Immobilized Netrin-1 but not BDNF or L1cam Polarizes Retinal Ganglion Cell Growth
Publications Category : Ophthalmology
Author(s): Lana J. Reaves, Katelyn M. Brown, Karl E. Kador
Project Description : Purpose : Retinal ganglion cells (RGCs), which grow randomly in vitro, respond to physical, secreted and ECM based guidance cues to create the nerve fiber layer (NFL) in vivo. Previously, we developed an electrospun scaffold capable of directing RGC neurite growth to match the radial alignment of the NFL but these scaffolds were not capable of polarizing this growth. In this study we will chemically immobilize the neural guidance factors Netrin-1, BDNF and L1cam in a gradient on electrospun scaffolds to determine if the combination of the physical guidance of the scaffold and the biochemical guidance of the individual factors can polarize RGC growth. Methods : Electrospun PLA scaffolds underwent CO2 plasma treatment to add carboxylic acid functional groups to the surface. Water contact angle measurements were conducted to determine the hydrophobicity, a measure of the degree of modification in order to optimize the reaction time. Netrin-1, BDNF and L1cam were immobilized on the treated scaffolds in dose response curves using EDC/NHS chemistry and the scaffolds seeded with RGCs isolated for early postnatal rats. Following two days in culture, the samples were fixed and stained for βIII-tubulin and imaged using a fluorescent microscope. IMAGEJ was used to analyze RGCs for their growth inward, outward, or bidirectional neurite growth. ANOVA was run for statistical analysis with post hoc testing. Results : Electrospun PLA scaffolds demonstrated a lower water contact angle following plasma reaction indicating an increase in hydrophilicity but degradation was observed after 5 minutes of reaction. Immobilization of BSA-FITC created a gradient as they adsorb across the scaffold. RGCs increased polarization following Netrin-1 immobilization with increasing concentration when samples were coated in fibronectin but samples coated in laminin were repulsive. However, samples cultured with PTEN knock out RGCs were observed to grow along the Netrin-1 gradient on both fibronectin and laminin coated scaffolds. Scaffolds containing gradients of BDNF and L1cam did not increase in RGC polarization compared to controls. Conclusions : PLA can be modified using CO2 to facilitate protein immobilization. The immobilization of Netrin-1 is able to polarize RGC growth to the center of biodegradable scaffolds indicating the incorporation of biochemical cues with physical guidance cues can better reproduce the organization seen in vivo.
Additional Informational : Presented at the 2025 ARVO Annual Meeting, held in Salt Lake City, Utah, May 4-8, 2025.