下颌神经切断术后降钙素基因相关肽在大鼠三叉神经运动核内表达的变化(英文)

本研究目的是观察下颌神经切断术后降钙素基因相关肽(CGRP)在大鼠三叉神经运动核(Vmo)内表达的变化。采用免疫组织化学和双重免疫荧光组织化学标记技术,观察了下颌神经切断术后3、7、14、21、28、35和42d时Vmo内CGRP和胶质纤维酸性蛋白(GFAP)免疫阳性物质表达的变化,并采用图像分析法计数了Vmo内存活的CGRP阳性运动神经元的数量。结果显示:神经切断能够引起手术侧Vmo内CGRP样免疫阳性物质的表达在术后3天和35天时呈"驼峰式"上调,且对照侧也有显著变化;定量分析显示神经切断后手术侧Vmo内运动神经元的数量几乎没有明显减少,但运动神经元周围GFAP的表达却大量增加。以上结果提示:Vmo内CGRP表达的上调可能对受损运动神经元的急性应激反应和恢复均具有营养作用,且激活的星形胶质细胞可能参与了其过程。     

地塞米松对大鼠两种不同功能运动神经元群树突的影响

目的:探讨地塞米松对大鼠趾长伸肌(EDL,快肌)、比目鱼肌(SOL,慢肌)两种不同功能运动神经元(Mn)群树突的影响.方法:大鼠分为注射生理盐水的对照组和注射地塞米松的实验组以霍乱毒素B亚单位结合辣根过氧化物酶(CB-HRP)逆行标记两Mn群,TMB法显示两运动神经元群被标记的树突;以免疫印迹法显示EDL及SOL糖皮质激素受体含量的高低.结果:与对照组比,实验组EDL-Mn群脊髓外侧索白质树突明显增多,其表面积密度比对照组增加了29.89%,平均树突长度比对照组增加了92.54%;SOL-Mn群脊髓外侧索白质树突表面积密度与对照组相比差异无统计学意义,平均树突长度比对照组增加32.26%;EDL糖皮质激素受体与β-actin免疫印迹条带相对吸光度比值明显高于SOL,为SOL的2.5倍.结论:地塞米松明显改变大鼠EDL-Mn群的树突构筑,这很可能与EDL含较高的糖皮质激素受体有关.     

鞘内注射强啡肽A（1-17）对不同功能运动神经元群树突的影响

目的:探讨强啡肽A对大鼠趾长伸肌(EDL,快肌)、比目鱼肌(SOL,慢肌)两种不同功能运动神经元(Mn)群树突的影响。方法:采用脊髓蛛网膜下隙给予强啡肽A,以霍乱毒素B亚单位结合辣根过氧化酶(CB-HRP)逆行标记EDL-Mn、SOL-Mn群,Mesulam-TMB法显示两运动神经元群被标记的树突。结果:与相应对照组比,强啡肽A致一过性后肢瘫大鼠用药后1h,位于腰4~5脊髓节段腹角的EDL-Mn、SOL-Mn群树突的分布范围减小,尤以EDL-Mn群为甚;EDL-Mn平均树突长度比对照缩短59.5%,而SOL-Mn平均树突长度比对照缩短35.6%。永久性后肢瘫大鼠用药后3h,EDL-Mn群仅见胞体和近端树突;与之相比,SOL-Mn群仍保留有较长的树突和较广的分布范围。结论:强啡肽A致一过性后肢瘫大鼠两群运动神经元树突明显受累,且以EDL-Mn群为甚,其差别可能与两群运动神经元所接受强啡肽A信息传入不同有关。     

