X-Message-Number: 1389
Date: 03 Dec 92 06:49:03 EST
From: Paul Wakfer <>
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 pulication.
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.
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