The Study On The Mechanism Of The Cerebrovascular Oxidase Activation And Apoptosis Induced By Long-Term Administration Of Ketamine In Cynomolgus Monkeys

HypothesisKetamine 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.ObjectiveThe 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.MethodsTwelve 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.Results1. 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.