Age-related Changes in Trigeminal Ganglion Macrophages Enhance Orofacial Ectopic Pain After Inferior Alveolar Nerve Injury.

BACKGROUND/AIM: The ectopic pain associated with inferior alveolar nerve (IAN) injury has been reported to involve macrophage expression in the trigeminal ganglion (TG). However, the effect of age-related changes on this abnormal pain conditions are still unknown. This study sought to clarify the involvement of age-related changes in macrophage expression and phenotypic conversion in the TG and how these changes enhance ectopic mechanical allodynia after IAN transection (IANX). MATERIALS AND METHODS: We used senescence-accelerated mouse (SAM)-prone 8 (SAMP8) and SAM-resistance 1 (SAMR1) mice, which are commonly used to study ageing-related changes. Mechanical stimulation was applied to the whisker pad skin under light anaesthesia; the mechanical head withdrawal threshold (MHWT) was measured for 21 d post-IANX. We subsequently counted the numbers of Iba1 (macrophage marker)-immunoreactive (IR) cells, Iba1/CD11c (M1-like inflammatory macrophage marker)-co-IR cells, and Iba1/CD206 (M2-like anti-inflammatory macrophage marker)-co-IR cells in the TG innervating the whisker pad skin. After continuous intra-TG administration of liposomal clodronate Clophosome®-A (LCCA) to IANX-treated SAMP8-mice, the MHWT values of the whisker pad skin were examined. RESULTS: Five days post-IANX, the MHWT had significantly decreased in SAMP8 mice compared to SAMR1-mice. Iba1-IR and Iba1/CD11c-co-IR cell counts were significantly increased in SAMP8 mice compared to SAMR1 mice 5 d post-IANX. LCCA administration significantly restored MHWT compared to control-LCCA administration. CONCLUSION: Ectopic mechanical allodynia of whisker pad skin after IANX is exacerbated by ageing, which involves increases in M1-like inflammatory macrophages in the TG.

P2Y14 receptor in trigeminal ganglion contributes to neuropathic pain in mice.

Trigeminal nerve injury is a common complication of various dental and oral procedures, which could induce trigeminal neuropathic pain but lack effective treatments. P2 purinergic receptors have emerged as novel therapeutic targets for such pain. Recent reports implied that the P2Y14 receptor (P2Y14R) was activated and promoted orofacial inflammatory pain and migraine. However, the role and mechanism of P2Y14R in trigeminal neuropathic pain remain unknown. We induced an orofacial neuropathic pain model by chronic constriction injury of the infraorbital nerve (CCI-ION). Von-Frey tests showed that CCI-ION induced orofacial mechanical hypersensitivity. The increased activating transcription factor 3 (ATF3) expression in the trigeminal ganglion (TG) measured by immunofluorescence confirmed trigeminal nerve injury. Immunofluorescence showed that P2Y14R was expressed in trigeminal ganglion neurons (TGNs) and satellite glial cells (SGCs). RT-qPCR and Western blot identified increased expression of P2Y14R in TG after CCI-ION. CCI-ION also upregulated interleukin-1ß (IL-1ß), interleukin-6 (IL-6), C-C motif chemokine ligand 2 (CCL2), and tumor necrosis factor-α (TNF-α) in TG. Notably, CCI-ION-induced mechanical hypersensitivity and pro-inflammatory cytokines production were decreased by a P2Y14R antagonist (PPTN). Trigeminal administration of P2Y14R agonist (UDP-glucose) evoked orofacial mechanical hypersensitivity and increased pro-inflammatory cytokines above in TG. Furthermore, CCI-ION induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 in TG, which also were reduced by PPTN. The inhibitors of ERK1/2 (U0126) and p38 (SB203580) decreased these upregulated pro-inflammatory cytokines after CCI-ION. Collectively, this study revealed that P2Y14R in TG contributed to trigeminal neuropathic pain via ERK- and p38-dependent neuroinflammation. Thus, P2Y14R may be a potential drug target against trigeminal neuropathic pain.

Extracellular ATP and cAMP signaling promote Piezo2-dependent mechanical allodynia after trigeminal nerve compression injury.

