Neuronal-immune axis alters pain and sensory afferent damage during dental pulp injury.

ABSTRACT: Dental pulp tissue is densely innervated by afferent fibers of the trigeminal ganglion. When bacteria cause dental decay near the pulpal tissue, a strong neuronal and immune response occurs, creating pulpitis, which is associated with severe pain and pulp tissue damage. Neuroimmune interactions have the potential to modulate both the pain and pathological outcome of pulpitis. We first investigated the role of the neuropeptide calcitonin gene-related peptide (CGRP), released from peptidergic sensory afferents, in dental pain and immune responses by using Calca knockout (Calca -/- ) and wild-type (Calca +/+ ) mice, in a model of pulpitis by creating a mechanical exposure of the dental pulp horn. We found that the neuropeptide CGRP, facilitated the recruitment of myeloid cells into the pulp while also increasing spontaneous pain-like behavior 20% to 25% at an early time point. Moreover, when we depleted neutrophils and monocytes, we found that there was 20% to 30% more sensory afferent loss and increased presence of bacteria in deeper parts of the tissue, whereas there was a significant reduction in mechanical pain response scores compared with the control group at a later time point. Overall, we showed that there is a crosstalk between peptidergic neurons and neutrophils in the pulp, modulating the pain and inflammatory outcomes of the disease.

Lipopolysaccharide-Induced TRPA1 Upregulation in Trigeminal Neurons is Dependent on TLR4 and Vesicular Exocytosis.

Pain from bacterial infection was believed to be the consequence of inflammation induced by bacterial products. However recent studies have shown that bacterial products can directly activate sensory neurons and induce pain. The mechanisms by which bacteria induce pain are poorly understood, but toll-like receptor (TLR)4 and transient receptor potential A1 (TRPA1) receptors are likely important integrators of pain signaling induced by bacteria. Using male and female mice we show that sensory neuron activation by bacterial lipopolysaccharides (LPS) is mediated by both TRPA1 and TLR4 and involves the mobilization of extracellular and intracellular calcium. We also show that LPS induces neuronal sensitization in a process dependent on TLR4 receptors. Moreover, we show that TLR4 and TRPA1 are both involved in sensory neurons response to LPS stimulation. Activation of TLR4 in a subset of sensory neurons induces TRPA1 upregulation at the cell membrane through vesicular exocytosis, contributing to the initiation of neuronal sensitization and pain. Collectively these data highlight the importance of sensory neurons to pathogen detection, and their activation by bacterial products like LPS as potentially important to early immune and nociceptive responses.SIGNIFICANCE STATEMENT Bacterial infections are often painful and the recent discovery that bacteria can directly stimulate sensory neurons leading to pain sensation and modulation of immune system have highlighted the importance of nervous system in the response to bacterial infection. Here, we showed that lipopolysaccharide, a major bacterial by-product, requires both toll-like receptor (TLR)4 and transient receptor potential A1 (TRPA1) receptors for neuronal activation and acute spontaneous pain, but only TLR4 mediates sensory neurons sensitization. Moreover, we showed for the first time that TLR4 sensitize sensory neurons through a rapid upregulation of TRPA1 via vesicular exocytosis. Our data highlight the importance of sensory neurons to pathogen detection and suggests that TLR4 would be a potential therapeutic target to modulate early stage of bacteria-induced pain and immune response.

Excess Growth Hormone Triggers Inflammation-Associated Arthropathy, Subchondral Bone Loss, and Arthralgia.

Growth hormone (GH) is a key mediator of skeletal growth. In humans, excess GH secretion due to pituitary adenoma, seen in patients with acromegaly, results in severe arthropathies. This study investigated the effects of long-term excess GH on the knee joint tissues. One year-old wild-type (WT) and bovine GH (bGH) transgenic mice were used as a model for excess GH. bGH mice showed increased sensitivity to mechanical and thermal stimuli, compared with WT mice. Micro-computed tomography analyses of the distal femur subchondral bone revealed significant reductions in trabecular thickness and significantly reduced bone mineral density of the tibial subchondral bone-plate associated with increased osteoclast activity in both male and female bGH compared with WT mice. bGH mice showed severe loss of matrix from the articular cartilage, osteophytosis, synovitis, and ectopic chondrogenesis. Articular cartilage loss in the bGH mice was associated with elevated markers of inflammation and chondrocyte hypertrophy. Finally, hyperplasia of synovial cells was associated with increased expression of Ki-67 and diminished p53 levels in the synovium of bGH mice. Unlike the low-grade inflammation seen in primary osteoarthritis, arthropathy caused by excess GH affects all joint tissues and triggers severe inflammatory response. Data from this study suggest that treatment of acromegalic arthropathy should involve inhibition of ectopic chondrogenesis and chondrocyte hypertrophy.

