经皮穿刺抽取大鼠脑脊液方法的改良

目的: 改进传统经皮穿刺抽取脑脊液的方法,提高采集大鼠脑脊液动物存活率。方法: 90只SD大鼠随机分为对照组、传统穿刺组和改良穿刺组(每组30只)。对照组不进行穿刺。传统穿刺组按反握抓持法进行穿刺取样。改良穿刺组采用左手固定大鼠头部,将其抬高至约135°,右手正握改良1 ml注射器,以枕骨隆突和第一颈椎为标志定位枕骨大孔,在枕骨大孔中心偏下平行进针,抽取脑脊液。分别于第1、4、7日抽取大鼠脑脊液,每次抽取记录取样时间、采集量、脑脊液性状,抽取后3 d观察大鼠存活情况,选取存活大鼠进行重复采样。结果: 传统经皮穿刺取样法采样成功率为63.33%,三次重复采样动物存活率为10%;改良经皮穿刺取样法采样成功率为86.67%,三次重复采样动物存活率为50%,相比传统采样有显著升高(P＜0.01)。结论: 改良经皮穿刺取样法操作简单,耗时少,成功率高。操作无需定位设备,单人可以完成。动物反复取样存活率高,方便进行脑脊液重复采样。     

家兔自发性蛛网膜下腔出血早期脑损伤模型的初步研究

目的:建立一种可重复的、有典型神经损伤症状的自发性蛛网膜下腔出血(SAH)动物模型,为研究蛛网膜下腔出血后早期脑损伤(EBI)的机制提供可靠的动物模型。方法:选用新西兰大耳白兔60只,随机分为实验组、对照组、空白组。采用枕大池穿刺一次注入自体动脉血法建立SAH模型。在4h、8h、12h、24h、48h、72 h时间点,观察比较行为学与脑组织形态学的变化。结果:(1)实验组枕大池和蛛网膜下腔内发现大量的血凝块和血液。(2)实验组光镜下可观察到蛛网膜下腔大量红细胞,神经元细胞水肿,电镜下可见胶质细胞空泡样改变,神经元细胞线粒体肿胀,髓鞘内存在空泡和板层分离现象。(3)实验组造模后均出现明显的神经系统损伤,对照组4h出现典型神经系统损伤表现,12h后恢复正常,空白组未见神经系统损伤表现。结论:枕大池一次注血法是一种简便、可重复的症状性自发性蛛网膜下腔出血早期脑损伤模型,适宜于研究早期脑损伤的致病机制。     

家兔自发性蛛网膜下腔出血早期脑损伤模型的初步研究

目的：建立一种可重复的、有典型神经损伤症状的自发性蛛网膜下腔出血（sAH）动物模型,为研究蛛网膜下腔出血后早期脑损伤（EBI）的机制提供可靠的动物模型。方法：选用新西兰大耳白兔60只,随机分为实验组、对照组、空白组。采用枕大池穿刺一次注入自体动脉血法建立SAH模型。在4h、8h、12h、24h、48h、72h时间点,观察比较行为学与脑组织形态学的变化。结果：（1）实验组枕大池和蛛网膜下腔内发现大量的血凝块和血液。（2）实验组光镜下可观察到蛛网膜下腔大量红细胞,神经元细胞水肿,电镜下可见胶质细胞空泡样改变,神经元细胞线粒体肿胀,髓鞘内存在空泡和板层分离现象。（3）实验组造模后均出现明显的神经系统损伤,对照组4h出现典型神经系统损伤表现,12h后恢复正常,空白组未见神经系统损伤表现。结论：枕大池一次注血法是一种简便、可重复的症状性自发性蛛网膜下腔出血早期脑损伤模型,适宜于研究早期脑损伤的致病机制。     

