Efficacy of thalidomide as immunossupressor in heterotopic heart transplantation

Introduction
The current success of organ transplantation is due to advances in immunosuppressive therapy and to the adminstration of a wide variety of pharmacological agents. This progress has permitted a change from an initially cytotoxic treatment with high morbidity to a treatment based on the regulation of the immune response and the induction of immunological tolerance. The search for new effective immunosuppressive drugs with lower toxicity and fewer side effects represents a challenge and has been the subject of intense research (1, 2, 3, 4, 5, 6, 7).
A medication that seems to have an immunosuppressive action is thalidomide. This drug has proved to be valid for the treatment of graft-versus-host disease observed in bone marrow transplantation. Experimental studies have demonstrated the action of thalidomide as an immunosuppressive and anti-inflammatory drug in organ transplants and in the treatment of a series of diseases. In rats, the combination of thalidomide with cyclosporine was found to be effective both for the treatment and for prevention of episodes of heart allograft rejection after initial induction with cyclosporine (8, 9, 10, 11, 12, 13, 14, 15, 16, 17).
Thus, thalidomide may represent a new immunosuppressive pharmacological option to be used for the control of heart allografts. This agent may eventually reduce the need for other medications such as corticosteroids and may be used in combination with other immunosuppressive drugs, with special emphasis on cyclosporine and FK-506 (tacrolimus) (3, 7, 8, 9).
Thus, the purpose of this study was to evaluate the immunosuppressive action of thalidomide alone or in combination with cyclosporine in heterotopic heart transplantation.

Material and Methods
This study was conducted according to the guidelines of the Brazilian Code for Animal Experimentation (1988) and the ethical principles for animal research proposed by the Ethics Committee for Animal Experimentation of The Federal University of Minas Gerais and was approved by the Ethics Committee of the Department of Surgery of this University (18, 19).
Fifty adult mongrel rabbits of both sexes (32 males and 18 females) were used, 25 of them as donors and 25 as receivers. They were randomly selected from the veterinary farm in order to diminish the possibility of relationship. Their weight ranged from 1.8 to 2.4 kg (2.1 ± 1.8).
The recipients were divided into five groups:
Group 1 (n = 5): control, no immunosuppressed.
Group 2 (n = 5): cyclosporine administered at the dose of 10 mg/kg/day.
Group 3 (n = 5): thalidomide administered at the dose of 100 mg/kg/day.
Group 4 (n = 5): cyclosporine administered at the dose of 5.0 mg/kg/day.
Group 5 (n = 5): cyclosporine administered at the dose of 5.0 mg/kg/day in combination with thalidomide at the dose of 50 mg/kg/day.
The medications were administered through an orogastric catheter on the day before surgery, on the day of surgery and on subsequent postoperative days. The animals were followed-up until their death or until the transplanted heart stopped beating.
The transplants were performed under general intravenous anesthesia. The anesthetic, sodium pentobarbital (Hypnol®, Cristália, Itapera, São Paulo, Brazil) was injected in the marginal ear vein at the dose of 30 mg/kg. Antisepsis was performed with a 2% alcoholic iodine solution after appropriate shaving of chest and abdomen.
In the donor, the thoracic and abdominal cavities were exposed through an incision made from the sternal manubrium to the pubic symphysis. The pericardium was opened, and the heart and its large vessels were dissected. The cranial and caudal vena cava were ligated with 2-0 silk sutures and sectioned between ligatures. The aorta and pulmonary trunk were dissected and a 27 G needle was introduced into the upper portion of the ascending aorta for injection of Saint Thomas cardioplegic solution at 4ºC and of 0.3 ml sodium heparin. After cardiac stop, the aorta and pulmonary trunk were ligated with 2-0 silk sutures 4.0 cm far from the heart. The heart was then removed with the large vessels. The pulmonary veins and arteries were ligated with 4-0 silk sutures close to the pulmonary hilus. The heart was separated from lungs and was stored in 0.9% sodium chloride solution at 4ºC.
The heterotopic heart transplant was performed by the technique of MANN et al. (1) (fig. 1). The abdominal cavity was opened through a median xyphopubic incision. The retroperitoneum was opened longitudinally with an incision in the posterior parietal peritoneum, and the abdominal aorta and caudal vena cava were dissected distally to renal vessels. After exposure of these vessels for a longitudinal extension of 6.0 cm, 0.3 ml sodium heparin was injected in the vena cava. After 2 minutes, two bull-dog clamps were placed proximally and distally to the site selected for the vascular anastomoses. The abdominal aorta and caudal vena cava were opened longitudinally and anteriorly along an extension corresponding to the caliber of the pulmonary trunk and of the aorta of the heart to be transplanted. The anastomoses were performed with the aid of a surgical magnifying lens at 2.5 magnification. The aorta of the donor heart was anastomosed with the abdominal aorta of the receiver with a running end-to-side prolene 7-0 sutures. The pulmonary trunk of the donor heart was anastomosed with the caudal vena cava of the receiver with end-to-side running prolene 7-0 sutures (fig. 1).
Respiratory movements, mucosal color, and heart beats in the original and transplanted hearts were evaluated at 5 minute intervals until the recovery of normal cardiac activity to an adequate rhythm and rate. The animals were then transferred to appropriate individual cages and kept warm with 100 Watts fluorescent lamps, with free access to ration and filtered water.
Three ml of blood were collected from the animals immunosuppressed with cyclosporine to measure the serum levels of the drug by radioimmunoassay (RIA).
During the postoperative period the animals were weighed daily for calculation of drugs dosages their general condition was checked and the presence of beats of the transplanted heart was identified by palpation. When the transplanted heart stopped beating, the animals were killed with intravenous injection of sodium pentabarbital. The transplanted heart, lungs and kidneys of the receiver rabbits were removed for histopathological examination and stained with hematoxylin and eosin. After sudden death of any animal the same histopathological studies were performed and the cause of death was investigated.
The pathological rejection score was determined according to criteria of Billingham et al. (1990) and criteria adopted by the International Society for Heart Transplantation, with the modifications shown in table 1 (20, 21).
Data are reported as means + standard error of mean (SEM). Since the responses measured were qualitative (degree of rejection), the results were analyzed statistically by the nonparametric Kruskal-Wallis test and Student t-test. The level of significance was set at p < 0.05.

