EFFECT OF HUMAN CRYOPRESERVATION PROTOCOL
ON THE ULTRASTRUCTURE OF THE CANINE BRAIN

by Michael Darwin, Sandra Russell, Paul Wakfer, Larry Wood, and Candy Wood

(Originally published by BioPreservation, Inc., as BPI Tech Brief 16 on CryoNet and sci.cryonics May 31, 1995)

I. Introduction
II. Materials and Methods
III. Effects of Closed Chest Cardioipulmonary Support
IV Effects of Glycerolization
V. Gross Effects of Cooling to and Rewarming From -90 C
VI. Effects of Cryopreservation on Brain Ultrastructure
VII. Summary and Discussion

Clinical human cryopreservation has the objective of the preservation of brain structures which encode personal identity sufficient to allow for resuscitation or reconstruction of the individual should molecular nanotechnology be realized (1,2). Aside from the pioneering work of Suda, et al (3,4) and three previous studies conducted by cryonics organizations (5,6,7) there has been virtually no systematic effort to examine the fidelity of the ultrastructural preservation of the brain: particularly at the level of the neuropil and synaptic and intra-synaptic structures following cryopreservation using clinical (human) cryopreservation (“cryonic suspension”) techniques in either an animal or cadaver model.

Previous ultrastructural studies conducted by Cryovita Laboratories in conjunction with the Alcor Life Extension Foundation in the mid-1980's and more recently by Pichugin, et al., under the auspices of the Cryonics Institute have shown massive disruption of ultrastructure at every level. (5,6)

A brief summary of the lesions observed in these studies disclosed the following kinds of injury occurring uniformly throughout both the white and gray matter of the cerebral cortex in both cats (Darwin, et al) and sheep (Pichugin, et al):

a) ultrastructural-level tearing and fraying of the ripped ends of nerve tracts by osmotic contraction of cells coupled with the push of extracellular ice creating debris-strewn gaps at intervals of 5 to 100 microns in width;

b) separation of capillaries from surrounding brain tissue (visible both in the frozen state with freeze-substitution and upon thawing following fixation and embedding);

c) physical disruption of the capillaries due to intracapillary ice formation, lysis of the endothelial cells with occasional adherent endothelial cell nuclei, and separation of the endothelial cells from capillary basement membrane;

d) Separation of myelin from axons, formation of gaps between the axon membrane and the myelin, unraveling of the myelin, and frequent loss of intraaxonal material, possibly as a result of disruption of the axolemma;

e) Extensive disruption of the neuropil and of the plasma membrane of both neuronal and glial cells with conversion of intracellular and synaptic membrane structure into amorphous debris or empty and/or debris-containing vesicles.

The principal objective of this study was to survey the effects of glycerolization to a much higher concentration than has been used in past, principally 7.4M glycerol versus 4 to 5 M glycerol, freezing to -90 C, storage at this temperature for a period of at least 1 year, and rewarming at varying rates, on gross structure, histology, and ultrastructure of the canine brain using a preparation protocol similar to the one now used on human cryopreservation patients by BioPreservation, Inc. of Rancho Cucamonga, California.

The work described in this paper was carried out from October of 1993 to May of 1995. The technique used for cryopreservation of animals in this study closely paralells that used by BioPreservation (8) and by the Alcor Life Extension Foundation of Phoenix, AZ (9) in preparation of human patients for long-term cryopreservation.

It should be noted that training of staff in procedures for transport (closed chest cardiopulmonary support), total body washout (TBW) and cryoprotective perfusion and freezing of human cryopreservation patients was an important second goal of this study.

Preperfusion Procedures

Five adult dogs weighing between 24 and 28 kg were used in this study. All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guide for the Care and Use of Laboratory Animals” prepared by the National Institutes of Health (NIH Publication No. 80-23, revised 1978). Prior to induction of anesthesia the animals were given 0.5 mg/kg acepromazine maleate and 0.25 mg atropine IM. Anesthesia in both groups was secured by the intravenous administration of 40 mg/kg of sodium pentobarbital. The animals were then intubated and placed on a volume-cycled MA-1 ventilator using a tidal volume of 15 cc/kg, PEEP of 5 cm H20 and an FiO2 of 21.

EKG was monitored throughout the procedure until cardiac arrest was induced. Rectal and esophageal temperatures were continuously monitored during perfusion using copper constantan 20 gauge thermocouple probes (Instrument Laboratories 53-20-507). A 14 Fr. x 48 cm double lumen Salem Sump gastric tube was passed esophageally into the stomach (positioning verified fluoroscopically) to facilitate alkalinization of stomach contents with Maalox (alluminum hydroxide suspension) and prevent erosion of gastric mucosa during subsequent periods of ischemia, hypoperfusion, and hypothermia.

Following placement of temperature probes, an IV was established in the medial foreleg vein and a drip of Normosol-R (pH 7.4) at rate of 50 cc/hr was begun to maintain the patency of the IV and support blood circulating volume during surgery.

