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Circulation. 1995;91:431-444

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(Circulation. 1995;91:431-444.)
© 1995 American Heart Association, Inc.

Articles

A New Solution for Life Without Blood

Asanguineous Low-Flow Perfusion of a Whole-Body Perfusate During 3 Hours of Cardiac Arrest and Profound Hypothermia

M. J. Taylor, PhD; J. E. Bailes, MD; A. M. Elrifai, MD; S.-R. Shih, MD; E. Teeple, MD; M. L. Leavitt, PhD; J. G. Baust, PhD; J. C. Maroon, MD

From the Cryobiology and Hypothermic Medicine Program (M.J.T., A.M.E), Neurosciences Research Center, Allegheny-Singer Research Institute, Departments of Neurosurgery (M.J.T., J.E.B., A.M.E., S.-R.S., M.L.L., J.C.M.) and Anesthesiology (E.T.), Allegheny General Hospital, and Medical College of Pennsylvania (Allegheny Campus), Pittsburgh, Pa; and Cryomedical Sciences Inc (J.G.B.), Rockville, Md.

Correspondence to Dr M.J. Taylor, Department of Neurosurgery Research, Allegheny-Singer Research Institute, 320 East North Ave, Pittsburgh, PA 15212.


*    Abstract
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Background The benefits of hypothermia for preventing ischemic injury are well known, but its application in surgery to protect the whole body during procedures requiring circulatory arrest is currently limited to <1 hour at 15°C using 50% hemodilution. In a significant departure from previous methods, we have developed a technique of asanguineous blood substitution with low-flow perfusion and cardiac arrest at <10°C in a canine model. Our approach has been to design a hypothermic blood substitute that would protect the brain and visceral organs during several hours of bloodless perfusion. Two different solutions have been designed to fulfill separate requirements in the procedure.

Methods and Results With the use of extracorporeal cardiac bypass, 14 adult dogs were exsanguinated during cooling; 11 dogs were blood substituted using in combination the "purge" and "maintenance" solutions (group 1), and 3 dogs were perfused throughout with the "purge" solution alone as controls (group 2). After cardiac arrest, the solutions were continuously circulated for 3 hours by the extracorporeal pump (flow rate, 40 to 85 mL · kg-1 · min-1; mean arterial blood pressure, 25 to 40 mm Hg). The temperature was maintained at <10°C (nadir, 6.6±0.1°C) for 3 hours, and the hematocrit was kept at <1% before controlled rewarming and autotransfusion. In the experimental group, the heart always started spontaneously in the temperature range of 11°C to 27°C, and 8 animals have survived long-term (current range, 14 to 110 weeks) without any detectable neurological deficit. In contrast, two control animals survived after extensive and aggressive cardiac resuscitation efforts; after surgery they exhibited transient motor and sensory deficits for approximately 1 week. Evaluation of biochemical and hematological parameters showed only a transient and inconsequential elevation in enzymes (eg, brain, liver, cardiac) in group 1 compared with the markedly greater elevations in group 2. For example, immediate postoperative values (mean±SEM) for lactate dehydrogenase were 114±10 for group 1 versus 490±210 for group 2 (P<.03); for SGOT, values were 93±18 for group 1 versus 734±540 for group 2 (P<.05). On day 1 for creatine kinase (CK), the group 1 value was 7841±2307 versus 71 550±2658 for group 2 (P=.03), and for CK-BB, the group 1 value was 108±22 versus 617±154 for group 2 (P=.03). Neurological evaluation using deficit scores (NDS) was based on a modification of the Glasgow Coma Scale score: 0, normal; 1, minimal abnormality; 2, weakness; 3, paralysis; 4, coma; and 5, death. At days 1 and 2 after surgery, NDS (mean±SEM) were 0±0 for the experimental group versus 1.5±0.5 for the control group. At days 3 and 7 after surgery, NDS were 0±0 for group 1 versus 1.0±1.0 for group 2.

Conclusions The faster neurological recovery of dogs treated with the "intracellular-type" maintenance solution supports the biochemical data showing the benefits of this type of blood substitute for extending the safe limits of hypothermic cardiac arrest procedures to >3 hours.


Key Words: hypothermia • blood substitutes • heart arrest • ischemia


*    Introduction
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 up arrowAbstract
 *Introduction
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Today, hypothermia is frequently used as an adjunct in a variety of surgical procedures by virtue of its inherent property to facilitate tissue preservation during an ischemic insult.1 Potential clinical benefits of hypothermia were recognized as early as 1939, when it was demonstrated that surface cooling of an ischemic limb in rats improved overall survival.2 However, it was not until the advent of cardiopulmonary bypass in the early 1950s that hypothermia became widely used clinically.3 4 Applications of hypothermia extend from the ex vivo preservation of a variety of donor organs before transplantation to the preservation of tissues in situ during surgical procedures. The success of certain techniques, predominantly in the fields of neurosurgery, cardiovascular surgery, and trauma medicine, depends on varying degrees of imposed hypothermia to minimize ischemic injury by slowing metabolism and thus reducing the demand for oxygen and energy substrates in tissues. Nevertheless, the safe limit of cardiac arrest at temperatures in the region of 15°C is <60 minutes if clinical sequelae, especially neurological deficits, are to be avoided.5 6 7 This severely limits surgical procedures that are otherwise technically feasible, and it remains a major goal for surgeons to extend the safe limits of hypothermic arrest beyond the present 1-hour limit.

