Optimized Minimally Invasive Transscleral Subretinal Injection Technique in Mouse

Summary

Subretinal injections are an indispensable technique for preclinical development of therapeutics for photoreceptor and RPE diseases. We describe an optimized transscleral subretinal injection technique in mice that improves intraoperative success rates and minimizes retinal damage.

Abstract

The conventional method of material delivery to the subretinal space in the mouse involves dual perforation of the neural retina, which causes extensive surgical damage. This leads to variability in the subsequent outcome measures of the visual function, such as electroretinogram (ERG) recordings or behavioral vision assays, which confound efficacy assessments of experimental therapeutics. To overcome these barriers, we optimized a transscleral minimally invasive subretinal injection technique in mice. In this technique, the superior fornix is accessed using a custom-made tungsten wire eyelid speculum to perform conjunctival peritomy and tenotomy. A pinpoint sclerotomy is made using a diamond knife without penetrating the retina, through which a fine glass needle is carefully inserted at a shallow angle. The payload is delivered to the subretinal space using a microinjection pump. Optical coherence tomography (OCT) is used to assess the size and position of the subretinal bleb immediately following injection. We compared the outcomes of this technique to the conventional transretinal method performed using a spring-loaded syringe with a 33G needle sclerotomy. ERG recordings indicated excellent preservation of retinal function in transsclerally-injected mice, comparable to that of uninjected control mice. In contrast, an appreciable ERG signal reduction was observed in the conventionally injected cohorts. In summary, we have optimized a minimally invasive technique for subretinal injection in mice. We demonstrate that this technique is a robust and efficient method for gene therapy administration that minimizes anatomic damage to the retina and has minimal impact on retinal function. This method lowers the threshold for the development of therapies targeting both the retinal pigmented epithelium and photoreceptors to treat a wide range of retinal and macular diseases.

Introduction

Subretinal injection is an ocular surgical procedure in which material is delivered to the potential space between the retina and the retinal pigmented epithelium (RPE). Compared to other methods of drug delivery to the posterior segment, such as intravitreal or suprachoroidal injections, subretinal injections achieve the most immediate access to both the photoreceptors and the RPE. For delivery of viral gene therapy particles, subretinal injection achieves much higher rates of photoreceptor and RPE transduction, which allows for lower viral titers and reduced risk of viral toxicity¹. Additionally, the subretinal space is anatomically confined,....

Protocol

All experiments were performed in accordance with the Duke University Institutional Animal Care and Use Committee and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Animal preparation

1. Administer an intraperitoneal injection of 1 mg/kg of atropine (in a total volume of 50 μL) 30 min prior to performing the subretinal injection. This step greatly increases success rates by preventing potentially lethal oculocardiac and oculorespiratory reflexes triggered by transient intraocular pressure elevation during the injection

Results

A successful injection using this transscleral technique generates a subretinal bleb observable on OCT immediately following the injection procedure (Figure 2A). Given the technical challenge of this technique, the imaging step is indispensable for identifying successfully injected animals. For researchers who plan to use this technique for gene therapy experiments, we recommend performing practice injections with adeno-associated virus (AAV) particles carrying a fluorescent reporter gene. A.......

Discussion

The technique described here introduces several improvements to the previously described transscleral injection approach⁴, which enhance the reliability of the procedure and further reduce surgical damage to the eye. One such improvement is the usage of a speculum to retract the upper eyelid, allowing for stable, unencumbered, and hands-free access to the eye. Commercial specula are often poorly optimized, even for adult mice, and outright unavailable for juveniles. To overcome this obstacle, we d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride Injection, USP	Hospira	PAA130066
1% Atropine Sulfate Ophthalmic Solution, USP	Akorn	NDC 17478-215-05
30° diamond knife	Katena	K2-6560
AmScope 6W LED dual gooseneck illuminator	AmScope	LED-11CR
Angled Vannas scissors	Roboz	RS-5618
BSS Sterile Irrigating Solution	Alcon	289010-0902
Cyclomydril dilating eyedrops	Alcon	0065-0359-02	0.2% cyclopentolate, 1% phenylephrine
epT.I.P.S. MicroloaderTM pipette tips	Eppendorf	5242 956.003
FemtoJet 4i Microinjector	Eppendorf	E5252000021	Discontinued product
GenTeal Tears lubricant eye ointment	Alcon	16027802
Glass microinjection needles	Clunbury Scientific	B100-58-50	30° bevel. Diameter: 50 µm outer, 29 µm inner
Isoflurane solution	Covetrus	29405
MICRON IV retinal imaging microscope with image-guided OCT	Phoenix	N/A
Stereomicroscope SteREO Discovery.V8	Zeiss	495015-0001-000
Stern-Castroviejo locking toothed forceps	Katena	K5-2552
Toothed forceps	Roboz	RS-5172
Tungsten cleaning wires for 23s gauge needles	Hamilton	18306
Ultracell PVA Eye Spears	BVI Medical	40400-8
Ultra-Fine2015 insulin syringes	BD	328438	3/10 mL, 8 mm (5/16”), 31G