The animals were then placed in a Portable Ice Bath (PIB) identical to that used for transport of human cryopreservation patients and the chest was stabilized in a specially fabricated padded holder to allow for stable mid-sternal application of a Michigan Instruments Model # 1004 Thumper (external cardiac compressor/ventilator). (Figure 1)

Surgical Protocol

Both groins and the right neck over the entire length of external jugular vein were shaved and prepped for surgery using povidone iodine solution (Betadine). Since these were sacrifice studies, sterile technique was not used. However gloves, gowns and masks were used to protect staff from infection and simulate actual working conditions with human cases. For the same reasons, Betadine was used to simulate the appearance of the preoperative skin and kill skin flora minimizing risk of infection in the event of sharps injury to staff.

An Edwards adult Swan-Ganz catheter was placed via open cutdown of the right jugular vein and was wedged in the pulmonary artery. The proximal line of the Swan-Ganz catheter was connected to a Bentley Trantec 800 pressure transducer for measurement of pulmonary artery diastolic (PAD) and wedge pressures, and the thermistor cable was connected to an American Edwards COM-1 thermodilution cardiac output computer to facilitate measurement of cardiac output (CO) and core blood temperature during post-cardiac arrest Thumper cardiopulmonary support. The position of the Swan-Ganz catheter in the pulmonary artery was verified both by evaluation of the pressure waveform on a Tektronix 414 physiologic monitor, and fluoroscopically with Siemans Siemens, Inc. Siremobile II C-arm fluoroscope.

Both groins were cut down to access the femoral arteries and veins. An Argyle 18 Fr. pressure monitoring catheter connected to a Cobe 4-way stopcock was placed in the right femoral artery and connected via a Cobe large-bore pressure monitoring line to a Trantec 800 pressure transducer and Tektronix 413 monitor for measurement of mean arterial pressure (MAP).

Venous return was achieved using USCI type 1967 cannulae of either 21 or 22 Fr. diameter which were placed in both femoral veins and positioned under fluoroscopy: the right venous cannula being advanced up the inferior vena cava (IVC) to approximately the level of the right atrium, the left venous cannula being advanced to approximately the level of the renal veins.

Arterial perfusion was via a Cardiovascular Instruments 4.0mm or 4.5 mm ID stainless-steel cannula placed in the right femoral artery. Typical cannula placement is shown in Figure 2.

Extracorporeal Circuit

The extracorporeal circuit for the cryoprotectant treated animals (Figure 3) consisted of 1/4” (arterial) and 3/8” (venous) medical grade polyvinyl chloride tubing. The circuit was comprised of two sections: a recirculating loop to which the animal was connected and a glycerol addition system. The recirculating system consisted of a 20 liter polyethylene reservoir positioned atop a magnetic stirrer with a floating lid to avoid entraining air (10), an arterial (recirculating) roller pump (Sarns 5000 heart-lung machine console), a Sarns pediatric 16267 hollow fiber membrane oxygenator/heat exchanger and a 40-micron Pall LP 1440 40 micron blood filter. The recirculating reservoir was continuously stirred with a 3” teflon-coated magnetic stir bar driven by a Thermolyne type 7200 magnetic stirrer. Temperature was continuously monitored at the arterial port of the oxygenator using a Sarns thermistor temperature probe and a YSI 73BTAX remote sensing digital thermometer. Glycerol concentrate was continuously added to the the recirculating system from a 60 liter polyethelene reservoir using a Drake-Willock 7401 hemodialysis pump. Glycerol ramp was monitored using an Atago hand-held sugar refractometer.

Three animals constituted the experimental group and were subjected to simulated transport, TBW, cryoprotectuve perfusion and freezing-thawing and fixation.

Fixative Perfused Controls

Two control animals were prepared as per the above with the following modifications: One of the animals was subjected to fixation after induction of anesthesia and placement of cannulae (i.e., normothermic, non-ischemic, beating-heart fixation). Fixation was achieved by first perfusing the animal with 3 liters of bicarbonate- buffered Lactated Ringer's containing 50 g/l Dextran-40 with an average molecular weight of 40,000 (Pharmachem) (pH adjusted to 7.4) to displace blood and facilitate good distribution of fixative. This Ringer's Dextran flush was followed immediately by perfusion of 20 liters of Trump's fixative (Composition given in Table I) to which 100 ml of Higgin's India Ink (colloidial carbon) had been added.

TABLE I


	    Composition Of Trump's Storage Fixative


     Component                                 g/l


     Paraformaldehyde                          40 g
     50% Glutaraldehyde                        20 ml
     Sodium Hydroxide                          2.7g
     Dibasic Sodium Phosphate .H2O             11.6 g
     Distilled Water                           980 ml


     pH adjusted to 7.4 with sodium hydroxide
	

Buffered Ringers-Dextran-40 perfusate and Trump's solution, (prior to addition of the India Ink) were filtered through 0.2 micron filters and delivered with the same extracorporeal circuit described above.

