Cryonics, November 1985
CASE REPORT: TWO CONSECUTIVE SUSPENSIONS, A COMPARATIVE STUDY IN EXPERIMENTAL HUMAN SUSPENDED ANIMATION
by Jerry D. Leaf, Mike Federowicz, and Hugh Hixon
The two suspensions reported on here were carried out in January of 1980 by Cryovita Laboratories. Both patients discussed in this report are now being maintained in long-term cryogenic storage by Trans Time Inc., of Emeryville, CA. Since these patients were placed into suspension within 24 hours of each other, essentially the same perfusion and cooling protocol was employed for each of them. There have been many changes in perfusion protocol since these patients were placed into suspension. Most of these changes are a direct result of information obtained during the course of these two patients' transport and perfusion.
The protocol used in these cases tested a number of hypotheses: 1) Glycerol, as a cryoprotective agent, should cause less edema than dimethyl sulfoxide (DMSO); 2) The "Smith Criterion" (Appendix A) can be approximated in ischemically injured patients; 3) An improved cryoprotective ramp can be achieved by using stepped recirculation rather than a single-pass technique; 4) Arterial/ Venous differences in cryoprotective agent concentration can be held low; 5) A surface thermistor probe on the surface of the brain can be used to indicate the freezing point of the cerebral cortex; 6) A cranial burr hole can be used to make observations of the CNS response to cryoprotective perfusion.
These hypotheses were tested, as well as other minor ones. The methods used as well as the results are described in the text of this paper. The answers to these inquiries have suggested future hypotheses and improved our current state of the art.
The age, medical history, and transport protocol of these patients differ greatly and these differences complicate evaluation of the significance of any one of these factors. Nevertheless, an attempt will be made to relate these factors to significant differences observed in the two patients, (degree of blood washout, development of edema, and increase in vascular resistance).
This case report is intended to inform the public about the nature of cryonic suspension procedures (experimental human suspended animation). This kind of reporting is a necessary part of a larger body of information that would constitute being informed about cryonics. Most people making cryonic suspension arrangements today are signing a consent form, which implies that they are informed about the experimental nature of suspension procedures. The concept of an "informed consent" is a reasonable standard of practice that we would like to achieve. A complete "informed consent" approach is also being advocated by legal counsel James Bianchi (Bianchi, 1983). It is hoped that this information will add to the individual readers' empirical knowledge of these procedures to the end that they will truly be more informed.
In keeping with Cryovita's ethical practices, the identities of these patients are being maintained in confidence and for purposes of identification they will be referred to as Suspension Patient One (SP1) and Suspension Patient Two (SP2) in this report.
In December of 1983, one of these patients, SP2, was removed from whole body cryogenic storage and converted to neuropreservation. The body of SP2 from below approximately the 6th cervical vertebra was thus available for postmortem gross and histological examination, and the results of this study will be discussed as they relate to perfusion and preparation for cryogenic storage. A complete discussion of the autopsy findings from this patient has been reported previously (Federowicz, Hixon, and Leaf, 1984).
SP1 was a male caucasian, 79 years old, weighing 59.1 kilograms. Details of past medical history are unknown because the next of kin has not provided medical records. Examination showed no evidence of previous chest or abdominal surgery. External examination was otherwise remarkable only in that the right arm had been amputated at approximately midforearm, reportedly as a result of an injury sustained during WWI.
Approximately 48 hours prior to deanimation SP1 had been admitted to a hospital in Wisconsin, at which time we were notified that his condition was probably terminal and clinical death was likely to occur within 72 hours. Suspension arrangements were made a few days prior to the patient's clinical death.
The patient's admitting diagnosis at the hospital was transient cerebral ischemia with probable recent stroke. Admitting chest films disclosed a large mediastinal mass which the attending physicians felt was a malignancy. Initial examination and laboratory work-up disclosed moderate cachexia with slight jaundice and elevated liver enzymes and bilirubin, indicative of hepatitis secondary to obstructive biliary disease. The obstruction was thought to be a result of either primary or secondary hepatic carcinoma. The patient had a history of adult onset diabetes which had been managed with diet and oral medications.
Patient stabilization and transport were carried out by Soma, Inc. of Indianapolis, Indiana (now merged with Cryovita Laboratories). Initially a team of five people was dispatched to undertake transport/stabilization. Owing to economic constraints, all of these individuals with the exception of one was allowed to return to Indianapolis after the first 48 hours. Transport and initial stabilization were thus carried out by one team member (Michael Federowicz) with assistance of the hospital staff and a local mortician whose facilities were also used to carry out external cooling during extended, mechanically assisted CPR.
At the time the rescue/stabilization team arrived the patient was in the Intensive Care Unit (ICU) and still alert and communicative. He appeared to be in moderate distress at the time he was examined by Federowicz and emesis showed moderate amounts of bile-tinged fluid during the course of the brief (10 minute) examination. Heart sounds were normal and breath sounds were remarkable only for slight rhonchi present bilaterally. The patient was febrile with a charted rectal temperature of 38°C. The patient appeared to be fully cognizant of the severity of his condition and expressed relief and gratitude that cryonics personnel were present.
During the following 24 hours the patient's condition deteriorated and he demonstrated only intermittent episodes of consciousness and responsiveness. Periods of unconsciousness were often accompanied by Cheyne-Stokes respiration. Pupillary reflexes were present and cerebral perfusion appeared intact until cardiac arrest occurred at 4:00 pm on January 15th.
Cardiac arrest was initially determined by monitoring the patient's EKG. The ICU staff had agreed to promptly initiate cardiopulmonary support and summon a physician for a prompt pronouncement of legal death. CPR was carried out for approximately four minutes prior to the arrival of the physician, at which time it was interrupted for approximately one minute to allow the physician to auscultate the chest, determine the absence of any electrocardiographic activity, and pronounce legal death. CPR was then promptly resumed, and the patient was intubated by a respiratory therapist and coupled to an MA-1 ventilator. The patient was hyperventilated using pure oxygen at a rate of 25 respirations per minute and a tidal volume of 1.5 liters while manual CPR was continued.
Concomitant with manual CPR, medications were administered through an intravenous line which had been previously placed in the left forearm. A heat exchange blanket had been placed under the patient prior to deanimation, and immediately after pronouncement of legal death this blanket was connected to a Blanketrol unit which had been preset to circulate coolant at 4°C.
