Deciphering the precise c-Fos connectome of ocular pain in mice

Abstract

Purpose : Chronic ocular pain dramatically affects the quality of life of patients and is difficult to treat given that the therapies are still unsatisfactory. The pathophysiological mechanisms are still poorly understood primarily due to our lack of knowledge of the ocular pain pathway.
To characterize the central corneal pain network, we used the 'c-Fos connectome' based on c-Fos neuronal staining, in a preclinical mouse model of chronic ocular pain.

Methods : The mouse model of chronic ocular pain was triggered by the use of benzalkonium chloride (BAC), a bactericidal preservative in ophthalmic preparation that promotes ocular surface damages and inflammation. Adult male mice received either a chronic topical instillation of 0.2% BAC (10µl, twice a day, 7 days; n=10) or no instillation was performed (naive animals; n=10). The chronic ocular pain was evaluated using behavioral responses (spontaneous ocular discomfort, von Frey mechanical sensitivity test) and cellular tests (slit lamp imaging).
Then the brainstem was cut in coronal sections and the rest of the brain in sagittal sections and c-Fos immunostaining was performed. All the sections were imaged with NanoZoomer and 1/5 of the total number of sections (n=22) in a brain was used to build the c-Fos connectome. These sections were aligned with the Allen Brain Atlas using ABBA plug-in in Image J software. c-Fos quantification was performed on each region of each section of a brain (for a mean of 820 regions per brain) using QuPath software and the c-Fos connectome was built using Cytoscape software.

Results : BAC mice developed epithelial corneal damage, ocular discomfort and corneal hypersensitivity after topical 0.2% BAC compared to naive mice. In the brainstem, a significantly higher number of c-Fos positive cells were observed in BAC compared to naive mice in two regions of the spinal trigeminal nucleus, Vc/C1 and V1/Vc, known to receive the central terminals of the corneal nociceptors and to contain the soma of the second order neurons. In the rest of the brain, regional quantification uncovered increased c-Fos positive cells in several brain regions associated with the pain pathways.

Conclusions : We develop a new and efficient pipeline of analysis of c-Fos staining in the entire brain that allows us to build a c-Fos connectome to characterize precisely the ocular pain pathways. This study is encouraging to provide valuable insights into the therapy to chronic corneal pain.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.