Photobiomodulation Activates Microglia/Astrocytes and Relieves Neuropathic Pain in Inferior Alveolar Nerve Injury.

Objective: This study aimed to determine microglial/astrocyte changes and their associated analgesic effect in inferior alveolar nerve injury (IANI) model rats treated with photobiomodulation therapy (PBMT) using a 940-nm diode laser. Background: Very few basic studies have investigated microglial/astrocyte dynamics following PBMT aimed at relieving neuropathic pain caused by IANI. Methods: Rats were divided into an IANI-PBM group, IANI+PBM group, and sham+PBM group. Observations were made on the day before IANI or the sham operation and on postoperative days 3, 5, 7, 14, and 28. PBMT was delivered for 7 consecutive days, with an energy density of 8 J/cm2. Behavioral analysis was performed to determine pain thresholds, and immunohistological staining was performed for the microglia marker Iba1 and astrocyte marker glial fibrillary acidic protein, which are observed in the spinal trigeminal nucleus. Results: Behavioral analysis showed that the pain threshold returned to the preoperative level on postoperative day 14 in the IANI+PBM group, but decreased starting from postoperative day 1 and did not improve thereafter in the IANI-PBM group (p ≤ 0.001). Immunological analysis showed that microglial and astrocyte cell counts were similar in the IANI+PBM group and IANI-PBM group shortly after IANI (day 3), but the expression area was larger (p ≤ 0.001) and hypertrophy of microglia and astrocyte cell bodies and end-feet extension (i.e., indicators of activation) were more prominent in the IANI+PBM group. Conclusions: PBMT after IANI prevented hyperalgesia and allodynia by promoting glial cell activation shortly after injury.

Metabotropic glutamate receptor 5-mediated inhibition of inward-rectifying K+ channel 4.1 contributes to orofacial ectopic mechanical allodynia following inferior alveolar nerve transection in male mice.

Inward-rectifying K+ channel 4.1 (Kir4.1), which regulates the electrophysiological properties of neurons and glia by affecting K+ homeostasis, plays a critical role in neuropathic pain. Metabotropic glutamate receptor 5 (mGluR5) regulates the expression of Kir4.1 in retinal Müller cells. However, the role of Kir4.1 and its expressional regulatory mechanisms underlying orofacial ectopic allodynia remain unclear. This study aimed to investigate the biological roles of Kir4.1 and mGluR5 in the trigeminal ganglion (TG) in orofacial ectopic mechanical allodynia and the role of mGluR5 in Kir4.1 regulation. An animal model of nerve injury was established via inferior alveolar nerve transection (IANX) in male C57BL/6J mice. Behavioral tests indicated that mechanical allodynia in the ipsilateral whisker pad lasted at least 14 days after IANX surgery and was alleviated by the overexpression of Kir4.1 in the TG, as well as intraganglionic injection of an mGluR5 antagonist (MPEP hydrochloride) or a protein kinase C (PKC) inhibitor (chelerythrine chloride); Conditional knockdown of the Kir4.1 gene downregulated mechanical thresholds in the whisker pad. Double immunostaining revealed that Kir4.1 and mGluR5 were co-expressed in satellite glial cells in the TG. IANX downregulated Kir4.1 and upregulated mGluR5 and phosphorylated PKC (p-PKC) in the TG; Inhibition of mGluR5 reversed the changes in Kir4.1 and p-PKC that were induced by IANX; Inhibition of PKC activation reversed the downregulation of Kir4.1 expression caused by IANX (p < .05). In conclusion, activation of mGluR5 in the TG after IANX contributed to orofacial ectopic mechanical allodynia by suppressing Kir4.1 via the PKC signaling pathway.

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.

The α2δ-1-NMDAR1 interaction in the trigeminal ganglion contributes to orofacial ectopic pain following inferior alveolar nerve injury.