The properties of hypothermia as applied to whole-body perfusion are not exclusively beneficial. Hypothermia is associated with an increase in blood viscosity that contributes to red cell sludging in the microvasculature as well as detrimental coagulopathies.8 9 10 11 Attempts to counteract these effects have required that patients are hemodiluted to varying degrees using a variety of solutions, but the choice of diluent and extent of hemodilution remain controversial.12 13 In view of the risks and limitations associated with deep hypothermia (<15°C) combined with hemodilution, the approach we favor and advocate for further development is to use ultraprofound hypothermia (<10°C) with complete blood replacement.14 15 Deeper hypothermia can provide more effective suppression of metabolism, thereby extending the tolerance to ischemia, and minimizes demand for oxygen to levels that can be adequately supplied in a cold aqueous solution without the need for oxygen-carrying molecules. Complete exsanguination ameliorates the complications associated with increased viscosity, coagulopathies, and erythrocyte clumping of cool blood. We have previously demonstrated the feasibility of this approach in a canine model,14 and we now report the design of aqueous blood substitutes that provides rapid and improved full neurological, physiological, and biochemical recovery after an extended period of >=3 hours of controlled ultraprofound hypothermia (<10°C).


*    Design of an Acellular, Aqueous Blood Substitute
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Developments in the field of ex vivo organ preservation have advanced during the past quarter century to the point where organs for transplantation can be safely stored for variable periods depending on the nature of the organ. A kidney, liver, and pancreas can be stored for days, but the clinically accepted limit for a heart is only <=6 hours16 17 and in practice it is <3 hours.17 Although the tolerance of neurological tissue to hypothermic storage has not been studied widely, cerebral recovery has been reported after 4-hour storage at 2±1°C,18 19 and thus development of a technique for universal preservation of all the tissues of the body for 3 hours would appear to be a reasonable objective on the basis of achievements for isolated tissue preservation. In contrast to the types of solutions that have been used historically as hemodiluents for clinical hypothermia, ie, normal physiological "extracellular" balance salt solutions, our approach has been to design aqueous blood substitutes for total body hypothermic perfusion that embody many of the principles now identified as contributory and important for successful organ preservation.20 21 22 For descriptive purposes, we refer to this new hypothermic blood substitute as Hypothermosol (HTS).

In practice, two solutions have been formulated to fulfill separate requirements during an established procedure for profound hypothermia with complete blood substitution.14 The principal solution is a hyperkalemic "intracellular-type" solution specifically designed to "maintain" cellular integrity during the hypothermic interval at the lowest temperature. This solution has therefore been designated the Hypothermosol maintenance solution (HTS-M). The second solution is an extracellular-type flush solution designed to purge the circulation of blood during cooling. In particular, it is important to remove erythrocytes, which can cause sludging and blockage of the microvasculature during anoxia and hypothermia. This "purge" solution, designated Hypothermosol purge solution (HTS-P), is also designed to flush the system of the hyperkalemic HTS-M solution during warming and to help flush out accumulated toxins and metabolic by-products that might promote oxidative stress and free radical injury on reperfusion.


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Preparation of Solutions
The compositions of the aqueous blood substitute solutions HTS-M and HTS-P are given in Table 1Down. The solutions were prepared by dissolving chemicals and ingredients of the highest purity available in ultrapure deionized and distilled water. Where possible, tissue culture–tested chemicals (Sigma Chemical Co) were used to maximize biocompatibility. All solutions were filter sterilized (0.22 µm, Gelman filters), dispensed into 1-L sterile plastic bottles (Nalgene, PETG), and stored in a cold room (4°C) before use.


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  		Table 1. Composition of Hypothermosol Blood Substitutes

Surgical Procedure and Cardiopulmonary Bypass
Adult mongrel dogs used in this study ranged in weight from 10 to 18 kg, and the study procedure was in accord with the guidelines and standards of the US Public Health Services for the use and care of laboratory animals and approved by the Institutional Animal Care and Use Committee of Allegheny-Singer Research Institute.

With an aseptic technique used throughout, animals were surgically prepared for anesthesia and extracorporeal cardiac bypass as previously described.14 15 In brief, dogs were preanesthetized with atropine (0.04 mg/kg IM) and pentothal (10 mg/kg IV) before the insertion of an endotracheal tube to maintain anesthesia using an azeotropic mixture of halothane and ether (flether). Standard ECG electrodes were placed for continuous recording of heart activity, and an intravenous catheter was inserted into the cephalic vein for infusion of a plasmolyte drip (60 mL/h). Brain, esophageal, and subcutaneous temperatures were measured continuously by appropriate placement of temperature transducers. Intracranial pressure was recorded with the use of an intraparenchymal transducer (Camino) placed in the right frontal lobe. The left femoral artery and right external jugular vein were cannulated to establish extracorporeal bypass circulation. The right femoral artery was cannulated to measure systemic arterial blood pressure and for arterial blood sampling. A 7F Swan-Ganz catheter was advanced to the pulmonary artery via the right femoral vein to monitor pulmonary artery wedge pressure and central venous pressure. The animal's blood was anticoagulated with heparin (100 U/kg) to achieve an activated clotting time of >300 seconds.

The circuit used for bypass was similar to that described previously14 15 but was modified to include a centrifugal pump (Medtronic, Biomedicus) and a membrane oxygenator. The circuit closely resembled a clinical bypass circuit except the following modifications were installed: a drain line was connected to the venous side of the circuit to facilitate exsanguination, and a port connecting the oxygenator to a funnel was introduced to permit the blood substitutes to be added to the circuit.

