艾司西酞普兰对慢性应激大鼠海马脑源性神经营养因子基因外显子表达及DNA甲基化的影响

目的 探讨艾司西酞普兰对成年慢性应激大鼠海马脑源性神经营养因子(BDNF)基因不同外显子表达及DNA甲基化的影响.方法 以慢性不可预测温和应激(chronic unpredictablemild stress,CUMS)建立应激抑郁模型并予艾司西酞普兰干预.56只雄性Sprague-Dawley大鼠随机分为CUMS+水组、CUMS+药组、对照+水组及对照+药组,每组14只,以蔗糖水偏好试验评估大鼠抑郁样行为;模型建立第3周后分别检测上述各组大鼠海马BDNF基因第Ⅰ、Ⅱ、Ⅳ、Ⅵ外显子mRNA及BDNF总mRNA(第Ⅸ外显子)表达和第Ⅳ启动子区DNA甲基化水平.结果 (1)蔗糖水偏好试验:模型建立第2,3周,CUMS+水组[(34±21)％,(63±21)％]蔗糖水偏好均低于对照+水组[(67±15)％,(80±15)％],差异均有统计学意义(事后检验,P均＜0.05);而CUMS+药组[(58士19)％,(80±14)％]与对照+水组间的差异均无统计学意义(事后检验,P均＞0.05).(2)BDNF外显子表达:模型建立第3周,第Ⅳ外显子mRNA为BDNF总mRNA(第Ⅸ外显子)表达中的最主要者.CUMS+水组BDNF第Ⅳ及Ⅸ外显子mRNA水平[(4.64±0.65)×10-3,(5.73±0.79) ×10-3]均低于对照+水组[ (6.14±0.87)×10-3,(6.82±0.35)×10-3],差异均有统计学意义(事后检验,P均＜0.05);而CUMS+药组[(5.69±0.18)×10-3,(6.91±0.98)×10-3]与对照+水组间的差异均无统计学意义(事后检验,P均＞0.05).(3)DNA甲基化:各组大鼠海马BDNF第Ⅳ启动子区DNA均未发生甲基化.结论 艾司西酞普兰主要调节BDNF第Ⅳ外显子转录阻止CUMS成年大鼠海马的该基因表达下降,艾司西酞普兰第Ⅳ启动子区DNA甲基化无影响.     

高血压对慢性脑缺血大鼠海马和大脑皮质的脑源性神经营养因子表达的影响

目的 探讨高血压对慢性脑缺血大鼠海马及大脑皮质部位脑源性神经营养因子（Brain-derivedneurotrophic factor,BDNF）表达的影响。方法 使用正常血压的WKY大鼠以及有高血压的SHR大鼠制作双侧总颈动脉永久阻塞的慢性脑缺血模型。应用原位杂交及免疫组织化学染色观察BDNF在缺血后第1～4周的变化,H&E染色比较缺血后第4周脑梗死范围的大小。结果 慢性脑缺血后,在SHR大鼠海马CA1和大脑皮质,BDNF的mRNA及免疫染色密度在缺血后第1~4周皆有显著的减少（P <0.05）,蛋白质印迹（Western-blot）实验也呈现了相同的结果。而WKY大鼠,只在缺血后第1周有短暂的减少（P <0.05）。在第4周,HE染色显示SHR大鼠比WKY大鼠有较大范围的脑组织受损（[ 12.40±4.26）% vs（0.41±0.17）%,P =0.026]。结论 在慢性脑缺血的情况下,长期的高血压会加重脑损伤,并且影响BDNF的mRNA及蛋白质的表达,尤其在缺血耐受性低的海马CA1及大脑皮质部位。     

Investigation of the functional brain-derived neurotrophic factor gene variant Val66MET in migraine

Experimental studies and investigations of the cerebrospinal fluid in migraineurs have suggested an involvement of brain-derived neurotrophic factor (BDNF) in migraine pathophysiology. In a case-control study approach, the functional Val66MET polymorphism (rs6265) of the BDNF gene was investigated in 265 migraine patients and 153 controls. Genotype and allele frequencies did not differ between healthy subjects and migraineurs. A subgroup analysis for the occurrence of aura or clinical characteristics, including the number of attacks, did not reveal a positive association for the investigated polymorphism. Our data argue against a role of this well characterized BDNF gene variant as a risk factor in migraine.     

