Corneal Stimulation Research Articles

Efficacy of the ketogenic diet in the 6‐Hz seizure test

Since the ketogenic diet is effective in drug-resistant epilepsies, we sought to determine whether it is active in the 6-Hz seizure test, which identifies agents with a broader spectrum of activity than conventional antiepileptic screening tests. Male (3-4 week old) NIH Swiss mice were fed a normal or ketogenic diet ad libitum for 2-21 days. The intensity of the corneal stimulation current required to elicit seizures in the 6-Hz test was measured. Blood glucose and beta-hydroxybutyrate were measured on the day of seizure testing. CC(50) (current intensity producing seizures in 50% of mice tested) was 50.6 mA and 15 mA in mice fed for 12 days with a ketogenic or normal diet, respectively (p < 0.001). CC(50) was elevated in separate experiments after 16, but not 2, 5, and 21 days of ketogenic diet exposure. CC(50) values of growing mice fed the normal diet does not differ, indicating CC(50) does not vary with mouse weight during a rapid growth phase. beta-Hydroxybutyrate was significantly higher, and glucose was significantly lower in mice fed the ketogenic diet than those fed the normal diet. Blood glucose and beta-hydroxybutyrate levels did not correlate with CC(50). The ketogenic diet significantly elevates the seizure threshold in the 6-Hz test in a time-specific manner. Protection from seizures in this model was not related to level of ketosis. CC(50) was insensitive to body weight in mice fed the normal diet, demonstrating that the 6-Hz model can assess anticonvulsant regimens where weight is a confounding factor.

The three-neuron corneal reflex circuit and modulation of second-order corneal responsive neurons

Neurons located in the border region between the interpolaris and caudalis subdivisions of the spinal trigeminal nucleus (Vi/Vc) are second order neurons of the corneal reflex, receiving corneal afferents and projecting to the lid closing, orbicularis oculi (OO) motoneurons. Recordings of Vi/Vc neurons identified by antidromic activation from stimulation of the facial nucleus and non-identified Vi/Vc neurons reveal two neuron types, phasic and tonic. Corneal stimulation elicits Adelta latency action potentials that occur early enough to initiate OO contraction and C-fiber latency action potentials that can modulate the end of the blink in phasic Vi/Vc neurons. Tonic Vi/Vc neurons exhibit a constant irregular, low frequency discharge as well as the cornea-evoked activity exhibited by phasic neurons. For both phasic and tonic neurons, blink amplitude increases with the total number of spikes evoked by the corneal stimulus. Peak firing frequency predicts peak orbicularis oculi EMG activity. Paradigms that suppress cornea-evoked blinks differentially affect Vi/Vc neurons. Microstimulation of the border region between the spinal trigeminal caudalis subdivision and the C1 spinal cord (Vc/C1) significantly reduces the number of spikes evoked by corneal stimulation and suppresses blink amplitude. In the paired stimulus paradigm, a blink evoked by a corneal stimulus 150 ms after an identical corneal stimulus is significantly smaller than the blink elicited by the first stimulus. Vi/Vc neuron discharge, however, is slightly larger for the second blink. Our data indicate that second-order Vi/Vc neurons do not determine the specific pattern of OO muscle activity; rather Vi/Vc neurons initiate OO motoneuron discharge and program the activity of another circuit that generates the late phase of the blink. The Vc/C1 suppression of Vi/Vc neurons suggests that the Vc/C1 region provides an "internal model" of the intended blink.

Increased Levels of Diadenosine Polyphosphates in Dry Eye

To analyze the levels of the diadenosine polyphosphates Ap4A and Ap5A in tears, in a set of control subjects and in groups of symptomatic and nonsymptomatic persons with dry eye. Ninety-seven subjects participated in the study. The subjects were divided into five experimental groups: control subjects; symptomatic patients with normal tear secretion; symptomatic patients with low tear secretion; forced blink; and corneal mechanical stimulation provided by a gas esthesiometer. The Schirmer I test was used to measure and collect tear secretions from each subject. All samples were processed by high pressure liquid chromatography (HPLC) and their Ap4A and Ap5A levels determined. The levels of Ap4A and Ap5A in tears were greater in all symptomatic patients than in control subjects, especially in symptomatic subjects with low tear secretion. Within the symptomatic subjects with normal tear secretion, significant differences in concentrations of Ap4A and Ap5A were found between men and women. In the forced blink experiments, concentrations of the Ap4A and Ap5A rose with increasing blink frequency. When the cornea was mechanically stimulated, the levels of Ap4A and Ap5A rose significantly during both moderate and high-flow rate tests. The increased levels of Ap4A and Ap5A in tears of patients with dry eye allow these dinucleotides to be used as objective biomarkers in dry eye conditions.

