Corneal Nociception Research Articles

Enhancing orofacial pain relief: α-phellandrene complexed with hydroxypropyl-β-cyclodextrin mitigates orofacial nociception in rodents.

Orofacial pain affects 10-15% of adults and can severely impact quality of life. Despite ongoing treatment challenges, monoterpene alpha-phellandrene (PHE) shows potential therapeutic benefits. This study aimed to develop and evaluate an inclusion complex of PHE with hydroxypropyl-beta-cyclodextrin (PHE-HPβCD) for treating orofacial pain. The PHE-HPβCD complex was created using physical mixing and characterized by differential scanning calorimetry (DSC) and high-performance liquid chromatography (HPLC) to determine encapsulation efficiency. The complex exhibited a 70.45% encapsulation efficiency. Male Swiss mice were used in models of orofacial pain induced by formalin, cinnamaldehyde, glutamate, and corneal nociception by hypertonic saline. Additionally, cytokine levels (TNF-α and IL-1β) were measured in the upper lip tissue of mice subjected to the formalin model. Both PHE and PHE-HPβCD showed significant antinociceptive effects at a 50mg/kg dose during formalin-induced pain, reducing both neurogenic and inflammatory phases of pain. PHE-HPβCD also reduced TNF-α and IL-1β levels. For cinnamaldehyde and glutamate-induced nociception, both treatments reduced pain behavior, but only PHE-HPβCD decreased eye wipes in corneal nociception. These results suggest that PHE, especially in complexed form, alleviates orofacial pain by potentially modulating pain-related receptors (TRPA1 and TRPV1), mediators, like glutamate, and reducing pro-inflammatory cytokines. Further research is needed to explore the precise mechanisms of PHE in chronic orofacial pain models, but the study indicates promising avenues for new pain treatments.

Neurosensory abnormalities and stability of a mouse model of dry eye disease

This study aimed to use a mouse model of dry eye disease (DED) induced by topical administration of benzalkonium chloride (BAK) and assess its stability and the presence of neurosensory abnormalities, including ocular pain. Eight-week-old C57BL6/6 N male mice were used in this study. Mice were treated with 10 μL of 0.2% BAK dissolved in artificial tears (AT), administered twice daily for 7 days. After one week, animals were randomized into two groups: one was administered with 0.2% BAK in AT once per day for 7 days, while the other was not further treated. Corneal epitheliopathy was quantified at days 0, 3, 7, 12, and 14. Moreover, tear secretions, corneal nociception, and corneal nerve integrity were measured after BAK treatment. After sacrifice, corneas were dissected to assess nerve density and leukocyte infiltration by immunofluorescence. Topical BAK instillation for 14 days significantly increased corneal fluorescein staining (p < 0.0001) compared to day 0. On the other hand, interruption of BAK instillation was associated with improvement of corneal epitheliopathy (day 12, p < 0.0001; day 14, p < 0.001). BAK treatment increased ocular pain (p < 0.0001) and resulted in a significant increase in leukocyte infiltration in the cornea (p < 0.01). Moreover, corneal sensitivity was reduced (p < 0.0001), together with corneal nerve density (p < 0.0001) and tear secretion (p < 0.0001). One week twice a day, followed by one additional week once a day, of 0.2% BAK topical administration induces stable clinical and histological signs of DED, which is associated with neurosensory abnormalities, including pain.

Limonene, a citrus monoterpene, non-complexed and complexed with hydroxypropyl-β-cyclodextrin attenuates acute and chronic orofacial nociception in rodents: Evidence for involvement of the PKA and PKC pathway