Glia and Orofacial Pain: Progress and Future Directions.

Orofacial pain is a universal predicament, afflicting millions of individuals worldwide. Research on the molecular mechanisms of orofacial pain has predominately focused on the role of neurons underlying nociception. However, aside from neural mechanisms, non-neuronal cells, such as Schwann cells and satellite ganglion cells in the peripheral nervous system, and microglia and astrocytes in the central nervous system, are important players in both peripheral and central processing of pain in the orofacial region. This review highlights recent molecular and cellular findings of the glia involvement and glia-neuron interactions in four common orofacial pain conditions such as headache, dental pulp injury, temporomandibular joint dysfunction/inflammation, and head and neck cancer. We will discuss the remaining questions and future directions on glial involvement in these four orofacial pain conditions.

Effects of Calcitonin Gene-related Peptide on Dental Pulp Stem Cell Viability, Proliferation, and Differentiation.

INTRODUCTION: Pulpitis is an inflammation of dental pulp caused by bacterial proliferation near or within pulpal tissues. In advanced stages, when the inflammation is associated with pulp necrosis, pulp preservation is dependent on dental pulp stem cells (DPSCs) that can differentiate into odontoblastlike cells and produce reparative dentin. In this study, we evaluated the influence of sensory neurons through calcitonin gene-related peptide (CGRP) on DPSC viability and proliferation and the ability of DPSCs to differentiate into mineralizing cells. METHODS: Commercially available DPSCs were treated with varying doses of CGRP, and metabolic activity, viability, proliferation, and cell death were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays, trypan blue staining, 5-bromo-2'-deoxyuridine cell proliferation assay, and caspase-3 staining, respectively. DPSC differentiation was assessed with alizarin red staining and by quantifying messenger RNA expression of odontoblast makers. RESULTS: CGRP induced a dose-dependent decrease of DPSC metabolic activity that was prevented by the CGRP receptor antagonist CGRP 8-37. The decrease in the proportion of live cells induced by CGRP is associated with a decrease of cell proliferation but not with caspase-3-dependent apoptosis. Interestingly, dexamethasone-induced DPSC differentiation into mineralizing cells was neither inhibited nor enhanced by CGRP treatment. CONCLUSIONS: The neuropeptide CGRP has an inhibitory effect on DPSC proliferation but does not enhance or inhibit the differentiation of DPSCs into mineralizing cells. This suggests that CGRP might negatively influence the ability of DPSCs to contribute to regenerative or tissue repair processes.

TRPM8 and TRPA1 do not contribute to dental pulp sensitivity to cold.

Sensory neurons innervating the dental pulp have unique morphological and functional characteristics compared to neurons innervating other tissues. Stimulation of dental pulp afferents whatever the modality or intensity of the stimulus, even light mechanical stimulation that would not activate nociceptors in other tissues, produces an intense pain. These specific sensory characteristics could involve receptors of the Transient Receptor Potential channels (TRP) family. In this study, we compared the expression of the cold sensitive receptors TRPM8 and TRPA1 in trigeminal ganglion neurons innervating the dental pulp, the skin of the cheek or the buccal mucosa and we evaluated the involvement of these receptors in dental pulp sensitivity to cold. We showed a similar expression of TRPM8, TRPA1 and CGRP in sensory neurons innervating the dental pulp, the skin or the mucosa. Moreover, we demonstrated that noxious cold stimulation of the tooth induced an overexpression of cFos in the trigeminal nucleus that was not prevented by the genetic deletion of TRPM8 or the administration of the TRPA1 antagonist HC030031. These data suggest that the unique sensory characteristics of the dental pulp are independent to TRPM8 and TRPA1 receptors expression and functionality.

