Apoptosis is induced by radiofrequency fields through the caspase-independent mitochondrial pathway in cortical neurons

Joubert, V., Bourthoumieu, S., Leveque, P. and Yardin, C. Apoptosis is Induced by Radiofrequency Fields through the Caspase-Independent Mitochondrial Pathway in Cortical Neurons. Radiat. Res. 169, 38-45 (2008). In the present study, we investigated whether continuous-wave (CW) radiofrequency (RF) fields induce neuron apoptosis in vitro. Rat primary neuronal cultures were exposed to a CW 900 MHz RF field with a specific absorption rate (SAR) of 2 W/kg for 24 h. During exposure, an increase of 2 degrees C was measured in the medium; control experiments with neurons exposed to 39 degrees C were then performed. Apoptosis was assessed by condensation of nuclei with 4',6-diamino-2-phenylindole (DAPI) staining observed with an epifluorescence microscope and fragmentation of DNA with TdT-mediated dUTP nick-end labeling (TUNEL) analyzed by flow cytometry. A statistically significant difference in the rate of apoptosis was found in the RF-field-exposed neurons compared to the sham-, 37 degrees C- and 39 degrees C-exposed neurons either 0 or 24 h after exposure using both methods. To assess whether the observed apoptosis was caspase-dependent or -independent, assays measuring caspase 3 activity and apoptosis-inducing factor (AIF) labeling were performed. No increase in the caspase 3 activity was found, whereas the percentage of AIF-positive nuclei in RF-field-exposed neurons was increased by three- to sevenfold compared to other conditions. Our results show that, under the experimental conditions used, exposure of primary rat neurons to CW RF fields may induce a caspase-independent pathway to apoptosis that involves AIF.     

Phospho-Rb Mediating Cell Cycle Reentry Induces Early Apoptosis Following Oxygen–Glucose Deprivation in Rat Cortical Neurons

The aim of this study was to investigate the relationship between cell cycle reentry and apoptosis in cultured cortical neurons following oxygen–glucose deprivation (OGD). We found that the percentage of neurons with BrdU uptake, TUNEL staining, and colocalized BrdU uptake and TUNEL staining was increased relative to control 6, 12 and 24 h after 1 h of OGD. The number of neurons with colocalized BrdU and TUNEL staining was decreased relative to the number of TUNEL-positive neurons at 24 h. The expression of phosphorylated retinoblastoma protein (phospho-Rb) was significantly increased 6, 12 and 24 h after OGD, parallel with the changes in BrdU uptake. Phospho-Rb and TUNEL staining were colocalized in neurons 6 and 12 h after OGD. This colocalization was strikingly decreased 24 h after OGD. Treatment with the cyclin-dependent kinase inhibitor roscovitine (100 μM) decreased the expression of phospho-Rb and reduced neuronal apoptosis in vitro. These results demonstrated that attempted cell cycle reentry with phosphorylation of Rb induce early apoptosis in neurons after OGD and there must be other mechanisms involved in the later stages of neuronal apoptosis besides cell cycle reentry. Phosphoralated Rb may be an important factor which closely associates aberrant cell cycle reentry with the early stages of neuronal apoptosis following ischemia/hypoxia in vitro, and pharmacological interventions for neuroprotection may be useful directed at this keypoint.     

Sevoflurane Induces Hippocampal Neuronal Apoptosis by Altering the Level of Neuropeptide Y in Neonatal Rats

