Dean Dessem

Trigeminal P2X3 receptor expression differs from dorsal root ganglion and is modulated by deep tissue inflammation

&NA; The distribution and modulation of the P2X3 receptor was studied in trigeminal ganglion neurons to provide insight into the role of ATP in craniofacial sensory mechanisms. Binding to the D‐galactose specific lectin IB4 was found in 73% of P2X3‐positive neurons while only 16% of IB4 neurons expressed P2X3. Neurons expressing P2X3 alone were significantly larger than IB4‐or IB4/P2X3‐positive neurons. Investigation of target‐specificity revealed that 22% of trigeminal ganglion muscle afferent neurons were positive for P2X3 versus 16% of cutaneous afferent neurons. Muscle P2X3 afferents were significantly smaller than the overall muscle afferent population while P2X3 cutaneous afferent neurons were not. Presumptive heteromeric (P2X2/3) muscle afferent neurons were also identified and comprised 77% of the P2X3 muscle afferent population. Muscle afferent neurons co‐expressed P2X3 with either calcitonin gene‐related peptide (15%) or substance P (4%). The number of P2X3‐positive muscle afferent neurons significantly increased one and four days following complete Freunds adjuvant‐induced masseter muscle inflammation, but significantly decreased after 12 days. These results indicate that within trigeminal ganglia: (1) the P2X3 receptor is expressed in both small and medium‐sized neurons; (2) the P2X3 receptor is not exclusively expressed in IB4 neurons; (3) P2X3 is co‐expressed with neuropeptides; (4) differences in the proportion of cutaneous versus muscle P2X3 afferents are not apparent. Trigeminal P2X3 neurons therefore differ markedly from dorsal root ganglion P2X3 afferents. This study also shows that deep tissue inflammation modulates expression of the P2X3 receptor and thus may warrant exploration as a target for therapeutic intervention.

Downregulation of selective microRNAs in trigeminal ganglion neurons following inflammatory muscle pain

Active regulation of gene expression in the nervous system plays an important role in the development and/or maintenance of inflammatory pain. MicroRNA (miRNA) negatively regulates gene expression via posttranscriptional or transcriptional inhibition of specific genes. To explore the possible involvement of miRNA in gene regulation during inflammatory pain, we injected complete Freunds adjuvant (CFA) unilaterally into the rat masseter muscle and quantified changes in neuron-specific mature miRNAs in the trigeminal ganglion (TG). Real-time reverse-transcription polymerase chain reaction revealed significant, but differential, downregulation of mature miR-10a, -29a, -98, -99a, -124a, -134, and -183 in the ipsilateral mandibular division (V3) of the TG within 4 hr after CFA. In contrast, levels of tested miRNAs did not change significantly in the contralateral V3 or the ipsilateral ophthalmic and maxillary divisions of the TG from inflamed rats, nor in the ipsilateral V3 of saline-injected animals. The downregulated miRNAs recovered differentially to a level equal to or higher than that in naive animals. Full recovery time varied with miRNA species but was at least 4 days. Expression and downregulation of some miRNAs were further confirmed by in situ hybridization of TG neurons that innervate the inflamed muscle. Although neurons of all sizes expressed these miRNAs, their signals varied between neurons. Our results indicate that miRNA species specific to neurons are quickly regulated following inflammatory muscle pain.

Deep tissue inflammation upregulates neuropeptides and evokes nociceptive behaviors which are modulated by a neuropeptide antagonist.

