Neuroprotective Strategies for Retinal Ganglion Cell Degeneration: Current Status and Challenges Ahead

Abstract

The retinal ganglion cells (RGCs) are the output cells of the retina into the brain. In mammals, these cells are not able to regenerate their axons after optic nerve injury, leaving the patients with optic neuropathies with permanent visual loss. An effective RGCs-directed therapy could provide a beneficial effect to prevent the progression of the disease. Axonal injury leads to the functional loss of RGCs and subsequently induces neuronal death, and axonal regeneration would be essential to restore the neuronal connectivity, and to reestablish the function of the visual system. The manipulation of several intrinsic and extrinsic factors has been proposed in order to stimulate axonal regeneration and functional repairing of axonal connections in the visual pathway. However, there is a missing point in the process since, until now, there is no therapeutic strategy directed to promote axonal regeneration of RGCs as a therapeutic approach for optic neuropathies.

Keywords: axonal regeneration; neurodegeneration; neuroprotection; optic neuropathies; retinal ganglion cells.

Figure 1

Schematic representation of the neural sensory retina, depicting the organization of the cells into nuclear and plexiform layers. The nuclei of photoreceptors, rods and cones, are located in the outer nuclear layer (ONL) and nuclei of interneurons, amacrine, bipolar and horizontal cells, are located predominately in the inner nuclear layer (INL). The cell bodies of RGCs are in the ganglion cell layer (GCL), and their axons run in the nerve fiber layer (NFL). There are two types of macroglia: Müller cells that span vertically the entire retina and astrocytes that are present in the GCL. Microglial cells are localized predominately in the inner retina and in the outer plexiform layer (OPL). IPL: inner plexiform layer; IS/OS: inner and outer segments of photoreceptors.

Figure 2

Neurite growth of RGCs in culture. (A) Schematic representation of the experimental design. Retinas were dissected from Wistar rats at PND 5 and nearly pure RGC cultures (~93% purity assessed with anti-RBPMS antibody; Abcam, Cat. # ab194213, 1:500) were obtained by sequential immunopanning, as previously described [8,9]. RGCs were cultured for 1 day in vitro (DIV1), DIV2 and DIV3, followed by fixation in paraformaldehyde and processed for immunocytochemistry. (B) RGCs were identified by immunolabeling for Brn3a (red, Millipore, Cat. # MAB1585, 1:500), a transcription factor expressed only by these cells in the retina. The neurites, labelled with an antibody that recognizes β-tubulin III (green, BioLegend, Cat. # 802001; 1:1000), extended during the period in culture. Nuclei were stained with DAPI (blue).

Figure 3

Elevated hydrostatic pressure (EHP) impacts neurite growth of RGCs. (A) Schematic representation of the experimental design. RGCs were purified from Wistar rats at PND 5 by sequential immunopanning, as previously described [8,9] and were cultured for DIV2. RGCs were challenged with EHP (+70 mmHg above atmospheric pressure) [78,79]) for 24 h and 48 h and then processed for immunocytochemistry as described in the legend of Figure 2. (C) RGCs were plated in a coverslip with a cloning cylinder and neurite extension was observed beyond the limit established by the cylinder (B, grey dashed circle). Exposure to EHP decreased the length of the neurites when compared with the control (CTR) condition (normal pressure). (D) Higher magnification. This effect on the neurites of RGCs is dependent on the duration of the exposure to EHP.

Figure 4

Schematic representation of the main strategies for RGC neuroprotection. Blue squares represent the therapies directed to RGCs and red squares represents the undirected therapies that culminates in RGCs neuroprotection.

Figure 5

Diagram summarizing the main interactions of Müller cells (blue) with RGCs (orange). Scheme showing the roles of Müller cells in RGC neuroprotection, such as glucose metabolism regulation, water and ion homeostasis, neurotransmitters uptake, antioxidant defense systems (GSH) against ROS, secretion of trophic factors. The role of Müller cells in inflammation by secretion of cytokines that may be detrimental for RGCs is also depicted (red arrow).