Although the transcriptional alterations inside the facial nucleus after facial nerve injury have been well studied,the gene expression changes in the facial nerve trunk after injury are still unknown.In this study,we...Although the transcriptional alterations inside the facial nucleus after facial nerve injury have been well studied,the gene expression changes in the facial nerve trunk after injury are still unknown.In this study,we established an adult rat model of facial nerve crush injury by compressing the right lateral extracranial nerve trunk.Transcriptome sequencing,differential gene expression analysis,and cluster analysis of the injured facial nerve trunk were performed,and 39 intersecting genes with significant variance in expression were identified.Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the 39 intersecting genes revealed that these genes are mostly involved in leukocyte cell-cell adhesion and phagocytosis and have essential roles in regulating nerve repair.Quantitative real-time polymerase chain reaction assays were used to validate the expression of pivotal genes.Finally,nine pivotal genes that contribute to facial nerve recovery were identified,including Arhgap30,Akr1b8,C5ar1,Csf2ra,Dock2,Hcls1,Inpp5d,Sla,and Spi1.Primary Schwann cells were isolated from the sciatic nerve of neonatal rats.After knocking down Akr1b8 in Schwann cells with an Akr1b8-specific small interfering RNA plasmid,expression levels of monocyte chemoattractant protein-1 and interleukin-6 were decreased,while cell proliferation and migration were not obviously altered.These findings suggest that Akr1b8 likely regulates the interaction between Schwann cells and macrophages through regulation of cytokine expression to promote facial nerve regeneration.This study is the first to reveal a transcriptome change in the facial nerve trunk after facial nerve injury,thereby revealing the potential mechanism underlying repair of facial nerve injury.This study was approved by the Animal Ethics Committee of Nantong University,China in 2018(approval No.S20180923-007).展开更多
Objective To investigate T cell activation following facial nerve axotomization and latent neuroimmunologic mechanisms in traumatic facial paralysis.Methods A murine model of facial nerve transaction was used.Lymphocy...Objective To investigate T cell activation following facial nerve axotomization and latent neuroimmunologic mechanisms in traumatic facial paralysis.Methods A murine model of facial nerve transaction was used.Lymphocytes from cervical and mesenteric lymph nodes in BABL/c mice at specific times were collected and expression rates of CD69 on T cells were assessed by flow cytometry.Results Infiltrating T cells were detected around the facial neurons in the facial nerve nucleus in mice whose facial nerve was transected.Immunofluorescent staining showed recruitment of activated T cells.Three days post-facial nerve transection,the expression rate of CD69 on T cells from cervical draining lymphoid nodes(CDLNs) was significantly different from that on T cells from mesenteric lymph nodes(MLNs)(P =0.0457),whereas the latter was similar to that in animals undergoing sham surgeries and that in blank control animals(p= 0.2817 and 0.2724,respectively).Two weeks post-nerve transection,the T cell CD69 expression rate from CDLNs remained at a higher level and than that in the sham-operation animals(p= 0.0007).At two weeks,CD69 expression rate on T cells from MLNs was also up-regulated and different compared with the sham-operation animals and with itself at three days post-operation(p= 0.0082 and 0.0133,respectively).Conclusion T cells appear to be activated and up-regulated in CDLNs following facial nerve transection.There is even evidence of T cell activation in MLNs at 2 weeks post-nerve transection.This suggestes an alteration of immune response from local to general immunity in the acute stage of facial nerve trauma,which may help coordinating and controlling the scales and orientation of the neuroimmune response during the pathogenesis and progression of facial nerve trauma.展开更多
Previous studies have demonstrated that muscarinic,and nicotinic receptors increase free Ca2+ levels in the facial nerve nucleus via various channels following facial nerve injury.However,intracellular Ca2+ overload c...Previous studies have demonstrated that muscarinic,and nicotinic receptors increase free Ca2+ levels in the facial nerve nucleus via various channels following facial nerve injury.However,intracellular Ca2+ overload can trigger either necrotic or apoptotic cell death.Gamma-aminobutyric acid(GABA),an important inhibitory neurotransmitter in the central nervous system,exists in the facial nerve nucleus.It is assumed that GABA negatively regulates free Ca2+ levels in the facial nerve nucleus.The present study investigated GABA type A(GABAA) receptor expression in the facial nerve nucleus in a rat model of facial nerve injury using immunohistochemistry and laser confocal microscopy,as well as the regulatory effects of GABAA receptor on nicotinic receptor response following facial nerve injury.Subunits α1,α3,α5,β1,β2,δ,and γ3 of GABAA receptors were expressed in the facial nerve nucleus following facial nerve injury.In addition,GABAA receptor expression significantly inhibited the increase in nicotinic receptor-mediated free Ca2+ levels in the facial nerve nucleus following facial nerve injury in a concentration-dependent fashion.These results suggest that GABAA receptors exhibit negative effects on nicotinic receptor responses following facial nerve injury.展开更多
BACKGROUND: Recently, many investigators have tried to use natural biomaterials, such as, artery, vein, decalcified bone, etc., as conduits for nerve repair. However, immunological rejection of conduits made of natura...BACKGROUND: Recently, many investigators have tried to use natural biomaterials, such as, artery, vein, decalcified bone, etc., as conduits for nerve repair. However, immunological rejection of conduits made of natural biomaterials limits their application. Therefore, it is essential to identify more suitable types of biomaterials. OBJECTIVE: To observe the characteristics of a bioengineering processing method using venous conduit as a stent for repairing facial nerve injury. DESIGN: A controlled observational experiment. SETTING: Animal Laboratories of the Third Hospital Affiliated to Sun Yat-sen University and the 157 Hospital. MATERIALS: Thirty-three male New Zealand rabbits of pure breed, weighing 1.5 to 2.0 kg, were provided by Medical Experimental Animal Room of Sun Yat-sen University. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Venous conduits and autogenous nerves were transplanted into the left and right cheeks, respectively. Eleven animals were chosen for anatomical observations at 5, 10 and 15 weeks after surgery. METHODS: This experiment was carried out in the Animal Laboratories of the Third Hospital Affiliated to Sun Yat-sen University and the 157 Hospital between May and November 2006. After animals were anesthetized, 15 mm of retromandibular vein was harvested for preparing a venous conduit. Approximately 3 cm of low buccal branch of facial nerve was exposed. A segment of 1.2 cm nerve was resected from the middle, and a gap of 1.5 cm formed due to bilateral retraction. The prepared venous conduit of 1.5 cm was sutured to the outer membrane of the severed ends of the nerve. Muscle and skin were sutured layer by layer. Using the same above-mentioned method, the low buccal branch of right autogenous facial nerve was resected, and the left facial nerve segment from the same animal was transplanted using end-to-end neurorrhaphy for control. MAIN OUTCOME MEASURES: ①Post-operatively, food intake, vibrissae activity and wound healing of each animal were observed daily. ② Animals were anesthetized at 5, 10 and 15 weeks after operation for observing the structural change of the venous conduit, the appearance of regenerated nerve, and the relationship between conduit and peripheral muscle tissue. ③ The action potential and latency of bilateral nerves of animals were measured by electrophysiologic examination, and nerve conduction velocity was calculated. ④Neural myelination and neurite growth were observed by histological staining using an optical microscope. RESULTS: Thirty-three New Zealand rabbits were involved in the final analysis. ①Immediately following the operation, vibrissae activity and orbicularis oris muscle activity of the upper lip on venous conduit side were more prominent, and their amplitudes of movement were larger as compared with autogenous nerve side. ② At postoperative 10 weeks, by visual inspection, we found that on the venous conduit side, the venous conduit exhibited membrane structure which encased regenerated nerve. Regenerated nerve adhered to the muscle edge of orbicularis oris muscle. Muscle and nerve could be separated with a forceps. The muscle of musculus orbicularis oris of rabbit was darker and thicker as compared with autogenous nerve side. After the venous conduit was longitudinally split, the regenerated nerve and nerves at two the severed ends were connected together. When compared with postoperative 5 weeks, the connected nerve was thickened, texture was tough and its middle part was thicker than its two ends. On the autogenous nerve side, the regenerated nerve stem was enwrapped by scar tissue. It was bulky and adhered to peripheral muscle. Its neural profile structure was unclear. The two stomas were obviously enlarged. ③At postoperative 10 weeks and 15 weeks, nerve action potentials could be elicited from both the venous conduit and autologous nerve side. The mean nerve conduction velocity on the venous conduit side was greater than that of the autologous nerve side. ④At postoperative 10 weeks, using histochemical staining, it was found that in the venous conduit, regenerated medullated nerve fibers were densely distributed, with well split facial nerve structure, while on the autologous nerve side, nerve fibers were sparsely scattered, with immature medullated nerve structure. CONCLUSION: Biological natural venous conduit processed by bioengineering technology overcomes the tissue inflammatory reactions and connective tissue reactions caused by natural biomaterials. It is more conducive to promote neural regeneration and functional recovery than autologous nerve transplantation.展开更多
