Corneal Stimulation Research Articles

Corneal Sensitivity Testing Procedure for Ophthalmologic and Optometric Patients.

The cornea is the most densely innervated structure in the human body, making it one of the most sensitive tissues. Changes in corneal nerve sensitivity can be observed in several ocular surface diseases. Nerve sensitivity may be increased, as is often seen in patients with a neuropathic component to ocular pain, or decreased, as is seen in patients with neurotrophic keratitis. Corneal sensitivity testing involves assessing a patient's reaction to brief corneal stimulation, yields insight into the health of the corneal nerves, and provides diagnostic value for evaluating the health of the nerves and the interplay with the ocular surface. Currently, there is limited published guidance on how to conduct corneal sensitivity testing in a clinical setting. This article presents a protocol for testing corneal sensitivity using easy-to-use, low-cost materials that are readily accessible to eye care providers (either a cotton swab, a piece of dental floss, or a finely tapered tissue). This protocol allows for qualitative assessment of corneal sensitivity in which responses to corneal stimulation are rated from 0 (no response) to 3 (hypersensitive response). This test can be performed quickly (in approximately 30 s). Given its diagnostic value and accessibility, corneal sensitivity testing should be included as part of the standard eye examination for any patient undergoing an ocular surface examination.

Trigeminal Sensitization in a Closed Head Model for Mild Traumatic Brain Injury.

Mild traumatic brain injury (mTBI) is often accompanied by neurological and ocular symptoms that involve trigeminal nerve pathways. Laser-induced shock wave (LISW) was applied to the skull of male rats as a model for mTBI, while behavioral and neural recording methods were used to assess trigeminal function. The LISW caused greater eye wiping behavior to ocular instillation of hypertonic saline (Sham = 4.83 ± 0.65 wipes/5 min, LISW = 12.71 ± 1.89 wipes/5 min, p < 0.01) and a marked reduction in the time spent in bright light consistent with enhanced periocular and intraocular hypersensitivity, respectively (Sham = 16.3 ± 5.6 s, LISW = 115.5 ± 27.3 s, p < 0.01). To address the early neural mechanisms of mTBI, single trigeminal brainstem neurons, identified by activation to corneal or dural mechanical stimulation, were recorded in trigeminal subnucleus interpolaris/caudalis (Vi/Vc) and trigeminal subnucleus caudalis/upper cervical cord (Vc/C1) regions. The LISW caused marked sensitization to hypertonic saline and to exposure to bright light in neurons of both regions (p < 0.05). Laser speckle imaging revealed an increase in meningeal arterial blood flow to bright light after LISW (Sham = 4.7 ± 2.0 s, LISW = 469.0 ± 37.9 s, p < 0.001). Local inhibition of synaptic activity at Vi/Vc, but not at Vc/C1, by microinjection of CoCl2, prevented light-evoked increases in meningeal blood flow in LISW-treated rats. By contrast, topical meningeal application of phenylephrine significantly reduced light-evoked responses of Vi/Vc and Vc/C1 neurons. These data suggested that neurons in both regions became sensitized after LISW and were responsive to changes in meningeal blood flow. Neurons at the Vi/Vc transition and at Vc/C1, however, likely serve different roles in mediating the neurovascular and sensory aspects of mTBI.

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)

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)

Fabrication and Characterization of a Flexible Thin-Film-Based Array of Microelectrodes for Corneal Electrical Stimulation.

The electric stimulation (ES) of the cornea is a novel therapeutic approach to the treatment of degenerative visual diseases. Currently, ES is delivered by placing a mono-element electrode on the surface of the cornea that uniformly stimulates the eye along the electrode site. It has been reported that a certain degree of correlation exists between the location of the stimulated retinal area and the position of the electrode. Therefore, in this study, we present the development of a sectioned surface electrode for selective electric stimulation of the human cornea. The proposed device consists of 16 independent microelectrodes, a reference electrode, and 18 contact pads. The microelectrodes have a size of 200 µm × 200 µm, are arranged in a 4 × 4 matrix, and cover a total stimulation area of 16 mm2. The proposed fabrication process, based on surface micromachining technology and flexible electronics, uses only three materials: polyimide, aluminum, and titanium, which allow us to obtain a simplified, ergonomic, and reproducible fabrication process. The fabricated prototype was validated to laboratory level by electrical and electrochemical tests, showing a relatively high electrical conductivity and average impedance from 712 kΩ to 1.4 MΩ at the clinically relevant frequency range (from 11 Hz to 30 Hz). Additionally, the biocompatibility of the electrode prototype was demonstrated by performing in vivo tests and by analyzing the polyimide films using Fourier transform infrared spectroscopy (FTIR). The resulting electrode prototype is robust, mechanically flexible, and biocompatible, with a high potential to be used for selective ES of the cornea.

