VX-induced cell death involves activation of caspase-3 in cultured rat cortical neurons

Exposure of cell cultures to organophosphorous compounds such as VX can result in cell death. However, it is not clear whether VX-induced cell death is necrotic or involves programmed cell death mechanisms. Activation of caspases, a family of cysteine proteases, is often involved in cell death, and in particular, caspase-3 activation appears to be a key event in programmed cell death processes including apoptosis. In this study, we investigated VX-induced neuronal cell death, as well as the underlying mechanism in terms of its effect on caspase-3 activity. Primary cortical neuronal cultures were prepared from gestational days 17 to 19 Sprague Dawley rat fetuses. At maturation, the cells were treated with varying concentrations of VX and cell death was evaluated by lactate dehydrogenase (LDH) release. VX induced an increase in LDH release in a concentration-dependent manner. Morphological VX-induced cell death was also characterized by using nuclear staining with propidium iodide and Hoechst 33342. VX induced a concentration- and time-dependent increase in caspase-3 activation. Caspase-3 activation was also confirmed by the proteolytic cleavage of poly(ADP-ribose)polymerase (PARP), an endogenous caspase-3 substrate. These data suggested that in rat cortical neurons, VX-induced cell death via a programmed cell death pathway that involves changes in caspase-3 protease.     

3-Nitropropionic acid induces cell death and mitochondrial dysfunction in rat corticostriatal slice cultures

Exposure of organotypic rat corticostriatal slice cultures to the mitochondrial toxin 3-nitropropionic acid (3-NP) resulted in concentration-dependent loss of cresylviolet-stained cells and increase of lactate dehydrogenase and lactate efflux into the culture medium, indicators for cell death and metabolic activity in the slices, respectively. The involvement of apoptosis in these slices was suggested by using the terminal transferase-mediated biotinylated-UTP nick end-labeling (TUNEL) technique, and immunohistochemistry for the apoptosis-related markers Bax and Bcl-2. In 3-NP-exposed slices, TUNEL-positive cells were observed in both the striatum and the cortex but in different forms: striatal neurons were either diffusely stained or showed nuclear fragmentation, cortical neurons only exhibiting nuclear fragmentation. In 3-NP-exposed slices, the pro-apoptotic protein Bax was abundantly expressed, whereas the anti-apoptotic protein Bcl-2 was not expressed in striatal neurons. We suggest that both apoptosis and necrosis are involved in the 3-NP-treated slices, apoptosis as well as necrosis in the striatum and apoptosis in the cortex.     

NMDA对新生大鼠皮层神经元的兴奋毒作用

目的:建立NMDA诱导原代培养皮层神经元兴奋毒损伤模型,探讨NMDA对NMDA受体过度活化诱导兴奋性神经毒的可能途径。方法:原代培养新生大鼠大脑皮层神经元,通过倒置显微镜形态学观察、细胞活力检测(MTT及LDH释放的检测)及胞内Ca2+的动态测定,探索NMDA诱导毒性作用的适当浓度及时间。通过对ROS、NO检测,分析NMDA诱导毒性作用于线粒体的损伤情况。结果:NMDA(100μmol/L/2 h)引起皮层神经元形态学改变,且引起神经元细胞活力时间和浓度依赖性的下降,由同时伴随LDH释放增加(P<0.05),ROS和NO的生成量明显增加(P<0.05),皮层神经元内Ca2+的快速升高,并维持在高水平。结论:NMDA诱发皮层神经元明显的细胞毒性作用,提示NMDA过度活化NMDA受体后通过神经元膜内Ca2+超载造成ROS和NO的生成量增加,导致皮层神经元产生毒性损伤。     

原代培养大鼠皮质神经元中β-Tubulin的表达

目的观察原代培养大鼠皮质神经元生长过程中-βTubulin的表达变化并进行图像分析。方法取孕13~14天胚鼠大脑皮质接种于覆有多聚赖氨酸的爬片上,培养液为添加了B27(v/v%:2%)和Glutamax(v/v%:1%)的Neurobasl Medium(Gibco)。细胞爬片进行免疫细胞化学染色,显微镜观察,LeicaD MR Q550图像分析系统进行图像分析。结果-βTubulin表达和分布随着神经元发育成熟而变化,生长初期主要分布于核周附近,随神经元的成熟散在分布于胞质及突起内,构成细胞骨架,维持细胞形态。结论在神经元生长过程中伴随有-βTubulin的表达变化,随神经元的成熟而构成细胞骨架,维持细胞形态。     

Beta-amyloid peptide fragment 31-35 induces apoptosis in cultured cortical neurons.