BACKGROUND: Studies have shown that agmatine can reduce inhibition of neuronal regeneration by in-creasing cyclic adenosine monophosphate and brain-derived neurotrophic factor (BDNF) in the hippocampusof morphine-depe...BACKGROUND: Studies have shown that agmatine can reduce inhibition of neuronal regeneration by in-creasing cyclic adenosine monophosphate and brain-derived neurotrophic factor (BDNF) in the hippocampusof morphine-dependent rats. The hypothesis that agmatine exerts similar effects on facial nerve injury de-serves further analysis. OBJECTIVE: To study the effects of peritoneal agmatine injection on BDNF levels in the rat brainstem af-ter facial nerve injury. DESIGN, TIME AND SETTING: A controlled animal experiment was performed at the Department ofOtolaryngology-Head and Neck Surgery at the Second Affiliated Hospital, Chongqing University of MedicalSciences (Chongqing, China), between October and December in 2007. MATERIALS: Twenty-four male Sprague-Dawley rats were randomly divided into a control, a lesion, andan agmatine treatment group, with eight rats in each group. Bilateral facial nerve anastomosis was induced inthe lesion and agmatine treatment groups, while the control group remained untreated. A rat BDNF En-zyme-linked immunosorbent assay kit was used to measure BDNF levels in the brainstem facial nucleus. METHODS: Starting on the day of lesion, the agmatine group received a peritoneal injection of 100 mg/kgagmatine, once per day, for a week, whereas rats in the lesion group received saline injections. MAIN OUTCOME MEASURES: BDNF levels in the brainstem containing facial nucleus were measuredby ELISA. RESULTS: Twenty-four rats were included in the final analysis without any loss. Two weeks after lesion,BDNF levels were significantly higher in the lesion group than in the control group (P < 0.01). A significantincrease was noted in the agmatine group compared to the lesion group (P < 0.01). CONCLUSION: Agmatine can substantially increase BDNF levels in the rat brainstem after facial nerveinjury.展开更多
BACKGROUND: Previous studies have demonstrated that postsynaptic density protein-95 (PSD-95) is widely distributed in the central nervous system and is related to the development of the CNS and sensory signal transmis...BACKGROUND: Previous studies have demonstrated that postsynaptic density protein-95 (PSD-95) is widely distributed in the central nervous system and is related to the development of the CNS and sensory signal transmission as well as acute or chronic nerve cell death following ischemic brain injury. OBJECTIVE: To semi-quantitatively determine the pathological changes of apoptotic facial neurons and the expression of PSD-95 in the facial nucleus following facial nerve injury of varying extents using immu- nohistochemical staining methods. DESIGN, TIME AND SETTING: Randomized, controlled animal experiments were performed in the Ul- trasonic Institute of the Second Affiliated Hospital of Chongqing University of Medical Sciences from Sep- tember to December 2007. MATERIALS: Sixty-five healthy, adult, Sprague-Dawley (SD) rats, both male and female, were used for this study. Rabbit anti-rat PSD-95 polyclonal antibody was purchased from Beijing Biosynthesis Biotech- nology Co., Ltd. METHODS: SD rats were randomly assigned into a control group with five rats and three injured groups with 20 rats per group. Exposure, clamp and cut for bilateral facial nerve trunks were performed in the rats of the injury groups, and no injury was inflicted on the rats of the control group. MAIN OUTCOME MEASURES: The brainstems of all the rats were excised on days 1, 3, 7, and 14 post injury, and then the facial nuclei were stained with hematoxylin-eosin to observe any pathological changes due to apoptosis in facial neurons. PSD-95 expression in facial nuclei was detected by immunohistochemistry and the number of PSD-95 positive cells was counted under a light microscope. RESULTS: The expression of PSD-95 in the facial nucleus and morphology of the facial neuron within the exposure group had no obvious changes at various points in time tested (P > 0.05). However, the expressions of PSD-95 in the facial nucleus of the clamp group and cut group increased on day 1 post injury (P < 0.05), and showed further increase on day 7 post injury (P < 0.01). This did not decrease until day 14 post injury. Facial neuron apoptosis was detected on day 3 post injury and this was even more obvious on day 7 and was maintained to day 14 post injury. The number of cells expressing PSD-95 and displaying severe degrees of facial neuron apoptosis were as follows: cut group > clamp group > exposure group. CONCLUSION: The apoptotic extent of facial neurons and the expression of PSD-95 in apoptotic facial neurons increased with the degree of aggravation of injured severity of facial nerve.展开更多
Muscarinic receptors and nicotine receptors can increase free calcium ion levels in the facial nucleus via different channels following facial nerve injury. In addition, γ-aminobutyric acid A (GABAA) receptors have b...Muscarinic receptors and nicotine receptors can increase free calcium ion levels in the facial nucleus via different channels following facial nerve injury. In addition, γ-aminobutyric acid A (GABAA) receptors have been shown to negatively regulate free calcium ion levels in the facial nucleus by inhibiting nicotine receptors. The present study investigated the influence of GABAA, γ-aminobutyric acid B (GABAB) and C (GABAC) receptors on muscarinic receptors in rats with facial nerve injury by confocal laser microscopy. GABAA and GABAB receptors exhibited significant dose-dependent inhibitory effects on increased muscarinic receptor-mediated free calcium ion levels following facial nerve injury. Results showed that GABAA and GABAB receptors negatively regulate muscarinic receptor effects and interplay with cholinergic receptors to regulate free calcium ion levels for facial neural regeneration.展开更多
BACKGROUND: Erythropoietin and recombinant human erythropoietin (rhEPO) inhibit apoptosis of motor neurons caused by spinal cord injury and brain damage in rats. However, it still remains to be shown whether rhEPO can...BACKGROUND: Erythropoietin and recombinant human erythropoietin (rhEPO) inhibit apoptosis of motor neurons caused by spinal cord injury and brain damage in rats. However, it still remains to be shown whether rhEPO can protect facial motoneurons (FMNs) as well. OBJECTIVE: To test the neuroprotective effects of rhEPO on injured FMNs, as well as the influence on Caspase-3 expression. DESIGN, TIME AND SETTING: Randomized, controlled, animal experiment. This study was performed at the Central Laboratory of Basic Medical College, Chongqing Medical University from January to October 2007. MATERIALS: Seventy-five female SD rats, weighing 210–230 g. rhEPO injection was provided by Sansheng pharmaceuticals company, Shenyang City, Liaoning Province, China, and the License number was HMLN S20010001. METHODS: A total of 75 female rats were randomly divided into rhEPO treatment, control, and sham operation groups, with 25 rats in each group. Rat models of facial nerve injury were established in the rhEPO treatment group and the control group by crushing the main trunk of the left facial nerve. Surgical microscopic observation of the facial nerve damage displayed perineurial disruption. The left stylomastoid foramen of the sham operation group were only exposed, but without nerve injury. The rhEPO treatment group was treated with rhEPO (5 000 U/kg, i.p.) once following injury and once a day for two weeks. The control and sham operation groups were treated with the same dose of normal saline (i.p.), once following injury and once a day for two weeks. MAIN OUTCOME MEASURES: Rats were sacrificed 3, 7, 14, 21, and 28 days after injury, FMN survival after facial nerve injury was analyzed by Toluidine blue staining, and then survival ratios (L/R) were calculated. The number of apoptotic profiles in the injured FMNs were evaluated by TUNEL staining. Expression of Caspase-3 in the facial nucleus was detected by immunohistochemistry methods. RESULTS: A total of 75 rats were included in the final analysis. FMN survival ratios, both in rhEPO treatment group and control group, decreased gradually between seven and 28 days; however, FMN survival ratios were significantly greater in the rhEPO treatment group compared to the control group (P < 0.05). No TUNEL-positive cells were observed three days after injury in the rhEPO treatment and control groups; however, by seven days after injury, apoptotic cells were observed and peaked by 14 days in the control group. Between seven and 21 days, apoptotic cell numbers were significantly lower in the rhEPO treatment group compared to the control group (P < 0.05). The expression of Caspase-3 increased three days after injury and peaked at 14 days in the control group. Nevertheless, Caspase-3 expression was significantly lower in the rhEPO treatment group compared to the control group at each time point (P < 0.05). CONCLUSION: Treatment with rhEPO can effectively protect facial motoneurons by reducing expression of Caspase-3 and inhibiting apoptosis.展开更多
Recently,we have shown that manual stimulation of paralyzed vibrissal muscles after facial-facial anastomosis reduced the poly-innervation of neuromuscular junctions and restored vibrissal whisking.Using gene knock ou...Recently,we have shown that manual stimulation of paralyzed vibrissal muscles after facial-facial anastomosis reduced the poly-innervation of neuromuscular junctions and restored vibrissal whisking.Using gene knock outs,we found a differential dependence of manual stimulation effects on growth factors.Thus,insulin-like growth factor-1 and brain-derived neurotrophic factor are required to underpin manual stimulation-mediated improvements,whereas FGF-2 is not.The lack of dependence on FGF-2 in mediating these peripheral effects prompted us to look centrally,i.e.within the facial nucleus where increased astrogliosis after facial-facial anastomosis follows "synaptic stripping".We measured the intensity of Cy3-fluorescence after immunostaining for glial fibrillary acidic protein(GFAP) as an indirect indicator of synaptic coverage of axotomized neurons in the facial nucleus of mice lacking FGF-2(FGF-2^(-/-) mice).There was no difference in GFAP-Cy3-fluorescence(pixel number,gray value range17-103) between intact wildtype mice(2.12± 0.37×10~7) and their intact FGF-2^(-/-) counterparts(2.12±0.27×10~7) nor after facial-facial anastomosis +handling(wildtype:4.06±0.32×10~7;FGF-2^(-/-):4.39±0.17×10~7).However,after facial-facial anastomosis,GFAP-Cy3-fluorescence remained elevated in FGF-2^(-/-)-animals(4.54±0.12×10~7),whereas manual otimulation reduced the intensity of GFAP-immunofluorescence in wild type mice to values that were not significantly different from intact mice(2.63±0.39×10).We conclude that FGF-2 is not required to underpin the beneficial effects of manual stimulation at the neuro-muscular junction,but it is required to minimize astrogliosis in the brainstem and,by implication,restore synaptic coverage of recovering facial motoneurons.展开更多
