大鼠脊髓全横断后神经营养素-3在红核表达的变化

目的:研究大鼠脊髓全横断后神经营养素-3(NT-3)在红核表达的变化,为进一步探索脊髓损伤后的再生修复机制和神经营养因子治疗脊髓损伤提供实验依据。方法:雄性SD大鼠36只随机分为3组,脊髓横断组:大鼠脊髓在胸10~11节段之间完全横断,按存活时间不同又分为术后1、3、7及14d组;假手术组(只行椎板切除术);正常对照组。动物到达存活时间点后,应用免疫组织化学ABC法和图像分析技术检测NT-3在红核的表达。结果:对照组和实验组红核有NT-3阳性细胞。脊髓全横断后红核NT-3的表达逐渐增高,于术后7d达高峰,后缓慢下降。结论:脊髓全横断后红核对NT-3的表达增加,内源性NT-3的增加可能有利于受损的红核内神经元的存活与再生。     

督脉电针对早期受损伤的脊髓神经营养素-3表达的影响

目的 观察督脉电针对早期受损伤的脊髓神经营养素-3(NT-3)表达的影响及其细胞定位.方法 成年雌性大鼠分为损伤组和电针加损伤组.全横断损伤两组大鼠的脊髓1d后,开始对电针+损伤组大鼠进行督脉电针,损伤组大鼠不做督脉电针.电针加损伤组在电针后1、3和7d时间点取出脊髓损伤区组织,损伤组也在相应时间点取出损伤区组织,用ELISA方法检测损伤区组织NT-3水平.再取电针后7d大鼠脊髓损伤区及其邻近组织切片做NT-3的免疫荧光组织化学双标染色.结果 电针加损伤组和损伤组的脊髓损伤区组织NT-3表达在时间上基本一致;在前3 d,NT-3水平是下降的,在后3 d,NT-3水平是增高的.但是,与损伤组相比,电针加损伤组的NT-3水平是明显增高(P＜0.05).免疫荧光双标染色结果显示,神经元、星形胶质细胞、少突胶质细胞和巨噬细胞都有NT-3的表达.结论 督脉电针可以促进受损伤早期的脊髓组织细胞合成和分泌内源性NT-3,这可能是督脉电针促进急性脊髓损伤修复的适宜微环境因素之一.     

大鼠面神经切断及修复后面神经核内STAT3的变化

目的：探讨面神经损伤及修复条件下，面神经核运动神经元STAT3 mRNA和蛋白的变化规律。方法：采用原位杂交和免疫组化染色法，观察成年大鼠面神经切断和即刻端端吻合后面神经核STAT3 mRNA和蛋白的时程变化。结果：面神经切断后损伤侧面神经核STAT3 mRNA信号的强度增加，在术后第21d和35d，面神经单纯切断组和面神经切断后端端吻合组间STAT3 mRNA信号的强度相差显著（P＜0．05）。结论：面神经损伤后，面神经核运动神经元内由STAT3介导的细胞因子的信号转导增加。在面神经无再生状态下，STAT3表达上调呈持续性；而在面神经再生状态下，随着面神经的成功再生，STAT3可逐渐恢复至正常水平。     

面神经颧支的解剖及临床应用进展

正面神经为混合性神经,包含4种纤维成分,其中特殊内脏运动纤维主要支配面肌运动,构成面神经颅外部分,面神经颧支是面神经五大分支之一,该神经主要支配颧大肌、颧小肌、眼轮匝肌等~([1])。熟悉面神经颧支的走行层次、分支及其与周围组织结构的关系,对于除皱术中避免面神经颧支损伤,以及在腮腺切除术和治疗面瘫时行跨面神经移植术中准确寻找颧支有重要意义。目前解剖学教材对颧支缺乏详细描述,国内外关于颧支的研究报道尚不多见且达成共识的方面较少~([2-3])。随着美容与整     