A synthetic fragment 31-35 of beta-amyloid peptide was used in cultured cortical neurons to examine whether this smaller sequence could trigger apoptotic degeneration in vitro by using morphological, biochemical and flow-cytometric examinations. The results showed that: (i) neurons treated with fragment 31-35 of beta-amyloid peptide exhibited membrane blebbing, compaction of nuclear chromatin, nuclear shrinkage and nuclear fragmentation; (ii) a typical DNA ladder was revealed by agarose gel electrophoresis following fragment 31-35 of beta-amyloid peptide exposure; (iii) the internucleosome DNA fragmentation was also detected by flow-cytometric examination following fragment 31-35 of beta-amyloid peptide exposure; and (iv) the DNA fragmentation induced by fragment 31-35 of beta-amyloid peptide in the above two examinations could be blocked by co-treatment with aurintricarboxylic acid or actinomycin D. It is suggested that fragment 31-35 of the beta-amyloid peptide may be a shorter sequence of beta-amyloid peptide responsible for triggering an apoptotic process in cultured neurons.     

On the mechanism of GABA-induced currents in cultured rat cortical neurons

 We applied the perforated-patch-clamp technique to cultured cortical neurons of the rat to characterize the ionic basis of membrane potential changes and membrane currents induced by γ-aminobutyric acid (GABA). Gramicidin was used as the membrane-perforating agent, to allow the recording of whole-cell currents without impairing the intracellular Cl^– concentration ([Cl^–]_i). In current-clamp experiments in the presence of 26 mM HCO₃ ^– the application of 50 μM GABA evoked changes in the membrane potential of neurons including depolarizations (19%), hyperpolarizations (38%) and biphasic changes in membrane potential (31%), characterized by a transient hyperpolarization followed by a sustained depolarization. Accordingly, GABA (50–200 μM) induced inward, outward or biphasic current responses under voltage-clamp. Inward and biphasic currents as well as depolarizations and biphasic membrane potential responses, respectively, occurred more frequently in the presence of 26 mM HCO₃ ^–. The second phase of the biphasic membrane potential or current responses was markedly reduced when the preparation was bathed in a HCO₃ ^–-free saline, indicating a contribution from HCO₃ ^–. The reversal potential of the GABA-induced currents (E _GABA) determined with the gramicidin-perforated-patch mode and in the nominal absence of HCO₃ ^– was –73 mV, while it was shifted to –59 mV in the presence of HCO₃ ^–. Combined patch-clamp and microfluorimetric measurements using the Cl^–-sensitive dye 6-methoxy-1-(3-sulphonatopropyl)quinolinium (SPQ) showed that GABA evoked an increase of [Cl^–]_i in 54% (n=13) of the neurons. We conclude that this increase of [Cl^–]_i in combination with the efflux of HCO₃ ^– results in a shift of E _GABA above the resting membrane potential that gives rise to GABA-mediated depolarizations. Received: 2 June 1998 / Received after revision: 4 August 1998 / Accepted: 10 September 1998     

Exogenous aluminum accumulates in the lysosomes of cultured rat cortical neurons

In order to study the intracellular localization of aluminum, 0.01% AlCl3 was added to rat cerebral organotypic cultures following 14 days incubation in a standard medium. The cultures were maintained in the aluminum (Al)-containing medium for 1 or 3 days. Subsequently, electron probe X-ray micro-analysis (EPXMA), was used to localize aluminum in the neurons. The Al was found in the cells as early as after 1 day of AlCl3 exposure. The Al was detected exclusively in the neuronal lysosomes, in 66% (1 day exposure) and 97% (3 days) of the measured lysosomes. This localization was confirmed by laser microprobe mass analysis (LAMMA) measurements. Our results demonstrate an Al localization in the neurons, exposed to exogenous Al, different from that in the brains of patients with Alzheimer's disease.     

