Message: #2567 - BPI Tech Brief #002
Date: 18 Jan 94 23:32:00 EST
From: Mike Darwin <>
Message-Subject: CRYONICS  BPI Tech Brief #002


     Recently there has been a great deal of discussion about the need for 
"cryonics," or brain cryopreservation research.  It is our opinion that a 
good place to start such research is with an investigation into exactly 
what the limitations of current brain cryopreservation technology are.  It 
is rather amazing that cryonics organizations are spending upward of 
$400,000 a year on operations (with another 200-300K being used/set aside 
for actual cryopreservation operations) with virtually NO direct evidence 
in-hand about what the quality is of the preservation they offering.  While 
much indirect evidence is cited, there is currently (to our knowledge) NO 
cryonics organization which offers ultrastructural studies documenting the 
level of preservation they are able to offer their clients under optimum 
(let alone sub-optimum) conditions.

     Soliciting people as clients for cryopreservation is reasonable, but 
we believe that it should be accompanied by evidence documenting what is 
REALLY being done to the client (patient) when the treatment is 
administered.  It is our position that there would be fewer clients and 
*considerably* more research on brain cryopreservation were such 
documentation available.

     However, ultimately it is not opinion that counts here, but rather 
facts.  BPI and Cryovita Laboratories are committed to doing this research 
and answering this question.  The research protocol which follows is the 
first step in that direction. During the coming year BPI and Cryovita hope 
to work together to conduct this research and get those answers.  Those 
wishing to join us in this effort are encouraged to do so.

BPI Protocol For Brain Cryopreservation Research


by Michael Darwin 


     The central problem of human cryopreservation is the preservation of 
the human brain in a sufficiently intact state to allow for future repair 
and recovery, with restoration of life and health in individuals so treated 
(1).  A necessary prerequisite to the possibility of future repair is that 
sufficient brain structure be preserved to allow the determination of the 
healthy, functioning state of the brain from the injured, non-functioning 
state resulting from illness, ischemia and cryopreservation (i.e.,the 
information-theoretic criterion) (2).  The ideal solution to this problem 
would be the development of a cryopreservation protocol that would allow 
for non-injurious, long-term suspended animation of the brain.

Current State-Of-The-Art

     The best available evidence, while far from complete, suggests that 
current organ cryopreservation techniques which have been adapted for use 
in human cryopreservation cause serious injury, largely as a result of 
toxicity (3) and mechanical injury to tissue from ice formed during 
freezing (4, 5, 6).  In-house studies conducted on adult cats subjected to 
cryopreservation protocols similar to those currently in use on human 
patients suggest that the brain may be one the most severely cryoinjured 
organ (7).  Electron microscopy of cerebral cortex taken from these animals 
following glycerol perfusion to 3M, cooling to -196*C at rates used in 
human suspensions, slow rewarming to just below 0*C, and fixation in the 
presence of cryoprotectant, has disclosed serious injury on several levels.

     On the macroscopic level, fracturing of the brain occurs. These 
fractures frequently completely penetrate the brain and result in 
fragmentation of the brain into two or more discrete pieces.  This injury 
is thought to result from the creation of internal strains associated with 
contraction on cooling following solidification of the system at the glass 
transition point (Tg).  There is both direct and theoretical evidence that 
fracturing may be safely avoided by not cooling the brain significantly 
below Tg.  This appears to be a biologically safe strategy, at least for 
intermediate periods of storage (years to decades) as a result of the 
substantial, if not complete, arrest of biochemical activity produced by 
both the low temperature and the cooling below Tg.

     On the histological level, there is widespread evidence of disruption 
of tissue architecture, presumably as a result of the formation of 
mechanically damaging amounts of ice.  The neuropil is uniformly peppered 
with ice cavities .5 to 1u in diameter at 2u to 5u intervals. There is also 
frequent separation of the neuropil from neuronal cell membranes.  
Occasionally there are large cavities on the order of 10u to 20u in 
diameter, which may be ice cavities or tears resulting from ice formation. 
Brain capillaries are frequently separated from basement membrane or from 
neuropil.  There is also evidence of the disruption of long fibers (such as 
axons), presumably as a result of ice formation.

     On an ultrastructural level, the damage is far more apparent and far 
more widespread.  There is often apparent loss, or alteration of ground 
substance, and damage to sub-cellular components.  In particular, the 
mitochondria and myelin appear to be severely damaged.  Mitochondria are 
often present only as dilated, debris-filled cavities with occassionally 
recognizable cristae. The myelin is often swollen, "shredded", or unraveled 
in appearance.  Ruptured endothelial cells and capillaries are littered 
with cellular debris, and ice cavities and interstitial spaces frequently 
contain debris.