BackgroundChronic orofacial pain is a serious public health problem with a prevalence of 7–11% in the population. This disorder has different etiologies and characteristics that make pharmacological treatment difficult. Natural products have been shown to be a promising source of treatments for the management of chronic pain, as an example the terpenes. PurposeThe aim of this study was to evaluate the anti-nociceptive and anti-inflammatory effects of one of these terpenes, d-limonene (LIM - a common monoterpene found in citrus fruits) alone and complexed with hydroxypropyl-β-cyclodextrin (LIM/HPβCD) in preclinical animal models. MethodsOrofacial pain was induced by the administration of hypertonic saline on the corneal surface, the injection of formalin into the temporomandibular joint (TMJ), or chronic constriction injury of the infraorbital nerve (CCI-IoN). The study used male Wistar rats and Swiss mice treated with LIM (50 mg/kg), LIM/HPβCD (50 mg/kg), vehicle (control), gabapentin or morphine, and eyes wiping (induced by hypertonic saline), face rubbing (formalin-induced in TMJ) or mechanical hyperalgesia (provoked by CCI-IoN) were assessed. Additionally, ELISA was used to measure TNF-α, and western blot analysis to assess levels of PKAcα, NFκB, p38MAPK and phosphorylated PKC substrates. Serum levels of aspartate aminotransferase (AST) and alanine transferase (ALT) were also evaluated. ResultsLIM and LIM/HPβCD significantly reduced (p < 0.001) corneal nociception and formalin-induced TMJ nociception. In addition, both substances attenuated (p < 0.001) mechanical hyperalgesia in the CCI-IoN model. The antinociceptive effect induced by LIM and HPβCD/LIM was associated with decreased TNF-α levels, downregulation of the NFκB and p38MAPK signalling pathways and reduced PKC substrate phosphorylation and PKA immunocontent. Moreover, the results demonstrated that complexation with HPβCD was able to decrease the therapeutic dose of LIM. ConclusionLIM was found to be a promising molecule for the treatment of orofacial pain due to its capacity to modulate some important mediators essential to the establishment of pain, and HPβCD can be a key tool to improve the profile of LIM.

Capsazepine decreases corneal pain syndrome in severe dry eye disease

BackgroundDry eye disease (DED) is a multifactorial disease of the ocular surface accompanied by neurosensory abnormalities. Here, we evaluated the effectiveness of transient receptor potential vanilloid-1 (TRPV1) blockade to alleviate ocular pain, neuroinflammation, and anxiety-like behavior associated with severe DED.MethodsChronic DED was induced by unilateral excision of the Harderian and extraorbital lacrimal glands of adult male mice. Investigations were conducted at 21 days after surgery. The mRNA levels of TRPV1, transient receptor potential ankyrin-1 (TRPA1), and acid-sensing ion channels 1 and 3 (ASIC1 and ASIC3) in the trigeminal ganglion (TG) were evaluated by RNAscope in situ hybridization. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous and stimulated (cold, heat, and acid) corneal nerve responsiveness in ex vivo eye preparations. DED mice received topical instillations of the TRPV1 antagonist (capsazepine) twice a day for 2 weeks from d7 to d21 after surgery. The expression of genes involved in neuropathic and inflammatory pain was evaluated in the TG using a global genomic approach. Chemical and mechanical corneal nociception and spontaneous ocular pain were monitored. Finally, anxiety-like behaviors were assessed by elevated plus maze and black and white box tests.ResultsFirst, in situ hybridization showed DED to trigger upregulation of TRPV1, TRPA1, ASIC1, and ASIC3 mRNA in the ophthalmic branch of the TG. DED also induced overexpression of genes involved in neuropathic and inflammatory pain in the TG. Repeated instillations of capsazepine reduced corneal polymodal responsiveness to heat, cold, and acidic stimulation in ex vivo eye preparations. Consistent with these findings, chronic capsazepine instillation inhibited the upregulation of genes involved in neuropathic and inflammatory pain in the TG of DED animals and reduced the sensation of ocular pain, as well as anxiety-like behaviors associated with severe DED.ConclusionThese data provide novel insights on the effectiveness of TRPV1 antagonist instillation in alleviating abnormal corneal neurosensory symptoms induced by severe DED, opening an avenue for the repositioning of this molecule as a potential analgesic treatment for patients suffering from chronic DED.

Modulating Ocular Surface Pain Through Neurokinin-1 Receptor Blockade.

