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Organ Preservation
Organ Transplantation
0039-6109/86 $0.00
+ .20
Organ Preservation D. E. Pegg, M.D. *
All effective methods of organ preservation rely upon reduction of temperature as the principal protective element. As soon as an organ becomes ischemic, its supply of metabolites ceases, productsj)f metabolism accumulate, and damage occurs, reversible at first but eventually irreversible. It may be assumed that the processes of deterioriation that accompany ischemia are mediated by chemical reactions, the rate of which is dependent upon temperature. Simple cooling has been remarkably successful in reducing damage, although it does have limitations, the reasons for which are gradually becoming clearer. This article briefly explores the mechanisms of ischemic damage and then considers the effects of cooling and other supplementary measures that can protect organs during storage. Clinical applications of this information will be considered, and likely avenues for future improvements in techniques for organ preservation will be discussed. THE NATURE OF ISCHEMIC INJURY
The cellular structural changes !hat accompany ischemia have been well described. 14 These affect mitochondria, nuclei, endoplasmic reticulum, lysosomes, and ultimately the cell membranes. The precise distinction between reversible and irreversible injury is difficult to determine, but certainly gross changes to the mitochondria are a prominent feature of early irreversible damage, and rupture of the cell membranes is conclusive. As is well known, different cells have different susceptibilities to ischemic injury. In the kidney, ischemic changes occur first and become most severe in the proximal convoluted tubule. After 30 minutes there are gross changes in some, but not all, tubules; at 60 minutes there is total necrosis of all segments of the majority of tubules. Nevertheless, 90 per cent of animals *Hp.ad of MRC Medical Cryobiology Group, University Department of Surgery, Cambridge, United Kingdom More detailed reviews of work on organ preservation have recently been published by the author, and this article is based on those publications. 24. l'.5
Surgical Clinics of North America-Vol. 66, No.3, June 1986
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10
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9
9
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FRESH C +W LI3=-'=-5_..:.1::..5_--=3..:.0_ _6,-0--" MINS
2
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WI T
°
FRESH C+W
1,-3_.5_ _ 15_ _ 3_0_ _6_0~1 MINS
WIT
Figure 1. Time course of loss of adenine nucleotides in rabbit kidneys subjected to increasing periods of warm ischemia (WIT). (FRESH = freshly isolated with negligible warm ischemia; C + W = cannulated and flushed with WF5PO solution, as described by Pegg, Wusteman, and Foreman 29 ; ATP = adenosine triphosphate; TAN = total adenine nucleotides; OW = dry weight of tissue.)
will survive 30 minutes of warm ischemia and 75 per cent even 60 minutes; thus much of this damage is recoverable. However, after 60 minutes of warm ischemia, there is evidence of permanent histologic injury. 1, 18 Inevitably, metabolic changes accompany ischemic injury, and in view of the severity of structural damage to mitochondria, the effects on energy metabolism have been most extensively studied. The earliest changes are due to lack of molecular oxygen for oxidative phosphorylation, which brings about an accumulation of NADH and depletion of ATP within the mitochondria. The mitochondrial membrane is impermeable to these molecules, which are normally translocated by enzyme-driven mechanisms, and consequently NADH accumulates and NAD+ levels fall. NAD+ and ATP are essential for the operation of the glycolytic pathway, the rate-limiting step of which (phosphofructokinase) is inhibited by hydrogen ions. Thus glycolysis, which is capable of trapping only about 5 per cent of the total energy of glucose and is the only source of ATP available to anoxic cells, is inhibited. The situation is relieved to a limited extent by the lactate dehydrogenase reaction, which generates lactate and NAD+ from pyruvate, NADH, and hydrogen ions. It is likely that both hydrogen ions and lactate are responsible for the production of structural damage in ischemia. The depletion of ATP is fundamental to ischemic injury. ATP levels drop extremely rapidly in rabbit kidneys, reaching 20 per cent of normal in only 15 minutes; ADP and AMP concentrations fall more slowly (Fig. 1).29 None of these nucleotides is able to diffuse across the cell membrane, but dephosphorylation of AMP to the corresponding nucleoside adenosine, which is due to the action of 5' -nucleotidase enzymes, leads to the loss of total adenine nucleotides since nucleosides can traverse the cell membrane. This loss of nucleosides is probably a very important factor in the failure of tissues subjected to prolonged ischemia to regenerate ATP after restoration
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of a normal blood supply. Adenosine is further degraded to inosine, hypoxanthine, xanthine, and uric acid. This loss of purine is important because the salvage of purine skeletons plays a dominant role in adenine nucleotide synthesis and is energetically advantageous in that the "salvage pathway" uses only 2 moles of ATP/mole synthesized, as opposed to 6 moles for de novo synthesis. The relationship between structural and metabolic changes is interesting. It is apparent that the speed at which ATP levels fall far exceeds the rate at which structural or gross functional damage can be observed. Hence low ATP levels cannot be damaging per se. It has been shown that mitochondria isolated from kidneys that have been subjected to 30 minutes of warm ischemia are able to phosphorylate efficiently, yet these kidneys are not able to regain normal ATP levels: this is probably due to the loss of total adenine nucleotides by the mechanism discussed earlier, rather than mitochondrial structural damage. The lost nucleotides can be replaced, however, and this will be discussed later. A prominent feature of ischemic injury is swelling of the cells. The sodium pump is responsible for maintaining both the peculiar intracellular ionic balance of cells and their normal cell volume; this is because the transport of ions and water is osmotically coupled. The detailed mechanisms involved have been discussed elsewhere;25 fundamentally, it is the impermeant nature of the bulk of the intracellular anion that is responsible for the net entry of solute and water when the sodium pump is arrested, whether this is caused by lack of ATP in ischemia or by reduction of temperature in hypothermic preservation. It has been shown that the vascular endothelial cells of ischemic kidneys double their thickness after 60 minutes of warm ischemia. 12 The precise role of cell swelling in ischemic injury is a matter of debate, but Jamart and Lambotte have compared the effect of a standard anoxic treatment with the effect of perfusion with a hypotonic solution that produces a similar degree of cell swelling. 19 They clearly demonstrated that hypotonic cell swelling is less damaging than a comparable degree of anoxic cell swelting, confirming that anoxia damages by mechanisms additional to cell swelling. Nonetheless, as will be elaborated subsequently, the reduction of cell swelling does go some way to mitigate anoxic injury. One of the striking features of an organ that has suffered a period of warm ischemia is the failure of blood flow to return when circulation is restored. 36 This "no reflow" phenomenon is of great importance in determining the fate of a transplanted organ, since intact parenchymal cells can be of no value in the absence of circulation. The addition of substances to which the cells are impermeable, such as mannitol, is able to reduce the degree of vascular endothelial swelling and improve the reflow of blood under some conditions,12 but this approach is unable to completely cure the problem. However, the addition of dextran 40, which inhibits red cell aggregation, improves blood flow dramatically. Other evidence indicates that the "no reflow" phenomenon is much worse if red blood cells are present in the organ during the ischemic period than otherwise; indeed, Wusteman found no reduction in perfusate flow after up to 90 minutes of ischemia when the kidneys did not contain red cells during the ischemic
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period and were reperfused with a bloodless perfusate. 45 Thus it seems unlikely that the increase in vascular resistance is due to endothelial cell swelling but rather is caused by red cell sludging. This may be partly due to the increase in red cell rigidity that occurs when the cells are depleted of ATP during ishemia. 43 Addition'ally, it is known that the capillaries become increasingly leaky to protein after more than 30 minutes of warm ischemia, which would lead to a loss of the oncotic pressure which retains fluid in the capillaries and hence to an increase in the hematocrit within the vessels. This in turn would increase viscosity dramatically and therefore lead to stagnation. It seems likely that vascular factors playa major part in the eventual failure to recover function in organs that have suffered significant ischemia.
CONCLUSIONS
Although the picture is still incomplete, it is clear that those biochemical changes that occur early in ischemia and are due to lack of oxygen are reversible and that hydrogen ions and lactate can accumulate to remarkably high levels and yet be compatible with complete recovery. Loss of important metabolites, for example, adenine nucleotides, is more serious, and in prolonged ischemia the gross accumulation of metabolites, including hydrogen ions and lactate, presumably does cause structural damage. One feature of ischemic injury that has received particular attention, cell swelling, is only one metabolic consequence of lack of ATP: in itself it appears to be only moderately harmful and the total damage of ischemia cannot be duplicated by an equivalent degree of cell swelling. In particular, the "no reflow" phenomenon, also a prominent feature of organs that have suffered ischemic damage, is not primarily due to cell swelling but rather is caused by the trapping of red cells in the microcirculation, probably because of an increase in their rigidity and leakiness of the capillaries to protein. The fundamental cause of ischemic injury lies in molecular changes in membranes. Ultrastructural studies are able to demonstrate this damage directly, and confirmation is provided by the observation that proteins leak from capillaries into the interstitial space, that intracellular enzymes leak into the vascular system, and that lysosomal enzymes leak into the cytosol. The ultimate cause of this membrane damage is still unclear, but it is apparent that the preservation of membrane integrity is fundamentally important for organs that are to be transplanted.