Influence of age, gender and iris color on mechanical and chemical sensitivity of the cornea and conjunctiva

The purpose of this study was to establish the influence of age, gender and iris color on the mechanical and chemical sensitivity of the cornea and the conjunctiva. In 57 healthy subjects (27 males, 30 females; ages between 23 and 71 years), sensory thresholds to mechanical and chemical stimulation were measured in the central cornea and the temporal conjunctiva using a Belmonte's gas esthesiometer. Mechanical stimulation consisted of warmed air pulses of 3s duration at different flow rates (40-200 ml/min). For chemical (acidic) stimulation, 3-s warmed gas pulses containing 10% to 80% CO(2) in air were applied, at a flow below mechanical threshold flow. Corneal and conjunctival thresholds to mechanical and chemical stimuli increased with age. Premenopausal women were more sensitive to corneal stimulation than men of similar ages but overall differences in mechanical and chemical threshold between men and women were not significant. Individuals with blue eyes had significantly lower corneal chemical thresholds than those with brown or green eyes. Multiple linear regression analysis evidenced that corneal mechanical threshold depends on age and iris color according to the equation 58.2 ml/min+1.3.(Age-23 years)-9.7 if blue eyes. For chemical threshold, the equation was 28.6%CO(2)+05.(Age-23 years)-12.5 if blue eyes. In the case of the conjunctiva, only age determined mechanical and chemical thresholds according to the equations: 78.2 ml/min+1.4.(Age-23 years) and 41.6%CO(2)+0.8.(Age-23 years), respectively. Therefore, normal values of mechanical and chemical thresholds of the cornea and conjunctiva measured with the Belmonte gas esthesiometer can be predicted according to age and iris color. Variations of sensitivity with age, iris color and gender may reflect differences in innervation density and neural responsiveness associated with the hormonal status.

The Inhibitory Interaction between Human Corneal and Conjunctival Sensory Channels

To explore human corneal and conjunctival sensory channels at suprathreshold level. Ten healthy human subjects participated in the study. The Belmonte pneumatic esthesiometer was used to apply mechanical and chemical stimuli to the central cornea and temporal conjunctiva of the left eye. Stimuli were applied in a paired and unpaired way for conjunctival stimulation. A 100-point visual analog scale (VAS) was used to rate the intensity of the stimulus. The magnitudes of the sensation evoked from the conjunctiva were different when using different methods for presenting stimuli to the ocular surface. When stimuli were applied to the conjunctiva alone, the magnitude of the sensation was stronger than when the stimuli were applied in pairs to the cornea and conjunctiva for both mechanical (P = 0.04) and chemical (P = 0.02) stimulation. The relatively strong discomfort evoked from the cornea appears to suppress partially the relatively weaker conjunctival stimulation. This manifested as the conjunctival sensory transducer function being shallower (less intense sensation) when immediately preceded by corneal stimulation than when the conjunctival sensory transducer functions were measured alone (unpaired). The underlying mechanism could be adaptation or some other inhibitory effect, such as diffuse noxious inhibitory control. At some level therefore, corneal and conjunctival sensory channels are not independent.

Changes in Mechanical, Chemical, and Thermal Sensitivity of the Cornea after Topical Application of Nonsteroidal Anti-inflammatory Drugs

