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This work studies some of the advances that have participated in the development of the liver transplantation-reperfusion injury. This lesion is a rather complex one that probably is not only associated with the production of free radicals, but also of other vasoactive substances such as, prostanoids, altered calcium related compounds, abnormal coagulation factors, as well as other important potentially harmful substances. Our interest since 1975, has resided in compounds that modify the xanthine oxidase pathway, such as allopurinol, that might either protect from the formation of free radicals or might act through other mechanisms such as, purine salvage and production of high energy compounds, among few of them. The pharmacological manipulation of the reperfusion injury, will require in this way, the use of various substances in the protection of the transplanted liver.

This study applies cyclosporin A as a donor pretreatment prior to organ harvesting or as a graft pretreatment during preservation of canine kidney allografts by hypothermic pulsatile perfusion or hypothermic storage. All recipients except those in Group IX received minimal immunosuppression with azathioprine after transplantation (5 to 2.5 mg. per kg. per day). No significant differences in survival (X +/- SD) were observed between the 3 control groups which were either 1) flushed with untreated Ringer's lactate solution and immediately transplanted (Group I, no. = 8, 14 +/- 3.33 days), 2) preserved by hypothermic pulsatile perfusion for 24 hours (Group II, no. = 7, 12.0 +/- 8.92 days), or 3) hypothermically stored for 24 hours (Group III, no. = 7 13.1 +/- 11.6 days). A trend towards improved survival was seen in the 2 groups of animals that received kidneys that had been graft pretreated with cyclosporin A (12.5 mg.) during 24 hours preservation by either hypothermic pulsatile perfusion (Group IV, no. = 10, 17.4 +/- 13.32 days, p less than .25) or hypothermic storage (Group V, no. = 6, 8 +/- 12.75 days, p less than .25). Survival of recipients in Groups VI (no. = 6) and VII (no. = 9) who received kidneys whose donors had been pretreated with 25 and 50 mg. per kg. respectively was dependent on the dosage of cyclosporin A used. Donor pretreatment at 50 mg. per kg. was deleterious to kidney function (Group VI, 2.16 +/- 1.47 days, p less than 0.0005). Donor pretreatment with 25 mg. per kg. did not significantly improve survival over control groups (15.66 +/- 12.9 days). Recipients in Groups VIII (no. = 10) and IX (no. = 6) were transplanted with kidneys from cyclosporin A pretreated donors (15 mg. per kg.). These kidneys also received cyclosporin A graft pretreatment (10 mg.) during 24 hours of hypothermic storage. The only difference between Groups VIII and IX was that the animals in Group IX received minimal amounts of cyclosporin A (5 mg. per kg. per day) after transplantation. Combined donor and graft pretreatment yielded improved kidney allograft survival (Group VIII, 21.7 +/- 13.36 days, p greater than .10, Group IX, 20.83 +/- 14.2 days, p greater than .10). However, there was no significant difference observed as a result of the different immunosuppressive protocols used in Groups VIII and IX. These results indicate a trend towards improved renal allograft survival under certain conditions, after donor and graft pretreatment with cyclosporin A.

Modification of graft immunogenicity using graft (GPTX) and donor pretreatment (DPTX) has been pursued in an attempt to modify allograft immunogenicity using various immunosuppressive agents. The murine skin allograft and canine renal allograft models were used to study the efficacy of Cyclosporine (Cy A) as a DPTX and GPTX prior to transplantation. Tail skin allografts from C3HHeN male mice were grafted to Balb/c female mouse recipients. Minimal immunosuppression was given to all skin graft recipients. Skin allograft were either GPTX with Cy A, DPTX with either Cy A, methylprednisolone (MP), or cyclophosphamide (CP), or Cy A GPTX and DPTX with the three drugs alone or in combination. Cy A GPTX alone of skin allografts did not significantly prolong survival. DPTX with Cy A significantly prolonged skin graft survival, however, CP or MP alone did not. The various combinations of MP, Cy A, and CP as DPTX and MP, Cy A, and CP DPTX used together with Cy A GPTX also significantly prolonged murine skin allograft survival. Kidney allografts used unrelated mongrel dogs as donors or recipients. Renal transplant experimental groups were either: Non-pretreated and immediately transplanted, nonpretreated and hypothermically stored (HS) for 24 hours in Collins (C-2) solution, GPTX with 12.5 mg Cy A during 24 hr. HS in C-2, DPTX with Cy A (25 mg/Kg), or Cy A DPTX (15 mg/kg) and GPTX during 24 hrs. HS in C-2. Cy A GPTX during HS was sometimes effective in prolonging kidney allograft survival greater than 30 days using only minimal immunosuppression with azathioprine. Cy A DPTX prolonged survival somewhat, but not significantly. Improved results were seen, however, when Cy A DPTX was used together with Cy A graft pretreatment. These results indicate the potential for the successful use of Cy A as a donor and/or graft pretreatment, however, further studies will be necessary to optimize the use of Cy A in these modalities.

