Rat Facial Nerve Research Articles

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

Prevention of denervated muscle atrophy with accelerated nerve-regeneration by babysitter procedure in rat facial nerve paralysis model.

The "babysitter" procedure is a reconstruction technique for facial nerve complete paralysis and uses the movement source from the healthy facial nerve with a cross-nerve graft. First, an end-to-side neurorrhaphy is performed between the affected facial nerve trunk and hypoglossal nerve for continuously delivering stimuli to the mimetic muscles for preventing the atrophy of mimetic muscles. Despite favorable clinical results, histological and physiological mechanisms remain unknown. This study attempted to establish a model for the "babysitter" procedure and find its efficacy in rats with facial nerve complete paralysis. A total of 16 Lewis rats were used and divided into 2 groups; cross nerve graft (n = 8) and babysitter groups (n = 8). The facial nerve trunk was transected in both groups. Babysitter group underwent a two-stage procedure. Cross nerve graft group underwent only the transfer of nerve graft from the healthy side to affected side. The animals were assessed physiologically by compound muscle action potential (CMAP), and the regenerated nerve tissues were evaluated histopathologically at 13 weeks after surgery. Facial nucleus stained with retrograde tracers proved the re-innervation of affected facial muscle by the babysitter procedure. In CMAP, the amplitude of babysitter group was significantly higher than that of the cross-facial nerve graft group (p < .05). Histological examination found a significant difference in myelin g-ratio between two groups (p < .05). This study investigated the "babysitter" procedure for rat facial nerve palsy. Babysitter procedure shortened the denervation period without mimic muscle atrophy.

The efficiency of ozone therapy and low-level laser therapy in rat facial nerve injury

PurposeComparison of low-level laser therapy (LLLT) and ozone therapy (OT) methods for the treatment of facial nerve injury (FNI) in rats, evaluated by histomorphometric measurement analysis. Materials and methodsThirty rats were randomly divided into control (C), LLLT, and OT groups. The left facial nerves (FNs) of all rats (n = 30) were used in this study. These were held in a surgical clamp for 30 s to create neuropathic damage. The non-injured right FNs of the rats in the control group formed the fourth, sham (S) group in this study (n = 10). Therefore the total number of evaluated samples was 40. The injured FNs of rats in the control group were left to heal spontaneously, whereas LLLT was applied for 21 consecutive days (output 100 mW/cm2 and wavelength 850 nm) and OT (2 ml; 80 μm/ml) once every 2 days for 21 days. ResultsAfter histomorphological evaluation, the OT group revealed statistically significant outcomes following FNI compared with the OT and control groups in terms of branching of nerve fibers (p = 0.003), nerve fiber diameters (p = 0.0398), nerve fiber areas (p = 0.042), and axon numbers (p = 0.0327). Although the LLLT group revealed a better healing process than the control group, the outcome was not statistically significant in terms of branching of nerve fibers (p = 0.6804), nerve fiber diameters (p = 0.7424), nerve fiber areas (p = 0.7048), and axon numbers (p = 0.7588). ConclusionsOT resulted in statistically significant differences in outcome when compared with the LLLT and control groups, and provided a safe and effective treatment for FNI in rats. OT could therefore be considered as an alternative treatment of FNI. Clinical studies should now be performed to establish whether comparable results can be achieved in humans.

Dedifferentiated fat cells in polyglycolic acid-collagen nerve conduits promote rat facial nerve regeneration.

