脊髓损伤及其继发损伤时的兴奋性氨基酸含量变化

采用Ａｌｌｅｎ’ｓ打击法以５０ｇ／ｃｍ和１００ｇ／ｃｍ致伤大鼠脊髓，于伤后１０分钟、１小时、２小时、４小时、８小时、２４小时取伤段脊髓，用氨基酸微量检测技术测定天冬氨酸（ＡＳＰ）和谷氨酸（Ｇｌｕ）含量。结果显示：脊髓损伤（ＳＣＩ）后１０分钟两个致伤组ＡＳＰ、Ｇｌｕ均较对照组（单纯椎板切除无损伤）明显升高；但伤后１～２４小时两组ＡＳＰ较对照组明显降低，其中２小时和２４小时降低最显著，ＡＳＰ在８小时虽也降低，但较２小时略回升；５０ｇ／ｃｍ组伤后１小时、４小时、８小时Ｇｌｕ含量较１０分钟时降低，但仍高于对照组。１００ｇ／ｃｍ组伤后２小时、２４小时Ｇｌｕ降低最明显。结果表明ＳＣＩ后兴奋性氨基酸ＥＡＡ存在两个高低不同的反应峰，提示ＥＡＡ参与了ＳＣＩ后的继发性损伤。     

半乳糖凝集素-1对急性脊髓损伤模型大鼠的保护作用及其机制

目的研究半乳糖凝集素-1(Galectin-1)对大鼠急性脊髓损伤的保护作用及其机制。方法 45只SPF级SD大鼠随机平均分为Galectin-1组、甲泼尼龙(MP)组和生理盐水对照组(生理盐水组)。以Allen's法打击脊髓造模,分别在术后1 d、7 d、14 d每组各取5只大鼠,先做BBB评分及斜板试验,然后处死大鼠取损伤处脊髓组织进行苏木精-伊红(HE)染色及胶质纤维酸性蛋白(GFAP)免疫组化染色检测,同时用酶联免疫吸附测定(ELISA)法检测血清白细胞介素-10(IL-10)的含量。结果术后1 d,3组大鼠BBB评分、斜板试验结果及血清IL-10含量的差异无统计学意义(均P0.05);术后7 d、14 d,MP组和Galectin-1组的BBB评分、斜板试验结果及血清IL-10含量均较生理盐水组高(均P0.05),而MP组与Galectin-1组之间的差异均无统计学意义(均P0.05)。HE染色显示术后各时间点,Galectin-1组和MP组脊髓损伤肿胀及出现核固缩或解离神经元的数量少于生理盐水组。术后1 d,3组大鼠脊髓星形胶质细胞(GFAP表达阳性)数量无明显区别;术后7 d、14 d,Galectin-1组及MP组明显多于生理盐水组,而Galectin-1组与MP组则无明显差别。结论Galectin-1对大鼠急性脊髓损伤具有一定的保护作用,其机制可能是通过增强机体IL-10的表达,从而抑制损伤后的炎症反应而发挥脊髓保护作用的。     

香芹酚对大鼠急性脊髓损伤的保护作用

目的 探讨香芹酚对大鼠脊髓损伤（SCI）后神经功能的影响及其机制。方法 将60只雄性SD大鼠（200~250 g）随机分为5组:假手术组（n=12）、SCI组（n=12）、香芹酚组（n=36）,香芹酚组根据香芹酚剂量分为低、中、高剂量3个亚组,每亚组12只。低、中、高剂量香芹酚组SCI后30 min腹腔注射香芹酚,剂量分别为10、20、40 mg/kg,每日一次;假手术组和SCI组每日腹腔注射等量生理盐水。采用Allen法建立大鼠SCI模型;假手术组只行椎板切除手术。SCI后24、48、72 h,采用BBB评分评估大鼠神经功能;SCI后72 h,采用ELISA法检测损伤脊髓组织丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽（GSH）、过氧化氢酶（CAT）、caspase-3活性;Western-blot法检测损伤脊髓组织Bax,Bcl-2蛋白表达水平。结果 SCI后,大鼠BBB评分均明显降低（P<0.05）,损伤脊髓组织水肿指数以及MDA、caspase-3和Bax水平均明显增高（P<0.05）,而SOD、GSH、CAT、Bcl-2水平均明显降低（P<0.05）;香芹酚能明显改善大鼠BBB评分（P<0.05）,明显降低水肿指数以及MDA、caspase-3和Bax水平（P<0.05）,而显著增加CAT、SOD、GSH、Bcl-2水平（P<0.05）。结论 香芹酚可通过减轻脊髓水肿、抑制氧化应激反应以及抗凋亡作用而对SCI大鼠发挥神经保护作用。     

