Exploiting Müller Glia Progenitor Cell Signaling Pathways for Retinal Regeneration in Chick Retinas Using Small Molecule Drug Cocktails
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Date
2025-03
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Abstract
Humans lack the capacity for retinal regeneration, making degenerative retinal diseases irreversible. However, animals like chickens and zebrafish exhibit partial or complete retinal regeneration, respectively, with Müller glia (MG) cells playing a pivotal role. MG can de-differentiate into Müller glia-derived progenitor cells (MGPCs), but only a small proportion of chick MGPCs develop into neurons. This study investigates methods for increasing regenerative neurogenesis rates in chickens, with the goal of identifying mechanisms that could be leveraged for therapeutic interventions. Post-hatch day 3 to 8 (P3–8) White Leghorn chicks received intraocular injections of damage-inducing N-methyl-D-aspartate (NMDA). EdU (5-ethynyl-2'-deoxyuridine), a lineage tracer for proliferating MGPCs, was co-injected. Three days after damage, chicks were injected with mixtures of small-molecule drug cocktails reported to promote neural fate in cell culture or a vehicle control. Small molecules tested were: Wnt pathway activators (CHIR-99201, ISX9), a BET (bromodomain and extra-terminal domain protein) inhibitor (I-BET151), an adenylate cyclase activator (Forskolin), a ROCK (rho-associated, coiled-coil containing protein kinase) inhibitor and a PKA (protein kinase A) activator (Bucladesine). After weeklong incubation, retinas were processed for HuC/D immunostaining and EdU incorporation visualization. MGPCs that differentiated into neurons (EdU+/HuC/D+ cells) were visualized via confocal microscopy and counted. Statistical analysis was performed using a two-tailed t-test. Cocktails containing Forskolin, I-BET151, and Wnt activators significantly enhanced neurogenesis. Retinas treated with Cocktail 1 (C1: Forskolin, I-BET151, CHIR-99201) showed significantly more new neurons than controls (Δx̄ = +1.39; p = 0.01). Adding ISX9 to C1 increased neurogenesis but had a smaller effect size (Δx̄ = +1.00; p = 0.04). Cocktails including a ROCK inhibitor and Bucladesine showed a non-significant increase (Δx̄ = +1.10; p = 0.06). These findings suggest the Wnt pathway and BET proteins play a role in the differentiation of MGPCs into new neurons.
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Health Under the Microscope (The Ohio State University Denman Undergraduate Research
Forum)