大鼠下丘脑催产素及加压素样神经元与脑啡肽样传入纤维末梢的关系

用免疫组织化学ABC双标记法,显示大鼠下丘脑各大细胞神经分泌核团中催产素样免疫反应(OT-li)、加压素样免疫反应(VP-li)神经元、亮-脑啡肽样免疫反应(L-ENK-li)和甲-脑啡肽样免疫反应(M-ENK-li)纤维末梢,并对不同神经分泌核团内脑啡肽样传入纤维末梢与OT-li及VP-li神经元的关系加以分析。结果发现,L-ENK-li及M-ENK-li纤维末梢在下丘脑各神经分泌核团中,与OT-li及VP-li神经元均有一定程度的接触关系。L-ENK-li与M-ENK-li纤维末梢在第三脑室周、前连合核、背内侧和背外侧副核、穹窿前和后核的OT-li及VP-li神经元周围最密集,在视上核、室旁核次之,在血管周细胞群内的OT-li及VP-li神经元周围密度较低。这些神经分泌性核团内L-ENK-li纤维末梢,均较M-ENK-li纤维末梢密集。表明脑啡肽能传入纤维末梢,与视上核、室旁核及各神经分泌副核(除血管周细胞群外)中的OT-li及VP-li神经元,均有不同程度的接触关系。因此,脑啡肽能传入纤维末梢可能在大细胞神经分泌系统OT-li及VP-li神经元分泌活动的调节中,起较广泛的重要作用。     

不同神经示踪剂组合对大鼠脊髓运动神经元的逆行标记效率比较

目的:探讨神经示踪剂荧光金(FG)、真蓝(TB)和荧光红(FR)两两组合对脊髓运动神经元的标记效率差异,为再生神经重支配准确性研究奠定基础.方法;采用大鼠胫神经示踪模型,采取神经内注射与神经横断后近侧断端浸泡(20 min)2种方式,分别对FG、TB和FR的两两组合进行示踪试验.示踪术后5d,取脊髓腰膨大段冷冻纵切,共聚焦显微镜进行显微成像和计数.结果:FG联合TB示踪标记的运动神经元数量最多,其次为FG联合FR,而FR联合TB组标记细胞数最少,双标比例也最小.神经断端浸泡方式使用示踪剂时标记效率仅为神经内注射的2/3左右.结论:FG联合TB以及FG联合FR示踪对脊髓运动神经元的标记效果较好,且神经内注射使用示踪剂效果优于持续20 min的神经断端浸泡.     

Clenbuterol，β_2-肾上腺素能受体激动剂促进成年大鼠脊髓半横切后红核脊髓束轴突再生和运动功能的恢复

目的研究Clenbuterol对于促进脊髓半横切(SCI)后轴突再生和运动功能恢复的功效。方法成年Wistar大鼠(n=10-12)接受硬脊膜内的第7颈段脊髓单侧半横切显微手术。Clenbuterol以9 mg/L的剂量加入饮用水中,而对照组未给予任何治疗。采用Cylinder试验和患肢抓力试验观察运动功能的恢复状况。行为学观察期(6周)结束后,大鼠接受第二胸段脊髓处椎板切开术,4%Fluorogold微量注射进入红核脊髓束,一周后被处死。结果运动功能逐渐恢复,从SCI后第3周开始,Clenbuterol治疗组显著优于对照组(最终患肢抓力:403.45±19.82g vs 335.13±13.48g,p<0.05);在Cylinder试验中,双侧前肢共同使用触壁的百分比,Clenbuterol组显著高于对照组(43.39%±5.85%vs 19.42%±6.61%,p<0.05)。Clenbuterol治疗显著地降低了SCI后继发性组织损伤,提高了pCREB阳性细胞核在整个SCI点的表达水平。与对照组相比,Clenbuterol显著地促进了红核脊髓束轴突的再生。结论脊髓损伤后,Clenbuterol可促进离断的轴突再生以及运动功能的恢复。     

