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     

神经干细胞移植促进鼠脊髓损伤后髓鞘结构的修复

目的 观察神经干细胞移植治疗对鼠脊髓损伤后髓鞘结构修复的作用并探讨其作用机制。方法 制备鼠T10脊髓损伤模型,体外培养、诱导鼠神经干细胞,定量评价神经干细胞移植对脊髓损伤后髓鞘结构修复的影响。结果 与对照组相比,神经干细胞移植组明显地增强了蛋白前脂蛋白信使核糖核酸(PLP mRNA)的表达,促进了髓鞘碱性蛋白(MBP)性的髓鞘再生和髓鞘结构的修复。结论 神经干细胞移植通过增强髓鞘的再生而促进了脊髓损伤后髓鞘结构的修复,是急性脊髓损伤一种有效的治疗方案。     

神经营养素与中枢神经系统损伤后突触的再生修复

突触的再生修复是细胞水平上中枢神经系统损伤后再生修复的重要组成部分之一.神经营养素能够通过多种机制调节突触的再生修复,增强突触的功效,促进中枢神经系统损伤的修复.深入了解神经营养素对突触的结构和功能的作用机制,探索最佳用药方法和途径,将有效促进损伤的中枢神经功能的恢复.     

阿司匹林促进大鼠缺血脑组织脑源性神经营养因子的表达

目的:探讨不同剂量阿司匹林(Asp)对脑缺血/再灌注(CI/RP)损伤大鼠的神经保护作用及其对脑源性神经营养因子(BDNF)表达的影响。方法:采用线栓法建立大鼠大脑中动脉CI/RP模型,对CI/RP后大鼠进行肢体神经功能缺损评分:1~3分的48只大鼠入选。入选大鼠分为对照组、Asp小剂量组(20 mg.kg-1)、Asp中剂量组(80 mg.kg-1)和Asp大剂量组(320 mg.kg-1),于CI/RP术后每日腹腔注射Asp或溶媒,并进行神经功能缺损评分,72 h后处死,测定脑梗死体积和BDNF免疫组化检测。结果:CI/RP后24、48和72 h各Asp组大鼠与对照组相比,神经缺损评分明显降低(P<0.05或P<0.01),梗死灶体积显著减小(P<0.01),缺血区域BDNF表达显著增加(P<0.05或P<0.01)。对BDNF表达的作用Asp大剂量组的作用并不优于Asp小剂量组和中剂量组。结论:Asp可以减少CI/RP大鼠的脑梗死体积;促进内源性BDNF的表达可能是其神经保护的机制之一,Asp对BDNF表达的作用并非随Asp剂量的加大而增强。     

脑源性神经营养因子与中枢神经修复再生

神经营养因子在保护神经元存活并促进其突起生长发育过程中,常出现基因表达的时相变异,对不同种类的神经元有明显的作用选择性.脑源性神经营养因子(BDNF)作为神经营养因子家族中的一员,广泛分布于大脑中,是一类可促进运动神经元、感觉神经元、基底节前脑胆碱能神经元、皮层神经元、海马神经元、多巴胺能神经元等的存活和生长发育并能防止它们受损死亡,改善神经元病理状态、促进受损伤神经元再生及分化成熟等生物效应的多肽或蛋白质,在中枢神经系统(CNS)的损伤修复中具有重要的作用.本文就其理化性质、生物学特性及在中枢神经修复与再生中的作用等进行综述.     

脑源性神经营养因子与亨廷顿病

脑源性神经营养因子(BDNF)支持多种神经元的生存、发育和分化。在基因转录水平,野生型huntingtin和突变型huntingtin有区别地调节BDNF的表达,以及BDNF的轴突转运功能发生障碍,使亨廷顿病患者和模型动物脑内BDNF减少,导致纹状体神经元的丢失。BDNF可以保护纹状体的易感性神经元,但是将BDNF用于治疗HD还存在许多困难。     

脑源性神经营养因子与中枢神经修复再生

神经营养因子在保护神经元存活并促进其突起生长发育过程中 ,常出现基因表达的时相变异 ,对不同种类的神经元有明显的作用选择性。脑源性神经营养因子 (BDNF)作为神经营养因子家族中的一员 ,广泛分布于大脑中 ,是一类可促进运动神经元、感觉神经元、基底节前脑胆碱能神经元、皮     