Numerous studies have shown that the inhaled general anesthetic sevoflurane imposes toxicity on the central nervous system during the developmental period but the underlying mechanisms remain unclear. Neuropeptide Y (NPY) was reported to have important neuroprotective effects, which can attenuate neuronal loss under pathological conditions. However, the effects of NPY on sevoflurane-induced hippocampal neuronal apoptosis have not been investigated. In this study, postnatal day 7 (PND7) Sprague–Dawley rats and primary cultured cells separated from hippocampi were exposed to sevoflurane (2.4% for 4 h) and the NPY expression levels after treatment were analyzed. Furthermore, neuronal apoptosis assay was conducted via immunofluorescence staining of cleaved caspase-3 and flow cytometry after exogenous NPY administration to PND7 rats as well as cultured hippocampal neurons to elucidate the role of NPY in sevoflurane-induced neurotoxicity. Our results showed the level of NPY gradually decreased within 24 h after sevoflurane exposure in both the hippocampus of PND7 rats and cultured hippocampal neurons, but not in cultured astrocytes. In the exogenous NPY pretreatment study, the proportion of cleaved caspase-3 positive cells in the CA1 region of the hippocampus was increased significantly at 24 h after sevoflurane treatment, while NPY pretreatment could reduce it. Similarly, NPY could also reverse the apoptogenic effect of sevoflurane on cultured neurons. Herein, our results showed that sevoflurane caused a significant decrease in NPY expression, whereas exogenous NPY supplementation could reduce sevoflurane-induced hippocampal neuronal apoptosis both in vivo and in vitro.

     

Neuritin Attenuates Neuronal Apoptosis Mediated by Endoplasmic Reticulum Stress In Vitro

Neuritin is an extracellular glycophosphatidylinositol-linked protein that promotes neuronal survival, differentiation, function, and repair, but the exact mechanism of this neuroprotective effect remains unclear. Meanwhile, endoplasmic reticulum stress (ERS) induced apoptosis is attracting increased attention. In this work, we hypothesized that neuritin inhibited ERS to protect cortical neurons. To check this hypothesis, we exposed primary cultured cortical neurons to oxygen and glucose deprivation (OGD) for 45 min followed by reperfusion (R) to activate ERS. We then performed resuscitation for 6, 12, 24, and 48 h. ERS-related factors such as glucose-regulated protein 78 (GRP78), caspase-12 and CHOP were detected by Western blotting and quantitative real-time polymerase chain reaction assay. Apoptosis was assessed by Annexin V binding and propidium iodide staining. Ultrastructural changes of endoplasmic reticulum were observed under a transmission electron microscope. Results showed that GRP78 expression significantly increased at 12, 24, and 48 h and peaked at 24 h. Caspase-12 and CHOP expression significantly increased in a time-dependent manner at 12, 24, and 48 h. GRP78, caspase-12 and CHOP expression as well as apoptosis rate of primary cultured neurons and the ultrastructural changes of endoplasmic reticulum in the OGD/R?+?neuritin group significantly improved compared with the OGD/R group. In conclusion, the neuroprotection function of neuritin may be involved in ERS pathways.     

TGF-beta1 inhibits caspase-3 activation and neuronal apoptosis in rat hippocampal cultures

The effect of TGF-beta1 on apoptosis varies depending on the cell type, the kind of stimulus and the experimental conditions. The present study attempted to identify whether TGF-beta1 can prevent neuronal apoptosis and interrupt caspase-3 activation in rat primary hippocampal cultures after staurosporine treatment. TGF-beta1 at the concentration of 1 and 10 ng/ml significantly reduced neuronal damage as detected by trypan blue exclusion. Nuclear staining with Hoechst 33258 and TUNEL-staining further demonstrated that TGF-beta1 at the same concentration range effectively diminished neuronal apoptosis 24 h after staurosporine treatment, whereas 0.1 ng/ml of TGF-beta1 did not. Furthermore, TGF-beta1 (1 and 10 ng/ml) markedly inhibited the activation of caspase-3 induced by staurosporine as demonstrated by both caspase-3 activity assay and Western blotting. This study provides evidence that TGF-beta1 is able to efficiently inhibit caspase-3 activation, and thereby protects cultured hippocampal neurons against apoptosis.     

