Inhibitory effect of lead on GABA A receptor-mediated currents and GABAergic synaptic transmission in rat cortical neurons / 中国药理学与毒理学杂志

OBJECTIVE To investigate the inhibitory effect and mechanism of lead(Pb2+)on γ-amino-butyric acid(GABA)A receptor-mediated currents(IGABA)and GABAergic synaptic transmission in rat cortical neurons.METHODS ①The cortical neurons from 0 d Sprague Dawley(SD)rats were cultured for experiments.The cultured cells(7-14 d)were recorded using the patch-clamp technique to analyze the effects of Pb2+ at different concentrations(1,5,10,50 and 100 μmol·L-1)on IGABA induced by GABA 100 μmol·L-1.② The effects of Pb2+ 50 μmol·L-1 on IGABA induced by GABA at different concentrations(1,10,50,100,500 and 100 μmol·L-1)were detected.③Brain slices(350 μm)were prepared from SD rats(15-19 d).The spontaneous inhibitory post-synaptic currents(sIPSCs),miniature inhibitory post-synaptic currents(mIPSCs)and current injection-induced action potential(AP)were recorded to detect the effects of Pb2+ 10 μmol·L-1 on the amplitude and frequency of sIPSCs and mIPSCs,and the frequency of AP.RESULTS ①Pb2+ inhibited IGABA in a concentration-dependent manner,and IC50 was(68±20)μmol·L-1.②Pb2+ also suppressed the maximum current induced by GABA(P<0.01),with a significant increase of the GABA′s EC50 from(20±6)μmol·L-1 to(87±39)μmol·L-1,indicating that Pb2+ might inhibit IGABA in a non-competitive mechanism.③Pb2+ 10 μmol·L-1 inhibited the frequency(P<0.01)rather than the ampli-tude of sIPSCs reversibly,but had no effect on eigher the frequency or amplitude of mIPSCs.In addi-tion,Pb2+ decreased the frequency of evoked AP by current injection(P<0.01)and reduced the overall excitability of rat cortical neurons.CONCLUSION Pb2+ can significantly inhibit IGABA in primary cultured neurons.In the brain slice experiment,Pb2+ may affect sIPSCs frequency by inhibiting the AP of cortical neurons,suggesting that there are different intrinsic mechanisms through which Pb2+ inhibits both IGABA in primary cultured neurons and the frequency of sIPSCs in brain slice neurons,which points to the complexity of the mechanism of Pb2+ poisoning.

Effects of ring finger and tryptophan-aspartic acid 2 on dendritic spines and synapse formation in cerebral cortex neurons of mice / 南方医科大学学报

OBJECTIVE@#To clarify the functional effects of differential expression of ring finger and tryptophan-aspartic acid 2 (RFWD2) on dendritic development and formation of dendritic spines in cerebral cortex neurons of mice.@*METHODS@#Immunofluorescent staining was used to identify the location and global expression profile of RFWD2 in mouse brain and determine the co-localization of RFWD2 with the synaptic proteins in the cortical neurons. We also examined the effects of RFWD2 over-expression (RFWD2-Myc) and RFWD2 knockdown (RFWD2-shRNA) on dendritic development, dendritic spine formation and synaptic function in cultured cortical neurons.@*RESULTS@#RFWD2 is highly expressed in the cerebral cortex and hippocampus of mice, and its expression level was positively correlated with the development of cerebral cortex neurons and dendrites. RFWD2 expression was detected on the presynaptic membrane and postsynaptic membrane of the neurons, and its expression levels were positively correlated with the length, number of branches and complexity of the dendrites. In cultured cortical neurons, RFWD2 overexpression significantly lowered the expressions of the synaptic proteins synaptophysin (P < 0.01) and postsynapic density protein 95 (P < 0.01), while RFWD2 knockdown significantly increased their expressions (both P < 0.05). Compared with the control and RFWD2-overexpressing cells, the neurons with RFWD2 knockdown showed significantly reduced number of dendritic spines (both P < 0.05).@*CONCLUSION@#RFWD2 can regulate the expression of the synaptic proteins, the development of the dendrites, the formation of the dendritic spines and synaptic function in mouse cerebral cortex neurons through ubiquitination of Pea3 family members and c-Jun, which may serve as potential treatment targets for neurological diseases.

