脑源性神经营养因子干预腰椎管狭窄症大鼠神经元活性、疼痛及相关因子的变化

背景：研究发现,脑源性神经营养因子缺失会导致中枢运动结构的神经退行性病变,从而导致多种运动神经疾病的发生,而腰椎管狭窄症作为慢性进行性神经功能障碍症候群,脑源性神经营养因子可能成为治疗该病的有效靶点。目的：探究脑源性神经营养因子对腰椎管狭窄症大鼠神经元活性、疼痛及缺氧诱导因子1α/血管内皮生长因子的影响。方法：选取40只SPF级SD雄性大鼠,随机取30只建立腰椎管狭窄模型,剩余10只为正常组。建模成功后将大鼠随机分为模型组、神经生长因子组及脑源性神经营养因子组。神经生长因子组大鼠腹腔注射1 500 U的神经生长因子,脑源性神经营养因子组大鼠鞘内注射20μL 10 mg/L的脑源性神经营养因子,均1次/d,持续30 d;正常组、模型组同期灌胃同体积生理盐水。给药结束后,观察并记录大鼠运动功能及体表疼痛情况,CT检测腰椎管密度,神经干动作点位传导速度测定仪检测大鼠神经传导速度,TUNEL法检测脊髓组织神经元活性,免疫印迹法检测缺氧诱导因子1α/血管内皮生长因子蛋白表达。结果与结论：(1)与正常组比较,模型组大鼠平板运动距离、体表疼痛值、神经传导速度均明显降低(P <0.05);与模型...     

坐骨神经损伤大鼠模型鞘内移植神经干细胞后脊髓背角和背根神经节脑源性神经营养因子的表达

背景：坐骨神经损伤模型可测试伤害性的热刺激和机械刺激所引发的痛觉过敏及冷、触觉异常。 目的：观察坐骨神经损伤模型大鼠鞘内移植神经干细胞后脊髓背角和背根神经节脑源性神经营养因子的表达。 方法：72只SD大鼠随机均分为假手术组、对照组和实验组。对照组和实验组制作坐骨神经损伤模型,假手术组仅暴露坐骨神经,不结扎。分别于造模后第3,10天进行鞘内移植,实验组注入30 μL的神经干细胞悬液,空白组和对照组注入30 μL的细胞培养液。 结果与结论：与假手术组相比,对照组和实验组移植后3 d机械痛阈和热痛阈逐渐降低,至移植后7 d降低至最低点(P < 0.01),于移植后21 d恢复至移植前水平;实验组移植后7,14 d机械痛阈和热痛阈较对照组明显上升(P < 0.01)。与对照组相比,假手术组移植后7,14,21 d各组大鼠脑源性神经营养因子的表达呈低水平(P < 0.05);移植后14,21 d,实验组脑源性神经营养因子的表达量高于对照组(P < 0.05)。提示鞘内移植神经干细胞可提高脊髓背角和背根神经节中脑源性神经营养因子的表达。从而抑制了周围神经损伤产生的神经病理性疼痛。 关键词：脑源性神经营养因子;神经干细胞;慢性限制损伤;脊髓背角;背根神经节 doi:10.3969/j.issn.1673-8225.2012.10.031     

脂肪源性干细胞促进大鼠受损坐骨神经传导及脊髓脑源性神经营养因子和睫状神经营养因子的表达

目的:探讨脂肪源性干细胞(ADSCs)对坐骨神经损伤大鼠神经传导功能以及脊髓脑源性神经营养因子(BDNF)和睫状神经营养因子(CNTF)表达的影响。方法:将第4代ADSCs移植入脱细胞神经移植物(ANA)中,构建组织工程神经。大鼠随机分为正常组、杜氏改良Eagle培养基营养混合物F12(DMEM)组和ADSC组。DMEM组和ADSC组均建立坐骨神经损伤模型,后用相应的组织工程神经桥接损伤神经的断端。术后6周采用神经电生理记录仪检测各组大鼠坐骨神经传导速度和波幅,采用免疫荧光和Real-time PCR检测各组大鼠脊髓脑源性神经营养因子(BDNF)、睫状神经营养因子(CNTF)蛋白和mRNA的表达。结果:ADSC组大鼠坐骨神经传导速度、波幅和脊髓BDNF和CNTF蛋白及mRNA表达均显著高于DMEM组。结论:ADSCs可增加坐骨神经传导速度和波幅、上调脊髓BDNF和CNTF的表达。     

钠离子通道阻滞剂对背根神经节交感芽生的抑制作用

目的 研究神经损伤即刻给予钠离子通道阻滞剂布比卡因对交感芽生症状产生的抑制作用.方法 将慢性压迫性损伤(CCI)模型大鼠随机分为对照组和实验组.实验组在坐骨神经轻度结扎后即刻于神经损伤区周围包埋布比卡因粉剂或胶溶剂,而对照组不给药.观察实验组大鼠背根神经节(DRG)内交感芽生的改变,并观察两组大鼠在行为学上的差异.结果 实验组大鼠DRG内交感芽生明显减少.热痛过敏症状被抑制.结论 神经损伤后即刻在损伤区给予布比卡因可有效预防交感芽生的产生.     

