树鼩海马微环境谷氨酸异常与rCBF相互作用的探讨

目的:探讨海马微环境内异常谷氨酸与局部脑血流(regional cerebral blood flow,rCBF)互动变化及其可能机制。方法:单泵等速微灌流系统分别行树鼩海马高、中、低谷氨酸微灌流4 h,通过激光多普勒血同时NM-DA-NR1表达增加流计测量海马CA1区rCBF含量;并用免疫组化观察海马CA1区血管内皮细胞NMDA-NR1表达。微灌流谷氨酸溶液后原位灌流其受体拮抗剂MK-801,并观测上述指标改变。结果:树鼩微环境内随着谷氨酸的增加海马rCBF逐渐降低(P<0.01),其中高浓度谷氨酸微灌流后的rCBF(PU)最低(6.9±0.3,P<0.01)同时NMDA-NR1表达增加(P<0.01),使用Glu受体NMDA拮抗剂MK-801后可显着升高rCBF,同时可降低NM-DA-NR1表达(P<0.01)。结论:海马神经元微环境中Glu增多时,可诱导局部脑血管内皮细胞损伤继而可能是诱发海马神经元损伤的原因之一。     

树鼩海马微灌流谷氨酸和钙所致线粒体应激及银杏内酯B的影响

目的观察树鼩海马微环境中谷氨酸(Glu)及钙(Ca2 )改变对细胞色素C(CytC)释放以及caspase凋亡基因激活的影响,探讨银杏内酯B(GB)干预海马神经元线粒体应激的分子机制。方法使用单泵等速微灌流系统行树鼩海马Glu及Ca2 微灌流,24h后用免疫组化法观察海马神经元CytC蛋白表达;免疫印迹法检测线粒体及胞质的CytC含量;实时荧光定量PCR技术检测海马caspase-3及caspase-9 mRNA的含量。微灌流Glu和Ca2 溶液后6h于舌下静脉注射银杏内酯B5mg/kg,24h后观察上述指标的改变。结果树鼩海马微灌流Glu和Ca2 溶液后24h,线粒体CytC含量显著下降,而胞质部分出现CytC;海马组织caspase-3、caspase-9 mRNA明显升高。GB治疗组线粒体CytC含量增多,胞质中仍有CytC存在,而海马组织caspase-9 mRNA显著降低,但caspase-3 mRNA无差异。结论海马微环境中Glu与Ca2 的大量堆积促进线粒体CytC的释放,可能是激活caspase级联反应而导致线粒体应激、神经元继发损伤的始动环节;GB的神经保护效应可能与其特异性拮抗血小板活化因子(PAF)受体,抑制Ca2 内流,部分阻滞CytC释放入胞质,从而减少caspase-9基因转录有关。     

银杏叶制剂对蛛网膜下腔出血 早期大鼠脑缺血的影响

目的:探讨银杏叶制剂(GBE)对蛛网膜下腔出血(SAH)早期缺血性脑损伤的防治作用。方法:对假手术组(SO组)、SAH组和SAH+GBE组大鼠检测24h内局部脑血流量(rCBF)、脑组织内皮素-1(ET-1)含量和Ca含量改变,并对海马CA1区组织作光镜检查。结果:SAH组于术后rCBF迅速而持续降低,脑组织ET-1含量和Ca含量在诱导SAH 1 h后均显著高于SO组,3 d后海马CA1区神经元明显受损。 SAH+GBE组的上述改变均较轻。结论:GBE可减轻SAH后缺血性脑损伤。     