Orofacial ectopic pain can often arise following nerve injury. However, the exact mechanism responsible for orofacial ectopic pain induced by trigeminal nerve injury remains unknown. The α2δ-1 and glutamate N-methyl-d-aspartic acid receptor (NMDAR) interactions have been demonstrated to participate in neuropathic pain regulation in the spinal cord. In this study, a rat model of inferior alveolar nerve transection (IANX) was used to investigate the role of α2δ-1-NMDAR1 interaction in the trigeminal ganglion (TG) in regard to the regulation of orofacial ectopic pain. Western blot (WB) analysis indicated that α2δ-1 and NMDAR1 in the TG were substantially higher in IANX rats than they were in sham/naive rats. Additionally, immunofluorescence (IF) results revealed that α2δ-1 and NMDAR1 were co-expressed and distributed within neurons and activated satellite glial cells in the TG. Co-immunoprecipitation (Co-IP) results indicated that α2δ-1-NMDAR1 complex levels in the TG were higher in IANX rats than they were in sham rats. Furthermore, the results of behavioral tests demonstrated that intra-TG injection of gabapentin (α2δ-1 inhibitory ligand) or memantine hydrochloride (NMDAR antagonist) reversed the decrease in mechanical head-withdrawal threshold (HWT) in IANX rats. Moreover, inhibition of α2δ-1 by intra-TG administration of gabapentin suppressed the upregulation of the NMDAR1 protein, and the inhibition of NMDAR by intra-TG administration of memantine hydrochloride inhibited the increased expression of α2δ-1 protein induced by IANX. In conclusion, the physical and functional interaction between α2δ-1 and NMDAR1 is critical for the development of orofacial ectopic pain, indicating that α2δ-1, NMDAR1, and the α2δ-1-NMDAR1 complex may represent potential targets for the treatment of orofacial ectopic 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.

Recovery of inferior alveolar nerve by photobiomodulation therapy using two laser wavelengths: A behavioral and immunological study in rat.

Postoperative sensory disturbances of inferior alveolar nerve (IAN) are major challenges in dental procedures. We aimed to investigate the effect of photobiomodulation therapy (PBMT) with 810 nm and 980 nm diode lasers on behavioral and immunological factors in a rat IAN crush model. Seventy-two rats were randomly assigned to the four groups of 810 nm laser (crush injury+810 nm laser; 6 J/cm2, 15 sessions, every 48 h), 980 nm laser (crush injury+980 nm laser; same protocol), control (crush injury without irradiation), and sham surgery (no crush injury and no irradiation). The neurosensory response of IAN was evaluated by Von Frey behavioral test before (baseline) and post-surgery in a period of one month. Changes of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), nuclear factor-kappa B (NF-κB), TNF-α, and IL-1ß, were assessed on days 2 and 30 post injury. Data were analyzed for significant differences by repeated measures and one-way ANOVA (p < .05). One day after surgery, all rats subjected to nerve injury showed significant increase in the withdrawal threshold of von Frey test compared to the baseline (p = .02 for control and p = .03 for laser groups). The threshold gradually returned to the baseline scores in 810 nm, 980 nm, and control groups from days 11, 17, and 29, respectively. There was a significant lower withdrawal threshold in 810 nm and 980 nm laser groups compared to the control group in days 11 to 19 and 9 to 23, respectively. At both time points, the levels of NGF and BDNF were significantly higher in 810 nm laser group compared to the control group. There was a significant difference between laser and control groups regarding NF-κB expression (all p values<.001). TNF-α and IL-1ß were significantly lower in laser groups compared to the control group (all p values < .001). PBMT with 810 and 980 nm diode laser protocol used in this study, promoted the neurosensory recovery of IAN after crush injury in rats. In addition, application of 810 nm diode laser was associated with more improvement in immunological responses compared to that of 980 nm laser.

Serotonin1B receptor-mediated presynaptic inhibition of proprioceptive sensory inputs to jaw-closing motoneurons.

