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.

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.

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.

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.

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.

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.

Effects of calcitonin gene-related peptide on the expression and activity of nitric oxide synthase during mandibular bone healing in rabbits: an experimental study.

PURPOSE: Previous studies have found that calcitonin gene-related peptide (CGRP) takes part in the local regulation of bone growth and metabolism. However, the detailed regulatory mechanism of CGRP in the bone healing has not been explored. The objective of this study was to investigate the effects and the regulatory mechanism of CGRP on the expression and activity of nitric oxide synthase (NOS) in bony callus during mandibular defect healing. MATERIALS AND METHODS: To determine the effect of CGRP on bony callus, a bone defect in the left mandible was created in 48 adult rabbits (divided randomly into experimental and control group) and half of them underwent inferior alveolar nerve amputation. The bony callus were collected 4, 7, 14, and 28 days after operation, and the expressions of CGRP and NOS in the paraffin slices were analyzed with immunohistochemical staining. The activity of calcium-dependent nitric oxide synthase (cNOS) and inducible nitric oxide synthase (iNOS) in the fresh specimens were measured with the NOS detecting kit. RESULTS: In the immunohistochemical analysis of bony callus, the immunohistochemical stain of CGRP was lower in experimental groups than in control groups from 4 to 14 days (P< .05 or P< .01), and the stain of eNOS showed the same phenomena from 4 to 7 days (P< .01), but the stain of iNOS did not show any statistical difference. In the NOS activity analysis, the activity of cNOS was lower in experimental groups than in control groups from 4 to 7 days (P< .05 or P< .01), and the activity of iNOS was lower in experimental groups than in control groups from 7 to 14 days (P< .01). CONCLUSION: The expression and activity of NOS has a positive correlation with CGRP expression during bone healing. CGRP may promote fracture healing via regulating the expression and the activity of NOS.

Co-expression of nerve growth factor and p75NGFR in the inferior alveolar nerve after mandibular distraction osteogenesis.

During mandibular distraction osteogenesis (DO), the inferior alveolar nerve (IAN) is damaged during distractor activation, but spontaneously recovers during consolidation. Although many neurotrophic factors are known to play critical roles, there have been few studies on the mechanism of peripheral nerve recovery after DO. The aim of this study was to observe the expression pattern of p75NGFR (low-affinity receptor of NGF) and to detect autocrine growth activity in IANs following mandibular DO. Unilateral mandibular distractions (0.5mm each, twice per day for 10 days) were conducted on eight mongrel dogs. Two each were killed at 7, 14, 28 and 56 days after completing distraction. The distracted IAN and contralateral control nerve were harvested. Immunohistochemical staining was performed to determine p75NGFR expression, and double immunofluorescent staining to detect NGF and p75NGFR co-expression. Levels of p75NGFR expression were found to be significantly elevated at 7 and 14 days in Schwann cells located in the outer layer of axon, but were almost undetectable at 28 and 56 days. In double immunofluorescent images, the co-expression of NGF and p75NGFR was also detected at 7 and 14 days. p75NGFR plays an important role in remyelination due to its abundant expression in Schwann cells of damaged nerves, and NGF is an autocrine growth factor present in distracted IANs during the early consolidation period after mandibular DO.

Expression of nerve growth factor and vascular endothelial growth factor in the inferior alveolar nerve after distraction osteogenesis.

The objective of this study was to evaluate changes occurring in the inferior alveolar nerve (IAN) subsequent to mandibular distraction osteogenesis, with regard to the expression of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF). Unilateral mandibular distractions (0.5mm each, twice per day for 10 days) were conducted on 8 mongrel dogs. Two animals were killed at 7, 14, 28 and 56 days after completion of distraction. The distracted IAN and contralateral control nerve were then harvested and analysed histologically and immunohistochemically. Signs of acute nerve injury, including demyelination, were observed in the distracted IAN on the 7th and 14th day after distraction. At 56 days, the histological features of the distracted IAN were similar to those of the control nerve. The levels of NGF and VEGF expression were significantly elevated on the 7th and 14th day after distraction. NGF was expressed in most of the distracted nerve tissues, but VEGF was primarily detected in Schwann cells and the neurovasorum. VEGF expression had returned to normal but NGF expression was still profoundly elevated 28 days after distraction. NGF expression returned to normal levels at 56 days after distraction. NGF and VEGF appeared to have been elicited from the Schwann cells and damaged nervous tissues, and they may play important roles in the initial healing of damaged nerves. VEGF expression returned to normal more quickly than did NGF expression. This may indicate that hypoxic conditions within the distracted nerve had recovered to normal during the early stages of consolidation. Micro-vessels in the distracted nerve may have recovered more rapidly than did the nerve tissue itself.