Molecular, cellular and behavioral changes associated with pathological pain signaling occur after dental pulp injury.

Abstract: Persistent pain can occur after routine dental treatments in which the dental pulp is injured. To better understand pain chronicity after pulp injury, we assessed whether dental pulp injury in mice causes changes to the sensory nervous system associated with pathological pain. In some experiments, we compared findings after dental pulp injury to a model of orofacial neuropathic pain, in which the mental nerve is injured. After unilateral dental pulp injury, we observed increased expression of activating transcription factor 3 (ATF3) and neuropeptide Y (NPY) mRNA and decreased tachykinin precursor 1 gene expression, in the ipsilateral trigeminal ganglion. We also observed an ipsilateral increase in the number of trigeminal neurons expressing immunoreactivity for ATF3, a decrease in substance P (SP) immunoreactive cells, and no change in the number of cells labeled with IB4. Mice with dental pulp injury transiently exhibit hindpaw mechanical allodynia, out to 12 days, while mice with mental nerve injury have persistent hindpaw allodynia. Mice with dental pulp injury increased spontaneous consumption of a sucrose solution for 17 days while mental nerve injury mice did not. Finally, after dental pulp injury, an increase in expression of the glial markers Iba1 and glial fibrillary acidic protein occurs in the transition zone between nucleus caudalis and interpolaris, ipsilateral to the injury. Collectively these studies suggest that dental pulp injury is associated with significant neuroplasticity that could contribute to persistent pain after of dental pulp injury.

Immunohistochemical comparison of astrocytic mGluR5 upregulation in infraorbital nerve- versus sciatic nerve-ligated rat.

The differential pharmacological responsiveness of cephalic and extra-cephalic neuropathic pain has been proposed to relate to distinct mechanisms that may involve neuroinflammatory reactions mediated by glial cells. Astrocytes are particularly important for neuronal sensitization in neuropathic pain, in part through modulation of glutamatergic transmission. Because the metabotropic glutamate receptor 5 (mGluR5) is involved in the astrocytic regulation of the glutamatergic system, we investigated modifications of its expression in models of cephalic versus extra-cephalic neuropathic pain. Adult male rats underwent unilateral chronic constriction injury (CCI) of either the infraorbital nerve (ION) or the sciatic nerve (SN). Seven days later, mGluR5 and the astrocyte marker GFAP (glial fibrillary acidic protein) were overexpressed and appeared localized mainly in the superficial lamina of the trigeminal nucleus in CCI-ION and the spinal cord dorsal horn in CCI-SN rats. In addition, colocalization of GFAP and mGluR5 strongly suggested an increase of astrocytic mGluR5 expression in nerve-injured rats compared to sham animals. The present data show an upregulation of astrocytic mGluR5 in central structures in both CCI-ION and CCI-SN. This suggests that the pharmacological modulation of mGluR5 could be a new approach to reduce both cephalic and extra-cephalic neuropathic pain.

Bilateral tactile hypersensitivity and neuroimmune responses after spared nerve injury in mice lacking vasoactive intestinal peptide.

Vasoactive intestinal peptide (VIP) is one of the neuropeptides showing the strongest up-regulation in the nociceptive pathway after peripheral nerve injury and has been proposed to support neuropathic pain. Nevertheless, the story may be more complicated considering the known suppressive effects of the peptide on the immune reactivity of microglial cells, which have been heavily implicated in the onset and maintenance of pain after nerve injury. We here used mice deficient in VIP and the model of spared nerve injury, characterized by persistent tactile hypersensitivity. While tactile hypersensitivity developed similarly to wild type mice for the ipsilateral hindpaw, only transgenic mice showed a mirror-image tactile hypersensitivity in the contralateral hindpaw. This exacerbated neuropathic pain phenotype appeared to be mediated through a local mechanism acting at the level of the lumbar spinal cord as a distant nerve lesion in the front limb did not lead to hindpaw hypersensitivity in VIP-deficient mice. Innocuous tactile hindpaw stimulation was found to increase a neuronal activation marker in the bilateral superficial laminae of the lumbar dorsal horn of VIP-deficient, but not wild type mice, after SNI. A deeper study into the immune responsiveness to the nerve lesion also proved that VIP-deficient mice had a stronger early pro-inflammatory cytokine response and a more pronounced microglial reactivity compared to wild type controls. The latter was also observed at four weeks after spared nerve injury, a time at which bilateral tactile hypersensitivity persisted in VIP-deficient mice. These data suggest an action of VIP in neuropathic states that is more complicated than previously assumed. Future research is now needed for a deeper understanding of the relative contribution of receptors and fiber populations involved in the VIP-neuropathic pain link.