Granulocyte-colony stimulating factor in experimental stroke and its effects on infarct size and functional outcome: A systematic review

Background: Granulocyte-colony stimulating factor (G-CSF) shows promise as a treatment for stroke. This systematic review assesses G-CSF in experimental ischaemic stroke. Methods: Relevant studies were identified with searches of Medline, Embase and PubMed. Data were extracted on stroke lesion size, neurological outcome and quality, and analysed using Cochrane Review Manager using random effects models; results are expressed as standardised mean difference (SMD) and odds ratio (OR). Results: Data were included from 19 publications incorporating 666 animals. G-CSF reduced lesion size significantly in transient (SMD − 1.63, p <   0.00001) but not permanent (SMD − 1.56, p =   0.11) focal models of ischaemia. Lesion size was reduced at all doses and with treatment commenced within 4 h of transient ischaemia. Neurological deficit (SMD − 1.37, p = 0.0004) and limb placement (SMD − 1.88, p =   0.003) improved with G-CSF; however, locomotor activity (≥ 4 weeks post-ischaemia) was not (SMD 0.76, p =   0.35). Death (OR 0.27, p < 0.0001) was reduced with G-CSF. Median study quality was 4 (range 0-7/8); Egger's test suggested significant publication bias (p <   0.001). Conclusions: G-CSF significantly reduced lesion size in transient but not permanent models of ischaemic stroke. Motor impairment and death were also reduced. Further studies assessing dose response, administration time, length of ischaemia and long-term functional recovery are needed.     

Human bone marrow stromal cell treatment improves neurological functional recovery in EAE mice

We investigated the treatment of remitting-relapsing experimental autoimmune encephalomyelitis (EAE) in mice with human bone marrow stromal cells (hBMSCs). hBMSCs were injected intravenously into EAE mice upon onset of paresis. Neurological functional tests were scored daily by grading clinical signs (score 0-5). Immunohistochemistry was performed to measure the transplanted hBMSCs, cell proliferation (bromodeoxyuridine, BrdU), oligodendrocyte progenitor cells (NG2), oligodendrocytes (RIP), and brain-derived neurotrophic factor (BDNF). The maximum clinical score and the average clinical scores were significantly decreased in the hBMSC-transplanted mice compared to the phosphate-buffered-saline-treated EAE controls, indicating a significant improvement in function. Demyelination significantly decreased, and BrdU(+) and BDNF(+) cells significantly increased in the hBMSC-treated mice compared to controls. Some BrdU(+) cells were colocalized with NG2(+) and RIP(+) immunostaining. hBMSCs also significantly reduced the numbers of vessels containing inflammatory cell infiltration. These data indicate that hBMSC treatment improved functional recovery after EAE in mice, possibly, via reducing inflammatory infiltrates and demyelination areas, stimulating oligodendrogenesis, and by elevating BDNF expression.     

Regulation of brain-derived neurotrophic factor (BDNF) in the chronic unpredictable stress rat model and the effects of chronic antidepressant treatment

Chronic unpredictable stress (CUS) is a widely used animal model of depression. The present study was undertaken to investigate behavioral, physiological and molecular effects of CUS and/or chronic antidepressant treatment (venlafaxine or imipramine) in the same set of animals. Anhedonia, a core symptom of depression, was assessed by measuring consumption of a palatable solution. Exposure to CUS reduced intake of a palatable solution and this effect was prevented by chronic antidepressant treatment. Moreover, chronic antidepressant treatment decreased depressive-like behavior in a modified forced swim test in stressed rats. Present evidence suggests a role for brain-derived neurotrophic factor (BDNF) in depression. BDNF mRNA levels in the ventral and dorsal hippocampus were assessed by in situ hybridization. Exposure to CUS was not correlated with a decrease but rather with an increase in BDNF mRNA expression in both the dentate gyrus of the dorsal hippocampus and the CA3 region of the ventral hippocampus indicating that there is no simple link between depression-like behaviors per se and brain BDNF levels in rats. However, a significant increase in BDNF mRNA levels in the dentate gyrus of the dorsal hippocampus correlated with chronic antidepressant treatment emphasizing a role for BDNF in the mechanisms underlying antidepressant activity.     