In addition to their well-known anti-inflammatory actions, some of the nonsteroidal anti-inflammatory drugs (NSAIDs) appear to have an analgesic effect. In human subjects, the changes in threshold and intensity of sensations evoked by mechanical, chemical, and thermal stimulation of the cornea induced by topical administration of two commercial NSAIDs, diclofenac sodium (Voltaren; Novartis, Basel, Switzerland) and flurbiprofen (Ocuflur; Allergan, Irvine, CA), were studied. Corneal sensitivity was measured in 10 young, healthy subjects with a gas esthesiometer. Chemical (10%-70% CO2 in air), mechanical (0-264 mL/min), and thermal (corneal temperature changes between -4.5 degrees C and +3 degrees C around the normal value) stimuli were applied to the center of the cornea. The intensity and perceived magnitude of the psychophysical attributes of the evoked sensation were scored at the end of the pulse in a 10-cm, continuous visual analog scale (VAS). The threshold was expressed as the stimulus intensity that evoked a VAS score >0.5. Sensitivity was measured in both eyes of each subject on two separate days, one without treatment and the other 30 minutes after topical application of 0.03% flurbiprofen (seven subjects) or 0.1% diclofenac sodium (six subjects). Diclofenac attenuated significantly all the sensation parameters evoked by high-intensity mechanical, chemical, and thermal stimuli. Flurbiprofen produced a slight reduction of the sensations evoked by mechanical and chemical stimulation that became significant only for the irritation caused by chemical stimuli of maximum intensity (70% CO2). None of the drugs modified significantly the detection threshold of the different stimuli. Flurbiprofen had a very limited effect on sensations evoked by corneal stimulation, whereas diclofenac reduced the intensity of sensations evoked by stimuli of different modality, suggesting a mild local anesthetic effect of this drug on all types of corneal sensory fibers. Such anesthetic action could explain the analgesic effect that has been reported after topical application of diclofenac in inflamed human eyes.

Activation of 5-HT(1B/1D) receptor in the periaqueductal gray inhibits nociception.

It is considered that the site of action of the abortive antimigraine compounds acting at serotonin, 5-HT(1B/1D,) receptors (triptans) is the trigeminovascular system. We tested whether there is a non-trigeminal site of action. The 5-HT(1B/1D) agonist, naratriptan, was microinjected into the ventrolateral periaqueductal gray (vlPAG), and activity in the trigeminal nucleus caudalis (TNC) was monitored. Recordings were made from 20 nociceptive neurons in the dorsal horn of the TNC that received convergent input from the dura mater and face. Responses of neurons to dural, facial cutaneous and corneal stimulation were studied before and after injection of naratriptan. Naratriptan decreased the excitability to electrical stimulation of the dura mater as the A-fiber response decreased by 24 +/- 4.1% (p < 0.001) and the C-fiber response decreased by 42 +/- 8.2% (p < 0.001). Spontaneous activity was decreased by 38 +/- 7.5% (p < 0.001). After injection, the mechanical thresholds of the dura mater increased from (n = 14, p < 0.01). Responses to stimulation of the face and cornea were not altered by injection of naratriptan. These results suggest that 5-HT(1B/1D) receptor activation in the vlPAG activates descending pain-modulating pathways that inhibit dural, but not facial and corneal nociceptive input. These findings have implications for the understanding of the action of triptans in migraine and cluster headache, suggesting that brain loci other than the trigeminal nucleus may play a role in the clinical action of triptans.

Allopregnanolone analogs that positively modulate GABA receptors protect against partial seizures induced by 6-Hz electrical stimulation in mice.

Low-frequency (6 Hz), long-duration (3 s) electrical stimulation in mice produces seizures characterized by immobility, focal clonus, and automatic behaviors reminiscent of human limbic epilepsy. Renewed interest has been expressed in this seizure model with the recognition that it is sensitive to a broad spectrum of anticonvulsants (AEDs) and may have distinct pharmacologic responsiveness from other in vivo tests of AED efficacy. Here we sought to determine whether the progesterone-derived neuroactive steroid allopregnanolone (5alpha,3alpha-P) and several structural analogues with varying degrees of activity as positive allosteric modulators of gamma-aminobutyric acid (GABA)A receptors are protective in the 6-Hz seizure model. Mice were pretreated with neuroactive steroids (15 min before) or clonazepam (CZP; 30 min before) to 6-Hz corneal stimulation (32 mA). Animals that failed to exhibit immobility were considered protected. The neuroactive steroids prevented 6-Hz seizures with rank order of potencies (ED50 values): ganaxolone (6.3 mg/kg) > 5alpha,3alpha-P (14.2 mg/kg) > or = 5beta,3alpha-P (14.4 mg/kg) > 5alpha,3beta-P (>100 mg/kg). CZP also was protective (ED(50) value, 0.075 mg/kg). The potencies of the neuroactive steroids and CZP are similar to their previously reported activities in the pentylenetetrazol (PTZ) seizure model. Neuroactive steroids have comparable potencies in the 6-Hz and PTZ models. Their structural specificity in both models corresponds with their activities as positive allosteric modulators of GABAA receptors, although ganaxolone is more potent than expected, probably because it has greater bioavailability. The 6-Hz model may be a valuable tool in drug development for the identification of GABAergic AEDs.

