Renal preservation by hypothermic perfusion. I. The importance of pressure-control

Rabbit kidneys were preserved by hypothermic perfusion at 5 °C using a perfusate containing an extracellular balance of ions, dextran and bovine serum albumin. Two groups were studied: in one, the pressure was kept constant at 40 mm Hg, while in the other the flow was maintained at 13 ml/min. The mean flows in the two groups were similar but the resistance of the kidneys perfused under constant-flow conditions was lower and more stable: the vascular resistance in the constant-pressure group showed considerable fluctuations throughout the 24 hr perfusion period. The function of the kidneys was assessed by autotransplantation with immediate contralateral nephrectomy. The constant-pressure group functioned better in all respects: the proportion of animals surviving was higher, the postoperative blood urea and creatinine levels were lower, and histological examination of the kidneys revealed less damage. It is concluded that constantpressure perfusion should be preferred to constant-flow perfusion. These experiments confirmed that there is a correlation between potassium release into the perfusate and subsequent function, and an unexpected inverse correlation was observed between the perfusate glucose level and subsequent function. Possible reasons for this are discussed.     

Renal preservation by hypothermic perfusion: 2. The influence of oxygenator design and oxygen tension

Rabbit kidneys have been perfused for 24 hr at 5 °C and tested by autotransplantation with immediate contralateral nephrectomy. The perfusate contained an extracellular balance of ions with dextran and bovine serum albumin. The circuit included a nonpulsatile pump, a 0.22-μm membrane filter, and an oxygenator.The efficiency of membrane, film, and bubble oxygenators was compared. Flat membrane oxygenators were found to be more efficient than tubing oxygenators, and although the bubble oxygenator was the most efficient it created problems with foaming. A simple film oxygenator provided the best compromise.Ten kidneys were perfused and transplanted in each of three experimental groups; a film oxygenator was used to provide partial pressures of oxygen of 676 mmHg, 137 mmHg, and 9 mmHg, respectively.There were no significant differences in behavior during perfusion, function after transplantation, or histology of transplanted kidneys in the three groups.The similarity of the results using a film oxygenator with those previously obtained with a membrane oxygenator indicates that complex membrane oxygenators are not necessary for preservation in our system. Moreover, the similarity of the results with oxygen tensions from 9 mmHg to 676 mmHg shows that deliberate oxygenation is also unnecessary.     

Renal preservation by hypothermic perfusion. IV. The use of gelatin polypeptides as the sole colloid

Rabbit kidneys were perfused with a solution of extracellular electrolyte composition, rendered hypertonic with glucose and containing 1.75% Haemaccel (Hoechst) as the sole colloid. Perfusions were carried out at 10 °C for 24 and 48 hr using perfusion pressures of 20 or 40 mm Hg. Function, was tested by autografting with immediate contralateral nephrectomy. All the kidneys perfused at 20 mm Hg for 24 hr showed excellent life-sustaining function and 7 of 10 survived in the 40 mm Hg group; the difference was not statistically significant. However, all the kidneys perfused for 48 hr failed to sustain life, and histological examination revealed extensive breakdown of the microcirculation. The 24 hr results were similar to those previously obtained with an albumin-based perfusate, but the 48-hr results were inferior: However, the obvious advantages of a well-standardised, cheap, and easily stored perfusate are such as to justify further study of gelatin-derived colloids for organ preservation.     

Forty-eight hours hypothermic pulsatile perfusion of canine hearts before transplantation.

Canine hearts preserved for 24 hr under hypothermic pulsatile perfusion had a good function after transplantation. The perfusate consisted of cryoprecipitated plasma that was modified by the addition of salt poor albumin, potassium chloride and glucose; the final osmolarity was 340 mOsm/L. Fresh allografts without perfusion survived for an average of two weeks after transplantation, and the 24 hr perfused hearts survived for more than 19 days after transplantation. When the perfusion was extended to 48 hr, the survival was decreased to 11 days. These data indicate that hypothermic pulsatile perfusion is completely safe and feasible for 24 hr without significant functional or histological impairment. The survival response of the hearts perfused for 48 hr was significantly decreased when compared to the hearts perfused for 24 hr.     

