Eccentric muscle contraction and stretching evoke mechanical hyperalgesia and modulate CGRP and P2X(3) expression in a functionally relevant manner.

Non-invasive, movement-based models were used to investigate muscle pain. In rats, the masseter muscle was rapidly stretched or electrically stimulated during forced lengthening to produce eccentric muscle contractions (EC). Both EC and stretching disrupted scattered myofibers and produced intramuscular plasma extravasation. Pro-inflammatory cytokines (IL-1beta, TNF-alpha, IL-6) and vascular endothelial growth factor (VEGF) were elevated in the masseter 24h following EC. At 48h, neutrophils increased and ED1 macrophages infiltrated myofibers while ED2 macrophages were abundant at 4d. Mechanical hyperalgesia was evident in the ipsilateral head 4h-4d after a single bout of EC and for 7d following multiple bouts (1 bout/d for 4d). Calcitonin gene-related peptide (CGRP) mRNA increased in the trigeminal ganglion 24h following EC while immunoreactive CGRP decreased. By 2d, CGRP-muscle afferent numbers equaled naive numbers implying that CGRP is released following EC and replenished within 2d. EC elevated P2X(3) mRNA and increased P2X(3) muscle afferent neuron number for 12d while electrical stimulation without muscle contraction altered neither CGRP nor P2X(3) mRNA levels. Muscle stretching produced hyperalgesia for 2d whereas contraction alone produced no hyperalgesia. Stretching increased CGRP mRNA at 24h but not CGRP-muscle afferent number at 2-12d. In contrast, stretching significantly increased the number of P2X(3) muscle afferent neurons for 12d. The sustained, elevated P2X(3) expression evoked by EC and stretching may enhance nociceptor responsiveness to ATP released during subsequent myofiber damage. Movement-based actions such as EC and muscle stretching produce unique tissue responses and modulate neuropeptide and nociceptive receptor expression in a manner particularly relevant to repeated muscle damage.

Nociceptive craniofacial muscle primary afferent neurons synapse in both the rostral and caudal brain stem.

Limited information is available on muscle afferent neurons with fine fibers despite their presumed participation in musculoskeletal disorders, including temporomandibular disorders. To study these neurons, intracellular recordings were made from the central axons of slowly conducting muscle afferent neurons in anesthetized rats. After intraaxonal impalement, axons were characterized by masseter nerve stimulation, receptive field testing, muscle stretching and intramuscular injection of hypertonic saline. Intracellular recordings were made from 310 axons (conduction velocity: 6.5-60(M)/s, mean = 27.3(M)/s; following frequency: 27-250 Hz, mean = 110Hz). No neurons responded to cutaneous palpation or muscle stretching. Some axons (n = 34) were intracellularly stained with biotinamide. These neurons were classified as group II/III noxious mechanoreceptors because their mechanical threshold exceeded 15 mN, and conduction velocities ranged from 12 to 40.2(M)/s (mean = 25.3(M)/s). Two morphological types were recognized by using an object-based, three-dimensional colocalization methodology to locate synapses. One type (IIIHTM(Vp-Vc)) possessed axon collaterals that emerged along the entire main axon and synapsed in the trigeminal principal sensory nucleus and spinal trigeminal subnuclei oralis (Vo), interpolaris (Vi), and caudalis (Vc). A second type (IIIHTM(Vo-Vc)) possessed axon collaterals that synapsed only in caudal Vo, Vi, and Vc. Our previous studies show that muscle spindle afferent neurons are activated by innocuous stimuli and synapse in the rostral and caudal brain stem; here we demonstrate that nociceptive muscle mechanoreceptor afferent axons also synapse in rostral and caudal brain stem regions. Traditional dogma asserts that the most rostral trigeminal sensory complex exclusively processes innocuous somatosensory information, whereas caudal portions receive nociceptive sensory input; the data reported here do not support this paradigm.

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 Freund's 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.

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

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.

