The involvement of Neuregulin-1 in the process of facial nerve injury repair through the utilization of dental pulp stem cells.

BACKGROUND: Facial nerve injury often results in poor prognosis due to the challenging process of nerve regeneration. Neuregulin-1, a human calmodulin, is under investigation in this study for its impact on the reparative capabilities of Dental Pulp Stem Cells (DPSCs) in facial nerve injury. METHODS: Lentivirus was used to transfect and construct Neuregulin-1 overexpressed DPSCs. Various techniques assessed the effects of Neuregulin-1: osteogenic induction, lipid induction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Counting Kit-8 assay, wound healing, immunofluorescence, Phalloidin staining, nerve stem action potential, Hematoxylin-eosin staining, transmission electron microscopy, and immunohistochemistry. RESULTS: Neuregulin-1 effectively enhanced the proliferation, migration, and cytoskeletal rearrangement of DPSCs, while simultaneously suppressing the expression of Ras homolog gene family member A (RhoA) and Microfilament actin (F-actin). These changes facilitated the neural differentiation of DPSCs. Additionally, in vivo experiments showed that Neuregulin-1 expedited the restoration of action potential in the facial nerve trunk, increased the thickness of the myelin sheath, and stimulated axon regeneration. CONCLUSION: Neuregulin-1 has the capability to facilitate the repair of facial nerve injuries by promoting the regenerative capacity of DPSCs. Thus, Neuregulin-1 is a significant potential gene in the reparative processes of nerve damage.

Morphofunctional regeneration by mesenchymal stem cell and IGF-1 inoculation in a model of facial nerve crush injury in rats.

OBJECTIVE: To analyze the morphofunctional regeneration process of facial nerve injury in the presence of insulin-like growth factor-1 and mesenchymal stem cells. METHODS: Fourteen Wistar rats suffered unilateral facial nerve crushing and were randomly divided into two groups. All received insulin-like growth factor-1 inoculation, but only half of the animals received an additional inoculation of mesenchymal stem cells. The animals were followed for 90 days and facial nerve regeneration was analyzed via spontaneous facial motor function tests and immunohistochemistry in the nerve motor nucleus. RESULTS: The group that received the growth factor and stem cells showed a statistically superior mean in vibrissae movements (p < 0.01), touch reflex (p = 0.05) and eye closure (p < 0.01), in addition to better immunohistochemistry reactivity. There was a statistically significant difference in the mean number of cells in the facial nerve nucleus between the experimental groups (p = 0.025), with the group that received the growth factor and stem cells showing the highest mean. CONCLUSION: The association between growth factor and stem cells potentiates the morphofunctional regeneration of the facial nerve, occurring faster and more effectively. LEVEL OF EVIDENCE: 4, degree of recommendation C.

Cellular Sources and Neuroprotective Roles of Interleukin-10 in the Facial Motor Nucleus after Axotomy.

Facial motoneuron (FMN) survival is mediated by CD4+ T cells in an interleukin-10 (IL-10)-dependent manner after facial nerve axotomy (FNA), but CD4+ T cells themselves are not the source of this neuroprotective IL-10. The aims of this study were to (1) identify the temporal and cell-specific induction of IL-10 expression in the facial motor nucleus and (2) elucidate the neuroprotective capacity of this expression after axotomy. Immunohistochemistry revealed that FMN constitutively produced IL-10, whereas astrocytes were induced to make IL-10 after FNA. Il10 mRNA co-localized with microglia before and after axotomy, but microglial production of IL-10 protein was not detected. To determine whether any single source of IL-10 was critical for FMN survival, Cre/Lox mouse strains were utilized to selectively knock out IL-10 in neurons, astrocytes, and microglia. In agreement with the localization data reflecting concerted IL-10 production by multiple cell types, no single cellular source of IL-10 alone could provide neuroprotection after FNA. These findings suggest that coordinated neuronal and astrocytic IL-10 production is necessary for FMN survival and has roles in neuronal homeostasis, as well as neuroprotective trophism after axotomy.

Inhibiting Matrix Metalloproteinases Protects Evoked Electromyography Amplitudes and Muscle Tension in the Orbicularis Oris Muscle in a Rat Model of Facial Nerve Injury.