Cooling and Controlled Exsanguination
Hypothermia was first obtained with surface cooling to 25°C. As shown in Fig 1Down, the average cooling rate in the range of 37° to 25°C was 0.2°C/min. Once esophageal (core) temperature fell to {approx}25°C or the heart rate slowed to 45 beats per minute, exsanguination was started. Drained blood was collected in sterile containers and maintained at 4°C. Extracorporeal circulation was initiated with the HTS-P solution to wash out the remaining blood, and the entire blood volume was exchanged with the blood substitute. Throughout this and subsequent exchanges, the operating table was raised to maintain a hydrostatic head of 40 in above the venous return reservoir, since preliminary experiments had shown this to be essential for effective drainage and control of fluid exchange necessary to avoid fluid retention. The HTS-P blood substitute was immediately exchanged with precooled, oxygenated (PO2 >500 mm Hg) HTS-M, which was also an effective cardioplegic solution. Immediately after the animal had cardiac arrest, the respirator was turned off, and the HTS-M blood substitute was continuously circulated for 3 hours at rates of 40 to 85 mL · kg-1 · min-1, yielding a systolic arterial fluid pressure of 25 to 40 mm Hg. The average cooling rate effected via the pump was 0.5°C/min between 25° and 10°C (Fig 1Down).



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  		Figure 1. A and B, Plots of temperature profiles for the two groups of dogs showing perfusion fluid exchanges during cooling and rewarming. HTS-M indicates Hypothermosol maintenance solution (M); HTS-P, Hypothermosol purge solution (P).

When the temperature had fallen to <10°C (nadir, 6.6±0.1°C), the hematocrit was measured at <1%, showing that complete blood substitution was achieved. During this phase, the perfusion fluid was completely exchanged twice at regular intervals with fresh precooled HTS-M solution.

Rewarming and Blood Replacement
After 3 hours of perfusion at an esophageal temperature <10°C, the rewarming regimen was initiated using both internal and external warming. At the commencement of warming from the nadir temperature, the HTS-M solution was replaced with HTS-P to purge the system of the hyperkalemic maintenance solution. The objective here was to reduce the potassium concentration to {approx}10 mmol/L or less, necessary to permit cardiac reactivation. When the esophageal temperature reached 10°C, the animal's own blood was introduced into the circuit and removal of the HTS-P continued until the whole blood volume was reintroduced.

During rewarming, the heart usually started and resumed normal sinus rhythm spontaneously between 11° and 20°C; otherwise, electroversion (150 to 200 J) was implemented. Respiration was resumed at between 24° and 34°C. The early phase of rewarming (10° to 28°C) was controlled via the pump at a rate of 0.7±0.1°C/min based on our previous observations.23 From 28°C, dogs were allowed to warm slowly (0.09°C/min) to normal temperature with the aid of external warming provided by a heating blanket and heat lamps. As rewarming progressed, anesthetic was given to smooth the transition from cold narcosis to recovery. The animals were then weaned from the pump, decannulated, and observed during unrestricted postoperative recovery for neurological function. Neurological evaluation was performed using Neurological Deficit Scale (NDS) scores based on a modified Glasgow Coma Scale score; in NDS, 0 equals normal; 1, minimal abnormality; 2, weakness; 3, paralysis; 4, coma; and 5, death.

In addition, blood and urine samples were collected at regular intervals during the days and weeks after surgery for evaluation of biochemical status and to determine organ function.

Experimental Groups
The first group was composed of 11 animals that were blood substituted with a combination of the HTS-P and HTS-M solutions as described above. A second group of three animals was treated identically, except the dogs were perfused throughout the hypothermic period with HTS-P alone and not substituted with the HTS-M solution. This group served as controls for evaluation of the merits of hypothermic perfusion with the intracellular-type maintenance solution per se. In the knowledge that perfusion with HTS-P alone was suboptimal, increasing the probability of postoperative complications, the control group was limited to 3 animals on the grounds of both humane and economic considerations.


*    Results
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As a pilot experiment in this study, the first dog in group 1 was maintained at 7°C for 120 minutes (cardiac arrest, 149 minutes), but for the remaining 10 animals the esophageal temperature was maintained at <10°C for 182±1 minutes during which time a nadir core temperature of 6.4±0.1°C and a brain nadir of 7.5±0.5°C were recorded. The cardiac arrest time for these animals was 215±3 minutes. The core (esophageal) temperature of the control group of animals (group 2) was maintained at <10°C (nadir, 6.4±0.1°C) for 175±6 minutes and the hearts were arrested for 193±1 minutes during the procedure.

Physiological and Neurological Recovery
During rewarming, animals were observed closely for signs of physiological and neurological recovery. All dogs in group 1 were successfully revived, with the first heart beat recorded in the temperature range of 11° to 27°C, regular heart beat and sinus rhythm resuming at 26±1°C, and respirations in the range of 21° to 32°C. Five dogs in this group required minimal intervention with electroversion and small doses of lidocaine to control cardiac ventricular arrhythmia (Table 2Down). In contrast, in 1 dog in group 2 (in the temperature range of 27° to 32°C), the heart initiated a few beats before lapsing into a persistent ventricular fibrillation that was refractory to interventional correction (eight doses at 150 J and three attempts using 200 J). Electromechanical dissociation was observed, and the animal died. The remaining 2 animals in this group demonstrated similar problems during the rewarming phase. However, resuscitative measures were revised in an aggressive attempt to stabilize the persistent ventricular fibrillation; these involved the administration of 15 to 30 mEq of KCl over 10 to 15 minutes in combination with 500 mg Ca2+ and repeated countershocks (100 to 200 J) to return the heart to sinus rhythm, a requirement noted previously by others.24 25 Subsequent doses of K+ and electroversion were required to correct frequent relapses into ventricular fibrillation. These abnormal and aggressive resuscitative measures were eventually successful in stabilizing the hearts of these 2 dogs during the rewarming phase. Table 2Down shows that a significantly greater number of doses of various cardiotonic and cardioversion measures were required in the group 2 dogs compared with group 1.


