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Trigeminal Pain Mechanisms

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MODULATION OF EXTEROCEPTIVE SUPPRESSION PERIODS IN THE HUMAN JAW-CLOSING MUSCLES INDUCED BY TOPICAL CAPSAICIN AND TACTILE STIMULATION.

Antonietta Romaniello*. Peter Svensson, Giorgio Cruccu, Lars Arendt-Nielsen. Orofacial Pain Laboratory, Center for Sensory-Motor Interaction, Univ of Aalborg, DK-9200, Denmark and Dept of Neurological Science, Univ "La Sapienza", Rome.

Aim: Convergence of afferent inputs onto brain stem neurons may play an important role for regulation oftrigeminal motor functions. This study examined the efficacy of painful and non-painful peri-oral stimulation for modulation of the two exteroceptive suppression periods (ES1 and ES2) in human jaw-closing muscles.

Methods: Ten healthy subjects (27.3 4.2 yrs) participated. The inhibitory jaw-reflexes, ES1 (15-17 ms onset) and ES2 (45-47 ms onset), were recorded with surface electrodes from masseter and temporalis muscles. Sixteen electrical stimuli were applied to the right mental nerve (0.1 ms pulses) while the subject was biting at 50% of the maximal force. Five conditions were compared: baseline, repetitive tactile stimulation on the right cheek (1 Hz), topical application ofcapsaicin (5%) on the right cheek, repetitive tactile stimulation plus capsaicin, and post-baseline. The perceived intensity of the electrical stimuli were evaluated by the subjects on a 10-cm VAS. For each condition, the 16 traces were averaged and rectified; duration and root-mean-square (RMS) of the ES1 and ES2 were measured as the EMG activity below 50% of the pre-stimulus EMG activity. Repeated measure ANOVA was used for statistical analysis.

Results: Repetitive tactile stimulation alone or capsaicin alone failed to induce significant changes of ES1 and ES2. In contrast, repetitive tactile stimulation plus capsaicin induced significantly less inhibition of ES1 (P < 0.05) and ES2 (P < 0.001) and significantly shorter duration of ES2 (P < 0.001). No differences were found between different muscles. The perceived intensity of the electrical stimuli was unchanged during the experimental conditions.

Conclusions: Our results suggest that convergence ofnociceptive and non-nociceptive inputs play an important role for modulation of the neural pathways involved in the short-latency ES1 and the long-latency ES2.

Acknowledgment: Supported by the Danish National Research Foundation.

FACILITATORY EFFECTS OF TONIC JAW-MUSCLE PAIN ON STRETCH REFLEXES IN SURFACE EMG RECORDINGS

Kelun Wang. Peter Svensson, Lars Arendt-Nielsen, Center for Sensory-Motor Interaction, Aalborg Univ, DK-9200, Denmark

Aim of Investigation: To investigate the effect of experimental muscle pain on the human jaw-stretch reflex.

Methods: Twelve subjects participated. In random order and separated by 1 week, 5% hypertonic or 0.9% isotonic saline was infused into the left masseter muscle for up to 15 min. Pain intensity was scored continuously by the subjects on a 10-cm VAS. Surface EMG activity was recorded from the left and right masseter and anterior temporalis muscles before infusion during the early (2 to 8 min) and late phases of infusion (9 to 15 min), and 15 min after infusion. Stretch reflexes (1 mm, 10 ms ramp) were evoked with an electromagnetic jaw stretcher. Subjects were given feedback to help them bite on the jaw-bar with their incisor teeth at 15% of maximal voluntary contraction. Twenty trials were recorded in each condition

Results: The pain from the hypertonic saline was moderate in the j early phase of infusion and significantly lower in the late phase. I The pre-stimulus EMG activity in the left masseter (feedback mus-J cle) was constant, whereas the EMG activity in right masseter was j significantly higher after hypertonic saline infusion (P < 0.05). The." normalized peak-to-peak amplitude of the stretch reflex in the left | masseter was significantly greater during early and late infusion I compared with pre- and post-infusion (P < 0.05). Isotonic saline j did not affect the amplitude of the stretch reflex

Conclusions: The increased stretch reflex measured in the surface 1 EMG (multi-unit recording) may be mediated by a differential fa- I cilitation of the fusimotor system by group III and IV afferent in- \ puts.

Acknowledgments: Supported by Danish National Research Foundation.

INHIBITORY EFFECTS OF TONIC JAW-MUSCLE | PAIN ON STRETCH REFLEXES IN SINGLE MOTOR UNll RECORDINGS

Peter Svensson. Timothy S. Miles*, Thomas Graven-Nielsen, Lars^ Arendt-Nielsen. Center for Sensory-Motor Interaction, Aalborg ^ Univ, Denmark; Dept of Physiology, Univ of Adelaide, Australia .1

Aim of Investigation: Muscle pain seems to facilitate the stretch reflex in surface EMG recordings. However, the response in single motor units (SMUs) is unknown.

Methods: Stretch reflexes were evoked in SMUs in the human masseter with an electromagnetic jaw stretcher. Stretch trials were obtained before, during and after pain. The action potentials of one SMU were discriminated with a computer-based device that matched the shape of the waveforms to pre-established templates. Thirteen subjects bit on the bars of the stretcher with a force that caused the motor unit to discharge around 10-12 Hz. When the duration of two consecutive ISIs fell within the range of 80-120 ms, the stretcher was triggered to apply a 1 mm, 10 ms ramp stretch. Fifty stretches were given. In successive trials, the stretches were given at precisely-timed delays ranging from 2 to 100 ms after the preceding action potential. Pain was induced by the infusion of 5% hypertonic saline into the right masseter.

Results: The average pain intensity was 6.8 0.5 on a 10-cm VAS. The stretch evoked a strong excitatory response in all SMUs at monosynaptic latency. During the pain, the probability of evoking a reflex response in a masseter SMU fell by about 21% (peak CUSUM: 17.2 1.6 counts) compared with before pain (22.7 1.7) and after pain (21.6 2.4) (P < 0.05).