PurposeThe purpose of this study was to test the role of substance P (SP) and its receptor neurokinin 1 (NK1R) on ocular surface pain.MethodsEight-week-old C57BL6/N (wild type [WT]) and B6.Cg-Tac1tm1Bbm/J (TAC1-KO) male mice were used. 5 M NaCl was topically applied on the cornea, followed by topical fosaprepitant 2, 10, and 50 mg/mL; 4 mg/mL oxybuprocaine chloride, or 0.1% diclofenac. Th eye wiping test was used to quantify ocular surface pain. SP content was quantified in the tear fluid and trigeminal ganglia (TG), and TAC1 mRNA was assessed in the cornea. Corneas were immunostained for β3-tubulin and NK1R, or CD45, to quantify leukocyte infiltration.ResultsTAC1-KO mice displayed a significant reduction of ocular pain (P < 0.001). Similarly, a single dose of 10 or 50 mg/mL fosaprepitant applied topically to WT mice reduced ocular pain as compared to vehicle (P < 0.001). Fosaprepitant 2 mg/mL, instead, induced corneal analgesia only when it was administered for 10 days, 6 times/day (P < 0.05). Diclofenac or oxybuprocaine reduced corneal nociception when compared to vehicle or fosaprepitant (P < 0.05). Fosaprepitant or oxybuprocaine groups showed lower SP content in tear secretions and TG (P < 0.05), and reduction in TAC1 mRNA (P < 0.05), and leukocyte infiltration (P < 0.05) in the cornea. Colocalization of NK1R and β3-tubulin was detected in mouse corneas.ConclusionsTopical administration of the NK1R antagonist fosaprepitant effectively reduces ocular surface nociception by decreasing SP release in the tear fluid and TG, and corneal leukocyte infiltration. Fosaprepitant repurposing shows promise for the treatment of ocular pain.

Antinociceptive effect of triterpene acetyl aleuritolic acid isolated from Croton zehntneri in adult zebrafish (Danio rerio)

Croton zehntneri is a plant known as canelinha de cunhã, prevalent in the northeast region of Brazil. Many constituents of the vegetable have already been studied, and their pharmacological properties have been proven, but this is the first study to analyze the antinociceptive effect in adult zebrafish (ZFa) of the triterpene acetyl aleuritolic acid (AAA) isolated from the stem bark. The animals (ZFa; n = 6/group) were treated intraperitoneally (ip; 20 μL) with AAA (0.1 or 0.3 or 1.0 mg/mL) or vehicle (0.9% saline; 20 μL), and submitted to the locomotor activity test, as well as 96 h acute toxicity. Other groups (n = 6/each) received the same treatments and underwent acute nociception tests (formalin, cinnamaldehyde, glutamate, acid saline, capsaicin, and hypertonic saline). Possible neuromodulation mechanisms were evaluated. AAA (0.1 or 0.3 or 1.0 mg/mL) reduced the nociceptive behavior induced by acid saline and capsaicin, as well as inhibited corneal nociception induced by hypertonic saline, both without altering the animals’ locomotor system and without toxicity. These analgesic effects of AAA were significantly (p > 0.05) similar to those of morphine, used as a positive control. The antinociceptive effect of AAA was inhibited by methylene blue, ketamine, camphor, ruthenium red, amiloride, and mefenamic acid. The antinociceptive effect of AAA on the cornea of animals was inhibited by capsazepine. Therefore, AAA showed pharmacological potential for the treatment of acute pain, and this effect is modulated by cGMP, NMDA receptors, transient receptor potential channels (TRPs), ASICs and has pharmacological potential for the treatment of corneal pain modulated by the TRPV1 channel.

Topical treatment with a mu opioid receptor agonist alleviates corneal allodynia and corneal nerve sensitization in mice

Corneal pain is considered to be a core symptom of ocular surface disruption and inflammation. The management of this debilitating condition is still a therapeutic challenge. Recent evidence supports a role of the opioid system in the management of corneal nociception. However, the functional involvement of the mu opioid receptor (MOR) underlying this analgesic effect is not known. We first investigated the expression of the MOR in corneal nerve fibers and trigeminal ganglion (TG) neurons in control mice and a mouse model of corneal inflammatory pain. We then evaluated the anti-nociceptive and electrophysiological effects of DAMGO ([D-Ala2,N-Me-Phe4,Gly5-ol] enkephalin), a MOR-selective ligand. MOR immunoreactivity was detected in corneal nerve fibers and primary afferent neurons of the ophthalmic branch of the TG of naive mice. MOR expression was significantly higher in both structures under conditions of inflammatory corneal pain. Topical ocular administration of DAMGO strongly reduced both the mechanical (von Frey) and chemical (capsaicin) corneal hypersensitivity associated with inflammatory ocular pain. Repeated instillations of DAMGO also markedly reversed the elevated spontaneous activity of the ciliary nerve and responsiveness of corneal polymodal nociceptors that were observed in mice with corneal pain. Finally, these DAMGO-induced behavioral and electrophysiological responses were totally blunted by the topical application of naloxone methiodide, an opioid receptor antagonist. Overall, these results provide evidence that topical pharmacological MOR activation may constitute a therapeutic target for the treatment of corneal pain and improve corneal nerve function to alleviate chronic pain.

