In vivo imaging demonstrates a time-line for new vessel formation in islet transplantation

Abstract

Vascularization of transplanted islets must be maintained to provide long-term graft function. In vivo assessment of new vessel formation in islet grafts has been poorly documented. The purpose of this study was to investigate whether neovascularization was detectable in vivo in a Feridex-labeled murine syngeneic subcapsular islet mass using DCE MRI over 180 days. Subcapsular transplants could be visualized at post-transplant days three, seven, 14, and 28 using T2-weighted MRI and at post-transplant day 180 by immunohistochemistry. Injection of the contrast agent gadolinium (Gd)-DTPA for DCE at three, seven, and 14 days showed increased signal in the transplant area consistent with new vessel formation. Areas under contrast enhancement curves suggested peak angiogenesis at 14 days. At 180 days, there was no observable change in signal intensity after contrast injection suggesting established vascularization or islet mass reduction. Immunohistochemistry confirmed MRI and DCE findings. These data suggest that islet angiogenesis occurs early after transplantation and is likely established after one month of transplantation. This study provides an in vivo time-line of neovascularization in subcapsular islet grafts. We anticipate that contrast extravasation captured by MRI may provide useful monitoring of graft angiogenesis if reproduced in a clinically relevant intraportal model.

Fig. 1

Representative T2-weighted imaging of Feridex labeled islets under the kidney capsule at three, seven, 14, 28, and 180 days post-transplantation. Islets are visible as hypointensities (white arrow) at all early time points, but not at day 180 post-transplantation.

Fig. 2

Representative immunohistochemistry of islet grafts. Kidneys with islet grafts were recovered on days 14, 28, and 180 post-transplantation, and consecutive sections were processed for H&E, PB for iron particles, and insulin for the presence of islets. PB reveals the iron-labeled islets in the subcapsular region of the kidney at days 14, 28, and 180. Unstained iron can be seen in H&E sections as brown regions at all three time points. Strong insulin immunostaining (red) shows the presence of islets at days 14 and 28 compared with weaker staining at day 180. Scale bar, 250 μm.

Fig. 3

Temporal evolution of DCE MRI. (a) DCE imaging of subcapsular islet graft reveals peak enhancement at 14 days post-transplantation. (b) AUC analysis shows a progressive increase in overall enhancement over the total imaging time of 32 min (ANOVA p = 0.03 with Tukey post hoc test, p = 0.02). Data were averaged from all animals at each time point, n = 3.

Fig. 4

Representative temporal vascularization of islet grafts. Kidneys with islet grafts were recovered on days 3, 7, 14, 28, and 180 post-transplantation, and immunohistochemistry of anti-vWF (brown; a marker of newly formed blood vessels) was performed. Day 3 shows disorganized staining of vWF. Day 7 shows some microvessels, which are more clearly delineated at day 14 (yellow arrows indicate vessels). Day 28 is similar to day 14 with apparently larger microvessels. Day 180 shows absence of vWF staining. [These sections were immediately adjacent to sections in Fig. 2. Hence the brown in day 180 indicates iron not vWF as can be seen in corresponding H&E section (Fig. 2)]. Scale bar, 250 μm.