Comparative Study Of The Cellular Triad - Müller Cells, Astrocytes, Vessels - Between Normal And Dystrophin 71 Deficient Mice Using An AAV Variant Targeting Müller Cells

Abstract

Purpose: : Dystrophin Dp71 is a membrane cytoskeletal protein expressed in Müller cells, astrocytes and pericytes. We have shown that in comparison with wild-type mice (WT), the absence of Dp71 induces a blood retinal barrier (BRB) breakdown (Sene, 2009). A highly permeable BRB has been considered as a characteristic of diabetic retinopathy. At this respect, Dp71-null mice could be a good model to approach the study of this pathology. It has been shown that the AAV variant ShH10-GFP, closely related to AAV serotype 6, is capable of efficient, selective Müller cells infection through intravitreal injection (IVT) in rat retina (Klimczak, 2009). Here, we tested the efficiency of ShH10-GFP in mice retina and studied Müller cells morphology and their relations with astrocytes and vessels in WT and Dp71-null mice.

Methods: : We performed IVT of ShH10-GFP at 4.10^11 vg/µl of 7-weeks-old C57BL/6J WT and Dp71-null mice. Every week until one month after injection, we have made fundus imaging and scanning laser ophtalmoscopy to follow ShH10-GFP expression. Mice were sacrificed one month after injection. Retinae were prepared for flatmounts or cryosections, and labeled with GFAP antibody and Lectin marker. Cellular morphology was observed by confocal imaging and analysed by Fiji software.

Results: : As in rat, ShH10-GFP transduced Müller cells in WT and Dp71-null mice. On fundus imaging, we have observed that ShH10-GFP is strongly expressed one week after injection and its expression level is constant to at least one month after injection. On cryosections, we have seen that transduced cells are specifically Müller cells. On flatmount retinae, transduced Müller cells appear to be more evenly transduced in Dp71-null mice than in WT. However, in absence of Dp71, (i) interactions between Müller cells and vessels seem to be less tight and Müller cells appear to be more evenly transduced in whole retina and (ii) astrocytes are dramatically reduced in number and have more elongated shapes.

Conclusions: : Here we demonstrate that (i) in mice, ShH10-GFP targets specifically Müller cells and that (ii) in Dp71-null mice, Müller cells are deficient and astrocytes are less well organized. This study also suggests the potential therapeutic role of ShH10 genetic tool. Indeed, ShH10 could be used to restore Dp71 expression in order to treat pathologies related to BRB breakdown.

Keywords: glia • adenovirus • gene transfer/gene therapy