二十二碳六烯酸降低β淀粉样蛋白25-35致大鼠皮质神经元损伤

目的观察二十二碳六烯酸(DHA)对β淀粉样蛋白25-35(Aβ25-35)致原代培养大鼠皮质神经元损伤的保护作用。方法原代培养Wistar大鼠皮质神经元,先后给予不同剂量的DHA(20、50和100μmol/L)及Aβ25-35(25μmol/L),用CCK-8比色法观察神经元存活率,用激光扫描共聚焦显微镜观察细胞内游离钙离子浓度。结果 1)与对照组相比,Aβ25-35使细胞存活率明显下降(31%±6%,P<0.05);使细胞内游离钙离子浓度明显升高(249%±12%,P<0.05);2)孵育DHA可降低Aβ25-35引起的神经元存活率明显下降及细胞内游离钙离子浓度升高。结论 Aβ致细胞内钙超载是Aβ产生神经毒作用的一个方面,而DHA可部分拮抗Aβ25-35的神经毒作用。     

二十二碳六烯酸降低β淀粉样蛋白25-35致大鼠皮层神经元损伤

 摘要：目的 观察二十二碳六烯酸（DHA）对β淀粉样蛋白25-35（Aβ25-35）致原代培养大鼠皮层神经元损伤的保护作用。方法 原代培养Wistar大鼠皮层神经元,先后给予不同剂量的DHA（20、50、100μmol/L）及Aβ25-35（25μmol/L）,用CCK-8比色法观察神经元存活率,用激光共聚焦显微镜观察细胞内游离钙离子浓度。结果：1）与对照组相比,Aβ25-35使细胞存活率明显下降（31± 6 ％,n=8,　p＜0.05）;使细胞内游离钙离子浓度明显升高（249±12 ％,n=8, p＜0.05）;2）孵育DHA可降低Aβ25-35引起的神经元存活率明显下降及细胞内游离钙离子浓度升高。结论：Aβ致细胞内钙超载是Aβ产生神经毒作用的一个方面而DHA可部分拮抗Aβ25-35的神经毒作用。     

淫羊藿苷对缺血/再灌致神经元损伤的保护作用研究

目的 :观察淫羊藿苷对体外模拟缺血 /再灌致神经元损伤的保护作用 ,初步探讨其可能的作用机制。方法 :原代培养大鼠乳鼠大脑皮层神经元 ,分别在缺氧和缺糖 (6h) /再复氧和复糖培养后不同时间点 (0、3、1 2、2 4h)检测细胞活度、LDH活性、细胞凋亡率和细胞内游离钙离子浓度。结果 :淫羊藿苷 0 .2 5、0 .5和1mg·L 1明显减轻缺氧和缺糖 /复氧和复糖致神经细胞损伤 ,提高神经元MTT值 ,减少乳酸脱氢酶释放 ,减少凋亡和抑制细胞内游离钙离子浓度的升高。结论 :淫羊藿苷具有保护神经元抵抗模拟缺血性损伤的作用 ;其阻止细胞内钙离子超载可能是…     

人参皂甙Rd对皮层神经元兴奋性毒性损伤后胞内游离钙的影响

目的:探讨人参皂甙Rd(Ginsenoside Rd,GSRd)对皮层神经元兴奋性毒性损伤后细胞内游离钙离子浓度变化的影响。方法:采用原代方法培养大鼠皮层神经元,免疫荧光染色鉴定神经元纯度。应用激光共聚焦显微镜,观察GSRd对谷氨酸(Glutamate,Glu)和N-甲基-D-天门冬氨酸(NMDA)刺激后神经元胞内游离钙离子浓度变化的影响。使用钙离子荧光探针Fluo-4,AM标记细胞内游离钙,以Fluo-4的荧光强度反映细胞内游离钙浓度变化。结果:空白对照组荧光强度没有明显变化,而高浓度Glu刺激可迅速升高神经元胞内的荧光强度;在给予GSRd干预时,荧光强度升高的幅度明显降低,与MK-801的作用相似;NMDA刺激亦可使神经元胞内荧光强度明显升高,而加入GSRd干预时,荧光强度升高的幅度较NMDA损伤组有明显减小。结论:GSRd能够抑制高浓度Glu和NMDA引起的大量钙内流,提示减轻兴奋性毒性损伤过程中的钙超载可能是GSRd神经保护作用的机制之一。     

