Protective effect and mechanism of baicalein on hypoxia-induced cortical neuron injury in rats / 中国药房

OBJECTIVE To explore the protective effect and possible mechanism of baicalein on hypoxia-induced cortical neuron injury in rats. METHODS The cortical neurons of rats （RN-C cells） were studied and cultured under hypoxic conditions （5%CO2， 94% N2， 1%O2） for 24 hours； the effects of different concentrations of baicalein （0.01， 0.1， 1， 10， 100 μmol/L） on the survival rate of hypoxic RN-C cells were investigated； the effects of baicalein （0.1 μmol/L） on the activities of lactate dehydrogenase （LDH） and superoxide dismutase （SOD）， the content of malondialdehyde （MDA）， migration rate， apoptotic rate， cell cycle and the expressions of cleaved caspase-3， B-cell lymphoma-2 （Bcl-2） and Bcl-2 X protein （Bax） were all detected. RESULTS Compared with control group， the survival rate of cells in the hypoxia group was significantly reduced （P＜0.01）； 0.01， 0.1 and 1 μmol/L baicalein could reverse survival rate of hypoxia-induced cortical neurons （P＜0.05 or P＜0.01）. Scratch experiments showed that baicalein significantly increased the migration rate of hypoxic RN-C cells （P＜0.01）. Compared with control group， the activity of LDH in the supernatant and the content of MDA in the cells， apoptotic rate and the proportion of cells in G1 phase， were significantly increased in the hypoxia group， while SOD activity and the proportion of cells in G2+S phase was decreased significantly （P＜0.01）. The protein expressions of cleaved caspase-3 were increased significantly， while the ratio of Bcl-2/Bax in cells was significantly reduced （P＜0.05 or P＜0.01）. Compared with hypoxia group， the above indexes were all reversed significantly in baicalein group （P＜0.01）. CONCLUSIONS Baicalein can promote the proliferation and migration of cortical neurons， improve hypoxia-induced cell apoptosis and cell cycle distribution， decrease the activity of LDH in supernatant and the level of cellular lipid peroxidation， and improve antioxidant levels. Its mechanism may be related to regulating the caspase- 3/Bax/Bcl-2 pathway.

Protective effect of thymosin ß4 on oxygen-glucose deprivation/reoxygenation injury in rat cortical neurons / 中国脑血管病杂志

Objective To investigate the protective effect and mechanisms of thymosin beta 4 (Tß4) on oxygen-glucose deprivation/reoxygenation (O G D / R) injury in rat cortical neurons. Methods Primary cultured cortical neurons were isolated and identified; Ischemia-reperfusion injury model of rat cortical neurons was established (oxygen glucose deprivation 6 h, reoxygenation 12 h) by OGD / R. The rats were divided into the control group,the model group and the treatment group (addition of thymosinß4 2 h before modeling); The cell counting Kit-8 (CCK8) was used to determine the optimal concentration of Tß4. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to detect apoptosis. Western blotting was used to detect the expression of 78 000 glucose-regulated protein 78 (GRP 78), CCAAT/enhancer binding protein-homologous protein (CHOP), B-cell lymphoma-2 (Bel-2) and Bax. Expression levels were compared among groups. One-way analysis of variance was used to compare the normally distributed measurement data among three groups with the SPSS19. 0 software. Results Cortical neurons were separated correctly. The optimal concentration of Tß4 was 10 (ig/L. Comparing the model group with the control group, the survival rate of cortical neurons decreased significantly (P = 0. 002), the apoptotic rate increased significantly (P < 0. 01), the expression of GRP78,CHOP and Bax was up-regulated significantly (P values were 0. 034,0 and 0. 045 .respectively),and the expression of Bcl-2 was reduced significantly (P = 0. 006). Comparing the treatment group(10 p.g/L exogenous Tß4) with the model group, cell viability increased significantly (P = 0. 008), the apoptotic rate decreased significantly (P = 0. 002),the expression of GRP78,CHOP and Bax decreased significantly (P values were 0. 032,0. 027 and 0. 019, respectively),and the expression of Bcl-2 increased significantly (P = 0.028) .The differences were statistically significant. Conclusions Tß4 inhibits OGD/R-induced endoplasmic reticulum stress-dependent apoptosis. These results provide a theoretical basis for the application of Tß4 in the treatment of cerebral ischemia-reperfusion injury.