THE EFFECTS OF COOLING Structural Cooling slows the rate at which the structural changes seen at 37° C develop, but there are also some qualitative differences. Cell swelling is much more prominent in hypothermia, and there is a more uniform effect upon different cell types, including, in the case of the kidney, the glomeruli,
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Table 1. Composition of Flush Solutions CONSTITUENT (PER LITER)
Na+ (mmol) K+ (mmol) Mg2- (mmol) Citrate (mmol) SO;- (mmol) H,P04 - (mmol) HPO/- (mmol) CI- (mmol) HC03 - (mmol) Mannitol (mmol) Glucose (mmol) Osmolality (mOsm/kg)
COLLINS' C 2
EURO-COLLlI\iS'
SOLUTION
SOLUTIOI\i
10,0 115,0 30,0
10,0 115,0
30,0 15,0 42,5 i5,0 10,0
HYPERTONIC CITRATE SOLUTION
83,7 79,5 40,6 54.4 40,6
15,0 42,5 15,0 10,0 185,5
140 360
194 375
400
which are remarkably resistant to warm ischemic lOJury. The exposure times required to produce damage at low temperatures far exceed those needed to produce warm ischemic injury and consequently there is no doubt that cooling is highly protective. However, the presence of additional lesions in hypothermia does suggest that additional damaging mechanisms exist at low temperatures. Biophysical We have already noted that potassium is lost and sodium and water are gained when cells are exposed to normothermic ischemia and that this is due to the limited amount of ATP available to drive the sodium pump. Under hypothermic conditions, however, even if sufficient ATP is present, the sodium pump becomes inoperative due to cooling and the cells therefore swell. Since the primary cause of swelling in the absence of the sodium pump is the impermeable nature of tht;, bulk of intracellular anion, balancing this osmotic pressure by external impermeant solutes can provide a means for controlling cell swelling. If a relatively high molecular weight polymer is used, for example, polyethyleneglycol of molecular weight 6000, then about 20 mOsm/kg is sufficient. 32 More commonly, lower molecular weight materials have been used for this purpose, and since these do penetrate the cells, albeit slowly, higher osmolatities are required for effective control of cell volume. Manipulation of the ion balance in the perfusing solution makes it possible to prevent the loss of intracellular potassium and gain of sodium. This approach was pioneered by Collins and colleagues9 and is now the most widespread method of renal preservation. Some examples of these so-called "intracellular" solutions are shown in Table 1. It will be apparent that not all of them mimic intracellular ion concentrations closely, but they have two features in common: they contain a significant amount of impermeant solute and a high concentration of potassium. Collins was able to achieve 30 hours' storage of canine kidneys flushed with his solution and stored at about 0° C. More recently, Andrews and Coffeyl have observed a considerable degree of proximal tubular damage and swelling of the ascending distal tubules in kidneys preserved
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with Collins' solution, but they found that an isosmotic phosphate-buffered saline that included 140 mmolll of sucrose gave superior ultrastructural appearances. Several groups have attempted to identify the crucial components of "intracellular" solutions and their mechanism of action, and it is clear that the inclusion of impermeant or slowly permeating solutes is of far greater importance than elevation of the potassium concentration. 15 Thus, although cell swelling is probably of only secondary significance in normothermic ischemia and is unimportant in the etiology of the "no reflow" phenomenon, its prevention is able to mitigate the injury that occurs in hypothermic storage and to prolong the storage times available. The importance of varying the ionic balance is more problematic. Most studies have found elevation of the potassium concentration to be advantageous and some, though not all, have found that increased magnesium concentrations are advantageous. We, however, have found elevation of magnesium concentrations to be damaging in some circumstances,21 and both experimental and clinical experience with the Euro-Collins' version of Collins' solution (which omits magnesium sulfate) have shown it to be highly effective. 10, 38 Probably calcium is also of considerable importance, although it has only recently received detailed attention. Cytosolic concentrations of calcium are normally of the order of 10-8 to 10-7 molar, whereas the extracellular fluid contains about 10-3 molar calcium. The accumulation of calcium, particularly in the mitochondria, is a universal finding in hypothermic cells, and in view of the importance of calcium as a second messenger in the regulation of numerous physiological processes, this accumulation is likely to have important effects. Calcium acts as a messenger by combining with and activating the regulatory protein calmodulin,7 and this can be blocked by the addition of phenothiazene compounds. Asari and associates 2 have shown the addition of one of these compounds (trifluoperazine) to Collins' solution improves the recovery of viable kidneys stored at 4-6° C for 72 hours from 33 per cent to 80 per cent. It would seem that this approach is worthy offurther study. Biochemical Metabolism during hypothermia is beginning to receive considerable attention, particularly with respect to energy metabolism. Free fatty acids are the principal fuel for many tissues, including renal cortex, under welloxygenated conditions at 37° C, although glucose is the preferred substrate for renal medulla. When renal tissue is cooled, for example to 10° C, fatty acids continue to be respired, but ketone bodies are then by far the most effective substrate. 17 However, glucose is also taken up by hypothermic kidneys at roughly the same rate as fatty acid, and the majority is metabolized to lactate. 3o Lactate continues to accumulate throughout hypothermic perfusion even when the perfusate is well oxygenated, which suggests that anaerobic glycolysis plays a major role in energy metabolism under these conditions. In our own laboratory, using rabbit kidneys perfused at 10° C, we have found that a perfusate p02 of 150 mm Hg causes glucose to be consumed and lactate to accumulate. The addition of caprylate increases both the glucose consumption rate and the lactate production rate. 29 Thus, anaerobic glycolysis is probably the principal source
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of energy under these perfusion conditions and the stimulatory effect of caprylate is probably due to its indirect stimulation of phosphofructokinase. We attempted to increase aerobic respiration by raising the p02 to approximately 650 mm Hg, which reduced glucose consumption and lactate production and improved the synthesis of high energy nucleotides. The further addition of hypoxanthine as a purine "salvage pathway" precursor produced a further significant improvement in high energy phosphate concentrations. In fact, the combination of glucose, caprylate, and hypoxanthine with a p02 of approximately 650 mm Hg restored all the adenine nucleotide indices to normal except for the ATP:ADP ratio, which at 80 per cent of normal was similar to that observed after only 2 minutes of normothermic ischemia. The most striking finding in this study was the observation that even a kidney that had suffered 60 minutes of warm ischemia, which reduces ATP levels to 10 per cent of control and total adenine nucleotides to 20 per cent of control, would recover normal adenine nucleotide indices under these perfusion conditions (except for the ATP:ADP ratio which was 50 per cent of normal, or equivalent to 5 or 6 minutes of warm ischemia). The data suggested that the energy for this resynthesis was partly glycolytic and partly aerobic. These findings have been confirmed in canine kidneys. 26 The impairment of oxidative phosphorylation at low temperatures points to a mitochondrial defect. Oxygen consumption by isolated mitochondria decreases with falling temperature, often with a change in the rate of decrease at about IS° C. 37 It appears from more detailed studies that the enzymes responsible for translocating adenine nucleotide (AN translocase) across the mitochondrial membrane become ineffective below the temperature at which the change in rate of oxygen consumption occurs. Even under normal physiologic conditions, AN translocase has negligible spare capacity20 and is therefore very likely to become rate-limiting at low temperatures. Similarly, the enzymes responsible for moving NADH across the mitochondrial membrane (the mal~te-aspartate shuttle) are ineffective under hypothermic conditions. It seems clear therefore that the failure of aerobic metabolism in hypothermia is due primarily to the inability of the mitochonrial transport enzymes to function effectively at storage temperatures. Nevertheless, providing that the appropriate substrates are present, it is possible for hypothermic kidneys to synthesize adenine nucleotides at the temperatures typically used for hypothermic organ preservation. An additional (or alternative) strategy may be used to support adenine nucleotide syntheSis. It has been mentioned that AMP is lost from the cells only after it has been dephosphorylated to adenosine, and it follows that the pharmacologic inhibition of 5' -nucleotidase would increase total adenine nucleotide levels. This has been achieved by the use of allopurinol, 1,3 dior 1,3,7 tri-methylxanthine,5 but this approach has not been extensively studied clinically. It seems reasonable to conclude that the maintenance of high ATP levels during hypothermic preservation, which is certainly possible, is also desirable. However, the reversible inhibition of transport enzymes that occurs during hypothermia eventually becomes irreversible and is probably one of the major factors limiting the duration of effective hypothermic storage.
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It has been mentioned that an increase in the oxygen tension in a perfusate used for the hypothermic preservation of kidneys results in improved ability to resynthesize adenine nucleotides during the storage period. However, oxygen can be supplied without resorting to continuous perfusion by using the technique of retrograde oxygen persuffiation, introduced by Fischer and colleagues. l l With this method, kidneys that have been flushed with a conventional flush solution are supplied with gaseous oxygen by a cannula attached to the renal vein and the gas is allowed to escape through numerous small needle pricks made over the surface of the organ. The gas flows through only a part of the venous system which then acts as an exchange surface from which oxygen can diffuse into the whole organ. Fischer himself used Collins' solution, an oxygen delivery pressure of 55 mm Hg, and a temperature of 6° C. Ross and EscottJ4 and we 33 have used a lower temperature, 0° C, a lower gas pressure, 10 to 12 mm Hg, and hypertonic citrate flush solution as shown in Table 1. 35 These experiments showed this maneuver to be quite effective in improving the preservation of ischemically injured kidneys: We were able to obtain 48 hours' preservation of canine kidneys damaged by 30 minutes of prior warm ischemia or 24 hours' preservation after 60 minutes of warm ischemia. The technique produces a high and uniform oxygen tension throughout the organ; air is less effective than pure oxygen, and inert gases are completely ineffective. Fischer was able to show some restoration of adenine nucleotide reserves during the hypothermic storage period but we were unable to confirm that finding. The difference is probably due to our use of a lower temperature at which, perhaps due to the effect of cooling on AN translocase activity, oxygen was unable to stimluate further ATP synthesis. The mechanism of this effect is unclear; it is possible that some stimulation of ATP synthesis (but too little to be detected by our chemical techniques) did occur, or alternatively it is conceivable that oxygen may have stabilized cytochrome oxidase and hence the inner mitochondrial membrane. Whatever the mechanism, the effectiveness of this quite simple technique does seem to be clearly established. Whether the procedure might have a role in the preservation of kidneys that have not suffered prior warm ischemia is unknown, but this remains a possibility.
