In Vitro and In Vivo Methods for Studying Retinal Ganglion Cell Survival and Optic Nerve Regeneration

Abstract

Glaucoma is marked by a progressive degeneration of the optic nerve and delayed loss of retinal ganglion cells (RGCs), the projection neurons of the eye. Because RGCs are not replaced and because surviving RGCs cannot regenerate their axons, the visual loss in glaucoma is largely irreversible. Here, we describe methods to evaluate treatments that may be beneficial for treating glaucoma using in vitro cell culture models (immunopanning to isolate neonatal RGCs, dissociated mature retinal neurons, retinal explants) and in vivo models that test potential treatments or investigate underlying molecular mechanisms in an intact system. Potentially, use of these models can help investigators continue to improve treatments to preserve RGCs and restore visual function in patients with glaucoma.

The extent of extra-axonal tissue damage determines the levels of CSPG upregulation and the success of experimental axon regeneration in the CNS

“…No clinical treatments are available to date to help those who suffer from loss of function due to axonal injuries associated with spinal cord injury 1 , stroke 2 , 3 , brain trauma 4 , 5 , and optic neuropathy 6 – 9 . The ONC rodent model of traumatic optic neuropathy (TON) is a well-established system for tackling the fundamental problem of long-distance axon regeneration failure in the CNS and for determining therapeutic potential of novel treatments 10 , 11 . For example, molecules such as Pten and Klf7 that were discovered to control axon growth in this model 12 , 13 , were later found to also regulate spinal cord axon regenereation 14 , 15 .…”

“…Because spontaneous axon regeneration failure in the CNS affects mammals, but not necessarily lower vertebrates, rodent models of optic nerve, spinal cord, and brain injuries have been developed to tackle this problem. For example, like other non-retinal CNS projection neurons, rodent retinal ganglion cells (RGCs) do not spontaneously regenerate axons disrupted by an optic nerve crush (ONC) injury 10 , 11 . Although modest sprouting near the injury site may occur, the axons do not regenerate over long distances without treatment.…”

Modeling of Retina and Optic Nerve Ischemia–Reperfusion Injury through Hypoxia–Reoxygenation in Human Induced Pluripotent Stem Cell-Derived Retinal Ganglion Cells

“…However, these animal studies significantly differ from the situation in which human RGCs are exposed to ischemia-reperfusion. In vitro, RGCs have been isolated and cultured from a rat retina [29,30]. Nevertheless, RGC isolation and culture from a rat retina are unstable and may not be suitable for a cell death analysis because they have short neurites but not long axons.…”

The Healthy and Diseased Retina Seen through Neuron–Glia Interactions

“…Additionally, acute injuries to the optic nerve, such as axotomy or transection, are induced in animal models to investigate the processes governing the death and survival of these cells. Various in vivo and in vitro models, including those developed by the authors of [445,446], have been instrumental in studying RGC survival and optic nerve regeneration [447]. In order to prevent or delay the death of these cells, different neuroprotective mechanisms have been identified, such as the secretion of neurotrophic factors, the activation of antioxidant enzymes, the increased expression of anti-apoptotic proteins, the induction of autophagy, and proteostasis regulation [448][449][450][451].…”