Effects of extracellular vesicles from mesenchymal stem cells on oxygen-glucose deprivation/reperfusion-induced neuronal injury / 世界急诊医学杂志（英文）

@#BACKGROUND: Small extracellular vesicles (sEVs) from bone marrow mesenchymal stem cells (BMSCs) have shown therapeutic potential for cerebral ischemic diseases. However, the mechanisms by which BMSC-derived sEVs (BMSC-sEVs) protect neurons against cerebral ischemia/reperfusion (I/R) injury remain unclear. In this study, we explored the neuroprotective effects of BMSC-sEVs in the primary culture of rat cortical neurons exposed to oxygen-glucose deprivation and reperfusion (OGD/R) injury. METHODS: The primary cortical neuron OGD/R model was established to simulate the process of cerebral I/R in vitro. Based on this model, we examined whether the mechanism through which BMSC-sEVs could rescue OGD/R-induced neuronal injury. RESULTS: BMSC-sEVs (20 μg/mL, 40 μg/mL) significantly decreased the reactive oxygen species (ROS) productions, and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx). Additionally, BMSC-sEVs prevented OGD/R-induced neuronal apoptosis in vivo, as indicated by increased cell viability, reduced lactate dehydrogenase (LDH) leakage, decreased terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining-positive cells, down-regulated cleaved caspase-3, and up-regulated Bcl-2/Bax ratio. Furthermore, Western blot and flow cytometry analysis indicated that BMSC-sEV treatment decreased the expression of phosphorylated calcium/calmodulin-dependent kinase II (p-CaMK II)/CaMK II, suppressed the increase of intracellular calcium concentration ([Ca2+]i) caused by OGD/R in neurons. CONCLUSIONS: These results demonstrate that BMSC-sEVs have significant neuroprotective effects against OGD/R-induced cell injury by suppressing oxidative stress and apoptosis, and Ca2+/CaMK II signaling pathways may be involved in this process.

The protective effects of gastrodin on methamphetamine-induced neurotoxicity / 中国药理学通报

Aim To compare the protective effects of gastrodin and melatonin on methamphetamine ( MA )-induced neurotoxicity of cortical neurons. Methods Cortical neuron cells were treated with different concentrations of methamphetamine for 24 h, then the cell viability was detected by CCK-8 kit to choose the optimal concentration of MA. The cells were treated with different concentrations of gastrodin or melatonin 2 h before the treatment of cells with optimal concentration of MA for 24 h. The optimal concentrations of gastrodin and melatonin were determined by CCK-8 kit as well. Cortical neuron cells were randomly divided into con trol group, MA group, gastrodin plus MA group, and melatonin plus MA group. Then the apoptotic rate of cells was detected by TUNEL method . The expression of Caspase-3 in cells was assessed by immunofluorescence. Results The optimal concentration of MA was 0. 5 mmol • L"1. After treating with MA, the numbers of cortical neurons decreased, the length of synapses became shorter, and the cell vacuolation, the number of dead cells increased. The optimal concentration of gastrodin was 25 mg • L'1, and the optimal concentration of melatonin was 10 jimol • L"1. Gastrodin treatment could reduce the apoptosis of cortical neurons induced by MA, and the expression of Caspase-3 decreased as well, and there was no significant difference compared with the intervention effect of gastrodin with melatonin. Conclusions MA has neurotoxic damage to cortical neurons. Gastrodin could attenuate MA-in-duced neurotoxicity via alleviating the apoptosis induced by MA.

The role of miR-132 in primary cortical neuron ischemic injury induced by oxygen-glucose-deprivation / 中风与神经疾病杂志

@#Objective　To investigate the role of miR-132 in oxygen-glucose-deprivation （OGD） induced ischemic injury model using primary cortical neurons.Methods　Cultured primary cortical neurons were exposed to oxygen-glucose-deprivation （OGD）for 2 h followed by 24 h reperfusion.The cells were randomly divided into sham operation group (Sham),OGD group,lentivirus control group (LV-control),miR-132 low expression group (LV-anti-miR-132),and miR-132 overexpression group (LV-miR-132).CCK-8,real-time PCR,and Western blot were used to study the effects and mechanisms of miR-132 on primary cortical neurons injured by OGD.Results　The expression levels of miR-132 were significantly reduced in the OGD-induced ischemic injury model (P<0.05).The reduced expression of miR-132 aggravated OGD injury and significantly reduced cell viability in OGD-induced primary cortical neuron ischemic injury.On the contrary,the increased expression levels of miR-132 reduced OGD injury and increased cell survival.Western blot results showed that overexpression of miR-132 upregulated the levels of synapsin-1 and PSD95.Conclusion　Overexpression of miR-132 can improve OGD-induced ischemic injury and increase the survival of primary cortical neurons by upregulating the synapsin-1 and PSD95.