MgSO_4对大鼠脑缺血-再灌注损伤的保护作用

目的验证MgSO4作为NMDA受体非竞争性拮抗剂对大鼠脑缺血再灌注后的保护作用及其与凋亡的关系。方法：用线栓法制作Wistar大鼠大脑中动脉栓塞模型．术后2小时予以再通．分别于再通的同时及再通后2小时给予不同剂量MgSO4治疗。术后21小时进行运动功能评估．测定脑组织含水量,观察病理组织形念学变化．并计算每视野下凋亡细胞数量。结果：早期大剂MgSO4组大鼠运动功能改善明显．并可减轻脑组织含水量．但早期给予MgSO4在短期内也不能减少凋亡细胞数目。结论：MgSO4作为兴奋性氨基酸受体（EAAs-R）非竞争性拮抗剂对大鼠脑缺血-再灌注有保护作用,但在短期内其作用与抑制细胞凋亡发生无关     

MgSO_4对大鼠脑缺血-再灌注损伤的保护作用

目的验证MgSO4作为NMDA受体非竞争性拮抗剂对大鼠脑缺血再灌注后的保护作用及其与凋亡的关系。方法：用线栓法制作Wistar大鼠大脑中动脉栓塞模型．术后2小时予以再通．分别于再通的同时及再通后2小时给予不同剂量MgSO4治疗。术后21小时进行运动功能评估．测定脑组织含水量,观察病理组织形念学变化．并计算每视野下凋亡细胞数量。结果：早期大剂MgSO4组大鼠运动功能改善明显．并可减轻脑组织含水量．但早期给予MgSO4在短期内也不能减少凋亡细胞数目。结论：MgSO4作为兴奋性氨基酸受体（EAAs-R）非竞争性拮抗剂对大鼠脑缺血-再灌注有保护作用,但在短期内其作用与抑制细胞凋亡发生无关     

大鼠实验性脊髓损伤后胶质瘢痕分布规律研究

目的观察慢性脊髓损伤胶质瘢痕形成的时间与空间分布特征。方法应用Allen′s法建立大鼠脊髓打击伤模型,通过神经功能评分、体感及运动诱发电位、组织病理学及免疫荧光双标记方法确定胶质瘢痕形成时间及分布。结果神经功能评分、诱发电位、组织病理学显示伤后4周进入相对稳定状态,伤后4周损伤区形成空洞,胶质瘢痕位于空洞壁。结论脊髓损伤后4周进入慢性期,研究伤后脊髓空洞及胶质瘢痕形态,为判断切除胶质瘢痕的界限提供形态学依据。     

乙酰左旋肉碱对大鼠脊髓损伤保护作用的实验研究

目的研究乙酰左旋肉碱对大鼠急性脊髓损伤后的干预效果,从行为学、组织形态学和线粒体结构探讨乙酰左旋肉碱对大鼠运动功能恢复的影响。方法将大鼠随机分为脊髓损伤组、乙酰左旋肉碱治疗组、假手术组3组,每组22只,构建大鼠脊髓损伤模型。于损伤后1 d、3 d、7 d、14 d、21 d、28 d和35 d评估大鼠后肢运动功能恢复情况;并取损伤后1 d、3 d的脊髓组织行HE染色,进行形态学观察;透射电镜观察脊髓损伤后24 h线粒体形态的变化。结果 Basso Beattie Bresnahan运动功能评分损伤后7 d、14 d、21 d、28 d、35 d,乙酰左旋肉碱组评分均高于SCI组,差异有统计学意义(P0.05);SCI+ALC组损伤后3 d,变性的神经元细胞结构逐渐清楚,细胞核清晰,核膜完整;神经元胞浆内线粒体含量较多且结构完整,线粒体空泡化也较损伤组减轻。结论乙酰左旋肉碱可能通过改善脊髓神经元线粒体功能,促进大鼠脊髓损伤后后肢运动功能的恢复。     