Respective pharmacological features of neuropathic-like pain evoked by intrathecal BDNF versus sciatic nerve ligation in rats.

Numerous reported data support the idea that Brain Derived Neurotrophic Factor (BDNF) is critically involved in both depression and comorbid pain. The possible direct effect of BDNF on pain mechanisms was assessed here and compared with behavioral/neurobiological features of neuropathic pain caused by chronic constriction injury to the sciatic nerve (CCI-SN). Sprague-Dawley male rats were either injected intrathecally with BDNF (3.0 ng i.t.) or subjected to unilateral CCI-SN. Their respective responses to anti-hyperalgesic drugs were assessed using the Randall-Selitto test and both immunohistochemical and RT-qPCR approaches were used to investigate molecular/cellular mechanisms underlying hyperalgesia in both models. Long lasting hyperalgesia and allodynia were induced by i.t. BDNF in intact healthy rats like those found after CCI-SN. Acute treatment with the BDNF-TrkB receptor antagonist cyclotraxin B completely prevented i.t. BDNF-induced hyperalgesia and partially reversed this symptom in both BDNF-pretreated and CCI-SN lesioned rats. Acute administration of the anticonvulsant pregabalin, the NMDA receptor antagonist ketamine, the opioid analgesics morphine and tapentadol or the antidepressant agomelatine also transiently reversed hyperalgesia in both i.t. BDNF injected- and CCI-SN lesioned-rats. Marked induction of microglia activation markers (OX42, Iba1, P-p38), proinflammatory cytokine IL-6, NMDA receptor subunit NR2B and BDNF was found in spinal cord and/or dorsal root ganglia of CCI-SN rats. A long lasting spinal BDNF overexpression was also observed in BDNF i.t. rats, indicating an autocrine self-induction, with downstream long lasting TrkB-mediated neuropathic-like pain. Accordingly, TrkB blockade appeared as a relevant approach to alleviate not only i.t. BDNF- but also nerve lesion-evoked neuropathic pain.

Modulation of spinal glial reactivity by intrathecal PPF is not sufficient to inhibit mechanical allodynia induced by nerve crush.

Spinal glial reactivity has been strongly implicated in pain that follows peripheral nerve injury. Among the many therapeutic agents that have been tested for anti-allodynia through immune modulation is the atypical methylxanthine propentofylline. While propentofylline shows a potent anti-allodynia effect after nerve transection injury, we here demonstrate that, when propentofylline is used intrathecally at the effective immune-modulatory dose, allodynia after rat nerve crush injury is completely preserved. Microglial/macrophage Iba-1 and astrocytic GFAP expression, increased in the dorsal horn of nerve crushed animals, was, however, effectively attenuated by propentofylline. Effective modulation of spinal glial reactivity is, thus, no assurance for anti-allodynia.

Differential pharmacological alleviation of oxaliplatin-induced hyperalgesia/allodynia at cephalic versus extra-cephalic level in rodents.

