Immunohistochemical analysis of gap junction protein connexin43 following retinal ischaemia and in the normal and diseased human eye

Abstract

Purpose Gap junctions are specialised intercellular conduits that link the cytoplasm of neighbouring cells. These channels permit the movement of small molecules between cells and play an important role in maintaining local homeostasis. Gap junctions are involved in the earliest cellular responses to injury and may modulate the response to central nervous system injury. This series of related studies aimed first to characterise the spatial and temporal expression of connexin43, the most abundant gap junction protein in the central nervous system, following retinal ischaemia-reperfusion injury and then to investigate connexin43 expression in the normal and diseased human eye. Methods Qualitative and quantitative analyses of connexin43 expression were performed using both an animal model and on donated human tissue. Unilateral retinal ischaemia-reperfusion injury was induced by elevating intraocular pressure to 120 mmHg for 60 minutes and then normalised in male Wistar rats. Post-mortem human eyes were obtained from the New Zealand National Eye Bank. Double-label fluorescent immunohistochemistry was used in combination with confocal microscopy to characterise the spatial and cell-specific expression of connexin43. To evaluate the relationship to astrocyte activation, glial fibrillary protein was assessed using immunohistochemistry and western blot analysis. In animal model studies, Evans blue dye leak from retinal vessels was used to assess vascular leakage and blood vessel integrity was examined using isolectin-B4 labelling. Retinal whole mounts and retinal ganglion cell counts were used to quantify neurodegeneration. Results Confocal microscopy generated high-quality images of retinal microstructure enabling precise cellular localisation of connexin43 antigen. Retinal ischaemia-reperfusion induced significant vascular leakage and disruption. Connexin43 immunoreactivity was significantly increased post ischaemia-reperfusion injury in both ischaemic and contralateral retinas, co-localising with activated astrocytes, Müller cells, and vascular endothelial cells. Subsequently, significant retinal ganglion cell loss was observed in the ischaemic eye with a trend toward loss in the contralateral eye. The subsequent studies established the pattern of connexin43 immunoreactivity in the normal and diseased human retina. Qualitative analysis using doublelabel fluorescent immunohistochemistry and confocal microscopy revealed connexin43 expression on glia, blood vessels, and epithelial cells in the normal human retina. Significant alterations were observed in eyes with primary open angle glaucoma. Key observations included increased connexin43 immunoreactivity at the level of the lamina cribrosa and in the peripapillary and mid-peripheral retina in association with glial activation. Conclusions These studies have provided important qualitative and quantitative data that add to our knowledge of the expression of connexin43 in health and disease.