Transcorneal stimulation of trigeminal nerve afferents to increase cerebral blood flow in rats with cerebral vasospasm: a noninvasive method to activate the trigeminovascular reflex.

The goal of this study was to investigate whether stimulation of trigeminal afferents in the cornea could enhance cerebral blood flow (CBF) in rats after they have been subjected to experimental subarachnoid hemorrhage (SAH). Cerebral vasospasm following SAH may compromise CBF and increase the risks of morbidity and mortality. Currently, there is no effective treatment for SAH-induced vasospasm. Direct stimulation of the trigeminal nerve has been shown to dilate constricted cerebral arteries after SAH; however, a noninvasive method to activate this nerve would be preferable for human applications. The authors hypothesized that stimulation of free nerve endings of trigeminal sensory fibers in the face might be as effective as direct stimulation of the trigeminal nerve. Autologous blood obtained from the tail artery was injected into the cisterna magna of 10 rats. Forty-eight and 96 hours later (five rats each) trigeminal afferents were stimulated selectively by applying transcorneal biphasic pulses (1 msec, 3 mA, and 30 Hz), and CBF enhancements were detected using laser Doppler flowmetry in the territory of the middle cerebral artery. Stimulation-induced changes in cerebrovascular parameters were compared with similar parameters in sham-operated controls (six rats). Development of vasospasm was histologically verified in every rat with SAH. Corneal stimulation caused an increase in CBF and blood pressure and a net decrease in cerebrovascular resistance. There were no significant differences between groups for these changes. Data from the present study demonstrate that transcorneal stimulation of trigeminal nerve endings induces vasodilation and a robust increase in CBF. The vasodilatory response of cerebral vessels to trigeminal activation is retained after SAH-induced vasospasm.

Integration of cornea and cardiorespiratory afferents in the nucleus of the solitary tract of the rat.

We determined the activity of neurons within the nucleus of the solitary tract (NTS) after stimulation of the cornea and assessed whether this input affected the processing of baroreceptor and peripheral chemoreceptor inputs. In an in situ, unanesthetized decerebrate working heart-brain stem preparation of the rat, noxious mechanical or electrical stimulation was applied to the cornea, and extracellular single unit recordings were made from NTS neurons. Cornea nociceptor stimulation evoked bradycardia and an increase in the cycle length of the phrenic nerve discharge. Of 90 NTS neurons with ongoing activity, corneal stimulation excited 51 and depressed 39. There was a high degree of convergence to these NTS neurons from either baroreceptors or chemoreceptors. The excitatory synaptic response in 12 of 19 baroreceptive and 10 of 15 chemoreceptive neurons was attenuated significantly during concomitant electrical stimulation of the cornea. This inhibition was GABA(A) receptor mediated, being blocked by pressure ejection of bicuculline. Thus the NTS integrates information from corneal receptors, some of which converges onto neurons mediating reflexes from baroreceptors and chemoreceptors to inhibit these inputs.

Pharmacological characterization of the 6 Hz psychomotor seizure model of partial epilepsy

Originally described as a model of ‘psychomotor seizures’ (J. Pharmacol. Exp. Ther. (1953) 107–273), the 6 Hz corneal stimulation model was abandoned shortly after its description because of its lack of sensitivity to phenytoin. This observation is the basis for the present study designed to validate the 6 Hz seizure as a model of therapy-resistant epilepsy. The pharmacological profile of the 6 Hz seizure was determined at varying current intensities using seven established AEDs (phenytoin, carbamazepine, clonazepam, phenobarbital, ethosuximide, trimethadione, valproic acid) and five second-generation AEDs (lamotrigine, levetiracetam, felbamate, tiagabine, topiramate). The immediate early gene c-Fos was used as a marker of seizure-induced neuronal activation to help define those brain structures that were activated by 6 Hz corneal stimulation. At the current intensity required to produce a seizure in 97% of the population (CC97=22 mA), the 6 Hz seizure did not discriminate between clinical classes of AEDs tested. Increasing the current intensity by 50% (i.e. 32 mA) decreased the sensitivity of the 6 Hz seizure to phenytoin and lamotrigine. At a current intensity of 2×CC97 (i.e. 44 mA), only two AEDs, levetiracetam and valproic acid, displayed complete protection against the 6 Hz seizure, though the efficacy of these drugs was reduced when compared to the lower stimulation intensities. Intense c-Fos staining from 6 Hz seizures induced by 22 and 32 mA stimulus intensities remained localized to the amygdala and piriform cortex. Increasing the stimulus intensity to 44 mA resulted in additional heavy staining of the dentate gyrus. This recruitment of the dentate gyrus may account for the decrease in potency of levetiracetam and valproic acid at 44 mA. The pharmacological results combined with the c-Fos immunohistochemistry suggest that the 6 Hz stimulation may provide a useful model of therapy-resistant limbic seizures.