CsA对树鼩海马微灌流谷氨酸和钙所致线粒体应激的影响

目的：观察环孢菌素A(CsA)对树鼩海马由谷氨酸（Glu）及钙（Ca²⁺）引起微环境改变所致线粒体应激的影响，并探讨其分子机制。方法:单泵等速微灌流系统行树鼩海马Glu及Ca²⁺微灌流，24 h后免疫组化法检测海马神经元细胞色素C（Cyt C）蛋白表达；低温差速离心分离海马神经元线粒体和胞质部分，免疫印迹（Western blotting）法检测Cyt C在胞内表达空间分布；实时荧光定量PCR技术检测海马caspase-3及caspase-9 mRNA的含量。微灌流Glu和Ca²⁺溶液后6 h于舌下iv CsA 40 mg/kg BW，24 h后观察上述指标的改变。结果:树鼩海马微灌流Glu和Ca²⁺溶液后24 h，海马神经元Cyt C表达增强，而线粒体Cyt C含量显著下降，同时胞质部分可见Cyt C；海马组织caspase-3、caspase-9mRNA明显升高；微灌流后6 h静脉注射CsA组, 海马神经元Cyt C表达显著减少，而线粒体Cyt C含量则显著增加，胞质部分未见Cyt C;海马组织caspase-3、caspase-9 mRNA降低。结论:海马微环境中Glu与Ca²⁺的大量堆积,可促进线粒体Cyt C释放，激活caspase级联反应而导致线粒体应激；CsA的神经保护效应可与其抑制线粒体通透性转导孔（MPT）开放，防止Cyt C释放及减少caspase-3和caspase-9的活化有关。     

海马内微量注射硫酸镁对马桑内酯诱发大鼠痫样放电的影响

目的和方法:采用皮层应用或海马内注射马桑内酯的方法致痫大鼠,用钙离子选择性微电极检测了致痫动物皮层、海马细胞外Ca2+浓度的变化,观察了海马CA2区注射硫酸镁对致痫大鼠脑电图和皮层、海马诱发电位的影响.结果:马桑内酯致痫时,皮层、海马细胞外Ca2+浓度分别降低了0.61 mmol·L-1和0.74 mmol·L-1,海马内注射硫酸镁能明显地抑制致痫动物皮层、海马诱发电位和脑电图痫样放电.结论: 镁盐的上述作用可能与抑制Ca2+流入神经元内有关.     

高血糖对树鼩脑皮层局灶性缺血时海马微环境离子稳态及神经元继发性损伤的影响

 目的：研究高血糖对树鼩脑皮层血栓性缺血时海马微环境离子稳态的影响,探讨高血糖在缺血后神经元继发性损伤中的作用及机制。方法：用链脲佐菌素复制树鼩高血糖模型,并用光化学方法诱导脑皮层局部血栓性缺血,用单泵等速微灌流系统和离子分析仪测定缺血4 h、24 h及72 h海马离子微环境（细胞外pH值、K+、Na+、Ca2+、Cl-）的动态变化,并观察脑组织的病理形态学改变及海马神经元密度变化。结果：树鼩脑皮层缺血后,其海马微环境内出现了pH值、Na+、Ca2+及Cl-含量的降低,K+含量升高,变化以缺血后4 h为著,24 h次之,72 h无显著差异;高血糖加缺血进一步加重离子稳态的紊乱,缺血后4 h的pH值、K+和Ca2+含量,以及缺血后24 h的pH值和Na+含量与正常血糖缺血组同期值相比,变化显著（P<0.05）。形态学观察显示,光化学反应后4 h照射区皮层可见梗死灶,且患侧海马CA1区也存在缺血损伤性改变;24 h病损达高峰;72 h伴随胶质细胞增生等修复性反应。相应时点高血糖加缺血组皮层及海马的损伤均大于缺血组,以缺血后24 h(P<0.01)和72 h(P<0.05)尤为显著。结论: 树鼩脑皮层血栓性缺血形成后,缺血中心区扩布所导致的微环境内酸碱平衡及离子稳态性异常可能是海马神经元继发性损伤的重要原因,高血糖可加剧缺血脑区离子微环境的紊乱。     