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

The distribution and modulation of the P2X(3) 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 P2X(3)-positive neurons while only 16% of IB4 neurons expressed P2X(3). Neurons expressing P2X(3) alone were significantly larger than IB4-or IB4/P2X(3)-positive neurons. Investigation of target-specificity revealed that 22% of trigeminal ganglion muscle afferent neurons were positive for P2X(3) versus 16% of cutaneous afferent neurons. Muscle P2X(3) afferents were significantly smaller than the overall muscle afferent population while P2X(3) cutaneous afferent neurons were not. Presumptive heteromeric (P2X(2/3)) muscle afferent neurons were also identified and comprised 77% of the P2X(3) muscle afferent population. Muscle afferent neurons co-expressed P2X(3) with either calcitonin gene-related peptide (15%) or substance P (4%). The number of P2X(3)-positive muscle afferent neurons significantly increased one and four days following complete Freund's adjuvant-induced masseter muscle inflammation, but significantly decreased after 12 days. These results indicate that within trigeminal ganglia: (1) the P2X(3) receptor is expressed in both small and medium-sized neurons; (2) the P2X(3) receptor is not exclusively expressed in IB4 neurons; (3) P2X(3) is co-expressed with neuropeptides; (4) differences in the proportion of cutaneous versus muscle P2X(3) afferents are not apparent. Trigeminal P2X(3) neurons therefore differ markedly from dorsal root ganglion P2X(3) afferents. This study also shows that deep tissue inflammation modulates expression of the P2X(3) receptor and thus may warrant exploration as a target for therapeutic intervention.

Neuronal activation in the medulla oblongata during selective elicitation of the laryngeal adductor response.

Swallow and cough are complex motor patterns elicited by rapid and intense electrical stimulation of the internal branch of the superior laryngeal nerve (ISLN). The laryngeal adductor response (LAR) includes only a laryngeal response, is elicited by single stimuli to the ISLN, and is thought to represent the brain stem pathway involved in laryngospasm. To identify which regions in the medulla are activated during elicitation of the LAR alone, single electrical stimuli were presented once every 2 s to the ISLN. Two groups of five cats each were studied; an experimental group with unilateral ISLN stimulation at 0.5 Hz and a surgical control group. Three additional cats were studied to evaluate whether other oral, pharyngeal, or respiratory muscles were activated during ISLN stimulation eliciting LAR. We quantified < or = 22 sections for each of 14 structures in the medulla to determine if regions had increased Fos-like immunoreactive neurons in the experimental group. Significant increases (P < 0.0033) occurred with unilateral ISLN stimulation in the interstitial subnucleus, the ventrolateral subnucleus, the commissural subnucleus of the nucleus tractus solitarius, the lateral tegmental field of the reticular formation, the area postrema, and the nucleus ambiguus. Neither the dorsal motor nucleus of the vagus, usually active for swallow, nor the nucleus retroambiguus, retrofacial nucleus, and the lateral reticular nucleus, usually active for cough, were active with elicitation of the laryngeal adductor response alone. The results demonstrate that the laryngeal adductor pathway is contained within the broader pathways for cough and swallow in the medulla.

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.

Selective suppression of late laryngeal adductor responses by N-methyl-D-aspartate receptor blockade in the cat.

Laryngeal adductor responses to afferent stimulation play a key role in airway protection. Although vital for protection during cough and swallow, these responses also must be centrally controlled to prevent airway obstruction by laryngospasm during prolonged stimulation. Our purpose was to determine the role of N-methyl-D-aspartate (NMDA) receptors in modulating early R1 responses (at 9 ms) and/or later more prolonged R2 responses (at 36 ms) during electrical stimulation of the laryngeal afferent fibers contained in the internal branch of the superior laryngeal nerve in the cat. The percent occurrence, amplitude, and conditioning of muscle responses to single superior laryngeal nerve (SLN) stimuli presented in pairs at interstimulus intervals of 250 ms were measured in three experiments: 1) animals that had ketamine as anesthetic premedication were compared with those who did not, when both were maintained under alpha-chloralose anesthesia. 2) The effects of administering ketamine in one group of animals were compared with increasing the depth of alpha-chloralose anesthesia without NMDA receptor blockade in another group of animals. 3) The effects of dextromethorphan (without anesthetic effects) were examined in another group of animals. In the first experiment, the occurrence of R2 responses were reduced from 95% in animals without ketamine premedication to 25% in animals with ketamine premedication (P = 0.015). No differences occurred in the occurrence, amplitude, latency, or conditioning effects on R1 responses between these groups. In the second experiment, the occurrence of R2 responses was reduced from 96 to 79% after an increase in the depth of anesthesia with alpha-chloralose in contrast with reductions in R2 occurrence from 98 to 19% following the administration of ketamine to induce NMDA receptor blockade along with increased anesthesia (P = 0.025). In the third experiment, R2 occurrence was reduced from 89 to 27% (P = 0.017) with administration of dextromethorphan while R1 response occurrence and amplitude did not change. In each of these experiments, NMDA receptor blockade did not have significant effects on cardiac or respiratory rates in any of the animals. The results demonstrate that NMDA receptors play an essential role in long latency R2 laryngeal responses to laryngeal afferent stimulation. On the other hand, early R1 laryngeal adductor responses are likely to involve non-NMDA receptor activation.