脊髓全横断模型大鼠的行为学与病理学对照

背景：理想的脊髓损伤模型应既能模拟人类脊髓损伤,又能排除影响疗效的干扰因素,并具有广泛可重复性,脊髓全横断模型是目前较理想的选择。但由于操作方法的多样性致使疗效差异较大,各研究结果之间缺乏可比较性。目的：通过建立标准化的大鼠脊髓全横断模型,对比分析脊髓全横断大鼠后肢行为学改变和病理学特征。方法：成年雌性SD大鼠60只,随机分为假手术组（n=12）、常规脊髓横断组（n=24）和显微脊髓横断组（n=24）,每组再随机分为造模后7,14,28 d组。以T9椎体为中心,假手术组行椎板切除;其他两组行脊髓全横断,造成脊髓急性损伤模型,其中常规脊髓横断组采用常规外科方法造模,显微脊髓横断组采用标准化显微操作技术造模。各组于造模后7,14和28 d行后肢运动功能BBB评分及横断处脊髓的组织病理学观察,观测脊髓横断处瘢痕组织厚度、脊髓残端间距、空洞横径和脑脊液囊腔形成情况,计算瘢痕指数、残端间距指数和空洞指数。结果与结论：假手术组术前、术后BBB评分和脊髓病理无明显变化。常规脊髓横断组和显微脊髓横断组大鼠造模后后肢完全性瘫痪,其中常规脊髓横断组大鼠后肢功能无恢复。造模后一至二周,显微脊髓横断组大鼠开始出现后肢运动功能自发性恢复,脊髓病理学检测指标值均明显低于常规脊髓横断组,差异有非常显著性意义（P〈0.01）。各组病理学观测指标与BBB评分之间无相关关系。提示标准化脊髓全横断造模方法有利于消除个体差异,更有利于对治疗效果的量化分析和研究比较。     

3-磷酸肌醇依赖性蛋白激酶-1在大鼠脊髓横断损伤后的表达研究

目的探讨3-磷酸肌醇依赖性蛋白激酶-1(PDKl)及磷酸化3-磷酸肌醇依赖性蛋白激酶-1(p-PDK1)在横断性脊髓损伤(tSCI)后的表达变化及意义。方法将30只成年SD大鼠随机分为假手术对照组和T9横断伤6 h、12 h、1 d和3 d组,每组6只。采用Western blot方法检测损伤后各时间段PDK1和p-PDK1蛋白水平在脊髓中的表达变化;采用免疫组织化学方法检测p-PDK1在假手术对照组以及损伤组脊髓中的分布和定位;最后通过免疫荧光标记法检测p-PDK1在细胞中的定位。结果 Western blot和免疫组化显示PDKl蛋白表达在SCI后无明显变化,p-PDK1蛋白在SCI后表达增加,12 h达到高峰,之后逐渐下降,并于3 d恢复到假手术水平。免疫荧光双标显示p-PDK1神经元及少突胶质细胞存在大量共定位。结论脊髓损伤后PDKl蛋白水平无变化而p-PDK1呈现明显的时空变化,提示在脊髓损伤后PDK1可能通过磷酸化参与了神经元及少突胶质细胞的病理生理过程,发挥抗凋亡的作用。     

Excitatory amino acid release in the spinal cord caused by plantar incision in the rat

