From: Mike Darwin <75120.575@compuserve.com>
Subject: BPI Tech Brief 16: Canine Brain Cryopreservation (1/2)
Date: 01 Jun 1995

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From: Mike Darwin <75120.575@compuserve.com>
Date: 31 May 95 00:03:14 EDT
Subject: SCI.CRYONICS BPI Tech Brief 16: Canine Brain Cryopreservation (1/2)

A Brief Lay-Level Summary of Biopreservation's Canine Brain
Cryopreservation Results
by Charles Platt

In the 1950s, experiments showed that the damage caused when
the cells of a mammal are frozen can be reduced if the cells
are first treated with a solution of glycerol.

More recently, work by Leaf, Darwin, et. al. suggested that
damage to cryonics patients might be further minimized if
perfusion with glycerol was carefully monitored and
controlled, using a solution whose concentration gradually
increased during the perfusion process to a very high concentration
where much less ice will form than is the case when no cryoprotectant
or lower levels of cryoprotectant are used.

Until now, there has been no systematic study to verify that
this kind of controlled perfusion of cryonics patients really
does result in less freezing damage than a simpler protocol.
In particular, no one ever treated lab animals with the exact
same protocol that is currently used on human cryonics
patients by BioPreservation or the Alcor Foundation. (Note:
ACS may use a different protocol in future, since it is no
longer employing BioPreservation to handle its cases, and The
Cryonics Institute (CI) has a long-standing policy of
minimizing all medical procedures on its patients. CI does do
some glycerolization, but it is typically applied by a
mortician with non-medical equipment, and the concentration is
not ramped up and monitored using equipment of the
type employed by BioPreservation and Alcor.)

More than a year ago, we decided to take several dogs through
our cryonics protocol, keep them frozen for 12 to 18 months at
relatively high temperatures (dry ice which is -79xC), rewarm them,
and then look for brain damage using light and electron microscopy.
The dogs were anesthetized and cardiac arrest was induced during
unconscioiusness.

The animals were then given a short period of warm ischemia (lack of
blood flow) at normal body temperature (37xC) simulating the "waiting
time" that a cryonics patient might experience after death is pronounced,
before cryonics protocols are applied. The dogs were then given
cardio-pulmonary support using a "thumper" of the same type that we
employ on cryonics patients, and our usual medications were administered.
Blood washout and perfusion with glycerol were identical to the procedures
that we use on human patients.

After freezing, storage for a year or more, and thawing, we sent out
samples of brain tissue for examination. The following paper reports
our results, which were much more encouraging than we had hoped.
In every case, damage was greatly reduced compared with either our
prior results in the mid 1980's using 3-4M glycerol cryoprotection)
or than results that were obtained (based on our examination of the CI
light and electron microscope pictures) last year by the Cryonics Institute,
which funded experiments where sheep brains were subjected to CI's simpler
perfusion protocol.

Our results have been examined by a leading cryobiologist,
and we now firmly believe that our perfusion protocol does
minimize damage that would otherwise occur.

We note however that in our model, we assumed that a cryonics
patient can receive care just five minutes after death is
pronounced. There have been many cases where this was not
possible (for example, where patients died suddenly and
unexpectedly), and we believe that longer periods of ischemic
time in such cases probably cause much greater damage to the
integrity of tissues in the brain.

-------------------------------------------------------------

HUMAN CRYOPRESERVATION

PROTOCOL ON THE ULTRASTRUCTURE OF THE CANINE BRAIN

by Michael Darwin, Sandra Russell, Larry Wood, and Candy Wood

     I.    Introduction
     II.   Materials and Methods
     III.  Effects of Closed Chest Cardioipulmonary Support
     IV    Effects of Glycerolization
     V.    Gross Effects of Cooling to and Rewarming From -90 C
     VI.   Effects of Cryopreservation on Brain Ultrastructure
     VII. Summary and Discussion


I. INTRODUCTION

Clinical human cryopreservation has the objective of the
preservation of brain structures which encode personal
identity sufficient to allow for resuscitation or
reconstruction of the individual should molecular
nanotechnology be realized (1,2). Aside from the pioneering
work of Suda, et al (3,4) and three previous studies
conducted by cryonics organizations (5,6,7) there has been
virtually no systematic effort to examine the fidelity of the
ultrastructural preservation of the brain: particularly at
the level of the neuropil and synaptic and intra-synaptic
structures following cryopreservation using clinical (human)
cryopreservation ("cryonic suspension") techniques in either
an animal or cadaver model.