HIE幼龄大鼠神经元自噬诱导线粒体功能障碍的研究

目的:探讨细胞自噬对缺氧缺血性脑病(HIE)幼龄大鼠神经元线粒体功能的影响。方法:随机选取10日龄SPF级SD大鼠30只,分为假手术(sham)组和HIE组,后者结扎单侧颈总动脉复制缺血缺氧模型。取脑组织行镜下病理观察,免疫组化分析活化型caspase-3和LC3B-II蛋白表达;体外实验中观察缺氧诱导的原代大鼠神经元的自噬过程,Western blot检测相关蛋白,并对大鼠神经元线粒体功能进行测试。结果:(1)与sham组相比,HIE组大鼠出现脑萎缩和脑室增宽;HIE组免疫组化显示活化型caspase-3和LC3B-II蛋白表达上调(P<0.01);(2)体外细胞实验发现,缺氧可诱导大鼠神经元出现自噬和凋亡;(3)与sham组相比,单纯缺氧的神经元内活性氧簇增加,线粒体超氧化物上调,线粒体跨膜电位降低(P<0.01)。结论:在HIE大鼠模型中,缺氧诱导的神经元线粒体功能障碍,可能与缺氧时神经元出现的自噬和凋亡有关。这一结果将为临床上开发细胞自噬因子类药物治疗HIE提供了新的思路。     

Angiotensin II induces protein overexpression of hyperpolarization-activated cyclic nucleotide-gated channels in primary cultured nodose neurons

Modulating ion channel function includes acutely affecting the kinetics of the ion channels and chronically changing the expression of ion channels. Our previous study showed that angiotensin II (Ang II) acutely increased hyperpolarization-activated cyclic nucleotide-gated (HCN) currents in nodose ganglion (NG) neurons via NADPH oxidase-superoxide signaling. Therefore, the present study was to measure chronic treatment with Ang II on protein expression of the HCN channels in the primary cultured rat NG neurons. Immunofluorescent staining data showed that HCN1 was expressed in the A-type NG neurons, and HCN2 was expressed in the C-type NG neurons. Chronic treatment of Ang II (100 nM, 12 h) induced the protein expression of HCN2 besides the overexpression of HCN1 in the A-type NG neurons; and the overexpression of HCN2 in the C-type NG neurons. An Ang II type I receptor antagonist (1 μM losartan), a NADPH oxidase inhibitor (100 μM apocynin), or a superoxide dismutase mimetic (1mM tempol) attenuated the effect of Ang II to increase the protein expression of the HCN channels in rat nodose neurons. Whole cell patch-clamp data further confirmed that chronic treatment of Ang II (100 nM, 12 h) significantly enhanced the density of HCN currents in A- and C-type NG neurons. Above three inhibitors significantly inhibited the Ang II-induced increase of the HCN channel density in rat NG neurons. These findings suggest that Ang II-NADPH oxidase-superoxide signaling chronically regulates the protein expression of the HCN channels in rat nodose neurons.     

Cyclophosphamide promotes cell survival via activation of intracellular signaling in cultured cortical neurons

Cyclophosphamide (CP) has been used as an antitumour agent or immunosuppressant clinically, though the potential biological role of CP in the central nervous system (CNS) has not been clarified. In the present study, we found that pretreatment with CP prevented neuronal cell death caused by serum deprivation in cultured cortical neurons. Interestingly, CP stimulated activation of PI3K (phosphatidylinositol 3 kinase) and MAPK/ERK (mitogen-activated protein kinase/extracellular signal-regulated kinase) pathways, which are known as survival-promoting intracellular signalings. Furthermore, CP increased the expression of Bcl2, an anti-apoptotic factor. In the presence of inhibitors for PI3K or MAPK/ERK pathways, the CP-dependent neuronal survival and Bcl-2 up-regulation were both abolished. Importantly, significant increase in BDNF (brain-derived neurotrophic factor) expression was induced by CP application, implying that BDNF up-regulation is involved in the CP effect. We propose that CP has a protective effect on CNS neurons via the activation of intracellular signalings, and up-regulation of Bcl2 and BDNF.     

