重组腺病毒载体AxCA-BDNF基因转染修复坐骨神经损伤*☆

背景：如何促进周围神经损伤修复与再生一直是基础与临床研究的热点。基因治疗有可能成为今后解决该问题的主要手段之一。 目的：观察携带小鼠脑源性神经营养因子(brain-derived neurotrophic factor,BDNF) cDNA表达片段的重组腺病毒载体AxCA-BDNF转染大鼠损伤坐骨神经后BDNF的表达,以及脊髓前角运动神经元的存活和神经生长情况。 方法：切除成年Wistar大鼠股中部10 mm长的坐骨神经,AxCA-BDNF转染组、BDNF组和对照组分别用硅胶管内置AxCA-BDNF原液,BDNF溶液或空白病毒稀释液桥接坐骨神经两断端。术后3,7,14 d,1,2,4个月应用原位杂交和免疫组织化学方法检测损伤坐骨神经及相应脊髓节段BDNF mRNA和蛋白的表达,并观察损伤坐骨神经的组织学及超微结构改变,再生的神经元及有髓神经纤维数目和髓鞘厚度。 结果与结论：术后3,7,14 d及1个月时,AxCA-BDNF转染组损伤坐骨神经近、远端神经干及脊髓(L3~6)中BDNF mRNA和蛋白水平明显高于BDNF组和对照组(P < 0.01)。光、电镜病理组织学检查和图像分析证实,BDNF基因转染后,脊髓前角运动神经元存活数量、新生神经纤维数目及其髓鞘厚度、神经联接的再形成均明显优于对照组(P < 0.01)。说明经腺病毒介导转染的BDNF基因可在大鼠坐骨神经内有效表达,并通过轴突逆行转运到了相应的脊髓神经元,不仅能促进损伤神经纤维再生,也能保护损伤的脊髓神经元。 关键词：坐骨神经损伤;重组腺病毒;脑源性神经营养因子;基因转染;免疫组织化学;原位分子杂交;神经再生     

Dexamethasone prevents vascular damage in early-stage non-freezing cold injury of the sciatic nerve

Non-freezing cold injury is a prevalent cause of peripheral nerve damage, but its pathogenic mechanism is poorly understood, and treatment remains inadequate. Glucocorticoids have anti-inflammatory and lipid peroxidation-inhibiting properties. We therefore examined whether dexamethasone, a synthetic glucocorticoid compound, would alleviate early-stage non-freezing cold injury of the sciatic nerve. We established Wistar rat models of non-freezing cold injury by exposing the left sciatic nerve to cold(3–5°C) for 2 hours, then administered dexamethasone(3 mg/kg intraperitoneally) to half of the models. One day after injury, the concentration of Evans blue tracer in the injured sciatic nerve of rats that received dexamethasone was notably lower than that in the injured sciatic nerve of rats that did not receive dexamethasone; neither Evans blue dye nor capillary stenosis was observed in the endoneurium, but myelinated nerve fibers were markedly degenerated in the injured sciatic nerve of animals that received dexamethasone. After dexamethasone administration, however, endoneurial vasculopathy was markedly improved, although damage to the myelinated nerve fiber was not alleviated. These findings suggest that dexamethasone protects the blood-nerve barrier, but its benefit in non-freezing cold injury is limited to the vascular system.     

Ursolic acid induces neural regeneration after sciatic nerve injury

In this study,we aimed to explore the role of ursolic acid in the neural regeneration of the injured sciatic nerve.BALB/c mice were used to establish models of sciatic nerve injury through unilateral sciatic nerve complete transection and microscopic anastomosis at 0.5 cm below the ischial tuberosity.The successfully generated model mice were treated with 10,5,or 2.5 mg/kg ursolic acid via intraperitoneal injection.Enzyme-linked immunosorbent assay results showed that serum S100 protein expression level gradually increased at 1-4 weeks after sciatic nerve injury,and significantly decreased at 8 weeks.As such,ursolic acid has the capacity to significantly increase S100 protein expression levels.Real-time quantitative PCR showed that S100 mRNA expression in the L4-6 segments on the injury side was increased after ursolic acid treatment.In addition,the muscular mass index in the soleus muscle was also increased in mice treated with ursolic acid.Toluidine blue staining revealed that the quantity and average diameter of myelinated nerve fibers in the injured sciatic nerve were significantly increased after treatment with ursolic acid.10 and 5 mg/kg of ursolic acid produced stronger effects than 2.5 mg/kg of ursolic acid.Our findings indicate that ursolic acid can dose-dependently increase S100 expression and promote neural regeneration in BALB/c mice following sciatic nerve injury.     