腺病毒介导的神经营养素-3基因能够在大鼠脊髓前角运动神经元内过表达神经营养素-3

目的观察腺病毒介导的神经营养素-3(NT-3)基因在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元的过表达。方法在坐骨神经内直接注射含有绿色荧光蛋白(GFP)基因(报告基因)的NT-3基因重组腺病毒(Ad-NT-3-GFP),7d后应用免疫荧光组织化学染色技术,在荧光显微镜下观察脊髓前角运动神经元的NT-3过表达。结果 GFP表达组(对照组)和NT-3加GFP表达组两组动物的L4和L5脊髓段横切片上,有绿色荧光蛋白阳性标记的细胞。在NT-3加GFP表达组,还可以观察到NT-3阳性标记的细胞,这种细胞能与绿色荧光蛋白阳性标记的细胞重合,是过表达NT-3的前角运动神经元。与GFP表达组的前角运动神经元形态比较,NT-3加GFP表达组的过表达NT-3的前角运动神经元呈现更富有分支的突起。结论腺病毒介导的NT-3基因能够在发出坐骨神经传出纤维的大鼠脊髓前角运动神经元内过表达NT-3,这为下一歩应用NT-3基因治疗策略修复实验性脊髓损伤提供初歩的实验资料。     

神经营养因子-3对大鼠脊髓半横断后背根神经节c-Jun表达的影响

目的：研究神经营养因子-3(NT-3)对脊髓半横断后背根神经节c-Jun表达的影响，探索NT-3促进脊髓修复的作用机制。方法：将实验动物分为：对照组，损伤组和NT-3注射组，应用荧光免疫组化法结合激光扫描共聚焦显微镜，观察各组背根神经节c-Jun的表达，并计数细胞核完整的神经元数目。结果：脊髓损伤后，背根神经节的细胞内c-Jun的表达上调；NT-3注射组脊髓损伤侧背根神经节神经元的c-Jun表达明显上调，背根神经节内细胞核呈完整状态的神经元数量明显增多。结论：(1)c-Jun在轴突损伤后表达上调。(2)NT-3对轴突损伤后的神经元有保护作用。(3)NT-3可能通过使c-Jun表达上调而发挥其促神经再生作用。     

神经营养素-3能够促进体外受损伤大鼠脊髓神经元的存活及其神经突起生长

目的探讨神经营养素-3(NT-3)对体外机械性损伤的大鼠脊髓神经元存活及其神经突起生长影响。方法将体外培养的大鼠脊髓神经元分为4组:正常组、对照组、20 ng/ml NT-3组和40 ng/ml NT-3组。培养4 d后,除正常组外,其余3组建立划痕损伤模型。在划痕损伤后,对照组不做处理,另外2组分别在培养液中加入20 ng/mlNT-3和40 ng/ml NT-3继续培养。直至培养第6 d,应用4%多聚甲醛固定4组细胞。固定后的细胞分别做转移酶介导的三磷酸脱氧鸟苷-生物素刻痕末端标记(TUNEL)、微管相关蛋白2(MAP2)和生长相关蛋白-43(GAP43)免疫荧光染色,检测划痕损伤的神经元凋亡及其神经突起生长情况。结果免疫荧光化学染色显示,与对照组相比,应用NT-3处理的2组可以显著降低划痕损伤后的脊髓神经元凋亡率,并促进其神经突起生长。尤其是40 ng/mlNT-3组的神经元凋亡率最低,其神经突起可穿过划痕损伤边界。结论 NT-3能够促进体外机械性损伤的大鼠脊髓神经元存活及其神经突起生长。     

灵芝孢子粉对戊四氮致痫大鼠脑组织GDNF与NT-3表达的影响

目的:观察和探讨灵芝孢子粉干预后戊四氮(PTZ)致痫大鼠皮质和海马区胶质细胞源性神经营养因子(GDNF)和神经营养因子-3(NT-3)的变化,进一步研究癫痫的发病机制及灵芝孢子粉的作用机制。方法:Wistar大鼠30只,随机分为正常对照组、癫痫模型组和灵芝孢子粉治疗组,每组10只。模型组和治疗组采用PTZ腹腔注射制作Wistar大鼠慢性点燃模型。实验后断头迅速取脑,采用免疫组化方法检测皮质和海马区GDNF和NT-3表达的变化;同时用Westernblotting检测海马各指标蛋白的变化。结果:灵芝孢子粉干预后,免疫组化显示:皮质和海马区GDNF和NT-3较癫痫模型组明显增加;Western blotting检测显示:海马中GDNF和NT-3表达较癫痫组显著增加。结论:灵芝孢子粉能够增强GDNF和NT-3的表达从而减轻癫痫发作给神经系统带来的损伤,所以灵芝孢子粉可能具有减轻痫性发作、保护神经元的作用。     

