Abstract
Purpose :
The cornea is the most densely innervated tissue in the body and is innervated by trigeminal ganglion (TG) neurons. In vitro studies currently use pup TG cultures to study neuronal function and mechanisms. However, it is not clear if the findings from pup TG can be translated to neuronal mechanisms in adult mice. Thus, the purpose of the study was to compare the expression of neuropeptides and neuropeptide receptors between pup and adult TG neurons in mice
Methods :
TG neurons were excised pooled and digested with collagenase D, DNase, and Dispase II from p10 and six-week-old adult C57BL/6 mice. Neurons were isolated by differential Percoll gradient. To assess gene expression, mRNA was isolated using SingleShot Lysis kit. cDNA was generated using iScript reverse transcriptase. cDNA was amplified with SsoPreamp and assayed with ssoAdvanced SYBR Green with IDT primers on CFX-Connect. We examined vasointestinal peptide (VIP), calcitonin gene-related peptide (CGRP), pro-opiomelanocortin (POMC), proenkephalin (PENK), and melanocortin receptor-4 (MC4) and kisspeptin receptor (KISS1R) mRNA levels normalized to GAPDH. For protein expression, TG neurons were stained with anti-KISS1R antibody and analyzed on a BD LSRII flow cytometer. Significance was determined by T test. P < 0.05 was considered significant. Experiments were repeated twice
Results :
We observed no significant difference in the mRNA levels of the neuropeptides VIP, CGRP, POMC, or PENK between pup and adult TG neurons (p > 0.05). We subsequently examined the relative expression of neuropeptide receptors MC4 and KISS1R between pup and adult TG neurons. We observed no differences in the mRNA expression of MC4 between the groups (p > 0.05). However, a statistically significant increase in mRNA expression of KISS1R (p < 0.02) was found in adult compared to pup TG neurons. Flow cytometry confirmed that adult TG neurons expressed higher levels of KISS1R protein compared to pups (2.4-fold increase in the percentage of KISS1R-expressing TG neurons)
Conclusions :
This data suggests gene expression in pup and adult TG neurons is not interchangeable. For VIP, CGRP, POMC and PENK pups and adult were no different. Yet, adult TG neurons express higher levels of the neuropeptide receptor KISS1R compared to pups. Functionally, expression of KISS1R by TG neurons may be involved in alteration in behavior after corneal insults
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.