IntroductionPolyglycolic acid (PGA) nerve conduits, an artificial biodegradable nerve regeneration-inducing tube currently used in clinical practice, are effective in regenerating peripheral nerves. Dedifferentiated fat (DFAT) cells differentiate into various cells including adipocytes, osteoblasts, chondrocytes, skeletal muscle cells, and myofibroblasts, when cultured in appropriate differentiation-inducing conditioned culture medium. This study made a hybrid artificial nerve conduit by filling a PGA conduit with DFAT cells, applied the conduit to a rat facial nerve defect model, and investigated the facial nerve regenerative ability of the conduit.MethodsUnder inhalational anesthesia, the buccal branch of the facial nerve in Lewis rats was exposed, and a 7-mm nerve defect was created. PGA nerve conduits were filled with DFAT cells, which were prepared from rat subcutaneous adipose tissue with type I collagen as a scaffold, and then grafted into the nerve defect sites in rats with a microscope (DFAT group) (n = 10). In other rats, PGA artificial nerve conduits alone were similarly grafted into the nerve defect sites (the control group) (n = 10). Reinnervation was confirmed at 13 weeks postoperatively by a retrograde tracer, followed by histological and physiological comparative studies.ResultsThe mean number of myelinated fibers was significantly higher in DFAT group (1605 ± 806.23) than in the control group (543.6 ± 478.66). Myelin thickness was also significantly lager in DFAT group (0.57 ± 0.17 μm) than in the control group.(0.46 ± 0.14 μm). Although no significant difference was found in the amplitude of compound muscle action potential (CMAP) between DFAT group (2.84 ± 2.47 mV) and the control group (0.88 ± 0.56 mV), whisker motion was lager in DFAT group (9.22° ± 0.65°) than in the control group (1.9° ± 0.84°).ConclusionsDFAT cell-filled PGA conduits were found to promote nerve regeneration in an experimental rat facial nerve defect model.

Effect of Motor versus Sensory Nerve Autografts on Regeneration and Functional Outcomes of Rat Facial Nerve Reconstruction

Cranial nerve injury is disabling for patients, and facial nerve injury is particularly debilitating due to combined functional impairment and disfigurement. The most widely accepted approaches for reconstructing nerve gap injuries involve using sensory nerve grafts to bridge the nerve defect. Prior work on preferential motor reinnervation suggests, however, that motor pathways may preferentially support motoneuron regeneration after nerve injury. The effect of motor versus sensory nerve grafting after facial nerve injury has not been previously investigated. Insights into outcomes of motor versus sensory grafting may improve understanding and clinical treatment of facial nerve paralysis, mitigating facial asymmetry, aberrant reinnervation, and synkinesis. This study examined motor versus sensory grafting of the facial nerve to investigate effect of pathway on regeneration across a 5-mm rodent facial nerve defect. We enrolled 18 rats in 3 cohorts (motor, sensory, and defect) and recorded outcome measures including fiber count/nerve density, muscle endplate reinnervation, compound muscle action potential, and functional whisker twitch analysis. Outcomes were similar for motor versus sensory groups, suggesting similar ability of sensory and motor grafts to support regeneration in a clinically relevant model of facial nerve injury.

Experimental Studies on Facial Nerve Regeneration

Insufficient recovery after injury of a peripheral motor nerve is due to (1) inappropriate pathfinding as a result of axonal regrowth to inappropriate targets, (2) excessive collateral axonal branching at the lesion site, and (3) polyinnervation of the neuromuscular junctions (NMJs). The rat facial nerve model is often used because of its simple and reliable readout to measure recovery of function (vibrissal whisking). Over the last decades scientists have concentrated their efforts to combat mostly NMJ polyinnervation, because it turned out to be very difficult to reduce collateral axonal branching and impossible to navigate thousands of axons toward the original fascicles. In the past, several groups of scientists concentrated their efforts to reduce the activity-dependent polyinnervation of NMJs by electrical stimulation of the muscles (square 0.1 msec pulses at 5 Hz). The results showed no recovery of functions and a severe reduction in the number of innervated NMJs to approximately one fifth of those observed in intact animals. More recent experiments, however, have shown that motor recovery improved significantly following mechanical stimulation of the denervated facial muscles (vibrissal and orbicularis oculi) and that restored functions could invariably be linked to reduced polyinnervation at the NMJ while the number of innervated NMJ remained the same. These results suggest that clinically feasible and effective therapies could be developed and tested in the near future. Anat Rec, 302:1287-1303, 2019. © 2019 Wiley Periodicals, Inc.

Mechanisms of the Immunomodulation Effects of Bone Marrow-Derived Mesenchymal Stem Cells on Facial Nerve Injury in Sprague-Dawley Rats.