Modulation of SOD1 Subcellular Localization by Transfection with Wild- or Mutant-type SOD1 in Primary Neuron and Astrocyte Cultures from ALS Mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by selective degeneration of motor neurons. Mutant superoxide dismutase 1 (SOD1) is often found as aggregates in the cytoplasm in motor neurons of various mouse models and familial ALS patients. The interplay between motor neurons and astrocytes is crucial for disease outcome, but the mechanisms underlying this phenomenon remain unknown. In this study, we investigated whether transient transfection with wild-type and mutant-type SOD1 may lead to amplification of mutant SOD1-mediated toxicity in cortical neurons and astrocytes derived from wild-type and mutant-type (human G93A-SOD1) mice. In transgenic mice expressing either wild- or mutant-type SOD1, we found that green fluorescent protein (GFP)-wtSOD1 was present in the cytoplasm and nuclei of wild-type cortical neurons and astrocytes, whereas GFP-mutant SOD1 was mainly cytoplasmic in wild- and mutant-type cortical neurons and astrocytes. These findings indicate that intracellular propagation of misfolding of GFP-wt or mtSOD1 are possible mediators of toxic processes involved in initiating mislocalization and aggregation. Here, we provide evidence that cytoplasmic aggregates induce apoptosis in G93A-SOD1 mouse cortical neurons and astrocytes and that the toxicity of mutant SOD1 in astrocytes is similar to the pathological effects of ALS on neurons in vitro. Collectively, our results indicate that mtSOD1 probably interacts with wtSOD1 via an unknown mechanism to produce augmented toxicity and may influence aggregate formation and apoptosis.

Protective effect of 17β-estradiol against propofol-induced apoptosis of primarily cultured cortical neurons / 医学研究生学报

Objective 17β-estradiol is known to have a neuroprotective effect.The aim of this study was to investigate the effects of 17β-estradiol on propofol-induced neuroapoptosis in primarily cultured cortical neurons. Methods Rat cortical neurons were primarily cultured for 7 days and randomly divided into groups A ( vehicle control) , B, and C, treated with equal volume of 20%intralipid, 500 μmol/L propofol, and 500 μmol/L propofol +0.1 μmol/L 17β-estradiol, respectively.At 12 hours after treatment, the morphology of the neurons was observed under the microscope, their survival rate calculated by MTT, their apoptosis was deter-mined by FCM assay, and their mitochondrial membrane potential measured by fluorescent dye rhodamine 123. Results Compared with group A, group B showed a significantly reduced number of neurons, lack of 3-dimensional appearance, unclear contour, and fractured neuron axons, but a remarkable improvement was observed in the propofol-induced morphological damage in group C.The survival rate of the neurons and the mitochondrial membrane potential were markedly decreased in group B ([52.3 ±5.2]% and [59.1 ± 5.3]%) as compared with groups A ( [99.9 ±3.6]%and [99.6 ± 5.8]%) and C ([90.1 ±7.2]%and [89.2 ±7.1]%) (both P<0.01 ) , while the rate of neuroapoptosis significantly increased in group B ([43.4 ±4.6]%) in comparison with A ([3.1 ±0.2]%) and C ([22.3 ±3.2]%) (both P<0.01). Conclusion 17β-es-tradiol can protect against propofol-induced apoptosis of primarily cul-tured neurons by inhibiting the reduction of their mitochondrial membrane potential.

Effect of ginsenoside Rg1 on JNK and oligomeric Aβ1-42-induced apoptosis / 中国药理学通报

Aim To explore the possible protective effect of ginsenoside Rg1 on oligomeric Aβ1-42 induced apoptosis and its possible mechanism. Methods The damage was induced by oligomeric Aβ1-42 in primary cortical neurons. Cells were incubated in the absence or presence of Aβ, or co-incubated in sp600125 with Aβ, or pre-incubated in ginsenoside Rg1 then co-incu-bated in Aβ. The p-JNK, JNK, caspase-3 activity and TUNEL-positive cells were detected. Results In Aβ1-42 treated group, the ratio of p-JNK/JNK level was increased more than that in non-treated group for 15 min. However, in neurons preincubated with (2. 5, 5, 10 μmol·L-1 ) ginsenoside Rg1 and then co-incuba-ted with 5 μmol·L-1 oligomeric Aβ1-42 , the p-JNK/JNK ratio, caspase-3 activity and TUNEL positive neu-rons were significantly decreased compared with those of Aβ1-42 treated group. Conclusion Ginsenoside Rg1 can attenuate the oligomeric Aβ1-42-induced apop-tosis by JNK pathway.

