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J Vis Exp. 2015 Apr 19;(98). doi: 10.3791/52451.

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease.

Author information

1
Department of Therapeutics, Institut de la Vision, Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968; INSERM, U968; CNRS, UMR_7210.
2
Department of Therapeutics, Institut de la Vision, Sorbonne Universtés, UPMC Univ Paris 06, UMR_S 968; INSERM, U968; CNRS, UMR_7210; deniz.dalkara@inserm.fr.

Abstract

Müller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling Müller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms. There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5 has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label Müller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

PMID:
25938717
PMCID:
PMC4541578
DOI:
10.3791/52451
[Indexed for MEDLINE]
Free PMC Article

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