Cyproheptadine Regulates Pyramidal Neuron Excitability in Mouse Medial Prefrontal Cortex / 神经科学通报·英文版

Cyproheptadine (CPH), a first-generation antihistamine, enhances the delayed rectifier outward K current (I) in mouse cortical neurons through a sigma-1 receptor-mediated protein kinase A pathway. In this study, we aimed to determine the effects of CPH on neuronal excitability in current-clamped pyramidal neurons in mouse medial prefrontal cortex slices. CPH (10 µmol/L) significantly reduced the current density required to generate action potentials (APs) and increased the instantaneous frequency evoked by a depolarizing current. CPH also depolarized the resting membrane potential (RMP), decreased the delay time to elicit an AP, and reduced the spike threshold potential. This effect of CPH was mimicked by a sigma-1 receptor agonist and eliminated by an antagonist. Application of tetraethylammonium (TEA) to block I channels hyperpolarized the RMP and reduced the instantaneous frequency of APs. TEA eliminated the effects of CPH on AP frequency and delay time, but had no effect on spike threshold or RMP. The current-voltage relationship showed that CPH increased the membrane depolarization in response to positive current pulses and hyperpolarization in response to negative current pulses, suggesting that other types of membrane ion channels might also be affected by CPH. These results suggest that CPH increases the excitability of medial prefrontal cortex neurons by regulating TEA-sensitive I channels as well as other TEA-insensitive K channels, probably I and inward-rectifier Kir channels. This effect of CPH may explain its apparent clinical efficacy as an antidepressant and antipsychotic.

Sustained Intracellular Acidosis Triggers the Na⁺/H⁺ Exchager-1 Activation in Glutamate Excitotoxicity

The Na⁺/H⁺ exchanger-1 (NHE-1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the brain, heart, and other organs. It is increased by intracellular acidosis through the interaction of intracellular H⁺ with an allosteric modifier site in the transport domain. In the previous study, we reported that glutamate-induced NHE-1 phosphorylation mediated by activation of protein kinase C-β (PKC-β) in cultured neuron cells via extracellular signal-regulated kinases (ERK)/p90 ribosomal s6 kinases (p90RSK) pathway results in NHE-1 activation. However, whether glutamate stimulates NHE-1 activity solely by the allosteric mechanism remains elusive. Cultured primary cortical neuronal cells were subjected to intracellular acidosis by exposure to 100 μM glutamate or 20 mM NH₄Cl. After the desired duration of intracellular acidosis, the phosphorylation and activation of PKC-β, ERK1/2 and p90RSK were determined by Western blotting. We investigated whether the duration of intracellular acidosis is controlled by glutamate exposure time. The NHE-1 activation increased while intracellular acidosis sustained for >3 min. To determine if sustained intracellular acidosis induced NHE-1 phosphorylation, we examined phosphorylation of NHE-1 induced by intracellular acidosis by transient exposure to NH₄Cl. Sustained intracellular acidosis led to activation and phosphorylation of NHE-1. In addition, sustained intracellular acidosis also activated the PKC-β, ERK1/2, and p90RSK in neuronal cells. We conclude that glutamate stimulates NHE-1 activity through sustained intracellular acidosis, which mediates NHE-1 phosphorylation regulated by PKC-β/ERK1/2/p90RSK pathway in neuronal cells.