Corneal stimulation of MMP-1, -9 and uPA by platelet-activating factor is mediated by cyclooxygenase-2 metabolites

Purpose. This study was undertaken to evaluate the significance of cyclooxygenase-2 (COX-2) activity on urokinase plasminogen activator (uPA) and matrix metalloproteinases (MMPs)-1 and -9 induction in cornea following platelet-activating factor (PAF) treatment. Methods. Corneal organ cultures were pre-treated with increasing concentrations of COX-2-specific inhibitors NS398 or nimesulide prior to PAF stimulation. To determine the effect of exogenous prostaglandins (PGs) on uPA, MMP-1 and MMP-9 levels, corneas were pre-treated with COX-2 inhibitors followed by the addition of 2.5 µM PGD 2, PGE 2 or PGF 2a. The levels of uPA and MMP-9 were assayed by casein and gelatin zymography, respectively. MMP-1 levels were determined by Western Blot analysis. Results. The increase in uPA, MMP-9 and MMP-1 levels detected in corneal organ cultures treated with 100 nM cPAF was blocked by 5 µM NS398 and 10 µM nimesulide, concentrations at which these inhibitors selectively inhibit COX-2 activity. Furthermore, pre-incubation with COX-2 inhibitors, followed by supplementation with PGD 2, PGE 2 or PGF 2a, increases uPA, MMP-9 and MMP-1 levels in corneas similar to and in some cases greater than that produced by cPAF treatment alone. Conclusions. During corneal injury and inflamation, PAF is an important factor in the activation of proteolytic cascades, which could lead to corneal epithelial defects and ultimately ulceration. One important goal in treating these defects is to modulate the activity of enzymes that destroy the extracellular matrix. Our results suggest that COX-2 induction following PAF stimulation and subsequent eicosanoid release may play a crucial role in the induction of uPA, MMP-1 and MMP-9 enzymes. Specific COX-2 inhibition could therefore block the actions of PAF when inflammation is sustained.

Responses of medullary dorsal horn neurons to corneal stimulation by CO(2) pulses in the rat.

Corneal-responsive neurons were recorded extracellularly in two regions of the spinal trigeminal nucleus, subnucleus interpolaris/caudalis (Vi/Vc) and subnucleus caudalis/upper cervical cord (Vc/C1) transition regions, from methohexital-anesthetized male rats. Thirty-nine Vi/Vc and 26 Vc/C1 neurons that responded to mechanical and electrical stimulation of the cornea were examined for convergent cutaneous receptive fields, responses to natural stimulation of the corneal surface by CO(2) pulses (0, 30, 60, 80, and 95%), effects of morphine, and projections to the contralateral thalamus. Forty-six percent of mechanically sensitive Vi/Vc neurons and 58% of Vc/C1 neurons were excited by CO(2) stimulation. The evoked activity of most cells occurred at 60% CO(2) after a delay of 7-22 s. At the Vi/Vc transition three response patterns were seen. Type I cells (n = 11) displayed an increase in activity with increasing CO(2) concentration. Type II cells (n = 7) displayed a biphasic response, an initial inhibition followed by excitation in which the magnitude of the excitatory phase was dependent on CO(2) concentration. A third category of Vi/Vc cells (type III, n = 3) responded to CO(2) pulses only after morphine administration (>1.0 mg/kg). At the Vc/C1 transition, all CO(2)-responsive cells (n = 15) displayed an increase in firing rates with greater CO(2) concentration, similar to the pattern of type I Vi/Vc cells. Comparisons of the effects of CO(2) pulses on Vi/Vc type I units, Vi/Vc type II units, and Vc/C1 corneal units revealed no significant differences in threshold intensity, stimulus encoding, or latency to sustained firing. Morphine (0.5-3.5 mg/kg iv) enhanced the CO(2)-evoked activity of 50% of Vi/Vc neurons tested, whereas all Vc/C1 cells were inhibited in a dose-dependent, naloxone-reversible manner. Stimulation of the contralateral posterior thalamic nucleus antidromically activated 37% of Vc/C1 corneal units; however, no effective sites were found within the ventral posteromedial thalamic nucleus or nucleus submedius. None of the Vi/Vc corneal units tested were antidromically activated from sites within these thalamic regions. Corneal-responsive neurons in the Vi/Vc and Vc/C1 regions likely serve different functions in ocular nociception, a conclusion reflected more by the difference in sensitivity to analgesic drugs and efferent projection targets than by the CO(2) stimulus intensity encoding functions. Collectively, the properties of Vc/C1 corneal neurons were consistent with a role in the sensory-discriminative aspects of ocular pain due to chemical irritation. The unique and heterogeneous properties of Vi/Vc corneal neurons suggested involvement in more specialized ocular functions such as reflex control of tear formation or eye blinks or recruitment of antinociceptive control pathways.