大鼠腰神经根受压后背根神经节及脊髓后角神经元型一氧化氮合酶阳性神经元的变化

目的探讨大鼠腰神经根受压后后背根神经节和脊髓后角神经元型一氧化氮合酶阳性神经元的变化规律.方法选用12只Wistar大鼠,随机分为实验组和正常组,采用免疫组织化学ABC法结合图像分析系统进行研究.结果大鼠腰神经根受压后4周,实验组背根神经节中神经元型一氧化氮合酶阳性神经元细胞数、平均面积明显增多,脊髓后角神经元型一氧化氮合酶阳性神经末梢也明显增多,与正常组相比均有显著性差异.神经元形态以中、小型细胞为主.结论大鼠腰神经根受压后感觉神经元神经元型一氧化氮合酶表达上调,提示神经元型一氧化氮合酶可能与根性腰腿痛的发生有关.     

大鼠下丘脑第三脑室周催产素样神经元与脑脊液关系的电镜研究

本研究用高浓度戊二醛灌注固定、包埋前ABC免疫酶染色方法,显示了大鼠下丘脑第三脑室周与脑脊液密切相关的室管膜内及室管膜下OT-LI神经元丛。结果发现:在室旁核后大细胞亚核水平,第三脑室腔的顶及底部常有一些OT-LI轴突终末突入第三脑室腔,与脑脊液相接触,其纤维来源不明;透射电镜下见:这些OT-LI轴突终末附于室管膜细胞之上或夹行于微绒毛之间,常与第三脑室室管膜形成一种类似突触的连接,绝大多数OT-LI轴突终末内含有大量线粒体,其轴突包膜均呈节段性增厚。在视前区至室间孔水平的第三脑室底部,可见到OT-LI神经元胞体突入室管膜细胞间,似部分与脑脊液直接接触,透射电镜下见此类OT-LI神经细胞仅隔一层极薄的室管膜细胞浆与脑脊液相接触。在第三脑室侧壁室管膜下常有较密集的室管膜下神经元树突丛,透射电镜下常见到紧贴室管膜下走行的OT-LI树突,而且往往有轴突与之形成轴-树突触。上述结果提示,室管膜下的OT-LI神经元及树突可能既接受脑脊液中各种信息物质的调控又受到一些传入神经轴突终末的突触性传入控制而实施其分泌活动;突入脑室腔的OT-LI辅突可能是脑脊液中OT的产生途径之一。     

大鼠脊髓广动力范围神经元早成分放电对晚成分放电的调控

采用细胞外记录方法 ,通过选择性阻断有髓粗纤维的传入 ,观察大鼠脊髓背角深层广动力范围神经元(WDR)早成分放电对晚成分放电及其潜伏期的影响。结果显示 :0 3mg蛇毒溶液注入到大鼠坐骨神经鞘膜下后 ,WDR神经元出现如下动态变化 :(1)诱发放电的早成分显著减少 ,每次刺激所诱发的早成分放电个数由 6 94± 0 4 8降至 0 75± 0 2 5 (P <0 0 5 ) ,15min左右几近完全消失。 (2 )随着早成分放电的减少 ,晚成分放电则逐渐增加 ,放电数从 9 94± 0 9增加到 4 5 94± 1 16 (P <0 0 5 )。此时轻刷WDR神经元的感受野不能引起其活动改变 ,但伤害性齿镊夹捏仍可引起WDR神经元放电增多。 (3)晚成分放电的潜伏期缩短 ,即宁静期的时程变短 ,由给蛇毒前的116 83± 3 6 7ms降至 4 6 86± 1 13ms(P <0 0 1)。提示 :A纤维传入冲动在调控WDR神经元对伤害性刺激传入的反应性和兴奋性上具有重要作用     

