draw-string closures and were then positioned atop a
6" high aluminum platform in an MVE TA-60 cryogenic dewar to which 2"-
3" of liquid nitrogen had been added. Over a period of approximately
48 hours the liquid nitrogen level was gradually raised until the
animal was submerged. A typical cooling curve to liquid nitrogen
temperature for animals in this study is shown in Figure 6. Cooling
rates to liquid nitrogen temperature were approximately 2*C per hour.
After cool-down animals were maintained in liquid nitrogen for a
period of 6-8 months until being removed and rewarmed for gross
structural, histological, and ultrastructural evaluation.
Rewarming
The animals in both groups were rewarmed to -2*C to -3*C by
removing them from liquid nitrogen and placing them in a precooled box
insulated on all sides with a 2" thickness of styrofoam and containing
a small quantity of liquid nitrogen. The animals were then allowed to
rewarm to approximately -20*C, at which time they were transferred to
a mechanical refrigerator at a temperature of 8*C. When the core
temperature of the animals had reached -2*C to -3*C the animals were
removed to a bed of crushed ice for post-mortem examination and tissue
collection for light and electron microscopy. A typical rewarming
curve is presented in Figure 7.
Modification of Protocol Due To Tissue Fracturing
After the completion of the first phase of this study (perfusion
and cooling to liquid nitrogen temperature) the authors had the
opportunity to evaluate the gross and histological condition of the
remains of three human cryonic suspension patients who were removed
from cryogenic storage and converted to neuropreservation (thus
allowing for post-mortem dissection of the body, excluding the head)
(10). The results of this study confirmed previous, preliminary, data
indicative of gross fracturing of organs and tissues in animals cooled
to and rewarmed from -196*C. These findings led us to abandon our
plans to reperfuse the animals in this study with oxygenated,
substrate-containing perfusate (to have been followed by fixative
perfusion for histological and ultrastructural evaluation) which was
to be have been undertaken in an attempt to assess post-thaw viability
by evaluation of post-thaw oxygen consumption, glucose uptake, and
tissue-specific enzyme release.
Rewarming and examination of the first animal in the study
confirmed the presence of gross fractures in all organ systems. The
scope and severity of these fractures resulted in disruption of the
circulatory system, thus precluding any attempt at reperfusion as was
originally planned.
Preparation of Tissue Samples For Microscopy
Fixation
Samples of four organs were collected for subsequent histological
and ultrastructural examination: brain, heart, liver and kidney.
Dissection to obtain the tissue samples was begun as soon as the
animals were transferred to crushed ice. The brain was the first
organ removed for sampling. The burrhole created at the start of
perfusion was rapidly extended to a full craniotomy using rongeurs
(Figure 8). The brain was then removed en bloc to a shallow pan
containing iced, modified Karnovsky's fixative containing 25% w/v
glycerol (see Table I for composition) sufficient to cover it.
Slicing of the brain into 5 mm thick sections was carried out with the
brain submerged in fixative in this manner. At the conclusion of
slicing a 1 mm section of tissue was excised from the visual cortex
and fixed in a separate container for electron microscopy. During
final sample preparation for electron microscopy care was taken to
avoid the cut edgdes of the tissue block in preparing the Epon
embedded sections.
The sliced brain was then placed in 350 ml of Karnovsky's
containing 25%w/v glycerol in a special stirring apparatus which is
illustrated in Figure 9. This fixation/deglycerolization apparatus
consisted of two plastic containers nested inside of each other atop a
magnetic stirrer. The inner container was perforated with numerous 3
mm holes and acted to protect the brain slices from the stir bar which
continuously circulated the fixative over the slices. The stirring
reduced the likelihood of delayed or poor fixation due to overlap of
slices or stable zones of tissue water stratification. (The latter
was a very real possibility owing to the high viscosity of the 25%w/v
glycerol-containing Karnovsky's.)