香草酸受体亚型Ⅰ与降钙素基因相关肽和植物凝集素结合位点在大鼠脊神经节内感觉神经元的共存

目的:研究香草酸受体亚型Ⅰ(VR1)在大鼠脊神经节(DRG)内感觉神经元的表达.及与降钙素基因相关肽(CGRP)、植物凝集素(IB4)结合位点的共存,为探讨VR1在伤害性感觉刺激信号传导中的作用提供形态学依据.方法:应用免疫荧光组织化学三标方法结合激光共聚焦扫描显微镜技术观察VR1与CGRP和IB4结合位点在DRG的分布及相互关系.结果:背根节内可见大量中、小型神经元胞体和神经纤维表达VR1,一群独特的VR1阳性特小神经元胞体(直径8～11 μm)呈荧光强阳性.荧光双标显示背根节内许多VR1阳性神经无与CGRP共存或结合IB4,而CGRP/IB4双标神经元数量稀少.有41.1%±3.2%VR1阳性细胞呈CGRP阳性,有54.9%±3.8%VR1阳性神经元结合IB4.VR1强荧光特小神经元胞体未见CGRP阳性标记或结合IB4.VR1/CGRP/IB4三标神经元数量较少,只有1.5%±1.1% VR1阳性神经元同时呈CGRP阳性并结合IB4.结论:背根节内可能存在VR1/CGRP与VR1/IB4两种不同的与伤害性刺激相关的VR1阳性神经元亚群.     

大鼠坐骨神经压榨损伤后早期降钙素基因相关肽的变化

目的：研究大鼠坐骨神经压榨损伤后早期降钙素基因相关肽(CGRP)的动态变化及与神经再生的关系。方法：SD大鼠坐骨神经压榨损伤后分别存活1d到21d,免疫组化技术观察CGRP分布和含量的变化。结果：(1)1d组神经CGRP大量堆积,压榨近端明显多于远端,随即下降,21d组基本消失。(2)1d组背根节、脊髓后角和前角CGRP开始增高,并分别在3～5d、5～7d和7d组达峰值,随后渐降,21d组脊髓前角CGRP阳性运动神经元仍明显高于假手术组和对照侧。结论：神经压榨损伤后CGRP表达变化呈明显的时空模式,可能参与了神经元保护并介导了损伤信号的传导。     

GDNF及HSV—GDNF对坐骨神经损伤大鼠脊髓运动神经元Bcl—2表达的影响

目的：观察胶质细胞源性神经营养因子（GDNF）及单纯疱疹病毒（HSV）载体介导的GDNF（HSV－GDNF）对坐骨神经损伤大鼠脊髓运动神经元了Bcl-2表达的影响。方法：分别取坐骨神经损伤后4d、7d和14d大鼠（）分成对照组、GDNF组和HSV－GDNF组）的腰段脊髓）L4－6）,行石蜡包埋,切片、;用抗Bcl-2抗血清进行免疫组织化学染色,观察Bcl-2免疫反应（Bcl-2－IR）神经元数目,并在图像分析仪Bcl-2－IR阳性神经元作光密度的色谱分析。结果：1．坐骨神经损伤后4d、7d,GDNF组和HSV－GDNF组损伤侧脊髓运动神经Bcl-2－IR阳性神经元的数量和平均光密度均明显高于对照组损伤测,2．坐骨神经损伤后14d时,对照组、GDNF组和HSV－GDNF组损伤侧脊髓运动神经元对Bcl-2的表达已无明显差别。结论GDNF与HSV－GDNF能够增强坐骨神经扣内务 大鼠脊髓运动神经元Bcl-2的表达,减少神经元的退化死亡。     

