Dual ROCK1/2–MYLK4 Kinase Inhibition Preserves Visual Function in a Rat Model of Neuromyelitis Optica Spectrum Disorder Optic Neuritis

Highlights: What are the main findings? Intravitreal administration of ITRI-E-(S)4046 (dual ROCK1/2–MYLK4 inhibitor) preserved visual-evoked potentials and retinal ganglion cell survival in an NMOSD rat optic neuritis model. TRI-ES suppressed optic nerve inflammation, demyelination, and glial activation while promoting anti-inflammatory (M2) macrophage polarization. What is the implication of the main finding? Dual ROCK/MYLK4 inhibition represents a novel neuroprotective strategy that may complement immunotherapy in NMOSD-related optic neuritis. This study supports the translation of kinase-targeting therapeutics aimed at preserving visual function in autoimmune demyelinating diseases. Background: Neuromyelitis optica spectrum disorder (NMOSD) causes severe optic nerve (ON) inflammation and vision loss. Current treatments remain limited, prompting exploration of new therapeutic strategies. This study evaluated the efficacy of ITRI-E-(S)4046 (ITRI-ES), a dual ROCK1/2 and MYLK4 kinase inhibitor, in a rat model of NMOSD optic neuritis. Methods: NMOSD-like optic neuritis was induced in rats by applying NMOSD patient serum-soaked sponges around the ON. Rats received intravitreal injections of either 0.2% ITRI-ES, phosphate-buffered saline (PBS), or intraperitoneal methylprednisolone (MP). Visual function was assessed using flash visual-evoked potentials (fVEP). Retinal ganglion cell (RGC) survival and apoptosis were quantified using FluoroGold retrograde labeling and TUNEL assay. ON inflammation and demyelination were evaluated via immunohistochemistry and Western blot analysis of aquaporin-4 (AQP4), myelin basic protein (MBP), glial fibrillary acidic protein (GFAP), and inflammatory markers. Results: ITRI-ES significantly preserved visual function, restoring fVEP amplitudes (~36 μV vs. ~21 μV in PBS-treated, p < 0.05) and RGC density (~85% of normal vs. ~37% PBS). RGC apoptosis was reduced (~2.3-fold lower vs. PBS, p < 0.05). PBS-treated rats showed decreased AQP4 and MBP (2.5–2.8-fold vs. sham) and increased GFAP (2.8-fold). ITRI-ES maintained higher AQP4 (~3.5-fold) and MBP (~1.5-fold) levels, suppressed GFAP (~5.5-fold vs. PBS), reduced NF-κB, IL-1β, TNF-α, microglia activation, and macrophage infiltration, and increased anti-inflammatory Arg1 and CD206 markers (~3-fold vs. PBS). Conclusions: ITRI-ES alleviates optic nerve inflammation, preserves retinal integrity, and maintains visual function in NMOSD-associated optic neuritis, underscoring kinase inhibition as a promising therapeutic strategy.