Trigeminal neuralgia (TN) is a type of severe paroxysmal neuropathic pain commonly triggered by mild mechanical stimulation in the orofacial area. Piezo2, a mechanically gated ion channel that mediates tactile allodynia in neuropathic pain, can be potentiated by a cyclic adenosine monophosphate (cAMP)-dependent signaling pathway that involves the exchange protein directly activated by cAMP 1 (Epac1). To study whether Piezo2-mediated mechanotransduction contributes to peripheral sensitization in a rat model of TN after trigeminal nerve compression injury, the expression of Piezo2 and activation of cAMP signal-related molecules in the trigeminal ganglion (TG) were detected. Changes in purinergic P2 receptors in the TG were also studied by RNA-seq. The expression of Piezo2, cAMP, and Epac1 in the TG of the TN animals increased after chronic compression of the trigeminal nerve root (CCT) for 21 days, but Piezo2 knockdown by shRNA in the TG attenuated orofacial mechanical allodynia. Purinergic P2 receptors P2X4, P2X7, P2Y1, and P2Y2 were significantly up-regulated after CCT injury. In vitro, Piezo2 expression in TG neurons was significantly increased by exogenous adenosine 5'-triphosphate (ATP) and Ca2+ ionophore ionomycin. ATP pre-treated TG neurons displayed elevated [Ca2+ ]i and faster increase in responding to blockage of Na+ /Ca2+ exchanger by KB-R7943. Furthermore, mechanical stimulation of cultured TG neurons led to sustained elevation in [Ca2+ ]i in ATP pre-treated TG neurons, which is much less in naïve TG neurons, or is significantly reduced by Piezo2 inhibitor GsMTx4. These results indicated a pivotal role of Piezo2 in peripheral mechanical allodynia in the rat CCT model. Extracellular ATP, Ca2+ influx, and the cAMP-to-Epac1 signaling pathway synergistically contribute to the pathogenesis and the persistence of mechanical allodynia.

Effects of trigeminal nerve injury on the expression of galanin and its receptors in the rat trigeminal ganglion.

In the spinal nervous system, the expression of galanin (GAL) and galanin receptors (GALRs) that play important roles in the transmission and modulation of nociceptive information can be affected by nerve injury. However, in the trigeminal nervous system, the effects of trigeminal nerve injury on the expression of GAL are controversy in the previous studies. Besides, little is known about the effects of trigeminal nerve injury on the expression of GALRs. In the present study, the effects of trigeminal nerve injury on the expression of GAL and GALRs in the rat trigeminal ganglion (TG) were investigated by using quantitative real-time reverse transcription-polymerase chain reaction and immunohistochemistry. To identify the nerve-injured and nerve-uninjured TG neurons, activating transcription factor 3 (ATF3, the nerve-injured neuron marker) was stained by immunofluorescence. The levels of GAL mRNA in the rostral half and caudal half of the TG dramatically increased after transection of infraorbital nerve (ION) and inferior alveolar nerve (IAN), respectively. Immunohistochemical labeling of GAL and ATF3 revealed that GAL level was elevated in both injured and adjacent uninjured small and medium-sized TG neurons after ION/IAN transection. In addition, the levels of GAL2R-like immunoreactivity were reduced in both injured and adjacent uninjured TG neurons after ION/IAN transection, while levels of GAL1R and GAL3R-like immunoreactivity remained unchanged. Furthermore, the number of small to medium-sized TG neurons co-expressing GAL- and GAL1R/GAL2R/GAL3R-like immunoreactivity was significantly increased after ION/IAN transection. In line with previous studies in other spinal neuron systems, these results suggest that GAL and GALRs play functional roles in orofacial neuropathic pain and trigeminal nerve regeneration after trigeminal nerve injury.

Microglia-Astrocyte Communication via C1q Contributes to Orofacial Neuropathic Pain Associated with Infraorbital Nerve Injury.

