Quantification and three-dimensional imaging of the insulitis-induced destruction of beta-cells in murine type 1 diabetes

Abstract

Objective: The aim of this study was to refine the information regarding the quantitative and spatial dynamics of infiltrating lymphocytes and remaining beta-cell volume during the progression of type 1 diabetes in the nonobese diabetic (NOD) mouse model of the disease.

Research design and methods: Using an ex vivo technique, optical projection tomography (OPT), we quantified and assessed the three-dimensional spatial development and progression of insulitis and beta-cell destruction in pancreata from diabetes-prone NOD and non-diabetes-prone congenic NOD.H-2b mice between 3 and 16 weeks of age.

Results: Together with results showing the spatial dynamics of the insulitis process, we provide data of beta-cell volume distributions down to the level of the individual islets and throughout the pancreas during the development and progression of type 1 diabetes. Our data provide evidence for a compensatory growth potential of the larger insulin(+) islets during the later stages of the disease around the time point for development of clinical diabetes. This is in contrast to smaller islets, which appear less resistant to the autoimmune attack. We also provide new information on the spatial dynamics of the insulitis process itself, including its apparently random distribution at onset, the local variations during its further development, and the formation of structures resembling tertiary lymphoid organs at later phases of insulitis progression.

Conclusions: Our data provide a powerful tool for phenotypic analysis of genetic and environmental effects on type 1 diabetes etiology as well as for evaluating the potential effect of therapeutic regimes.

FIG. 1.

Islet β-cell distribution over time in NOD.H-2b and NOD mice. A–J′: Isosurface rendered OPT images of representative NOD.H-2b splenic (A–E), duodenal (A′–E′), and NOD splenic (F–J) and duodenal (F′–J′) pancreata labeled for insulin (red). The pancreas outline (gray) is based on the signal from tissue autoflourescense. In contrast to NOD.H-2b mice (A–E′), the expected progressive destruction in islet β-cell volume is clearly observed in NOD mice (F–J′). Scale bar corresponds to 2.3 mm in I′; 2.1 mm in C; 2 mm in D, D′, J, J′, and G′; 1.9 mm in B and H; 1.8 mm in I and H′; 1.7 mm in C′ and E′; 1.6 mm in A, E, and F′; 1.5 mm in G; 1.4 mm in A′ and B′; and 1.2 mm in F. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

NOD β-cell decay by islet β-cell volume size categories. A–E: Isosurface rendered OPT images of representative islet β-cell distributions in 3-, 6-, 8-, 12-, and 16-week-old NOD mice (splenic lobe). Individual islet β-cell volumes are reconstructed based on the signal from insulin-specific antibodies and have been pseudo colored to highlight the distribution of large (>5 × 106 μm³ [yellow]), intermediate (1–5 × 106 μm³ [red]), and small (<1 × 106 μm³ [white]) islets. Scale bar in corresponds to 2 mm in E, 1.9 mm in C, 1.8 mm in D, 1.5 mm in B, and 1.2 mm in A. F: Graph illustrating the progressive loss of Ins+ islets in 3- to 16-week NOD pancreas (duodenal and splenic) broken down to size categories. The values on the y-axis corresponds the 10 log value of the average number of Ins+ islets within each size category ± SE. The numbers on each bar corresponds to the average number of Ins+ islets within each size category. G: Graph illustrating individual variations in total pancreatic β-cell volume in NOD mice at 3, 6, 8, 12, and 16 weeks. Relatively unaffected individuals (black bars) with a total β-cell volume over 12 × 109 μm³ (red line) can be identified at both 12 and 16 weeks. n = 5 for 3, 6, 8, and 16 weeks and n = 4 for 12 weeks. Significance levels for the decay of β-cell volume and number of islets are indicated. *P < 0.05; **P < 0.01. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

A and D: Graphs showing the average whole-pancreas volume (duodenal and splenic) at 3, 8, and 16 weeks and in NOD.H-2b mice (A) and in NOD mice (D) at 3, 6, 8, 12, and 16 weeks. B and E: Average total pancreatic β-cell volume in NOD.H2-b (B) and NOD (E) mice. C and F: Average islet number in NOD.H2-b (C) and NOD (F) mice. n = 5 for NOD at 3, 6, 8, and 16 weeks, n = 4 for NOD at 12 weeks, and n = 3 for NOD.H-2b at 3, 8, and 16 weeks. Values are given ± SE. Significance levels for the decay of β-cell volume and number of islets are indicated. *P < 0.05; **P < 0.01.

