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

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. 




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 


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. 

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 

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 

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. 


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. 


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. 


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 

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: 

                   Mc =  --------  Mp
                          Vc + Vp


     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 


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. 


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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