Conclusions: The inhibitory effects of tonic pain in this population of low-threshold single motor units may be due to a decrease in the afferent volley as the result of a differential action on the gamma drive by group III and IV afferent inputs.

Acknowledgments: Supported by the Danish National Research Foundation

THALAMIC PROJECTIONS OF CORNEAL-RESPONSIVE NEURONS FROM MEDULLARY DORSAL HORN IN THE RAT

DA Bereiter. DF Bereiter*, AP Benetti* and H Hirata*, Depts. Surgery & Neuroscience RI Hospital/Brown Univ., Providence, RI, 02903 USA

Aim of Investigation: To determine the efferent projections of cor-neal-responsive neurons found at the trigeminal subnucleus inter-polaris/caudalis transition (Vi/Vc) and at laminae I-II of the subnucleus caudalis/upper cervical cord transition (Vc/Cl).

Methods: In male rats (Harlan, Sprague-Dawley) under barbiturate anesthesia, the retrograde tracer Fluoro-Gold (FG) was injected into medial (n. submedius, SM) or lateral (ventroposteromedial, VPM or posterior n., PO) regions ofcontralateral. thalamus 1 wk prior to comeal stimulation by mustard oil. C-fos immunostaining was used as a marker for comeal activation. In recording studies, single neurons responsive to mechanical stimulation of the cornea were tested for antidromic activation from sites in medial or lateral thalamus or superior salivatory nucleus/facial motor nucleus (SS/VII).

Results: C-fos/FG double-labeled cells were found in Vc/Cl and Vi/Vc after injections into PO, but none were seen after injections into VPM. FG injection into SM labeled Fos-positive neurons only in Vi/Vc. Recording results confirmed that Vc/Cl and Vi/Vc cor-neal neurons were antidromically activated from PO, but not from sites in VPM. Stimulation ofSM or SS/VII activated only Vi/Vc comeal units.

Conclusions: These results suggest that Vc/Cl and Vi/Vc units mediate different aspects of comeal pain. Based on stimulus encoding, response to morphine and efferent projections to PO, Vc/Cl units likely serve a sensory-discriminative function. By contrast, Vi/Vc units may mediate ocular-specific responses such as eye blink or tear formation and projections to SM may contribute to affective aspects of cornea! pain or recruitment of descending pain control pathways. Unexpectedly, neither Vc/Cl nor Vi/Vc comeal units projected to VPM, the main sensory relay in thalamus for repre-sentatioJi of other craniofacial structures supplied by the trigeminal nerve.

Acknowledgments: Supported by NIH grant NS26137.

LOCATION OF AXONAL AND NEURONAL LABELING IN THE TRIGEMINAL GANGLION FOR WGA-HRP PLACED IN THE MAXILLARY SINUS OF RABBITS

B.Hu and K.C. Kajander''2, Depts. of Oral Science' and Cell Biology & Neuroanatomy2, and Graduate Program in Neuroscience2, Univ of Minnesota, Minneapolis, MN 55455, USA.

Aim of Investigation: We describe the location within the trigeminal (Gasserian) ganglion of axons and cell bodies labeled with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) retro-gradely transported from the maxillary sinus of rabbits.

Methods: Adult, male New Zealand White rabbits (2.5 - 3.5 kg, n=5) were anesthetized and the maxillary sinus was exposed from the dorsal surface. The maxillary ostium was sealed, and 2 mg of WGA-HRP crystals were placed into the sinus cavity. The bone was repaired; the skin was sutured; and the rabbit was allowed to recover. Five days later, the rabbit was perfused transcardially with 4% paraformaldehyde and the trigeminal ganglion was removed. The ganglion was sectioned (30 urn), and tissue was processed for WGA-HRP labeling. Sections were evaluated using confocal microscopy.

Results: The vast majority of labeled cells (80%) were located in the anterior one-third of the ganglion. Fewer cells (20%) were located in the middle one-third, and no labeled cells were observed in the posterior one-third of the ganglion. In addition, most labeled cells (70%) were found in the medial half. Finally, 75% of labeled neurons were located in the dorsal half of the ganglion. Labeled axons were observed only in the maxillary division. Conclusions: The vast majority of cells (and all axons) labeled with WGA-HRP from the maxillary sinus are located in the maxillary division of the trigeminal ganglion. These data suggest that the maxillary nerve may provide exclusive innervation to the maxillary sinus of the rabbit.

Acknowledgments: This research was supported by grants from the National Insts of Health (NS33908) and the State of Minnesota.

ON THE CONVERGENCE OF FACIAL AND CERVICAL AFFERENTS; IN THE TRIGEMINAL SUBNUCLEUS CAUDALIS: A C-FOS EXPRESSION STUDY IN RATS.

G.A. Lucas'. M.I. Nogueira''2', J C. Bittencourt''2 , Neuroscience Research Center' and Dept. Anatomy2, Univ ofSao Paulo, SP, 05508-900, Brazil

Aim of Investigation: The heavy projection of trigeminal and cervical afferents to overlapping areas in the trigeminal subnucleus caudalis (Vsc) and upper spinal cord has been claimed as the anatomical basis for facial pain stemming from the cervical region. The purpose of this study was to investigate a functional aspect of these projections throughout/o.s-protein labeling in the Vsc following facial and cervical noxious stimulation.

Methods: Noxious stimulation was achieved by 150 ul ofs.c. 4% formalin injection in the left supraocular area, close to the upper eyelid. In a separated group, the same stimulus was applied to the cervical area. Two hours later, the lower medulla and the upper cervical spinal cord segments (C1-C3) were removed and processed immunohistochemically for/as' protein according to the ABC method.

Results: Unilateral painful stimulation of the supraorbital region showed ybs-immunoreactive neurons (fos-ir) in the ventro-lateral region of the rostral-most segment of the Vsc and in the first segment of spinal cord (Cl). The laminar analysis showed/a.?-ir occurred mainly in the superficial lamina (I-II). Labeled cells in deep layers were almost absent except in lamina X. The immunoreactive cells at C2 were scarce and no labeled neurons were found at C3 spinal segment. Otherwise, following cervical nociceptive stimulation most labeled neurons were found in the laminae I-II of the dorsal horn at C2 and C3 segments of the spinal cord. Labeled cells were also found in lamina IV-V and around the central canal. Few cells were labeled in the upper half of C2 and none at the Cl level or in the rostral-most segment of the Vsc.