The proprioceptive sensory inputs from neurons in the mesencephalic trigeminal nucleus (MesV) to masseter motoneurons (MMNs) play an important role in regulating masseter muscle activity during mastication. Several histological studies have shown that serotonin (5-HT) fibers densely innervate both the MesV and the trigeminal motor nucleus. However, the functional roles of 5-HT in the regulation of the excitatory synaptic inputs from MesV afferents to MMNs remain to be clarified. Thus, using the whole-cell recording technique in brainstem slice preparations from juvenile Wistar rats aged between postnatal days 8 and 12, we examined the effects of 5-HT on the excitatory synaptic inputs from MesV afferents to MMNs. Bath application of 5-HT reduced the peak amplitude of excitatory postsynaptic potentials evoked in MMNs by electrical stimulation of the MesV afferents (eEPSPs), and this inhibitory effect of 5-HT on eEPSPs was replicated with the 5-HT1B receptor agonist CP-93129 but not by the 5-HT1A receptor agonist 8-OH-DPAT. Moreover, the 5-HT1B receptor antagonist SB-224289 but not the 5-HT1A receptor antagonist WAY-100635 antagonized the inhibitory effect of 5-HT on eEPSPs. CP-93129 increased the paired-pulse ratio and decreased the frequency of miniature excitatory postsynaptic currents (mEPSCs), while it did not alter the mEPSC amplitude. These results suggest that presynaptic 5-HT1B receptors are involved in the inhibition of the excitatory synaptic inputs from MesV afferents to MMNs. Such inhibition may regulate MesV afferent activity during mastication.

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.

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.

Neurochemical effects of photobiostimulation in the trigeminal ganglion after inferior alveolar nerve injury.

Orofacial pain is associated with peripheral and central sensitization of trigeminal nociceptive neurons. Nerve injury results in release of chemical mediators that contribute to persistent pain conditions. The activation of the transient receptor potential vanilloid 1 (TRPV1), promotes release of calcitonin gene-related peptide (CGRP) and substance P (SP) from trigeminal nerve terminals. CGRP and SP contribute to the development of peripheral hyperalgesia. The expression of SP and CGRP by primary afferent neurons is rapidly increased in response to peripheral inflammation. CGRP receptor activation promotes activation of AMPA receptors, leading to increased firing of neurons which is reflected as central sensitization. In this study we investigated whether inferior alveolar nerve (IAN) injury influences AMPA receptors, CGRP, SP and TRPV1 expression in the trigeminal ganglion (TG). The relative expression of the protein of interest from naive rats was compared to those from injured rats and animals that received low level laser therapy (LLLT). IAN-injury did not change expression of GluA1, GluA2 and CGRP, but increased the expression of TRPV1 and SP. LLLT increases GluA1 and GluA2 expression and decreases TVPV1, SP and CGRP. These results, together with previous behavioral data, suggest that IAN-injury induced changes in the proteins analyzed, which could impact on nociceptive threshold. These data may help to understand the molecular mechanisms of pain sensitization in the TG.

Neuropeptide expression and morphometric differences in crushed alveolar inferior nerve of rats: Effects of photobiomodulation.

Inferior alveolar nerve (IAN) injuries may occur during various dental routine procedures, especially in the removal of impacted lower third molars, and nerve recovery in these cases is a great challenge in dentistry. Here, the IAN crush injury model was used to assess the efficacy of photobiomodulation (PBM) in the recovery of the IAN in rats following crushing injury (a partial lesion). Rats were divided into four experimental groups: without any procedure, IAN crush injury, and IAN crush injury with PBM and sham group with PBM. Treatment was started 2 days after surgery, above the site of injury, and was performed every other day, totaling 10 sessions. Rats were irradiated with GaAs Laser (Gallium Arsenide, Laserpulse, Ibramed Brazil) emitting a wavelength of 904 nm, an output power of 70 mWpk, beam spot size at target ∼0.1 cm2, a frequency of 9500 Hz, a pulse time 60 ns, and an energy density of 6 J/cm2. Nerve recovery was investigated by measuring the morphometric data of the IAN using TEM and by the expression of laminin, neurofilaments (NFs), and myelin protein zero (MPZ) using Western blot analysis. We found that IAN-injured rats which received PBM had a significant improvement of IAN morphometry when compared to IAN-injured rats without PBM. In parallel, all MPZ, laminin, and NFs exhibited a decrease after PBM. The results of this study indicate that the correlation between the peripheral nerve ultrastructure and the associated protein expression shows the beneficial effects of PBM.