皮质酮对原代培养的海马神经元及其Ca~(2+)/CaMKII的影响

在培养液中加入 10 7、10 6和 10 5浓度(mol/L)的皮质酮,采用MTT染色测定、Fura 2 /AM的荧光标记以及Western印迹的方法,观察了不同浓度的皮质酮作用下海马神经元形态学和细胞存活率的变化以及胞浆内游离钙离子浓度 [Ca2+ ]i和CaMKII表达的变化规律, 探讨其对原代培养的大鼠海马神经元及其Ca2+ /CaMKII的影响和可能的机制。结果发现: 10 6、10 5浓度的皮质酮对海马神经元的形态学影响较大,与对照组比较,细胞存活率明显降低; [Ca2+ ]i分别为 113. 1022±16. 9716、155.3794±20. 7727;CaMKII的表达也明显减少;三者的变化均显著 (P<0. 01 ),而 10 7浓度的皮质酮对上述指标影响不大 (P>0.05)。此外,相关性分析表明: [Ca2+ ]i和CaMKII的表达呈现负相关(P<0. 05)。以上结果提示,皮质酮对大鼠海马神经元的作用存在浓度依赖效应,浓度越高,对大鼠海马神经元的损伤越大,同时也验证了皮质酮是啮齿类动物的主要的应激激素。     

后适应对树鼩不同脑缺血模型同一"时间窗"海马神经元保护的比较

目的: 观察缺血后适应(PC)对血栓性脑缺血(TC)及大脑中动脉闭塞(MCAO)2种脑缺血模型同一"时间窗"的脑保护效果,并探讨其可能机制。方法: 建立树鼩TC及MCAO脑缺血模型,于脑缺血后4 h夹闭同侧颈总动脉实施缺血PC。用TTC染色显示皮层梗塞面积,采用电镜及激光多普勒(LD)技术分别观察脑缺血后4 h、24 h及72 h海马CA1区神经元超微结构及局部脑血流(rCBF)的变化,比较PC对2种中风模型海马 rCBF和神经元超微结构的影响。结果: 脑缺血后海马CA1区神经元的超微结构改变以线粒体肿胀、嵴断裂以及内质网扩张为主,MCAO的梗塞面积和细胞损伤程度明显高于TC组,且PC缓减TC组海马线粒体损伤的作用较MCAO的明显。TC和MCAO后海马rCBF明显降低,TC组rCBF的降低以24 h明显(P＜0.01),而MCAO组rCBF的降低则以72 h明显(P＜0.01)。脑缺血后4 h实施PC可改善TC组海马rCBF(P＜0.01)和减轻线粒体损伤,而MCAO组rCBF及神经元形态学改善不明显。结论: 脑缺血后4 h实施缺血PC能有效对抗缺血性脑损伤,其脑保护效果与rCBF的增加有关。缺血PC对TC组的脑保护作用较MCAO组明显。     

树鼩脑缺血后适应升高海马区rCBF及VEGF的变化

目的 探讨缺血后适应(PC)缓解海马rCBF与血管内皮生长因子(VEGF)的变化及其机制.方法 建立树鼩血栓性局部脑缺血模型,通过激光多普勒血流计测量海马CA1区rCBF含量;用免疫组化法测定海马VEGF的表达.结果 树鼩脑缺血时海马rCBF逐渐降低,以24 h的改变最显著,脑缺血后海马CA1区VEGF阳性细胞数增多,12 h表达最强(P<0.01);缺血PC可显著影响缺血所致的改变:rCBF逐渐增加,72 h最显著(P<0.01),与此同时VEGF的表达除8 h外均比血栓性缺血组增强(P<0.01),12 h组最明显;电镜显示缺血24 h血栓性缺血组的海马线粒体应激及内质网池形成最明显,给予PC后得以缓解.结论 缺血12 h内PC通过明显增强VEGF的表达可能与其改善rCBF有关,从而延长治疗的时间窗.     