The purpose of this control was to serve as a reference on our basic fixation and EM preparation technique essentially demonstrating that fixation and microscopy in our hands yielded normal appearing tissues thus ruling out artifact from fixation and preparation for microscopy.

The second control animal was subjected to cryoprotective perfusion to 7.4M glycerol (end arterial concentration) per the protocol below, and immediately thereafter perfused with 20 liters of Trump's fixative prepared in 7.0M glycerol (also filtered through a 0.2 micron Pall prebypass filter) with 200 ml of India ink added after filtration. Glycerol-fixative was perfused at a temperature of 8.0 C.

Immediately following fixative perfusion the animals were dissected and 4-5 mm thick coronal sections of organs were cut, placed in glass screw-cap jars containing pre-cooled (4 C) Trump's fixative or Trump's fixative containing 7.4M glycerol (as appropriate), refrigerated to 4 C, and transported, as detailed below, for electron microscopy. Tissue from the glycerol perfused-fixed animal was deglycerolized at 4 C following cutting of the tissue blocks for electron microscopy using two protocols of deglycerolization: fast and slow.

Preparation and Post Cardiac Arrest Support of Cryopreserved Animals

Following placement of cannulae, baseline CO and EtCO2 measurements were made. CO was 1.3 to 1.5 liters/min and EtCO2 was 5% in all animals. Mean arterial pressure (MAP) was 80mmHg to 90mmHg.

Cardiac arrest was induced by the administration of 1 mEq/kg potassium chloride via the distal port of the Swan-Ganz catheter. Cardiac arrest occurred uniformly within 3-15 seconds. A period of 5 minutes of normothermic ischemia was then allowed to elapse before closed chest cardiopulmonary support (CCCS) using the Thumper was initiated. Esophageal temperature at the time of cardiac arrest in the animals varied between between a low of 37.4 C and a high of 38.2 C.

At the start of CCCS the following medications were given via the peripheral IV and the proximal line of the Swan-Ganz catheter for the purpose of minimizing both ischemic and reperfusion-trickle flow injury.

Medications

Epinephrine: 0.20 mg/kg given every 10 minutes, IV push for 30 minutes Nimodipine: 10 micrograms/kg followed by 10 micrograms/kg every 10 minutes by slow IV push for 30 minutes THAM (tromethamine) 0.3M 250 mg/kg IV infusion Deferoxamine: 500 mg HCl IV push Sodium Citrate: 120 mg/kg via slow IV push Trolox: 45 mg/kg slow IV push Heparin: 420 IU/kg IV push Methylprednisolone 1 g via IV infusion Metubine Iodide: 2 mg IV push Maalox, 30cc was also given via the gastric tube and the gastric tube flushed with 20 cc of tap water.

Simultaneous with the start of CCCS the animals were covered with crushed ice and 10 gallons of water were added to the portable ice bath. A recirculating water pump connected to both a perforated tubing array (which was draped over the animal) and to a cooling blanket placed under the animal, was used to facilitate induction of hypothermia via external (immersion-simulated) cooling.

The protocol for CCCS using Thumper support consisted of 80 compressions per minute with a compression to relaxation ration of 50:50. Pressure cycled ventilation, delivered between every fifth chest compression using the Thumper ventilator at a peak airway pressure of 30 cmH20, and an FiO2 of 80% was used throughout CCCS. Efficacy of CCCS was evaluated by measurement of CO, end-tidal CO2, (Nellcor Easy Cap) and pulse oximetery (using the tongue as the measuring site) (CSI Model 503 Pulseoximeter). CCCS was continued for 30 minutes before starting extracorporeal support and total body washout (TBW).

Animals were placed on closed-circuit cardiopulmonary bypass using the recirculating loop of the cryoprotective perfusion circuit. This circuit was primed with approximately 3 liters of asanguineous solution consisting of 1 liter of Dextran 40 in normal saline, 2 liters of Normosol-R (ph7.4) and 25 mEq sodium bicarbonate. Following pump-oxygenator-heat exchanger cooling to approximately 15 C, animals were subjected to total body washout (TBW) by open-circuit perfusion of 10 liters of MHP-2 perfusate containing 5% v/v glycerol. (see Table II for composition) The extracorporeal circuit was then closed and addition of 65% v/v glycerol- containing MHP-2 perfusate at a rate of approximately 400 mM/min. was begun. Cryoprotective perfusion continued until the target concentration of glycerol was reached.