灵芝孢子对大鼠脊神经腹根切断后脊髓运动神经元存活及其NT-3、NOS表达的影响

目的探讨灵芝孢子（萌动激活赤灵芝孢子）对大鼠脊髓受损伤运动神经元存活和表达神经营养素-3（NT-3）及一氧化氮合酶（NOS）的影响.方法对单侧腹根切断后的大鼠胃饲不同剂量的灵芝孢子,计算受损伤运动神经元存活率;用免疫组织化学及原位杂交方法检测NT-3的表达;用酶组织化学方法检测NOS的活性.结果腹根切断后35 d,对照组运动神经元存活率为47.32%,灵芝孢子低、中、高剂量组的运动神经元存活率分别为67.11%、72.67%和81.67%;腹根切断后高剂量组存活的运动神经元NT-3和NOS的表达都高于对照组. 结论灵芝孢子促进大鼠脊髓前角受损伤的运动神经元存活,其存活率与用药剂量有关;灵芝孢子促进大鼠脊髓存活的运动神经元表达NT-3和NOS.     

应用Sihler氏染色研究人喉神经分支分布类型

采用Sihler氏染色,经过染色后,在立体显微解剖镜下、可清晰地观察到喉内肌肌肉神经支配类型及神经间的联系,结果:(1)喉返神经支配除环甲肌以外的所有喉内肌,环甲肌由喉上神经外支支配.(2)喉上神经内支不仅支配声门上粘膜,也支配杓间肌(包括的横肌和杓斜肌)和声门下粘膜.(3)在每侧杓间肌肉.喉返神经和喉上神经内支之间百多个吻合支.(4)喉返神经的杓间肌神经和环杓后肌神经之间常有交通支.     

Expression of alpha-cardiac and alpha-skeletal actin mRNAs in relation to innervation in regenerating and non-regenerating rat skeletal muscles.

The expression of alpha-cardiac and alpha-skeletal actin mRNA in regenerating muscle was examined. Changes in mRNA levels were analyzed in autografted extensor digitorum longus (EDL) muscles in rats using alpha-isoform specific synthetic oligonucleotides and beta-actin cDNA as probes. After autografting, the expression of alpha-cardiac actin mRNA was induced; concomitantly that of alpha-skeletal actin mRNA was reduced. The pattern of alpha-actin mRNA expression appeared to be similar to that seen in embryonic skeletal muscle. In order to evaluate the effects of innervation on alpha-actin mRNA expression in regenerating muscle, nerveless, standard, and nerve-intact autografted muscles were examined. More complete innervation facilitated the recovery of alpha-skeletal actin mRNA to control levels, but had little effect on the amount of alpha-cardiac actin mRNA. We found that regenerating muscle shows that embryonic pattern of alpha-actin mRNAs in the early stage and concluded that the recovery of alpha-skeletal actin mRNA expression to the adult pattern is influenced by innervation, while alpha-cardiac actin mRNA expression is nerve independent.     

Reinnervation of Müller's smooth muscle by atypical sympathetic pathways following neonatal ganglionectomy in the rat: structural and functional investigations of enhanced neuroplasticity

Müller's extraocular smooth muscle is reinnervated by sympathetic nerves following denervation by ipsilateral superior cervical ganglionectomy in neonates but not in older animals. Experiments were performed to determine: (1) the source and extent of reinnervation, (2) the role of impulse activity in sympathetic outgrowth and (3) the effects of reinnervation on smooth muscle maturation. Müller's muscles were evaluated structurally (muscle volume, catecholamine histochemistry, retrograde labeling of sympathetic neurons) and functionally (contractile responses to electrical stimulation of postganglionic innervation and adrenoceptor agonist) in control preparations and in muscles following neonatal ipsilateral superior ganglionectomy, ipsilateral decentralization, ipsilateral superior ganglionectomy combined with contralateral decentralization of chemical (guanethidine) sympathectomy. Fluorescent tracer injections of muscles in adult control rats labeled cells in the ipsilateral superior (98%) and middle cervical ganglia. Acute ipsilateral superior ganglionectomy produced complete degeneration of sympathetic innervation of Müller's muscle in neonatal and adult rats. In preparations denervated neonatally and maintained chronically, muscles were reinnervated by neurons in both the contralateral superior and ipsilateral middle cervical ganglia. The total number of neurons reinnervating the muscle was one half that of controls. Sectional density of innervation was 45% of control. Electrical stimulation of postganglionic axons in the contralateral pathway produced muscle contractions with a prolonged time course. Reinnervation alleviated, in part, deficits in muscle volume and contraction which occurred following sustained denervation by chemical sympathectomy. Decentralization decreased ipsilateral muscle volume but did not affect numbers of neurons projecting to or nerve density within the muscle. Stimulation frequencies required to produce a 50% maximum contraction were reduced in these preparations. Decentralization of the contralateral ganglion did not impede sprouting into the denervated muscle, as nerve density and number of labeled cells were comparable to muscles reinnervated by contralateral ganglia with intact preganglionic innervation. However, maximum contraction to electrical stimulation was reduced. Comparisons with ipsilaterally decentralized muscles revealed that increased stimulation frequencies were required for 50% maximum contraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