RT-SHIV感染中国恒河猴及体内传代

目的了解RT-SHIV感染中国恒河猴的感染特点,研究RT-SHIV在中国恒河猴中传代特点;建立RT-SHIV中国恒河猴动物模型,为评价HIV-1药物有效性提供动物平台。方法选择4只健康恒河猴,其中两只动物经上肢静脉感染RT-SHIV病毒,感染急性期采取外周血分离CD8-PBMC,扩增病毒,将新制备的病毒静脉感染另外两只中国恒河猴,通过监测血浆病毒载量,CD4＋/CD8＋比值,CD4＋T淋巴细胞和B淋巴细胞的绝对数,了解实验猴的感染状态,同时分析病毒RT基因变异情况。结果 4只动物均获得系统性感染,且传代动物急性期表现更为强烈,RT基因在感染和传代的过程中共观察到3个氨基酸的改变。结论本研究为RT-SHIV中国恒河猴模型的建立提供了基础信息。     

SARS-CoV动物模型研究之中国现状

目的综合对比SARS-CoV感染的恒河猴、布氏田鼠及Lewis大鼠的病理学、免疫学以及病毒的复制与外排情况的变化,来探讨此三种动物在建立SARS模型上的特点。方法SARS病毒感染8只恒河猴、9只Lewis大鼠和20只布氏田鼠,在感染后不同时间安乐死动物,应用光镜对动物的各脏器进行病理观察研究;用病毒分离和RT-PCR方法检测病毒外排与复制的情况;用ELISA法检测动物产生特异性抗体情况。结果在SARS-CoV感染恒河猴、Lewis大鼠和布氏田鼠后,肺组织均出现一定的与人类SARS疾病相似的病理改变,在动物体内均可检测到活病毒或病毒核酸,并可检测到特异性IgG抗体的存在。在病死率上布氏田鼠最高;在病毒的复制与外排方面恒河猴的检出率最高,持续时间最长;在抗体产生情况上恒河猴与Lewis大鼠基本相似;在病理变化上恒河猴病变最重且最为复杂,与人类SARS疾病的病理变化最为接近。结论布氏田鼠,Lewis大鼠,特别是恒河猴动物模型可以用于SARS发病机制、疫苗和药物的研发,恒河猴动物模型是目前研究SARS疾病最理想的动物模型。     

中国恒河猴Mamu-A*01基因筛查及其对抗原肽p11C的特异性CTL反应

目的 筛查中国恒河猴Mamu-A*01基因,比较中国恒河猴和印度恒河猴的Mamu-A*01基因序列和功能是否相同.方法 PCR方法检测128只中国恒河猴,用特异性引物扩增Mamu-A*01基因,将PCR扩增后的产物克隆测序后与印度恒河猴的Mamu-A*01基因进行同源比对;酶联免疫斑点检测 (ELISPOT) 方法分别检测5只Mamu-A*01基因阳性和5只阴性恒河猴针对SIV、SHIV抗原肽p11C的特异性CTL反应.结果 共筛查出5 只Mamu-A*01基因阳性恒河猴 (3.91%),经测序分析后与印度恒河猴的同源性可达99.1 %.这5只均为SIV/SHIV感染恒河猴,其中四只SIV感染的猴的ELISPOT结果显示针对p11C的高频CTL反应,斑点数在500-1400/106 PBMCs之间,而另1只SHIV感染的恒河猴及5只阴性猴没有斑点出现.结论 中国恒河猴含有Mamu-A*01基因,基因频率有区域性差异,中国恒河猴的Mamu-A*01可提呈特异性抗原肽p11C.     

SHIV-KB9感染中国恒河猴动物模型的建立

目的为了进一步确证SHIV-KB9感染中国恒河猴的病毒浓度范围,测试动物对病毒的适应性,明确该动物模型的可重复性。方法实验前采集猴血清并进行血清学检查。选出4只无SIV、STLV、SRV/D和B病毒感染的恒河猴,分别用10倍系列稀释的病毒液静脉感染实验猴,使用流氏细胞术、血常规、病毒分离、DNA-PCR和RT-PCR等方法确定实验猴是否被感染,以及感染后恒河猴体内病毒复制和免疫细胞损伤情况。结果实验猴的血浆病毒载量、病毒分离结果、CD4＋/CD8＋比值和CD4＋T细胞数等证实,4.8×105 copies/mL以上浓度的SHIV-KB9病毒液能成功感染中国恒河猴。结论本研究进一步明确了SHIV-KB9感染中国恒河猴的有效病毒浓度范围,确定了SHIV-KB9病毒感染中国恒河猴的病毒学、免疫学的测定指标,成功的建立了SHIV-KB9/中国恒河猴动物模型。     

