24-hour rabbit heart storage with UW solution. Effects of low-flow perfusion, colloid, and shelf storage.

A new technique for 24-hr cardiac preservation is described utilizing very low flow perfusion (microperfusion) with a cold flush solution. Rabbit hearts were arrested with UW solution and then perfused with the same solution through the aortic root at 0 degrees C at a rate of 3-6 ml/gm heart weight/24 hr. When tested on an ex vivo working heart model, the cardiac output (CO) was 28.72 +/- 7.69 ml/g/min compared with fresh UW flushed controls of 26.48 +/- 2.25 ml/g/min. Both oxygenated highflow perfusion with a more conventional perfusate and 24-hr ice storage with UW led to inferior results. Omission of the colloid, hydroxyethyl starch (HES), from the UW solution or prolonged shelf storage were also significantly detrimental. When a previously untested colloid, polyethylene glycol 20,000, was substituted for HES for microperfusion, excellent cardiac function was obtained. In fact, the mean CO of this group, 31.91 +/- 5.70, was significantly above that of fresh HES-UW unstored controls. The suggestion that the UW solution might be improved by this substitution warrants further study.

Impairment of renal slice function following brain death, with reversibility of injury by hormonal therapy.

The effects of the agonal period and subsequent donor management on renal slice function, using the K+ - Na+ ratio, have been studied in the pig. Brain ischaemia or death resulted in a reduction in renal slice function, whether the pig was maintained normovolemic or hypovolemic by i.v. fluid and dobutamine therapy. This deterioration in function was, however, reversed or prevented by a period of therapy with thyroxine (T3), insulin, and cortisol. A period of 24 hr storage of the kidney slice in a low ionic strength solution in ice resulted in a further deterioration in slice function in all groups studied.

Loss of myocardial viability following hypothermic perfusion storage from contaminating trace elements in the perfusate.

Two groups (A and B) of isolated baboon hearts were preserved by continuous hypothermic perfusion storage for 48 hours using perfusates that, according to the manufacturers, differed only in the concentrations of the contaminating trace elements iron, lead, and arsenic. Storage with the perfusate containing the higher concentration of these elements (perfusate B) led to significantly less gain in heart mass, a greater reduction in coronary flow, coronary sinus effluent lactate, and myocardial arteriovenous oxygen difference and a greater increase in coronary sinus effluent lactate dehydrogenase, when compared with perfusate A. Group B hearts totally failed to support the circulation following orthotopic transplantation, whereas group A hearts showed excellent function. Group B hearts had undergone the typical changes of enhanced resting myocardial tension during the storage period (before warm blood reperfusion); we proposed that these changes were brought about by the production of superoxide anions and radicals by the higher relative concentration of iron, or a combination of contaminating trace elements, in perfusate B. To confirm that these perfusates did differ significantly in the concentration of these trace elements, in particular with regard to iron, the superoxide anion activity in both solutions was measured and was found to be significantly higher in perfusate B. The addition of superoxide dismutase to both solutions inhibited superoxide anion activity by more than 80%.

Calcium antagonists in sodium lactobionate sucrose solution for rat liver preservation.

The effects of the calcium antagonists, chlorpromazine (CPZ), nisoldipine (NIS), trifluoperazine (TFP), and nicardipine (NIC) were compared in rat livers following either 20- or 30-hr ice storage in sodium lactobionate sucrose solution (SLS). Survivals beyond 7 days after orthotopic liver transplantation following 20-hr cold storage were 1/14 in the University of Wisconsin solution, 4/14 in SLS, 4/8 in UW+CPZ, 7/8 in SLS+CPZ. Survivals beyond 7 days after OLT following 30-hr cold storage were 3/8 in SLS+CPZ, 3/8 in SLS+NIS, 2/8 in SLS+TFP, 0/8 in SLS+NIC, and 0/8 in SLS alone. Survival rates were significantly (P less than 0.05) better in both SLS+CPZ and SLS+NIS than in UW and SLS alone. The effluent lactate dehydrogenase (LDH) levels and pH changes were measured at the time of OLT. After 20 hr, LDH levels were 525 +/- 78 IU/L (mean +/- SEM) in UW, 492 +/- 44 in SLS, 322 +/- 35 in UW+CPZ, and 290 +/- 39 in SLS+CPZ. After 30 hr, LDH values were 416 +/- 40 in SLS+CPZ, 450 +/- 25 in SLS+NIS, 448 +/- 21 in SLS+TFP, 573 +/- 18 in SLS+NIC, and 614 +/- 68 in SLS. The LDH levels for SLS+CPZ and SLS+NIS were significantly lower than those of SLS and UW (P less than 0.01). The pH changes in the effluent were significantly less in both the CPZ and NIS groups (P less than 0.01). This study demonstrated improved liver preservation by the use of a simplified colloid-free lactobionate solution containing sodium as the principal cation. The addition of CPZ or NIS to the solution demonstrated the same potency for significant improvement in efficacy of this solution, while NIC was ineffective.

