Light-Sheet Imaging of Intact Retinal Vasculature and Quantitative Analysis in Murine Model

Abstract

Purpose : Oxygen induced retinopathy (OIR) is one of the leading causes of childhood blindness. The conventional means studying the OIR in the murine model is limited in 2-D imaging and analysis.We hereby integrate the dual-illumination light-sheet fluorescence microscopy and modified passive CLARITY for retina (MPCR) to reveal complete primary and deep capillary plexus in the intact 3-D retinal vascular system.

Methods : Retinas were dissected from C57BL/6 mice at P12 were fixed and transferred to monomer solution (4% Acrylamide (wt/vol), 0.05% Bis-Acrylamide (wt/vol), and 0.25% VA-044 initiator (wt/vol) in PBS). After thorough incubation, we placed the sample in 37°C incubator for hydrogel polymerization. The retinas were then placed into a clearing solution comprising 4% w/v sodium dodecyl sulfate (SDS) and 1.25% w/v boric acid (pH 8.5). After clearing, the retinas were immunostained with Isolectin (IB4) conjugated with Alexa-488 to complete the vascular staining. Filament tracing was applied to the entire 3-D vascular network for quantifying the vascular connectivity.

Results : Our MPCR method optimized the tissue-hydrogel hybrids to support the intact hemispheric structure of the retina (Fig. 1A). We revealed the vascular network of the entire retina, including supeficial capillary and deep vascular plexuses distributed from the nerve fiber layer (NFL) to outer plexiform layer (OPL), bypassing the conventional flat-mount sample preparation (Fig. 1B). One volume of interest (VOI) was quantified to reveal the vascular connectivity; thus, demonstrating the feasibility to analyze the morphological and topological difference in vascular development and diseases (Fig. 1C).

Conclusions : Overall, the integration of optical clearing and dual-illumination LSFM elucidate structural phenotypes to advance the field of micro-vascular development and injury.

This is a 2020 ARVO Annual Meeting abstract.

 

Figure 1. A₁. Anatomy structure of mouse eye. Postnatal (12 days) mouse retinas (A₂) before and (A₃) after MPCR. B₁. Intact 3-D retinal vascular network and VOI (blue box). B₂. Magnified views of Primary and secondary plexuses were presented. C₁. 3-D rendering and C₂.Filament tracing as well as connectivity quantification for one VOI. Scale bars: (A) 1 mm, (B₁) 500 µm, (B₁) 200 µm, (C) 100 µm.