Over-expression of PGC-1α reverses mitochondrial function reduction and apoptosis in OGD/R-induced neurons / 中国病理生理杂志

AIM:To investigate the effect of over-expression of peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) on mitochondrial morphology and cell apoptosis in the cortical neurons with oxygen glucose depriva-tion/reoxygenation(OGD/R). METHODS:The whole gene sequence of PGC-1α was obtained from the cerebral cortex of C57BL/6 mice by RT-PCR and cloned into the eukaryotic expression vector pEGFP-N1. The pEGFP-N1-PGC-1α was iden-tified by PCR,and transfected into cortical neurons. The level of PGC-1α expression was identified by Western blot. The cortical neurons transfected with pEGFP-N1 and pEGFP-N1-PGC-1α vectors were treated with OGD/R. The mitochondrial mass,reactive oxygen species (ROS) and ATP production,cell apoptosis and changes of cleaved caspase-3 were detected by MitoTracker Red staining,flow cytometry,ATP metabolic assay kit and TUNEL. RESULTS:Over-expression of PGC-1α inhibited the decrease in mitochondrial biogenesis capacity and the ROS formation of OGD/R neurons(P<0.05),en-hanced the ability of ATP synthesis (P<0.01),inhibited neuronal apoptosis (P<0.01) and decreased the activation of caspase-3 (P<0.01). CONCLUSION:PGC-1α over-expression inhibits neuronal apoptosis with OGD/R treatment by promoting mitochondrial biogenesis,inhibiting the production of ROS and maintaining mitochondrial function. PGC-1α may be used as a target for the development of cerebral ischemia/reperfusion injury drugs.

Study of anti-aging protective effect of catalpol on cultured cortical neurons from rats / 中国药理学通报

Aim To investigate the anti-aging effect of catalpol on normal cortical neurons and its possible mechanism. Methods Primary cultured cortical neu-rons from 24 h newly born rats were dissociated and cultured. The cells were divided into normal group, catalpol (0. 1 mg · L - 1 )group,catalpol (1 mg · L - 1 )group and catalpol (10 mg·L - 1 )group. Neu-rons were cultured for 13d,and the cell morphology was observed by microscope. Cell activity was detected by MTT assay. p-S6 and Map-2 of 13d neurons were detected by immunofluorescence to reveal cell viability and the axon growth. GAP-43,p-S6,PI3K,p-PI3K, Akt,p-Akt,mTOR and p-mTOR protein expression of 13d neurons were detected by Western blot. Results Compared with normal group,different concentrations of catalpol could delay the aging of neurons,promote their survival and increase cell viability. The nμmber of p-S6 positive cells increased. Different concentra-tions of catalpol could promote axon growth. GAP-43, p-S6,PI3K,p-PI3K,Akt,p-Akt,mTOR and p-mTOR protein expression of 13d neurons increased. Conclusions Catalpol has anti-aging effects on cere-bral cortical neurons,and its mechanism may be relat-ed to regulating PI3K/ Akt/ mTOR signaling pathway and enhancing neuronal growth activity.

Effect of CREB-shRNA on mitochondrial morphology and cell apoptosis in OGD/R-induced cortical neurons / 中国病理生理杂志

AIM: To construct recombinant lentiviral vector with short hairpin RNA (shRNA) of CREB gene, and to investigate the effect of CREB gene silencing on mitochondrial morphology and cell apoptosis in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cortical neurons.METHODS: Three lentiviral vectors pLentiLox3.7 (PLL) inserted shRNA fragments targeting CREB gene were co-transfected with the packaging plasmids psPAX2 and pMD2.G to the 293T cells, and the virus particles, which was infected with the primary cortical neurons, was encapsulated.The protein expression of CREB was detected by Western blot.The mitochondrial morphology, cell apoptosis and the expression of Bcl-2 and Bax were evaluated by the methods of MitoTracker red, TUNEL and Western blot in OGD/R induced cortical neurons after CREB gene silencing.RESULTS: The pLL-CREB-shRNA1 was the most effective shRNA, which inhibited 80% CREB gene expression in the cortical neurons.The mitochondrial was appeared dot and fragment morphology in OGD/R induced cortical neurons with transfected pLL-CREB-shRNA1 plasmid.In addition, the expression of Bcl-2 was decreased, the expression of Bax, and the apoptosis of the neurons were increased by tranfected with pLL-CREB-shRNA1.CONCLUSION: CREB shRNA recombinant lentiviral vector specifically inhibits the expression of CREB gene.CREB gene silencing promotes the cell apoptosis and mitochondrial morphological changes in the cortical neurons induced by OGD/R.