Optimal cardioplegia and 24-hour heart storage with simplified UW solution containing polyethylene glycol.

Recent work has shown that UW may be better than standard cardioplegic solutions for short-term heart preservation. In this study we have used a rabbit heart model to evaluate a simplified UW solution in which penicillin, dexamethasone, insulin, allopurinol, and adenosine were omitted and 5% polyethylene glycol (PEG20M) was substituted for hydroxyethyl starch. The test systems consisted of 4-hr cardioplegic storage at 15 degrees C with repeated flushing every 30 min for 2 hr and 24-hr hypoxic low-flow microperfusion (3 ml/g/24 hr) at 0 degrees C. Control groups were arrested with a 15-25 ml flush in iced saline and immediately tested. Cardiac output (CO)* after preservation was measured in a working heart model using an acellular perfusate at 37 degrees C at an aortic pressure of 100 cm H2O. The CO (ml/g heart wt/min) were as follows--Controls: St. Thomas II 20.5 +/- 8.3 (5), UW 34.7 +/- 11.7 (16), PEG20M 41.8 +/- 4.4 (14); 4-hr cardioplegia: St. Thomas II 17.4 +/- 0.9 (4), Bretschneider HTK 14.9 +/- 7.0 (4), UW 25.2 +/- 11.5 (9), PEG20M 41.1 +/- 7.8 (8); 24-hr microperfusion: UW 25.4 +/- 11.1 (18), PEG20M 37.1 +/- 8.2 (18). Following cardioplegic or microperfusion preservation, PEG20M hearts functioned at control levels (P greater than 0.05) and were significantly superior to all other solutions, with approximately double the CO (P less than 0.05, all other groups). We conclude that for heart preservation, 5 components can be eliminated from UW and substitution of PEG20M for HES appears to have improved its performance.

Twenty-four-hour preservation of the pig heart by a portable hypothermic perfusion system.

A portable hypothermic perfusion system for storage of hearts has been developed. The system uses the airlift pump principle, whereby the flow of gas maintains circulation of the perfusate through the heart; no other energy source is required. Performance on ex vivo functional testing of 10 pig hearts stored for 20 to 24 hr using this system (group 3) was compared with that of freshly excised hearts (group 1) and hearts stored simply in the perfusate under hypothermic conditions, but not perfused (group 2). Group 2 hearts performed less well on functional testing than those of groups 1 and 3 which showed little statistical difference, suggesting good preservation by hypothermic perfusion. This has been confirmed by orthotopic transplantation of similarly preserved baboon hearts with survival until rejection at a mean of 27 days. The importance of the various constituents of the perfusate and the significance of weight gain during the storage and reperfusion periods are discussed.

Modification of rejection by polyethylene glycol in small bowel transplantation.

The use of polyethylene glycol (PEG) in preservation solutions has been associated with a decreased incidence of rejection in clinical and experimental organ transplantation. In this study, we examined the effect of PEG with different molecular configurations on rejection of small bowel allografts in the rat. Male ACI and LEW rats were used as donors and recipients, respectively. Orthotopic small bowel transplantation was performed using the following preservation solutions: lactated Ringer's solution (n = 7), University of Wisconsin solution (n = 7), University of Wisconsin solution without hydroxyethyl starch (sUW; n = 7), sUW with PEG20M (n = 9), sUW with PEG8000 (n = 6), and sUW with PEG20L (n = 7). No immunosuppression was given. In orthotopic small bowel transplantation, only groups with a high molecular weight PEG, PEG20M and PEG20L, demonstrated longer survival (P < 0.01 and P < 0.001, respectively) and delayed onset of unkempt appearance (P < 0.05 and P < 0.001, respectively). In heterotopic small bowel transplantation, sUW was compared with sUW with PEG20L. Rejection occurred later and its progression was slower in the sUW with PEG20L than in the sUW alone. Our observations suggest that the onset and progression of rejection after small bowel transplantation were influenced by the molecular weight and configuration of the PEG molecule. The mechanism is unclear, but high molecular weight PEG appears to reduce or change the immunogenicity of the small bowel allograft.