Nitric oxide-induced increase of excitatory amino acid levels in the trigeminal nucleus caudalis of the rat with tactile hypersensitivity evoked by the loose-ligation of the inferior alveolar nerves.

To investigate whether or not N-methyl-D-aspartate (NMDA)/nitric oxide (NO) pathway in the trigeminal system is involved in the development and/or maintenance of such pathological pain states as the hyperalgesia and allodynia observed after dental surgery, we examined the alteration patterns of excitatory amino acid (EAA) level in the superficial layer of subnucleus caudalis of the brain-stem trigeminal sensory nuclear complex (SpVc-I,II) by in vivo microdialysis. A very high EAA release response was observed immediately after the start of the perfusion in ligated animals compared with sham-operated rats. The EAA level evoked by application of the 40-V tooth pulp-stimulation or 1% capsaicin cream was significantly higher in the ligated animals than those in the sham-operated animals. This increase of EAA level induced by capsaicin cream was inhibited by adding carboxy-PTIO (100 microM) to the perfusate. The applications of SNAP (2 mM) into the perfusate enhanced the level of EAAs in ligated animals and sham-operated animals. However, SNAP-evoked EAA levels in ligated animals were not significantly different compared with those of sham-operated animals. These results suggest that alterations in the stimulus-evoked raised EAA levels that occur in the site of the first synaptic relay of the dental pain pathway and which are expressed via endogenous NO, and which play an important role in development and/or maintenance of pathological pain states following dental peripheral nerve injury.

Neuropeptide expression following constriction or section of the inferior alveolar nerve in the ferret.

Some of the sensory abnormalities that follow peripheral nerve injury may result from the development of ectopic discharge from the damaged axons. Previous studies in our laboratory have shown that, following tight ligation of the inferior alveolar nerve (IAN), there is a close association between the time-course of this neural activity and the accumulation of neuropeptides at the injury site. In this study we investigated whether the type of injury has any effect on the time-course or level of neuropeptide expression. In 36 adult ferrets, the IAN was either loosely constricted or sectioned, and the animals left to recover for 3 days, 3 weeks, or 3 months. The tissue was processed using indirect immunofluorescence and image analysis was used to quantify levels of substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, enkephalin, galanin, and neuropeptide Y. Immunoreactivity to all of the neuropeptides was present within the injured nerve 3 days after both types of injury, and decreased to lower levels by 3 weeks and 3 months. Comparisons between the levels of neuropeptide immunoreactivity in each group revealed that the pattern of accumulation was similar following loose constriction or section, and also similar to that found in our previous study on tight ligation. For each injury the time-course of neuropeptide expression was similar to that of the spontaneous activity we had previously recorded. These data support the suggestion that neuropeptide accumulation may be linked to the development of ectopic neural activity but indicate that the type of injury has little effect on the extent of expression.

Haemodynamic and immunohistochemical studies of rat incisor pulp after denervation and subsequent re-innervation.

The effects of injury to the inferior alveolar nerve on the distribution of neuropeptides and neurogenic blood-flow reactions were studied in rat mandibular dental pulp. In normal incisor pulps, calcitonin gene-related peptide (CGRP)-like immunoreactivity was common, while substance P- and neurokinin (NKA)-positive nerve fibres were much less abundant. There were no signs of vasoactive intestinal peptide-like, neuropeptide Y-like or 5-hydroxytryptamine-like immunoreactivity. In normal pulps, electrical stimulation (100 microA, 5 ms, 15 Hz for 30 s) of the tooth crown resulted in transient vasoconstriction followed by vasodilation, which was enhanced after alpha-adrenoceptor blockade. At 3 days-4 weeks after unilateral nerve section there were no signs of CGRP-, substance P- and NKA-immunoreactivity, and there was no vasodilation in response to tooth stimulation. The vasoconstrictor response was also absent during this period but at 4 weeks postoperatively a weak response was obtained and after 7 weeks the vasoconstrictor response had regained normal amplitude. At 7 weeks postoperatively, a large number of CGRP-positive fibres had reappeared and at 11 weeks the pattern of CGRP-immunoreactivity was normal. However, substance P- and NKA-immunoreactivity were not found at 7 or 11 weeks after surgery. Vasodilator responses appeared at 7 weeks, and showed normal amplitude at 11 weeks after the creation of the nerve lesion. The results show that during nerve regeneration, sympathetic vasoconstriction was regained earlier than neurogenic vasodilation in rat incisor teeth. The reappearance of neurogenic vasodilation after nerve injury was temporarily associated with the presence of CGRP-immunoreactivity in regenerating trigeminal afferent nerves.