Objective:To explore the mechanism of electroacupuncture(EA) in promoting recovery of the facial function with the involvement of autophagy,glial cell line-derived neurotrophic factor(GDNF),and phosphatidylinositol-3-...Objective:To explore the mechanism of electroacupuncture(EA) in promoting recovery of the facial function with the involvement of autophagy,glial cell line-derived neurotrophic factor(GDNF),and phosphatidylinositol-3-kinase(PI3K)/mammalian target of rapamycin(mTOR) signaling pathway.Methods:Seventy-two male Sprague-Dawley rats were randomly allocated into the control,sham-operated,facial nerve injury(FNI),EA,EA+3-methyladenine(3-MA),and EA+GDNF antagonist groups using a random number table,with 12 rats in each group.An FNI rat model was established with facial nerve crushing method.EA intervention was conducted at Dicang(ST 4),Jiache(ST 6),Yifeng(SJ 17),and Hegu(LI 4) acupoints for 2 weeks.The Simone’s 10-Point Scale was utilized to monitor the recovery of facial function.The histopathological evaluation of facial nerves was performed using hematoxylin-eosin(HE) staining.The levels of Beclin-1,light chain 3(LC3),and P62 were detected by immunohistochemistry(IHC),immunofluorescence,and reverse transcriptionpolymerase chain reaction,respectively.Additionally,IHC was also used to detect the levels of GDNF,Rai,PI3K,and mTOR.Results:The facial functional scores were significantly increased in the EA group than the FNI group(P<0.05 or P<0.01).HE staining showed nerve axons and myelin sheaths,which were destroyed immediately after the injury,were recovered with EA treatment.The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats(P<0.01);however,EA treatment reversed these abnormal changes(P<0.01).Meanwhile,EA stimulation significantly increased the levels of GDNF,Rai,PI3K,and mTOR(P<0.01).After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist,the repair effect of EA on facial function was attenuated(P<0.05 or P<0.01).Conclusions:EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI.EA may exert this neuroreparative effect through mediating the release of GDNF,activating the PI3K/mTOR signaling pathway,and further regulating the autophagy of facial nerves.展开更多
Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide ...Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.展开更多
The effect of platelet-rich plasma on nerve regeneration remains controversial.In this study,we established a rabbit model of sciatic nerve small-gap defects with preserved epineurium and then filled the gaps with pla...The effect of platelet-rich plasma on nerve regeneration remains controversial.In this study,we established a rabbit model of sciatic nerve small-gap defects with preserved epineurium and then filled the gaps with platelet-rich plasma.Twenty-eight rabbits were divided into the following groups(7 rabbits/group):model,low-concentrati on PRP(2.5-3.5-fold concentration of whole blood platelets),medium-concentration PRP(4.5-6.5-fold concentration of whole blood platelets),and high-concentration PRP(7.5-8.5-fold concentration of whole blood platelets).Electrophysiological and histomorphometrical assessments and proteomics analysis we re used to evaluate regeneration of the sciatic nerve.Our results showed that platelet-rich plasma containing 4.5-6.5-and 7.5-8.5-fold concentrations of whole blood platelets promoted repair of sciatic nerve injury.Proteomics analysis was performed to investigate the possible mechanism by which platelet-rich plasma promoted nerve regeneration.Proteomics analysis showed that after sciatic nerve injury,platelet-rich plasma increased the expression of integrin subunitβ-8(ITGB8),which participates in angiogenesis,and differentially expressed proteins were mainly enriched in focal adhesion pathways.Additionally,two key proteins,ribosomal protein S27 a(RSP27 a)and ubiquilin 1(UBQLN1),which were selected after protein-protein interaction analysis,are involved in the regulation of ubiquitin levels in vivo.These data suggest that platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury by affecting angiogenesis and intracellular ubiquitin levels.展开更多
Research has shown that long-chain noncoding RNAs(lncRNAs) are involved in the regulation of a variety of biological processes, including peripheral nerve regeneration, in part by acting as competing endogenous RNAs. ...Research has shown that long-chain noncoding RNAs(lncRNAs) are involved in the regulation of a variety of biological processes, including peripheral nerve regeneration, in part by acting as competing endogenous RNAs. c-Jun plays a key role in the repair of peripheral nerve injury. However, the precise underlying mechanism of c-Jun remains unclear. In this study, we performed microarray and bioinformatics analysis of mouse crush-injured sciatic nerves and found that the lncRNA Pvt1 was overexpressed in Schwann cells after peripheral nerve injury. Mechanistic studies revealed that Pvt1 increased c-Jun expression through sponging miRNA-214. We overexpressed Pvt1 in Schwann cells cultured in vitro and found that the proliferation and migration of Schwann cells were enhanced, and overexpression of miRNA-214 counteracted the effects of Pvt1 overexpression on Schwann cell proliferation and migration. We conducted in vivo analyses and injected Schwann cells overexpressing Pvt1 into injured sciatic nerves of mice. Schwann cells overexpressing Pvt1 enhanced the regeneration of injured sciatic nerves following peripheral nerve injury and the locomotor function of mice was improved. Our findings reveal the role of lncRNAs in the repair of peripheral nerve injury and highlight lncRNA Pvt1 as a novel potential treatment target for peripheral nerve injury.展开更多
We previously prepared nerve growth factor poly-lactide co-glycolid sustained-release microspheres to treat rat sciatic nerve injury using the small gap sleeve technique.Multiple growth factors play a synergistic role...We previously prepared nerve growth factor poly-lactide co-glycolid sustained-release microspheres to treat rat sciatic nerve injury using the small gap sleeve technique.Multiple growth factors play a synergistic role in promoting the repair of peripheral nerve injury;as a result,in this study,we added basic fibroblast growth factors to the microspheres to further promote nerve regeneration.First,in an in vitro biomimetic microenvironment,we developed and used a drug screening biomimetic microfluidic chip to screen the optimal combination of nerve growth factor/basic fibroblast growth factor to promote the regeneration of Schwann cells.We found that 22.56 ng/mL nerve growth factor combined with 4.29 ng/mL basic fibroblast growth factor exhibited optimal effects on the proliferation of primary rat Schwann cells.The successfully prepared nerve growth factor-basic fibroblast growth factor-poly-lactide-co-glycolid sustained-release microspheres were used to treat rat sciatic nerve transection injury using the small gap sleeve bridge technique.Compared with epithelium sutures and small gap sleeve bridging alone,the small gap sleeve bridging technique combined with drug-free sustained-release microspheres has a stronger effect on rat sciatic nerve transfection injury repair at the structural and functional level.展开更多
The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor(GDNF) has been shown to protect ne...The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor(GDNF) has been shown to protect neurons by binding to N-cadherin. Our previous results have shown that electroacupuncture could increase the expression of N-cadherin mRNA in facial neurons and promote facial nerve regeneration. In this study, the potential mechanisms by which electroacupuncture promotes nerve regeneration were elucidated through assessing the effects of electroacupuncture on GDNF and N-cadherin expression in facial motoneurons of rabbits with peripheral facial nerve crush injury. New Zealand rabbits were randomly divided into a normal group(normal control, n = 21), injury group(n = 45) and electroacupuncture group(n = 45). Model rabbits underwent facial nerve crush injury only. Rabbits in the electroacupuncture group received facial nerve injury, and then underwent electroacupuncture at Yifeng(TE17), Jiache(ST6), Sibai(ST2), Dicang(ST4), Yangbai(GB14), Quanliao(SI18), and Hegu(LI4; only acupuncture, no electrical stimulation). The results showed that in behavioral assessments, the total scores of blink reflex, vibrissae movement, and position of apex nasi, were markedly lower in the EA group than those in the injury group. Hematoxylin-eosin staining of the right buccinator muscle of each group showed that the cross-sectional area of buccinator was larger in the electroacupuncture group than in the injury group on days 1, 14 and 21 post-surgery. Toluidine blue staining of the right facial nerve tissue of each group revealed that on day 14 post-surgery, there was less axonal demyelination and fewer inflammatory cells in the electroacupuncture group compared with the injury group. Quantitative real time-polymerase chain reaction showed that compared with the injury group, N-cadherin mRNA levels on days 4, 7, 14 and 21 and GDNF mRNA levels on days 4, 7 and 14 were significantly higher in the electroacupuncture group. Western blot assay displayed that compared with the injury group, the expression of GDNF protein levels on days 7, 14 and 21 were significantly upregulated in the electroacupuncture group. The histology with hematoxylin-eosin staining and Nissl staining of brainstem tissues containing facial neurons in the middle and lower part of the pons exhibited that on day 7 post-surgery, there were significantly fewer apoptotic neurons in the electroacupuncture group than in the injury group. By day 21, there was no significantly difference in the number of neurons between the electroacupuncture and normal groups. Taken together, these results have confirmed that electroacupuncture promotes regeneration of peripheral facial nerve injury in rabbits, inhibits neuronal apoptosis, and reduces peripheral inflammatory response, resulting in the recovery of facial muscle function. This is achieved by up-regulating the expression of GDNF and N-cadherin in central facial neurons.展开更多
