| Aim:To observe neuroprotective effect of tea polyphenol (TP) (1, 3 and 10mg/L) and its relating mechnisms on mouse brain slices subjected to oxygen–glucose deprivation (OGD) and glutamate (Glu).Method:OGD cortical and hippocampal slices were induced by 15/10min of OGD followed by 60min of re-oxygenation, while Glu slices were induced by adding 1mM of Glu into incubation solution lasting for 30min. Neuronal damage was assessed by using TTC staining method and measurement of LDH release. Besides, SOD activity in OGD cortical and hippocampal slices was assessed to evaluate antioxidant ability of brain tissue. And the effects of magnesium-contained/free ACSF, TP (10mg/L) and MK801 (0.03mM) on Glu injury in the whole brain slices were compared as well.Results:1. Effect of TP on TTC staining and LDH release in mouse cortical and hippocampal slices subjected to OGD: 490nm absorbance (A490) of TTC staining of control cortical and hippocampal slices was 0.35±0.02 (n=8), 0.18±0.01 (n=10), and LDH release of that was 187.42±14.17 U/mg?tissue (n=8), 106.32±8.61 U/mg?tissue (n=8) respectively. And OGD for 15 or 10 min followed by 60 min of re-oxygenation induced a significant reduce in A490 of TTC staining of cortical slices (0.13±0.01, p<0.01, n=8) and of hippocampal slices (0.08±0.01, p<0.01, n=10). While, LDH release experienced a sharply growth, which was 271.00±18.24 U/mg?tissue (p<0.01, n=8) in TP (1, 3 and 10mg/L) greatly grew A490 of TTC staining of slices subjected to OGD and greatly decreased OGD-induced LDH release as well.2. Effect of TP on SOD activity in mouse cortical and hippocampal slices subjected to OGD: SOD activity of control cortical and hippocampal slices was 115.58±5.19 U/mgprot (n=8), 114.31±4.82 U/mgprot (n=8). OGD for 15 or 10 min followed by 60 min of re-oxygenation induced a significant decline in SOD activity, which was 74.30±6.42 U/mgprot (p<0.01, n=8) and 59.38±4.50 U/mgprot (p<0.01, n=8) respectively. And TP (1, 3 and 10mg/L) dose-dependently attenuated OGD-induced descent of SOD activity markedly. Noticeably, TP (10mg/L) rose SOD activity of cortical and hippocampal slices to 158.17±10.62 U/mgprot (n=8) and 126.52±2.39 U/mgprot (n=8) respectively, which was even higher than that of control slices (p<0.01 in cortical slices, p<0.05 in hippocampal slices).3. Effect of TP on TTC staining and LDH release in mouse cortical and hippocampal slices subjected to Glu: A490 of TTC staining of control cortical and hippocampal slices was 0.20±0.01 (n=8), 0.13±0.005 (n=8), and LDH release of that was 196.15±29.49 U/mg?tissue (n=8), 119.94±9.93 U/mg?tissue (n=8) respectively. 1mM of Glu injuring for 30min dramatically descended A490 of TTC staining, which was 0.13±0.01 (p<0.01, n=8) and 0.08±0.005 (p<0.01, n=8) respectively and dramatically ascended LDH release as well, which was 368.73±30.14U/mg?tissue (p<0.01, n=8) and 217.19±8.32U/mg?tissue (p<0.01, n=8) respectively. TP (1, 3 and 10mg/L) steadily increased A490 of TTC staining of slices subjected to Glu and greatly decreased Glu-induced LDH release as well. Especially, its effect on A490 of TTC staining of cortical slices followed a dose-dependent manner.4. Effects of magnesium-contained/free ACSF, TP and MK801 on the whole brain slices subjected to Glu: 1mM or 3mM of Glu injuring for 30min dose-dependently decreased A490 of TTC staining of the whole brain slices. A490 of TTC staining of magnesium-contained control slices was 0.48±0.02, which dropped to 0.40±0.02 (vs. magnesium-contained control, p<0.05, n=6) and 0.31±0.03 (vs. magnesium-contained control, p<0.01, n=6) when 1mM and 3mM of Glu added. Moreover, the viability of brain slices incubated with magnesium-free ACSF was constantly worse than that incubated with magnesium-contained ACSF (p<0.01). TP (10mg/L) and MK801 (0.03mM) produced similar significant effects on 3mM of Glu slices. In addition, the neuroprotective rates of TP (10mg/L) on 3mM of Glu slices were 1.40±0.07 (magnesium-contained group) and 1.44±0.07 (magnesium-free group), the difference between which did not reach statistical significance (p>0.05). And in the same way, the difference of the neuroprotective rates of MK801 (0.03mM) between magnesium-contained group and magnesium-free group did not reach statistical significance as well (p>0.05).Conclusion:1. TP can exert significant neuroprotection against OGD/Glu induced-injury in mouse brain slices, which is probably partly through enhancing levels of SOD and through attenuating excitotoxicity mediated by NMDA receptor.2. The ionic environment supplied by magnesium-contained ACSF is more appropriate for slices to survive. And the effect of 10mg/L of TP on slices suffering from Glu injury is fitly correspondent to that of 0.03mM of MK801 in vitro in brain slices, and their neuroprotective effects are both affected little by magnesium-contained/free ACSF. |
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