Deglycerolization of Samples
To avoid osmotic shock all tissue samples were initially immersed
in Karnovsky's containing 25%w/v glycerol at room temperature and were
subsequently deglycerolized prior to staining and embedding by
stepwise incubation in Karnovsky's containing decreasing
concentrations of glycerol (see Figure 10 for deglycerolization
protocol).
To prepare tissue sections from heart, liver, and kidney for
microscopy, the organs were first removed en bloc to a beaker
containing an amount of ice-cold fixative containing 25% w/v glycerol
sufficient to cover the organ. The organ was then removed to a room
temperature work surface at where 0.5 mm sections were made with a
Stadie-Riggs microtome. The microtome and blade were pre-wetted with
fixative, and cut sections were irrigated from the microtome chamber
into a beaker containing 200 ml of room-temperature fixative using a
plastic squeeze-type laboratory rinse bottle containing fixative
solution. Sections were deglycerolized using the same procedure
previously detailed for the other slices.
Osmication and Further Processing
At the conclusion of deglycerolization of the specimens all
tissues were separated into two groups; tissues to be evaluated by
light microscopy, and those to be examined with transmission electron
microscopy. Tissues for light microscopy were shipped in glycerol-
free modified Karnovsky's solution to American Histolabs, Inc. in
Rockville, MD for paraffin embedding, sectioning, mounting, and
staining.
Tissues for electron microscopy were transported to the
facilities of the University of California at San Diego in glycerol-
free Karnovsky's at 1* to 2*C for osmication, Epon embedding, and EM
preparation of micrographs by Dr. Paul Farnsworth.
Due to concerns about the osmication and preparation of the
material processed for electron microscopy by Farnsworth, tissues from
the same animals were also submitted for electron microscopy to
Electronucleonics of Silver Spring, Maryland.
- **Electronucleonics results are not covered here since another
investigator has yet to provide the necessary information and we do
not have access to the pictures.
III. EFFECTS OF GLYCEROLIZATION
Perfusion of FGP Animals
Blood washout was rapid and complete in the FGP animals and
vascular resistance decreased markedly following blood washout.
Vascular resistance increased steadily as the glycerol concentration
increased, probably as a result of the increasing viscosity of the
perfusate.
Within approximately 5 minutes of the beginning of the
cryoprotective ramp, bilateral ocular flaccidity was noted in the FGP
animals. 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 "caved-in" 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 (Figure 11), and a "waxy-
leathery" appearance and texture to both cut skin and skeletal muscle.
Tissue water evaluations conducted on ileum, kidney, liver, lung, and
skeletal muscle confirmed and extended the gross observations.
Preliminary observation suggest that water loss was in the range of
30% to 40% in most tissues. As can be seen in Table III, total body
water losses attributable to dehydration, while typically not as
profound, were still in the range of 18% to 34%. The gross appearance
of the heart suggested a similar degree of dehydration, as evidenced
by modest shrinkage and the development of a "pebbly" surface texture
and a somewhat translucent or "waxy" appearance.
Examination of the cerebral hemispheres through the burr hole
(Figure 12) 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 waxy appearance that was
characteristic of muscle, skin, and brain.
Oxygen consumption (determined by measuring the arterial/venois
difference) throughout perfusion was fairly constant and did not
appear to be significantly impacted by glycerolization, as can be seen
Figure 12.
Perfusion of FIGP Animals
As previously noted, the ischemic animals had far lower flowrates
at the same perfusion pressure as FGP animals and demonstrated
incomplete blood washout. Intravascular clotting was serious a
barrier to adequate perfusion. Post-thaw dissection demonstrated
multiple infarcted areas in virtually all organ systems; areas where
blood washout and glycerolization were incomplete or absent. In
contrast to the even color and texture changes observed in the FGP
animals, the skin of the FIGP animals developed multiple, patchy,
nonperfused areas which were clearly outlined by surrounding,
dehydrated, amber-colored glycerolized areas.