The expression of cylindromatosis in oxygen and glucose deprivation and reperfusion induced primary cortical neuron necroptosis / 中华行为医学与脑科学杂志

Objective To investigate the location of CYLD in the neurons and explore the expression of CYLD in OGD/reperfusion-induced neuronal necroptosis.Methods Primary cortical neurons were cultured for 6days and neuronal purity was observed by double staining immunofluorescence of β3-tubulin and DAPI.The location of CYLD was identified by double staining immunofluorescence of NeuN,DAPI and CYLD using primary cortical neurons cultured for 14 days.Then,primary cortical neurons were divided into 8 groups:Control,EBSS,DMSO,OGD/reperfusion(0 h,2 h,6 h,8 h,12 h).Neurons were pretreated with zVAD-fmk for 30 min,OGD for 2 h and the levels of CYLD were evaluated after reoxygenation at different time points.The peak value(8 h) was chosen as reoxygenation time point.Neurons were divided into two groups as Control and OGD.The levels of CYLD were determined in both cytoplasm and nucleus after OGD 2 h and reoxygenation 8 h.Results The double staining immunofluorescence showed that neuronal cultured purity was about 70％ and the CYLD strongly expressed in nucleus but weakly in cytoplasm.The levels of CYLD increased gradually with different reoxygenation time and arrived at peak value after reoxygenation for 8 h (P ＜ 0.05),which was in accordance with the change of LDH (P ＜0.05) (Control (1.00±0.00),EBSS (1.07 ±0.03),DMSO (1.09 ±0.03),0h (1.40±0.12),2 h (1.74±0.08),6 h (2.25 ± 0.12),8 h (2.97 ± 0.15),12 h (3.01 ± 0.08)).The level of cytoplasm CYLD increased significantly in the OGD group (reoxygenation for 8 h)than that in control group (P＜0.05).But the level of nucleus CYLD had no difference between OGD and control group (P ＞ 0.05),which was in accordance with the results of immunofluorescence.Conclusion The CYLD in neurons cytoplasm is involved in necroptosis induced by OGD/deprivation and downregulating of CYLD has a protective effect on the brain injury resulted from ischemia/ reperfusion.

Human telomerase reverse transcriptase protects human embryonic cortical neurons / 中国组织工程研究

BACKGROUND: Telomerase can maintain the telomere length and avoid cel replicative senescence and apoptosis in somatic cells. Its catalytic subunit cal ed telomerase reverse transcriptase has roles in mediating cellsurvival and anti-apoptotic functions. OBJECTIVE: To evaluate the effects of human telomerase reverse transcriptase on amyloid β1-40-induced human embryonic cortical neurons injury. METHODS: Human cortical neurons derived from 12-16 weeks old aborted fetuses were transfected with recombinant adenovirus vector encoding human telomerase reverse transcriptase. Expression of human telomerase reverse transcriptase was evaluated by immunocytochemical staining. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol enzyme-linked immunosorbent assay kit. Human embryonic cortical neurons were treated with 10 μmol/L ol/L amyloid β1-40 after transfected for 3 days. Cel viability, reactive oxygen species levels and glutathione contents in human embryonic cortical neurons were respectively detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate and chromatometry. RESULTS AND CONCLUSION: Expression of human telomerase reverse transcriptase reached peak at 3 days after transfection, and the telomerase activity was rebuilt; 10 μmol/L amyloid β1-40 could significantly reduce the cel viability of neurons and glutathione content (P < 0.05 and P < 0.01), and increase the reactive oxygen species levels (P < 0.05). The neurons transfected with human telomerase reverse transcriptase gene could be significantly against the toxicity of amyloid β1-40 and increase the cel viability and glutathione content (P < 0.05 and P < 0.01), and decrease the reactive oxygen species levels (P < 0.05). The results indicate that human telomerase reverse transcriptase can protect amyloid β1-40-induced human embryonic cortical neurons injury