肝细胞生长因子对氧糖剥夺/再灌注神经元的保护作用

本文旨在探讨肝细胞生长因子（hepatocyte growth factor,HGF）对神经元氧糖剥夺/再灌注损伤的影响。取原代培养12d的Sprague-Dawley大鼠大脑皮层神经元,无糖、无氧（95%N2＋5%CO2）孵育2h后,换含25mmol/L葡萄糖的培养液、常氧培养0-24h,以MTT比色法检测细胞活力、乳酸脱氢酶（lactate dehydrogenase,LDH）漏出率作为细胞损伤指标,建立体外氧糖剥夺/再灌注损伤细胞模型;用流式细胞仪和Hoechst33258染色分析细胞凋亡率;用RT-PCR和Western blot分别检测大鼠脑皮层神经元HGF受体c-Met mRNA和蛋白的表达。于氧糖剥夺2h/再灌注24h处理前2h,加入不同终浓度（5-120ng/mL）的HGF,观察HGF对皮层神经元的影响。结果显示,c-Met表达于皮层神经元,氧糖剥夺2h/再灌注24h后,c-Met mRNA和蛋白表达均显著上调,神经元细胞活力明显降低,LDH漏出率和细胞凋亡率显著增高。HGF预处理明显促进氧糖剥夺/再灌注损伤神经元的存活,降低LDH漏出率,最大效应剂量为80ng/mL。流式细胞术和Hoechst33258染色结果均显示,HGF（80ng/mL）显著降低氧糖剥夺/再灌注神经元的细胞凋亡率。此外,c-Met抑制剂SU11274（5μmol/L）完全阻断HGF的神经保护作用。结果表明,HGF对皮层神经元氧糖剥夺/再灌注损伤具有直接的保护作用,呈一定的剂量依赖关系,并能有效对抗神经元凋亡。     

Protective effect of panaxydol and panaxynol on sodium nitroprusside-induced apoptosis in cortical neurons

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. The protective effect of panaxydol (PND) and panaxynol (PNN) on sodium nitroprusside (SNP)-induced neuronal apoptosis and potential mechanism were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN for 24 h following 1 mM SNP, an exogenous NO donor, exposure for 1 h, resulted significantly in reduction of cell death induced by SNP determined by MTT assay, LDH release and Hoechst staining. 5 μM PND and PNN also reduced the up-regulation of the pro-apoptotic gene, Bax, down-regulation of the anti-apoptotic gene, Bcl-2. The observations demonstrated that PND and PNN protect neurons against SNP-induced apoptosis via regulating the apoptotic related genes. The results raise the possibility that PND and PNN reduce neurodegeneration in the Alzheimer's brain.     

Toxicity of tunicamycin to cultured brain neurons: ultrastructure of the degenerating neurons.

Our previous study has shown that tunicamycin irreversibly downregulates the expression of GABA(A)R and causes cell death in cultured brain neurons by biochemical and light microscopic methods. In this study, we examined mechanisms underlying the degeneration of the neurons mainly employing electron microscopic analysis. Cultured neurons derived from embryonic chicken brains were incubated with 5 microg/ml of tunicamycin (TM) for 24 h, followed by continual incubation or removal of TM for additional 3 h or 24 h. Neurons treated with TM for 24 h showed dilated rough endoplasmic reticulum (rER), nuclear envelope and components of Golgi apparatus, in addition to the degranulation of rER and disaggregation of ribosomal rosettes. In neurons subjected to the prolonged incubation, some ribosomes reattached to the membranes of rER; the polyribosomes reappeared, and the swelling of Golgi apparatus subsided. However, the distention of rER persisted, and an uncommon spindle-like structure appeared in the perikarya. This structure is implicated to involve the neuronal degeneration. Moreover, extracellular cell debris was increased with time of incubation. The ratio of the light neurons, defined as containing lower cytoplasmic matrix density than the untreated control, decreased from 28% at 3 h to 3% at 24 h after the removal of TM, and 45% at further 3 h to 6% at further 24 h incubation of TM, whereas dense neurons only appeared in the two 24 h groups, as 44% and 34%. The light neurons resemble necrotic cells, but the dense neurons exhibit distinct morphological features from necrosis and apoptosis. The gel electrophoresis assay revealed the absence of DNA fragmentation in all cultures. In addition, whole cell recordings exhibited a 40% decrease of the GABA-elicited current in the neurons exposed to TM for 24 h. The results indicate irreversible toxicity of chronic TM treatment to the neurons and suggest differential mechanisms for the neuronal death among various populations of cells. It is evident that the N-glycosylation plays a critical role for neuronal survival.     

Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

胍丁胺对体外培养脊髓神经元的影响及在谷氨酸诱导损伤条件下的作用

研究胍丁胺(agmatine,AG)对培养脊髓神经元的作用及谷氨酸(glutamate,Glu)损伤后的影响,探讨其对神经元的毒性作用及其可能的机理.采用原代细胞培养法,分离培养胚胎大鼠脊髓神经元,3d后加入不同浓度的AG(0.5～80mg/L)继续培养12、24、36h,接着采用四甲基氮唑蓝法和中性红法分别测定胍丁胺对细胞存活率以及毒性的影响.在加入AG的同时给予2mmol/LGlu对细胞进行损伤,建立体外的损伤模型.然后进行细胞形态学观察、NeuN免疫细胞化学染色、四甲基氮唑蓝法测定细胞存活率、Hoechst33342和PI染色检测细胞凋亡和坏死率,同时对神经元中丙二醛(malondialdehyde,MDA)含量的变化检测,最后对各组进行比较和统计分析.结果发现,AG作用浓度低于40mg/L对正常培养的脊髓神经元的存活没有明显的影响,而80mg/L的AG对神经元生长有毒副作用.在给予Glu损伤后,细胞生长状态较差,细胞出现退化,而且细胞存活率显著下降,细胞坏死和凋亡率显著增高(P<0.05或0.01),而同时给予AG干预(AG-Glu组),细胞生长较正常培养的细胞生长状态略差,但明显优于Glu组,而且细胞凋亡和坏死率降低,细胞存活率增高,MDA含量减少(P<0.05).结果提示AG作为一种新发现的神经递质或调质,在正常生理情况下对神经元的生长没有毒副作用,而在Glu诱导损伤条件下,能够抑制Glu诱导的损伤.其发生机制可能与AG通过拮抗N-甲基-M-天冬氨酸(N-methyl-D-aspartate,NMDA)受体,阻断或抑制Glu的氧化毒性的级链反应有关.     

Oleic acid causes apoptosis and dephosphorylates Bad

There is increasing evidence showing the involvement of unsaturated free fatty acids in cell death pathways, particularly in the context of apoptotic signalling. Our previous in vitro study has demonstrated that oleic acid, a monounsaturated fatty acid, reduces phosphorylation of proapoptotic Bad through activation of protein phosphatase type 2Cbeta. In the present study, we attempted to investigate the role of oleic acid in neuronal apoptosis using different types of cell cultures, and, furthermore, to explore the underlying mechanism with regard to its effect on Bad expression. As revealed by nuclear staining, oleic acid caused a concentration- and time-dependent damage with typical apoptotic features in cortical and hippocampal cultures from embryonic and neonatal rats, respectively, as well as in human neuroblastoma SH-SY5Y cells. In mixed hippocampal cultures, nearly all neurons were damaged at 24 h after the treatment, while damage of astrocytes was detected 48 h after adding this fatty acid, suggesting that neurons were more vulnerable than astrocytes. Nile blue staining showed that oleic acid and oleic acid methyl ester were both taken up by the neurons within 30 min. In contrast to oleic acid, oleic acid methyl ester did not change cell viability demonstrating that oleic acid-induced cell death was not due to an overload of the cells with lipids. Caspase-3 activity was not increased by oleic acid in cultured hippocampal cells. Western blot analysis of phospho-Ser112 Bad and the total Bad in cultured hippocampal cells revealed a significant decrease in the ratio of phospho-Ser112 Bad to total Bad in a time- and concentration-dependent manner after the exposure with oleic acid. We conclude that oleic acid induces neuronal apoptosis through a caspase-3-independent mechanism involving dephosphorylation of Bad.     

p38 mitogen-activated protein kinase regulates low potassium-induced c-Jun phosphorylation and apoptosis in cultured cerebellar granule neurons