There is extensive evidence that spinal excitatory amino acids (EAAs) like glutamate (Glu) and aspartate (Asp) are important in the processing of nociceptive behaviors caused by incisions. To better understand EAA-induced dorsal horn sensitization caused by surgery, we examined the time course and extent of spinal amino acid (AA) release during and after a plantar incision utilizing in vivo microdialysis. We also examined the role of primary afferent input and axonal conduction by measuring spinal EAAs in rats after hindpaw denervation and in rats treated with spinal tetrodotoxin (TTX).In halothane-anesthetized rats, a microdialysis filament (200 microm diameter, 45,000 MW cut off, Hospal AN69) was passed transversely through the deep dorsal horn of the spinal cord (L4-L6). After 18-24h, the dialysis filament was perfused with artificial cerebrospinal fluid (ACSF), dialysate samples collected and analyzed for AAs (Glu, Asp, asparagine, glutamine, serine, and glycine). Rats underwent anesthetic induction with halothane followed by a plantar incision (n=8) or sham operation (n=8). AAs were also measured in incised rats that underwent hindpaw denervation (n=4), in rats that had the filament placed outside the L4-L6 spinal segments (n=7), in rats with a microdialysis catheter placed in the ventral horn at L4-L6 (n=7) and in rats treated with spinally administered TTX (n=5). AAs were measured during recovery from anesthesia and for the next 8h.In the sham-operated group, Asp and Glu did not change throughout the experiment. In rats undergoing plantar incision, Asp and Glu increased from 10 to 30 min after incision to 200+/-30 and 138+/-12 percentage of control, respectively. The EAAs returned to baseline by 1h after incision. For the other AAs, only serine and asparagine increased after incision. No increase in AA release by incision was observed after hindpaw denervation, TTX treatment or placement of the filament outside the L4-L6 segments. In rats with a filament implanted in the ventral horn (L4-L6), EAAs increased during halothane induction and sham preparation. Thus, the EAA release required an intact afferent nerve barrage and segmental excitatory nerve transmission.The incision-induced nociceptive afferent barrage increased the release of Glu and Asp in the lumbar dorsal horn for 45 min. The concentrations of AAs returned to baseline by 1h. The percentage increase is in some cases less and for a shorter period of time compared to other models of persistent pain, perhaps because the incision injury is less severe compared to others models. This profile of EAA release further explains why models of inflammation and chemical irritation do not translate well to human postoperative pain.     

Nogo-A在脊髓损伤大鼠脊髓组织中的动态表达

目的:观察大鼠脊髓损伤后神经生长抑制因子Nogo-A在脊髓组织中的动态表达变化,探讨Nogo-A蛋白在神经再生过程中的意义和作用。方法:选用108只SD大鼠随机分成正常组、假手术组和模型组,每组36只,A组不做任何处理,B组咬除T_9-T_(11)棘突及椎板,避免损伤脊髓;C组按照改良Allen法造模。分别于干预后24h、第3天、第7天、第14天处死大鼠,每组9只,以免疫组化及Western Blot检测各组大鼠脊髓组织中Nogo-A蛋白的表达变化,以荧光定量PCR检测Nogo-A mRNA表达变化。结果:正常组、假手术组各时间点Nogo-A蛋白和mRNA无显著变化(P0.05)。模型组在损伤后24h Nogo-A蛋白和mRNA表达较低,3d后下降至最低,7d后迅速上升达到高峰,至14d逐渐下降,但仍高于假手术组;与假手术组比较,模型组在损伤后7d、14d,Nogo-A蛋白和mRNA表达均明显增高,差异均有显著性意义(P0.05)。结论:大鼠脊髓损伤后,Nogo-A蛋白在早期一过性下降后可持续保持高水平表达,可能是造成脊髓损伤后中枢神经系统神经再生困难的重要原因之一。     

CD34+细胞移植后脊髓全横断大鼠脊髓功能恢复的形态学及行为学观察

目的:观察脊髓全横断SD大鼠模型骨髓CD34 细胞移植术后,脊髓功能恢复情况。方法:实验于2004-11/2005-07在昆明医学院神经科学研究所完成。①采用Percoll细胞分离液离心 贴壁分离法对绿色荧光蛋白转基因小鼠骨髓细胞进行粗筛后,收集骨髓悬浮细胞进行体外培养,密切观察细胞生长情况。②收集培养至第3周的悬浮细胞,采用免疫细胞化学法进行CD34单克隆抗体鉴定。③选健康雌性SD大鼠30只,采用随机数字法分为移植组和对照组,每组15只(1d,1,2,4和8周各3只)。将经CD34单克隆抗体鉴定后的CD34 细胞移植入脊髓全横断大鼠模型脊髓横断处尾侧。④术后密切观察大鼠后肢运动功能恢复情况,分别在术后1d,2,4和8周行BBB评分检测大鼠后肢运动功能的恢复。⑤同时行冰冻切片于荧光显微镜下观察绿色荧光细胞的分布情况,并用免疫组织化学法检测CD34 细胞的存活。结果:对照组大鼠第22天死亡1只,进入结果分析29只。①免疫荧光镜下见,培养至两三周的骨髓悬浮细胞中,圆形绿色荧光细胞可达90%以上。②免疫细胞化学染色结果显示,培养至两三周后的骨髓悬浮细胞多数呈CD34阳性,阳性率达(74.6±1.7)%。③两组大鼠BBB评分结果显示,移植组大鼠后肢自主运动功能的恢复明显优于对照组。④移植后2,4和8周时移植组脊髓横断处头尾两侧均可见绿色荧光细胞,且多分布于灰质中,散在或聚集成片;免疫组织化学法可见移植组脊髓横断处头尾两侧切片中均有CD34 细胞散在。结论:移植骨髓CD34 细胞可在脊髓全横断大鼠脊髓中存活并迁移,其大鼠后肢自主运动功能得到部分恢复。     