Conclusions: The overlapping sites occupied hyfos-n neurons in the Vsc following supra-ocular and cervical noxious stimulation are markedly reduced compared to the overlapping areas described by tract-tracing techniques suggesting that the central projections from cervical and trigeminal sensory ganglia are not activated indiscriminately after acute noxious stimulation.

CHARACTERISING NEURONS IN THE RAT CAUDAL SPINAL TRIGEMINAL NUCLEUS (CSTN) INVOLVED IN HYPERALGESIA

LiuHai-Ping'. Yeo Jinn-Fel2, and Leong Seng-Kee*', Dept of Anatomy', Dept of Oral & Maxillofacial Surgery2, NUS, Singapore 119260

Aim of Investigation: To investigate whether neurons in the CSTN involved in orofacial hyperalgesia express glutamate receptors and nitric oxide synthase (NOS) and whether such neurons project to the thalamus.

Methods: Hyperalgesia was induced by subcutaneous injection of 4% formalin into the lateral face ofWistar rats. The resulting c-fos expressing neurons in the CSTN were localized by Fos immuno-histochemistry. Double immunofluorescence was performed for colocalization study. Thalamus projecting neurons in the CSTN were traced by the injection oftetramethylrhodamine-dextran (TMR-D) into the thalamus or tregeminothalamic tract.

Results: Many Fos positive nuclei were induced in the superficial laminae of the iosilateral CSTN following formalin stimulation.

Confocal laser scanning microscope revealed that almost all neurons containing Fos immunofluorescent nuclei were colocalised with NMDAR1, 94% with GluR2/3 and a maximum of 14% with NOS. Some Fos neurons were very close to NOS positive neurons. Also some of the Fos positive neurons were labelled by TMR-D.

Conclusions: The results suggested that activation of NMDA and AMPA receptors upon glutamate release in response to noxious stimulation to orofacial region might mediate c-fos expression in neurons involved in hyperalgesia. The expression ofc-/o.s- in the neurons could also be mediated by NO produced from the same as well as neighbouring neurons upon nociception. Some of the Fos positive neurons in the CSTN are thalamus projecting neurons.

Acknowledgments: Supported by grants RP940320 and RP960331 from the NUS.

BEHAVIORAL AND CARDIOVASCULAR EFFECTS OF TEMPOROMANDIBULAR JOINT INFLAMMATION IN THE RAT.

A.C. Hartwig* and G.F. Gebhart, Depts. of Pharmacology (GFG) and Oral & Maxillofacial Surgery (ACH), The Univ of Iowa, Iowa City, IA, 52242, USA.

Aim of Investigation: Despite years of clinical experience and basic research, head and neck pain continues to challenge the dental, medical, and scientific communities. This project describes a behavioral model ofTMJ pain in the rat which is complemented by cardiovascular data and quantification of changes in c-Fos expression in trigeminal and cardiovascular brainstem centers.

Methods: Mustard oil (1-20%) was used to inflame the TMJ of briefly anesthetized rats; they were then observed for two to five hours while specific facial grooming behaviors were counted. Heart rate and blood pressure were measured via previously inserted femoral arterial lines. c-Fos was visualized in brainstem sections of rats fixed with 4% paraformaldehyde using a polyclonal antibody (Sigma) and DAB/peroxide.

Results: Mustard oil caused a concentration-dependent increase in behaviors, as well as in c-Fos expression in the trigeminal caudalis. Behaviors were maximal 2 hours following inflammation and returned to baseline by 4 hours. Behaviors were dose-dependently reduced by systemic morphine, and c-Fos expression was reduced by preemptive intraarticular bupivacaine or morphine. TMJ inflammation caused transient but significant increases in blood pressure and heart rate; these changes were blocked or reversed by intraarticular morphine. c-Fos-reactive nuclei were identified in increased numbers in the trigeminal subnucleus caudalis, nucleus of the solitary tract, and lateral parabrachial nuclei following TMJ inflammation when compared with anesthesia only controls.

Conclusions: This model provides a foundation for further studies of TMJ pain using conscious rats, which may improve our understanding of trigeminal pain mechanisms and thereby improve the treatment of patients with head and neck pain.

Acknowledgments: Supported by NIH DA 02879 and DE021801100.

EFFECTS OF CASTRATION OR OVARIECTOMY ON THE OROFACIAL FORMALIN TEST IN THE RAT.

J. Pajot*, A. Woda, and P. Pionchon Faculte de Chirurgie Dentaire, 63000 Clermont-Ferrand, France.

Aim of Investigation: To investigate the relationship between sex hormones and orofacial nociception.

Methods: Twenty male and twenty female Sprague-Dawley rats, about 7 weeks old, were divided into six groups. At the start of the experiment, gonadectomy was performed in 8 males and 8 females, a sham operation was undertaken on 6 males and 6 females and 12 rats constituted one male and one female non surgical groups. The nociceptive tests took place 15 (tl) and 90 (t2) days after gonadectomy. The duration of rubbing after formalin injection (50ul, 1.5%) into the upper lip was measured and analysed (t test) for the time interval between the 12th and the 39th minute after the injection.

Results: Comparison between the mean rubbing duration of sham operated and non surgical groups showed no difference. As a consequence, they were grouped as controls. 1) At tl but not at t2, females of the control group displayed more nociceptive reactions than males (p<0.05). 2) Male gonadectomy did not modify the rubbing duration at either tl (p=0.52) or at t2 (p=0.47). 3) In both genders, ageing decreased rubbing duration of control animals (p<0;001 for females, p<0.05 for males). This was also true for castrated males (p<0.05).4)0variectomy decreased the nociceptive score at tl (p<0.01) but increased it at t2 (p<0.05).

