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