C亚型SHIVCHN19P4强毒株在中国恒河猴体内的传代研究

目的研究C亚型SHIVCHN19P4强毒株在中国恒河猴体内传代中病毒学和免疫学等反应的变化特点,分离制备SHIVCHN19P4中国恒河猴传代适应株病毒。方法选择4只健康成年恒河猴,其中两只经后肢静脉感染SHIVCHN19P4病毒,60 d后,分别采集EDTA抗凝全血静脉途径传代至另两只猴,使用流式细胞术、PCR、结合抗体检测和序列分析等方法研究传代动物病毒学、免疫学和序列变异特点。选择性从传代动物感染急性期外周血中分离PBMC,CD8＋T细胞敲除后与正常PBMC共培养分离病毒。结果 4只传代动物均获得系统性感染,且传代后病毒毒力明显增强,序列分析发现SHIVCHN19P4病毒序列在传代过程中发生适应性改变;同时,成功分离制备SHIVCHN19P4传代适应性病毒株。结论 SHIVCHN19P4在中国恒河猴体内适应性传代研究为进一步建立C亚型SHIV强毒株/SAIDS模型奠定了良好的实验基础,为研究C亚型HIV-1流行株致病特点以及预防性黏膜疫苗和杀微生物的有效性评价提供了数据支持。     

改良导管固定法在硬膜外阻滞大鼠模型构建中的应用

目的探讨改良硬膜外导管固定法应用于构建硬膜外阻滞动物模型的可行性及效果。方法采用随机数字法将24只雄性Wistar大鼠分为实验组和对照组,每组12只,用于构建硬膜外阻滞大鼠模型。实验组在切开置管后导管固定使用创新性关卡,采用不透气无菌医用胶带封闭导管;对照组则采用单纯缝线环绕法固定,并保留导管接头固定于后颈部。术后每天硬膜外腔注射0.1%罗哌卡因30 uL,共28 d。对比观察两组术后发生感染率,硬膜外阻滞模型有效时间及硬膜外导管深度变化等情况。结果术后感染发生率：两组间差异无统计学意义（P〉0.05）;模型有效时间：实验组长于对照组（26.2±1.7d＆18.5±3.0d,P〈0.05）;硬膜外腔内导管深度：实验组与对照组比,其均值和离散程度的差异均具有显著性（1.81±0.07＆1.44±0.55,P〈0.05）。结论应用改良导管固定法构建大鼠硬膜外阻滞模型,有助于提高其稳定性和成功率。     

兔、犬、猴硬脊膜外腔及椎管内置管法

硬脊膜外腔及椎管内用药是目前临床镇痛和麻醉药物的常用施药途径,在镇痛麻醉药物临床前研究中需要对动物施以硬脊膜外与椎管内置管手术以便进行药效与毒性研究。本研究建立一种实用于兔,犬,猴等较大动物的硬脊膜外及蛛网膜下腔的短期（数天）和长期（数月）置管方法。该方法结合临床穿刺和造影术定位,并根据动物的不同种类和差异进行适配和改进。上述置管法应用于虎纹镇痛肽（HWAP－I）的临床前药效和毒理研究,经200多例次兔,犬和猴动物实验证明,这是一种定位准确稳定可靠的置管法。     

一种Beagle犬腰椎穿刺置管术介绍

目的建立一种新的Beagle犬脑脊液连续留取的方法。方法通过氯胺酮麻醉进行腰椎穿刺后置管可连续留取脑脊液。结果腰椎穿刺置管后成功连续留取清亮脑脊液超过48h。结论腰椎穿刺置管连续留取脑脊液简单、有效。     