Background:The underlying mechanism of sciatic nerve injury(SNI)is a common motor functional disorder,necessitates further research.Methods:A rat model of SNI was established,with the injury group subjected to compres...Background:The underlying mechanism of sciatic nerve injury(SNI)is a common motor functional disorder,necessitates further research.Methods:A rat model of SNI was established,with the injury group subjected to compressive injury of the right sciatic nerve exposed at the midpoint of the thigh and the sham surgery group undergoing the same surgical procedure.An oxygen-glucose deprivation model was employed to simulate in vitro SNI in PC12 cells.Following data acquisition and quality control,differentially expressed proteins(DEPs)in each model were identified through differential analysis,and enrichment analysis was used to explore the potential functions and pathways of the DEPs.Venn diagrams were drawn,and DEPs from both in vivo and in vitro SNI models were imported into the STRING database to construct a protein-protein interaction network and screen for hub proteins.Results:After the peptide segments obtained from rat nerve blockade and PC12 cells met quality requirements,258 DEPs were identified in rat nerve samples,and 119 DEPs were screened in PC12 cells.Enrichment analysis revealed that DEPs in the rat model were predominantly concentrated in biological functions such as myogenic cell proliferation and signaling related to lipid and energy metabolism.DEPs in the in vitro model were mainly enriched in biological processes such as phagocytosis and were associated with lipid transport and metabolism.Two hub proteins,amyloid precursor protein(APP)and fibronectin 1(FN1),were identified through MCC,MCODE,and Degree scoring.Both PC12 cells and external validation sets showed relatively higher expression of APP and FN1 in injured samples.Results of gene set enrichment analysis indicated that these two proteins were associated with metabolic pathways,such as biosynthesis of glycosaminoglycan chondroitin sulfate and biosynthesis of unsaturated fatty acids.Conclusion:APP and FN1 are potential key molecules involved in SNI and are associated with various metabolic pathways in nerve repair.These findings provide a theoretical basis for the development of therapeutic targets for SNI.展开更多
Objective To provide useful information for selecting the most appropriate peripheral nerve injury model for different research purposes in nerve injury and repair studies,and to compare nerve regeneration capacity an...Objective To provide useful information for selecting the most appropriate peripheral nerve injury model for different research purposes in nerve injury and repair studies,and to compare nerve regeneration capacity and characteristics between them.Methods Sixty adult SD rats were randomly divided into two groups and underwent crush injury alone(group A,n=30)or transection injury followed by surgical repair(group B,n=30)of the right hind paw.Each group was subjected to the CatWalk test,gastrocnemius muscle evaluation,pain threshold measurement,electrophysiological examination,retrograde neuronal labeling,and quantification of nerve regeneration before and 7,14,21,and 28 days after injury.Results Gait analysis showed that the recovery speed in group A was significantly faster than that in group B at 14 days.At 21 days,the compound muscle action potential of the gastrocnemius muscle in group A was significantly higher than that in group B,and the number of labeled motor neurons in group B was lower than that in group A.The number of new myelin sheaths and the g-ratio were higher in group A than in group B.There was a 7-day time difference in the regeneration rate between the two injury groups.Conclusion The regeneration of nerve fibers was rapid after crush nerve injury,whereas the transection injury was relatively slow,which provides some ideas for the selection of clinical research models.展开更多
Monocytes,including monocyte-derived macrophages and resident microglia,mediate many phases of optic nerve injury pathogenesis.Resident microglia respond first,followed by infiltrating macrophages which regulate neuro...Monocytes,including monocyte-derived macrophages and resident microglia,mediate many phases of optic nerve injury pathogenesis.Resident microglia respond first,followed by infiltrating macrophages which regulate neuronal inflammation,cell proliferation and differentiation,scar formation and tissue remodeling following optic nerve injury.However,microglia and macrophages have distinct functions which can be either beneficial or detrimental to the optic nerve depending on the spatial context and temporal sequence of their activity.These divergent effects are attributed to pro-and anti-inflammatory cytokines expressed by monocytes,crosstalk between monocyte and glial cells and even microglia-macrophage communication.In this review,we describe the dynamics and functions of microglia and macrophages in neuronal inflammation and regeneration following optic nerve injury,and their possible role as therapeutic targets for axonal regeneration.展开更多
Accurate localization of cranial nerves and responsible blood vessels is important for diagnosing trigeminal neuralgia(TN)and hemifacial spasm(HFS).Manual delineation of the nerves and vessels on medical images is tim...Accurate localization of cranial nerves and responsible blood vessels is important for diagnosing trigeminal neuralgia(TN)and hemifacial spasm(HFS).Manual delineation of the nerves and vessels on medical images is time-consuming and labor-intensive.Due to the development of convolutional neural networks(CNNs),the performance of medical image segmentation has been improved.In this work,we investigate the plans for automated segmentation of cranial nerves and responsible vessels for TN and HFS,which has not been comprehensively studied before.Different inputs are given to the CNN to find the best training configuration of segmenting trigeminal nerves,facial nerves,responsible vessels and brainstem,including the image modality and the number of segmentation targets.According to multiple experiments with seven training plans,we suggest training with the combination of three-dimensional fast imaging employing steady-state acquisition(3D-FIESTA)and three-dimensional time-of-flight magnetic resonance angiography(3DTOF-MRA),and separate segmentation of cranial nerves and vessels.展开更多
Traumatic brain injury is one of the main causes of mortality and disability worldwide.Traumatic brain injury is characterized by a primary injury directly induced by the impact,which progresses into a secondary injur...Traumatic brain injury is one of the main causes of mortality and disability worldwide.Traumatic brain injury is characterized by a primary injury directly induced by the impact,which progresses into a secondary injury that leads to cellular and metabolic damages,starting in the first few hours and days after primary mechanical injury.To date,traumatic brain injury is not targetable by therapies aimed at preventing and/or limiting the outcomes of secondary damage but only by palliative therapies.Nerve growth factor is a neurotrophin targeting neuronal and non-neuronal cells,potentially useful in preventing/limiting the outcomes of secondary damage in traumatic brain injury.This potential has further increased in the last two decades since the possibility of reaching neurotrophin targets in the brain through its intranasal delivery has been exploited.Indeed,molecules intranasally delivered to the brain parenchyma may easily bypass the blood-brain barrier and reach their therapeutic targets in the brain,with favorable kinetics,dynamics,and safety profile.In the first part of this review,we aimed to report the traumatic brain injury-induced dysfunctional mechanisms that may benefit from nerve growth factor treatment.In the second part,we then exposed the experimental evidence relating to the action of nerve growth factor(both in vitro and in vivo,after administration routes other than intranasal)on some of these mechanisms.In the last part of the work,we,therefore,discussed the few manuscripts that analyze the effects of treatment with nerve growth factor,intranasally delivered to the brain parenchyma,on the outcomes of traumatic brain injury.展开更多
文摘Although the transcriptional alterations inside the facial nucleus after facial nerve injury have been well studied,the gene expression changes in the facial nerve trunk after injury are still unknown.In this study,we established an adult rat model of facial nerve crush injury by compressing the right lateral extracranial nerve trunk.Transcriptome sequencing,differential gene expression analysis,and cluster analysis of the injured facial nerve trunk were performed,and 39 intersecting genes with significant variance in expression were identified.Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analyses of the 39 intersecting genes revealed that these genes are mostly involved in leukocyte cell-cell adhesion and phagocytosis and have essential roles in regulating nerve repair.Quantitative real-time polymerase chain reaction assays were used to validate the expression of pivotal genes.Finally,nine pivotal genes that contribute to facial nerve recovery were identified,including Arhgap30,Akr1b8,C5ar1,Csf2ra,Dock2,Hcls1,Inpp5d,Sla,and Spi1.Primary Schwann cells were isolated from the sciatic nerve of neonatal rats.After knocking down Akr1b8 in Schwann cells with an Akr1b8-specific small interfering RNA plasmid,expression levels of monocyte chemoattractant protein-1 and interleukin-6 were decreased,while cell proliferation and migration were not obviously altered.These findings suggest that Akr1b8 likely regulates the interaction between Schwann cells and macrophages through regulation of cytokine expression to promote facial nerve regeneration.This study is the first to reveal a transcriptome change in the facial nerve trunk after facial nerve injury,thereby revealing the potential mechanism underlying repair of facial nerve injury.This study was approved by the Animal Ethics Committee of Nantong University,China in 2018(approval No.S20180923-007).