尼莫地平对急性颅脑外伤患者血浆中内皮素含量的影响

尼莫地平是一种钙通道拮抗剂，对神经细胞和脑血管内此细胞膜上的钙通道有特异性的阻滞作用。^[1]。为此，本文测定了37例急性颅脑外伤患者尼莫地平治疗前，后血浆内皮素（ET）水平变化，旨在观察尼莫地平对急性颅脑外伤患者血浆ET水平的影响。     

马桑内酯对大鼠海马神经元细胞内游离钙离子浓度的影响

以荧光探针Fluo-3/AM为胞浆内游离钙特异性指示剂,通过激光扫描共聚焦显微镜动态监测急性分离SD大鼠(出生后7～14 d)海马神经元细胞内游离钙离子浓度([Ca2 ]i)的变化情况,以了解钙离子及钙通道在癫痫发病中的作用,探讨马桑内酯(coriaria lactone, CL)调节神经细胞内钙稳态的机理.实验发现,CL能使急性分离大鼠海马神经元[Ca2 ]i增加;nifedipine和低浓度NiCl2虽可延迟,但不能完全阻断这种作用.除促使电压依赖的T-、L-型钙离子通道开放外,CL尚可通过其他途径增加海马神经元内[Ca2 ]i,改变细胞兴奋性而致痫.     

尼古丁对脐静脉内皮细胞蛋白激酶C和Ras蛋白活性及胞浆钙浓度的影响

为研究尼古丁对血管内皮细胞内游离钙、蛋白激酶C（PKC）和Ras蛋白作用,用激光共聚焦显微镜观察细胞内游离钙离子浓度的变化,用γ-^32P-ATP掺入法检测PKC活性,用GST－RBD沉淀并纯化活化的Ras蛋白和Western印迹杂交法检测Ras蛋白含量。观察到（1－5）10^4-mol/L尼古丁使人脐静脉内皮细胞[Ca^2 ]i迅速增加,峰值约超过基础值50％－200％,钙通道阻滞剂尼莫地平可拮抗尼古丁的此种效应;10^-4mol/L尼古丁作用1－12h后胞浆PKC活性明显降低;而胞膜PKC活性则升高。10^-4-10^-3mol/L尼古丁作用于人脐静脉内皮细胞10－30min可使活化的Ras蛋白含量增加。以上结果提示尼古丁可使人脐静脉内皮细胞[Ca^2 ]i增加和活化内皮细胞的PKC及Ras蛋白。     

实验性颅脑火器伤的脑电图研究

观察实验性颅脑火器伤瞬间的ＥＥＧ改变，发现伤时瞬间ＥＥＧ主要由极高波幅损伤电流和长时程的损伤电位构成，损伤电流时程（ＩＣＴ）反应脑损伤的严重程度。ＩＣＴ越长，脑损伤越严重，反之越轻，伤后ＥＥＧ主要为慢波和低波幅改变。     

骨髓间充质干细胞立体定向移植联合亚低温治疗大鼠脑损伤

背景：临床研究证明,亚低温(33~35 ℃)能有效减轻继发性脑和脊髓损伤,对中枢神经损伤有确切的保护作用。 目的：检测是否可以通亚低温治疗的方法提高骨髓间充质干细胞立体定向移植对重型颅脑损伤大鼠的治疗效果。 方法：采用液压颅脑损伤仪,给予Wistar大鼠2.53.31~303.98 kPa液压冲击力,制成重型液压颅脑损伤大鼠模型,将其随机分为脑损伤组,骨髓间充质干细胞移植组,亚低温+骨髓间充质干细胞组。后2组伤后6 h将体外培养的SD大鼠骨髓间充质干细胞立体定向移植到脑损伤灶内,亚低温+骨髓间充质干细胞组同时给予低温治疗。伤后第3天用Western Blot检测脑组织中AQP4蛋白合成的变化,采用干湿比重法测脑组织含水量。伤后24 h,3 d及伤后1,2 周行动物神经学缺损评分,2周后处死行免疫组织化学和苏木精-伊红染色。 结果与结论：亚低温治疗后,与脑损伤组和骨髓间充质干细胞移植组相比,亚低温+骨髓间充质干细胞组AQP4蛋白表达量及脑水肿程度也明显降低(P < 0.05),移植后1和2周,亚低温+骨髓间充质干细胞组的大鼠神经学缺损评分明显低于其他两组;且其脑组织切片中的神经元数量较其他两组明显增多(P < 0.01)。提示骨髓间充质干细胞立体定向移植联合亚低温治疗大鼠脑损伤可明显改善重型颅脑损伤后大鼠的神经学功能。     