Nitric Oxide Is an Essential Mediator for Neuronal Differentiation of Rat Primary Cortical Neuron Cells

Nitric oxide (NO) regulates proliferation, differentiation and survival of neurons. Although NO is reported to involve in NGF-induced differentiation of PC12 cells, the role of NO has not been characterized in primary neuron cells. Therefore, we investigated the role of NO in neuronal differentiation of primary cortical neuron cells. Primary cortical neuron cells were prepared from rat embryos of embryonic day 18 and treated with NMMA (NOS inhibitor) or PTIO (NO scavenger). Neurite outgrowth of neuron cells was counted and the mRNA levels of p21, p27, c-jun and c-myc were measured by RT-PCR. Neurite outgrowth of primary cortical neuron cells was inhibited a little by NOS inhibitor and completely by NO scavenger. The mRNA levels of p21 and p27, differentiation-induced growth arrest genes were increased during differentiation, but they were decreased by NOS inhibitor or NO scavenger. On the other hand, the level of c-jun mRNA was not changed and the level of c-myc mRNA was increased during differentiation differently from previously reported. The levels of these mRNA were reversed in NOS inhibitor- or NO scavenger-treated cells. The level of nNOS protein was not changed but NOS activity was inhibited largely by NOS inhibitor or NO scavenger. These results suggest that NO is an essential mediator for neuronal differentiation of primary cortical neuron cells.

The effect of caffeine on the primarily cultured cortical neuron apoptosis in neonate mice / 中国法医学杂志

Objective To examine the effect of Caffeine on the cultured cortical neuron apoptosis in neonatal rats.Methods The primary cerebral cortex neurons for cultures were obtained from neonatal mice 2-3 days after birth,Caffeine reconstituted at final concentrations 300μmol/L and 1 000μmol/L was added to the cell cultures and continuously co-incubated for 6-36 h,respectively after the cortical neurons were continuously cultivated 7 days after incubation under temperature of 37℃ incubator with 5% CO_2 and 100% relative humidity,the intracellular calcium concentration,mitochondrial membrane potential and apoptosis rate were determined by the flow cytometry.The activity of Caspase-9 was assayed by enzyme-labeled instrument,and Caspase-9 activity by the enzyme-1inked analyzer.Cell morphological changes were observed under electron microscope and fluorescent microscope after being stained with Hoechst 33258 fluorescent dye.Results Compared with the control group,the average increase in intracellular calcium fluorescence intensity was most significant(P<0.05),which elevated from the normal value 43.13±2.02 to 45.28±1.16 and 46.92±1.99,respectively at 6 h;mitochondrial membrane potentials were reduced most significandy(P<0.05).from the base value 443.58 ±11.77 down to 289.53±16.47 and 165.14±14.72,respectively at 8h.Caspase-9 activity was peaked(P<0.05),from the normal value 1.00±0.000 to 5.33±1.02 and 8.33±0.92,respectively at 10 h.The neuronal apoptosis ratio was increased significantly (P<0.05),from the normal value 4.94±1.74 to 15.98±2.03 and 18.70±2.09,at 36h.The apoptotic bodies were observed at 24 h after administration of 300 μmol/L and 1000 μmol/L Caffeine.Conclusion Caffeine may promote neuronal apoptosis in neonate mice.

Effects of Hypoxia on the Ubiquitin-proteasome System in Primary Cortical Neuronal Cell Cultures / 체질인류학회지

The ubiquitin-proteasome system is crucial in maintaining cellular growth and metabolism. Dysfunction of this system may contribute to neurodegenerative diseases, such as Parkinson's disease. But its effects on primary neurons are largely unknown. In the present study, we investigated the effects of proteasome inhibitor and hypoxia on primary neuronal cultures to determine whether proteasomal malfunction induces neuronal death. Neuronal apoptosis increased in primary cultured cortical neurons with treatment of proteasomal inhibitor in normoxic condition and in the presence or absence of proteasomal inhibitor in hypoxic condition. Also expression of PARP and activated caspase 3 increased. NF-kappaB, a key transcription factor in this system expression increased in hypoxic condition and proteasomal inhibition. Interestingly, hypoxic condition induced an expression and accumulation of alpha-synuclein in neuron, one of components of Lewy body in Parkinson's disease. Our findings determine that hypoxic condition may affect the ubiquitin-proteasome system. Furthermore, it suggests that hypoxic condition and proteasomal inhibitors are involved, at least in parts, in neurodegeneration of mouse model for Parkinson's disease.