Vascular In view of the importance of vascular effects in warm ischemic injury, it is relevant to consider the effect of hypothermia on the vascular system. The cell swelling caused by hypothermia affects the vascular endothelium as it does other cells, yet for a given period of ischemia, cooling alone is highly effective in protecting against the "no reflow" phenomenon. This is further evidence that "no reflow" is not caused by swelling of endothelial cells. Scanning electron microscopy, carried out in Belzer and Southard's laboratory, indicates little structural change after storage periods in Collins' solution that are associated with functional renal recovery, and even at 7 days the endothelium showed a continuous sheet. Epithelial podocytes also showed strikingly good preservation even after 7 days' storage. In continuously perfused canine kidneys, Belzer and colleagues3 have shown intact endothelium after 3 days' storage, but severe damage at 7 days, and their
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recent scanning electron micrographs have shown that the glomerular podocytes develop numerous microvillous projections into Bowman's capsule, the foot processes acquire protruberances and the cell bodies become indistinct. Thus the vascular system does seem to be less well preserved in continuously perfused than in flushed organs. We have found that the choice of colloid has an important influence on the generation of vascular injury; of the protein substitutes, the gelatin polypeptide Haemaccel (Hoechst) was the best,28 but the most effective colloid is probably purified albumin. In a clinical study of kidneys perfused with cryoprecipitated plasma, Hill and colleagues 16 observed disruption of the glomerular endothelium, which caused platelet and fibrin deposition on the exposed basement membrane after transplantation. These changes were seen even in kidneys perfused for less than 20 hours (although they were more severe after longer preservation), but they were minimal or absent in kidneys perfused with purified albumin and were reduced if the perfusion pressure was lowered. This damage is often, but not invariably, associated with an increase in the total vascular resistance of the organ. Vascular injury, whether augmented by perfusion or not, is undoubtedly a major factor determining the time limit of effective hypothermic renal preservation. Nevertheless this damage can be ameliorated in the various ways. There is substantial evidence that, under normal physiologic circumstances, blood cells and endothelial cells interact in complex ways to maintain an intact vascular system, and platelets probably play a crucial role in this phenomenon. This "nurturing" action is probably the mechanism that was responsible for the effectiveness of intermediate normothermic blood perfusion in prolonging the total hypothermic preservation time of canine kidneys in the experiments of van der Wijk and associates and Rijkmans and coworkers. 31. 44 In these experiments, kidneys were perfused with an albumin solution for 6 days at 6° C and grafted. Only 2 out of 14 animals survived in this experiment, whereas interrupting the hypothermic perfusion for 3 to 4 hours on the !.hird day for a period of ex vivo normothermic blood perfusion enabled·11 out of 12 animals to survive, and they had much lower peak creatinine levels. Blood perfusion with a heartlung machine was equally effective. These results strongly argue in favor of a vascular repair mechanism. It is therefore not surprising that measures to prevent the adherence of platelets and deposition of fibrin within damaged vessels after transplantation, and thereby allow time for endothelial repair, are able to prolong the time for which organs can be stored. Recently, a long-acting analogue of prostacyclin, the most powerful inhibitor of platelet aggregation known, has been shown to be highly protective against the vascular and functional effects of 120 minutes of normothermic renal ischemia in the dog, and prostacyclin has been used in the hypothermic preservation of livers. 23 Similarly urokinase, a plasminogen activator that prevents fibrin deposition, has been reported to increase the preservation time obtainable with Collins' solution and the canine kidney to 96 hours with 100 per cent survival, and even to 120 hours (5 days) with 50 per cent survival. 22
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CONCLUSIONS
Cooling retards the development of ischemic injury but adds other specific damage. Anoxic cell swelling is actually accentuated by cooling, although it can be prevented by the inclusion of impermeant solutes in preservation solutions. Intracellular ionic balance can be improved and the damaging effects of calcium loading reduced by appropriate changes in the composi.tion of the preservation solution. At the temperatures commonly used for perfusion preservation, significant metabolism continues, but that metabolism is altered from normal physiologic biochemistry. If appropriate precursors are provided, ATP levels can be maintained, or even improved where there has been prior warm ischemia. For this reason, continuous perfusion methods that can support metabolism seem attractive, yet continuous perfusion incurs the risk of greater vascular injury. The provision of a high oxygen tension is advantageous in its own right, particularly when dealing with kidneys that have suffered significant warm ischemia, but the mechanism of this effect is unclear. As with normothermic ischemia, the fundamental limiting factor in hypothermic organ preservation is probably damage to cell membranes, and progress in preservation techniques will be dependent upon methods for combating these membrane changes. It is distinctly possible, but not yet proven, that oxygen free radicals may play an important part in the generation of low temperature membrane damage. These highly toxic agents are generated by the stepwise addition of the four electrons that are normally added to oxygen in a single step by cytochrome oxidase. The enzymes superoxide dismutase, catalase, and glutathione peroxidase are normally responsible for converting these radicals into harmless water, and many compounds-including mannitol, glycerol, and dimethylsulfoxide, for example-have free radical scavenging properties. There is no doubt that free radicals do attack membrane lipids, releasing lipid peroxides and seriously damaging the cells, and because free radical reactions have a low activation energy, they are depressed to a lesser extent by decreasing temperature than the enzymic reactions that normally remove them. Consequently, it is entirely possible that oxygen free radicals are important in hypothermic injury. There is already some evidence that the addition of radical scavengers to preservation media may improve the viability of organs. OPTIMAL TECHNIQUES FOR HYPOTHERMIC PRESERVATION The relative merits of continuous perfusion and simple cold storage have been actively debated for many years, and to this debate must now be added the arguments in favor of retrograde oxygen persuffiation. It is not proposed to repeat all the arguments here but more briefly to consider the relative merits of the three approaches. Continuous perfusion makes it possible to support metabolism, but this is achieved at the expense of bulky, complicated, and expensive equipment. Flush preservation, on the other hand, is limited to replacement
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of the vascular contents with a chosen solution and exchanging the intracellular fluid of the orgap. with that solution to a very limited degree. However, the method is simple, convenient, and inexpensive. The important question is whether the more complex approach secures any significant advantages. It is clear that metabolism does occur at the temperatures generally used for storage, and this certainly provides a stong theoretical argument in favor of perfusion. Continuous perfusion is probably the only technique that will adequately support hypothermic metabolism. There is strong evidence that the metabolic difficulties experienced by hypothermic organs are largely due to the inhibition of membrane-bound transport enzymes, which suggests that higher temperatures than those used hitherto might be beneficial. If so, metabolism will be greatly accelerated, and continuous perfusion is then likely to be indispensable. However, continuous perfusion places additional stresses upon the vascular system that are avoided by simple flush preservation, and once the inability of flush preservation to support metabolism is acknowledged, it becomes sensible to use the lowest possible temperature to minimize metabolic activity. The choice of technique really depends upon the objective: If kidneys with no prior ischemia are to be preserved for less than 48 hours, then flush preservation, for example with one of the solutions shown in Table 1, is the method of choice. If, however, there has been significant prior warm ischemia, or if much longer preservation is needed, then continuous perfusion will be more effective. The longest period of renal preservation so far reported (8 days) was by Cohen, who used a continuous perfusion method. 8 If organs with significant warm ischemia are to be used, then the relatively trivial additional technical complication of retrograde oxygen persuffiation seems worthwhile. In many countries, however, the use of kidneys damaged by warm ischemia has been almost eliminated, and the value of retrograde oxygen persuffiation in the absence of prior warm ischemia is largely unstudied.
PRACTICAL TECHNIQUES FOR TRANSPLANT ORGANS Kidney24 Preservation of the cadaveric kidney commences before its removal from the donor. Pretreatment of the donor with heparin, an alpha-adrenergic blocker, and the production of a good diuresis are all desirable. Once removed, the kidney should be flushed with one of the "intracellular" solutions that are available and probably not significantly different in effectiveness: Collins C2, the so-called Euro-Collins solution, or hypertonic or isotonic citrate solutions are all acceptable. A pressure of not less than 60 mm Hg should be used to secure effective removal of blood, and the temperature should be about 5° C. Flushing is usually continued until at least 1 liter has flowed through the kidney and the effiuent is completely clear of blood. A decision must then be made between the three approaches to preservation that are available. When the period of ischemia has been
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PRESSURE TRANSDUCER
FLAKED
ICE
WATERPROOF PLASTIC BAG
ORG~
CONTAINER _ _
RENAL
VEIN--
Figure 2. System used for retrograde oxygen persuffiation of human kidney grafts. (From Pegg, D. E.: Organ preservation at temperatures above 0° C. In CaIne, R. Y. (ed.): Transplantation Immunology: Clinical and Experimental. Oxford, Oxford University Press, 1984, p. 355, with permission.)
brief (less than 15 minutes) and the required period of preservation is less than 48 hours, then storage in the flush solution within a sterile container surrounded by ice is probably the method most commonly used at the present time. If the warm ischemic period has exceeded 45 minutes, then continuous perfusion is probably a better choice, although we are now using retrograde oxygen persufflation on a trial basis in these circumstances (Fig. 2). With intermediate warm ischemia time (15 to 45 minutes), simple flush storage will usually be satisfactory so long as the intended storage period is less than 24 hours, but if continuous perfusion is available, it may be wiser to use it. It should be noted, however, that continuous perfusion of the kidney or any other organ requires trained personnel with adequate experience if satisfactory results are to be obtained. Heart In experimental studies, prOViding there has been minimal warm ischemia, up to 24 hours' preservation seems to be achievable, either by flush preservation with an "intracellular" solution or by continuous hypothermic perfusion. 24 At present there are no satisfactory methods available for dealing with hearts damaged by warm ischemia or for obtaining greater than 24 hours' preservation. In clinical practice, however, storage periods have generally been limited to 6 hours, and standard cardioplegic solutions have been used: there does seem to be a need for better flush preservation methods. Current work in our laboratory, in collaboration with Papworth Hospital, Cambridge, suggests that modifications to the standard cardioplegic solutions may result in significant improvement in storage time. 13 These methods have yet to be tested in the clinical situation. Liver Continuous hypothermic perfusion has been shown to permit 20 to 24 hour preservation of the liver but it is generally felt that the technique is
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too complex for routine use and both of the major groups that are now carrying out clinical liver transplant programs use simple flush methods. The Denver-Pittsburgh group uses Collins' solution,4 whereas the Cambridge-King's College group uses a plasma protein solution with an elevated potassium concentration (modified PPF). The technique is described in detail by Calne. 6 Most recently in this laboratory a comparison has been made in the rat liver among Collins' solution, PPF, hypertonic citrate solution, and isotonic citrate solution for flush preservation. The isotonic citrate solution proved to be superior to the others. This !/olution has yet to be evaluated clinically. Additionally, it has been found that a perfusion solution based upon the isotonic citrate formulation, but with additional Haemaccel, and fluorocarbon emulsion, has made it possible to achieve 24 to even 48 hours' preservation of rat liver. Whether perfusion techniques will ever prove acceptable for clinical use is essentially a technical and financial question, but it does seem likely that the present flush solutions are capable of significant improvement. Pancreas The flush preservation approach has yielded effective storage of the pancreas for 24 hours using a wide range of solutions. 21 Forty-eight hour preservation has also been reported with a hyperosmolar flush solution containing albumin,4O but this result has not been confirmed. Continuous perfusion seems to be no more effective. In our own experiments, an elevated magnesium concentration in the flush solution was found to be deleterious. 21 Lung24 This organ has a very limited ability to tolerate either warm ischemia or deflation; either inflation or ventilation of the ischemic lung significantly prolongs its survival even if nitrogen is used as the inflating gas. 3~, 41 Preservation is possible for 24 hours by flushing with an intracellular solution, inflation with 40 per cent oxygen plus 60 per cent nitrogen, and storage at 4° C,42
FUTURE POSSIBILITIES It is likely that minor refinements of the existing flush preservation methods will continue to produce useful improvements in post-preservation function, but really significant improvements will require a considerable increase in our understanding of hypothermic physiology and biochemistry, It is my opinion that the complexity and expense of continuous hypothermic perfusion makes it rather unlikely that it will be more widely used than at present. The technique of retrograde oxygen persuffiation is certainly promising but limited in scope-it requires further study before it can be confidently advocated for clinical use. At the moment it is being used only for kidneys, but if it should prove applicable to other organs, it might conceivably increase the uncomfortably short preservation times that are currently available for hearts and livers, without incurring the expense and
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complication of continuous perfusion. The fundamental limiting factor in hypothermic preservation is probably damage to cell membranes, and an increase in understanding of the mechanism of such damage should prove invaluable. Oxygen free radicals may be involved, but it is too soon to say whether or to what extent practical procedures based upon free radical scavenging will be useful in clinical practice. Truly indefinite storage will be possible, if at all, only by the use of temperatures well below 0° C, but this raises problems of an altogether different order. Although rpuch success has attended efforts to cryopreserve isolated cells, and even some simple tissues, the long-term subzero preservation of organs has so far proved impossible. 25, 27 Much scientific work remains to be done before organ cryopreservation enters the realm of practical application.