Maximal tear secretion evoked by controlled stimulation of corneal sensory nerves in healthy individuals and dry eye subjects.

To measure, the tear flow changes evoked in healthy subjects and dry eye disease (DED) patients by controlled sensory stimulation of the eye surface with i-Onion™, a new stimulation device. Sensory corneal nerves were stimulated with an instrument (i-Onion™) that ejects puffs of CO2 gas (99.9%) at 200 ml.min-1 for 3s, delivered 5 mm from the cornea. Using Schirmer test strips, tear volumes were measured over 3 min in the cornea of one eye before (basal tear volume -BTV) and in the other eye after stimulation of the sensory nerves with CO2 (stimulated tear volume -STV). These measurements were obtained from a control group of adults of either sex (17 students aged 20-30 and 29 subjects without signs of dry eye aged 25-61), a cohort of DED patients (aged 34-75) that included 12 asymptomatic, suspected DED subjects (Schirmer <7 mm and/or TBUT <10s), and 30 Sjögren's syndrome (SS) patients. CO2 stimulation significantly increased the tear volume (BTV = 14.6 ± 1.0 mm, STV = 19.0 ± 1.1 mm: n = 46) in 78% of control subjects, reflecting a mean tear reserve volume (TRV = STV-BTV) of 4.4 ± 0.8 mm. Individual differences were wide, and while no increase in reflex tearing was evoked in 30% of subjects with a BTV >10 mm, the remaining 70% responded vigorously to stimulation, even those with a BTV >18 mm. Asymptomatic DED subjects displayed weaker responses to CO2 stimulation, with lower STVs. Both the BTV and STV of SS patients were low, significantly below those of the healthy controls. Measuring the rise in reflex tearing volume evoked by controlled corneal stimulation provides objective information about the tear glands' secretory capacity in health and disease.

The Efficient Regeneration of Corneal Nerves via Tunable Transmembrane Signaling Channels Using a Transparent Graphene-Based Corneal Stimulation Electrode.

The efficient regeneration of corneal nerves is of limited success in the field of ophthalmology. This work reports the use of a non-invasive electrical stimulation technique that uses a transparent graphene-based corneal stimulation electrode and that can achieve efficient regeneration of corneal nerves. The corneal stimulation electrode is prepared using electroactive nitrogen-containing conducting polymers such as polyaniline functionalized graphene (PAG). This composite can carry a high capacitive current. It can be used to tune transmembrane signaling pathways including calcium channels and the MAPK signaling pathway. Tuning can lead to the efficient regeneration of corneal damaged nerves after the surgery of laser in-situ keratomileusis (LASIK). The composite and its application reported have the potential to provide a new way to treat nerve-related injuries.

Antiseizure Effects of Fully Characterized Non-Psychoactive Cannabis sativa L. Extracts in the Repeated 6-Hz Corneal Stimulation Test.

Compounds present in Cannabis sativa L. preparations have recently attracted much attention in the treatment of drug-resistant epilepsy. Here, we screened two olive oil extracts from a non-psychoactive C. sativa variety, fully characterized by high-performance liquid chromatography and gas chromatography. Particularly, hemp oils with different concentrations of terpenes were administered at the same dose of cannabidiol (25 mg/kg/day orally), 1 h before the 6-Hz corneal stimulation test (44 mA). Mice were stimulated once a day for 5 days and evaluated by video-electrocorticographic recordings and behavioral analysis. Neuronal activation was assessed by FosB/ΔFosB immunoreactivity. Both oils significantly reduced the percentage of mice experiencing convulsive seizures in comparison to olive oil-treated mice (p < 0.050; Fisher’s exact test), but only the oil enriched with terpenes (K2) significantly accelerated full recovery from the seizure. These effects occurred in the presence of reduced power of delta rhythm, and, instead, increased power of theta rhythm, along with a lower FosB/ΔFosB expression in the subiculum (p < 0.050; Duncan’s method). The overall findings suggest that both cannabinoids and terpenes in oil extracts should be considered as potential therapeutic agents against epileptic seizures and epilepsy.