Normal facial nerve (FN) function is very important for human being. However, if injured, FN function is difficult to restore completely. Recently, many studies reported the immune regulation function of stem cells (SCs). However, the immunomodulation function of SCs on FN injury is still unclear. Our study aims to explore the mechanism of immunomodulation effect of Sprague-Dawley rat bone marrow-derived SCs (BMSCs) on FN injury and specially focus on the regulation of Th17 and the protection effects of BMSCs on central facial motor neurons (FMNs). First, rat FNs were harvested. FN and BMSCs were cultured together or separately and levels of transforming growth factor (TGF)-β1, interleukin (IL)-6, hepatocyte growth factor (HGF), inducible nitric oxide synthase (iNOS), and prostaglandin E2 (PGE2) in supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Then, after treating with or without local BMSCs injection, the proportion of Th17 in neck lymph nodes (LNs) was investigated in rat FN injury models. Furthermore, the apoptotic index of FMNs was studied in rat FN injury models that were treated with or without BMSCs. We found that BMSCs could secrete high levels of IL-6, HGF, PGE2, iNOS, and TGF-β1 in culture. The percentage of Th17 of neck LNs in BMSCs-treated group was significantly lower than that in the control group. The apoptotic index of FMNs in BMSCs-treated group was significantly lower than that in the control group. In conclusion, our research indicates BMSCs could independently secrete cytokines IL-6, HGF, PGE2, iNOS, and TGF-β1, and these cytokines could regulate the balance among subsets of CD4+ T cells and could protect FMNs by inhibiting neuron apoptosis.

Surgical Technique and Other Protocols Used Probably Did Not Induce Polyethylene Glycol Fusion of Rat Facial Nerves.

Surgical Technique and Other Protocols Used Probably Did Not Induce Polyethylene Glycol Fusion of Rat Facial Nerves.

Stem Cells from Human Exfoliated Deciduous Teeth (SHED) Differentiate in vivo and Promote Facial Nerve Regeneration

Post-traumatic lesions with transection of the facial nerve present limited functional outcome even after repair by gold-standard microsurgical techniques. Stem cell engraftment combined with surgical repair has been reported as a beneficial alternative. However, the best association between the source of stem cell and the nature of conduit, as well as the long-term postoperative cell viability are still matters of debate. We aimed to assess the functional and morphological effects of stem cells from human exfoliated deciduous teeth (SHED) in polyglycolic acid tube (PGAt) combined with autografting of rat facial nerve on repair after neurotmesis. The mandibular branch of rat facial nerve submitted to neurotmesis was repaired by autograft and PGAt filled with purified basement membrane matrix with or without SHED. Outcome variables were compound muscle action potential (CMAP) and axon morphometric. Animals from the SHED group had mean CMAP amplitudes and mean axonal diameters significantly higher than the control group (p < 0.001). Mean axonal densities were significantly higher in the control group (p = 0.004). The engrafted nerve segment resected 6 weeks after surgery presented cells of human origin that were positive for the Schwann cell marker (S100), indicating viability of transplanted SHED and a Schwann cell-like phenotype. We conclude that regeneration of the mandibular branch of the rat facial nerve was improved by SHED within PGAt. The stem cells integrated and remained viable in the neural tissue for 6 weeks since transplantation, and positive labeling for S100 Schwann-cell marker suggests cells initiated in vivo differentiation.

Adipose-derived stem cells and the stromal vascular fraction in polyglycolic acid-collagen nerve conduits promote rat facial nerve regeneration.