Conclusions: Ovariectomy had differential effects on the orofacial formalin test. These differential effects may result either from the age at the time of the nociceptive tests or from the duration of oestrogen deprivation.

INTRACISTERNAL/HIGH CERVICAL INTRATHE-CAL ADMINISTRATION OF MORPHINE, CLONIDINE OR BACLOFEN ARE ANTINOCICEPTIVE IN THE OROFACIAL FORMALIN TEST IN THE RAT.

TheodoreS. Grabow'. Han-Rong Weng2, Peter S. Staats', Patrick M. Dougherty2, DeptofAnesthesiology', DeptofNeurosurgery2, Johns Hopkins Univ, Baltimore, MD, 21287, USA

Aim of Investigation: Head and neck pain can be difficult to treat clinically. The goal of this study is to combine a novel method of intracistemal/high cervical intrathecal delivery of analgesics with an animal model of acute and chronic pain of cranial origin.

Methods: Male Wistar rats were implanted with intrathecal catheters that terminated at the high cervical (C1-C4) spinal cord by cephalad advancement through a guide cannula inserted at the lumbar level. Only those rats demonstrating full neurological recovery were used in the experiment. The orofacial formalin test was utilized to assess antinociception. Vehicle (saline) or drug was administered through the intrathecal catheter followed ten minutes later by an injection of 2.5% (50ul) formalin solution into the vi-brissal pad. The amount of time spent grooming the ipsilateral face was recorded for fifteen three-minute intervals for a total time of forty-five minutes. Catheter location was verified post-mortem after cervical laminectomy.

Results: Injection of formalin produced a characteristic biphasic behavioral response. High cervical/ intracistemal injection of morphine (10 ug), clonidine (3 ug), and baclofen (0.1 ug) decreased the second phase but produced little effect on first phase responses. No motor dysfunction or obvious behavioral toxicity was observed at these doses.

Conclusions: In summary, intrathecal catheters that are inserted at the lumbar level and advanced to more cephalad sites can be utilized to deliver analgesics in an animal model of phasic and tonic pain of cranial origin. The application of this technique toother animal models of cranial pain will be required to determine the utility of this method of drug administration for the management of chronic head and neck pain in humans.

Acknowledgments: Supported by NS 32386.

UBIQUITOUS BUT DIVERSE RESPONSIV1TY OF TRIGEMINAL SENSORY NEURONS TO CHOL1NERGIC STIMULATION: AN IN VITRO SLICE STUDY IN THE RAT.

K.A. Kohlmeier'*. M.P. Kristensen2, and P.J. Soja2, "Kinsman Laboratory and faculty of Pharmaceutical Sciences, Univ. of British Columbia, Vancouver, BC, V6T 1Z3, Canada.

Aim of Investigation: Acetylcholine has been implicated in spinal antinociception (Brain Res. 311:267,1984). Cholinergic mechanisms may mediate similar effects on the trigeminal system, since muscarinic receptors are found in this region (Am. J. Physiol. 273:R896, 1997), however cholinoceptive properties of trigeminal sensory cells are not well understood. The purpose of the current study was to evaluate electrophysiological consequences of choli-nergic stimulation of cells located in the principal sensory trigeminal nucleus (PSTN).

Methods: Brain stem slices were prepared from 6-17 day old Wistar rats. Whole cell patch recordings of PSTN cells were obtained in bridge mode or voltage clamp (at -60 mV) prior to and following carbachol application.

Results: All examined PSTN cells responded to bath applied carbachol [10mM] with transient shifts in membrane potentials/currents and/or conductance changes. The majority of cells (70%) depo-larized (AVn, = 14%; n=l,1) or displayed inward currents (I== 176 pA; n = 22), while 20% hyperpolarized (AVn, = 5%; n = 4) or exhibited outward currents (I = 251 pA; n = 5), and 10% showed a complex, biphasic response of an outward, followed by an inward current. Responses were reproducible for each cell and persisted in the presence ofTTX. Estimation of reversal potentials from current -voltage plots (a -80 mV) suggest that a portion ofhyperpolarizing and depolarizing responses may be caused by cholinergically induced increases and decreases of iC-conductances, respectively. The ionic bases for responses in cells that displayed a depolariza-tion accompanied by resistance decreases are unclear, since no intercept of current-voltage plots appeared within the tested range (-50to-130mV).

Conclusions: These data indicate that most, if not all, PSTN cells are cholinoceptive and suggest that cholinergic receptor activation exerts disparate effects directly on PSTN neurons. This may allow cholinergic afferents to different! ally modulate PSTN throughput and potentially afford selective gating of transmission of sensory modalities, including pain, via ascending trigeminal tracts.

ROLE OF NEURONAL NICOTINIC RECEPTORS IN THE ACTIVATION OF NEURONS IN TRIGEMINAL SUB-NUCLEUS CAUDALIS BY NICOTINE DELIVERED TO THE ORAL MUCOSA.

E. Carstens. C.T. Simons, J.-M. Dessirier*, M. lodi-Carstens* and S.L. Jinks. Neurobiology, Physiology & Behavior, Univ. of California, Davis, CA 95616 USA.

Aim of Investigation: The method ofc-fos immunohistochemistry was used to characterize the role of neuronal nicotinic acetylcho-line receptors (nAChRs) in the activation of trigeminal nociceptive neurons by intraoral nicotine.

Methods: Anesthetized rats received one of the following stimuli delivered to the dorsal anterior tongue: (1) 0.9% NaCI followed by nicotine 1%= 61 mM, (2) the nAChR antagonist, mecamylamine 0.1%= 4.9 mM + nicotine, (3) the muscarinic antagonist atropine 0.1%= 1.46 mM + nicotine, (4) atropine 1%= 14.6 mM + nicotine, (5) 0.9% NaCI control, or (6) no stimulus control. Two hr later animals were perfused with 3% paraformaldehyde. Brainstem sections were immunohistochemically processed for fos-like immu-noreactivity (FLI) and examined under the light microscope.