自身免疫神经保护:MBP主动免疫对脊髓损伤后二次损害的有益作用

目的　研究髓鞘碱性蛋白 (MBP)主动免疫 ,对大鼠脊髓损伤 (spinalcordinjurySCI)后二次损害的治疗作用 ,探讨自身免疫T细胞对中枢神经系统损伤的神经保护机制。方法　用不完全福式佐剂 (IFA)乳化的MBP、全脊髓匀浆 (WSCH)、卵白蛋白 (OVA)分别免疫三组SD大鼠 ,1周后使用OHBASIKI打击器制作脊髓损伤模型 (SCI)。每周进行一次斜板试验及BBB评分 ,2 8天后标本取材行光镜及电镜检查。结果　用MBP或WSCH免疫鼠神经功能康复明显优于OVA免疫鼠 ,斜板实验及BBB评分与后者相比差异显著 (P <0 0 1)。形态学检查前两组大鼠损伤脊髓组织大部分恢复 ,形态基本正常 ,后者仍破坏严重。结论　MBP或WSCH主动免疫能中断SCI后二次损害的破坏作用 ,被激活的自身免疫T细胞对中神经系统损伤具有神经保护功能。     

丁酸钠对脊髓损伤大鼠模型的神经保护作用及机制

目的 探讨丁酸钠对脊髓损伤大鼠模型的神经保护作用及机制.方法 SD大鼠36只,随机分为丁酸钠组和对照组.用NYU撞击仪击打T8脊髓制作脊髓损伤模型,并立即给予丁酸钠组大鼠丁酸钠600 mg/(kg·d)腹腔注射,连续7 d.制模前、后1～6周给予大鼠Basso-Beattie-Bresnahan(BBB)评分.制模后1周时,用免疫荧光染色法检查脊髓组织凋亡细胞和炎症细胞,制模后6周时进行神经传导功能检查.结果 脊髓损伤后4～6周时,丁酸钠组BBB评分显著高于对照组(均P＜0.05);在脊髓损伤后1周时,丁酸钠组脊髓的凋亡细胞和炎症细胞数显著少于对照组(均P＜0.05);脊髓损伤后6周时,丁酸钠组运动诱发电位的潜伏期明显短于、波幅明显高于对照组(均P＜0.05).结论 丁酸钠对脊髓损伤大鼠具有神经保护作用,其机制可能与抗凋亡和抗炎作用有关.     

大鼠脊髓损伤后巢蛋白表达

目的探讨成年大鼠脊髓损伤后损伤区局部巢蛋白（nestin）的表达及意义。方法应用Allen＇s法建立大鼠脊髓拟伤模型，行为学评分采用BBB评分（Basso，Beattie＆Bresnahan locomotor rating scale，BBB scale），观察局部病理组织学改变及用免疫荧光组织化学方法检测局部脊髓在不同时段nestin的阳性表达变化。结果伤后1w BBB评分最低，随后增加，到4w以后达到最高并进入平台期。常规病理学检查显示拟伤模型类似于临床常见的脊髓损伤。损伤后1W，可见损伤区附近nestin表达升高，2W达高峰，4W后nestin表达明显下调。结论脊髓损伤可诱导损伤区周围短暂的nestin阳性表达，后者可能存在脊髓损伤后的再生与修复中起重要作用。     

Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis

The spinal cord of 20 patients with amyotrophic lateral sclerosis (ALS) and 5 patients with lower motor neuron disease (LMND) were investigated immunohistochemically using anti-human excitatory amino acid transporter 1 (EAAT1) and EAAT2 antibodies which are the astrocytic transporters. The purpose of the study was to examine relationships between EAAT1 and EAAT2 immunoreactivity and degeneration of anterior horn neurons. Specimens from 20 patients without any neurological disease served as controls. In controls, spinal cord gray matter was densely immunostained by antibodies, whereas the white matter was generally not immunostained. In motor neuron disease (MND) patients, EAAT1 immunoreactivity was relatively well preserved in the gray matter despite neuronal loss of anterior horn cells. On the other hand, EAAT2 immunoreactivity in anterior horns correlated with the degree of neuronal loss of anterior horn cells: in the patients with mild neuronal depletion, anterior horns were densely immunostained by the antibody, whereas in the patients with severe neuronal loss, EAAT2 expression was markedly reduced. Degenerated anterior horn cells frequently showed a much denser EAAT1 and EAAT2 immunoreactivity around the surface of the neurons and their neuronal processes than that observed in normal-appearing neurons. There was no difference in the expression of EAAT1 and EAAT2 immunoreactivity between LMND and ALS patients. These findings suggest that in the early stage of degeneration of anterior horn cells, EAAT1 and EAAT2 immunoreactivity is preserved in the astrocytic foot directly attached to normal-appearing neurons, whereas levels of EAAT1 and EAAT2 protein rather increase in the astrocytic foot directly attached to degenerated anterior horn neurons; the latter effect most probably reduces the elevated glutamate level, compensates for the reduced function of astroglial glutamate transporters, or represents a condensation of EAAT1 and EAAT2 immunoreactivity secondary to loss of neurites and greater condensation of astrocytic processes. Thus, we demonstrate a difference in EAAT1 and EAAT2 immunoreactivity in different stages of progression in ALS, as a feature of the pathomechanism of this disease. Received: 8 September 1999 / Revised, accepted: 28 October 1999     

Participation of AMPA- and NMDA-type excitatory amino acid receptors in the spinal reflex transmission,in rat

Classical in vitro and in vivo models and electrophysiological techniques were used to investigate the role of AMPA- and NMDA-type glutamate receptors in various components of spinal segmental reflex potentials. In the rat hemisected spinal cord preparation, the AMPA antagonists NBQX and GYKI 52466 abolished the monosynaptic reflex (MSR) potential but caused only partial inhibition of the motoneuronal population EPSP. NMDA antagonists had no noticeable effect on the MSR in normal medium, but markedly depressed the late part of EPSP. However, an NMDA receptor antagonist sensitive monosynaptic response was recorded in magnesium-free medium at complete blockade of the AMPA receptors. In spinalized rats, the AMPA antagonists completely blocked all components of the dorsal root stimulation evoked potential. MK-801 (2mg/kg, i.v.) reduced monosynaptic responses in a frequency dependent way, with no effect at 0.03 Hz and 22% inhibition at 0.25 Hz. The reduction of the di- and polysynaptic reflex components was about 30% and did not depend on stimulation frequency. Long-latency reflex discharge responses, especially when evoked by train stimulation, were more sensitive to MK-801 than the polysynaptic reflex.These results suggest that glutamate activates MSR pathways through AMPA receptors. However, under certain conditions, NMDA receptors can modulate this transmission through plastic changes in the underlying neuronal circuits. AMPA and NMDA receptors play comparable roles in the mediation of longer latency reflex components.     

Activation of 5-HT1C/2 receptors depresses polysynaptic reflexes and excitatory amino acid-induced motoneuron responses in frog spinal cord