基金supported by grants from the National "Tenth-Five" Scientific & Technological Problem-Tackling Project(№:2004BA720A18-01)Social Welfare Foundation of Scientific Research Institutes of Chinese Ministry of Science & Technology(№:2002DB40097)
文摘Objective To investigate T cell activation following facial nerve axotomization and latent neuroimmunologic mechanisms in traumatic facial paralysis.Methods A murine model of facial nerve transaction was used.Lymphocytes from cervical and mesenteric lymph nodes in BABL/c mice at specific times were collected and expression rates of CD69 on T cells were assessed by flow cytometry.Results Infiltrating T cells were detected around the facial neurons in the facial nerve nucleus in mice whose facial nerve was transected.Immunofluorescent staining showed recruitment of activated T cells.Three days post-facial nerve transection,the expression rate of CD69 on T cells from cervical draining lymphoid nodes(CDLNs) was significantly different from that on T cells from mesenteric lymph nodes(MLNs)(P =0.0457),whereas the latter was similar to that in animals undergoing sham surgeries and that in blank control animals(p= 0.2817 and 0.2724,respectively).Two weeks post-nerve transection,the T cell CD69 expression rate from CDLNs remained at a higher level and than that in the sham-operation animals(p= 0.0007).At two weeks,CD69 expression rate on T cells from MLNs was also up-regulated and different compared with the sham-operation animals and with itself at three days post-operation(p= 0.0082 and 0.0133,respectively).Conclusion T cells appear to be activated and up-regulated in CDLNs following facial nerve transection.There is even evidence of T cell activation in MLNs at 2 weeks post-nerve transection.This suggestes an alteration of immune response from local to general immunity in the acute stage of facial nerve trauma,which may help coordinating and controlling the scales and orientation of the neuroimmune response during the pathogenesis and progression of facial nerve trauma.
基金a Grant from the Youth Research Foundation of Qingdao University,No.2007
文摘Previous studies have demonstrated that muscarinic,and nicotinic receptors increase free Ca2+ levels in the facial nerve nucleus via various channels following facial nerve injury.However,intracellular Ca2+ overload can trigger either necrotic or apoptotic cell death.Gamma-aminobutyric acid(GABA),an important inhibitory neurotransmitter in the central nervous system,exists in the facial nerve nucleus.It is assumed that GABA negatively regulates free Ca2+ levels in the facial nerve nucleus.The present study investigated GABA type A(GABAA) receptor expression in the facial nerve nucleus in a rat model of facial nerve injury using immunohistochemistry and laser confocal microscopy,as well as the regulatory effects of GABAA receptor on nicotinic receptor response following facial nerve injury.Subunits α1,α3,α5,β1,β2,δ,and γ3 of GABAA receptors were expressed in the facial nerve nucleus following facial nerve injury.In addition,GABAA receptor expression significantly inhibited the increase in nicotinic receptor-mediated free Ca2+ levels in the facial nerve nucleus following facial nerve injury in a concentration-dependent fashion.These results suggest that GABAA receptors exhibit negative effects on nicotinic receptor responses following facial nerve injury.
基金Science and Technology Bureau of Guangdong Province, No.2004B33801007Scienceand Technology Bureau of Guangzhou City, No.2007Z3-D2031
文摘BACKGROUND: Recently, many investigators have tried to use natural biomaterials, such as, artery, vein, decalcified bone, etc., as conduits for nerve repair. However, immunological rejection of conduits made of natural biomaterials limits their application. Therefore, it is essential to identify more suitable types of biomaterials. OBJECTIVE: To observe the characteristics of a bioengineering processing method using venous conduit as a stent for repairing facial nerve injury. DESIGN: A controlled observational experiment. SETTING: Animal Laboratories of the Third Hospital Affiliated to Sun Yat-sen University and the 157 Hospital. MATERIALS: Thirty-three male New Zealand rabbits of pure breed, weighing 1.5 to 2.0 kg, were provided by Medical Experimental Animal Room of Sun Yat-sen University. The protocol was carried out in accordance with animal ethics guidelines for the use and care of animals. Venous conduits and autogenous nerves were transplanted into the left and right cheeks, respectively. Eleven animals were chosen for anatomical observations at 5, 10 and 15 weeks after surgery. METHODS: This experiment was carried out in the Animal Laboratories of the Third Hospital Affiliated to Sun Yat-sen University and the 157 Hospital between May and November 2006. After animals were anesthetized, 15 mm of retromandibular vein was harvested for preparing a venous conduit. Approximately 3 cm of low buccal branch of facial nerve was exposed. A segment of 1.2 cm nerve was resected from the middle, and a gap of 1.5 cm formed due to bilateral retraction. The prepared venous conduit of 1.5 cm was sutured to the outer membrane of the severed ends of the nerve. Muscle and skin were sutured layer by layer. Using the same above-mentioned method, the low buccal branch of right autogenous facial nerve was resected, and the left facial nerve segment from the same animal was transplanted using end-to-end neurorrhaphy for control. MAIN OUTCOME MEASURES: ①Post-operatively, food intake, vibrissae activity and wound healing of each animal were observed daily. ② Animals were anesthetized at 5, 10 and 15 weeks after operation for observing the structural change of the venous conduit, the appearance of regenerated nerve, and the relationship between conduit and peripheral muscle tissue. ③ The action potential and latency of bilateral nerves of animals were measured by electrophysiologic examination, and nerve conduction velocity was calculated. ④Neural myelination and neurite growth were observed by histological staining using an optical microscope. RESULTS: Thirty-three New Zealand rabbits were involved in the final analysis. ①Immediately following the operation, vibrissae activity and orbicularis oris muscle activity of the upper lip on venous conduit side were more prominent, and their amplitudes of movement were larger as compared with autogenous nerve side. ② At postoperative 10 weeks, by visual inspection, we found that on the venous conduit side, the venous conduit exhibited membrane structure which encased regenerated nerve. Regenerated nerve adhered to the muscle edge of orbicularis oris muscle. Muscle and nerve could be separated with a forceps. The muscle of musculus orbicularis oris of rabbit was darker and thicker as compared with autogenous nerve side. After the venous conduit was longitudinally split, the regenerated nerve and nerves at two the severed ends were connected together. When compared with postoperative 5 weeks, the connected nerve was thickened, texture was tough and its middle part was thicker than its two ends. On the autogenous nerve side, the regenerated nerve stem was enwrapped by scar tissue. It was bulky and adhered to peripheral muscle. Its neural profile structure was unclear. The two stomas were obviously enlarged. ③At postoperative 10 weeks and 15 weeks, nerve action potentials could be elicited from both the venous conduit and autologous nerve side. The mean nerve conduction velocity on the venous conduit side was greater than that of the autologous nerve side. ④At postoperative 10 weeks, using histochemical staining, it was found that in the venous conduit, regenerated medullated nerve fibers were densely distributed, with well split facial nerve structure, while on the autologous nerve side, nerve fibers were sparsely scattered, with immature medullated nerve structure. CONCLUSION: Biological natural venous conduit processed by bioengineering technology overcomes the tissue inflammatory reactions and connective tissue reactions caused by natural biomaterials. It is more conducive to promote neural regeneration and functional recovery than autologous nerve transplantation.