Transport medication consisted of intravenous administration of the following:
Heparin, 507 IU/kg
Mannitol, 2.12 g/kg
Anectine, 0.88 mg/kg
Cimedtidine, 5.0 mg/kg
Thorazine, 50 mg
Methylprednisolone, 250 mg
THAM (Tromethamine), 212 mg/kg (250 cc)
(All the above medications were given as boluses. Another 250 cc of THAM were given by continuous IV infusion over the next 3 hours.)
During administration of transport medications, the hospital staff began packing the patient in ice with special attention to the head, neck, groin, and axilla. After administration of transport medications was complete, the patient was coupled to a Travenol/Brunswick HLR 50-90 for continued cardiac support. Transportation from the ICU to the mortuary was via hearse, and HLR-supported CPR was continued throughout transport and subsequent external cooling. Immediately prior to removal of the patient from the ICU, respiratory support was transferred from the MA-1 to a bag- type manual ventilator. Hyperventilation using pure oxygen was continued by manual ventilation until the patient reached the mortuary.
Upon arrival at the mortuary, the patient was transferred from the ambulance cot to an embalming table, where he was completely covered in bags of crushed ice. HLR support was not interrupted during this transfer. The patient's temperature descent was monitored via a rectal thermistor (YSI Type 401) employing a YSI 42SL Telethermometer. Respiratory support during cooling alternated between hyperventilation with a manual resuscitator and use of the HLR respirator (which automatically imposes one ventilation between each five compressions) as dictated by operator fatigue. The patient's temperature descent during HLR supported external cooling is shown in Figure 1.
FIGURE 1. SP1 Cooling, 37-7° C.
Six hours following the start of the procedure, an IV drip consisting of 50,000 IU of sodium heparin in 1,000 cc of normal saline was begun. This additional heparin was administered as a safeguard against loss of anticoagulation due to acidosis-induced degradation of previously administered heparin.
Six and a half hours following the start of the procedure, phlebotomy was successfully performed on a peripheral leg vein which was distended using a tourniquet. Blood collected in a red-stopper Vacutainer demonstrated grossly visible clumping or agglutination of red cells which was initially thought to be possible intravascular coagulation. This clumping disappeared as the sample warmed to room temperature, indicating the presence of cold agglutinins.
Nine hours and twenty minutes after the beginning of the procedure the patient's rectal temperature was 13°C and it was noted that the jaws appeared to be in rigor mortis. Shoulders and limbs did not appear to be affected.
At 3:05 am on January 16th, CPR was discontinued at a rectal temperature of 12øC. The patient was then placed in a rubberized canvas "body bag," transferred to a metal Ziegler case nested in a styrofoam insulated box, and completely covered over in Zip-Loc plastic bags containing crushed ice. Transportation to Los Angeles was via commercial air freight at approximately 8:00 AM (CST) that morning.
SURGICAL AND PERFUSION PROTOCOL
Upon arrival at Cryovita Laboratories that evening, the suspension patient was placed on the operating table and fresh ice packs applied to maintain hypothermia. External examination of the patient showed no remarkable pathology relevant to the impending surgical procedure. A Foley catheter was in place and was connected to a urinometer. A rectal temperature probe was already in place from the transport. The temperature on arrival was 7°C.
Five surgical sites were prepared: sternum, right neck, scalp, and the middle anterior thighs, bilaterally. All sites were preped as previously described (Leaf, 1979, 1981). Plastic 3M drapes were used on all operative sites. The top of the scalp, left of the midline, was draped with surgical towels and an incision 3 cm long was made down to the periosteum. A Wietlaner retractor was placed to expose the periosteum. A periosteal elevator was used to expose the skull, approximately 1.5 cm left of the midline. A 19 mm burr hole was made with a neuro burr and drill. The dura and pia mater were opened to expose the surface of the brain.
The burr hole allowed direct observation of the brain during cryoprotective perfusion. To our knowledge this was the first time the human brain has been directly visualized during cryoprotective perfusion. This procedure was undertaken to determine what, if any, volume changes the brain undergoes during exposure to increasing cryoprotective agent (CPA) concentration, to determine if there is complete blood washout from the surface vessels of the brain, and to provide exposure for placement of a temperature probe directly on the surface of the brain (it was hoped that by doing this we would be better able to determine the cortical freezing point during subsequent subzero cooling and thus determine cortical CPA concentration).
A screen (Cohen Frame) and patient drapes were applied as previously described (Leaf, 1979, 1981). After sternotomy an arterial cannula was placed through a purse string suture in the aortic root for arterial perfusion. The venous cannula was placed in the right atrium through a purse string suture for venous return (Leaf, 1979). (Plates 1 and 2) A right jugular dissection was done and a 30 cm x 0.062 cm I.D. catheter (10) was introduced through a veinotomy and advanced as far toward the brain as possible for effluent sampling. This technique represents a mixed venous sample, but provides an approximation of CPA equilibration in the brain which can be compared to central venous CPA equilibration for the whole body.
Plate 1. Cannulation of the vessels of the heart to connect the patient's circulatory system to the heart-lung machine for perfusion. The sternum has been divided for access to the heart, and is being held open by a retractor (R).
Plate 2. SP1 with the connections to the heart-lung machine in place. Arterial line (A), Venous line (V), Tubing holder (H), Sternal retractor (R), Arterial cannula (AC), Venous cannula (VC), Drape (D).
Bilateral incisions, 2 cm long, were made in the middle anterior thigh, into the rectus femorus muscle. These provided fluid sampling sites for comparing the symmetry of perfusion in the extremities, using osmolality as an indicator. (This was not repeated in the suspension of SP2.) Venous samples were taken by venipuncture of the superficial foot veins to monitor the hematocrit during total body washout (TBW), and CPA perfusion. This technique provided a good indication of the mean arterial pressure (MAP) necessary to perfuse the extremities in order to achieve blood washout. Forty minutes into the perfusion, these samples showed higher hematocrits than the central circulation, 7 Hct vs. 2 Hct, indicating that blood washout occurred much more slowly in the limbs.
Perfusion to achieve TBW was done using a base perfusate containing 5% v/v glycerol, the base perfusate composition is given in Table 1. Medical Grade USP Water for Injection was used as the solvent in the perfusate. The specifiations for USP Grade water are given in Table 2. Methylprednisolone, 250 mg/liter, was added as a membrane stabilizer. The extracorporeal circuit (Figure 2 & Plate 3) is modified from previous designs, the primary difference being the addition of a pre-filter/pre-cooling capability at the heart-lung machine.