     Also in evidence is the poor permeability of the brain to glycerol.  
Many axons are surrounded by large shrinkage cavities with the axon inside 
being dehydrated in appearance and electron dense.  Often these periaxonal 
shrinkage spaces contain unraveled myelin or other debris.

The Nature Of The Problem

     The histological and ultrastructural studies performed by Cryovita and 
Alcor, as well organ cryopreservation studies reported in the literature, 
suggest that adequate cryoprotection is not being achieved.  The especially 
poor results with the brain (the kidney and heart were far better preserved 
in the in-house work) are deeply disturbing.  Most current theories of 
memory posit encoding of learned behavior in either connections between 
neurons (8, 9, 10), or in individual neurons in the form of altered 
morphology of the synapses (11).  Widespread disruption of brain 
architecture on every level, including damage to the ground substance, may 
degrade memory storage mechanisms sufficiently to prevent recovery of 
individuals subjected to cryopreservation today.  

     Clearly, a protocol of cryoprotection which inflicts less injury to 
the brain is highly desirable.  Such a protocol must, at a minimum, achieve 
the following:

     1) be applicable to the human brain,

     2) be affordable (defined here as costing not more than 2-3 times the 
current cost of human cryopreservation as practiced by the Alcor Foundation 
of Riverside, CA circa 1992),

     3) offer a significant reduction in histological and ultrastructural 
disruption of the brain,

     4) reduce or eliminate poor cerebral penetration of cryoprotectant 
agent, and

     5) have an acceptable level of toxicity.

     Fahy et al. (12, 13) have demonstrated reasonable histological 
preservation of brain tissue using concentrations of glycerol as low as 3M 
with superior preservation observed at 6M glycerol.  However, dehydration 
was observed when 6M glycerol was introduced at 10*C versus 25*C.
     A modest amount of work has been conducted evaluating other 
cryoprotectants for the brain, but in general these agents suffer from one 
or more of the same problems as glycerol: poor permeability and/or the need 
to introduce very high (and no doubt toxic) concentrations in order to hold 
ice formation to a point compatible with survival of the organ.  The work 
of Smith (14) and Storey (15) indicates that, while vertebrates can 
tolerate a large fraction of organ or total body water converted into ice, 
the upper limit would seem to be about 60%, with 50% being the more 
conservative limit.

     In order to achieve this limit on ice formation during 
cryopreservation with glycerol it would be necessary to replace 
approximately 35% of the tissue water on a volume/volume basis with 
glycerol (16).  Further, because of the large mass of the human brain, it 
would be necessary to cool it slowly.  This, in turn, would result in the 
brain being exposed to the equilibrium freezing point throughout cooling, 
exposing it to a terminal concentration of glycerol of > or = 68% (v/v).  
The situation is the same with other commonly used cryoprotectants such as 
1,2 propanediol and Me2SO (17).

The Need for Additional Research

     The results observed in the Alcor/Cryovita study conducted in the mid-
1980's were subject to a number of important caveats and limitations:

     1) No histology or electron microscopy was performed on glycerolized 
brains perfused with fixative prior to cryopreservation.  Thus it is 
impossible to determine which histological and ultrastructural changes were 
the result of the effects of glycerolization and which were the result of 

     2)  Tissue was not deglycerolized after cryopreservation and prior to 
fixation, making it impossible to determine which changes were as a result 
of dehydration secondary  to glycerolization, and which were as a result of 
cryopreservation.  Also, the "compressed" appearance of the tissue, 
presumably a result of glycerol-related dehydration, made effective 
evaluation of ultrastructure impossible.  A series of experiments in which 
animals are glycerolized and deglycerolized in the absence of 
cryopreservation are critical to understanding the cause(s) of the changes 

     3) Tissue was not stained and examined at the light microscopy level 
and then subjected to electron microscopy in order to allow for  
correlation between light and EM examinations, nor was a histologically 
precise area of the cerebral cortex examined repeatedly.

     4) Gross fracturing of the brain, which occurred upon cooling to below 
Tg, made reperfusion with fixative impossible, thus introducing the 
possibility of autolytic tissue changes as a result of poor fixation of the 
brain tissue block; a phenomenon known to occur in central nervous tissues 
prepared in this way for electron microscopy.

     5) There have been major changes in the cryopreservation protocols 
used for humans since the Alcor/Cryovita study was performed. The most 
relevant of these changes was the introduction of 6M glycerol vs. 3M 
glycerol used in the early and mid-1980's, and complete redesign of the 
base perfusate, including the use of sucrose, a known membrane 
cryoprotectant, as the impermeant osmotic agent.

     Given the problems with the study delineated above and the major 
changes in the suspension protocol since this study was conducted, there is 
clearly a need for additional research to establish the degree of 
histological and ultrastructural preservation of the mammalian brain being 
achieved with the human cryopreservation protocols currently in use.