Cultured rat cerebellar granule neurons are widely used as a model system for studying neuronal apoptosis. After maturation by culturing in medium containing 26 mm potassium (high K(+)), changing to medium containing 5 mm potassium (low K(+); LK) rapidly induces neuronal apoptosis. Then over 50% of granule cells die within 24 h. However, the molecular mechanisms by which the LK-induced apoptosis occurs in cultured cerebellar granule cells remain unclear. In the present study, we found that p38 MAP kinase (p38) was an important factor for LK-induced apoptosis. Three hours after changing to LK medium, p38 was markedly activated. In addition, SB203580, a specific inhibitor of p38, strongly inhibited the phosphorylation and expression of c-Jun in LK-induced apoptosis of cultured cerebellar granule cells. In vitro kinase assay using glutathione S-transferase-c-Jun as a substrate showed that p38 directly phosphorylated c-Jun. Furthermore, in the presence of SB203580, about 80% of neurons survived. These results indicate that p38 regulates LK-induced apoptosis of cerebellar granule neurons.     

Loss of the Xeroderma Pigmentosum Group A Gene (XPA) Enhances Apoptosis of Cultured Cerebellar Neurons Induced by UV but Not by Low-K+ Medium

Abstract: To study the involvement of the xeroderma pigmentosum group A gene ( XPA ) in neuronal apoptosis, we cultured cerebellar neurons from mice lacking XPA gene ( XPA ^−/−) and induced apoptosis by exposure to UV irradiation or medium containing a low concentration of potassium (low-K⁺ medium). When cerebellar neurons from postnatal days 15–16 wild-type mice were treated with UV irradiation, apoptotic neuronal death was observed after 24–48 h. About 60% of neurons survived 48 h after UV irradiation at a dose of 5 J/m². On the other hand, neurons from XPA ^−/− mice showed a significantly increased vulnerability to UV irradiation, and >90% of neurons died 48 h after UV irradiation at a dose of 5 J/m². In contrast, low-K⁺ medium induced apoptosis of neurons from mice of each genotype with the same kinetics. These results suggest that the XPA gene is involved in neuronal DNA repair and that it thereby influences apoptosis induced by DNA damage in cultured cerebellar neurons.     

Ginkgolide B preconditioning on astrocytes promotes neuronal survival in ischemic injury via up-regulating erythropoietin secretion

Although ischemic preconditioning (IP) can provide powerful protection on brain against ischemic insult, it is rarely used in clinic to prevent the occurrence of ischemic stroke because of safety concerns. It is therefore necessary to seek the safer stimuli to initiate pharmacological preconditioning. Our previous work demonstrated that ginkgolide B (GB) could protect neurons against ischemia-induced apoptosis. Astrocytes are the most numerous cells in mammalian central nervous system and there is a close bi-directional communication between neurons and astrocytes in brain. Besides neurons, whether GB can exert the role of preconditioning on astrocytes through which to further improve neuronal survival under ischemic condition is not yet known. In the present study, primary cultured astrocytes were treated with GB for 24 h or short-term ischemia (ischemia for 3 h, as ischemic preconditioning/IP), and then cultured back to normoxia and normal medium for 24 h to induce the preconditioning response. Astrocyte-conditioned medium (ACM) was then collected and used to incubate the cultured neurons for 24 h before neurons were subjected to severe ischemia. Our results demonstrated that not only GB and IP increased astrocytic viability in ischemia, but also the conditioned medium from astrocytes treated with GB or IP increased cell viability and decreased the number of apoptosis of neurons in ischemia. We also found that GB and IP significantly stimulated astrocytes to express and secrete erythropoietin (EPO) into ACM, and the addition of anti-EPO antibody blocked the protective effect of GB or IP-treated astrocytes culture medium on neurons in ischemia. Further study of above protection revealed that ACM from astrocytes treated with GB or IP induced the inactivation of proapoptotic factor Bad by phosphorylation at serine 136 and 112 (¹³⁶p-Bad and ¹¹²p-Bad) in neurons. Together, our results suggest that GB is capable of preconditioning on astrocytes as IP and then protects neurons against ischemia-induced apoptosis, which is mediated by EPO.     