Adipose-derived stem cells (ADSCs) and the stromal vascular fraction (SVF) promote nerve regeneration. Biodegradable nerve conduits are used to treat peripheral nerve injuries, but their efficiencies are lower than those of autologous nerve grafts. This study developed biodegradable nerve conduits containing ADSCs and SVF and evaluated their facial nerve regenerating abilities in a rat model with a 7-mm nerve defect. SVF and ADSCs were individually poured into nerve conduits with polyglycolic acid-type I collagen as a scaffold (ADSCs and SVF groups). The conduits were grafted on to the nerve defects. As the control, the defect was bridged with polyglycolic acid-collagen nerve conduits without cells. At 13 weeks, after transplantation, the regenerated nerves were evaluated physiologically and histologically. The compound muscle action potential of the SVF group was significantly higher in amplitude than that of the control group. Electron microscopy showed that the axon diameter of the SVF group was the largest, followed by the ADSC group and control group with significant differences among them. The SVF group had the largest fiber diameter, followed by the ADSC group and control group with significant differences among them. The ADSC group had the highest myelin thickness, followed by the SVF group and control group with significant differences among them. Identical excellent promoting effects on nerve regeneration were observed in both the ADSC and SVF groups. Using SVF in conduits was more practical than using ADSCs because only the enzymatic process was required to prepare SVF, indicating that SVF could be more suitable to induce nerve regeneration.

Effects of the Number of Muscle-Nerve-Muscle Grafts on Rat Facial Nerve Functional Recovery.

Facial nerve denervation can be devastating for patients. Primary neurorrhaphy and interposition (IP) nerve grafting are common reinnervation techniques. Muscle-nerve-muscle (MNM) grafting is a lesser known alternative. After previously demonstrating significant return of function using MNM grafting in a rat facial nerve model, the authors compare the use of multiple MNM nerve grafts with that of single MNM and IP nerve grafts. Thirty-six male rats were randomized into 4 groups: (1) repair with IP grafts, (2) 1 MNM graft, (3) 3 MNM grafts, and (4) control with no intervention. All groups had the lower zygomatic, buccal, and marginal mandibular branches of the right facial nerve removed. Return of movement and snout symmetry was measured over 16 weeks. Axonal regeneration and muscle atrophy were assessed and quantified. All intervention groups had significantly improved movement and snout symmetry compared with control. Rats in the IP group had significantly increased axon density compared with those in the MNM groups but with smaller axonal diameter than control rats. No difference in axon density or diameter was observed between MNM groups. Use of 3 MNM grafts and IP grafts resulted in preservation of similar muscle mass compared with the control and 1-MNM groups. MNM grafting may be an alternative when other reanimation techniques are not possible. NA.

Olfactory ensheathing cells in facial nerve regeneration

IntroductionOlfactory ensheathing cell is a unique kind of glia cells, which can promote axon growth. Little is known about the differences between olfactory mucosa olfactory ensheathing cells and olfactory bulb olfactory ensheathing cells in the capability to promote nerve regeneration. ObjectiveTo study the recovery of the rat facial nerve after olfactory ensheathing cells transplantation, and to compare the differences between the facial nerve regeneration of olfactory mucosa-olfactory ensheathing cells and olfactory bulb olfactory bulb olfactory ensheathing cells transplantation. MethodsInstitutional ethical guideline was followed (201510129A). Olfactory mucosa-olfactory ensheathing cells and olfactory bulb olfactory ensheathing cells were cultured and harvested after 7 days in vitro. 36 Sprague Dawley male rats were randomly divided into three different groups depending on the transplanting cells: Group A: olfactory mucosa-olfactory ensheathing cells; Group B: olfactory bulb olfactory ensheathing cells; Group C: DF-12 medium/fetal bovine serum. The main trunk of the facial nerve was transected and both stumps were inserted into a polylactic acid/chitosan conduit, then the transplanted cells were injected into the collagen in the conduits. After 4 and 8 weeks after the transplant, the rats of the three groups were scarified and the facial function score, facial nerve evoked potentials, histology analysis, and fluorescent retrograde tracing were tested and recorded, respectively, to evaluate the facial nerve regeneration and to analysis the differences among the three groups. ResultsOlfactory ensheathing cells can promote the facial nerve regeneration. Compared with olfactory bulb olfactory ensheathing cells, olfactory mucosa olfactory ensheathing cells were more effective in promoting facial nerve regeneration, and this difference was more significant 8 weeks after the transplantation than 4 weeks. ConclusionWe discovered that olfactory ensheathing cells with nerve conduit could improve the facial nerve recovery, and the olfactory mucosa olfactory ensheathing cells are more effective for facial nerve regeneration compared with olfactory bulb olfactory ensheathing cells 8 weeks after the transplantation. These results could cast new light in the therapy of facial nerve defect, and furnish the foundation of auto-transplantation of olfactory mucosa olfactory ensheathing cells in periphery nerve injury.

