Cold Thermoreceptors Research Articles

The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

The mechanistic target of rapamycin (mTOR) signalling pathway is a key regulator of cell growth and metabolism. Its deregulation is implicated in several diseases. The macrolide rapamycin, a specific inhibitor of mTOR, has immunosuppressive, anti-inflammatory and antiproliferative properties. Recently, we identified tacrolimus, another macrolide immunosuppressant, as a novel activator of TRPM8 ion channels, involved in cold temperature sensing, thermoregulation, tearing and cold pain. We hypothesized that rapamycin may also have agonist activity on TRPM8 channels. Using calcium imaging and electrophysiology in transfected HEK293 cells and wildtype or Trpm8 KO mouse DRG neurons, we characterized rapamycin's effects on TRPM8 channels. We also examined the effects of rapamycin on tearing in mice. Micromolar concentrations of rapamycin activated rat and mouse TRPM8 channels directly and potentiated cold-evoked responses, effects also observed in human TRPM8 channels. In cultured mouse DRG neurons, rapamycin increased intracellular calcium levels almost exclusively in cold-sensitive neurons. Responses were markedly decreased in Trpm8 KO mice or by TRPM8 channel antagonists. Cutaneous cold thermoreceptor endings were also activated by rapamycin. Topical application of rapamycin to the eye surface evokes tearing in mice by a TRPM8-dependent mechanism. These results identify TRPM8 cationic channels in sensory neurons as novel molecular targets of the immunosuppressant rapamycin. These findings may help explain some of its therapeutic effects after topical application to the skin and the eye surface. Moreover, rapamycin could be used as an experimental tool in the clinic to explore cold thermoreceptors.

Role of corneal sensory nerves in tearing and blinking

The cornea is innervated trigeminal neurons classified in three different functional types: polymodal nociceptor, mechano‐nociceptor and cold thermoreceptor neurons. Corneal sensory nerves are responsible of the conscious sensations evoked by corneal stimulation, and are also involved in the trophic maintenance of the tissue. Besides, corneal nerve activity is also involved in the regulation of the protective functions of the ocular surface such as tearing and blinking.Regulation of reflex and basal tearing is integrated at the brainstem and, in both cases, under the influence of the sensory inflow provided by several types of corneal sensory nerves. While reflex tearing is induced selectively by the stimulation of corneal polymodal nociceptors, basal tearing rate depends on the spontaneous activity of cold thermoreceptors. Thus, when cold thermoreceptors are silenced by environmental warming, basal tearing is compromised.Voluntary, reflex and spontaneous (basal) blinking present differences in their characteristics and regulation mechanisms. Voluntary blinking depends on the direct instructions generated at the brain cortex, acting over the motor neurons located at the facial nucleus. Reflex blinking is integrated at the brainstem, being induced by the stimulation of the cornea and periocular skin, and even by loud sounds. Spontaneous blinking is claimed to be controlled by a CNS centre that established a blinking pattern. This pattern is modified under different conditions such as cognitive activity, being for example reduced during visual attentional tasks in order not to lose relevant visual information. When the activity of corneal cold thermoreceptors is abolished or reduced by ocular instillation of local anaesthetics, the frequency and characteristics of the spontaneous blinking is not modified. However, when the activity of cold thermoreceptors is increased for prolonged periods (for instance after topical instillation of F6H8 drops, which reduces the ocular surface temperature), blinking pattern become more regular and an increased number of blinks of larger the amplitude and shorter duration are induced. This increased blinking frequency produced when the activity of cold thermoreceptors is increased is sufficient to even counteract the reduction of blinking frequency induced during visual tasks performance. The increase in blink frequency driven by corneal cold thermoreceptors' activation may constitute a protective mechanism to prevent excessive desiccation of the ocular surface.(Supported by PID2020‐115934RB‐I00 from DOI: MCIN/AEI/10.13039/50110001103, and CIPROM/2021/48 from the Generalitat Valenciana, Spain)

Conscious sensations and protective reflexes driven by eye surface nerves in health and disease

The trigeminal primary sensory neurons that innervate the ocular surface, especially the cornea, have been classified in three functional types: polymodal nociceptors, mechano‐nociceptors and cold thermoreceptors. These sensory nerves are responsible for the conscious sensations evoked in response to ocular surface stimulation as well as for the regulation of tearing and blinking, and they are also involved in the trophic maintenance of the ocular tissues.Depending on the transducing channels they express in their membrane, sensory nerves from the ocular surface respond to different types of stimulus and, after this information is processed by the CNS, different conscious sensations are evoked: corneal mechano‐nociceptors' activation led to pricking pain; polymodal nociceptors evoke burning irritation, and cold thermoreceptors' activation may evoke freshness/cold/dryness/pain depending on the subtype of cold thermoreceptor neuron activated by the stimulus. While reflex blinking and tearing are driven by the activation of polymodal nociceptors as the afferent arm of the reflex, basal blinking and tearing rate are under the control of cold thermoreceptors' activity.During inflammation and after injury (such as UV‐induced keratitis, allergic keratoconjunctivitis, corneal surgery, tear‐deficient dry eye) the activity of corneal sensory nerves is modified in both eyes, even when inflammation or damage affects only one eye. The abnormal corneal sensory nerve activity constitutes the basis for the discomfort, dryness and pain sensations experienced under these conditions. Also, protective mechanisms depending on ocular nerve activity such as blinking and tearing become altered.(Supported by PID2020‐115934RB‐I00 from DOI: MCIN/AEI/10.13039/50110001103, and CIPROM/2021/48 from the Generalitat Valenciana, Spain)