GDNF激活星形胶质细胞保护PD模型大鼠黑质多巴胺能神经元

探索在胶质细胞源性神经营养因子(GDNF)保护受损多巴胺(DA)能神经元过程中,星形胶质细胞的可能作用。成年大鼠右侧纹状体内注射6-羟多巴胺(6-OHDA)制备Parkinson病(PD)模型。动物模型右侧黑质内注射GDNF,于注射后第7、14和21d进行动物行为学分析,行黑质节段连续冠状石蜡切片,以抗酪氨酸羟化酶(TH)、胶质原纤维酸性蛋白(GFAP)抗体免疫组化染色,分别显示DA能神经元和激活的星形胶质细胞,光镜观察并进行细胞计数。用免疫印迹法分析注射后第21d黑质内TH、GFAP蛋白表达量,蛋白条带用图像处理仪扫描,LabWorks软件分析处理。结果显示:动物在注射GDNF后第21d,行为学功能出现显著性恢复;TH阳性神经元数量显著增加;在检测的各时间点均可观察到大量的GFAP阳性细胞;TH、GFAP的蛋白表达量显著高于对照组。以上结果提示,黑质内注射GDNF可能通过激活了的星形胶质细胞保护PD模型大鼠黑质DA能神经元。     

Tet-Off对GDNF重组腺相关病毒载体表达的调控作用

胶质细胞源性神经营养因子(GDNF)对多巴胺能神经元、运动神经元等都具有神经营养作用,是帕金森病等中枢神经系统退行性病变常见的治疗基因。以腺相关病毒(AAV)为载体将 GDNF 导入尾状核头部等功能核团能缓解帕金森病症状、促进多巴胺能神经元再生。本研究在 AAV 介导 GDNF 体内高效表达的基础上,在 GDNF 上游插入 Tet-Off 反式激活子及其反应元件启动子复合物, 从而降低转基因后可能的致瘤作用及其它潜在危险。     

GDNF及HSV-GDNF对体外培养大鼠脊髓运动神经元受损后凋亡的影响

目的　观察胶质细胞源性神经营养因子 (GDNF)及单纯疱疹病毒载体介导的 GDNF(HSV- GDNF)对体外培养的胎鼠脊髓运动神经元在划痕损伤后凋亡的影响。　方法　对培养 12 d神经元行划痕损伤 ,并将其分成 4组 (无血清对照组、血清组、HSV- GDNF组和 GDNF组 ) ,给予不同培养液 ,定期观察各组的运动神经元存活数。分别于划痕损伤第 4d和第 7d时对神经元作 TU NEL 染色 ,检测运动神经元凋亡数 ,并在图像分析仪上对凋亡神经元作平均光密度的色谱分析。　结果　各组内运动神经元存活数与培养时间成反比。从对照组、HSV- GDNF组到 GDNF组 ,运动神经元凋亡数和凋亡神经元的平均光密度均依次减少 ,但对照组和血清组、GDNF组和 HSV-GDNF组间的运动神经元凋亡数以及凋亡神经元平均光密度均无显著差异。　结论　 GDNF和 HSV- GDNF能挽救生长发育过程中受损的脊髓运动神经元的凋亡 ,对体外培养的受损脊髓运动神经元具有一定的保护作用。     

GDNF对局灶性脑缺血MRI及皮质和尾壳核神经干细胞增殖和分化的影响

观察胶质细胞源性神经营养因子(GDNF)对大鼠局灶性脑缺血磁共振成像(MRI)及皮质和尾壳核神经干细胞(NSCs)增殖和分化的影响,并探讨GDNF对内源性NSCs增殖分化的作用机制。制作右侧局灶性脑缺血模型,左侧脑室注射GDNF,5-溴脱氧尿核苷(BrdU)标记DNA合成期(S期)细胞,Y迷宫检测大鼠学习记忆能力,MRI观察脑部影像学变化,免疫组化法观察正常组、假手术组、缺血组、生理盐水组和GDNF组大鼠局灶性脑缺血90min后再灌注不同时间(3、7、14、21、28d)皮质和尾壳核内BrdU/nestin、BrdU/NeuN、BrdU/GFAP阳性双标细胞。GDNF组对学习记忆的恢复较模型组和生理盐水组明显;MRI检查T2WI上缺血区信号明显增高和轻微脑肿胀,GDNF组缺血后3d,缺血区出现小面积信号增高影,14d信号强度明显下降;GDNF组Br-dU/nestin双标细胞数明显增加;新生细胞分化结果显示28d时,GDNF组BrdU/NeuN(58.23%±15.30%)、BrdU/GFAP(11.29%±4.30%),与其它组相比均有显著性差异(P<0.05)。以上结果证实局灶性脑缺血激活皮质和尾壳核内的NSCs,而GDNF可促进内源性NSCs增殖、分化,从而促进学习记忆能力的恢复。     