大鼠下丘脑催产素样和加压素样神经元与5-羟色胺及儿茶酚胺能传入纤维末梢的关系

用免疫组织化学ABC双标记法,显示下丘脑各神经分泌核区中催产素样免疫反应(oxytocin-like immunoreactivity,OT-li)、加压素样免疫反应(vasopressin-like immunoreaetivity,VP-li神经元及5-羟色胺样免疫反应(serotonin-like immunoreactivity,5-HT-li)和儿茶酚胺能,即酪胺酸羟化酶免疫反应(tyrosine hydroxylase-like immunoractivity,TH-li)纤维末梢,对这两种单胺能纤维末梢在视上核、室旁核及各种神经分泌副核中的分布,及其与OT-li和VP-li神经元相关的特点及异同加以分析。结果显示,5-HT-li与TH-li纤维末梢在下丘脑视上核、室旁核及各神经分泌副核中,与OT-li及VP-li神经元相接触的多寡程度多不相同。视上核、室旁核内有极高密度的TH-li纤维末梢,与OT-li及VP-li神经元相接触,并在多数神经分泌副核的OT-li及Vp-li神经元周围,也有多寡不等的分布;而中等至高密度的5-HT-li纤维末梢,主要与各神经分泌副核内的OT-li或VP-li神经元相接触。说明5-HT-li和儿茶酚胺能传入纤维末梢,可能分别侧重调控不同神经分泌核区的大细胞神经分泌细胞群。     

基于有限元-神经元模型的大鼠硬膜外脊髓电刺激仿真研究

临床研究证实,硬膜外脊髓电刺激（ESCS）能促进脊髓损伤后的运动功能恢复,但基本神经机制则需通过动物实验进行研究。在建立大鼠ESCS有限元-神经元组合模型的基础上,仿真L2节段单阴极刺激条件下脊髓组织内的电势分布,确定L1到S2节段背根、腹根纤维和不同深度背柱纤维激活阈值的变化规律,分析纤维位置和刺激脉宽对神经纤维激活阈值的影响。仿真结果发现,距电极最近的背根纤维激活阈值最低为041 V,浅层背柱纤维的激活阈值略高为047 V,最近腹根纤维的激活阈值最高为078 V,减小电极-纤维距离有利于脊髓纤维的选择性激活;不同深度背柱纤维的激活阈值随刺激脉宽增加而减小,但脉宽过大导致背柱纤维激活阈值的降幅变小,过长的脉宽使得激活阈值的降幅趋缓,合理选择刺激脉宽有利于激活深层背柱纤维,增加脊髓组织的激活区域,提高ESCS对于深层背柱纤维的募集能力。该仿真结果可为动物实验研究中合理选择刺激参数、提高刺激选择性能提供理论指导。     

雌激素对大鼠红核神经元保护作用的体视学定量

目的 观察雌激素（E₂）替代治疗对去卵巢SD大鼠脊髓损伤后红核神经元逆行性损伤的影响。方法 成年雌性SD大鼠80只, 随机分为正常组、红核脊髓束(RST)损伤组、E2替代治疗组、雌激素受体拮抗剂（ICI）治疗组和E₂+ICI联合治疗组。SD大鼠去势后1周采用选择性切断脊髓C3～C4左侧背外侧索制作单侧红核脊髓束（RST）横断损伤模型,给予不同条件治疗后1周、2周和4周对各组大鼠采用前肢支撑探测实验进行行为学评价,用红色荧光金（FR）逆行荧光示踪及体视学定量分析法,观察红核神经元的形态及数目变化。结果 前肢支撑探测实验结果显示,各时间点E2替代治疗组的左前肢使用率均高于除正常组外其他各组,但无统计学意义（P >0.05）;形态学检测结果可见各组大鼠中脑右侧FR阳性红核神经元数目除正常组外均有不同程度减少,尤以4周时最为明显。E2替代治疗组右侧中脑FR阳性红核神经元与RST损伤组、ICI治疗组和E₂+ICI 联合治疗组相比胞体饱满、轮廓清晰、突起长,体视框计数结果显示,E₂替代治疗组红核FR阳性神经元数目明显多于其余各组（P<0.05）,但RST损伤组、ICI治疗组和E₂+ICI联合治疗组之间右侧中脑FR阳性红核神经元数目未见明显差异（P>0.05）。结论 大鼠脊髓横断损伤后中脑红核神经元发生逆行性损伤,E₂替代治疗可减轻脊髓损伤引发的继发性红核神经元损伤。     