Influencing Factors and Optimizing Cultivating Conditions of Primary Cortical Neurons of Rat in Serum-Free Culture / 实用儿科临床杂志

Objective To explore the influencing factors for the purity and viability of primary cultured cortical neurons of rat,and optimize the separating and cultivating conditions of the cortical neurons.Methods The primary cotical neurons were cultured in a serum-free culture system of B27-supplemented neurobasal medium.The differences in purity and viability of primary cultured neurons between embr-yonic rat group and newborn rat [postnatal 24 h and 5 d] group were evaluated by morphology,immunocytochemistry of neuron-specific enolase(NSE) and trypan blue staining.The changes of neurons purity and viability in different trypsin digestion time(0 min,5 min and 15 min) at different environment temperatures(20 ℃ and 30 ℃) were assessed by immunocytochemistry and trypan blue staining.Results The primary cultured neurons from fetal and newborn rats grew well.There was no significant difference in embryonic rat and postnatal 1 d newborn rat group[(91.30?1.03)%,(89.50?1.78)% respectively in purity;and(98.20?0.58)%,(97.10?0.98)% respectively in viability].The neurons from 5-days newborn rat were inferior to that from fetal and 1-day newborn rat in purity and viability[(82.00?1.25)% and(92.87?1.56)% respectively].Shortening operation time and adjusting digestion time according to the environment temperatures could improve neuronal viability:A digestion for 15min at the environment temperature of 20 ℃ or for 5 min at 30 ℃ could acquired cells with higher viability [(98.20?0.58)% and(96.70?0.64)% respectively].Conclusions It is an easy,practical choice to culture priamry cortical neurons from postnatal 1 d newborn rats.Optimizing the separating and cultivating condition(environment temperatures,digest time,et al.) will improve the neurons purity and viability.

Effects of Bone Marrow Mescenchymal Stem Cells Conditioned-Medium on Cortical Neuronal Apoptosis Induced by Hypoxia-Reoxygenation in vitro in Rats / 实用儿科临床杂志

Objective To observe the effects of bone marrow mesenchymal stem cells(BMMSCs) conditioned-medium (B-CM) on apoptosis in cultured cortical neurons after hypoxia-reoxygenation in vitro in rats.Methods BMMSCs were isolated,cultured and expanded,the culture me-dium was substituted by new medium that contain no serum when cells grew to 90% confluence.After cultured for 24 hours,the medium was collected as B-CM.Cortical neurons of mouse cultured for 8 days were divided into three groups:normal control groups,hypoxic group,B-CM disposed groups.Cell viability were detected by 3-(4,5-dimethlthiazd-2-yl)-2,5-diphenyltetra-zoliu bromidel (MTT) methods and the apoptosis of neurons was examined by flow cytometry and telemicroscopy on hypoxia for 0,6 hours and hypoxia-reoxygenation for 24 hours,respectively.Results Compared with control group,hypoxia for 6 hours could decrease cell viability,hypoxia-reoxygenation for 24 hours increased the cell apoptosis ratio remarkably(P

Influence of Conditioned Medium of Rat Brain Microvascular Endothelial Cells on Activity of Cortical Neurons and Protective Effect of Tong Luo Jiu Nao Injection / 中华中医药杂志