Combination therapy using evening primrose oil and electrical stimulation to improve nerve function following a crush injury of sciatic nerve in male rats

Peripheral nerve injuries with a poor prognosis are common. Evening primrose oil (EPO) has beneficial biological effects and immunomodulatory properties. Since electrical activity plays a major role in neural regeneration, the present study investigated the effects of electrical stimulation (ES), combined with evening primrose oil (EPO), on sciatic nerve function after a crush injury in rats. In anesthetized rats, the sciatic nerve was crushed using small haemostatic forceps followed by ES and/or EPO treatment for 4 weeks. Functional recovery of the sciatic nerve was assessed using the sciatic functional index. Histopathological changes of gas-trocnemius muscle atrophy were investigated by light microscopy. Electrophysiological changes were assessed by the nerve conduction velocity of sciatic nerves. Immunohistochemistry was used to determine the remy-elination of the sciatic nerve following the interventions. EPO + ES, EPO, and ES obviously improved sciatic nerve function assessed by the sciatic functional index and nerve conduction velocity of the sciatic nerve at 28 days after operation. Expression of the peripheral nerve remyelination marker, protein zero (P0), was in-creased in the treatment groups at 28 days after operation. Muscle atrophy severity was decreased significantly while the nerve conduction velocity was increased significantly in rats with sciatic nerve injury in the injury+ EPO + ES group than in the EPO or ES group. Totally speaking, the combined use of EPO and ES may pro-duce an improving effect on the function of sciatic nerves injured by a crush. The increased expression of P0 may have contributed to improving the functional effects of combination therapy with EPO and ES as well as the electrophysiological and histopathological features of the injured peripheral nerve.     

Guinea pigs as an animal model for sciatic nerve injury

The overwhelming use of rat models in nerve regeneration studies is likely to induce skewness in treatment outcomes.To address the problem,this study was conducted in 8 adult guinea pigs of either sex to investigate the suitability of guinea pig as an alternative model for nerve regeneration studies.A crush injury was inflicted to the sciatic nerve of the left limb,which led to significant decrease in the pain perception and neurorecovery up to the 4th weak.Lengthening of foot print and shortening of toe spread were observed in the paw after nerve injury.A 3.49 ± 0.35 fold increase in expression of neuropilin 1(NRP1) gene and 2.09 ± 0.51 fold increase in neuropilin 2(NRP2) gene were recorded 1 week after nerve injury as compared to the normal nerve.Ratios of gastrocnemius muscle weight and volume of the experimental limb to control limb showed more than 50% decrease on the 30 th day.Histopathologically,vacuolated appearance of the nerve was observed with presence of degenerated myelin debris in digestion chambers.Gastrocnemius muscle also showed degenerative changes.Scanning electron microscopy revealed loose and rough arrangement of connective tissue fibrils and presence of large spherical globules in crushed sciatic nerve.The findings suggest that guinea pigs could be used as an alternative animal model for nerve regeneration studies and might be preferred over rats due to their cooperative nature while recording different parameters.     

Boric acid reduces axonal and myelin damage in experimental sciatic nerve injury

hTe aim of this study was to investigate the effects of boric acid in experimental acute sciatic nerve injury. Twenty-eight adult male rats were randomly divided into four equal groups (n = 7): control (C), boric acid (BA), sciatic nerve injury (I) , and sciatic nerve injury + boric acid treatment (BAI). Sciatic nerve injury was generated using a Yasargil aneurysm clip in the groups I and BAI. Boric acid was given four times at 100 mg/kg to rats in the groups BA and BAI atfer injury (by gavage at 0, 24, 48 and 72 hours) but no injury was made in the group BA.In vivo electrophysiological tests were performed at the end of the day 4 and sciatic nerve tissue samples were taken for histopathological examination. The amplitude of compound action potential, the nerve conduction velocity and the number of axons were signiifcantly lower and the myelin structure was found to be broken in group I compared with those in groups C and BA. However, the amplitude of the compound action potential, the nerve conduction velocity and the number of axons were signiifcantly greater in group BAI than in group I. Moreover, myelin injury was signiifcantly milder and the intensity of nuclear factor kappa B immunostaining was signiifcantly weaker in group BAI than in group I. hTe results of this study show that administration of boric acid at 100 mg/kg atfer sciatic nerve injury in rats markedly reduces myelin and axonal injury and improves the electrophysiological function of injured sciatic nerve possibly through alleviating oxidative stress reactions.     