五味子酮保护大鼠皮层神经元对抗氯胺酮损伤的实验研究

目的研究五味子酮是否具有保护神经元免受氯胺酮损伤的作用。方法原代培养的神经元分别加入不同浓度的氯胺酮、五味子酮后培养24 h,观察神经元形态特征和检测神经元存活率。结果神经元经不同浓度氯胺酮处理后胞体立体感消失,颜色变暗,细胞轮廓不清,较高浓度氯胺酮组神经元轴突断裂,部分死亡。与对照组比较随着氯胺酮剂量的增加神经元存活率逐渐下降（P〈0.05）。20μmol/L氯胺酮分别加入30、50、70μmol/L五味子酮处理组神经元存活率明显高于20μmol/L氯胺酮组（P〈0.05）,且70μmol/L五味子酮组神经元存活率高于其他两组（P〈0.05）。结论氯胺酮具有损伤体外培养神经元的作用且成剂量依赖性,五味子酮具有保护神经元免受氯胺酮损伤的作用。     

Identification of an ATP-sensitive K+ channel in rat cultured cortical neurons

To determine whether membranes of mammalian central neurons contain an ATP-sensitive K⁺ (K_ATP) channel similar to that present in pancreatic cells, the patch-clamp technique was applied to cultured neurons prepared from the neonatal rat cerebral cortex and hippocampus. In whole-cell experiments with hippocampal neurons, extracellular application of 0.5 mM diazoxide (a K_ATP channel activator) elicited a hyperpolarization concomitant with an increase in membrane conductance, whereas application of 0.5 mM tolbutamide (a K_ATP channel blocker) induced a depolarization with a decrease in conductance. Similar results were obtained with cortical neurons. In outside-out patch experiments with cortical neurons, a K⁺ channel sensitive to these drugs was found. The channel was completely blocked by 0.5 mM tolbutamide and activated by 0.5 mM diazoxide. The single-channel conductance was 65 pS under symmetrical 145 mM K⁺ conditions and 24 pS in a physiological K⁺ gradient. In inside-out patch experiments, this channel was demonstrated to be inhibited by an application of 0.2–1 mM ATP to the cytoplasmic surface of the patch membrane. These results indicate that the membranes of rat cortical neurons contain a K_ATP channel that is quite similar to that found in pancreatic cells. It is also suggested that the same or a similar K⁺ channel may exist in membranes of hippocampal neurons.     

Asiatic acid derivatives protect cultured cortical neurons from glutamate-induced excitotoxicity.

Asiatic acid, a triterpene of Centella asiatica (L.) Urban (Umbelliferae), has been patented as a treatment for dementia and an enhancer of cognition by the Hoechst Aktiengesellschaft (EP 0 383 171 A2). We modified the chemical structure of asiatic acid and obtained 36 derivatives of asiatic acid in an attempt to prepare neuroprotective compounds that were more efficacious than asiatic acid itself. The neuroprotective activities of these derivatives were evaluated using primary cultures of rat cortical neurons insulted with the neurotoxin, glutamate, as an in vitro screening system. Among the semi-synthesized derivatives, three derivatives significantly mitigated the neurotoxicity induced by glutamate in this screening system. The neuroprotective activities of these 3 derivatives appeared to be more powerful than that of asiatic acid itself. These 3 derivatives significantly attenuated decreases in the levels of glutathione, glutathione peroxidase and other enzymes, which participate in the cellular defense mechanisms blunting oxidative stress. Furthermore, they significantly reduced the overproduction of NO induced by glutamate. These results showed that these derivatives of asiatic acid exerted significant neuroprotective effects on cultured cortical cells by their potentiation of the cellular oxidative defense mechanism. Therefore, these agents may prove to be efficacious in protecting neurons from the oxidative damage caused by exposure to excess glutamate.     

Spatio-temporal cholinergic modulation in cultured networks of rat cortical neurons: spontaneous activity

Activation of the cholinergic innervation of the cortex has been implicated in sensory processing, learning, and memory. At the cellular level, acetylcholine both increases excitability and depresses synaptic transmission, and its effects on network firing are hard to predict. We studied the effects of carbachol, a cholinergic agonist, on network firing in cultures of rat cortical neurons, using electrode arrays to monitor the activity of large numbers of neurons simultaneously. These cultures show stable spontaneous synchronized burst firing which propagates through dense synaptic connections. Carbachol (10-50 microM), acting through muscarinic receptors, was found to induce a switch to asynchronous single-spike firing and to result in a loss of regularity and fragmentation of the burst structure. To obtain a quantitative measure of cholinergic actions on cortical networks, we applied a cluster Poisson-process model to sets of paralleled spike-trains in the presence and absence of carbachol. This revealed that the time series can be well-characterized by such a simple model, consistent with the observed 1/f(b)-like spectra (0.04相似文献   