Previous ultrastructural studies conducted by Cryovita
Laboratories in conjunction with the Alcor Life Extension
Foundation in the mid-1980's and more recently by Pichugin,
et al., under the auspices of the Cryonics Institute have
shown massive disruption of ultrastructure at every level.
(5,6)

A brief summary of the lesions observed in these studies
disclosed the following kinds of injury occurring uniformly
throughout both the white and gray matter of the cerebral
cortex in both cats (Darwin , et al) and sheep (Pichugin, et al):

a) ultrastructural-level tearing and fraying of the ripped
ends of nerve tracts by osmotic contraction of cells coupled
with the push of extracellular ice creating debris-strewn
gaps at intervals of 5 to 100 microns in width;

b) separation of capillaries from from surrounding brain
tissue (visible both in the frozen state with freeze-
substitution and upon thawing following fixation and
embedding);

c)physical disruption of the capillaries due to
intracapillary ice formation, lysis of the endothelial cells
with occassional adherent endothelial cell nuclei, and
separation of the endothelial cells from capillary basement
membrane;

d) Separation of myelin from axons, formation of gaps between
the axon membrane and the myelin, unravelling of the myelin,
and frequent loss of intraaxonal material, possibly as a
result of disruption of the axolemma;

e) Extensive disruption of the neuropil and of the plasma
membrane of both neuronal and glial cells with conversion of
intracellular and synaptic membrane structure into amorphous
debris or empty and/or debris-containing vesicles.

The principal objective of this study was to survey the effects of
glycerolization to a much higher concentration than has been used
in past, principally 7.4M glycerol versus 4 to 5 M glycerol,
freezing to -90 C, storage at this temperature for a period of at
least 1 year, and rewarming at varying rates, on gross structure,
histology, and ultrastructure of the canine brain using a preparation
protocol similar to the one now used on human cryopreservation
patients by BioPreservation, Inc. of Rancho Cucamonga, California.

The work described in this paper was carried out from October of 1993
to May of 1995. The technique used for cryopreservation of animals in
this study closely paralells that used by BioPreservation (8) and by
the Alcor Life Extension Foundation of Phoenix, AZ (9) in preparation
of human patients for long-term cryopreservation.

It should be noted that training of staff in procedures for
transport (closed chest cardiopulmonary support), total body
washout (TBW) and cryoprotective perfusion and freezing of
human cryopreservation patients was an important second goal
of this study.


II. MATERIALS AND METHODS

Preperfusion Procedures

Five adult dogs weighing between 24 and 28 kg were used in
this study. 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 Publicoation No. 80-23,
revised 1978). Prior to induction of anesthesia the animals
were given 0.5 mg/kg acepromazine maleate and 0.25 mg
atropine IM. Anesthesia in both groups was secured by the
intravenous administration of 40 mg/kg of sodium
pentobarbital. The animals were then intubated and placed on
a volume-cycled MA-1 ventilator using a tidal volume of 15
cc/kg, PEEP of 5 cm H2O and an FiO2 of 21.

EKG was monitored throughout the procedure until cardiac arrest was
induced. Rectal and esophageal temperatures were
continuously monitored during perfusion using copper
constantan 20 gauge thermocouple probes (Instrument
Laboratories 53-20-507) . A 14 Fr. x 48 cm double lumen
Salem Sump gastric tube was passed esophageally into the
stomach (positioning verified fluoroscopically) to facilitate
alkalinization of stomach contents with Maalox (alluminum hydroxide
suspension) and prevent erosion of gastric mucosa during subsequent
periods of ischemia, hypoperfusion, and hypothermia.