50Hz磁场长期暴露对大龄大鼠学习记忆及脂质过氧化反应的影响

目的探讨长期0.1mT 50Hz磁场暴露对大龄大鼠学习记忆及海马组织的超氧化物歧化酶(SOD)、Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性与丙二醛(MDA)水平的影响.方法应用Morris水迷宫方法测定动物的空间学习记忆能力;应用试剂盒分别测定SOD、Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性及MDA水平.结果 10d 0.1mT 50Hz磁场暴露对大龄大鼠的学习记忆功能无明显影响.长期(4个月或8个月)0.1 mT 50Hz磁场暴露能使大鼠的逃避潜伏期明显延长,穿环系数显著减少,表明人鼠的空间学习记忆能力受损.长期磁场暴露也使大龄人鼠海马组织及其线粒体的SOD活件降低,MDA水平升高,Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性降低.结论长期0.1 mT 50Hz磁场暴露可损伤大龄大鼠的学习记忆能力,此作用可能与其增强海马组织的脂质过氧化反应有关.     

Activation of the intracerebral cholinergic nerve fibers originating in the basal forebrain increases regional cerebral blood flow in the rat's cortex and hippocampus

In the rat, activation of the intracerebral cholinergic system originating in the basal forebrain and projecting to the cortex and hippocampus releases acetylcholine in the cortex and hippocampus, which results in vasodilation and an increase in regional cerebral blood flow (rCBF) in the cortex and hippocampus. The augmentation of rCBF is independent of both systemic blood pressure and regional metabolism. The intracerebral cholinergic fibers are able to act as autonomic nerve fibers for the regulation of cortical and hippocampal blood flow.     

Anteroposterior perfusion heterogeneity in human hippocampus measured by arterial spin labeling MRI

Measurements of blood flow in the human hippocampus are complicated by its relatively small size, unusual anatomy and patterns of blood supply. Only a handful of arterial spin labeling (ASL) MRI articles have reported regional cerebral blood flow (rCBF) values for the human hippocampus. Numerous reports have found heterogeneity in a number of other physiological and biochemical parameters along the longitudinal hippocampal axis. There is, however, only one ASL study of perfusion properties as a function of anteroposterior location in the hippocampus, reporting that rCBF is lower and the arterial transit time (ATT) is longer in the anterior hippocampus than in the posterior hippocampus of the rat brain. The purpose of this article was to measure ATT and rCBF in anterior, middle and posterior normal adult human hippocampus. To better distinguish anteroposterior perfusion heterogeneity in the hippocampus, a modified ASL method, called Orthogonally Positioned Tagging Imaging Method for Arterial Labeling with Flow‐sensitive Alternating Inversion Recovery (OPTIMAL FAIR), was developed that provides high in‐plane resolution with oblique coronal imaging slices perpendicular to the long axis of the hippocampus to minimize partial volume effects. Perfusion studies performed with this modified FAIR method at 3 T indicated that anterior, middle and posterior human hippocampus segments have unique transit time and rCBF values. Of these three longitudinal hippocampal regions, the middle hippocampus has the highest perfusion and the shortest transit time and the anterior hippocampus has the lowest perfusion and the longest transit time. Copyright © 2013 John Wiley & Sons, Ltd.     

