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% o....

Protocol

All experiments were approved by the University of Southern California Institutional Animal Care and Use Committee (IACUC) and were performed following the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals and The Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research. A total of 12 Royal College of Surgeon (RCS) male rats were used in the present study. Animals were bred in the animal facility and included in the study once they reached the age of 28 ± 1 days postnatal. A complete eye exam was performed to verify the lack of eye abnormalities. The subretina....

Results

Implantation of a subretinal implant in RCS rats (N = 12) demonstrated the feasibility and reproducibility of the surgical technique for subretinal delivery in rats. In this study, the right eye was the treated eye (N = 12) with the implant. In the clinical assessment conducted at the end of the procedure using the surgical microscope, nine of the 12 treated eyes demonstrated a subretinal localization of the implant (75.00%), two eyes (16.67%) were identified as an intraretinal placement of the implant, and in one eye (8.......

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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