REFERENCES 1. Andrews, P. M., and Coffey, A. K.: Factors that improve the preservation of nephron morphology during cold storage. Lab. Invest., 46: 100-120, 1982. 2. Asari, H., Anaise, D., Bachvaroff, R. J., et aI.: Preservation techniques for organ transplantation 1, Protective effects of calmodulin inhibitors in cold preserved kidneys. Transplantation, 37:113-114, 1984. 3. Belzer, F. 0., Hoffman, R, Huang, J., et al.: Endothelial damage in perfused dog kidney and cold sensitivity of vascular Na-K-ATPase. Cryobiology, 9:457-460, 1972. 4. Benichou, J., Halgrimson, C. G., Wei!, R, III, et al.: Canine and human liver preservation for 6-18 hours by cold perfusion. Transplantation, 24:407-411, 1977. 5. Buhl, M. R., and Jensen, M. H.: The role of 5'-nucleotidase in purine depletion of ischaemic renal tissue. In Pegg, D. E., Jacobsen, I. A. (eds.): Organ Preservation II. Edinburgh, Churchill Livingstone, 1979, pp. 239-246. 6. Caine, R Y.: Liver. In Karow, A. M., Jr., and Pegg, D. E. (eds.): Preservation of Organs for Transplantation, Ed. 2. New York, Marcel Dekker, 1981, pp. 617-624. 7. Cheung, W. Y.: Calmodulin plays a pivotal role in cellular regulation. Science, 207:1927, 1980. 8. Cohen, G. L.: Eight-day kidney preservation. Ch. M. Thesis, University of Liverpool, 1983. 9. Collins, G. M., Bravo-Shugarman, M., and Terasaki, P. I.: Kidney preservation for transportation. Lancet, 2:1219-1222, 1969. 10. Dreikorn, K., and Horsch, R.: 48 to 96 hour preservation of canine kidneys by initial perfusion and hypothermic storage using the Euro-Collins solution. In Marberger, M., and Dreikorn, K. (eds.): Renal Preservation. Baltimore, Williams and Wilkins, 1983, pp. 261-266. 11. Fischer, J. H., Czerniak, A., Hauer, U., et al.: A new simple method for optimal storage of ischa~mically damaged kidneys. Transplantation, 25:43-49, 1978. 12. Flores, J., Di Bona, D. R, Frega, N., et al.: Cell volume regulation and ischaemic tissue damage. J. Membr. BioI., 10:331-343, 1972. 13. Foreman, J., Pegg, D. E., and Armitage, W. J.: Solutions for preservation of the heart at O°C. J. Thorac. Cardiovasc. Surg., 89:867-871, 1985. 14. Glaumann, B., Glaumann, H., Berezesky, I. K., et al.: Studies on the pathogenesis of isch;lemic cell injury II. Morphologicill changes of the pars convoluta (PI and P2) of the proximal tubule of the rat kidney made ischaemic in vivo. Virchows Archiv (Cell Pathol.), 19:281-302, 1975. 15. Green, C. J., and Pegg, D. E.: Mechanism of action of "in.tracellular" renal preservation solutions. World J. Surg., 3:115-120, 1979. 16. Hill, G. S., Light, J. A., and Perloff, L. J.: Perfusion-related injury in renal transplantation. Surgery, 79:440-447, 1976. 17. Huang, J. S., Downes, G. L., Childress, G. L., et al.: Oxidation of1'C-labelied substrates by dog kidney cortex at 10 and 38°C. Cryobiology, 11:387-394, 1974.
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18. Jablonski, P., Howden, B. 0., Rae, D. A., et al.: An experimental model for assessment of renal recovery from warm ischaemia. Transplantation, 35:198-204, 1983. 19. Jamart, J., and Lambotte, L.:. Differential effect of swelling and anoxia on kidney function and its consequences on the mechanisms of action of intracellular organ preservation solutions. Transplantation, 34:176-182, 1982. 20. Kemp, A., Jr., Groot, G. S. P., and Reitsma, H. J.: Oxidative phosphorylation as a function of temperature. Biochim. Biophys. Acta, 180:28-34, 1969. 21. Klempnauer, J., Pegg, D. E., Taylor, M. J., et al.: Hypothermic preservation of the rat pancreas. In Pegg, D. E., Jacobsen, I. A., and Halasz, N. A. (eds.): Organ Preservation: Basic and Applied Aspects. Lancaster, MTP Press, 1982, pp. 191-198. 22. Masaki, Y., Uchida, H., Osakabe, T., et al.: Successful 96- and 120-hour preservation of the canine kidney by simple surface cooling with high units of urokinase and modified Collins' solution. Transplant. Proc., 17:1449-1453, 1985. 23. Monden, M., and Fortner, J. G.: Twenty-four and 48-hour canine liver preservation by simple hypothermia with prostacyclin. Ann. Surg., 196:38-42, 1982. 24. Pegg, D. E.: Organ preservation at temperatures above O°C. In CaIne, R. Y. (ed.): Transplantation Immunology: Clinical and Experimental. Oxford, Oxford University Press, 1984, pp. 349-359. 25. Pegg, D. E.: Principles of tissue preservation. In Morris, P. J., and Tilney, N. L. (eds.): Progress in Transplantation, Volume II. Edinburgh, Churchill Livingstone, 1985, pp. 69-105. 26. Pegg, D. E., Foreman, J., and Rolles, K.: Metabolism during preservation and viability of ischaemically injured canine kidneys. Transplantation, 38:78-81, 1984. 27. Pegg, D. E., and Jacobsen, I. A.: Current status of cryopreservation of whole organs with particular reference to the kidney. In Marberger, M., Dreikorn, K. (eds.): Renal Preservation. Baltimore, Williams and Wilkins, 1983, 301-322. 28. Pegg, D. E., Jacobsen, 1. A., and Walter, C. A.: Hypothermic perfusion of rabbit kidneys with solutions containing gelatin polypeptides. Transplantation, 24:29-38, 1977. 29. Pegg, D. E., Wusteman, M. C., and Foreman, J.: The metabolism of normal and ischaemically injured rabbit kidneys during perfusion for 48 hours at 10°C. Transplantation, 32:437-443, 1981. 30. Petterson, S., Claes, G., and Schersten, T.: Fatty acid and glucos~ utilization during continuous hypothermic perfusion of dog kidney. Eur. Surg. Res., 6:79-94, 1974. 31. Rijkmans, B. G., Buurman, W. A., and Kootstra, G.: Six day canine kidney preservation. Hypothermic perfusion combined with isolated blood perfusion. Transplantation, 37:130-134, 1984. 32. Robinson, J. R.: Control of water content of non-metabolizing kidney slices by sodium chloride and polyethylene glycol (PEG 6000). J. Physiol., 213:227-234, 1971. 33. Rolles, K., Foreman, J., and Pegg, D. E.: :Preservation of ischaemically injured canine kidneys by retrograde oxygen persufRation. Transplantation, 38:102-106, 1984. 34. Ross, H., and Escott, M. L.: Gaseous oxygen perfusion of the renal vessels as an adjunct in kidney preservation. Transplantation, 28:362-364, 1979. 35. Ross, H., Marshall, V. c., and Escott, M. L.: 72-hour canine kidney preservation without continuous perfusion. Transplantation, 21 :498-501, 1976. 36. Sheehan, H. L., and Davis, J. c.: Renal ischaemia with failed rellow. J. Pathol. Bacteriol., 78:105-120, 1959. 37. Southard, J. H., Van der Laan, N. c., Lutz, M., et al.: Comparison of the effect of temperature on kidney cortex mitochondria from rabbit, dog, pig and human: Arrhenius plots of ADP-stimulated respiration. Cryobiology, 20:395-400, 1983. 38. SquifRet, J. P., and Alexandre, G. P. J.: Clinical results of renal preservation witl) simple hypothermic storage using Euro-Collins solution. In Marberger, M., Dreikorn, K. (eds.): Renal Preservation. Baltimore, Williams and Wilkins, 1983, pp. 267-275. 39. Toledo-Pereyra, L. H., and Condie, R. M.: Lung transplantation: Hypothermic storage for 24 hours in a colloid hyperosmolar solution. J. Thorac. Cardiovasc. Surg., 76:846852, 1978. 40. Toledo-Pereyra, L. H., Chee, M., Condie, R. M., et al.: Forty-eight hours hypothermic storage of whole canine pancreas allografts. Improved preservation with a colloid hyperosmolar solution. Cryobiology, 16:221-228, 1979. 41. Veith, F. J., Sinha, S. B. P., Graves, J. S., et al.: Ischaemic tolerance of the lung. J. Thorac. Cardiovasc. Surg., 61 :804-810, 1971.
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42. Veith, F. J., Crane, R., Torres, M., et al.: Effective preservation and transportation of lung transplants. J. Thorac. Cardiovasc. Surg., 72:97-105, 1976. 43. Weed, R. I., La CelIe, P. L., and Merrill, E. W.: Metabolic dependence of red cell deformability. J. Clin. Invest., 48:795-809, 1969. 44. van der Wijk, Rijkmans, B. G., Kootstra, G.: Six-day kidney preservation in a canine model. Influence of a 1-4 hour ex vivo perfusion interval. Transplantation, 35:408-411, 1983. 45. Wusteman, M. C.: The effect of warm ischaemia on the function of rabbit kidneys measured by isolated normothermic perfusion. J. Surg. Res., 23:332-338, 1977.
