Abstract
Purpose :
In patients with dry eyes, increased friction between eyelids and ocular surface while blinking has been reported to cause corneal inflammation and pain. To test the hypothesis that friction affects neurons and elicits pain responses, fluid shear stress was experimentally applied to a human neuroblastoma SH-SY5Y cell line, and neurite morphology and expression of factors involved in pain transmission pathways were evaluated.
Methods :
SH-SY5Y cells were seeded into ibidi μ-Slide IV (ibidi GmbH) (1.2 x 106 cells / cm2) and incubated at 37 ° C for 72 h in a 5% CO2 environment. The cells were then subjected to shear stress (59 dyn / cm2) for 24 h using a bi-directional water flow operated by ibidi Pump System (ibidi GmbH). Neurite morphology was observed over time using light microscopy and manual measurement of the total neurite length per image was performed. The gene expression levels of Nav1.7 sodium channel (Scn9a), protachykinin-1 (tac1), and cyclic AMP-dependent transcription factor (atf3) were measured using real-time PCR. The protein expression and localization of Nav1.7 was evaluated using immunostaining.
Results :
Focal bead-like swelling in neurite was initiated as early as 5 min following shear stress loading, which resulted in a significant decrease in the total neurite length at 10 min in comparison to pre-shear stress loading（1.96±1.61 vs 6.48±0.90 µm / image, p <0.001）. In gene expression analysis, 1 h of frictional shear stress significantly upregulated the expression of tac1 (17.9-fold, p < 0.001) and atf3 (2.30-fold, p < 0.001) and the increase was maintained until 24 h post shear stress loading. Scn9a increased in a time-dependent manner from 3 h and the highest expression was observed at 24 h following shear stress loading (2.66-fold, p < 0.001). Protein expression of Nav1.7 localized in the cytoplasm and neurites was increased at 24 h following shear stress loading.
Conclusions :
Shear stress loading alters neurite structure, which may result in upregulating the expression of atf3. Subsequently, this may upregulate the expression of tac1 and Scn9a. These results indicate that frictional shear stress injures neurons and activates pain transmission pathways, suggesting that friction during blinking may play an important role in development of corneal pain.
This is a 2020 ARVO Annual Meeting abstract.