Proposed Research

     It is proposed that a study be conducted by Cryovita Laboratories to 
evaluate the effectiveness of the current human clinical cryopreservation 
protocol employed by Alcor.  This study involves 5 related experiments as 

1) Pre-glycerolization fixation.  Two animals in this group are subjected 
to anesthesia, surgery, and cannulation per the same protocol used for all 
other animals in the study, followed by perfusion with fixative and 
preparation of tissues for light and electron microscopy. The concentration 
of fixative (modified Karnovsky's) is increased linearly to terminal 
concentration over a 30-minute period to prevent cellular dehydration from 
osmotic effects.

2) Glycerolized to 6M at 10*C.   Three animals are perfused to a terminal 
concentration of 6M glycerol at a rate of 30 mM/min. at 10*C, after which 
they are perfused with 6M glycerol perfusate containing fixative.   The 
concentration of fixative (modified Karnovsky's) is increased linearly to a 
terminal concentration over a 30-minute period to prevent cellular 
dehydration from osmotic effects.  Tissue is then prepared for histological 
and ultrastructural examination.

3) Glycerolized to 6M at 20*C.   Three animals are perfused to a terminal 
concentration of 6M glycerol at a rate of 30 mM/min. at 20*C after which 
they are perfused with 6M glycerol perfusate containing fixative.  The 
concentration of fixative (modified Karnovsky's) is increased linearly to a 
terminal concentration over a 30-minute period to prevent cellular 
dehydration from osmotic effects.  Tissue is then prepared for histological 
and ultrastructural examination.

4) Glycerolized/deglycerolized.  Three animals are glycerolized to 6M at 
the temperature determined to be optimum in experiment 3 and 4 after which 
they are are deglycerolized using an appropriate protocol of controlled 
removal of glycerol employing an osmotic antagonist (mannitol).  Following 
deglycerolization, the brains are perfused with fixative and prepared for 
histological and ultrastructural examination. The concentration of fixative 
(modified Karnovsky's) is increased linearly to terminal concentration over 
a 30-minute period to prevent cellular dehydration from osmotic effects.

5)  Glycerolized and cooled to -80*C  Three animals are glycerolized to 6M, 
cooled to -80* at 4*C per hour, re-warmed from -80*C at 4*C per hour and 
perfused with fixative at 4*C.  The concentration of fixative (modified 
Karnovsky's) is increased linearly to a terminal concentration over a 30-
minute period to prevent cellular dehydration from osmotic effects.

6) Glycerolized, cooled to -80*C and deglycerolized.  Three animals are are 
glycerolized to 6M, cooled to -80*C, deglycerolized upon rewarming and 
fixative perfused, followed by histological and ultrastructural 
examination.  The concentration of fixative (modified Karnovsky's) is 
increased linearly to terminal concentration over a 30-minute period to 
prevent cellular dehydration from osmotic effects.

7) Freeze substitution at -80*C following glycerolization and freezing.  
Three animals are glycerolized to 6M, cooled to -80*C, sectioned in 5mm 
slabs and freeze substituted in methanol-osmium for light and electron 

     During glycerolization and deglycerolization of all groups the 
following laboratory studies are performed:

     *determination of oxygen uptake
     *determination of glucose uptake
     *arterial and venous pH
     *perfusate lactate levels
     *perfusate CK, CKMB, and LDH levels
     *perfusate sodium, potassium, chloride, and calcium levels
     These experiments will serve to expand understanding of the metabolic 
effects of glycerolization and cryopreservation on the mammalian central 
nervous system.


     The purposes of this study are to determine the histological, 
ultrastructural and metabolic effects of a cryopreservation protocol 
currently being used by Alcor. 

     Rabbits are the animal of choice for this study for the following 

     1) A significant body of previous work on brain cryopreservation has 
been carried out on rabbits, which provides a baseline of information.

     2) The cost of base perfusate ingredients and cryoprotective agents 
makes application of this research to a larger animal model impractical.

     3) Due to logistic constraints the use of smaller animals is not 

     It is anticipated that 15 animals will be used in the study as 
detailed under the Proposed Research above. 

     These numbers were chosen to allow for some confidence in 

Animal Welfare and the Utility of the Proposed Investigation

No Needless Duplication

     A careful review of the literature has been conducted using both 
MEDLINE and manual methods to determine if there is any alternative to 
using animals to obtain the information sought this study.  It is the 
determination of the Principal Investigator (and outside consultants) that 
the work proposed herein does not duplicate any experiments reported in the 
literature at the time the evaluation was conducted.