Trigeminal nerve injury causes a distinct time window of glial activation in the trigeminal spinal subnucleus caudalis (Vc), which are involved in the initiation and maintenance phases of orofacial neuropathic pain. Microglia-derived factors enable the activation of astrocytes. The complement component C1q, which promotes the activation of astrocytes, is known to be synthesized in microglia. However, it is unclear whether microglia-astrocyte communication via C1q is involved in orofacial neuropathic pain. Here, we analyzed microglia-astrocyte communication in a rat model with infraorbital nerve injury (IONI). The orofacial mechanical hypersensitivity induced by IONI was significantly attenuated by preemptive treatment with minocycline. Immunohistochemical analyses revealed that minocycline inhibited the increase in c-Fos immune-reactive (IR) cells and the fluorescence intensity of both Iba1 and glial fibrillary acidic protein (GFAP) in the Vc following IONI. Intracisternal administration of C1q caused orofacial mechanical hypersensitivity and an increase in the number of c-Fos-IR cells and fluorescence intensity of GFAP. C1q-induced orofacial mechanical hypersensitivity was completely abrogated by intracisternal administration of fluorocitrate. The present findings suggest that the enhancement in the excitability of Vc nociceptive neurons is produced by astrocytic activation via the signaling of C1q released from activated microglia in the Vc following IONI, resulting in persistent orofacial neuropathic pain.

Involvement of Satellite Cell Activation via Nitric Oxide Signaling in Ectopic Orofacial Hypersensitivity.

: The mechanical head-withdrawal threshold (MHWT) was significantly reduced following inferior alveolar nerve transection (IANX) in rats. Nitrate and nitrite synthesis was dramatically increased in the trigeminal ganglion (TG) at 6 h after the IANX. The relative number of neuronal nitric oxide synthase (nNOS)-immunoreactive (IR) cells was significantly higher in IANX rats compared to sham-operated and N-propyl-L-arginine (NPLA)-treated IANX rats. On day 3 after NPLA administration, the MHWT recovered considerably in IANX rats. Following L-arginine injection into the TG, the MHWT was significantly reduced within 15 min, and the mean number of TG cells encircled by glial fibrillary acidic protein (GFAP)-IR cells was substantially higher. The relative number of nNOS-IR cells encircled by GFAP-IR cells was significantly increased in IANX rats. In contrast, after NPLA injection into the TG, the relative number of GFAP-IR cells was considerably reduced in IANX rats. Fluorocitrate administration into the TG significantly reduced the number of GFAP-IR cells and prevented the MHWT reduction in IANX rats. The present findings suggest that following IANX, satellite glial cells are activated via nitric oxide (NO) signaling from TG neurons. The spreading satellite glial cell activation within the TG results in mechanical hypersensitivity of face regions not directly associated with the trigeminal nerve injury.

Increase in IGF-1 Expression in the Injured Infraorbital Nerve and Possible Implications for Orofacial Neuropathic Pain.

Insulin-like growth factor-1 (IGF-1) is upregulated in the injured peripheral nerve bundle and controls nociceptive neuronal excitability associated with peripheral nerve injury. Here, we examined the involvement of IGF-1 signaling in orofacial neuropathic pain following infraorbital nerve injury (IONI) in rats. IONI promoted macrophage accumulation in the injured ION, as well as in the ipsilateral trigeminal ganglion (TG), and induced mechanical allodynia of the whisker pad skin together with the enhancement of neuronal activities in the subnucleus caudalis of the spinal trigeminal nucleus and in the upper cervical spinal cord. The levels of IGF-1 released by infiltrating macrophages into the injured ION and the TG were significantly increased. The IONI-induced the number of transient receptor potential vanilloid (TRPV) subfamily type 4 (TRPV4) upregulation in TRPV subfamily type 2 (TRPV2)-positive small-sized, and medium-sized TG neurons were inhibited by peripheral TRPV2 antagonism. Furthermore, the IONI-induced mechanical allodynia was suppressed by TRPV4 antagonism in the whisker pad skin. These results suggest that IGF-1 released by macrophages accumulating in the injured ION binds to TRPV2, which increases TRPV4 expression in TG neurons innervating the whisker pad skin, ultimately resulting in mechanical allodynia of the whisker pad skin.

Lipopolysaccharide-mediated inflammatory priming potentiates painful post-traumatic trigeminal neuropathy.

