大鼠脊髓半横断损伤后硫酸软骨素蛋白多糖Versican的变化

目的观察大鼠脊髓半横断损伤(hSCI)位点头尾段硫酸软骨素蛋白多糖Versican的表达变化,探讨其与脊髓损伤修复的关系。方法20只成年健康SD大鼠随机分为假手术对照组和脊髓半横断损伤3d、7d和14d组。建立成年SD大鼠脊髓半横断(T9～T10)模型。取损伤位点头尾段T9、T10节段制作冰冻切片,运用Versican抗血清进行免疫组化ABC法染色。分别观察并计数各组前角中Versican阳性细胞数,采用计算机图像分析技术测量免疫阳性产物的平均光密度(A)值。结果Versican阳性产物主要分布于细胞外基质中,前角神经元和胶质细胞的胞浆中也有分布;损伤后前角中Versican阳性神经元和胶质细胞数较对照组明显减少(P<0.05),免疫阳性产物平均A值较对照组明显增加(P<0.05)。结论脊髓半横断损伤后,表达增加的Versican很可能参与损伤后轴突生长被抑制的过程。     

低血钙大鼠脊髓腹角钙结合蛋白免疫反应神经元的变化

为观察低血钙对神经元内钙结合蛋白的影响，用免疫细胞化学法，研究了低血钙大鼠髓腹角内钙结合蛋白免疫反应神经 的变化。     

大鼠腰骶髓后连合核SP受体免疫阳性神经元与SP免疫阳性终末的突触联系

应用免疫组织化学双重反应的方法研究了大鼠腰骶髓后连合核内ＳＰ受体免疫阳性神经元在光、电镜下的特征及其与ＳＰ免疫阳性终末的突触联系。结果证明，ＳＰ免疫阳性终末和ＳＰ受体阳性胞体及树突均密集分布于后连合核区。在光镜下双重免疫反应阳性结果显示二者重叠分布，且可见大量的ＳＰ样免疫阳性终扣与ＳＰ受体阳性胞体或树突紧密接触。电镜下证明ＳＰ受体免疫阳性产物不仅位于突触后部位而且分布于胞体和树突的非突触部位，有４２％的ＳＰ受体阳性树突与ＳＰ阳性终末形成突触连接。在此未发现ＳＰ受体免疫阳性终末。通过一系列实验证明：腰骶髓后连合核神经元接受由ＳＰ介导的外周伤害性信息的传入，且其主要为盆腔脏器内脏初级传入成分。     

SPS模型大鼠前额皮质分子伴侣钙网蛋白的表达变化

目的观察创伤后应激障碍(PTSD)大鼠前额皮质钙网蛋白(calretinculin,CRT)的表达变化,旨在揭示PTSD部分发病机制。方法取健康成年雄性Wistar大鼠75只,随机分为连续单一刺激(singleprolonged stress,SPS)模型1d、4d、7d、14d和正常对照组,用免疫组织化学法、免疫印迹法和RT-PCR法检测PTSD内侧前额皮质(media prefrontal cortex,mPFC)CRT的表达变化。结果在正常大鼠脑mPFC神经细胞中有CRT的表达,与对照组相比SPS刺激后CRT表达有增高趋势,mRNA和蛋白水平从SPS1d开始增多,SPS4d达到高峰,SPS7d组和SPS14d组有降低趋势,但仍高于正常组。结论 CRT在SPS大鼠mPFC过表达提示其可能参与PTSD大鼠记忆情绪改变的病理生理学机制。     

BDNF对体外培养的大鼠脊髓前角神经元内突触素Ⅰ与突触囊泡素表达的影响

探讨BDNF对体外培养的大鼠脊髓前角神经元内突触素I与突触囊泡素(SYN)表达的影响。取孕14d大鼠子宫内胎鼠的脊髓腹侧部分神经元,体外有血清培养。在培养7d后,随机分成对照组、BDNF组和抗BDNF组。BDNF组培养液中加入BDNF(20ng/ml),抗BDNF组培养液中加入BDNF抗体(20μg/ml),对照组加入等量Hanks液。3d后在倒置显微镜下计数三组神经元存活数,并用NF200、MAP2、NSE的免疫组化反应对神经细胞进行鉴定。行突触素I与SYN免疫组化反应,对部分细胞行突触素ImRNA原位杂交反应,运用图像分析系统对突触素I与SYN免疫组织反应阳性产物以及突触素I原位杂交反应阳性产物作光密度分析。结果发现有血清培养时各组脊髓前角神经元的存活数无显著差异(P>0.05);BDNF组突触素I与SYN免疫反应阳性产物的平均光密度值高于其它两组,抗BDNF组最低(P<0.01)。BDNF组突触素ImRNA阳性产物的平均光密度值明显高于其它两组,抗BDNF组突触素ImRNA阳性产物的平均光密度值最低(P<0.01)。本研究结果提示BDNF对有血清培养时脊髓前角神经元的存活没有明显影响,但BDNF可明显上调培养的脊髓前角神经元内突触素I与SYN的表达。     