Avoidance of Pain/Discomfort
     All procedures used in this study will avoid or minimize pain, 
discomfort, and distress to the animals.  Since this is a sacrifice study 
no animal will recover from the initial anesthesia. All surgical procedures 
will be conducted under general anesthesia.  Pain/discomfort will be 
confined to restraint of the animal in a standard rabbit restrainer, and 
the ear-stick used to obtain venous access to facilitate induction of 

Living Conditions/Housing and Personnel

     Animals will be acquired from the supplier the night before the 
experiment and housed in stainless steel cages for less than 24 hours prior 
to onset of the experiment.  The animal cages comply with Part 3, 
Subchapter A of the Animal Welfare Act.  

     Personnel conducting procedures on the animals have been appropriately 
trained to carry them out competently and with minimum discomfort to the 
animal.  Personnel and their qualifications are listed below:

     Gregory M. Fahy, Ph.D.:  Dr. Fahy is a professional cryobiologist with 
extensive experience in studying the effects of cryopreservation of the 
rabbit central nervous system.

     Steven B. Harris, M.D.: Dr. Harris is a physician with 10 years of 
experience in small animal research including rabbits at the UCLA Medical 
Center in Los Angeles.  

     Michael Darwin, C.R.T.: Mr. Darwin is a hemodialysis technician and 
non-certified cardiopulmonary perfusionist with 12 years of experience in 
dog and rabbit research.

Veterinary Care

     Veterinary medical care for animals will be provided through our 
consulting veterinarian. 

Materials and Methods General

Experimental Animal

     The rabbit has been the animal used in most published and unpublished 
work on brain cryopreservation. Further, the investigators have extensive 
experience with the care, handling, anesthesia, surgical management, and 
the effects of cryopreservation on this animal.  For these reasons, and due 
to the need to contain costs (rabbits are both inexpensive and have small-
volume heads/brains with a resultant decrease in volume requirements of 
cryoprotectant), it is proposed that the rabbit be used for this study, and 
in particular the New Zealand White rabbit.

Experimental Preparation

     While the target organ for this study is the rabbit brain, practical 
considerations dictate that the brain be maintained within the head 
(cephalon) during the cryopreservation protocol.  Working with an intact 
head preparation has the advantages of: 1) providing thermal and mechanical 
protection of the brain during surgical manipulation, perfusion, cooling, 
rewarming and initial reperfusion/evaluation, 2) reducing the surgical time 
and surgical skill level required to carry out the work, and 3) most 
closely approximating the human whole-body and neurosuspension models.  The 
cephalon model will also have an added advantage in that it will generate 
modest amounts of other tissues subjected to perfusion/cryopreservation, 
which may be fixed and examined later if funding permits.

Surgical Protocol

     Anesthesia is induced in New Zealand White rabbits weighing at least 4 
kg by intravenous administration of 5 mg ketamine, 0.04 mg xylazine, and 
0.2 mg atropine per kg via the marginal ear vein.  Anesthesia is maintained 
by continuous IV infusion of Xylazine and ketamine into the marginal ear 

     Once anesthesia is secured, the ventral surface of the animal is 
shaved, a tracheostomy is performed, and the animal is maintained on a 
respirator.  Type T thermocouple probes are then placed in the rectum and 
the oral pharynx and secured with surgical staples or suture to preclude 
movement or dislodging.  The animal is positioned in a tub of crushed ice 
prior to the start of surgery.

     A 3 cm incision is made at the lateral surface of the neck beginning 
at the caudal edge of the transverse process of the 1st cervical vertebra.  
The subcutaneous tissues are incised and the splenius muscle  is identified 
and reflected dorsally to expose the intratransversarius cervicus dorsalis 
muscle.  The intratransversarius cervicus dorsalis and the 
intertransversarius intermedius muscles are separated to expose the 
vertebral artery as it passes out of the transverse foramen of the second 
cervical vertebra.  The artery is identified and ligated with a silk tie.  

     Next, the carotid artery is isolated from the nerve trunks that are 
adjacent to it in the carotid sheath, and two ligatures are placed around 
it so that it can be cannulated. The internal jugular veins  are handled 
similarly.  Two-thirds of a 12-inch length of umbilical tape is placed into 
a space created between the cervical vertebra dorsally and the trachea 
ventrally.  The remaining one-third is left protruding from the incision.  
The wound is covered with saline soaked gauze, the rabbit is rolled over, 
and the surgical procedure is repeated on the other side.  

     Once the vertebral arteries are ligated and the carotid arteries 
isolated, the previously buried umbilical tape is brought out of the skin 
incision.  The right jugular vein is cannulated with a 2 cm length of 2 mm 
diameter polyethylene tubing, which is heat-sealed at the end opposite from 
that which is inserted into the vessel.  The umbilical tape ligature is 
then tightened by tying it around the perivertebral muscles at the level of 
the third cervical vertebrae, thereby occluding muscular branches 
anastomosing with the occipital branch of the vertebral artery.

