In vitro model to study degeneration and neuroprotection of corneal nerve diseases.

Abstract

Purpose : Corneal sensory innervation plays a critical role in maintaining ocular surface homeostasis. It participates in the corneal defensive response to injury and regulates ocular surface hydration. Corneal sensory nerve terminals are exposed to the effect of oxidative damage associated with aging or pathology and visual function may be compromised. Little is known about the effect of neuroprotectants on corneal innervation. The aim of this study is to develop an in vitro model to study neuroprotective treatments of ocular surface sensory nerves as preventive therapies for the onset of age-related oxidative pathologies such as dry eye disease.

Methods : The SH-SY5Y line was used to develop an in vitro model to study and validate the short amplitude wavelength light stimulus (blue light, 470 nm, 400 lux, 18 N/m2) as a generator of oxidative stress and mitochondrial dysfunction in neurites. Sodium azide (NaN3) and rotenone were used as control oxidizing agents. Once replicable models of neuronal degeneration were obtained, neuroprotective strategies were tested. Cultures were pretreated with Rapamycin, N-acetyl cysteine (NAC) and crocin from saffron (Crocus sativus). Immunofluorescence techniques with specific antibodies against β-tubulin III, phosphorylated tau, CaMKII and Heme oxygenase-1 were performed to analyze degeneration and oxidative stress.

Results : The results showed that overexposure to blue light induced distal nerve degeneration in cultures, characterized by the disorganization of neurite microtubules and reduced cell viability. Neuroprotectants showed significant beneficial effects compared with untreated neurons in this model, including a lower level of HO-1 enzyme activation, maintenance of mitochondrial membrane integrity and reduction of CaMKII expression and subsequent microtubule smoothness and stability. In addition, neuroprotection resulted in increased cell viability compared to untreated neurons.

Conclusions : This in vitro system is an sensituve and effective model for the study of the degenerative processes that can occur in corneal sensory nerves. Treatment with antioxidants and Rapamycin had neuroprotective effects on neuronal nerve fibers, suggesting a possible effect in preventing oxidative damage to the innervation of the ocular surface.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.