JNK and p38 were involved in hypoxia and reoxygenation-induced apoptosis of cultured rat cerebellar granule neurons

As a model of the reperfusion injury found in stroke, we treated cerebellar granule neurons (CGNs) with hypoxia followed by reoxygenation. Hypoxia for 3 h followed by 24 h reoxygenation (H/R) induced a typical apoptosis of CGNs. CGNs exposed to H/R responded by activating JNK, increasing the expression of p38 and ultimately caused CGNs dying. Furthermore, apoptosis of CGNs induced by H/R was inhibited by pre-treatment with SB203580 or SP600125, and the inhibitory effect of SB203580 was greater than that of SP600125. Additionally, we also found that H/R temporally activated Akt and inactivated glycogen synthesis kinase-3β (GSK-3β), two proteins the functions of which were important in cell survival and energy metabolism. These findings demonstrated that H/R-induced apoptosis in CGNs by enhancing JNK and p38 activity, which contributed at least in part to H/R-induced apoptosis of CGNs.     

Novel Na-independent and adenine-specific transport system for adenine in primary cultured rat cortical neurons

Endogenous adenine is an important modulator of cell survival and activity in the central nervous system. In the present study, we examined the transport mechanisms for adenine in primary cultured rat cortical neurons and astrocytes. [³H]Adenine was time-dependently taken up into neurons, but not into astrocytes. In kinetic analysis, the [³H]adenine uptake by neurons was observed to be saturable, and an Eadie–Hofstee plot showed that a single component was involved in the uptake, with kinetic parameters of K_m = 6.09 μM and V_max = 0.340 nmol/mg protein per min. In inhibition assaying by nucleobases and nucleosides, and inhibitors for equilibrative nucleoside transporters, organic ion transporters and peptide transporters, which were reported to transport nucleobases and their analogues, the [³H]adenine uptake by neurons was found to be significantly inhibited by excess concentrations of adenine, hypoxanthine and adenosine, and was greatly reduced only by the addition of adenine. Therefore, it was indicated that adenine in the extracellular fluid in the central nervous system is taken up into neurons, but not into astrocytes, and that neurons may present a novel Na⁺-independent and adenine-specific transport system.     

Spatio-temporal cholinergic modulation in cultured networks of rat cortical neurons: evoked activity

We studied the effects of carbachol, a cholinergic agonist, on extracellularly evoked firing of networks in mature cultures of rat cortical neurons, using multi-electrode arrays to monitor the activity of large numbers of neurons simultaneously. These cultures show evoked burst firing which propagates through dense synaptic connections. When a brief voltage pulse was applied to one extracellular electrode, spiking electrical responses were evoked in neurons throughout the network. The response had two components: an early phase, terminating within 30-80 ms, and a late phase which could last several hundreds of milliseconds. Action potentials evoked during the early phase were precisely timed, with only small jitter. In contrast, the late phase characteristically showed clusters of electrical activity with significant spatio-temporal fluctuations. The late phase was suppressed by applying a relatively small amount of carbachol (5 microM) in the external solution, even though the spontaneous firing rate was not significantly changed. Carbachol increased both the spike-timing precision and the speed of propagation of population spikes, and selectively increased the firing coincidence in a subset of neuron pairs in the network, while suppressing late variable firing in responses. Hence, the results give quantitative support for the idea that cholinergic activation in the cortex has a general role of focusing or enhancing significant associative firing of neurons.     