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  		Table 2. Cardiac Resuscitation Measures Administered During Rewarming

Eight dogs in group 1 quickly regained full consciousness within 315±45 minutes of termination of cardiopulmonary bypass and have achieved long-term survival (current range, 14 to 110 weeks) with no apparent neurological deficits. Of the remaining 3 dogs in this group, 2 survived the procedure and regained consciousness but did not achieve long-term survival; 1 died on the second postoperative day from uncertain causes but pancreatitis and possible hypokalemia from vomiting and diarrhea were suspected. A second dog was killed 4 days after surgery due to the occurrence of seizures that autopsy revealed were possibly induced by the placement of the intracerebral pressure and temperature transducers. The third animal regained neurological reflexes but did not regain consciousness and died on the second postoperative day. It was observed in this animal that the intracranial pressure was high throughout, and autopsy revealed hemorrhage in the subarachnoid space, brain stem, and spinal cord as well as in other tissues. Excess heparin was suspected as a contributory factor in this one case. All survivors from group 1 were generally active with complete recovery of motor function within 24 hours as defined by their ability to stand, walk, eat, and drink. Three dogs showed extremely rapid recovery and were able to stand and walk within 12 hours of the procedure (the fastest was 5 hours, and the mean for the group was 20.2±4.0 hours). Moreover, these animals were all able to walk normally within 2 days and did not show any signs of hindlimb weakness, a problem frequently noticed and reported by others26 and by us in a previous series of experiments.

In group 2, the 2 animals that were successfully revived using the aggressive resuscitative measures described above have achieved long-term survival (18 and 26 weeks before elective killing), but their physiological and neurological recovery was noticeably slower. They demonstrated some mild to moderate neurological deficits such as hindlimb weakness and decreased vision consistent with observations from a previous study using alternative blood substitutes. These deficits appear to resolve within 1 week.

NDS scores were 0 at days 1 and 2 after surgery for the experimental group (group 1) compared with 1.5±0.5 for the control group (group 2); at 1 week after surgery, NDS scores were 0±0 versus 1.0±1.0 for the two groups, respectively.

Hematology and Biochemistry
Blood samples were collected and analyzed for a wide range of biochemical and hematological parameters before and after exposure to the hypothermic blood substitution procedure as we have previously described.27 28 Samples were also collected on days 1, 2, and 3 and weeks 1, 2, and 3 after surgery. Where appropriate, statistical comparisons between groups were made by comparing mean values using the Mann-Whitney test with 95% confidence limits.

Hematocrit, hemoglobin, and red cell counts were slightly suppressed for 2 weeks after surgery in all surviving dogs; however, these parameters were not as depressed as we have reported previously in a former series of experiments using the previous generation of blood substitute.14 28 Platelet counts were decreased after surgery but were normal by 1 week. Prothrombin time was not elevated and fibrinogen concentrations were normal except during the immediate posthypothermia interval.

All dogs, irrespective of the experimental treatment, had normal electrolyte levels after the procedure. Only magnesium concentrations deviated from normal levels during the immediate postoperative period for group 1, possibly due to the elevated Mg2+ content in the HTS-M solution, but serum levels had returned to normal by the first postoperative day. Similarly, serum glucose levels were elevated in the first postoperative samples from animals in both groups but were in the normal canine range by postoperative day 1. Indications of hepatorenal functions such as blood urea nitrogen, creatinine, and bilirubin remained within the normal ranges in all dogs throughout the postoperative follow-up. Cholesterol, triglyceride, and amylase levels were also normal.

Measurements of enzymes that might reveal any injury in vital tissues such as muscle, liver, heart and brain showed the following changes: lactate dehydrogenase did not exceed normal values at any point in group 1 animals perfused with HTS-M. However, as shown in Fig 2Down, lactate dehydrogenase values for dogs perfused with HTS-P alone were highly elevated and were significantly greater (P<.05) at each sampling point within the first 2 postoperative weeks. Other diagnostic enzymes were elevated in animals in both groups, but as shown in Table 3Down, the extent of increase in group 1 animals perfused with HTS-M was generally modest compared with the enormous rises measured in group 2 animals perfused with HTS-P alone.



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  		Figure 2. Bar graph of presurgical (Pre) and postsurgical (Post) serum levels of lactate dehydrogenase (LDH) showing mean±SEM values for group 1 animals perfused during hypothermia with the combination of HTS-P and HTS-M, as described in the text, and mean±SEM values for the group 2 dogs perfused during hypothermia with HTS-P alone. Upper limit of the normal range for dogs is shown by the dotted line. HTS-P indicates Hypothermosol purge solution; HTS-M, Hypothermosol maintenance solution.


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  		Table 3. Proportional Increase in Postsurgical Serum Enzyme Levels Relative to Presurgical Levels as a Diagnostic Indicator of Tissue Stability

Serum levels of creatinine kinase (CK) and its isozymes are used clinically as sensitive indicators of injury and disease in specific organs and tissues. Fig 3Down shows that increases in the levels of CK and the isozymes for skeletal muscle (CK-MM), brain (CK-BB), and heart (CK-MB) were transiently and moderately elevated in group 1 animals compared with the huge and more prolonged increases recorded in the surviving control animals of group 2.



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  		Figure 3. This and facing page. Bar graphs of comparative changes in levels of serum creatine kinase (CK) and the isozymes for muscle (CK-MM), brain (CK-BB), and heart (CK-MB) before (Pre) and after (Post) hypothermic blood replacement showing mean±SEM values for group 1 dogs perfused during hypothermia with a combination of HTS-P and HTS-M (striped bars) and mean±SEM values for group 2 dogs perfused during hypothermia with HTS-P alone (cross-hatched bars). HTS-P indicates Hypothermosol purge solution; HTS-M, Hypothermosol maintenance solution.

