Ocular Therapeutic Delivery and Advanced Tissue Retrieval in Adult Rats

The scope of the research is to develop injection techniques to deliver drugs to the back of the eye to treat various ocular pathologies. We also wanted to develop a technique to visualize the entire optic track en bloc.

The current literature lacks a standardized methodology for the retrobulbar injection in the adult rat. This protocol is to establish that methodology while also providing a top-down procedure for getting en bloc tissue samples from the entire neuro-visual track.

Compared to other techniques, this injury protocol minimizes potential postmortem injuries and yields en bloc globes and nerves. On the other hand, our injection protocol details efficacious needle sizes for future repeatability.

These findings will expand the literature on retrobulbar injections in adult rats and enable the therapeutic intervention and en bloc analysis of posterior ocular tissue in small animal models.

Future research will focus on using these techniques to apply treatments to posterior ocular injuries in small animal models. We also want to use this dissection technique to extract the tissues intact without damaging them.

To begin, place the ophthalmic surgery platform with the anesthetized animal under a live operating microscope. Using a 10-microliter syringe fitted with a 33 gauge needle, draw up 0.9% bacteriostatic sodium chloride. Flush the syringe and needle with the saline, then dip the needle into a hot bead sterilizer. After the needle has cooled, use it to draw up the therapeutic of interest to the desired amount. Using fine ophthalmic forceps with teeth, gently grasp the scleral tissue at the limbus to stabilize the eye. Identify the faint red ring around the iris and use it as a landmark. Insert the needle with the bevel side down and inject the therapeutic into the posterior eye. Slowly remove the needle. Close the eye and hold pressure for 10 to 15 seconds. Flush the area with saline. Place the ophthalmic surgery platform with the anesthetized animal under a live operating microscope. Using a 0.5 milliliter insulin syringe with a 28 gauge needle, draw up the therapeutic of interest to a desired volume. Using fine ophthalmic forceps with teeth, gently grasp the lower eyelid to stabilize. Insert the needle bevel side down at an angle between six and seven o'clock along the lower orbital rim until the back of the ocular socket is felt. Pull the needle back slightly and slowly inject the therapeutic. Gently and slowly remove the needle. Close the eye and apply pressure for 10 to 15 seconds. Then flush the eye with saline. After gently breaking through the lateral skull from the foramen magnum to the temporal region on both sides, use a flat spatula to gently reflect the brain rostrally to expose the optic nerves. With the optic nerves visible, take a pair of medium micro scissors and make a small incision across the optic chiasm to sever both nerves from the brain. Using a pair of small iris scissors and toothed ophthalmic micro forceps, carefully remove excess tissue, such as eyelids and connective tissue, from around the eye. Using hemostats and small iris scissors, cut through the ocular orbit carefully, avoiding damage to the optic nerve. Break away the bone at the back of the ocular socket using the hemostats, and for precision, use the small dissection scissors. After bone removal, delicately cut around the ocular orbit with micro scissors and fine forceps to remove fat pads, glands, and extraocular muscles. Now, remove the connective tissue surrounding the nerve on the lower skull ridge. Further clean the optic nerve and eye of any remaining excess tissue, such as the dural sheath. This figure presents the results of flash visual evoked potential or FVEP testing used to assess the safety of retrobulbar injection on visual function over time. The FVEP amplitudes did not show any significant difference between right eyes injected with saline and untreated left eyes at baseline and day seven, confirming the safety of the retrobulbar injection protocol. FVEP waveforms of the saline-treated right eyes remained consistent between baseline and day seven, indicating no disruption in visual pathway function. The full optic nerve and eye were successfully isolated and processed and immunofluorescence staining revealed intact optic nerve head structure with clear labeling of neuronal, glial, and nuclear components. This table summarizes the detection of various neuroprotective therapeutics in retinal and optic nerve tissues 24 hours after administration. Cyclosporine was present in both tissues via both injection routes, while tauroursodeoxycholic acid and ibudilast were mostly undetected and anakinra was only detected in the retina after intravitreal injection.