Lauren Brasile

Hypothermia – a Limiting Factor in Using Warm Ischemically Damaged Kidneys

A study was performed to determine the limiting factors to expanding the donor pool with warm ischemically (WI) damaged kidneys. Canine kidneys were damaged by 30 min of WI, and then either cold stored (CS) in ViaSpan (4 °C) for 18 h, or warm perfused with exsanguineous metabolic support (EMS) technology (32 °C) for 18 h, or subjected to combinations of both techniques. The kidneys were autotransplanted with contralateral nephrectomy. In kidneys with WI and CS alone, the mean peak serum creatinine value was 6.3 mg/dL and took 14 days to normalize. In contrast, kidneys where renal metabolism was resuscitated ex vivo during 18 h of warm perfusion demonstrated mild elevations in the serum chemistries (2.6 mg/dL). The damage in kidneys CS for 18 h was ameliorated with 3 h of subsequent warm perfusion and eliminated by 18 h of warm perfusion. In contrast, reversing the order with CS following WI and 18 h of warm perfusion resulted in a time‐dependent increase in damage. These results identify hypothermia as a major limiting factor to expanding indications for kidney donation. While hypothermia represents the foundation of preservation in the heart‐beating donor, its use in WI damaged organs appears to represent a limiting factor.

NOS: The Underlying Mechanism Preserving Vascular Integrity and During Ex Vivo Warm Kidney Perfusion

Research involving metabolically active and functioning organs, maintained ex vivo in culture‐like conditions, could provide numerous opportunities for medical innovations and research. We report successful perfusion of isolated canine and human kidneys ex vivo at near physiologic temperature for 48 h. During the perfusions parameters of metabolism and function remained stable. Nitric oxide synthase (NOS) was identified as the underlying mechanism preserving vascular integrity. Most importantly, when the canine kidneys were reimplanted there was immediate normal renal function. This report highlights the potential significance of whole organ culture using a warm temperature ex vivo perfusion and discusses medical applications that could be developed.

ORGAN PRESERVATION WITHOUT EXTREME HYPOTHERMIA USING AN OXYGENT~~ SUPPLEMENTED PERFUSATE

All methods of organ preservation depend upon hypothermia to depress metabolism during storage. Yet, hypothermia may represent the rate-limiting factor in organ preservation. A new perfusate has been developed which supports organ preservation without extreme hypothermia. The perfusate consists of a complex fluid supplemented with an oxygen carrying perfluorocarbon emulsion, Oxygent (Alliance Pharmaceutical Corp,). The perfusate was used to preserve canine kidney autografts using pulsatile preservation at 32 degrees C and static storage at 25 degrees C. Upon autografting the dogs produced urine within minutes of reperfusion. These results indicate the new perfusate may have significant potential in organ preservation without extreme hypothermia.

II : Ex vivo viability testing of kidneys after postmortem warm ischemia

Future approaches to expand the organ donor pool with marginal and nonheartbeating donors, will be dependent upon prospective organ evaluation. Restoration of metabolism by preservation at warmer temperatures could potentially provide the window for such evaluation. Using a small bovine model, kidneys were subjected to either < 15, < 30 or < 60 minutes of warm ischemia (WI) followed by cold ischemia (CI) in ViaSpan. After WI and CI, kidneys were transitioned to a warm temperature perfusion (30 degrees C to 32 degrees C) using exsanguinous metabolic support (EMS) technology. Restored renal metabolism and function was assessed by oxygen consumption, glucose consumption, urine production, glomerular filtration rate, and hemodynamic characteristics. The results of this study suggest that it is feasible to distinguish viable from nonviable organs ex vivo by assessing renal metabolism and function during warm preservation using EMS technology.

Delayed skin allograft rejection following matrix membrane pretreatment

INTRODUCTION No solution has been offered to induce long-term skin allograft survival in burn patients. We investigated whether transplant acceptance could be improved by a nonsystemic pretreatment of the graft and recipient wound surfaces with a bioengineered interface consisting of an acellular matrix membrane. METHODS Group 1 (n=30): Crosstransplants of untreated skin grafts between BALB/c and C57BL/6 mice. Group 2 (n=30): Crosstransplants of matrix-treated skin grafts between BALB/c and C57BL/6 mice. Group 3 (n=30): Retransplantation of skin grafts from the original donor on to the sensitised recipients. Sensitisation was accomplished by prior transplantation of an untreated skin allograft from the same donor (Group 1 mice). Two skin grafts were transplanted: one treated and one untreated. RESULTS Rejection occurred in the untreated group after a mean of 6.8 days (+/-1.5 days). In contrast, treatment with the bioengineered matrix membrane was found to substantially prolong allograft survival with a mean of 28 days (+/-3.8 days). Graft survival between the two groups reached statistical significance (P<0.05). In the sensitised mice, the untreated skin regrafts were all rejected in an accelerated fashion with an onset of less than 4 days (mean+/-1 days). However, the matrix membrane-treated skin regrafts were maintained for a mean of 18 days (+/-3 days). CONCLUSION These results show that treatment with the bioengineered matrix membrane greatly delays the onset of acute allograft rejection. The described topical application to the wound surfaces of both the graft and the recipient may offer a new and readily available source of wound coverage in patients with extensive burns.

The feasibility of organ preservation at warmer temperatures.