Abstract Promising recent developments in the therapeutic value of neuropeptide antagonists have generated renewed importance in understanding the functional role of neuropeptides in nociception and inflammation. To explore this relationship we examined behavioral changes and primary afferent neuronal plasticity following deep tissue inflammation. One hour following craniofacial muscle inflammation ipsilateral as well as contralateral head withdrawal thresholds and ipsi‐ and contralateral hindpaw withdrawal thresholds were lowered and remained reduced for 28 days. Elevated levels of calcitonin gene‐related peptide (CGRP) within the trigeminal ganglion temporally correlated with this mechanical allodynia. Inflammation also induced an increase in the number of CGRP and substance P (SP)‐immunopositive trigeminal ganglion neurons innervating inflamed muscle but did not evoke a shift in the size distribution of peptidergic muscle afferent neurons. Trigeminal proprioceptive muscle afferent neurons situated within the brainstem in the mesencephalic trigeminal nucleus did not express CGRP or SP prior to or following inflammation. Intravenous administration of CGRP receptor antagonist (8‐37) two minutes prior to adjuvant injection blocked plasma extravasation and abolished both head and hindlimb mechanical allodynia. Local injection of CGRP antagonist directly into the masseter muscle prior to CFA produced similar, but less pronounced, effects. These findings indicate that unilateral craniofacial muscle inflammation produces mechanical allodynia at distant sites and upregulates CGRP and SP in primary afferent neurons innervating deep tissues. These data further implicate CGRP and SP in deep tissue nociceptive mechanisms and suggest that peptide antagonists may have therapeutic potential for musculoskeletal pain.

Central connections of trigeminal primary afferent neurons: topographical and functional considerations.

This article reviews literature relating to the central projection of primary afferent neurons of the trigeminal nerve. After a brief description of the major nuclei associated with the trigeminal nerve, the presentation reviews several early issues related to theories of trigeminal organization including modality and somatotopic representation. Recent studies directed toward further definition of central projection patterns of single nerve branches or nerves supplying specific oral and facial tissues are considered together with data from intraaxonal and intracellular studies that define the projection patterns of single fibers. A presentation of recent immunocytochemical data related to primary afferent fibers is described. Finally, several insights that recent studies shed on early theories of trigeminal input are assessed.

Chemical phenotypes of muscle and cutaneous afferent neurons in the rat trigeminal ganglion.

Retrograde labeling was combined with cytochemistry to investigate phenotypic differences in primary afferent neurons relaying sensory information from deep and superficial craniofacial tissues. Calcitonin gene‐related peptide (CGRP), substance P (SP), somatostatin (SOM) immunoreactivity and isolectin IB4, and cholera toxin B (ChTB) binding were examined for trigeminal masticatory muscle and cutaneous afferent neurons. Somata labeled from muscle were larger than cutaneous afferent neurons. Muscle afferent neurons exhibited positive staining as follows: 22% CGRP, 5% SP, 0% SOM; 18% ChTB, 5% IB4. The somata of CGRP‐ and SP‐positive muscle afferent neurons were smaller than that of the overall muscle afferent population. Size differences were not detected between IB4‐ or ChTB‐binding muscle afferent neurons and the total muscle afferent population. The following distribution was found for cutaneous afferent neurons: 26% CGRP, 7% SP, 1% SOM, 26% ChTB, 44% IB4. Cutaneous afferent neurons positive for SP were smaller, while ChTB‐binding cutaneous afferents were larger than the overall cutaneous afferent population. No size differences were found between cutaneous CGRP‐, SOM‐, or IB4‐positive neurons and the total cutaneous afferent population. Target‐specific differences exist for SOM and IB4. The percentage of cutaneous afferent neurons positive for SOM and IB4 exceeds that for SOM‐ or IB4‐positive muscle afferents. The number of retrogradely labeled neurons never differed between sexes. The percentage of retrogradely labeled muscle afferent neurons that were CGRP‐positive was greater in males than females. These data indicate the presence of phenotypic, target, and sex differences in trigeminal ganglion primary afferent neurons. J. Comp. Neurol. 460:167–179, 2003.

