Evaluation of porcine pancreatic islets transplanted in the kidney capsules of diabetic mice using a clinically approved superparamagnetic iron oxide (SPIO) and a 1.5T MR scanner

Abstract

Objective: To evaluate transplanted porcine pancreatic islets in the kidney capsules of diabetic mice using a clinically approved superparamagnetic iron oxide (SPIO) and a 1.5T MR scanner.

Materials and methods: Various numbers of porcine pancreatic islets labeled with Resovist, a carboxydextran-coated SPIO, were transplanted into the kidney capsules of normal mice and imaged with a 3D FIESTA sequence using a 1.5T clinical MR scanner. Labeled (n = 3) and unlabeled (n = 2) islets were transplanted into the kidney capsules of streptozotocin-induced diabetic mice. Blood glucose levels and MR signal intensities were monitored for 30 days post-transplantation.

Results: There were no significant differences in viability or insulin secretion between labeled and unlabeled islets. A strong correlation (r(2) > 0.94) was evident between the number of transplanted islets and T(2) relaxation times quantified by MRI. Transplantation with labeled or unlabeled islets helped restore normal sustained glucose levels in diabetic mice, and nephrectomies induced the recurrence of diabetes. The MR signal intensity of labeled pancreatic islets decreased by 80% over 30 days.

Conclusion: The transplantation of SPIO-labeled porcine islets into the kidney capsule of diabetic mice allows to restore normal glucose levels, and these islets can be visualized and quantified using a 1.5T clinical MR scanner.

Keywords: Diabetes; Magnetic resonance (MR); Porcine pancreatic islet; Superparamagnetic iron oxide; Xenotransplantation.

Fig. 1

Accumulation of superparamagnetic iron oxide in porcine islets. A. Graph shows dose-dependent increase of iron accumulation in islets labeled with Resovist. Islet cells labeled with Resovist (100 µg Fe/mL) were found to contain 13.2 ± 3.2 pg of total iron per cell, assuming that pancreatic islets contained 2000 cells on average. Values are expressed as means ± standard deviations of three independent experiments. Differences were considered significant at p values ≤ 0.05. ^*p ≤ 0.05 versus unlabeled. B. Prussian blue staining of labeled islets shows intracellular superparamagnetic iron oxide s (blue color) in all labeled islets. Islets were counterstained with nuclear fast red (red color). C. Transmission electron microscopy of labeled islets shows lysosome or endosome containing superparamagnetic iron oxides (arrows). G = dense granule, ISG = insulin secreting granule, M = mitochondria, N = nucleus

Fig. 2

Islet viability and insulin secretion. A. Viability of labeled and unlabeled islets determined using MTT assays were similar. B. Insulin secretion stimulated by treatment with glucose (3 mM or 16.7 mM) and theophylline (5 mM) showed no difference between labeled and unlabeled islets (p > 0.05). Values are expressed as means ± standard deviations of four independent experiments.

Fig. 3

In vitro MRI of superparamagnetic iron oxide-labeled porcine pancreatic islets. A. Gradient echo T₂*-weighted images of 500 islets suspended in 1% agarose phantom show decreased signal intensity of labeled islets compared to unlabeled islets. B. T₂ relaxation rates (1/T₂) of labeled and unlabeled islets were 12.98 ± 0.01 sec^-1 and 1.002 ± 0.24 sec^-1, respectively (^*p = 0.001). Islets were labeled for 48 hours in medium containing 100 µg Fe/mL of Resovist.

Fig. 4

MRI quantification of transplanted islets in normal mice. Different numbers (100, 250, 1000, and 2500) of porcine islets were transplanted into kidney capsules. A. Gradient echo T₂*-weighted axial images show areas of hypointensity (arrows) in kidney capsules corresponding to different numbers of transplanted islets. B. Graph shows linear correlation (r² = 0.9942) between transplanted islet number and graft volume as quantified on gradient echo T₂*-weighted images. C. Graph shows linear correlation (r² = 0.9356) between islet number and T₂* relaxation time as measured on gradient echo T₂*-weighted images.

Fig. 5

MRI and blood glucose changes in diabetic mice after transplantation of labeled and unlabeled islets. A. Gradient echo T₂*-weighted coronal MR images show hypointense area (arrow in left panel) in left kidney containing 2500 transplanted, labeled islets. No signal drop is seen in kidney with unlabeled islets (arrow in right panel). B. Graph shows blood glucose levels in diabetic mice measured after labeled or unlabeled islet transplantation. Blood glucose levels in diabetic mice returned to normal (≤ 150 mg/dL) 24 hours after transplantation and remained normal for 30 days, but nephrectomy performed at 30 days post-transplantation caused recurrence of elevated blood glucose levels. C. Graph shows results of intraperitoneal glucose tolerance tests performed at 28 days post-transplantation. No significant differences were observed between labeled and unlabeled islets in terms of restoration of normal blood glucose levels. Values are expressed as mean ± standard deviation.

Fig. 6

Correlation between MR signal intensity changes and blood glucose levels in diabetic mice with labeled islets. A. Gradient echo T₂*-weighted axial images show low signal intensity areas (arrows) in capsules of left kidneys of normoglycemic and hyperglycemic mouse at 8 and 25 days of post-transplantation. B. Blood glucose levels measured 25 days after transplantation of labeled islets in normoglycemic mice (n = 2) and hyperglycemic mouse (n = 1) were 120 ± 4.3 mg/dL and 308 mg/dL, respectively. C. Decreases in MRI signal intensities measured 25 days after transplantation of labeled islets in normoglycemic mice (n = 2) and hyperglycemic mouse (n = 1) were 81 ± 1.0% and 68%, respectively.

Fig. 7

Ex vivo MRI and histological analysis of diabetic mouse kidneys containing transplanted islets. A. Ex vivo MRI of kidneys excised 30 days after transplantation shows labeled pancreatic islets (arrow in upper panel) as hypointense areas. B. Prussian blue staining shows labeled pancreatic islets transplanted into kidney capsule (blue in upper panel). Insulin immunostaining shows well-preserved transplanted islets (brown in lower panel).