Facial nerve injury results in degradation of the neuromuscular junction (NMJ) and blocks neurotransmission between the pre- and postsynaptic structures, which are separated by a synaptic cleft. Matrix metalloproteinases (MMPs), enzymes that degrade and modify the extracellular matrix, play critical roles in regulating NMJ remodeling. We previously demonstrated that MMP1, MMP2, MMP3, MMP7, and MMP9 are overexpressed in facial nerve-innervated orbicularis oris muscle after facial nerve injury in a rat model. In the present study, the MMP inhibitor prinomastat was administered to rats after facial nerve injury. The MMP levels, agrin expression, and muscle-specific kinase (MuSK) phosphorylation were evaluated. Variations in evoked electromyography (EEMG) amplitude were also recorded. Compared with the control group, MMP expression in the orbicularis oris after facial nerve injury was significantly reduced in the prinomastat group. Inhibition of MMP expression maintained agrin expression and MuSK phosphorylation; the NMJ morphology was also protected after the injury. Moreover, prinomastat treatment sustained EEMG amplitude and muscle tension after the injury. These findings indicate that inhibiting MMPs can protect the function and morphology of the NMJ and demonstrate the need for protection of the NMJ at early stages after facial nerve injury.

Identification of genes involved in neuronal cell death and recovery over time in rat axotomy and neurorrhaphy models through RNA sequencing.

Facial nerves are frequently injured during cosmetic or other types of facial surgery. However, information on the genes involved in the damage and recovery of the facial nerves is limited. Here, we aimed to identify the genes affected by facial nerve injury and repair using next-generation sequencing. We established a rat axotomy model and a parallel epineurial neurorrhaphy model, in which gene expression was analyzed from 3 days to 8 weeks after surgery. We discovered that ARRB1, SGK1, and GSK3B genes associated with neuronal cell death were upregulated in the axotomy model. In contrast, MFRP, MDK, and ACE genes involved in neural recovery and regeneration exhibited higher expression in the neurorrhaphy model. In the present study, the analysis of the big data obtained from the next-generation sequencing (RNA-seq) technology reveals that the expression of genes involved in neuronal cell death is induced during nerve damage, and those associated with neural recovery are more abundantly expressed during repair processes. These results are considered to be useful for the establishment of the treatment of related diseases and basic research in various neuroscience fields by utilizing damage and recovery mechanism of facial nerves.

Hedgehog signaling promotes endoneurial fibroblast migration and Vegf-A expression following facial nerve injury.

BACKGROUND: Peripheral nerve injuries are a common clinical problem which may result in permanent loss of motor or sensory function. A better understanding of the signaling pathways that lead to successful nerve regeneration may help in discovering new therapeutic targets. The Hedgehog (Hh) signaling pathway plays significant roles in nerve development and regeneration. In a mouse model of facial nerve injury, Hedgehog-responsive fibroblasts increase in number both at the site of injury and within the distal nerve. However, the role of these cells in facial nerve regeneration is not fully understood. We hypothesize that the Hh pathway plays an angiogenic and pro-migratory role following facial nerve injury. METHODS: Hedgehog pathway modulators were applied to murine endoneurial fibroblasts isolated from the murine facial nerve. The impact of pathway modulation on endoneurial fibroblast migration and cell proliferation was assessed. Gene expression changes of known Hedgehog target genes and the key angiogenic factor Vegf-A were determined by qPCR. In vivo, mice were treated with pathway agonist (SAG21k) and injured facial nerve specimens were analyzed via immunofluorescence and in situ hybridization. RESULTS: Hedgehog pathway activation in facial nerve fibroblasts via SAG21k treatment increases Gli1 and Ptch1 expression, the rate of cellular migration, and Vegf-A expression in vitro. In vivo, expression of Gli1 and Vegf-A expression appears to increase after injury, particularly at the site of nerve injury and the distal nerve, as detected by immunofluorescence and in situ hybridization. Additionally, Gli1 transcripts co-localize with Vegf-A following transection injury to the facial nerve. DISCUSSION: These findings describe an angiogenic and pro-migratory role for the Hedgehog pathway mediated through effects on nerve fibroblasts. Given the critical role of Vegf-A in nerve regeneration, modulation of this pathway may represent a potential therapeutic target to improve facial nerve regeneration following injury.