Instillation of Ophthalmic Formulation Containing Nilvadipine Nanocrystals Attenuates Lens Opacification in Shumiya Cataract Rats.

We developed ophthalmic formulations based on nilvadipine (NIL) nanocrystals (NIL-NP dispersions; mean particle size: 98 nm) by using bead mill treatment and investigated whether the instillation of NIL-NP dispersions delivers NIL to the lens and prevents lens opacification in hereditary cataractous Shumiya cataract rats (SCRs). Serious corneal stimulation was not detected in either human corneal epithelial cells or rats treated with NIL-NP dispersions. The NIL was directly delivered to the lens by the instillation of NIL-NP dispersions, and NIL content in the lenses of rats instilled with NIL-NP dispersions was significantly higher than that in the ophthalmic formulations based on NIL microcrystals (NIL-MP dispersions; mean particle size: 21 µm). Moreover, the supply of NIL prevented increases in Ca2+ content and calpain activity in the lenses of SCRs and delayed the onset of cataracts. In addition, the anti-cataract effect in the lens of rats instilled with NIL-NP dispersions was also significantly higher than that in NIL-MP dispersions. NIL-NPs could be used to prevent lens opacification.

Xanthotoxin enhances the anticonvulsant potency of levetiracetam and valproate in the 6-Hz corneal stimulation model in mice.

Xanthotoxin (8-methoxypsoralen; XANT) is a furanocoumarin that has many biological properties, including antiepileptic activity. This study evaluated the effect of XANT on the ability of classical and novel antiepileptic drugs to prevent seizures evoked by the 6-Hz corneal stimulation-induced seizure model, which is thought to be an experimental model of psychomotor (limbic) seizures in humans. XANT (50 mg/kg, administered i.p.) significantly potentiated the anticonvulsant activity of levetiracetam and valproate, decreasing their median effective dose (ED50 ) values from 19.37 to 2.83 mg/kg (P < 0.01) for levetiracetam and from 92.89 to 44.44 mg/kg (P < 0.05) for valproate. Neither XANT (50 mg/kg) alone nor its combination with the anticonvulsant drugs (at their ED50 values from the 6-Hz test) affected motor coordination; skeletal muscular strength and long-term memory, as determined in the chimney; and grip strength and passive avoidance tests, respectively. Measurement of total brain antiepileptic drug concentrations revealed that XANT (50 mg/kg) had no impact on levetiracetam total brain concentrations, indicating the pharmacodynamic nature of interaction between these antiepileptic drugs in the mouse 6-Hz model. However, XANT (50 mg/kg, i.p.) significantly increased total brain concentrations of valproate (P < 0.01), indicating the pharmacokinetic nature of interactions between drugs. XANT in combination with levetiracetam exerts beneficial anticonvulsant pharmacodynamic interactions in the 6-Hz mouse psychomotor seizure model.