Calcitonin gene-related peptide-like immunoreactivity in motoneuron pools innervating different hind limb muscles in the rat

The content of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) in motoneurons was studied in four motor pools supplying muscles in the rat hind limb subserving different types of motor activity. The motor pools were identified by retrograde labeling with horseradish peroxidase or fluorophore-conjugated dextran amines, which were injected into the soleus, tibialis anterior, lateral gastrocnemius, or abductor digiti minimi muscles. After processing for immunohistochemistry, a semiquantitative evaluation was carried out to estimate the proportion of strongly, intermediately, and weakly labeled motoneurons, as well as motoneurons totally lacking CGRP staining. This revealed a considerable diversity in the intensity of CGRP labeling even for motoneurons in the same motoneuron pool. Thus, strongly labeled cells, as well as cells devoid of CGRP label, were found in all four motoneuron pools. However, a difference was found in the distribution of motoneurons innervating muscles with a dominant composition of fast and slow motor units, respectively, in that a larger fraction of the latter type lacked CGRP-LI. Moreover, generally motoneurons in the small motor units of the abductor digiti minimi muscle displayed weaker staining, and a larger proportion of cells was totally devoid of CGRP-LI (16%) compared with larger motor units of the other three muscles (1–10%). Small-sized cells within the -motoneuron size range were weakly stained or, more frequently, totally devoid of CGRP label (50%) as compared to larger cells, presumably representing -motoneurons (1–16%). Five days after axotomy all four studied motoneuron pools displayed stronger CGRP labeling than corresponding unlesioned pools. However, a considerable variation in CGRP labeling persisted also among axotomized motoneurons. These results indicate that motoneurons normally display a great variation in CGRP-LI levels, but that motoneurons of small and slow-twitch motor units in general have lower levels than motoneurons of large and fast-twitch motor units, respectively. After axotomy, CGRP-LI increases in lesioned motoneuron pools compared with normal, but in a fraction of the axotomized motoneurons the increase seems to be discrete or even absent. The possible physiological implications of these findings are discussed.     

Quantitative analyses of expression of GDNF and neurotrophins during postnatal development in rat skeletal muscles

Neurotrophic factors are thought to be critically involved in formation and maintenance of the neuromuscular system. To know precise expression levels of these factors in the muscles during the postnatal period, we developed competitive RT-PCR and two-site enzyme immunoassay and quantitatively measured neurotrophic factors in the rat gastrocnemius and soleus muscles during the postnatal development. mRNAs of glial cell line-derived neurotrophic factor (GDNF) in the gastrocnemius and the soleus muscles were expressed in the highest amount among the neurotrophic factors at birth and dramatically decreased in the first 3 months, while GDNF proteins substantially existed at 3 months of age. Neurotrophin-3 and brain-derived neurotrophic factor in the gastrocnemius muscle kept constant expression in mRNA and protein during the postnatal period. In contrast, mRNA of neurotrophin-4 increased in the first 2 weeks. In the soleus muscles all the neurotrophic factor proteins increased with age for the first month, contrasting with their expressions in the gastrocnemius. The present results showed that GDNF is constitutively supplied to the neuromuscular junction (NMJ) during postnatal development and into adulthood, suggesting its importance in maintenance of the NMJ. Expression of other neurotrophins was also regulated independently during development possibly according to their own roles in the neuromuscular circuit.     

