Innovative 3D approach to evaluate extracellular matrix in pancreatic islets using Light Sheet Fluorescence Microscopy

Session

Poster Session Two

Authors

Matias Ramirez Acosta (2), Pierre Gianello (2), Caroline Bouzin (1)

Affiliations

1. IREC Imaging Platform / Université catholique de Louvain
2. Laboratory of Experimental Surgery and Transplantation / Université catholique de Louvain

Keywords

Light sheet fluorescence microscopy

3D image analysis

Pancreatic islets

Type 1 diabetes

Extracellular matrix

Hypoxia

Abstract text

Summary

We developed a protocol for clearing, multiplex immunofluorescence, 3D light sheet fluorescence microscopy (LSFM) and digital image analysis (LSM) to evaluate changes in pancreatic islets’ extracellular matrix (ECM), in the context of islet transplantation.

Introduction

Pancreatic islets can be transplanted to treat type 1 diabetes mellitus. However, this approach is limited by reduced survival and function of islets, mainly due to hypoxia and anoikis, an apoptotic process triggered by elimination of ECM during islet isolation.

To evaluate the impact of hypoxia on ECM, we developed an innovative imaging approach, based on tissue clearing and 3D microscopy, to visualize and quantify up to four concomitant immunofluorescence (IF) stains. We evaluated three ECM proteins, collagen 4, fibronectin and laminin, and E-cadherin, an adhesion protein involved in anoikis.

Materials and methods

We cultured human islets in normoxic (O2 21%) or hypoxic (O2 1%) conditions. After fixation, they were submitted to an IF protocol consisting in blockage of unspecific epitopes using BSA, followed by 3-day antibody incubations, using different combinations of antibodies targeting insulin, glucagon, carbonic anhydrase 9 (CA9), collagen 4, fibronectin, laminin, E-cadherin, and Hoechst as a nuclear stain. Islets were then cleared using an adaptation of the CUBIC technique, mounted in agarose rods, and imaged using a LSFM. Imaris® was used for 3D rendering, morphological evaluation, and quantification, using ad-hoc protocols.

Results/Discussion

Insulin and glucagon allowed islet identification, and the hypoxia marker CA9 certified hypoxic conditions.

Volumetric analysis of target proteins showed no significant differences amongst experimental groups. However, 3D projections permitted the identification of specific morphological features differentiating islets cultured in normoxic and hypoxic conditions. 

In hypoxia, we could highlight areas or scattered nuclei and clusters of ECM proteins, evoking scarring tissue (Figure 1). Interestingly, E-cadherin was absent in these areas (Figure 2), in agreement with what is described in the literature.

Under normoxic conditions, areas of scarring were also identified, although smaller than in hypoxia. Collagen 4 was found coating blood vessels (Figure 3).

Taken together, our results show that employing a three-dimensional method for morphological evaluation offers comprehensive understanding of protein distribution, revealing distinctions between groups that cannot be grasped through simple total protein quantification. Indeed, hypoxic islets presented similar quantities of proteins than normoxic islets, although their distribution corresponded to tissue scarring. This is in line with the diminution of functional mass shown by transplanted islets, which suffer from a period of low oxygenation immediately after grafting.

Conclusion

Using 3D assessment of islets structure, we highlighted ECM organizational changes during hypoxia, providing morphological evidence of anoikis. This methodology can support the development of new strategies to improve islet transplantation.

Figures

Figure 1. Slice view of an islet, showing a central scar, characterized by condensed fibres of ECM (fibronectin, in green), and scarce nuclei (white), which present altered morphology, like irregular shape and small size.

Figure 2. Slice view of an islet, showing e-cadherin-positive cells (orange), localized in the periphery of the islet. A central accumulation of CA9-positive cells (cyan) suggests a region of cellular hypoxia.

 

Figure 3 – 3D projection of Collagen 4 IF, where we can identify scattered areas of signal (yellow) and structures consistent with blood vessels (light blue).

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