Table 1. Base Perfusate Composition.
Component Conc., mM ---------------------------- Dextrose 180.16 Potassium chloride 26.3 Potassium phosphate 7.2 Sodium bicarbonate 10.0 Glutathione 5.0 Magnesium chloride 2.0 Calcium chloride 1.0 Adenine 1.0 Polyvinylpyrrolidinone 6% w/v Glycerol 0.54 to 3.0 Molar -----------------------------
Table 2. Specifications, USP Water for Injection.
Not to Exceed ------------------------------ Total Solids 10 mg/L Heavy Metals 1.0 mg/L Chloride 0.3 mg/L Sulfate 0.3 mg/L Ammonia 3.0 mg/L Calcium 0.3 mg/L Oxidizable Substances 0.8 mg/L pH 5.0-7.0 Culture STERILE Pyrogens FREE ------------------------------
Figure 2. Extracorporeal perfusion circuit.
1. Main (Circulating) Reservoir
|8. Glycerol Ramp Pump
9. 0.20 Micron Pall Filter
10. Prefiltration and Precooling Pump
11. Travenol Miniprime Heat Exchanger
12. 40 Micron Pall Prefilter
13. Cardiotomy Sucker
14. Cardiotomy Pump
Plate 3. The heart-lung machine. Roller pumps (P), Pump controls (C), Gas flowmeters (F), Telethermometers (T), Thermometer switch (S), 40-micron blood filter (F), Oxygenator reservoir (R), Heat Exchanger (H).
Eighty liters of perfusate were used, havng the following volumes and concentrations:
5% glycerol perfusate, 30 liters
10% glycerol perfusate, 10 liters
15% glycerol perfusate, 10 liters
20% glycerol perfusate, 20 liters
50% glycerol perfusate, 10 liters
Cryoprotective perfusion was achieved by gradual stepped increases in glycerol concentration up to 3.0 molar. Electrolyte and other solute concentrations were kept constnt at all glycerol concentrations. A closed circuit perfusion system was used. The glycrol ramp was achieved by maintaining a contant volume, as measured in the oxygenator reservoir, while adding higher glycerol conentration perfusate. The use of glycerol, instead of DMSO, allowed longer perfusion time with a more gradual increase of CPA concentration. Previous suspensions in this laboratory were carried out using single pass stepped increases in perfusate, with recirclation at the terminal CPA con centration. Closed circuit systems will be used in the future due to the efficacy of this procedure as established during this perfusion.
Perfusate temperature averaged 4°C +- 1°C, and esophageal temperature averaged 6°C +- 2°C. Glycerol was selected as the CPA of choice, based on experience with previous animal and human perfusions. Continuous perfusion for 6.75 hours at 50 mm Hg arterial pressure was used. The average pump flow rate was 0.8 liters/min. Refractometer (temperature-compensated Atago, Model N-1, Cat #311- N) measurements of arterial and venous samples were used as an indicator for increasing CPA concentration. (Table 3) Termination of the perfusion was determined by direct observation of the brain volume, as described below. Terminal arterial glycerol concentration was 2.85 Molar and venous concentration was 2.67 Molar. The jugular effluent, as an indicator of brain equilibration, and vena cava (body) effluent samples at the final CPA concentration showed no difference, 2.67 molar for both (Table 3). Molar glycerol concentrations were derived by measuring osmolality, subtracting the estimated osmotic contribution of the base perfusate, and using the empirically compiled graph shown in Figure 3, after first subtracting the estimated osmotic contribution of the base perfusate (Table 3). The glycerol concentration data is plotted in Figure 4.
Table 3. SP1 - Glycerol Concentration.
Time (min) 15 113 200 500 --------------------------------------------- Arterial Brix No. (1) 11.0 16.2 19.4 23.1 Moles (2) 0.68 1.13 1.71 2.85 Percent (3) 5.8 10.1 15.0 24.8 Time (min) 15 213 288 504 --------------------------------------------- Venous Brix No. (1) 7.48 11.5 13.8 20.9 Moles (2) 0.296 0.712 1.26 2.32 Percent (3) 2.5 6.1 11.0 20.1 ---------------------------------------------1) Measurements made at the time of perfusion with a hand-held refractometer, temperature compensated, Atago Model N-1, Cat. #311-N.
2) Molar glycerol concentration calculated at a later date on the same samples that the Brix measurements were made on. Measurements made with a Micro-Osmette Model 5004 freezing point osmometer.
3) Conversion of glycerol concentration, molar to w/w percent, from Handbook of Chemistry and Physics, 60th Ed., CRC Press, 1979.
Figure 3. Glycerol concentration conversion. Molar <--> Osmoles/Kg.
Figure 4. SP1 Glycerol Concentration vs. Perfusion Time
Changes in peripheral vascular resistance (PVR) were calculated from the standard formula,
(MAP-CVP) PVR = --------- Flow
where MAP is mean arterial pressure in mm Hg, measured at the aortic root, CVP is central venous pressure in mm Hg measured in the vena cava near the heart, and Flow is in liters per minute, measured with a calibrated occlusive arterial pump. Pressure measurement was done with Statham P23Db transducers. Figure 5 shows changes in PVR over the course of the perfusion. The initial rise in PVR reflects the reistance to washout of blood cellular components at low temperature. Of special relevance to PVR was the observed cold agglutination of red cells, particularly in the peripheral circulation. The identification of cold agglutination as a problem was first made during these suspensions. Specific changes in transport and perfusion protocols have been made to help eliminate cold agglutination problems (Leaf, 1984). The rise of PVR after it reached its lowest point was probably caused by increased viscosity of the perfusate, i.e., higher CPA concentration, and/or increased tissue edema.
Figure 5. SP1 Peripheral Vascular Resistance (PVR).