TABLE II

Perfusate Composition
FORMULA FOR MHP-2 BASE PERFUSATE


Component          Molar Concentration  Grams/Liter  Grams/20 Liters mM


Mannitol               170.0 (MW 182.20)      30.97         619.40

Adenine HCl            0.94 (MW 180.6)         0.17           3.4

D-Ribose               0.94 (MW 150.2)         0.14           2.82

Sodium Bicarbonate     10.00 (MW 84.0)         0.84          16.8

Potassium Chloride     28.3 (MW 74.56)         2.11          42.2
	 
Calcium Chloride
   10% (w/v) soln.     1 (MW 111)             0.28 ml        5.6 ml

Magnesium Chloride
   20% (w/v) soln.     1 (MW 95.2)             1.0 ml        20.0 ml

Sodium HEPES           15 (MW 260.3)           3.90          78.0

Glutathione (free acid) 3 (MW 307.3)           0.92          18.44

Hydroxyethyl Starch     ----                  50.00       1,000.00

Glucose                 5 (MW 180.2)           1.80          36.0

Heparin                 ----               1,000 IU      20,000 IU

Insulin (Humulin U-100)                       40 IU         800 IU
	

Perfusate

The perfusate used for cryoprotective perfusion was an intracellular formulation which employed sodium HEPES, glucose and mannitol as the impermeant species and hydroxyethyl starch (HES, McGaw Pharmaceuticals, Irvine, CA; av. MW 400,000 - 500,000) as the colloid. The composition of the base perfusate is given in Table I.I The pH of the perfusate was adjusted to 8.0 + or - 0.3 with potassium hydroxide or hydrochloric acid (rarely required) where needed. A pH of 8.0 was selected because it was deemed “appropriate” to the degree of hypothermia experienced during cryoprotective perfusion (11).

Perfusate components were reagent or USP grade and were dissolved in USP grade water for injection. Perfusate was through a Pall 0.2 micron prebypass filter prior to loading into the extracorporeal circuit.

Cryoprotective Perfusion

Cryoprotective perfusion of the animals was begun by carrying out total body washout (TBW) with the base perfusate containing 5% v/v glycerol. Washout was typically achieved within 4-6 minutes of the start of open circuit perfusion at a flow rate of 1.5 to 1.7 L/min and a mean arterial pressure (MAP)of 60 mmHg. TBW was considered complete when the hematocrit was unreadable and the venous effluent was pink-tinged or clear. This typically was achieved after perfusion of 7 to 8 liters of 5% v/v glycerol containing MHP-2 perfusate.

The arterial pO2 of the animals was maintained between 300 mmHg and 500 mmHg throughout TBW and subsequent glycerol perfusion. Arterial pH during cryoprotective perfusion was between 7.4 and 7.7 with terminal arterial pH typically being between 7.6 and 7.7. Venous pH was typically between 7.3 and 7.5 with terminal venous pH being between 7.45 and 7.55

Introduction of glycerol was by constant rate addition of base perfusate containing 65 v/v glycerol to a recirculating reservoir containing approximately 15 liters of 5% v/v glycerol-in MHP-2 base perfusate. The target terminal tissue glycerol concentration was 7.4M in the venous effluent and the target time course for completion of the cryoprotectant ramp was 2 hours. The volume of 65% v/v glycerol concentrate required to reach a terminal concentration in the recirculating system (and thus presumably in the animal) was calculated as follows:

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

Mc = Molarity of glycerol in animal and circuit.

Mp = Molarity of glycerol concentrate.

Vc = Volume of circuit and exchangeable volume of animal (assumes an exchangeable water volume of 60% of the preperfusion weight of the animal).

Vp = Volume of perfusate added.

Glycerolization of the animals was carried out starting at an esophageal temperature of 15 C with more or less linear reduction of temperature as glycerol concentration was increased, with perfusion typically terminating at 6 C.

Cryoprotective perfusion began at a MAP of 40 mmHg and at an esophageal temperature of 15 C. MAP rose steadily as glycerol concentration was increased and MAP at conclusion of perfusion was typically between 130mm Hg and 160mm Hg

Following termination of the cryoprotective ramp, the animals were removed from bypass and the arterial cannula, with a short length of PVC tubing left attached, was plugged using a foley catheter plug. The venous cannulae were also left in place and cross-connected to each other with a Cobe 3/8” straight connector, taking care to exclude air from both the cannulae and connector. Cannulae were left in place to facilitate prompt reperfusion upon rewarming, The margins of the groin wounds were loosely approximated using surgical staples and the endotracheal tube was plugged with a rubber laboratory stopper. To prevent entry of cooling bath media into the lungs should the shielding plastic bag leak during cooling to -79C.

The rectal and esophageal thermocouple probes used to monitor core temperature during perfusion were augmented with two external thermocouple probes of the same type for monitoring cooling to -79 C. One of these external probes was stapled to the skin at the midline of the scalp and the other was stapled to the abdomen, also at the midline, approximately 4 cm below the Xyphoid process.

Cooling to -79 C

Cooling to -79 C was carried out by placing the animals within a 6 mil polyethylene bag from which air was evacuated with a shop-type vacuum cleaner and then submerging them in an n-propanol bath which had been precooled to -40 C. Animal temperatures at the time of placement in the cooling bath were typically 5-6 C esophageal, 7-9 C rectal, and 8-9 C surface. Bath temperature was slowly reduced to -79 C by the periodic addition of dry ice. A typical cooling curve obtained in this fashion is shown in Figure 4. Cooling was at a rate (averaged) of approximately 4 C per hour.

