Optic Nerve Crush in Mice to Study Retinal Ganglion Cell Survival and Regeneration

In diseases such as glaucoma, the failure of retinal ganglion cell (RGC) neurons to survive or regenerate their optic nerve axons underlies partial and, in some cases, complete vision loss. Optic nerve crush (ONC) serves as a useful model not only of traumatic optic neuropathy but also of glaucomatous injury, as it similarly induces RGC cell death and degeneration. Intravitreal injection of adeno-associated virus serotype 2 (AAV2) has been shown to specifically and efficiently transduce RGCs in vivo and has thus been proposed as an effective means of gene delivery for the treatment of glaucoma. Indeed, we and others routinely use AAV2 to study the mechanisms that promote neuroprotection and axon regeneration in RGCs following ONC. Herein, we describe a step-by-step protocol to assay RGC survival and regeneration in mice following AAV2-mediated transduction and ONC injury including 1) intravitreal injection of AAV2 viral vectors, 2) optic nerve crush, 3) cholera-toxin B (CTB) labeling of regenerating axons, 4) optic nerve clearing, 5) flat mount retina immunostaining, and 6) quantification of RGC survival and regeneration. In addition to providing all the materials and procedural details necessary to execute this protocol, we highlight its advantages over other similar published approaches and include useful tips to ensure its faithful reproduction in any modern laboratory.