The effects of claudin 14 during early Wallerian degeneration after sciatic nerve injury

Claudin 14 has been shown to promote nerve repair and regeneration in the early stages of Wallerian degeneration(0–4 days) in rats with sciatic nerve injury, but the mechanism underlying this process remains poorly understood. This study reported the effects of claudin 14 on nerve degeneration and regeneration during early Wallerian degeneration. Claudin 14 expression was up-regulated in sciatic nerve 4 days after Wallerian degeneration. The altered expression of claudin 14 in Schwann cells resulted in expression changes of cytokines in vitro. Expression of claudin 14 affected c-Jun, but not Akt and ERK1/2 pathways. Further studies revealed that enhanced expression of claudin 14 could promote Schwann cell proliferation and migration. Silencing of claudin 14 expression resulted in Schwann cell apoptosis and reduction in Schwann cell proliferation. Our data revealed the role of claudin 14 in early Wallerian degeneration, which may provide new insights into the molecular mechanisms of Wallerian degeneration.     

PEMF fails to enhance nerve regeneration after sciatic nerve crush lesion

Abstract The use of electromagnetic fields has been reported to enhance peripheral nerve regeneration. This study aimed to identify the effects of a prolonged protocol of low‐frequency pulsed electromagnetic field (PEMF) on peripheral nerve regeneration. Thirty‐four male Swiss mice (Mus musculus) were divided into PEMF (n = 17) and control (n = 17) groups. All animals underwent a unilateral sciatic‐crush lesion, and the PEMF group was exposed to a 72‐Hz, 2‐G electromagnetic field for 30 min, five days a week, for three weeks. Functional analysis was carried out weekly. After three weeks, the animals were euthanized, and histological, morphometric, oxidative stress, and TGF‐β1 analyses were performed. Functional analysis showed no differences between the groups. Histological appearance was similar between PEMF and control nerves. Morphometric assessment showed that the PEMF nerves trended toward decreased regeneration. The levels of free radicals were more pronounced in PEMF nerves, but were not associated with an increase in the content of the TGF‐β1/Smad signaling pathway. Prolonged PEMF regimen leads to delayed histological peripheral nerve regeneration and increased oxidative stress but no loss of function recovery.     

Mice lacking tissue plasminogen activator and urokinase plasminogen activator genes show attenuated matrix metalloproteases activity after sciatic nerve crush

Plasminogen activators (PAs), tissue PA (tPA) and urokinase PA (uPA), have been shown to be induced in sensory neurons after sciatic nerve crush. These findings suggested that PAs facilitate peripheral nerve regeneration by digesting adhesive cell contacts and by activation of other proteases, thereby initiating a proteolytic cascade. Both tPA and uPA activate some matrix metalloproteases (MMPs), indirectly via plasminogen activation or directly, such as the uPA activation of MMP-2. In this study, we demonstrated, by using tPA and uPA knockout mice, that a lack of a plasminogen activator affected MMP-9 and MMP-2 activity after crushing of the sciatic nerve. These findings show that the PAs are important for MMP-9 and MMP-2 activity at the crush site.     