实验用猴脑脊液采集技术方法的创新

脑脊液在艾滋病的研究中有着重要的意义。近年来脑脊液的检测逐步成为SIV/SHIV感染猴模型研究和应用中的重要指标。传统的采集方法不易学习和掌握。针对上述情况我们优化了脑脊液的采集方法,优化后的方法明显缩短穿刺时间,显著提高成功率。     

Calculating lumbar puncture depth in children

Lumbar puncture was performed in 195 children and the depth of needle was recorded. Our results show that the depth of lumbar puncture necessary to obtain uncontaminated cerebrospinal fluid correlates best with the child's weight. The simple formula: mean depth of insertion (cm) = 1.3 + 0.07 x body weight (kg), can be used to estimate the depth of lumbar puncture of children older than 3 months. The depths of lumbar puncture of children younger than 3 months are mostly 1.0-1.5 cm.     

In vivo influences on cerebrospinal fluid amino acid levels

The concentration of 19 amino acids and ethanolamine in two independent groups (n = 36, n = 19) of normal human cerebrospinal fluid (CSF) samples were measured by high performance liquid chromatography. Age, sex, time of lumbar puncture, position during lumbar puncture, protein and glucose content of the CSF were monitored and the influence of these parameters on CSF amino acid levels was determined. Hypotheses formulated after observing measurements from the first group of CSF samples were tested against the second group of CSF samples using conservative statistics. The main finding was a positive correlation between CSF glucose and CSF glutamate levels.     

Spinal puncture headache

Headache is the commonest complication of spinal puncture. There is no significant difference in the incidence of headache after lumbar puncture, whether or not the puncture is followed by injection of an anesthetic agent. The sequence of events leading to postlumbar puncture headaches is probably (1) decreased volume of cerebrospinal fluid with lowered pressure; (2) increased differential between the pressure of the cerebrospinal fluid and the intracranial venous pressure; (3) dilation of venous structures with increase in brain volume; and (4) production of tension on the pain sensitive areas in the cranium. Prevention of postlumbar puncture headache consists largely in attempts to avoid the development of the pressure differential between that of the cerebrospinal fluid and intracranial venous pressure. Treatment consists of analgesics, hydration and attempts to restore normal cerebrospinal fluid pressure.     

脑脊液检查在早产儿及足月儿细菌性脑膜炎的诊断价值

目的:评价脑脊液检查在早产儿及足月儿细菌性脑膜炎诊断中的价值。方法:选取2014年6月1日至2016年12月31日上海市儿童医院新生儿科收治的行腰椎穿刺检查的447例新生儿,回顾性分析新生儿的一般资料、脑脊液常规生化、培养等指标,根据胎龄将患儿分为早产儿167例与足月儿280例,再根据有无患发细菌性脑膜炎分为早产儿细菌性脑膜炎27例(早产儿观察组)、早产儿非细菌性脑膜炎140例(早产儿对照组)、足月儿细菌性脑膜炎38例(足月儿观察组)、足月儿非细菌性脑膜炎242例(足月儿对照组),采用受试者工作特征(ROC)曲线评估蛋白定量、白细胞计数、葡萄糖对早产儿及足月儿细菌性脑膜炎的诊断价值。结果:与同组对照组相比,足月儿观察组和早产儿观察组蛋白定量和白细胞计数均明显升高,而葡萄糖含量显著下降,且差异均具有统计学意义(P0.05);本研究65例细菌性脑膜炎患儿脑脊液培养分离出11株细菌(16.9%)。足月儿脑脊液白细胞计数、蛋白定量以及葡萄糖诊断细菌性脑膜炎的ROC曲线下面积分别为0.995、0.846、0.703。早产儿脑脊液白细胞计数、蛋白定量以及葡萄糖诊断细菌性脑膜炎ROC曲线下面积分别为0.970、0.711、0.705。结论:脑脊液白细胞计数、蛋白定量在足月儿和早产儿细菌性脑膜炎中具有较高的诊断价值。     

Effect of naloxone administration upon the diurnal concentrations of oxytocin in the cerebrospinal fluid of rhesus and cynomolgus monkeys.

