<em>In Vivo</em> Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Abstract

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period. The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch's membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch's membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ. This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

Combination of AIBP, apoA-I, and Aflibercept Overcomes Anti-VEGF Resistance in Neovascular AMD by Inhibiting Arteriolar Choroidal Neovascularization

“… 43 The retinal thickness in CNV lesion areas was measured from nerve fiber layer to RPE. 44 , 45 Three to four measurements from different locations from each CNV lesion were taken.…”

A New Experimental Model of Krypton Laser-Induced Choroidal Neovascularization in Tree Shrew

“…Symantas et al recently proposed that SD-OCT provides a detailed CNV pathology within retinal tissue. SD-OCT images of CNV formation, liquid accumulation, and scarring tissue on mouse CNV model were displayed in his research 49 . In our study, liquid accumulation was not observed in the SD-OCT of tree shrew CNV lesions (Fig.…”

Neovascular Progression and Retinal Dysfunction in the Laser-Induced Choroidal Neovascularization Mouse Model

“…The choroidal neovascularization was induced in mice’s retinas (n = 30 LI-CNV mice and n = 6 control mice) by applying a green laser to rupture Bruch’s membrane as described [ 44 , 45 , 46 , 47 , 48 ]. Briefly, once the animal was anesthetized, pupils were dilated with Tropicamide eyedrops (and the animal was placed in the Micron III platform (Phoenix Research Labs., Pleasanton, CA, USA), putting the lens in contact with the cornea.…”