Subretinal injection is the best approach to deliver viral vectors directly to targeting cells in the subretinal space. which provides a new therapeutic method for vitreoretinal diseases. In this protocol, we here present the experimental procedure of subretinal injection in the mouse as follows. Preparation
●Before surgery, mouse is anesthetized by intraperitoneal injection of 2 % sodium pentobarbital (50 mg/kg). Wait until the mouse becomes motionless (~5 min).
●Position the rodent onto its side so that the eye that will be injected is facing the ceiling. Dilate pupils with an eye drop of phenylephrine 0.5% and tropicamide 0.5%.
Target coordinates and injection
●Prepare the syringe containing with solution to be injected. Under a dissecting microscope, open the eyelid and protrude the eye to expose the equator for injection. 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.
●Apply a drop of ophthalmic viscoelastic solution to the corneal surface. Place a small round cover slide on the top of the cornea to visualize the retina
●Puncture a small hole at slight posterior to the limbus using a sterile 30 G 1/2 needle for the further subretinal injection. Make the hole inferior for the right eye, and superior for the left eye for the convenience.
●Place the 33 G blunt needle of microliter syringe through the pre-punctured hole and approach the needle into the subretinal space until the point when mild resistance is felt.
●Inject the viral vectors (i.e., 1 x 10⁶ TU/µl) gently into the subretinal space without tremor to avoid unwanted tissue damage and withdraw the needle gently. Hold the eyeball firmly during the injection.
Post-injection care
●Observe the formation of subretinal bleb after the injection under operating microscope to make sure there is no retinal bleeding.
●Gently close the eyelid to cover the injection site for self-sealing. Return the mice to home cage and keep alive until the evaluation.

Reference
For more details please refer to the literature “Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium”

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