ANXA1 directs Schwann cells proliferation and migration to accelerate nerve regeneration through the FPR2/AMPK pathway.

Many factors are involved in the process of nerve regeneration. Understanding the mechanisms regarding how these factors promote an efficient remyelination is crucial to deciphering the molecular and cellular processes required to promote nerve repair. Schwann cells (SCs) play a central role in the process of peripheral nerve repair/regeneration. Using a model of facial nerve crush injury and repair, we identified Annexin A1 (ANXA1) as the extracellular trigger of SC proliferation and migration. ANXA1 activated formyl peptide receptor 2 (FPR2) receptors and the downstream adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling cascade, leading to SC proliferation and migration in vitro. SCs lacking FPR2 or AMPK displayed a defect in proliferation and migration. After facial nerve injury (FNI), ANXA1 promoted the proliferation of SCs and nerve regeneration in vivo. Collectively, these data identified the ANXA1/FPR2/AMPK axis as an important pathway in SC proliferation and migration. ANXA1-induced remyelination and SC proliferation promotes FNI regeneration.

Accelerated outgrowth in cross-facial nerve grafts wrapped with adipose-derived stem cell sheets.

In this study, we devised a novel cross-facial nerve grafting (CFNG) procedure using an autologous nerve graft wrapped in an adipose-derived stem cell (ADSC) sheet that was formed on a temperature-responsive dish and examined its therapeutic effect in a rat model of facial palsy. The rat model of facial paralysis was prepared by ligating and transecting the main trunk of the left facial nerve. The sciatic nerve was used for CFNG, connecting the marginal mandibular branch of the left facial nerve and the marginal mandibular branch of the right facial nerve. CFNG alone, CFNG coated with an ADSC suspension, and CFNG wrapped in an ADSC sheet were transplanted in eight rats each, designated the CFNG, suspension, and sheet group, respectively. Nerve regeneration was compared histologically and physiologically. The time to reinnervation, assessed by a facial palsy scoring system, was significantly shorter in the sheet group than in the other two groups. Evoked compound electromyography showed a significantly higher amplitude in the sheet group (4.2 ± 1.3 mV) than in the suspension (1.7 ± 1.2 mV) or CFNG group (1.6 ± 0.8 mV; p < .01). Toluidine blue staining showed that the number of myelinated fibers was significantly higher in the sheet group (2,450 ± 687) than in the suspension (1,645 ± 659) or CFNG group (1,049 ± 307; p < .05). CFNG in combination with ADSC sheets, prepared using temperature-responsive dishes, promoted axonal outgrowth in autologous nerve grafts and reduced the time to reinnervation.

CD4+ T cell expression of the IL-10 receptor is necessary for facial motoneuron survival after axotomy.

BACKGROUND: After peripheral nerve transection, facial motoneuron (FMN) survival depends on an intact CD4+ T cell population and a central source of interleukin-10 (IL-10). However, it has not been determined previously whether CD4+ T cells participate in the central neuroprotective IL-10 cascade after facial nerve axotomy (FNA). METHODS: Immunohistochemical labeling of CD4+ T cells, pontine vasculature, and central microglia was used to determine whether CD4+ T cells cross the blood-brain barrier and enter the facial motor nucleus (FMNuc) after FNA. The importance of IL-10 signaling in CD4+ T cells was assessed by performing adoptive transfer of IL-10 receptor beta (IL-10RB)-deficient CD4+ T cells into immunodeficient mice prior to injury. Histology and qPCR were utilized to determine the impact of IL-10RB-deficient T cells on FMN survival and central gene expression after FNA. Flow cytometry was used to determine whether IL-10 signaling in T cells was necessary for their differentiation into neuroprotective subsets. RESULTS: CD4+ T cells were capable of crossing the blood-brain barrier and associating with reactive microglial nodules in the axotomized FMNuc. Full induction of central IL-10R gene expression after FNA was dependent on CD4+ T cells, regardless of their own IL-10R signaling capability. Surprisingly, CD4+ T cells lacking IL-10RB were incapable of mediating neuroprotection after axotomy and promoted increased central expression of genes associated with microglial activation, antigen presentation, T cell co-stimulation, and complement deposition. There was reduced differentiation of IL-10RB-deficient CD4+ T cells into regulatory CD4+ T cells in vitro. CONCLUSIONS: These findings support the interdependence of IL-10- and CD4+ T cell-mediated mechanisms of neuroprotection after axotomy. CD4+ T cells may potentiate central responsiveness to IL-10, while IL-10 signaling within CD4+ T cells is necessary for their ability to rescue axotomized motoneuron survival. We propose that loss of IL-10 signaling in CD4+ T cells promotes non-neuroprotective autoimmunity after FNA.