以脑源性神经营养因子等干预视神经损伤大鼠视神经拉伸力学的特性分析

以拉伸力学性能指标研判以人脐血干细胞、脑源性神经营养因子干预视神经损伤动物模型的效果。以钳夹法制备视神经损伤大鼠模型,于视神经损伤大鼠模型造模7 d后,分别以人脐血干细胞、脑源性神经营养因子进行干预治疗,对各组大鼠于造模30 d后取出眶内段视神经进行组织形态观察和拉伸实验。视神经组织形态观察结果表明,视神经损伤动物模型以BDNF干预组一部分神经纤维排列较整齐,胶质细胞可见少量细胞空泡,一部分细胞核排列不规则,但视神经轴突直径改变不明显。视神经损伤动物模型以h UCBSC干预组视神经纤维排列较密集,胶质细胞核增多,多数轴突形态正常。各组动物视神经拉伸实验结果表明,视神经损伤动物模型组视神经的最大载荷、最大应力、最大应变、弹性限度载荷、弹性限度应力小于视神经损伤模型以h UCBSC、以BDNF干预组,差异显著(P0.05),视神经损伤模型以BDNF干预组的最大载荷、最大应力、最大应变、弹性限度载荷、弹性限度应力小于以h UCBSC干预组,差异显著(P0.05)。视神经损伤动物模型以BDNF和以h UCBSC干预后动物视神经的拉伸力学性能指标得到了显著提高。提示,视神经损伤模型大鼠通过BDNF、h UCBSC干预治疗后,具有明显的疗效。     

施万样细胞对大鼠脊神经节NGF和BDNF表达的影响

目的观察施万样细胞对坐骨神经损伤(sciatic nerve injury,SNI)大鼠脊神经节NGF和BDNF表达的影响,初步探讨施万样细胞对脊神经节的保护作用。方法先将脂肪源性干细胞(adipose-derived stem cells,ADSCs)诱导分化为施万样细胞并对后者进行鉴定,后将二者分别植入脱细胞神经移植物(ANA)中,构建组织工程神经。大鼠随机分为正常对照组、ADSC组和施万样细胞组。后两组建立SNI模型并用相应的组织工程神经桥接损伤的神经。术后4周采用Western Blot和Real-time PCR检测各组大鼠脊神经节神经生长因子(nerve growth factor,NGF)和脑源性神经营养因子(brainderived neurotrophic factor,BDNF)蛋白和m RNA的表达。结果 ADSCs能够诱导分化为施万样细胞并表达施万细胞标记物S100β和GFAP蛋白。施万样细胞组大鼠脊神经节内NGF和BDNF蛋白及m RNA表达量均高于ADSC组(P0.05)。结论施万样细胞可上调脊神经节NGF和BDNF的表达,对SNI所致的脊神经节内神经元损伤有保护作用。     

肉毒毒素A对大鼠慢性神经源性疼痛中P物质和炎性反应的影响

目的探索肉毒毒素A(Bo NT-A)对慢性神经源性疼痛的镇痛效应和作用机制。方法将大鼠随机分为对照组、假手术组、疼痛模型组(结扎左侧L5、L6脊神经,结扎3 d后于同侧足底皮下注射0.9%氯化钠溶液)、肉毒毒素干预组(结扎左侧L5、L6脊神经,结扎3 d后同侧足底皮下注射Bo NT-A 30 U/kg)。根据处死时间不同将各组分为1 d、3 d和1周(1 w)组。HE染色观察组织形态学变化,免疫组化检测P物质(SP)、白细胞介素6(IL-6)、肿瘤坏死因子(TNF-α)蛋白表达情况,原位杂交及实时定量PCR法测定其基因表达水平。结果 SNL后大鼠脊髓炎性细胞浸润,并随着结扎时间的增长浸润程度加重。与对照组比较,疼痛模型组1 d、3 d和1周时SP、IL-6和TNF-α的蛋白、mRNA表达均显著上升(P0.05);与疼痛模型组比较,肉毒毒素干预组1 d、3 d和1周时SP、IL-6和TNF-α的蛋白、mRNA表达均显著下降(P0.05)。结论 Bo NT-A对慢性神经源性疼痛的镇痛机制可能是通过抑制神经递质SP和炎性介质IL-6、TNF-α的表达实现的。     