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.

Changes in expression of Slit1 and its receptor Robo2 in trigeminal ganglion and inferior alveolar nerve following inferior alveolar nerve axotomy in adult rats: a pilot study.

The objective of this study was to analyze changes in expression pattern of Slit1 and Robo2, and to clarify the relationship between these changes and functional recovery of the axotomized inferior alveolar nerve (IAN) without repair using a rat IAN axotomy model. Slit1 and Robo2 were weakly expressed in samples taken from trigeminal ganglion (TG) and IAN of sham surgery rats. In axotomized rats, expression levels increased significantly from day 2 to day 28 post-axotomy, with peaks on days 14 (Slit1) and 7 (Robo2) after axotomy (relative to sham: Slit1 in TG P<0.0005, Slit1 in IAN P = 0.003, Robo2 in TG P<0.0005, and Robo2 in IAN P<0.0005). Over-expressed Slit1 and Robo2 in both the TG and IANs of axotomized rats did not return to sham levels during the 28-day observation period of this study. The regeneration and functional recovery of axotomized IAN was evaluated by jaw opening reflex (JOR) recorded before and after axotomy. JOR occurrence (0% on day 7, 35% on day 14, and 85% on day 28) increased gradually, and the relative threshold of electrical stimulation eliciting JOR decreased gradually (1000.0 ± 0.0% on day 7, 854.3 ± 132.5% on day 14, and 302.6 ± 92.3% on day 28). On day 28 after axotomy, JOR occurrence and the relative JOR threshold had almost returned to those of sham rats. These findings suggest that Slit1 and Robo2 are involved in the regeneration and functional recovery of the axotomized IAN.

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.

Inferior alveolar nerve transection enhanced formalin-induced nocifensive responses in the upper lip: systemic buprenorphine had more antinociceptive efficacy over morphine.

This study was designed to investigate the efficacy of a partial µ-opioid agonist, buprenorphine, against the formalin-induced hyperalgesia in the upper lip in chronically inferior alveolar nerve (IAN)-transected rats. Subcutaneous injection of diluted formalin into the upper lip in the IAN-transected rats showed an increased number of pain-related behavior (PRB; face-rubbing behavior) in every phase up to 45 min (p < 0.01) compared with that in the nontransected sham control rats. The numbers of c-Fos-immunoreactive (IR) cells in the superficial layers of the trigeminal nucleus caudalis (VcI/II) at the rostral (0-0.7 mm caudal to the obex) and middle levels (1.4-2.2 mm caudal to the obex) 2 h after the formalin injection in the IAN-transected rats were significantly increased compared with those in the control rats. The PRB in phases 1 and 2 (0-15 and 15-30 min after formalin injection) in rats with preadministration of morphine (3 mg/kg i.p.) or buprenorphine (100 µg/kg i.p.) was significantly (p < 0.05) smaller than those in the control rats. There was no significant difference in the efficacy between morphine and buprenorphine at these doses. The antinociceptive efficacy in phase 2 of buprenorphine (100 µg/kg) was higher (p < 0.05) than that of morphine (3 mg/kg) in the IAN-transected rats. The number of c-Fos-IR cells in the VcI/II at every level (0-3.6 mm caudal to the obex) after formalin injection was significantly decreased (p < 0.01) with preadministration of morphine (3 mg/kg) or buprenorphine (100 µg/kg) in the control rats. In the IAN-transected rats, the number of c-Fos-IR cells in the caudal VcI/II (2.2-3.6 mm caudal to the obex) after formalin injection was significantly decreased (p < 0.01) with preadministration of buprenorphine (100 µg/kg) but not so much (2.2-2.9 mm caudal to the obex, p < 0.05; 2.9-3.6 mm caudal to the obex, p > 0.05) with preadministration of morphine (3 mg/kg). These results indicate that IAN transection enhanced formalin-induced nocifensive responses in the upper lip, the dermatome of the intact nerve neighboring the IAN. Systemic preadministration of buprenorphine had more antinociceptive effects on the formalin-induced nocifensive behavior in the upper lip compared with morphine in the IAN-transected rats.

