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

Summary

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

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.

Introduction

Müller cells are the major glial component of the retina. Morphologically, they span the retina radially and their endfeet, in contact with the vitreous, face the ILM and secret components of the latter. The ILM is a basement membrane composed of about ten different extracellular matrix proteins (laminin, agrin, perlecan, nidogen, collagen and several heparin sulfate proteoglycans). During development, its presence is indispensable for retinal histogenesis, navigation of optic axons, and survival of ganglion cells^1-3. However, ILM is unessential in adult retina and can be surgically removed in certain pathologies without causing retinal damage

Protocol

All animals used in this study were cared for and handled according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Production of Recombinant AAV (rAAV) by Transient Transfection of HEK-293 Cells^17,18

NOTE: See McClure C, JoVE (2011)¹⁹.

1. Purify a large-scale plasmid preparation (at least 1 mg/ml) of the AAV vector plasmids. Use 3 plasmids. The AAV helper plasmid carrying the replication and capsid genes without the AAV inverted terminal repeats ITRs, the transgene expression cassette carrying the AAV ITR referred to as the transfer plasmid, ....

Results

We expect increased retinal transduction of Müller glial cells using ShH10 if the animal model shows perturbations in the structure of the ILM (Figure 2A-B). For example, we have shown that in absence of Dp71, ShH10 targets specifically but more efficiently Müller glial cells by intravitreal injection, indicating increased permeability of the ILM in this mouse line compared to wild-type mice¹⁶ (Figure 2C-F).

AAV5 ca.......

Discussion

The BRB regulates the exchange of molecules between blood and retina. Its breakdown is associated with various diseases such as diabetic retinopathy or age-related macular degeneration (AMD). We recently showed that in a dystrophin knock-out mouse, which displays permeable BRB, the retina becomes more permissive to gene delivery mediated by adeno-associated viral vectors (AAV). However, despite BRB permeability AAV particles injected intraocularly stay confined to the ocular compartment in this model. Our results indicat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C57BL6J mice strain	JANVIER LABS		mice
Ketamine 500	Virbac France		anesthetic
Xylazine Rompun 2%	Bayer Healthcare		anesthetic
Neosynephrine 5% Faure	Europhta		dilatant
Mydriaticum 0.5%	Thea		dilatant
Sterdex	Novartis		anti-inflammatory
Cryomatrix embedding resin	Thermo Scientific	6769006
Superfrost Plus Adhesion Slides	Thermo Scientific	10143352	slides
anti-laminin	Sigma	L9393	antibody
anti-rhodopsin clone 4D2	Millipore	MABN15	antibody
anti-glutamine synthetase clone GS-6	Millipore	MAB302	antibody
Anti-Glial Fibrillary Acidic Protein	Dako	334	antibody
PNA Lectin	Invitrogen	L32459	probe
Alexa fluor conjugated secondary antibodies	Invitrogen		antibody
Fluorsave reagent	Calbiochem	345789	mounting medium
QIAmp DNA Micro Kit	QIAGEN	56304
GoTaq DNA polymerase	Promega	M3001
Evans Blue dye	Sigma	E2129	dye
5 µm filter	Millipore
Sodium citrate	Sigma	S1804
Citric acid	Sigma	C1909-2.5KG
Formamide spectrophotometric	Sigma	295876-2L
Fluorescein	Sigma	F2456	dye
Micron III	Phoenix Research Labs		Microscopy system based on 3-CCD color camera, frame grabber, and off-the-shelf software enables researchers to image mouse retinas.
Insulin Syringes	Terumo	SS30M3109
Syringe 10 µl Hamilton	Dutscher	74487	Seringue 1701
Needle RN G33, 25 mm, PST 2	Fisher Scientific	11530332	Intravitreal Injection
UltraMicroPump UMP3	World Precision Instruments	UMP3	Versatile injector uses microsyringes to deliver picoliter volumes
UltraMicroPump (UMP3) (one) with SYS-Micro4 Controller	UMP3-1	Digital controller
Binocular magnifier SZ76	ADVILAB	ADV-76B2	Zoom 0.66 x 5 x LEDs with stand epi and dia / Retinas dissection
Spring scissors straight - 8.5 cm	Bionic France S.a.r.l	15003-08	Retinas dissection
curved Vanna scissor	15004-08
Pince Dumont 5	11254-20
Veriti 96-Well Thermal Cycler	Life technologies	4375786	Thermocycler
Ultrasonic cleaner	Laboratory Supplies	G1125P1T
Nanosep 30k omega tubes	VWR
Speedvac	Fisher Scientific	SC 110 A
Spectrofluorometer	TECAN	infinite M1000