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

The overall goal of this procedure is to investigate the blood retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease. This is accomplished by first injecting A A V particles containing A GFP transgene into the vitreous or into the bloodstream. The second step is to sample blood at different time points after the injections.

The blood sampling is complimented by weekly in vivo fundus imaging. The final step is to perform immunohistochemistry on retinal flap mounts and retinal cryo sections. Ultimately, confocal microscopy and PCR are used to show the transduction pattern indicator of the ILM integrity and to detect a a v particles in the bloodstream, thus demonstrating permeability of the blood retinal barrier.

The main advantage of this technique, our existing methods, is that the AAV paste method gives information on the blood retinal barrier permeability not provided by other cells. The implications of this technique extend toward AAV mediad gene therapy because it helps to ensure the blood retinal barrier in permeability to AAV particles, thus avoiding any undesirable side effects. This method can provide insight into the inner limiting membrane integrity in animal models of other retinal diseases such as diabetic rats.

We first had the ID for this method when we saw that the retina of our DP 71 non-use model showing your blood retina barrier breakdown was more efficiently transduced than the wild tap. Right now Begin by anesthetizing C 57 black six J mice. Check for the loss of three reflexes, the writing reflex, the withdrawal reflex, and tail pinch response.

Loss of these reflexes confirms anesthetization next dilate the pupils by applying 5%nesrine and 0.5%mire atum to the cornea from a dropper. Then use a veterinary ointment to lubricate the eyes so they do not dry too much. Now load an ultra fine 30 gauge disposable needle with stock solution containing one to four times 10 to the 11th particles of A-A-V-G-F-P.

Generally, people new to this method will struggle because they have to perform the safest intravitreal injection without touching the lens or the retina. Then pass an ultra fine 30 gauge disposable needle through the equator and next to the limbus into the vitreous cavity. After introducing the needle to the eye, inject a microliter of solution while observing the needle at the center of the vitreous cavity.

Use one eye as a control for inner limiting membrane experiments and inject both eyes per blood retinal barrier experiments. After the injection, apply an anti-inflammatory and an antibacterial topical treatment to each injected eye. One week later dilate the pupils with nesrine and MyUM and perform fundus examinations with an eye fundus camera.

Track the GFP expression with observations at one week, two weeks, one month, and two months post injection. After harvesting the eyes from a euthanized injected animal, dissect the eyes to remove the lens and cornea. Then immersion.

Fix the dissected eye in 4%para formaldehyde for an hour. After an hour, cryo protect the eyes in 10%sucrose for another hour at room temperature. Then soak the eyes in 20%sucrose for one third of an hour at room temperature.

Finally, leave the eyes in 30%sucrose solution overnight at four degrees Celsius. The next day, embed and freeze the eye cups in an embedding resin such as cryo matrixx. Then using the cryostat, make 10 micron sections and mount them to slides prepared for frozen tissue.

Now perme the sections on the slides with detergent for five minutes. Follow this with two brief washes in PBS. Then block the eyes for an hour at room temperature for retinal flat mounts.

Facilitate the dissection by fixing the enucleated eyes in 4%para formaldehyde for 15 to 30 minutes. This fixation and the fixation following the dissection must be performed properly as this is critical to preserve the GFP fluorescence Within 15 to 30 minutes of the fixation period. Dissect the eyes, remove the cornea and lens.

Separate the retina from the retinal pigment epithelium and from the sclera by cutting around the oral serrata and the optic nerve. After the dissection, immerse the retinas in 4%Para formaldehyde for another 30 minutes to fix the tissue and the fluorescent protein in transduced retinal cells. After the fixation, wash the specimen for five minutes in sterile PBS at pH 7.4.

Then change the PBS to blocking buffer and incubate the retinas for four hours at room temperature or overnight at four degrees Celsius. Begin the labeling step by incubating the samples with the primary antibodies in blocking buffer for either four hours at room temperature or overnight at four degrees celsius. After applying the primary antibody, wash the tissues three times with PBS, then incubate tissues in a one to 500 dilution ofor conjugated antibodies in blocking buffer.

Incubate for one hour for cryo sections or two hours for flat mounts. Do this at room temperature and protect it from light. After the secondary has been applied, remove it with three washes in PBS.

As before now, make relieving cuts to the retina so that it can be mounted flatly onto a glass slide with either the photoreceptors or retinal ganglion cells facing upwards. Apply an aqueous mounting medium and a cover slip. The slides can be stored at four degrees Celsius prior to confocal microscopy imaging.

Begin with generating a positive control for circulating a a v particles. First, take a 10 to 20 microliter blood sample before injecting the A A v. Then once anesthetized inject 100 microliters of the stock viral solution into the bloodstream.

The easier way to inject substances into the bloodstream is the penile vein. In this video capture, we inject blue dye as a visual control of the injection over the next three days. Continue to take a small blood sample periodically and then sacrifice the mouse from a tissue sample.

Extract the genomic DNA from the samples using a commercial kit, using the DNA perform A PCR amplification with a primer pair to GFP run 30 cycles with 45 second extensions and 55 degrees Celsius. A kneeling the transduction pattern of SHH 10 vector was expected to increase in the DP 71 NU mice. If the animal model showed perturbations in the structure of the inner limiting membrane DP 71 null mice have compromised retinal barriers and are hence a useful comparison in the DP 71 null mice intravitreal injection of S SHH 10 targets Miller glial cells more efficiently.

This indicates a destabilization of the inner limiting membrane in this transgenic mouse line as compared to wild type controls. Injecting A A V 5G FP demonstrated inner limiting membrane permeability as well. AAV five was an effective by intravitreal injection in wild type mice, but became strongly effective in gene delivery to photoreceptors in mice with compromised retinal barriers such as with the DP 71 null mice.

By PCR analysis. The blood retinal barrier in DP 71 null mice remains selective to AAV particles since no trace of a a v was found in their blood samples after intravitreal injection. After watching this video, you should have a good understanding of how to use adeno associated virus in order to test the inner limiting membrane integrity or the blood biopsy permeability by analyzing the transduction pattern of the retina or the AV particle presence into the bloodstream.