This study continues our investigations in the area of canine islet cell cryopreservation. Islet cell allografts were frozen using a simple method to −196 °C with rapid freezing rates and dimethyl sulfoxide as the cryoprotectant. Good in vitro viability was observed using trypan blue dye exclusion. After intrasplenic transplantation, grafts which did not reject were able to maintain normoglycemia for periods of greater than 60 days. The use of either Cy A as an immunosuppressant or ALG as a graft pretreatment contributed to prolongation of allograft survival in these long-term surviving recipients. These results encourage further studies in this area for potential future clinical application of this technique to human pancreas.

Isolated-perfused canine heart-lungs were used as a model to measure the effects of 24-hour hypothermic storage on cardiopulmonary function and metabolism. Heart-lungs were stored at 4–7°C in Euro-Collins solution ( n = 6) or TP-V ( n = 6). a hyperosmolar colloid solution containing dextrose, sucrose. ATP and MgCl2. Lung inflation was maintained with 100% nitrogen. Following preservation, the heart-lungs were perfused with an albumin-mannitol perfusate for three hours at 37°C. for functional and laboratory determinations. Cold storage with TP-V soiution resulted in significantly lower enzyme activity for CPK ( p < 0·0005) and LDH ( p < 0·01) at 0, 1, and 3 hours of normothermic isolated perfusion. A significant reduction in lactate production ( p < 0·001) was also seen in the heart-lungs stored in TP-V. No apparent differences were seen in the pH, PCO2. and PO2 among the two groups, nor were there any significant haemodynamic changes. Histological specimens revealed that TP-V was less damaging to both cardiac and pulmonary tissue, as only moderate oedema and congestion was apparent. These results indicate that 24 hour hypothermic storage with TP-V may be a more appropriate preservation solution for canine heart-lungs.

The development of optimal methods for preservation is important for the advancement of liver transplantation. This study compares hypothermic storage (HS) and hypothermic pulsatile perfusion (HPP) with various solutions, using an isolated normothermic perfusion model (LIPM). Canine livers were removed from mongrel dogs without warm ischemia and flushed with either heparinized Ringer's lactate (control and HPP-preserved groups) or the solution used for hypothermic storage (TP-V or modified Collins). The type of preservation and solution for each of the experimental groups was as follows: group I (n = 7), no preservation, fresh; group II (n = 7), 24-h HS with TP-V (a hyperosmolar colloid solution containing sucrose, dextrose, and ATP-MgCl2); group III (n = 7), 24-h HS with modified Collins (C-2), an intracellular crystalloid solution; group IV (n = 5), 24-h HP with TP-V; group V (n = 6), 24-h HPP with Belzer solution, containing ATP-MgCl2; group VI (n = 3), 24-h HPP with albumin. After the preservation period, livers were placed on HPP at 37 degrees C with albumin-mannitol solution for 3-h testing in an LIPM. Perfusate samples were taken at 1-h intervals to assess liver function. LDH, SGOT, alkaline phosphatase, lactic acid, LAP, GGT, pO2, pCO2, pH, osmolarity, AMP, ADP, and ATP were studied. Histologic studies were performed, as were representative HIDA scans. Using the LIPM, livers preserved by HS and HPP with TP-V solution appeared to be superior to those preserved with modified Collins, Belzer, and albumin solutions. In these non-TP-V groups, the greatest cellular and organ damage was observed. TP-V HPP appeared to give the best overall liver functional response and histologic results and is recommended as the preferred method for 24-h liver preservation.

The sensitivity of liver allografts to even minimal periods of ischemia currently limits the duration of hepatic preservation prior to liver transplantation. This study evaluates the role of activated carbon hemoperfusion (ACH) for assisting the recovery of canine livers ischemically damaged by a 20-min occlusion of the portal vein and hepatic artery prior to organ harvesting. Animals in Group I (n = 5) receiving damaged liver allografts without ACH survived a mean (+/- SD) of 18.0 +/- 13.5 h. One ACH treatment given to recipients immediately after liver transplantation in Group II (n = 5) resulted in improved survival to a mean of 3.8 +/- 2.16 days (p less than 0.05). The best survival was obtained after three ACH treatments in Group III (n = 6) on days 0, 1, and 2 (26.6 +/- 27.1 days) (p less than 0.05). These results indicate that ACH may be helpful in assisting the recovery of ischemically damaged liver allografts after transplantation.

Development of techniques for cryopreservation of pancreatic islets of Langerhans could potentially allow for increased freedom from the time restrictions presently affecting viability in islet cell transplantation. While several investigators have attempted islet cell freezing and have obtained favorable in vitro results after thawing, there have been few reported in vivo successes with islets transplanted after freezing. We have developed a simple system for freezing islet cell pancreatic fragments to −196 °C and have either stored them in liquid nitrogen for 24 hr or immediately thawed the islets prior to transplantation. In addition, antilymphoblast globulin has been used as graft pretreatment modality in order to modify islet cell immunogenicity. We found that ALG was effective in prolongation of graft survival after freezing as well as on fresh nonfrozen transplants. The use of freezing and ALG appears, therefore, to have a favorable effect on the immunogenicity of the pancreatic islet cell allograft.

