AAV-Mediated Gene Delivery for Visualization of Compromised Retinal Membrane Integrity

Abstract

Source: Vacca, O., et al. Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease. J. Vis. Exp. (2015).

This video demonstrates the method of AAV-mediated gene delivery in a transgenic mouse retina, followed by tissue dissection and staining to assess inner limiting membrane integrity.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Immunohistochemistry

1. For retinal flatmounts (Figure 1), fix enucleated eyes in 4% paraformaldehyde for 15 min to facilitate the dissection.
   1. In 15-30 min, dissect the eyes to remove the cornea and lens. Separate the retina from the retinal pigment epithelium (RPE) and sclera by cutting around the ora serrata and the optic nerve. Immerse in 4% paraformaldehyde for another 30 min to fix the retina and the fluorescent protein in transduced retinal cells (fixation must not exceed 24 hr). Wash 5 min in sterile PBS, pH 7.4.
   2. Change the phosphate-buffered saline (PBS) to blocking buffer (PBS with 1% bovine serum albumin (BSA), 2% normal goat or donkey serum, 0.5% Triton X-100), and incubate in blocking buffer for either 4 hr at room temperature or 4 °C overnight.
2. Incubate tissues with primary antibodies in a blocking buffer for either 4 hr at room temperature or at 4 °C overnight. Wash 3x with PBS.
   NOTE: The antibodies used are as follows: anti-laminin (1/1,000) labeling the ILM (inner limiting membranes), anti-rhodopsin clone 4D2 (1/500) labeling rods, anti-glutamine synthetase clone GS-6 (1/1,500) labeling Müller cells, Peanut agglutinin (PNA) Lectin (1/40) labeling cones.
3. Incubate tissues with 1:500 dilution of alexa fluor conjugated secondary antibodies in blocking buffer for 1 hr (cryosections) or 2 hr (flatmounts) at room temperature and protected from light. Wash 3x with PBS.
4. Make relieving cuts to the retina and flat-mount it onto a glass slide with either the photoreceptor or retinal ganglion cell (RGC) side facing upwards. Add aqueous mounting medium, apply coverslip and store at 4 °C.
5. Perform confocal microscopy on a laser-scanning confocal microscope. Acquire images sequentially, line by line, to reduce excitation and emission crosstalk. Define step size according to the Nyquist–Shannon sampling theorem. Use exposure settings that minimize oversaturated pixels in the final images. Process 12-bit images with FIJI, project Z-sections on a single plane using maximum intensity under the Z-project function, and finally convert to 8-bit RGB color mode.

Results

Figure 1: Schematic representation of the general protocol. After AAV production, particles are injected into the vitreous or into the penile vein to anesthetize adult mice. Fundus images with a Micron III camera are performed to follow the GFP (Green fluorescent protein) expression. The GFP expression pattern is analyzed on retinal cryosections and flat-mounted retinas thanks to immunohistochemistry and confocal images. The AAV passage through the BRB is assessed by PCR analysis on blood samples coming from intravitreally or intrapenally injected mice, in order to detect the presence of the GFP sequence, an indicator of the AAV particles presence into the bloodstream.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
Evans Blue dye	Sigma	E2129	Dye
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.
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	Retinal dissection