Sucrose gap recordings from the ventral roots of isolated, hemisected frog spinal cords were used to evaluate the effects of high concentrations of serotonin (5-HT) and alpha-methyl-5-HT (alpha-Me-5-HT) on the changes in motoneuron potential produced by dorsal root stimulation and by excitatory amino acids and agonists. Bath application of 5-HT in concentrations of 10 microM or greater produced a concentration-dependent motoneuron depolarization. Polysynaptic ventral root potentials evoked by dorsal root stimuli were reduced in both amplitude and area by 5-HT or alpha-Me-5-HT (both 100 microM). This may result from a reduction of the postsynaptic sensitivity of motoneurons to excitatory amino acid transmitters because 5-HT significantly depressed motoneuron depolarizations produced by addition of L-glutamate and L-aspartate to the superfusate. Similarly, 5-HT reduced depolarizations produced by the excitatory amino acid agonists N-methyl-D-aspartate (NMDA), quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA), and kainate. alpha-Me-5-HT reduced NMDA depolarizations. Tetrodotoxin (TTX) did not affect the ability of 5-HT to attenuate NMDA or kainate depolarizations, but did eliminate the 5-HT-induced attenuation of quisqualate and AMPA depolarizations. The glycine receptor site associated with the NMDA receptor did not appear to be affected by 5-HT because saturation of the site by excess glycine did not alter the 5-HT-induced depression of NMDA responses. The 5-HT1C/2 antagonist ketanserin and the 5-HT1A/2 antagonist spiperone significantly attenuated the 5-HT-induced depression of NMDA-depolarizations.(ABSTRACT TRUNCATED AT 250 WORDS)     

胚胎脊髓移植在恢复损伤脊髓传导功能中的作用

目的：探讨胚胎脊髓移植在恢复损伤脊髓传导功能中的作用。方法：将Ｅ１４胚胎脊髓植入成鼠损伤脊髓后３０、４５、６０天时，用单位放电记录技术观察了正常脊髓神经元和移植物神经元的自发放电活动，及其对刺激坐骨神经、红核和同时刺激的反应。结果：正常脊髓神经元的自发单位放电多是一个低频的单发脉冲活动。无论选择那种刺激方式，都可见兴奋、抑制和无反应三种反应。术后３０天时，胚胎神经元的自发单位放电以高频电脉冲活动为主，簇状放电所占比例较大，对刺激有反应的放电单位数也较少；随着动物存活时间的延长，这些单位放电的情况逐渐向着低频、单脉冲以及高反应率的方向发展。至术后６０天时，胚胎神经元单位放电的频率、形式以及对刺激的反应情况都变得和正常神经元的相似。结论：胚胎神经元移植后经历了一个逐渐发育分化过程，在这个过程中它们有可能逐渐和宿主神经元形成了功能性突触连接。     

Altered metabolism of excitatory amino acids, N-acetyl-aspartate and N-acetyl-aspartylglutamate in amyotrophic lateral sclerosis

Since recent studies provided evidence for abnormal glutamate metabolism in amyotrophic lateral sclerosis, we measured amino acid levels in the fasting plasma of 52 ALS patients and an equal number of controls of a similar age. In addition, the content of amino acids, N-acetyl-aspartate (NAA) and N-acetyl-aspartyl-glutamate (NAAG) were measured in spinal cord and brain tissue obtained at autopsy from patients dying of ALS. Results showed significant elevations (by about 70%) in the plasma levels of glutamate in the ALS patients as compared to controls. In contrast, glutamate levels were significantly decreased in all CNS regions studied of ALS patients (by 21–40%), with the greatest changes occurring in the spinal cord. The ratio of glutamine to glutamate was altered significantly in the spinal cord ALS tissue. In addition, reductions in the levels of aspartate (by 32–35%), NAA, and NAAG (by 40–48%) were found in the spinal cord of ALS patients. These results are consistent with a generalized defect in the metabolism of neuroexcitotoxic amino acids. An altered distribution of these compounds may occur between their intracellular and extracellular pools with resultant abnormal potentiation of excitatory transmission mediated by glutamate receptors and selective degeneration of motor neurons.     