     At this time, the rabbit's temperature will have been reduced by 
external cooling to approximately 30*C.  Sodium heparin, 30 mg/kg is given 
IV. The right carotid artery is cannulated using a custom, right angle 
polyethylene catheter, the heat-sealed end is cut from the tip of the  
right jugular catheter, and perfusion of base perfusate pre-chilled to 
10*C, of the composition shown in Table I, is begun at a pressure of 60 mm 
Hg by connecting the carotid artery cannula to one arm of a "Y" connector 
which is in turn connected to the perfusion apparatus.  Once the venous 
effluent is clear, the temperature of the perfusate is reduced to 1*C to 

     Concurrent with the initiation of hypothermic perfusion, the 
contralateral carotid artery and jugular vein are ligated and cannulated.  
Once the second carotid artery is cannulated, the cannula is connected to 
the other arm of the "Y" connector, the jugular cannula opened  and 
perfusion continued through both carotid arteries until the venous effluent 
is clear and the oral temperature is 5*C or below.  

     The arterial and venous cannulae are firmly secured to adjacent muscle 
using silk suture, and the vessels are severed between the ligatures and 
the cannula.  The surgical wound is expanded laterally and all structures, 
muscular and cutaneous, are severed by scalpel just below the level of the 
4th cervical vertebra.  The vertebral column and spinal cord are severed 
with Mayo Scissors and the isolated head is placed into the perfusion 

Materials and Methods - Preglycerolization Fixation

     After preparation of the cephalon for perfusion using the procedure 
above, it is positioned in the perfusion apparatus, which has been primed 
with modified Karnovosky's fixative. Fixative is introduced at a pressure 
of 60 mmHg and a temperature of 10*C.  Fixation and preparation of sections 
for light and electron microscopy is discussed below.

Materials and Methods - Glycerolized/Deglycerolized

Cryoprotective Loading/Unloading Perfusion Apparatus

     The perfusion apparatus consists of two 2-liter reservoirs; one for 
cryoprotectant concentrate and one for the recirculating system.  The 
recirculating reservoir is positioned atop a stir-table and stirred 
continuously with a teflon coated magnet.  The glycerol concentrate 
reservoir is connected to the recirculating reservoirs by a section of 3/8" 
diameter Tygon tubing.  The recirculating reservoir supplies a perfusion 
circuit consisting of a Drake-Willock Model 7401 hemodialysis pump, a CD 
Medical 90 SCE low priming volume hollow fiber dialyzer (serving as an 
oxygenator), a Gish extracorporeal aluminum heat exchanger, a Pall ECF-40 
40u Ultipor pediatric extracorporeal blood filter, and a "Y" connector with 
luer port capable of accepting the tubing connections to the carotid artery 
cannula and the monitoring line of a pressure transducer. Tubing on the 
arterial side of the system is medical grade PVC throughout.  

     Venous effluent is collected in a funnel positioned under the head 
from 2 cm long stent cannula in the jugular veins and any drainage from the 
head stump, and returned to the recirculating system.  The funnel is 
connected to the recirculating reservoir with 3/8" silastic tubing.

     The entire perfusion assembly is enclosed within a temperature control 
cabinet consisting of a Fisher Scientific 148G Chromatography refrigerator, 
with sliding glass doors to permit continuous observation of the system and 
reach-through capability to allow adjustment and manipulation of the 
controls without loss of the temperature controlled environment surrounding 
the perfusion apparatus.

     The perfusion circuit is instrumented with a type T thermocouple probe 
placed immediately before the 40 u arterial filter and a Trantec 800 
pressure transducer connected to the luer port of the arterial "Y" 
connector and monitored by a Tetronix Model 414 monitor.  The transducer 
and monitor is external to the refrigerated compartment of the apparatus.

     A Drake-Willock Model 7401 dialysis pump is connected by a T to both 
the recirculating reservoir and the venous return line.  This pump is then 
adjusted to remove perfusate from the recirculating system at a 
predetermined rate, causing glycerol concentrate to flow into the 
recirculating system at the same rate.

Cryoprotective Perfusion Protocol

     The flushed, chilled cephalon is placed within the refrigerated 
cabinet of the perfusion apparatus and positioned with a Stoddard clamp, 
stump down, over the venous return funnel.  Closed circuit perfusion is 
initiated at a pressure of 60 mm Hg and a temperature of 10*C.  

     When stable, closed circuit perfusion is achieved, the cryoprotective 
ramp is begun and the concentration of glycerol in the recirculating system 
is increased at a rate of 30 mM per minute up to the desired terminal 
concentration of 6M glycerol.  Cephalons to be perfused at 20*C are warmed 
by circulation of perfusate at 20*C. When the target concentration of 
glycerol is reached, the temperature of the cephalon is reduced to 2*C by 
reducing the perfusate and air temperature in the cabinet.  When the 
cephalon has reached 2*C, glycerol perfusion is discontinued. 