Results: Nicotine evoked significant increases in FLI compared to controls in: dorsomedial and ventrolateral trigeminal caudalis, nucleus of the solitary tract (NTS), ventrolateral medulla, and area postrema. Mecamylamine significantly reduced nicotine-evoked FLI in dorsomedial and ventrolateral caudalis, ventrolateral medulla, and area postrema. Atropine 1% significantly reduced FLI in each of the same areas and also NTS, while 0.1% atropine significantly increased FLI in NTS with no significant effect in other regions.

Conclusions: These results are consistent with the idea that nicotine activates nAChRs residing on nociceptive fibers innervating the tongue which, in turn, excite neurons in trigeminal caudalis and other brainstem regions.

Acknowledgments: Supported by grants from the California Tobacco Disease-Related Research Program # 6RT-0231 and NIH NS-35778.

C-FOS EXPRESSION IN TRIGEMINAL NUCLEUS CAUDALIS NEURONS EVOKED BY APPLICATION OF CARBONATED WATER TO THE TONGUE IS REDUCED BY BLOCKERS OF CARBONIC ANHYDRASE.

C.T. Simons, S.L. Jinks, J.-M. Dessirier*, M. lodi Carstens* andE, Carstens. Neurobiology, Physiology & Behavior, Univ. of California, Davis, CA. 95616 USA

Aim of investiqation: We used c-fos immunohistochemistry to determine if the activation of brainstem trigeminal neurons by carbonated water delivered to the tongue is reduced by pre-treatment with the carbonic anhydrase blockers, acetazolamide and dorzolamide.

Methods: In anesthetized rats, carbonated water (50 ml/10 min) was flowed over the tongue which had been pretreated with topical application of(l)isotonic NaCI, (2) acetazolamide (1%), or (3) dorzolamide (2%). NaCl-treated or untreated rats not receiving carbonated water, and rats receiving flat (non-carbonated) water, served as controls. Two hr later rats were perfused with 3% para-formaldehyde. Brainstem sections were histochemically processed and examined under the light microscope for fos-like immunoreac-tivity (FLI).

Results: Animals receiving carbonated water + saline showed a significant increase in FLI above saline and flat-water controls in superficial laminae of the dorsomedial aspect trigeminal nucleus caudalis. Carbonated water also evoked significantly higher FLI compared to unstimulated controls in the nucleus of the solitary tract, ventrolateral caudalis, and ventrolateral medulla. Pretreat-ment with either acetazolamide or dorzolamide significantly reduced FLI in dorsomedial caudalis evoked by carbonated water, compared to animals receiving carbonated water + saline.

Conclusions: The results support the hypothesis that carbonated water activates intraoral nociceptors by conversion of CO; to carbonic acid. The nociceptors in turn excite trigeminal neurons involved in signaling oral irritation.

Acknowledgments: Supported by grants from the Calif. Tobacco Disease-Related Research Program and NIH NS-35778, and a gift from Danone Corporation, France.

RESPONSES OF NEURONS IN TRIGEMINAL NUCLEUS CAUDALIS TO INTRAORAL APPLICATION OF CARBONATED WATER ARE REDUCED BY DORZOLAMIDE, A BLOCKER OF CARBONIC ANHYDRASE

J.-M. Dessirier*, C.T. Simons, J.H. Eisele, Jr. and E. Carstens. Neurobiology, Physiology & Behavior, Univ. of California, Davis, CA 95616 USA.

Aim of Investigation: We used electrophysiological methods to determine if single neurons in trigeminal nucleus caudalis of anesthetized rats respond to application of carbonated water on the tongue in a manner that is reduced by the carbonic anhydrase blocker, dorzolamide.

Methods: Electrophysiological methods were used to record from single wide dynamic range (WDR) neurons that responded to application of noxious heat as well as carbonated water to the tongue. Neurons were histologically localized to superficial laminae in the dorsomedial aspect oftrigeminal caudalis. Results: Each of 10 WDR neurons responded to carbonated water flowed over the tongue (6 ml/45 sec; pH = 4.7), and some of the neurons tested also responded to HC1 (pH = 1). Following topical pretreatment of the tongue with dorzolamide (2%), the mean response of the 10 neurons to carbonated water was significantly reduced, with recovery over the ensuing 45 min. Dorzolamide did not affect neuronal responses to noxious heat or HC1 when tested.

Conclusions: These data support the hypothesis that irritation elicited by carbonated water results from chemical activation ofintra-oral nociceptors by carbonic acid, which is formed from CO^ in a reaction catalyzed by carbonic anhydrase. These nociceptors, in turn, excite neurons in trigeminal nucleus caudalis which constitute a relay in central pathways mediating oral irritation.

Acknowledgments: Supported by a gift from the Danone Group, France and by grants from the California Tobacco Disease-Related Research Program and NIH NS-35778.

WINDUP OF SPINAL TRIGEMINAL CONVERGENT NEURONS CAN BE BLOCKED BY MORPHINE.

C. Duale'*. P. Raboisson2, J.-L. Molat'*, R. Dallel', 'Lab. ofOro-facial Physiology, Fac. of Dentistry, 63000 Clermont-Ferrand, France, ^Astra Pain Control AB, Discovery Division, 14157 Hud-dinge Sweden.

Aim of the investigation: To study the effects of morphine on the windup of spinal trigeminal nucleus oralis (Sp50) convergent neurons.

Methods: Convergent neurons were recorded in the Sp50 using extracellular micropipettes in halothane-anesthetized rats. Windup of the C-fiber-evoked response was triggered by trains of 15 electrical shocks (0.5 Hz, 2 ms duration) applied into the neuron receptive field, at 3-times the threshold for obtaining a C-fiber-evoked response. Windup was calculated as the total number of spikes recorded at a C-fiber latency during a train, minus the input. The input was calculated as the number of spikes produced by the first stimulation multiplied by 15. The slope of the windup curve was also analyzed. After 2 controls, morphine (6 mg/kg) was administered i.v. and 2 tests were performed 5 and 10 min after injection. The specificity of the observed effects was tested by naloxone (0.4 mg/kg, i.v.).