Corneal sensory nerve function: An overview

Somatosensory innervation of the cornea is provided by the peripheral axons of a small number of primary sensory neurons located in the ophthalmic region of the trigeminal ganglion. Corneal sensory nerve fibres are heterogeneous and may express different neurotransmitters and ion channels involved in transduction and coding of the stimuli acting on the tissue. Based on their activation by one or several types of physical and chemical stimuli, corneal nerve fibres are classified as polymodal nociceptors, mechanonociceptors and cold thermoreceptors. Impulse activity originated at corneal sensory nerves reaches second‐order neurons located at the lower brainstem, from where sensory information travels to the contralateral posterior thalamus and, then, to the brain cortex.After processing, the activation of the different classes of corneal neurons results in different sensations that depend on the type of primary sensory neuron initially activated by the stimulus, although pain is the main sensation elicited by corneal stimulation. Sensations originated at the cornea are explored by esthesiometry, allowing to quantify the threshold, intensity and quality of the evoked sensations. Sensitivity of the ocular surface is reduced with age and altered by several pathological processes that affect sensory innervation, either ocular (such as allergic keratoconjunctivitis, infectious keratitis, eye surgery, hereditary corneal dystrophies, dry eye, etc.) or systemic diseases, such as diabetes and peripheral neuropathies.A prominent feature of ocular nociceptors is their sensitization by the inflammatory mediators released after noxious stimulation or tissue injury, while cold thermoreceptor activity is inhibited by inflammatory substances. Both the spontaneous and the stimulus‐evoked activity increases in sensitized nociceptors, which causes sustained pain and hyperalgesia. After corneal sensory nerves damage (produced as a consequence of an ocular or systemic disease, or by accidental or surgical injury), part of sensory nerves degenerates leading to reduced sensitivity to natural stimulation in the affected area. At the same time, axons start to regenerate and to recover the ability to transduce and encode the stimulus. During this regeneration process, cold thermoreceptor and nociceptor nerves may become hyperexcitable due to changes in their expression/activity of ion channels, which give rise to dryness sensation and ocular neuropathic pain.(Supported by PID2020‐115934RB‐I00 from DOI: MCIN/AEI/10.13039/50110001103, and CIPROM/2021/48 from the Generalitat Valenciana, Spain)

Corneo‐conjunctival sensory neuron: Types, transduction channels and codification of nerve impulses

The cornea and the conjunctiva are innervated by the peripheral projections of a small number of primary sensory neurons whose somata are located at the ophthalmic region of the trigeminal ganglion. Most trigeminal neurons innervating the ocular surface are small and medium size, and contain neuropeptides such as SP and CGRP. Functionally, ocular surface neurons have been classified into five main neuronal types, based on their response to multiple modalities of stimuli (mechanical forces, heat, cold, H+, chemical substances, etc.). Polymodal nociceptors (activated by several modalities of stimulus, mechanical energy, heat, H+, and chemical irritants), mechanonociceptors (activated by mechanical energy), cold thermoreceptors (activated by temperature reductions and hyperosmolarity) are found in the cornea, the sclera and the conjunctiva. Besides, low‐threshold mechanoreceptors (activated by mechanical forces of low intensity) are found in the corneoscleral limbus and the lid border, and pruriceptors (activated by pruritogen substances) have been described in the tarsal conjunctiva, close to the lid margin.The ability to respond to the different modalities of stimulus lies in the types of transducing channels that each type of neuron expresses at its peripheral nerve endings. Sensory transduction involves the conversion of the physical or chemical stimuli acting on the nerve terminal into electrical energy, that is, into a local change of membrane potential that, if it is large enough, will generate action potentials that will travel along the axon to be processed by the CNS. So that, nerve terminals with TRPV1 channels are directly gated by heat, H+, and several chemical irritants such as capsaicin; nerve terminals with Piezo2 channels will be activated by mechanical forces; TRPM8 channels provide nerve terminals sensitivity to cold and osmolarity increases; et cetera.Ocular sensory neurons also express other ion channels that contribute to coding, that is to help the sensory neuron to establish a correspondence between the characteristics of the stimulus (intensity, temporal course, etc.) and specific parameters of the firing it induces. Voltage‐gated Ca2+, Na+ and K+ channels as well as cyclic nucleotide‐gated channels, among others, contribute to define the firing frequency and pattern to encode the traits of the stimulus. Thus, the ion channels present in the nerve terminals of each class of ocular sensory neuron provide the neurons the ability to detect and encode one or several modalities of stimulus, which constitutes the origin of the different sensations evoked on the ocular surface.(Supported by PID2020‐115934RB‐I00 from DOI: MCIN/AEI/10.13039/50110001103, and CIPROM/2021/48 from the Generalitat Valenciana, Spain)