Trichostatin A induces cell death at the concentration recommended to differentiate the RGC-5 cell line

Supplementation with Trichostatin A (TSA) has been described as the method of choice for differentiating the RGC-5 cell line into cells with neuronal properties. However, TSA is known to induce apoptosis. We therefore investigated whether TSA at the recommended concentration for differentiation (500 nM) and at three additional concentrations (40, 150 and 2000 nM) induces apoptosis or cell death in the RGC-5 cell line. Morphological changes of the RGC-5 cells occurred after 24 and 48 hours (h) of treatment with 500 and 2000 nM TSA. Differentiation of RGC-5 cell began at 150 nM. A decrease in the cell count was observed from 150 nM TSA onwards compared to controls. Five hundred nanomolar of TSA reduced the amount of cells to 51% (p<0.005) after 24h and to 24% (p<0.005) after 48 h compared to controls on crystal violet staining. At 500 nM TSA a massive induction of apoptosis after 24 and 48 h was noted. Supplementation of 500 nM TSA increased caspase 3/7 activity 5.0-fold (p<0.005). Furthermore, 27× more TUNEL-positive cells were found and the cleaved caspase 3/caspase 3 ratio was 1.8-fold (p<0.1) higher 24h after the addition of 500 nM TSA. The Bax/Bcl-2 ratio was 3.4-fold (p<0.05) higher after 48 h. Cell viability decreased to 70% (p<0.005) and to 35% (p<0.005) after 24 and 48 h, respectively. Moreover, 103× (p<0.05) more dead cells (via propidium iodide staining) were found after 48 h of treatment with 500 nM TSA. In conclusion, TSA induces cell death and apoptosis at the concentration recommended for differentiation. The induction of apoptosis occurred dose and time dependently and already at even lower concentrations of TSA which did not lead to differentiation induced apoptosis. Thus, studies with RGC-5 cells should not be performed within the first 48 h after supplementation with TSA.     

Mild hypothermia protects hippocampal neurons from oxygen-glucose deprivation injury through inhibiting caspase-3 activation

Mild hypothermia (MH) is thought to be one of the most effective therapeutic methods to treat hypoxic-ischemic encephalopathy (HIE) after cardiac arrest (CA). However, its precise mechanisms remain unclear. In this research, hippocampal neurons were cultured and treated with mild hypothermia and Ac-DEVD-CHO after oxygen-glucose deprivation (OGD). The activity of caspase-3 was detected, in order to find the precise concentration of Ac-DEVD-CHO with the same protective role in OGD injury as MH treatment. Western blot and immunofluorescence staining were conducted to analyze the effects of MH and Ac-DEVD-CHO on the expressions of caspase-3, caspase-8, and PARP. The neuronal morphology was observed with an optical microscope. The lactic acid dehydrogenase (LDH) release rate, neuronal viability, and apoptotic rate were also detected. We found that MH (32 °C) and Ac-DEVD-CHO (5.96 μMol/L) had equal effects on blocking the activation of caspase-3 and the OGD-induced cleavage of PARP, but neither had any effect on the activation of caspase-8, which goes on to activate caspase-3 in the apoptotic pathway. Meanwhile, both MH and Ac-DEVD-CHO had similar effects in protecting cell morphology, reducing LDH release, and inhibiting OGD-induced apoptosis in neurons. They also similarly improved neuronal viability after OGD. In conclusion, caspase-3 serves as a key intervention point of the key modulation site or regulatory region in MH treatment that protects neuronal apoptosis against OGD injury. Inhibiting the expression of caspase-3 had a protective effect against OGD injury in MH treatment, and caspase-3 activation could be applied to evaluate the neuroprotective effectiveness of MH on HIE.     