Miconazole enhances nerve regeneration and functional recovery after sciatic nerve crush injury

Introduction: Improving axonal outgrowth and remyelination is crucial for peripheral nerve regeneration. Miconazole appears to enhance remyelination in the central nervous system. In this study we assess the effect of miconazole on axonal regeneration using a sciatic nerve crush injury model in rats. Methods: Fifty Sprague‐Dawley rats were divided into control and miconazole groups. Nerve regeneration and myelination were determined using histological and electrophysiological assessment. Evaluation of sensory and motor recovery was performed using the pinprick assay and sciatic functional index. The Cell Counting Kit‐8 assay and Western blotting were used to assess the proliferation and neurotrophic expression of RSC 96 Schwann cells. Results: Miconazole promoted axonal regrowth, increased myelinated nerve fibers, improved sensory recovery and walking behavior, enhanced stimulated amplitude and nerve conduction velocity, and elevated proliferation and neurotrophic expression of RSC 96 Schwann cells. Discussion: Miconazole was beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Muscle Nerve 57 : 821–828, 2018     

Strain and stress variations in the human amniotic membrane and fresh corpse autologous sciatic nerve anastomosis in a model of sciatic nerve injury

A 10-mm long sciatic nerve injury model was established in fresh normal Chinese patient cadavers. Amniotic membrane was harvested from healthy maternal placentas and was prepared into multilayered,coiled,tubular specimens.Sciatic nerve injury models were respectively anastomosed using the autologous cadaveric sciatic nerve and human amniotic membrane.Tensile test results showed that maximal loading,maximal displacement,maximal stress,and maximal strain of sciatic nerve injury models anastomosed with human amniotic membrane were greater than those in the autologous nerve anastomosis group.The strain-stress curves of the human amniotic membrane and sciatic nerves indicated exponential change at the first phase,which became elastic deformation curves at the second and third phases,and displayed plastic deformation curves at the fourth phase,at which point the specimens lost their bearing capacity.Experimental findings suggested that human amniotic membranes and autologous sciatic nerves exhibit similar stress-strain curves, good elastic properties,and certain strain and stress capabilities in anastomosis of the injured sciatic nerve.     

Craniocerebral injury promotes the repair of peripheral nerve injury

The increase in neurotrophic factors after craniocerebral injury has been shown to promote fracture healing. Moreover, neurotrophic factors play a key role in the regeneration and repair of peripheral nerve. However, whether craniocerebral injury alters the repair of peripheral nerve injuries remains poorly understood. Rat injury models were established by transecting the left sciatic nerve and using a free-fall device to induce craniocerebral injury. Compared with sciatic nerve injury alone after 6–12 weeks, rats with combined sciatic and craniocerebral injuries showed decreased sciatic functional index, increased recovery of gastrocnemius muscle wet weight, recovery of sciatic nerve ganglia and corresponding spinal cord segment neuron morphologies, and increased numbers of horseradish peroxidase-labeled cells. These results indicate that craniocerebral injury promotes the repair of peripheral nerve injury.     

维拉帕米与神经生长因子促进大鼠坐骨神经再生的协同作用

背景：实验证明周围神经损伤时,轴突的变性与神经元凋亡都与Ca2+的超载有着极其密切的关系。 目的：利用大鼠坐骨神经损伤模型观察L型钙离子通道阻滞剂维拉帕米联合神经生长因子促进周围神经再生的协同作用。 设计、时间及地点：随机对照动物实验,于2007-04/2008-11在辽宁医学院手外科实验室完成。 材料：同系健康雄性SD大鼠32只,体质量220~260 g;维拉帕米为辽宁卫星制药厂产品,国药准字H21022847;神经生长因子为sigma公司产品。 方法：同系SD大鼠32只随机分为4组,每组8只,分别在右侧梨状肌下缘5 mm切断坐骨神经后立即原位缝合造成坐骨神经损伤模型。①维拉帕米+神经生长因子组：腹腔注射维拉帕米4 mg/(kg•d),术侧腓肠肌肉注射神经生长因子0.6 μg/d。②维拉帕米组：腹腔注射维拉帕米4 mg/(kg•d),术侧腓肠肌注射等量生理盐水。③神经生长因子组：术侧腓肠肌注神经生长因子0.6 μg/d,并腹腔注射等量生理盐水。④空白对照组：分别腹腔,肌注等量生理盐水。以左侧坐骨神经为正常对照。 主要观察指标：术后12周对各组再生神经进行大体观察,神经电生理测定,组织学观察及有髓神经纤维计数。 结果：术后12周,维拉帕米+神经生长因子组足部溃疡的出现与愈合以及展抓反射出现的时间均早于其他各组。神经传导速度恢复率和有髓神经纤维计数恢复率分析表明：维拉帕米+神经生长因子组>维拉帕米组>神经生长因子组>空白对照组。光镜和电镜下可见：维拉帕米+神经生长因子组再生的神经纤维最多,轴突较为粗大。有髓神经纤维多,髓鞘完整,优于其他3组。神经纤维直径恢复率分析表明：维拉帕米+神经生长因子组>神经生长因子组>维拉帕米组>空白对照组。 结论：维拉帕米与神经生长因子对促进周围神经形态结构和功能的恢复均具有明显的协同作用。     