Electrophysiological assessment of a peptide amphiphile nanofiber nerve graft for facial nerve repair.

Facial nerve injury can cause severe long-term physical and psychological morbidity. There are limited repair options for an acutely transected facial nerve not amenable to primary neurorrhaphy. We hypothesize that a peptide amphiphile nanofiber neurograft may provide the nanostructure necessary to guide organized neural regeneration. Five experimental groups were compared, animals with (1) an intact nerve, (2) following resection of a nerve segment, and following resection and immediate repair with either a (3) autograft (using the resected nerve segment), (4) neurograft, or (5) empty conduit. The buccal branch of the rat facial nerve was directly stimulated with charge balanced biphasic electrical current pulses at different current amplitudes whereas nerve compound action potentials (nCAPs) and electromygraphic responses were recorded. After 8weeks, the proximal buccal branch was surgically reexposed and electrically evoked nCAPs were recorded for groups 1-5. As expected, the intact nerves required significantly lower current amplitudes to evoke an nCAP than those repaired with the neurograft and autograft nerves. For other electrophysiologic parameters such as latency and maximum nCAP, there was no significant difference between the intact, autograft, and neurograft groups. The resected group had variable responses to electrical stimulation, and the empty tube group was electrically silent. Immunohistochemical analysis and transmission electron microscopy confirmed myelinated neural regeneration. This study demonstrates that the neuroregenerative capability of peptide amphiphile nanofiber neurografts is similar to the current clinical gold standard method of repair and holds potential as an off-the-shelf solution for facial reanimation and potentially peripheral nerve repair.

3D bio-printed scaffold-free nerve constructs with human gingiva-derived mesenchymal stem cells promote rat facial nerve regeneration

Despite the promising neuro-regenerative capacities of stem cells, there is currently no licensed stem cell-based product in the repair and regeneration of peripheral nerve injuries. Here, we explored the potential use of human gingiva-derived mesenchymal stem cells (GMSCs) as the only cellular component in 3D bio-printed scaffold-free neural constructs that were transplantable to bridge facial nerve defects in rats. We showed that GMSCs have the propensity to aggregate into compact 3D-spheroids that could produce their own matrix. When cultured under either 2D- or 3D-collagen scaffolds, GMSC spheroids were found to be more capable of differentiating into both neuronal and Schwann-like cells than their adherent counterparts. Using a scaffold-free 3D bio-printer system, nerve constructs were printed from GMSC spheroids in the absence of exogenous scaffolds and allowed to mature in a bioreactor. In vivo transplantation of the GMSC-laden nerve constructs promoted regeneration and functional recovery when used to bridge segmental defects in rat facial nerves. Our findings suggest that GMSCs represent an easily accessible source of MSCs for 3D bio-printing of scaffold-free nervous tissue constructs with promising potential application for repair and regeneration of peripheral nerve defects.

Electrical stimulation promotes regeneration and re-myelination of axons of injured facial nerve in rats

ObjectiveTo investigate the effects of electrical stimulation (ES) on the nerve regeneration and functional recovery of facial expression muscles in facial nerve defect rats.MethodsSixty rats were surgically introduced with a 1-cm defect on the right facial nerves and evenly divided into the Surgery group (Group A, the main trunk of the right facial nerve was surgically cut-off with a 1.0 cm at the foramina stylomastoideum) and the Surgery + ES group (Group B). Twenty normal rats were as normal control group (without receiving surgery or ES). For rats in group B, the orbicularis oris muscle of the right paralyzed face was stimulated with an electrical pulse of 3 V, 20 Hz and 0.3 mA for 1 h each day. The effects of ES on the facial muscle movement, compound muscle action potentials (CMAPs), histological structure, and the expression levels of S100B and NF200 proteins were comparatively studied.ResultsIn group A, facial paralysis scores were slightly improved from day 1 to 28; the facial nerve trunks had swelled and malformed till day 14; and CMAPs could be induced in fewer animals and were abnormal, resulting in a slow recovery of the facial muscle movement. In group B, facial paralysis scores were improved from 4 to 2.6 during the 4 weeks; more rats showed a higher amplitude and shorter latency of CMAPs from day 14 to 28 after surgery; and increased axons and the expression of S100B and NF200 proteins and gradually decreased swelling in the injured facial nerve.ConclusionES promotes outgrowth and myelination of axons and a partial functional recovery of facial muscles in injured facial nerve rats.