MRC Medical Cryobiology Group University Department of Surgery Douglas House, Trumpington Road Cambridge, United Kingdom CB22AH
0039-6109/86 $0.00
+ .20
Organ Preservation D. E. Pegg, M.D. *
All effective methods of organ preservation rely upon reduction of temperature as the principal protective element. As soon as an organ becomes ischemic, its supply of metabolites ceases, productsj)f metabolism accumulate, and damage occurs, reversible at first but eventually irreversible. It may be assumed that the processes of deterioriation that accompany ischemia are mediated by chemical reactions, the rate of which is dependent upon temperature. Simple cooling has been remarkably successful in reducing damage, although it does have limitations, the reasons for which are gradually becoming clearer. This article briefly explores the mechanisms of ischemic damage and then considers the effects of cooling and other supplementary measures that can protect organs during storage. Clinical applications of this information will be considered, and likely avenues for future improvements in techniques for organ preservation will be discussed. THE NATURE OF ISCHEMIC INJURY
The cellular structural changes !hat accompany ischemia have been well described. 14 These affect mitochondria, nuclei, endoplasmic reticulum, lysosomes, and ultimately the cell membranes. The precise distinction between reversible and irreversible injury is difficult to determine, but certainly gross changes to the mitochondria are a prominent feature of early irreversible damage, and rupture of the cell membranes is conclusive. As is well known, different cells have different susceptibilities to ischemic injury. In the kidney, ischemic changes occur first and become most severe in the proximal convoluted tubule. After 30 minutes there are gross changes in some, but not all, tubules; at 60 minutes there is total necrosis of all segments of the majority of tubules. Nevertheless, 90 per cent of animals *Hp.ad of MRC Medical Cryobiology Group, University Department of Surgery, Cambridge, United Kingdom More detailed reviews of work on organ preservation have recently been published by the author, and this article is based on those publications. 24. l'.5
Surgical Clinics of North America-Vol. 66, No.3, June 1986
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10
10
9
9
8
8
~7
~7 ~
~
o 6
o 6
....~... 0 .
z «
>-
3
·····
2
......
'
°
.'
"
FRESH C +W LI3=-'=-5_..:.1::..5_--=3..:.0_ _6,-0--" MINS
2
.......
n·· ..
WI T
°
FRESH C+W
1,-3_.5_ _ 15_ _ 3_0_ _6_0~1 MINS
WIT
Figure 1. Time course of loss of adenine nucleotides in rabbit kidneys subjected to increasing periods of warm ischemia (WIT). (FRESH = freshly isolated with negligible warm ischemia; C + W = cannulated and flushed with WF5PO solution, as described by Pegg, Wusteman, and Foreman 29 ; ATP = adenosine triphosphate; TAN = total adenine nucleotides; OW = dry weight of tissue.)
will survive 30 minutes of warm ischemia and 75 per cent even 60 minutes; thus much of this damage is recoverable. However, after 60 minutes of warm ischemia, there is evidence of permanent histologic injury. 1, 18 Inevitably, metabolic changes accompany ischemic injury, and in view of the severity of structural damage to mitochondria, the effects on energy metabolism have been most extensively studied. The earliest changes are due to lack of molecular oxygen for oxidative phosphorylation, which brings about an accumulation of NADH and depletion of ATP within the mitochondria. The mitochondrial membrane is impermeable to these molecules, which are normally translocated by enzyme-driven mechanisms, and consequently NADH accumulates and NAD+ levels fall. NAD+ and ATP are essential for the operation of the glycolytic pathway, the rate-limiting step of which (phosphofructokinase) is inhibited by hydrogen ions. Thus glycolysis, which is capable of trapping only about 5 per cent of the total energy of glucose and is the only source of ATP available to anoxic cells, is inhibited. The situation is relieved to a limited extent by the lactate dehydrogenase reaction, which generates lactate and NAD+ from pyruvate, NADH, and hydrogen ions. It is likely that both hydrogen ions and lactate are responsible for the production of structural damage in ischemia. The depletion of ATP is fundamental to ischemic injury. ATP levels drop extremely rapidly in rabbit kidneys, reaching 20 per cent of normal in only 15 minutes; ADP and AMP concentrations fall more slowly (Fig. 1).29 None of these nucleotides is able to diffuse across the cell membrane, but dephosphorylation of AMP to the corresponding nucleoside adenosine, which is due to the action of 5' -nucleotidase enzymes, leads to the loss of total adenine nucleotides since nucleosides can traverse the cell membrane. This loss of nucleosides is probably a very important factor in the failure of tissues subjected to prolonged ischemia to regenerate ATP after restoration
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of a normal blood supply. Adenosine is further degraded to inosine, hypoxanthine, xanthine, and uric acid. This loss of purine is important because the salvage of purine skeletons plays a dominant role in adenine nucleotide synthesis and is energetically advantageous in that the "salvage pathway" uses only 2 moles of ATP/mole synthesized, as opposed to 6 moles for de novo synthesis. The relationship between structural and metabolic changes is interesting. It is apparent that the speed at which ATP levels fall far exceeds the rate at which structural or gross functional damage can be observed. Hence low ATP levels cannot be damaging per se. It has been shown that mitochondria isolated from kidneys that have been subjected to 30 minutes of warm ischemia are able to phosphorylate efficiently, yet these kidneys are not able to regain normal ATP levels: this is probably due to the loss of total adenine nucleotides by the mechanism discussed earlier, rather than mitochondrial structural damage. The lost nucleotides can be replaced, however, and this will be discussed later. A prominent feature of ischemic injury is swelling of the cells. The sodium pump is responsible for maintaining both the peculiar intracellular ionic balance of cells and their normal cell volume; this is because the transport of ions and water is osmotically coupled. The detailed mechanisms involved have been discussed elsewhere;25 fundamentally, it is the impermeant nature of the bulk of the intracellular anion that is responsible for the net entry of solute and water when the sodium pump is arrested, whether this is caused by lack of ATP in ischemia or by reduction of temperature in hypothermic preservation. It has been shown that the vascular endothelial cells of ischemic kidneys double their thickness after 60 minutes of warm ischemia. 12 The precise role of cell swelling in ischemic injury is a matter of debate, but Jamart and Lambotte have compared the effect of a standard anoxic treatment with the effect of perfusion with a hypotonic solution that produces a similar degree of cell swelling. 19 They clearly demonstrated that hypotonic cell swelling is less damaging than a comparable degree of anoxic cell swelting, confirming that anoxia damages by mechanisms additional to cell swelling. Nonetheless, as will be elaborated subsequently, the reduction of cell swelling does go some way to mitigate anoxic injury. One of the striking features of an organ that has suffered a period of warm ischemia is the failure of blood flow to return when circulation is restored. 36 This "no reflow" phenomenon is of great importance in determining the fate of a transplanted organ, since intact parenchymal cells can be of no value in the absence of circulation. The addition of substances to which the cells are impermeable, such as mannitol, is able to reduce the degree of vascular endothelial swelling and improve the reflow of blood under some conditions,12 but this approach is unable to completely cure the problem. However, the addition of dextran 40, which inhibits red cell aggregation, improves blood flow dramatically. Other evidence indicates that the "no reflow" phenomenon is much worse if red blood cells are present in the organ during the ischemic period than otherwise; indeed, Wusteman found no reduction in perfusate flow after up to 90 minutes of ischemia when the kidneys did not contain red cells during the ischemic
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period and were reperfused with a bloodless perfusate. 45 Thus it seems unlikely that the increase in vascular resistance is due to endothelial cell swelling but rather is caused by red cell sludging. This may be partly due to the increase in red cell rigidity that occurs when the cells are depleted of ATP during ishemia. 43 Addition'ally, it is known that the capillaries become increasingly leaky to protein after more than 30 minutes of warm ischemia, which would lead to a loss of the oncotic pressure which retains fluid in the capillaries and hence to an increase in the hematocrit within the vessels. This in turn would increase viscosity dramatically and therefore lead to stagnation. It seems likely that vascular factors playa major part in the eventual failure to recover function in organs that have suffered significant ischemia.
CONCLUSIONS
Although the picture is still incomplete, it is clear that those biochemical changes that occur early in ischemia and are due to lack of oxygen are reversible and that hydrogen ions and lactate can accumulate to remarkably high levels and yet be compatible with complete recovery. Loss of important metabolites, for example, adenine nucleotides, is more serious, and in prolonged ischemia the gross accumulation of metabolites, including hydrogen ions and lactate, presumably does cause structural damage. One feature of ischemic injury that has received particular attention, cell swelling, is only one metabolic consequence of lack of ATP: in itself it appears to be only moderately harmful and the total damage of ischemia cannot be duplicated by an equivalent degree of cell swelling. In particular, the "no reflow" phenomenon, also a prominent feature of organs that have suffered ischemic damage, is not primarily due to cell swelling but rather is caused by the trapping of red cells in the microcirculation, probably because of an increase in their rigidity and leakiness of the capillaries to protein. The fundamental cause of ischemic injury lies in molecular changes in membranes. Ultrastructural studies are able to demonstrate this damage directly, and confirmation is provided by the observation that proteins leak from capillaries into the interstitial space, that intracellular enzymes leak into the vascular system, and that lysosomal enzymes leak into the cytosol. The ultimate cause of this membrane damage is still unclear, but it is apparent that the preservation of membrane integrity is fundamentally important for organs that are to be transplanted.