The efficacy of three flushing techniques on subsequent liver function was assessed using the in vivo isolated liver perfusion model (ILPM). Livers from brain-dead mongrel dogs were flushed with cold Euro-Collins as follows: Group I, rapid in situ flushing (10 min); Group II, regular in situ flushing (45 min); Group HI, ex vivo flushing (10 min). All livers were then heterotopically transplanted into recipients, using the ILPM, by anastomosis of the portal vein, vena cava, and hepatic artery to the recipient's portal vein, iliac vein, and iliac artery. Reperfusion followed for 30 min. Laboratory samples collected at 0, 5, 15, and 30 min showed that hepatic function was not altered by ex vivo flushing and was only slightly altered by rapid in situ flushing. Regular in situ flushing proved to be damaging to livers. Histological analysis confirmed these findings. Therefore, either rapid in situ or ex vivo flushing can be safely used by the transplant specialist.

Etude sur 62 transplantations de rein de cadavre. Confirmation de l'effet benefique de transfusions sanguines administrees au donneur peu avant la nephrectomie sur la survie du greffon a long terme

The function of kidneys stored for 48 to 72 hours in hypertonic crystalloid, intracellular solution, Euro-Collins (360 mOsm./l.), was compared with those stored in a new hyperosmolar (520 mOsm./l.) colloid solution designated as TP-II. The TP-II solution contained less K2HPO4 (1.05 gm./l.) and KH2PO4 (317 gm./l.) and more glucose (40 gm./l.) than the Euro-Collins, and also had an albumin concentration of 4.3 gm./dl. Kidneys obtained from beagle dogs were flushed with either Euro-Collins or TP-II solution (4C). Hypothermic storage followed for either 48 or 72 hours, prior to autotransplantation into the iliac fossa and contralateral nephrectomy. Four experimental groups were transplanted as follows: group 1 (n = 8) and group 2 (n = 7) received kidneys that were flushed with 250 ml. of Euro-Collins or TP-II solution, respectively, prior to 48 hours hypothermic storage. Group 3 (n = 5) and group 4 (n = 5) received kidneys that were flushed in the same way as those in groups 1 and 2, respectively, but were stored for a 72-hour period. TP-II appears to be superior to Euro-Collins for hypothermic storage of kidneys for periods as long as 48 hours. When hypothermic storage times are extended to 72 hours, the number of kidneys with normal function after transplantation is reduced for both solutions, however, TP-II solution has a slight advantage over Euro-Collins solution. Further studies will hopefully clarify this issue and lead to the application of TP-II in the clinical setting.

Islet cell transplantation has been associated with ultimate graft rejection. This preliminary study investigates the use of the renal subcapsular region as a site for placement of canine islet cell allografts. A new noncollagenase mechanical technique was used for preparation of the allografts. Animals in group I (N = 6) died of hyperglycemia in 4.0 +/- 1.89 days (X +/- SD) after pancreatectomy without subsequent islet cell transplant. Normoglycemia and excellent survival (greater than 60 days) was obtained in pancreatectomized animals in group II (N = 6) and in group III (N = 6), who received an islet cell allograft to the renal subcapsular site. Group II recipients were given no immunosuppression, and animals in group III received minimal immunosuppression with azathioprine. Dependence on the islet cell allograft for maintenance of normoglycemia was confirmed in the majority of the recipients by nephrectomy, to remove the graft, with resulting hyperglycemia and death. One normoglycemic animal in group II died on day 6 from peritonitis. One recipient in group II was normoglycemic at greater than 1 mo after removal of the first graft by nephrectomy, followed by retransplantation of islet cells from a third-party donor. Two other recipients are being studied on a long-term basis, and have been normoglycemic for greater than 6 mo and greater than 4 mo after transplantation. These studies encourage further investigation in this area for application of islet cell transplantation in man, and elucidation of the possible mechanisms for prolongation of islet cell allograft survival at the renal subcapsular site.

The increased use of long-distance procurement for donor livers has recently created a dire need for prolonged hepatic preservation. To date, a variety of experimental liver preservation techniques have been studied; however, consistent success for more than 12 hours has yet to be achieved.1–3 Methods for testing the efficacy of preservation solutions that successfully and safely prolong the cold preservation time are needed and might offer a realistic solution to this problem. Toledo-Pereyra et al.4,5 and Lambotte et al.6 have suggested that the use of hyperosmolar perfusates may be beneficial in achieving prolonged hepatic cold-storage times. In addition, Toledo-Pereyra et al4 and Fuller et al 7 have reported on enhanced hypothermic preservation of donor livers following the use of pulsatile perfusion with specially made perfusates. Nevertheless, the vulnerability of the liver to both warm and cold ischemic damage and reperfusion injury remains as the major obstacle to immediate posttransplantation graft function.