Molecular and cellular changes underlying plasticity and regeneration of intact, injured and aged cold corneal nerves

The peripheral nerve endings from trigeminal neurons that innervate the cornea are key neural structures to detect and encode the features of the physical and chemical external stimuli at the ocular surface. These neurons are functionally diverse and have been classified into polymodal nociceptors, mechano‐nociceptors and cold thermoreceptors. Each functional type evokes qualitative different conscious sensations. The sensory modality that is generated from a nerve ending is determined by the type and specific characteristics of the ion channels involved in stimulus transduction and encoding. Among them, TRPM8 channels transduce both cold and osmotic stimuli in corneal cold nerve terminals. These cold thermoreceptors mediate sensations of ocular surface cooling and dryness, and play an important role in the regulation of basal tearing and blinking. Morphologically, cold thermosensitive neurons display a more complex branching of their peripheral nerve endings innervating the cornea in comparison to the other functional classes of sensory neurons.With aging, TRPM8‐expressing cold sensory neurons innervating the mouse cornea experience morphological and functional alterations. These changes occur earlier and are more prominent at the nerve terminal than at the neuron cell body. A drastic reduction of the nerve terminal branching complexity in addition to lower background impulse activity is observed in cold nerves of aged mice. Similarly, after performing photorefractive keratectomy (PRK) in the adult mouse cornea, both the nerve terminal branching and background activity are reduced in a fraction of the cold nerve terminals. An altered morphological configuration along with an aberrant transducing and coding ion channels expression at corneal cold nerve terminals are likely to contribute to the discomfort sensations experienced after corneal surgery and to the age‐related abnormal tearing in the elderly.(Supported by PID2021‐124460OB‐I00 from DOI: MCIN/AEI/ 10.13039/501100011033, and CIPROM/2021/48 from the Generalitat Valenciana, Spain)

Cyclosporine A Decreases Dryness-Induced Hyperexcitability of Corneal Cold-Sensitive Nerve Terminals.

Cyclosporine A (CsA) is used for the treatment of dry eye (DE) with good clinical results, improving tear secretion and decreasing subjective symptoms. These effects are attributed to the improved tear film dynamics, but there are no data on the effect of CsA on the abnormal sensory nerve activity characteristic in DE. Our purpose was to evaluate the CsA effect on the enhanced activity of corneal cold thermoreceptors in a tear-deficient DE animal model using in vitro extracellular recording of cold thermoreceptors nerve terminal impulses (NTIs) before and in the presence of CsA. NTI shape was also analyzed. Blinking frequency and tearing rate were also measured in awake animals before and after topical CsA. CsA increased the tearing and blinking of treated animals. CsA significantly decreased the peak response to cold of cold thermoreceptors. Neither their spontaneous NTIs discharge rate nor their cooling threshold were modified. CsA also seemed to reverse some of the changes in NTI shape induced by tear deficiency. These data suggest that, at least in part, the beneficial clinical effects of CsA in DE can be attributed to a direct effect on sensory nerve endings, although the precise mechanisms underlying this effect need further studies to be fully clarified.

The Contribution of TRPA1 to Corneal Thermosensitivity and Blink Regulation in Young and Aged Mice.

The role of TRPA1 in the thermosensitivity of the corneal cold thermoreceptor nerve endings was studied in young and aged mice. The contribution of the TRPA1-dependent activity to basal tearing and thermally-evoked blink was also explored. The corneal cold thermoreceptors' activity was recorded extracellularly in young (5-month-old) and aged (18-month-old) C57BL/6WT (WT) and TRPA1-/- knockout (TRPA1-KO) mice at basal temperature (34 °C) and during cooling (15 °C) and heating (45 °C) ramps. The blink response to cold and heat stimulation of the ocular surface and the basal tearing rate were also measured in young animals using orbicularis oculi muscle electromyography (OOemg) and phenol red threads, respectively. The background activity at 34 °C and the cooling- and heating-evoked responses of the cold thermoreceptors were similar in WT and TRPA1-KO animals, no matter the age. Similar to the aged WT mice, in the young and aged TRPA1-KO mice, most of the cold thermoreceptors presented low frequency background activity, a low cooling threshold, and a sluggish response to heating. The amplitude and duration of the OOemg signals correlated with the magnitude of the induced thermal change in the WT but not in the TRPA1-KO mice. The basal tearing was similar in the TRPA1-KO and WT mice. The electrophysiological data suggest that the TRPA1-dependent nerve activity, which declines with age, contributes to detecting the warming of the ocular surface and also to integrating the thermally-evoked reflex blink.