脊髓全横断大鼠神经生长因子和脑源性神经营养因子表达及三七皂苷的干预效应

目的:神经生长因子和脑源性神经营养因子同属神经营养素家族,在神经系统发育及维持正常神经元功能中有重要作用。实验拟证实脊髓全横断损伤三七皂苷对大鼠神经生长因子及脑源性神经营养因子蛋白水平的表达变化产生了影响。方法:实验于2005-06/09在昆明医学院神经科学研究所完成。①实验材料:清洁级健康雌性SD大鼠72只,质量(200±20)g;三七皂苷由云南植物药业提供。②分组及实验过程:大鼠被随机分为4组:假手术组、单纯脊髓全横断损伤组、脊髓全横断损伤 生理盐水(0.5mL/次)组、脊髓全横断损伤 三七皂苷(100mg/kg/次)组,每组18只。于T10水平横断大鼠脊髓,假手术组仅剪开硬脊膜而不损伤脊髓。后2组于术后30min,4,24,48,72h腹腔注射给药各1次。③实验评估:各组于术后3,7及21d分别取6只大鼠L1～2段脊髓制作冰冻切片,采用免疫组织化学ABC法染色。观察并计数脊髓腹角神经生长因子、脑源性神经营养因子蛋白的表达变化;常规苏木精伊红染色观察脊髓的组织病理变化。结果:72只大鼠全部进入结果分析:①脊髓全横断损伤后脊髓出现明显的神经变性坏死、炎性浸润等病理变化,三七皂苷可减轻这些变化。②神经生长因子蛋白主要分布于灰质神经元胞浆及胶质细胞胞核中。在正常脊髓有少量表达,脊髓损伤后7d表达明显升高,直到伤后21d仍高于假手术组(P<0.05);三七皂苷可明显促进其表达,伤后3,7d均明显高于其他各组(P<0.05),在21d时下降但仍高于假手术组(P<0.05)。③脑源性神经营养因子蛋白主要分布于腹角的运动神经元胞浆中,胶质细胞未见着色。在正常脊髓有少量表达,脊髓损伤后7d表达明显升高(P<0.05),伤后21d已下降,同假手术组相比差异无显著性(P>0.05);三七皂苷可明显促进其表达,伤后3,7,21d均明显高于所有对照组(P<0.05)。结论:三七皂苷可减轻脊髓横断性损伤后继发损害,增加神经生长因子、脑源性神经营养因子表达量及提前神经生长因子、脑源性神经营养因子表达时间,提示其可以促进脊髓损伤早期修复。     

脊髓伤后脊髓组织中一氧化氮合酶活性变化与兴奋性氨基酸释放间的联系

目的探明脊髓伤后脊髓组织中一氧化氮合酶(NOS)活性变化与兴奋性氨基酸(EAA)释放间的关系.方法首先通过蛛网膜下腔注射谷氨酸(GLU)观测其对大鼠脊髓组织NOS活性动态变化的影响;然后通过微透析技术及高效液相色谱荧光法动态地探测不同NOS活性水平对伤段脊髓局部EAA含量变化的影响.结果外源性GLU迅速激活了脊髓组织NOS的活性,而抑制NOS活性则明显降低了伤段脊髓组织EAA的浓度.结论脊髓伤后脊髓组织中一氧化氮(NO)与EAA的释放相互促进,因而在继发性脊髓损伤中形成级联放大的神经毒性因子释放.     