Accommodative response to ocular surface pain

ABSTRACT Clinical relevance The discovery of an accommodative response to ocular surface stimulation could inform clinicians and patients that optical effects may occur due to ocular discomfort and perhaps an assessment of the accommodative system after carrying out interventions impacting the ocular surface, may be warranted. Background There have been no previous reports evaluating the effect of noxious stimulation on accommodation. Here, the accommodative response of healthy participants after the application of noxious corneal stimulation is characterised. Methods A computerised Belmonte pneumatic esthesiometer was used to determine detection thresholds (using ascending method of limits), and to randomly deliver mechanical and chemical stimuli from levels of detection threshold to twice the threshold in 50% steps, to the central cornea of 15 healthy subjects. For each suprathreshold stimulus, accommodative and pupil responses were measured with a validated eccentric infrared photorefractor. Quantitative differences in accommodative/pupil response, stimulus modality/intensity and left/right eye were analysed using repeated measures ANOVA. Tukey HSD tests were used for all post hoc analyses. Results Accommodation increased from baseline as the corneal apical stimulus intensity increased. This happened regardless of whether mechanical or chemical stimulation occurred (ANOVA, p < 0.05). At 200% threshold, accommodative response was greater than all stimulus intensities (Tukey HSD, all p < 0.05). There was no difference in pupil response between the stimulation intensities (100%, 150% and 200% threshold). There was no difference in accommodative response between the left and right eye for mechanical (ANOVA, p > 0.05) and chemical stimulation (ANOVA, p > 0.05). Conclusion Noxious stimulation of the cornea seems to produce a dose-dependent increase in the accommodative response in the eyes but not a dose-dependent pupil response.

Thymic stromal lymphopoietin participates in the TLR2-and TLR4-dependent immune response triggered by Aspergillus fumigatus in human corneal cells

Fungal keratitis constitutes a serious vision-threatening disease. Toll-like receptors (TLRs) comprise key mediators of innate immunity triggered by Aspergillus fumigatus (AF) in the cornea, but the messenger between innate and adaptive immunity remained unknown. Thymic stromal lymphopoietin (TSLP) represents a critical factor of adaptive immunity. Here we investigated the expression of TSLP in corneal epithelial and stromal cells challenged by AF and its relationship with TLRs. We stimulated corneal cells with TLR ligands zymosan or lipopolysaccharide (LPS), human recombinant TSLP, or AF hyphae for various periods, with or without prior TLR2, TLR4, or TSLP inhibition. TLR2, TLR4, TSLP, IL-8, and TNF-α release and expression were measured via enzyme-linked immunosorbent analysis, quantitative polymerase chain reaction, or western blot. Corneal cell stimulation with zymosan or LPS induced up-regulated TSLP expression. Enhanced TSLP expression was associated with AF treatment in human corneal cells; TLR2 or TLR4 inhibition impaired the AF-induced TSLP levels. Human recombinant TSLP augmented TLR2 and TLR4 expression; RNA interference of TSLP attenuated TLR, IL-8, and TNF-α expression stimulated by AF hyphae. These findings indicated that TSLP participates in the immune response of corneal cells triggered by AF, which is closely related to TLR function, and the innate immunity mediated by TLRs could be enhanced by TSLP. Innate immunity may therefore transmit inflammatory signals to adaptive immunity through activation of TSLP; in turn, adaptive immunity likely exerts certain regulatory effects on innate immunity via TSLP. That is, TSLP could interact with innate immunity mediated by TLR2 and TLR4 in human corneal cells challenged by AF and thus may serve as a messenger between the innate and adaptive immune responses in AF keratitis.

Cannabidiolic acid exhibits entourage-like improvements of anticonvulsant activity in an acute rat model of seizures

ObjectivesCannabidiolic acid (CBDa) is pharmacologically unique from cannabidiol (CBD), but its chemical instability poses challenges for potential clinical utility. Here, we used magnesium ions to stabilize two cannabidiolic acid-enriched hemp extracts (Mg-CBDa and Chylobinoid, the latter of which also contains minor cannabinoid constituents) and compared their anticonvulsant activities with CBD in the maximal electroshock seizure test (MES) in rats. MethodsSprague-Dawley rats received intraperitoneal (i.p.) injections of Chylobinoid, Mg-CBDa, or CBD at varying doses at discrete time points. Rats were challenged with a 0.2 s, 60 Hz, 150 mA corneal stimulation and evaluated for resultant hindlimb tonic extension. Dose-response relationships were calculated using Probit analysis and statistical significance was assessed with a two-sample z-test. ResultsMedian effective doses (ED50) and 95 % confidence intervals were calculated for each compound and adjusted according to percentage of CBDa (w/w): Chylobinoid: 76.7 (51.7–109.2) mg/kg. Mg-CBDa: 115.4 (98.8–140.9) mg/kg. CBD: 68.8 (56.6–80.0) mg/kg. SignificanceCBDa-enriched hemp extracts exhibited dose-dependent protection in the MES model at doses comparable, but not more effective than, CBD. Chylobinoid was more effective than Mg-CBDa despite lower CBDa content. Test compounds should be compared by sub-chronic dosing in the MES test in order to assess safety and pharmacokinetic profiles. CBDa should be evaluated in pharmacoresistant and chronic animal models of epilepsy.