Dopaminergic Modulation of Orofacial Mechanical Hypersensitivity Induced by Infraorbital Nerve Injury.

While the descending dopaminergic control system is not fully understood, it is reported that the hypothalamic A11 nucleus is its principle source. To better understand the impact of this system, particularly the A11 nucleus, on neuropathic pain, we created a chronic constriction injury model of the infraorbital nerve (ION-CCI) in rats. ION-CCI rats received intraperitoneal administrations of quinpirole (a dopamine D2 receptor agonist). ION-CCI rats received microinjections of quinpirole, muscimol [a gamma-aminobutyric acid type A (GABAA) receptor agonist], or neurotoxin 6-hydroxydopamine (6-OHDA) into the A11 nucleus. A von Frey filament was used as a mechanical stimulus on the maxillary whisker pad skin; behavioral and immunohistochemical responses to the stimulation were assessed. After intraperitoneal administration of quinpirole and microinjection of quinpirole or muscimol, ION-CCI rats showed an increase in head-withdrawal thresholds and a decrease in the number of phosphorylated extracellular signal-regulated kinase (pERK) immunoreactive (pERK-IR) cells in the superficial layers of the trigeminal spinal subnucleus caudalis (Vc). Following 6-OHDA microinjection, ION-CCI rats showed a decrease in head-withdrawal thresholds and an increase in the number of pERK-IR cells in the Vc. Our findings suggest the descending dopaminergic control system is involved in the modulation of trigeminal neuropathic pain.

Rat Facial Nerve Regeneration with Human Immature Dental Pulp Stem Cells.

Facial paralysis can result in severe implications for the patients. However, stem cell biology has become an important field in regenerative medicine since the discovery and characterization of mesenchymal stem cells. Our aim was to evaluate the regeneration after facial nerve crush injury and application of human immature dental pulp stem cells (iDPSC). For this study 70 Wistar rats underwent a unilateral facial nerve crush injury and were divided into two groups: Group I (GI): Crushed; Group II (GII): Crushed and iDPSC, and distributed into study periods of 3, 7, 14, 21, and 42 postoperative days. Facial nerve regeneration was analyzed via functional recovery of whisker movement, histomorphometric analysis, and immunoblotting assay. The results show that GII had complete functional recovery at 14 days, while GI recovered after 42 days. Also, regarding the facial nerve trunk, GII presented histological improvement, evidencing better axonal and structural organization of the myelin sheath, and exhibited statistically higher values for the outer and inner perimeters and g-ratio. Nevertheless, GI exhibited statistically higher values for the thickness of myelin sheath. In the buccal branch, no differences were observed for all parameters between groups. At 42 days, both groups GI and GII were close to the levels observed for the control group. Concerning nerve growth factor expression, GII exhibited statistically greater values (p < 0.05) compared with the control group at 7 days. In summary, a single injection of human iDPSC promoted a positive effect on regeneration of the facial nerve trunk after 14 days and provided an alternative to support regeneration following peripheral nerve injury.

Nicotinic acetylcholine receptor subunit expression in the gastrocnemius and in the orbicularis oris before and after facial nerve injury in rats.