Three of the five enzymes measured had returned to normal levels within 1 week in the HTS-M group, whereas only alkaline phosphatase had returned to normal in the same period in the HTS-P group. Although CK was the only enzyme to remain above normal levels for more than 2 weeks after surgery in the HTS-M group, the actual extent of elevation was very modest compared with the values for surviving control dogs. Table 3Up shows that after 7 days, total CK was only 1.8±0.5 times greater than presurgical values in the experimental group compared with 6.3±2.5 times higher for the controls. Measurements of the CK isozymes revealed that the mean proportional increases in isozyme release were greater for muscle (CK-MM) and heart (CK-MB) than for brain (CK-BB), which showed only a threefold increase on day 1 in the HTS-M group. For the control dogs (HTS-P), the mean proportional increase in the brain fraction of serum CK was an order of magnitude greater at day 1. Table 3Up also shows that heart and skeletal muscle fractions of CK had returned to presurgical levels (factor approximately 1.0) within 1 week in the experimental group but remained fourfold to fivefold higher in the control group.

The faster return to normalcy of tissue biochemistry in group 1, as judged by the measured release of a variety of enzymes, correlates with the noticeably quicker neurological recovery of dogs in the experimental group (HTS-M) compared with the revived dogs from the control group.

The two surviving control dogs and two of the long-term surviving animals from the experimental group were killed at 18, 26, 78, and 77 weeks, respectively. Gross postmortem examination revealed no abnormalities in any of the tissues from dogs in either group except in the hearts from the control animals. In both cases, severe scar tissue was prevalent in the right and left ventricles, indicative of repair of multiple myocardial infarcts. Histopathology showed multiple microscopic lesions and myocardial fibrosis. These observations are consistent with the enzyme profiles that showed that serum CK-MB (heart fraction) was only marginally elevated in the HTS-M dogs and normal values were recorded within 1 week after surgery. This contrasts markedly with the surviving control dogs, in which there was 146±19-fold increase in CK-MB on the first day and a persistent fivefold elevation after 1 week.


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Need for Profound Hypothermia in Surgery
Safe techniques for bloodless surgery have long been a goal to facilitate a variety of complex surgical procedures. Moreover, total circulatory arrest is widely acknowledged to be an ultimate, yet rarely used, tool of the surgeon. It has been recognized since the early 1950s that attainment of these objectives will rely on hypothermia as a potentially safe and practical method of protecting the brain and vital organs during ischemia and anoxia. The theoretical basis for this has been reviewed by others1 9 29 and, in essence, relies principally on the effect of temperature reduction on metabolism and oxygen demand. It is well established that within the range of 0° to 42°C, oxygen consumption in tissues decreases by 50% for each 10°C fall in temperature.30 For the brain, oxygen consumption at 5°C is estimated to be 6% of the normothermic rate, and Bering postulated that the brain may tolerate ischemic periods for up to 3 hours at temperatures below 5°C.31 It is also known that myocardial tissue can be preserved during 3 hours of global ischemia at 4°C.32 33 Since it is well established that other vital organs can tolerate anoxia for much longer periods than the heart and the brain, it has been anticipated that whole-body protection may be possible during 3 hours of total circulatory arrest if body temperature is maintained as low as 5°C.34 Nevertheless, one theoretical calculation based on adult oxygen reserves and decreases in oxygen consumption with hypothermia predicted a safe arrest time of 56 minutes at 10°C.35 This is supported by the clinically determined "safe limits" in cardiopulmonary bypass, which for the past three decades have been set at approximately 1 hour using a lower temperature limit of 12° to 15°C as we and others have reviewed previously.1 12 14 29 These limits greatly restrict surgical times and potential application in other areas besides the surgical repair of congenital heart defects, which is currently the area of greatest clinical application of hypothermia. Extended hypothermic arrest would be of immense benefit for interventional surgery in a variety of conditions that are otherwise inoperable; these include complex neurosurgical problems such as giant cerebral aneurysms, other vascular lesions, the treatment of aneurysms of the aortic arch, and the excision of tumors in the proximity of major blood vessels.

Lower temperatures have been explored experimentally in a rational attempt to extend the protective period of hypothermic circulatory arrest. With profound hypothermia (<10°C), arrest times of 90 to 120 minutes have been reported in dogs without evidence of neurological damage, but a significant proportion of animals died or had serious problems relating to hemorrhage, pulmonary edema, or other detrimental events.24 25 26 Moreover, early clinical use of profound hypothermia during cardiopulmonary bypass resulted in neurological complications even without circulatory arrest.29 After these experiences in the early 1960s, profound hypothermia was not exploited clinically, and accepted limits for circulatory arrest have remained at approximately 60 minutes with temperatures no lower than 15°C.1 12 14 29

Rationale for Complete Blood Replacement During Profound Hypothermia
Throughout the history of the use of hypothermia in relation to clinical procedures, attempts to extend the safe limits of cardiac arrest have focused on a variety of important aspects of the technique; these have been reviewed by Hickey and Anderson.1 29 Nevertheless, it is surprising that relatively little attention has been devoted to the composition of the whole-body perfusate. Logically, it has been assumed that blood-based perfusates provide the best medium for vascular perfusion during clinical hypothermic procedures, and this is justified by the principal requirement for continued and substantial demands for oxygen during mild or moderate hypothermia. However, it is well established that cooling induces detrimental changes to various properties of blood that are not effectively ameliorated by simple hemodilution; these include dramatic increases in blood viscosity, coagulopathies, and the deformability and clumping of erythrocytes, which contributes significantly to the problem of multifocal blockage of the microvasculature and formation of tissue infarcts.36 The concept of totally removing the blood and replacing it with a suitable acellular substitute solution is a novel approach, the feasibility of which we have demonstrated experimentally for several hours of cardiac arrest.14 This technique provides a number of potential benefits; in addition to total vascular and capillary washout and the removal of harmful catabolic products, blood substitution provides the opportunity to control the extracellular environment directly and the intracellular milieu indirectly. Solutes can be added to maintain ionic and osmotic balance at the cellular and tissue levels during hypothermia. Biochemical and pharmacological reagents can help sustain tissue integrity in a variety of ways, including efficient vascular flushing, membrane stabilization, free radical scavenging, and providing substrates for the regeneration of high energy compounds during rewarming and reperfusion.20 21 22

