Objective: Since apoptosis almost always accompanies CAR, and offers cell membrane signatures of the death process in form of asymmetric exposure of phosphatidylserine and phosphatidylethanolamine (PE), we evaluated the usefulness of radiolabeled PE-targeting agent- Technetium-99m-labeled duramycin (D) by microSPECT/CT imaging.
Methods: 16 mice received abdominal heterotopic cardiac allografts in 4 groups of 4 animals each. Group 1: BALB/c (H-2d) donor to B6 (H-2b) recipient (allogeneic transplant, ALO). Group 2: B6 donor to B6 recipient (syngeneic control transplant, CON). Group 3: BALB/c donor to B6 recipient treated with CTLA4-IG (allogeneic transplant and immunosuppressed, IMS). Group 4: Bm12 H-2bm12) donor to B6 recipient (class II MHC disparate transplant to test for chronic CAR, CHR). ALO and CON were sacrificed 6-7 days following transplant. IMS animals were sacrificed 14-15 days following transplant. CHR animals were sacrificed 21 days following transplant (all grafts were beating at the time of harvest). MicroSPECT/CT imaging of the heterotopic transplant was performed in vivo and ex vivo after intravenous D administration. The transplant was collected for quantitative injected dose/gram uptake (%ID/g) of radiotracer. Hearts were then pathologically characterized using the ISHLT grading rubric.
Results: The in vivo and ex vivo imaging, and the quantitative uptake (expressed in mean %ID/g ±SD) of the radiotracer demonstrated the most intense uptake in ALO (5.8±2.2 %ID/g), followed by CHR (1.8±1.5 %ID/g), then IMS (1.2±0.4 %ID/g), and finally CON (0.90±0.04 %ID/g). One-way ANOVA with Tukey post-hoc test revealed ALO had significantly higher uptake than that of the CON and IMS groups (P<0.05). Pathology demonstrated ISHLT Grade 3R rejection in ALO (3 out of 4 animals), no rejection in CON and Grade 1-2R in IMS and CHR groups. Chronic vasculopathy was also observed in the CHR group.
Conclusion: Noninvasive molecular imaging of apoptosis in CAR is feasible with radiolabeled D, which can identify various shades of graft rejection verified by histopathological characterization.