Immunolocalization of Gap Junction Protein Connexin 43 (GJA1) in retina and Glial Müller Cells in WT and Dp-71 null mice

Abstract

Purpose : Dp71, the main product of the Duchenne muscular dystrophy gene expressed in retina, is essential for glial cell functions such as retinal water homeostasis and maintenance of the inner blood–retinal barrier (iBRB). Tight junction complexes at the iBRB contain a large number of proteins sucha as connexin 43 (Cx43) among many others. Downregulation of Cx43 expression provokes vascular cell death and increases vascular permeability. We have previously shown that Dp71 is localized in Müller glial cells (MGC) and astrocytes, together with endothelial cells and pericytes surround the retinal vessels to form the iBRB. The purpose of this study is to determine the expression of Cx43 in WT and Dp71-null mice retina in order to explore the role of Dp71 in the maintaining of the iBRB.

Methods : According with the ARVO guidelines for animal use for research, 8 weeks WT and Dp71 null mice were used. The expression of Cx43 was determined by qRT-PCR in WT and Dp71 null mice. Sagittal 12-micras cryosections of retinas and isolated retinal cells were used for double-label immunohistochemistry using anti Connexin-43 (Cx43), Glial Fibrillary Acidic Protein (GFAP) and Glutamine synthase (GS) antibodies to determine the expression pattern and the localization of Cx43 in WT and Dp71 null mice retina, astrocytes and MGC.

Results : A significant decrease (p=0057) was observed in mRNA expression of Cx43 in Dp71-null mice retina comparing with WT mice. In WT mice retina, Cx43 immunoreactivity was detected on GFAP-positive astrocytes, in the retinal ganglion cell layer and on GS-labeled Müller cells. We observed a impressive decrease of Cx43 expression in MGC and astrocytes of Dp71-null mice retina when compared with WT strain. Moreover, Dp71 deficiency was also associated with an impaired clustering of Cx43. Cx43 immunostaining decreased around blood vessels and in the inner limiting membrane.

Conclusions : To our knowledge, this is the first report that explores connexin 43 expression in Dp71-null mice retina. Taken together the results suggest that the absence of Dp71 affects the intercellular tight junctions in mice retina, affecting the expression and localization of Cx43; this impairment could be, at least in part responsible of the abnormalities observed in Dp71 null mice retina, specifically on the increase of iBRB permeability.

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