文摘BACKGROUND: Studies have shown that agmatine can reduce inhibition of neuronal regeneration by in-creasing cyclic adenosine monophosphate and brain-derived neurotrophic factor (BDNF) in the hippocampusof morphine-dependent rats. The hypothesis that agmatine exerts similar effects on facial nerve injury de-serves further analysis. OBJECTIVE: To study the effects of peritoneal agmatine injection on BDNF levels in the rat brainstem af-ter facial nerve injury. DESIGN, TIME AND SETTING: A controlled animal experiment was performed at the Department ofOtolaryngology-Head and Neck Surgery at the Second Affiliated Hospital, Chongqing University of MedicalSciences (Chongqing, China), between October and December in 2007. MATERIALS: Twenty-four male Sprague-Dawley rats were randomly divided into a control, a lesion, andan agmatine treatment group, with eight rats in each group. Bilateral facial nerve anastomosis was induced inthe lesion and agmatine treatment groups, while the control group remained untreated. A rat BDNF En-zyme-linked immunosorbent assay kit was used to measure BDNF levels in the brainstem facial nucleus. METHODS: Starting on the day of lesion, the agmatine group received a peritoneal injection of 100 mg/kgagmatine, once per day, for a week, whereas rats in the lesion group received saline injections. MAIN OUTCOME MEASURES: BDNF levels in the brainstem containing facial nucleus were measuredby ELISA. RESULTS: Twenty-four rats were included in the final analysis without any loss. Two weeks after lesion,BDNF levels were significantly higher in the lesion group than in the control group (P < 0.01). A significantincrease was noted in the agmatine group compared to the lesion group (P < 0.01). CONCLUSION: Agmatine can substantially increase BDNF levels in the rat brainstem after facial nerveinjury.
文摘BACKGROUND: Previous studies have demonstrated that postsynaptic density protein-95 (PSD-95) is widely distributed in the central nervous system and is related to the development of the CNS and sensory signal transmission as well as acute or chronic nerve cell death following ischemic brain injury. OBJECTIVE: To semi-quantitatively determine the pathological changes of apoptotic facial neurons and the expression of PSD-95 in the facial nucleus following facial nerve injury of varying extents using immu- nohistochemical staining methods. DESIGN, TIME AND SETTING: Randomized, controlled animal experiments were performed in the Ul- trasonic Institute of the Second Affiliated Hospital of Chongqing University of Medical Sciences from Sep- tember to December 2007. MATERIALS: Sixty-five healthy, adult, Sprague-Dawley (SD) rats, both male and female, were used for this study. Rabbit anti-rat PSD-95 polyclonal antibody was purchased from Beijing Biosynthesis Biotech- nology Co., Ltd. METHODS: SD rats were randomly assigned into a control group with five rats and three injured groups with 20 rats per group. Exposure, clamp and cut for bilateral facial nerve trunks were performed in the rats of the injury groups, and no injury was inflicted on the rats of the control group. MAIN OUTCOME MEASURES: The brainstems of all the rats were excised on days 1, 3, 7, and 14 post injury, and then the facial nuclei were stained with hematoxylin-eosin to observe any pathological changes due to apoptosis in facial neurons. PSD-95 expression in facial nuclei was detected by immunohistochemistry and the number of PSD-95 positive cells was counted under a light microscope. RESULTS: The expression of PSD-95 in the facial nucleus and morphology of the facial neuron within the exposure group had no obvious changes at various points in time tested (P > 0.05). However, the expressions of PSD-95 in the facial nucleus of the clamp group and cut group increased on day 1 post injury (P < 0.05), and showed further increase on day 7 post injury (P < 0.01). This did not decrease until day 14 post injury. Facial neuron apoptosis was detected on day 3 post injury and this was even more obvious on day 7 and was maintained to day 14 post injury. The number of cells expressing PSD-95 and displaying severe degrees of facial neuron apoptosis were as follows: cut group > clamp group > exposure group. CONCLUSION: The apoptotic extent of facial neurons and the expression of PSD-95 in apoptotic facial neurons increased with the degree of aggravation of injured severity of facial nerve.
基金the Youth Research Foundation of Qingdao University, No. 2007
文摘Muscarinic receptors and nicotine receptors can increase free calcium ion levels in the facial nucleus via different channels following facial nerve injury. In addition, γ-aminobutyric acid A (GABAA) receptors have been shown to negatively regulate free calcium ion levels in the facial nucleus by inhibiting nicotine receptors. The present study investigated the influence of GABAA, γ-aminobutyric acid B (GABAB) and C (GABAC) receptors on muscarinic receptors in rats with facial nerve injury by confocal laser microscopy. GABAA and GABAB receptors exhibited significant dose-dependent inhibitory effects on increased muscarinic receptor-mediated free calcium ion levels following facial nerve injury. Results showed that GABAA and GABAB receptors negatively regulate muscarinic receptor effects and interplay with cholinergic receptors to regulate free calcium ion levels for facial neural regeneration.
文摘BACKGROUND: Erythropoietin and recombinant human erythropoietin (rhEPO) inhibit apoptosis of motor neurons caused by spinal cord injury and brain damage in rats. However, it still remains to be shown whether rhEPO can protect facial motoneurons (FMNs) as well. OBJECTIVE: To test the neuroprotective effects of rhEPO on injured FMNs, as well as the influence on Caspase-3 expression. DESIGN, TIME AND SETTING: Randomized, controlled, animal experiment. This study was performed at the Central Laboratory of Basic Medical College, Chongqing Medical University from January to October 2007. MATERIALS: Seventy-five female SD rats, weighing 210–230 g. rhEPO injection was provided by Sansheng pharmaceuticals company, Shenyang City, Liaoning Province, China, and the License number was HMLN S20010001. METHODS: A total of 75 female rats were randomly divided into rhEPO treatment, control, and sham operation groups, with 25 rats in each group. Rat models of facial nerve injury were established in the rhEPO treatment group and the control group by crushing the main trunk of the left facial nerve. Surgical microscopic observation of the facial nerve damage displayed perineurial disruption. The left stylomastoid foramen of the sham operation group were only exposed, but without nerve injury. The rhEPO treatment group was treated with rhEPO (5 000 U/kg, i.p.) once following injury and once a day for two weeks. The control and sham operation groups were treated with the same dose of normal saline (i.p.), once following injury and once a day for two weeks. MAIN OUTCOME MEASURES: Rats were sacrificed 3, 7, 14, 21, and 28 days after injury, FMN survival after facial nerve injury was analyzed by Toluidine blue staining, and then survival ratios (L/R) were calculated. The number of apoptotic profiles in the injured FMNs were evaluated by TUNEL staining. Expression of Caspase-3 in the facial nucleus was detected by immunohistochemistry methods. RESULTS: A total of 75 rats were included in the final analysis. FMN survival ratios, both in rhEPO treatment group and control group, decreased gradually between seven and 28 days; however, FMN survival ratios were significantly greater in the rhEPO treatment group compared to the control group (P < 0.05). No TUNEL-positive cells were observed three days after injury in the rhEPO treatment and control groups; however, by seven days after injury, apoptotic cells were observed and peaked by 14 days in the control group. Between seven and 21 days, apoptotic cell numbers were significantly lower in the rhEPO treatment group compared to the control group (P < 0.05). The expression of Caspase-3 increased three days after injury and peaked at 14 days in the control group. Nevertheless, Caspase-3 expression was significantly lower in the rhEPO treatment group compared to the control group at each time point (P < 0.05). CONCLUSION: Treatment with rhEPO can effectively protect facial motoneurons by reducing expression of Caspase-3 and inhibiting apoptosis.
基金financially supported by the Koln Fortune Programmthe Jean-Uhrmacher FoundationAkdeniz University Research Fund
文摘Recently,we have shown that manual stimulation of paralyzed vibrissal muscles after facial-facial anastomosis reduced the poly-innervation of neuromuscular junctions and restored vibrissal whisking.Using gene knock outs,we found a differential dependence of manual stimulation effects on growth factors.Thus,insulin-like growth factor-1 and brain-derived neurotrophic factor are required to underpin manual stimulation-mediated improvements,whereas FGF-2 is not.The lack of dependence on FGF-2 in mediating these peripheral effects prompted us to look centrally,i.e.within the facial nucleus where increased astrogliosis after facial-facial anastomosis follows "synaptic stripping".We measured the intensity of Cy3-fluorescence after immunostaining for glial fibrillary acidic protein(GFAP) as an indirect indicator of synaptic coverage of axotomized neurons in the facial nucleus of mice lacking FGF-2(FGF-2^(-/-) mice).There was no difference in GFAP-Cy3-fluorescence(pixel number,gray value range17-103) between intact wildtype mice(2.12± 0.37×10~7) and their intact FGF-2^(-/-) counterparts(2.12±0.27×10~7) nor after facial-facial anastomosis +handling(wildtype:4.06±0.32×10~7;FGF-2^(-/-):4.39±0.17×10~7).However,after facial-facial anastomosis,GFAP-Cy3-fluorescence remained elevated in FGF-2^(-/-)-animals(4.54±0.12×10~7),whereas manual otimulation reduced the intensity of GFAP-immunofluorescence in wild type mice to values that were not significantly different from intact mice(2.63±0.39×10).We conclude that FGF-2 is not required to underpin the beneficial effects of manual stimulation at the neuro-muscular junction,but it is required to minimize astrogliosis in the brainstem and,by implication,restore synaptic coverage of recovering facial motoneurons.