Design of Hypothermic Blood Substitutes
The new whole-body perfusates developed in the present study were designed taking account of the current principles established for the preservation of isolated organs by cold storage.20 The rationale for the formulation of HTS-M was to provide the optimal concentration of ions and colloids to maintain ionic and osmotic balance within body tissues during hypothermia. In particular, an effective impermeant anion is included to partially replace chloride in the extracellular space and prevent osmotic cell swelling (ie, to balance the fixed ions inside cells that are responsible for the oncotic pressure leading to osmotic cell swelling and eventual lysis during ischemia and hypothermia). Lactobionate (formula weight, 358 D) was selected as a proven effective impermeant in hypothermic preservation solutions21 37 38 39 40 and because it is known to be a strong chelator of calcium and iron and may therefore contribute to minimizing cell injury due to calcium influx and free radical formation.41 The osmoticum of HTS-M is supported by the inclusion of sucrose and mannitol, the latter of which also has properties as a hydroxyl radical scavenger and reduces vascular resistance by inducing a prostaglandin-mediated vasodilatation, which may be of additional benefit.29 42 Clinical-grade dextran is included as a colloid for oncotic support to balance the hydrostatic pressure of perfusion and help prevent interstitial edema. It has long been known that dextran can improve the efficiency of the removal of erythrocytes from the microvasculature of cooled organs by inhibiting red cell clumping, increasing intravascular osmotic pressure, and reducing vascular resistance.43 44 45 46 Dextran is widely used clinically as a plasma expander and is readily and rapidly excreted by the kidneys.47 Moreover, there is ample recent evidence that dextran-40 is an effective and well-tolerated colloid in modern cold storage solutions for organ preservation.48 49 50

The ionic balance, notably the Na+/K+ and Ca2+/Mg2+ ratios, is adjusted to restrict passive diffusional exchange at low temperatures when ionic pumps are inactivated. In the area of cardioplegia and myocardial preservation, there is good evidence for improved survival using elevated concentrations of magnesium and very low but not zero calcium to avoid the putative calcium paradox.51 52 53 Some glucose is included in these hypothermic solutions as a substrate, but the concentration is low to prevent exogenous overload during hypothermia. This can potentiate lactate production and intracellular acidosis by anaerobic glycolysis.54

Acidosis is a particular hazard during hypothermia, and attention has been given to the inclusion of a pH buffer that will be effective under nonphysiological conditions that prevail at low temperatures. HEPES was selected as one of the most widely used biocompatible sulfonic acid buffers, which have been shown to possess superior buffering capacity at low temperatures,55 56 and has been included as a major component of other hypothermic tissue preservation media.55 57 58 Synthetic zwitterionic buffers such as HEPES also contribute to osmotic support in the extracellular compartment by virtue of their molecular size (HEPES, 238 D). Adenosine is a multifaceted molecule and is included in the hypothermic blood substitutes not only as an essential substrate for the regeneration of ATP during rewarming but also as a vasoactive component to facilitate efficient vascular flushing by vasodilatation.59 60 Glutathione is included as an important cellular antioxidant and hydroxyl radical scavenger as well as a cofactor for glutathione peroxidase, which enables metabolism of lipid peroxides and hydrogen peroxide.21 61 62

The companion purge solution is a modified extracellular-type medium with a plasmalike ionic component that is typical of other established balance salt solutions such as Krebs' buffer and Ringer's lactate. For the reasons explained, the present purge solution also contains the colloid dextran, HEPES buffer, adenosine, and glutathione. Because the role of the purge solution in this hypothermic blood substitution technique is to remove blood from the circulation during cooling and to flush out the hyperkalemic maintenance solution during warming, it is anticipated that additional benefits could be achieved by using this formulation as a vehicle solution for pharmacological agents that would protect against membrane destabilization and reperfusion injury. The development of the Carolina Rinse solutions to inhibit reperfusion injury in livers subjected to ex vivo preservation has demonstrated the merits of such an approach,63 64 but details of this aspect of the design of hypothermic blood substitutes for whole-body perfusion remain to be investigated.

Improved Outcome Using an Intracellular-Type Solution
It is clear from the present study that the purge solution alone does not provide optimal tissue preservation during whole-body hypothermic perfusion. Nevertheless, the fact that some animals perfused with the HTS-P alone were successfully revived and achieved long-term survival indicates that this solution has some protective properties as a hypothermic blood substitute. The outcome of the control experiments corroborates previous studies showing that whole-body exsanguination and washout with an extracellular-type solution such as Ringer's lactate, or other commonly used hemodiluents, can result in surviving animals but with variable quality of survival and speed of recovery to physiological and neurological normality. In sharp contrast, the present study demonstrates that the combined use of the Hypothermosol purge and maintenance solutions provides a less severe biochemical disturbance with a more rapid return to normality and more reproducible long-term survival.