Following placement of temperature probes, an IV was established
in the medial foreleg vein and a drip of Normosol-R (pH 7.4) at
rate of 50 cc/hr was begun to maintain the patency of the IV and
support blood circulating volume during surgery.

The animals were then placed in a Portable Ice Bath (PIB) identical
to that used for transport of human cryopreservation patients and the
chest was stabilized in a specially fabricated padded holder to allow
for stable mid-sternal application of a Michigan Instruments Model # 1004
Thumper (external cardiac compressor/ventilator). (Figure 1)


Surgical Protocol

Both groins and the right neck over the entire length of
external jugular vein were shaved and prepped for surgery
using povidone iodine solution (Betadine). Since these were
sacrifice studies, sterile technique was not used. However
gloves, gowns and masks were used to protect staff from
infection and simulate actual working conditions with human
cases. For the same reasons, Betadine was used to simulate
the appearance of the preoperative skin and kill skin flora
minimizing risk of infection in the event of sharps injury
to staff.

An Edwards adult Swan-Ganz catheter was placed via open
cutdown of the right jugular vein and was wedged in the
pulmonary artery. The proximal line of the Swan-Ganz
catheter was connected to a Bentley Trantec 800 pressure
transducer for measurement of pulmonary artery diastolic
(PAD) and wedge pressures, and the thermistor cable was
connected to an American Edwards COM-1 thermodilution
cardiac output computer to facilitate measurement of cardiac
output (CO) and core blood temperature during post-cardiac
arrest Thumper cardiopulmonary support. The position of the
Swan-Ganz catheter in the pulmonary artery was verified both
by evaluation of the pressure waveform on a Tektronix 414
physiologic monitor, and fluoroscopically with Siemans
Siemens, Inc. Siremobile II C-arm fluoroscope.

Both groins were cut down to access the femoral arteries and
veins. An Argyle 18 Fr. pressure monitoring catheter
connected to a Cobe 4-way stopcock was placed in the right
femoral artery and connected via a Cobe large-bore pressure
monitoring line to a Trantec 800 pressure transducer and
Tektronix 413 monitor for measurement of mean arterial
pressure (MAP).

Venous return was achieved using USCI type 1967 cannulae of
either 21 or 22 Fr. diameter which were placed in both
femoral veins and positioned under fluoroscopy: the right
venous cannula being advanced up the inferior vena cava
(IVC) to approximately the level of the right atrium, the
left venous cannula being advanced to approximately the level
of the renal veins.

Arterial perfusion was via a Cardiovascular Instruments
4.0mm or 4.5 mm ID stainless-steel cannula placed in the
right femoral artery. Typical cannula placement is shown in
Figure 2.


Extracorporeal Circuit

The extracorporeal circuit for the cryoprotectant treated animals
(Figure 3) consisted of 1/4" (arterial) and 3/8" (venous) medical
grade polyvinyl chloride tubing. The circuit was comprised of two
sections: a recirculating loop to which the animal was connected
and a glycerol addition system. The recirculating system consisted
of a 20 liter polyethylene reservoir positioned atop a magnetic
stirrer with a floating lid to avoid entraining air (10), an
arterial (recirculating) roller pump (Sarns 5000 heart-lung machine
console), a Sarns pediatric 16267 hollow fiber membrane oxygenator/heat
exchanger and a 40-micron Pall LP 1440 40 micron blood filter.
The recirculating reservoir was continuously stirred with a 3"
teflon-coated magnetic stir bar driven by a Thermolyne type 7200
magnetic stirrer. Temperature was continuously monitored at the
arterial port of the oxygenator using a Sarns thermistor temperature
probe and a YSI 73BTAX remote sensing digital thermometer.
Glycerol concentrate was continuously added to the the recirculating
system from a 60 liter polyethelene reservoir using a Drake-Willock
7401 hemodialysis pump. Glycerol ramp was monitored using an Atago
hand-held sugar refractometer.