Previous data showed that neuropathic pain induced by mechanical lesion of peripheral nerves responds differently to alleviating drugs at cephalic versus extracephalic level. Because neuropathic pain evoked by anti-cancer drugs differs from that triggered by mechanical nerve lesion, we investigated whether differences between cephalic and extracephalic levels could also be characterized in rodents rendered neuropathic by treatment with the anti-cancer platinum derivative oxaliplatin. C57BL/6J mice received two injections and Sprague-Dawley rats three injections of oxaliplatin (10 mg/kg, i.p.) or its vehicle, with three days intervals. Supersensitivity to mechanical (von Frey filaments), cold (acetone drop) and chemical/inflammatory (formalin) stimulations was assessed in vibrissae and hindpaw territories. Transcripts of neuroinflammatory markers were quantified by real-time RT-qPCR in rat ganglia and central tissues. Oxaliplatin induced mechanical allodynia, cold hyperalgesia and chemical/inflammatory supersensitivity at both hindpaw and vibrissal levels in mice and rats. Acute treatment with gabapentin (30 mg/kg i.p.), morphine (3 mg/kg s.c.) or the 5-HT1A receptor agonist 8-OH-DPAT (0.16 mg/kg s.c.) significantly reduced oxaliplatin-induced supersensitivity in hindpaw but not vibrissal territory. In contrast, the antimigraine drugs naratriptan (0.1 mg/kg s.c.) and olcegepant (0.6 mg/kg i.v.) decreased oxaliplatin-induced supersensitivity in vibrissal territory only. Among the various markers investigated, only TRPA1 transcript was upregulated in ganglia of oxaliplatin-treated rats. These data showed that oxaliplatin induced supersensitivity to various stimuli in both cephalic and extra-cephalic territories in rodents. Regional differences in the efficacy of drugs to alleviate oxaliplatin-induced allodynia/hyperalgesia further support the idea that mechanisms underlying neuropathic pain have peculiarities at cephalic versus extra-cephalic level.

Multiple roles of serotonin in pain control mechanisms--implications of 5-HT7 and other 5-HT receptor types.

Among monoamine neurotransmitters, serotonin (5-HT) is known to play complex modulatory roles in pain signaling mechanisms since the first reports, about forty years ago, on its essentially pro-nociceptive effects at the periphery and anti-nociceptive effects when injected directly at the spinal cord level. The discovery of multiple 5-HT receptor subtypes allowed possible explanations to this dual action at the periphery versus the central nervous system (CNS) since both excitatory and inhibitory effects can be exerted through 5-HT activation of different 5-HT receptors. However, it also appeared that activation of the same receptor subtype at CNS level might induce variable effects depending on the physiological or pathophysiological status of the animal administered with agonists. In particular, the marked neuroplastic changes induced by nerve lesion, which account for sensitization of pain signaling mechanisms, can contribute to dramatic changes in the effects of a given 5-HT receptor agonist in neuropathic rats versus intact healthy rats. This has notably been observed with 5-HT7 receptor agonists which exert a pronociceptive action in healthy rats but alleviate hyperalgesia consecutive to nerve lesion in neuropathic animals. Analysis of cellular mechanisms underlying such dual 5-HT actions mediated by a single receptor subtype indicates that the neuronal phenotype which expresses this receptor also plays a key role in determining which modulatory action 5-HT would finally exert on pain signaling mechanisms.

GABA, but not opioids, mediates the anti-hyperalgesic effects of 5-HT7 receptor activation in rats suffering from neuropathic pain.

Among receptors mediating serotonin actions in pain control, the 5-HT(7)R is of special interest because it is expressed by primary afferent fibers and intrinsic GABAergic and opioidergic interneurons within the spinal dorsal horn. Herein, we investigated whether GABA and/or opioids contribute to 5-HT(7)R-mediated control of neuropathic pain caused by nerve ligation. Acute administration of 5-HT(7)R agonists (AS-19, MSD-5a, E-55888) was found to markedly reduce mechanical and thermal hyperalgesia in rats with unilateral constriction injury to the sciatic nerve (CCI-SN). In contrast, mechanical hypersensitivity caused by unilateral constriction injury to the infraorbital nerve was essentially unaffected by these ligands. Further characterization of the anti-hyperalgesic effect of 5-HT(7)R activation by the selective agonist E-55888 showed that it was associated with a decrease in IL-1ß mRNA overexpression in ipsilateral L4-L6 dorsal root ganglia and lumbar dorsal horn in CCI-SN rats. In addition, E-55888 diminished CCI-SN-associated increase in c-Fos immunolabeling in superficial laminae of the lumbar dorsal horn and the locus coeruleus, but increased c-Fos immunolabeling in the nucleus tractus solitarius and the parabrachial area in both control and CCI-SN rats. When injected intrathecally (i.t.), bicuculline (3 µg i.t.), but neither phaclofen (5 µg i.t.) nor naloxone (10 µg i.t.), significantly reduced the anti-hyperalgesic effects of 5-HT(7)R activation (E-55888, 10 mg/kg s.c.) in CCI-SN rats. These data support the idea that 5-HT(7)R-mediated inhibitory control of neuropathic pain is underlain by excitation of GABAergic interneurons within the dorsal horn. In addition, 5-HT(7)R activation-induced c-Fos increase in the nucleus tractus solitarius and the parabrachial area suggests that supraspinal mechanisms might also be involved.