Arterial and venous perfusate pH values were measured during the course of perfusion. The results are shown in Table 4. The persistent fall in venous pH, despite use of a high arterial perfusate pH, appears to be the result of both respiratory and metabolic acidosis. The respiratory component was secondary to not oxygenating the perfusate. Since the perfusate provided abundant substrate for anaerobic metabolism, lactic acid production was the other major component of the acidosis (Lehninger, 1979, pp. 421-2, 485). A later measurement of lactate demonstrated 3.3 times the expected lactate content of human blood (Lehninger, 1979, p. 831). The presence of high levels of lactate is evidence of active cellular metabolism, and is unexpected in view of the deep hypothermia employed. It seems likely that the persistently high lactate levels observed during perfusion were a result of metabolic activity rather than as a result of metabolic uncoupling. Metabolic uncoupling as a result of exposure to toxins or cellular death would have resulted in rapid loss of lactate. These data are a positive indication of biological viability. Subsequent animal experiments at Cryovita Laboratories using glycerol perfusion have demonstrated that continuous oxygenation of CPA containing perfusate (or other perfusates rich in metabolic substrates) is essential to control pH and that active metabolism continues in deep hypothermia even at terminal concentrations of 3.0 molar glycerol. In future cryonic suspension operations patients should be perfused with oxygenated perfusates, and perfusate pH and blood gases monitored, if adequate metabolic support and control of pH is to be achieved.
Table 4. SP1 Arterial/Venous Perfusate pH.
Time (min) 10 124 184 268 388 426 ________________________________________________________ Arterial 7.79 8.00 7.71 7.85 7.90 7.25 Venous 7.35 7.39 7.09 7.15 7.18 7.15
During the course of perfusion, an attempt was made to maintain a close arterial/venous (A/V) glycerol concentration in order to minimize osmotic stress as much as possible. At the outset of perfusion, a target terminal glycerol concentration of 3.0 M was selected. Unfortunately, due to increasing vascular resistance and the development of both generalized and cerebral edema, this goal was not achieved (Figure 4). The development of edema and the subsequent increase in PVR was probably to a large extent due to the patient's condition at the time of perfusion, as will be discussed later. In future operations of this kind the addition of pulsatile perfusion may help achieve better peripheral distribution of CPA. Research is currently underway to determine the effectiveness of such an approach. Lowering PVR will achieve better CPA equilibration by improving peripheral perfusion and consequently removing more body water by CPA exchange.
The deciding factor in terminating the perfusion was the development of cerebral edema as determined by direct observation of the brain. Early in the course of the perfusion, cerebral volume decreased by an estimated 20% as a consequence of osmotic removal of water with increasing glycerol concentration. The degree of reduction in brain volume observed was comparable to that seen in a clinical setting where mannitol is employed to reduce cerebral volume for neurosurgical procedures. The subsequent re-expansion of the brain indicates that at some point in the perfusion, significant amounts of glycerol began to cross the blood-brain barrier. Osmotic dilution of cellular contents plus glycerol then produced the swelling.
Decannulation and sternal and skin closures were accomplished in the usual fashion (Leaf, 1979). (Plate 4) A YSI Style 729 disk temperature probe was placed on the surface of the cerebral cortex approximately 1.5 cm to the right of the burr hole margin. Bone wax was used to fill the burr hole, and the temperature probe lead was securely sutured in place with subcutaneous scalp sutures. The scalp incision was closed with continuous 2-0 Ticron suture and additional stay sutures were used to anchor the probe lead wires to the surface of the skin. Parke-Davis spray-on bandage was used on all suture lines.
Plate 4. Skin closure with surgical stapler (S) at the end of the perfusion. The sternum has previously been approximated with stainless steel wire. Stapleline (L), Cardiotomy sucker (C).
Copper-constantan thermocouple temperature probes were placed in the esophagus and rectum, and on the foot, and the patient placed in double vinyl ethyl acetate plastic bags, as previously reported (Leaf, 1981). The patient was then placed in a dry ice/alcohol bath at -22°C for slow cooling to -77°C. Figure 6 shows the temperature measured at probes in the esophagus and rectum, on the surface of the brain, and in the alcohol bath. The time required for cooling to a core temperature of -70°C was 28 hours at an average cooling rate of 2.5°C per hour. Following cooling to -77°C, the patient was covered with dry ice for shipping in an insulated container. Liquid nitrogen vapor phase cooling and encapsulation for storage in liquid nitrogen was carried out in Emeryville, CA at a later date.
Figure 6. SP1 Dry ice cooling.
SP2 was a female caucasian, age 36, weight approximately 65 kg. The following history from records was provided by the next of kin and represents only an outline of what was a very complicated and protracted course of illness.
In 1972, SP2 presented with a nasopharyngeal mass which biopsy subsequently demonstrated to be a squamous cell carcinoma. A concomitant finding was severe cervical lymphadenopathy. SP2 had a history of frequent respiratory infections and general poor health, including several surgeries for removal of benign breast masses.
A course of radiation treatment was decided upon as the best method to manage the patient's oropharyngeal malignancy. Radiotherapy resulted in complete regression of the tumor, and elimination of associated adenopathy Unfortunately, shortly after completion of this therapy the patient developed a moderately severe myelopathy and retinopathy which initially manifested as a loss of fine motor skills and bladder control, and ended in confinement to a wheelchair. Clinical findings were consistent with radiation myelopathy. There was a developing temporo-mandibular fibrosis, restricting the patient's ability to open her mouth. Lacrimal atrophy resulted in "dry cornea" and frequent eye infections. Brain stem and inner ear damage, presumably also a result of radiation therapy, caused vertigo and episodic severe nausea and vomiting.
The character and location of the malignancy, in addition to the long- standing history of frequent respiratory infections suggested the possibility of chronic Epstein-Barr Virus (EBV) infection. Subsequent evaluation of serum and cerebrospinal fluid confirmed the presence of chronic EBV infection. As a probable consequence of EBV infection and radiotherapy, the patient's immune status continued to deteriorate and her course over the following years was one characterized by moderately severe immunosuppression and numerous secondary bacterial and viral infections, many of which required hospitalization. The last few years of the patient's life were spent at home in strict reverse isolation in attempt to reduce the frequency of infection. Followup neurological examination revealed a pancerebellar syndrome characterized by truncal ataxia and marked limb ataxia affecting both the upper and lower extremities.
Other remarkable medical history was hypothyroidism of long duration and a total hysterectomy, the reason for and date of which were not available in the medical records provided us.
During the course of her illness, the patient was managed with a wide range of medications. In the period immediately prior to her deanimation the following medications were being taken: Ruvert for vertigo, 2 grains thyroid extract daily for hypothyroidism, multivitamin tablet, Percodan for pain, and antibiotics, including Keflex.
Additional details of the patient's condition will be described in the section on the surgical protocol. After a long course of chronic illness with steady deterioration of her condition, SP2 succumbed to an acute aspiration during a seizure episode which was felt to be secondary to sepsis. SP2 had been a Suspension Member of the Bay Area Cryonics Society for several years prior to her deanimation.
