Optimized Minimally Invasive Transscleral Subretinal Injection Technique in Mouse

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.