Ex-vivo study of flow dynamics and endothelial cell structure during extended hypothermic machine perfusion preservation of livers

Liver transplantation is often the only effective treatment for end stage liver diseases resulting from cirrhosis, hepatitis, progressive jaundice, and biliary atresia. Hypothermic machine perfusion (HMP) preservation may enhance donor pool by extending preservation time and reclaiming marginal donor livers including those from non-heart beating donors (NHBD), as demonstrated in the kidney. However, current HMP protocols have not been successful in improving extended preservation of livers and the major cause of preservation injury remains unknown. An intravital microscopy study was conducted to understand the flow dynamics of sinusoidal perfusion during 24h HMP with cold modified University of Wisconsin (UW) solution. Fluorescein isothiocynate (FITC) labeled albumin was utilized to visualize microvascular space and FITC labeled red blood cells (RBCs) were used to visualize flow dynamics during HMP. A heterogeneous flow pattern with regions of red cell stasis was observed after 24-h HMP. To examine the cause of red cell stasis, intravital and confocal microscopy studies of endothelial cells (ECs) structure labeled with DiI acetylated low-density lipoprotein (DiI acLDL) were conducted. These studies suggest that morphological changes in EC structures occurred during 24h HMP, which may cause obstruction to the sinusoidal flow. Histological findings confirm these results. As a result, heterogeneous flow pattern, red cell stasis, and edema occur, which may lead to the failure of these tissues following extended HMP.     

The assessment of renal preservation by normothermic bloodless perfusion.

A method of estimating renal function by normothermic perfusion in vitro has been developed. In this paper, its application to the study of different methods of hypothermic renal preservation in the rabbit is described. Groups of kidneys were stored at 4 °C for 24 hr by surface cooling alone, by initial perfusion followed by storage (washout perfusion), and by continuous perfusion. Renal function was found to be severely compromised after surface cooling alone or after washout perfusion with an isotonic solution resembling extracellular fluid. Washout with a solution containing sufficient additional glucose to raise the osmolality to 400 mosm/kg gave greatly improved function, but increasing the concentration of magnesium from 2 to 72 mequiv/litre failed to confer any additional benefit, and increasing the concentration of potassium from 4 to 74 mequiv/ litre depressed function. Continuous perfusion with a solution containing albumin and dextran gave results that were inferior to the best washout method, but increasing the osmolality of the perfusate with glucose again resulted in a very significant improvement in function, which however was still inferior to the best washout method of storage. The further use of this test system to study methods of renal preservation is advocated.     

Continuous hypothermic perfusion of rabbit kidneys.

Addition of cryoprotective agents to whole organs is possible only by vascular perfusion with the cryoprotectant dissolved in a suitable perfusion fluid.Vascular resistance, organ weight gain, release of lactate dehydrogenase (LDH), and post-transplant function was studied during and after hypothermic perfusion at +6 °C of rabbit kidneys with six different perfusion fluids. A mixture of dextran and bovine serum albumin (BSA), BSA alone in various concentrations, and human serum albumin were tested as colloids, and the effect of perfusate osmolality was investigated.The dextran-BSA mixture was found to be superior to 4.5 and 6.0% BSA alone in terms of better perfusion characteristics, better post-transplant function, and lower LDH release. Perfusion characteristics during perfusion with human serum albumin and subsequent graft function were not different from those observed in experiments with dextran-BSA, but the LDH release was lower.Perfusate osmolality was increased by the addition of glucose or mannitol. Perfusion characteristics during perfusion with the hypertonic perfusates were not different from those observed during isotonic perfusion, but post-transplant function seemed to be better after perfusion with the fluid made hypertonic with glucose, whereas addition of mannitol seemed to be deleterious.Thus a perfusion fluid of extracellular electrolyte composition, containing human serum albumin as a colloid and made hypertonic with glucose, can be used as a vehicle for cryoprotectants during their addition to rabbit kidneys.     

