长期使用氯胺酮导致食蟹猴大脑血管氧化应激及凋亡的机制研究
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摘要
研究背景
氯胺酮是N-甲基-D-天门冬氨酸(N-methyl-D-aspartate,NMDA)受体的非竞争性拮抗剂,长期滥用会导致学习、记忆等认知功能的损害,但其机制尚不完全清楚。大量研究认为,长期应用麻醉剂量的氯胺酮可致神经细胞调亡,从而引发与记忆密切相关的脑区如海马功能紊乱,继而出现记忆障碍。最近的研究结果表明,氯胺酮作为新兴毒品被长期滥用,除导致记忆功能损伤外,还引起人情绪和人格出现异常,提示氯胺酮的中枢损伤效应存在多元性。学习记忆障碍是脑血管性疾病的主要症状,由脑血管损伤引发的缺血缺氧是导致学习记忆障碍的主要原因之一。但氯胺酮长期使用是否对脑血管产生影响尚没有系统研究。本实验通过观察长期静脉应用氯胺酮的食蟹猴行为学、大脑动脉形态学及蛋白质组学的改变来研究氯胺酮引起食蟹猴大脑血管氧化应激及凋亡的可能机制。
研究目的
观察氯胺酮连续给药对食蟹猴行为学、大脑动脉(动脉环交通支)形态、氧化应激及凋亡的影响。
研究方法
随机将12只健康雄性食蟹猴分为氯胺酮组和生理盐水组,氯胺酮组8只,每天股静脉注射盐酸氯胺酮注射液,剂量为1mg/kg。生理盐水组4只,每天股静脉注射生理盐水,剂量为1mL/kgo分别在造模的第1、3、7、14、56、112、183、184、185天,于自然状态下用摄像机记录注射氯胺酮或生理盐水前后15分钟的行为变化,连续观察6个月。持续给药6个月后牺牲动物,冰上剥离食蟹猴脑血管(动脉环交通支),部分置-80℃保存,剩余部分制备石蜡切片。HE染色观察大脑动脉的形态学变化；原位末端脱氧核糖核苷酸转移酶标记技术(Tunnel法)检测脑动脉细胞凋亡水平：用比色法测定H2O2水平用以衡量血清和血管组织内的活性氧(reactive oxygen species,ROS)水平；采用免疫组织化学染色和Western blot检测血管中NADPH氧化酶的表达。
研究结果
1.与对照组比较,氯胺酮组行为学未出现明显变化。
2.连续给药6个月后,氯胺酮组动脉环交通支中出现明显的血栓,血栓成分主要是血小板、红细胞、白细胞和纤维蛋白,呈混合血栓的特征,另外还可发现血管内膜增厚；而对照组其血管壁光滑,无异物出现。此外,氯胺酮组动脉环交通支外膜有明显的炎性细胞浸润；而对照组血管壁无白细胞浸润。
3.连续给药6个月后,氯胺酮组和对照组动脉环交通支细胞均未出现明显的凋亡现象。
4.连续给药6个月后,氯胺酮组血清和血管组织中活性氧水平明显高于对照组,说明氯胺酮长期滥用后可引起组织中抗氧化能力降低、细胞功能受损。
5.连续给药6个月后,氯胺酮组免疫组织化学和Western blot检测动脉环交通支组织NADPH亚基p47phox蛋白的表达明显高于对照组,差异有统计学意义。
研究结论
1.长期连续使用氯胺酮不会引起食蟹猴明显的行为学改变。
2.长期连续使用氯胺酮不会引起食蟹猴脑血管细胞凋亡。
3.长期连续使用氯胺酮可激活脑血管内皮细胞NADPH氧化酶,后者诱发血管内皮细胞氧化应激,导致血管炎症和血栓形成。
Hypothesis
Ketamine is an antagonist of the N-methyl-d-aspartate receptor (NMDAR). Long-termed abuse of ketamine might result in the dysfunction in learning and memory. However, to date, the underlying mechanism remains unclear. Nowadays, more and more evidence have suggested that ketamine could cause neuronal apoptosis. If it occurred in the hippocampus, which is an important region responsible for learning and memory in the central nervous system, damaged ability of learning and memory were also paralleled as a consequence of behavioral change. Recent evidence showed that ketamine was abused as a new drug and caused a lot of issues related to emotion and personality besides dysfunction of learning and memory, suggesting that ketamine may have multiple negative effects on the different regions in the central nervous system. Given the fact that cerebrovascular diseases can lead to hypoxia and ischemia, which may be the factors contributing to the dysfunction in learning and memory. However, till now, it is still to be elucidated that weather long-term administration of ketamine affects cerebrovascular system.
Objective
The aim of the present study was to explore the effects of long-term administration of ketamine on behavior in macaca fascicularis. Further more, we examined weather ketamine could have an effect on morphology of arterial circle of Willis and the underlying mechanisms relevant to NADPH oxidase activation and apoptosis of endothelial of cerebrovascular system.