Cooling to and Storage at -90 C

Following cooling to -79 C, the plastic bags used to protect the animals from alcohol were rapidly swabbed off using cloth towels, the animals were placed inside nylon sleeping bags with draw-string closures and were then positioned atop three 6”x 12” styrofoam blocks inside a two-stage Rheem Ultra Low, -90 C mechanical freezer. Cooling to -90 C from -77 (typical dry ice-alcohol endpoint) was complete in approximately 6 hours. After cool-down to -90 C animals were maintained at temperatures between -80 C and -90 C for a period of 12 to 18 months until being removed and rewarmed for gross structural, histological, and ultrastructural evaluation. Dry ice was used as thermal ballast in the mechanical freezer to guard against warming due to mechanical failure or power disruption.

Rewarming

Animals were rewarmed to -10 C to -8 C by removing them from -90 C freezer and placing them in a well stirred n-propanol bath which had been precooled to 0 C. Bath temperature typically declined to approximately -15 C and rose slowly towards 0 C. When the temperature of the bath reached 0 C it was maintained at 0 C + or - 3 C by addition of dry ice to the alcohol bath until the animal's core temperature reached -10 C. Rewarming was at an average rate of 10 C per hour to -10 C at which point no ice could be detected in the tissues by external palpation. A typical rewarming curve is shown in Figure 5.

When the animals' core temperatures reached -6 C they were removed from the alcohol bath, the 6 mil plastic bags were removed, and the animals were placed atop a bed of Zip-Loc plastic bags filled with crushed ice and covered over with crushed ice containing Zip-Loc bags on the operating room table. The animals were re-connected to a simplified extracorporeal circuit for perfusion of fixative. The arterial pressure monitoring catheter was also reconnected to the pressure transducer to allow for pressure monitoring during fixative perfusion.

Note: great difficulty was encountered in measuring perfusion pressure in the first two animals due to failure to flush the monitoring lines with 7.4M glycerol prior to freezing. The lines were instead filled with saline from the Intraflow set-up used to prevent clotting prior to heparinization of the animal (3cc normal saline per hour flowing through the catheter). Since the greatest length of the catheter was deep within the animal, and the core temperature of the animal was well below the freezing point of saline, it required great ingenuity to free the lumen of the pressure monitoring catheters from ice; this was finally achieved by slowly advancing a heated copper wire through the catheter.

Fixation

After positioning on the operating table a midline incision was made from sternal notch to the symphisis pubis. The thorax was opened via a median sternotomy and the abdomen via a mid-ventral laparotomy. The thoracic and abdominal incisions were retracted open to allow visualization of the viscera when fixative perfusion commenced (Figure 6).

When the core temperature (esophageal) reached -6 C perfusion of fixative perfusion was begun. Precooled fixative (1-2 C) was delivered at a temperature of 4 C using an open circuit consisting of a roller pump, a Gish pediatric heat exchanger, and a Pall 1440 40 micron filter. Venous return was via the femoral venous cannula to which was attached a (primed) 3/8” Y-connector and several feet of 3/8”x3/32” line which was allowed to drain into a covered pail with 1” of corn oil in the bottom (to minimize exposure of staff to formalin). Approximately 15-20 liters of Trump's storage fixative containing 7.0 M glycerol (to which 100 ml of India Ink was added) was then perfused open-circuit.

Following fixative perfusion the animal was immediately dissected and samples of heart, lung, liver, pancreas, spleen, kidney, and skeletal muscle were collected for subsequent histological and ultrastructural examination. Samples of these organs were immediately immersed in chilled glycerol containing Trump's fixative. All organs were multiply sectioned both sagitally and coronally to evaluate the degree of reperfusion, as indicated by distribution of India Ink.

The brain was then removed en bloc to a flask containing 300- 400 ml of fixative with 7M glycerol (sufficient to cover the brain completely). The brain was momentarily removed from this fixative bath and each hemisphere was sectioned (incompletely) both coronally and sagitally to evaluate distribution of fixative/ink and the integrity of the capillary bed. The brain was then returned to the glycerol containing fixative and refrigerated overnight prior to the cutting of sections for microscopy.

The following day coronal sections of the left cerebral hemisphere at the level of the hippocampus of varying thickness (from 5 mm to to 1 mm) were cut, placed in fresh glycerol containing Trump's and shipped on ice to an academic facility for processing by a professional electron microscopist using standard techniques. Prior to normal preparative procedures for EM the tissue was subjected to multiple washings with Trump's fixative containing progressively lower concentrations of glycerol over a period of about 1 week until all the glycerol was washed out. During final sample preparation for electron microscopy, care was taken to avoid using the cut edges of the tissue sections in preparing the Epon embedded sections.