Jaw-muscle spindle afferent Pathways to the trigeminal motor nucleus in the rat

Neural pathways conveying proprioceptive feedback from the jaw muscles were studied in rats by combining retrograde and intracellular neuronal labeling. Initially, horseradish peroxidase was iontophoresed unilaterally into the trigeminal motor nucleus (Vmo). Two days later, 1–5 jaw‐muscle spindle afferent axons located in the mesencephalic trigeminal nucleus were physiologically identified and intracellularly stained with biotinamide. Stained mesencephalic trigeminal jaw‐muscle spindle afferent axon collaterals and boutons were predominantly distributed in the supratrigeminal region (Vsup), Vmo, dorsomedial trigeminal principal sensory nucleus (Vpdm), parvicellular reticular formation (PCRt), alpha division of the parvicellular reticular formation (PCRtA), and dorsomedial portions of the spinal trigeminal subnuclei oralis (Vodm), and interpolaris (Vidm). Numerous neurons retrogradely labeled with horseradish peroxidase from the trigeminal motor nucleus were found bilaterally in the PCRt, PCRtA, Vodm, and Vidm. Retrogradely labeled neurons were also present contralaterally in the Vsup, Vpdm, Vmo, peritrigeminal zone, and bilaterally in the dorsal medullary reticular field. Putative contacts between intracellularly stained mesencephalic trigeminal jaw‐muscle spindle afferent boutons and trigeminal premotor neurons retrogradely labeled with horseradish peroxidase were found in the ipsilateral Vodm, PCRtA, and PCRt, as well as the contralateral Vsup, Vmo, Vodm, PCRt, and PCRtA. Thus, multiple disynaptic jaw‐muscle spindle afferent‐motoneuron circuits exist. These pathways are likely to convey long‐latency jaw‐muscle stretch reflexes and may contribute to stiffness regulation of the masticatory muscles. J. Comp. Neurol. 435:341–353, 2001.

MUSCLE INFLAMMATION INDUCES A RAPID INCREASE IN CALCITONIN GENE-RELATED PEPTIDE (CGRP) mRNA THAT TEMPORALLY RELATES TO CGRP IMMUNOREACTIVITY AND NOCICEPTIVE BEHAVIOR

Recent data support an important role for calcitonin gene-related peptide (CGRP) in deep tissue nociceptive processing. Using real-time reverse transcriptase polymerase chain reaction (RT-PCR), radioimmunoassay, immunohistochemistry and behavioral testing, we studied the early time course of CGRP mRNA and protein expression as well as nociceptive behavior following muscle inflammation. A rapid and significant increase in CGRP mRNA occurred in the mandibular division (V3) of the ipsilateral trigeminal ganglion at 30 minutes, 4 and 24 h after the injection of complete Freunds adjuvant as an inflammatory agent into rat masseter muscle. No change in mRNA occurred in the ipsilateral ophthalmic and maxillary divisions (V1/V2) or in the contralateral V3. The levels of immunoreactive calcitonin gene-related peptide (iCGRP) in the ipsilateral V3 significantly increased at 1, 4 and 24 h following muscle inflammation. In contrast, no change occurred in iCGRP levels in either the ipsilateral V1/V2 or contralateral V3. When saline was injected into the masseter muscle, the levels of mRNA or iCGRP did not change in the ipsilateral V3 suggesting that the biochemical changes are specific to CFA-induced muscle inflammation. The number of muscle afferent neurons immunoreactive for CGRP was significantly reduced compared with control at 1, 4 and 24 h in the ipsilateral but not in the contralateral trigeminal ganglion following inflammation. This decrease in the ipsilateral ganglion may indicate a loss of intrasomatic CGRP as a result of increased axonal transport away from the neuronal cell body and/or release of CGRP. Behavioral testing showed a reduction in head withdrawal thresholds bilaterally from 30 min through 24 h following muscle inflammation. Thus upregulation of CGRP mRNA and iCGRP levels are temporally related to the development of inflammation and lowered pain thresholds. The present data support the hypothesis that CGRP is upregulated during deep tissue inflammation and suggest that gene transcription is involved in this upregulation.

Injection of adjuvant but not acidic saline into craniofacial muscle evokes nociceptive behaviors and neuropeptide expression.

