Imaging inflammation of the pancreatic islets in type 1 diabetes

Abstract

Type 1 diabetes is the clinical manifestation of aberrant leukocytic infiltration of the pancreatic islets; it is usually diagnosed only very late in disease progression, after the critical autoimmune phenomena have mostly played out. A noninvasive means of directly monitoring the evolution of islet infiltrates would have important research and clinical applications. We have exploited fluorescence and MRI of long-circulating magnetofluorescent nanoparticles to visualize micro-vascular leakage, as an indicator of inflammation, in pancreata of mouse models of type 1 diabetes ex vivo or in vivo. We could detect the onset and evolution of insulitis in vivo and in real time, permitting us to study the natural history of diabetes in individual animals.

Figures

Fig. 1.

Ex vivo confocal microscopic imaging of microvascular leakage accompanying insulitis in BDC2.5 TCR tg mice. We injected 4-wk-old female BDC2.5/NOD or NOD/RAG–/– mice with 10 mg/kg CMFN diluted with PBS to 150 μl or with PBS only. Twenty-four hours later, they were i.v. injected with 25 mg/kg DTZ, and, after 5 min, the whole pancreas was removed and imaged by confocal microscopy using a ×10 objective. Islets were identified by DTZ staining (red), and signal from the CMFN probe (green) was visualized and quantitated. (a) CMFN probe accumulation, reflective of microvascular leakage, as an accompaniment to islet infiltration. Images are representative of islets from at least three animals of each type in each condition. (b) Procedure to quantitate probe accumulation. Custom-designed image-analysis software was used to measure the MFI of CMFN over ROI that represent islets (identified by DTZ staining) or exocrine tissue (an area selected as distinct from islets and major vessels). (c) Quantitative comparison of insulitic and noninsulitic pancreata. We took 13 4-wk-old BDC2.5/NOD and age-matched NOD-RAG–/– animals. Symbols represent the MFI of individual animals calculated as the average MFI of all of the islets (–10) acquired per animal. Bar represents the average value.

Fig. 2.

CMFN probe uptake by macrophages in the vicinity. We injected 4-wk-old female BDC2.5/NOD and Ea16/NOD mice with CMFN, as described in the legend to Fig. 1. Twenty-four hours later, animals were anesthetized and i.v. injected with 25 mg/kg Hst to stain nuclei, and after 5 min, they were perfused through the left ventricle with PBS/heparin, followed by 5 ml of 10% neutral-buffered formalin. The pancreas was further fixed overnight in 10% neutral-buffered formalin. It was placed in glycerol-based mounting medium and kept for 1 wk until transparent enough to visualize. (a) A noninfiltrated vs. an infiltrated islet. Images are representative of those from at least two animals of each genotype. CMFN, green; Hst, blue. The mass of blue nuclei corresponds to lymphocytic infiltrate, as identified histologically. A ×25 objective was used. (b) Higher magnification. Images reveal the uptake of CMFN (green) by cells of macrophage-like morphology. Nuclei of infiltrating, mostly lymphoid, cells are stained blue with Hst. A ×40 objective was used. (c) Identification of the cellular repository by flow cytometry. A group of three 4-wk-old female BDC2.5/NOD mice was used per sample. One group was left uninjected, whereas the other group was i.v. injected with 10 mg/kg CMFN and was otherwise treated as described above except that no counterstain or fixative was administered. After perfusion, the pancreas was treated with collagenase, washed, and made into a single-cell suspension for staining. CMFN-positive cells were gated as shown in Upper, and staining by anti-CD11b and CD11c reagents is shown.

Fig. 3.

More aggressive insulitis leads to more probe accumulation. (a) At initiation of insulitis. Two- or 3-wk-old female BDC2.5/NOD or NOD-RAG–/– mice were injected with CMFN (or PBS) and DTZ, and their pancreas were imaged ex vivo, as described in the legend to Fig. 1. A 10× objective is shown. (b) Through the evolution of insulitis. The procedures were the same as described above except that animals ranged in age from 2 to 15 wk. MFIs were calculated as described in the legends to Fig. 1 b and c. Symbols represent values for individual islets (i) or averages of all islet values for individual animals (ii). Average MFIs of CMFN signals for pancreata of individual animals were correlated with the fraction of the animal's islets that were infiltrated, which was assessed from hematoxylin–eosin (H&E)-stained sections of the same pancreas used for imaging. (c and d) According to the composition and appearance of the infiltrate. Pancreata were obtained from CMFN/DTZ-injected NOD variants protected from insulitis because of a lack of α/β T and B cells (ci) or to an augmentation of regulatory T cell numbers or activity (cii), or from CMFN/DTZ-injected BDC2.5 TCR tg mice on different genetic backgrounds, promoting greater insulitis aggressivity in the following order: BDC2.5/NOD (ciii) < BDC2.5/B6.H-2g7g7 (civ) < BDC2.5/NOD-RAG–/– (cv). Images were taken with a 10× objective. Images were quantitated and MFIs surrounding the islets plotted (di) and correlated with insulitis intensity revealed by H&E histological analysis (dii).

Fig. 4.

Ex vivo imaging of leakage accompanying insulitis in NOD mice. (a and b) MFIs of CMFN signal associated with pancreatic islets of insulitic NOD vs. noninsulitic NOD-RAG–/– female mice. Insulitis becomes evident in NOD mice at 6–8 wk. Animals were injected and pancreata processed for ex vivo confocal microscopic imaging as described in the legend to Fig. 1. Values reflect signal over individual islets (a) or the average signal over all of an animal's4–10 scored islets (b). P ≤ 0.005 at 6–10 wk, and P < 0.00001 at 12–15 wk in a; and P = 0.0002 at 12–15 wk in b. (c) Correlation between the average MFIs of CMFN signal over the islets and the fraction of infiltrated islets in individual NOD mice sorted according to age. Values were calculated as described in the legend to Fig. 3biii.

Fig. 5.

In vivo visualization of infiltrated pancreata. We anesthetized 4-wk-old female insulitic BDC2.5/NOD or noninsulitic Eα16/NOD mice and immediately “preimaged” them by MRI; i.v. injected them with 40 mg/kg MION-47; and, after 24 h, anesthetized and “postimaged” them by MRI. ROI were drawn around different organs. (a) Comparison of preimage and 24-h postimage values for the two strains. Symbols represent values for individual pancreata, and their correlation is explained in Methods. P values were significantly different between the 24-h postimages of BDC2.5/NOD and Eα16/NOD mice (P = 0.0001) and the preimages and 24-h postimages of BDC2.5/NOD mice (P < 0.00001). (b) Parallel comparisons with muscle tissue. Upper shows delineation of ROIs, and Lower shows the values for muscle and pancreas. (c) Correlation between degree of islet infiltration, calculated as described in the legend to Fig. 3biii, and accumulation of MION probe.

Fig. 6.

A noninvasive, real-time longitudinal study of islet infiltration. Imaging of MION probe accumulation in organs of individual BDC2.5/NOD or Eα16/NOD female mice, as described in the legend to Fig. 5. Values reflect the ratio of relaxation rate measured over ROIs encompassing the pancreas and muscle. Values for individual animals (a) or average values for all individuals of each strain (b) are given.