THE EFFECTS OF COOLING Structural Cooling slows the rate at which the structural changes seen at 37° C develop, but there are also some qualitative differences. Cell swelling is much more prominent in hypothermia, and there is a more uniform effect upon different cell types, including, in the case of the kidney, the glomeruli,
621
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Table 1. Composition of Flush Solutions CONSTITUENT (PER LITER)
Na+ (mmol) K+ (mmol) Mg2- (mmol) Citrate (mmol) SO;- (mmol) H,P04 - (mmol) HPO/- (mmol) CI- (mmol) HC03 - (mmol) Mannitol (mmol) Glucose (mmol) Osmolality (mOsm/kg)
COLLINS' C 2
EURO-COLLlI\iS'
SOLUTION
SOLUTIOI\i
10,0 115,0 30,0
10,0 115,0
30,0 15,0 42,5 i5,0 10,0
HYPERTONIC CITRATE SOLUTION
83,7 79,5 40,6 54.4 40,6
15,0 42,5 15,0 10,0 185,5
140 360
194 375
400
which are remarkably resistant to warm ischemic lOJury. The exposure times required to produce damage at low temperatures far exceed those needed to produce warm ischemic injury and consequently there is no doubt that cooling is highly protective. However, the presence of additional lesions in hypothermia does suggest that additional damaging mechanisms exist at low temperatures. Biophysical We have already noted that potassium is lost and sodium and water are gained when cells are exposed to normothermic ischemia and that this is due to the limited amount of ATP available to drive the sodium pump. Under hypothermic conditions, however, even if sufficient ATP is present, the sodium pump becomes inoperative due to cooling and the cells therefore swell. Since the primary cause of swelling in the absence of the sodium pump is the impermeable nature of tht;, bulk of intracellular anion, balancing this osmotic pressure by external impermeant solutes can provide a means for controlling cell swelling. If a relatively high molecular weight polymer is used, for example, polyethyleneglycol of molecular weight 6000, then about 20 mOsm/kg is sufficient. 32 More commonly, lower molecular weight materials have been used for this purpose, and since these do penetrate the cells, albeit slowly, higher osmolatities are required for effective control of cell volume. Manipulation of the ion balance in the perfusing solution makes it possible to prevent the loss of intracellular potassium and gain of sodium. This approach was pioneered by Collins and colleagues9 and is now the most widespread method of renal preservation. Some examples of these so-called "intracellular" solutions are shown in Table 1. It will be apparent that not all of them mimic intracellular ion concentrations closely, but they have two features in common: they contain a significant amount of impermeant solute and a high concentration of potassium. Collins was able to achieve 30 hours' storage of canine kidneys flushed with his solution and stored at about 0° C. More recently, Andrews and Coffeyl have observed a considerable degree of proximal tubular damage and swelling of the ascending distal tubules in kidneys preserved
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with Collins' solution, but they found that an isosmotic phosphate-buffered saline that included 140 mmolll of sucrose gave superior ultrastructural appearances. Several groups have attempted to identify the crucial components of "intracellular" solutions and their mechanism of action, and it is clear that the inclusion of impermeant or slowly permeating solutes is of far greater importance than elevation of the potassium concentration. 15 Thus, although cell swelling is probably of only secondary significance in normothermic ischemia and is unimportant in the etiology of the "no reflow" phenomenon, its prevention is able to mitigate the injury that occurs in hypothermic storage and to prolong the storage times available. The importance of varying the ionic balance is more problematic. Most studies have found elevation of the potassium concentration to be advantageous and some, though not all, have found that increased magnesium concentrations are advantageous. We, however, have found elevation of magnesium concentrations to be damaging in some circumstances,21 and both experimental and clinical experience with the Euro-Collins' version of Collins' solution (which omits magnesium sulfate) have shown it to be highly effective. 10, 38 Probably calcium is also of considerable importance, although it has only recently received detailed attention. Cytosolic concentrations of calcium are normally of the order of 10-8 to 10-7 molar, whereas the extracellular fluid contains about 10-3 molar calcium. The accumulation of calcium, particularly in the mitochondria, is a universal finding in hypothermic cells, and in view of the importance of calcium as a second messenger in the regulation of numerous physiological processes, this accumulation is likely to have important effects. Calcium acts as a messenger by combining with and activating the regulatory protein calmodulin,7 and this can be blocked by the addition of phenothiazene compounds. Asari and associates 2 have shown the addition of one of these compounds (trifluoperazine) to Collins' solution improves the recovery of viable kidneys stored at 4-6° C for 72 hours from 33 per cent to 80 per cent. It would seem that this approach is worthy offurther study. Biochemical Metabolism during hypothermia is beginning to receive considerable attention, particularly with respect to energy metabolism. Free fatty acids are the principal fuel for many tissues, including renal cortex, under welloxygenated conditions at 37° C, although glucose is the preferred substrate for renal medulla. When renal tissue is cooled, for example to 10° C, fatty acids continue to be respired, but ketone bodies are then by far the most effective substrate. 17 However, glucose is also taken up by hypothermic kidneys at roughly the same rate as fatty acid, and the majority is metabolized to lactate. 3o Lactate continues to accumulate throughout hypothermic perfusion even when the perfusate is well oxygenated, which suggests that anaerobic glycolysis plays a major role in energy metabolism under these conditions. In our own laboratory, using rabbit kidneys perfused at 10° C, we have found that a perfusate p02 of 150 mm Hg causes glucose to be consumed and lactate to accumulate. The addition of caprylate increases both the glucose consumption rate and the lactate production rate. 29 Thus, anaerobic glycolysis is probably the principal source
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of energy under these perfusion conditions and the stimulatory effect of caprylate is probably due to its indirect stimulation of phosphofructokinase. We attempted to increase aerobic respiration by raising the p02 to approximately 650 mm Hg, which reduced glucose consumption and lactate production and improved the synthesis of high energy nucleotides. The further addition of hypoxanthine as a purine "salvage pathway" precursor produced a further significant improvement in high energy phosphate concentrations. In fact, the combination of glucose, caprylate, and hypoxanthine with a p02 of approximately 650 mm Hg restored all the adenine nucleotide indices to normal except for the ATP:ADP ratio, which at 80 per cent of normal was similar to that observed after only 2 minutes of normothermic ischemia. The most striking finding in this study was the observation that even a kidney that had suffered 60 minutes of warm ischemia, which reduces ATP levels to 10 per cent of control and total adenine nucleotides to 20 per cent of control, would recover normal adenine nucleotide indices under these perfusion conditions (except for the ATP:ADP ratio which was 50 per cent of normal, or equivalent to 5 or 6 minutes of warm ischemia). The data suggested that the energy for this resynthesis was partly glycolytic and partly aerobic. These findings have been confirmed in canine kidneys. 26 The impairment of oxidative phosphorylation at low temperatures points to a mitochondrial defect. Oxygen consumption by isolated mitochondria decreases with falling temperature, often with a change in the rate of decrease at about IS° C. 37 It appears from more detailed studies that the enzymes responsible for translocating adenine nucleotide (AN translocase) across the mitochondrial membrane become ineffective below the temperature at which the change in rate of oxygen consumption occurs. Even under normal physiologic conditions, AN translocase has negligible spare capacity20 and is therefore very likely to become rate-limiting at low temperatures. Similarly, the enzymes responsible for moving NADH across the mitochondrial membrane (the mal~te-aspartate shuttle) are ineffective under hypothermic conditions. It seems clear therefore that the failure of aerobic metabolism in hypothermia is due primarily to the inability of the mitochonrial transport enzymes to function effectively at storage temperatures. Nevertheless, providing that the appropriate substrates are present, it is possible for hypothermic kidneys to synthesize adenine nucleotides at the temperatures typically used for hypothermic organ preservation. An additional (or alternative) strategy may be used to support adenine nucleotide syntheSis. It has been mentioned that AMP is lost from the cells only after it has been dephosphorylated to adenosine, and it follows that the pharmacologic inhibition of 5' -nucleotidase would increase total adenine nucleotide levels. This has been achieved by the use of allopurinol, 1,3 dior 1,3,7 tri-methylxanthine,5 but this approach has not been extensively studied clinically. It seems reasonable to conclude that the maintenance of high ATP levels during hypothermic preservation, which is certainly possible, is also desirable. However, the reversible inhibition of transport enzymes that occurs during hypothermia eventually becomes irreversible and is probably one of the major factors limiting the duration of effective hypothermic storage.
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It has been mentioned that an increase in the oxygen tension in a perfusate used for the hypothermic preservation of kidneys results in improved ability to resynthesize adenine nucleotides during the storage period. However, oxygen can be supplied without resorting to continuous perfusion by using the technique of retrograde oxygen persuffiation, introduced by Fischer and colleagues. l l With this method, kidneys that have been flushed with a conventional flush solution are supplied with gaseous oxygen by a cannula attached to the renal vein and the gas is allowed to escape through numerous small needle pricks made over the surface of the organ. The gas flows through only a part of the venous system which then acts as an exchange surface from which oxygen can diffuse into the whole organ. Fischer himself used Collins' solution, an oxygen delivery pressure of 55 mm Hg, and a temperature of 6° C. Ross and EscottJ4 and we 33 have used a lower temperature, 0° C, a lower gas pressure, 10 to 12 mm Hg, and hypertonic citrate flush solution as shown in Table 1. 35 These experiments showed this maneuver to be quite effective in improving the preservation of ischemically injured kidneys: We were able to obtain 48 hours' preservation of canine kidneys damaged by 30 minutes of prior warm ischemia or 24 hours' preservation after 60 minutes of warm ischemia. The technique produces a high and uniform oxygen tension throughout the organ; air is less effective than pure oxygen, and inert gases are completely ineffective. Fischer was able to show some restoration of adenine nucleotide reserves during the hypothermic storage period but we were unable to confirm that finding. The difference is probably due to our use of a lower temperature at which, perhaps due to the effect of cooling on AN translocase activity, oxygen was unable to stimluate further ATP synthesis. The mechanism of this effect is unclear; it is possible that some stimulation of ATP synthesis (but too little to be detected by our chemical techniques) did occur, or alternatively it is conceivable that oxygen may have stabilized cytochrome oxidase and hence the inner mitochondrial membrane. Whatever the mechanism, the effectiveness of this quite simple technique does seem to be clearly established. Whether the procedure might have a role in the preservation of kidneys that have not suffered prior warm ischemia is unknown, but this remains a possibility.