Healing of periodontal defects and calcitonin gene related peptide expression following inferior alveolar nerve transection in rats.

The roles of nerve and neuropeptides in the process of bone formation and remolding have been studied previously. However, the effects of nervous system and neuropeptide on periodontal alveolar bone formation remained unknown. The aim of this study was to assess the effect of innervation on regeneration of alveolar bone and expression levels of calcitonin gene related peptide (CGRP) in periodontal tissues of rats, so as to have a better understanding of the effect of nerve and its related neuropeptide on periodontal tissue regeneration. Rats received transection of the left inferior alveolar nerve and a surgery to produce bilateral periodontal defect, then the alveolar tissue was obtained from animals of each group at week 1, 2, 4, 6 and 8 weeks after operation, respectively. Hematoxylin and eosin staining, and Masson staining were performed to evaluate the ability to restore and repair periodontal tissues at 4, 6 and 8 after surgery. Then new bone formation area and mineralized area were quantified using imagepro-plus6.0 software after pictures were taken under the microscope and SPSS17.0 was used for statistical analysis. Immunohistochemical staining was applied to investigate the expression of CGRP at 1, 2, 4, 6 and 8 weeks. Rats received transection of the left inferior alveolar nerve surgery and were then sacrificed at day 1, 3, 7, 14, 21, 28 after the operation. The change of CGRP expression in periodontal tissue was detected using immunohistochemical methods. The results showed that the volume of new bone formation was not significantly difference between the experimental and control groups, but the mineralized new bone area between the two groups was statistically significant. The level of CGRP expression was lower than normal at week 1, and then it began to rise in the next stage. The plateau, at higher than normal level, was reached at 6 weeks post-surgery. Results of transection of the left inferior alveolar nerve demonstrated the expression of CGRP was decreased in early stage; it reached the lowest level at day 7. Then the expression level began to increase until it returned to normal level at day 28. The results of this study suggest that nerve and its related neuropeptide CGRP are the important factors that can affect the quality of regenerated alveolar bone by reducing bone density during the mineralization process.

Genome-wide association study of sensory disturbances in the inferior alveolar nerve after bilateral sagittal split ramus osteotomy.

BACKGROUND: Bilateral sagittal split ramus osteotomy (BSSRO) is a common orthognatic surgical procedure. Sensory disturbances in the inferior alveolar nerve, including hypoesthesia and dysesthesia, are frequently observed after BSSRO, even without distinct nerve injury. The mechanisms that underlie individual differences in the vulnerability to sensory disturbances have not yet been elucidated. METHODS: The present study investigated the relationships between genetic polymorphisms and the vulnerability to sensory disturbances after BSSRO in a genome-wide association study (GWAS). A total of 304 and 303 patients who underwent BSSRO were included in the analyses of hypoesthesia and dysesthesia, respectively. Hypoesthesia was evaluated using the tactile test 1 week after surgery. Dysesthesia was evaluated by interview 4 weeks after surgery. Whole-genome genotyping was conducted using Illumina BeadChips including approximately 300,000 polymorphism markers. RESULTS: Hypoesthesia and dysesthesia occurred in 51 (16.8%) and 149 (49.2%) subjects, respectively. Significant associations were not observed between the clinical data (i.e., age, sex, body weight, body height, loss of blood volume, migration length of bone fragments, nerve exposure, duration of anesthesia, and duration of surgery) and the frequencies of hypoesthesia and dysesthesia. Significant associations were found between hypoesthesia and the rs502281 polymorphism (recessive model: combined χ² = 24.72, nominal P = 6.633 × 10⁻7), between hypoesthesia and the rs2063640 polymorphism (recessive model: combined χ² = 23.07, nominal P = 1.563 × 10⁻6), and between dysesthesia and the nonsynonymous rs2677879 polymorphism (trend model: combined χ² = 16.56, nominal P = 4.722 × 10⁻5; dominant model: combined χ² = 16.31, nominal P = 5.369 × 10⁻5). The rs502281 and rs2063640 polymorphisms were located in the flanking region of the ARID1B and ZPLD1 genes on chromosomes 6 and 3, whose official names are "AT rich interactive domain 1B (SWI1-like)" and "zona pellucida-like domain containing 1", respectively. The rs2677879 polymorphism is located in the METTL4 gene on chromosome 18, whose official name is "methyltransferase like 4". CONCLUSIONS: The GWAS of sensory disturbances after BSSRO revealed associations between genetic polymorphisms located in the flanking region of the ARID1B and ZPLD1 genes and hypoesthesia and between a nonsynonymous genetic polymorphism in the METTL4 gene and dysesthesia.

