Abstract
Purpose :
Dry eye disease is marked by symptoms such as discomfort, pain, and visual changes that may impact daily living. With pain being a significant symptom of this disease, it is paramount to elucidate the neural pathways activated by corneal pain and alterations induced by dry eye. The aim of this study was to genetically label activated neurons using the transgenic mouse line Fos-TRAP2 following corneal stimulation in control and dry eye animals.
Methods :
Targeted recombination in activated populations (TRAP) mice were used to identify corneal stimulation-activated neurons. Male and female TRAP2 mice (Fos2A-iCreER, Jackson Laboratory) were crossed with the CAG-Sun1/sfGFP reporter line to visualize neurons in the trigeminal nucleus caudalis (TNC) and the lateral parabrachial nucleus (LBPN) that were activated by corneal hypertonic saline (HS) application in control and lacrimal gland excision (LGE)-induced dry eye mice. 4-OH-Tamoxifen (4OHT) (75 mg/kg, intraperitoneal (i.p.)) was injected 15 min prior to either corneal HS (5M NaCl, 5 applications, once every 3 min), no stimulation (mock application of HS), or no treatment (remained in holding chamber after i.p. injection). Lacrimal gland excision was performed two weeks prior to 4OHT. Two weeks after 4OHT, all animals received corneal HS and were perfused 90 min later. Immunohistochemistry for GFP and c-Fos protein was performed on free floating 50um sections.
Results :
Corneal HS increased Sun1-GFP and c-Fos-positive neurons in two distinct regions of the TNC, one anterior and the other posterior, and in the lPBN. In superficial laminae of the posterior TNC, HS produced a greater number of Sun1-GFP positive neurons in LGE animals compared to intact animals. In the anterior TNC, LGE animals without HS application had a greater number of Sun1-GFP positive neurons compared to intact animals. In the LPBN, HS increased the number of Sun1-GFP positive neurons in both LGE and intact animals.
Conclusions :
These results support the use of the Fos-TRAP2;Sun1/sfGFP mouse to identify neurons activated with corneal stimulation. Future studies will isolate Sun1-GFP-positive cell nuclei and perform single nucleus RNA sequencing to identify expression patterns in corneal activated neurons and alterations following dry eye.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.