Effects of cysteinyl receptor agonist and antagonists on rat primary cortical neurons / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the effect of cysteinyl receptor agonist leukotriene D(4) (LTD(4)) and its antagonists on rat primary neurons.</p><p><b>METHODS</b>In the primarily cultured rat cortical neurons, the neuron injury was evaluated by measuring intracellular calcium, 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) reduction, and propidium iodide (PI) and Hoechst 33258 staining. The in vitro ischemic injury was induced by oxygen-glucose deprivation (OGD) for 1.5 h and reperfusion for 24 h.</p><p><b>RESULT</b>LTD(4) (0.01-1 micromol/L) did not induce the elevation of intracellular calcium, neuron viability changes and neuron death. OGD-induced injury was not significantly ameliorated by the CysLT(1) receptor antagonists, pranlukast (0.2-10 micromol/L) and montelukast (0.2-10 micromol/L), as well as by the CysLT(1)/CysLT(2) receptor non-selective antagonist, BAY u9773 (0.02-1 micromol/L).</p><p><b>CONCLUSION</b>Neither agonist nor antagonists of cysteinyl receptors have the effects on the viability and ischemic-like injury in rat primary neurons.</p>

Homeostatic conditions affect the protective effect of edaravone on ischemic injury in neurons / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine whether homeostatic conditions (pH, glycine or ion concentration) affect the protective effects of edaravone on ischemic injury in rat cortical neurons.</p><p><b>METHODS</b>In cultured rat cortical neurons, the compositions in the experimental solutions were changed to mimic the disturbance of homeostasis after cerebral ischemia. In vitro ischemic injury was induced by oxygen-glucose deprivation (OGD) for 3 h and reperfusion for 12 h, and the neuron injury was evaluated by 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyl tetrazolium bromide (MTT) reduction assay and lactate dehydrogenase (LDH) release. Effect of edaravone on OGD injury was observed in different experimental solutions.</p><p><b>RESULT</b>In weak alkalified solution (pH 7.8) or the solution containing glycine (10 micromol/L), OGD injury became more serious; but in weak acidic (pH 6.5) or higher Mg(2+) (1.8 mmol/L) solutions, OGD injury was attenuated. Edaravone (1 micromol/L) reversed the injury in the solutions with pH 6.1,7.4 and 7.8 or the solution containing glycine, but did not show protective effect in the solution with pH 6.5 and the higher Mg(2+) or lower Ca(2+) solution.</p><p><b>CONCLUSION</b>The changes of homeostatic conditions affect the severity of ischemic injury of neurons and the protective effect of edaravone.</p>

GM1 stabilizes expression of NMDA receptor subunit 1 in the ischemic hemisphere of MCAo/reperfusion rat / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>To determine the protective effect of monosialoganglionside (GM1) and evaluate the influence of GM1 on expression of N-methyl-D-aspartate receptor subunit 1 (NMDAR1) in Sprague-Dawley (SD) rats with focal cerebral ischemia-reperfusion (I/R).</p><p><b>METHODS</b>Left middle cerebral artery (MCA) was occluded by an intraluminal suture for 1 h and the brain was reperfused for 72 h in SD rats when infarct volume was measured, GM1 (10 mg/kg) was given ip (intraperitoneally) at 5 min (group A), 1 h (group B) and 2 h (group C) after MCA occlusion (MCAo). Expression of NMDAR1 was detected by Western blot at various time after reperfusion (4 h, 6 h, 24 h, 48 h and 72 h) in ischemic hemispheres of the rats with or without GM1 administered.</p><p><b>RESULTS</b>(1) Adjusted relative infarct volumes of groups A and B were significantly smaller than that of group C and the control group (P<0.01 and P<0.05, respectively). (2) Expression level of NMDAR1 was temporally high at 6 h after reperfusion, and dipped below the normal level at 72 h after reperfusion. GM1 at 5 min after MCAo significantly suppressed the expression of NMDAR1 at 6 h after reperfusion (P<0.05 vs the control). At 72 h after reperfusion, the NMDAR1 expression level of rats treated with GM1 administered (at 5 min or 2 h after MCAo) was significantly higher than that of the control (P<0.05).</p><p><b>CONCLUSION</b>GM1 can time-dependently reduce infarct volume in rats with focal cerebral I/R partly through stabilizing the expression of NMDAR1.</p>