Objectives: To observe the expression of nicotinic acetylcholine receptor (AChR) subunits in normal orbicularis oris and gastrocnemius muscles and to explore the relationships between the expression of AChR subunits and the severity of facial nerve injury. Methods: Gene and protein expression of AChR subunits in the orbicularis oris and gastrocnemius muscles of male Sprague-Dawley rats was measured by reverse transcription polymerase chain reaction and western blotting, respectively, 1-90 days after graded facial nerve injury. Results: Expression of ε-AChR in the normal orbicularis oris was significantly higher than that in the gastrocnemius, whereas no γ subunit expression was observed. Expression of α, ß, δ, ε, and γ subunits was upregulated in the orbicularis oris and was positively correlated with the degree of facial nerve injury. Discussion: We demonstrated the higher expression of the AChR subunits in the orbicularis oris, compared to gastrocnemius muscles. The differences in expression of these subunits between muscles innervated by the facial nerve and somatic nerves and the correlation of AChR subunit expression with the degree of facial nerve injury yield insights into the sensitivity to muscle relaxants during intraoperative facial nerve monitoring.

Two Pathways Regulate Differential Expression of nAChRs Between the Orbicularis Oris and Gastrocnemius.

BACKGROUND: We previously demonstrated differential expression of nicotinic acetylcholine receptors (nAChRs) in the facial nerve-innervated orbicularis oris and somatic nerve-innervated gastrocnemius, which contribute to different sensitivities to muscle relaxants. Furthermore, the orbicularis oris exhibits less sensitivity to muscle relaxants after facial nerve injury, which is also related to upregulation of nAChRs. Here, we explored the regulatory mechanism for the different expression patterns. Because the agrin/Lrp4/MuSK/rapsyn and neuregulin1/ErbB signaling pathways are indispensable for maintaining the expression of nAChRs, we examined the activity of these two signaling pathways in gastrocnemius and orbicularis oris innervated by normal or injured facial nerves. MATERIALS AND METHODS: A quantitative analysis of these two signaling pathways was realized by immunofluorescence, and immunoprecipitation was applied to detect the level of phosphorylated MuSK in the gastrocnemius and orbicularis oris innervated by normal or injured facial nerves in adult rats. RESULTS: ErbB and the phosphorylated MuSK were expressed more in orbicularis oris than in gastrocnemius (P < 0.05). No significant difference was found in the expression of agrin/Lrp4/MuSK/rapsyn. After facial nerve injury, the level of agrin and the percentage of phosphorylated MuSK decreased significantly, although the expression levels of MuSK, rapsyn, and neuregulin1/ErbB were highly upregulated. CONCLUSIONS: Differential expression of the neuregulin1/ErbB signaling pathway may account for the different expression patterns of nAChRs at the neuromuscular junctions of the orbicularis oris and gastrocnemius. Overexpression of MuSK and rapsyn may contribute to upregulation of nAChRs after facial nerve injury.

Collagen/ß-TCP nerve guidance conduits promote facial nerve regeneration in mini-swine and the underlying biological mechanism: A pilot in vivo study.

This study aimed to evaluate the efficiency of new nerve guidance conduits (NGCs) in bridging facial nerve gaps, and investigate the underlying biological mechanisms implicated in the regeneration process. A collagen/ß-TCP conduit was prepared and applied to a facial nerve gap in a mini-swine model. Functional recovery and axonal regeneration were further evaluated by electrophysiological and histological examinations at 3 months after surgery. Furthermore, the global transcriptomic profiles of regenerated and normal tissues were analyzed by gene microarray to identify the differentially expressed genes at day three and seven, postoperatively. Subsequently, associated biological processes were analyzed by gene ontology (GO) enrichment analysis. The electrophysiological examination and morphological analysis revealed that significant nerve regeneration effects were achieved in the Col/ß-TCP group (p < 0.05). Transcriptional analysis revealed that at day three post-surgery, the majority of overexpressed genes were associated with inflammatory, immune and stimuli response, accompanied by angiogenesis, while at day seven, the majority of overexpressed genes were associated with cell, tissue and organ regeneration and development, synaptic transmission, neurogenesis, and neuronal differentiation, as well as the WNT, MAPK/ERK, and JAK/STAT signaling pathways. In conclusion, the present results suggest that collagen/ß-TCP NGCs provide a promising tubular micro-environment for nerve regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1122-1131, 2019.