Results: I.v. morphine produced a decrease of the total C-fiber-evoked response (405% of initial value), the input (6023%), and the windup (365%). The slope of the windup curve was shifted rightward and significantly decreased by morphine. Naloxone reversed preferentially morphine effects on the windup (959% of initial value).

Conclusion: The present study shows that contrary to what has been reported before, "windup" a form of central sensitization, can be blocked by morphine acting both on segmental (pre and post-synaptic opioid receptors) and suprasegemental levels.

INVOLVEMENT OF GLYCINE RECEPTORS IN THE MODULATION OF SPINAL TRIGEMINAL CONVERGENT NEURONS ACTIVITIES

C. Ressot. V. Collado, R. Dallel (SPON: W. Mesnay). Lab. ofOro-facial Physiology, Fac. of Dentistry, 63000 Clermont-Ferrand, France

Aim of Investigation: The processing oftrigeminal sensory inputs is under an inhibitory control mediated in great part by the aminoacids, GABA and glycine. The purpose of this study was to examine the effect of strychnine (STR), a glycine receptor antagonist, on the A-fiber-evoked responses of spinal trigeminal nucleus oralis (Sp50) convergent neurons, to transcutaneous electrical stimulation.

Methods: Convergent neurons were recorded in the Sp50 using extracellular micropipettes in halothane-anesthetized rats. The neurons were characterized by their responses to both mechanical and electrical stimulations, applied to their receptive field. The experimental procedure consisted of sequences of 15 electrical shocks (0.1 Hz, O.lms duration) applied at the threshold for A-fiber activation. In the first part of the study, we examined the influence of i.v. administration of STR (0.2, 0.4 or 0.8 mg/kg) on the A-fiber-evoked response. In the second part, STR was microinjected (2 nM in 0.5ul of saline) in the spinal trigeminal nucleus caudalis (Sp5C).

Results: I.v. STR increased the A-fiber-evoked responses of Sp50 convergent neurons in a dose-related fashion. The effects were nearly maximal 5 min after injection and lasted approximately 40 min. The responses were increased to 135 19%, 153 12 % and 220 15% of the initial value, following 0.2, 0.4 and 0.8 mg/kg of STR, respectively. STR microinjected into the Sp5C also increased the A-fiber-evoked responses of Sp50 neurons to 143 14% of the initial value, 5 min after application.

Conclusion: This study highlights the role of glycine as a modulator of the A-fiber-evoked responses of Sp50 convergent neurons, and suggests that STR may act at least on glycine receptors located in the Sp5C.

SELECTIVE ANTAGONISM OF INFLAMMATORY HYPEREXCITABILITY IN A SUBPOPULATION OF MDH NOCICEPTIVE NEURONS BY A GLYCINE SITE NMDA RECEPTOR ANTAGONIST AND A SODIUM CHANNEL BLOCKER

Kenji Miki. Ronald Dubner, Ke Ren, Dept ofOCBS, Univ. Maryland, Baltimore MD 21201, USA

Aim of Investigation: To investigate the effect of a glycine site N-methyl-D-aspartate (NMDA) receptor antagonist, GV 196771 A, and a sodium channel blocker, 4030W92, on nociceptive neuronal activity in trigeminal subnucleus caudalis after deep or cutaneous orofacial tissue inflammation.

Methods: Single neurons were isolated extracellularly from trigeminal subnucleus caudalis of the rat. Deep or cutaneous orofacial tissue inflammation was induced 24 h before the recordings by injecting complete Freund's adjuvant (CFA) into the temporoman-dibular joint (TMJ) capsule or the perioral (PO) skin. Cumulative doses ofGV 196771A and 4030W92 were injected i.p. during the study of the response characteristics of single neurons.

Results: Two populations of nociceptive neurons with large (76721 mm2, n=27) and small (1359 mm2, n=29) receptive fields were identified in inflamed rats, while all nociceptive neurons in naive rats had small receptive fields. GV 196771A (1 mg/kg) and 4030W92 (20 mg/kg) produced a selective reduction in receptive field size in neurons with large receptive fields in TMJ-inflamed rats (74% and 88%, respectively) (PO.05). The responses to noxious mechanical stimuli were also significantly reduced in these neurons. The two agents produced suppression of nociceptive neurons in PO-inflamed rats only at much higher doses and had no effects in naive rats.

Conclusions: These results indicate that orofacial deep tissue inflammation results in hyperexcitability in a subpopulation of neurons that are sensitive to NMDA receptor and sodium channel blockade. Compared to PO cutaneous inflammation, NMDA receptor activation appears to play a more important role in neuronal plasticity after TMJ inflammation.

Acknowledgments: Supported by GlaxoWellcome and NIDCR 'Grant DEI 1964.

EFFECTS OF MICROINJECTION OF MK801 IN LATERAL MEDULLARY DORSAL HORN (SP5C) ON THE NOCICEPTIVE RESPONSES OF CONVERGENT TRI-GEMINAL NEURONES.

Alain Woda. C. Parada' and P. Luccarini', Faculte de Chirurgie Dentaire, 63000 Clermont-Ferrand, France. Universidade de estadual de Campinas, Unicamp, Brazil

Aim of Investigation: To investigate the control ofnociceptive responses of convergent neurones by NMDA receptors in the lateral Sp5C.

Methods: Extracellular recordings of convergent neurones oftri-geminal oralis subnucleus (Sp50) were performed in paralysed and anaesthetised rats. MK801 (0.06,0.12,0.25 and 0.5ug in 0.5ul) was microinjected in Sp5C.The total number of spikes occurring in the C fibber range following 30 successive suprathreshold electrical stimulations of the receptive field was recorded before (control) and after the injection. The effects ofmicroinjection were considered as excitatory or inhibitory if the modification was more than 20% of the control.