Protective effect of notoginsenoside R1 on neuron injury induced by OGD/R through ATF6/Akt signaling pathway / 中国中药杂志

Notoginsenoside R1(NGR1),a critical compound in traditional herb Panax notoginseng, is a kind of estrogen receptor agonist.It is reported to exhibit anti-apoptotic,anti-oxidative and anti-inflammatory properties activity, so it is widely used for treatment of various diseases.In order to investigate the potential neuroprotective effect of NGR1 in hypoxic-ischemic brain damage(HIBD), primary cortical neurons were used in this study to establish oxygen-glucose deprivation/reoxygenation(OGD/R) injury models. They were treated with NGR1 and estrogen receptor inhibitor ICI-182780 respectively, then the neuronal survival, cell membrane integrity and apoptosis were assessed by MTT assay,lactate dehydrogenase test(LDH) and Hoechst 33342 stain respectively, while the protein expression levels of ATF6α,p-Akt,Akt,Bax and Cleaved Caspase-3 were measured by Western blotting. Results indicated that as compared with the blank control group,OGD/R could induce cell injury and apoptosis(P<0.05), reduce relative integrity of cell membrane(P<0.05), decrease protein expression of ATF6α,p-Akt(P<0.05), and increase protein expression of Bax and Cleaved Caspase-3(P<0.05) in the primary cortical cells. After NGR1 treatment, the expression levels of ATF6α,p-Akt were obviously increased, and the expression levels of Bax and Cleaved Caspase-3 and the apoptosis of neuron were decreased(P<0.05). However, these neuroprotective properties of NGR1 against ODG/R-induced cell damage could be blocked by ICI-182780. This finding indicated that NGR1 may protect the primary cortical neurons against OGD/R induced injury,and the mechanism may be associated with accelerating the activation of the ATF6/Akt signaling pathway via estrogen receptors.

Expression and significance of connexin Cx36 in hippocampus and cortical neurons of febrile seizure rats / 中华实用儿科临床杂志

Objective To explore the potential changes of connexin Cx36 in hippocampus and cortical neurons of rats with hyperthermia -induced convulsion.Methods Rats were divided into 2 groups according to the random number table method:normal control group and experimental group.Febrile convulsion model was elicited through im-mersion in warm water.The experimental group was generated following febrile convulsion model:hyperthermia group and febrile convulsion group.Among normal control group,hyperthermia group and febrile convulsion group,western blot analysis and immunofluorescence labeling techniques were used to examine the expression of Cx36 protein in the hippo-campus and cortex area.One -Way ANOVA was used to compare the mean of multiple sample,the LSD test was used to compare the two means.Tamhane′s test was used when variance were uneven.Results The incubation period,seizure duration and temperature were (4.39 ±0.08)min,(5.38 ±0.07)min,(41 .87 ±0.06)℃ after hyperthermia-in-duced convulsion,respectively.Western blot analysis showed that the expression of Cx36 protein in the hippocampus and cortex area decreased gradually after 1 0 times of seizure in normal control group,hyperthermia group and febrile convulsion group,and the febrile convulsion group decreased most obviously.Compared with normal control group and hyperthermia group,respectively,in febrile convulsion group Cx36 expression obviously decreased in the hippocampus and cortex in rats with 1 ,5,1 0 seizure times induced by hyperthermia,and with the increase of number of induced con-vulsion,the expression of Cx36 was significantly decreased in the cortex (0.1 04 ± 0.01 2)and CA1 (0.091 ± 0.01 1 ),CA3 (0.090 ±0.01 1 )and DG (0.092 ±0.01 2)areas of hippocampal neurons compared with the normal control group (0.21 2 ±0.01 7,0.1 67 ±0.01 3,0.1 59 ±0.01 4,0.1 71 ±0.01 3)and the hyperthermia group (0.1 89 ± 0.006,0.1 44 ±0.008,0.1 29 ±0.005,0.1 65 ±0.01 1 )(all P <0.05).Furthermore,the extent of reduction in Cx36 expression seemed to correlate with the number of seizures.Conclusion With the increase of thermal seizure frequen-cy,Cx36 expression of rats was decreased obviously which may lower convulsion threshold and lead to recurrent seizures.