缺血后适应对树鼩海马CA1区脑血流及星形胶质细胞活化的影响及可能机制

目的：研究缺血后适应（PC）对树鼩局部脑缺血海马CA1区脑血流（rCBF）与星形胶质细胞（AS）活化的影响，探讨缺血PC影响AS表达胶质纤维酸蛋白（GFAP)的可能机制。方法：建立树鼩血栓性局部脑缺血及缺血PC模型，通过激光多普勒（LD）血流计测量脑缺血后4 h、8 h、12 h、24 h及72 h海马CA1区rCBF的改变；用免疫组化法测定脑缺血上述时间海马GFAP的表达，并用图像分析系统测定其平均灰度值。于脑缺血后4 h重复3次夹闭缺血侧颈总动脉实施缺血PC，并观察其对海马CA1区rCBF和AS活化以及GFAP表达的影响。结果：树鼩脑缺血后4h海马CA1区GFAP阳性细胞数增多，AS表达GFAP增强，24h可见AS胀亡；72h海马CA1区AS表达GFAP达高峰（120.0±2.1，P＜0.01）。脑缺血时海马CA1区rCBF逐渐降低，以24 h的改变最显著,为(2.55±0.28) PU，P＜0.01；72 h时海马CA1区rCBF略有增加，为(9.84±1.22) PU。实施缺血PC后，海马CA1区rCBF逐渐增加，72 h最显著,为(18.74±1.60) PU，P＜0.01；此时海马GFAP表达进一步增强（111.0±1.3),P＜0.01。但AS胀亡的病理改变基本消失。结论：多次短暂的闭塞动物的颈动脉可延长树鼩脑缺血治疗的“时间窗”；缺血PC的脑保护机制可能与增加海马rCBF、调控AS活化及改善海马微环境有关。     

Association between major depressive symptoms in heart failure and impaired regional cerebral blood flow in the medial temporal region: a study using 99m Tc-HMPAO single photon emission computerized tomography (SPECT)

BACKGROUND AND PURPOSE: Depressive symptoms are frequently associated with heart failure (HF), but the brain mechanisms underlying such association are unclear. We hypothesized that the presence of major depressive disorder (MDD) emerging after the onset of HF would be associated with regional cerebral blood flow (rCBF) abnormalities in medial temporal regions previously implicated in primary MDD, namely the hippocampus and parahippocampal gyrus. METHOD: Using 99mTc-SPECT, we measured rCBF in 17 elderly MDD-HF patients, 17 non-depressed HF patients, and 18 healthy controls, matched for demographic variables. Group differences were investigated with Statistical Parametric Mapping. RESULTS: Significant rCBF reductions in MDD-HF patients relative to both non-depressed HF patients and healthy controls were detected in the left anterior parahippocampal gyrus and hippocampus (ANOVA, p=0.008 corrected for multiple comparisons) and the right posterior hippocampus and parahippocampal gyrus (p=0.005 corrected). In the overall HF group, there was a negative correlation between the severity of depressive symptoms and rCBF in the right posterior hippocampal/parahippocampal region (p=0.045 corrected). CONCLUSIONS: These findings are consistent with the notion that the medial temporal region is vulnerable to brain perfusion deficits associated with HF, and provide evidence that such functional deficits may be specifically implicated in the pathophysiology of MDD associated with HF.     

Positron emission tomography reveals changes in global and regional cerebral blood flow during noxious stimulation of normal and inflamed elbow joints in anesthetized cats

In cats the global (gCBF) as well as the regional cerebral blood flow (rCBF) and blood pressure were measured before, during, and after noxious inward and outward rotations of normal and inflamed elbow joints. The animals were anesthetized with halothane and immobilized by gallamine triethiodide. The gCBF as well as the rCBF were measured using positron emission tomography (PET) with a camera specifically designed for use in small animals. Slow intravenous bolus injections of ¹⁵O-labeled water were followed by 3-min acquisition of regional radioactivity starting at the time of injection. In all experiments the gCBF as well as the blood pressure were increased by noxious inward-outward rotations of the normal and of the inflamed joint, whereas the blood pressure and the rCBF remained unchanged during bolus injections under control conditions (without any joint movement). Movements of the inflamed joint evoked significantly greater increases in blood pressure and gCBF than corresponding ones of the normal joint. These increases in gCBF were paralleled by increases in rCBF along the complete anterior to posterior axis of the brain. Again, the increases in rCBF were larger, more extensive and more uniform following the stimulation of the inflamed joint relative to the results obtained with stimulation of the normal joint. No significant laterality was seen, but when an atlas-based region of interest (ROI) analysis was carried out and when the individual variations in rCBF were removed with two-way ANOVA, significant differences were disclosed in rCBF between the stimulated condition and the resting condition in a large number of brain regions. In particular, noxious rotation of the normal (right) elbow joint induced a significant increase in rCBF over the cerebral cortex and in the right thalamus and hippocampus. The same stimulation of the (left) inflamed joint induced a significant increase in rCBF throughout the brain; the biggest increase being over the right posterior cortex. It is concluded that under the conditions of the present experiments the generally accepted autoregulation of the cerebral blood flow is not fully functioning, and various factors that may be responsible for this failure (which obscures rCBF differences) are discussed. The more pronounced increases in rCBF when moving inflamed joints instead of normal ones is thought to be a direct consequence of the peripheral sensitization of the articular nociceptors and the consequent central hyperexcitability induced in the articular nociceptive pathways. Received: 2 June 1997 / Accepted: 12 August 1997     

