PurposePersistent ocular surface pain occurs in moderate to severe dry eye disease (DE); however, the mechanisms that underlie this symptom remain uncertain. The aim of this study was to determine if the transient receptor potential vanilloid ion channels play a role in hypertonic saline (HS)-evoked corneal reflexes in a model for aqueous tear deficient DE.MethodsEye wipe behavior and orbicularis oculi muscle activity (OOemg) were measured after ocular instillation of HS, capsaicin, or menthol 14 days after exorbital gland removal. Total RNA and protein were measured from anterior eye segment and trigeminal ganglia of sham and DE rats.ResultsEye wipe behavior was enhanced in DE rats after HS and capsaicin instillation, but not after menthol when compared to sham rats. DE rats displayed greater OOemg activity after HS and capsaicin, but not after menthol, compared to sham rats. HS-evoked OOemg activity was reduced by selective TRPV1 antagonists and by coapplication of capsaicin plus QX-314, a charged lidocaine derivative. Menthol did not affect OOemg activity; however, selective antagonism of TRPM8 reduced HS-evoked OOemg activity. TRPV1 protein levels were increased in anterior eye segment and trigeminal ganglion samples from DE rats, whereas TRPM8 levels were not affected.ConclusionsThese results suggest that TRPV1 plays a significant role in mediating enhanced nocifensive behavior in DE, while TRPM8 may play a lesser role. Strategies to target specific transducer molecules on corneal nerves may prove beneficial as adjunct therapies in managing ocular pain in moderate to severe cases of DE.

Persistent tear deficiency was sufficient to cause sensitization of neurons at multiple regions of the trigeminal brainstem and enhanced orbicularis oculi muscle activity.

Dry eye (DE) disease is commonly associated with ocular surface inflammation, an unstable tear film and symptoms of irritation. However, little is known about the role of central neural mechanisms in DE. This study used a model for persistent aqueous tear deficiency, exorbital gland removal, to assess the effects of mustard oil, a TRPA1 agonist, on eyeblink and eyewipe behavior and Fos-like immunoreactivity (Fos-LI) in the trigeminal brainstem of male rats. Spontaneous tear secretion was reduced by about 50% and spontaneous eyeblinks were increased more than 100% in DE rats compared to sham rats. Mustard oil (0.02–0.2%) caused dose-related increases in eyeblink and forelimb eyewipe behavior in DE and sham rats. Exorbital gland removal alone was sufficient to increase Fos-LI at the ventrolateral pole of trigeminal interpolaris/caudalis (Vi/Vc) transition region, but not at more caudal regions of the trigeminal brainstem. Under barbiturate anesthesia ocular surface application of mustard oil (2–20%) produced Fos-LI in the Vi/Vc transition, in the mid-portions of Vc and in the trigeminal caudalis/upper cervical spinal cord (Vc/C1) region that was significantly greater in DE rats than in sham controls. Mustard oil caused an increase in Fos-LI ipsilaterally in superficial laminae at the mid-Vc and Vc/C1 regions in a dose-dependent manner. Smaller, but significant, increases in Fos-LI also were seen in the contralateral Vc/C1 region in DE rats. TRPA1 protein levels in trigeminal ganglia from DE rats ipsilateral and contralateral to gland removal were similar. Persistent tear reduction enhanced the behavioral and trigeminal brainstem neural responses to ocular surface stimulation by mustard oil. These results suggested that TRPA1 mechanisms play a significant role in the sensitization of ocular-responsive trigeminal brainstem neurons in this model for tear deficient DE.

Cornea-evoked eyeblinks maintain tear film integrity on the ocular surface in response to dryness and protect the eye from real or potential damage. Eyelid movement following electrical stimulation has been well studied in humans and animals; however, the central neural pathways that mediate protective eyeblinks following natural nociceptive signals are less certain. The aim of this study was to assess the role of the trigeminal subnucleus interpolaris/caudalis (Vi/Vc) transition and subnucleus caudalis/upper cervical cord (Vc/C1) junction regions on orbicularis oculi electromyographic (OOemg) activity evoked by ocular surface application of hypertonic saline or exposure to bright light in urethane anesthetized male rats. The Vi/Vc and Vc/C1 regions are the main sites of termination for trigeminal afferent nerves that supply the ocular surface, while hypertonic saline (saline = 0.15-5M) and bright light (light = 5-20k lux) selectively activate ocular surface and intraocular trigeminal nerves, respectively, and excite second-order neurons at the Vi/Vc and Vc/C1 regions. Integrated OOemg activity, ipsilateral to the applied stimulus, increased with greater stimulus intensities for both modalities. Lidocaine applied to the ocular surface inhibited OOemg responses to hypertonic saline, but did not alter the response to light. Lidocaine injected into the trigeminal ganglion blocked completely the OOemg responses to hypertonic saline and light indicating a trigeminal afferent origin. Synaptic blockade by cobalt chloride of the Vi/Vc or Vc/C1 region greatly reduced OOemg responses to hypertonic saline and bright light. These data indicate that OOemg activity evoked by natural stimuli known to cause irritation or discomfort in humans depends on a relay in both the Vi/Vc transition and Vc/C1 junction regions.

Altered corneal reflex activity is a common feature of dry eye disease (DE). Trigeminal sensory nerves supply the ocular surface and terminate at the trigeminal interpolaris/caudalis (ViVc) transition and spinomedullary (VcC1) regions. Although both regions contribute to corneal reflexes, their role under dry eye conditions is not well defined. This study assessed the influence of local inhibitory and excitatory amino acid neurotransmission at the ViVc transition and VcC1 regions on hypertonic saline (HS) evoked orbicularis oculi muscle activity (OOemg) in urethane-anesthetized male rats after exorbital gland removal (DE). HS increased the magnitude of long duration OOemg activity (OOemgL, >200 ms) in DE compared to sham rats, while short duration OOemg activity (OOemgS, <200 ms) was similar for both groups. Inhibition of the ViVc transition by muscimol, a GABAA receptor agonist, greatly reduced HS-evoked OOemgL activity in DE rats, whereas injections at the VcC1 region had only minor effects in both groups. Blockade of GABAA receptors by bicuculline methiodide at the ViVc transition or VcC1 region increased HS-evoked OOemgL activity in DE rats. Blockade of N-methyl-D-aspartate (NMDA) receptors at either region reduced HS-evoked OOemgL activity in DE and sham rats. GABAαβ3 receptor density was reduced at the ViVc transition, while NMDA receptor density was increased at both regions in DE rats. Loss of GABAergic inhibition at the ViVc transition coupled with enhanced NMDA excitatory amino acid neurotransmission at the ViVc transition and the VcC1 region likely contribute to altered corneal reflexes under dry eye conditions.