Three animals constituted the experimental group and were subjected to
simulated transport, TBW, cryoprotectuve perfusion and freezing-
thawing and fixation.


Fixative Perfused Controls

Two control animals were prepared as per the above with the following
modifications: One of the animals was subjected to fixation after
induction of anesthesia and placement of cannulae (i.e., normothermic,
non-ischemic, beating-heart fixation). Fixation was achieved
by first perfusing the animal with 3 liters of bicarbonate-
buffered Lactated Ringer's containing 50 g/l Dextran-40 with
an average molecular weight of 40,000 (Pharmachem) (pH
adjusted to 7.4) to displace blood and facilitate good
distribution of fixative. This Ringer's Dextran flush was
followed immediately by perfusion of 20 liters of Trump's
fixative (Composition given in Table I) to which 100 ml of
Higgin's India Ink (colloidial carbon) had been added.

Buffered Ringers-Dextran-40 perfusate and Trump's solution,
(prior to addition of the India Ink) were filtered through
0.2 micron filters and delivered with the same extracorporeal
circuit described above.

The purpose of this control was to serveas a reference on our basic
fixation and EM preparation technique essentially demonstrating that
fixation and microscopy in our hands yielded normal appearing tissues
thus ruling out artifact from fixation and preparation for microscopy.

The second control animal was subjected to cryoprotective
perfusion to 7.4M glycerol (end arterial concentration) per
the protocol below, and immediately thereafter perfused with
20 liters of Trump's fixative prepared in 7.0M glycerol (also
filtered through a 0.2 micron Pall prebypass filter) with 200
ml of India ink added after filtration. Glycerol-fixative was
perfused at a temperature of 8.0 C.

Immediately following fixative perfusion the animals were
dissected and 4-5 mm thick coronal sections of organs were
cut, placed in glass screw-cap jars containing pre-cooled (4
C) Trump's fixative or Trump's fixative containing 7.4M
glycerol (as appropriate), refrigerated to 4 C, and
transported, as detailed below, for electron microscopy.
Tissue from the glycerol perfused-fixed animal was
deglycerolized at 4 C following cutting of the tissue blocks
for electron microscopy using two protocols of
deglycerolization : fast and slow.

Preparation and Post Cardiac Arrest Support of Cryopreserved Animals

Following placement of cannulae, baseline CO and EtCO2
measurements were made. CO was 1.3 to 1.5 liters/min and
EtCO2 was 5% in all animals. Mean arterial pressure (MAP)
was 80mmHg to 90mmHg.

Cardiac arrest was induced by the administration of 1 mEq/kg
potassium chloride via the distal port of the Swan-Ganz
catheter. Cardiac arrest occurred uniformly within 3-15
seconds. A period of 5 minutes of normothermic ischemia was
then allowed to elapse before closed chest cardiopulmonary
support (CCCS) using the Thumper was initiated. Esophageal
temperature at the time of cardiac arrest in the animals
varied between between a low of 37.4 C and a high of 38.2 C.

At the start of CCCS the following medications were given
via the peripher IV and the proximal line of the Swan-Ganz
catheter for the purpose of minimizing both ischemic and
reperfusion-trickle flow injury.

Medications

Epinephrine: 0.20 mg/kg given every 10 minutes, IV push for
 30 minutes
Nimodipine: 10 micrograms/kg followed by 10 micrograms/kg
 every 10 minutes by slow IV push for 30 minutes
THAM (tromethamine) 0.3M 250 mg/kg IV infusion
Deferoxamine: 500 mg HCl IV push
Sodium Citrate: 120 mg/kg via slow IV push
Trolox: 45 mg/kg slow IV push
Heparin: 420 IU/kg IV push
Methylprednisolone 1 g via IV infusion
Metubine Iodide: 2 mg IV push
Maalox, 30cc was also given vuia the gastric tube and the
 gastric tube flushed with 20 cc of tap water.