A diurnal pattern in oxytocin concentrations is present in cerebrospinal fluid (CSF) removed from the spinal subarachnoid space of monkeys, with elevated levels occurring in the early light hours. In order to investigate the possible role of endogenous opioid peptides in the generation of this oxytocin rhythm, we administered naloxone (0.4 mg/kg/h x 48 h) to rhesus and cynomolgus monkeys and examined the effects on the diurnal pattern of oxytocin in CSF collected from the lumbar subarachnoid spinal space. Monkeys maintained on jacket/tether/swivel systems and in a 12 h light: 12 h dark cycle (lights on 07.00-19.00 h) were implanted with temporary spinal subarachnoid catheters. CSF was continuously collected from the lumbar subarachnoid space and assayed for oxytocin. Oxytocin concentrations in CSF showed a diurnal variation with peak and nadir concentrations during light and dark hours, respectively. The lumbar CSF concentrations of oxytocin were not significantly different during naloxone vs. saline infusion. Plasma oxytocin concentrations, measured in the same animals, displayed no diurnal variation and were not significantly different during naloxone vs. saline infusion. We conclude that naloxone administration for 48 h does not perturb the diurnal variation in oxytocin concentrations in the CSF of monkeys. Mu opioid receptors are unlikely to be involved in modulating the diurnal rhythm of oxytocin in the CSF of monkeys.     

重复经颅磁刺激对创伤性颅脑损伤患者脑脊液中兴奋性氨基酸含量的影响

目的：探讨重复经颅磁刺激（rTMS）对急性颅脑损伤患者脑脊液中兴奋性氨基酸（EAA）含量的影响。方法：30例创伤性颅脑损伤（TBI）病人按格拉斯哥昏迷评分分为轻型组（rTMS3）、中型组（rTMS2）、重型组（rTMS1）,每组10例,各组病人分别随机分为rTMS对照亚组（A组）及治疗亚组（B组）,每亚组5例。于TBI后第15天行腰椎穿刺采集脑脊液,采用高效液相色谱法测定脑脊液中谷氨酸（ASP）及门冬氨酸（GLU）含量。结果：脑脊液ASP和GLU水平随着脑损伤程度的加重而升高,各rTMS治疗组与相应各对照组的EAA相比,rTMS治疗组EAA的水平均低于相应对照组。结论：rTMS可通过降低TBI后脑脊液EAA水平发挥脑保护作用。脑脊液EAA的含量变化可作为TBI严重程度的生化指标。     

Taurine in plasma and CSF: a study in healthy male volunteers

In order to explore the interrelationship between plasma and cerebrospinal fluid taurine concentrations, three consecutive 6-ml fractions of cerebrospinal fluid were drawn from 30 healthy male volunteers in the early morning after 8 h in the fasting condition. Repeated plasma samples were drawn over 24 h the day before lumbar puncture. Taurine in plasma and cerebrospinal fluid was determined by high performance liquid chromatography. The subjects were categorized as extensive or poor metabolizers with respect to the cytochrome P450 2D6 genotype. The taurine cerebrospinal fluid/plasma ratio at 8 a.m. was negatively influenced by the plasma taurine concentration at 4 p.m. the previous day. It was also negatively influenced by body mass index and positively by the intraspinal pressure. Three poor metabolizers of cytochrome P450 2D6 had higher plasma taurine areas under the curve than 27 extensive metabolizers. Hypothetically, cytochrome P450 2D6 influences the transport of taurine across the blood–brain barrier.     

Cerebrospinal fluid cyclic AMP levels in rhesus monkeys: daily fluctuations.

Daily fluctuations in cerebrospinal fluid cyclic AMP levels were found in neurologically intact rhesus monkeys. When animals were maintained in continuous dim light as opposed to a 12L/12D regimen, the fluctuations in cerebrospinal fluid cyclic AMP levels appeared to be reduced. Knowledge of these daily fluctuations may be helpful for protocols of future experiments concerning measurements of CSF cAMP.