The expression of aquaporin-4 is regulated based on innervation in skeletal muscles

Aquaporin-4 (AQP4) is a selective water channel, which expresses on the plasma membrane of myofibers and regulates the osmotic pressure, energy metabolism and morphological changes in myofibers by modulating water transport across sarcolemma in skeletal muscles. Although the physiological roles of AQP4 have been gradually clarified in skeletal muscles, the regulatory mechanisms of AQP4 expression have been poorly understood in skeletal muscles. Recently, it was reported that the expression of AQP4 decreased in atrophied skeletal muscles following sciatic nerve transection, but not tail-suspension. Therefore, expecting that the nerve supply to myofibers would be one of the major regulatory factors regulating AQP4 expression in skeletal muscles, we investigated whether the expression patterns of AQP4 were changed in skeletal muscles by denervation and subsequent reinnervation. As a result, while the APQ4 expression levels were significantly decreased by sciatic nerve freezing-induced denervation, subsequently the expression levels of AQP4 were fully restored during reinnervation in skeletal muscles (p?<?0.05, respectively). On the other hand, the expression levels of α1-syntrophin and AQP1, which are respectively structural and functional related AQP4 factors, were stably maintained during the denervation and subsequent reinnervation. Therefore, the present study demonstrated that the expression of AQP4 may be regulated depending on the innervation to skeletal muscles. Moreover, AQP4 regulatory mechanisms may be fundamentally different to those of AQP1 in skeletal muscles.     

Repeated stimuli for axonal growth causes motoneuron death in adult rats: the effect of botulinum toxin followed by partial denervation

Axons of motoneurons to tibialis anterior and extensor digitorum longus muscles of adult rats were induced to sprout by injecting botulinum toxin into them, by partial denervation or by a combination of the two procedures. Ten weeks later, the number of motoneurons innervating the control and operated tibialis anterior and extensor digitorum longus muscles was established by retrograde labelling with horseradish peroxidase. In the same preparations, the motoneurons were also stained with a Nissl stain (gallocyanin) to reveal motoneurons in the sciatic pool. Examination of the spinal cords from animals treated with botulinum toxin showed that the number of retrogradely labelled cells and those stained with gallocyanin in the ventral horn on the treated compared to the control side was unchanged. In rats that had their L4 spinal nerve sectioned on one side, the number of retrogradely labelled cells on the operated side was 48+/-3% (n = 5) of that present in the control unoperated ventral horn. Thus, just over half the innervation was removed by cutting the L4 spinal nerve. Counts made from gallocyanin-stained sections showed that 94+/-4% (n = 5) of motoneurons were present in the ventral horn on the operated side. Thus, section of the L4 spinal nerve did not lead to any death of motoneurons. In rats that had their muscles injected with botulinum toxin three weeks prior to partial denervation, the number of retrogradely labelled cells was reduced from 48+/-3% (n = 5) to 35+/-4% (n = 5). Moreover, only 67+/-5% (n = 5) of motoneurons stained with gallocyanin, suggesting that a proportion of motoneurons died after this combined procedure. This result was supported by experiments in which motor unit numbers in extensor digitorum longus muscles were determined by measurements of stepwise increments of force in response to stimulation of the motor nerve with increasing stimulus intensity. In partially denervated extensor digitorum longus muscles, 16.6+/-0.7 (n = 5) motor units could be identified, and in animals treated with botulinum toxin prior to partial denervation only 13.3+/-0.9 (n = 3) motor units were present. Taken together, these results show that treatment with botulinum toxin followed by partial denervation causes motoneuron death in adult rats.     

