Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Foveal trauma during retinal pigment epithelial graft transplantation can result in poor visual outcomes. This proposed method can reduce the likelihood of foveal tears when compared to conventional techniques. The proposed method addresses multiple factors contributing to foveal trauma including the release of subretinal adhesions, better visualization of retinal layers, and tangential shear forces during bleb formation.

This technique, which involves intravitreal plasminogen pretreatment, microscope integrated OCT-guided retinal bleb formation, and the perfluorocarbon liquid vitreous tamponade using a dual bore cannula produces the likelihood of foveal trauma with a gentler procedure. Demonstrating the procedure will be Dr.Zenping Liu, a senior research fellow, and Mr.Daniel Soo Lin Wong, a research assistant, both from a laboratory. Professor Gopal Lingam, a senior consultant, will also demonstrate the operation procedures.

To begin, use the Vannas scissors for incising the conjunctiva near the limbus for a 360 degree conjunctival peritomy and perform a blunt dissection to enlarge the peritomy. Use a 25 gauge micro vitreoretinal blade to make a three millimeter incision in the sclera at eight o'clock in the right eye or four o'clock in the left eye. Insert a 25 gauge custom side port infusion cannula and suture using a 7-0 Vicryl suture.

After confirming the intravitreal location, start the balanced salt solution infusion and set the system to maintain the intraocular pressure of 20 milliliters of mercury. Use a 25 gauge flathead trocar to incise the sclera at two o'clock in the right eye or 10 o'clock in the left eye. Insert a 25 gauge chandelier light into the flathead trocar and secure the light with sticky tape.

Adjust the light source to approximately 60%Perform another sclerotomy at 10 o'clock for the right eye or two o'clock for the left eye as demonstrated. Place a U-shaped Vicryl 7-0 suture around the sclerotomy without tying the knots. Insert the vitrectomy trocar through the sclerotomy.

Perform the vitrectomy around the entry port, followed by a short core vitrectomy with a maximum of 5, 000 cuts per minute and maximum aspiration at 400 millimeters of mercury. Inject 20 to 50 microliters of triamcinolone for better vitreous visualization. Separate the vitreous body from the retina to induce a posterior vitreous detachment.

Position the vitrector above the optic disc to allow gentle induction of the posterior vitreous detachment. Keep the vitrector only on aspiration at a maximum of 400 millimeters of mercury without involving any cutting. If required, use the 25 gauge intraocular forceps to tear the vitreous cortex for facilitating the detachment.

Open the posterior hyaloid membrane with the cutter and remove the detached vitreous skirt up to the vitreous base. Aspirate any remaining triamcinolone on the retinal surface. Inject one to two milliliters of perfluorocarbon liquid to cover the posterior pole up to the anterior and mid peripheral retina.

Enter the eye with a subretinal injection cannula. Use either the 25 by 41 gauge customized dual bore subretinal injection cannula or 25 by 38 gauge subretinal injection cannula connected to a 250 microliter syringe for subretinal injection. Set the intraocular pressure to zero to four millimeters of mercury.

Gently perform a subretinal injection of BSS to induce a localized retinal detachment. Once the bleb crosses the fovea, stop injecting BSS. Create a second bleb from a separate direction.

Merge both the blebs to fully detach the fovea. Enable the intraoperative optical coherence tomography function to visualize bleb formation, ensuring that the line and cube scans are in high definition mode to acquire an image at the fovea. Enlarge the incision of the retina to 1.5 millimeters with a pair of vertical 25 gauge vitreoretinal scissors to allow access to the subretinal space for transplantation.

Set the intraocular pressure to 50 millimeters of mercury. Use a brushed silicone tip cannula to remove the perfluorocarbon liquid. Use a 1.4 millimeter incision knife to extend the sclerotomy.

Enter a 20 gauge extensible loop instrument and remove the submacular host retinal pigment epithelia by scraping. Insert the tip of the shooter device through the sclerotomy and pass through the vitreous cavity at an intraocular pressure of 20 millimeters of mercury. Inject the implant towards the subretinal space via the retinotomy edge created from the retinal surface with the cell carrier side facing the Brooks membrane and the xenograft facing the photoreceptors.

Visualize the implant location by enabling the intraoperative optical coherence tomography function. Adjust the implant position with the subretinal injection cannula or a 25 gauge curved intraocular scissor to ensure the implant is resting flat on the Brooks membrane in the subretinal space with an intact overlying retina, as well as located distantly from the created retinotomy and not impinged on the retinotomy site. Use a brushed silicone tip cannula for the fluid-air exchange and gently aspirate the subretinal fluid from the bleb retinal detachment and retinotomy edge apposition until the retina is reattached over the implant.

Then to finish the surgery, close the working port sclerotomy using the pre-placed 7-0 Vicryl suture. Administer 0.05 milliliter per two milligrams of intravitreal preservative-free triamcinolone at the eight o'clock sclerotomy. Remove the chandelier and infusion cannula.

Close these sclerotomies with Vicryl sutures. Palpate the eye to ensure that the intraocular pressure is within the acceptable range. Inject filtered air or BSS via a 30 gauge needle if required.

An in vivo imaging of the left eye submacular retinal pigment epithelial graft transplant on various imaging modalities was conducted for up to three months. The fundus photography indicated that the transplanted graft at the fovea did not migrate over time. The fundus auto-fluorescence imaging showed minimal changes in hyper auto-fluorescence overlapping the retinal pigment epithelial graft.

The early and late phase fundus fluorescein angiography did not show any leakage surrounding the graft. The macular optical coherence tomography images showed the preservation of outer retinal layers over the graft as time progressed, suggesting the functionality of the retinal pigment epithelial cells. The HE staining showed intact retinal layers with no evidence of micro tears.

The intraoperative optical coherence tomography allows the visualization of the bleb dimensions and the potential foveal tear intraoperatively during foveal detachment. The presence of tear can interfere with bleb formation. The success of correct bleb formation was indicated by the absence of tear.

For the functional assessment of the retina, full field electroretinogram assessments of the retinal pigment epithelial xenografted eye were performed. The absence of significant deterioration in the electroretinogram waveforms suggests that the function of both rod and cone photoreceptors was maintained with the xenografts. During the creation of the localized retinal bleb, do it very gently as any sudden change in jet velocity can lead to foveal tears.

Performing this step slowly is key to reducing foveal injury. Multimodal imaging including fundus auto-fluorescence, fluorescein angiography, and optical coherence tomography along with electrophysiology studies can provide greater insight on the survivability of the graft and factors that affect it.