Antonio Marni

A four-technique comparative study of orthotopic liver transplantation in the rat

A four-technique controlled study was conducted on rat liver isografting to compare the microsurgical technique (Group 1), the two-cuff technique (Group 2), the three-cuff technique (Group 3), and a newly developed splinting suture technique (Group 4). The 60 day survival rates were significantly better in Groups 2 and 4 than in Groups 1 and 3. The anhepatic phase was significantly shorter with the cuff and splinting techniques than with the microsurgical technique, and total hepatic ischemic time was significantly shorter in the splint group than in the other three groups. Hepatic failure and shock had a higher incidence in those groups displaying a longer total hepatic ischemia time and a longer anhepatic phase. The results of this study show that the two-cuff technique and the splinting technique have substantial advantages over the microsurgical technique and the three-cuff technique. In particular, the splinting technique is preferable to the two-cuff technique because total hepatic ischemia time is shorter, and it is faster for a well-trained microsurgeon to learn this procedure than to learn the cuff technique de novo.

METABOLIC FUNCTION OF GRAFTED LIVER IN RATS

We studied the metabolic variations in grafted livers at different times after transplant by measuring the hepatic energy and redox states. Five groups of rats were studied: control ungrafted Wistar (RT1y) rats (group 1), ungrafted Wistar rats with ligature of the hepatic artery (group 2), isografted Wistar rats (group 3), allografted Wistar rats with livers from ACI (RT1a) donors (group 4, long-term surviving rat strain combination), and allografted Wistar rats with livers from BN (RT1n) rats (group 5, rejector rats). The metabolism of grafted livers was studied for 7 days in groups 2 and 3, for 2 months in group 4, and at the time of rejection in group 5. Adenine nucleotide levels (ATP, ADP, AMP) were significantly impaired at 24 hr and at 48 hr from grafting in isografted and in allografted livers, and the reestablishment of normal values began at the 7th day from grafting. Cytoplasmic NAD+/NADH ratios were lowered at 24 hr from grafting in isografted and in allografted livers. Mitochondrial NAD+/NADH ratios were lowered at 24 hr in isografted livers and at 24 hr and 48 hr from grafting in allografted livers. The metabolic studies performed for 2 months revealed a significant correlation between well-maintained metabolic functions and transplant survival. On the contrary, an important energy loss was evidenced in livers of group 5, at the time of rejection.

Protection of rat heart from damage due to ischemia-reperfusion during procurement and grafting by defibrotide

We studied the efficacy of defibrotide, a prostacyclin-stimulating agent, in preventing ischemia reperfusion injury in Wistar rat heart by using three experimental models: (1) hearts from donors were perfused with the drug (32 mg/kg/hr) during 15, 30, 45, and 60 min of cold ischemia following 5, 10, and 15 min of warm ischemia; (2) hearts from donors treated with the drug were cold-stored for 12 or 24 hr; and (3) procured hearts perfused with the drug were isografted, after 30 or 60 min of warm ischemia, in recipient rats treated daily with defibrotide. Hearts perfused with saline and/or vehicle of the drug were used as controls. At the end of established ischemia times, and after 30 min, and 2, 4, 7 and 14 days from transplantation, hearts were rapidly cooled in liquid nitrogen. ATP, ADP, AMP, cAMP contents, and NAD+/NADH ratios were evaluated in prepared tissue extracts. Cardiac ATP and ADP levels and NAD+/NADH ratios were significantly higher in defi-brotide-treated organs than in controls. Isografted defibrotide-treated hearts were also significantly preserved, with respect to controls, from the loss of ATP levels until rejection occurred. Our results demonstrate the protective activity of the drug against the myocardial metabolic damage due to ischemia-reperfusion.

Effect of cyclosporine and aminophylline on streptozotocin-induced diabetes in rats

Diabetes induced in rats by multiple low doses of streptozotocin is thought to mimic type 1 disease in man. We tested the effect of concomitant treatment with immunomodulator drugs in this diabetic experimental model. Administration of cyclosporine resulted in a rapid appearance of hyperglycemia, perhaps by a potentiation of the direct cytotoxic action of streptozotocin on beta cells. By contrast, aminophylline administration protected the animals from the diabetogenic action of streptozotocin. Concomitant treatment with aminophylline and cyclosporine failed to protect the rats from the hyperglycemia induced by streptozotocin.

The effect of cyclosporine on nucleotide content of rat lymphocytes.

