Differential Activation of Colonic Afferents and Dorsal Horn Neurons Underlie Stress-Induced and Comorbid Visceral Hypersensitivity in Female Rats.

Chronic Overlapping Pain Conditions, including irritable bowel syndrome (IBS) and temporomandibular disorder (TMD), represent a group of idiopathic pain conditions that likely have peripheral and central mechanisms contributing to their pathology, but are poorly understood. These conditions are exacerbated by stress and have a female predominance. The presence of one condition predicts the presence or development of additional conditions, making this a significant pain management problem. The current study was designed to determine if the duration and magnitude of peripheral sensitization and spinal central sensitization differs between restraint stress-induced visceral hypersensitivity (SIH) and chronic comorbid pain hypersensitivity (CPH; stress during pre-existing orofacial pain). SIH in female rats, as determined by the visceromotor response, persisted at least four but resolved by seven weeks. In contrast, CPH persisted at least seven weeks. Surprisingly, colonic afferents in both SIH and CPH rats were sensitized at seven weeks. CPH rats also had referred pain through seven weeks, but locally anesthetizing the colon only attenuated the referred pain through four weeks, suggesting a transition to colonic afferent independent central sensitization. Different phenotypes of dorsal horn neurons were sensitized in the CPH rats seven weeks post stress compared to four weeks or SIH rats. The current study suggests differential processing of colonic afferent input to the lumbosacral spinal cord contributes to visceral hypersensitivity during comorbid chronic pain conditions. PERSPECTIVE: Chronic Overlapping Pain Conditions represent a unique challenge in pain management. The diverse nature of peripheral organs hinders a clear understanding of underlying mechanisms accounting for the comorbidity. This study highlights a mismatch between the condition-dependent behavior and peripheral and spinal mechanisms that contribute to visceral pain hypersensitivity.

Peripheral mechanisms contribute to comorbid visceral hypersensitivity induced by preexisting orofacial pain and stress in female rats.

BACKGROUND: Stress exacerbates many chronic pain syndromes including irritable bowel syndrome (IBS). Among these patient populations, many suffer from comorbid or chronic overlapping pain conditions and are predominantly female. Nevertheless, basic studies investigating chronic psychological stress-induced changes in pain sensitivity have been mostly carried out in male rodents. Our laboratory developed a model of comorbid pain hypersensitivity (CPH) (stress in the presence of preexisting orofacial pain inducing chronic visceral pain hypersensitivity that significantly outlasts transient stress-induced pain hypersensitivity (SIH)) facilitating the study of pain associated with IBS. Since CPH and SIH are phenotypically similar until SIH resolves and CPH persists, it is unclear if underlying mechanisms are similar. METHODS: In the present study, the visceromotor response (VMR) to colorectal distention was recorded in the SIH and CPH models in intact females and ovariectomized rats plus estradiol replacement (OVx + E2). Over several months, rats were determined to be susceptible or resilient to stress and the role of peripheral corticotrophin-releasing factor (CRF) underlying in the pain hypersensitivity was examined. KEY RESULTS: Stress alone induced transient (3-4 weeks) visceral hypersensitivity, though some rats were resilient. Comorbid conditions increased susceptibility to stress prolonging hypersensitivity beyond 13 weeks. Both models had robust peripheral components; hypersensitivity was attenuated by the CRF receptor antagonist astressin and the mast cell stabilizer disodium cromoglycate (DSCG). However, DSCG was less effective in the CPH model compared to the SIH model. CONCLUSIONS AND INFERENCES: The data indicate many similarities but some differences in mechanisms contributing to comorbid pain conditions compared to transient stress-induced pain.

A clinically relevant animal model of temporomandibular disorder and irritable bowel syndrome comorbidity.

UNLABELLED: Temporomandibular disorder and irritable bowel syndrome are comorbid functional chronic pain disorders of unknown etiology that are triggered/exacerbated by stress. Here we present baseline phenotypic characterization of a novel animal model to gain insight into the underlying mechanisms that contribute to such comorbid pain conditions. In this model, chronic visceral hypersensitivity, a defining symptom of irritable bowel syndrome, is dependent on 3 factors: estradiol, existing chronic somatic pain, and stress. In ovariectomized rats, estradiol replacement followed by craniofacial muscle injury and stress induced visceral hypersensitivity that persisted for months. Omission of any 1 factor resulted in a transient (1 week) visceral hypersensitivity from stress alone or no hypersensitivity (no inflammation or estradiol). Maintenance of visceral hypersensitivity was estradiol dependent, resolving when estradiol replacement ceased. Referred cutaneous hypersensitivity was concurrent with visceral hypersensitivity. Increased spinal Fos expression suggests induction of central sensitization. These data demonstrate the development and maintenance of visceral hypersensitivity in estradiol-replaced animals following distal somatic injury and stress that mimics some characteristics reported in patients with temporomandibular disorder and comorbid irritable bowel syndrome. This new animal model is a powerful experimental tool that can be employed to gain further mechanistic insight into overlapping pain conditions. PERSPECTIVE: The majority of patients with temporomandibular disorder report symptoms consistent with irritable bowel syndrome. Stress and female prevalence are common to both conditions. In a new experimental paradigm in ovariectomized rats with estradiol replacement, masseter inflammation followed by stress induces visceral hypersensitivity that persists for months, modeling these comorbid pain conditions.