Simultaneous with the start of CCCS the animals were covered
with crushed ice and 10 gallons of water were added to the
portable ice bath. A recirculating water pump connected to
both a perforated tubing array (which was draped over the
animal) and to a cooling blanket placed under the animal, was
used to facilitate induction of hypothermia via external
(immersion-simulated) cooling.

The protocol for CCCS using Thumper support consisted of 80
compression per minute with a compression to relaxation
ration of 50:50. Pressure cycled ventilation, delivered
between every fifth chest compression using the Thumper
ventilator at a peak airway pressure of 30 cmH2O, and an
FiO2 of 80% was used throughout CCCS. Efficacy of CCCS was
evaluated by measurement of CO, end-tidal CO2, (Nellcor Easy
Cap) and pulse oximetery (using the tongue as the measuring
site) (CSI Model 503 Pulseoximeter). CCCS was continued for
30 minutes before starting extracorporeal support and total
body washout (TBW).

Animals were placed on closed-circuit cardiopulmonary bypass
using the recirculating loop of the cryoprotective perfusion
circuit. Thie circuit was primed with approximately 3 liters
of asanguineous solution consisting of 1 liter of Dextran
40 in normal saline, 2 liters of Normosol-R (ph7.4) and 25
mEq sodium bicarbonate. Following pump-oxygenator-heat
exchanger cooling to approximately 15 C, animals were
subjected to total body washout (TBW) by open-circuit
perfusion of 10 liters of MHP-2 perfusate containing 5% v/v
glycerol. (see Table II for composition) The extracorporeal
circuit was then closed and addition of 65% v/v glycerol-
containing MHP-2 perfusate at a rate of approximately 400 mM/min. was
begun. Cryoprotective perfusion continued until the target
concentration of glycerol was reached.

Perfusate

The perfusate used for cryoprotective perfusion was an intracellular
formulation which employed
sodium HEPES, glucose and mannitol as the impermeant species
and hydroxyethyl starch (HES, McGaw Pharmaceuticals, Irvine,
CA; av. MW 400,000 - 500,000) as the colloid. The
composition of the base perfusate is given in Table I.I The
pH of the perfusate was adjusted to 8.0 + or - 0.3 with
potassium hydroxide or hydrochloric acid (rarely required)
where needed. A pH of 8.0 was selected because it was
deemed "appropriate" to the degree of hypothermia
experienced during cryoprotective perfusion (11).

Perfusate components were reagent or USP grade and were
dissolved in USP grade water for injection. Perfusate was
through a Pall 0.2 micron prebypass filter prior to loading
into the extracorporeal circuit.


Cryoprotective Perfusion

Cryoprotective perfusion of the animals was begun by carrying
out total body washout (TBW) with the base perfusate
containing 5% v/v glycerol. Washout was typically achieved
within 4-6 minutes of the start of open circuit perfusion at
a flow rate of 1.5 to 1.7 L/min and a mean arterial
pressure (MAP)of 60 mmHg. TBW was considered complete when
the hematocrit was unreadable and the venous effluent was
pink-tinged or clear. This typically was achieved after
perfusion of 7 to 8 liters of 5% v/v glycerol containing MHP-
2 perfusate.

The arterial pO2 of the animals was maintained between 300
mmHg and 500 mmHg throughout TBW and subsequent glycerol
perfusion. Arterial pH during cryoprotective perfusion was
between 7.4 and 7.7 with terminal arterial pH typically
being between 7.6 and 7.7. Venous pH was typically between
7.3 and 7.5 with terminal venous pH being between 7.45 and
7.55

Introduction of glycerol was by constant rate addition of
base perfusate containing 65 v/v glycerol to a recirculating
reservoir containing approximately 15 liters of 5% v/v
glycerol-in MHP-2 base perfusate. The target terminal tissue
glycerol concentration was 7.4M  in the venous effluent and the target
time course
for completion of the cryoprotectant ramp was 2 hours. The
volume of 65% v/v 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 animals was carried out starting at an
esophageal temperature of 15 C with more or less linear
reduction of temperature as glycerol concentration was
increased, with perfusion typically terminating at 6 C.