Molecular determinants of TRPM8 function: key clues for a cool modulation.

Cold thermoreceptor neurons detect temperature drops with highly sensitive molecular machinery concentrated in their peripheral free nerve endings. The main molecular entity responsible for cold transduction in these neurons is the thermo-TRP channel TRPM8. Cold, cooling compounds such as menthol, voltage, and osmolality rises activate this polymodal ion channel. Dysregulation of TRPM8 activity underlies several physiopathological conditions, including painful cold hypersensitivity in response to axonal damage, migraine, dry-eye disease, overactive bladder, and several forms of cancer. Although TRPM8 could be an attractive target for treating these highly prevalent diseases, there is still a need for potent and specific modulators potentially suitable for future clinical trials. This goal requires a complete understanding of the molecular determinants underlying TRPM8 activation by chemical and physical agonists, inhibition by antagonists, and the modulatory mechanisms behind its function to guide future and more successful treatment strategies. This review recapitulates information obtained from different mutagenesis approaches that have allowed the identification of specific amino acids in the cavity comprised of the S1-S4 and TRP domains that determine modulation by chemical ligands. In addition, we summarize different studies revealing specific regions within the N- and C-terminus and the transmembrane domain that contribute to cold-dependent TRPM8 gating. We also highlight the latest milestone in the field: cryo-electron microscopy structures of TRPM8, which have provided a better comprehension of the 21years of extensive research in this ion channel, shedding light on the molecular bases underlying its modulation, and promoting the future rational design of novel drugs to selectively regulate abnormal TRPM8 activity under pathophysiological conditions.

Unilateral corneal insult also alters sensory nerve activity in the contralateral eye

AbstractWhen the ocular surface of one eye is inflamed or injured, its corneal sensory nerve activity is modified, which in turn evokes ocular discomfort, irritation, and pain sensations referred to the affected eye. Some patients with unilateral ocular inflammation, infection, or surgery also report discomfort and pain in the contralateral eye. Are these sensations experienced at the fellow eye due to alterations of its sensory nerve activity?To test this hypothesis, we recorded in vitro the activity of corneal mechanonociceptor, polymodal nociceptor and cold thermoreceptor nerves of both eyes of guinea pigs previously subjected unilaterally to three different experimental inflammatory and lesion conditions: UV‐induced photokeratitis, microkeratome corneal surgery, and chronic tear deficiency caused by removal of the main lacrimal gland. Recordings from the eyes of naïve animals were used as control.Compared with the naïve guinea pigs, animals subjected to unilateral UV‐induced mild corneal inflammation, showed inhibition of the spontaneous and stimulus‐evoked activity of cold thermoreceptors, and increased activity (sensitization) of nociceptors affecting both the ipsilateral and the contralateral eye. Unilateral microkeratome surgery affected the activity of nociceptors mostly, inducing sensitization in both eyes. Chronic eye dryness by removal of the main lacrimal gland increased cold thermoreceptor activity in both eyes. These results show that after unilateral eye damage, corneal sensory nerve activity is altered in both eyes, although to a lesser degree in the contralateral eye.This is the first direct demonstration that unilateral corneal nerve insult, and especially ocular surface inflammation, functionally affects the activity of the different types of corneal sensory nerves in both the ipsilateral and contralateral eyes. The mechanisms underlying the changes of contralateral sensory nerve activity remain to be determined, although available data support the involvement of the immune system. The present results have two main implications: (a) contralateral eyes cannot be considered as a control in experimental designs of both preclinical and clinical studies; and, (b) clinicians must consider the convenience of treating both eyes of patients with unilateral ocular conditions to avoid pain and secondary undesirable effects in the fellow eye.

Validation of the CD11c‐DTR transgenic mouse as a model to study neuroimmune interactions in the cornea