Effects of chronic exercise and imipramine on mRNA for BDNF after olfactory bulbectomy in rat

We examined the effects of chronic activity wheel running and antidepressant treatment on brain-derived neurotrophic factor (BDNF) messenger RNA (mRNA) in multiple brain regions-hippocampal formation (HF), ventral tegmental area/substantia nigra (VTA/SN), nucleus accumbens (NAc), and piriform cortex (PFx)-after bilateral olfactory bulbectomy (OBX). Male, Long-Evans rats (n=72) underwent either sham or OBX surgery and were randomly divided into eight experimental groups in a 2 (sham vs. OBX) x 2 (sedentary vs. activity wheel)x2 (saline vs. imipramine) factorial design. Animals were killed after 21 days of treatment. Drug x exercise interaction effects were observed for HF (P=0.006-0.023) and VTA/SN (P=0.021); exercise increased BDNF mRNA in the saline treated animals but not in the imipramine treated animals. OBX did not affect BDNF mRNA in the HF or VTA/SN (P>0.05). BDNF mRNA levels in the PFx were not altered by exercise, drug, or OBX (P>0.05). These results suggest that the effect of exercise on BDNF mRNA extends beyond the HF to the mesolimbic ventral tegmental area and that the potentiation of BDNF mRNA by exercise and antidepressant pharmacotherapy, reported by other investigators, is time limited.     

Cognitive and functional outcome after intravenous recombinant tissue plasminogen activator treatment in patients with a first symptomatic brain infarct

Objective To examine whether intravenous recombinant tissue plasminogen activator (rt–PA) treatment given in the acute phase of ischaemic stroke has a favourable effect on cognitive and functional outcome at six months post–stroke. Methods The present study included 92 patients with a first–ever symptomatic infarct, of whom 25 (27%) were subjected to rt–PA treatment in the first three hours post–stroke. Multivariate logistic regression analyses adjusted for stroke severity, education, age, and sex were performed to examine whether rt–PA treatment influenced cognitive outcome (assessed with a neuropsychological examination covering 7 cognitive domains), basic ADL independence (modified Barthel Index ≥ 19), and instrumental ADL independence (Frenchay Activities Index ≥ 15) after six months. Results The adjusted odds ratio for intact cognition was 1.0 (95% CI 0.2 to 4.3), that for basic ADL outcome 13.5 (95 % CI 1.4 to 129.4) and for instrumental ADL 7.1 (95 % CI 1.2 to 42.2). Conclusion Our findings suggest that rt–PA treatment is associated with a favourable basic and instrumental ADL outcome, but not with a beneficial cognitive outcome after 6 months.     