脊髓半横断损伤后大鼠皮层神经元细胞骨架蛋白基因表达的变化

目的　探讨神经再生过程中细胞骨架蛋白的作用机制 ,了解中枢和外周神经系统神经元应答神经损伤和再生的差异。　方法　用原位分子杂交方法观察了大鼠T10～ 11脊髓半横断损伤后 1、3、5、7、10、14、2 1、2 8、5 6d皮层感觉运动区皮层神经元中α 管蛋白和 3个神经丝蛋白亚基mRNA的表达变化。　结果　脊髓半横断损伤后损伤侧皮层感觉运动区皮层神经元中α 管蛋白、神经丝 L、 M、 HmRNA表达明显下调。α 管蛋白mRNA表达水平在损伤后 1d降低 ,而神经丝亚基mRNA表达水平直到下一个时间点 (损伤后 3d)才下调 ,至损伤后 5 6d均无恢复趋势。结论　皮层神经元损伤后α 管蛋白mRNA的表达被抑制 ,提示皮层神经元和外周神经元应答损伤的信号不同     

去卵巢后雌性大鼠下丘脑视前区NOS阳性神经元形态学和突触素表达的变化

目的：为解释围绝经期的精神、神经症状提供实验依据。方法：采用免疫细胞化学方法显示去卵巢雌性大鼠下丘脑视前内侧区(MPA)和视前外侧区(LPA)内NOS阳性神经元,形态学改变以及突触素表达。结果：(1)去卵巢组和对照组大鼠的视前内、外区内可见大量NOS阳性神经元;(2)去卵巢动物的下丘脑内NOS阳性神经元突起长度比对照组的明显变短,分支明显变少;(3)去卵巢组动物下丘脑内突触素表达比对照组的明显减少。结论：大鼠去卵巢后,由于雌激素分泌减少,导致了NOS阳性神经元的突起减少和突触减少,结果NO介导的神经元信号传导失常,这些变化可能是引发围绝经期的精神、神经症状的原因之一。     

大鼠腰骶髓后连合核内P物质受体阳性神经元与GABA能轴突终末的突触联系

用包埋前免疫组织化学方法结合包埋后免疫金电镜技术，研究了大鼠腰骶髓后连合核内Ｐ物质受体（ＳＰＲ）免疫反应神经元与ＧＡＢＡ能终末的突触联系。ＳＰＲ免疫反应产物主要分布于神经元胞体和树突的胞膜下。双重标记电镜结果表明，约有４６％的ＳＰＲ阳性树突和胞体与ＧＡＢＡ能终末形成对称性突触。其中绝大部分为轴树突触，个别为轴体突触。本研究结果提示，大鼠腰骶髓后连合核ＳＰＲ阳性神经元可能接受ＧＡＢＡ能终末的调控。     

大鼠延髓背角内5-羟色胺、脑啡肽、γ-氨基丁酸、甘氨酸或P-物质能终末与钙结合蛋白阳性神经元间的联系

CB(calbindin-D28k),CR(calretinin)和PV(parvalbumin)是最常见的3种钙结合蛋白(calcium-binding proteins,CaBPs)。本研究首先观察了面口部给予伤害性刺激诱发大鼠延髓背角(又称三叉神经脊束核尾侧亚核)神经元表达FOS蛋白的状况;然后通过免疫荧光组织化学技术检测这些神经元内是否含有CaBPs(CB、CR和PV);最后通过免疫荧光和免疫电镜染色技术观察5-HT、GABA、甘氨酸转运体2(glycine transporter 2,GlyT2)、脑啡肽(enkephalin,ENK)或SP与CaBPs/FOS双标神经元间的联系。在光镜下可观察到:(1) FOS阳性神经元在延髓背角各层均有分布,以Ⅱ层最为密集;(2)大多数CB、CR或PV阳性神经元位于Ⅱ层,余者分布在Ⅰ层和Ⅲ层;(3) 5-HT、GABA、GlyT2,ENK及SP阳性纤维和终末主要位于延髓背角浅层(4)部分FOS阳性神经元同时呈CB、CR或PV阳性;(5) 5-HT、GABA、GlyT2或ENK阳性终末分别与FOS/CB、FOS/CR或FOS/PV双标神经元形成密切接触;(6) SP阳性终末与5-HT、GABA、GlyT2或ENK阳性终末同时与CB、CR或PV阳性神经元形成密切接触。在电镜下观察到5-HT、GABA、GlyT2或ENK阳性终末与CB、CR或PV阳性神经元主要形成对称型(抑制性)突触联系。这些结果提示在大鼠延髓背角,5-HT、GABA、甘氨酸或ENK可能通过抑制含钙结合蛋白的伤害性感受神经元来调节面口部伤害性信息的传递。     