Molecular, physiological and behavioral characterization of the heterozygous Df[h15q13]/+ mouse model associated with the human 15q13.3 microdeletion syndrome

The human 15q13.3 microdeletion syndrome (DS) is caused by a heterozygous microdeletion (MD) affecting six genes: FAN1; MTMR10; TRPM1; KLF13; OTUD7A; and CHRNA7. Carriers are at risk for intellectual disability, epilepsy, autism spectrum disorder, and schizophrenia. Here we used the Df[h15q13]/+ mouse model with an orthologous deletion to further characterize molecular, neurophysiological, and behavioral parameters that are relevant to the 15q13.3 DS. First, we verified the expression and distribution of the α7 nicotinic acetylcholine receptor (nAChR), a gene product of the CHRNA7, in cortical and subcortical areas. Results revealed similar mRNA distribution pattern in wildtype (WT) and heterozygous (Het) mice, with about half the number of α7 nAChR binding sites in mutants. Hippocampal recordings showed similar input/output responses of field excitatory post-synaptic potentials and theta-burst induced long-term potentiation in WT and Het mice. Het males exhibited impaired spatial learning acquisition in the Barnes Maze. Indicative of increased seizure susceptibility, Het mice developed secondary seizures after 6-Hz corneal stimulation, and had significantly increased sensitivity to the chemoconvulsant pentylenetetrazol resulting in increased spiking in hippocampal EEG recordings. Basal mRNA expression of brain derived neurotrophic factor and activity regulated immediate early genes (c-fos, Arc, Erg-1 and Npas4) during adolescence, a critical period of brain maturation, was unaffected by genotype. Thus, the MD did not show gross neuroanatomical, molecular, and neurophysiological abnormalities despite deficits in spatial learning and increased susceptibility to seizures. Altogether, our results verify the phenotypic profile of the heterozygous Df[h15q13]/+ mouse model and underscore its translational relevance for human 15q13.3 DS.

Pupil response to noxious corneal stimulation.

PurposeOcular somatosensory-autonomic reflexes play critical roles in maintaining homeostasis of the eye. The purpose of this study was to investigate the pupil response to nociceptive corneal stimuli.MethodsA Waterloo-Belmonte pneumatic esthesiometer was used to determine detection thresholds and randomly deliver mechanical and chemical stimuli from levels of detection threshold to twice the threshold in 50% steps to the central cornea of 15 healthy subjects. For each stimulus, imaging of the stimulated/unstimulated eye was performed using two modified/calibrated Logitech c920 digital cameras for 4 seconds each, pre/post stimulus capture. The data were processed with a custom segmentation algorithm to help identify the pupils and pupil diameter was measured using ImageJ software. Pupil dilation response differences between the ipsi- and contralateral eye was analyzed using dependent t-tests. The effect of stimulus intensity, modality and sex of subjects were analyzed using repeated measures.ResultsIn mechanical and chemical stimulation experiments, there was no difference in pupil responses between the stimulated eye and the unstimulated eye, (all dependent T-test p > 0.05). On average, pupil diameter increased from baseline as the corneal stimulus intensity increased. This happened regardless of whether mechanical or chemical stimulation occurred (ANOVA p < 0.05). At 200% threshold, pupil diameter was greater than at all stimulus intensities (Tukey HSD, all p < 0.05). Based on stimulus intensity, females had greater pupil diameters than males at levels of 150% threshold and 200% threshold (ANOVA p < 0.05, all Tukey HSD p < 0.05).ConclusionThis study serves as a basis for the characterization of the local stimulus-response neural circuitry relating nociceptive stimuli to autonomic responses and in combination with our work on completely separate autonomic circuits of bulbar conjunctival vessel dilation and reflex tearing suggests that the monotonic measurements of redness, tearing and pupils provide accurate, separable responses that reflect painful stimulus intensity.