基金Supported by the National Natural Science Foundation of China (No.81603706)。
文摘Objective:To explore the mechanism of electroacupuncture(EA) in promoting recovery of the facial function with the involvement of autophagy,glial cell line-derived neurotrophic factor(GDNF),and phosphatidylinositol-3-kinase(PI3K)/mammalian target of rapamycin(mTOR) signaling pathway.Methods:Seventy-two male Sprague-Dawley rats were randomly allocated into the control,sham-operated,facial nerve injury(FNI),EA,EA+3-methyladenine(3-MA),and EA+GDNF antagonist groups using a random number table,with 12 rats in each group.An FNI rat model was established with facial nerve crushing method.EA intervention was conducted at Dicang(ST 4),Jiache(ST 6),Yifeng(SJ 17),and Hegu(LI 4) acupoints for 2 weeks.The Simone’s 10-Point Scale was utilized to monitor the recovery of facial function.The histopathological evaluation of facial nerves was performed using hematoxylin-eosin(HE) staining.The levels of Beclin-1,light chain 3(LC3),and P62 were detected by immunohistochemistry(IHC),immunofluorescence,and reverse transcriptionpolymerase chain reaction,respectively.Additionally,IHC was also used to detect the levels of GDNF,Rai,PI3K,and mTOR.Results:The facial functional scores were significantly increased in the EA group than the FNI group(P<0.05 or P<0.01).HE staining showed nerve axons and myelin sheaths,which were destroyed immediately after the injury,were recovered with EA treatment.The expressions of Beclin-1 and LC3 were significantly elevated and the expression of P62 was markedly reduced in FNI rats(P<0.01);however,EA treatment reversed these abnormal changes(P<0.01).Meanwhile,EA stimulation significantly increased the levels of GDNF,Rai,PI3K,and mTOR(P<0.01).After exogenous administration with autophagy inhibitor 3-MA or GDNF antagonist,the repair effect of EA on facial function was attenuated(P<0.05 or P<0.01).Conclusions:EA could promote the recovery of facial function and repair the facial nerve damages in a rat model of FNI.EA may exert this neuroreparative effect through mediating the release of GDNF,activating the PI3K/mTOR signaling pathway,and further regulating the autophagy of facial nerves.
基金supported by the National Natural Science Foundation of China,No.32371048(to YK)the Peking University People’s Hospital Research and Development Funds,No.RDX2021-01(to YK)the Natural Science Foundation of Beijing,No.7222198(to NH)。
文摘Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.
基金supported by grants from the Department of Technology of Jilin Province,Nos.3D5195941430(to YSW),20190201087(to ZCK)the Department of Finance of Jilin Province,No.3D517DV93429(to ZCK)。
文摘The effect of platelet-rich plasma on nerve regeneration remains controversial.In this study,we established a rabbit model of sciatic nerve small-gap defects with preserved epineurium and then filled the gaps with platelet-rich plasma.Twenty-eight rabbits were divided into the following groups(7 rabbits/group):model,low-concentrati on PRP(2.5-3.5-fold concentration of whole blood platelets),medium-concentration PRP(4.5-6.5-fold concentration of whole blood platelets),and high-concentration PRP(7.5-8.5-fold concentration of whole blood platelets).Electrophysiological and histomorphometrical assessments and proteomics analysis we re used to evaluate regeneration of the sciatic nerve.Our results showed that platelet-rich plasma containing 4.5-6.5-and 7.5-8.5-fold concentrations of whole blood platelets promoted repair of sciatic nerve injury.Proteomics analysis was performed to investigate the possible mechanism by which platelet-rich plasma promoted nerve regeneration.Proteomics analysis showed that after sciatic nerve injury,platelet-rich plasma increased the expression of integrin subunitβ-8(ITGB8),which participates in angiogenesis,and differentially expressed proteins were mainly enriched in focal adhesion pathways.Additionally,two key proteins,ribosomal protein S27 a(RSP27 a)and ubiquilin 1(UBQLN1),which were selected after protein-protein interaction analysis,are involved in the regulation of ubiquitin levels in vivo.These data suggest that platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury by affecting angiogenesis and intracellular ubiquitin levels.
基金supported by the National Natural Science Foundation of China,No. 81801213 (to BP)Xuzhou Special Fund for Promoting Scientific and Technological Innovation,Nos. KC21177 (to BP),KC21195 (to HF)Science and Technology Project of Yili Kazak Autonomous Prefecture,No. YZ2019D006 (to HF)。
文摘Research has shown that long-chain noncoding RNAs(lncRNAs) are involved in the regulation of a variety of biological processes, including peripheral nerve regeneration, in part by acting as competing endogenous RNAs. c-Jun plays a key role in the repair of peripheral nerve injury. However, the precise underlying mechanism of c-Jun remains unclear. In this study, we performed microarray and bioinformatics analysis of mouse crush-injured sciatic nerves and found that the lncRNA Pvt1 was overexpressed in Schwann cells after peripheral nerve injury. Mechanistic studies revealed that Pvt1 increased c-Jun expression through sponging miRNA-214. We overexpressed Pvt1 in Schwann cells cultured in vitro and found that the proliferation and migration of Schwann cells were enhanced, and overexpression of miRNA-214 counteracted the effects of Pvt1 overexpression on Schwann cell proliferation and migration. We conducted in vivo analyses and injected Schwann cells overexpressing Pvt1 into injured sciatic nerves of mice. Schwann cells overexpressing Pvt1 enhanced the regeneration of injured sciatic nerves following peripheral nerve injury and the locomotor function of mice was improved. Our findings reveal the role of lncRNAs in the repair of peripheral nerve injury and highlight lncRNA Pvt1 as a novel potential treatment target for peripheral nerve injury.
基金supported by the National Key Research and Development Program of China, No. 2016YFC1101603 (to DYZ)the National Natural Science Foundation of China, Nos. 31640045 (to YHW), 81901251 (to ML)the Natural Science Foundation of Beijing of China, No. 7204323 (to ML)
文摘We previously prepared nerve growth factor poly-lactide co-glycolid sustained-release microspheres to treat rat sciatic nerve injury using the small gap sleeve technique.Multiple growth factors play a synergistic role in promoting the repair of peripheral nerve injury;as a result,in this study,we added basic fibroblast growth factors to the microspheres to further promote nerve regeneration.First,in an in vitro biomimetic microenvironment,we developed and used a drug screening biomimetic microfluidic chip to screen the optimal combination of nerve growth factor/basic fibroblast growth factor to promote the regeneration of Schwann cells.We found that 22.56 ng/mL nerve growth factor combined with 4.29 ng/mL basic fibroblast growth factor exhibited optimal effects on the proliferation of primary rat Schwann cells.The successfully prepared nerve growth factor-basic fibroblast growth factor-poly-lactide-co-glycolid sustained-release microspheres were used to treat rat sciatic nerve transection injury using the small gap sleeve bridge technique.Compared with epithelium sutures and small gap sleeve bridging alone,the small gap sleeve bridging technique combined with drug-free sustained-release microspheres has a stronger effect on rat sciatic nerve transfection injury repair at the structural and functional level.
