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Marie Darche, Morgane Belle, Alain Chedotal, Jose Courty, Michel Paques, Ilaria Cascone; 3D visualization and analysis of vascular and perivascular networks in the eye using light sheet microscopy. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4699. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Pathological microvascular remodeling and/or angiogenesis are involved in a variety of ocular pathologies. It has recently been shown that light-sheet fluorescence microscopy (LSFM) provides high speed imaging, high signal-to-noise ratio and low level of photobleaching on large (centimetric) tissue samples. In this work, we demonstrate the feasibility of this approach for imaging the mouse eye in toto, exemplified by the 3D visualization of the entire ocular vascular network.
We combined LSFM with the tissue preparation technique called iDISCO+ clearing (adapted from Renier et al. 2016) on intact albino mouse eyes. The acquired serial images were then reconstructed in 3D and analyzed using Imaris software. To obtain optimal clearing and tissue preservation, we tested different protocols of sample preparation, bleaching, fixation and clearing. Several antibodies and co-immunostaining parameters were tested in order to find the best markers for the vascular (anti-collagen IV, anti-CD31 and anti-Meca32) and perivascular (anti-NG2, anti-PDGFr and anti-αSMA) networks.We determined the best imaging process to successfully stain non-dissected mouse eyes and perform fast acquisitions (20 minutes to acquire an entire eye in two wavelengths).
Vascular networks of the iris, the ciliary body, the retina, the optic nerve and hyaloid vessels were reconstructed in 3D with a resolution allowing the observation of capillaries and pericytes. Modelization of the different structures allowed the morphometric analysis of the networks.
We showed that LSFM and iDISCO+ clearing combination is an interesting tool to study vascular and perivascular networks in the intact eye. Contrary to traditional imaging techniques, this method is rapid, non-destructive, and can be used to analyze networks in their natural spatial organization. This method will facilitate the exploration of ocular vascular diseases.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
3D reconstruction of the iris and ciliary body vascular networks (stained with anti-MECA 32) and the retinal vascular network (stained with anti-collagen IV), acquired using LSFM and iDISCO+ clearing.
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