Baclofen, gamma-aminobutyric acidB receptors and substance P in the mouse spinal cord

Antinociceptive effects of baclofen, a gamma-aminobutyric acidB (GABAB) agonist, were studied in mice along with other GABAergic agents, all administered intrathecally (i.t.): i.e., muscimol (GABAA agonist), bicuculline (GABAA antagonist) and 5-aminovaleric acid (GABAB antagonist). After i.t. administration, none of the four compounds increased the withdrawal latency in the tail-flick test. With the intradermal hypertonic saline (6% saline) behavioral test, baclofen decreased the number of behaviors in a dose-dependent and 5-aminovaleric acid-reversible manner, whereas i.t. administered muscimol was ineffective. With the i.t. substance P (SP) behavioral test, muscimol was again ineffective, whereas the SP-induced behaviors were differentially modified by baclofen depending on the temporal order of their i.t. administration. Although baclofen, coadministered with SP, decreased the number of SP-induced behaviors, baclofen pretreatment (2-100 min before i.t. administration of SP) increased the number of behaviors in a dose-dependent and 5-aminovaleric acid-reversible manner. Two minutes after several fixed doses of baclofen were administered i.t., dose-response curves for induction of behaviors by SP (i.t.) were shifted progressively to the left by increasing doses of baclofen, suggesting that hypersensitivity to SP had developed during this time frame. Decreased responsiveness to a peripheral noxious stimulus (hypertonic saline-induced behavior) is therefore associated with hypersensitivity to i.t. applied SP (SP behavioral test). The selective action of a GABAB agonist on neurokinin-elicited behaviors shown in this study is in clear contrast to the selective action of a GABA agonist against excitatory amino acid spinal activity noted in the following paper.     

Mefloquine enhances nigral gamma-aminobutyric acid release via inhibition of cholinesterase

Mefloquine, a widely used antimalarial drug, has many neuropsychiatric effects. Although the mechanisms underlying these side effects remain unclear, recent studies show that mefloquine enhances spontaneous transmitter release and inhibits cholinesterases. In this study, we examined the effect of mefloquine on GABA receptor-mediated, spontaneous inhibitory postsynaptic currents (sIPSCs) of dopaminergic neurons, mechanically dissociated from the substantia nigra pars compacta of rats aged 6 to 17 postnatal days. Mefloquine (0.1-10 microM) robustly and reversibly increased the frequency of sIPSCs with an EC50 of 1.3 microM. Mefloquine also enhanced the frequency of miniature inhibitory postsynaptic currents in the presence of tetrodotoxin but without changing their mean amplitude. This suggests that mefloquine acts presynaptically to increase GABA release. Mefloquine-induced enhancement of sIPSCs was significantly attenuated in medium containing low Ca2+ (0.5 mM) or following pretreatment with 1,2-bis (2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester (30 microM), a membrane-permeable Ca2+ chelator. In contrast, 100 microM Cd2+ did not alter the action of mefloquine. This suggests that mefloquine-induced facilitation of GABA release depends on extracellular and intraterminal Ca2+ but not on voltage-gated Ca2+ channels. Mefloquine-induced enhancement of sIPSCs was significantly attenuated in the presence of the anticholinesterase agent physostigmine or blockers of non-alpha7 nicotinic acetylcholine receptors. Taken together, these data suggest that mefloquine enhances GABA release through its inhibition of cholinesterase. This allows accumulation of endogenously released acetylcholine, which activates neuronal nicotinic receptors on GABAergic nerve terminals. The resultant increase of Ca2+ entry into these terminals enhances vesicular release of GABA. This action may contribute to the neurobehavioral effects of mefloquine.     

Spontaneous and evoked release of met-enkephalin-like material from the spinal cord of arthritic rats in vivo

Perfusion of the intrathecal space with artificial CSF was achieved in control and arthritic rats under halothane anaesthesia in order to collect the met-enkephalin-like material (MELM) released from the whole spinal cord. On the fourth week following the intradermal injection of Freund's adjuvant to induce arthritis, a marked reduction (-56%) in the spontaneous outflow of MELM was noted in arthritic rats. This effect did not involve changes in the degradation process of MELM, since it persisted when kelatorphan was added to the perfusing fluid in order to inhibit completely the peptidases acting on met-enkephalin. Raising the K+ concentration in the perfusing fluid from 2.4 to 40 mM, as well as moving the hind paws, produced a significant enhancement of MELM release which was (at least) as pronounced in arthritic as in control rats. These results suggest that the basal activity of spinal enkephalinergic neurones, but not that triggered by various stimuli, is reduced in arthritic rats.     