文摘The efficacy of electroacupuncture in the treatment of peripheral facial paralysis is known, but the specific mechanism has not been clarified. Glial cell-derived neurotrophic factor(GDNF) has been shown to protect neurons by binding to N-cadherin. Our previous results have shown that electroacupuncture could increase the expression of N-cadherin mRNA in facial neurons and promote facial nerve regeneration. In this study, the potential mechanisms by which electroacupuncture promotes nerve regeneration were elucidated through assessing the effects of electroacupuncture on GDNF and N-cadherin expression in facial motoneurons of rabbits with peripheral facial nerve crush injury. New Zealand rabbits were randomly divided into a normal group(normal control, n = 21), injury group(n = 45) and electroacupuncture group(n = 45). Model rabbits underwent facial nerve crush injury only. Rabbits in the electroacupuncture group received facial nerve injury, and then underwent electroacupuncture at Yifeng(TE17), Jiache(ST6), Sibai(ST2), Dicang(ST4), Yangbai(GB14), Quanliao(SI18), and Hegu(LI4; only acupuncture, no electrical stimulation). The results showed that in behavioral assessments, the total scores of blink reflex, vibrissae movement, and position of apex nasi, were markedly lower in the EA group than those in the injury group. Hematoxylin-eosin staining of the right buccinator muscle of each group showed that the cross-sectional area of buccinator was larger in the electroacupuncture group than in the injury group on days 1, 14 and 21 post-surgery. Toluidine blue staining of the right facial nerve tissue of each group revealed that on day 14 post-surgery, there was less axonal demyelination and fewer inflammatory cells in the electroacupuncture group compared with the injury group. Quantitative real time-polymerase chain reaction showed that compared with the injury group, N-cadherin mRNA levels on days 4, 7, 14 and 21 and GDNF mRNA levels on days 4, 7 and 14 were significantly higher in the electroacupuncture group. Western blot assay displayed that compared with the injury group, the expression of GDNF protein levels on days 7, 14 and 21 were significantly upregulated in the electroacupuncture group. The histology with hematoxylin-eosin staining and Nissl staining of brainstem tissues containing facial neurons in the middle and lower part of the pons exhibited that on day 7 post-surgery, there were significantly fewer apoptotic neurons in the electroacupuncture group than in the injury group. By day 21, there was no significantly difference in the number of neurons between the electroacupuncture and normal groups. Taken together, these results have confirmed that electroacupuncture promotes regeneration of peripheral facial nerve injury in rabbits, inhibits neuronal apoptosis, and reduces peripheral inflammatory response, resulting in the recovery of facial muscle function. This is achieved by up-regulating the expression of GDNF and N-cadherin in central facial neurons.
基金funded by the Project of 2022 Health Commission Technology Plan of Zhejiang Province(Project Number:2022KY1169)the 2022 Ningbo Natural Science Foundation Young Doctoral Project(Project Number:2022J027).
文摘Background:The underlying mechanism of sciatic nerve injury(SNI)is a common motor functional disorder,necessitates further research.Methods:A rat model of SNI was established,with the injury group subjected to compressive injury of the right sciatic nerve exposed at the midpoint of the thigh and the sham surgery group undergoing the same surgical procedure.An oxygen-glucose deprivation model was employed to simulate in vitro SNI in PC12 cells.Following data acquisition and quality control,differentially expressed proteins(DEPs)in each model were identified through differential analysis,and enrichment analysis was used to explore the potential functions and pathways of the DEPs.Venn diagrams were drawn,and DEPs from both in vivo and in vitro SNI models were imported into the STRING database to construct a protein-protein interaction network and screen for hub proteins.Results:After the peptide segments obtained from rat nerve blockade and PC12 cells met quality requirements,258 DEPs were identified in rat nerve samples,and 119 DEPs were screened in PC12 cells.Enrichment analysis revealed that DEPs in the rat model were predominantly concentrated in biological functions such as myogenic cell proliferation and signaling related to lipid and energy metabolism.DEPs in the in vitro model were mainly enriched in biological processes such as phagocytosis and were associated with lipid transport and metabolism.Two hub proteins,amyloid precursor protein(APP)and fibronectin 1(FN1),were identified through MCC,MCODE,and Degree scoring.Both PC12 cells and external validation sets showed relatively higher expression of APP and FN1 in injured samples.Results of gene set enrichment analysis indicated that these two proteins were associated with metabolic pathways,such as biosynthesis of glycosaminoglycan chondroitin sulfate and biosynthesis of unsaturated fatty acids.Conclusion:APP and FN1 are potential key molecules involved in SNI and are associated with various metabolic pathways in nerve repair.These findings provide a theoretical basis for the development of therapeutic targets for SNI.
基金supported by the National Natural Science Foundation of China:Investigation on central cortical functional compensation and re-organization after hypoglossal-facial neurorrhaphy for facial nerve injury using repetitive transcranial magnetic stimulation combined with biofeedback peripheral electrical stimulation[Grant No.82171364]the Beijing Municipal Health Commission:Study on repair mechanism of nervous system injury[Grant No.PXM2020_026280_000002].
文摘Objective To provide useful information for selecting the most appropriate peripheral nerve injury model for different research purposes in nerve injury and repair studies,and to compare nerve regeneration capacity and characteristics between them.Methods Sixty adult SD rats were randomly divided into two groups and underwent crush injury alone(group A,n=30)or transection injury followed by surgical repair(group B,n=30)of the right hind paw.Each group was subjected to the CatWalk test,gastrocnemius muscle evaluation,pain threshold measurement,electrophysiological examination,retrograde neuronal labeling,and quantification of nerve regeneration before and 7,14,21,and 28 days after injury.Results Gait analysis showed that the recovery speed in group A was significantly faster than that in group B at 14 days.At 21 days,the compound muscle action potential of the gastrocnemius muscle in group A was significantly higher than that in group B,and the number of labeled motor neurons in group B was lower than that in group A.The number of new myelin sheaths and the g-ratio were higher in group A than in group B.There was a 7-day time difference in the regeneration rate between the two injury groups.Conclusion The regeneration of nerve fibers was rapid after crush nerve injury,whereas the transection injury was relatively slow,which provides some ideas for the selection of clinical research models.
基金supported by NIH Center Core Grant P30EY014801a Research to Prevent Blindness Unrestrictea Grant+3 种基金partially supported by the Walter G.Ross Foundationpartly supported by the Gutierrez Family Research Fundthe Camiener Family Glaucoma Research Fundthe National Natural Science Foundation of China(No.82201170 to XL)。
文摘Monocytes,including monocyte-derived macrophages and resident microglia,mediate many phases of optic nerve injury pathogenesis.Resident microglia respond first,followed by infiltrating macrophages which regulate neuronal inflammation,cell proliferation and differentiation,scar formation and tissue remodeling following optic nerve injury.However,microglia and macrophages have distinct functions which can be either beneficial or detrimental to the optic nerve depending on the spatial context and temporal sequence of their activity.These divergent effects are attributed to pro-and anti-inflammatory cytokines expressed by monocytes,crosstalk between monocyte and glial cells and even microglia-macrophage communication.In this review,we describe the dynamics and functions of microglia and macrophages in neuronal inflammation and regeneration following optic nerve injury,and their possible role as therapeutic targets for axonal regeneration.
文摘Accurate localization of cranial nerves and responsible blood vessels is important for diagnosing trigeminal neuralgia(TN)and hemifacial spasm(HFS).Manual delineation of the nerves and vessels on medical images is time-consuming and labor-intensive.Due to the development of convolutional neural networks(CNNs),the performance of medical image segmentation has been improved.In this work,we investigate the plans for automated segmentation of cranial nerves and responsible vessels for TN and HFS,which has not been comprehensively studied before.Different inputs are given to the CNN to find the best training configuration of segmenting trigeminal nerves,facial nerves,responsible vessels and brainstem,including the image modality and the number of segmentation targets.According to multiple experiments with seven training plans,we suggest training with the combination of three-dimensional fast imaging employing steady-state acquisition(3D-FIESTA)and three-dimensional time-of-flight magnetic resonance angiography(3DTOF-MRA),and separate segmentation of cranial nerves and vessels.
基金funded by the Italian Ministry of Health Grant:RF-2018-12366594“Nerve growth factor in paediatric severe traumatic brain injury:translational and clinical studies on a candidate biomarker and therapeutic drug”(to AC)。
文摘Traumatic brain injury is one of the main causes of mortality and disability worldwide.Traumatic brain injury is characterized by a primary injury directly induced by the impact,which progresses into a secondary injury that leads to cellular and metabolic damages,starting in the first few hours and days after primary mechanical injury.To date,traumatic brain injury is not targetable by therapies aimed at preventing and/or limiting the outcomes of secondary damage but only by palliative therapies.Nerve growth factor is a neurotrophin targeting neuronal and non-neuronal cells,potentially useful in preventing/limiting the outcomes of secondary damage in traumatic brain injury.This potential has further increased in the last two decades since the possibility of reaching neurotrophin targets in the brain through its intranasal delivery has been exploited.Indeed,molecules intranasally delivered to the brain parenchyma may easily bypass the blood-brain barrier and reach their therapeutic targets in the brain,with favorable kinetics,dynamics,and safety profile.In the first part of this review,we aimed to report the traumatic brain injury-induced dysfunctional mechanisms that may benefit from nerve growth factor treatment.In the second part,we then exposed the experimental evidence relating to the action of nerve growth factor(both in vitro and in vivo,after administration routes other than intranasal)on some of these mechanisms.In the last part of the work,we,therefore,discussed the few manuscripts that analyze the effects of treatment with nerve growth factor,intranasally delivered to the brain parenchyma,on the outcomes of traumatic brain injury.