AbstractPurpose: CD11c‐DTR+ mice have a transgene encoding for a simian diphtheria toxin (DT) receptor under the CD11c promoter that makes dendritic cells (DCs) sensitive to DT, which causes DC depletion. Preliminary results using this mouse suggest that there is a functional interaction between DCs and corneal sensory nerves, as DC depletion modifies nerve activity and nociceptive behaviour. The aim of this work was to validate the CD11c‐DTR+ mouse model proving that the observed effects are not due to the DT itself, using littermates negative for the transgene and therefore resistant to DC depletion by DT.Methods: CD11c‐DTR+ and CD11c‐DTR‐ mice (5 months, both sexes) were used. Both groups received single or repetitive (every 2 days for 8 days) subconjunctival injections of DT or its vehicle (PBS). Basal tearing (in mm) was measured using phenol red threads placed in the temporally side of the eye for 30 s in isoflurane‐anaesthetised mice. To quantify spontaneous eye pain, the eye closure ratio was calculated by dividing the height by the width of the lid opening measured in photographs of freely moving animals. Nerve terminal impulse (NTI) activity of corneal cold thermoreceptor endings was recorded ex vivo in superfused eyes; spontaneous and stimulus‐evoked activity was explored.Results: Basal tearing was higher in DT‐injected CD11c‐DTR‐ animals, especially after repetitive injections (Day 0: 2 ± 0.16 mm vs. day 2: 2.8 ± 0.37 mm vs. day 8: 4.7 ± 0.36 mm, n = 5, p ≤ 0.001, RM ANOVA). Repetitive PBS injections caused similar effects in CD11c‐DTR+ and CD11c‐DTR‐ mice tearing (DTR−: 4.7 ± 0.36 mm, n = 5 vs. DTR+: 3.7 ± 1.48 mm, n = 3, p = 0.399, t‐test). No signs of spontaneous pain were observed in DT‐injected CD11c‐DTR‐ mice (Day 0: 0.91 ± 0.01 vs. 48 h: 0.93 ± 0.01 vs. day 8: 0.95 ± 0.01, n = 5, p = 0.065, RM ANOVA). No significant differences were found in spontaneous and stimulus‐evoked NTI activity recorded after repetitive injections of DT in CD11c‐DTR− mice or PBS in CD11c‐DTR+ mice.Conclusions: CD11c‐DTR+ mice are a valid model to study neuroimmune interactions. DT has no effects on pain behaviour or NTI activity of animals lacking the DT receptor. The changes in tearing appear to be consequence of the subconjunctival injection process per se.Reference PID2020‐115934RB‐I00 from DOI: MCIN/AEI/10.13039/50110001103

Effects of contact lens wearing on corneal sensitivity and reflex functions

AbstractPurpose: To study the effects of contact lens (CL) use on corneal sensory nerve activity, exploring corneal sensitivity and ocular functions regulated by sensory input in asymptomatic (CL‐A) and symptomatic (CL‐S) CL wearers.Methods: CL wearers (23.8 ± 1.0 years; n = 31) participating in the study were classified as CL‐S when OSDI ≥13. Sex and age eye glasses (EG) users (24.5 ± 0.8 years, n = 29) participated as control group. Corneal mechanical, chemical and cold sensitivity was explored using a gas esthesiometer and 4°C‐saline drops; intensity, irritation and cold components of the evoked sensation were measured with Visual Analogue Scales (VASs). Tear volume (with phenol red threads), tear stability (TBUT), basal blinking frequency (BF), ocular surface temperature (OST; by IR thermography) were also assessed.Results: CL wearers scored higher irritation VAS values than EG wearers for mechanical (2.5 ± 0.5 vs. 1.4 ± 0.4 for 160 ml/min air flow, CL vs EG, p = 0.09) and chemical stimuli (1.8 ± 0.5 vs. 0.7 ± 0.3 for 40% CO2, p < 0.05), and lower intensity values for cold stimulation (2.2 ± 0.4 vs. 4.5 ± 0.7, p < 0.05); tear volume was similar (27.0 ± 2.2 vs. 27.2 ± 1.6 mm), TBUT lower (8.3 ± 0.5 vs. 11.7 ± 0.5 s, p < 0.001), BF was higher (25.1 ± 2.4 vs. 14.3 ± 1.9 blinks/min, p < 0.001), and OST was lower (34.4 ± 0.2 vs. 35.2 ± 0.2°C, p < 0.01). VAS values for cold stimulation was slightly lower in CL‐S than in CL‐A (2.0 ± 0.4 vs. 4.0 ± 0.8, p = 0.06), while OST was higher (35.0 ± 0.2 vs. 33.9 ± 0.3°C, p < 0.01).Conclusions: CL wearing affects corneal sensory nerve functions, changing corneal sensitivity, blinking rate, tearing stability, and OST. The changes found in corneal sensitivity fully agree with those described in neural activity of corneal sensory nerves after injury or inflammation using animal models: sensitization of corneal nociceptors and inhibition of cold thermoreceptors. The differences between CL‐S and CL‐A wearers could be due to the presence of subclinical ocular surface inflammation in symptomatic subjects.

Transient Receptor Potential Channels: Important Players in Ocular Pain and Dry Eye Disease.

Dry eye disease (DED) is a multifactorial disorder in which the eyes respond to minor stimuli with abnormal sensations, such as dryness, blurring, foreign body sensation, discomfort, irritation, and pain. Corneal pain, as one of DED’s main symptoms, has gained recognition due to its increasing prevalence, morbidity, and the resulting social burden. The cornea is the most innervated tissue in the body, and the maintenance of corneal integrity relies on a rich density of nociceptors, such as polymodal nociceptor neurons, cold thermoreceptor neurons, and mechano-nociceptor neurons. Their sensory responses to different stimulating forces are linked to the specific expression of transient receptor potential (TRP) channels. TRP channels are a group of unique ion channels that play important roles as cellular sensors for various stimuli. These channels are nonselective cation channels with variable Ca2+ selectivity. TRP homologs are a superfamily of 28 different members that are subdivided into 7 different subfamilies based on differences in sequence homology. Many of these subtypes are expressed in the eye on both neuronal and non-neuronal cells, where they affect various stress-induced regulatory responses essential for normal vision maintenance. This article reviews the current knowledge about the expression, function, and regulation of TRPs in ocular surface tissues. We also describe their implication in DED and ocular pain. These findings contribute to evidence suggesting that drug-targeting TRP channels may be of therapeutic benefit in the clinical setting of ocular pain.