Two factors responsible for the development of denervation hypersensitivity

1. Innervated adult skeletal muscle is sensitive to acetylcholine at the end-plate region only. After denervation the entire muscle membrane becomes chemosensitive. The period of greatest increase in sensitivity in rat soleus muscles following section of the sciatic nerve in the thigh is between 48 and 72 hr post-operatively.2. Direct electrical stimulation was found to prevent the onset of the development of denervation hypersensitivity during the first 2-3 days after nerve section. Thereafter, electrical stimulation only reduced the sensitivity of denervated muscles to acetylcholine (ACh).3. The period of greatest increase in sensitivity follows loss of transmission and degeneration of the nerve terminals. Once this degeneration is under way, electrical stimulation is no longer as effective in preventing the development of denervation hypersensitivity.4. Hypersensitivity is also seen in muscles on which a small piece of thread or degenerating nerve has been placed. Hypersensitivity following these procedures declines within a few days, unlike denervation hypersensitivity which persists until innervation is restored.5. The present results suggest that activity alone cannot prevent the development of hypersensitivity in the presence of degenerating nerve fibres, or muscle damage. Activity does however counteract increased sensitivity. It is suggested that two factors interact to produce denervation hypersensitivity; the presence of degenerating nerve tissue and concomitant cellular changes bring about changes in the muscle fibre membrane causing it to become hypersensitive; and the loss of muscle activity, resulting in the persistence of hypersensitivity until innervation is restored.     

Effect of thyroid hormones on acetylcholinesterase mRNA levels in the slow soleus and fast extensor digitorum longus muscles of the rat

In the rat, the level of acetylcholinesterase messenger RNA in the typical slow soleus muscles is only about 20-30% of that in the fast extensor digitorum longus muscles. The expression of contractile proteins in muscles is influenced by thyroid hormones and hyperthyroidism makes the slow soleus muscle faster. The influence of thyroid hormones on the levels of acetylcholinesterase messenger RNA level in the slow soleus and fast extensor digitorum longus muscle of the rat was studied in order to examine the effect of thyroid hormones on muscle acetylcholinesterase expression. Hyperthyroidism was induced in rats by daily thyroid hormone injection or thyroid hormone releasing tablet implantation. Hind-limb suspension was applied to produce muscle unloading. Muscle denervation or reinnervation was achieved by sciatic nerve transection or crush. Acetylcholinesterase messenger RNA levels were analyzed by Northern blots and evaluated densitometrically. Hyperthyroidism increased the levels of acetylcholinesterase messenger RNA in the slow soleus muscles close to the levels in the fast extensor digitorum longus. The effect was the same in the unloaded soleus muscles. Acetylcholinesterase expression increased also in the absence of innervation (denervation), in the presence of changed nerve activation pattern (reinnervation), and under enhanced tonic neural activation of the soleus muscle (electrical stimulation). However, the changes were substantially smaller than those observed in the control soleus muscles. Enhancement of acetylcholinesterase expression in the soleus muscles by the thyroid hormones is, therefore, at last in part due to hormonal effect on the muscle itself. On the contrary, increased level of the thyroid hormones had no influence on acetylcholinesterase expression in the normal fast extensor digitorum longus muscles. However, some enhancing influence was apparent whenever the total number of nerve-induced muscle activations per day in the extensor digitorum longus muscle was increased. Thyroid hormones seem to be an independent extrinsic factor of acetylcholinesterase regulation in the slow soleus muscle.     

Neurotrophin-3 attenuates galanin expression in the chronic constriction injury model of neuropathic pain

We have recently shown that exogenous neurotrophin-3 (NT-3) acts antagonistically to nerve growth factor (NGF) in regulation of nociceptor phenotype in intact neurons and suppresses thermal hyperalgesia and expression of molecules complicit in this behavioral response induced by chronic constriction injury (CCI) of the sciatic nerve. The present study examines whether there is a global influence of NT-3 in mitigating alterations in peptide and NGF receptor expression; molecules believed to also contribute to CCI-associated pain. Thus, the influence of NT-3 on phenotypic changes in dorsal root ganglion (DRG) neurons in rats coincident with CCI was examined using in situ hybridization. Seven days following injury, the incidence of expression of the neuropeptides galanin and pituitary adenylate cyclase-activating polypeptide (PACAP) was increased in L5 sensory neurons ipsilateral to the injury from 12% to 60% and 16% to 37% respectively, in addition to an increased level of expression. In contrast, there was no consistent significant change in tropomyosin-related kinase A (trkA) expression following CCI. Intrathecal infusion of NT-3 globally mitigated both the increased incidence and elevated levels of galanin messenger RNA (mRNA) expression observed following CCI, reducing the former from 60% to 39%. NT-3 infusion resulted in a limited reduction in the incidence and level of neuronal PACAP in medium to large size, but not small size, DRG neurons. NT-3 had no significant net effect on CCI-induced alterations in trkA mRNA expression.