Reversing effect of histamine on neuron death induced by N-methyl-D-aspartate / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the effect of histamine on N-methyl-D-aspartate (NMDA) induced neuron death and to elucidate its mechanism.</p><p><b>METHODS</b>The primary cortical cell culture was adopted. Neuron morphology and MTT assay were used to evaluate the drugs effects.</p><p><b>RESULT</b>Histamine at doses of 10(-4) 10(-6) 10(-7) 10(-8) mol/L reversed the neuron death induced by NMDA (50 micromol/L) for 3 h. The protection of histamine peaked at doses of 10(-4) mol/L and 10(-7)mol/L. The effect of histamine of 10(-7) mol/L was reversed only by cimetidine an H(2)receptor antagonist. However, the effect of histamine of 10(-4) mol/L was reversed only by pyrilamine but not cimetidine.</p><p><b>CONCLUSION</b>Histamine could reduce neuron death induced by NMDA; its protection at a low dose might be mediated by H(2)receptor, and at a high dose by H(1)receptor.</p>

Study on protective effect of monosialoganglioside (GM1) on injury induced by oxygen glucose deprivation/reperfusion in rat hippocampal slices / 中国应用生理学杂志

<p><b>AIM</b>To investigate the protective effect of monosialoganglioside (GM1) on injury induced by oxygen glucose deprivation/reperfusion (OGD/Rep) in rat hippocampal slices.</p><p><b>METHODS</b>The protective effects of GM1 on hippocampal slices after OGD/Rep were observed by detecting the light transmittance (LT) changes of rat hippocampal slices and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining of rat hippocampal slices.</p><p><b>RESULTS</b>(1) In four groups treated with 0 (control), 0.1, 1.0, 10 micromol/L GM1, the peak of light transmittance (LT) in the slices treated with 1.0 micromol/L GM1 was significantly lower than that of the control and the group treated with 0.10 micromol/L GM1 (P < 0.01, ANOVA), while the peak of LT in the slices treated with 10.0 micromol/L GM1 was significantly lower than that of the other groups (P < 0.01, ANOVA). The time to reach the peak of LT in four groups was significantly different from each other (P < 0.05, Kruskal-Wallis test). The time to reach the peak of LT in the group treated with 1 micromol/L GM1 was the significantly longer than that in the control (P < 0.01, Mann-Whitney U test). (2) There was characteristic dose-response relationship between GM1 and TTC staining of rat hippocampal slices. In the five groups, treated with 0 (control), 0.01, 0.1, 1.0, 10 micromol/L GM1 respectively, TTC staining in the group treated with 1 micromol/L GM1 was the deepest (P < 0.05 vs. control, 0.01 and 0.1 micromol/L GM1 group, ANOVA), and the next was in the group treated with 10 micromol/L GM1 (P < 0.05 vs. control and 0.01 micromol/L GM1 group, ANOVA).</p><p><b>CONCLUSION</b>GM1 could protect injury induced by OGD/Rep in rat hippocampal slices effectively in vitro.</p>

An improved quantitative method for evaluation of ischemic injury and neuroprotection in mouse brain slices / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a simpler and more accurate method for evaluating in vitro ischemic injury and neuroprotective effects of drugs through improving experimental instrument and quantitative index in mouse brain slices.</p><p><b>METHODS</b>An incubation instrument was developed and its application tested. 2,3,5-triphenyltetrazolium chloride (TTC) was used as a substrate to biosynthesize formazan standard in mouse brain slices, and formazan was isolated, purified and identified. Ischemic injury of mouse brain slices was induced by oxygen/glucose deprivation (OGD), the produced formazan from TTC in the cortex and striatum was measured at 490 nm spectrophotometrically. Edaravone and ONO-1078 were added into the incubation medium to observe their neuroprotective effects.</p><p><b>RESULT</b>The incubation instrument worked well for incubating brain slices and obtaining stable results efficiently. Standard formazan was biosynthesized and purified with a purity of 99.3%, and showed a linear range of 0.05 - 1 mg/ml in absorbance at 490 nm (r=0.9997). OGD decreased formazan production in the cortex and striatum in a duration-dependent manner. Edaravone (0.01 to 1 micromol/L) recovered OGD-induced decrease of formazan production, but ONO-1078 showed no effect.</p><p><b>CONCLUSION</b>The incubation instrument and quantitative measurement of formazan developed in this study are efficient,accurate and simple for evaluating ischemic injury and neuroprotection,which can be used in screening of neuroprotective drugs in vitro.</p>