Significance of terminal rinse for rat liver preservation.

A terminal rinse (TR) is standard practice in liver preservation with University of Wisconsin solution (UW) to avoid a potassium load. The fact that sodium lactobionate sucrose solution (SLS) is an effective organ preservation solution with a low potassium provided an opportunity to evaluate rat liver preservation without the TR step. Its importance was investigated in 122 rat liver preservation experiments. In study 1, UW and a hydroxyethyl starch-free, modified UW (UWm) were used for 20-hr liver preservation followed by either no TR or Ringer's lactate TR. The 1-week survival was: UW-TR, 2/14; UW-no TR, 1/6; UWm-TR, 0/6; UWm-no TR, 5/5 (P < 0.01). In study 2, livers were stored for 30 hr in SLS, UW, UWm, and UWm + chlorpromazine 5 mg/L, all without a TR. Nine of 11 rats survived 7 days after SLS, but there were no survivors in the other groups (P < 0.05). Study 3 compared no TR with TR with SLS, Ringer's lactate (RL), or a modified Carolina rinse (CRm) after 30-hr SLS preservation. Survival, serum aspartate aminotransferase and alanine aminotransferase, and histology were assessed. One-week survival of 9/11 rats in no TR was significantly better than in the other groups (3/14 in TR-SLS, 0/8 in TR-RL, and 0/14 in TR-CRm, P < 0.01). The values of aspartate aminotransferase (mean +/- SE) 3 hr after transplantation were 1862 +/- 439 U/L, 3334 +/- 817 U/L, 6591 +/- 1944 U/L, and 7028 +/- 1704 U/L, respectively, in no TR, TR-SLS, TR-RL, and TR-CRm. There were significant differences both in aspartate aminotransferase and alanine aminotransferase between no-TR and each of TR-RL and TR-CRm (P < 0.05). Liver specimens from rats killed 3 hr after OLT showed only mild injury in the no TR group and severe injury in the remaining groups. We conclude that a terminal rinse is harmful in rat liver preservation.

The effects of brain death and 24 hours' storage by hypothermic perfusion on donor heart function in the pig.

The effects on the myocardium of the agonal period and subsequent management have been studied in the pig. Acute ischemia of the brain led to major temporary hemodynamic changes. Brain death, with or without hemodynamic support of the circulation, led to a significant reduction in subsequent myocardial function, associated with some depletion of the myocardial high-energy phosphate and glycogen reserves, although the rate of this depletion was reduced by anaerobic glycolysis. Although 24 hours' storage by continuous hypothermic perfusion of hearts taken from control animals led to only a minimal reduction in myocardial function, storage increased the reduction in function associated with brain death when intravenous fluid and dobutamine support had been given to maintain the brain dead pig in a normotensive state. Storage, however, reduced the anaerobic metabolism seen in hearts functioning in hypotensive brain dead pigs and led to replenishment of the glycogen stores.

Twenty-four-hour ice storage of rabbit heart.

Although cardioplegia is limited to 4 hours of ice storage, University of Wisconsin solution has successfully extended this period to approximately 12 hours. In this study we have substituted polyethylene glycol for hydroxyethyl starch in a simplified University of Wisconsin solution (Cardiosol). Rabbit hearts were ice stored for 24 hours at 0 degrees C in either University of Wisconsin solution or Cardiosol (containing either 5% or 10% polyethylene glycol). Fresh control hearts were tested immediately after cardiectomy. Function was evaluated in an in vitro working heart model for 1 hour with aortic afterload at 100 cm H2O. Total cardiac output or the proportion of hearts reaching 100 cm H2O were compared. Hearts stored in University of Wisconsin solution for 24 hours functioned at 6% of control levels at 15 minutes of observation. None reached 100 cm H2O or deteriorated further with time (p < 0.05). By contrast, hearts stored in 5% Cardiosol showed progressive recovery during the 1-hour observation. Of the 13 hearts, 11 reached 100 cm H2O with a mean cardiac output of 51% of the control value. Increasing the concentration of polyethylene glycol to 10% improved cardiac output at all observation times, reaching 80% of control heart performance at 1 hour (control > 10% > 5% > University of Wisconsin solution [p < 0.05]). We concluded that 10% polyethylene glycol significantly improved 24-hour ice storage and, hence, viability to a functional level that matched our previously reported microperfusion results.

Cardiotonic effects of reduced sulfhydryl amines after preservation of rabbit hearts.

