Delivery of a Fluorescent Dye into the Subretinal Space of a Mouse

Take an anesthetized mouse and trim the whiskers to facilitate eye visualization.

Place the mouse under a microscope on a warming pad to maintain body temperature.

Dilate the pupil to enable retina visualization, then lubricate the eye to prevent dryness.

Pinch the conjunctiva, a transparent membrane, and make an incision.

Incise the underlying Tenon’s capsule and remove the connective tissue layer while rotating the eye.

This exposes the sclera, the outer layer of the eye, and provides access to the injection site near the optic nerve.

Make a scleral incision and insert a syringe containing a fluorescent dye into the subretinal space between the retinal photoreceptor layer and retinal pigment epithelium.

Inject the dye to create a controlled retinal detachment, then withdraw the needle.

Rinse the eye and apply an antibiotic to prevent infection.

Allow the mouse to recover and administer pain relief. The mouse is ready for imaging to confirm successful subretinal injection.

To begin this procedure, trim the whiskers of an anesthetized mouse to facilitate visualization and maintain the animal's body temperature at 37 degrees Celsius with a circulating water pad. Next, dilate its pupils with 2.5% phenylephrine eye drops.

Then apply methylcellulose eye drops to prevent dryness and minimize anesthetic induced transient cataracts. Sterilize the instruments prior to surgery. Afterward, prepare the diluted fluorescein in a biosafety cabinet. Fill the syringe with the appropriate amount of fluorescein.

Next, perform a toe pinch to ensure the animal is deeply anesthetized and position the mouse so that the eye to be injected is facing up and clearly visible in the dissecting microscope. Gently pinch the temporal conjunctiva with a pair of fine tipped forceps.

Then make a circumferential incision of approximately 90 degrees using the curved Vannas scissors. Repeat the procedure on the underlying Tenon's capsule. After that, resect the surrounding connective tissue with fine tip forceps while rotating the globe nasally. Work towards the injection site at approximately 0.5 millimeters away from the optic nerve and take great care to avoid disrupting the retro orbital sinus.

A critical step is exposing the injection site, which requires rotation of the eye without damaging the eye itself or associated structures, particularly avoiding damage to the orbital blood sac.

In this procedure, make a small incision at the sclera of the injection site by gently scratching the eye cup with a 22.5 degree ophthalmic blade. This incision should just be large enough to allow the tip of the needle to pass through the sclera.

Next, insert the beveled 33 gauge needle into the sclerotomy with the bevel facing and angled parallel to the retina. Inject the desired amount of 0.01% fluorescein by pressing the plunger slowly with even pressure.

Note that when the needle is in the subretinal space, a slight resistance will be felt while pressing the plunger. There will be no resistance if the needle punctures the retina, but high resistance if the needle does not penetrate the sclera or RPE.

Another critical step is the injection targeting the subretinal space. This must be accomplished without penetrating through the neurosensory retina to the vitreous cavity.

Wait several seconds before withdrawing the needle to minimize backflow.

Subsequently, rinse the eye with sterile saline and ensure the eye has rotated back to its normal position. Then apply a thick coat of triple antibiotic ophthalmic cream to the corneal surface of the injected eye.

Afterward, place the mouse in a clean, solitary cage for recovery. Monitor its respiration and temperature during anesthesia recovery and check that it can maintain sternal recumbency. Perform additional appropriate postoperative monitoring and treatment, including a subcutaneous injection of carprofen for post-surgical pain management.