Carvedilol Repurposing in Orofacial Pain: Preclinical Findings in Adult Zebrafish

Carvedilol is a non‐selective β‐adrenergic antagonist that is used in the treatment of heart diseases. β‐blockers are known to have an anti‐inflammatory effect that contributes to its analgesic effect. Here we show the acute orofacial antinociceptive effect of carvedilol in adult zebrafish (Danio rerio). Acute nociception was induced by capsaicin (4.93 μM; TRPV1 agonist), formalin (0.1 %; TRPA1 agonist) or menthol (1.2 μM; TRPM8 agonist) into in the upper lip (5.0 μL) of adult wild (short‐fin phenotype) zebrafish. Corneal nociception was induced with hypertonic saline (TRPV1 agonist; 5.0 M NaCl; 5.0 μL) applied into the surface of the right or left eye of the fishes. Then, animals (n=6/group) were placed in a glass Petri dish (10 × 15 cm), divided into quadrants, and the nociceptive response was registered after capsaicin (10 – 20 min), formalin (0 – 5 and 15 – 30 min), menthol (0 – 10 min) or hypertonic saline (0 – 5 min) application. Zebrafish were pretreated by gavage (20 μL) with vehicle (Control; 3 % Tween 80) or carvedilol (0.03; 0.1 or 0.3 mg/mL) 60 min before induction. The effect of carvedilol on zebrafish locomotor behavior was evaluated with the open field test (0 – 5 min). Naive groups (n=6) were included. The interaction between carvedilol and TRPV1 was analyzed using molecular docking. The HEX software was used for molecular docking calculations and the direct action of carvedilol on TRPV1 was viewed using PyMOL v1.4.7. The experimental protocols followed the ethical guidelines of CONCEA (Brazilian Council for the Control of Animal Experimentation) and were approved by the UECE Animal Research Ethics Committee (#7210149/2016). Pre‐treatment with carvedilol (0.03 and 0.1 mg/mL) was associated with a reduction in nociceptive behavior induced by capsaicin and hypertonic saline (TRPV1 agonists; (*p&lt;0.05 vs Control), but not by formalin, or menthol. Carvedilol per se promoted no alteration on the locomotor activity of zebrafish. In the docking experiments, the interaction between carvedilol and the TRPV1 channel showed high reproducibility. It was possible to observe a direct action of carvedilol on TRPV1 amino acids (Asn687, Asn687, Asn687, Asn687, Ala680 and Gly683). The results indicate the potential clinical application of carvedilol an inhibitor of orofacial nociception and that this effect may be due to the modulation of TRPV1 channel.Support or Funding InformationNational Council for Scientific and Technological Development ‐ CNPqCoordination for the Improvement of Higher Education Personnel ‐ CapesResearch Support Foundation of Ceará State ‐ FuncapEdson Queiroz Foundation ‐ FEQThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of corneal injury on ciliary nerve fibre activity and corneal nociception in mice: A behavioural and electrophysiological study.