银杏内酯B对谷氨酸诱导脑皮质神经元凋亡及Ca2+超载的影响

目的：观察银杏内酯B对谷氨酸诱导培养脑皮质神经元凋亡的拮抗作用，并探讨这种作用与神经细胞内游离Ca2+浓度改变的关系。方法： 采用改良的方法原代培养胎小鼠脑皮质神经元，用噻唑兰（MTT）法检测神经元的存活情况；细胞凋亡采用形态学观察、DNA琼脂糖凝胶电泳法和Hoechst 33258核染色方法进行分析；用Fura-2/AM荧光指示剂法测定细胞内Ca2+浓度。结果： 谷氨酸（0.8 mmol/L）能诱导神经细胞凋亡和胞内Ca2+超载，银杏内酯B（10-250 μmol/L）能减轻谷氨酸所致的细胞损伤，表现为神经元存活率提高，细胞形态的恢复和DNA断裂减少。结论： 银杏内酯B可拮抗谷氨酸所致的神经细胞毒性作用，这可能与其能竞争PAF受体并降低神经细胞内［Ca2+］从而抑制谷氨酸诱导的神经元凋亡有关。     

Astrocyte-derived estrogen enhances synapse formation and synaptic transmission between cultured neonatal rat cortical neurons

Recent in vitro studies have found that astrocytes exert powerful control over the number of neuronal synapses, leading us to consider why glia can exert this control and what the underlying mechanism(s) may be. To understand the potential possibility, we studied the formation of synapses and synaptic function in primary rat cortical neurons. We found that primary cultured neonatal rat cortical astrocytes modulate synaptogenesis and synaptic function through producing and secreting estradiol into culture medium. The concentration of estradiol produced by pure cultured astrocytes increased in correspondence with the days of culture and the number of proliferating astrocytes, which peaked at 266+/-22 ng/l around day 14 of culture. When astrocyte-conditioned medium (ACM) was added into pure cultured cortical neurons, the number of synapses formed between cortical neurons increased by nearly sixfold. The mean frequency and the amplitude of mini-postsynaptic currents (mPSCs) increased from 13+/-4 events/min and 20.5+/-2 pA to 73+/-16 events/min and 29.1+/-3 pA, respectively. In the meantime, the level of estrogen receptor-alpha (ER-alpha) expressed on neonatal rat cortical neurons was significantly up-regulated. Moreover, the effect of ACM on synaptic formation and transmission was blocked by tamoxifen (estrogen receptor antagonist) in culture. After the treatment of tamoxifen, the number of synapses on neurons decreased from 79+/-9 to 32+/-3. The mean amplitude and frequency of mPSCs were also dropped to 24.5+/-2 pA and 35+/-10/min, respectively. Unexpectedly, exogenic estradiol can mimic the effect of ACM on synaptic formation and transmission. Finally, to understand whether astrocyte-derived estradiol regulates the synaptic transmission via presynapse, the release of presynaptic vesicle from neuron was monitored by FM 4-64 assay. The results showed that when ACM or exogenic estradiol was added into neurons, the kinetics of vesicle release speed are similar to that of neuronal cultured with astrocytes, which were faster than that of just pure neuronal cultures. These observations suggest that estrogen synthesized and secreted by astrocytes can regulate synapse formation and synaptic transmission.     

灯盏花素干预体外培养大鼠脑皮质神经元的存活与生长

背景：神经生长因子在维持中枢神经系统神经细胞的存活、分化,促进其生长、发育,维持其功能方面具有不可替代的作用。 目的：观察灯盏花素对SD大鼠大脑皮质体外培养神经元生长的影响,并初步探讨其机制。 方法：取新生SD大鼠胚胎,取其大脑皮质后,对神经元进行原代培养,随机分为3组,其中灯盏花素组加入10 g/L灯盏花素,对照组加入等量生理盐水,正常组不作任何处理,各组又分为24 h、48 h亚组。对24孔板中各组细胞各时间点采集图像,之后采用RT-PCR技术检测神经生长因子、酪氨酸激酶A mRNA的表达变化。对96孔板中各组细胞不同时间点采用MTT检测神经元生长活力。 结果与结论：正常组和对照组在各时间点细胞数、胞体面积、突起长度及细胞活力无明显差异(P > 0.05),但正常组及对照组均有随时间点延长,3个指标均上调(P < 0.05),灯盏花素组细胞数、胞体面积、突起长度及细胞活力均较正常组及对照组升高(P < 0.05)。神经生长因子及TrkA mRNA的表达在正常组和对照组在各时间点无明显差异(P > 0.05),灯盏花素组各时间点较正常组和对照组上调(P < 0.05)。提示灯盏花素促进SD大鼠脑源性神经元的存活、生长,其机制可能是通过上调神经生长因子及其受体TrkA的表达发挥作用。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：