氧反常心肌细胞内游离钙浓度的变化及丹参素和SOD的影响

目的：观察心肌细胞织反常时细胞内的游离钙浓度的变化,探讨丹参素和SOD对细胞内的游离钙浓度的影响。方法：体外分离成年大鼠心肌细胞复制氧反常模型,缺氧40min后给氧20min,用钙荧光探针（Fluo－3）分别测定了正常大鼠心肌细胞（Control,n=11）、缺氧40min（H40min,n=11）以及缺氧40min再给氧20min（H40minR20min,n=12）的大鼠心肌细胞内的游局钙浓度,并观察了丹参素（H40minR20min＋DS,n＝10)和SOD（H40minR20min＋SOD,n=11）对氧反常的心肌细胞内的游离钙浓度的影响,并用显微镜观察了细胞杆形率的改变。结果：氧反常组的细胞内的游离钙浓度（596．71±20．29nmol/L）明显比正常对照和单纯缺氧40min的高（158．67±5．01和328,51±13．86nmol/LP＜0.01）。加入丹参素和SOD,可明显降低氧反常细胞内的游离钙的浓度（分别为245.29±8．83和300．04±13.04nmol/L,P＜0．01）。细胞杆形率亦有类似的改变。结论：氧反常时心肌细胞发生明显的钙超载,丹参素和SOD对细胞内钙超载具有一定的防治作用。     

Alterations in neuronal calcium levels are associated with cognitive deficits after traumatic brain injury

Traumatic brain injury (TBI) survivors often suffer from a post-traumatic syndrome with deficits in learning and memory. Calcium (Ca(2+)) has been implicated in the pathophysiology of TBI-induced neuronal death. However, the role of long-term changes in neuronal Ca(2+) function in surviving neurons and the potential impact on TBI-induced cognitive impairments are less understood. Here we evaluated neuronal death and basal free intracellular Ca(2+) ([Ca(2+)](i)) in acutely isolated rat CA3 hippocampal neurons using the Ca(2+) indicator, Fura-2, at seven and thirty days after moderate central fluid percussion injury. In moderate TBI, cognitive deficits as evaluated by the Morris Water Maze (MWM), occur after injury but resolve after several weeks. Using MWM paradigm we compared alterations in [Ca(2+)](i) and cognitive deficits. Moderate TBI did not cause significant hippocampal neuronal death. However, basal [Ca(2+)](i) was significantly elevated when measured seven days post-TBI. At the same time, these animals exhibited significant cognitive impairment (F(2,25)=3.43, p<0.05). When measured 30 days post-TBI, both basal [Ca(2+)](i) and cognitive functions had returned to normal. Pretreatment with MK-801 blocked this elevation in [Ca(2+)](i) and also prevented MWM deficits. These studies provide evidence for a link between elevated [Ca(2+)](i) and altered cognition. Since no significant neuronal death was observed, the alterations in Ca(2+) homeostasis in the traumatized, but surviving neurons may play a role in the pathophysiology of cognitive deficits that manifest in the acute setting after TBI and represent a novel target for therapeutic intervention following TBI.     

长春新碱诱导的自噬性凋亡与细胞内游离Ca~(2+)浓度的相关性研究

目的　研究长春新碱 (VCR)诱导的L 0 2细胞自噬性凋亡时细胞内游离钙离子浓度 ([Ca2 +]i)的变化 ,以及自噬特异性抑制剂 3 methyladenine(3MA)对此自噬性凋亡和 [Ca2 +]i的影响。方法　应用已建立的VCR诱导L 0 2细胞自噬性凋亡模型 ,使用电镜、流式细胞术检测细胞凋亡 ;用Fluo 3/AM荧光探针经流式细胞仪测定L 0 2细胞平均 [Ca2 +]i。结果　电镜及流式细胞术检测证实VCR诱导L 0 2细胞发生了自噬性凋亡 ,此凋亡过程中 [Ca2 +]i明显升高 ,以凋亡早期更为明显 :3MA可显著抑制VCR所致的 [Ca2 +]i升高并能降低凋亡细胞比例。结论　在体外条件下VCR可以诱导L 0 2细胞自噬性凋亡 ,其发生可能与VCR导致 [Ca2 +]i升高有关 ;3MA可能通过抑制 [Ca2 +]i升高而抑制自噬及VCR所致的自噬性凋亡。     