Corneal surface temperature change as the mode of stimulation of the non-contact corneal aesthesiometer.

The non-contact corneal aesthesiometer (NCCA) assesses corneal sensitivity by using a controlled pulse of air, directed at the corneal surface. The purpose of this paper was to investigate whether corneal surface temperature change was a component in the mode of stimulation. Thermocouple experiment: A simple model corneal surface was developed that was composed of a moistened circle of filter paper placed on a thermocouple and mounted on a glass slide. The temperature change produced by different stimulus pressures was measured for five different ambient temperatures. Thermal camera experiment: Using a thermal camera, the corneal surface temperature change was measured in nine young, healthy subjects after exposure to different stimulus air pulses. Pulse duration was set at 0.9 s but was varied in pressure from 0.5 to 3.5 millibars. Thermocouple experiment: An immediate drop in temperature was detected by the thermocouple as soon as the air flow was incident on the filter paper. A greater temperature change was produced by increasing the pressure of the incident air flow. A relationship was found and a calibration curve plotted. Thermal camera experiment: For each subject, a drop in surface temperature was detected at each stimulus pressure. Furthermore, as the stimulus pressure increased, the induced reduction in temperature also increased. A relationship was found and a calibration curve plotted. The NCCA air-pulse stimulus was capable of producing a localized temperature change on the corneal surface. The principal mode of corneal nerve stimulation, by the NCCA air pulse, was the rate of temperature change of the corneal surface.

Frontal versus transcorneal stimulation to induce maximal electroshock seizures or kindling in mice and rats.

Frontal stimulation, i.e. electrical stimulation where electrodes are pressed on the skin of the intact frontal skull of mice or rats, may represent a more humane alternative to the widely used transcorneal stimulation to induce electroshock seizures. The aim of this work was to directly compare transcorneal and frontal stimulation in eliciting maximal electroshock-induced seizures (MES) in mice and the anticonvulsant effect of carbamazepine (CBZ) and phenytoin (PHT) on thus produced seizures. In addition, we stimulated mice and rats repeatedly via transcorneal and frontal electrodes to see whether kindling is produced by this procedure. Two electroshock tests were used in mice, i.e. maximal electroshock seizure threshold (MEST) test and MES generated by supramaximal stimulation (50 mA). Frontal stimulation resulted in lower convulsive threshold than in the case of corneal stimulation. Both CBZ and PHT produced dose-dependent increases in seizure threshold for both sites of stimulation, i.e. transcorneal and frontal. As regards type of electrodes, higher doses of PHT were required to increase seizure threshold in the case of frontal than transcorneal stimulation. Supramaximal stimulation (50 mA) yielded comparable ED50 values regardless of the site of stimulation. Furthermore, once-daily stimulation of mice, regardless of the placement of electrodes, did not induce any changes in convulsive threshold. We also attempted to kindle mice and rats via corneal and frontal electrodes by repetitive electrical stimulation using currents which initially did not produce generalized clonic seizures. Mice were stimulated once daily for 2 s with 3 mA (corneal electrodes) or 2 mA (frontal electrodes) and rats were stimulated twice daily for 4 s at 8 mA (corneal electrodes) or 5 mA (frontal electrodes). With corneal stimulation in rats there was a clear progression of kindling development which was not the same in nature when compared with corneally-stimulated mice. Frontal stimulation did not produce kindling. Moreover, corneal stimulation was better tolerated by rats, while in mice high mortality was seen after either method of current delivery. Our data indicate that frontal electrodes can be used as an alternative to transcorneal stimulation to produce MES by supramaximal or threshold current intensities as screening procedures in antiepileptic drug (AED) development. Nevertheless, this type of stimulation cannot be used to produce minimal electroshock seizures and seems not to be useful to produce kindling in rats and mice.