Effects of chronic treatment with a cyclic AMP-selective phosphodiesterase inhibitor, rolipram, on excitatory amino acid neurotransmission systems in young and aged rat brains

Summary Rolipram selectively inhibits cyclic AMP-specific phosphodiesterase, and leads to an increase in cyclic AMP levels in the brain. In this study, we investigated the effects of chronic rolipram treatment on excitatory and inhibitory amino acid neurotransmission systems in young and aged Wistar rat brains. We used in vitro autoradiography with [³H]MK-801, [³H]glycine, D-[³H]aspartate, and [³H]muscimol to label N-methyl-D-aspartate (NMDA) receptors, glycine modulatory sites, glutamate transport sites, and -aminobutyric acid-A (GABA) receptors, respectively. Rolipram (0.01 or 0.1 mg/kg, per os) or its vehicle (distilled water) was administered once a day for 4 weeks. The highest binding of [³H]MK-801, [³H]glycine, and d-[³H]aspartate was seen in the hippocampus in vehicle-treated rats. No significant differences in these binding activities were seen between young and aged rat brains. [³H]Muscimol binding was the highest in the cerebellum, and decreased in many brain regions in aged rats. The chronic rolipram treatment resulted in (1) an increase in [³H]MK-801 binding in the dentate gyrus in both young and aged rats, (2) remarkable reductions in D-[³H]aspartate binding in many regions of both young and aged rats, and (3) no or minimal changes in [³H]glycine and [³H]muscimol binding. These results suggest that the chronic rolipram treatment modifies the excitatory amino acid neurotransmission system.     

高压氧对脑外伤大鼠脑内兴奋性氨基酸水平的影响

目的研究高压氧对脑外伤大鼠脑内兴奋性氨基酸水平的影响。 方法通过制作大鼠脑外伤模型,设计随机实验分组：模型对照组、高气压组、高浓度氧组和高压氧组给予脑外伤大鼠治疗;同时设空白对照组。分别于第3天、第6天、第9天取大鼠全脑测量谷氨酸（Glu）和天冬氨酸（Asp）的含量。 结果大鼠脑损伤后脑组织中Glu、Asp含量明显升高,高压氧可明显下调Glu、Asp含量,与模型对照组比较差异具有统计学意义（P<0.05）,而高气压组、高浓度氧组不能明显抑制Glu、Asp表达,与模型对照组比较差异无统计学意义（P>0.05）。 结论高压氧可以显著抑制脑外伤大鼠脑组织中兴奋性氨基酸释放从而减轻脑外伤后继发性脑损害,而高气压、高浓度氧不能明显抑制兴奋性氨基酸的释放。     

尼莫的平对Alzheimer病大鼠海马和额叶细胞外液氨基酸的影响

目的 研究尼莫的平对Ａｌｚｈｅｉｍｅｒ病大鼠海马和额叶兴奋性氨基酸的影响。方法 在立体定位下于迈内特基底核（ＮＢＭ）注入β淀粉样蛋白（β－ＡＰ）１０μｇ建立Ａｌｚｈｅｉｍｅｒ病模型,对照组在ＮＢＭ注入生理盐水,治疗组在建立模型后腹腔注射尼莫的平,每天注射１次至实验结束,模型组不给任何药物。脑内微透析技术采集大鼠海马、额叶细胞外液、反相高效液相色谱技术测定氨基酸类神经递质。结果 模型组海马和额叶细胞外液４种氨基酸浓度均较对照组升高,谷氨酸最明显。尼莫的平治疗后谷氨酸明显降低。结论 由β－ＡＰ产生的Ａｌｚｈｅｉｍｅｒ病大鼠痴呆与兴奋毒性有关,其作用可能是由钙离子介导。     

Illness-induced hyperalgesia is mediated by spinal neuropeptides and excitatory amino acids

The spinal cord dorsal horn contains neural mechanisms which can greatly facilitate pain. We have recently shown that ‘illness’-inducing agents, such as intraperitoneally administered lipopolysaccharide (LPS; bacterial endotoxin), can produce prolonged hyperalgesia. This hyperalgesic state is mediated at the level of the spinal cord via activation of the NMDA-nitric oxide cascade. However, prolonged neuronal depolarization is required before such a cascade can occur. The present series of experiments were aimed at identifying spinal neurotransmitters which might be responsible for creating such a depolarized state. These studies show that LPS hyperalgesia is mediated at the level of the spinal cord by substance P, cholecystokinin and excitatory amino acids acting at non-NMDA sites. No apparent role for serotonin or kappa opiate receptors was found.