CXCL12 has therapeutic value in facial nerve injury and promotes Schwann cells autophagy and migration via PI3K-AKT-mTOR signal pathway.

Facial nerve injury is a clinically common disease accompanied by demyelination of damaged nerves. The remyelination of damaged nerves and the unsatisfactory function recovery are problems that have been plaguing people for a long time. The role that CXCL12 plays after facial nerve injury remains unknown. Our experiments found that the expression of CXCL12 was up-regulated in the early stage of facial nerve injury and decreased after two weeks. Further research found that CXCL12 had no effect on Schwann cells proliferation, apoptosis and cell cycle, while significantly promoted Schwann cells migration. Treatment with CXCL12 decreased the phosphorylation of PI3K, AKT and mTOR, but increased autophagy marker LC3II/I. The CXCL12-induced Schwann cells migration was significantly attenuated by inhibition of autophagy and activation of PI3K pathway through pretreatment with 3-MA and IGF-1 respectively, and this effect was enhanced by PI3K pathway inhibitor LY294002. Animal experiment also confirmed that CXCL12 could improve facial nerve function and myelin regeneration. The findings of this study indicate that CXCL12 can promote the migration of Schwann cells and potentially become a key molecule in the repair of facial nerve injury.

The role of mutated SOD1 gene in synaptic stripping and MHC class I expression following nerve axotomy in ALS murine model.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motoneuron death. Several cellular pathways have been described to be involved in ALS pathogenesis; however, the involvement of presynaptic stripping and the related MHC class I molecules in mutant SOD1 motoneurons remains to be clarified. To this purpose, we here investigated, for the first time, the motoneurons behavior, di per seand after facial axonal injury, in terms of synaptic stripping and MHC class I expression in wild-type (Wt) mice and in a murine model of ALS, the SOD1(G93A) mice, at the presymptomatic and symptomatic stage of the disease. Concerning Wt animals, we found a reduction in synaptophysin immunoreactivity and an increase of MHC class I molecules in facial motoneurons after axotomy. In uninjured motoneurons of SOD1(G93A) mice, an altered presynaptic framework was evident, and this phenomenon increased during the disease course. The alteration in the presynaptic input is related to excitatory fibers. Moreover, after injury, a further decrease of excitatory input was not associated to an upregulation of MHC class I molecules in motoneuron soma. This study demonstrates, for the first time, that the presence of mutated SOD1 protein affects the MHC class I molecules expression, altering the presynaptic input in motoneurons. Nevertheless, a positive MHC class I immunolabeling was evident in glial cells around facial injured motoneurons, underlying an involvement of these cells in synaptic stripping. This study contributes to better understand the involvement of the mutated SOD1 protein in the vulnerability of motoneurons after damage.

Sustained delivery of glial cell-derived neurotrophic factors in collagen conduits for facial nerve regeneration.

Facial nerve injury caused by traffic accidents or operations may reduce the quality of life in patients, and recovery following the injury presents unique clinical challenges. Glial cell-derived neurotrophic factor (GDNF) is important in nerve regeneration; however, soluble GDNF rapidly diffuses into body fluids, making it difficult to achieve therapeutic efficacy. In this work, we developed a rat tail derived collagen conduit to connect nerve defects in a simple and safe manner. GDNF was immobilized in the collagen conduits via chemical conjugation to enable controlled release of GDNF. The GDNF delivery system prevented rapid diffusion from the site without impacting bioactivity of GDNF; degradation of the collagen conduit was inhibited owing to the chemical conjugation. The artificial nerve conduit was then used to examine facial nerve regeneration across a facial nerve defect. Following transplantation, the artificial nerve conduits degraded gradually without causing dislocations and serious inflammation, with good integration into the host tissue. Functional and histological tests indicated that the artificial nerve conduits were able to guide the axons to grow through the defect, reaching the distal stumps. The degree of nerve regeneration in the group that was treated with the artificial nerve conduit approached that of the autograft group, and exceeded that of the other conduit grafted groups. STATEMENT OF SIGNIFICANCE: In this study, we developed artificial nerve conduits consisting of GDNF immobilized on collagen, with the aim of providing an environment for nerve regeneration. Our results show that the artificial nerve conduits guided the regeneration of axons to the distal nerve segment. GDNF was immobilized stably in the artificial nerve conduits, and therefore retained a sufficient concentration at the target site to effectively promote the regeneration process. The artificial nerve conduits exhibited good biocompatibility and facilitated nerve regeneration and functional recovery with an efficacy that was close to that of an autograft, and better than that of the other conduit grafted groups. Our approach provides an effective delivery system that overcomes the rapid diffusion of GDNF in body fluids, promoting peripheral nerve regeneration. The artificial nerve conduit therefore qualifies as a putative candidate material for the fabrication of peripheral nerve reconstruction devices.