Dendritic cell depletion modifies corneal nerve activity and mice nociceptive behaviour

AbstractPurposeTo determine the influence of resident dendritic cells (DC) in modulating corneal sensory nerve activity and its possible implications in basal tearing and pain.MethodsC57BL/6 CD11c‐DTR mice (both sexes, 5–6 months old) were used. Short‐term (48 hr, STD) and long‐term (8 days, LTD) DC depletion was done by bilateral subconjunctival injections of diphtheria toxin (DT) under anesthesia. Naïve mice (no DT‐injections) served as control. Background and stimulus‐evoked (cooling and heating ramps) activity of cold thermoreceptor nerve terminals was recorded ex vivo. Basal tearing rate was measured using commercial phenol red threads during 30 seconds in isoflurane‐anesthetised animals. For monitoring the presence of spontaneous eye pain, the eye closure ratio (calculated by dividing the height by the width of the palpebral fissure measured in photographs) was used.ResultsSTD produced a significant decrease in heating threshold (37.7 ± 0.7°C, n = 10 versus naïve, 41.6±1.8°C, n = 4 and LTD, 42.3±1°C, n = 8; p = 0.003, ANOVA). After LTD, lower temperature values were needed to reach both cooling threshold and peak frequency during cooling ramps (30.7 ± 0.5°C, n = 17 versus naïve, 32.3 ± 0.3°C, n = 20, and STD, 32.2±0.4°C, n = 17; p = 0.005, ANOVA). Basal tearing was slightly reduced in STD (2.8 ± 0.6 mm before DT versus 1.2 ± 0.38 mm at 48 hr, n = 6; p = 0.119, paired t‐test) and signs of spontaneous pain were observed in both STD and LTD, being the eye closure ratio lower than before DT‐injections (STD: 0.92 ± 0.01 before DT versus 0.18 ± 0.02 at 48 hr, n = 23, p ≤ 0.001, paired t‐test; LTD: 0.9 ± 0.03 before DT versus 0.17 ± 0.02 at 48 hr versus 0.46 ± 0.07 at day 8, n = 3, p ≤ 0.001, RM One‐way ANOVA).ConclusionsDifferences observed in nerve impulse activity and the subsequent changes in basal tearing and signs of spontaneous pain suggest a crucial role of DC in corneal nerve activity and ocular surface homeostasis.Supported by SAF2017‐83674‐C2‐1‐R and ‐2‐R (AEI‐ERDF), PROMETEO/2018/114 and ACIF/2017/169 (Generalitat Valenciana).

Combining Topical Agonists With the Recording of Event-Related Brain Potentials to Probe the Functional Involvement of TRPM8, TRPA1 and TRPV1 in Heat and Cold Transduction in the Human Skin

TRP channels play a central role in the transduction of thermal and nociceptive stimuli by free nerve endings. Most of the research on these channels has been conducted in vitro or in vivo in nonhuman animals and translation of these results to humans must account for potential experimental biases and interspecific differences. This study aimed at evaluating the involvement of TRPM8, TRPA1 and TRPV1 channels in the transduction of heat and cold stimuli by the human thermonociceptive system. For this purpose, we evaluated the effects of topical agonists of these 3 channels (menthol, cinnamaldehyde and capsaicin) on the event-related brain potentials (ERPs) elicited by phasic thermal stimuli (target temperatures: 10°C, 42°C, and 60°C) selected to activate cold Aδ thermoreceptors, warm sensitive C thermoreceptors and heat sensitive Aδ polymodal nociceptors. Sixty-four participants were recruited, 16 allocated to each agonist solution group (20% menthol, 10% cinnamaldehyde, .025% capsaicin and 1% capsaicin). Participants were treated sequentially with the active solution on one forearm and vehicle only on the other forearm for 20 minutes. Menthol decreased the amplitude and increased the latency of cold and heat ERPs. Cinnamic aldehyde decreased the amplitude and increased the latency of heat but not cold ERPs. Capsaicin decreased the amplitude and increased the latency of heat ERPs and decreased the amplitude of the N2P2 complex of the cold ERPs without affecting the earlier N1 wave or the latencies of the peaks. These findings are compatible with previous evidence indicating that TRPM8 is involved in innocuous cold transduction and that TRPV1 and TRPA1 are involved in noxious heat transduction in humans. PerspectiveBy chemically modulating TRPM8, TRPA1 and TRPV1 reactivity (key molecules in the transduction of temperature) and assessing how this affected EEG responses to the activation of cold thermoreceptors and heat nociceptors, we aimed at confirming the role of these channels in a functional healthy human model.

