There is increasing evidence that alterations in the choroidal vasculature play a key role in the early pathogenesis of age-related macular degeneration (AMD) (Seddon et al., 2016; Mullins et al., 2011; McLeod et al., 2009). The understanding of pathologic processes in the choroidal blood vessels requires an understanding of normal choroidal vascular structure and angioarchitecture. The choroidal vasculature has been extensively investigated in human and in experimental animals by various methods including histology, India ink injections, flat preparations, neoprene latex, and vascular corrosion casts, and alkaline phosphatase staining (Weiter and Ernest, 1974; Hayreh, 1975, 1990; Yoneya and Tso, 1987; Bhutto and Amemiya, 2001; McLeod and Lutty, 1994; Jiao et al., 2020). Clinically, in vivo imaging includes conventional techniques using both fluorescein (FA) and indocyanine green angiography (ICGA) (Gelisken et al., 1998; Yannuzzi, 2011). In addition, optical coherence tomography angiography (OCTA) (de Carlo et al., 2015a, 2015b; Wong et al., 2011) and spectral domain optical coherence tomography (SD-OCT) are now routinely used in the clinic (Margolis and Spaide, 2009; Mrejen and Spaide, 2013; Ferrara et al., 2016; Koh et al., 2017; Wang et al., 2018). Although, the general distribution and pattern of the choroidal vasculature of primates and other species including rodents has been described, a reliable method for the qualitative and quantitative histological assessment of choroidal vasculature in pigmented mice would improve and benefit from the removal of melanin pigment. Here we describe a simple, reliable method for visualizing the choroidal vasculature in whole mounts of pigmented mouse eyes using a pre-bleaching technique compatible with immunostaining with anti-podocalyxin antibody. While the method was initially developed using normal pigmented mouse eyes (Bhutto et al., 2020), we subsequently compared the morphometric results with choroidal flat-mounts from a subretinal sodium iodate (NaIO3) retinal pigmented epithelial (RPE) atrophy model and a laser-induced choroidal neovascularization (CNV) model.

Currently, the NaIO3-induced RPE atrophy model and laser-induced CNV model are among the most widely used experimental mouse models of dry and wet AMD. The laser-induced CNV model, which leads to the growth of new blood vessels from the choroid into the subretinal space, mimics the main characteristic of the exudative form of human AMD (Tobe et al., 1998; Pennesi et al., 2012; Lambert et al., 2013) and provides an opportunity to explore the molecular mechanisms of CNV. The NaIO3-induced retinal degeneration model is universally used as a preclinical model of atrophic AMD and geographic atrophy (GA) (Machalinska et al., 2010; Jian et al., 2015; Chowers et al., 2017). NaIO3 is thought to directly affect the RPE cells with secondary effects on photoreceptors and the CC. The effects of systemic NaIO3 injection on retinal degeneration has been described in different mammalian species including swine, rabbit, rat and mouse with varying doses and routes of administration (Mones et al., 2016; Petrus-Reurer et al., 2017; Koh et al., 2019; Machalinska et al., 2010; Chowers et al., 2017). The systemic delivery, however, results in sporadic, widespread RPE degeneration. We recently reported a novel model in which a single 1 μl subretinal injection of NaIO3 created a focal area of RPE atrophy which was followed by photoreceptor cell loss, subretinal glial membrane formation, and CC degeneration in Brown Norway pigmented rats (Bhutto et al., 2018). We report herein the choroidal vascular changes in the mice given subretinal NaIO3 injections at 3 weeks following injection. This timepoint was selected based on atrophic progression in the rat model which includes RPE loss by three days, extensive photoreceptor loss by seven days and choriocapillaris changes by 14 days post-injection (Bhutto et al., 2018).

The aim of this study was to develop a simple reliable method to stain choroidal vasculature in highly pigmented normal mouse eyes and to use the technique to perform semi-quantitative morphometric analysis of pathologic changes in choroidal vasculature in two murine models of AMD. Lastly, we compared the morphometric features in these models to previous results from human choroids with GA and neovascular AMD (nAMD).