许旺细胞-海藻酸钠凝胶移植修复大鼠脊髓损伤

目的:观察许旺细胞-海藻酸钠凝胶移植对大鼠脊髓损伤后细胞凋亡、Bcl-2表达及下肢运动功能恢复的影响.方法:清洁级SD大鼠随机分为4组:正常对照组、单纯损伤组、许旺细胞组、许旺细胞海藻酸钠凝胶组.后3组制作脊髓全横断损伤模型.正常对照组、单纯损伤组不进行移植处理,许旺细胞组植入吸附许旺细胞悬液的明胶海绵块、许旺细胞-海藻酸钠凝胶组植入许旺细胞-海藻酸钠凝胶.分别于12 h,1,3,7,21 d对动物进行BBB评分后处死,取损伤区脊髓节段制成石蜡切片进行TUNEL、Bcl-2染色,观察脊髓内凋亡细胞、Bcl-2细胞的数量及分布变化.结果:正常对照组仅有少量淡染Bcl-2阳性细胞;单纯损伤组神经元Bcl-2免疫反应阳性细胞表达的高峰在第3天,14 d时Bcl-2免疫反应阳性细胞表达接近正常水平.许旺细胞-海藻酸钠凝胶移植后损伤脊髓细胞Bcl-2免疫反应阳性细胞表达具有显著增高(P＜0.05),7 d高度表达并持续2周以上.单纯损伤组脊髓内细胞凋亡最多,并于损伤后1,7 d形成两个高峰,多分布于白质中.许旺细胞-海藻酸钠凝胶组BBB评分较单纯损伤组及许旺细胞组明显提高(P＜0.05).结论:许旺细胞-海藻酸钠凝胶移植能抑制大鼠脊髓损伤后脊髓细胞凋亡、促进Bcl-2的表达,提高了脊髓运动功能的恢复,但未达到正常水平.     

大鼠脊髓损伤动物模型的建立

目的:建立一个稳定、损伤程度适中的脊髓损伤大鼠模型的技术及方法学特征。方法:实验于2004-09/2005-01在承德医学院临床技能实验室完成。选取30只Wistar大鼠,体质量230～260g,纵行切开大鼠背部皮肤及皮下组织,剥离棘突旁肌肉,切除T7棘突及椎板,暴露脊髓硬膜。根据打击物(重10g)调节高度的不同将30只大鼠分为3mm高度组、5mm高度组、8mm高度组,每组10只。高度使重物自然下落,打击T7段脊髓,迅速移开打击重物及打击头,逐层缝合伤口,喂养8周后行病理学检查。选取通过脊髓最大径的切片在低倍镜下进行显微摄影,照片上侧量并计算空洞或瘢痕的最大直径与同水平脊髓直径的比例。结果:纳入大鼠30只,死亡5只,存活率83.3%;其中3mm高度组死亡1只,5mm高度组死亡1只,8mm高度组死亡3只。3mm高度组3例标本镜下可见损伤段脊髓中有不规则的空腔,6例标本于脊髓内形成不规则瘢痕,损伤部位瘢痕最大直径与同水平脊髓直径比例为(10.0±5.2)%;5mm高度组9例镜下均可见损伤段脊髓中有一个长圆形或不规则的空腔,损伤部位瘢痕最大直径与同水平脊髓直径比例为(46.0±3.8)%;8mm高度组存活的7例镜下均可见损伤段脊髓全部中断,形成瘢痕与硬膜外组织分界不清。结论:5mm高度组存活的大鼠的脊髓均产生位于脊髓中央部或近中央部的空洞,损伤的位置和程度较恒定,是较为理想的损伤模型。     

Up-regulation of renal adenylate cyclase-coupled vasopressin receptors after chronic administration of vasopressin antagonists to rats.

