Intraoperative Visualization of Subretinal Injection and Retinal Detachment in Rats

I am exploring mechanisms of cell death in retinal detachments, and my goal is to ensure traceability of in vitro data to in vivo experiments. In our team, we demonstrated the involvement of different cell death mechanisms during photoreceptor degeneration. There are many ways that have been described to induce retinal detachment in rodents.

This protocol was designed to answer to two challenges, reproducibility and safety. Providing a reproducible and safe protocol in retinal detachment will ensure efficiency in this kind of research. To begin, mount a non-beveled 10-millimeter 30-gauge cannula metal tip cemented on a 25-microliter syringe onto a micro injector.

To induce long-term retinal detachment, use 2%hydroxypropyl methylcellulose or 1%to 5%sodium hyaluronate. Load the prepared solution into the syringe. Next, apply one drop of 0.5%tropicamide on one eye of an anesthetized rat to obtain mydriasis.

Apply one drop of oxybuprocaine in the eye to be operated. Use an ophthalmic microscope connected to a foot switch to perform the surgery. To expose the sclera, anchor two eyelid pulling sutures at the external quarter of the palpebral margin at the upper and lower eyelids.

Pull the sutures to achieve gentle bulging of the eye. With a 30-gauge needle, create a scleral channel through the temporal bulbar conjunctiva approximately one to two millimeters from the corneal limbus. Apply tear gel as a lens eye interface throughout the procedure.

To visualize the retinal plate, place a flat contact lens with an eight millimeter diameter on the eye surface. Use the foot switch of the microscope to focus on the retinal plane. Now, insert the cannula vertically through the preformed scleral channel.

Slowly approach the retina and gently press it until retinal whitening is observed. Maintain the tip stability while injecting through the retinotomy formed by the injection flow After injection, remove the tip from the retinotomy. Then, gently withdraw the entire cannula from the vitreous cavity, ensuring the lens is not touched.

Ensure the pupillary red reflex is altered by the retinal detachment without observing blood flow within the vitreous cavity. Remove the eyelid sutures, then swab any bleeding carefully. Briefly assess intraocular pressure manually.

For postoperative care, apply chloramphenicol retinol eye ointment to the operated eye. Then, administer one milliliter of 5%glucose monohydrate intraperitoneally. Inject 0.9 milligrams per kilogram atipamezole subcutaneously.

Finally, transfer the animal to a temperature-controlled chamber and monitor it until it wakes up. Successful subretinal injection was verified by clear subretinal fluid in optical coherence tomography, and the absence of subretinal hemorrhage in fundus photography. And a distinct retinal detachment was observed in both modalities.

Minor intravitreal hemorrhages were occasionally observed, primarily near the injection site, but did not extend into the subretinal space.