Cryoprotective perfusion began at a MAP of 40 mmHg and at an
esophageal temperature of 15 C. MAP rose steadily as
glycerol concentration was increased and MAP at conclusion
of perfusion was typically between 130mm Hg and 160mm Hg

Following termination of the cryoprotective ramp, the animals
were removed from bypass and the arterial cannula, with a short
length of PVC tubing left attached, was plugged using a foley
catheter plug. The venous cannulae were also left in place
and cross-connected to each other with a Cobe 3/8" straight
connector, taking care to exclude air from both the cannulae
and connector. Cannulae were left in place to facilitate prompt
reperfusion upon rewarming, The margins of the groin wounds
were loosely approximated using surgical staples and the
endotracheal tube was plugged with a rubber laboratory stopper. to
prevent entry of cooling bath media into the lungs should the
sheilding plastic bag leak during cooling to -79C.

The rectal and esophageal thermocouple probes used to
monitor core temperature during perfusion were augmented with
two external thermocouple probes of the same type for
monitoring cooling to -79 C. One of these external probes
was stapled to the skin at the midline of the scalp and the
other was stapled to the abdomen, also at the midline,
approximately 4 cm below the Xyphoid process.


Cooling to -79 C

Cooling to -79 C was carried out by placing the animals
within a 6 mil polyethylene bag from which air was evacuated
with a shop-type vacuum cleaner and then submerging them in
an n-propanol bath which had been precooled to -40 C. Animal
temperatures at the time of placement in the cooling bath
were typically 5-6 C esophageal, 7-9 C rectal, and 8-9 C
surface. 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 4. Cooling was
at a rate (averaged) of approximately 4 C per hour.


Cooling to and Storage at -90 C

Following cooling to -79 C, the plastic bags used to protect
the animals from alcohol were rapidly swabbed off using
cloth towels, the animals were placed inside nylon sleeping
bags with draw-string closures and were then positioned atop
three 6"x 12" styrofoam blocks inside a two-stage Rheem Ultra
Low, -90 C mechanical freezer. Cooling to -90 C from -77
(typical dry ice-alcohol endpoint) was complete in
approximately 6 hours. After cool-down to -90 C animals were
maintained at temperatures between -80 C and -90 C for a
period of 12 to 18 months until being removed and rewarmed
for gross structural, histological, and ultrastructural
evaluation.  Dry ice was used as thermal ballast in the mechanical
freezer to guard against warming due to mechanical failure or power
disruption.


Rewarming

Animals were rewarmed to -10 C to -8 C by removing them from
-90 C freezer and placing them in a well stirred n-propanol
bath which had been precooled to 0 C. Bath temperature
typically declined to approximately -15 C and rose slowly
towards 0 C. When the temperature of the bath reached 0 C it
was maintained at 0 C + or - 3 C by addition of dry ice to
the alcohol bath until the animal's core temperature reached
-10 C. Rewarming was at an average rate of 10 C per hour to
-10 C at which point no ice could be detected in the tissues
by external palpation. A typical rewarming curve is shown in
Figure 5.

When the animals' core temperatures reached -6 C they were
removed from the alcohol bath, the 6 mil plastic bags were
removed, and the animals were placed atop a bed of Zip-Loc
plastic bags filled with crushed ice and covered over with
crushed ice containing Zip-Loc bags on the operating room
table. The animals were re-connected to a simplified
extracorporeal circuit for perfusion of fixative. The
arterial pressure monitoring catheter was also reconnected to
to the pressure transducer to allow for pressure monitoring
during fixative perfusion.