Objective: To study the influence of conditioned medium of rat brain microvascular endothelial cells on the activity of cortical neurons and the protective effect of Tong Luo Jiu Nao Injection.Methods: We collected the conditioned media from 4 different cultured endothelial cell groups,which were normal endothelial cells,the normal ones and treated with Tong Luo Jiu Nao Injection,the injured ones damaged by simulated cerebral ischemia,as well as the injured ones and treated with Tong Luo Jiu Nao Injection,respectively(N-CM,NT-CM,I-CM,IT-CM).Then,the conditioned medium was added into the cultures of the normal neurons and the damage ones which are injured by simulated cerebral ischemia as well,respectively.The effect of each type of conditioned medium on the activities of neurons was determined through the measurement of MTT and the transduction rate of LDH.Results:(1) N-CM has no obvious effect on the normal neurons,but does show some protective effect on the injured ones by increasing its activity significantly;(2) I-CM could decrease the activity of the normal neurons as well as aggravate the damage on the injured ones,while this injury effect can be reversed remarkably by IT-CM.Conclusion: The paracrine secretion of the brain microvascular endothelial cells might be one of the vital mechanisms in cerebral ischemic injury,indicating that the brain microvascular endothelial cells could be the therapeutic targets of Chinese medicine,which are not able to permeate through the Blood-Brain Barrier.

Protective effects of huang lian jie du tang active fraction on ischemia injury in cerebral cortical neuronal cultures / 中国药理学通报

Aim To investigate the protective effects of huang lian jie du tang active fraction(HLJDTAF) on ischemia injury in cerebral cortical neuronal cultures. Methods The cortical neurons of rat fetal were cultured in vitro. The protective effects of HLJDTAF on ischemia injury were observed by treating cells with sodium dithionite in glucose free medium. Results HLJDTAF 610 mg?kg -1 increased the activity of neuron, 305 mg?kg -1 reduced the leakage rate of LDH; HLJDTAF 610, 305, 153 mg?kg -1 reduced the content of NO,MDA and increased the activity of SOD. Conclusion HLJDTAF protected the cerebral cortical neurons from ischemia injury by reducing the contents of NO and suppressing the generation of lipidperoxide.

Protective effects of imipramine on ischemic injury in cultured rat cerebral neurons / 中国临床药理学与治疗学

AIM: To study the protective effects of Imipramine (Imi) on ischemic injury in cultured rat cerebral cortical neurons. METHODS: Cortical neurons of fetal rat were cultured in vitro. The protective effects of Imi on ischemic injury in cultured rat cerebral neurons were observed. RESULTS: Imi ( 10 -5, 10 -6, and 10 -7 mol?L -1) reduced the number of cell death, lowered LDH,NO,and MDA content, and increased of activity of SOD. CONCLUSION: Imi can protect rat cerebral cortical neurons from ischemic injury and toxicity of Glu. And it maybe attribute its to antioxidation and calcium antagonism.

Effect of panaxynol on rat primary cultured neuron injured by H_2O_2 / 中草药

Objective To investigate the neuroprotective effect of panaxynol on primary cultured cortical neuron against oxidative stress. Methods Viability of panaxynol acted on neuron oxidative stress was monitored by MTT assay and FCM method. Scavenging effects of panaxynol on free radicals were observed in vitro. Effects of panaxynol on SOD activity and GSH-Px, and MDA content in primary neuron injured by H_2O_2 were also determined. Results Panaxynol (2—16 ?mol/L) could dose-dependently protect neuron from oxidative stress induced by H_2O_2; 8 ?mol/L of panaxynol could decrease necrosis and apoptosis rate of neuron significantly (P

Effects of melatonin on cellular viability and injury induced by oxygen and glucose deprivation at different phases of co-cultured neuron and glia / 中国药理学通报

AIM To observe the effects of melatonin on acute and chronic injuries induced by oxygen and glucose deprivation (OGD) in co-cultured neuron and glia and to explore the probable mechanisms of melatonin in antagonizing the injuries. METHODS The injury model of cultured neuron and co-cultured neuron and glia was made by administration of sodium dithionite and glucose-deprived Earles solution. In neuron and glia co-culture, two different models, acute injury model at the phase of OGD and chronic injury model after 'reperfusion' were established. The levels of nitric oxide (NO) and the activity of lactate dehydrogenase (LDH) were measured by Griess reagent and LDH kits respectively. The content of malondialdehyde (MDA) was determined by TBA method. Cell viability was analyzed using colorimetric MTT assay. RESULTS Melatonin increased the level of NO at the concentration of 10 -6 , 10 -7 mol?L -1 and decreased the level of MDA content elevated by OGD at the concentration of 10 -6 , 10 -7 , 10 -8 mol?L -1 in vitro cultured cortical neurons. In the chronic injury model after 'reperfusion' melatonin (10 -6 , 10 -7 , 10 -8 mol?L -1 ) significantly decreased LDH activity and increased MTT value in neurons and glia co-cultured. But in the acute injury model, melatonin obviously increased LDH activity and decreased MTT value. CONCLUSION Melatonin protection for neuron from injuries induced by oxygen and glucose deprivation may be related to increase in the level of NO and decrease in the content of MDA. Melatonin can antagonize the injury in the chronic injury model after 'reperfusion', but exaggerate the injury in the acute injury model. These may be all related to its antioxidant action. Our results also suggest that melatonin may probably inhibit activation of microglia.