Myocardial protection by propolis during prolonged hypothermic preservation

Oxidative stress is involved in the pathogenesis of ischemia-reperfusion during myocardial transplantation. Therefore, graft preservation solutions may be improved by supplementation with antioxidants to minimize graft dysfunction caused by cold ischemic injury. Propolis is a polyphenol-rich substance which has an important antioxidant activity. The protective effect of propolis against oxidative stress induced by prolonged cold preservation of heart was investigated. Mice were subjected to a hypothermic model of ischemia in which hearts were preserved for 24 h at 4 °C in Krebs-Hensleit (KH) solution in the absence or presence of propolis concentrations (50, 150 and 250 μg/ml). Levels of released Lactate dehydrogenase (LDH), Creatine phosphokinase (CPK) and Troponine-I (Trop I) were assessed in the preservation solution and histological assessement of heart ischemia injuries was performed. Oxidative stress biomarkers malondialdehyde (MDA) and advanced oxidation protein products (AOPP) and antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) were assessed in cardiac tissue. Mitochondria were isolated from stored hearts and production of reactive oxygen species (ROS) was tested. Propolis supplementation protected efficiently hearts during preservation by reducing significantly levels of lipids and proteins oxidation and restoring activities of antioxidant enzymes. Also, propolis preserved tissue integrity altered by hypothermic ischemia in a concentration-dependent manner. Propolis reduced significantly the rate of H₂O₂ produced by mitochondrial respiration, the best antioxidant effect being obtained at the highest propolis concentration (250 μg/ml). Algerian propolis is a non-temperature sensitive scavenger that protects heart from oxidative damage induced by prolonged hypothermic ischemia.     

The influence of out-of-plane geometry on pulsatile flow within a distal end-to-side anastomosis

We present an experimental and computational investigation of time-varying flow in an idealized fully occluded 45 degrees distal end-to-side anastomosis. Two geometric configurations are assessed, one where the centerlines of host and bypass vessels lie within a plane, and one where the bypass vessel is deformed out of the plane of symmetry, respectively, termed planar and non-planar. Flow experiments were conducted by magnetic resonance imaging in rigid wall models and computations were performed using a high order spectral/hp algorithm. Results indicate a significant change in the spatial distribution of wall shear stress and a reduction of the time-averaged peak wall shear stress magnitude by 10% in the non-planar model as compared to the planar configuration. In the planar geometry the stagnation point follows a straight-line path along the host artery bed with a path length of 0.8 diameters. By contrast in the non-planar case the stagnation point oscillates about a center that is located off the symmetry plane intersection with the host artery bed wall, and follows a parabolic path with a 0.7 diameter longitudinal and 0.5 diameter transverse excursion. A definition of the oscillatory shear index (OSI) is introduced that varies between 0 and 0.5 and that accounts for a continuous range of wall shear stress vector angles. In both models, regions of elevated oscillatory shear were spatially associated with regions of separated or oscillating stagnation point flow. The mean oscillatory shear magnitude (considering sites where OSI>0.1) in the non-planar geometry was reduced by 22% as compared to the planar configuration. These changes in the dynamic behavior of the stagnation point and the oscillatory shear distribution introduced by out-of-plane graft curvature may influence the localization of vessel wall sites exposed to physiologically unfavorable flow conditions.     

Preservation of guinea pig hearts by hypothermic gas perfusion

The lack of a satisfactory method for long-term preservation of hearts during transport limits the source of human hearts for transplant to the geographic vicinity of the transplant center. Experimentally, reduction of myocardial oxygen requirements with hypothermia and cardioplegia prolong storage time to 48 h, but always with some evidence of myocardial damage. In this study, the combination of hypothermia with a procedure known to increase oxygen tension in cardiac muscle, gas perfusion, preserved contractile activity in guinea pig hearts for 24 h and did not cause edema. Cardioplegia or gas perfusion at temperatures below 10 degrees C or above 20 degrees C resulted in failure of hearts to contract upon rewarming. Contracture, dehydration, elevation of tissue calcium, reduced perfusate flow, and elevated creatine kinase levels occurred if liquid reperfusion was begun at 15 degrees C but not 25 degrees C. The results suggest that under the appropriate conditions, hypothermic gas perfusion is a potentially useful means of extending storage time of hearts for transplant.     