Effects of propofol treatment on neuroapoptosis in primary cultured cortical neurons / 临床麻醉学杂志

Objective To investigate the effect of propofol exposure on neuroapoptosis in pri-mary cultured cortical neurons and its mechanisms.Methods Cortical neurons were primarily cultured for seven days,then divided into two groups:control group (treated with equal volume of 20% in-tralipid),propofol-treated group (treated with 500 μmol/L propofol).The neurons were treated for 12 h.The neuron viability was determined by MTT.Neuroapoptosis was identified by Hoechest 33 258 dying.Mitochondrial membrane potential was measured by the fluorescent dye rhodamine 123 (Rh123).Western blot was performed to detect the level of cyt-c and cleaved-caspase-3.Results Neu-rons survival rate (54.4%±6.4%)in the propofol group was significantly lower than that of control group (99.8% ± 4.1%) (P < 0.05 ), the rate of neuronal apoptosis (46.5% ± 5.3%) was significantly higher than that of control group (7.2%±0.9%)(P <0.05),mitochondrial membrane potential (59.6%±4.3%)was significantly lower than that of the control group (99.9% ± 5.7%) (P <0.05 ),cyt-C protein level (0.38 ± 0.03 )was significantly higher than that of control group (0.1 5±0.02)(P < 0.05 ),level of cleaved-caspase-3 protein level (0.46 ± 0.04)was significantly higher than that of control group (0.13±0.02)(P <0.05).Conclusion Propofol induces neuroapo-tosis in primary cultured cortical neurons,which is associated with the decreased level of MPP and the increase levels of cyt-c and cleaved-caspase-3.

Cadmium Activates Reactive Oxygen Species-dependent AKT/mTOR and Mitochondrial Apoptotic Pathways in Neuronal Cells / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To examine the role of Cd-induced reactive oxygen species (ROS) generation in the apoptosis of neuronal cells.</p><p><b>METHODS</b>Neuronal cells (primary rat cerebral cortical neurons and PC12 cells) were incubated with or without Cd post-pretreatment with rapamycin (Rap) or N-acetyl-L-cysteine (NAC). Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3'-kinase/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays.</p><p><b>RESULTS</b>Cd-induced activation of Akt/mTOR signaling, including Akt, mTOR, p70 S6 kinase (p70 S6K), and eukaryotic initiation factor 4E binding protein 1 (4E-BP1). Rap, an mTOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/mTOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein (Bcl-2/Bax) ratio, release of cytochrome c, cleavage of caspase-3 and poly(ADP-ribose) polymerase (PARP), and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease G (Endo G).</p><p><b>CONCLUSION</b>Cd-induced ROS generation activates Akt/mTOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that mTOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.</p>

Comparison and improvement of three methods for primary culture of rat embryonic cortical neurons / 中国实验动物学报

Objective To compare three methods for culture of fetal cortical neurons of SD rats and find out the suitable culture conditions of fetal cortical neurons in vitro.Methods The cortex of 16-18-day embryonic rat was used for culture in this study.Mechanical dissociation, trypsin digestion and papain digestion were applied respectively to the neuron culture.The morphological characteristics of neuronal cells at different time points were observed and neuron purity was identified by immunofluorescence staining assay.Results High purity of the fetal rat cortical neurons was successfully achieved by all the three culture methods, and each had distinct morphological characteristics at different time points.The purity of neurons was 96.28%, 95.63%and 97.34%, respectively, with no significant differences among the three groups (P>0.05).Conclusions The three culture methods are improved in our study.Stable neurons with high purity can be obtained by all the three methods respectively, and each of these methods has distinct characteristics.

Neuroprotective Effect of Clonidine on Primary Cultured Cortical Neurons in Rats Subjected to Oxygen-glucose Deprivation Injury / 医药导报