Cannabinoid 1 receptors are expressed by nerve growth factor- and glial cell-derived neurotrophic factor-responsive primary sensory neurones

Expression of the cannabinoid 1 (CB1) receptor and its regulation were studied in the different nociceptive and non-nociceptive sub-populations of cultured primary sensory neurones of adult rats. Bandairaea simplicifolia isolectin B4 (IB4) binding and calcitonin gene-related peptide (CGRP) immunostaining were used to distinguish between the glial cell-derived neurotrophic factor (GDNF)- and nerve growth factor (NGF)-responsive nociceptive and the non-nociceptive primary sensory neurones while a specific CB1 receptor antibody was used to study the expression of the CB1 receptor protein. About half of the total number of primary sensory neurones (47±3.2%) cultured for 1 day in the presence of both neurotrophic factors (50 ng/ml each) showed CB1 receptor-like immunostaining, whereas 21.8±3.3% and 32.7±5.6% of the neurones showed CGRP-like immunopositivity and IB4 binding, respectively. A proportion of the CB1 receptor-like immunopositive neurones was immunostained for CGRP (31.7±5%) and IB4 (48.2%±7.5), with a minimal (1%) co-expression of CGRP and IB4 binding. About a fifth of the CB1 receptor-like immunopositive neurones did not show either CGRP-like immunostaining or IB4 binding.

To find out whether CB1 receptor expression in nociceptive primary sensory neurones is regulated by GDNF or NGF, cultures were grown in the presence or absence of the neurotrophic factors for 7 days. Vanilloid receptor 1 (VR1) immunostaining was used as a control marker to monitor the effect of the neurotrophins. In cultures maintained in the presence of both factors (50 ng/ml each) 51±2.6% and 42.4±1.2% of the cells showed CB1 receptor-like and VR1-like immunostaining, respectively. In cultures grown for 7 days in the absence of either of the neurotrophic factors the relative number of VR1-like immunopositive cells decreased to 13.4±2.7%, whereas the relative number of CB1 receptor-like immunopositive neurones was unchanged (50.6±1.1%).

Our data suggest that the CB1 receptor is expressed in all of the three major sub-populations of primary sensory neurones and that the CB1 receptor expression is not regulated by either NGF or GDNF.     

Non-viral delivery of the gene for glial cell line-derived neurotrophic factor to mesenchymal stem cells in vitro via a collagen scaffold

Recent advances in tissue engineering that combine an extracellular matrix-like scaffold with therapeutic molecules, cells, DNA encoding therapeutic proteins, or a combination of the three hold promise for treating defects in the brain resulting from a penetrating injury or tumor resection. The purpose of this study was to investigate a porous sponge-like collagen scaffold for non-viral delivery of a plasmid encoding for glial cell line-derived neurotrophic factor (pGDNF) to rat marrow stromal stem cells (also referred to as mesenchymal stem cells, MSCs). The effects of the following parameters on GDNF synthesis in the three-dimensional (3D) constructs were evaluated and compared with results in monolayer culture: initial plasmid load (2-50 microg pGDNF), ratio of a lipid transfection reagent to plasmid (5:10), culture environment during the transfection (static and dynamic), and cell density. The level of gene expression in the collagen scaffolds achieved therapeutic levels that had previously been found to support survival of dopaminergic and trigeminal neurons in vitro. For the highest loading of plasmid (50 microg), the level of GDNF protein remained six to seven times above the control level after 2 weeks, a significant difference. Cell density in the scaffold was of importance for an early increase in GDNF production, with accumulated GDNF being approximately 60% greater after 9 days of culture when scaffolds were initially seeded with 2 million rat MSCs compared to 500,000 cells. Application of orbital shaking during the 4 h of transfection had a positive effect on the production of GDNF on 3D constructs but not of the same magnitude as reported in monolayer studies. Overall, these results demonstrate that the combination of tissue engineering and non-viral transfection of MSCs for the over-expression of GDNF is a promising approach for the long-term production of GDNF and probably for neurotrophic factors in general.     

