Optimized Minimally Invasive Transscleral Subretinal Injection Technique in Mouse

Subretinal injections are an important tool for preclinical investigations of therapies for inherited retinal diseases. In this video, we present an optimized, minimally invasive subretinal injection technique in mice. Transscleral subretinal injections are an excellent way to deliver material to photoreceptors and RPE.

However, this technique is technically challenging and calls for developing an optimized protocol to increase its reproducibility and accessibility. Our improvements include the development of a mouse eyelid speculum, preoperative administration of atropine, creation of a pinpoint sclerotomy using a diamond knife and optimization of needle-size and injection approach. We hope that our method's consistency, reproducibility, excellent transduction coverage, and minimal surgical damage will improve the accessibility of subretinal injections to the scientific community.

Our optimizations make it easier and faster to perform subretinal injections in mice, reduce experimental variability, and improve injection success rates. This technique will facilitate future preclinical translational retina research. To begin, administer one milligram per kilogram of atropine intraperitoneal in a total volume of 50 microliters 30 minutes before performing the subretinal injection.

Load a glass microneedle with the desired volume of the injection solution. Attach the needle to the micro injector and set the compensation pressure to five hectopascals to counteract capillary action. Place the anesthetized mouse under a dissecting stereomicroscope.

Apply a drop of 0.5%proparacaine to the eye to achieve topical anesthesia. Apply a drop of a mixture containing 0.2%cyclopentolate and 1%phenylephrine to the eye to dilate the pupil for postoperative imaging of the subretinal bleb and to reduce intraoperative bleeding. Using a custom miniature wire speculum, expose the superior fornix and immobilize the speculum to enable hands-free upper eyelid retraction.

Using angled Vannas scissors, make a pair of limbal incision approximately one millimeter posterior to the limbus in the superior conjunctiva and Tenon's capsule to expose the superior sclera. Carefully remove any remnants of Tenon's capsule over the injection site. Now grasp the anterior edge of the conjunctival peritomy and tenotomy with locking toothed forceps.

Then gently inferoduct the globe to expose the superior sclera. Immediately apply a drop of balanced buffered saline to the eye to keep the bare sclera hydrated and to achieve greater optical magnification. Using a diamond knife, make a pinpoint sclerotomy at approximately 12 o'clock and one to two millimeters from the limbus.

Positioning the blade tangentially to the sclera to ensure that the sclerotomy is small and shallow. Use a surgical sponge to gently remove all balanced buffered saline from the surface of the eye. Hold the needle bevel down at a shallow angle to the sclera and slowly insert the tip into the sclerotomy, advancing just 0.5 to one millimeter to access the subretinal space without penetrating the retina.

While holding the needle steady, initiate injection at 500 hectopascals using the foot pedal and maintain continuous pressure for 15 seconds. Remove the needle and observe a partial reflux of the injected solution through the sclerotomy site, indicating successful delivery as the subretinal space cannot accommodate the full volume of the solution injected. Carefully remove the locking forceps and the eyelid speculum without applying pressure to the globe to avoid additional reflux.

Immediately after injection, apply a generous amount of lubricant eye ointment to the eye to assist with subretinal bleb visualization. A subretinal bleb was clearly visible on OCT immediately after transscleral injection, confirming successful delivery. To evaluate retinal pigment epithelium, or RPE, targeting AAV-8 dystrophin 1-GFP was injected, resulting in widespread green fluorescence across the epithelium layer, indicating efficient and broad RPE transduction using the transscleral approach.

AAV-8 rhodopsin GFP led to a strong green fluorescent signal in rod photoreceptors, visible in both cross-sections and fundus views, indicating successful targeting and expression in photoreceptor cells. AAV8-CAG-mCherry produced broad red fluorescence across retinal layers, demonstrating the ability of this technique to support generalized transgene expression beyond specific cell types. Western blotting of retinal lysates also showed strong green fluorescent protein expression with the highest levels corresponding to the center of the injection bleb, validating photoreceptor transduction with AAV8 rhodopsin GFP.

AAV titer optimization using immunofluorescence analysis of retinal sections allows for the selection of the viral titer that achieves the transduction of approximately 90%of target cells. For example, in this case, a one to 10 dilution of a AAV8 rhodopsin GFP viral stock produced transgene expression in almost all photoreceptors. By contrast, a one to 100 dilution resulted in an insufficient transduction rate.

Electroretinogram showed that transscleral injections preserved normal retinal function while transretinal injections led to significantly reduced A wave and B wave responses, indicating retinal damage.