Repeated muscle injury as a presumptive trigger for chronic masticatory muscle pain.

skeletal muscles sustain a significant loss of maximal contractile force after injury, but terminally damaged fibers can eventually be replaced by the growth of new muscle (regeneration), with full restoration of contractile force over time. After a second injury, limb muscles exhibit a smaller reduction in maximal force and reduced inflammation compared with that after the initial injury (i.e., repeated bout effect). In contrast, masticatory muscles exhibit diminished regeneration and persistent fibrosis, after a single injury; following a second injury, plasma extravasation is greater than after a single injury and maximal force is decreased more than after the initial injury. Thus, masticatory muscles do not exhibit a repeated bout effect and are instead increasingly damaged by repeated injury. We propose that the impaired ability of masticatory muscles to regenerate contributes to chronic muscle pain by leading to an accumulation of tissue damage, fibrosis, and a persistent elevation and prolonged membrane translocation of nociceptive channels such as P2X(3) as well as enhanced expression of neuropeptides including CGRP within primary afferent neurons. These transformations prime primary afferent neurons for enhanced responsiveness upon subsequent injury thus triggering and/or exacerbating chronic muscle pain.

Physiological, morphological and neurochemical characterization of neurons modulated by movement.

The role of individual neurons and their function in neuronal circuits is fundamental to understanding the neuronal mechanisms of sensory and motor functions. Most investigations of sensorimotor mechanisms rely on either examination of neurons while an animal is static or record extracellular neuronal activity during a movement. While these studies have provided the fundamental background for sensorimotor function, they either do not evaluate functional information which occurs during a movement or are limited in their ability to fully characterize the anatomy, physiology and neurochemical phenotype of the neuron. A technique is shown here which allows extensive characterization of individual neurons during an in vivo movement. This technique can be used not only to study primary afferent neurons but also to characterize motoneurons and sensorimotor interneurons. Initially the response of a single neuron is recorded using electrophysiological methods during various movements of the mandible followed by determination of the receptive field for the neuron. A neuronal tracer is then intracellularly injected into the neuron and the brain is processed so that the neuron can be visualized with light, electron or confocal microscopy (Fig. 1). The detailed morphology of the characterized neuron is then reconstructed so that neuronal morphology can be correlated with the physiological response of the neuron (Figs. 2,3). In this communication important key details and tips for successful implementation of this technique are provided. Valuable additional information can be determined for the neuron under study by combining this method with other techniques. Retrograde neuronal labeling can be used to determine neurons with which the labeled neuron synapses; thus allowing detailed determination of neuronal circuitry. Immunocytochemistry can be combined with this method to examine neurotransmitters within the labeled neuron and to determine the chemical phenotypes of neurons with which the labeled neuron synapses. The labeled neuron can also be processed for electron microscopy to determine the ultrastructural features and microcircuitry of the labeled neuron. Overall this technique is a powerful method to thoroughly characterize neurons during in vivo movement thus allowing substantial insight into the role of the neuron in sensorimotor function.

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.

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 Freund's 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.

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.

Classification of muscle spindle afferents innervating the masseter muscle in rats.

Taylor et al. [Taylor, A., Durbaba, R., Rodgers, J.F., 1992a. The classification of afferents from muscle spindles of the jaw-closing muscles of the cat. J Physiol 456, 609-628] developed a method to classify muscle spindle afferents using succinylcholine (Sch) and ramp and hold stretches. They demonstrated that cat jaw muscle spindle afferents show high proportion of intermediate responses to ramp and hold jaw stretch. Together with observations on the responses to Sch their data suggests that the majority of jaw muscle spindle afferents are influenced by a combination of nuclear bag(2) and nuclear chain fibres. Relatively few are influenced solely by nuclear bag(1) fibres. The purpose of this study was to categorize jaw muscle spindle afferent in rodents in response to ramp and hold stretches. Several measures were used to classify spindle afferents including (1) conduction velocity, (2) coefficient of variation (C.V.) of the interspike interval during jaw opening, and (3) the dynamic sensitivity and the initial discharge of spindle afferents before and after succinylcholine infusion (Sch, 100mg/kg, i.v.). Consistent with observations in the cat jaw muscles, the distribution of the conduction velocity and the C.V. of Vmes masseter afferents were unimodal. Therefore, these parameters were of little value in functional classification of spindle innervation. Succinylcholine injection either markedly increased the dynamic sensitivity or produced no change in Vmes afferents. Unlike cat jaw muscle spindle afferents, the effect of Sch on the initial discharge was not clearly separable from those responding or not responding to Sch. These results suggest that rat jaw muscle spindle afferents, have physiological properties that are primarily intermediate in nature and are likely to reflect a predominance of influence from nuclear bag(2) and chain fibres. However, the distinction between bag(2) and chain fibres influences is not as clearly defined in the rat compared to the cat.

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.

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.