Ocular surface diseases are among the most frequent ocular pathologies. Ocular pain following corneal injury is frequently observed in clinic. Corneal sensory innervation is supplied by ciliary nerves derived from ophthalmic division of the trigeminal ganglion. Extracellular activity of the mouse ciliary nerve was first used to investigate the corneal responsiveness to chemical, mechanical and thermal stimulations in order to specifically study the responses of polymodal nociceptors, mechano-nociceptors and cold thermoreceptor in a control cornea. Then, in two models of corneal injury (repeated instillations of 0.02% benzalkonium chloride and corneal scraping), we first measured the corneal sensitivity to chemical (eye-wiping test) and mechanical (von Frey filaments) stimulation. Thereafter, we evaluated whether these corneal injuries modified the spontaneous and chemical stimulation-evoked activity of the ciliary nerve. Both models of injury induced a significant corneal chemical hypersensitivity correlated with an increase of the spontaneous activity of the ciliary nerve and a faster response of the ciliary nerve after a chemical stimulation. Overall, this study provides new insights into the functional aspects of corneal nerve fibre activity in mice after corneal injury. The increase in ciliary nerve activity may thus contribute to the development of ocular pain after corneal damage. This study highlights the parallel increase in ciliary nerve activity and corneal sensitivity after corneal injury in mice. The strategy of combining ex vivo electrophysiological recordings of the ciliary nerve in mice and corneal sensitivity measurements therefore helps to uncover the functional aspects of corneal pain.

Adult Zebrafish (Danio rerio) As a Model for the Study of Corneal Antinociceptive Compounds.

Zebrafish is an excellent model that can be utilized as an adjunct to current rodent models for studies of eye diseases because the anatomy and ultrastructural characterization of its cornea show much similarity with the human cornea. Therefore, we developed a behavioral model of corneal nociception using the adult zebrafish (Danio rerio). We analyzed the nociceptive effect of hypertonic saline (0.15-5.0 M sodium chloride [NaCl]) applied to the surface of the right or left cornea, on the animals' gender and locomotor activity through the open-field test. The behavioral model of corneal nociception was characterized by the antinociceptive effect of morphine (8.0 or 16 mg/kg; intraperitoneally [i.p.]), an opioid analgesic, and capsazepine, an antagonist of transient receptor potential vanilloid type 1 channels. We also tested whether the corneal antinociceptive effect of morphine could be modulated by naloxone, an opioid antagonist. Finally, we used the light and dark test to assess the anxiolytic effect of hypertonic saline (5.0 M NaCl; 5 μL) applied to the right or left cornea of the animals. As a result, hypertonic saline significantly increased (p < 0.01 vs. control) the corneal nociceptive behavior of adult zebrafish (D. rerio). Morphine significantly inhibited (p < 0.01 vs. 5.0 M NaCl) the hypertonic saline-induced corneal nociception and this effect was blocked by naloxone. Capsazepine (20 mg/kg; i.p.) significantly inhibited (p < 0.05 vs. control) the corneal nociception induced by hypertonic saline. Hypertonic saline, applied to the surface of the right or left cornea of the animals, induced nociception and did not cause a presumptive anxiolytic effect. Gender and site of application did not affect the profile of response to hypertonic saline. The results suggest that the adult zebrafish can also be used as a behavioral model of corneal nociception, with the advantages of significant homology with the human genome and low cost.

Orofacial antinociceptive effect of Mimosa tenuiflora (Willd.) Poiret

Mimosa tenuiflora (Willd.) Poiret, popularly known in Brazil as “jurema-preta” is widely used against bronchitis, fever, headache and inflammation. Its antioxidant, anti-inflammatory and antinociceptive potential has already been reported. To assess the orofacial antinociceptive effect of M. tenuiflora, ethanolic extracts of M. tenuiflora (leaves, twigs, barks and roots) were submitted to in vitro tests of antioxidant activity. The extract with the highest antioxidant potential was partitioned and subjected to preliminary chemical prospecting, GC–MS, measurement of phenolic content and cytotoxicity tests of the fraction with the highest antioxidant activity. The nontoxic fraction with the highest antioxidant activity (FATEM) was subjected to tests of acute and chronic orofacial nociception and locomotor activity. The possible mechanisms of neuromodulation were also assessed. The EtOAc fraction, obtained from the ethanolic extract of M. tenuiflora barks, was the one with the highest antioxidant potential and nontoxic (FATEM), and Benzyloxyamine was the major constituent (34.27%). FATEM did not alter the locomotor system of mice and reduced significantly the orofacial nociceptive behavior induced by formalin, glutamate, capsaicin, cinnamaldehyde or acidic saline compared to the control group. FATEM also inhibited formalin- or mustard oil-induced temporomandibular nociception. In addition, it also reduced mustard oil-induced orofacial muscle nociception. However, FATEM did not alter hypertonic saline-induced corneal nociception. Neuropathic nociception was reversed by treatment with FATEM. The antinociceptive effect of FATEM was inhibited by naloxone, L-NAME and glibenclamide. FATEM has pharmacological potential for the treatment of acute and neuropathic orofacial pain and this effect is modulated by the opioid system, nitric oxide and ATP-sensitive potassium channels. These results lead us to studies of isolation and characterization of bioactive principles.