Selective blockade of substance P or neurokinin A receptors reduces the expression of c-fos in trigeminal subnucleus caudalis after corneal stimulation in the rat

Stimulation of the cornea activates neurons in two distinct regions of the spinal trigeminal nucleus: at the transition between trigeminal subnucleus interpolaris and subnucleus caudalis and at the transition between trigeminal subnucleus caudalis and the upper cervical spinal cord as estimated by expression of the immediate early gene, c-fos. To determine if receptors for substance P or neurokinin A, neurokinin 1 and neurokinin 2 receptors, respectively, contribute to the production of Fos-positive neurons in these brainstem regions, receptor-selective antagonists were given intracerebroventricularly 15min prior to stimulation of the cornea in anesthetized rats. The number of Fos-positive neurons produced in superficial laminae at the trigeminal subnucleus caudalis/cervical cord transition by application of the selective small fiber excitant, mustard oil, to the corneal surface was reduced by the neurokinin 1 receptor antagonist, CP99,994 (5–100nmol, i.c.v.) and the neurokinin 2 receptor antagonist, MEN10,376 (0.01–1.0nmol, i.c.v.). Combined pretreatment with CP99,994 and the competitive N-methyl-d-aspartate receptor antagonist, CPP, caused a greater reduction in c-fos expression at the subnucleus caudalis/cervical cord transition than after either drug alone suggesting interaction between receptors for glutamate and substance P. Tachykinin receptor antagonists did not reduce the number of Fos-positive neurons produced at the subnucleus interpolaris/subnucleus caudalis transition. The elevation in plasma concentration of adrenocorticotropin, but not the increases in arterial pressure or heart rate, evoked by corneal stimulation was prevented by pretreatment with CP99,994 or MEN10,376 at doses lower than those needed to reduce c-fos expression.The results indicate that receptors for substance P and neurokinin A contribute to the transmission of sensory input from corneal nociceptors to brainstem neurons in trigeminal subnucleus caudalis and to increased activity of the hypothalamo–pituitary axis that accompanies acute stimulation of the cornea.

Gabapentin Reverses the Allodynia Produced by the Administration of Anti-GD2 Ganglioside, an Immunotherapeutic Drug

Systemically administered, the anti-GD2 antibody produces allodynia demonstrated by decreased mechanical withdrawal threshold. Electrophysiologic recordings indicate a probable neuropathic origin, as small-diameter sensory fibers develop continuous high-frequency discharge after antibody administration. Gabapentin (GBP) is a gamma-aminobutyric acid analog originally synthesized for its anticonvulsant actions. Several open-label clinical studies, as well as a wealth of anecdotal evidence, suggest that GBP may be beneficial for the treatment of neuropathic pain. This study examined the effects of GBP given as a posttreatment after induction of an anti-GD2-associated allodynia. Anti-GD2 (1 mg/kg intravenously [i.v.]) administered to Sprague-Dawley rats reduced the mean withdrawal threshold from 14.71 to 4.95 g (P < 0.001), as measured by using von Frey hairs. This was reversed by GBP in a dose-dependent fashion; the minimal effective dose was between 3 and 30 mg/kg i.v. The maximal percent analgesic effect of GBP was 76% and 93% at doses of 30 and 100 mg/kg, respectively (P < 0.001). With these doses, side effects were minimal and were manifested as slightly decreased spontaneous movement and startle response. No changes were seen in reflex responses to corneal or pinna stimulation, and no motor deficits were observed. These data support the use of GBP as an effective therapy for neuropathic pain. After the administration of anti-GD2 antibody, rats display an escape reaction to light touch, increased blood pressure, and aberrant firing in nerve fibers associated with pain transmission. Systemic gabapentin reduced or eliminated the escape response and reversed the hypertension with minimal side effects. This suggests that gabapentin blocked the antibody-associated (neuropathic) pain.

