Corneal neuroepithelial compartmentalized microfluidic chip model for evaluation of toxicity-induced dry eye.

Part of the lacrimal functional unit, the cornea protects the ocular surface from numerous environmental aggressions and xenobiotics. Toxicological evaluation of compounds remains a challenge due to complex interactions between corneal nerve endings and epithelial cells. To this day, models do not integrate the physiological specificity of corneal nerve endings and are insufficient for the detection of low toxic effects essential to anticipate Toxicity-Induced Dry Eye (TIDE). Using high-content imaging tool, we here characterize toxicity-induced cellular alterations using primary cultures of mouse trigeminal sensory neurons and corneal epithelial cells in a compartmentalized microfluidic chip. We validate this model through the analysis of benzalkonium chloride (BAC) toxicity, a well-known preservative in eyedrops, after a single (6h) or repeated (twice a day for 15 min over 5 days) topical 5.10-4% BAC applications on the corneal epithelial cells and nerve terminals. In combination with high-content image analysis, this advanced microfluidic protocol reveal specific and tiny changes in the epithelial cells and axonal network as well as in trigeminal cells, not directly exposed to BAC, with ATF3/6 stress markers and phospho-p44/42 cell activation marker. Altogether, this corneal neuroepithelial chip enables the evaluation of toxic effects of ocular xenobiotics, distinguishing the impact on corneal sensory innervation and epithelial cells. The combination of compartmentalized co-culture/high-content imaging/multiparameter analysis opens the way for the systematic analysis of toxicants but also neuroprotective compounds.

Benzalkonium chloride-induced direct and indirect toxicity on corneal epithelial and trigeminal neuronal cells: proinflammatory and apoptotic responses in vitro.

Benzalkonium chloride (BAK), a quaternary ammonium compound widely used as disinfecting agent as well as preservative in eye drops is known to induce toxic effects on the ocular surface with inflammation and corneal nerve damage leading to dry eye disease (DED) in the medium-to-long term. The aim of this study was to evaluate in vitro the toxicity of a conditioned medium produced by corneal epithelial cells previously exposed to BAK (BAK-CM) on trigeminal neuronal cells. A human corneal epithelial (HCE) cell line was exposed to 5.10-3% BAK (i.e. 0.005% BAK) for 15 min and let recover for 5 h to prepare a BAK-CM. This BAK concentration is the lowest one found in eye drops. After this recovery period, BAK effect on HCE cells displayed cytotoxicity, morphological alteration, apoptosis, oxidative stress, ATP release, CCL2 and IL6 gene induction, as well as an increase in CCL2, IL-6 and MIF release. Next, a mouse trigeminal ganglion primary culture was exposed to the BAK-CM for 2 h, 4 h or 24 h. Whereas BAK-CM did not alter neuronal cell morphology, or induced neuronal cytotoxicity or oxidative stress, BAK-CM induced gene expression of Fos (neuronal activation marker), Atf3 (neuronal injury marker), Ccl2 and Il6 (inflammatory markers). Two and 4 h BAK-CM exposure promoted a neuronal damage (ATF-3, phospho-p38 increases; phospho-Stat3 decreases) while 24 h-BAK-CM exposure initiated a prosurvival pathway activation (phospho-p44/42, phospho-Akt increases; ATF-3, GADD153, active Caspase-3 decreases). In conclusion, this in vitro model, simulating paracrine mechanisms, represents an interesting tool to highlight the indirect toxic effects of BAK or any other xenobiotic on corneal trigeminal neurons and may help to better understand the cellular mechanisms that occur during DED pathophysiology.

Formaldehyde Gas Exposure Increases Inflammation in an In Vitro Model of Dry Eye.

Dry eye (DE) is a multifactorial ocular surface disease whose incidence continues to rise. Various environmental stresses such as low air humidity and pollution are known to be involved in epithelial alterations inducing ocular discomfort. However, no experimental study assessing the combined effects of dry air and polluted atmospheres has been conducted so far. Formaldehyde (FA) is a ubiquitous pollutant present in the living spaces where humans spend most of their time. Using an in vitro DE model, we evaluated the cytotoxic and inflammatory responses of a conjunctival cell line exposed at the air-liquid interface (ALI) conditions to various controlled atmospheres combining low humidity (LH), airflow (AF), and formaldehyde gas (FG). Conjunctiva-derived cells grown onto transwell inserts were directly exposed to LH conditions without AF, with AF or with FG flow at 100 or 1200 µg/m3 for 15-30 min. Cell viability assays revealed an increase in cell death after a 15-min exposure to FG at 100 or 1200 µg/m3, whatever the recovery period. After a 1-h recovery period, an increase in IL-6 and CXCL8/IL-8 gene expression was observed with the 15-min exposure at 100 µg/m3 FG and with 30 min of exposure at 1200 µg/m3 FG. After 24 h of recovery, we also noted increased secretion of the proinflammatory cytokine MIF with 100 µg/m3 FG exposure and CXCL8/IL-8 at 1200 µg/m3, for both exposure periods. Together, these findings suggest that the exposure to FG at environmental levels aggravates cell death and inflammation observed in dry air conditions. This in vitro model of DE seems to be a relevant tool to study and explain the inflammatory responses observed in dry eye patients when exposed to combined environmental disturbances such as LH, airflow, and the presence of airborne pollutants.