Subretinal Injection of Viral Vectors into the Retinal Pigment Epithelium in a Mouse Model

Abstract

Source: Park, S. W., et al. Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium. J. Vis. Exp. (2015)

The video describes the method for subretinal injection of viral vectors into the retinal pigment epithelium (RPE) in a mouse model. It details the procedure from anesthesia and eye preparation to precise lentiviral vector delivery, ensuring gene integration and expression in the RPE.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparing Injection Kit and Viral Vectors

1. Prepare the microliter syringe equipped with a 33 G blunt needle sterilized using ethylene oxide gas. Dilute the viral vectors in PBS (Phosphate buffered saline) for adequate titer in the microtube (i.e., 1 x 10⁶ TU/µl). Flush the syringe several times with viral vectors to remove any dead space in the syringe.

2. Subretinal Injection of Viral Vectors

1. Anesthetize the adult mice (i.e., 6 - 8 weeks old) with an intraperitoneal injection of the mixture of tiletamine and zolazepam (1:1, 2.25 mg/kg body weight) and xylazine hydrochloride (0.7 mg/kg body weight) or an alternative suitable anesthesia regimen.
2. Dilate pupils with an eye drop of phenylephrine 0.5% and tropicamide 0.5%.
3. Prepare the microliter syringe by loading it with 1.5 - 2 µl of viral vectors.
4. Open the eyelid and protrude the eye to expose the equator for convenient injection. Focus on it under the operating microscope. Maintain the protruded eye position until finishing the injection, or displacement of the needle can occur during the injection. To hold the eyeball firmly, place the fingers outside the orbital rim.
5. Apply a drop of ophthalmic viscoelastic solution to the corneal surface.
6. Place a small round cover slide on the top of the cornea to visualize the retina.
7. Puncture a small hole at the slight posterior to the limbus using a sterile 30 G 1/2 needle for further subretinal injection. For convenience, make the hole inferior for the right eye and superior for the left eye.
   NOTE: If the hole is made at the temporal or nasal, it’s hard to be covered by the eyelid after the injection. The initial puncture should be made slightly posterior to the limbus to avoid the limbal vessels that run along the limbus and can be easily recognized. Be careful to avoid hitting the lens with a needle while making the initial puncture. Do not insert the entire bevel of the needle to avoid lens puncture.
8. Place the 33 G blunt needle of the microliter syringe through the pre-punctured hole and approach the needle into the subretinal space until mild resistance is felt.
   NOTE: For the subretinal injection, the best approach angle of the needle is about 45 degrees against the iris plane, and the blunt needle should be pushed posteriorly toward the peripapillary area. The dotted square indicates the suggested needle pathway across the vitreous cavity for the subretinal injection in Figure 1.
   NOTE: There will be no resistance felt when piercing (or passing) the retinal layers as they are very soft. Thus, the first feeling of mild resistance indicates that the needle has already touched the RPE (Retinal pigment epithelium) layer, and the insertion of the needle should be stopped. Be careful not to penetrate the scleral tissue with excessive pressure because the needle should be placed in the potential subretinal space. If the needle punctures the scleral tissue by excessive power, it enters into the extraocular orbital spaces without resistance. (This step is critical)
9. Inject the viral vectors (e.g., 1 x 10⁶ TU/µl) gently into the subretinal space without a tremor to avoid unwanted tissue damage. Then, withdraw the needle gently. Hold the eyeball firmly during the injection, as described in 2.4.
10. Observe the formation of subretinal blebs after the injection under an operating microscope to ensure there is no retinal bleeding.

Results

Figure 1. Schematic Diagram of the Subretinal Injection. H&E (Hematoxylin and Eosin...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tweezers DUMONT #5 11cm DUMOSTAR 0.1 x 0.06 mm tips	WPI	500233
VANNAS Scissors S/S, 105mm	WPI	555583S
33G Blunt needle	WPI	NF33BL-2
NanoFil Syringe, 10 microliter	WPI	NANOFIL
RPE-KIT	WPI	RPE-KIT	For easy one hand injection
30Gx1/2 (0.3mmx 13mm) BD PrecisionGlideTM Needle	BD	305107	Initial puncture for subretinal injection
Microscope Cover Glasses (No. 1 3 mm diameter)	Warner Instruments	64-0720 (CS-3R)
Leica operating microscope	Leica	LM M80
Fluoresecein microscope	Nikon	Eclipse 80i
Lentivirus	Thermo scientific	TMO.LV-Ctr	Used to dilute vectors
PBS	Gibco	10010-015	Used to dilute vectors
Troperin (Phenylephrin 0.5%-Tropicamide 0.5%)	Hanmi		For dilation
Proparacaine Hydrochloride Ophthalmic Solution USP, 0.5% (Sterile)	Bausch&Lomb		For topical anesthesia
Healon GV OVD	Abbott Medical Optics Inc.
Zoletil 50 (tiletamine hypochloride and zolazepam hypochloride)	Virbac		For general anesthesia
Rompun® injection (Xylazine HCl)	Bayer		For general anesthesia