FIG. 4.

A: Graph showing the average volume of the 10 largest islet-cell volumes in 6- and 8- and 12- and 16-week-old NOD mice (in the latter group only animals with a total pancreatic β-cell volume over 12 × 109 μm³ indicated by filled bars in Fig. 2G were included). The average size of the 10 largest islet β-cell volumes increase from 6 to 16 weeks (P = 0.011). n = 5 for 6 and 8 weeks and n = 4 for 12 and 16 weeks. B: Graph showing the average volume of the 10 largest islet-cell volumes in 8- and 12- and 16-week-old NOD.H-2b mice.

FIG. 5.

Comparison of overt diabetic and nondiabetic NOD sibling pairs. A and B′: Isosurface rendered OPT images of representative diabetic (A and A′) and nondiabetic (B and B′) splenic (A and B) and duodenal (A′ and B′) pancreata labeled for insulin (red). The pancreas outline (gray) is based on the signal from tissue autoflourescense. C: Graph showing the average total β-cell volume in overt diabetic and nondiabetic NOD sibling pairs (n = 5 for each group, pairs analyzed at 14, 14, 18, 18, and 23 weeks) and in NOD.H 2b mice at 16 weeks (n = 3) ± SE. Scale bar is 2.2 mm in A′ and 2 mm in A, B, and B′. Significance levels are indicated, *P < 0.05; **P < 0.01. (A high-quality color representation of this figure is available in the online issue.)

FIG. 6.

Spatial assessment of the progression of autoimmune insulitis in the NOD mouse. A–E: Isosurface rendered OPT images of representative pancreata (duodenal) from NOD mice at 3, 6, 8, 12, and 16 weeks. Ins+ islets (red) are reconstructed based on the signal from insulin-specific antibodies and infiltrating T-cells (green) based on the signal from CD3-specific antibodies. A′–E′′, insets: High-magnification views corresponding to the enclosed boxes in A, D, and E, respectively. n = 3 for 3, 6, 8, and 12 weeks; n = 5 for 16 weeks. Ins, insulin. F and G: Sections of a pancreata from a 14-week-old female NOD mouse stained with DAPI, anti-CD3 (green), and anti-CD19 (red) (F) or anti-CD3 (green) and anti-MAdCAM-1 (red) (G). F: B-cell areas (arrow head) and T-cell areas (arrow) are indicated by arrow head. G: High endothelial venules (arrow head). The scale bar in E corresponds to 2 mm in E, 1.5 mm in C, 1.4 mm in B, 1.3 mm in D, and 1.0 mm in A. The scale bar in E″ corresponds to 1 mm in E″, E′, D″, D′, and 0.76 mm in A″, A′. Scale bar in G corresponds to 100 μm in F and G. Scale bar in I corresponds to 100 μm in H and I. (A high-quality color representation of this figure is available in the online issue.)

FIG. 7.

Comparision of α- and β-cell distribution in 12w NOD and NODH2b mice. A: and F: Volume renderings of β-cell distribution. Cryosections in B–E and G–J correspond to red pseudocolor in A and F, respectively. Sections were counterstained against glucagon (C, E, H, and J) and DAPI (D and I). Note the remaining Glu+ cells in the highly infiltrated NOD islets (C and E). B and G: Ins+ β-cells. Scale bar in A and F corresponds to 1 mm. Scale bar in J corresponds to 100 μm in B–E and G–J. (A high quality color representation of this figure is available in the online issue.)