Methods
Twelve healthy male macaca fascicularis were randomly divided into two groups:4 in control group which was treated with normal saline in a intravenous manner; 8 in experimental group administered with ketamine in 1 mg/kg/day.The duration of the experiment were 6 months. Behavior data including climb, movement, jump and walk was recorded by a video camera 15 minute before and after administration of the drug or vehicle. At the end of the experiment, the monkeys were sacrificed and trunk blood was collected. In addition, the brain was removed and cerebrovascular system were dissected on ice and stored at-80℃for further study. Morphology of arterial circle of Willis was examined by HE staining and apoptosis of cerebrovascular endothelium was also assessed by TdT-mediated dUTP nick end labeling (TUNEL) assay. Serum level of H2O 2 was taken as an indicator of reactive oxygen species (ROS). Immunohistochemistry and Western blotting were performed to observe the NADPH oxidase activation.
Results
1. Compared to the control group, monkeys in the experimental goup did not show significant difference in behavioral changes.
2. Six months after ketamine administration, cerebrovascular endothelial thrombosis was markedly evidenced by HE staining in the experimental group compared to the control group. The main components of thrombus were composed of platelet, red blood cells (RBC), white blood cells (WBC) and fibrin, which is the characteristic pattern of mixed thrombus. In addition, cerebrovascular intima hyperplasia was also observed. There were also a large number of inflammatory cells infiltration in cerebrovascular adventitia in the experimental group, while the control group did not show the corresponding changes.
3. There were also no cerebrovascular endothelial apoptosis observed both in the control and experimental group after 6 months of administration of ketamine.
4. In the experimental group, serum and cerebrovascular levels of reactive oxygen species were higher than those in the control group, indicating that long-termed use of ketamine might result in the decrease of the antioxidant capacity and cellular dysfunction.
5. Six months after ketamine administration, by Immunohistochemistry and Western blotting, there were also higher level of expression of p47phox, one of subunits of NADPH, in the experimental group compared to the control group.
Conclusion
First, long-term(6 months) administration of ketamine had no effect on the behavioral changes.
Second, after long-term(6 months) administration of ketamine cerebrovascular endothelial apoptosis was not observed in macaca fascicularis.
Last, long-term(6 months) administration of ketamine was able to activate cerebrovascular endothelial NADPH oxidase, which could further induce the imbanlace of reactive oxygen species (ROS) and result in the cerebrovascular inflammation and formation of thrombosis in macaca fascicularis.
氯胺酮是N-甲基-D-天门冬氨酸(N-methyl-D-aspartate,NMDA)受体的非竞争性拮抗剂,长期滥用会导致学习、记忆等认知功能的损害,但其机制尚不完全清楚。大量研究认为,长期应用麻醉剂量的氯胺酮可致神经细胞调亡,从而引发与记忆密切相关的脑区如海马功能紊乱,继而出现记忆障碍。最近的研究结果表明,氯胺酮作为新兴毒品被长期滥用,除导致记忆功能损伤外,还引起人情绪和人格出现异常,提示氯胺酮的中枢损伤效应存在多元性。学习记忆障碍是脑血管性疾病的主要症状,由脑血管损伤引发的缺血缺氧是导致学习记忆障碍的主要原因之一。但氯胺酮长期使用是否对脑血管产生影响尚没有系统研究。本实验通过观察长期静脉应用氯胺酮的食蟹猴行为学、大脑动脉形态学及蛋白质组学的改变来研究氯胺酮引起食蟹猴大脑血管氧化应激及凋亡的可能机制。
研究目的
观察氯胺酮连续给药对食蟹猴行为学、大脑动脉(动脉环交通支)形态、氧化应激及凋亡的影响。
研究方法
随机将12只健康雄性食蟹猴分为氯胺酮组和生理盐水组,氯胺酮组8只,每天股静脉注射盐酸氯胺酮注射液,剂量为1mg/kg。生理盐水组4只,每天股静脉注射生理盐水,剂量为1mL/kgo分别在造模的第1、3、7、14、56、112、183、184、185天,于自然状态下用摄像机记录注射氯胺酮或生理盐水前后15分钟的行为变化,连续观察6个月。持续给药6个月后牺牲动物,冰上剥离食蟹猴脑血管(动脉环交通支),部分置-80℃保存,剩余部分制备石蜡切片。HE染色观察大脑动脉的形态学变化；原位末端脱氧核糖核苷酸转移酶标记技术(Tunnel法)检测脑动脉细胞凋亡水平：用比色法测定H2O2水平用以衡量血清和血管组织内的活性氧(reactive oxygen species,ROS)水平；采用免疫组织化学染色和Western blot检测血管中NADPH氧化酶的表达。
研究结果
1.与对照组比较,氯胺酮组行为学未出现明显变化。
2.连续给药6个月后,氯胺酮组动脉环交通支中出现明显的血栓,血栓成分主要是血小板、红细胞、白细胞和纤维蛋白,呈混合血栓的特征,另外还可发现血管内膜增厚；而对照组其血管壁光滑,无异物出现。此外,氯胺酮组动脉环交通支外膜有明显的炎性细胞浸润；而对照组血管壁无白细胞浸润。
3.连续给药6个月后,氯胺酮组和对照组动脉环交通支细胞均未出现明显的凋亡现象。
4.连续给药6个月后,氯胺酮组血清和血管组织中活性氧水平明显高于对照组,说明氯胺酮长期滥用后可引起组织中抗氧化能力降低、细胞功能受损。
5.连续给药6个月后,氯胺酮组免疫组织化学和Western blot检测动脉环交通支组织NADPH亚基p47phox蛋白的表达明显高于对照组,差异有统计学意义。
研究结论
1.长期连续使用氯胺酮不会引起食蟹猴明显的行为学改变。
2.长期连续使用氯胺酮不会引起食蟹猴脑血管细胞凋亡。
3.长期连续使用氯胺酮可激活脑血管内皮细胞NADPH氧化酶,后者诱发血管内皮细胞氧化应激,导致血管炎症和血栓形成。
Hypothesis
