全身麻醉机制与星形胶质细胞突触传递调控作用的相关性研究

Reveal the mechanism of general anesthesia induced loss of consciousness is crucial in the study of anesthetic effect. Study found that neuronal activity and synaptic transmission can be regulated by a variety of neural active substance released from astrocytes, which participating in the brain information integration. Astrocytes may take part in the general anesthesia induced loss of consciousness, however, there is no related report. This project will be based on the experience of previous anesthesia mechanism research work and choose the rats cortical astrocytes as the main object of observation, utilizing the brain slice patch clamp technique, calcium imaging and laser confocal microscopy to investigate the effects of three intravenous anesthetics (propofol, etomidate and ketamine) on astrocytes calcium ion excitatory and the information exchange between astrocytes and neurons, while exploring the similarities and differences of drugs effect. This project will also use the vivo brain micro-injection and Western blot technology to confirm weather the glutamate and ATP release from astrocytes take part in the consciousness loss induced by three anesthetics, and analysis the related mechanisms. This project focus on the astrocytes calcium excitability, ion channel receptors and glial neurotransmitters release, expounds the role of astrocytes in general anesthesia induced loss of consciousness, which providing new experimental evidence for mechanism study of general anesthesia.

揭示全身麻醉致意识消失的机制是全麻药作用研究的关键。研究发现星形胶质细胞能释放多种神经活性物质调控神经元活性和突触传递，参与大脑信息整合，推测星形胶质细胞可能参与全麻药物的致意识消失作用，但未见相关报道。本项目拟在前期全麻机制相关研究的基础上，将大鼠皮层内星形胶质细胞作为主要观察对象，通过脑片膜片钳技术、脑片钙成像和激光共聚焦技术，考察三种静脉全麻药异丙酚、依托咪酯和氯胺酮对星形胶质细胞钙离子兴奋性及其与神经元间信息交流的影响，并探讨药物对星形胶质细胞功能影响的异同；进而借助活体大鼠脑区微注射和Western blot等技术，分析星形胶质细胞释放的谷氨酸和ATP是否参与三种全麻药的致意识消失作用及相关机制。本项目将从星形胶质细胞钙离子兴奋性、离子通道受体和胶质递质分泌等角度，探索星形胶质细胞在全麻意识消失中所发挥的作用，为全麻机制的研究提供新的实验依据。

星形胶质细胞释放出多种神经递质至突触间隙，并作用于突触前膜及突触后膜。星形胶质细胞被认为是中枢突触信息传递及调控过程的重要组成部分。全身麻醉药已经被发现可以抑制星形细胞的活动，但目前尚不清楚临床浓度范围内的全身麻醉药是否影响星形细胞介导的突触调节。.主要研究内容.1. 反射荧光成像系统动态观察全身麻醉药物丙泊酚、氯胺酮以及右旋美托咪啶对二磷酸腺苷诱发星形胶质细胞胞内钙瞬变的影响.2. 应用ELISA法检测胞内三磷酸肌醇的水平变化.3. 应用液相色谱技术，观察全麻药物作用下皮层星形胶质细胞释放谷氨酸，γ-氨基丁酸，丝氨酸的情况.4. 使用M型乙酰胆碱受体受体阻断剂与N-甲基-D-天冬氨酸受体阻断剂，明确全麻药物对星形胶质细胞作用的靶点受体。.5. 采用大鼠急性前额叶皮质脑片膜片钳全细胞记录法，研究了异丙酚、右美托咪定和氯胺酮在临床有效范围内对慢内向电流（SICs）和抑制性突触后电流的影响。.重要结果.1. 氯胺酮可以有效抑制ADP诱发的星形胶质细胞钙瞬变，且呈浓度依赖趋势；.2. 氯胺酮不能有效影响培养星形胶质细胞释放Glu和Ser。.3. 临床浓度氯胺酮通过作用于突触外Glun2b受体，抑制星形胶质细胞与神经元间的谷氨酸传递。.关键数据.氯胺酮呈剂量依赖性的抑制星形胶质细胞介导的慢内向电流，既抑制星形胶质细胞与神经元间的谷氨酸传递。30μM氯胺酮抑制配对神经元慢内向电流的同步率（从42.5 ± 1.4%降至 9.6 ± 0.8%），30μM氯胺酮使配对神经元慢内向电流的同步率将为0。GluN2B受体特异拮抗剂ifenprodil增加30μM氯胺酮对慢内向电流的抑制作用。低浓度氯胺酮（3 μM）能抑制慢内向电流的频率，但不能抑制神经元自发兴奋性突触后电流。30 μM氯胺酮对慢内向电流的抑制率要明显高于自发兴奋性突触后电流（44.5 ± 3%对28.3 ± 6%）。.科学意义.本研究证明了在氯胺酮、丙泊酚和右美托咪定中，只有氯胺酮可以对星形胶质细胞的钙兴奋性和谷氨酸传递产生影响。提示星形胶质细胞参与了氯胺酮的中枢作用，为进一步阐明氯胺酮的全身麻醉机制提供了实验支持。