At 1:30 AM, shortly after SP1 had begun cooling to dry ice temperature, we were notified of SP2's deanimation and were asked to prepare for her cryonic suspension. Arrangements were made for SP2 to be flown from Northern California, where she had deanimated, to the Orange County Airport by air ambulance.
Of necessity, the majority of the suspension team was released to get some sleep, during which time preparations for the perfusion of SP2 were carried out by three supervisory personnel. All of the team members present for the suspension for SP1 remained for the perfusion of SP2. In some instances personnel remained actively working without sleep in excess of 72 hours.
Negotiation with hospital personnel and arrangement of transport for SP2 were carried out by Art Quaife. In brief, SP2 had a respiratory arrest after aspiration of food following a grand mal seizure. At the time of her arrival in the hospital emergency room (ER) the patient was in full cardiac arrest. The ER staff agreed to administer such transport medications as they had on hand while giving CPR. A note attached to the patient when she arrived at Cryovita Laboratories listed the following medications as having been administered:
mannitol, 500 cc
Anectine, 60 mg
Tagamet, 300 mg
Solu-Medrol, 250 mg
Heparin was verbally reported as given, but was omitted from the list of medications accompanying the patient. CPR was continued for 15 minutes following administration of the last medication, after which the patient was transferred to the refrigerated morgue of the hospital and packed in ice.
External examination of SP2 upon arrival showed remarkable pathology, the details of which will be discussed in the next section.
SURGICAL AND PERFUSION PROTOCOL
After the patient's arrival, surface cooling with crushed ice was maintained until preparations were completed for the perfusion. The patient's temperature upon arrival was measured using YSI Type 401 thermistor probes. Temperatures were as follows: vaginal, 12.5øC; esophageal, 12.0øC; and oral, 8.0øC. Vaginal rather than rectal temperature was taken owing to the presence of rigor and our inability to position or turn the patient in the ice-filled shipping container in which she arrived. Cursory examination of the patient disclosed the presence of fully developed rigor mortis. A more detailed examination revealed several lesions on the lower extremities which penetrated deeply into subcutaneous fat and in one instance into muscle. These lesions had been packed with iodophor gauze tape and 4x4 gauze sponges. The etiology of these lesions is unknown, but they presented the appearance of chronically infected decubiti. Bilateral fibrotic scarring and numerous vesicles filled with clear fluid were observed on both legs and the lower abdomen. The abdomen was markedly distended and palpation (which was complicated by the presence of rigor and hypothermic stiffening) revealed a very firm, large, and immobile mass that was originally thought to be a large fibroma or other neoplasm, but was later discovered to be a massive enlargement of the liver (Federowicz, Hixon, and Leaf, 1984).
Following external examination, the patient was moved onto the operating table and thermistor temperature probes were placed in the esophagus and vagina. The procedure for surgical preparation was essentially the same as that employed for SP1: scalp incision with burr hole, right jugular cannula placement, and median sternotomy. No mid-thigh incisions were made, since the refractometer measurements of fluid filtrate on SP1 provided no significant information compared to arterial/venous differences.
When the median sternotomy was retracted open, the right diaphragm was found to protrude approximately 20 cm upward into the pleural space. The diaphragm was displaced by the massively enlarged liver, which compressed the right lower lobe of the lung.
Despite this anomaly, arterial and venous cannulation proceeded as described for SP1. However, an examination of the descending aorta and inferior vena cava, at the level of the diaphragm, indicated they were running through a solid mass. The inferior venous cannula was advanced beyond the diaphragm to confirm patency, then withdrawn to the normal position, above the diaphragm.
The perfusion protocol was the same as was used for SP1 in regard to extracorporeal circuit, base perfusate (except Solu-Medrol was omitted), and choice of glycerol as the cryoprotective agent. The average arterial perfusate temperature was 3.4°C, with a range of 3.0°C to 5.0°C. Esophageal temperature averaged 8.8°C, with a range of 4.2°C to 11.5°C. Continuous perfusion for 4 hours and 6 minutes at an average MAP of 40 mm Hg was used. Average pump flow rate was 0.7 liters per minute. Clumps of red blood cells typical of cold agglutination were also noted in this patient during the initial blood washout. A total of 80 liters of perfusate was used, with the following quantities and compositions:
5% glycerol perfusate, 25 liters
10% glycerol perfusate, 10 liters
15% glycerol perfusate, 10 liters
20% glycerol perfusate, 10 liters
25% glycerol perfusate, 10 liters
50% glycerol perfusate, 15 liters
Refractometry of paired arterial and venous perfusate samples was used to determine how rapidly glycerol concentration could be increased. An attempt was made to confine the A-V difference in glycerol concentration to a range of 150 to 200 mM. The data on glycerol concentration is tabulated in Table 5 and plotted in Figure 7. As can be seen, the objective of maintaining a 200 mM or less spread between arterial and venous glycerol concentrations was achieved. Introduction of glycerol was also achieved employing a substantially shorter perfusion time than was required for SP1. As with SP1, termination of perfusion was determined by observation of brain swelling through the burr hole in the skull. Terminal arterial glycerol concentration was 2.95 M, and venous glycerol concentration was 2.87 M.
Table 5. SP2 - Glycerol Concentration.
Time (min) 5 34 108 133 -------------------------------------------- Arterial Brix No. (1) 11.7 10.3 16.2 20.0 Moles (2) 0.608 0.610 1.39 1.95 Percent (3) 5.2 5.2 12.0 17.0 Time (min) 5 38 133 235 -------------------------------------------- Venous Brix No. (1) 9.0 10.6 17.2 26.0 Moles (2) 0.432 0.616 1.62 2.87 Percent (3) 4.0 5.5 14.1 25.0 --------------------------------------------1) Measurements made at the time of perfusion with a hand-held refractometer, temperature compensated, Atago Model N-1,Cat. #311-N.
2) Molar glycerol concentration calculated at a later date on the same samples that Brix measurements were made on. Measurements made with a Micro Osmette Model 5004 freezing point osmometer.
3) Conversion of glycerol concentration, molar to w/w percent, from Handbook of Chemistry and Physics, 60th Ed., CRC Press, 1979.
Figure 7. SP2 Glycerol Concentration vs. Perfusion Time.
Changes in peripheral vascular resist ance (PVR) are shown in Figure 8. The significantly lower PVR probably made most of the difference in perfusion time requirements between SP1 and SP2, i.e., good peripheral perfusion was achieved, compared to SP1.