Note: great difficulty was encountered in measuring perfusion
pressure in the first two animals due to failure to flush the
monitoring lines with 7.4M glycerol prior to freezing. The
lines were instead filled with saline from the Intraflow set-
up used to prevent clotting prior to heparinization of the
animal (3cc normal saline per hour flowing through the
catheter). Since the greatest length of the catheter was
deep within the animal, and the core temperature of the
animal was well below the freezing point of saline, it
required great ingenuity to free the lumen of the pressure
monitoring catheters from ice; this was finally achieved by
slowly advancing a heated copper wire through the catheter.


Fixation

After positioning on the operating table a midline incision
was made from sternal notch to the symphisis pubis. The
thorax was opened via a median sternotomy and the abdomen via
a mid-ventral laparotomy. The thoracic and abdominal
incisions were retracted open to allow visualization of the
viscera when fixative perfusion commenced (Figure 6).

When the core temperature (esophageal) reached -6 C perfusion
of fixative perfusion was begun. Precooled fixative (1-2 C)
was delivered at a temperature of 4 C using an open circuit
consisting of a roller pump, a Gish pediatric heat
exchanger, and a Pall 1440 40 micron filter. Venous return
was via the femoral venous cannula to which was attached a
(primed) 3/8" Y-connector and several feet of 3/8"x3/32" line
which was allowed to drain into a covered pail with 1" of
corn oil in the bottom (to minimize exposure of staff to
formalin). Approximately 15-20 liters of Trump's storage
fixative containing 7.0 M glycerol (to which 100 ml of India
Ink was added) was then perfused open-circuit.

Following fixative perfusion the animal was immediately
dissected and samples of heart, lung, liver, pancreas,
spleen, kidney, and skeletal muscle were collected for
subsequent histological and ultrastructural examination.
Samples of these organs were immediately immersed in chilled
glycerol containing Trump's fixative. All organs were
multiply sectioned both sagitally and coronally to evaluate
the degree of reperfusion, as indicated by distribution of
India Ink.

The brain was then removed en bloc to a flask containing 300-
400 ml of fixative with 7M glycerol (sufficient to cover
the brain completely). The brain was momentarily removed
from this fixative bath and each hemisphere was sectioned
(incompletely) both coronally and sagitally to evaluate
distribution of fixative/ink and the integrity of the
capillary bed. The brain was then returned to the glycerol
containing fixative and refrigerated overnight prior to the
cutting of sections for microscopy.

The following day coronal sections of the left cerebral
hemisphere at the level of the hippocampus of varying
thickness (from 5 mm to to 1 mm) were cut, placed in fresh
glycerol containing Trump's and shipped on ice to an academic
facility for processing by a professional electron
microscopist using standard techniques. Prior to normal
preparative procedures for EM the tissue was subjected to
multiple washings with Trump's fixative containing
progressively lower concentrations of glycerol over a period
of about 1 week until all the glycerol was washed out. During
final sample preparation for electron microscopy, care was
taken to avoid using the cut edges of the tissue sections in
preparing the Epon embedded sections.


Deglycerolization of Samples

As noted above, in order to avoid osmotic shock all tissue
samples were initially perfused with and immersed in Trump's
fixative containing 7M glycerol and were subsequently
deglycerolized prior to staining and embedding by stepwise
incubation in Trump's containing decreasing concentrations of
glycerol. The need to use such an approach on presumably
well-fixed and thus presumably osmotically "desensitized"
tissues may seem without foundation. However, both we and
other investigators have found a significant injurious effect
of simply immersing fixed tissue loaded with multimolar
concentrations of glycerol into glycerol free (and thus by
comparison very hypo-osmolar) fixative (12).


  <<< To be Continued >>

From: Mike Darwin <75120.575@compuserve.com>
Subject: BPI Tech Brief 16: Canine Brain Cryopreservation (2/2)
Date: 02 Jun 1995

    ---- CryoNet Message Auto-Forwarded by <kqb@cryonet.org> ----

From: Mike Darwin <75120.575@compuserve.com>
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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