We explored the molecular and behavioral effects of a perineural Lipopolysaccharide (LPS)-mediated inflammatory priming on the development and maintenance of painful post-traumatic trigeminal neuropathy (PPTTN) following infra-orbital nerve chronic constriction injury (CCI-IoN) in rats. Rats were pretreated with repetitive perineural injections in the vicinity of the IoN of either LPS or vehicle (Vhcl) before being submitted to CCI-IoN. Orofacial pain-like behaviors (response to Von Frey Filament testing and spontaneous isolated face grooming) were measured during the period of LPS injections (three weeks) and following CCI-IoN surgery (two weeks). Local LPS administration induced an early pain-like behavior (i.e. an increase in spontaneous pain [SP] or mechanical static allodynia [MSA]) in both conditions, and following CCI-IoN, MSA and SP developed earlier and more severely in LPS-pretreated rats than in the control group. Ipsilateral increases of key neuropathic pain mRNA markers in the IoN parenchyma, trigeminal ganglia (TG) and spinal trigeminal nucleus caudalis (Sp5C) were observed in CCI-IoN injured animals as compared to controls. Although no significant molecular differences could be observed within the IoN parenchyma between LPS and Vhcl-pretreated animals, a significant increase of key inflammatory cytokine Interleukin 1 beta (IL - 1ß) could be found in the TG of LPS-pretreated CCI-injured animals versus controls. Finally, a higher increase of inducible nitric oxide synthase (iNOS) in ipsilateral Sp5C of LPS-pretreated animals was observed as compared to Sp5C of Vhcl-pretreated animals. These results suggest a key role of inflammatory priming in the development and maintenance of PPTTN implicating IL-1ß/iNOS-dependent central sensitization mechanisms.

A Novel Role for Lymphotactin (XCL1) Signaling in the Nervous System: XCL1 Acts via its Receptor XCR1 to Increase Trigeminal Neuronal Excitability.

Chemokines are known to have a role in the nervous system, influencing a range of processes including the development of chronic pain. To date there are very few studies describing the functions of the chemokine lymphotactin (XCL1) or its receptor (XCR1) in the nervous system. We investigated the role of the XCL1-XCR1 axis in nociceptive processing, using a combination of immunohistochemical, pharmacological and electrophysiological techniques. Expression of XCR1 in the rat mental nerve was elevated 3 days following chronic constriction injury (CCI), compared with 11 days post-CCI and sham controls. XCR1 co-existed with neuronal marker PGP9.5, leukocyte common antigen CD45 and Schwann cell marker S-100. In the trigeminal root and white matter of the brainstem, XCR1-positive cells co-expressed the oligodendrocyte marker Olig2. In trigeminal subnucleus caudalis (Vc), XCR1 immunoreactivity was present in the outer laminae and was colocalized with vesicular glutamate transporter 2 (VGlut2), but not calcitonin gene-related peptide (CGRP) or isolectin B4 (IB4). Incubation of brainstem slices with XCL1 induced activation of c-Fos, ERK and p38 in the superficial layers of Vc, and enhanced levels of intrinsic excitability. These effects were blocked by the XCR1 antagonist viral CC chemokine macrophage inhibitory protein-II (vMIP-II). This study has identified for the first time a role for XCL1-XCR1 in nociceptive processing, demonstrating upregulation of XCR1 at nerve injury sites and identifying XCL1 as a modulator of central excitability and signaling via XCR1 in Vc, a key area for modulation of orofacial pain, thus indicating XCR1 as a potential target for novel analgesics.

The Sonic Hedgehog signaling pathway regulates inferior alveolar nerve regeneration.

Activation of Shh signaling is known to be observed following injury of the peripheral nerves such as the sciatic nerve. However, the precise role of Shh signaling during peripheral nerve regeneration is not fully understood. The inferior alveolar nerve (IAN) is most commonly injured during oral surgery. Unlike the sciatic nerve, the IAN is isolated from other craniofacial tissues, as it resides in a long bony canal within the mandible. The IAN is thus an excellent experimental model for investigating peripheral nerve regeneration. In this study, the role of Shh signaling in peripheral nerve regeneration was investigated using the mouse IAN transection model. During regeneration, Shh signaling was activated within the entire distal region of the IAN and proximal stumps. Inhibition of Shh signaling by cyclopamine application at the transection site led to abnormal axon growth in random directions, a reduced number of macrophages, and an increase in myelin debris within the distal region. Shh signaling is thus involved in peripheral nerve regeneration via the regulation of myelin degradation.

Role of KCNQ2 channels in orofacial cold sensitivity: KCNQ2 upregulation in trigeminal ganglion neurons after infraorbital nerve chronic constrictive injury.