脑源性神经营养因子和脑缺血

有证据表明BDNF在脑缺血的保护中起重要作用。BDNF保护缺血引起的神经细胞损伤的机制为(1)稳定细胞内钙离子水平,减少兴奋性氨基酸引起的损伤;(2)拮抗NO介导的细胞毒作用;(3)调节自由基代谢;(4)对损伤神经元具有修复作用。     

BDNF基因修饰神经干细胞移植治疗脊髓损伤的实验研究

目的研究BDNF基因修饰神经干细胞移植对脊髓损伤后神经细胞凋亡的影响。方法采用电控大鼠脊髓损伤打击装置制作大鼠脊髓损伤模型。120只SD大鼠随机分为4组:假手术组(Sham组),脊髓损伤组(SCI组),神经干细胞组(NSC组),BDNF基因修饰神经干细胞组(NSC-BDNF组)。通过免疫组化法检测大鼠脊髓BDNF、Bax、Bcl-2的表达,流式细胞仪检测大鼠脊髓细胞凋亡率。结果NSC-BDNF组中BDNF免疫阳性细胞光密度值较NSC、SCI组增加明显(P<0.05),表达时间及表达高峰延长,且Bcl-2的表达较其他组在各个时间点上均增高(P<0.05),而Bax的表达较其他组在各个时间点上均降低(P<0.05),凋亡率亦明显低于NSC和SCI组(P<0.01)。结论BDNF基因修饰神经干细胞移植可引起BDNF在损伤脊髓内有效表达,且明显的促进了脊髓损伤后Bcl-2的高表达,抑制了Bax的表达,从而降低了神经细胞的凋亡率。     

高海拔地区骨髓源神经干细胞及BDNF联合移植对大鼠缺血再灌注模型的治疗

目的 探讨高海拔地区大鼠骨髓源神经干细胞(bone marrow mesenchymal stem cells-derived neural stem cells,BMSCs-NSCs)及脑源性神经生长因子(Brain-derived neurotrophic factor,BDNF)联合移植对大鼠脑缺血再灌注模型的疗效及其相关机理。方法 60只Wistar雄性大鼠,置西宁地区正常饲养,制备大鼠脑缺血-再灌注损伤模型; 模型制备完毕后立体定向下进行细胞移植治疗,将大鼠分为3组,即A组:大鼠骨髓源性神经球组(BMSCs-NSCs组,n=20); B组:大鼠骨髓源性神经球联合BDNF组(BMSCs-NSCs+BDNF组,n=20),注射大鼠骨髓源性神经球细胞的同时,联合注射100 ng BNDF; C组:对照组(仅注射DMEM/F12培养基,n=20); 术后对其神经功能进行评定,并于术后24 d取脑组织,行Nestin、GFAP、Map2免疫荧光检测。结果 细胞移植后第3 d,各组间神经功能评分无显著性差异; 细胞移植后第14 d BMSCs-NSCs+BDNF组神经功能评分显著优于BMSCs-NSCs组,BMSCs-NSCs组优于对照组; 免疫组化检测发现,BMSCs-NSCS+BDNF组Nestin、GFAP、Map2的IOD值均显著高于BMSCs-NSCs组; BMSCs-NSCs+BDNF组、BMSCs-NSCs组各检测指标水平均高于对照组; Nestin、GFAP、Map2的表达主要集聚于脑梗死灶与正常脑组织交界处。结论 在西宁地区联合移植大鼠骨髓源神经干细胞及BDNF可显著促进大鼠大脑中动脉闭塞再灌注损伤模型的神经功能恢复。     

BDNF abolishes the survival effect of NT-3 in axotomized Clarke neurons of adult rats

Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) have previously been shown to support survival and axonal regeneration in various types of neurons. Also, synergistic neuroprotective effects of these neurotrophins have been reported in descending rubrospinal neurons after cervical spinal cord injury (Novikova et al., [2000] Eur. J. Neurosci. 12:776-780). The present study investigates the effects of intrathecally delivered NT-3 and BDNF on the survival and atrophy of ascending spinocerebellar neurons of Clarke nucleus (CN) after cervical spinal cord injury in adult rats. At 8 weeks after cervical spinal cord hemisection, 40% of the axotomized CN neurons had been lost, and the remaining cells exhibited marked atrophy. Microglial activity was significantly increased in CN of the operated side. Intrathecal infusion of NT-3 for 8 weeks postoperatively resulted in 91% cell survival and a reduction in cell atrophy, but did not reduce microglial activity. In spite of the fact that the CN neurons expressed both TrkC and TrkB receptors, only NT-3 had a neuroprotective effect, whereas BDNF was ineffective. Furthermore, when a combination of BDNF and NT-3 was administered, the neuroprotective effect of NT-3 was lost. The present results indicate a therapeutic potential for NT-3 in the treatment of spinal cord injury, but also demonstrate that in certain neuronal populations the neuroprotection obtained by a combination of neurotrophic factors may be less than that of a single neurotrophin.     

Correlation of brain-derived neurotrophic factor to cognitive impairment following traumatic brain injury in rats

BACKGROUND: In vitro and in vivo studies have confirmed that brain-derived neurotrophic factor (BDNF) can promote survival and differentiation of cholinergic, dopaminergic and motor neurons, and axonal regeneration. BDNF has neuroprotective effects on the nervous system. OBJECTIVE: To explore changes in BDNF expression and cognitive function in rats after brain injury DESIGN, TIME AND SETTING: The neuropathology experiment was performed at the Second Research Room, Department of Neurosurgery, Fujian Medical University (China) from July 2007 to July 2008. MATERIALS: A total of 72 healthy, male, Sprague Dawley, rats were selected for this study. METHODS: Rat models of mild and moderate traumatic brain injury were created by percussion, according to Feeney's method (n = 24, each group). A bone window was made in rats from the sham operation group (n = 24), but no attack was conducted. MAIN OUTCOME MEASURES: At days 1,2, 4 and 7 following injury, BDNF expression in the rat frontal lobe cortex, hippocampus and basal forebrain was examined by immunohistochemistry (streptavidin-biotin-peroxidase complex method). Changes in rat cognitive function were assessed by the walking test, balance-beam test and memory function detection. RESULTS: Cognitive impairment was aggravated at day 2, and recovered to normal at days 3 and 7 in rats from the mild and moderate traumatic brain injury groups. BDNF expression in the rat frontal lobe cortex, hippocampus and basal forebrain was increased at 1 day, decreased at day 2, and then gradually increased in the mild and moderate traumatic brain injury groups. BDNF expression was greater in rats from the moderate traumatic brain injury group than in the sham operation and mild traumatic brain injury groups (P < 0.05). CONCLUSION: BDNF expression in the rat frontal lobe cortex, hippocampus and basal forebrain is correlated to cognitive impairment after traumatic brain injury. BDNF has a protective effect on cognitive function in rats following injury     

Brain-derived neurotrophic factor,stress and depression: A minireview

Brain-derived neurotrophic factor (BDNF) is a member of the nerve growth factor family, and is widely expressed in the adult mammalian brain. Besides its well known neuroprotective activity after traumatic brain injury the evidences regarding its activity dependent release by the pathophysiology of major depression are rapidly replicating. Considering the data that stress plays an important role by the development of depression which is characterized with prominent hippocampal cell death, as well as the well known neuroprotective effects of BDNF, we aimed to investigate the link between the BDNF, stress and depression. Thus we prepared a minireview in order to evaluate the neuroprotective role of BDNF by psychiatric disorders which are characterized with prominent neuronal cell death.     

Neuroprotective Role of Exogenous Brain-Derived Neurotrophic Factor in Hypoxia–Hypoglycemia-Induced Hippocampal Neuron Injury via Regulating Trkb/MiR134 Signaling

Hypoxic–ischemic brain injury is an important cause of neonatal mortality and morbidity. Brain-derived neurotrophic factor (BDNF) has been reported to play a neuroprotective role in hypoxic–ischemic brain injury; however, the specific effects and mechanism of BDNF on hypoxic–hypoglycemic hippocampal neuron injury remains unknown. The current study investigated the action of BDNF in regulating cerebral hypoxic-ischemic injury by simulating hippocampal neuron ischemia and hypoxia. We found that BDNF, p-Trkb, and miR-134 expression levels decreased, and that exogenous BDNF increased survival and reduced apoptosis in hypoxic–hypoglycemic hippocampal neurons. The results also show that BDNF inhibits MiR-134 expression by activating the TrkB pathway. Transfection with TrkB siRNA and pre-miR-134 abrogated the neuroprotective role of BDNF in hypoxic–hypoglycemic hippocampal neurons. Our results suggest that exogenous BDNF alleviates hypoxic–ischemic brain injury through the Trkb/MiR-134 pathway. These findings may help to identify a potential therapeutic agent for the treatment of hypoxic–ischemic brain injury.     