Anesthetic and supportive management during experimental pulsatile flow perfusion studies in calves

The purpose of this study was to determine the factors influencing successful experimental cardiopulmonary bypass studies using pulsatile flow perfusion and the medications and methodology necessary to produce successful bypass in calves. In six calves showing no cardiopulmonary pathology prior to bypass procedures, successful anesthesia and surgical intervention was accomplished. Animals were maintained on 5 hours of pulsatile flow bypass perfusion. Successful recovery from the procedures was accomplished. In two calves with pre-existing pulmonary pathology, anesthetic and surgical intervention was accomplished with the utilization of extensive anesthetic management and cardiac supportive medications until the animals could be initiated into 5 hours of pulsatile flow bypass perfusion, in spite of major pulmonary dysfunction. In these two animals, attempts to resuscitate upon termination of pulsatile flow perfusion were unsuccessful due to pre-existing excessive lesions in the lungs. This study shows a contrast between complete success of a pulsatile flow system in normal subjects versus the ultimate failure in experimental animals with pre-existing pulmonary pathology. The inability of experimental calves with a diseased lung to resume spontaneous cardiopulmonary function after the challenges of thoracic intervention indicates the unsuitability of animals with marked pre-existing pulmonary disease status for use in cardiopulmonary bypass studies.     

Organ vessel functions by pulsatile flow

It was found that changes of resistance, capacity and exchange vessel functions of the skeletal muscle depended on values of amplitude and frequency of perfusion blood flow in acute experiments on cats. Amplitude-frequency characteristics of hemodynamic parameters of these functions were nonlinear. Ranges of amplitude and frequency variation with significant shifts of these parameters were determined. Conditions of perfusion (constant flow or constant pressure) changed volume and direction of haemodynamic parameter shifts under the amplitude-frequency modulation of the pulsatile blood flow. The pulsatile blood flow oscillations exerted maximum influence on hemodynamic parameters of exchange vessel function. The author has proposed the wave regulation conception of organ vessel functions.     

Prediction of viability of rabbit kidneys preserved by hypothermic perfusion

The results of a range of tests carried out during renal preservation by hypothermic perfusion were examined to see whether they could be used to predict the viability of rabbit kidneys. Kidneys were perfused at +5 °C for 24 hr and measurements were made of weight gain and change in vascular resistance as well as changes in the composition of the perfusate. Renal function was assessed by autografting and postoperatively measurements of blood urea and serum creatinine were made; an index of renal function was obtained by integrating the blood urea concentration with respect to time during the first 14 postoperative days. Two sets of statistical correlations were made, the first was between the tests and survival or nonsurvival, and the second was between the test results and the excellence of function in the surviving group. The traditional indices of weight gain and resistance change during perfusions were not found to have any predictive value. Of the biochemical parameters measured only the GOT activity of the perfusate proved to be of significance both in terms of prediction of viability and of subsequent renal function. The results obtained, however, were not decisive enough to support a firm recommendation that GOT measurements should be used in clinical practice to decide whether or not to transplant a particular kidney. Nevertheless, a high GOT concentration is a feature of damaged kidneys and it may be that with longer perfusion periods and longer warm ischaemia times, the test may have greater discriminatory value.     

The influence of storage temperature during machine perfusion on preservation quality of marginal donor livers

Background

Although non-heart-beating donors have the potential to increase the number of available organs, the livers are used very seldom because of the risk of primary non-function. There is evidence that machine perfusion is able to improve the preservation of marginal organs, and therefore we evaluated in our study the influence of the perfusate temperature during oxygenated machine perfusion on the graft quality.

Methods

Livers from male Wistar rats were harvested after 60-min warm ischemia induced by cardiac arrest. The portal vein was cannulated and the liver flushed with Lifor® (Lifeblood Medical, Inc.) organ preservation solution for oxygenated machine perfusion (MP) at 4, 12 or 21 °C. Other livers were flushed with HTK and stored at 4 °C by conventional cold storage (4 °C-CS). Furthermore two groups with either warm ischemic damage only or without any ischemic damage serve as control groups. After 6 h of either machine perfusion or cold storage all livers were normothermic reperfused with Krebs–Henseleit buffer, and functional as well as structural data were analyzed.

Results

Contrary to livers stored by static cold storage, machine perfused livers showed independently of the perfusate temperature a significantly decreased enzyme release of hepatic transaminases (ALT) during isolated reperfusion. Increasing the machine perfusion temperature to 21 °C resulted in a marked reduction of portal venous resistance and an increased bile production.

Conclusions

Oxygenated machine perfusion improves viability of livers after prolonged warm ischemic damage. Elevated perfusion temperature of 21 °C reconstitutes the hepatic functional capacity better than perfusion at 4 or 12 °C.