Glycerolized And Cooled to -80*C

     Following perfusion to 6M glycerol per the procedure above, the 
carotid and venous cannula connecting tubes are clamped, the cannula 
disconnected, and the cephalon placed within a 3-mil polyethylene bag and 
submerged in a bath of 5 cs. polydimethylsiloxane oil (Silcool) precooled 
to -30*C, 4*C below the freezing point of a 6M glycerol solution, with 
approximately 300 mOsm of salts.  The cephalon is seeded with ice and held 
at this bath temperature until the core temperature of the cephalon (as 
measured by the TC probe in the oral pharynx) reaches -26*C.  The cephalon 
is then cooled at a rate of 4*C/hr. to -80*C, where it will remain for 4 

     Following glycerolization and cooling to -80*C, the cephalon is 
removed from the -80*C bath and transferred to another bath at -15*C where 
it is held until the core temperature reaches -20*C, at which point it is 
transferred to a bath at 0*C.  When the core temperature reaches 0*C, the 
cephalon is removed from the bath, placed within the perfusion apparatus, 
and the arterial and venous cannulae are reconnected.  The arterial cannula 
and lines are cleared of air and perfusion with the terminal concentration 
of glycerol reached during cryoprotective perfusion is initiated.  Fixation 
is achieved by perfusion of modified Karnovsky's in glycerol perfusate of 
this same concentration.  Introduction of fixative is by linear gradual 
addition during a period of 30-minutes. 

Glycerolization, Cooling to -80*C, and Deglycerolization

     Following glycerolization and cooling to -80*C and rewarming to 0*C, 
the cephalon is transferred to the perfusion apparatus and the arterial and 
venous cannulae reconnected.  The arterial cannula and lines are cleared of 
air and perfusion with the terminal concentration of glycerol reached 
during cryoprtective perfusion is initiated.  

     Glycerol concentration is then reduced in the circulating perfusate 
(using the reverse of same technique employed to introduce it) at a rate of 
approximately 30 mM glycerol per minute, using 300mM mannitol  as the 
osmotic antagonist (18). Deglycerolization is carried out at 10*C.  
Following deglycerolization, the cephalon is perfused with modified 
Karnovsky's fixative (fixative slowly introduced) in base perfusate and 
prepared for light and electron microscopy.

Data Acquisition Protocol

     All significant milestones are noted as they occur.  Below are listed 
critical milestones which are recorded for each animal.  
*  The following data are recorded on the Operative Data Collection Sheet:

     General Condition
     Time Baseline Lactate Drawn
     Time Baseline Blood Chemistries Drawn
     Baseline Serum Sodium, Potassium, Calcium, and Chloride Readings
     Pre and Intraoperative Medications
     Time and Dose of Anesthetics
     Time of Anesthetic Induction
     Baseline Temperature Readings (Esoph. and Rectal)
     Baseline Pulse Oximetery Readings
     Time Surgery Commences
     Time of Cannulation 
     Cannula Size and Location
     Baseline Arterial, CV Pressures
     Baseline Blood Gases 
     Time Cryoprotective Perfusion Starts
* Once perfusion is begun, the following data are recorded at 15 minute 
intervals on the Perfusion Data Collection Sheet. (Please note that all 
gases and electrolyte data are printed by the Nova Stat 5 at the time the 
test is conducted).

     Esoph Temp.
     Surgical Pulse Oximetery Readings
     Arterial Temp.
     Arterial Pressure
     Arterial Flow Rate
     Arterial pH
     Arterial HCO3
     PvO2 and 
     Venous pH
     Venous HCO3
     Base Excess
     Serum Sodium, Potassium, Calcium and Chloride
     arterial glycerol concentration (by refractometry)
     venous glycerol concentration (by refractometry)
     % O2 in gas being delivered to oxygenator
     Flowrate of gas to oxygenator

*  Perfusate samples in serum separator tubes are collected at the start of 
perfusion and at 30-minute intervals until the conclusion of perfusion. 
Samples are promptly spun down after collection.  

*  The time perfusion is started and completed is clearly noted, along with 
flowrates into the animal, and arterial and CV pressures during perfusion.  
Vascular resistance is calculated from arterial pressure and flowrate.

Laboratory Tests

      For both whole blood and perfusate, sodium, potassium, calcium, 
chloride, glucose pH, pO2 and pCO2 determinations are made with a Nova Stat 
5 blood gas and electrolyte analyzer (Nova Biomedical, Boston, MA).  Tissue 
oxygen saturation levels are measured using a CSI 503 pulse oximeter.  

     Blood/perfusate samples are drawn by syringe and analyzed immediately.  
Chemistry analysis samples are collected in serum separator or red stopper 
vacutainers (Becton-Dickinson, Rutherford, NJ) as appropriate, and analyzed 
using a Kodak Ektachem DT clinical chemistry analyzer.  Back-up blood gas 
equipment is an Instrumentation Laboratories IL 1302.  Back-up equipment 
for sodium and potassium analysis is a Nova 1 sodium/potassium analyzer.