Laser therapy and pain-related behavior after injury of the inferior alveolar nerve: possible involvement of neurotrophins.

Nerve-related complications have been frequently reported in dental procedures, and a very frequent type of occurrence involves the inferior alveolar nerve (IAN). The nerve injury in humans often results in persistent pain accompanied by allodynia and hyperalgesia. In this investigation, we used an experimental IAN injury in rats, which was induced by a Crile hemostatic clamp, to evaluate the effects of laser therapy on nerve repair. We also studied the nociceptive behavior (von Frey hair test) before and after the injury and the behavioral effects of treatment with laser therapy (emitting a wavelength of 904 nm, output power of 70 Wpk, a spot area of ∼0.1 cm², frequency of 9500 Hz, pulse time 60 ns and an energy density of 6 J/cm²). As neurotrophins are essential for the process of nerve regeneration, we used immunoblotting techniques to preliminarily examine the effects of laser therapy on the expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The injured animals treated with laser exhibited an improved nociceptive behavior. In irradiated animals, there was an enhanced expression of NGF (53%) and a decreased BDNF expression (40%) after laser therapy. These results indicate that BDNF plays a locally crucial role in pain-related behavior development after IAN injury, increasing after lesions (in parallel to the installation of pain behavior) and decreasing with laser therapy (in parallel to the improvement of pain behavior). On the other hand, NGF probably contributes to the repair of nerve tissue, in addition to improving the pain-related behavior.

Increased expression of interleukin-18 in the trigeminal spinal subnucleus caudalis after inferior alveolar nerve injury in the rat.

Interleukin-18 (IL-18) is an important regulator of innate and immune responses, and is known to be expressed in various types of cells and upregulated in pathological conditions including tissue injury and inflammation, suggesting it has both proinflammatory and compensatory roles. Here we show that IL-18 was increased in microglia in the trigeminal spinal subnucleus caudalis (Vc) after peripheral nerve injury. We used a trigeminal neuropathic pain model in which the withdrawal threshold of maxillary whisker pad skin was significantly decreased after inferior alveolar nerve transection, and observed a striking increase in IL-18 expression in the Vc around the obex area from 3d and continued until 14d after nerve injury. The IL-18 labeled cells were largely colocalized with Iba1, suggesting this upregulation occurred in hyperactive microglia. We also found that the IL-18 induction coexisted with phosphorylated p38 MAPK, indicating a possible role of p38 in the regulation of IL-18. Our findings are the first report that injury of trigeminal nerve induced IL-18 upregulation in activated microglia in the Vc, suggesting a possible role of IL-18 in orofacial neuropathic pain.

Qualitative effect on mRNAs of injury-associated proteins by cell phone like radiation in rat facial nerves.

Rats were exposed to cell phone radiation for 6 hours per day for 18 weeks. The buccal and mandibular branches of the facial nerve were evaluated for this study. The mRNA levels of four proteins that are usually up regulated when an injury has occurred were investigated; included were Calcium ATP-ase, Endothelin, Neural Cell Adhesion Molecule, and Neural Growth Factor. These isolated mRNAs were subjected to RT-PCR and all four were up regulated. The mandibular nerve showed a higher and broader level of up regulation than the buccal nerve. All four mRNA up regulations for the mandibular nerve and two for the buccal nerve were also statistically significant. These specific injury-related findings were mild. As the use of these cell phones continues, there most likely will be permanent damage to these tissues over the years and the likelihood of tumors, cancers, and system failures will potentially increase.