生长激素释放肽对全脑缺血／再灌注损伤大鼠海马组织的保护作用及对谷氨酸／γ-氨基丁酸敏感神经元放电活动的影响

Ghrelin protects against hippocampal injury after global cerebral ischemia/reperfusion and regulate glutamic acid/γ-aminobutyric acid sensitive neuron discharge

目的探讨生长激素释放肽（Ghrelin）对全脑缺血／再灌注（I／R）大鼠海马神经元的保护效应及其作用机制。方法按随机数字表法将雄性sD大鼠分为假手术（Sham）组、I／R组、生理盐水（Ns）＋I/R组和Ghrelin＋I／R组，每组42只。夹闭双侧颈内动脉15min、再灌注60min制备全脑I／R模型；Sham组仅暴露颈动脉，不做任何处理。Ghrelin＋I／R组、NS＋I／R组于I／R前经侧脑室注射Ghrelin或NS 1μL。实验结束后取部分大鼠脑组织，采用化学比色法测定海马组织丙二醛（MDA）、髓过氧化物酶（MPO）和谷胱甘肽（GSH）水平，测量梗死面积，观察组织病理学改变。其余大鼠采用单细胞外记录神经元放电方法，分别观察海马CAl区谷氨酸（Glu）和γ-氨基丁酸（GABA）敏感神经元（Glu—N、GABA—N）的放电活动。结果与Sham组比较，I／R模型大鼠海马组织MDA、MPO显著增高，GSH显著降低，海马组织梗死面积明显增加，固缩神经元细胞数明显增多；而NS＋I／R组与I／R组各指标比较差异均无统计学意义。与NS＋I／R组比较，Ghrelin＋I／R组海马组织MDA、MPO显著降低[MDA（nmol／g）：16．4±4．2比24．5±6．7，MPO（nmol／g）：6．4±1．8比10．2±2．9，均P〈0．05]，GSH显著升高（μmol／g：2．65±0．72比1．66±0．50，P〈0．05），海马组织梗死面积明显缩小[（43．9±9．5）％比（77．0±12．7）％，P〈0．01]，固缩神经元细胞数明显减少（个：36．2±4．5比47．1±6．1，P〈0．01）。电生理研究结果显示，I／R模型大鼠海马CA1区Glu—N和GABA—N放电频率较Sham组明显增加；NS＋I／R组Glu—N和GABA—N放电活动与I／R组比较差异无统计学意义。与NS＋UR组比较，Ghrelin＋I／R组海马CAl区Glu—N放电频率（Hz）显著降低（缺血时：3．81±0．67比4．98±0．33，再灌注时：3．01±0．37比3．77±0．41，均P〈0．05），GABA—N放电频率（Hz）显著增加（缺血时：5．62±0．54比3．62±0．39，再灌注时：4．81±0．48比3．71±0．21，均P〈0．05）。结论在I／R损伤过程中，Ghrelin对海马组织可能具有神经元保护效应，该效应可能与降低神经元兴奋性有关。

Objective To observe the protective effect of ghrelin on hippocampal injury induced by global cerebral ischemia/reperfusion （I/R） and explore its effect mechanisms. Methods The male Sprague-Dawley （SD） rats were randomly divided into four groups, namely sham group, I/R group, normal saline （NS）＋I/R group and Ghrelin＋I/R group, with 42 rats in each group. The model of I/R was reproduced by clipping bilateral carotid artery of rats 15 minutes and then releasing them for 60 minutes. There were no challenges for rats in sham group, just exposed their carotid artery. Ghrelin＋ldR group and NS＋I/R group were challenged by injecting 1μL ghrelinor NS into lateral ventricle before I/R. Some of brain tissue in the rats was harvested after experiment to determine the levels of malonaldehyele （MDA）, myeloperoxidase （MPO） and glutathione （GSH） in hippocampus by using chemical colorimetry and observe infarct sizes and histopathology. Single extracellular neuron discharge in other rats was recorded to observe the activity of glutamic sensitive neurons （Glu-N） and γ-aminobutyric acid （GABA） sensitive neurons （GABA-N） in hippocampus CA1 region of rats suffered I/R. Results Compared with sham group, the levels of MDA and MPO in hippocampus of rats in the I/R group were raised markedly, the level of GSH was decreased significantly, the infarct sizes was increased significantly and pycnosis neurons were increased markedly. All sorts of indexes between NS＋I/R group and I/R group showed no significantly statistical significance. Compared with NS＋I/R group, the levels of MDA and MPO in hippocampus of rats in the Ghrelin＋I/R group were decreased significantly [MDA （nmol/g）： 16.4 ± 4.2 vs. 24.5 ± 6.7, MPO （nmol/g）： 6.4 ± 1.8 vs. 10.2 ± 2.9, both P 〈 0.05], the activity of GSH was risen remarkably （μmol/g： 2.65 ± 0.72 vs. 1.66 ± 0.50, P 〈 0.05）, the infarct sizes of hippocampus were reduced markedly [（43.9 ± 9.5）% vs. （77.0 ± 12.7）%, P 〈 0.01], the number of pycnosis neuron was reduced markedly （cells： 36.2 ±4.5 vs. 47.1 ± 6.1, P 〈 0.01）. The results of electrophysiology showed that the discharge frequency of GIu-N and GABA-N in hippocampus CA1 region of rats in I/R group increased markedly as compared with sham group, and no significant difference in the discharge frequency of Glu-N and GABA-N between NS＋I/R group and I/R group. Compared with NS＋I/R group, injected ghrelin could make the discharge frequency of Glu-N in hippocampus CA1 region of rats decreased markedly （Hz： 3.81 ±0.67 vs. 4.98 ±0.33 at ischemia, 3.01 ± 0.37 vs. 3.77 ±0.41 at reperfusion, both P 〈 0.05）, and the discharge frequency of GABA-N increased markedly （Hz： 5.62 ± 0.54 vs. 3.62±0.39 at ischemia, 4.81±0.48 vs. 3.71±0.21 at reperfusion, both P 〈 0.05）. Conclusion Ghrelin might protect hippocampal neuron after I/R injury, and neuron excitability decrease might be related.