Estrogen Modulates Corneal Nociception and Maintains Corneal Homeostasis in Rat Eye.

To evaluate the role of estrogen in corneal nociception, its influence on lacrimal secretion, and development of dry eye. Ovariectomy was performed in normal healthy female rats (OVX). Estrogen replacement was performed in a population of these rats (OVX+E). Tests for dry eye and corneal sensitivity were performed and compared with rats in proestrus (PRO) as controls. Gene expression of neuropeptides such as substance P, calcitonin gene receptor-like protein (CGRP), estrogen receptor α, TRPV1, and TRPM8 was evaluated in the cornea and trigeminal ganglion. Expression of substance P and CGRP in the cornea was also examined by immunohistochemistry. The response of the cornea to capsaicin and menthol was evaluated to identify the activity of receptors TRPV1 and TRPM8, respectively. There was a significant decrease in tear formation (4.2 ± 0.6 mm/min vs. 6.6 ± 0.42 mm/min), corneal sensitivity (2.2 ± 0.17 cm vs. 6 ± 0 cm), and increase in fluorescein staining in corneas after ovariectomy compared with controls. There was a significant decrease in gene expression of CGRP, substance P, TRPV1, and TRPM8 in the ovarioectomized cornea. A significant decrease in tear formation (3.17 ± 0.30 mm/min vs. 7.17 ± 0.87 mm/min) and eye wipe response (10.5 ± 1.99 wipes vs. 18.33 ± 1.05 wipes) after treatment with menthol and capsaicin in OVX rats was observed. Estrogen replacement significantly enhanced tear formation (4.02 ± 0.6 mm/min vs. 6.7 ± 0.80 mm/min), corneal sensitivity (2.2 ± 0.17 cm vs. 3.2 ± 0.17 cm), and response to capsaicin (10.5 ± 1.99 eye wipes vs. 24.5 ± 0.92 wipes) and menthol (3.17 ± 0.30 mm/min vs. 6.5 ± 0.22 mm/min) and increased expression of neuropeptides, TRPV1 and TRPM8. This study demonstrates the role of estrogen in corneal nociception and its deficiency as a cause of dry eye.

Antinociceptive effect of (-)-α-bisabolol in nanocapsules

This study aimed to develop and to evaluate the antinociceptive effect of a drug delivery system containing (-)-α-bisabolol (BISA). Nanocapsules containing BISA (BISA-NC) were prepared using acetylated galatomannan. Particle size distribution was determined by atomic force microscopy, zeta potential measurement and photon correlation spectroscopy. Corneal nociception was induced by topical application of 5M NaCl and the nociceptive behavior was characterized by eye wiping in mice. Molecular docking was conducted on the TRPV1 channel. Nanocapsules showed mean particle sizes between 94.44 and 105.44nm and the zeta potential of was -1.34mV. Animals pretreated with BISA-NC (200mg/mL) had a significant reduction (**p<0.01) in the number of nociceptive behaviors. Docking study indicated an interaction between BISA and TRPV1. This study indicates that BISA-NC may be useful for producing eye drops for the treatment of ocular pain.

Acute and neuropathic orofacial antinociceptive effect of eucalyptol.