The design of the present study permits some conclusions to be drawn concerning the quality of whole-body protection during hypothermic perfusion with aqueous blood substitute solutions. The combined strategic use of an extracellular-type flush solution (HTS-P) and intracellular-type maintenance solution (HTS-M) not only minimized biochemical and physiological disturbances during the acute phase of recovery but also led to a more rapid return to normalcy with fewer neurological sequelae in the long-term survivors. By comparison, animals treated identically but perfused throughout with the extracellular-type purge solution could not be resuscitated without unusual, aggressive interventional steps directed principally at reactivating and stabilizing the heart. Nevertheless, this was achieved in two of the three control animals, thereby providing the means to compare directly the effects of the nature of the hypothermic perfusates on a variety of postoperative parameters in our standardized procedure. Successful resuscitation of these control dogs after 3 hours of cardiac arrest and perfusion with HTS-P alone was in itself an achievement since others found it necessary to use thoracotomy to permit manual cardiac massage for resuscitation of dogs subjected to 2 hours of circulatory arrest at 11°C.26

The Hypothermosol purge solution retains some features of a former generation of hypothermic blood substitutes that were used in our previous studies,14 together with some additional components such as adenosine and glutathione, in common with the newly formulated Hypothermosol maintenance solution. The nature of the recovery of the control dogs is entirely consistent with previous observations using this type of solution for profound hypothermia and blood substitution.14 The most significant observation in the present study, however, is that whole-body perfusion with the intracellular-type HTS-M solution using precisely the same procedure led to markedly improved and consistent outcome.

The work of Kondo et al in 197426 provides a useful reference study for comparison with our experiments since their study is one of the few that has attempted >2 hours of circulatory arrest using profound hypothermia. Moreover, Kondo et al tried to improve their method of prolonged cardiac arrest with profound hypothermia by perfusing the vital organs such as brain, heart, and lungs in dogs with an intracellular-type solution. Using Collins' kidney preservation solution containing high concentrations of potassium, magnesium, phosphate, and glucose, they reported the successful resuscitation of a group of dogs after 2 hours of circulatory arrest at 11°C. However, several animals died immediately, with evidence of respiratory distress, and autopsy revealed pulmonary congestion, edema, and alveolar hemorrhage. Only 60% of the animals survived >48 hours, and these were reported to suffer from a variety of postsurgical complications, including severe but transient metabolic derangements, hindlimb weakness and loss of vision that either took 3 weeks to disappear or did not resolve, and various degrees of pathological lesions that persisted in the central nervous system. These observations led Kondo et al to conclude that although improvements had been realized in their technique using Collins' intracellular solution, 2 hours of circulatory arrest remained beyond the "safe time limits" of hypothermic cardiac arrest.

In marked contrast, we demonstrate that 3 hours of cardiac arrest is not beyond the safe limit when a procedure that maintains low-flow perfusion of an appropriate whole-body washout solution is used. The tolerance to total circulatory arrest has not yet been tested in our model, but we contend that by using a bloodless system the necessity for absolute circulatory arrest is circumvented. Moreover, there is a growing body of evidence that even in a hemodiluted blood-based system, low-flow perfusion is superior to circulatory arrest, resulting in fewer complications.65 66 67 Comparisons of our present findings with those reported by others such as Kondo et al,26 and even comparisons between the two groups within the present study clearly demonstrate a striking improvement in the quality and speed of recovery of animals resuscitated after low-flow hypothermic perfusion with the Hypothermosol maintenance solution.

It is our opinion that although there are a number of characteristics and components of HTS-M that will theoretically contribute to the observed benefits of this solution, the most important component is the presence of an impermeant ion to suppress cell swelling during hypothermia and anoxia. It is now well established from the design of preservation solutions for ex vivo storage of isolated organs that the inclusion of an impermeant ion such as lactobionate is the crucial component for effective preservation under hypothermic conditions.20 21 22 Although a wide variety of organ preservation solutions, differing markedly in the details of their compositions, have been devised, the presence of an impermeant molecule is an underlying characteristic of all successful formulations. Moreover, in recent years it has been firmly established that for general or universal tissue preservation, the choice of impermeant species is important because molecules such as glucose or mannitol that have proven to be effective in solutions for preservation of single organs such as the kidney are not effective for other organs such as the liver or pancreas; this is due to organ-specific metabolic differences.20

It is important to note that the so-called intracellular solution used by Kondo et al in their attempts to extend the safe periods of circulatory and cardiac arrest during profound hypothermia did not contain effective impermeant species that would suppress cell swelling. We contend, on the basis of our findings, that it is extremely important to include components in the hypothermic blood substitute that will effect control of tissue hydration at both the cellular and vascular levels: Collins' original solution used by Kondo et al26 contained neither an impermeant ion to control cell swelling generally nor a colloid to raise the oncotic pressure of the intravascular space and restrict interstitial edema. This lack of control of hydration during hypothermic exposure would undoubtedly have contributed to poor preservation of cellular integrity and deleterious increases in tissue edema, particularly in the lungs.

The lungs are especially vulnerable to congestion and edema during cardiopulmonary bypass, even without adjunctive hypothermia, and the dog is particularly sensitive to this problem, rendering the canine model a critical test for strategies designed to avoid these complications.68 In reviewing the historical development of cardiopulmonary bypass and the role of hypothermia, Sealy identified that early lengthy delays in the adoption of experimental techniques for clinical practice were due largely to the peculiar response of dogs (as the experimental model) to extracorporeal circulation. It has been a long-standing observation that survival rates of >50% to 60% for normal dogs after cardiopulmonary bypass have been difficult to achieve.68 Against this background, it is highly encouraging that respiratory distress was not observed in the present study for dogs perfused with the Hypothermosol solutions. Moreover, autopsy of dogs that were killed immediately for various reasons showed no signs of pulmonary edema by gross examination. Control of fluid balance during exchanges in the procedure, by maintaining an adequate hydrostatic pressure head between the animal on the operating table and the venous return on the pump, was also considered to be contributory to avoiding the problem of fluid retention at the gross level often observed in previous studies.27