Cooperative roles of BDNF expression in neurons and Schwann cells are modulated by exercise to facilitate nerve regeneration

After peripheral nerve injury, neurotrophins play a key role in the regeneration of damaged axons that can be augmented by exercise, although the distinct roles played by neurons and Schwann cells are unclear. In this study, we evaluated the requirement for the neurotrophin, brain-derived neurotrophic factor (BDNF), in neurons and Schwann cells for the regeneration of peripheral axons after injury. Common fibular or tibial nerves in thy-1-YFP-H mice were cut bilaterally and repaired using a graft of the same nerve from transgenic mice lacking BDNF in Schwann cells (BDNF(-/-)) or wild-type mice (WT). Two weeks postrepair, axonal regeneration into BDNF(-/-) grafts was markedly less than WT grafts, emphasizing the importance of Schwann cell BDNF. Nerve regeneration was enhanced by treadmill training posttransection, regardless of the BDNF content of the nerve graft. We further tested the hypothesis that training-induced increases in BDNF in neurons allow regenerating axons to overcome a lack of BDNF expression in cells in the pathway through which they regenerate. Nerves in mice lacking BDNF in YFP(+) neurons (SLICK) were cut and repaired with BDNF(-/-) and WT nerves. SLICK axons lacking BDNF did not regenerate into grafts lacking Schwann cell BDNF. Treadmill training could not rescue the regeneration into BDNF(-/-) grafts if the neurons also lacked BDNF. Both Schwann cell- and neuron-derived BDNF are thus important for axon regeneration in cut peripheral nerves.     

A dose-dependent facilitation and inhibition of peripheral nerve regeneration by brain-derived neurotrophic factor

The time-dependent decline in the ability of motoneurons to regenerate their axons after axotomy is one of the principle contributing factors to poor functional recovery after peripheral nerve injury. A decline in neurotrophic support may be partially responsible for this effect. The up-regulation of BDNF after injury, both in denervated Schwann cells and in axotomized motoneurons, suggests its importance in motor axonal regeneration. In adult female Sprague-Dawley rats, we counted the number of freshly injured or chronically axotomized tibial motoneurons that had regenerated their axons 1 month after surgical suture to a freshly denervated common peroneal distal nerve stump. Motor axonal regeneration was evaluated by applying fluorescent retrograde neurotracers to the common peroneal nerve 20 mm distal to the injury site and counting the number of fluorescently labelled motoneurons in the T11-L1 region of the spinal cord. We report that low doses of BDNF (0.5-2 microg/day for 28 days) had no detectable effect on axonal regeneration after immediate nerve repair, but promoted axonal regeneration of motoneurons whose regenerative capacity was reduced by chronic axotomy 2 months prior to nerve resuture, completely reversing the negative effects of delayed nerve repair. In contrast, high doses of BDNF (12-20 microg/day for 28 days) significantly inhibited motor axonal regeneration, after both immediate nerve repair and nerve repair after chronic axotomy. The inhibitory actions of high dose BDNF could be reversed by functional blockade of p75 receptors, thus implicating these receptors as mediators of the inhibitory effects of high dose exogenous BDNF.     

