A mouse model for studying intrahepatic islet transplantation

Abstract

Intrahepatic human islet transplantation has raised hopes for a cure for diabetes mellitus, especially in patients with type 1 diabetes; however, the need for a substantial amount of islets and, in many instances, repeated transplantations demonstrates underlying problems with this procedure, such as failure of angiogenesis and immunologic rejection. Studies using rodent models may be helpful in improving the success of islet transplantation. However, most of the studies using rodents for islet transplantation have been under the kidney capsule rather than the liver. Using islets from transgenic mice expressing green fluorescent protein under the control of mouse insulin I promoter, the authors have developed a method with which to visualize histologic and pathologic changes in intraportally transplanted islets and surrounding hepatic tissue using reflected light confocal imaging. Initial events 24 hr after islet transplantation in the liver include beta-cell loss and hepatic ischemic injuries.

FIGURE 1

Macroscopic visualization of MIP-GFP islets in the liver 24 hr posttransplantation. (left) Bright-field images; (middle) GFP fluorescence; (right) merged images. (A) Intact liver showing transplanted islets in all lobes. Note that bile in the gallbladder (gb) shows strong autofluorescence. Scale bar = 3 mm. (B) Ventral view of the liver in situ showing ischemic regions in the peripheral lobes (left). The autofluorescence from the gallbladder is evident. Scale bar = 3 mm. (C) The right medial lobe of the liver in A. Merged image (right) shows islets lodged in the blood vessels. Scale bar = 1 mm. (D) Proximity of some islets and necrotic regions suggest embolism caused by the islets. Scale bar = 0.5 mm.

FIGURE 2

Visualization of the site of islet transplantation. (A) Orthogonal views through merged laser scanning confocal images of GFP fluorescence (green) and reflected light (red). (crosshairs) Same locations in face (XY) and lateral views through the tissue volume (XZ and YZ image reconstructions). Note the detailed morphology of the surrounding hepatic tissue at the site of islet implantation including the blood vessel (v) and nuclei (n) that are visualized as dark structures. Scale bar = 50 μm. (B) Optical sectioning showing top-down view (0-, 20-, and 34-μm slices are shown) of the site of islet implantation in A. Note the formation of a thrombus (t) (see also Supplement 1 and Video 1). (C) Three-dimensional reconstruction using a stack of images shown in B. Note that the signals in reflected light images (red in B) were inverted and the branches of blood vessels can be seen throughout the site of the islet transplant. (D) The z-axis of the three-dimensional reconstructed image showing the descending blood vessel (v) in which the islet is lodged. Nuclei appear as red circles. Scale bar = 50 μm (see also Video 2).

FIGURE 3

Pathologic changes of a transplanted islet and surrounding tissue. (A) Transmitted light image merged with fluorescent image showing the transplanted MIP-GFP islet in green (black arrow). The islet appears to be trapped in the blood vessel and to have disrupted the downstream blood supply. The necrotic region in the hepatic tissue shows a wedge-shaped rough surface (yellow arrows) compared with the smooth texture of the center of the lobe. Scale bar = 200 μm. (B) A merged confocal laser scanning images of a fluorescent image (green) and a reflected light image (red) showing the site of transplantation of the islet in A. The islet-caused embolism shows the clear boundary where the blood supply has been blocked (upper right; v, vasculature). The hepatocytes in the ischemic region show abnormal cell shapes and texture. The β-cells in the islets show fragmentation caused by the loss of blood supply, suggesting that the islet was undergoing apoptosis or degenerative necrosis (inset). Scale bar = 50 μm. (C) A confocal image from top-down view of the site of islet implantation in A and B (see also Supplement 2 and Video 3) showing the complete blockage of the blood vessel by the islet. Scale bar = 25 μm. (D) The islet-caused embolism is visualized in a three-dimensional reconstruction of the top-down view of the site of islet implantation. Note that the brightness in reflected light images (red in C, asterisk) was inverted to show the blood vessels and nuclei in bright red. Necrotic cells reflect more laser light. (E) Animated image created by capturing real-time motion of image reconstructions using image stacks shown in C. The branched blood vessels were clearly seen in the right side of the site of islet implant but disappeared in the necrotic region (see also Videos 4 and 5). (F) Three-dimensional reconstruction of a tiny islet lodged in the vasculature of the liver. This islet contained only three GFP-expressing β-cells. Scale bar = 50 μm.