External and internal examination of the brain and spinal cord
revealed surprisingly good blood washout of the central nervous
system. While grossly visible infarcted areas were noted, these were
relatively few and were generally no larger than 2 mm to 3 mm in
diameter. With few exceptions, the pial vessels were free of blood
and appeared empty of gross emboli. One striking difference which was
consistently observed in FIGP animals was a far less profound
reduction in brain volume during glycerolization (Figure 13). This
may have been due to a number of factors: lower flow rates, higher
perfusion pressures, and the increased capillary permeability and
perhaps increased cellular permeability to glycerol.
Whereas edema was virtually never a problem during
glycerolization of FGP animals, edema was universal in the FIGP
animals after as little as 30 minutes of perfusion. In the central
nervous system this edema was evidenced by a "rebound" from initial
cerebral shrinkage to frank cerebral edema, with the cortices,
restrained by the dura, often abutting or slightly projecting into the
burrhole. Marked edema of the nictating membranes, the lung, the
intestines, and the pancreas was also a uniform finding at the
conclusion of cryoprotective perfusion. The development of edema in
the central nervous system sometimes closely paralleled the beginning
of "rebound" of ocular volume and the development of ocular turgor and
frank ocular edema.
In contrast to the relatively good blood washout observed in the
brain, the kidneys of FIGP animals had a very dark and mottled
appearance. While some areas (an estimated 20% of the cortical
surface) appeared to be blood-free, most of the organ remained blood-
filled throughout perfusion. Smears of vascular fluid made from renal
biopsies which were collected at the conclusion of perfusion (for
tissue water determinations) revealed the presence of many free and
irregularly clumped groups of crenated and normal-appearing red cells,
further evidence of the incompleteness of blood washout. Microscopic
examination of recirculating perfusate revealed some free, and a few
clumped red cells. However, the concentration was low, and the
perfusate microhematocrit was unreadable at the termination of
perfusion (i.e., less than 1%).
The liver of FIGP animals appeared uniformly blood-filled
throughout perfusion, and did not exhibit even the partial blood
washout evidenced by the kidneys. However, despite the absence of any
grossly apparent blood washout, tissue water evaluations in one FIGP
animal were indicative of osmotic dehydration and thus of some
perfusion.
The mesenteric, pancreatic, splanchic, and other small abdominal
vessels were largely free of blood by the conclusion of perfusion.
However, blood-filled vessels were not uncommon, and examination
during perfusion of mesenteric vessels performed with an
ophthalmoscope at 20X magnification revealed stasis in many smaller
vessels, and irregularly shaped small clots or agglutinated masses of
red cells in most of the mesenteric vessels. Nevertheless, despite
the presence of massive intravascular clotting, perfusion was
possible, and significant amounts of tissue water appear to have been
exchanged for glycerol.
One immediately apparent difference between the FGP and FIGP
animals was the accumulation in the lumen of the ileum of large
amounts of perfusate or perfusate ultrafiltrate by the ischemic
animals. Within approximately 10 minutes of the start of reperfusion,
the ileum of the ischemic animals that had been laparotomized was
noticed to be accumulating fluid. By the end of perfusion, the
stomach and the small and large bowel had become massively distended
with perfusate. Figure 14 shows both FIGP and FGP ileum at the
conclusion of glycerol perfusion. As can be clearly seen, the FIGP
intestine is markedly distended. Gross examination of the gut wall
was indicative of tissue-wall edema as well as intraluminal
accumulation of fluid. Often by the end of perfusion, the gut had
become so edematous and distended with perfusate that it was
impossible to completely close the laparotomy incision. Similarly,
gross examination of gastric mucosa revealed severe erosion with the
mucosa being very friable and frankly hemorrhagic.
Escape of perfusate/stomach contents from the mouth (purging)
which occurs during perfusion in ischemically injured human suspension
patients did not occur, perhaps due to greater post-mortem competence
of the gastroesophageal valve in the cat.
Oxygen consumption in the two ischemic cats in which it was
measured was dramatically impacted, being only 30% to 50% of control
and deteriorating throughout the course of perfusion (Figure 12).
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.
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.
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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
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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