Deglycerolization of Samples

As noted above, in order to avoid osmotic shock all tissue samples were initially perfused with and immersed in Trump's fixative containing 7M glycerol and were subsequently deglycerolized prior to staining and embedding by stepwise incubation in Trump's containing decreasing concentrations of glycerol. The need to use such an approach on presumably well-fixed and thus presumably osmotically “desensitized” tissues may seem without foundation. However, both we and other investigators have found a significant injurious effect of simply immersing fixed tissue loaded with multimolar concentrations of glycerol into glycerol free (and thus by comparison very hypo-osmolar) fixative (12).

III EFFECTS OF CLOSED CHEST CARDIOPULMONARY SUPPORT

At the start of Thumper support MAP was between 25mmHg and 30 mmHg and increased to between 35mmHg to 45mmHg with the administration of initial bolus of high dose (0.2 mg/kg) epinephrine. End-tidal CO2 at this time was 1-2% and cardiac output was 0.5 to 0.7 liters per minute (LPM). After 30 minutes of CCCS, MAP had declined to 30mmHg to 35mmHg with a corresponding decrease in responsiveness to each bolus of epinephrine. End-tidal CO2 declined to 1% to 0.5% and CO declined to 0.3 to 0.5 LPM. Esophageal temperature at the end of CCCS and immediately prior to the start of bypass had declined to 21 to 28 C depending on the mass of the animal and the amount of subcutaneous fat covering the animal (subcutaneous fat served as a good insulator and greatly slowed cooling, somewhat independent of total body mass).

IV. EFFECTS OF GLYCEROLIZATION

Blood washout was rapid and complete in all the animals. MAP rose sharply as glycerol concentration increased, probably as a result of the increasing viscosity of the perfusate as is shown in Figure 7.

Within approximately 5 minutes of the beginning of the cryoprotective ramp, bilateral ocular flaccidity was noted. As the perfusion proceeded, ocular flaccidity progressed until the eyes had lost approximately 30% to 50% of their volume. Gross examination of the eyes revealed that initial water loss was primarily from the aqueous humor, with more significant losses from the posterior chamber of the eyes apparently not occurring until later in the course of perfusion. Within 15 minutes of the start of glycerolization the corneal surface became dimpled and irregular and the eyes had developed a concave appearance.

Dehydration was also apparent in the skin and skeletal muscles and was evidenced by a marked decrease in limb girth, profound muscular rigidity, cutaneous wrinkling, a “waxy- leathery” texture and a mummified appearance of both cut skin and skeletal muscle. Tissue water evaluations conducted on ileum, kidney, liver, lung, and skeletal muscle confirmed the gross observations. Preliminary observations suggest that water loss was in the range of 30% to 40% in most tissues as was previously observed both in previous animal studies (5,6) and in humans undergoing cryopreservation using a similar protocol. (13)

Examination of the cerebral hemispheres upon cranitomy revealed an estimated 30% to 50% reduction in cerebral volume, presumably as a result of osmotic dehydration secondary to glycerolization. The cortices also had the “waxy” amber appearance previously observed as characteristic of glycerolized brains.

The gross appearance of the kidneys, spleen, mesenteric and subcutaneous fat, pancreas, and reproductive organs (where present) were unremarkable. The ileum and mesentery appeared somewhat dehydrated, but did not exhibit the dense mummified/waxy appearance that was characteristic of muscle, skin, and brain.

Oxygen consumption (determined by measuring the arterial/venous difference) throughout perfusion was fairly constant to about 3M glycerol and then dropped off sharply as 6M glycerol concentration was approached (the high viscosity of the perfusate above 6M made measurement by the Nova Stat 5 Profile blood gas-electrolyte system used in these experiments impossible. Oxygen consumption versus glycerol concentration is shown in Figure 8. Arterial and venous pH, PO2, PCO2, and electrolytes are shown in Figures 9, 10, 11, and 12 respectively.

IV. GROSS EFFECTS OF COOLING TO AND REWARMING FROM -90 C

The gross appearance of the animals' skin, thoracic and abdominal viscera was surprisingly good (Figure 13). In contrast to subtle post-thaw alterations in the appearance of the tissues of cryopreserved animals in our previous studies, the tissue colors were “normal”; i.e., normal for organs and tissues subjected to TBW with MHP-2 (a survivable procedure). Particularly absent was the previously observed (14, 15) altered texture of the tissues following thawing, with no pulpy material coating gloves or instruments on sectioning. Also, in contrast to prior post-cryopreservation evaluation of both humans (14) and animals, the vasculature contained perfusate in noticeable amounts after thawing and the “filling time” required to achieve venous return was far shorter.

Peerhaps most striking was the excellent reperfusion of virtually every organ system in the animals (Figures 13, 14, 15) with the exception of the spleen (Figure 16), which failed to perfuse almost completely. Distribution of carbon was uniform, occurred rapidly and evenly after the start of perfusion, and venous return was excellent. In fact, MAP dropped steadily during the first 5-10 minutes of reperfusion from 140 mmHg to 80mmHg to 90mm Hg, before beginning to rise, presumably as fixation took place rendering the capillaries both rigid and freely permeable to colloid. Fixative flow rates were in the range of 800cc/min to 1.2 LPM.