Table 6 shows arterial and venous pH during the course of the perfusion. We believe these values are indicative of the presence of active anaerobic glycolysis.
Table 6. SP2 Arterial/Venous Perfusate pH.
Time (min) 10 40 142 164 ------------------------------------------------- Arterial 7.80 7.90 7.10 7.09 Venous 6.80 6.89 6.99 7.07
Observation of the cerebral cortex through the burr hole during perfusion revealed the same pattern of initial cerebral dehydration followed by cerebral edema as was observed in SP1 as glycerol penetrated the blood-brain barrier. Perfusion of both SP1 and SP2 was terminated at the point cerebral edema became pronounced enough to result in slight bulging of the cortex into the burr hole without injury to the brain. Values for PVR over the course of the perfusion are plotted in Figure 8. There was a significant correlation between terminal PVR values and maximum brain volume in both patients. These results and their importance to future cryonic suspension operations will be discussed in more detail below.
Figure 8. SP2 Peripheral Vascular Resistance vs. Perfusion Time.
Decannulation and closure of surgical wound sites was accomplished (Plate 5). External copper-constantan thermocouples were placed in the trachea and vagina, and on the foot, the patient was placed in double ethylene vinyl acetate plastic bags, and cooling begun in a dry ice/alcohol bath precooled to - 26°C (Plate 6). Figure 9 shows the tracheal, brain, and alcohol bath temperatures during cooling. Approximately the same total time as for SP1, 28 hours, was used to achieve -70°C. (At the time, it was thought that a probe was being placed in the esophagus. Postmortem examination of SP2's remains following conversion to neuropreservation in 1983 disclosed that the "esophageal" probe had in reality entered the trachea and was not in close contact with tissue, which accounts for the slow change in the tracheal probe temperature seen in Figure 9 (Federowicz, Hixon, and Leaf, 1984)).
Plate 5. Closing the sternum with stainless steel wire. The wire has been driven through the sternum with a needle holder (H), and is being pulled tight with Kocher clamps (C).
Plate 6. SP2 being cooled in an alcohol-dry ice bath in a foam insulated box. Telethermometer (T) and Temperature probe switch (S).
Figure 9. SP2 Dry Ice Cooling.
SP1 and SP2 were transported together to Emeryville, California for liquid nitrogen vapor phase cooling and placement in long term cryogenic storage.
A list of factors are presented in Table 7 for comparison. Differences in age were probably the most important. SP1 was 79, and SP2 was 36. The general condition of the vascular system and the multiplicity of other age- related changes are probably of significance. Based on greater age alone, the presence of a less compliant vascular system, narrowing of the inner diameter of major arterial vessels, reduced permeability of vessel walls, and greater antibody activity (i.e., increased cold agglutination) would be anticipated. These are certainly major contributing factors to differences in the final glycerol concentration achieved and the duration of perfusion.
Table 7. Comparisons: SP1 and SP2.
SP1 SP2 --------------------------------------------------------------------- Age 79 years 36 years Place of death Hospital Emergency Room Cause of death Cancer/MSOF Cancer/Acute aspiration Transport HLR/Drugs/Hypothermia Drugs/Hypothermia Weight 59.1 Kg. 65 Kg. Ischemic time 28 hours 24 hours Av. Temp. (Trach.) 6.0øC +- 2øC 8.8øC +- 4øC Perfusion MAP 50 mm Hg 40 mm Hg Perf. flow rate 0.8 l/min 0.7 l/min PVR 160 peak, 70 final 72 peak, 70 final Perfusion time 446 min 240 min CPA Equilib. Conc. 2.67 Molar 2.85 Molar Metabolism less lactate than SP2 lactate Cold agglutination Positive Positive ---------------------------------------------------------------------
Both SP1 and SP2 were in clinical environments when they were pronounced dead, and were not exposed to long periods of postmortem deterioration at ambient temperatures without transport medications and surface cooling. SP2 received only manual chest compression during the course of administration of the pre-transport pharmaceuticals. The patient was then packed in ice for transport to Cryovita. There was no metabolic support provided SP2 during surface cooling.
SP1 received a clearly better transport regimen. It is not possible to assess the relative contributions made by the differences in the administered transport protocols. The long distances these patients had to be transported would have made continuous support of circulation virtually impossible. HLR support is not well suited to lengthy transports due to low efficiency and marginal cardiac output. Only ECMO transport (Extracorporeal Membrane Oxygenator transport using a portable heart-lung machine), would have been adequate. ECMO capability will be available in the near future.
SP1 was a thin man with low body fat. Since muscle tissue is more vascularized than fat, we would expect a higher flow of perfusate and better distribution of glycerol in SP1. SP2 had a greater amount of fat, which is natural in women compared to men, but also a much greater amount due to the sedentary life style she led as a result of her long illness. Considering these factors, we would have predicted a better glycerol distribution and shorter perfusion time for SP1 compared to SP2. However, as shown by their respective glycerol equilibration graphs, SP1 required significantly longer perfusion time and achieved a slightly lower final glycerol concentration than did SP2, which is the opposite of what would have been expected if there were no other contributing factors.
As previously shown, peripheral vascular resistance (PVR) was calculated for both patients over the course of the perfusion. Figure 10 shows comparative vascular resistance, in terms of PVR, for SP1 and SP2. The areas under the curves, for four hours of perfusion, were measured by planimeter and compared. The PVR of SP1 was 47.7% greater than SP2, quite significantly different. Factors contributing to the observed difference in PVR are as follows:
Figure 10. Comparison of Peripheral Vascular Resistance, SP1 and SP2.
1) Normal aging pathology of arteries, including medial fibrosis and subsequent thickening of the endoarterial layer (Wolstenholme and Freeman, 1954, p.17). Arterial wall thickening is at the expense of inside vessel diameter. Resistance to flow is related to vessel length and inversely related to vessel diameter, e.g., a small change in inside vessel diameter has a large effect on resistance to flow. Additionally, this kind of vessel pathology reduces vessel elasticity and further restricts flow, compared to the normally more compliant arterial vessels of younger individuals.
2) Rheology of the blood. Because of its cellular components, blood is a non-Newtonian fluid, and therefore there is a non-linear relationship between pressure and flow. Reduced blood flow thus causes increased viscosity and vascular resistance, in general. However, this would not be a significant factor after blood washout.