Procedure for Fixation

     Cephalons to be prepared for light and electron microscopy are 
perfused with modified Karnovsky's of the Composition shown in Table II.  

     In those instances where fixation is to occur before the tissue is 
unloaded of glycerol, Karnovsky's is prepared with appropriate 
concentrations of glycerol and introduced using linear gradual addition 
during a period of 30 minutes to avoid osmotic shock.

     Immediately following perfusion with this fixative or glycercol-
fixative solution, the brain is dissected from the head and submerged in a 
minimum of 250 cc of the same solution, freshly prepared.  Brains are held 
at least overnight in this solution before cutting tissue blocks for 

     Tissue blocks for evaluation by electron microscopy will be cut from 
the right frontal lobe of the cerebral cortex and from the CA1 area of the 
hippocampus. Tissue blocks are 5 mm thick to minimize the possibility of 
sectioning artifacts.

Freeze Substitution Protocol

     Cephalons to be evaluated by freeze substitution  are thermally 
equilibrated at -80*C and transferred to a mixture of 95% ethanol and dry 
ice for sectioning.  A Dremel craft tool is then used to cut 5mm coronal 
sections of the brain, keeping the blade immersed in the ethanol bath 
during sectioning.  Sections are then transferred to anhydrous methanol for 
freeze substitution at -80*C.  The methanol bath is at least 20 times 
greater in volume than the tissue block and is changed after 24 and 48 
hours, and at intervals of 1 week during the four weeks the sections are 
maintained at -80*C.  Samples for electron microscopy are fixed by the 
addition of 1% OsO4.  Samples for light microscopy are substituted in 
anhydrous methanol only.

Light and Electron Microscopy

     Tissue blocks for light and electron microscopy are prepared by making 
5 mm thick coronal sections through both cerebral hemispheres so as to 
include the CA1 area of the hippocampus. Sections are sent to American 
Histolabs of Rockville, MD and Electron Microscopy Consultants of Tuscon, 
AZ. A variety of stains are used for light microscopy including Bodian and 
Hematoxylin and Eosin.

     Electron Microscopy Consultants (EMC) prepare some samples in such a 
way that they can be examined by both light microscopy and transmision 
electron microscopy (TEM).  EMC will sample the coronal sections for TEM in 
a uniform way, dehydrate the samples in anyhydrous ethanol, embed them in 
Epon, and cut thin sections using a Reichert Ultracut Ultramicrotome.  Thin 
sections are stained with uranyl acetate and examined using a JEOL 100 CX 

     Procedures for preparation of freeze substituted samples for light and 
transmission electron microscopy follow those of Hunt (19).  Sections to be 
prepared for light microscopy are transferred to precooled ethanol at -40*C 
and warmed slowly to room temperature.  Slices are then fixed in 1% HgCl2 
in ethanol for 1-hour, washed in 0.5% I2 in ethanol, and embedded in wax.  
Sections are stained with Bodian's stain and examined by light microscopy.

     After freeze-substitution, the samples to be prepared for TEM  are 
allowed to warm slowly to 4*C before transfer to anhydrous ethanol.  
Samples are then taken by punch biopsy under ethanol, and embedded in Epon.  
Thin sections are cut using a Reichert Ultracut Ultramicrotome, stained 
with uranyl acetate and examined using a JEOL 100 CX TEM.

Future Research

     It is emphasized that the primary objective of this study is to assess 
the efficacy of the human cryopreservation protocol now in clinical use at 
Alcor in preserving brain histology and ultrastructure. This protocol is 
not likely to contribute significantly to improving the the outcome, but 
will most likely be useful in defining the problem.

     In the event that cryopreservation following equilibration with 6M 
glycerol results (as expected) in significant histological, 
ultrastructural, or metabolic disruption, further research will be needed 
to discover less injurious techniques.  Screening of alternative 
cryoprotectants and/or mixtures of cryoprotectants using a brain slice 
model as proposed by Fahy (20) is probably the most effective next step.

     In the event that good histological and ultrastructural preservation 
is observed with 6M glycerol, with evidence of the conservation of 
reasonable metabolism (i.e., glucose and oxygen utilization), the next step 
will be to carry out functional evaluations of the brain by evaluating 
tissue Na++/K+ ratios, electroencepholography (EEG) and EEG with visual and 
auditory evoked potentials.