Terpenes have a wide range of pharmacological properties, including antinociceptive action. The anti-inflammatory and antinociceptive effects of eucalyptol are well established. The purpose of this study was to evaluate the antinociceptive effect of eucalyptol on acute and neuropathic orofacial pain in rodent models. Acute orofacial and corneal nociception was induced with formalin, capsaicin, glutamate and hypertonic saline in mice. In another series, animals were pretreated with capsazepine or ruthenium red to evaluate the involvement of TRPV1 receptors in the effect of eucalyptol. In a separate experiment, perinasal tissue levels of IL-1β, TNF-α and IFN-γ were measured. Rats were pretreated with eucalyptol before induction of temporomandibular joint pain with formalin or mustard oil. In another experiment, rats were submitted to infraorbital nerve transection (IONX) to induce chronic pain, followed by induction of mechanical hypersensitivity using Von Frey hairs. Locomotor performance was evaluated with the open-field test, and molecular docking was conducted on the TRPV1 channel. Pretreatment with eucalyptol significantly reduced formalin-induced nociceptive behaviors in all mouse strains, but response was more homogenous in the Swiss strain. Eucalyptol produced antinociceptive effects in all tests. The effect was sensitive to capsazepine but not to ruthenium red. Moreover, eucalyptol significantly reduced IFN-γ levels. Matching the results of the experiment in vivo, the docking study indicated an interaction between eucalyptol and TRPV1. No locomotor activity changes were observed. Our study shows that eucalyptol may be a clinically relevant aid in the treatment of orofacial pain, possibly by acting as a TRPV1 channel antagonist.

Burning Eye Syndrome: Do Neuropathic Pain Mechanisms Underlie Chronic Dry Eye?

Dry eye is a multi-factorial disorder that manifests with painful ocular symptoms and visual disturbances, which can only be partly attributed to tear dysfunction. This disorder may also involve neuroplasticity in response to neuronal injury. This review will emphasize the key characteristics of dry eye pain and its pathologic mechanisms, making the argument that a subset of dry eye represents a neuropathic pain disorder of the eye, more appropriately called "burning eye syndrome." A literature review was conducted using a PubMed search focusing on dry eye, corneal nociception, and neuropathic pain. Articles were reviewed and those discussing clinical course, pathophysiology, and neuronal regulation of chronic ocular pain as related to dry eye were summarized. We found that there is a discordance between ocular pain and dryness on the ocular surface. Although tear dysfunction may be one of the initial insults, its persistence may be associated with repeated ocular sensory nerve injury leading to an acute-to-chronic pain transition associated with neuropathologic changes (peripheral and central sensitization), neuronal dysfunction, and spontaneous ocular pain. Dry eye is becoming a major health concern due to its increasing incidence, significant morbidity, and economic burden. Recent evidence suggests that a subset of dry eye may be better represented as a chronic neuropathic pain disorder due to its features of dysesthesia, spontaneous pain, allodynia, and hyperalgesia. Future therapies targeted at the underlying neuroplasticity may yield improved efficacy for patients with this subset of dry eye, which we term "burning eye syndrome."

Progress of research on corneal transient receptor potential channel superfamily

Cornea senses thermal, mechanical and chemical stimuli via various nociceptors. In the early years, knowledge of the mechanism of corneal nociception was merely restricted to neurophysiology, which classifies nociceptors according to the clusters of nerve fibers which transmit identical stimulation signals. During the past decade, benefiting from the promptly developing molecular biology and technology, classification of the nociceptors has been modified into the level of cell sensors, among which, transient receptor potential (TRP) channel superfamily weighs the most. Researches have shown that there are a couple of TRP superfamily members expressed in cornea, which play major roles in the process of corneal pain, neurotrophic keratopathy, epithelial wound healing and immuno-inflammation. Some TRPs have even been recognized as targets of management of keratopathy.

Effects of subcutaneous and intracerebroventricular injection of physostigmine on the acute corneal nociception in rats

The present study investigated the effects of subcutaneous (sc) and intracerebroventricular (icv) injections of physostigmine (a cholinesterase inhibitor), atropine (an antagonist of muscarinic cholinergic receptors) and hexamethonium (an antagonist of nicotinic cholinergic receptors) on the acute corneal nociception in rats. Local application of 5M NaCl solution on the corneal surface of the eye produced a significant nociceptive behavior, characterized by eye wiping. The number of eye wipes was counted during the first 30s. The sc (0.25, 0.5 and 1mg/kg) and icv (1.25, 2.5, 5 and 10µg) injections of physostigmine significantly (p<0.05) decreased the number of eye wipes. Atropine and hexamethonium at (2mg/kg, sc and 20µg, icv) had no effects when used alone, however, atropine, but not hexamethonium prevented the antinociception induced by physostigmine (sc and icv). The results of this study indicate that the central muscarinic, but not nicotinic receptors might be involved in the antinociceptive effect of physostigmine in the acute corneal model of pain in rats.