Intramuscular nerve sprouting induced by CNTF is associated with increases in CGRP content in mouse motor nerve terminals

It is known that motor nerve terminal sprouting induced by either nerve injury or muscle paralysis is associated with an increase in calcitonin gene-related peptide (CGRP) content in the soma of motoneurons and in motor endplates. In the present study, CGRP-like immunoreactivity (CGRP-LI) was determined in motor endplates of animals in which nerve terminal sprouting had been induced by exogenous application of ciliary neurotrophic factor (CNTF). After 18 days of CNTF treatment we observed a significant increase in CGRP-LI in motor endplates. The results indicate that CGRP is upregulated when motor nerve outgrowth is induced, even in the absence of muscle paralysis or nerve lesion.     

胶质细胞源性神经营养因子基因修饰骨髓基质干细胞移植脑出血大鼠 神经营养因子的表达

背景：研究证实,细胞移植和神经营养因子相结合治疗脑损伤能促进大鼠神经功能的恢复。 目的：观察移植胶质细胞源性神经营养因子基因修饰的骨髓基质干细胞对大鼠脑出血后神经营养因子表达的影响。 方法：通过脑立体定位仪向SD大鼠脑尾壳核注射胶原酶和肝素建立脑出血动物模型,将48只模型鼠随机分为3组,骨髓基质干细胞组、胶质细胞源性神经营养因子/骨髓基质干细胞组和对照组于建模后第3天在脑出血部位分别移植骨髓基质干细胞、胶质细胞源性神经营养因子/骨髓基质干细胞以及生理盐水。 结果与结论：与对照组和骨髓基质干细胞组相比,胶质细胞源性神经营养因子/骨髓基质干细胞组大鼠神经功能恢复更好;与对照组相比,移植后1,2周其他2组各神经营养因子表达均显著增加(P < 0.05)。提示胶质细胞源性神经营养因子基因修饰的骨髓基质干细胞移植治疗脑出血大鼠比单纯骨髓基质干细胞有更好的神经保护作用。     

GDNF基因体内转染对大鼠面神经运动神经元的保护作用

目的：研究GDNF基因体内转染对大鼠面神经损伤后运动神经元的保护作用，为基因治疗周围神经损伤提供依据。方法：用脂质体介导pEGFP-GDNFcDNA基因转染受损面神经所支配的面肌，荧光显微镜和免疫组化法分别检测面肌和面神经运动神经元内GDNF的表达。计算损伤侧面神经元的存活率。结果：转染侧面肌细胞内有绿色荧光。面神经损伤后的第7、14、21和28d时间段，对照组面神经运动神经元的存活率分别为93．06％、76．06％、72．38％和70．69％，转染组分别为94．00％、84．00％、79．25％和78．06％。转染组面神经运动神经元GDNF的表达高于对照组。结论：面神经损伤后经脂质体介导GDNF基因体内转染靶器官后，对受损面神经运动神经元有保护作用。     

Effect of increased physical activity on growth and differentiation of regenerating rat soleus muscle

Our purpose was to determine the effect of physical exercise on growth and differentiation during regeneration of a slow-twitch muscle. Degeneration/regeneration of the left soleus muscles of Wistar female rats was induced by injection of a snake venom. Muscular differentiation was studied by monitoring the sequential expression of the various myosin heavy chain isoforms (MHCs). Rats were assigned to one of two groups: cage sedentary (n?=?14) or exercised (n?=?16). The exercise programme began 1-day post-injection and the rats ran 1?h/day on a motorized treadmill. Then, 9 and 25 days after venom treatment, the soleus MHC phenotype as determined by immunohistology, electrophoresis and immunoblotting, was studied. At 25 days the expression of MHCs by regenerating soleus was not changed by the increased level of physical activity (P? >?0.05). Exercised and sedentary regenerating muscles contained similar numbers of type-I fibres (100% of total fibres), levels of MHC-1 (85.4 and 89.5% of total MHCs), MHC-2a and M?/?HC-2x/d and their fibres expressed MHC-1 (100% of total fibres) and MHC-2 (45–50%) in the same way. Moreover, the masses of regenerating and nonregenerating soleus were significantly increased by physical exercise (P? 相似文献