The overall goal of this study is to describe a Novel subretinal injection technique that can be used to target viral vectors, pharmacological agents, or induce pluripotent stem cells to the subretinal space in mice. This method can answer key questions in the vision science and ophthalmology fields, such as assessing the efficacy of Novel therapeutic interventions for retinal degenerations. The main advantage of this technique is that it can be used to deliver material to the subretinal space, with high efficacy, minimal damage, and fast recovery of retinal structure and function.
Demonstrating the procedure will be Sachin P, a technician from my laboratory. To begin this procedure, trim the whiskers of an an anesthetised mouse to facilitate visualization, and maintain the animal's body temperature at 37 degrees Celsius with a circulating water pad. Next, dilate its pupils with 2.5%phenylephrine eye drops.
Then, apply methylcellulose eye drops to prevent dryness, and minimize anesthetic induced transient cataracts. Sterilize the instruments prior to surgery. Afterward, prepare the diluted fluorescein in a biosafety cabinet.
Fill the syringe with the appropriate amount of fluorescein. Next, perform a toe pinch to ensure the animal is deeply anesthetised, and position the mouse so that the eye to be injected is facing up and clearly visible in the dissecting microscope. Gently pinch the temporal conjunctiva with a pair of fine-tipped forceps.
Then, make a circumferential incision of approximately 90 degrees using the curved Vannas'scissors. Repeat the procedure on the underlying Tenon's capsule. After that, resect the surrounding connective issue with fine-tipped forceps, while rotating the globe nasally.
Work towards the injection site at approximately 0.5 millimeters away from the optic nerve and take great care to avoid disrupting the retro-orbital sinus. A critical step is exposing the injection site which requires rotation of the eye, without damaging the eye itself, or associated structures, particularly avoiding damage to the orbital blood sac. In this procedure, make a small incision at the sclera of the injection site, by gently scratching the eye cup with a 22.5 degree ophthalmic blade.
This incision should just be large enough to allow the tip of the needle to pass through the sclera. Next, insert the beveled 33 gauge needle into the sclerotomy with the bevel facing and angled parallel to the retina. Inject the desired amount of 0.01%fluorescein by pressing the plunger slowly with even pressure.
Note that when the needle is in the subretinal space, a slight resistance will be felt while pressing the plunger. There will be no resistance if the needle punctures the retina, but high resistance if the needle does not penetrate the sclera or RPE. Another critical step is the injection targeting subretinal space.
This must be accomplished without penetrating through the neurosensory retina, to the vitreous cavity. Wait several seconds before withdrawing the needle, to minimize backflow. Subsequently, rinse the eye with sterile saline, and ensure the eye has rotated back to its normal position.
Then, apply a thick coat of triple antibiotic ophthalmic cream to the corneal surface of the injected eye. Afterward, place the mouse in a clean solitary cage for recovery. Monitor its respiration and temperature during anesthesia recovery, and check that it can maintain sternal recumbency.
Perform additional appropriate post-operative monitoring and treatment, including a subcutaneous injection of carprofen, for post-surgical pain management. Here is a 3D reconstruction of the dome-shaped bleb at the injection site. The dome-shaped bleb is then filled in dense white to show the extent and margins of the retinal detachment.
Retinal cross-sections from the OCT can be seen in white, while the fluid-filled space at the level of the RPE and photoreceptors appears black. And in this figure, the representative OCTB scans at the site of maximal retinal detachment are shown for pre-injection, 10 minutes post-injection, and four weeks post-injection. This image shows the formation and resolution of a flat bleb from a 0.3 microliter injection.
And this image shows the formation and resolution of a domed bleb from a 0.5 microliter injection, whereas this image shows the formation and resolution of a domed bleb from a one microliter injection. Lastly, here is an example of severe choroidal scarring and retinal thinning at the injection site. Retinas retain normal function after bleb resolution.
At nine illumination intensities, the waveforms for scotopic rod mediated responses are shown for pre-injection and four weeks post-injection for 0.3 microliter, 0.5 microliter, and 1 microliter injections. Once mastered, this technique can be completed in 10 to 15 minutes per eye. While attempting this procedure, it is important to evaluate the health of the eyes before commencing.
Exclude eyes with corneal or lens opacities, or deep-set eyes for which this procedure would be very difficult. Following this procedure, other methods such as OCT and fundus imaging can be performed in order to evaluate the quality of the injection.
