Bone marrow-derived chimerism in non-irradiated, cyclosporin-treated rats receiving microvascularized limb transplants: evidence for donor-derived dendritic cells in recipient lymphoid tissues.

Tolerance to donor transplantation antigens develops when recipients are made chimeric with donor bone marrow. To establish chimerism, the haemopoietic system of recipients typically is severely compromised. We report on a system in which chimerism develops without ablative therapies. Immunosuppression with cyclosporin A allowed the lower limb of a rat to be replaced by a microvascularized transplant from a fully allogeneic donor. Many donor-derived cells populated recipient lymph nodes and spleen, and most had the large size, irregular shape and strong major histocompatibility complex (MHC) class II expression that typify dendritic cells. Donor cells were not found in the macrophage-rich regions of lymphoid tissues, but instead occupied splenic white pulp and lymph node cortex. The donor cells were derived from radiosensitive marrow precursors, as chimerism was abolished if the grafted limb was irradiated, or if muscle and skin flaps devoid of bone were grafted. Donor cells were rare or not detectable in blood, thymus and liver. Whereas lymphoid chimerism was prominent following limb transfer, donor cells were not detected 1-2 weeks after an injection of two femur equivalents of a marrow suspension. We suggest that dendritic cells that undergo rapid turnover in lymphoid organs are replaced from allogeneic precursors in bone grafts. The combination of cyclosporin and vascularized bone provides a means for inducing chimerism in lymphoid tissues of non-irradiated recipients.

Harvesting of the latissimus dorsi muscle: a small animal model for seroma formation.

A common complication of soft tissue dissection and muscle harvesting is seroma formation. In order to manage and understand the formation of seromas, we developed a small animal model for seromas in the Sprague Dawley rat. Skin flaps and subcutaneous tissue were elevated and the latissimus dorsi muscle was harvested in 20 animals. Eighteen of the 20 rats (90%) formed clinically significant seromas. Sixteen animals had associated skin flap necrosis and 12 required serial drainage for recurrent seromas. At necropsy, gross capsular formation occurred in all animals who developed seromas. Microscopically, a fibrous capsule enveloping the seroma was seen associated with a local chronic inflammatory cell infiltrate. We conclude: (1) Elevation of the latissimus dorsi muscle in the rat is a reliable and practical animal model for seroma formation; (2) Sequelae of clinically significant seromas are often as severe as skin flap necrosis; (3) An inflammatory reaction may be associated with seromas.

Donor leukocyte migration following extremity transplantation in an experimental model.

In an effort to further define the immunologic mechanisms leading to acute composite-tissue allograft rejection, the migratory patterns of donor leukocytes were evaluated. Using a rat model, 52 orthotopic vascularized hindlimb transplants were performed in strains representing major histocompatibility mismatches. In order to evaluate the effect of allogeneic skin on limb rejection, all donor skin was removed in a second group of allografts. Recipient lymphoid organs were examined during the week following transplantation for antigen-presenting cells using a donor-specific class II monoclonal antibody. Donor leukocytes, with dendritic cell morphology, were identified in recipient spleen and lymph nodes draining the allograft. Significantly higher numbers of donor leukocytes were present during postoperative days 1 through 4 for both groups. Association of these important passenger leukocytes with host T-lymphocytes may represent the site of initiation of the immune response.

Migration of donor leukocytes from limb allografts into host lymphoid tissues.

This study was undertaken in an initial effort to characterize the immunology of extremity transplantation by examining the pattern and kinetics of leukocyte migration from rat limb transplants. Migration of donor leukocytes was evaluated by examining recipient lymphoid tissues with a donor-specific, anti-major histocompatibility complex, class I monoclonal antibody. Double-antibody, two-color labeling was used to localize donor cells to specific regions within these tissues. Donor leukocytes, with dendritic cell morphology, were found in the T-cell-rich areas of lymph nodes draining the allograft and spleen. The donor cells were present on postoperative days 1 through 3 but were not present on days 5 to 7. Donor leukocytes were not present in distant lymph nodes or liver. These findings indicate a migration of leukocytes, most likely the highly immunogenic dendritic cell, from rat limb transplants to the draining lymphoid tissues. Migration occurs shortly after transplantation and may lead to the sensitization of alloreactive T-cells.

The effects of preservation on microvascular vein grafts in rats.

Segments 15 mm in length were excised from the femoral veins of rats and preserved by refrigeration at 4 C in lactated Ringer's solution for periods up to 21 days. The findings show that veins can be preserved for up to seven days and successfully grafted to recipients. Although there was some success in preserving vein segments for more than seven days, a high rate of thrombosis occurred after implantation in the recipients. It is generally accepted that damaged endothelium causes thrombosis. The light and electron microscopic observations in this study, however, suggest that the condition of the endothelium may not be the only important factor in the patency of small vessels. A thickened and prominent elastic lamina may also play a role in keeping the lumen open.

The effects of short-term preservation on microvascular free groin flaps in rats.

Free groin flaps taken from rats were preserved by refrigeration at 4 degrees C in either lactated Ringers solution or tissue culture medium for various periods of time. The results indicate that a high survival rate can be expected at periods up to 72 hours, but there was no success in preserving flaps longer than 72 hours. These preliminary experimental findings suggest that, clinically, a high survival rate can be achieved in free flaps following excision from donor sites even if the microvascular transfer must be postponed for a period up to but not exceeding 48 hours.

The microvascular technique of vein grafting in rats as a training and experimental model.

Techniques for obtaining and implanting vein grafts in the femoral arteries of rats are described. Grafts 5 mm in length can be removed from the femoral vein without ligating any side branches; a 15-mm segment is the maximum graft that can be obtained from the femoral vein in a rat. This requires ligation and division of all the branches between the inguinal ligament and the great saphenous vein. The superficial epigastric vein also can be used as a source of grafts to be used in the femoral artery. In this study, neither the femoral nor the superficial epigastric vein appeared to have functioning valves. Therefore, reversing the vein graft before implantation was not necessary.

Prolongation of skin allograft survival by Valium (diazepam).

The survival of rat skin allografts was significantly increased by repeated intraperitoneal injections of 2.5 mg Valium (diazepam) into hosts. In addition, in vivo pretreatment of the donor animals at this dose level decreased the intensity of the recipients' immune responses to skin allografts. In vitro exposure of skin grafts to Valium also prolonged allograft survival.

Inosculation of vessels of skin graft and host bed: a fortuitous encounter.

From the combined procedures of skin stereomicroscopy of in situ grafts in rats, graft removal and supravital intracardiac injection of a contrast medium, the data suggest that the revascularisation of skin grafts is an orderly sequence of events which include: active invasion of the graft dermis by the ingrowing host capillary sprouts; development of anastomoses between the graft and host vasculatures; entry of blood into the graft through the vascular anastomoses by 48 hours after transplantation.