Hypothermia has represented the foundation of organ preservation, since it was demonstrated that hypothermia reduces the metabolic needs of an organ. At approximately 4°C, the utilization of oxygen is only 5% of that at normothermia. The inability to supply oxygen lead to our current reliance on hypothermia. A new perfusate has been developed which can be used without traditional hypothermia. An increased PO2 is provided by a perfluorochemical emulsion (PFOB, Alliance Pharmaceutical Corp.). The available oxygen can be more effectively utilized because preservation at warmer temperatures (25°C & 32°C) means the cell membranes are in a more normal fluid state. We have demonstrated that kidneys can be preserved at warmer temperatures with stable flow dynamics, O2-consumption and diuresis for time periods of at least 18 hours. METHODS-Fifty canine and 40 bovine kidneys have been studied, at two temperatures (25°C & 32°C) for time periods ranging from 3 to 18 hours. The kidneys were pumped on a modified Mox-100 at mean pressures ranging from 60-90 mmHg, resulting in vascular flow rates of 80-150cc/min. The renal perfusions were conducted at PO2 of 170–185 Torr to deliver oxygen at 7.7m1/min. RESULTS-The kidneys pumped at 25°C and 32°C demonstrated stable flow dynamics and diuresis for time periods of at least 18 hours. The renal metabolism occurring at the two temperatures was monitored as described below.

Ex vivo evaluation of organ function after cold ischemia.

An ex vivo perfusion of kidneys was performed at 34 degrees C after cold ischemia of 24, 48, 72, and 96 hours to evaluate organ function prospectively. The prospective evaluation of organ function followed static hypothermic storage of the kidneys in a solution representative of clinical organ preservation. The warm perfusion was performed with an acellular solution that supports oxidative metabolism of sufficient magnitude to restore urine flow ex vivo. The parameters of organ function evaluated included oxygen consumption, vascular resistance, urine flow, and glomerular filtration rates, which were correlated with the histologic findings. The results of this study suggest that kidneys exposed to 24 and 48 hours of cold ischemia demonstrated oxygen consumption rates and vascular dynamics similar to control kidneys without exposure to cold ischemia, indicating cell viability. When the cold ischemic period was increased beyond 48 hours of preservation, substantially reduced rates of oxygen consumption and increased vascular resistances were observed, representing a loss of viability confirmed histologically. However, organ function was found to be impaired after exposure to cold ischemia at every time point. These results suggest that cold ischemic exposure had a negative impact on immediate renal function once oxidative metabolism was restored, which was exacerbated as the cold ischemic period was extended. Furthermore, these findings suggest that although the renal cells were viable after cold ischemic exposure, the viability status did not result in immediate function. Therefore, assessment of an organ based solely on cell viability may falsely indicate a functional organ. It will be necessary to identify parameters of organ function that can distinguish reversibility from non-reversibility of cellular impairment to distinguish permanent functional disturbances. The ability to predict organ function prospectively will be an important aspect of any effective future expansion of the organ donor pool.

Postmortem Organ Salvage Using an Oxygent™ Supplemented Perfusate

The world-wide shortage of organs for clinical transplantation is caused by the limited existing donor pool of heartbeating cadavers. Attempts to expand into the nonheartbeating cadaver population have been hindered by warm ischemic damage. We evaluated if a new Oxygent (Alliance Pharmaceutical Corp.) supplemented perfusate could be used to salvage canine kidneys postmortem. The kidneys preserved in the Oxygent perfusate could be maintained in situ for time points ranging from one-eight hours postmortem; enough time to declare death and obtain consent for organ donation. In contrast, the control kidneys yielded abnormal histologic findings and impaired flow dynamics. These results suggest that new perfusate may have significant potential to expand the existing organ donor pool.

In Situ Preservation Without Traditional Hypothermia

The shortage of organs for transplantation has lead to an expansion of the donor pool into the nonheartbeating cadaver (NHBCAD) population. The ability to in situ preserve organs from NHBCAD at warm temperatures could present the opportunity to perform in situ flushing without the current use of extreme hypothermia with solutions containing high K+ concentrations. We have developed a preservation solution which can be used to preserve kidneys without traditional hypothermia. The perfusate consists of a modified tissue culture medium and is supplemented with a perfluorochemical emulsion (PFOB, Alliance Pharmaceutical Corp.). We evaluated if the perfusate could be used to in situ preserve renal auto-and allografts at near physiologic temperatures. METHODS-Model 1: An in situ model was developed which provided for kidney preservation while allowing for animal survivaL The limitation to the model was that the aorta and vena cava below the renal vessels would be ischemic. Therefore, this in situ model could be used for only three hours of in situ preservation. Using an abdominal approach, two dogs were anesthetized and cannulas were placed in the aorta and vena cava. The vasculature above and below the kidneys was occluded. The vasculature was flushed with the new perfusate until the hematocrit was <1%. The perfusate was then recirculated during the period of in situ preservation at 37oC using a pulsatile pump. The cannulas were then removed and the blood vessels were repaired. Model 2: To extend the period of in situ preservation past three hours, an allotransplant model was used. The allotransplant model involved in situ preservation of the kidneys in two euthanized dogs. Following a period of six hours, one kidney from each was allotransplanted into a canine recipient. This involved an additional 30 minutes of warm ischemia without perfusion during reanastomosis. The native kidneys were nephrectomized at the time of allotransplantation. The control experiment entailed two hours of warm ischemia at normothermia without in situ perfusion. RESULTS-Diuresis continued throughout the in situ preservation in all four test dogs. The results with both models demonstrated excellent renal function following three and six hours of in situ preservation. A control kidney experiencing two hours of warm ischemia, without perfusion, was irreversibly damaged.