Chronic administration of vasopressin [antidiuretic hormone (ADH)] antagonists has been shown to produce a paradoxical antidiuresis in both ADH-replete and ADH-deplete (diabetes insipidus) rats. The antidiuretic effect is progressive, reaching maximal levels in 4 to 5 days, and sustained, persisting for at least 24 hr after cessation of treatment. The antidiuretic profiles associated with these antagonists do not coincide with the profiles of potent ADH agonists, arginine vasopressin and 1-deamino-8-D-arginine vasopressin. To investigate the mechanism of the antidiuretic effect of ADH antagonists, male diabetes insipidus rats were administered antagonists selective for the renal [adenylate cyclase-coupled (V2)] or pressor (phosphytidyl inositol-coupled) vasopressin receptor and urine output (volume and osmolality) and renal vasopressin receptor properties (concentration and affinity) were determined and compared to rats treated with arginine vasopressin or 1-deamino-8-D-arginine vasopressin. After acute administration, only the V2-acting antagonists were antidiuretic, but were 3 orders of magnitude less potent than 1-deamino-8-D-arginine vasopressin. Following chronic administration, all of the antagonists were antidiuretic, but the level of antidiuresis achieved with the phosphytidyl inositol-coupled vasopressin receptor-selective antagonist was 2- to 3-fold lower than for analogs with V2 activity. Maximal antidiuretic effects were realized in 5 days and were apparent at 24 hr after cessation of treatment. The antidiuretic activities and potencies of the ADH antagonists appeared to be increased following chronic antagonist administration. Finally, renal vasopressin receptor concentration was significantly elevated 24 hr after cessation of antagonist treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Muscimol, gamma-aminobutyric acidA receptors and excitatory amino acids in the mouse spinal cord

These experiments examined the effects of intrathecally administered gamma-aminobutyric acid (GABA) agonists on the effects of intrathecally administered excitatory amino acid (EAA) agonists: N-methyl-D-aspartic acid (NMDA), quisqualic acid and kainic acid. We have found that muscimol, a GABAA receptor agonist, but not baclofen, a GABAB receptor agonist, dose-dependently inhibited caudally directed biting and scratching behavior induced by all three EAA agonists. This nonselective blockade of the expression of effects mediated by all three types of EAA receptor is in marked contrast to the selective blockade of NMDA effects seen previously in the case of mu opioids and phencyclidine receptor agonists. Inhibition by muscimol was blocked with the GABAA receptor antagonist, bicuculline. Decreased latency or hyperalgesia in the tail-flick test, found previously to be induced selectively by NMDA and blocked by an NMDA receptor antagonist, was similarly affected by muscimol but not baclofen, each given intrathecally. However, muscimol prolonged the tail-flick latency only after presentation of NMDA suggesting a possible antinociceptive effect of GABAA agonists in the presence of agonists at NMDA receptors. This study together with the preceding paper resolves GABA-mediated spinal antinociception into two components: a GABAA agonist selectively blocks nociception involving EAA receptors whereas a GABAB agonist selectively blocks substance P spinal activity (the preceding paper).     

人胚胎嗅鞘细胞植入鼠半横断脊髓对脊髓轴突再生的影响

目的:嗅鞘细胞(olfactoryensheathingcells,OECs)植入脊髓后可促进再生的轴突长入远段脊髓,观察人胚胎嗅鞘细胞移植对成鼠脊髓轴突再生的影响。方法:实验于2003-04/2004-10在中山大学第二附属医院实验中心脐血库完成。20只清洁级雌性SD大鼠被随机分为两组,实验组10只,将分离、纯化、培养2周的人胚胎OECs,移植于大鼠半横断胸髓(T10)的两端;对照组10只同样方法给予等量的DMEM。6周后组织切片,行苏木精-伊红染色、嗜银染色以及髓鞘碱性蛋白(MBP)和抗神经生长因子受体抗体(NGFR)免疫组化检查。结果:OECs移植组6周后脊髓大体标本显示初步愈合现象;组织学观察,OECs呈NGFR阳性表达,显示OECs仍然存活,且与宿主组织整合良好;部分再生轴突长入断端间组织,呈MBP阳性表达。结论:人胚胎OECs对成鼠胸髓损伤后的轴突再生有促进作用。     

Ethanol potentiation of GABAergic transmission in cultured spinal cord neurons involves gamma-aminobutyric acidA-gated chloride channels