3) Low temperature, increasing blood cell rigidity and consequently reducing flow at the capillary level, further contributing to viscosity increases. Again, after blood washout this should not be a problem.
4) Long ischemic times produced very low blood pH's. SP2 showed an arterial blood pH of 6.1 before the beginning of perfusion. Red cell rigidity, as well as rouleaux formation (stacks of blood cells), is increased by low pH.
5) The presence of cold agglutinins. Under some circumstances, which were present in both patients, blood cells stick together at reduced temperatures. A more complete discussion of these factors has been presented in the article on vascular obstruction and cold agglutinins published in the March, 1984 issue of CRYONICS (Leaf, 1984). We believe that Total Body Washout (TBW) before deep hypothermia would have been beneficial in both cases, in terms of preventing cold agglutination, but it clearly would have been of much more benefit in the case of SP1.
One final observation can be made concerning PVR. While there were considerable differences in PVR between SP1 and SP2 over the course of perfusion, there is one similarity. Both patients' perfusions were terminated at 70 PVR units. The criterion for termination of perfusion was brain size, without regard to PVR data, which was only calculated at a later date. However, if there were a reliable correlation between PVR and brain size, we could possibly use vascular resistance as an indication for perfusion termination. More data establishing the significance of this correlation will have to be acquired in humans before it can be validated. In any event, direct observation of the brain for developing edema will continue to be absolutely necessary, until other reliable criteria can be established.
Both patients' medical histories show chronic ailments. However, SP1 would be classed as a chronic death, and SP2 as acute. SP1 had active metastatic cancer that affected more than one organ system over an unknown period of time. The final stress of these failing organs resulted in cardiac arrest. SP2 had been successfully treated for cancer, but developed extensive fibrotic lesions, neurological deficits, and systemic infections. Death was acutely due to respiratory failure followed by cardiac arrest. Quantification of influences from the differences of pathology are not possible at this time. Only ischemic time is quantifiable, 28 hours for SP1 and 24 hours for SP2. To our knowledge, these are not significantly different.
Perfusion parameters, as shown in Table 7, are similar in terms of MAP and perfusate flow rates. Hypoxic perfusion time is quite different, SP1, 448 minutes, compared to SP2, 240 minutes. The effects of added hypoxia for SP1 at the temperature of perfusion are unknown, since we do not have comparable morphological data. In any case, it is extremely doubtful that hypoxic effects could be separated from other complications.
The final CPA concentrations given in Table 3 were achievable because we used glycerol rather than DMSO. No suspension patient has tolerated perfusion times close to these when DMSO was used. The added perfusion time of SP1 was well tolerated, in terms of edema, compared to DMSO- perfused patients, or compared to SP2.
Tables 3 and 5 show comparisons of arterial and venous determinations of glycerol concentration from samples taken from SP1 and SP2 respectively. The measure of CPA concentration in arterial and venous samples is important in determining how fast CPA concentration can be increased to the final value desired. Too rapid an introduction of CPA can cause cellular dehydration and damage. Figures 3 and 11 are the graphs used to convert osmolality and percent glycerol to moles, for the data given in Tables 3 and 5.
Figure 11. Glycerol Concentration. Molar <--> w/w%.
Table 8 lists some of the new information gained, and modifications in suspension protocol we shall institute, if possible, in future cryonic suspensions undertaken at Cryovita Laboratories.
Table 8. Conclusions.
1) Control surface cooling
The presence of cold agglutination in both of these patients demonstrates the importance of controlling surface cooling. Ice packs should only be used as long as necessary to achieve safe core body temperatures for transport. Unrestricted application of ice packs results in blood sludging, cold agglutination, high peripheral vascular resistance, restricted perfusate flow, poor CPA distribution, and prolonged perfusion time, (i.e., unnecessary exposure to high concentrations of CPA).
ECMO transport on portable heart-lung machines should be used when possible, in preference to HLR support. If suspension patients cannot be transported with circulatory support, they should undergo total body washout before being packed in ice and shipped to a facility for cryoprotective perfusion. TBW before shipping would also be desirable because: 1) Platelets and granulocytes could cause problems even with the stabilization medications present: and 2) Plasma is a poor cellular support compared to artificial solutions. If TBW is not possible, ice packs used in shipping should be removed from patients before TBW, and flushing perfusate temperature should be between 16-20°C to avoid inducing cold agglutination (Leaf, 1984). Administration of Dextran-40 and methylprednisolone in normal clinical amounts during transport will help reduce the degree of cold agglutination experienced.
The presence of persistent and pronounced lactic acidosis indicates the importance of oxygenating perfusates employed during ECMO transport, TBW, and cryoprotective perfusions. For short-duration transports (those 4 hours or less) or for TBW followed by air shipping, a bubble-type oxygenator such as the Shiley S-100A is preferred. A bubble-type oxygenator should always be employed where there is any possibility of intravascular coagulation, as the porous defoaming column can act as a coarse prefilter of large surface area. For transports involving extended periods of extracorporeal support (greater than 4 hours) a membrane type oxygenator such the Sci-Med spiral-wound silicone membrane is preferred, because of this oxygenator's ability to preserve cellular blood components for extended periods of perfusion. Another means of controlling pH is the use of perfusates containing only physiological levels of glucose.
The long perfusion times possible, and the high CPA concentrations achieved with these patients, as contrasted with previous perfusions carried out in this laboratory employing DMSO (Leaf, 1979, 1981), indicate the clear superiority of glycerol as the cryoprotective agent of choice for perfusion of ischemically injured suspension patients. Cryoprotective perfusion should be carried out at 8-12oC to allow for adequate intracellular penetration of glycerol and to minimize cellular dehydration.
