DED results in imbalance of neuropeptides, neurotrophins in the cornea and trigeminal ganglion

Reviewed by Flavia Leao Barbosa, PhD, and Pedram Hamrah, MD

As background for this talk, Dr. Barbosa explained that the cornea is the most densely innervated tissue in the human body, due to the great number of sensory nerve endings.

Cornea nerve fibers release neuromediators that provide trophic support to the ocular surface tissues, stimulate wound healing, and maintain the anatomic integrity of the cornea.

Dysfunction of the sensory innervation for many reasons causes breakdown of the corneal epithelium.

Impairment of corneal sensory innervation causes reduction of both protective reflexes and trophic neuromodulators that are essential for the vitality, metabolism, and wound healing of the ocular surface.

Dry eye disease
Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation, pain, and nerve abnormalities.

The ophthalmic division of trigeminal nerve provides sensory innervation to the cornea. The role of neuropeptides has been elucidated.

Many neuropeptides are expressed at the ocular surface, where they facilitate the crosstalk between the immune and nervous systems, and participate in suppressing inflammation in the cornea in order to preserve vision.

Any corneal injury or corneal nerve abnormalities may trigger molecular, cellular, and functional change within the trigeminal ganglion (TG). It has previously been demonstrated that dry eye disease results in decreased corneal nerve density.

Study purpose and design
The authors undertook a study to evaluate if DED results in alterations of neuropeptides (NPs) and neurotrophins in the cornea and TG.

They used a murine model of dry eye disease. Female mice ages 6 and 8 weeks were injected with 0.5mg of scopolamine hydrobromide 3 times a day and kept in a controlled environment chamber for 14 days and compared to control mice kept in standard housing.

At that time point clinical evaluations were performed, including cotton thread test for tear secretion volume, corneal fluorescein staining for slit-lamp microscopy, corneal mechanical sensitivity for blink reflex (using a Cochet-Bonnet aesthesiometer), and eye wipes for neural dysfunction.

mRNA 11 molecular targets of neuropeptides and neurotrophins were assessed by qRT-PCR and ELISA for neurotrophins in corneas and TG.

Nerve density was assessed by immunohistochemistry of corneal whole-mounts for Beta III Tubulin and imaged by confocal microscopy.

Results
Clinical evaluation showed that tear volume decreased in mice exposed to desiccating stress compared to naïve controls at Day 14. Quantification of fluorescein staining scores revealed a significant increase in corneal staining in mice exposed to desiccating stress compared to naïve controls. Corneal mechanical sensitivity decreased in DED and nerve dysfunctions markedly exacerbated the response in DED compared to the control.

Using whole mount corneas stained with neuron-specific beta III Tubulin and imaged by confocal, the authors found a significant decrease in nerve density at Day 14 in DED compared to control.

Results of the qRT-PCR analysis for neurotrophins in corneas showed there was downregulation of nerve growth factor (NGF) (3.1-fold), brain-derived neurotrophic factor (BDNF) (2.4-fold), neurotrophin-3 (NT-3) (2.9-fold).

Results were presented at fold changes normalized to the naïve controls group.

Confirming by ELISA analysis for neurotrophins in corneas, results showed protein expression of NGF, BDNF, and NT3 decreased significantly at day 14 in DED compared to control.

For neuropeptides in the cornea, results of qRT-PCR analysis again show downregulation of VIP (2.5-fold), PACAP (3.3-fold), SST (2.0-fold), NTS (5.0-fold) and NPY (5.0-fold). mRNA was significantly up-regulated at day 14 in SP (3.4-fold) and CGRP (2.5-fold).

In trigeminal ganglion, qRT-PCR analysis for neuropeptides and neurotrophins showed the neuropeptides SP and SST were up-regulated compared to controls (1.7 fold and 2.2-fold respectively). TG demonstrated upregulation of NGF (4.5-fold) and NT4/5 (6.3-fold).

Conclusions
Clinical assessment revealed that mice exposed to desiccating stress developed clinical signs of DED, and corneal nerve density decreased in DED at day 14.

The authors’ findings in corneas by mRNA expression demonstrated that neurotrophins were downregulated and, confirmed by ELISA, protein expression decreased significantly in DED at day 14.

There was downregulation by mRNA expression for neuropeptides in corneas, and a significant upregulation in SP and CGRP may reflect the alternation of innervation in DED.

Changes in mRNA expression were also found in TG following corneal nerve damage implicated in the development of neurogenic inflammation.

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Flavia Leao Barbosa, PhD
flaleao10@gmail.com
This article is based on Dr. Barbosa’s presentation at the ARVO 2021 virtual annual meeting. Dr. Barbosa has no financial disclosures.