Abstract
Purpose :
Mouse models have greatly increased our understanding of retinal biology and the contributing cellular populations in disease. Transgenic manipulation in mice is challenging however and only reliably permits labelling of up to three proteins simultaneously with fluorophores such as GFP. This restriction limits the linkage of in vivo features with multicellular changes in the retina or the detection of rare cell sub-populations. Here, we introduce a technique that facilitates the alignment of multimodal imaging and retinal OCT with immunohistochemistry, incorporating up to nine distinct markers.
Methods :
The laser choroidal neovascularisation (CNV) model was initiated in wild-type 10-week-old C57BL/6J mice using the Phoenix Micron V image-guided laser system. All experiments were performed in accord with the ARVO statement for the use of animals in ophthalmic and vision research. A single burn per eye was made with spot size 75μm, 150mW and 0.07s duration and assessed 3 days later. A custom-built multimodal system was then used to image the CNV lesion with en face OCT and OCT-angiography. The corresponding retinal flat mount underwent immunohistochemistry with Alexa Fluor (AF) fluorophores followed by tissue clearing with Ce3D reagent (Biolegend). The retinal lesion was imaged on a Stellaris 8 confocal microscope (Leica Microsystems).
Results :
The combination of en face OCT with 3D immunohistochemistry is feasible using OCT-angiography, then alignment against vascular staining on cleared retinal tissue. Imaging nine distinct markers is facilitated by the white light laser capability of the Stellaris 8. We have successfully generated datasets including the following cell types with markers for nuclei (DAPI), cones (PNA, AF488), activated Muller glia (GFAP, AF532), microglia (Iba1, AF555), vascular endothelium (Isolectin B4, AF568), leukocytes (CD45, AF594), vasculature (Collagen IV, AF647), outer limiting membrane (CD44, AF680) and arteries (aSMA, AF750). Rendering of 3D imaging datasets was performed in Imaris v10.1 software.
Conclusions :
This approach allows the alignment of nine immunohistological markers on mouse retinal flat mounts with pathological features seen on OCT imaging to better characterise multicellular tissue changes. Ongoing refinements in highly multiplexed immunohistochemistry may allow this limit to be increased in the future.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.