Abstract
Purpose :
The ability to predict ocular side effects of topically delivered drugs is an important challenge. It may be of utmost importance to develop novel models that could assess corneal toxicity based on two major ocular surface partners, nerve endings and epithelial cells.
Aims: To design a compartmentalized coculture between murine primary trigeminal neurons and corneal epithelial cells (MPCE) in a microfluidic device to reflect corneal anatomy. To validate the microfluidic coculture model using benzalkonium chloride (BAC), a common preservative in eyedrops known as a cause of Toxicity-Induced Dry Eye (TIDE).
Methods :
Trigeminal ganglia (TG) were dissected from adult male C57/bl6 mice and dissociated TG cells were cultured in the proximal compartment (PC) of the microfluidic device. Seven days after, MPCE were added to distal compartment (DC) containing trigeminal nerve endings. Microfluidic coculture was then exposed to a BAC low concentration (5.10-4%) for 6h (acute exposure) or BID 15min for 5 days (repeated exposure) in DC. Neuronal network, Activating Transcription Factor (ATF) 3/6, protein kinase R (PKR)-like endoplasmic reticulum kinase (pERK) and tight-junction protein ZO1 were studied by immunofluorescence after BAC and PBS (control) exposure. Images were acquired with CQ1 Confocal Quantitative Image Cytometer and analyzed with CellpathFinder Software.
Results :
Microfluidic device allows high-resolution imaging and quantification of axonal network, neuronal and epithelial cell alterations following BAC exposure. After acute exposure, in the DC, 30% of the neuronal network and ZO1 intensity were altered without significant decrease of MPCE number. In the PC, indirectly affected TG cells showed a slight increase (20%) of ATF3/6 factors. After BID exposure, axonal network and MPCE number were decreased by 80% and 50%, respectively. While acute exposure did not affect pERK, BID exposure triggered pERK intensity by 100% in DC and 30% in PC.
Conclusions :
This innovative microfluidic coculture system combined with a high-content quantitative analysis allows the detection of both direct toxicity on nerve endings and epithelial cells and indirect toxicity on TG and could represent a new attractive tool to assess ocular surface toxicity and evaluation of nerve regeneration therapies.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.