Sensitivity to cooling temperatures often becomes heightened in orofacial regions leading to orofacial cold allodynia/hyperalgesia after chronic trigeminal nerve injury. KCNQ2 channels are involved in controlling excitability of primary afferent neurons and thereby regulate sensory functions under both physiological and pathological conditions. In the present study, we sought to determine whether KCNQ2 channels in trigeminal nerves are involved in regulating orofacial operant behavioral responses to cooling stimulation. We also sought to examine whether chronic trigeminal nerve injury may alter KCNQ2 channel expression in trigeminal ganglions. Using the orofacial operant tests, animals show cold allodynia/hyperalgesia in orofacial regions following infraorbital nerve chronic constrictive injury (ION-CCI), which could be alleviated by subcutaneous administration of retigabine, a KCNQ2 activator. In contrast, subcutaneous administration of the KCNQ2 inhibitor XE991 directly elicits cold allodynia/hyperalgesia in sham animals. Using immunostaining, we show that KCNQ2 channels are primarily expressed in small-sized TG neurons. Interestingly, KCNQ2 channel expression becomes significantly upregulated in TG neurons following the ION-CCI. Our results suggest that KCNQ2 channels are involved in regulating orofacial cold sensitivity. Upregulation of KCNQ2 channels may be a compensatory change in attempting to limit injury-induced trigeminal hyperexcitability.

Combination Drug Therapy of Pioglitazone and D-cycloserine Attenuates Chronic Orofacial Neuropathic Pain and Anxiety by Improving Mitochondrial Function Following Trigeminal Nerve Injury.

OBJECTIVES: The study aim was to determine how peripheral trigeminal nerve injury affects mitochondrial respiration and to test efficacy of combined treatment with 2 Federal Drug Administration approved drugs with potential for improving mitochondrial bioenergetics, pain and anxiety-related behaviors in a chronic orofacial neuropathic pain mouse model. METHODS: Efficacy of (R)-(+)-4-amino-3-isoxazolidinone (D-cycloserine, DCS), an N-Methyl-D-aspartate antagonist/agonist, and Pioglitazone (PIO), a selective agonist of nuclear receptor peroxisome proliferator-activated receptor gamma was investigate in the trigeminal inflammatory compression (TIC) neuropathic nerve injury mouse model. Combined low doses of these drugs (80 mg/kg DCS and 100 mg/kg PIO) were given as a single bolus or daily for 7 days post-TIC to test ability to attenuate neuropathic nociceptive and associated cognitive dependent anxiety behaviors. In addition, beneficial effects of the DCS/PIO drug combination were explored ex vivo in isolated cortex/brainstem mitochondria at 28 weeks post-TIC. RESULTS: The DCS/PIO combination not only attenuated orofacial neuropathic pain and anxiety-related behaviors associated with trigeminal nerve injury, but it also improved mitochondrial bioenergetics. DISCUSSION: The DCS/PIO combination uncoupled mitochondrial respiration in the TIC model to improve cortical mitochondrial dysfunction, as well as reduced nociceptive and anxiety behaviors present in mice with centralized chronic neuropathic nerve injury. Combining these drugs could be a beneficial treatment for patients with depression, anxiety, or other psychological conditions due to their chronic pain status.

Bovine lactoferrin reduces extra-territorial facial allodynia/hyperalgesia following a trigeminal nerve injury in the rat.

There is an urgent clinical need for an effective therapeutic agent to treat neuropathic pain. This study explored whether intrathecal administration of bovine lactoferrin (bLF), in combination with signal transduction pathway inhibition or an inflammatory cytokine production, results in reduced allodynia/hyperalgesia in the whisker pad area following mental nerve transection (MNT) in rats. Rats were intrathecally infused with bLF, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS), an antagonist of Toll-like receptor 4 (TLR4), or interleukin (IL)-18 binding protein (BP). bLF attenuated allodynia/hyperalgesia and blocked upregulation of phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK), p-nuclear factor (NF)-κB p65, p-IκB kinase, and IL-18 in the trigeminal subnucleus caudalis (Vc). Microglia expressed p-p38 and astrocytes expressed p-NF-κB p65 in the Vc following MNT. LPS-RS had the same effects as bLF, except for attenuation of p-NF-κB p65. IL-18BP attenuated allodynia/hyperalgesia and IL-18 upregulation in the Vc. These results suggest that bLF suppresses IL-18 production, which is involved in allodynia/hyperalgesia following MNT, by inhibiting TLR4-derived p38 MAPK activation in microglia. Additionally, binding of bLF to tumor necrosis factor receptor-associated factor 6 might result in inhibition of p38 MAPK and NF-κB activation. The findings suggest that bLF could serve as a potent therapeutic agent for neuropathic pain.