大鼠侧脑室注射脂多糖选择性减少黑质脑源性神经营养因子的表达

目的:探讨脑内慢性炎症对大鼠黑质部位脑源性神经营养因子(BDNF)表达的影响以及是否具有选择性。方法:将脂多糖(LPS)单次注射入SD大鼠侧脑室,造成全脑慢性炎症帕金森病(PD)模型,在注射后的不同时间点分离出不同部位的脑组织并提取蛋白,通过Western Blot方法检测BDNF在黑质和皮层的表达。结果:LPS注射后1周时黑质中BDNF的蛋白表达显著低于对照组,2周和4周时无显著差异,从8周开始显著低于对照组并且一直持续到24周;而皮层中实验组BDNF的表达与对照组相比在2周、4周、12周和24周时显著升高。结论:脑内慢性炎症选择性、进行性减少黑质中BDNF的表达,提示可能与脑内慢性炎症选择性、进行性损伤黑质多巴胺能神经元相关。     

TRPM2在损伤性神经性疼痛过程中参与急性疼痛向慢性疼痛的转化

目的 探究TRPM2在损伤性神经性疼痛中的作用和可能机制.方法 建立坐骨神经慢性缩窄性损伤(CCI)大鼠模型,体外培养原代背根神经节(DRG)细胞并完成siRNA的转染,CCI大鼠早期(CCI术后1～4 d)或晚期(CCI术后7～10 d)每天给予阴性对照或siTRPM2处理.RT-PCR检测CCI大鼠DRG和脊髓中T...     

巨噬细胞迁移抑制因子抑制剂ISO-1对脑缺血再灌注大鼠的影响

目的:探讨巨噬细胞迁移抑制因子(MIF)对大鼠脑缺血再灌注损伤(I/R)后神经损伤及可能涉及的机制.方法:将42只雄性SD大鼠随机分为假手术组(sham)、缺血再灌注组(I/R)和MIF抑制剂ISO-1处理组(ISO-1).利用大脑中动脉阻塞(MCAO)制备大鼠I/R模型.ISO-1组大鼠于再灌注同时腹腔注射ISO-1...     

Upregulation of matrix metalloproteinase-9 dependent on hyaluronan synthesis after sciatic nerve injury

Using a rat chronic constriction injury (CCI) model, we analyzed the expression and activity of matrix metalloproteinase-9 (MMP-9) after sciatic nerve injury and its relationship with hyaluronan (HA). MMP-9 expression and activity were induced after the nerve injury. We found that CCI rats with oral administration of 4-methylunbelliferone (4-MU), which was a hyaluronan synthases inhibitor, exhibited reduced MMP-9 activity and mRNA expression compared with CCI rats that were not given 4-MU. MMP modulatory factors, such as TIMPs and uPA, did not change after the 4-MU was administered. This result indicated that the upregulation of MMP-9 after nerve injury in CCI rats was dependent on HA synthesis and was useful for the treatment of nerve injuries.     

1,8-桉叶素对背根神经节内P2X3受体介导神经病理痛的作用

目的:探讨1,8-桉叶素对大鼠背根神经节(DRG)神经元P2X3受体介导神经病理痛的作用。方法:建立大鼠坐骨神经慢性压迫性损伤模型(CCI)。SD大鼠随机分为假手术(Sham)组,坐骨神经慢性压迫性损伤(模型组,CCI)组、低剂量1,8-桉叶素治疗组、高剂量1,8-桉叶素治疗组、二甲亚砜对照组。检测大鼠术后7、14 d机械缩足反射(MWT)及热缩足反射潜伏期(TWL),观察大鼠行为学变化。免疫组织化学和原位杂交观察神经病理痛大鼠第4~5腰(L_(4-5))DRG神经元P2X3受体表达变化。结果:术后第7和14天,模型组大鼠MWT和TWL明显低于假手术组,低、高剂量治疗组较模型组明显升高,二甲亚砜组与模型组比较无差别;L_(4-5)DRG内P2X3受体表达模型组明显高于假手术组,低、高剂量治疗组较模型组均明显降低,二甲亚砜组与模型组比较无明显区别。结论:1,8-桉叶素抑制CCI大鼠L_(4-5)DRG神经元P2X3受体过表达,从而缓解神经病理性疼痛症状。     