End-to-end and end-to-side neurorrhaphy between thick donor nerves and thin recipient nerves: an axon regeneration study in a rat model.

During nerve reconstruction, nerves of different thicknesses are often sutured together using end-to-side neurorrhaphy and end-to-end neurorrhaphy techniques. In this study, the effect of the type of neurorrhaphy on the number and diameter of regenerated axon fibers was studied in a rat facial nerve repair model. An inflow-type end-to-side and end-to-end neurorrhaphy model with nerve stumps of different thicknesses (2:1 diameter ratio) was created in the facial nerve of 14 adult male Sprague-Dawley rats. After 6 and 12 weeks, nerve regeneration was evaluated in the rats using the following outcomes: total number of myelinated axons, average minor axis diameter of the myelinated axons in the central and peripheral sections, and axon regeneration rate. End-to-end neurorrhaphy resulted in a significantly greater number of regenerated myelinated axons and rate of regeneration after 6 weeks than end-to-side neurorrhaphy; however, no such differences were observed at 12 weeks. While the regenerated axons were thicker at 12 weeks than at 6 weeks, no significant differences in axon fiber thickness were detected between end-to-end and end-to-side neurorrhaphy. Thus, end-to-end neurorrhaphy resulted in greater numbers of regenerated axons and increased axon regeneration rate during the early postoperative period. As rapid reinnervation is one of the most important factors influencing the restoration of target muscle function, we conclude that end-to-end neurorrhaphy is desirable when suturing thick nerves to thin nerves.

A method package for electrophysiological evaluation of reconstructed or regenerated facial nerves in rodents

Compound muscle action potential (CMAP) recording via reconstructed or regenerated motor axons is a critical examination to evaluate newly developed surgical and regeneration techniques. However, there is currently no documentation on technical aspects of CMAP recordings via reconstructed or regenerated facial nerves. We have studied new techniques of plastic surgery and nerve regeneration using a rat facial nerve defect model for years, standardizing an evaluation pipeline using CMAP recordings. Here we describe our CMAP recording procedure in detail as a package including surgical preparation, data acquisition, analysis and troubleshooting. Each resource is available in public repositories and is maintained as a version control system. In addition, we demonstrate that our analytical pipeline can not only be applied to rats, but also mice. Finally, we show that CMAP recordings can be practically combined with other behavioral and anatomical examinations. For example, retrograde motor neuron labeling provides anatomical evidence for physical routes between the facial motor nucleus and its periphery through reconstructed or regenerated facial nerves, in addition to electrophysiological evidence by CMAP recordings from the same animal.•Standardized surgical, recording and analytical procedures for the functional evaluation of reconstructed or regenerated facial nerves of rats, extended to mice.•The functional evaluation can be combined with anatomical evaluations.•The methods described here are maintained in public repositories as version control systems.

ERK/MAPK and PI3K/AKT signal channels simultaneously activated in nerve cell and axon after facial nerve injury.