Locally Produced BDNF Promotes Sclerotic Change in Alveolar Bone after Nerve Injury.

Brain-derived neurotrophic factor (BDNF), which is released due to nerve injury, is known to promote the natural healing of injured nerves. It is often observed that damage of mandibular canal induces local sclerotic changes in alveolar bone. We reported that peripheral nerve injury promotes the local production of BDNF; therefore, it was possible to hypothesize that peripheral nerve injury affects sclerotic changes in the alveolar bone. This study aimed to evaluate the effect of BDNF on osteogenesis using in vitro osteoblast-lineage cell culture and an in vivo rat osteotomy model. MC3T3-E1 cells were cultured with BDNF and were examined for cell proliferative activity, chemotaxis and mRNA expression levels of osteoblast differentiation markers. For in vivo study, inferior alveolar nerve (IAN) injury experiments and mandibular cortical osteotomy were performed using a rat model. In the osteotomy model, exogenous BDNF was applied to bone surfaces after corticotomy of the mandible, and we morphologically analyzed the new bone formation. As a result, mRNA expression of osteoblast differentiation marker, osteocalcin, was significantly increased by BDNF, although cell proliferation and migration were not affected. In the in vivo study, osteopontin-positive new bone formation was significantly accelerated in the BDNF-grafted groups, and active bone remodeling, involving trkB-positive osteoblasts and osteocytes, continued after 28 days. In conclusion, BDNF stimulated the differentiation of MC3T3-E1 cells and it promoted new bone formation and maturation. These results suggested that local BDNF produced by peripheral nerve injury contributes to accelerating sclerotic changes in the alveolar bone.

Trigeminal nervous system sensitization by infraorbital nerve injury enhances responses in a migraine model.

Background Although the peripheral and central sensitizations of trigeminal nervous system may be one of the important factors of migraine, the precise mechanism is not fully understood. In this study, we examined the influence of the sensitization of the second division of the trigeminal nerve (V2) by chronic constriction injury (CCI) of the infraorbital nerve (ION) on migraine headache, using the capsaicin-induced migraine model. Methods Male Sprague-Dawley rats were assigned to four groups: (a) sham surgery and topical-dural vehicle application (Sham + Vehicle) group, (b) CCI-ION and topical-dural vehicle application (CCI-ION + Vehicle) group, (c) sham surgery and topical-dural capsaicin application (Sham + Capsaicin) group, (d) CCI-ION and topical-dural capsaicin application (CCI-ION + Capsaicin) group. Behavioral testing and immunohistochemical staining were performed. Results In the behavioral test, the Sham + Capsaicin group showed significantly longer duration of immobilization and shorter duration of exploration compared with the Sham + Vehicle group, which is similar to clinical features of migraine patients. Moreover, CCI-ION enhanced these effects in the CCI-ION + Capsaicin group. Immunohistochemical staining for phospho-extracellular signal-related kinase (pERK) in the trigeminal ganglion (TG) containing first and second divisions of the trigeminal nerve and the trigeminocervical complex (TCC) revealed that pERK expression was significantly increased in the CCI-ION + Capsaicin group compared with the other groups. However, comparing between effects of the peripheral and central sensitizations (in the TG and TCC), from our results, peripheral sensitization would play a much less or not significant role. Conclusions These data demonstrate that the sensitization of V2 could influence the activation and the sensitization of the first division of the trigeminal nerve in the TCC, subsequently exacerbating pain sensation and pain-related behaviors. We have shown for the first time that the existence of the central sensitization of V2 can be an exacerbating factor for migraine related nociceptive thresholds/activation.

Hedgehog Pathway-Mediated Vascular Alterations Following Trigeminal Nerve Injury.