The interaction of ethanol with gamma-aminobutyric acid (GABA)-mediated 36-Cl-influx and its modulation by various drugs was investigated in C57 mice spinal cord cultured neurons. Ethanol (5-100 mM) potentiated the effect of GABA on 36Cl-influx; whereas at concentrations greater than or equal to 50 mM ethanol activated Cl- channels directly. The effect of ethanol was specific for GABAA receptor-gated Cl- channels, as ethanol did not potentiate glycine-induced 36Cl-influx in the same neurons. Both the enhancing and direct effects of ethanol on 36Cl-influx were blocked by GABA antagonists like bicuculline, picrotoxinin and inverse agonists of the benzodiazepine site like the imidazodiazepine R015-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5 alpha], [1,4]benzodiazepine-3-carboxylate) and N-methyl-beta-carboline-3-carboxamide (FG-7142). Ethanol potentiating effect of GABA-induced 36Cl-influx was also reversed by methyl-6,7-dimethyl-4-ethyl-beta-carboline-3-carboxylate. The effects of the inverse agonists were blocked by the benzodiazepine receptor antagonist R015-1788. Both R015-4513 and FG-7142 reversed direct and GABA potentiating effects of ethanol effect at concentrations lower than those that exhibit inverse agonistic activity in the 36Cl-influx assay in cultured neurons. These results suggest that ethanol facilitation of GABAAergic transmission involves GABA receptor-gated Cl- channels and that this interaction may be responsible for some of the pharmacological effects of ethanol.     

脊髓全横断大鼠模型的构建

背景:脊髓全横断模型在造模时常难以保证神经纤维的完全离断。目的:构建大鼠脊髓全横断损伤模型。方法:将大鼠随机分为模型组和假手术组。模型组构建脊髓T10节段全横断模型;假手术组动物仅打开椎管与硬脊膜而后缝合,但不损伤脊髓。建模后1,3,5,7d分别进行BBB评分以评估后肢运动功能,检测其体感诱发电位和运动诱发电位来评估神经传导通路的完整性,并行形态学观察来评估脊髓肉眼观病理形态。结果与结论:与假手术组相比,模型组大鼠在建模后1,3,5,7d时,其BBB评分降低(P<0.01),未检测出体感和运动诱发电位。形态学观察结果显示模型组大鼠脊髓完全横断,而假手术组脊髓形态完整。结果提示实验成功构建了大鼠脊髓全横断模型。     

脊髓全横断大鼠模型的构建

背景：脊髓全横断模型在造模时常难以保证神经纤维的完全离断。目的：构建大鼠脊髓全横断损伤模型。方法：将大鼠随机分为模型组和假手术组。模型组构建脊髓T10节段全横断模型;假手术组动物仅打开椎管与硬脊膜而后缝合,但不损伤脊髓。建模后1,3,5,7d分别进行BBB评分以评估后肢运动功能,检测其体感诱发电位和运动诱发电位来评估神经传导通路的完整性,并行形态学观察来评估脊髓肉眼观病理形态。结果与结论：与假手术组相比,模型组大鼠在建模后1,3,5,7d时,其BBB评分降低（P〈0.01）,未检测出体感和运动诱发电位。形态学观察结果显示模型组大鼠脊髓完全横断,而假手术组脊髓形态完整。结果提示实验成功构建了大鼠脊髓全横断模型。     

急性脊髓损伤模型大鼠蛋白质组学研究：iTRAQ技术的验证

背景：同位素标记相对和绝对定量（Isobaric tags for relative and absolute quantitation,iTRAQ）质谱分析技术依据串联质谱中信号离子表达质荷比峰值的不同来研究相关对应蛋白质的信息。目的：建立急性脊髓损大鼠模型,观察其脑脊液差异蛋白谱,从微观分子水平研究急性脊髓损伤后继发性损伤的机制及有效治疗方法。方法：建立SD大鼠急性脊髓损伤模型,取脑脊液应用iTRAQ技术鉴定SD大鼠急性脊髓损伤后脑脊液的差异蛋白质。结果与结论：共鉴定蛋白数722个,差异表达蛋白107个：下调的差异蛋白有63个,上调的差异蛋白数是44个。其中相关神经再生的差异蛋白19个：上调14个,下调5个;调节神经再生的差异蛋白7个。实验中检测到的多种差异蛋白及表达明显的神经再生因子可能作为急性脊髓损伤的生物标记物或可能作为临床管理监测急性脊髓损伤的损伤进程、靶向治疗及评估疗效的强有力证据。