In vivo microscopic assessment of cremasteric microcirculation during hindlimb allograft rejection in rats

Experimental and clinical studies of vascular allogenic extremity transplan tation have yielded disappointing results and have not been clinically usef ul. with recent advances in transplantation immunology, considerable intere st has focused on the understanding of leukocyte-endothelial interaction at the microcirculatory level. The objective of this study was to characteriz e the alterations in leukocyte-endothelial interaction in the early stages of rat hindlimb allograft rejection. To study the changes at the microcircu latory level, a new microsurgical model was developed; the cremaster muscle was incorporated into the transplanted hindlimb. The purpose of this study was to report on the microcirculatory changes during rat hindlimb allograf t rejection. A total of 24 transplantations were performed among the four experimental g roups. In a control group, 12 rat hindlimb-cremaster grafts were transplant ed between genetically identical animals, Lewis to Lewis. Microcirculatory measurements of graft survival were taken at 24 hours (group 1A, n = 6) and at 72 hours (group 1B, n = 6). In the rejection control group, 12 transpla ntations were performed across a major histocompatibility barrier between L ewis-Brown Norway and Lewis rats. Microcirculatory measurements were taken at 24 (group 2A, n = 6) and 72 hours (group 2A, n = 6) as above. The follow ing parameters were evaluated to discover the leukocyte-endothelial interac tion: endothelial edema index and the number of rolling, adherent, and tran smigrating leukocytes and lymphocytes in the postcapillary venule. Physical signs of limb rejection, such as edema, erythema, scaling, plaque formatio n on the skin, hair loss, and skin surface temperature, were monitored. Microcirculatory signs of rejection included the following. There was a sig nificant increase in the number of adherent leukocytes in allograft transpl ants at both 24 hours (205 percent; 2.05 +/- 0.38) and 72 hours (431 percen t; 9.11 +/- 3.41) when compared with isograft controls (1.00 +/- 0.89 at 24 hours; 2.11 +/- 0.34 at 72 hours) (p < 0.05), The activation of leukocyte transmigration in creased more than 7-fold in muscle allografts at 24 hours (0.55 +/- 0.25 versus 4.16 +/- 1.89) and more than 6-fold at 72 hours (0.7 2 +/- 0.38 versus 4.38 +/- 1.28) after transplantation (p < 0.05). Endothel ial edema index, a measure of endothelial swelling and cellular deposit acc umulation, increased more than 119 percent in the allograft group 72 hours after transplantation (1.23 +/- 0.07 versus 1.46 +/- 0.09) (p < 0.05). The first clinical signs of limb rejection were scaling of the skin or hair los s; they were observed between the seventh and ninth postoperative days. The composite rat hindlimb-cremaster model presented in this study introduc es a new in vivo approach to monitor acute graft rejection using the intrav ital microscopy system. This is a valuable model for defining the timing, s equence, and correlation between immunologic events and clinical signs duri ng the acute phase of allograft-rejection.