Another characteristic of intracellular-type preservation solutions that has been proposed, tested, and debated has been the ionic balance. Conceptually, it has been argued that a hyperkalemic extracellular solution would be beneficial by inhibiting the passive exchange of monovalent cations across cell membranes during hypothermic exposure when active membrane pumps are suppressed. On this basis, many of the tissue and organ preservation solutions in common use are hyperkalemic solutions (UW, Eurocollins, CPTES). With reference again to the earlier attempts by Kondo et al26 to use an intracellular-type solution as a hypothermic blood substitute, the solution contained 107 mmol/L potassium, which is the principal feature of the solution that justifies its designation as an intracellular-type solution. These researchers reported improvements using this solution in a modified technique in which the extracorporeal circuit was simplified for left-side heart bypass only. The stated rationale for this technique was that total body perfusion with an intracellular solution, although theoretically desirable, requires too much perfusate and results in uncontrollable hyperkalemia during the rewarming process. The requirement for ventricular defibrillation as a standard part of the technique probably reflects the irritability of the myocardium induced by the high potassium environment. The use of very high potassium organ preservation solutions such as the UW solution (125 mmol/L K+) and Eurocollins (115 mmol/L K+) for ex vivo myocardial preservation is a topic of current debate because of concerns for the possible development of contraction band necrosis,69 enhanced entry of calcium into the cells,70 evidence of harmful effects on endothelium,71 and the likelihood of vasospasm and cardiac irregularities on reperfusion after transplantation. In the context of whole-body perfusion, there is no question that hyperkalemia used to suppress ionic imbalances in cooled, or anoxic cells, or simply as a component of cardioplegia, must be controlled during rewarming. With our technique, the HTS-M solution contains 42.5 mmol K+, which provides very effective cardioplegia and is sufficiently high to restrict the passive loss of intracellular potassium. The present study also demonstrates that the hyperkalemic state that is desirable during hypothermia is controllable and can be quickly and efficiently reversed by flushing with the normokalemic (3 mmol/L K+ for dogs) purge solution during warming. We have established that during the early rewarming phase, the circulation should be flushed with sufficient HTS-P to reduce the potassium concentration to <10 mmol/L before the blood is replaced. Under these circumstances, we encountered very little difficulty in reactivating the arrested heart or reestablishing normal sinus rhythm after 3 hours of arrest.

Conclusions
The present study clearly establishes that this hypothermic procedure with cardiac arrest and low-flow perfusion of a blood substitute is tolerated for at least 3 hours without significant or irreversible ischemic injury. Deep hypothermia can provide effective suppression of metabolism and cellular energy requirements, thereby extending the tolerance to ischemia by minimizing the demand for oxygen to levels that can be adequately supplied in a cold aqueous solution without the need for hemoglobin or other oxygen-carrying molecules. Oxygen dissociation from hemoglobin stops at temperatures of <12°C.72 Moreover, complete exsanguination facilitates the removal of blood-borne mediators of ischemia-and-reperfusion injury and permits closer control of the extracellular environment by perfusion with a solution appropriately designed for the hypothermic conditions. The superior results obtained using the maintenance blood substitute demonstrate the significance of having a specifically designed solution to act as an in situ universal preservation solution and successfully extend the safe time limits for hypothermic cardiac arrest procedures. Clinical application of this technique would open new avenues for therapeutic intervention through the prolonged suppression of cerebral metabolic activity.73 If this approach can be successfully transferred to humans, it is anticipated that major clinical benefits will be realized for cerebrovascular and cardiopulmonary procedures, endovascular techniques, and resuscitation from traumatic injury. In addition to providing a bloodless field for surgery, circulatory arrest could be intermittently used to provide vascular collapse and minimize the risk of catastrophic aneurysm rupture. Moreover, with this procedure, the demands for absolute circulatory arrest are diminished because precious blood is replaced completely with dispensable substitute. Low-flow perfusion could therefore be maintained, providing benefits of continuous vascular flushing while still achieving a clear bloodless field for surgical intervention. Clinical justification for this approach is strengthened by recent reports showing that a strategy involving predominantly circulatory arrest during hypothermic cardiopulmonary bypass was associated with a higher incidence of neurological sequelae in infants undergoing open-heart surgery compared with a strategy of low-flow cardiopulmonary bypass. Infants operated on using predominantly circulatory arrest showed a higher likelihood of clinical and EEG seizures, a longer time to recovery of normal brain activity, and a greater release of the BB isoenzyme of CK in the immediate postsurgical period.7

In an era of growing risks from the dangers of blood-borne transmissible diseases, it is recognized that application of hypothermic blood substitution would minimize, if not completely avoid, the need for using large quantities of donor blood. Furthermore, the totally synthetic formulations proposed here avoid the necessity of including any blood-based products that have previously been incorporated in experimental blood-substitute solutions.74

In conclusion, we advocate this approach for further study as a promising solution to support the concept of "life without blood."75


*    Acknowledgments
 
This work was supported in part by grants from Cryomedical Sciences, Inc, and NIH/SBIR (grant IR 43 HL-49028-01). Kim Klein, Cecilia Devenyi, and Babak Bazmi provided excellent technical assistance. We also gratefully acknowledge the help of Steve Gerun, Leslie Arelt, and their assistants in the Surgical Research and Animal Care facility at ASRI. We thank Dr Richard E. Clark for his continuing support, helpful advice, and critical reading of the manuscript.


*    Footnotes
 
As noted in "Acknowledgments," this work was sponsored in part by Cryomedical Sciences, Inc, which has a commercial interest in developing the bloodless solutions that we discuss. Also, Dr Bailes, Dr Shih, Dr Leavitt, and Dr Baust have equity or financial interest in Cryomedical Sciences, Inc.

Received May 11, 1994; accepted August 15, 1994.


*    References
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*References
 

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