Development of a Surgical Technique for Subretinal Implants in Rats
Summary
The present protocol describes the scleral approach for subretinal device implantation, a feasible surgical technique for implementation in animal models of retinal diseases in research.
Abstract
Retinal degeneration, such as age-related macular degeneration (AMD), is a leading cause of blindness worldwide. A myriad of approaches have been undertaken to develop regenerative medicine-based therapies for AMD, including stem cell-based therapies. Rodents as animal models for retinal degeneration are a foundation for translational research, due to the broad spectrum of strains that develop retinal degeneration diseases at different stages. However, mimicking human therapeutic delivery of subretinal implants in rodents is challenging, due to anatomical differences such as lens size and vitreous volume. This surgical protocol aims to provide a guided method for transplanting implants into the subretinal space in rats. A user-friendly comprehensive description of the critical steps has been included. This protocol has been developed as a cost-efficient surgical procedure for reproducibility across different preclinical studies in rats. Proper miniaturization of a human-sized implant is required prior to conducting the surgical experiment, which includes adjustments to the dimensions of the implant. An external approach is used instead of an intravitreal procedure to deliver the implant to the subretinal space. Using a small sharp needle, a scleral incision is performed in the temporal superior quadrant, followed by paracentesis to reduce intraocular pressure, thereby minimizing resistance during the surgical implantation. Next, a balanced salt solution (BSS) injection through the incision is carried out to achieve focal retinal detachment (RD). Lastly, insertion and visualization of the implant into the subretinal space are conducted. Post-operative assessment of the subretinal placement of the implant includes imaging by spectral domain optical coherence tomography (SD-OCT). Imaging follow-ups ascertain the subretinal stability of the implant, before the eyes are harvested and fixated for histological analysis.
Introduction
Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. The number of people affected with AMD in 2020 was estimated at 196 million, and this is projected to increase to about 288 million by 20401. Over the past decade, several therapeutics have been developed to mitigate the visual changes associated with the late stages of AMD, mainly to treat the development and progression of the choroidal neovascularization observed in wet AMD. Conversely, the treatment of dry AMD, where dysfunction and loss of retinal pigment epithelium (RPE) cell progress to RPE and retinal atrophy, has been estimated to account for 85% to 90% of AMD, with a prevalence of 0.44% worldwide1,2. AMD has been described as a multifactorial disease with, age, genetic, and environmental factors contributing to the onset and progression of the disease; several therapies are in development to address the different pathophysiological pathways associated with this disease3.
Stem cell-based therapy has been developed as a novel therapeutic option to replace the failing RPE in dry AMD4. Although the usage of pluripotent stem cells is still in early clinical trials, safety has been demonstrated in several clinical trials5,6,7. To date, there are two main routes to deploy stem cells into the subretinal space: suspension or inserting a monolayer patch seeded on a biocompatible implant8,9,10,11,12. New strategies using stem cell-based therapies in preclinical studies require animal models where the stem cell-based therapeutics can be delivered to the same targeted site as intended in humans. The difference in anatomy might mandate minor changes to the procedures, surgical equipment, and approach compared to those used with the final human product13,14. Modifying the ocular surgical techniques is one of the required changes that has been widely described as a successful approach for use across different animal models15,16,17.
Although previous publications have mentioned surgical techniques for subretinal implants in rats, there are no comprehensive descriptions of such techniques to overcome the technical difficulties researchers may encounter. Therefore, there is a need to properly describe the surgical techniques in detail, provide best practices and lessons learned to avoid, and, if needed, address problems during critical steps throughout the procedure. The purpose of this manuscript is to provide a comprehensive guideline for surgical implantation of the implant into the subretinal space in rats.
Protocol
Representative Results
Discussion
Although the procedure has been previously described with slight variations, the scope of this manuscript is to provide a comprehensive description of a surgical procedure for subretinal implants in rats to be followed while learning the technique and to overcome the surgical challenges and potential complications that investigators may encounter. The surgical protocol outlined here includes the usage of the ultrathin parylene membrane that has been widely utilized in our lab for several years9<su…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This study was supported by CIRM DT3 (MSH) and Research to Prevent Blindness (USC Roski Eye Institute). We want to thank Fernando Gallardo and Dr. Ying Liu for their technical assistance.
The sponsor had no role in the design or conduct of this research.
Materials
| 1 cc syringe | VWR | BD309659 | |
| 27 G needle 1/2'' | VWR | BD305109 | |
| 30 G needle 1/2'' | VWR | BD305106 | |
| 32 G Blunt needle – Small hub RN | Hamilton | 7803-04 | |
| 4-0 Perma Hand silk black 1X18" PC-5 | Ethicon | 1984G | |
| 6'' sterile cotton tips | VWR | 10805-154 | |
| Betadine 5% sterile ophthalmic prep solution | Alcon | 8007-1 | |
| BSS irrigating solution 15 mL | Accutome | Ax17362 | |
| Buprenorphine ER | ZooPharm | N/A | |
| Castroviejo Caliper | Storz | E2405 | |
| Castroviejo suturing forceps 0.12 mm | Storz | E1796 | |
| Clayman-Vannas scissors straight | Storz | E3383S | |
| Cover glass, square | WVR | 48366-227 | |
| EPS Polystyrene block | Silverlake LLC | CFB8x12x2 | |
| Gonak 15 mL | Accutome | Ax10968 | Eye lubricant |
| Halstead straight hemostatic mosquito forceps non-magnetic | Storz | E6772 | |
| Hamilton syringe 700 series 100 µL | Hamilton | 7638-01 | |
| HEYEX Software | Heidelberg | N/A | an image management software |
| Kelman-McPherson tying forceps angled | Storz | E1815 AKUS | |
| Ketamine (100 mg/mL) | MWI | 501072 | |
| Needle holder 9mm curved fine locking | Storz | 3-302 | |
| Neomycin/Polymyxin B sulfactes/Bacitracin zinc ointment 3.5 g | Accutome | Ax0720 | |
| Ophthalmic surgical microscope | Zeiss | SN: 233922 | |
| Phenylephrine 2.5% 15 mL | Accutome | Ax0310 | |
| Spectralis SD-OCT | Heidelberg | SPEC-CAM-011210s3600 | |
| Sterile Drape | VWR | 100229-300 | |
| Sterile surgical gloves | VWR | 89233-804 | |
| T-Pump heating system | Gaymar | TP650 | |
| Tropicamide 1% 15 mL | Accutome | Ax0330 | |
| Ultrathin membranes made from Parylene C and coated with vitronectin | Mini Pumps LLC, CA | specifically designed for this study | used as subretinal implants |
| Xylazine (100 mg/mL) | MWI | 510650 |
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