Objective To determine the neuroprotective effect of clonidine on primary cultured cortical neurons in rats exposed to oxygen-glucose deprivation ( OGD) injury. Methods Cortical neurons cultured for 8 days were randomly assigned to the three groups: normal control group, model control group, and clonidine pretreatment group. OGD injury model was established by chemical hypoxia and glucose deprivation in incubation liquid for 4 h. Clonidine (1. 0, 3. 0, 10 μmol·L-1 ) was added 24 h before OGD injury. Neuronal injury was evaluated by MTT staining and the release of lactate dehydrogenase ( LDH) . Results Under the microscope, primary cultured cortical neurons in normal control group presented great density, round size, smooth edge, and high diopter,The suvival rate of neurons and the percentage of LDH releasing were (100. 00±32. 12)% and (100. 00 ± 37. 51 )%, respectively. After exposure to OGD injury, cortical neurons showed karyopyknosis, incomplete cell membranes, low diopters and a significant reduction in optical density of MTT staining. In addition, the suvival rate of neurons and the percentage of LDH releasing were (53. 61±7. 62)% and (166. 07±9. 65)% separately compared with normal control group. In the group with pretreatment of different concentrations of clonidine (1. 0, 3. 0, 10μmol·L-1), morphological changes induced by OGD injury were significantly reversed and optical density of MTT staining was dose-dependently raised. The percentages of survival neurons much higher than that of model control group were [(67. 53±10. 54)%, (71. 50±9. 79)% and (87. 48±5. 29)%, separately] and the obvious reductions of LDH releasing were [(136. 45±25. 72)%, (130. 92±24. 94)%and (121. 63±32. 68)%, respectively]. Conclusion Clonidine can exert neuroprotection against OGD-induced injury in primary cultured cortical neurons in rats.

Dexmedetomidine protects cortical neurons from ketamine-induced apoptosis by activating the PI3 K-Akt signaling pathway / 医学研究生学报

Objective Dexmedetomidine is known to have a neuroprotective effect.The aim of this study was to investigate the effects of dexmedetomidine on ketamine-induced apoptosis of primarily cultured cortical neurons and its action mechanisms. Methods Rat cortical neurons were primarily cultured for 7 days and treated with ketamine (100μmol/L) and different concentrations of dexmedetomi-dine (0.001, 0.01, 0.1, and 1 μmol/L) for 24 hours, followed by measurement of the viability of the neurons by MTT assay.The neurons were divided into four groups:vehicle control, ketamine ( trea-ted with 100 μmol/L ketamine), dexmedetomidine+ketamine (DD+K, treated with 0.1 μmol/L DD and 100 μmol/L ketamine), and LY294002 ( treated with 0.1 μmol/L DD, 100 μmol/L ketamine, and 10 μmol/L LY294002) .After 24 hours of treatment, the apoptosis rate of the neurons was determined by Hoechst33258 staining, and the expressions of pAkt and cleaved-caspase-3 in the neu-rons detected by Western blot. Results The apoptosis rate of neurons was dramatically increased in the LY294002 and ketamine groups in comparison with the vehicle control and DD+K groups ([36.8 ±4.4] and [43.4 ±4.5]%vs [7.5 ±1.1] and [16.4 ± 3.6]%, P<0.01), the pAkt level remarkably decreased (0.26 ±0.02 and 0.15 ±0.01 vs 0.61 ±0.05 and 0.50 ±0.04, P<0.01), and the expression of cleaved caspase-3 significantly upregulated in the former two as compared with the latter two groups (0.40 ±0.02 and 0.65 ±0.03 vs 0.10 ±0.02 and 0.12 ±0.01, P<0.01). Conclusion Dexmedetomidine exerts a neuroprotec-tive effect against ketamine-induced apoptosis of neurons by activating the PI3K-Akt signaling pathway.

Activation of α7 nicotinic acetylcholine receptor alleviates cerebral cortical neuron injury induced by oxygen- glucose deprivation / 第二军医大学学报

Objective To investigate the effect of activatinga7 nicotinic acetylcholine receptor (α7nAchR) on cerebral cortical neurons injury induced by oxygen-glucose deprivation (OGD) and the possible mechanism. Methods Cerebral cortical neurons cultured for 7 d were randomly divided into three groups; control group, OGD group (Cells experienced a 12 h oxygen- glucose deprivation) and OGD group treated with PNU-282987 (Cells experienced a 12 h oxygen-glucose deprivation with PNU- 282987 pretreatment for 24 h). Cell viability was determined by CCK-8 assay, lactate dehydrogenase (LDH) was examined to reflect cell injury, apoptosis and reactive oxygen species (ROS) production were analyzed by flow cytometry, and expression of hemeoxygenase-1 (HO-1) and hypoxia inducible factor-1a (HIF-1a) were detected by Western blotting analysis. Results OGD resulted in cell death, LDH increase, and cell apoptosis. Compared with the OGD group, PNU-282987 pretreated group had significantly increased cell survival (P α protein expression was significantly reduced in PNU-282987 pretreated group compared with the OGD group (Pα7nAchR can protect cerebral cortical neurons against OGD-induced injury, which may be related to the anti-oxidative stress.