In two of the animals an area of obvious failed perfusion occurred (Figure 17) in the dependent part of the stomach as evidenced by the normal whitish pink appearance of an island of tissue as contrasted with the uniform black of the reperfused areas. Upon opening the stomach it was discovered that stomach fluid/contents were partially frozen over the area of failed reperfusion.

The logical explanation for this is that dilution of cryoprotectant concentration in the stomach wall underlaying the stomach contents, by diffusion of water from the stomach contents during the long time-course of cooling reduced the tissue glycerol concentration to a low enough level to compromise vascular integrity. Presumably such dilution would have resulted in more ice formation in the affected tissue and thus greater cryoinjury with subsequent compromise of the capillary bed.

The chamber of the left ventricle which is sequestered behind the aortic valve was uniformly found to contain large ice crystals in a slushy mass (Figure 18) with associated failed perfusion of the endocardium (again, presumably as a result of dilution of cryoprotectant to below the threshold required to provide capillary protection). This left ventricular ice was observed to have a strong pink cast and many red cell ghosts were observed when the ice was melted and examined under the light microscope.

Perhaps most importantly, there was no evidence of cracking or fracturing, even though these animals were cooled to near Tg for glycerol water solutions and rewarmed by transfer from -90 C to a 0 C liquid bath creating a large surface to core thermal differential. In order to explore the fragility and ductility of animals loaded with 7.4M glycerol and cooled to -90 one animal was loaded with 30 kilos of dry ice placed across the thorax and abdomen with the animal suspended (head and hindquarters) on two blocks of styrofoam (without supports between). This static loading was maintained for 48 hours at -90 C with no evidence of sagging, flexion or cracking at either the gross, histological, or ultrastructural levels.

Particularly striking was uniform fixative perfusion of the brain. (Figures 19, 20, 21) An advantage of carbon particle marker over dye is that it is possible to demonstrate not only filling of large vessels, but of perfusion of the capillaries as well, as evidenced by uniform darkening of the tissue to black or charcoal gray. A drawback of dyes is that they rapidly diffuse out of vessels into areas of failed perfusion. Solid particles of carbon (1-2 microns in diameter) cannot do this and thus remain where they are deposited during perfusion (14).

IV. EFFECTS OF CRYOPRESERVATION ON BRAIN ULTRASTRUCTURE

In sharp contrast to all of the previously cited studies, the high degree of ultrastructural preservation observed in this series of animals is unprecedented. In order to better characterize both the degree of preservation and the degree of injury, the discussion of these two facets of the results will be handled in seperate sections, beginning with an overview of the injury/alterations in brain tissue ultrastructure which were observed.

Injury and Alterations of Ultrastructure

There are basically four classes of lesions or alterations in appearance of ultrastructure observed in these animals: The first are changes seen in both glycerolized-fixed (but not frozen) animals and those observed in animals which were subjected to glycerolization, freezing, thawing and fixation. In both groups of animals there are characteristic changes in the density of the cytoplasm and ground substance that we associate with dehydration; there are packs of “stacked” ribosomes occupying large fractions of the cytoplasm (Figure 22), small mitochondria with dense cristae (Figure 23), and shrunken nucleoli. (Figure 24) The density of the ground substance appears enhanced in both groups, and some non- neuronal cells (possibly astrocytes) appear to have lost plasma membrane integrity and appear as naked nuclei surrounded by vesicular debris (Figure 24).

There are also alterations in nuclear density in both groups suggestive of either loss or redistribution of nuclear material. The nuclear membranes appear crisp and intact in both groups, so it is difficult to draw conclusions from this. In both frozen and nonfrozen glycerolized gray and white matter there is a modest increase in the inter-cellular space (Figure 25, 26) as compared to the unglycerolized control perfused with a beating heart (Figure 27). These increases in inter-cellular space are probably also as a result of dehydration secondary to glycerolization.

Finally, at least five other changes both groups have in common when compared to the beating-heart fixed control are partial unraveling of the myelin,(Figure 28, 29) shrinkage of the axoplasm within the myelin, dehydration of the mitochondria and nucleoli, the presence of occasional debris strewn “tears” in the tissue (Figure 30, 31), and increased difficulty in discerning plasma membranes. These tears are very uncommon in the glycerolized non-frozen controls and more common in the frozen-thawed controls; although they still occur infrequently in the frozen-thawed group as well.

Further, the etiology of these tears appears different between the two groups; in the frozen thawed groups the fissures or tears are relatively neat edged, the spaces contain minimal debris and the edges appear complementary, like two halves of a torn piece of paper. Perhaps the degree of “match” between the sides of these fissures could be best characterized by the degree of “match” observed in orbital photographs of continents experiencing millions of years of continental drift; that the patterns are related is obvious, but the match is not precise.