In vivo administration of cyclosporin A (CyA) was found to determine some variations in nucleotide content of rat lymphocytes. ATP levels were reduced by CyA treatment, and the effect was more evident in peripheral blood than in spleen lymphocytes. In contrast, cAMP values were increased upon pharmacologic treatment with the same major evidence at the blood lymphocyte level. Intralymphocytic phosphodiesterase enzyme activity became detectable during CyA administration, whereas the intracellular redox state (NAD+/NADH ratios) did not vary significantly. These results were amplified by increasing CyA concentration.

Prolonged survival of experimental heart transplantation induced by theophylline

High levels of cyclic adenosine monophosphate (cAMP) in lymphocytes are associated with a reduction in many immunological functions. Theophylline, a drug effective in elevating lymphocyte cAMP levels, was employed as a single immunomodulator treatment in rats submitted to heterotopic heart allotransplantation. In 3 strain combinations, the graft survival was consistently prolonged in treated animals in comparison with untreated controls.

Prevention of impaired liver metabolism due to ischemia in rats: Efficacy of defibrotide administration

The effect of defibrotide treatment in protecting liver metabolism from ischemic damage was studied. The drug was administered to male Wistar rats as a bolus (30 mg/kg body weight) at the beginning of 60 min ischemia and then continuously during 60 min of postischemic reperfusion at a dose of 30 mg/kg body weight. This dose was previously identified as useful to protect against myocardial ischemia induced in the cat. ATP and ADP intrahepatic levels were significantly higher in drug-treated rats than in untreated animals. The liver cytoplasmic NAD+/NADH ratio in defibrotide-treated rats was no different from that observed in sham-operated rats. The mitochondrial NAD+/NADH ratio in the liver was also improved by defibrotide treatment. Our data suggest that defibrotide may exert protective activity on hepatocytes useful for inducing a rapid restoration of their metabolism. Such a restoration is possibly related to improvement of microcirculation through an increase in prostaglandin I2 production or oxygen delivery due to drug administration.

Simultaneous transplantation of kidney and duct-ligated segmental pancreas in the rat. En bloc technique using two vascular anastomoses

A new surgical technique of simultaneous kidney and pancreas transplantation is described here. It was used to perform 28 isografts in inbred male Wistar ( RT1y ) rats: 3 animals died within 3 days from early complications and 4 had long-term complications (2 severe pancreatitis and 2 urinary tract complications); 1 animal was killed at 3 months, and its organs were examined histologically. At greater than 4 months after transplantation 2 animals died normoglycemic from undetermined causes. In 3 animals the isografted pancreas was removed at 6 months to assess the effectiveness of the transplant. Currently, 15 animals are alive and normoglycemic greater than 6 months after transplantation. Morphological aspects of simultaneously isografted organs are unchanged when compared with separately transplanted organs. In our opinion this technique provides a useful experimental model to study several technical and immunological problems still present in kidney and pancreas transplantation in diabetic patients.

Increased cAMP levels in lymphocytes of allografted rats: Possible relationship to delayed graft rejection

We studied the levels of nucleotides in peripheral blood and in spleen lymphocytes obtained from allografted rats. Four types of allografts were performed by using ACI rats (RT1a) as donors and Lewis rats (RT1l) as recipients: heart, pancreas, kidney and liver. Ungrafted Lewis rats were used as controls. Rejection time without immunosuppression occurred at 6.7 +/- 0.25 days from transplant in heart-allografted, at 7.3 +/- 0.25 days in pancreas-grafted, at 6.8 +/- 0.24 days in kidney-grafted, and at 20 +/- 3 days in liver-grafted rats. Biochemical assays on lymphocytes were performed on the 7th day after operation by measuring the intracellular content of APT, ADP, AMP, cAMP, NAD+ and NADH. Our results showed that the prolonged survival of allografts in liver-grafted rats correlated with the higher content of cAMP in their lymphocytes than in those of controls. In addition, ATP and ADP levels in lymphocytes from heart-and pancreas-allografted rats were significantly lower than those of lymphocytes from liver-allografted rats.

Heart and kidney transplantation in the rat using a mixed cuffstent microsurgical technique

A method has been described for heart and kidney transplantation in rats using a mixed cuff-stent microsurgical technique. The advantages of the method include reduction of total ischemia time to an average of 11 minutes for heart grafting and 6 minutes for kidney grafting, provided donor and recipient operations are performed simultaneously; ease of performance and short training period, which allows the method to be employed by transplantation novices and to be extended to laboratories that lack experienced microsurgeons; and the possibility of applying the method to transplantation of other vascularized organs in the rat.