Conjunctival Redness Response to Corneal Stimulation.

The conjunctiva is an integral component of the ocular surface, and its vasculature forms a terminal vascular bed of the human internal carotid artery. No research on the response of conjunctival vasculature to ocular surface stimulation exists; however, it is essential to understand the local physiological and pathological responses to such a stimulus. The purpose of this study was to characterize the ocular redness levels in healthy participants after the application of noxious corneal stimulation. A computerized Belmonte pneumatic esthesiometer was used to determine detection thresholds (using ascending method of limits) and to randomly deliver mechanical and chemical stimuli from levels of detection threshold to twice the threshold in 50% steps to the central cornea of 15 healthy subjects. For each suprathreshold stimulus, a spectrophotometer was used to measure ipsilateral and contralateral conjunctival redness before and after delivery of the corneal stimulus. Redness between the stimulated and unstimulated eyes was analyzed using dependent t tests. The effects of stimulus intensity and modality on conjunctival redness were analyzed using repeated-measures analysis of variance (ANOVA). Tukey honestly significant difference tests were used for all post hoc analyses. P ≤ .05 was considered statistically significant. In mechanical and chemical stimulation experiments, the stimulated eye became redder than the unstimulated eye (all t tests, P > .05). On average, redness increased from baseline as the corneal stimulus intensity increased with corneal stimulation (ANOVA, P < .05). Chemical stimulation produced greater conjunctival redness than did mechanical stimulation at all stimulation levels (ANOVA, P < .05; all Tukey honestly significant difference tests, P < .05). Stimulation of the central cornea by noxious mechanical and chemical stimuli evokes a dose-dependent autonomic conjunctival redness response. Chemical stimulation of the cornea seems to evoke a greater response compared with mechanical stimulation. This study serves as a basis for the characterization of the local stimulus-response neural circuitry relating nociceptive ocular surface stimuli to autonomic responses.

Progesterone Application to the Rat Forehead Produces Corneal Antinociception.

PurposeOcular pain and discomfort are the most defining symptoms of dry eye disease. We determined the ability of topical progesterone to affect corneal sensitivity and brainstem processing of nociceptive inputs.MethodsProgesterone or vehicle gel was applied to the shaved forehead in male Sprague Dawley rats. As a site control, gel also was applied to the cheek on the side contralateral to corneal stimulation. Corneal mechanical thresholds were determined using the Cochet-Bonnet esthesiometer in intact and lacrimal gland excision–induced dry eye animals. Eye wipe behaviors in response to hypertonic saline and capsaicin were examined, and corneal mustard oil-induced c-Fos immunohistochemistry was quantified in the brainstem spinal trigeminal nucleus.ResultsProgesterone gel application to the forehead, but not the contralateral cheek, increased corneal mechanical thresholds in intact and lacrimal gland excision animals beginning <30 minutes after treatment. Subcutaneous injection of the local anesthetic bupivacaine into the forehead region before application of progesterone prevented the increase in corneal mechanical thresholds. Furthermore, progesterone decreased capsaicin-evoked eye wipe behavior in intact animals and hypertonic saline evoked eye wipe behavior in dry eye animals. The number of Fos-positive neurons located in the caudal region of the spinal trigeminal nucleus after corneal mustard oil application was reduced in progesterone-treated animals.ConclusionsResults from this study indicate that progesterone, when applied to the forehead, produces analgesia as indicated by increased corneal mechanical thresholds and decreased nociceptive responses to hypertonic saline and capsaicin.

Corneomandibular reflex in a patient with pontine hemorrhage without impaired consciousness.

A 40-year-old man was admitted because of sudden loss of consciousness. CT scan showed a bilateral pontine hemorrhage, predominantly right (figure). Neurologic examination 19 days after onset showed ophthalmoplegia, anarthria, and tetraparesis. A prominent unilateral left corneomandibular reflex was observed and the patient was conscious and responsive to the examiner (video 1). This reflex consists of contralateral jaw deviation produced by corneal stimulation and has mostly been described in patients in coma.1 It helps to distinguish structural from metabolic processes in comatose patients. We emphasize that this sign can also be elicited in alert patients with destructive brainstem damage.