The fissures observed in the glycerolized non-frozen tissue (both grey and white matter) appear less clean and more debris strewn. The etiology of these tears remains more of a mystery.

Lesions observed exclusively or more extensively in the frozen-thawed brains are as follows:

a) Areas at high magnification (40,000 x) where the myelin appears to have lost its lamellar structure and presents an amorphous or disintegrated appearance, as if a coarse charcoal line-drawing of tightly concentric rings had been smeared or smudged (Figure 31).

b) Loss or alteration of nucleoplasm which is evident at both low magnification (6700x) and higher magnifications (40,000x). This change is not uniformly observed in all nuclei, but is very common (Figure 32).

c) Pericapillary holes or spaces (Figure 33) occasionally strewn with vesicles or debris (Figures 34, 35) are still present; these have been observed in previous work with cats and rabbits and their location and appearance correlate well with the observed presence of ice in freeze-substituted grey and white matter (Figures 36, 37). However, it should be noted that these “ice holes” occur with far less frequency in the 7.4M glycerolized brains than has been observed in brains cryopreserved with 3M glycerol, (or lower concentrations) (Figures 38,39).

Preservation of Ultrastructure

The most striking difference between this work and previous brain cryopreservation studies is the overall recognizability, inferrability and even “normality” which is present in the micrographs. (Figures 40, 41, 42) Examination of neuropil, individual synapses and axons at magnifications from 40,000x to 80,000x reveal excellent preservation of fine structure (Figures 43, 44, 45). Synapse morphology is normal in appearance and synaptic vesicles, membrane structure and general appearance are almost indistinguishable from unglycerolized, nonfrozen control, (Figure 46) and are virtually indistinguishable from glycerolized-fixed non-frozen controls (Figure 47). The relationship of the neurons to each other and of fine processes such as dendritic spines seems very well preserved with exception of the occasional 5-10 micron tears or fissures.

Capillary integrity is excellent with intact endothelial cell membranes, clearly visible intra-endothelial cell ultrastructure and intact basement membranes. Capillary lumens are either clear or show occasional dark black particles of carbon (Figure 48). Very rarely, small vesicles or bits of membrane material well under 0.2 micron in diameter can be observed in the lumen of the capillary adjacent to an endothelial cell (Figure 49). Blood washout appears to be complete as there are no red cells or other formed elements of the blood present in the capillaries in any micrograph.

Intracellular organelles while somewhat dehydrated in appearance are readily identifiable; the endoplasmic reticulum, mitochondria, golgi apparatus, lysosomes and the fine structure of the axoplasm are all well preserved. Mitochondria are rarely swollen, show (dehydrated, compressed) cristae, and are absent of calcium crystals. Similarly, the polyribosomes appear normal in architecture and are nondissociated.

SUMMARY

We believe this study demonstrates, for the first time, preservation of brain ultrastructure in sufficient detail to provide, in a qualified fashion, an evidentiary basis for reconstruction of cryopreserved humans using the information- theoretic criterion (15). Without a full understanding of how memory, personality and identity are encoded in the human brain it is not possible to state with certainty that these functions are being preserved, even with the comparatively good ultrastructural preservation reported here, and this remains the major “qualifier” on the optimism expressed above.

While there is much ultrastrucural and histological preservation in evidence in the micrographs obtained in this series, there is also evidence of considerable damage. Particularly disturbing are the continued presence of large (5 to 15 micron diameter in cross section) tears of unknown “depth” in both the grey and white matter. Dehydration of structures and the presence of what appear to be free nuclei and lysed glial cells are also disturbing.

Another important caveat to consider in the context of the comparatively positive results demonstrated in this study is the relatively benign pre-mortem (i.e., pre cardiac arrest) and post cardiac arrest insult that these animals were exposed to. Complete normothermic ischemia was brief and at the margin of contemporary clinical reversibility. The post arrest Thumper support (even with the use of high dose epinephrine) was grossly inadequate as indicated by low CO, EtCO2 aMAP and SaO2. This period of trickle-flow due to the failure of CCCS to deliver adequate CO was brief compared to the typical clinical cryonics patients' course. At a minimum, this study confirms the poverty of circulatory support provided by closed chest cardiopulmonary resuscitation and it can be reliably presumed that it was only the unrealistic brevity of this period of inadequate circulation and ventilation which prevented even more ischemic injury from occurring. Clearly, more effective means of circulatory support are needed to bridge the gap between pronouncement (cardiac arrest) and vascular access and the beginning of extracorporeal circulatory support.

Thus, while this study demonstrates substantial preservation of brain ultrastructure and histology, it also points out that much remains to done before either reversible brain cryopreservation can be achieved or there can be a high degree of confidence that the structures responsible for memory and personality remain sufficiently intact to allow recovery of cryopreserved patients on a reasonable time scale (50 to 150 years).