Whereas neurovascular interactions in spinal neuropathic pain models have been well characterized, little attention has been given to such neurovascular interactions in orofacial neuropathic pain models. This study investigated in male Sprague-Dawley rats the vascular changes following chronic constriction injury (CCI) of the infraorbital nerve (IoN), a broadly validated preclinical model of orofacial neuropathic pain. Following IoN-CCI, an early downregulation of tight junction proteins Claudin-1 and Claudin-5 was observed within the endoneurium and perineurium, associated with increased local accumulation of sodium fluorescein (NaFlu) within the IoN parenchyma, as compared with sham animals. These events were evidence of local blood-nerve barrier disruption and increased vascular permeability. A significant upregulation of immunocytes (CD3, CD11b) and innate immunity (TLR2, TLR4) mRNA markers was also observed, suggestive of increased local inflammation. Finally, a significant downregulation of Hedgehog pathway readouts Patched-1 and Gli-1 was observed within the IoN after CCI and local injections of cyclopamine, a Hedgehog pathway inhibitor, replicated in naïve rats the molecular, vascular, and behavioral changes observed following IoN-CCI. These results suggest a major role of Hedgehog pathway inhibition in mediating local increased endoneurial and perineurial vascular permeability following trigeminal nerve injury, thus facilitating immunocytes infiltration, neuroinflammation development, and neuropathic pain-like aversive behavior.

GABA-A receptor activity in the noradrenergic locus coeruleus drives trigeminal neuropathic pain in the rat; contribution of NAα1 receptors in the medial prefrontal cortex.

Trigeminal neuropathic pain is described as constant excruciating facial pain. The study goal was to investigate the role of nucleus locus coeruleus (LC) in a model of chronic orofacial neuropathic pain (CCI-ION). The study examines LC's relationship to both the medullary dorsal horn receiving trigeminal nerve sensory innervation and the medial prefrontal cortex (mPFC). LC is a major source of CNS noradrenaline (NA) and a primary nucleus involved in pain modulation. Although descending inhibition of acute pain by LC is well established, contribution of the LC to facilitation of chronic neuropathic pain is also reported. In the present study, a rat orofacial pain model of trigeminal neuropathy was induced by chronic constrictive injury of the infraorbital nerve (CCI-ION). Orofacial neuropathic pain was indicated by development of whisker pad mechanical hypersensitivity. Hypersensitivity was alleviated by selective elimination of NA neurons, including LC (A6 cell group), with the neurotoxin anti-dopamine-ß-hydroxylase saporin (anti-DßH-saporin) microinjected either intracerebroventricularly (i.c.v.) or into trigeminal spinal nucleus caudalis (spVc). The GABAA receptor antagonist, bicuculline, administered directly into LC (week 8) inhibited hypersensitivity. This indicates a valence shift in which increased GABAA signaling ongoing in LC after trigeminal nerve injury paradoxically produces excitatory facilitation of the chronic pain state. Microinjection of NAα1 receptor antagonist, benoxathian, into mPFC attenuated whisker pad hypersensitivity, while NAα2 receptor antagonist, idazoxan, was ineffective. Thus, GABAA-mediated activation of NA neurons during CCI-ION can facilitate hypersensitivity through NAα1 receptors in the mPFC. These data indicate LC is a chronic pain generator.

The relationship between changes in the expression of growth associated protein-43 and functional recovery of the injured inferior alveolar nerve following transection without repair in adult rats.

OBJECTIVE: The objective of this study was to analyze the changes in the expression of growth associated protein-43 (GAP-43) in trigeminal ganglions (TGs) and in the distal stumps of transected inferior alveolar nerves (IANs), and to clarify the relationship between these changes and functional recovery of the transected IAN without repair using a rat IAN axotomy model. MATERIAL AND METHODS: Following transection, GAP-43 expression was measured at multiple time points. The functional recovery of the transected IAN was evaluated based on the compound muscle action potentials recorded from the digastric muscle. RESULTS: GAP-43 expression in TGs was significantly higher at 2, 7, 14, 28, and 56 days following IAN transection compared to that in samples from sham-operated rats (p < 0.0005, p < 0.0005, p < 0.0005, p = 0.007, and p = 0.023, respectively). GAP-43 expression in the distal stumps of transected IANs was significantly higher at 2, 7, 14, and 28 days following IAN transection compared to that in samples taken from sham rats (p < 0.0005, p < 0.0005, p < 0.0005, and p = 0.009, respectively). GAP-43 expression in the distal stumps of transected IANs returned nearly to sham levels by day 56 following IAN transection. On days 7, 14, 28, and 56 following transection, the amplitude of the compound muscle action potential gradually increased, the latency gradually decreased, and the duration gradually increased. The amplitude, latency, and duration of the compound muscle action potentials nearly returned to sham levels on post-transection day 56. CONCLUSIONS: Time-dependent changes in the expression of GAP-43 in both TGs and distal stumps of transected IANs without repair are synchronously consistent with the regeneration and functional recovery of the transected IAN. The recovery of the amplitude, latency, and duration of the compound muscle action potentials indicates increased myelination and increased axon density of the regenerated nerve fibers.