Results: The effects were dose dependent. The number of inhibited, excited or non affected cells amounted: 14, 0 and 1 after injection of0.5ug, 6,4 and 4 after injection of0.25ug, 3, 11 and 1 for 0.12ug, and 0, 0 and 3 for 0.06ug respectively. These effects were time dependent since the mean number of spikes were significantly ': (ANOVA, all pairwise multiple comparison) modified at 15,25 ' and 35 minutes after injection. The effects depended on the injection site since, apart 3 non affected cells, all of them were inhibited after injection of 0.5 (n=5), 0.25 (n=4) or 0.12ug (n=4) in the medial part of Sp5C. Injections of either saline (n=4) or less active enantiomere MK801 (n=7) did not modify the Sp50 responses.

Conclusions: A biphasic effect of MK801 on the Sp50 convergent neurones could be observed after microinjection in the lateral but not in the medial part of Sp5C.

SP CONTENT IN FLUID PERFUSING CEREBRAL VENTRICLES DURING ELICITATION AND INHIBITION OF TRIGEMINO-HYPOGLOSSAL REFLEX.

Maria Zubrzycka. Wladyslaw Z. Traczyk., Dept of Physiology, Inst of Physiology and Biochemistry, Medical Univ of Lodz, Poland

Aim of Investigation: To establish whether PAG stimulation significantly affects the release of SP into the fluid perfusing the cerebral ventricles in rats.

Methods: In rats under chloralose anaesthesia (n=l 1), the content of substance P. (SP) in the fluid perfusing the cerebral ventricles was determined during incisor pulp stimulation with electrical impulses inducing nociceptive trigemino-hypoglossal reflex and then during inhibition of the reflex by stimulating midbrain periaque-ductal gray (PAG). Perfusion of the cerebral ventricles was carried out using artificial cerebrospinal fluid (aCSF). SP content in pg was determined in the samples by means ofradioimmunoassay (RIA). 0 - in cerebrospinal fluid (CSF); then 30-min perfusates were collected: 1 - during perfusion of the cerebral ventricles without stimulation; 2 - during perfusion of the cerebral ventricles and incisor pulp stimulation; 3 - during perfusion of the cerebral ventricles and simultaneous incisor pulp and inhibition of trigemino-hypoglossal reflex by PAG stimulation.

Results: SP content in samples was: in 0 - 458 pg; in 1-18,8 pg/30 min; 2 - 207,8 pg/30 min; 3 - 12,8 pg/30 min. Stimulation of incisor pulp led to increased release of SP into the fluid perfusing the cerebral ventricles. Stimulation of PAG reduced the release of SP into the cerebral ventricular system to the values obtained before tooth pulp stimulation.

Conclusions: The results indicate that PAG significantly inhibits the release of SP into the rat cerebral ventricular system and may be involved in the inhibition of trigemino-hypoglossal reflex.

Acknowledgments: The study was granted by the Medical Univ of Lodz.

CHALLENGE WITH SUBSTANCE P IN THE RAT INCREASES SCRATCHING BEHAVIOUR AND THE RELEASE OF SUBSTANCE P IN THE CENTRAL NERVOUS SYSTEM.

J.Carleson. B. Appelgren*, M. Jansson* and T. Lundeberg. Dept of Physiology & Pharmacology and Dept of Surgery and Rehabilitation, Karolinska Inst, S-171 77 Stockholm, Sweden.

Aim of Investigation: To investigate the acute effects of an in-tra-articular injection of 0,01 ml substance P (10"5 M) into the tem-poromandibular joint.

Methods: Scratching behaviour was used to assess the behavioural effects of an intra-articular injection of substance P. Microdialysis of central nervous system structures was carried out and analysed by radio immune assay.

Results: Intra-articular injection of substance P resulted in a significant increase in scratching behaviour (2314 sec) as compared to saline controls (68 sec). Increased substance P - like immune-reactivity concentrations were detected in hippocampus (p<0.05) and hypothalamus (p<0.01).

Conclusion: The increased scratching behaviour in rats subjected to an intra-articular injection of substance P may implicate that substance P has a strong pro-nociceptive role in the periphery which leads to scratching behaviour. The increase of substance P-like immunoreactivity in hippocampus, and hypothalamus suggests a role of substance P in the central mechanisms involved in the regulation of scratching behaviour. Peripherally released SP may contribute to the central adaptive reactions seen following inflammatory events.

Acknowledgments: Karolinska Institute Foundation. Swedish Dental Association.

CENTRAL EFFECTS OF THE ANTIMIGRAINE DRUG NARATR1PTAN IN A RAT MODEL OF MENIN-GEAL NOCICEPTION.

Jens Ellrich and Karl Messlinger, Inst of Physiology & Exp. Pathophysiology, Friedrich-Alexander Univ, 91054 Eriangen, Germany

Aim of Investigation: 5HTI agonists such as Naratriptan (Nara) may not only act via receptors located on peripheral trigeminovas-cular afferents but also via central neurons. Neurons of special importance for the pathophysiology of headache are located in the medullary dorsal horn (MDH) and the rostral ventromedial medulla (RVM) which is part of the endogenous pain control system (EPS). In this study the modulatory effect of Nara on MDH and RVM neurons was investigated in the rat.

Methods: The activity of MDH neurons with meningeal receptive fields was extracellularly recorded before and after intravenous (iv) administration of 200 ug/kg Nara (n=8) or saline (n=8). 30 min after administration of Nara, 1 mg/kg Naloxone (Nalox) was iv applied (n=7). Likewise, the activity of RVM neurons was extracellularly recorded before and after iv administration of 200 ug/kg Nara(n=ll).

Results: After Nara, neuronal activity of all MDH neurons decreased (p<0,001) and activity partly recovered in 3 MDH neurons after Nalox. Activity of RVM neurons increased in 3 of 4 off-cells and I of 3 neutral cell after Nara, decreased in 2 of 4 on-cells and in I neutral cell, and remained unchanged in 1 off-cell, 2 on-cells, and 1 neutral cell.

Conclusions: Nara is a strong inhibitor of central trigeminal MDH neurons with input from the meninges. This effect may not only be caused by activation of5HTl receptors on trigeminal neurons but also via an activation of EPS because (1) Nalox partly antagonizes the inhibitory effect of Nara on MDH neurons with meningeal input, and (2) the modulation ofRVM neuronal activity by Nara resembles the effect of morphine.

