YouTube video by arvoinfo

YouTube video by arvoinfo

YouTube video by arvoinfo

Aims/hypothesis This study used serial block-face scanning electron microscopy (SBF-SEM), a nanoscale imaging technique in x-y-z planes, to investigate 3D ultrastructural changes in the retinal neurovascular unit (NVU) associated with diabetes. We hypothesised that this approach would reveal previously uncharacterised pathological alterations that contribute to the development of diabetic retinal disease (DRD). Methods Retinas from male diabetic and non-diabetic mice, as well as from human male donors with and without diabetes, were prepared for SBF-SEM imaging. Retinal tissue was microdissected, fixed and embedded for serial sectioning and 3D reconstruction. Ultrastructural analysis of the NVU was performed in capillary regions exclusively within the superficial vascular plexus of both mouse and human retinas. Image stacks were processed using Microscopy Image Browser for contrast normalisation and segmentation, with 3D visualisation performed in Amira software. Quantitative analyses were conducted on pericyte–endothelial cell peg-and-socket formations, cell–basement membrane (BM) interactions, endothelial tubule formation and vascular BM thickness. Results SBF-SEM revealed novel 3D ultrastructural changes in the retinal NVU of diabetic mice and humans, including: (1) partial detachment and reduced frequency of pericyte–endothelium peg-and-socket formations (p<0.05–0.001); (2) localised detachment of endothelial cells and pericytes from the vascular BM (p<0.05–0.01), along with macroglial cell retraction from the outer vascular BM; and (3) increased formation of endothelial tubules (p<0.01–0.001). These changes were observed in the absence of any obvious vascular BM thickening, as no significant differences in mean or maximum BM thickness were found between diabetic and non-diabetic retinal capillaries analysed in this study. Conclusions/interpretation This study provides new insights into the early ultrastructural changes in the retinal NVU in DRD, offering a basis for a better understanding of the pathological processes that contribute to the development of this disease. Data availability Links to all raw image stacks analysed in this article are available at https://doi.org/10.5281/zenodo.15210333 . The MATLAB vascular BM thickness measurement script is available at the GitHub link https://github.com/Curtis-WWIEM/BM_thickness . Graphical Abstract

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