Unilateral Corneal Insult Also Alters Sensory Nerve Activity in the Contralateral Eye.

After the unilateral inflammation or nerve lesion of the ocular surface, the ipsilateral corneal sensory nerve activity is activated and sensitized, evoking ocular discomfort, irritation, and pain referred to the affected eye. Nonetheless, some patients with unilateral ocular inflammation, infection, or surgery also reported discomfort and pain in the contralateral eye. We explored the possibility that such altered sensations in the non-affected eye are due to the changes in their corneal sensory nerve activity in the contralateral, not directly affected eye. To test that hypothesis, we recorded the impulse activity of the corneal mechano- and polymodal nociceptor and cold thermoreceptor nerve terminals in both eyes of guinea pigs, subjected unilaterally to three different experimental conditions (UV-induced photokeratitis, microkeratome corneal surgery, and chronic tear deficiency caused by removal of the main lacrimal gland), and in eyes of naïve animals ex vivo. Overall, after unilateral eye damage, the corneal sensory nerve activity appeared to be also altered in the contralateral eye. Compared with the naïve guinea pigs, animals with unilateral UV-induced mild corneal inflammation, showed on both eyes an inhibition of the spontaneous and stimulus-evoked activity of cold thermoreceptors, and increased activity in nociceptors affecting both the ipsilateral and the contralateral eye. Unilateral microkeratome surgery affected the activity of nociceptors mostly, inducing sensitization in both eyes. The removal of the main lacrimal gland reduced tear volume and increased the cold thermoreceptor activity in both eyes. This is the first direct demonstration that unilateral corneal nerve lesion, especially ocular surface inflammation, functionally affects the activity of the different types of corneal sensory nerves in both the ipsilateral and contralateral eyes. The mechanisms underlying the contralateral affectation of sensory nerves remain to be determined, although available data support the involvement of neuroimmune interactions. The parallel alteration of nerve activity in contralateral eyes has two main implications: a) in the experimental design of both preclinical and clinical studies, where the contralateral eyes cannot be considered as a control; and, b) in the clinical practice, where clinicians must consider the convenience of treating both eyes of patients with unilateral ocular conditions to avoid pain and secondary undesirable effects in the fellow eye.

Deciphering the Action of Perfluorohexyloctane Eye Drops to Reduce Ocular Discomfort and Pain.

Perfluorohexyloctane (F6H8) eyedrops have been recently introduced in Europe as a product to treat dry eye disease, based on its ability to reduce tear film instability in Meibomian gland dysfunction and evaporative dry eye disease, although its mechanism of action is still unknown. In the present pilot study, we evaluated the effects of the ocular instillation of a single drop of commercial F6H8 eyedrops in 20 healthy humans (9 women/11 men), measuring: (a) Corneal surface temperature (CST) from infrared video images; (b) tear volume using phenol red threads; (c) blinking frequency; and (d) ocular surface sensations (cold, dryness, pricking, foreign body, burning, itching, gritty, eye fatigue, watering eyes, and light-evoked discomfort sensations; scored using 10 cm Visual Analog Scales), before and 5–60 min after F6H8 or saline treatment. CST decreased and tearing and blinking frequency increased significantly after F6H8 but not after saline solution. When applied unilaterally, CST decreased only in the F6H8-treated eye. No sensations were evoked after F6H8 or saline. The corneal surface temperature reduction produced by topical F6H8 does not evoke conscious ocular sensations but is sufficient to increase the activity of corneal cold thermoreceptors, leading to an increased reflex lacrimation and blinking that may relieve dry eye condition thus reducing ocular discomfort and pain.

Constitutive Phosphorylation as a Key Regulator of TRPM8 Channel Function

In mammals, environmental cold sensing conducted by peripheral cold thermoreceptor neurons mostly depends on TRPM8, an ion channel that has evolved to become the main molecular cold transducer. This TRP channel is activated by cold, cooling compounds, such as menthol, voltage, and rises in osmolality. TRPM8 function is regulated by kinase activity that phosphorylates the channel under resting conditions. However, which specific residues, how this post-translational modification modulates TRPM8 activity, and its influence on cold sensing are still poorly understood. By mass spectrometry, we identified four serine residues within the N-terminus (S26, S29, S541, and S542) constitutively phosphorylated in the mouse ortholog. TRPM8 function was examined by Ca2+ imaging and patch-clamp recordings, revealing that treatment with staurosporine, a kinase inhibitor, augmented its cold- and menthol-evoked responses. S29A mutation is sufficient to increase TRPM8 activity, suggesting that phosphorylation of this residue is a central molecular determinant of this negative regulation. Biophysical and total internal reflection fluorescence-based analysis revealed a dual mechanism in the potentiated responses of unphosphorylated TRPM8: a shift in the voltage activation curve toward more negative potentials and an increase in the number of active channels at the plasma membrane. Importantly, basal kinase activity negatively modulates TRPM8 function at cold thermoreceptors from male and female mice, an observation accounted for by mathematical modeling. Overall, our findings suggest that cold temperature detection could be rapidly and reversibly fine-tuned by controlling the TRPM8 basal phosphorylation state, a mechanism that acts as a dynamic molecular brake of this thermo-TRP channel function in primary sensory neurons.SIGNIFICANCE STATEMENT Post-translational modifications are one of the main molecular mechanisms involved in adjusting the sensitivity of sensory ion channels to changing environmental conditions. Here we show, for the first time, that constitutive phosphorylation of the well-conserved serine 29 within the N-terminal domain negatively modulates TRPM8 channel activity, reducing its activation by agonists and decreasing the number of active channels at the plasma membrane. Basal phosphorylation of TRPM8 acts as a key regulator of its function as the main cold-transduction channel, significantly contributing to the net response of primary sensory neurons to temperature reductions. This reversible and dynamic modulatory mechanism opens new opportunities to regulate TRPM8 function in pathologic conditions where this thermo-TRP channel plays a critical role.