Haloperidol-induced neuronal apoptosis: role of p38 and c-Jun-NH(2)-terminal protein kinase

We examined patterns and mechanisms of cell death induced by haloperidol. Cortical cell cultures exposed to 10-100 microM: haloperidol for 24 h underwent neuronal death without injuring glia. The degenerating neurons showed hallmarks of apoptosis, featuring cell body shrinkage, nuclear chromatin condensation and aggregation, nuclear membrane disintegration with intact plasma membrane, and prominent internucleosomal DNA fragmentation. Neither glutamate antagonists nor antioxidants prevented the haloperidol-induced neuronal apoptosis. The c-Jun-NH(2)-terminal protein kinase and p38 mitogen-activated protein kinase were activated within 1 h and were sustained over the next 3 h following exposure of cortical neurons to 30 microM haloperidol. Haloperidol-induced neuronal apoptosis was partially attenuated by 10-30 microM PD169316, a selective inhibitor of p38 mitogen-activated protein kinase. Inclusion of 1 microg/ml cycloheximide, a protein synthesis inhibitor, or 100 ng/ml insulin prevented activation of both kinases and subsequent neuronal death. The present study demonstrates that cortical neurons exposed to haloperidol undergo apoptosis depending on activation of p38 mitogen-activated protein kinase and c-Jun-NH(2)-terminal protein kinase sensitive to cycloheximide and insulin.     

溶血磷脂酸对β-淀粉样蛋白片段AβP31-35所致小鼠大脑皮层离体神经元凋亡的神经保护作用

已报道低浓度溶血磷脂酸(lysophosphatidic acid,LPA)对去血清培养所致的神经元凋亡有神经保护作用.为了进一步观察LPA是否对β-amyloid peptide fragment 31-35(AβP31-35)所致的神经元凋亡也起类似的作用,本研究应用DNA电泳分析、HO33342和TUNEL染色法等技术,对培养的小鼠大脑皮层神经元进行了观察.结果显示,只有使用较低浓度的LPA(1～10μmol/L)、并且将此剂量的LPA比AβP31-35提前12～24 h加入培养液时,才可看到LPA明显削弱了AβP31-35所致的神经元凋亡.以上结果表明,适当浓度的LPA在长时间预作用的条件下,可对AβP31-35所致的皮层神经元凋亡起保护因子或抗凋亡因子的作用,但其作用途径可能较在去血清培养所致的凋亡时更为复杂,因为在去血清的同时加入LPA就能制止去血清所致的凋亡.     

Apoptosis Induced via AMPA-Selective Glutamate Receptors in Cultured Murine Cortical Neurons

Abstract: We have investigated the mechanisms of cell death induced by long-term exposure to the glutamate receptor agonist ( S )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate [( S )-AMPA]. Using primary cultures of pure neurons (95%) grown in serum-free conditions, we found that 24-h exposure to ( S )-AMPA (0.01–1,000 µ M ) induced concentration-dependent neuronal cell death (EC₅₀ = 3 ± 0.5 µ M ) with cellular changes including neurite blebbing, chromatin condensation, and DNA fragmentation, indicative of apoptosis. ( S )-AMPA induced a delayed cell death with DNA fragmentation occurring in ∼50% of cells at concentrations between 100 and 300 µ M detected using terminal transferase-mediated dUTP nick end-labeling (TUNEL) and agarose gel electrophoresis. Apoptotic chromatin condensation was detected using 4,6-diamidino-2-phenylindole, a fluorescent DNA binding dye. Cell death induced by ( S )-AMPA was attenuated by the AMPA receptor-selective antagonist LY293558 (10 µ M ) and the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 µ M ), yielding EC₅₀ values of 73 ± 5 and 265 ± 8 µ M , respectively, and was unaffected by the NMDA receptor antagonist MK-801 (10 µ M ). The number of apoptotic nuclei induced by 300 µ M ( S )-AMPA (57%) was also reduced substantially by the antagonists LY293558 and CNQX, with only 20% and 18% of neurons, respectively, staining TUNEL-positive at 24 h. In addition, cycloheximide (0.5 µg/ml) also inhibited ( S )-AMPA-induced DNA fragmentation and cell death. Our results show that long-term exposure to AMPA can induce substantial neuronal death involving apoptosis in cultured cortical neurons, suggesting a wide involvement of AMPA-sensitive glutamate receptors in excitotoxic injury and neurodegenerative pathologies.