Immunosuppressive protocols with tacrolimus after cryopreserved aortal allotransplantation in rats

Abstract

Objectives and design: The aim of our study was to simulate in rats all aspects and techniques used in our new clinical program of cryopreserved alloarterial transplantation and investigate the influence of two immunosuppressive protocols with tacrolimus on acute rejection of these allografts.

Materials and methods: Cryopreserved abdominal aortic grafts were transplanted between Brown-Norway and Lewis rats. Tacrolimus (0.2 mg/kg daily) was administered from day 1 to day 30 (TAC1) or from day 7 to day 30 (TAC7), respectively. No immunosuppressed isogeneic (ISO) and allogeneic (ALO) rats combination served as control. Aortal wall infiltration by immunocompetent cells (MHC II+ cells of recipient origin) was studied on day 30 after transplantation. Flow cytometry was used for the analysis of day 30 sera for the presence of donor specific anti-MHC class I and II antibodies.

Results: The aortal allografts in both immunosuppressed groups showed regular morphology of aortal wall with no depositions of immunoglobulin G on day 30. The adventitial infiltration of non-immunosuppressed aortal allografts by MHC class II positive cells of recipient origin was significantly higher (ALO 20.7±6.7 cells, P<0.001) compared to both immunosuppressed groups (TAC1 5.9±5.5 cells, TAC7 6.1±5.1 cells). Day 30 sera from the allogeneic non-immunosuppressed animals decreased significantly the binding of fluorescence-labelled MHC class I (46.9±19.4%) and class II (65.8±11.9%) antibody to donors spleen cells compared with day 30 sera from both immunosuppressed groups (TAC1, anti-MHC class I 102.4±4.2%, p < 0.001, anti-MHC class II 102.6±6.0%), (TAC7, anti-MHC class I 79.9±3.3%, p < 0.001, anti-MHC class II 80.9±2.7%).

Conclusion: Both immunosuppressed protocols with tacrolimus (administration from day 1 or from day 7 following transplantation) were able to suppress acute cell- and antibody-mediated rejection of cryopreserved abdominal aortic allografts processed in accordance with our new standardized clinical protocol.

Fig 1. Weight increase on day 30 after transplantation of aortic grafts expressed as a percentage of preoperative weight.

No immunosuppressed animals (group ISO, group ALO) showed significantly higher weight increase on day 30 when compared to from day 1 immunosuppressed animals (group TAC1). However, the weight increase in from day 7 immunosuppressed animals (group TAC7) was compared to animals without immunosuppression (group ISO, group ALO).

Fig 2. a, b, c. Representative light microscopic features of immunosuppressed cryopreserved aortal allografts obtained at 30 days following transplantation.

a—The allografts showed normal histological feature of abdominal aorta with clear differentiation of all three basic anatomical layers, with no signs of intimal hyperplasia, smooth muscle cells necrosis or higher adventitial cellular infiltration. (Haematoxilin-Eosin, original magnification x 100). b—The luminal surface of allografts (arrow) was covered by monolayer of endothelial cells (stained brown). (Anti-Von Willebrand factor antibody, original magnification x 100). c—No deposition of immunoglobulins G was detected in the medial or intimal layer of cryopreserved allografts. (Anti-IgG fluorescein isothiocyanate-conjugated antibody, original magnification x 40).

Fig 3. a1, a2, b1, b2. Representative light microscopic histological features of adventitial infiltration of cryopreserved allografts by mononuclear cell at 30 days following transplantation.

The adventitial infiltration of Brown-Norway cryopreserved aortal grafts by MHC class II positive cell of Lewis origin (stained brown) (Fig 3 a1) was significantly reduced by both types of immunosuppressive protocols with tacrolimus (Fig 3 a2). The adventitial infiltration of Brown-Norway aortal grafts by CD4+ cell (Fig 3 b1) was significantly reduced by both immunosuppressive protocols with tacrolimus as well (Fig 3 b2). Original magnification ×400.

Fig 4. Anti MHC class I antibodies in serum.

The percentage of binding of the anti-MHC class I antibody (anti-RT1.Ac, OX-27) to quiescent BN splenocytes in the presence of sera obtained from recipient rats pretransplant (POD 0) and on day 30 after transplantation (POD 30). ISO—isogeneic group with no immunosuppression. ALO—allogeneic group with no immunosuppression. TAC1—from day 1 immunosuppressed allogeneic group. TAC7—from day 7 immunosuppressed allogeneic group.

Fig 5. Anti MHC class II antibodies in serum.

The percentage of binding of the anti-MHC class II antibody (anti-RT1.D, OX-17) to quiescent BN splenocytes in the presence of sera obtained from recipient rats pretransplant (POD 0) and on day 30 after transplantation (POD 30). ISO—isogeneic group with no immunosuppression. ALO—allogeneic group with no immunosuppression. TAC1—from day 1 immunosuppressed allogeneic group. TAC7—from day 7 immunosuppressed allogeneic group.