大鼠膈神经传出和传入神经成分在脊髓内的分布

采用HRP追踪法,对成年大鼠膈神经运动纤维的起源和初级传入神经元胞体的位置及在其中枢突脊髓的投射进行了观察。结果表明,膈神经运动纤维在脊髓标记(膈核)主要分布在注射侧C_3-C_5脊髓节段的前角。隔神经感觉纤维初级传入标记的神经元见于注射侧C_3-C_6后根节,其中C_4标记的细胞数最多。     

There is no loss of motor neurons in the rat spinal cord during postnatal maturation

Motor neurons are lost during embryonic development, but it remains controversial whether motor neuron cell death occurs during postnatal life. In this study we investigated the effect of postnatal maturation on the number of intact spinal motor neurons in the rat using retrograde labelling with model-based counting, and an unbiased stereological counting technique. To determine the number of motor neurons innervating a specific forelimb muscle in rats of different postnatal ages FluoroGold was injected into the flexor carpi radialis. Before postnatal day 21 there were higher numbers of retrogradely labelled motor neurons than in adult rats, suggesting a 'loss' with postnatal maturation. This loss may be attributed to tracer diffusion to adjacent muscles and to the permeability of the muscle spindle capsule in younger animals. To obtain an unbiased estimate of the number of motor neurons in the C7 and C8 segments of the postnatal rat cervical spinal cord the fractionator/optical disector counting technique was used. This method did not show a loss of spinal motor neurons between birth and adulthood. The main conclusion from this study is that there is no loss of spinal motor neurons during postnatal maturation.     

Propriospinal fibers reaching the lumbar enlargement in the rat

Propriospinal fibers reaching the lumbar enlargement were investigated in rat by means of retrograde transport of wheat germ agglutinin-horseradish peroxidase conjugate coupled or not coupled with gold particles. Unilateral or medial bilateral injections were done. Identification of projection cells was done by tetramethylbenzidine histochemistry or gold-silver intensification procedures. Unilateral injections resulted in bilateral labeling, with patterns and density related to the spinal segments of origin. Sacral, lumbar and thoracic afferents showed identical patterns. Ipsilateral connections originated laterally from dorsal, intermediate and ventral horns. Contralateral connections originated medially from laminae VII and VIII and laterally from the reticular extension of the neck of the dorsal horn. Cervical afferents were symmetrical, arising from both lamina VIII and the reticular extension of the neck of the dorsal horn. Lamina X projection cells were seen at all levels when injection sites involved this area. Laminae III and IV were almost totally devoid of projection cells. Superficial layer cells (laminae I and II) showed some labeling when injections were situated dorsally. The organization of these tracts in rat is similar to that in cat and monkey. Their origin is discussed in relation to those of long ascending pathways reaching supraspinal levels.     

Vasoactive intestinal polypeptide (VIP)-containing neurons in the spinal cord of the rat and their projections

The distribution of vasoactive intestinal polypeptide (VIP)-like immunoreactive structures in the rat spinal cord and their projections were investigated by means of an immunofluorescent method. In the normal rat, a small number of VIP-positive fibers were observed in the superficial layer of the dorsal horn and in the lateral funiculus. With colchicine pretreatment, VIP-positive neurons were demonstrated in the lateral spinal nucleus (lsn) and in the lamina X (Rexed). Transections of the spinal cord at various levels revealed that some of the VIP neurons in the lsn might project to supraspinal areas via lateral funiculus.     

Somatotopic organization of lumbar muscle-innervating neurons in the ventral horn of the rat spinal cord

The ventral horn of the rat spinal cord was investigated with respect to the somatotopic organization of the motor neurons that innervate the lumbar muscles. Neurotracer 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI) was applied to specific sites in lumbar muscles. Spinal cord segments at L1 through L4 levels were cut into 40‐μm serial transverse sections. Labeled neurons were located in the ventromedial nucleus (VM) and lateromedial nucleus (LM) nuclei of Rexed’s lamina IX. Motor neurons innervating the m. interspinales lumborum and m. multifidus were without exception present in the VM, whereas all motor neurons innervating the m. rectus abdominis were present in the LM. Forty percent of motor neurons innervating the m. quadratus lumborum were present in the VM and the other 60% were in the LM. Although most of the motor neurons innervating the m. psoas major were present in the LM, a few labeled neurons existed in the VM. These results suggest that the border zone demarcating the areas of innervation of the dorsal and ventral rami of spinal nerves crosses the m. quadratus lumborum.     

