A bioengineering strategy mimicking electrical stimulation to promote neurite outgrowth of retinal ganglion cells through glutamate signaling

Abstract

Purpose : Being a part of the central nervous system, retinal and optic nerve injuries caused by trauma or diseases often lead to progressive degeneration of retinal ganglion cells (RGCs) and permanent vision loss that cannot be restored. The purpose of this study is to develop a biocompatible polyelectrolyte scaffold that supports RGC survival and directs axon growth to promote optic nerve regeneration.

Methods : Retinal explants or primary RGCs were isolated from postnatal day 0-3 C57BL/6 mouse pups and cultured in the presence or absence of glutamate blockers targeting ionotropic or metabotropic glutamate receptors, respectively. Cultures were treated with or without electrical stimulation (ES) at 20 Hz, 50 μA for 15 min or plated on top of polyelectrolyte scaffolds that were fabricated by electrospinning and incubated for 1-5 days. The rate of cell survival, neurite outgrowth, neurite lengths, and angles of neurite extensions were quantified by immunocytochemistry. Statistical significance was calculated using Student's t-test or one-way analysis of variance (ANOVA), and a value of p < 0.05 was considered statistically significant.

Results : We found ES significantly promoted the number and lengths of RGC neurites. This effect of ES was largely diminished in the presence of glutamate receptor blockers targeting ionotropic N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors, or metabotropic receptors. Glutamate-conjugated polyelectrolyte biomimetic isotropic or aligned electrospun scaffolds were thus generated, featuring similar 3D fibrous structures and physical properties, to mimic continuous glutamate stimulation. The glutamate-containing polyelectrolyte scaffolds supported RGCs survival and promoted robust growth of long neurites in both retinal explants and dissociated cell cultures (p<0.01). The aligned scaffolds also drove directed nerve elongations along the direction of the fibers, guiding neurites growing towards the same orientation.

Conclusions : ES enhances RGCs growing long neurites through glutamate signaling. By constructing a glutamate-rich polymer scaffold, it presents a permissive biomaterial that drives robust and directed axon growth in primary cultured RGCs as well as retinal explants. Such polyelectrolyte scaffolds can be beneficial for future nerve repair and regeneration.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.