        TABLE I - Formula for SHP-1 Base Perfusate
Component                 Molar Concentration   Grams/Liter
Sucrose                   170.0  (MW 342.30)       58.19            
Adenine HCl                 0.94 (MW 180.6)         0.17       
D-Ribose                    0.94 (MW 150.2)         0.14             
Sodium Bicarbonate         10.00 (MW 84.0)          0.84  
Potassium Chloride         28.3  (MW 74.56)         2.11 
Calcium Chloride                                 
10% (w/v) soln.             1    (MW 111)           0.11 
Magnesium Chloride                               
20% (w/v) soln.             1    (MW 95.2)          0.095            
Sodium HEPES               15    (MW 260.3)         3.90             
Glutathione (free acid)     3    (MW 307.3)         0.92             
Hydroxyethyl Starch       ----                     50.00             
Glucose                    10    (MW 180.2)         1.8              
Heparin                   ----                    1,000 IU           
Total Osmolality: 303

pH is adjusted to 8.0 with potassium hydroxide. 

TABLE II - Composition Of Modified Karnovsky's Solution

Component                g/l

Paraformaldehyde           40
Glutaraldehyde             20
Sodium Chloride            0.2
Sodium Phosphate           1.42
Calcium Chloride           2.0 mM

pH adjusted to 7.4 with sodium hydroxide.

Table III - Proposed Budget

Quantity                       Item                          Ext. Price
1            Rabbits, New Zealand White                          $35.00
1            Shipping (for rabbits)                                5.00
1            24-hour housing                                       2.00
1            Filter, 0.2 micron,                                   5.00
1            Oxygen, 20 cu. ft.                                    2.00
1            Temperature Monitor, TC, use                          5.00
2            Probes, TC, use                                        .50
1            Heater-Cooler, Blanketrol, use                       10.00
1            Tracheostomy Equipment, use                           1.00
1            Tape, Assorted                                         .50
1            Probe, Temperature, Blood Path, YSI, use               .50
1            Batteries, assorted, use                               .50
3            Monitors, Tektronix, use                             15.00
1            Oxygenator, (Dialyzer, Travenol 14:11),              22.50
1            Reservoirs, use                                       1.00
1            Filter, Arterial                                     34.00
1            Ventilator, Harvard                                   5.00
1            Tubing Set, Extracorporeal                           15.00
1            Instruments, Surgical                                10.00
2            Cannula, Venous, Type 1967                            2.00
1            Solutions, Calibration, Blood Gas                    15.00
1            Dome, Pressure Monitoring, Trantec                    1.00
1            Dome, Pressure Monitoring, Statham                    1.00
3            Transducer, Statham, Trantec, use                    10.00
1            Stopcock, 3-gang                                      6.76
1            Cuvette, Temperature Sci-Med Temp                     1.20
2            Monitor Line, M/M, 4 ft.                               .40  
10           Perfusate, Glycerol/SHP-1, liters                   150.00
2            Monitor Lines, M/F, 6 ft.                             1.08
1            Catheter, Arterial Pressure                           2.25
6            Gloves, Exam                                          4.70
1            Ringer's Solution, 500 cc, Viaflex                    4.25
2            Scalpel Blades, #10 & #11                             2.34
1            Suture, 0 Silk                                        3.75
2            Suture, #2 Silk                                       3.00
2            Saline, Irrigating, 500 cc                            3.50
1            Rompun/Ketalar, dose                                   .50
1            Heparin, 5,000 IU                                     1.00
4            Gauze, 4"x4", sterile, 10 ea.                         9.00
2            Angiocath, 14 ga                                      8.00
1            Robinson Catheter                                     2.25
1            Sets, I.V., Administration                            2.00
50           Syringes, 3 cc, Monoject                              4.00
20           Syringes, 5 cc                                        4.00
5            Syringes, 20 cc, Terumo                               1.75
1            Refrigerant, Dry Ice                                 28.00  
1            Cooler, Controlled Rate, use                         20.00
1            Silcool, use                                         10.00
1            Solvent, Freeze Substitution                         24.00
1            Freezer, -80*C, use                                  25.00
1            Fiaxtives (Formalin, Karnovsky's)                     5.00
5            Evaluation, PAL                                     180.00
6            Evaluation, Lactate, Ektachem                        24.00
6            Evaluation, Chemistry, Misc., Ektachem               24.00
1            Shipping, Federal Express                            18.00
2            Evaluation, Histology/Electron Microscopy           400.00
1            Forms, Data Sheets                                    5.00
1            Secretarial, Data/Manuscript Preparation             32.50
                                            TOTAL             $1,209.73


Researcher                                                       200.00
Research Assistant                                               100.00
                                             TOTAL            $  300.00

                                            GRAND TOTAL       $1,509.73

       TOTAL COST OF PROJECT:   $1,509.73 x 20 Experiments = $30,194.60

Note on Disposables Costs: Many of the disposable items, the costs of
which are specified above, are items which will be re-used.  Thus, the
specified cost reflects both the charge for reprocessing and
amortization of the new purchase price for the item since indefinite
re-use is usually not possible.


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