X-Message-Number: 4468
From: Mike Darwin <>
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 H20 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 yeilded 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 cmH20, 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 >>
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