脑室内灌注神经肽Y对戊四氮致痫大鼠海马结构内脑源性神经营养因子表达的影响

目的 :探讨脑室内注入神经肽 Y(Neuropeptide Y,NPY)对戊四氮 (PTZ)致痫大鼠海马结构内脑源性神经营养因子 (Brain- derived Neurotrophic Factor,BDNF)表达的影响。方法 :将 18只健康雄性 Wistar大鼠随机分为 NPY实验组 (n=10 )和对照组 (n=8)。 NPY实验组 ,于 PTZ造模前给予侧脑室注射 NPY(6 nmol/ 10μl) ,对照组给予等容量生理盐水。侧脑室注射后 5分钟 ,腹腔注射 PTZ(6 0 mg/ kg) ,观察痫性发作持续时间并于痫性发作后 2小时处死动物。用免疫组化方法观察海马齿状回 (DG)和 CA1区 BDNF的免疫反应性。结果 :NPY实验组痫性发作时间短于对照组 (P<0 .0 0 1)。实验组 DG颗粒细胞层、分子层的 BDNF免疫反应性高于对照组相应各层 ,各组比较有显著性差异。实验组CA1区锥体细胞层、放射层及腔隙层免疫反应性均高于对照组相应各层 ,各组比较有显著性差异。实验组 DG分子层和 CA1区放射层及腔隙层免疫反应性分别高于颗粒细胞层和锥体细胞层免疫反应性 ,各组比较有显著性差异。 CA1区分子层未见 BDNF免疫反应性表达。结论 :脑室内注射 NPY可缩短痫性发作时间 ,并促进致痫大鼠海马内 BDNF的表达。     

Release of immunoreactive brain-derived neurotrophic factor in the spinal cord of the rat following sciatic nerve transection

Using the antibody microprobe method, the sites of spinal release of immunoreactive brain-derived neurotrophic factor (BDNF) was studied in normal rats, and rats with prior sciatic nerve transection. In normal rats, a significant basal release of immunoreactive BDNF was found in the superficial dorsal horn. Following sciatic nerve transection (performed 14 days previously), release of BDNF was found throughout the whole of the dorsal horn, extending into deeper laminae. Electrical stimulation of the ipsilateral sciatic nerve at a strength adequate to excite either A fibres (20 Hz at 2x threshold voltage) or A and C fibres (2 Hz at 20x threshold voltage) did not alter the basal release of immunoreactive BDNF in normal or in nerve-injured rats. The results suggest that BDNF is released from the central terminals of primary afferent fibres, but such release is not solely dependent upon action potential invasion of these terminals. The increased extent of release following nerve transection is consistent with the hypothesis that BDNF plays a role in the central response to peripheral nerve injury.     

基因重组BDNF蛋白在大肠杆菌中的表达纯化与功能鉴定

目的构建脑源性神经营养因子（BDNF）基因的原核表达载体，并在大肠杆菌中进行表达，以获取高产量、低成本、高纯度且具有生物学活性的BDNF蛋白。方法以人的全长BDNF cDNA为模板，用PCR方法扩增成熟区BDNF的cDNA，应用基因重组技术将人BDNF cDNA克隆到质粒pET-30a（＋）中，进行限制性内切酶酶切分析和DNA测序鉴定。将重组质粒转化大肠杆菌BL21（DE3）LysS，经IPTG诱导表达后，用Ni-NTA亲和层析纯化获取蛋白，用SDS-PAGE和western blot方法鉴别，噻唑蓝（MTT）法检测重组蛋白对PC12细胞增殖的影响。结果扩增出的人BDNF cDNA片段克隆进了原核表达载体，经酶切和核酸测序鉴定，得到了正确的重组质粒pET-BDNF，并在大肠杆菌中获得了表达。纯化后的蛋白经考马氏亮蓝染色呈单一条带；用抗BDNF的抗体进行western blot分析证明目的蛋白获得了表达。基因重组BDNF蛋白能够促进PC12细胞增殖。结论本研究成功构建了表达基因重组人BDNF的原核表达载体，基因重组人BDNF蛋白在大肠杆菌中获得了表达和纯化，所获得的基因重组蛋白具有较好的生物学活性。     

The roles of BDNF in the pathophysiology of major depression and in antidepressant treatment