The deciding factor in terminating the perfusion was the direct observation of brain size. As CPA concentration increased, the brain began to lose volume due to osmotic removal of water. Brain volume reduction was approximately 20%, and had the same appearance as in neurosurgical patients one of us (Leaf) has seen in the UCLA operating rooms after the patient was given 1-2 gr./kg. IV mannitol. This technique is used for neurosurgery when the optic nerves must be exposed. While the brain can tolerate considerable volume reduction, it cannot tolerate high pressures created by expansion against the constraining cranial vault. When the blood-brain barrier is opened by high enough CPA concentration, the brain assumes its normal size. If cerebral edema occurs during perfusion, then the obvious point to terminate perfusion will be before the brain is subject to elevated intracranial pressure with the associated risks of no-reflow and herniation of the brain stem or of the cortex through the burr hole. It would in any case be useless to continue perfusion, in terms of trying to increase CPA concentration in the brain, because cerebral perfusion will cease as the intracranial pressure exceeds the mean arterial pressure. Therefore, observation of brain size is the best method for determining the end point of perfusion for suspension patients. This is the criterion we used for SP1 and SP2. This is also now our standard criterion for termination of perfusion. The second standard will remain achievement of the desired terminal CPA concentration. Since brain volume decreases promptly when perfusion is discontinued after the development of cerebral edema, it has been proposed that higher terminal CPA concentrations might be achieved by reinitiating perfusion after a "rest period" to allow spontaneous reversal of brain swelling. Given enough stop-start cycles, the proper CPA concentration may be achievable despite the development of cerebral edema.
The most likely solution to the high PVR encountered in SP1, aside from controlling cold agglutination, is the application of pulsatile perfusion during TBW and cryoprotective perfusion. This is a known, effective method employed during clinical perfusion of patients for reducing PVR (Taylor et al, 1978). Cannulation, through median sternotomy, of the inferior and superior vena cava for perfusate return, and the aortic arch for perfusate delivery are desirable, particularly if pulsatile flow is to be used. In the future we will employ left ventricular venting in an effort to improve heart and lung perfusion by reducing left atrial pressure and potential left ventricular distension.
A technique of recirculating perfusate is superior to single pass stepped increases in CPA concentration for establishing a more controlled cryoprotective ramp. Several types of mixing systems that would provide a continuous ramp are available. We have used a system for animal work with a large reservoir, to which perfusate of high glycerol concentration is added continuously. This produces a ramp that increases quickly at the beginning, and slower and slower as more concentrated perfusate is added. Other more complicated systems that produce linear and upward-curving ramps are also possible. These might allow much better control of the brain edema.
Inability to monitor cryoprotectant concentration continuously interferes seriously with any attempt to actively control edema. Monitoring may be done either with osmometry or refractometry. Our experience with osmometry since these suspensions has been unsatisfactory in that they balk at measuring cryoprotectant solutions with high concentrations of colloid and cryoprotectant. Hand-held refractometers have been unsatisfactory because they are usually designed for a single application and work in a fairly narrow range. A good general purpose bench refractometer would be much more useful. Even better would be a recording system with process refractometers in the arterial and venous lines that could monitor cryoprotectant concentration continuously.
As noted at the beginning of this article, observations made during the course of these suspensions have been implemented both in animal work and the human suspension carried out since that time.
The goal of cryoprotective perfusion has been to replace enough water with cryoprotective agents to keep the amount of water available in tissue for freezing to approximately 50%. There is evidence from whole mammal research that this amount of ice is the maximum tolerable, or at least significantly more injury occurs if more than 50% of available water is frozen (Lovelock and Smith, 1956; Noble, 1980).
The changes in technique discussed above are all aimed at improving the primary goal of good cryoprotective perfusion. Every case of experimental human suspended animation, like every scientific experiment, offers its grain of truth that will hopefully lead to new understanding and knowledge that can be employed to achieve better perfusion of cryonic suspension patients.
We would like to thank Brenda Peters and Al Lopp for major help in entering and proofreading this paper.
Bianchi, J., (1983), Cryonics and the Law, Part II, CRYONICS, #40, (Nov, (1983), p. 24.
Federowicz, M., H. Hixon, and J. D. Leaf, (1984), Postmortem Examination of Three Cryonic Suspension Patients, CRYONICS, #30, (Sept, 1984), p. 16.
Leaf, J. D., (1979), Cryonic Suspension of Sam Berkowitz: Technical Report, Long Life Magazine, 3, 30-35, (March/April, 1979).
Leaf, J. D., (1981), Case Study: KVM Suspension, CRYONICS, #13, (Aug, 1981).
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Lovelock, J. E., and Audrey U. Smith, (1956), Studies in Golden Hamsters During Cooling To and Rewarming From Body Temperatures Below 0øC. Part III: Biophysical Aspects and General Discussion, Proc. Roy. Soc., Ser. B, 154, 427-42 (1956).
Noble, C., (1980), Letter to CRYO-LETTERS (Dr. Felix Franks), THE IMMORTALIST, 11(10), 2-5 (Oct, 1985).
Taylor, K. M., W. H. Bain, K. J. Maxted, M. N. Hutton, W. Y. McNab, and P. K. Caves, (1978), Comparative Studies of Pulsatile and Non-Pulsatile Flow During Cardiopulmonary Bypass, Journal of Thoracic and Cardiovascular Surgery, 75, 569-73 (1978).
Wolstenholme, G., and J. Freeman, Eds., (1954), Peripheral Circulation in Man, A CIBA Foundation Symposium, J.&A. Churchill, Ltd., London, 1954, p.174
Audrey Smith's hamsters survived if: 1) <= 50% of total body water froze; or, 2) <= 60% (rounded up from high 50's) of brain water froze.
This suggests that if 40-50% of liquid volume remains (NOT liquid water), mechanical injury from ice will be avoided. (Smith's results do not speak to the effect of dehydration injury, i.e., the effect of replacing water by glycerol. Perhaps this is a good thing, because it is impossible to prevent 50% of the water from freezing, since even a 50% glycerol solution will freeze! So will a 60% glycerol solution.)
Accepting the goal of preventing mechanical injury then, and forgetting about "solution effects" injury, we compute the following:
Let Vv = the concentration of glycerol (in %v/v) that is unfreezable.
Let Vs = the concentration of glycerol (in %v/v) one starts with.
During freezing, we let half the volume of the solution be converted into ice, during which time Vs --> Vv. Since the volume has changed by a factor of two, and the volume of glycerol is unchanged, the concentration of glycerol as %v/v has doubled. Therefore, Vs = 1/2 Vv. According to Rasmussen and Luyet, a 73% w/w glycerol solution will not freeze (Biodynamica, 10, 329 (1969)).
Converting to %v/v units gives:
73 --> 57.94 ml = 57.94 = 69.0% -- ----------------- ----- 73 --> 57.94 + 26 --> 26 83.94
Therefore Vs = 69/2 = 34.5% v/v ÷ 4.72 Molar.
If we relax the "Smith Criterion" from the body's 50% requirement to the brain's ~ 60% requirement, then:
Vs = (40/100) Vv = 0.4 Vv = 27.6% v/v ÷ 3.78 Molar.