Chronic Tear Deficiency Sensitizes Transient Receptor Potential Vanilloid 1-Mediated Responses in Corneal Sensory Nerves.

Chronic tear deficiency enhances the excitability of corneal cold-sensitive nerves that detect ocular dryness, which can lead to discomfort in patients with dry eye disease (DED). However, changes in corneal nerve excitations through the polymodal nociceptor “transient receptor potential vanilloid 1” (TRPV1) and the potential link between this receptor and symptoms of DED remain unclear. In this study, we examined the firing properties of corneal cold-sensitive nerves expressing TRPV1 and possible contributions of chronic tear deficiency to corneal nerve excitability by TRPV1 activation. The bilateral excision of lacrimal glands in guinea pigs decreased the tear volume and increased the frequency of spontaneous eyeblinks 1–4 weeks after surgery. An analysis of the firing properties of the cold-sensitive nerves was performed by single-unit recordings of corneal preparations 4 weeks after surgery in both the sham-operated and gland-excised groups. Perfusion of the TRPV1 agonist, capsaicin (1 μM), transiently increased the firing frequency in approximately 46–48% of the cold-sensitive nerves characterized by low-background activity and high threshold (LB-HT) cold thermoreceptors in both groups. Gland excision significantly decreased the latency of capsaicin-induced firing in cold-sensitive nerves; however, its magnitude was unchanged. Calcium imaging of cultured trigeminal ganglion neurons from both groups showed that intracellular calcium elevation of corneal neurons induced by a low concentration of capsaicin (0.03 μM) was significantly larger in the gland excision group, regardless of responsiveness to cold. An immunohistochemical study of the trigeminal ganglion revealed that gland excision significantly increased the proportion of corneal neurons enclosed by glial fibrillary acidic protein (GFAP)-immunopositive satellite glial cells. Topical application of the TRPV1 antagonist, A784168 (30 μM), on the ocular surface attenuated eye-blink frequency after gland excision. Furthermore, gland excision enhanced blink behavior induced by a low concentration of capsaicin (0.1 μM). These results suggest that chronic tear deficiency sensitizes the TRPV1-mediated response in the corneal LB-HT cold thermoreceptors and cold-insensitive polymodal nociceptors, which may be linked to dry eye discomfort and hyperalgesia resulting from nociceptive stimuli in aqueous-deficient dry eyes.

Morphological and Functional Changes of Corneal Nerves and Their Contribution to Peripheral and Central Sensory Abnormalities.

The cornea is the most densely innervated and sensitive tissue in the body. The cornea is exclusively innervated by C- and A-delta fibers, including mechano-nociceptors that are triggered by noxious mechanical stimulation, polymodal nociceptors that are excited by mechanical, chemical, and thermal stimuli, and cold thermoreceptors that are activated by cooling. Noxious stimulations activate corneal nociceptors whose cell bodies are located in the trigeminal ganglion (TG) and project central axons to the trigeminal brainstem sensory complex. Ocular pain, in particular, that driven by corneal nerves, is considered to be a core symptom of inflammatory and traumatic disorders of the ocular surface. Ocular surface injury affecting corneal nerves and leading to inflammatory responses can occur under multiple pathological conditions, such as chemical burn, persistent dry eye, and corneal neuropathic pain as well as after some ophthalmological surgical interventions such as photorefractive surgery. This review depicts the morphological and functional changes of corneal nerve terminals following corneal damage and dry eye disease (DED), both ocular surface conditions leading to sensory abnormalities. In addition, the recent fundamental and clinical findings of the importance of peripheral and central neuroimmune interactions in the development of corneal hypersensitivity are discussed. Next, the cellular and molecular changes of corneal neurons in the TG and central structures that are driven by corneal nerve abnormalities are presented. A better understanding of the corneal nerve abnormalities as well as neuroimmune interactions may contribute to the identification of a novel therapeutic targets for alleviating corneal pain.