BackgroundThe in-vitro study indicated that ERK/MAPK and PI3K/AKT signal channels may play an important role in reparative regeneration process after peripheral nerve injury. But, relevant in-vivo study was infrequent. In particular, there has been no report on simultaneous activation of ERK/MAPK and PI3K/AKT signal channels in facial nerve cell and axon after facial nerve injury.ResultsThe expression of P-ERK enhanced in nerve cells at the injury side on the 1 d after the rat facial nerve was cut and kept on a higher level until 14 d, but decreased on 28 d. The expression of P-AKT enhanced in nerve cells at the injury side on 1 d after injury, and kept on a higher level until 28 d. The expression of P-ERK enhanced at the near and far sections of the injured axon on 1 d, then increased gradually and reached the maximum on 7 d, but decreased on 14 d, until down to the level before the injury on 28 d. The expression of P-AKT obviously enhanced in the injured axon on 1 d, especially in the axon of the rear section, but decreased in the axon of the rear section on 7 d, while the expression of axon in the far section increased to the maximum and kept on till 14 d. On 28 d, the expression of P-AKT decreased in both rear and far sections of the axon.ConclusionThe facial nerve simultaneously activated ERK/MAPK and PI3K/AKT signal channels in facial nerve cells and axons after the cut injury, but the expression levels of P-ERK and P-AKT varied as the function of the time. In particular, they were quite different in axon of the far section. It has been speculated that two signal channels might have different functions after nerve injury. However, their specific regulating effects should still be testified by further studies in regenerative process of peripheral nerve injury.

Response of the GABAergic System to Axotomy of the Rat Facial Nerve.

The responses of inhibitory neurons/synapses to motoneuron injury in the cranial nervous system remain to be elucidated. In this study, we analyzed GABAA receptor (GABAAR) and GABAergic neurons at the protein level in the transected rat facial nucleus. Immunoblotting revealed that the GABAARα1 protein levels in the axotomized facial nucleus decreased significantly 5-14 days post-insult, and these levels remained low for 5 weeks. Immunohistochemical analysis indicated that the GABAARα1-expressing cells were motoneurons. We next examined the specific components of GABAergic neurons, including glutamate decarboxylase (GAD), vesicular GABA transporter (VGAT) and GABA transporter-1 (GAT-1). Immunoblotting indicated that the protein levels of GAD, VGAT and GAT-1 decreased transiently in the transected facial nucleus from 5 to 14 days post-insult, but returned to the control levels at 5 weeks post-insult. Although GABAARα1 protein levels in the transected nucleus did not return to their control levels for 5 weeks post-insult, the administration of glial cell line-derived neurotrophic factor at the cut site significantly ameliorated the reductions. Through these findings, we verified that the injured facial motoneurons suppressed the levels of GABAARα1 protein over the 5 weeks post-insult, presumably due to the deprivation of neurotrophic factor. On the other hand, the levels of the GAD, VGAT and GAT-1 proteins in GABAergic neurons were transiently reduced in the axotomized facial nucleus at 5-14 days post-insult, but recovered at 4-5 weeks post-insult.

PI3K/Akt and ERK/MAPK Signaling Promote Different Aspects of Neuron Survival and Axonal Regrowth Following Rat Facial Nerve Axotomy.

The ERK/MAPK and PI3K/Akt signaling pathways play important role in neuronal survival and axonal regeneration after peripheral nerve injury. However, the relative importance and degree of functional overlap of the two pathways are still debated due to lack of in-vivo data. We used rats which underwent a facial nerve axotomy, and examined subsequent ERK/MAPK and PI3K/Akt signaling activity by quantifying phosphorylation of ERK and Akt. We also assessed the survival rate of facial neurons, number of regenerated axons, and the length of axonal regrowth in axotomized animals treated with an inhibitor of ERK/MAPK (U0126) or PI3K/Akt (LY294002) phosphorylation, or with vehicle. Axotomy increased phosphorylation of ERK and Akt in the facial nucleus 7 days after injury. The inhibition of ERK phosphorylation significantly reduced the length of regenerated axons, but not the other parameters. Inhibition of Akt phosphorylation significantly reduced the survival rate of facial neurons and the number of new axons, as well as the length of regenerated axons. The results indicate that facial nerve injury activates the ERK/MAPK and PI3K/Akt signaling pathways in the facial nerve nucleus and its axons. However, the pathways promoted aspects of regeneration with only slight overlap: PI3K/Akt signaling improved the survival of neurons, as well as axonal growth and branching, whereas ERK/MAPK signaling promoted only axonal extension.