Vascular In view of the importance of vascular effects in warm ischemic injury, it is relevant to consider the effect of hypothermia on the vascular system. The cell swelling caused by hypothermia affects the vascular endothelium as it does other cells, yet for a given period of ischemia, cooling alone is highly effective in protecting against the "no reflow" phenomenon. This is further evidence that "no reflow" is not caused by swelling of endothelial cells. Scanning electron microscopy, carried out in Belzer and Southard's laboratory, indicates little structural change after storage periods in Collins' solution that are associated with functional renal recovery, and even at 7 days the endothelium showed a continuous sheet. Epithelial podocytes also showed strikingly good preservation even after 7 days' storage. In continuously perfused canine kidneys, Belzer and colleagues3 have shown intact endothelium after 3 days' storage, but severe damage at 7 days, and their
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recent scanning electron micrographs have shown that the glomerular podocytes develop numerous microvillous projections into Bowman's capsule, the foot processes acquire protruberances and the cell bodies become indistinct. Thus the vascular system does seem to be less well preserved in continuously perfused than in flushed organs. We have found that the choice of colloid has an important influence on the generation of vascular injury; of the protein substitutes, the gelatin polypeptide Haemaccel (Hoechst) was the best,28 but the most effective colloid is probably purified albumin. In a clinical study of kidneys perfused with cryoprecipitated plasma, Hill and colleagues 16 observed disruption of the glomerular endothelium, which caused platelet and fibrin deposition on the exposed basement membrane after transplantation. These changes were seen even in kidneys perfused for less than 20 hours (although they were more severe after longer preservation), but they were minimal or absent in kidneys perfused with purified albumin and were reduced if the perfusion pressure was lowered. This damage is often, but not invariably, associated with an increase in the total vascular resistance of the organ. Vascular injury, whether augmented by perfusion or not, is undoubtedly a major factor determining the time limit of effective hypothermic renal preservation. Nevertheless this damage can be ameliorated in the various ways. There is substantial evidence that, under normal physiologic circumstances, blood cells and endothelial cells interact in complex ways to maintain an intact vascular system, and platelets probably play a crucial role in this phenomenon. This "nurturing" action is probably the mechanism that was responsible for the effectiveness of intermediate normothermic blood perfusion in prolonging the total hypothermic preservation time of canine kidneys in the experiments of van der Wijk and associates and Rijkmans and coworkers. 31. 44 In these experiments, kidneys were perfused with an albumin solution for 6 days at 6° C and grafted. Only 2 out of 14 animals survived in this experiment, whereas interrupting the hypothermic perfusion for 3 to 4 hours on the !.hird day for a period of ex vivo normothermic blood perfusion enabled·11 out of 12 animals to survive, and they had much lower peak creatinine levels. Blood perfusion with a heartlung machine was equally effective. These results strongly argue in favor of a vascular repair mechanism. It is therefore not surprising that measures to prevent the adherence of platelets and deposition of fibrin within damaged vessels after transplantation, and thereby allow time for endothelial repair, are able to prolong the time for which organs can be stored. Recently, a long-acting analogue of prostacyclin, the most powerful inhibitor of platelet aggregation known, has been shown to be highly protective against the vascular and functional effects of 120 minutes of normothermic renal ischemia in the dog, and prostacyclin has been used in the hypothermic preservation of livers. 23 Similarly urokinase, a plasminogen activator that prevents fibrin deposition, has been reported to increase the preservation time obtainable with Collins' solution and the canine kidney to 96 hours with 100 per cent survival, and even to 120 hours (5 days) with 50 per cent survival. 22
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Cooling retards the development of ischemic injury but adds other specific damage. Anoxic cell swelling is actually accentuated by cooling, although it can be prevented by the inclusion of impermeant solutes in preservation solutions. Intracellular ionic balance can be improved and the damaging effects of calcium loading reduced by appropriate changes in the composi.tion of the preservation solution. At the temperatures commonly used for perfusion preservation, significant metabolism continues, but that metabolism is altered from normal physiologic biochemistry. If appropriate precursors are provided, ATP levels can be maintained, or even improved where there has been prior warm ischemia. For this reason, continuous perfusion methods that can support metabolism seem attractive, yet continuous perfusion incurs the risk of greater vascular injury. The provision of a high oxygen tension is advantageous in its own right, particularly when dealing with kidneys that have suffered significant warm ischemia, but the mechanism of this effect is unclear. As with normothermic ischemia, the fundamental limiting factor in hypothermic organ preservation is probably damage to cell membranes, and progress in preservation techniques will be dependent upon methods for combating these membrane changes. It is distinctly possible, but not yet proven, that oxygen free radicals may play an important part in the generation of low temperature membrane damage. These highly toxic agents are generated by the stepwise addition of the four electrons that are normally added to oxygen in a single step by cytochrome oxidase. The enzymes superoxide dismutase, catalase, and glutathione peroxidase are normally responsible for converting these radicals into harmless water, and many compounds-including mannitol, glycerol, and dimethylsulfoxide, for example-have free radical scavenging properties. There is no doubt that free radicals do attack membrane lipids, releasing lipid peroxides and seriously damaging the cells, and because free radical reactions have a low activation energy, they are depressed to a lesser extent by decreasing temperature than the enzymic reactions that normally remove them. Consequently, it is entirely possible that oxygen free radicals are important in hypothermic injury. There is already some evidence that the addition of radical scavengers to preservation media may improve the viability of organs. OPTIMAL TECHNIQUES FOR HYPOTHERMIC PRESERVATION The relative merits of continuous perfusion and simple cold storage have been actively debated for many years, and to this debate must now be added the arguments in favor of retrograde oxygen persuffiation. It is not proposed to repeat all the arguments here but more briefly to consider the relative merits of the three approaches. Continuous perfusion makes it possible to support metabolism, but this is achieved at the expense of bulky, complicated, and expensive equipment. Flush preservation, on the other hand, is limited to replacement
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of the vascular contents with a chosen solution and exchanging the intracellular fluid of the orgap. with that solution to a very limited degree. However, the method is simple, convenient, and inexpensive. The important question is whether the more complex approach secures any significant advantages. It is clear that metabolism does occur at the temperatures generally used for storage, and this certainly provides a stong theoretical argument in favor of perfusion. Continuous perfusion is probably the only technique that will adequately support hypothermic metabolism. There is strong evidence that the metabolic difficulties experienced by hypothermic organs are largely due to the inhibition of membrane-bound transport enzymes, which suggests that higher temperatures than those used hitherto might be beneficial. If so, metabolism will be greatly accelerated, and continuous perfusion is then likely to be indispensable. However, continuous perfusion places additional stresses upon the vascular system that are avoided by simple flush preservation, and once the inability of flush preservation to support metabolism is acknowledged, it becomes sensible to use the lowest possible temperature to minimize metabolic activity. The choice of technique really depends upon the objective: If kidneys with no prior ischemia are to be preserved for less than 48 hours, then flush preservation, for example with one of the solutions shown in Table 1, is the method of choice. If, however, there has been significant prior warm ischemia, or if much longer preservation is needed, then continuous perfusion will be more effective. The longest period of renal preservation so far reported (8 days) was by Cohen, who used a continuous perfusion method. 8 If organs with significant warm ischemia are to be used, then the relatively trivial additional technical complication of retrograde oxygen persuffiation seems worthwhile. In many countries, however, the use of kidneys damaged by warm ischemia has been almost eliminated, and the value of retrograde oxygen persuffiation in the absence of prior warm ischemia is largely unstudied.
PRACTICAL TECHNIQUES FOR TRANSPLANT ORGANS Kidney24 Preservation of the cadaveric kidney commences before its removal from the donor. Pretreatment of the donor with heparin, an alpha-adrenergic blocker, and the production of a good diuresis are all desirable. Once removed, the kidney should be flushed with one of the "intracellular" solutions that are available and probably not significantly different in effectiveness: Collins C2, the so-called Euro-Collins solution, or hypertonic or isotonic citrate solutions are all acceptable. A pressure of not less than 60 mm Hg should be used to secure effective removal of blood, and the temperature should be about 5° C. Flushing is usually continued until at least 1 liter has flowed through the kidney and the effiuent is completely clear of blood. A decision must then be made between the three approaches to preservation that are available. When the period of ischemia has been
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628
PEGG
PRESSURE TRANSDUCER
FLAKED
ICE
WATERPROOF PLASTIC BAG
ORG~
CONTAINER _ _
RENAL
VEIN--
Figure 2. System used for retrograde oxygen persuffiation of human kidney grafts. (From Pegg, D. E.: Organ preservation at temperatures above 0° C. In CaIne, R. Y. (ed.): Transplantation Immunology: Clinical and Experimental. Oxford, Oxford University Press, 1984, p. 355, with permission.)
brief (less than 15 minutes) and the required period of preservation is less than 48 hours, then storage in the flush solution within a sterile container surrounded by ice is probably the method most commonly used at the present time. If the warm ischemic period has exceeded 45 minutes, then continuous perfusion is probably a better choice, although we are now using retrograde oxygen persufflation on a trial basis in these circumstances (Fig. 2). With intermediate warm ischemia time (15 to 45 minutes), simple flush storage will usually be satisfactory so long as the intended storage period is less than 24 hours, but if continuous perfusion is available, it may be wiser to use it. It should be noted, however, that continuous perfusion of the kidney or any other organ requires trained personnel with adequate experience if satisfactory results are to be obtained. Heart In experimental studies, prOViding there has been minimal warm ischemia, up to 24 hours' preservation seems to be achievable, either by flush preservation with an "intracellular" solution or by continuous hypothermic perfusion. 24 At present there are no satisfactory methods available for dealing with hearts damaged by warm ischemia or for obtaining greater than 24 hours' preservation. In clinical practice, however, storage periods have generally been limited to 6 hours, and standard cardioplegic solutions have been used: there does seem to be a need for better flush preservation methods. Current work in our laboratory, in collaboration with Papworth Hospital, Cambridge, suggests that modifications to the standard cardioplegic solutions may result in significant improvement in storage time. 13 These methods have yet to be tested in the clinical situation. Liver Continuous hypothermic perfusion has been shown to permit 20 to 24 hour preservation of the liver but it is generally felt that the technique is
629
ORGAN PRESERVATION
too complex for routine use and both of the major groups that are now carrying out clinical liver transplant programs use simple flush methods. The Denver-Pittsburgh group uses Collins' solution,4 whereas the Cambridge-King's College group uses a plasma protein solution with an elevated potassium concentration (modified PPF). The technique is described in detail by Calne. 6 Most recently in this laboratory a comparison has been made in the rat liver among Collins' solution, PPF, hypertonic citrate solution, and isotonic citrate solution for flush preservation. The isotonic citrate solution proved to be superior to the others. This !/olution has yet to be evaluated clinically. Additionally, it has been found that a perfusion solution based upon the isotonic citrate formulation, but with additional Haemaccel, and fluorocarbon emulsion, has made it possible to achieve 24 to even 48 hours' preservation of rat liver. Whether perfusion techniques will ever prove acceptable for clinical use is essentially a technical and financial question, but it does seem likely that the present flush solutions are capable of significant improvement. Pancreas The flush preservation approach has yielded effective storage of the pancreas for 24 hours using a wide range of solutions. 21 Forty-eight hour preservation has also been reported with a hyperosmolar flush solution containing albumin,4O but this result has not been confirmed. Continuous perfusion seems to be no more effective. In our own experiments, an elevated magnesium concentration in the flush solution was found to be deleterious. 21 Lung24 This organ has a very limited ability to tolerate either warm ischemia or deflation; either inflation or ventilation of the ischemic lung significantly prolongs its survival even if nitrogen is used as the inflating gas. 3~, 41 Preservation is possible for 24 hours by flushing with an intracellular solution, inflation with 40 per cent oxygen plus 60 per cent nitrogen, and storage at 4° C,42
FUTURE POSSIBILITIES It is likely that minor refinements of the existing flush preservation methods will continue to produce useful improvements in post-preservation function, but really significant improvements will require a considerable increase in our understanding of hypothermic physiology and biochemistry, It is my opinion that the complexity and expense of continuous hypothermic perfusion makes it rather unlikely that it will be more widely used than at present. The technique of retrograde oxygen persuffiation is certainly promising but limited in scope-it requires further study before it can be confidently advocated for clinical use. At the moment it is being used only for kidneys, but if it should prove applicable to other organs, it might conceivably increase the uncomfortably short preservation times that are currently available for hearts and livers, without incurring the expense and
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PECC
complication of continuous perfusion. The fundamental limiting factor in hypothermic preservation is probably damage to cell membranes, and an increase in understanding of the mechanism of such damage should prove invaluable. Oxygen free radicals may be involved, but it is too soon to say whether or to what extent practical procedures based upon free radical scavenging will be useful in clinical practice. Truly indefinite storage will be possible, if at all, only by the use of temperatures well below 0° C, but this raises problems of an altogether different order. Although rpuch success has attended efforts to cryopreserve isolated cells, and even some simple tissues, the long-term subzero preservation of organs has so far proved impossible. 25, 27 Much scientific work remains to be done before organ cryopreservation enters the realm of practical application.