ISOLATION,CULTURE AND PURIFICATION OF CEREBRAL CORTICAL NEURONS AND GLIAL CELLS FROM THE EMBRYONIC GEKKO / 解剖学报

Objective To establish a method of neurons and glial cell culture from embryonic Gekko japonicus cerebral cortex. Methods Embryonic(E15) pallium was dissociated and digesting by trypsin.After counting,cells were seeded in culture flask.The glial cells were obtained by using differential adhesion potential combined with successive passage purified methods,and neurons were obtained by using neurobasal medium supplemented with B27.The cells were fixed and analyzed with immunohistochemic assay. Results After tetra-generation,GFAP positive cells were more than 95% in glial cells cultured condition;Neurons grew well in neurobasal medium,and NF and MAP-2 positive signals co-localized on neurons.After cultured for 10 days,the percentage of neurons was more than 95%.Conclusion The methods of isolation,culture and purification for embryonic Gekko japonicus cortical neurons and glial cells were established and it might be a valuable cell model to further investigate the central nerve system in Gekko japonicus.

ACTIVATION OF caspase-3 DURING APOPTOSIS OF RAT CORTICAL NEURONS INDUCED BY ?-AP_(1-40) / 解剖学报

Objective To investigate the effects of caspase\|3 on ? ?? 1 40 induced apoptosis in rat cortical neurons. Methods Apoptosis was induced by 40?mg\5L -1 ?\|AP 1 40 .The apoptotic neurons were observed by TUNEL staining and DNA gel electrophoresis.The activity and mRNA expression of caspase\|3 was measured by fluorescent spectrofluorometer and RT\|PCR.The cleaved caspase\|3 was detected with immunocytochemistry. Results After treatment with ?\|AP 1\|40 ,the rat cortical neurons showed DNA fragmentation.The expression of caspase\|3 mRNA ratio to ? actin was 0^78,0^85 and 0^39 respectively after treatment for 12,24 and 48?h.Caspase\|3 activity was 133 24?7 47,192 16?11 03,88 87?4 24 MFI\5?g protein -1 \5h -1 .The cleaved caspase\|3 was observed within cytoplasm.Specific inhibitor of caspase\|3 Ac\|DEVD\|CHO inhibited caspase\|3 activation and blocked cortical neurons apoptosis markedly.Conclusion\ Caspase\|3 might be an executor during ? AP 1 40 induced apoptosis in rat cortical neurons.[

CHANGES OF CYTOSKELETAL GENES EXPRESSION IN CORTICOSPINAL NEURONS AFTER SPINAL CORD SEMITRANSECTION INJURY IN RAT / 解剖学报

Objective To investigate the pattern and the role of cytoskeletal genes expression during nerve regeneration. Methods We examined the changes of expression of ? tubulin(? Tub)and three neurofilament(NF)subunits mRNA in rat corticospinal neurons of sensorimotor cortex on the 1st,3rd,5th,7th,10th,14th,21st,28th and 56th days after spinal cord semitransection injury by using in situ hybridization. Results Levels of ? Tub,NF L,NF M and NF H mRNA were dramatically downregulated within large sized pyramidal neurons in layer V of the sensorimotor cortex of side lateral after spinal cord injury.The level of ? Tub mRNA was decreased as early as 1 day postinjury,whereas the levels of NF subunits mRNA were not decreased until later time(on the 3rd days postinjury),and both of them did not return to control levels on the 56th days following the lesion. Conclusion ? Tub gene expression if inhibited in CNS neurons postinjury. The axotomy signeal and the continuance downregulation of NF mRNA expressioon following spinal cord injury may contribute to the ineffective regeneration response of CNS neurons.