Craniofacial muscle pain including muscular temporomandibular disorders accounts for a substantial portion of all pain perceived in the head and neck region. In spite of its high clinical prevalence, the mechanisms of chronic craniofacial muscle pain are not well understood. Injection of acidic saline into rodent hindlimb muscles produces pathologies which resemble muscular pathologies in chronic pain patients. Here we investigated whether analogous transformations occur following repeated injections of acidic saline into the rat masseter muscle. Injection of acidic saline (pH 4) into the masseter muscle transiently lowered i.m. pH to levels comparable to those reported for rodent hindlimb muscles. Nevertheless, repeated unilateral or bilateral injections of acidic saline (pH 4) into the masseter muscle failed to alter nociceptive behavioral responses as occurs in the hindlimb. Changing the pH of injected saline to pH 3.0 or 5.0 also did not evoke nocifensive behavior. Acid sensing ion channel 3 receptors, which are implicated in transformations following acidification of hindlimb muscles, were found on trigeminal ganglion muscle afferent neurons via combined neuronal tracing and immunocytochemistry. In contrast to the acidic saline, injection of complete Freunds adjuvant (CFA) into the masseter muscle induced mechanical allodynia for 3 weeks, thermal hyperalgesia for 1 week and an increase in the number of calcitonin gene-related peptide (CGRP)-immunoreactive muscle afferent neurons in the trigeminal ganglion. Although pH may alter CGRP release in primary afferent neurons, the number of CGRP-muscle afferent neurons did not change following i.m. injection of acidic saline. Further, there was no change in ganglionic iCGRP levels at 1, 4 or 12 days after i.m. injection of acidic saline. While these findings extend our earlier reports that CFA-induced muscle inflammation results in behavioral and neuropeptide changes they further suggest that i.m. acidification in craniofacial muscle evokes different responses than in hindlimb muscle and imply that disparate proton sensing mechanisms underlie these discrepancies.

Inflammation of craniofacial muscle induces widespread mechanical allodynia

The modulation of behavioral responses evoked by local and distant nociceptive stimuli following a discrete somatic injection of complete Freunds adjuvant (CFA) was examined in rats. Inflammation of one craniofacial muscle evoked mechanical allodynia not only in the region of inflammation but also secondary mechanical allodynia in the contralateral head, ipsilateral hindpaw, and contralateral hindpaw. In contrast to this, CFA-induced inflammation of either the hindpaw or gastrocnemius muscle evoked mechanical allodynia restricted to the hindlimb region. The widespread modulation of nocifensive behavior evoked by inflammation of deep craniofacial tissue found in this study resembles the widespread deep tissue pain reported in fibromyalgia, whiplash injury and some temporomandibular disorders and thus may provide insight into the mechanisms of these musculoskeletal pathologies.

Inflammation increases the excitability of masseter muscle afferents.

Temporomandibular disorder is a major health problem associated with chronic orofacial pain in the masticatory muscles and/or temporomandibular joint. Evidence suggests that changes in primary afferents innervating the muscles of mastication may contribute to temporomandibular disorder. However, there has been little systematic study of the mechanisms controlling the excitability of these muscle afferents, nor their response to inflammation. In the present study, we tested the hypotheses that inflammation increases the excitability of sensory neurons innervating the masseter muscle of the rat and that the ionic mechanisms underlying these changes are unique to these neurons. We examined inflammation-induced changes in the excitability of trigeminal ganglia muscle neurons following intramuscular injections of complete Freunds adjuvant. Three days after complete Freunds adjuvant injection acutely dissociated, retrogradely labeled trigeminal ganglia neurons were studied using whole cell patch clamp techniques. Complete Freunds adjuvant-induced inflammation was associated with an increase in neuronal excitability marked by a significant decrease in rheobase and increase in the slope of the stimulus response function assessed with depolarizing current injection. The increase in excitability was associated with significant decreases in the rate of action potential fall and the duration of the action potential afterhyperpolarization. These changes in excitability and action potential waveform were associated with significant shifts in the voltage-dependence of activation and steady-state availability of voltage-gated K(+) current as well as significant decreases in the density of voltage-gated K(+) current subject to steady-state inactivation. These data suggest that K(+) channel subtypes may provide novel targets for the treatment of pain arising from inflamed muscle. These results also support the hypothesis that the underlying mechanisms of pain arising from specific regions of the body are unique suggesting that it may be possible, if not necessary to treat pain originating from different parts of the body with specific therapeutic interventions.