REFERENCES 1. Andrews, P. M., and Coffey, A. K.: Factors that improve the preservation of nephron morphology during cold storage. Lab. Invest., 46: 100-120, 1982. 2. Asari, H., Anaise, D., Bachvaroff, R. J., et aI.: Preservation techniques for organ transplantation 1, Protective effects of calmodulin inhibitors in cold preserved kidneys. Transplantation, 37:113-114, 1984. 3. Belzer, F. 0., Hoffman, R, Huang, J., et al.: Endothelial damage in perfused dog kidney and cold sensitivity of vascular Na-K-ATPase. Cryobiology, 9:457-460, 1972. 4. Benichou, J., Halgrimson, C. G., Wei!, R, III, et al.: Canine and human liver preservation for 6-18 hours by cold perfusion. Transplantation, 24:407-411, 1977. 5. Buhl, M. R., and Jensen, M. H.: The role of 5'-nucleotidase in purine depletion of ischaemic renal tissue. In Pegg, D. E., Jacobsen, I. A. (eds.): Organ Preservation II. Edinburgh, Churchill Livingstone, 1979, pp. 239-246. 6. Caine, R Y.: Liver. In Karow, A. M., Jr., and Pegg, D. E. (eds.): Preservation of Organs for Transplantation, Ed. 2. New York, Marcel Dekker, 1981, pp. 617-624. 7. Cheung, W. Y.: Calmodulin plays a pivotal role in cellular regulation. Science, 207:1927, 1980. 8. Cohen, G. L.: Eight-day kidney preservation. Ch. M. Thesis, University of Liverpool, 1983. 9. Collins, G. M., Bravo-Shugarman, M., and Terasaki, P. I.: Kidney preservation for transportation. Lancet, 2:1219-1222, 1969. 10. Dreikorn, K., and Horsch, R.: 48 to 96 hour preservation of canine kidneys by initial perfusion and hypothermic storage using the Euro-Collins solution. In Marberger, M., and Dreikorn, K. (eds.): Renal Preservation. Baltimore, Williams and Wilkins, 1983, pp. 261-266. 11. Fischer, J. H., Czerniak, A., Hauer, U., et al.: A new simple method for optimal storage of ischa~mically damaged kidneys. Transplantation, 25:43-49, 1978. 12. Flores, J., Di Bona, D. R, Frega, N., et al.: Cell volume regulation and ischaemic tissue damage. J. Membr. BioI., 10:331-343, 1972. 13. Foreman, J., Pegg, D. E., and Armitage, W. J.: Solutions for preservation of the heart at O°C. J. Thorac. Cardiovasc. Surg., 89:867-871, 1985. 14. Glaumann, B., Glaumann, H., Berezesky, I. K., et al.: Studies on the pathogenesis of isch;lemic cell injury II. Morphologicill changes of the pars convoluta (PI and P2) of the proximal tubule of the rat kidney made ischaemic in vivo. Virchows Archiv (Cell Pathol.), 19:281-302, 1975. 15. Green, C. J., and Pegg, D. E.: Mechanism of action of "in.tracellular" renal preservation solutions. World J. Surg., 3:115-120, 1979. 16. Hill, G. S., Light, J. A., and Perloff, L. J.: Perfusion-related injury in renal transplantation. Surgery, 79:440-447, 1976. 17. Huang, J. S., Downes, G. L., Childress, G. L., et al.: Oxidation of1'C-labelied substrates by dog kidney cortex at 10 and 38°C. Cryobiology, 11:387-394, 1974.
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18. Jablonski, P., Howden, B. 0., Rae, D. A., et al.: An experimental model for assessment of renal recovery from warm ischaemia. Transplantation, 35:198-204, 1983. 19. Jamart, J., and Lambotte, L.:. Differential effect of swelling and anoxia on kidney function and its consequences on the mechanisms of action of intracellular organ preservation solutions. Transplantation, 34:176-182, 1982. 20. Kemp, A., Jr., Groot, G. S. P., and Reitsma, H. J.: Oxidative phosphorylation as a function of temperature. Biochim. Biophys. Acta, 180:28-34, 1969. 21. Klempnauer, J., Pegg, D. E., Taylor, M. J., et al.: Hypothermic preservation of the rat pancreas. In Pegg, D. E., Jacobsen, I. A., and Halasz, N. A. (eds.): Organ Preservation: Basic and Applied Aspects. Lancaster, MTP Press, 1982, pp. 191-198. 22. Masaki, Y., Uchida, H., Osakabe, T., et al.: Successful 96- and 120-hour preservation of the canine kidney by simple surface cooling with high units of urokinase and modified Collins' solution. Transplant. Proc., 17:1449-1453, 1985. 23. Monden, M., and Fortner, J. G.: Twenty-four and 48-hour canine liver preservation by simple hypothermia with prostacyclin. Ann. Surg., 196:38-42, 1982. 24. Pegg, D. E.: Organ preservation at temperatures above O°C. In CaIne, R. Y. (ed.): Transplantation Immunology: Clinical and Experimental. Oxford, Oxford University Press, 1984, pp. 349-359. 25. Pegg, D. E.: Principles of tissue preservation. In Morris, P. J., and Tilney, N. L. (eds.): Progress in Transplantation, Volume II. Edinburgh, Churchill Livingstone, 1985, pp. 69-105. 26. Pegg, D. E., Foreman, J., and Rolles, K.: Metabolism during preservation and viability of ischaemically injured canine kidneys. Transplantation, 38:78-81, 1984. 27. Pegg, D. E., and Jacobsen, I. A.: Current status of cryopreservation of whole organs with particular reference to the kidney. In Marberger, M., Dreikorn, K. (eds.): Renal Preservation. Baltimore, Williams and Wilkins, 1983, 301-322. 28. Pegg, D. E., Jacobsen, 1. A., and Walter, C. A.: Hypothermic perfusion of rabbit kidneys with solutions containing gelatin polypeptides. Transplantation, 24:29-38, 1977. 29. Pegg, D. E., Wusteman, M. C., and Foreman, J.: The metabolism of normal and ischaemically injured rabbit kidneys during perfusion for 48 hours at 10°C. Transplantation, 32:437-443, 1981. 30. Petterson, S., Claes, G., and Schersten, T.: Fatty acid and glucos~ utilization during continuous hypothermic perfusion of dog kidney. Eur. Surg. Res., 6:79-94, 1974. 31. Rijkmans, B. G., Buurman, W. A., and Kootstra, G.: Six day canine kidney preservation. Hypothermic perfusion combined with isolated blood perfusion. Transplantation, 37:130-134, 1984. 32. Robinson, J. R.: Control of water content of non-metabolizing kidney slices by sodium chloride and polyethylene glycol (PEG 6000). J. Physiol., 213:227-234, 1971. 33. Rolles, K., Foreman, J., and Pegg, D. E.: :Preservation of ischaemically injured canine kidneys by retrograde oxygen persufRation. Transplantation, 38:102-106, 1984. 34. Ross, H., and Escott, M. L.: Gaseous oxygen perfusion of the renal vessels as an adjunct in kidney preservation. Transplantation, 28:362-364, 1979. 35. Ross, H., Marshall, V. c., and Escott, M. L.: 72-hour canine kidney preservation without continuous perfusion. Transplantation, 21 :498-501, 1976. 36. Sheehan, H. L., and Davis, J. c.: Renal ischaemia with failed rellow. J. Pathol. Bacteriol., 78:105-120, 1959. 37. Southard, J. H., Van der Laan, N. c., Lutz, M., et al.: Comparison of the effect of temperature on kidney cortex mitochondria from rabbit, dog, pig and human: Arrhenius plots of ADP-stimulated respiration. Cryobiology, 20:395-400, 1983. 38. SquifRet, J. P., and Alexandre, G. P. J.: Clinical results of renal preservation witl) simple hypothermic storage using Euro-Collins solution. In Marberger, M., Dreikorn, K. (eds.): Renal Preservation. Baltimore, Williams and Wilkins, 1983, pp. 267-275. 39. Toledo-Pereyra, L. H., and Condie, R. M.: Lung transplantation: Hypothermic storage for 24 hours in a colloid hyperosmolar solution. J. Thorac. Cardiovasc. Surg., 76:846852, 1978. 40. Toledo-Pereyra, L. H., Chee, M., Condie, R. M., et al.: Forty-eight hours hypothermic storage of whole canine pancreas allografts. Improved preservation with a colloid hyperosmolar solution. Cryobiology, 16:221-228, 1979. 41. Veith, F. J., Sinha, S. B. P., Graves, J. S., et al.: Ischaemic tolerance of the lung. J. Thorac. Cardiovasc. Surg., 61 :804-810, 1971.
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42. Veith, F. J., Crane, R., Torres, M., et al.: Effective preservation and transportation of lung transplants. J. Thorac. Cardiovasc. Surg., 72:97-105, 1976. 43. Weed, R. I., La CelIe, P. L., and Merrill, E. W.: Metabolic dependence of red cell deformability. J. Clin. Invest., 48:795-809, 1969. 44. van der Wijk, Rijkmans, B. G., Kootstra, G.: Six-day kidney preservation in a canine model. Influence of a 1-4 hour ex vivo perfusion interval. Transplantation, 35:408-411, 1983. 45. Wusteman, M. C.: The effect of warm ischaemia on the function of rabbit kidneys measured by isolated normothermic perfusion. J. Surg. Res., 23:332-338, 1977.
MRC Medical Cryobiology Group University Department of Surgery Douglas House, Trumpington Road Cambridge, United Kingdom CB22AH