Time course of segmental reflex changes after chronic spinal cord hemisection in the rat

In a companion paper (Hultborn & Malmsten 1983a) it was described that, in the cat, direct excitatory reflexes became larger on the side of a chronic spinal hemisection than on the other side. In this paper the time course for change of the size of excitatory reflexes after spinal cord hemisection in the rat is described. Ipsilateral reflexes obtained by stimulation of cut dorsal roots L 4 and L 5 were recorded in the corresponding cut ventral roots. It was found that reflexes became larger on the lesioned (left) side than on the opposite side. For both mono-and polysynaptic reflexes there was a marked peak in left/right ratio of reflex size at 2 to 6 days post lesion. The ratio then returned to normal but increased again from about 21 days until at least 135 days. In control rats reflexes were larger on the right side, in agreement with findings in the cat.     

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.     

嗅鞘细胞移植对大鼠脊髓半横断损伤后轴突再生及后肢功能恢复的作用

为了对嗅鞘细胞移植治疗大鼠脊髓损伤(spinal cord injury, SCI)后轴突再生及后肢功能恢复进行综合性的评价,本实验采用22只雌性成年SD大鼠,并随机分成A、B、C、D 4组。其中A、B、D组行左侧T11 ～T12节段脊髓半横断手术,然后A组移植嗅鞘细胞(olfactory ensheathing cells,OECs)悬液;B组移植DMEM/F12培养液;D组移植核荧光试剂(Hoechst33342)标记的嗅鞘细胞,用于鉴定嗅鞘细胞在体内的存活情况;C组作为正常对照组。术后6周内,对大鼠进行BBB评分、IP斜板试验并观察皮质体感诱发电位(cortical somatosensory evoked potential, CSEP)、运动诱发电位(motor evoked potential, MEP)的潜伏期。将辣根过氧化物酶(HRP)注射到横断面尾段脊髓,逆行追踪观察横断面头段脊髓及对侧中脑红核内HRP逆标细胞数,并观察左侧后肢小腿三头肌肌细胞横截面积及直径的变化。结果显示:(1)移植后的OECs数量未见明显减少,细胞核形态较好,细胞未向头、尾侧迁移;(2)移植3周后BBB评分及IP斜板,试验A、B组较C组明显低(P<0.05),但A组明显高于B组(P<0.05);(3)A、B组的CSEP及MEP潜伏期较C组明显延长(P<0.05),但A组要比B组明显缩短(P<0.05);(4)HRP逆行追踪观察到对侧中脑红核大细胞区及脊髓T9 ～T11节段逆标细胞的数量,A组明显多于B组,但A、B组均少于C组;(5)左侧后肢小腿三头肌肌细胞的横截面积及直径,A、B组均较C组减少,但A组减小的幅度明显小于B组。以上结果表明OECs移植能促进半横断脊髓轴突的再生及后肢功能的恢复。     

Induced auxotonic muscle contractions in animals with lateral spinal cord hemisection under various cooling regimens

We studied the effects of long-term moderate hypothermia on induced auxotonic contractions of dog hindlimb extensor muscle after lateral hemisection of the spinal cord at the thoracolumbar region (T_XII-L_I–II) during the early and late postoperative periods (1–2 and 3–5 weeks, respectively). The mechanisms of antispastic effects of moderate hypothermia are different: the immediate effect was due to cold blockade of spinal neurons, while delayed effects resulted from activation of descending bilateral oversegmental motor apparatus. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 270–273, September, 1999     

GAP-43治疗大鼠脊髓横断后神经中丝NF200表达的变化

通过制备成年SD大鼠完全性脊髓损伤模型,研究生长相关蛋白(GAP-43)治疗大鼠脊髓损伤后神经中丝(NF200)表达的变化,探讨GAP-43在再生修复中的作用,为临床治疗提供实验依据。实验采用雌性8周龄SD大鼠75只,制成脊髓损伤模型后随机分为三组:GAP-43抗体组、GAP-43抗原组和对照组,每组25只。使用直接注射法将GAP-43抗原和GAP-43多克隆抗体分别注入抗原组和抗体组的大鼠脊髓的断端,对照组仅切断脊髓而不给药,最后观察各组大鼠肢体功能的恢复情况。用BBB评分法对不同时段大鼠的行为学表现进行评分,用HE染色及免疫组化染色观察NF200的表达,并对其进行相关性分析。结果发现对照组在不同的时间段行为学评分最低和抗原组评分最高,脊髓损伤区病理改变明显好转,NF200的表达呈进行性增高,且前角神经元NF200的表达早于后角神经元。抗体组早期恢复出现明显的停滞状态,但停药后能很快恢复。说明GAP-43能促进损伤脊髓的恢复,而抗体对损伤脊髓恢复的影响是可逆的,这对于脊髓再生的研究是一种值得探讨的新方法,对进一步探索脊髓损伤的治疗具有重要的意义。     

