Simultaneous comparison of mouse laser-induced choroidal neovascularization histology with visible-light optical coherence tomography angiography

Abstract

Purpose : Choroidal neovascularization (CNV) is the hallmark of neovascular AMD. We applied visible-light optical coherence tomography angiography (vis-OCTA) to longitudinally monitor laser-induced CNV in mice over the span of fourteen days after induction and compare our angiograms with post-mortem immunohistological stains.

Methods : Twenty-five adult pigmented mice of mixed background were anesthetized and subjected to laser induced CNV following established protocols. Four lesions were created around the optic nerve. Laser shots that did not successfully rupture Bruch’s membrane were excluded from analysis. To monitor the development and regression of the CNV lesion, retinal blood vessels were imaged in three dimensions with vis-OCTA from days 2 through 14 post laser injury. To compare ex vivo histology with the in vivo angiograms, we performed two sets of immunohistological stains to highlight mature and immature vessels. To highlight mature, perfused vessels, we performed intracardiac injection with isolectin B₄ 488 immediately before euthanasia. After euthanasia, the eyes were enucleated, fixed in paraformaldehyde, and to highlight all the endothelial cells in the area and, therefore, all the immature vessel sprouts, we stained the choroids with isolectin B₄ 594. The final double-stained choroids were imaged with confocal microscopy.

Results : Comparing the in vivo images obtained from vis-OCTA (Figure A) to the ex vivo immunostained flat mount (Figure B) shows consistency between the two techniques. The vessels visualized by the vis-OCTA are representative of the vessels visualized via immunohistological staining of the flat mount. We compared measured areas of the CNV lesion between the vis-OCTA and immunostained images. From this, we found that vis-OCTA can reliably detect CNV at five days post laser-injury.

Conclusions : Vis-OCTA of CNV correlates well with the true immunohistological morphology seen on choroidal flat mounts. Because the mouse laser induced CNV model is a widely accepted model for human neovascular AMD, vis-OCTA holds great promise for further study of the pathophysiology of CNV in rodents.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

A) Vis-OCTA of a CNV lesion Day 7 post laser induction colored by depth (see color bar). B) Ex vivo confocal microscopy of immunostained choroidal flat mount of the same CNV lesion. Arrows highlight same areas on both images. Scale bars: 100 um.