| The injury after brain ischemia/reperfusion during the perioperative period is the concerned question of clinical anesthesiology and recovery all the time. According to foreign reports, within the first weeks of adult heart surgery minor nerve function or mental injury incidence can be as high as 53%.Six months later some patient's function improved. But 24% of the patients are still left diminished mental acts. In 42% of the patients even continued five years. Lots of researches have already proved that brain ischemia/reperfusion injury is a malignant cascade of damage with multifactor and mechanism.How to prevent brain ischemia/reperfusion injury during the perioperative period became a urgent issue.Tetramethylpyrazine(TMP) is usually used in the treatment of ischemic cerebrovascular disease and its sequela in clinical, however, we are discord and controversy on the mechanism.We plan to culture rat hippocampal neurons in vitro, then to observe the effect of TMP on rat hippocampal neurons damage induced by anoxia-reoxygenation. If we can clarify TMP brain protection mechanism from cell and molecular level, it will create a new idea for TMP used for brain protection, and provide theoretical basis, alleviate social and economic burden.Objective:①To observe the effects of Tetramethylpyrazine(TMP) on the morphology and cell viability in cultured rat hippocampal neurons,then get the initial message about TMP interfere in neurons;②To study the protective effects and mechanism of action about TMP on cultured rat hippocampal neurons damage induced by anoxia-reoxygenation.Methods:①Rat hippocampal neurons were cultured in vitro and stained immunocytochemically with NSE antibody respectively on 8th-10th day.②Rat hippocampal neurons were cultured with 30,60,90,200,800,1000μg/ml TMP for 24h on 8th-10th day.③Neurons were exposed to TMP in three different concentration (60,200,800μg/ml) and control/normal groups was set in each experiments. After 1 hour place in an incubator with 90%N2+10%CO2 for 2 hour, then place in an incubator with 5% CO2 and 37℃for 24 hours to established rat hippocampal neurons damage by anoxia-reoxygenation.④After cultivation of 7-9 days of rat hippocampal neuronal damage induced by anoxia-reoxygenation, morphological changes were observed under phase contrast microscope, mitochondrial ultrastructure were observe under transmission electron microscope, cell viability was examined by MTT assay, MDA,SOD,LDH in culture solution was detected by spectrophotometer, apoptosis ratio were detected by flow cytometry, HSP70 protein and c-fos protein were examined by immunocytochemically, for assessing the effect on TMP in cultured rat hippocampal neurons damage induced by anoxia-reoxygenation.Results:①Perikaryons under CPM present metuliform or polygon, boles and branches of synapses extended and thickening obviously,conspicuous halation, refraction strengthen, to form crowded network. Immunocytochemical test shows that most of the cultured rat hippocampal neurocytes were positively stained with neuron specific enolase (NSE) antibody, and glial neurocytes were stained with a less degree. Cubic capacity of neurons is larger, cellular nucleus and cytoplasm is bigger comparatively, light gray, well-distributed chromoplasm, integrated double-deck nuclear membrane, abundant mitochondria, cytolysosome and ER were observed by TEM.②Rat hippocampal neurons were cultured with 30,60,90,200,800,1000μg/ml TMP for 24h on 7th-9th day, there were no conspicuous change in neurons cultured with 30,60,90,800μg/ml, compare with control, perikaryons of neurons cultured with 200μg/ml are larger, impaired cells were observed on neurons cultured with 1000μg/ml TMP.③Neurons on 7th-9th day damage induced by anoxia/reoxygenation, lower outstanding refraction, parts cell rounding, shrinkage,defluxion, flagment under CPM; Round neclei, smooth completed nuclear membrane, aggregate chromoplasm, swollen cytomicrosome, expanded ER were observe by TEM. There were no conspicuous change under CPM in neurons on 7th-9th day cultured with 60,200,800μg/ml TMP respectively for 1h before induced injury, no much ultramicrostructure change expect small amounts aggregate chromoplasm, swollen cytomicrosom and expanded ER slightly were observed by TEM in neurons cultured with 200μg/ml TMP for 1h before induced injury.④After neurons with 60,200,800μg/ml TMP on damage induced by anoxia-reoxygenation,their cell survival,MDA/SOD content, LDH leakage respectively are(0.59±0.09,0.72±0.04,0.60±0.07),(3.75±0.26,2.43±0.86,3.45±0.47)nmol/ml,(30.76±4.28,41.53±3.28,33.54±5.07)U/ml,(623.71±102.83,276.63±75.59,537.80±109.18) U/L, compare to control (P<0.05). The differences of 800μg/ml TMP is strongest.⑤After neurons with 60,200,800μg/ml TMP on damage induced by anoxia-reoxygenation, the apoptosis rates,c-fos protein expression are lower than control group, and the HSP70 protein expression is higher than the control. But the 200μg/ml group is better than 60,800μg/ml group.Conclusion:①High purity rat hippocampal neurons can be obtained by this cultured method in this study,which facilitates revealing neurons, ultrastructural neural pharmacology research.②Rat hippocampal neurons cultured with TMP within normal limit are promoted, nerve cell bodies larger, intensive synapsis.③After neurons with 60,200,800μg/ml TMP on damage induced by anoxia-reoxygenation, the MDAcontent,LDH leakage,apoptosis rates,c-fos protein expression are lower than control group, and the cell survival,SOD content,HSP70 protein expression is higher than the control. So,TMP has obvious neural protective action. The protective function of TMP preconditioning is concentration dependent. Under the suitable density, the protective function of TMP preconditioning is strongest. |
PDF Full Text Request