Acknowledgments: Supported by the Deutsche Forschungsgemein-schaft (SFB 353/B3).

THE POSSIBLE CAUSES AND MECHANISMS OF TRIGEMINAL NERVE PAINS

Smoliar Efim*. Smoliar Albert*, Heiman Alia*, Belkin Victor*. Dept. of Anatomy and Anthropology, Sackler Faculty of Medicine, Tel Aviv Univ; Ramat Aviv, 69978 Israel.

Am of Investigation: To study the trigeminal nerve sheaths, their blood vessels and innervation from clinical aspect.

Methods: Trigeminal nerves (CNV) were obtained from a samples of 83 human cadavers, 0 to 90 years old. Differentiation ofintra-neural blood vessels and nerve elements was done after silver nitrate impregnation.

Results: All elements ofmicrocirculatory bed (arterioles, precapil-laries, capillaries, postcapillaries, venules) are situated in epineu-rium and perineurium of ophthalmic, maxillary, and mandibular nerves, in capsule and stroma of the trigeminal ganglion. Only capillary nets are in endoneurium. The walls of arterioles have muscular cells, which sometimes form circular cuffs. The muscular cells gatherings form sphincter-like structures, seen at the places of precapillar exit from arterioles. The receptors innervation of the connective tissue and blood vessels of the CNV sheaths was comprised mostly of dense and diffuse polyvalent nonencapsulated endings within neural stroma and blood vessels wall. These nerve endings derivate from thin myelinated fibers of CNV itself. The great amount ofadrenergic plexuses exists on the wall ofintraneu-ral vessels. The neuro-vascular concentration is reached in maxillary and mandibular nerve sheaths.

Conclusions: The observed circular gatherings of muscular cells within intraneural blood vessels wall and adrenergic (sympathetic) innervation give possibility for spasm of blood vessels. The receptors within CNV can be considered as mechanoreceptors and chemoreceptors, which play significant role in genesis of trigeminal pain. In the clinical practice, there is a need to pay attention on places of concentration of neural endings along CNV.

GANGLIONIC LOCAL OPIOID ANALGESIA (GLOA) AS WELL AS SALINE INJECTION REDUCES PAIN IN TRIGEMINAL NEURALGIA

Anna Spacek'. Franz X. Neiger'*, Fritz Orlicek'*, Christian Wober2 *, Birgit Frommer'*, Wemer Brannath3*, Peter Wessely2, Hans G. Kress', Depts. ofAnaesthesiology and General Intensive Care (B) ', Neurology2 and Medical Statistics3'Univ of Vienna, A-1090 Vienna, AUSTRIA

Introduction: In this prospective, randomised, double-blind, placebo-controlled cross-over study, the analgesic efficacy of GLOA versus saline injections was compared in patients suffering from refractory trigeminal neuralgia.

Methods: A total of 20 patients, who were refractory to a continued carbamazepine therapy, were treated once a day for two 5-day periods with a series of either GLOA or saline injections, respectively, using a cross-over design. All applications were performed at the ganglion cervicale superius (GCS) of the affected side. The verum-GLOA consisted of 0.045 mg buprenorphine in 1.5 ml 0.9% NaCI, the placebo-GLOA of 1.5 ml 0.9% NaCI only. Pain reduction was measured with a visual analogue scale (VAS) before and after each injection. Data were analysed using analysis of variance.

Results: A significant pain relief occurred in both groups within the first week of treatment. Following a 2-day treatment-free interval, the second treatment period did not show any significant additional effect on the constant, lasting pain relief.

Conclusion: The application of verum-GLOA (with 0.045 mg buprenorphine) or of pure saline (placebo-GLOA) at the ganglion cervicale superius on five consecutive days was indistinguishably effective in patients that did not show sufficient pain relief under standard carbamazepine medication.

ANOREXIA INDUCED BY INTRACRANIAL PAIN IS MEDIATED BY PARABRACHIAL NEURONS CONTAINING CCK AND VENTROMEDIAL HYPOTHALAMIC NEURONS EXPRESSING LEPTIN RECEPTOR.

Amy Malick. Moshe Jakubowski', Marcella Mousavi and Rami Burstein112 (Sponsor), 'Dept. of Anesthesia, Bern Israel Deaconess Medical Center; ^Dept. ofNeurobiology, Harvard Medical School, Boston, MA 02115.

Aims of Investigation: Migraine attacks are invariably associated with gastrointestinal symptoms and alterations in appetite such as nausea, vomiting, and most notably, anorexia. The aims of the present study are to (1) determine whether the induction of intracranial pain in the rat can cause alterations in feeding behavior similar to the anorexia experienced by patients during migraine attacks, and (2) identify brain areas involved in producing these changes. Methods: A combination of behavioral (feeding), immunocyto-chemical (Fos protein), and in situ hybridization (CCK peptide, and CCK and leptin receptors) techniques were used to (1) establish an animal model of migraine induced anorexia and (2) identify possible brain regions and neuropeptides involved in relaying nocicep-tive trigeminovascular information to areas that regulate appetite suppression.

Results: In conscious rats, noxious stimulation of the meninges and trigeminovasculature caused (1) reduction in food intake and (2) neuronal activation within: (a) ventrolatcral ophthalmic neurons of trigeminal nucleus caudalis, (b) CCK peptide containing neurons within the lateral parabrachial nucleus, and (c) ventromedial hy-pothalamic nucleus neurons expressing leptin and/or CCK receptors.

Conclusions: The suppression of feeding behavior by intracranial pain is mediated by activation of parabrachial neurons that contain the anorectic peptide CCK, and ventromedial hypothalamic neurons that exhibit receptors for CCK and/or the satiety hormone leptin.

Acknowledgments: Supported by NIH grants DE-10904 and NS-35611-01.

9th WORLD CONGRESS ON PAIN, 1999, Vienna, Austria, p. 538 - 544

   

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