Differential effects of calcitonin gene-related peptide receptor blockade by olcegepant on mechanical allodynia induced by ligation of the infraorbital nerve vs the sciatic nerve in the rat.

Previous studies showed that 5-hydroxytryptamine (5-HT)(1B/1D) receptor stimulation by triptans alleviates neuropathic pain caused by chronic constriction injury to the infraorbital nerve (CCI-ION) but not the sciatic nerve (CCI-SN) in rats. To assess whether such differential effects in the cephalic vs extracephalic territories is a property shared by other antimigraine drugs, we used the same models to investigate the effects of olcegepant, which has an antimigraine action mediated through calcitonin gene-related peptide (CGRP) receptor blockade. Adult male rats underwent unilateral CCI to the ION or the SN, and subsequent allodynia and/or hyperalgesia were assessed in ipsilateral vibrissal territory or hindpaw, respectively, using von Frey filaments and validated nociceptive tests. c-Fos expression was quantified by immunohistochemistry and interleukin 6 and activating transcription factor 3 (ATF3) mRNAs by real-time quantitative reverse transcriptase-polymerase chain reaction. Like naratriptan (0.1 to 0.3mg/kg, subcutaneously), olcegepant (0.3 to 0.9mg/kg, intravenously) markedly reduced mechanical allodynia in CCI-ION rats. In contrast, in CCI-SN rats, mechanical allodynia was completely unaffected and hyperalgesia was only marginally reduced by these drugs. A supra-additive antiallodynic effect was observed in CCI-ION rats treated with olcegepant (0.3mg/kg intravenously) plus naratriptan (0.1mg/kg subcutaneously), whereas this drug combination remained inactive in CCI-SN rats. Olcegepant (0.6mg/kg, intravenously) significantly reduced the number of c-Fos immunolabeled cells in spinal nucleus of the trigeminal nerve and upregulation of ATF3 transcript (a marker of neuron injury) but not that of interleukin-6 in trigeminal ganglion of CCI-ION rats. These findings suggest that CGRP receptor blockade might be of potential interest to alleviate trigeminal neuropathic pain.

Differential anti-neuropathic pain effects of tetrodotoxin in sciatic nerve- versus infraorbital nerve-ligated rats--behavioral, pharmacological and immunohistochemical investigations.

Several voltage-gated sodium channels are expressed in primary sensory neurons where they control excitability and participate in the generation and propagation of action potentials. Peripheral nerve injury-induced alterations in both tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels have been proposed to contribute to neuropathic pain caused by such lesion. We herein investigated whether the blockade of TTX-sensitive channels could reduce pain-related behaviors and evoked c-Fos immunoreactivity in rats with neuropathic pain produced by chronic unilateral constriction injury to either the sciatic nerve or the infraorbital nerve. Acute as well as subchronic administration of TTX (1-6 mug/kg s.c.) was found to suppress for up to 3 h allodynia and hyperalgesia in sciatic nerve-ligated rats. In contrast, TTX was only moderately effective in rats with ligated infraorbital nerve. In sciatic nerve-ligated rats, TTX administration prevented the increased c-Fos immunoreactivity occurring in the dorsal horn of the lumbar cord and some supraspinal areas in response to light mechanical stimulation of the nerve-injured hindpaw. The anti-allodynia/antihyperalgesia caused by TTX in these neuropathic rats was promoted by combined treatment with naloxone (0.5 mg/kg s.c.) but unaffected by the 5-HT(1B) receptor antagonist F11648 (0.5 mg/kg s.c.) and the alpha(2)-adrenergic receptor antagonist idazoxan (0.5 mg/kg i.v.). In contrast, the anti-allodynic and anti-hyperalgesic effects of TTX were significantly attenuated by co-administration of morphine (3 mg/kg s.c.) or the cholecystokinin(2)-receptor antagonist CI-1015 (0.1 mg/kg i.p.). These results indicate that TTX alleviates pain-related behaviors in sciatic nerve-lesioned rats through mechanisms that involve complex interactions with opioidergic systems.