Protection of corticospinal tract neurons after dorsal spinal cord transection and engraftment of olfactory ensheathing cells

Transplantation of olfactory ensheathing cells (OECs) into the damaged rat spinal cord leads to directed elongative axonal regeneration and improved functional outcome. OECs are known to produce a number of neurotrophic molecules. To explore the possibility that OECs are neuroprotective for injured corticospinal tract (CST) neurons, we transplanted OECs into the dorsal transected spinal cord (T9) and examined primary motor cortex (M1) to assess apoptosis and neuronal loss at 1 and 4 weeks post-transplantation. The number of apoptotic cortical neurons was reduced at 1 week, and the extent of neuronal loss was reduced at 4 weeks. Biochemical analysis indicated an increase in BDNF levels in the spinal cord injury zone after OEC transplantation at 1 week. The transplanted OECs associated longitudinally with axons at 4 weeks. Thus, OEC transplantation into the injured spinal cord has distant neuroprotective effects on descending cortical projection neurons.     

Endogenous BDNF is required for myelination and regeneration of injured sciatic nerve in rodents

Following a peripheral nerve injury, brain-derived neurotrophic factor (BDNF) and the p75 neurotrophin receptor are upregulated in Schwann cells of the Wallerian degenerating nerves. However, it is not known whether the endogenous BDNF is critical for the functions of Schwann cells and regeneration of injured nerve. Treatment with BDNF antibody was shown to retard the length of the regenerated nerve from injury site by 24%. Histological and ultrastructural examination showed that the number and density of myelinated axons in the distal side of the lesion in the antibody-treated mice was reduced by 83%. In the BDNF antibody-treated animals, there were only distorted and disorganized myelinated fibres in the injured nerve where abnormal Schwann cells and phagocytes were present. As a result of nerve degeneration in BDNF antibody-treated animals, subcellular organelles, such as mitochondria, disappeared or were disorganized and the laminal layers of the myelin sheath were loosened, separated or collapsed. Our in situ hybridization revealed that BDNF mRNA was expressed in Schwann cells in the distal segment of lesioned nerve and in the denervated muscle fibres. These results indicate that Schwann cells and muscle fibres may contribute to the sources of BDNF during regeneration and that the deprivation of endogenous BDNF results in an impairment in regeneration and myelination of regenerating axons. It is concluded that endogenous BDNF is required for peripheral nerve regeneration and remyelination after injury.     

hTerapeutic potential of brain-derived neurotrophic factor (BDNF) and a small molecular mimics of BDNF for traumatic brain injur y

Traumatic brain injury (TBI) is a major health problem worldwide. Following primary mechanical insults, a cascade of secondary injuries otfen leads to further neural tissue loss. hTus far there is no cure to rescue the damaged neural tissue. Current therapeutic strategies primarily target the secondary injuries focusing on neuroprotection and neuroregeneration. hTe neurotrophin brain-derived neurotrophic factor (BDNF) has signiifcant effect in both aspects, promoting neuronal survival, synaptic plasticity and neurogenesis. Recently, the lfavonoid 7,8-dihydroxylfavone (7,8-DHF), a small TrkB agonist that mimics BDNF function, has shown similar effects as BDNF in promoting neuronal survival and regeneration following TBI. Com-pared to BDNF, 7,8-DHF has a longer half-life and much smaller molecular size, capable of penetrating the blood-brain barrier, which makes it possible for non-invasive clinical application. In this review, we sum-marize functions of the BDNF/TrkB signaling pathway and studies examining the potential of BDNF and 7,8-DHF as a therapy for TBI.     

Therapeutic potential of brain-derived neurotrophic factor(BDNF) and a small molecular mimics of BDNF for traumatic brain injury

Traumatic brain injury(TBI) is a major health problem worldwide.Following primary mechanical insults,a cascade of secondary injuries often leads to further neural tissue loss.Thus far there is no cure to rescue the damaged neural tissue.Current therapeutic strategies primarily target the secondary injuries focusing on neuroprotection and neuroregeneration.The neurotrophin brain-derived neurotrophic factor(BDNF) has significant effect in both aspects,promoting neuronal survival,synaptic plasticity and neurogenesis.Recently,the flavonoid 7,8-dihydroxyflavone(7,8-DHF),a small Trk B agonist that mimics BDNF function,has shown similar effects as BDNF in promoting neuronal survival and regeneration following TBI.Compared to BDNF,7,8-DHF has a longer half-life and much smaller molecular size,capable of penetrating the blood-brain barrier,which makes it possible for non-invasive clinical application.In this review,we summarize functions of the BDNF/Trk B signaling pathway and studies examining the potential of BDNF and 7,8-DHF as a therapy for TBI.