Edema and brain trauma

Brain edema leading to an expansion of brain volume has a crucial impact on morbidity and mortality following traumatic brain injury (TBI) as it increases intracranial pressure, impairs cerebral perfusion and oxygenation, and contributes to additional ischemic injuries. Classically, two major types of traumatic brain edema exist: "vasogenic" due to blood-brain barrier (BBB) disruption resulting in extracellular water accumulation and "cytotoxic/cellular" due to sustained intracellular water collection. A third type, "osmotic" brain edema is caused by osmotic imbalances between blood and tissue. Rarely after TBI do we encounter a "hydrocephalic edema/interstitial" brain edema related to an obstruction of cerebrospinal fluid outflow. Following TBI, various mediators are released which enhance vasogenic and/or cytotoxic brain edema. These include glutamate, lactate, H(+), K(+), Ca(2+), nitric oxide, arachidonic acid and its metabolites, free oxygen radicals, histamine, and kinins. Thus, avoiding cerebral anaerobic metabolism and acidosis is beneficial to control lactate and H(+), but no compound inhibiting mediators/mediator channels showed beneficial results in conducted clinical trials, despite successful experimental studies. Hence, anti-edematous therapy in TBI patients is still symptomatic and rather non-specific (e.g. mannitol infusion, controlled hyperventilation). For many years, vasogenic brain edema was accepted as the prevalent edema type following TBI. The development of mechanical TBI models ("weight drop," "fluid percussion injury," and "controlled cortical impact injury") and the use of magnetic resonance imaging, however, revealed that "cytotoxic" edema is of decisive pathophysiological importance following TBI as it develops early and persists while BBB integrity is gradually restored. These findings suggest that cytotoxic and vasogenic brain edema are two entities which can be targeted simultaneously or according to their temporal prevalence.     

耳蜗外毛细胞两种胞内钙库的初步探讨

为探讨毛细胞胞内钙库的种类 ,本文观察了在无钠、无钙和含镧液体中 ,在不受胞外 Ca2 +内流和质膜上 Ca2 +转运机制影响的条件下 ,和在三磷酸肌醇敏感钙库的工具药 thapsigargin和 ryanodine敏感钙库的工具药 caffeine作用下 ,毛细胞胞内游离钙([Ca2 + ] i)的变化过程。分离的豚鼠耳蜗外毛细胞经钙敏荧光染料 5μmol/L fluo-3染色后 ,用激光扫描共聚焦显微镜监测 ,以 fluo-3荧光相对值指示毛细胞 [Ca2 + ] i的高低。 3 0 nmol/L thapsigargin使外毛细胞 [Ca2 + ] i由静态值 1.0增至 1.64± 0 .76,再加入 10mmol/L caffeine后更增至 2 .45± 1.5 9(x± s,n=11,F=7.90 ,P<0 .0 1)。Q值检验示两种试剂引起外毛细胞 [Ca2 + ] i增高程度的差别有极显著意义 (P<0 .0 1) ,表明毛细胞内有对三磷酸肌醇敏感和对 ryanodine敏感的两种钙库参与了胞内 Ca2 + 释放机制     

3H]acetylcholine release and the change in cytosolic free calcium level induced by high K+ and ouabain in rat brain synaptosomes

High K+ (50 mM) increased both [3H]acetylcholine ([3H]ACh) release and cytosolic free calcium level ([Ca2+]i) in rat brain synaptosomes in the presence of extracellular Ca2+. Ouabain (5 x 10(-8) to 5 x 10(-4) M) also caused a dose-dependent increase in [3H]ACh release, but not in [Ca2+]i, in the absence of Ca2+. The effects of high K+ and ouabain on [3H]ACh and/or [Ca2+]i, were inhibited by the intracellular Ca2+ antagonist TMB-8 (10(-4) M). These results suggest that unlike high K+, ouabain increases transmitter release from nerve endings through a mechanism which is independent of [Ca2+]i, but sensitive to TMB-8.     