This investigation was designed to examine the role of glutathione and other reduced sulfhydryl amines during reperfusion of the ischemic rabbit heart. To identify the biochemical features of reduced sulfhydryl amines contributing toward improved myocardial function, we investigated several chemical agents sharing a common property with glutathione (L-leucine, L-glycine, ascorbate, oxidized glutathione, L-cysteine). After a period of 24-hour hypothermic storage of the rabbit heart in a modified University of Wisconsin solution containing polyethylene glycol, the hearts were functionally evaluated on a Langendorff working heart model. The agents were then injected as a bolus (60 mumol/L) during reperfusion, and coronary flow and aortic output were measured. Control hearts were untreated. Reduced sulfhydryl amines (glutathione, L-cysteine) significantly improved coronary flow (p < 0.005) and cardiac output (p < 0.005). Ascorbate, L-leucine, L-glycine, and oxidized glutathione all failed to influence cardiac function.

Successful 20-hour rat liver preservation with chlorpromazine in sodium lactobionate sucrose solution.

We investigated the effect of the addition of chlorpromazine to a new, simplified organ preservation solution, sodium lactobionate sucrose (SLS), for 20-hour hypothermic rat liver preservation. Survival beyond 7 days after orthotopic transplantation of the stored liver was eight of eight rats in control groups (immediate transplantation, less than 1-hour preservation), one of 14 rats with the University of Wisconsin (UW) solution, four of 14 rats with SLS, seven of eight rats with SLS + chlorpromazine, 1 mg/L, and seven of eight rats with SLS + chlorpromazine, 10 mg/L. The differences is survival between UW and SLS and between SLS and SLS + chlorpromazine were significant (p less than 0.05). Lactic dehydrogenase levels in the effluent after reflushing through the portal vein at the time of transplantation were 145 +/- 20 IU/L (mean +/- SEM) in the controls, 525 +/- 78 IU/L in UW, 492 +/- 44 IU/L in SLS, 290 +/- 39 IU/L in SLS + chlorpromazine, 1 mg/L, 290 +/- 11 IU/L in SLS + chlorpromazine, 10 mg/L. The values for the SLS + chlorpromazine were significantly lower than for SLS and UW (p less than 0.05). The pH of the effluent was 7.10 +/- 0.10 in controls, 6.42 +/- 0.12 in UW, 6.64 +/- 0.18 in SLS, and 7.07 +/- 0.02 in SLS + chlorpromazine, 1 mg/L and 10 mg/L. The pH drop was significantly greater in the groups without chlorpromazine (p less than 0.01). This study shows that superior rat liver preservation was achieved with a simplified lactobionate solution containing sodium as the principal cation, sucrose in place of raffinose, and omitting the colloid and several of the other UW components. The addition of low concentrations of chlorpromazine further enhanced the effectiveness of this solution, without the need for donor pretreatment.

New organ preservation solutions.

In 1969 we described a method for kidney preservation that used a brief flush with a new "intracellular" solution followed by ice storage. This paper stimulated research into optimizing solution composition culminating in the UW solution which is now the accepted standard. Further developments in the design of solutions for hypothermic organ preservation have proceeded along several paths, including: (1) modification and simplification of UW solution, (2) investigation of organ specific requirements, (3) addition of pharmacologic agents particularly calcium antagonists to flush solutions, (4) the concept of "microperfusion" for control of acidosis, (5) the use of solutions containing polyethylene glycol, and (6) the use of a terminal rinse solution. Broadly speaking, the results of these studies have shown that it is possible to improve upon the UW solution by simplification, eliminating several of the components, and that sodium variants, and pharmacological additives, such as chlorpromazine, may yield better results in experimental and clinical trials. It has also been found that there are special requirements for individual organs, rendering the concept of a universal solution unlikely. Of the promising new ideas, microperfusion and polyethylene glycol have been found to be very effective for heart preservation yielding for the first time virtually perfect 24-hour preservation. The concept of a terminal rinse to diminish reperfusion injury has strong experimental support and awaits clinical evaluation.

Initial experimental experience with a "replaceable" cardiac valve prosthesis.

An easily "replaceable" cardiac valve prosthesis has been designed. It consists of two parts: (1) a sewing ring incorporating a circlip and (2) a functioning valve (either mechanical or tissue). The circlip is encased in a sewing ring, which is sutured into the natural valve annulus, and grips the functional part of the prosthesis, thereby preventing dislodgment. A simple instrument has been designed to open the circlip a few millimeters to allow easy removal or insertion of the functional element. This sewing ring/circlip with the functional element of a Björk-Shiley prosthesis was used in 10 baboons undergoing mitral valve replacement. Removal and replacement of the functional element was carried out at a second operation between 1 and 12 weeks later. There were no operative deaths. Baboons were electively killed one day to twelve months after the second operation. There were no complications related to the prosthesis; cardiac catheterization showed normal hemodynamics before and after the second operative procedure.

