A Step by Step Protocol for Subretinal Surgery in Rabbits

The overall goal of this surgical intervention is to replace dysfunctional retina pigment epithelium, or RPE, to find a possible cure for RPE-related diseases, such as age-related macular degeneration. This method can help answer key questions in the optometric field about cell-based therapy and age-related macular degeneration. The main advantage of this technique is that it may provide a transport modality for cell replacement substrate or drug-delivery devices to the subretinal space.

Though this method may provide insight into vitrectomy and subretinal surgery in rabbits, it may also be used on other model organisms, such as pigs, monkeys, or humans. Generally, individuals new to this method will struggle, as not only intraocular surgery is complex, but also the personnel, organization, and technology for this procedure must be well synchronized. Demonstrating the instrument-loading procedure will be Fabian Thieltges, a post-doc, and Ralf Brinken, a surgical scrub nurse, both from my laboratory.

Before beginning the surgery, confirm the appropriate level of anesthesia and apply eye ointment to the other unoperated eye of the animal. Then, place the rabbit under a surgical microscope on its left side, with the right eye facing up and the nose slightly elevated by a blanket fold. Disinfect the eye for one minute with a few drops of povidone iodine.

Then, rinse the eye with sterile BSS, and cover the animal's head with a sterile drape with a pre-cut eye opening. Place a 12 by 17 centimeter piece of sticky surgical incision drape over the eye and use scissors to expose the eye with a midline horizontal incision. Using inverted calipers, displace the eye into proptosed position and secure the tissue with a 3-0 silk suture.

Then, use a pair of Vannas scissors to incise the conjunctiva close to the limbus, approximately one millimeter from the adjacent blood vessels, and enlarge the peritomy parallel to the limbus. Bluntly separate the conjunctiva, followed by a perpendicular six to seven millimeter vertical incision to make a T"in the conjunctiva. Make another small peritomy around the 2 o'clock position.

Then, carefully insert the tip of a 23-gauge microvitreoretinal blade through the sclera, pointing towards the posterior pole of the eye at 3.5 millimeters from the limbus in the 8 o'clock position. To avoid enlarging the incision, Slowly retract the blade in the same direction and pre-place a 7-0 Vicryl suture. Then, insert custom side port-infusion cannula into the scleral opening, and use the pre-placed suture to secure the cannula.

Set the intraocular pressure at 20 to 24 millimeters of mercury. Then, use 25-gauge flat head trocar to pierce the sclera at 2 o'clock position, and insert a 25-gauge chandelier light into the port. Secure the light with tape, and adjust it to about 30%power, then adjust individually.

Use a 20 scalpel to remove the edematous corneal epithelium to facilitate better intraocular visualization. Now, perform another sclerotomy with the 23-gauge microvitreoretinal blade at 10 o'clock and place a U-shaped 7-0 Vicryl suture around the incised sclera without completely tying the knot. Insert the tip of a high-speed vitrector into the incision and begin the vitrectomy around the entry port.

Cut the vitreous humor into small pieces at a maximum of 2, 000 to 3, 000 cuts per minute, continuing the vitrectomy over the optic disk and the fibrae medullares, and aspirating at a maximum of 200 millimeters of mercury. To gently separate the vitreous humor from the retina, hold the vitrector over the posterior pole and inferior of the disk and continue to aspirate without cutting. When the posterior vitreous detachment is complete, intravitreally inject approximately 50 microliters of the compound of interest to visualize the floating vitreous during its near total removal.

When the eye has been prepared, rinse the implantation cell culture three times with unsupplemented ophthalmic-grade BSS. Next, fill a 100 by 20 millimeter cell culture dish with 10 milliliters of ophthalmic-grade BSS and add the cell culture insert to the dish. Center the dish under a light microscope, and use a sharp oval custom-made hollow needle to punch a flat, bean-shaped 2.4 by 1.1 millimeter substrate with two long edge and two round edges out of the cell culture.

