DETECTION OF LIVER ALLOGRAFT APOPTOSIS USING AN IN VIVO IMAGING TECHNIQUE

Abstract

1031 Numerous studies have indicated that apoptosis occurs during allograft rejection. Detection of apoptotic cells commonly utilizes the TUNEL method, which detects DNA fragmentation, the end stage of apoptosis. However, these in vitro techniques are not relevant to the clinical setting. To determine if there is a correlation between on-going allograft rejection and graft apoptosis, we utilized 99mTc-labeled-annexin-V for in vivo imaging of cell death. Liver grafts from DA or Lewis rats were orthotopically transplanted to Lewis rats as allogeneic (n=4) and syngeneic (n=4) donor-recipient combinations. On days 2, 4 and 7 post-transplantation, the recipient rats were injected intravenously with 99mTc-annexin-V. One hour later, image analysis was performed. There was a significant increase in the uptake of 99mTc-annexin-V on days 4 and 7, compared with syngeneic grafts. Representative images of syngeneic and allogeneic grafts on day 7 are shown in Figure 1.Figure 1: Syngeneic (left) and allogeneic (right)We have determined in this model that activation of the caspase cascade occurs specifically during allograft rejection. To determine if we could block apoptosis in allografts, recipient rats were treated with 1.0 mg of the broad-spectrum caspase inhibitor, zVAD-fmk, on day 0, 2, 4 and 6 post-transplant. Treatment with zVAD-fmk decreased graft apoptosis as determined by 99mTc-annexin-V distribution in 2 of 3 allograft recipients (Figure 2). Importantly, body weight loss on day 7 was substantially decreased in zVAD-fmk treated allograft recipients as compared to untreated rats (14.6% for zVAD-fmk treated vs. 21.6% for untreated; recipients of syngeneic grafts lose 14.6% of body weight).Figure 2: 99mTc-annexin-V imagingIn conclusion, our data indicates that 99mTc-annexin-V imaging is useful for in vivo monitoring of graft damage, perhaps in clinical practice. Furthermore, treatment with zVAD-fmk may improve the general health of graft recipients and help reduce graft damage by inhibition of the caspase cascade.