The three dimensional (3D) ultrastructure of VEGF driven choroidal neovascularisation in the rat

Abstract

Purpose : A rat model for choroidal neovascularisation (CNV), based on VEGF overexpression in the retinal pigment epithelium (RPE), was used to investigate the ultrastructure of pathological angiogenesis as it also occurs in wet age related macular degeneration (AMD). Although CNV gross structure can be investigated with modern ophthalmic techniques, subcellular features of the neovessels and the extracellular matrix formation are not yet understood.

Methods : 2x10⁹ AAV-VEGF vector particles, diluted in 2 µl PBS were subretinally injected in 88 eyes of female Long Evans rats to establish the rat model of CNV. CNV formation was monitored by a multimodal imaging approach spanning from in vivo funduscopy, optical coherence tomography and angiography to immunohistochemistry and electron microscopy (EM). Serial sectioning and focused ion beam scanning EM tomography (Zeiss xb550) revealed the 3D structure of the newly formed vessel system. Segmentation and reconstruction of individual features were performed with Amira and Image J/Fiji software tools.

Results : The vasculature of choroid, choriocapillaris and CNV were each followed through the whole region of interest. RPE reorganization, branching of individual vessels and e.g. the location of their blood components (red and white blood cells, fibrin, etc.) can be localized and correlated to the pathology. By segmentation and 3D reconstruction particular areas of interest like suspicious RPE vacuolization, lipofuscinogenesis and pigment clustering occurring only in the CNV area were visualized.

Conclusions : The 3D projection of the CNV body allows investigating the vessel “tree” as it is growing through its bed of matrix and cells, e.g. proliferating RPE. The high lateral resolution of the EM images allows the investigation of endothelial junctions, fenestrations and possible origins of leakage in relation to their direct neighbourhood and the gross picture of the CNV depicted by serial light microscopy. The VEGF rat model is based on the same growth factor overexpression that is involved in human CNV formation and reflects many of its ultrastructural features, like pathological angiogenesis originating from the choriocapillaris, RPE proliferation and immense matrix formation, especially collagen in over 90 % of transduced eyes. It is therefore ideal for the investigation of new drug candidates in wet AMD therapy.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.