Sensory denervation leads to deregulated protein synthesis in the lacrimal gland.

The secretory function of the lacrimal gland is influenced by the release of classic and peptide neurotransmitters from sensory, sympathetic, and parasympathetic nerve terminals in the gland. These neuronal pathways constitute an integrated system that regulates the integrity of the front of the eye, particularly the optical qualities of the cornea. In everyday experience, this relationship is evidenced by the rapid tearing response to corneal stimulation. The release of neuromodulators resulting from activation of these pathways leads to receptor activation, mobilization of intracellular second messengers, exocytosis, and synthesis of secretory proteins. Consequently, the processes of cell signaling and synthesis of new secretory material must be regulated in coordination with secretion to maintain the normal exocrine function of the gland.

Morphine and somatostatin analogue reduce c-fos expression in trigeminal subnucleus caudalis produced by corneal stimulation in the rat

The influence of morphine and somatostatin on nociceptor-evoked activation of central trigeminal neurons and cardiovascular reflex responses was assessed in barbiturate-anaesthetized rats. Morphine or the somatostatin analogue, octreotide, was given intracerebroventricularly 20 min prior to application of mustard oil to the corneal surface. The expression of the immediate early gene, c-fos, was used to estimate neuronal activation within the spinal trigeminal nucleus. Morphine reduced the number of Fos-positive neurons produced at the transition region between trigeminal subnucleus caudalis and the upper cervical spinal cord, whereas c-fos expression at the subnucleus interpolaris/caudalis transition was not affected significantly. Morphine also reduced the arterial pressure and heart rate responses to corneal stimulation in proportion to the dose of morphine and required a threshold dose similar to that which reduced c-fos expression. Naloxone prevented the morphine-induced inhibition of c-fos expression and cardiovascular reflex responses to corneal stimulation. Somatostatin analogue reduced the number of Fos-positive neurons at the subnucleus caudalis/cervical cord transition, but not at the subnucleus interpolaris/caudalis transition, an effect that was not prevented by naloxone. Somatostatin analogue did not blunt the cardiovascular responses evoked by corneal stimulation. A subthreshold dose of morphine plus a threshold dose of somatostatin analogue caused a greater inhibition of Fos-positive neurons at the subnucleus caudalis/cervical cord transition, but not in reflex-evoked autonomic responses, than the same dose of either drug alone. Intracerebroventricular administration of morphine and somatostatin analogue inhibit corneal activation of neurons within the superficial laminae at the subnucleus caudalis/cervical cord transition through opioid and non-opioid-dependent neural pathways, respectively. By contrast, the low sensitivity of corneal-responsive neurons at the subnucleus interpolaris/caudalis transition to analgesics suggests that these neurons are not simply a rostral extension of the medullary dorsal horn. Correlation analyses suggest that morphine-induced inhibition of cardiovascular responses to corneal stimulation depend on the activity of neurons at the subnucleus caudalis/cervical cord transition and not on those at the subnucleus interpolaris/caudalis transition region.

Role of cerebellum in adaptive modification of reflex blinks.

We investigated the involvement of the cerebellar cortex in the adaptive modification of corneal reflex blinks and the regulation of normal trigeminal reflex blinks in rats. The ansiform Crus I region contained blink-related Purkinje cells that exhibited a complex spike 20.4 msec after a corneal stimulus and a burst of simple spike activity correlated with the termination of orbicularis oculi activity. This occurrence of the complex spike correlated with trigeminal sensory information associated with the blink-evoking stimulus, and the burst of simple spike activity correlated with sensory feedback about the occurrence of a blink. Inactivation of the inferior olive with lidocaine prevented all complex and significantly reduced simple spike modulation of blink-related Purkinje cells, but did not alter orbicularis oculi activity evoked by corneal stimulation. In contrast, both acute and chronic lesions of the cerebellar cortex containing blink-related Purkinje cells blocked adaptive increases in orbicularis oculi activity of the lid ipsilateral but not contralateral to the lesion. These data are consistent with the hypothesis that the cerebellum is part of a trigeminal reflex blink circuit. Changes in trigeminal signals produce modifications of the cerebellar cortex, which in turn, reinforce or stabilize modifications of brainstem blink circuits. When the trigeminal system does not attempt to alter the magnitude of trigeminal reflex blinks, cerebellar input has little or no effect on reflex blinks.