英语晨读 ·
山东省立医院疼痛科英语晨读已经坚持10余年的时间了，每天交班前15分钟都会精选一篇英文文献进行阅读和翻译。一是可以保持工作后的英语阅读习惯，二是可以学习前沿的疼痛相关知识。我们会将晨读内容与大家分享，助力疼痛学习。
本次文献选自Francesca Guidaa，Monica Iannottaa，Gabriella Misso, et al. Pain. 2022.本次学习由李芸主治医师主讲。
Microdialysis experiments were performed in awake and freely moving mice. In brief, mice were anaesthetized with pentobarbital (50 mg/kg, i.p.) and stereotaxically implanted with concentric microdialysis probes into the DG using the coordinates: AP: -2.1 mm from bregma, L: 1.5 mm from midline, and V: 1.2 mm below dura. Dialysis probes were constructed with 25 G (0.3 mm inner diameter, 0.5 mm outer diameter) stainless steel tubing (A-M Systems, Sequim, WA). Inlet and outlet cannulae (0.04 mm in inner diameter, 0.14 mm in outer diameter) consisted of fused silica tubing (Scientific Glass Engineering). The probe had a tubular dialysis membrane (Enka AG, Wuppertal, Germany), which was 1.3 mm in length. After a recovery period of 24 hours, dialysis was commenced with ACSF (NaCl 147 mM, CaCl2 2.2 mM, and KCl 4 mM; pH 7.2) perfused at a rate of 1 μL/minute by a Harvard Apparatus infusion pump. After a 60-minute equilibration period, 6 consecutive 30-minute dialysate samples were collected. At the end of experiments, mice were anaesthetized, and their brains were perfused and fixed through the left cardiac ventricle with heparinized paraformaldehyde saline (4%). The brains were dissected out and fixed in a 10% formaldehyde solution for 2 days. The brain was cut into 40-mm thick slices and observed under a light microscope to identify the probe locations. Dialysates were analyzed through a high-performance liquid chromatography method. The system comprised a Varian ternary pump (mod. 9010), a C18 reverse phase column, a Varian refrigerated autoinjector (mod. 9100), and a Varian fluorimetric detector. Dialysates were precolumn derivatized with o-pthaldialdehyde-N-acetylcysteine (OPA-NAC) (10 μL of dialysate +5μL of OPA-NAC + 10 μL of 10% borate buffer), and amino acid conjugates were re-solved using a gradient separation. The mobile phase consisted of 2components: (1) 0.2 M sodium phosphate buffer and 0.1 M citric acid (pH 5.8) and (2) 90% acetonitrile and 10% distilled water. Gradient composition was determined using an Apple microcomputer installed with Gilson gradient management software. Data were collected using a Dell Corporation PC system 310 interfaced to the detector through a Drew data collection unit. The mean dialysate concentration of amino acids in the 6 samples represented the basal release, and the results were expressed as the mean ± SEM of the pmol in 10 μL of perfusate.
2.7. 微透析实验
微透析实验是使用清醒和自由活动的小鼠进行的。简而言之，用戊巴比妥（50毫克/千克，静脉注射）麻醉小鼠，并将同心微透析探针立体植入齿状回DG，其坐标为前后：距前囟门-2.1毫米，旁开：距中线1.5毫米，深度：硬膜下1.2毫米。透析探针是用25G（内径0.3毫米，外径0.5毫米）不锈钢管制成的。进口和出口插管（内径0.04毫米，外径0.14毫米）由熔融石英管组成。探头有一个管状透析膜，长度为1.3毫米。经过24小时的恢复期后，开始用ACSF透析液（氯化钠147 mM，氯化钙2.2 mM，氯化钾4 mM；酸碱度7.2）进行透析，由输液泵以每分钟1μL的速度灌注。在60分钟的平衡期后，收集连续6个30分钟的透析液样本。实验结束后，对小鼠进行麻醉，用肝素化的多聚甲醛盐水（4%）通过左心室灌注以固定脑组织。剖开大脑，在10%的甲醛溶液中固定2天。将大脑切成40毫米厚的切片，在光学显微镜下观察，以确定探针的位置。透析液通过高效液相色谱法进行分析。该系统包括一个瓦里安三元泵（mod. 9010）、一个C18反相柱、一个瓦里安冷藏自动注射器（mod. 9100）和一个瓦里安荧光检测器。透析液用邻苯二甲酸-N-乙酰半胱氨酸（OPA-NAC）进行柱前衍生（10μL透析液+5μL OPA-NAC+10μL 10%硼酸盐缓冲液），并使用梯度分离法重新溶解氨基酸共轭物。流动相由两部分组成：(1）0.2M磷酸钠缓冲液和0.1M柠檬酸（pH 5.8）和（2）90%乙腈和10%蒸馏水。梯度组成是通过安装有Gilson梯度管理软件的苹果微机来确定的。使用戴尔公司的PC系统310通过德鲁数据收集单元与检测器连接来收集数据。6个样品中氨基酸的平均透析浓度代表基础释放，结果以10μL灌流液中pmol的平均值±SEM表示。
往期回顾：
长期神经病理性疼痛行为学改变与突触可塑性和边缘回路改变的相关性研究：一项小鼠的对照观察研究（八）
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