Vitamin B complex and vitamin B12 levels after peripheral nerve injury

The aim of the present study was to evaluate whether tissue levels of vitamin B complex and vitamin B12 were altered after crush-induced peripheral nerve injury in an experimental rat model. A total of 80 male Wistar rats were randomized into one control (n = 8) and six study groups (1, 6, 12, 24 hours, 3, and 7 days after experimental nerve injury;n = 12 for each group). Crush-induced peripheral nerve injury was per-formed on the sciatic nerves of rats in six study groups. Tissue samples from the sites of peripheral nerve injury were obtained at 1, 6, 12, 24 hours, 3 and 7 days after experimental nerve injury. Enzyme-linked immunosorbent assay results showed that tissue levels of vitamin B complex and vitamin B12 in the injured sciatic nerve were signiifcantly greater at 1 and 12 hours after experimental nerve injury, while they were signiifcantly lower at 7 days than in control group. Tissue level of vitamin B12 in the injured sciatic nerve was signiifcantly lower at 1, 6, 12 and 24 hours than in the control group. These results suggest that tissue levels of vitamin B complex and vitamin B12 vary with progression of crush-induced peripheral nerve injury, and supplementation of these vitamins in the acute period may be beneficial for acceleration of nerve regeneration.     

7, 8-dihydroxycoumarin improves neurological function in a mouse model of sciatic nerve injury

In the present study, a mouse model of sciatic nerve injury was treated with intraperitoneal injection of 7, 8-dihydroxycoumarin (10, 5, or 2.5 mg/kg per day). Western blot and real-time PCR results showed that growth associated protein 43 expression was significantly increased in the L4-6 seg-ments of the spinal cord. The amplitude and velocity of motor nerve conduction in the sciatic nerve were significantly increased in model mice. In addition, the appearance of the myelin sheath in the injured sciatic nerve was regular, with an even thickness and clear outline, and the surrounding fi-broplasia was not obvious. Our results indicate that 7, 8-dihydroxycoumarin can promote the repair of injured nerve by upregulating growth associated protein 43 expression in the corresponding spinal cord segments of mice with sciatic nerve injury.     

Gene expression profiling of the rat sciatic nerve in early Wallerian degeneration after injury

Wallerian degeneration is an important area of research in modern neuroscience.A large number of genes are differentially regulated in the various stages of Wallerian degeneration,especially during the early response.In this study,we analyzed gene expression in early Wallerian degeneration of the distal nerve stump at 0,0.5,1,6,12 and 24 hours after rat sciatic nerve injury using gene chip microarrays.We screened for differentially-expressed genes and gene expression patterns.We examined the data for Gene Ontology,and explored the Kyoto Encyclopedia of Genes and Genomes Pathway.This allowed us to identify key regulatory factors and recurrent network motifs.We identified 1 546 differentially-expressed genes and 21 distinct patterns of gene expression in early Wallerian degeneration,and an enrichment of genes associated with the immune response,acute inflammation,apoptosis,cell adhesion,ion transport and the extracellular matrix.Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed components involved in the Jak-STAT,ErbB,transforming growth factor-β,T cell receptor and calcium signaling pathways.Key factors included interleukin-6,interleukin-1,integrin,c-sarcoma,carcinoembryonic antigen-related cell adhesion molecules,chemokine(C-C motif) ligand,matrix metalloproteinase,BH3 interacting domain death agonist,baculoviral IAP repeat-containing 3 and Rac.The data were validated with real-time quantitative PCR.This study provides a global view of gene expression profiles in early Wallerian degeneration of the rat sciatic nerve.Our findings provide insight into the molecular mechanisms underlying early Wallerian degeneration,and the regulation of nerve degeneration and regeneration.