Morphological and functional changes in regenerated primary afferent fibres following mental and inferior alveolar nerve transection.

BACKGROUND: It is important to know the mechanisms underlying pain abnormalities associated with inferior alveolar nerve (IAN) regeneration in order to develop the appropriate treatment for orofacial neuropathic pain patients. However, peripheral mechanisms underlying orofacial pain abnormalities following IAN regeneration are not fully understood. METHODS: Head withdrawal threshold (HWT), jaw opening reflex (JOR) thresholds, single-fibre recordings of the regenerated mental nerve (MN) fibres, calcitonin gene-related peptide (CGRP), isolectin B4 (IB4), peripherin, neurofilament-200 (NF-200) and transient receptor potential vanilloid 1 (TRPV1) expression in trigeminal ganglion (TG) cells, and electron microscopic (EM) observations of the regenerated MN fibres were studied in MN- and IAN-transected (M-IANX) rats. RESULTS: HWT to mechanical or heat stimulation of the mental skin was significantly lower in M-IANX rats compared with sham rats. Mean conduction velocity of action potentials recorded from MN fibres (n = 124) was significantly slower in M-IANX rats compared with sham rats. The percentage of Fluoro-Gold (FG)-labelled CGRP-, peripherin- or TRPV1-immunoreactive (IR) cells was significantly larger in M-IANX rats compared with that of sham rats, whereas that of FG-labelled IB4- and NF-200-IR cells was significantly smaller in M-IANX rats compared with sham rats. Large-sized myelinated nerve fibres were rarely observed in M-IANX rats, whereas large-sized unmyelinated nerve fibres were frequently observed and were aggregated in the bundles at the distal portion of regenerated axons. CONCLUSIONS: These findings suggest that the demyelination of MN fibres following regeneration may be involved in peripheral sensitization, resulting in the orofacial neuropathic pain associated with trigeminal nerve injury.

Temporal mismatch between pain behaviour, skin Nerve Growth factor and intra-epidermal nerve fibre density in trigeminal neuropathic pain.

BACKGROUND: The neurotrophin Nerve Growth factor (NGF) is known to influence the phenotype of mature nociceptors, for example by altering synthesis of neuropeptides, and changes in NGF levels have been implicated in the pathophysiology of chronic pain conditions such as neuropathic pain. We have tested the hypothesis that after partial nerve injury, NGF accumulates within the skin and causes 'pro-nociceptive' phenotypic changes in the remaining population of sensory nerve fibres, which could underpin the development of neuropathic pain. RESULTS: Eleven days after chronic constriction injury of the rat mental nerve the intra-epidermal nerve fibre density of the chin skin from had reduced from 11.6 ± 4.9 fibres/mm to 1.0 ± 0.4 fibres/mm; this slowly recovered to 2.4 ± 2.0 fibres/mm on day 14 and 4.0 ± 0.8 fibres/mm on day 21. Cold hyperalgesia in the ipsilateral lower lip was detectable 11 days after chronic constriction injury, although at this time skin [NGF] did not differ between sides. At 14 days post-injury, there was a significantly greater [NGF] ipsilaterally compared to contralaterally (ipsilateral = 111 ± 23 pg/mg, contralateral = 69 ± 13 pg/mg), but there was no behavioural evidence of neuropathic pain at this time-point. By 21 days post-injury, skin [NGF] was elevated bilaterally and there was a significant increase in the proportion of TrkA-positive (the high-affinity NGF receptor) intra-epidermal nerve fibres that were immunolabelled for the neuropeptide Calcitonin Gene-related peptide. CONCLUSIONS: The temporal mismatch in behaviour, skin [NGF] and phenotypic changes in sensory nerve fibres indicate that increased [NGF] does not cause hyperalgesia after partial mental nerve injury, although it may contribute to the altered neurochemistry of cutaneous nerve fibres.