T lymphocytes play a role in neuropathic pain following peripheral nerve injury in rats

A catastrophic consequence of peripheral nerve injury is the development of abnormal, chronic neuropathic pain. The inflammatory response at the injury site is believed to contribute to the generation and maintenance of such persistent pain. However, the physiological significance and potential contribution of T cells to neuropathic pain remains unclear. Here we show that T cells infiltrate injured sciatic nerves following chronic constriction injury (CCI), but not uninjured nerves. Congenitally athymic nude rats, which lack mature T cells, developed a significantly reduced mechanical allodynia and thermal hyperalgesia following CCI, compared with their heterozygous littermates. To understand further the role played by different T-cell subsets, we generated polarized populations of type 1 and type 2 T cells, with different cytokine secretion profiles, from spleens of sciatic nerve-injured heterozygous rats. Passive transfer of type 1 T cells, which produce proinflammatory cytokines, into nude rats enhanced the recipients' pain hypersensitivity to a level similar to that of heterozygous donor rats. In contrast, passive transfer of polarized type 2 T cells, which produce anti-inflammatory cytokines, into heterozygous rats modestly though significantly attenuated their pain hypersensitivity. Thus, injection of type 1 and type 2 T-cell subsets produces opposing effects on neuropathic pain. These findings suggest the modulation of the T-cell immune response as a potential target for the treatment of neuropathic pain.     

Antihyperalgesic and Anti-inflammatory Effects of Atorvastatin in Chronic Constriction Injury-Induced Neuropathic Pain in Rats

Atorvastatin is a 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitor used in treatment of hypercholesterolemia and prevention of coronary heart disease. The aim of this study is to investigate the antihyperalgesic and anti-inflammatory effects of atorvastatin (3, 10, and 30 mg/kg by oral gavages for 14 days) in chronic constriction injury (CCI) model of neuropathic pain in rats. CCI caused significant increase in tumor necrosis factor-α, interleukin 1 beta, prostaglandin E2, along with matrix metalloproteases (MMP-2) and nerve growth factor (NGF) levels in sciatic nerve and spinal cord concomitant with mechanical and thermal hyperalgesia, which were significantly reduced by oral administration of atorvastatin for 14 days as compared to CCI rats. Our study demonstrated that atorvastatin attenuates neuropathic pain through inhibition of cytokines, MMP-2, and NGF in sciatic nerve and spinal cord suggesting that atorvastatin could be an additional therapeutic strategy in management of neuropathic pain.     

Comparison of sympathetic sprouting in sensory ganglia in three animal models of neuropathic pain

Sympathetic postganglionic fibers sprout in the dorsal root ganglion (DRG) after peripheral nerve injury. Therefore, one possible contributing factor of sympathetic dependency of neuropathic pain is the extent of sympathetic sprouting in the DRG after peripheral nerve injury. The present study compared the extent of sympathetic sprouting in the DRG as well as in the injured peripheral nerve in three rat neuropathic pain models: (1) the chronic constriction injury model (CCI); (2) the partial sciatic nerve ligation injury model (PSI); and (3) the segmental spinal nerve ligation injury model (SSI). All three methods of peripheral nerve injury produced behavioral signs of ongoing and evoked pain with some differences in the magnitude of each pain component. The density of sympathetic fibers in the DRG was significantly higher at all examined postoperative times than controls in the SSI model, while it was somewhat higher than controls only at the last examined postoperative time (20 weeks) in the CCI and PSI models. Therefore, data suggest that, although sympathetic changes in the DRG may contribute to neuropathic pain syndromes in the SSI model, other mechanisms seem to be more important in the CCI and PSI models at early times following peripheral nerve injury.     

Analgesic effect of intrathecally γ-aminobutyric acid transporter-1 inhibitor NO-711 administrating on neuropathic pain in rats

To investigate the analgesic effect of intrathecally administered γ-aminobutyric acid (GABA) transporter-1 inhibitor NO-711 on the sciatic nerve chronic constriction injury (CCI) rats. 5 days after intrathecal catheter placement, neuropathic pain model was established by CCI of sciatic nerve on rats. Withdrawal thresholds for mechanical allodynia and latency for thermal hyperalgesia were measured in all animals. All rats operated upon for CCI displayed decreased withdrawal thresholds for mechanical allodynia and latency for thermal hyperalgesia, which has significant difference compared with sham groups. After intrathecal NO-711 administration, withdrawal thresholds and latency were significantly increased on CCI rats compared with control group after 1 day. The results show that GABA transporter-1 inhibitor could effectively develop analgesic effect in sciatic nerve CCI rats’ model.     

Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn after peripheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors

We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize 5HT and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for 5HT and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter γ-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1–8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered 5HT levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.     

Neuropathic pain in aged rats: behavioral responses and astrocytic activation

We used the Bennett and Xie (1988) model of chronic neuropathic pain to study the effect of age on thermal and tactile sensitivity and on astrocytic activation in the dorsal horn of the spinal cord after nerve injury. Fischer 344 FBNF1 hybrid rats in three age groups, 4-6, 14-16, and 24-26 months, were studied. Rats were either unligated (day 0, control) or the left sciatic nerve was loosely ligated to cause a chronic constriction injury (CCI). CCI causes a neuropathic pain condition characterized by tactile allodynia and thermal hyperalgesia. Rats were behaviorally assessed for tactile and thermal sensitivity of their ligated and unligated hind paws up to 35 days postligation. Rats were sacrificed before or at various days postligation, and activated astrocytes were identified at the L4-L5 levels of their spinal cords by use of an antibody to glial fibrillary acid protein (GFAP). The number of GFAP-ir astrocytes in the dorsal horn of the spinal cord in the control, uninjured condition decreased with age (P < or = 0.001) but increased after CCI in all three age groups. After CCI, astrocytic activation in the cord was less robust in aged rats than in younger ones (P < or = 0.01). Not all the CCI rats displayed hyperalgesia to touch and to heat. Rats with an increased sensitivity to heat had increased levels of GFAP-ir in their cords; however, rats with decreased thermal sensitivity also displayed increased GFAP-ir. Thus the presence of activated astrocytes was not correlated with a single behavioral manifestation of neuropathic pain.     

Intrathecally administered Sema3A protein attenuates neuropathic pain behavior in rats with chronic constriction injury of the sciatic nerve

Semaphorins, one of the repulsive axonal guidance factors during development, are produced under pathological conditions in adult animals. In the neuropathic pain state associated with peripheral nerve injury, synaptic reorganization occurs in spinal cord dorsal horn. In the present study, we investigated the roles of intrathecal administration of Sema3A, a secreted semaphorin, in the spinal cord of chronic constriction injury (CCI) model rat. Neuropilin 1 (NPR1) and Plexin A (PlexA), co-receptors of Sema3A, were expressed in the dorsal horn of na?ve rats. NPR1, and not PlexA, protein expression increased in the dorsal spinal cord of CCI rats. Recombinant Sema3A protein attenuated mechanical allodynia and heat hyperalgesia in CCI rats, whereas heat-inactivated Sema3A had no effect. Immunohistochemistry revealed that Sema3A partially restored the decrease of isolectin B4-positive unmyelinated nerve terminals in lamina II of the ipsilateral dorsal horn of CCI rats. Contrary to our expectations, Sema3A did not change the distribution of myelinated fibers in lamina II at 7 days after CCI. Those results suggested that the suppressive role for Sema3A in the development of neuropathic pain associated with peripheral nerve injury in adult rats, which seemed to be independent from prevention of the myelinated fiber sprouting into lamina II.     

Effects of repeated administered ghrelin on chronic constriction injury of the sciatic nerve in rats

Chronic constriction injury (CCI) is a peripheral mononeuropathic pain model that is caused by an injury to the peripheral nervous system and refractory to available conventional treatment. Mechanisms involved in neuropathic pain are still unclear. Previous studies reveal that proinflammatory cytokines contribute to CCI-induced peripheral nerve pathology. Ghrelin, a novel identified gastric peptide, has been shown to have antinociceptive activity and also anti-inflammatory properties by decreasing proinflammatory cytokines. Therefore, the aim of the present study was to investigate the effects of ghrelin on the CCI and its relationship with proinflammatory cytokines in rats. Wistar rats underwent sciatic nerve ligation to induce CCI fallowed by repeated ghrelin administrations (50 and 100 μg/kg i.p., once daily) for a period of 14 days. Mechanical hyperalgesia was assessed before surgery and at day 14 after CCI. TNF-α, IL-1β and IL-6 were measured in blood and spinal cord. The changes of sciatic nerve was assessed histologically by both light and electron microscopy. Ghrelin attenuated mechanical hyperalgesia, reduced spinal TNF-α and IL-1β levels and enhanced sciatic nerve injury with correlated morphometric recovery. These results indicate that the protective effect by ghrelin in the spinal cord is mediated through the suppression of TNF-α and IL-1β. Thus ghrelin may be a promising peptide in the management of neuropathic pain.