Technique of endoscopic biopsy of islet allografts transplanted into the gastric submucosal space in pigs

Abstract

Currently, islet cells are transplanted into the liver via portal vein infusion. One disadvantage of this approach is that it is not possible to adequately biopsy the islets in the liver to assess for rejection. Islet transplantation (Tx) into the gastric submucosal space (GSMS) can be performed endoscopically and has the potential advantage of histological evaluation by endoscopic biopsy. The aim of this study was to determine whether a representative allograft sample could be obtained endoscopically. We performed islet Tx into the GSMS in nonimmunosuppressed pigs using simple endoscopic submucosal injection. Islets were transplanted at four sites. Endoscopic ultrasonography and biopsy of the transplanted islets at two sites by modified endoscopic submucosal dissection were carried out successfully in all pigs 5 days after islet Tx. Tissue obtained at both biopsy and necropsy (including full-thickness sections of the gastric wall around the sites of the remaining islets and biopsies) were examined by histology and immunohistochemistry to confirm the presence of the islet grafts and any features of rejection. Representative allograft sampling was successfully obtained from all biopsy sites. All biopsies included islets with insulin-positive staining. There was significant CD3(+) and CD68(+) cell infiltration in the islet masses obtained at biopsy and from sections taken at necropsy, with similar histopathological features. Endoscopic biopsy of islet allografts in the GSMS is feasible, provides accurate histopathological data, and would provide a significant advance if translated into clinical practice.

Figure 1

Endoscopic view of the submucosal bleb created as donor islets are injected into the GSMS of a recipient pig.

Figure 2

(A) Endoscopic appearance of the gastric submucosa at site of islet mass (black arrow) 5 days after islet Tx. (B) Endoscopic ultrasound identifying the islet mass (white arrows) in the GSMS 5 days after islet Tx.

Figure 3

Endoscopic view as a biopsy of an islet mass is taken 5 days after islet Tx. (A) After identification of the site of the islet mass to be biopsied, saline (2–5ml) was injected to create a submucosal bleb. The raised mucosal bleb has been incised with a Flex knife to allow access to the submucosal space. An IT2 knife was then used to cut around the site of the graft. (B) A biopsy of the graft is being taken by snaring the dissected tissue, including the submucosal tissue. (C) The biopsied tissue is being removed with forceps. (D) The removed specimen was 23×12 mm.

Figure 4

Histopathological appearance of an area of severe cellular infiltration of biopsies taken 5 days after islet Tx into the GSMS. (A) Islet mass in the GSMS (H&E, x40). (B) Polymorphonuclear and mononuclear cell infiltrate in islet mass (H&E, x100). (C) Few insulin-positive beta cells were identified in an area of severe cellular infiltration of the islet mass. Islets were infiltrated particularly by (D) CD3⁺ and (E) CD68⁺ cells (x200).

Figure 5

Histopathological appearance of an area of severe cellular infiltration of remaining non-biopsied islets (at necropsy, in the same recipient pig as in Figure 4) 5 days after islet Tx into the GSMS. (A) Islet mass in the GSMS (H&E, x40). (B) Polymorphonuclear and mononuclear cell infiltrate in islet mass (H&E, x100). (C) Few insulin-positive beta cells were identified whenever severe cell infiltration of the islet mass was present. Islets were infiltrated particularly by (D) CD3⁺ and (E) CD68⁺ cells (x200). Similar histopathological findings were detected between biopsied islets (Figure 4) and remaining non-biopsied islets.

Figure 6

Histopathological appearance of an area of mild cellular infiltration of islet biopsies and non-biopsied islets5 days after islet Tx into the GSMS (x200). (A) Insulin-positive beta cells and (B) glucagon-positive beta cells in an area of mild cellular infiltration in a biopsy of an islet mass in the GSMS. (C) Insulin-positive beta cells and (D) glucagon-positive cells in an area of mild cellular infiltration of an islet mass in the GSMS that had not been biopsied. Multiple insulin- and glucagon-positive cells were present in areas of mild cell infiltration compared to areas of severe cell infiltration.