Cloning and Characterization of a Mouse Model for Retinal Vasculopathy: Implications of Astrocyte Involvement

Abstract

Purpose:: The Neuromutagenesis Facility (NMF) at The Jackson Laboratory generated a mouse mutant, using N-ethyl-N-nitrosourea-induced mutagenesis, which demonstrates vitreal fibroplasia and hypervascularization of the retina. The present study aimed to identify the gene mutated in these mice while also charactering the observed clinical and histological phenotypes.

Methods:: Affected mice were first identified using indirect ophthalmoscopy between three and six weeks of age. After progeny testing demonstrated a recessive inheritance pattern, mice were crossed to DBA/2J for high resolution mapping. The critical region was identified and candidate genes screened by sequencing.The clinical phenotype of these mice was studied longitudinally using indirect ophthalmoscopy, fluorescein angiography, and electroretinography. The vascularization defect was investigated further using ADPase staining. Hematoxylin and eosin staining, immunohistochemistry, and electron microscopy were used for histological assessment of retinal and astrocyte morphology.

Results:: The gene mutated in NMF 223 mice lies on chromosome 17. Fundus examination of NMF 223 mice showed retinal spotting and vessel tortuosity in addition to vitreal fibroplasia and hypervascularization. Although normal at three months of age, ERG b-wave amplitudes were reduced at six months compared to wildtype mice and worsened slightly thereafter. This clinical phenotype corresponds to the progressive thinning observed in the peripheral inner nuclear layer beginning at three months of age. Histological assessment also demonstrated the presence of abnormal vitreal cells and vitreal vessels in mice of all ages. These vitreal vessels, which were more clearly visualized with ADPase staining, were endostatin-positive, suggesting they are hyaloid remnants. Electron microscopy revealed the disruption of the inner limiting membrane as well as the presence of astrocytes in the vitreous. Immunohistochemical staining of glial fibrillary acidic protein demonstrates areas devoid of astrocytes as well as astrocytic hyperstellation.

Conclusions:: NMF 223 is a potential model for studying both normal and pathological retinal angiogenesis as well as astrocyte development.

Keywords: retinal glia • retinal development • gene mapping