X-Message-Number: 2567
Date: 18 Jan 94 23:32:00 EST
From: Mike Darwin <>
Subject: CRYONICS BPI Tech Brief #002
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
PROPOSAL TO EVALUATE 6M GLYCEROL AS A
CRYOPROTECTANT FOR THE MAMMALIAN BRAIN
by Michael Darwin
Introduction
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
cryopreservation.
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
observed.
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
follows:
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
microscopy.
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.
Rationale
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
reasons:
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
feasible.
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
repeatability.
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
anesthesia.
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
vein.
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
2*C.
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
apparatus.
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
weeks.
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:
Weight
Age
Breed
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).
Time
Esoph Temp.
Surgical Pulse Oximetery Readings
Arterial Temp.
Arterial Pressure
CVP
Arterial Flow Rate
PaO2
PaCO2
Arterial pH
Arterial HCO3
PvO2 and
PvCO2
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
Remarks
* 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
microscopy.
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
TEM.
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
mM
-----------------------------------------------------------
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
Personnel
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.
-----------------------------------------------------------------------
References
1) Wowk, B., Darwin, M. Cryonics: reaching for tomorrow. Alcor. 12327
Doherty Street, Riverside, CA 92503. USA, 1991.
2) Merkle, R.C. The technical feasibility of cryonics. Medical Hypotheses
1992;36:6-12.
3) Clark, P., Fahy, G.M., and Karow A.W. Factors influencing renal
cryopreservation. II. Toxic effects of three cryoprotectants in
combination with three vehicle solutions in nonfrozen rabbit cortical
slices. Cryobiology 1984;21:274.
4) Pegg, D.E., Jacobsen, I.A., Armitage, W.J., and Taylor, M.J., Mechanisms
of cryoinjury in organs. In Pegg, D.E. and Jacobsen, I.A. (eds): "Organ
Preservation, Vol. 2." Churchill Livingstone, Edinburgh, pp. 132-33, 1979.
5) Fahy, G.M., Analysis of "solution effects" injury: cooling rate
dependence of the functional and morphological sequellae of freezing in
rabbit renal cortex protected with dimethyl sulfoxide, Cryobiology 18:550-
570 (1981).
6) Pegg, D.E., Ice crystals in tissues and organs. In Pegg, D.E., and
Karow, Jr., A.M., (eds): "The Biophysics of Organ Cryopreservation" Plenum
Press, New York, pp. 117-136, 1987.
7) Darwin, M., Leaf, J., Hixon Jr., H. The effects of cryopreservation on
the cat. In preparation.
8) Kandel, E.R., Schwartz, J.H., Principles of Neural Science. 2nd ed.
Elsevier, 1985.
9) Greenough, W.T., Bailey, C.H., The anatomy of memory: convergence of
results across a diversity of tests. Trends in Neurosci. 1988;11:4:142-47.
10) Lynch, G., Synapses, Circuits and the Beginnings of Memory. MIT Press,
1986.
11) Bailey, C.H., Chen, M. Morphological basis of long-term habituation and
sensitization in Aplysia. Science 1983;220:91-93.
12) Fahy, G.M., Takahashi, T., and Crane, A.M. Histological cryoprotection
of rat and rabbit brains, Cryoletters 1984;5:33-46.
13) Fahy, G.M., Crane, A.M. Histological cryoprotection of rabbit brain
with 3M glycerol. Cryobiology 1984;21:704
14) Smith, A.U. Studies on golden hamsters during cooling to and rewarming
from body temperatures below 0*C. II. Observations during and after
resuscitation. Proc. Royal Soc., Biology, Lond. Series B 1957;147:517.
15) Storey, K.B., Storey, J.M. Frozen and alive. Scientific American
1990;263:92-97.
16) Fahy, G.M., Levy, D.I., and Ali, S.E., Some emerging principles
underlying the physical properties, biological actions, and utility of
vitrification solutions. Cryobiology 1987;24:196-213.
17) Luyet, B.J., On the amount of water remaining amorphous in frozen
aqueous solution. Biodynamica 1969;10:277-291.
18) Pegg, D.E., Wusteman, M.C. Perfusion of rabbit kidneys with glycerol
solutions at 5*C. Cryobiology 1977;14:168-78.
19) Hunt, C.J. Studies on Cellular structure and ice location in frozen
organs and tissues: the use of freeze substitution and related techniques.
Cryobiology 1984;21:385-402.
20) Fahy, G.M. An approach to brain cryopreservation: a research agenda.
In preparation.
Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=2567