Ketamine is an antagonist of the N-methyl-d-aspartate receptor (NMDAR). Long-termed abuse of ketamine might result in the dysfunction in learning and memory. However, to date, the underlying mechanism remains unclear. Nowadays, more and more evidence have suggested that ketamine could cause neuronal apoptosis. If it occurred in the hippocampus, which is an important region responsible for learning and memory in the central nervous system, damaged ability of learning and memory were also paralleled as a consequence of behavioral change. Recent evidence showed that ketamine was abused as a new drug and caused a lot of issues related to emotion and personality besides dysfunction of learning and memory, suggesting that ketamine may have multiple negative effects on the different regions in the central nervous system. Given the fact that cerebrovascular diseases can lead to hypoxia and ischemia, which may be the factors contributing to the dysfunction in learning and memory. However, till now, it is still to be elucidated that weather long-term administration of ketamine affects cerebrovascular system.
Objective
The aim of the present study was to explore the effects of long-term administration of ketamine on behavior in macaca fascicularis. Further more, we examined weather ketamine could have an effect on morphology of arterial circle of Willis and the underlying mechanisms relevant to NADPH oxidase activation and apoptosis of endothelial of cerebrovascular system.
Methods
Twelve healthy male macaca fascicularis were randomly divided into two groups:4 in control group which was treated with normal saline in a intravenous manner; 8 in experimental group administered with ketamine in 1 mg/kg/day.The duration of the experiment were 6 months. Behavior data including climb, movement, jump and walk was recorded by a video camera 15 minute before and after administration of the drug or vehicle. At the end of the experiment, the monkeys were sacrificed and trunk blood was collected. In addition, the brain was removed and cerebrovascular system were dissected on ice and stored at-80℃for further study. Morphology of arterial circle of Willis was examined by HE staining and apoptosis of cerebrovascular endothelium was also assessed by TdT-mediated dUTP nick end labeling (TUNEL) assay. Serum level of H2O 2 was taken as an indicator of reactive oxygen species (ROS). Immunohistochemistry and Western blotting were performed to observe the NADPH oxidase activation.
Results
1. Compared to the control group, monkeys in the experimental goup did not show significant difference in behavioral changes.
2. Six months after ketamine administration, cerebrovascular endothelial thrombosis was markedly evidenced by HE staining in the experimental group compared to the control group. The main components of thrombus were composed of platelet, red blood cells (RBC), white blood cells (WBC) and fibrin, which is the characteristic pattern of mixed thrombus. In addition, cerebrovascular intima hyperplasia was also observed. There were also a large number of inflammatory cells infiltration in cerebrovascular adventitia in the experimental group, while the control group did not show the corresponding changes.
3. There were also no cerebrovascular endothelial apoptosis observed both in the control and experimental group after 6 months of administration of ketamine.
4. In the experimental group, serum and cerebrovascular levels of reactive oxygen species were higher than those in the control group, indicating that long-termed use of ketamine might result in the decrease of the antioxidant capacity and cellular dysfunction.
5. Six months after ketamine administration, by Immunohistochemistry and Western blotting, there were also higher level of expression of p47phox, one of subunits of NADPH, in the experimental group compared to the control group.
Conclusion
First, long-term(6 months) administration of ketamine had no effect on the behavioral changes.
Second, after long-term(6 months) administration of ketamine cerebrovascular endothelial apoptosis was not observed in macaca fascicularis.
Last, long-term(6 months) administration of ketamine was able to activate cerebrovascular endothelial NADPH oxidase, which could further induce the imbanlace of reactive oxygen species (ROS) and result in the cerebrovascular inflammation and formation of thrombosis in macaca fascicularis.
引文
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2. Taniguchi T, Shibata K, Yamamoto K. Ketamine inhibitsendotozxine induced shock in rats [J]. Anesthesiology.2001,95:928-932.
3. Larsen B, Hoff G, Wilhelm W, et al. Effect of intravenousanes thetics on sponlancous and endotoxin stimuliated cytokine response in cultured buman whole blood [J]. Anesthesiology.1998,89:1218-1227.
4.胡春旭,高崇荣.亚麻醉剂量氯胺酮用于治疗急性严重哮喘9例报道[J].临床麻醉学杂志.2000,16：1100-1106.
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