Long-term measures of free testosterone predict regional cerebral blood flow patterns in elderly men

We previously reported that high circulating free testosterone (T) was associated with better performance on tests of memory, executive function, and spatial ability, and with a reduced risk for Alzheimer's disease. In this study, we report that free T levels, measured on multiple occasions over 14 years, predict regional cerebral blood flow (rCBF) measured by PET in 40 older men. Voxel-based regression, indicated that higher Free T was associated with increased rCBF in the hippocampus bilaterally (extending to the parahippocampal gyrus on the right), anterior cingulate gyrus, and right inferior frontal cortex. Total T concentrations were positively correlated with rCBF in the left putamen, bilateral thalamus, and left inferior frontal cortex and negatively correlated with amygdala rCBF bilaterally. These findings suggest that endogenous T influences brain physiology in regions critical for memory and attention and provide one mechanism through which T may affect cognitive function.     

尼莫地平对大鼠脑血管痉挛缺血性脑损害的防治作用

目的：探讨尼莫地平（ＮＤ）对蛛网膜下腔出血（ＳＡＨ）后脑血管痉挛（ＣＶＳ）缺血性脑损害的保护作用。方法：应用非开颅大鼠模型,观察ＳＡＨ组和ＮＤ处理组２４ｈ内微区脑血流量（ＣＢＦ）和海马组织Ｃａ含量动态变化及３天后海马ＣＡ１区形态学改变。结果：在ＳＡＨ后２４ｈ内,ＳＡＨ组ＣＢＦ明显而持续降低,海马组织Ｃａ含量逐渐增加,３天后海马ＣＡ１区神经元明显受损。ＮＤ使上述改变均减轻。结论：ＮＤ通过对微循环的改善增加ＳＡＨ后ＣＶＳ时ＣＢＦ,通过阻断脑缺血时的有害代谢环节而减轻ＣＶＳ缺血性神经元损伤。     

低氧对家兔局部脑血循环及脑组织中血管活性肠肽含量的影响

本实验观察了急性低氧及低氧习服家兔暴露于模拟海拔5000米低氧环境时局部脑血流量变化及脑组织中血管活性肠肽(简称VIP)含量变化。动物分为急性低氧组,低氧习服组和常压对照组。用脑电阻图法测定了家兔大脑皮层、下丘脑和海马三个部位的血流变化,用放射免疫分析法测定了以上部位组织中VIP的含量变化。结果表明,急性低氧可引起局部脑血流量增加,其幅度为42.6%～78.8%(P<0.05),但低氧习服动物三个部位血流量均无显著变化。急性低氧组脑组织VIP含量较对照有明显增加,大脑皮层VIP含量自对照107.9±8.3ng/s组织增至120.8±16.9ns/g组织,下丘脑自12.1±1.1ng/g组织增至21.1±2.9ng/g组织(P<0.05),海马VIP含量自对照的35.7±2.6ng/gTiss增至45.9±1.7ng/g组织(P<0.01),低氧习服组的VIP含量无显著变化。本实验结果提示,急性低氧可引起家兔脑组织中VIP含量增加,这一变化很有可能参与低氧时脑血流量的调节过程。