Lamotrigine-resistant corneal-kindled mice: A model of pharmacoresistant partial epilepsy for moderate-throughput drug discovery.

Despite numerous treatments for epilepsy, over 30% of patients remain resistant to available antiseizure drugs (ASDs). Thus, there is a strong need for more effective ASDs for these individuals. Early ASD discovery has historically relied on acute invivo seizure models (maximal electroshock, subcutaneous pentylenetetrazol, 6Hz), which lack the pathophysiology that defines chronic epilepsy. Etiologically relevant rodent models of pharmacoresistant epilepsy exist (eg, phenytoin (PHT)- and lamotrigine (LTG)-resistant amygdala-kindled rat and focal kainic acid mouse), but these models are resource- and labor-intensive and thus unsuitable for frontline ASD discovery. We adapted the LTG-resistant amygdala-kindled rat protocol to the 60Hz corneal-kindled mouse (CKM) to develop a medium-throughput model of pharmacoresistant chronic seizures. Male CF-1 mice were administered either vehicle (VEH; 0.5% methylcellulose) or LTG (8.5mg/kg, ip) 30minutes prior to each twice-daily corneal stimulation until mice achieved kindling criterion. Prototype ASDs were then evaluated in fully kindled mice. Compounds with specific mechanisms of action of interest to epilepsy (fluoxetine, minocycline, and celecoxib) were also evaluated. LTG did not modify kindling acquisition. A challenge dose of 17mg/kg (ip) LTG did not block the secondarily generalized kindled seizure in LTG-kindled mice (mean seizure score [MSS]±standard error of the mean: 5.67±0.14), whereas VEH-treated mice were sensitive (MSS: 2.25±0.30); confirming LTG-resistance. LTG-resistant CKM were also resistant to carbamazepine, retigabine, and valproic acid at doses that significantly reduced MSS in VEH-treated kindled mice. Fluoxetine, minocycline, and celecoxib were ineffective at the doses tested in either kindled cohort. Finally, the behavioral phenotype of LTG-resistant CKM was also characterized. CKM demonstrated exacerbated hyperexcitability and increased anxiety-like behavior in an open field relative to sham-kindled mice regardless of LTG sensitivity. The pharmacoresistant LTG-resistant CKM provides an etiologically relevant moderate-throughput platform that is suitable for early compound discovery before advancing to more resource-intensive models of epilepsy.

Evaluation of anticonvulsant and analgesic activity of new hybrid compounds derived from N-phenyl-2-(2,5-dioxopyrrolidin-1-yl)-propanamides and –butanamides

Epilepsy is a chronic neurological disorder that is associated with various types of recurrent seizures, which are drug-resistant in about one third of patients. Moreover, anticonvulsant drugs are used to treat a wide range of non-epileptic conditions, including chronic pain. Here, we investigated the anticonvulsant activity of six new hybrid compounds based on the pyrrolidine-2,5-dione scaffold in the 6 Hz corneal stimulation test with 44 mA stimulus intensity in mice, which is the model of pharmacoresistant seizures. We demonstrated that two molecules, DK-10 (11) and DK-14 (14) show higher anticonvulsant activity and similar safety profile in comparison with valproic acid and much higher in comparison with levetiracetam in the aforementioned test. The second aim of this study was to examine analgesic activity of these compounds. For this purpose, the hot plate test, the formalin test, and the oxaliplatin-induced peripheral neuropathy model were performed. Among tested agents DK-11 (12) revealed prominent antinociceptive activity at non-sedative doses in the second (inflammatory) phase of the formalin test, which is the model of tonic pain and antiallodynic activity in the oxaliplatin-induced neuropathic pain, the model of painful chemotherapy-induced peripheral neuropathy. No cytotoxic effect on hepatoma cells was observed. Compound DK-10 (11) had high affinity for voltage-gated sodium channels, whereas compound DK-11 (12) showed weak binding toward sodium and calcium voltage-gated channels and the NMDA receptor. As a result, hybrid compounds reported herein seem to be very promising broad spectrum anticonvulsant molecules with collateral analgesic activity.