Cardiac transplantation following storage of the donor heart by a portable hypothermic perfusion system.

Four patients have undergone heterotopic heart transplantation with donor hearts stored by a portable hypothermic perfusion system. Total ischemic periods ranged from 6 hours 55 minutes to 16 hours 50 minutes. One heart, transplanted into a patient who had acutely rejected a previous graft, suffered accelerated, irreversible, acute rejection within five days, associated with strong antibody formation; donor heart function was never good. In the 3 remaining patients, donor heart function was good after initially being poor for a few hours in 2 patients. One patient died of long-term rejection after 6 months and 1 of tuberculous meningitis after 10 months; 1 remains alive at 15 months. Neither preservation of the donor heart for periods in excess of approximately 4 hours nor the use of continuous hypothermic perfusion as a method of preservation appears to have been reported previously in the context of the clinical situation.

Pathophysiology of pulmonary edema following experimental brain death in the chacma baboon.

Systemic and pulmonary hemodynamics have been studied during the induction of brain death in the chacma baboon. In 11 animals brain death was induced by acute intracranial hypertension. Continuous recording of blood flow through both the pulmonary artery and the aorta was obtained by electromagnetic flow meters placed around these vessels. Mean arterial, central venous, pulmonary arterial, and left atrial pressures were recorded continuously. Systemic and pulmonary vascular resistances were calculated. During the agonal period marked sympathetic activity occurred, with significant increases in circulating catecholamines and systemic vascular resistance. The great increase in systemic resistance resulted in acute left ventricular failure. Mean left atrial or pulmonary capillary wedge pressure rose above the mean pulmonary arterial pressure in 9 animals. As the systemic vascular resistance rose, a significant difference between pulmonary artery and aortic blood flows occurred, leading to blood pooling within the lungs. A mean of 72% of the total blood volume of the animal accumulated within these organs. The increase of left atrial pressure to levels higher than pulmonary artery pressure indicated a state of pulmonary capillary blood flow arrest. This, associated with the blood pooling within the lungs, almost certainly resulted in disruption of the anatomic integrity of the pulmonary capillaries (blast injury); 4 animals developed pulmonary edema, with alveolar septal interstitial hemorrhage.

Prevention of myocardial injury during brain death by total cardiac sympathectomy in the Chacma baboon.

In a previous study, structural myocardial damage was found to occur in 60% of baboons after brain death had been induced by a rapid increase in intracranial pressure. In the present study, we attempt to clarify the causative mechanisms involved in the development of such injury. Three groups of baboons were subjected to brain death: group A, the control; group B, those with previous surgical or pharmacological cardiac sympathectomy or cardiac denervation; and group C, those with bilateral vagotomy, incomplete sympathectomy, or bilateral adrenalectomy. Electrocardiographic and hemodynamic responses to brain death were greatly modified in group B baboons compared with responses in groups A and C. Groups A and C showed a high incidence of myocardial necrosis, whereas no myocyte damage was seen in the hearts of group B baboons. The histological appearance of innervated hearts following brain death (groups A and C) may closely resemble that seen during an acute rejection episode following cardiac transplantation. We suggest that myocardial damage occurring during the process of dying may be related to endogenous catecholamine release (possibly resulting in increased calcium uptake by the myocardial cells), inducing various forms of myocyte necrosis. This may result in early failure in a donor heart following cardiac transplantation.

The pathophysiological effects of brain death on potential donor organs, with particular reference to the heart.

Major electrocardiographic, haemodynamic, and histopathological changes take place during the development of brain death; myocardial and pulmonary injury may result. Significant depletion of certain circulating hormones occurs, resulting in an inhibition of mitochondrial function, leading to reduced aerobic metabolic oxidative processes, affecting the body as a whole. Major organ energy stores are therefore diminished, leading to deterioration of function. Replacement of the depleted hormones, in particular triiodothyronine (T3), cortisol, and insulin, leads to rapid replacement of organ energy stores, associated with a return to normal function. T3 alone leads to reactivation of the mitochondria, stimulating aerobic metabolism. Hormonal therapy to brain-dead potential organ donors has been shown to lead to metabolic and haemodynamic stability, resulting in no wastage of organs, and in improved function after transplantation.