低氧与缺血诱导培养海马和皮质神经元钙应答反应的比较

目的：钙作为重要信使参与多种生理和病理代谢过程,而且在缺血性神经元损伤机制中起着重要的作用。本实验模拟脑缺血的病理生化改变,用激光扫描共聚焦显微镜（LSCM）和Fluo-3荧光探针标记技术,观察低氧缺血状态下体外培养的海马,皮质神经元内钙浓度的变化。方法：用100μmol/L氰化钠造成细胞低氧;100μmol/L氰化钠和3.5mmol/L碘醋酸盐模拟在体完全性脑缺血;1mmol/LL-谷氨酸模拟在体脑缺血时兴奋性氨基酸大量释放;无葡萄糖介质剥夺细胞能量代谢的底物。结果：低氧使海马神经元[Ca^2 ]i显著升高,但有两种不同的钙振荡现象,谷氨酸引起海马神经元[Ca^2 ]i持续升高,但峰值低于低氧组,缺血组未见[Ca^2 ]i大幅升高,葡萄糖缺如不引起[Ca^2 ]i升高,结论：低氧和谷氨酸引起的神经元损害是能量依赖性的,轻度酸中毒可阻止胞内Ca^ 升高,糖对神经元具有保护作用,但单纯无糖引起的神经元损害与Ca^2 超载机制无关。     

弱氧化修饰低密度脂蛋白诱导ECV304组织因子基因表达及银杏内酯B的抑制作用

为研究弱氧化修饰低密度脂蛋白（mmLDL)对ECV304组织因子（TF）蛋白含量和mRNA水平的影响、对胞浆游离钙离子[Ca^2 ]i的影响，以及银杏内酯B等的干预作用。采用ELISA法检测细胞内TF蛋白含量，用RT－PCR法检测细胞内TF mRNA水平，用激光共聚焦显微镜观察[Ca^2 ]i的变化。结果显示，40mg/L mmLDL作用ECV304 4h，能使TF蛋白含量及mRNA水平显著增加，并迅速升高[Ca^2 ]i。抗氧化剂银杏内酯B及PKC抑制剂Staurosporine可拮抗mmLDL的上述诱导反应。以上结果提示，mmLDL可诱导ECV304 TF表达，Ca^2 与PKC参与mmLDL诱导ECV304 TF基因表达过程，并且银杏内酯B可抑制mmLDL的此种效应。     

Calcium and voltage-dependent alterations of cell volume in neuroblastoma×glioma hybrid NG108-15 cells

Intracellular calcium ([Ca2+](i)), cell volume, membrane potential and currents were measured in neuroblastomaxglioma hybrid cells to gain insight into how [Ca2+](i) controls cell volume. [Ca2+](i) was increased by fluid shear stress, mechanical stimulation of the cells, the Ca2+ ionophore A23187, caffeine and thapsigargin. The increase in [Ca2+](i) induced by mechanical stimulation was decreased by about 50% by caffeine and abolished after incubation of the cells in a Ca2+-free solution. Mechanical stimulation by stirring the cell suspension induced cell shrinkage that was abolished by caffeine, but induced cell swelling in Ca2+-free solution. In the presence of caffeine, A23187 induced cell shrinkage whereas thapsigargin induced cell swelling. Both cell volume changes were inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid. The cells were hyperpolarized by fluid shear stress and A23187 and depolarized by caffeine, thapsigargin and intracellular EGTA. Under all these conditions, the membrane input resistance was decreased. Voltage-clamp experiments suggested that, in addition to an increased anionic current, fluid shear stress and A23187 increased a K+ current, whereas caffeine and intracellular Ca2+ chelation increased a non-selective cation current and thapsigargin increased both a K+ and a non-selective cation current. Taken together, these results suggest that, if cell volume is closely dependent on [Ca2+](i) and the activity of Cl- channels, its relative value is dependent on the ionic selectivity of co-activated channels and the membrane potential.     

Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis.

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.     

Expression of c-fos mRNA following moderate lateral fluid percussion brain injury in rats

Expression of c-fos mRNA following moderate lateral fluid percussion brain injury in rats