Changes in the expression and localization of signaling molecules in mouse facial motor neurons during regeneration of facial nerves.

After injury, peripheral axons usually re-extend toward their target, and neuronal functions recover. Previous studies have reported that expression of various molecules are transiently altered in motor neurons after nerve injury, but the time course of these changes and their relationship with functional recovery have not been clearly demonstrated. We used the mouse facial nerve transection and suturing model, and examined the changes in expression of five molecules, choline acetyl transferase (ChAT), galanin, calcitonin gene-related protein (CGRP), gephyrin, and potassium chloride co-transporter 2 (KCC2) in the facial motor neurons after surgery until recovery. Number of ChAT-positive neurons was markedly decreased at days 3 and 7, and recovered to the normal level by day 60, when facial motor functions recovered. Localization of two neuropeptides, CGRP and galanin, was increased in the perikarya and axons during regeneration, and returned to the normal levels by days 60 and 28, respectively. Expression of two postsynaptic elements of γ-amino butyric acid synapses, gephyrin and KCC2, was decreased at days 3 and 7, and recovered by day 60. These results suggest that ChAT, CGRP, and KCC2 may be objective indicators of regeneration, and altering their expression may be related to the functional recovery and axonal re-extension.

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.

Neural stem/progenitor cells on collagen with anchored basic fibroblast growth factor as potential natural nerve conduits for facial nerve regeneration.

Introducing neural stem/progenitor cells (NS/PCs) for repairing facial nerve injuries could be an alternative strategy for nerve gap reconstruction. However, the lack of success associated with current methods of applying NS/PCs to neurological disease is due to poor engraftment following transplantation into the host tissue. In this work, we developed rat-tail collagen-based nerve conduits to repair lengthy facial nerve defects, promoting NS/PC proliferation in the natural nerve conduits with anchored bFGF to improve the therapeutic effects of cell transplantation. In vitro studies showed that heparinized collagen prevented leakage of bFGF and NS/PCs expended in the rat-tail collagen gel with the anchored bFGF. The natural nerve conduits were implanted to connect 8-mm facial nerve defects in rats. The repair outcomes including vibrissae movements, electrophysiological tests, immunohistochemistry and remyelination analysis of regenerated nerve were evaluated. At 12weeks after implantation, only natural nerve conduits treated group showed Hoechst labeled NS/PCs. Besides, the natural nerve conduit significantly promoted functional recovery and nerve growth, which was similar to those of the gold standard, an autograft. The animal experiment results suggesting that the natural nerve conduits were valuable for facial nerve reconstruction. STATEMENT OF SIGNIFICANCE: Neural stem/progenitor cells (NS/PCs) were beneficial for the treatment of nervous system diseases. However, after transplantation, the beneficial was limited because the number of living NS/PCs decreased rapidly due to insufficient signaling molecules, such as growth factors, in the microenvironments surrounding transplanted cells. In the present study, we constructed collagen-based nerve conduit with anchored bFGF to achieve higher numbers of NS/PCs for repairing facial nerve injury. Compared with other methods involving neutral salt treatment or dialysis, the fabrication method of collagen scaffolds was simple, low-cost and safe, requiring a relatively short time to prepare. At 12weeks after transplantation, the functional and histological results of natural nerve conduits treated group showed significant similarities to the gold standard method of nerve autografting.