Effect and mechanism of dexmedetomidine on propofol-induced apoptosis of cortical neurons in rats / 中华行为医学与脑科学杂志

Objective To investigate the mechanisms of the protective effects of dexmedetomidine against the propofol-induced neuroapoptosis in primary cultured cortical neurons.Methods The neurons were cultured 7days and then divided into four groups: vehicle-control group (treated with equal volume of intralipid), propofoltreated group (treated with 500 μmol/L propofol), propofol plus dexmedetomidine treated group (treated with 500 μmol/L propofol and 0.1 μmol/L dexmedetomidine), and LY294002 pretreated group (treated with 500 μmol/L propofol ,0.1 μ mol/L dexmedetomidine and 10 μmol/L LY294002).12 hours after different treatments, neuron viability was measured by MTT assay,neuroapoptosis was detected by Hoechst33258 staining, and the levels of pAkt and Bcl-2 protein were detected by Western blot.Results Compared with the vehicle-reduced group,propofol reduced neuron viability greatly((53.4±4.2)％ vs (99.9±6.3)％;P＜0.01), but increased neuroapoptosis greatly((44.6±4.3)％ vs (5.8±0.4)％;P＜0.01).The levels of pAkt((0.41±0.03) vs (0.86±0.07))and Bcl-2 ((0.15±0.02) vs (0.72±0.03)) were decreased greatly (both P＜0.01).Compared with propofol treatment group, the neuron viability of propofol plus dexmedetomidine group were increased greatly((86.4±5.3) ％ , P＜0.01) ,the neu roapoptosis was decreased greatly ((23.1 ± 3.5) ％, P＜ 0.01), and the levels of pA kt (0.8 ± 0.03) and Bc1-2 (0.52 ±0.05) were increased greatly (both P＜0.01).Compared with propofol plus dexmedetomidine treated group,LY294002 inhibited the protective effects of dexmedetomidine, decreased neuron viability greatly ((64.3±5.1) ％ ,P＜0.01), increased the number of apoptotic neurons((38.8±4.9) ％, P＜0.01), and reduced the levels of pAkt (0.52±0.04) and Bcl-2(0.31±0.02) significantly (P＜0.01).Conclusion Dexmedetomidine exerts the neuroprotective effects against propofol-induced neuroapoptosis by activating the PI3K-Akt-Bcl-2 signalling pathway.

Effects of 17β-estradiol on propofol-induced rat cortical neuroapoptosis / 中国比较医学杂志

Objective To investigate the protective effects and the mechanisms of 17β-estradiol on the propofol-induced neuroapoptosis in primary cultured rat cortical neurons.Methods The neurons were cultured for 7 days and then divided into three groups: vehicle-control group ( treated with equal volume of 20% intralipid ) , propofol-treated group ( treated with 500μmol/L propofol) , and propofol plus 17β-estradiol treated group ( treated with 500μmol/L propofol and 0.1 μmol/L 17β-estradiol).12 hours after the treatment, neuroapoptosis was detected by Hoechst 33258 staining and TUNEL assay, and the levels of Bcl-2, Bax and cleaved caspase-3 proteins were detected by Western blot.Results Compared with the vehicle-control group, the neuroapoptosis increased greatly ( P<0.01 ) , Bcl-2 level reduced ( P <0.01), Bax and cleaved caspase-3 levels increased greatly (P<0.01), and Bcl-2/Bax ratio reduced significantly (P<0.01).Compared with the propofol-treatment group, the neuroapoptosis decreased greatly ( P <0.01), Bcl-2 level increased ( P<0.01 ) , Bax and cleaved caspase-3 levels reduced greatly ( P <0.01 ) , and Bcl-2/Bax ratio increased greatly ( P <0.01 ) . Conclusions 17β-estradiol can protect cortical neurons against propofol-induced cortical neuroapoptosis by regulating the expression of Bcl-2 and Bax.