Results
Survival was greater in Groups 2, 3 and 5 than in Groups 1 and 4 (table 2). There was no significant difference in survival between Groups 2, 3 and 5. The immunosuppressive agents used for these groups alone or in combination were effective in increasing the graft survival (table 2).
Rejection was lower in Groups 2, 3 and 5, without difference from one to another. The three treatments were effective in controlling rejection (p < 0.01) (table 2).
Thalidomide at the dose of 100 mg/kg/day or in combination with cyclosporine was effective in controlling rejection (table 2 and fig. 2 and 3). Cyclosporine at the dose of 5.0 mg/kg/day (Group 4) was also effective in controlling rejection compared to Group 1 (control, non-immuno-suppressed animals), but was not effective in controlling rejection compared to the remaining drugs at the doses used, alone or in combination (table 2 and fig. 4).

Discussion
The technique of heterotopic transplant of the heart allograft in an abdominal position proposed by Mann et al. in 1933 (1) has been used as a model for the study of the pharmacological action of medications and hormones in order to verify the intensity of myocardial damage secondary to reperfusion and the histological pattern of heart allograft rejection (10, 11, 12, 13, 14, 15, 16, 17, 23, 24, 25, 26, 27). This method is simple and is relatively easy to be reproduced in various animals.
The use of different doses of thalidomide and cyclosporine establishes a pattern of rejection and the survival time of the transplanted rabbits. The use of lower doses with acceptable results in terms of reduction of the histopathological rejection score and the corresponding increase in survival may represent a considerable decrease in the cost of transplant procedures and the occurrence of less intense adverse effects with the use of immunosuppressive drugs.
The combination of thalidomide, which is an inexpensive drug, with cyclosporine at lower than therapeutic doses was as effective as the use of cyclosporine alone at immunosuppressive doses. The mechanisms related to the synergism of cyclosporine with thalidomide remain obscure but there is evidence suggesting that the two medications act selectively on different aspects of the host-immune response, with lower adverse effects induced by these drugs.
The mechanism of action of thalidomide on humoral and cellular immunity has not been fully understood. It is possible that its anti-inflammatory effects are associated with the inhibition of neutrophil chemotaxis (8, 21, 29, 30, 31). The action of thalidomide on the survival of skin grafts in rabbits (11, 12) and its efficacy in the prevention and treatment of graft-versus-host disease in mice in the presence of bone marrow transplantation are well known (13, 14, 15, 16). In the present study, thalidomide in combination with cyclos-porine reduced the endocardiac interstitial inflammatory infiltrate and perivascular mononuclear inflammatory process (fig. 2).
In conclusion, the longer survival after thalidomide administration indicates a possible effect of this drug in delaying the rejection process. The effectiveness of the combination of thalidomide and cyclosporine may represent a contribution in terms of a possible clinical application in the field of pharmacological immunosuppression in organ transplants.
Further studies are needed to determine the immunological mechanisms responsible for the synergism of thalidomide and cyclosporine and to establish the role in the pharmacotherapy of human organ transplantation.

Acknowledgment
The authors thank the National Council of Science and Technology (CNPq) and the Foundation for Assistance to Research of Minas Gerais State (FAPEMIG) for the financial support.

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