Regional distribution of a novel pituitary protein (7B2) in the rat spinal cord: effect of neonatal capsaicin treatment and thoracic cord transection

Using a specific radioimmunoassay, a novel pituitary protein (7B2) was shown to be distributed throughout the rat spinal cord. The concentration of 7B2-like immunoreactivity (7B2-IR) was significantly higher in dorsal lumbar than in whole lumbar cord, and the highest level was found in the sacral region. On both gel and high-performance liquid chromatography, 7B2-IR in rat spinal cord eluted in the same position as 7B2-IR extracted from porcine pituitary gland. Neonatal capsaicin treatment did not change dorsal lumbar spinal cord 7B2-IR concentrations, suggesting that it is not predominantly present in unmyelinated primary afferent fibres. Thoracic cord transection, however, resulted in significant increases of 7B2-IR in lumbar and especially sacral regions, providing evidence of a 7B2-containing ascending pathway originating in the lumbosacral spinal cord.     

Morphofunctional characteristics of the lumbar enlargement of the spinal cord in rats

The topography of the lumbar enlargement of the spinal cord in rats was studied; an immunohistochemical method was used to determine the distribution of synaptophysin — a membrane protein of synaptic vesicles. Synaptophysin-immunoreactive structures were detected in the gray matter of all Rexed laminae, around most neurons and in the neuropil. Previously undescribed subpial synaptic contacts were detected immunohistochemically in the white matter and confirmed by electron microscopy. A non-myelinated component of the corticospinal tract, including axonal varicosities and synaptic contacts, was observed in the dorsal part of the white matter of the lumbar enlargement of the spinal cord. __________ Translated from Morfologiya, Vol. 132, No. 5, pp. 33–37, September–October, 2007.     

Changes in segmental reflexes following chronic spinal cord hemisection in the cat:

The effect of a chronic spinal cord hemisection on segmental reflex transmission was studied in cats. Recordings of ventral root responses were made after a terminal transection below the initial lesion to eliminate descending influence through the intact spinal half. Procedures to ensure comparability between sides, that are lacking in earlier work on this experimental model, were introduced in the present work. It was demonstrated that there was an increase in reflex size on the lesioned (left) side, relative to the other side, of both mono-and polysynaptic reflexes. The reflex changes were found at all survival times studied (from 2 to 515 days).In control animals mono-and polysynaptic reflexes were found to be larger on the right side. It is discussed that side symmetry of reflex size is not to be presupposed even in a normal population. Detailed clinical examinations of lesioned animals were not performed, but a tendency for enhancement of the ipsilateral knee-jerk was found. There was no syndrome of spasticity and general motor recovery was very good.     

丹曲林对急性脊髓损伤大鼠脊髓白质的保护作用

目的:探讨丹曲林(dantrolene,DA)对大鼠急性脊髓损伤后脊髓白质的保护作用。方法:采用钳夹法制作大鼠急性脊髓损伤模型(SCI)。将48只成年雄性Sprague-Dawley大鼠体重280~310 g,随机分成3组,每组16只,假手术组(Sham组):只行椎板切除术;SCI组:术前1 d腹腔注射生理盐水;丹曲林处理组(DA组):术前1 d腹腔注射10 mg/kg丹曲林。SCI损伤后3、7、14、21、28 d利用BBB评分量表评估大鼠后肢运动功能恢复情况;术后24 h,利用抗NF200单克隆抗体的免疫荧光组织化学法标记轴突,观察各组轴突的形态和数量变化;Western Blot方法检测各组脊髓损伤部位的髓鞘碱性蛋白(Myelin basic protein,MBP)和环核苷酸磷酸二酯酶(2’,3’-cyclic nucleotide phosphodiesterase,CNPase)的蛋白表达水平。结果:不同时间点SCI组大鼠的BBB评分显著低于Sham组,提示SCI模型制备成功。注射丹曲林后,不同时间点SCI动物的BBB评分明显升高(P0.05),提示丹曲林可促进大鼠后肢运动功能的恢复。NF200免疫荧光组织化学法显示:Sham组轴突长,排列有序、分布均匀;SCI后24 h,NF200+纤维断裂、数量减少、排列紊乱、部分甚至卷曲;给予丹曲林后,NF200+纤维数量显著增多且较长,排列较规则。Western Blot结果显示:SCI组CNPase,MBP蛋白的表达量较Sham组显著降低(P0.01),给予丹曲林后可明显升高两者的表达(P0.01)。结论:丹曲林对急性脊髓损伤大鼠白质具有一定的保护作用。