Gently flood the needle through the second port with BSS to flush the implant into the BSS-filled cell culture dish. To obtain a third edge, make a 0.5 milliliter incision in one of the round ends of the implant and use 23-gauge membrane-peeling forceps to transfer the implant into the custom-made loading station. Make sure the implant is in the upright position, then use the forceps to gently push the implant completely into the shooter instrument until all of the tissue is secured inside the tip and place the shooter tip into the loading station under BSS until the cells are implanted.

To enable implantation of the cells under the retina, approach the neural retina with an extendable 41-gauge subretinal injection needle connected to a gas-tight syringe and subretinally inject BSS to create a bleb retinal detachment. When the bleb is about 2 to 3 disc diameter, use vertical 23-gauge VR-scissors to enlarge the retinotomy to 1.5 millimeters and use a 1.4 millimeter incision knife to precisely extend the 10 o'clock sclerotomy to a 20-gauge approach. Next, attempt to pass a 20-gauge shooter dummy through the sclerotomy, enlarging the incision as necessary to ensure a smooth but snug transition of the loaded shooter.

Then, pass the loaded shooter through the scleral incision at 20 to 24 millimeters of mercury to the retinotomy edge and inject the implant subretinally from an epiretinal position. The shooter should transition through the sclera swiftly on the first try, so ensure a sclerotomy of optimal size. The RPE cells should then be implanted subretinally at a shallow angle to avoid entrapment of the implant leading edge at Bruch's membrane.

Adjust the implant so that it is placed under the retina reasonably away from the retinotomy. Then, remove the port from the sclerotomy and inject 25 microliters of triamcinolone through the chandelier port. Using 7-0 sutures, close the 2 and 10 o'clock sclerotomies and check for leakage.

Remove the infusion cannula and immediately close the last sclerotomy with the pre-placed 7-0 Vicryl suture. Using a 30-gauge needle, inject BSS to adjust intraocular pressure as necessary, while palpating. Finally, suture the conjunctiva with 7-0 Vicryl and remove the proptosing silk, taking care to avoid the deep orbital venous plexus.

Engraftment under the retina has a surgical success rate of approximately 60%when a core vitrectomy is performed, which increases to over 75%when a posterior vitreous detachment is induced. Here, a scanning laser ophthalmoscope infrared reflectance image of xenotransplanted cultured retinal pigment epithelia on a polyester membrane after an uncomplicated procedure is shown, with the photoreceptor atrophy resulting from the immune reaction appearing as a halo around the implant. In this corresponding spectral domain optical coherence tomography image, outer nuclear layer thinning as a response to RPE's xenotransplantation can be observed, mainly in the retina with a visible hyper-reflective band above the implant.

The neural retina adjacent to the implant, however, exhibits near-normal reflection bands, suggesting an atraumatic delivery of the implant. As observed by H/E staining, subretinal scarring, and outer nuclear layer atrophy occur in xenotransplanted non-immunosuppressed rabbits. The Bruch's membrane beneath the implant also appears to be contiguous, with the choriocapillaris containing some scattered erythrocytes, further confirming the lack of trauma incurred during the implant delivery.

Once mastered, this technique can be completed in one hour if no complications are encountered. While attempting this procedure, it is important to remember to create a retinal bleb and retinotomy just big enough to fit the implant. Also, a faster surgical intervention will typically yield better outcomes.

Following this procedure, other methods such as optical coherence tomography or histology may be used to answer additional questions, such as success of the transplantation, subretinal biocompatibility, and the survival of the transplanted cells. After its development, this technique paved the way for researchers in the fields of stem cell biology and biomaterials to explore subretinal delivery of stem cell derivatives and/or biofunctionalized carrier substrates for retinal disease, particularly the RPE. After watching this video, you should have a good understanding of how to perform vitrectomy and subretinal implantation surgery in rabbits.

Don't forget that working with the lens, vitreoretinal, and subretinal interface can be extremely hazardous and that precautions, such as proper hand-positioning, good visualization, stable intraocular pressure should always be taken while performing this procedure.