Neurotrophic factors are critical regulators of the formation and plasticity of neuronal networks. Brain-derived neurotrophic factor (BDNF) is abundant in the brain and periphery, and is found in both human serum and plasma. Animal studies have demonstrated that stress reduces BDNF expression or activity in the hippocampus and that this reduction can be prevented by treatment with antidepressant drugs. A similar change in BDNF activity occurs in the brain of patients with major depression disorder (MDD). Recently, clinical studies have indicated that serum or plasma BDNF levels are decreased in untreated MDD patients. Antidepressant treatment for at least four weeks can restore the decreased BDNF function up to the normal value. Therefore, MDD is associated with impaired neuronal plasticity. Suicidal behavior can be a consequence of severe impaired neuronal plasticity in the brain. Antidepressant treatment promotes increased BDNF activity as well as several forms of neuronal plasticity, including neurogenesis, synaptogenesis and neuronal maturation. BDNF could also play an important role in the modulation of neuronal networks. Such a neuronal plastic change can positively influence mood or recover depressed mood. These alterations of BDNF levels or neuronal plasticity in MDD patients before and after antidepressant treatment can be measured through the examination of serum or plasma BDNF concentrations. BDNF levels can therefore be useful markers for clinical response or improvement of depressive symptoms, but they are not diagnostic markers of major depression.     

The response of human and rat fetal ventral mesencephalon in culture to the brain-derived neurotrophic factor treatment

Brain-derived neurotrophic factor (BDNF) has been shown to increase the survival of dopaminergic neurons in rodent mesencephalic cultures. The mRNAs of BDNF and trk B receptor have been found to be expressed in the substantia nigra of rat. In this study, the action of BDNF was studied on the survival and transmitter-specific differentiation of dopaminergic neurons of fetal human CNS aged 9–10-week in vitro. Dopaminergic neuron viability and phenotypic expression were monitored by tyrosine hydroxylase (TH) immunohistochemistry and measurement of dopamine (DA) content with HPLC, respectively. After seven days of treatment with BDNF there were 2.2-fold greater number of TH⁺ neurons surviving than in untreated cultures. Although very low levels of DA were detectable in human tissue, considerable amounts of DA was found in the culture medium from around 13 days in vitro (DIV), indicating that DA in human fetal tissue tended to be synthesised and released into the incubation medium more readily than from cultured rat fetal tissue during the same period. The content of DA in the BDNF-treated cultures was approximately double that of untreated cultures after 7 days. In rat fetal tissue, the capacity of each TH⁺ neuron to produce DA was not changed in the BDNF-treated cultures (7 DIV) compared with control cultures, suggesting that BDNF does not up-regulate the production of DA but rather acts to reduce cell death rates. Ciliary neurotrophic factor (CNTF) treatment of rat mesencephalic culture failed to improve the period of survival of fetal dopaminergic neurons and had no effect on the production of DA in cultures. Taken together, our results suggest that BDNF has potent trophic effect on both rat and human fetal mesencephalic dopaminergic neurons in culture and has a potential application in the treatment of Parkinson's disease.     

银杏叶提取物对脑缺血大鼠脑源性神经营养因子的影响

目的　观察银杏叶提取物 (GBE)对局灶脑缺血大鼠脑源性神经营养因子 (BDNF)表达的影响 ,探讨GBE与缺血损伤神经元可塑性的关系。方法　制作大鼠大脑中动脉闭塞 (MCAO)模型 ,应用免疫组化方法观察不同缺血时间 GBE治疗组及脑缺血组 BDNF阳性细胞数 ,并进行图像分析。结果　坏死灶中心区 GBE组及缺血组BDNF阳性神经元均消失 ,但在坏死灶周围区 ,两组 BDNF阳性细胞均显著增加。两组细胞形态无明显不同 ,但GBE治疗组阳性细胞数又显著高于相应缺血对照组。结论　银杏叶提取物可提高大鼠局灶脑缺血半暗带区 BDNF的表达水平 ,促进神经元的修复及重塑。     

Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord

We have investigated the effects of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) on the intraspinal regeneration of anterogradely labeled axotomized ascending primary sensory fibers in the adult rat. These fibers were allowed to grow across a predegenerated peripheral nerve graft and back into the thoracic spinal cord. In control animals that had been infused with vehicle for two weeks into the dorsal column, 3 mm rostral to the nerve graft, essentially no fibers had extended from the nerve graft back into the spinal cord. The number of sensory fibers in the rostral end of the nerve graft was not significantly different between control and neurotrophin-infused animals. With infusion of NGF, 37+/-2% of the fibers at the rostral end of the graft had grown up to 0.5 mm into the dorsal column white matter, 30+/-2% up to 1 mm, 19+/-3% up to 2 mm and 8+/-2% up to 3 mm, i.e., the infusion site. With infusion of NT-3, sensory fiber outgrowth was similar to that seen with NGF, but with BDNF fewer fibers reached farther distances into the cord. Infusion of a mixture of all three neurotrophins did not increase the number of regenerating sensory fibers above that seen after infusion of the individual neurotrophins. These findings suggest that injured ascending sensory axons are responsive to all three neurotrophins and confirm our previous findings that neurotrophic factors can promote regeneration in the adult central nervous system.     

Involvement of a spinal brain-derived neurotrophic factor/full-length TrkB pathway in the development of nerve injury-induced thermal hyperalgesia in mice

Partial sciatic nerve ligation in mice caused a marked and persistent decrease in the latency of paw withdrawal from a thermal stimulus only on the ipsilateral side. This thermal hyperalgesia was abolished by repeated intrathecal pretreatment with a specific antibody to brain-derived neurotrophic factor (BDNF), but not neurotrophin-4, just before and after the nerve ligation. These results provide direct evidence that BDNF within the spinal cord may contribute to the development of thermal hyperalgesia caused by nerve injury in mice. We previously reported that protein level of full-length TrkB, which contains the cytoplasmic protein tyrosine kinase domain, were clearly increased on the ipsilateral side of spinal cord membranes obtained from sciatic nerve-ligated mice. In the present study, we further demonstrated that the increased in the protein level of full-length TrkB is completely reversed by concomitant intrathecal injection of BDNF antibody. Furthermore, thermal hyperalgesia induced by nerve ligation was completely suppressed by repeated intrathecal injection of a specific antibody to full-length TrkB and an inhibitor of the protein tyrosine kinase activity for the neurotrophin receptor, K-252a. However, repeated intrathecal injection of a specific antibody to truncated TrkB, which lacks the cytoplasmic protein tyrosine kinase domain, failed to reverse thermal hyperalgesia observed in nerve-ligated mice. These findings suggest the possibility that the binding of BDNF to full-length TrkB and subsequent its activation may play a critical role in the development of neuropathic pain-like thermal hyperalgesia induced by nerve injury in mice.     

A functional polymorphism of the brain derived neurotrophic factor gene and cortical anatomy in autism spectrum disorder

Autism Spectrum Disorder (ASD) is associated with both (i) post-mortem and neuroimaging evidence of abnormal cortical development, and (ii) altered signalling in Brain Derived Neurotrophic Factor (BDNF) pathways - which regulate neuroproliferative and neuroplastic processes. In healthy controls genotype at a single nucleotide polymorphism that alters BDNF signalling (Val66met) has been related to regional cortical volume. It is not known however if this influence on brain development is intact in ASD. Therefore we compared the relationship between genotype and cortical anatomy (as measured using in vivo Magnetic Resonance Imaging) in 41 people with ASD and 30 healthy controls. We measured cortical volume, and its two sole determinants - cortical thickness and surface area - which reflect differing neurodevelopmental processes. We found "Group-by-Genotype" interactions for cortical volume in medial (caudal anterior cingulate, posterior cingulate) and lateral (rostral middle, lateral orbitofrontal, pars orbitalis and pars triangularis) frontal cortices. Furthermore, within (only) these regions "Group-by-Genotype" interactions were also found for surface area. No effects were found for cortical thickness in any region. Our preliminary findings suggest that people with ASD have differences from controls in the relationship between BDNF val66met genotype and regional (especially frontal) cortical volume and surface area, but not cortical thickness. Therefore alterations in the relationship between BDNF val66met genotype and surface area in ASD may drive the findings for volume. If correct, this suggests ASD is associated with a distorted relationship between BDNF val66met genotype and the determinants of regional cortical surface area - gyrification and/or sulcal positioning.