T-激肽原在大鼠腰骶髓及背根节的分布

目的 探讨T-激肽原在神经系统的定位及作用.方法 应用免疫细胞化学方法,结合光镜观察.本实验研究了T-激肽原在大鼠腰骶髓及L_(4-6)背根节的分布.结果T-激肽原分布于L_(4-6)背根节及腰骶髓灰质的第Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅸ层神经元及脊髓白质的神经胶质细胞和神经纤维.结论 在上述部位的神经元和神经胶质细胞,T-激肽原和高分子量激肽原、低分子量激肽原及其代谢产物可能共存.     

Labeled corticospinal neurons one year after spinal cord transection

One year after a T9 spinal cord transection, horseradish peroxidase was inserted into the spinal cord at T3-T4. Only about 7% of the number of corticospinal neurons labeled in control rats were labeled in exactly matched transected rats. This long-term loss of labeled neurons makes cell death the most likely explanation for the failure to identify corticospinal neurons in spinal-cord-transected rats.     

大鼠脊髓星形胶质细胞培养方法的改良

目的:改进大鼠脊髓星形胶质细胞的体外培养方法,为研究脊髓星形胶质细胞的生物学作用及脊髓相关疾病提供实验模型.方法:新生1～2d的SD乳鼠,无菌操作下游离整段脊柱,将脊髓从椎管中冲出,机械吹打制成单细胞悬液,差速黏附处理以去除成纤维细胞成分.利用GFAP免疫荧光显色对培养细胞进行鉴定.结果:经原代和传代培养,获得的星形胶质细胞纯化率高达99%以上.结论:采用本实验室改进的方法,操作简便快捷,培养的大鼠脊髓星形胶质细胞生长良好,纯度高,为后续实验对脊髓星形胶质细胞的生物学作用研究奠定实验基础.     

Regeneration of the rat spinal cord after thoracic segmentectomy: Growth and restoration of nerve conductors

The results presented in our previous report (Morfologiya, 127, No. 2 (2005)) provided evidence that consolidation of the spinal cord (SC) after thoracic segmentectomy in rats occurs as a result of the formation of a connective tissue scar, which is quicker when the defect is filled with collagen gel. The present report describes analysis of semithin sections and transmission electron microscopy studies demonstrating that regenerating nerve conductors traverse connective tissue in structures whose organization is identical to that of peripheral nerves. In the zone of SC rarefaction caudal to the trauma site, myelination of growing axons is mediated by glial cells without the formation of nerve trunks. Large numbers of fine regenerating conductors were seen at the sites of degenerated myelin fibers in the fasciculi of the white matter in the lumbar segments of the SC. __________ Translated from Morfologiya, Vol. 129, No. 1, pp. 30–38, January–February, 2006.     

大鼠脊髓慢性压迫性损伤实验模型的建立

目的：建立一种新型大鼠脊髓慢性压迫实验动物模型，为探索脊髓受压后的病理生理机制奠定基础。方法：根据大鼠脊柱解剖结构特点自行设计一种大鼠脊髓压迫器，用以制作大鼠慢性压迫模型。运用行为学、影像学、TTC、HE、Tunnel等方法，了解动物行为学变化及受压节段脊髓病理学改变，以评价模型的可靠性。结果：脊髓压迫后渐次出现肌力减退、行动瘫痪；TTC结果显示，在各时段可见脊髓缺血范围与压迫时间及压迫强度相关；压迫后，脊髓出现组织水肿、神经元空泡化、白质疏网状改变及退行性变，以及神经元和胶质细胞的凋亡。结论：（1）用大鼠脊髓压迫器制作的大鼠脊髓慢性压迫缺血性损伤模型，具有方法简单、科学、重复性强等特点；（2）脊髓压迫程度可根据实验目的不同进行调节；（3）本实验为脊髓压迫性损伤机制的研究提供了一种理想的动物试验模型。