Topical cannabinoid agonist, WIN55,212-2, reduces cornea-evoked trigeminal brainstem activity in the rat.

Cannabinoids act at receptors on peripheral and central neurons to modulate diverse physiological functions and produce analgesia. Corneal sensory nerves express the CB1 cannabinoid receptor and project to two spatially discrete regions of the lower brainstem, the trigeminal interpolaris/caudalis (Vi/Vc) transition and subnucleus caudalis/upper cervical cord (Vc/C1) junction region. The function of CB1 expression on corneal nerves is not known. To determine if cannabinoid receptors in the anterior eye affect the activity of trigeminal brainstem neurons at the Vi/Vc and Vc/C1 the CB1 agonist, WIN55,212-2 (WIN-2), was applied topically prior to chemical excitation of corneal afferent fibers. In the first series of experiments WIN-2 was applied topically prior to excitation of corneal nociceptors by mustard oil (MO). WIN-2 reduced significantly the number of Fos-like immunoreactive neuronal nuclei (Fos-LI) at the Vi/Vc transition (-46.7+/-8.2%, P<0.05), while smaller non-significant reductions occurred at the Vc/C1 junction region (-20.3+/-7.6%). The selective CB1 antagonist, SR141716A (1mg/kg, i.v.), prevented WIN-2-evoked reduction in Fos-LI after MO. Systemic administration of WIN-2 (1 or 10mg/kg, i.p.) or SR141716A (1mg/kg, i.v.) or topical corneal application of morphine sulfate did not affect Fos-LI produced by MO. In parallel experiments, topical WIN-2 reduced the magnitude of single unit activity recorded at the Vi/Vc transition (-80+/-7%, P<0.025), but not at the Vc/C1 junction region (-34+/-30%) evoked by CO(2) pulses applied to the cornea. Topical morphine did not alter CO(2)-evoked unit activity at either recording location. These results indicated that cannabinoid receptor agonists acted, at least in part, at CB1 receptors in the anterior eye to reduce corneal stimulation-evoked trigeminal brainstem neural activity. Corneal nociceptor-evoked activity at the Vi/Vc transition was reduced significantly by topical WIN-2, while activity at the Vc/C1 junction region displayed only minor decreases. These findings were consistent with the hypothesis that CB1 receptors affect the activity of corneal-responsive neurons that preferentially contribute to homeostasis of the anterior eye and/or reflexive aspects of nociception rather than the sensory-discriminative aspects of corneal nociception.

Is substance P necessary for corneal nociception?

Substance P (SP)-containing nerve fibers are few in the rabbit cornea, which is richly supplied with acetylcholinesterase-positive nerve fibers, presumably sensory in nature. Treatment with capsaicin given by retrobulbar injection 48 h prior to sacrifice caused SP to disappear from the SP nerves in the cornea without affecting the acetylcholinesterase-positive nerve fibers. The corneal sensitivity to a tactile stimulus (wetted cotton swabs) and to a chemical stimulus (local application of capsaicin) and the disruption of the blood-aqueous barrier after local injury (infrared irradiation of the iris) were tested after pretreatment with capsaicin (retrobulbar injection) the SP antagonist [D-Pro2,D-Trp7,9]SP (single topical application or long-term application twice daily for 2 months), the local anaesthetic oxibuprocaine (topical application), or the neuronal blocker tetrodotoxin (intravitreal injection). All these treatments abolished or reduced the disruption of the blood-aqueous barrier after local injury but only oxibuprocaine and tetrodotoxin abolished the corneal sensitivity to tactile and chemical stimuli. It is suggested that 1.(1) SP nerve fibers constitute a minor proportion of the sensory nerve supply to the cornea,2.(2) that the neurogenic mechanisms involved in the response to ocular injury differ from those involved in corneal nociception, and3.(3) that uveal SP is involved in the response to ocular trauma and that corneal SP is probably not necessary for corneal nociception.