| The high-altitude areas cover a quarter of land area in China. The special natureenvironment of cold and hypoxia in Qinghai-Tibet Plateau, which has an average elevationof4000m, seriously endangers people’s physical and mental health. Brain is one of themost sensitive organs to cold and hypoxia. Previous studies paid much attention aboutacute mountain sickness (AMS) which was caused by cerebral edema during acuteexposure of altitude. However, the impairments of higher-order brain functions caused by cold and hypoxia complex environment were rarely reported. Recently, the cognitiveability of learning and memory, reaction capacity, reasoning and judgments were reportedto be impaired during acute altitude exposure, which was becoming the focus in thisresearch field. The impairment of cognition may exert widespread impacts on the lifequality and working efficacy of peoples who were living in the high land or taking part inthe military assignments. So, how to prevent people from cognitive impairment duringacute cold and hypoxia exposure was one of the most important questions.The mechanisms leading to cognitive impairment and specific chemoprophylaxisduring acute cold and hypoxia exposure remain largely unknown. As its limitedexperimental conditions of temperature and atmospheric pressure control, most studiesabout altitude exposure using hypobaric chamber for animal and human experiments.However, the results from such studies under simulated environment may not provide awhole picture of actual situation because there are many more factors that may haveeffects on cognitive performance in high-altitude areas. It was reported that some analepticand steroids such as amphetamine and dexamethasone could improve cognitiveperformance during altitude exposure. However, in view of the side effects of these drugs,they could not be used as conventional medications. As preferred prophylactic drug inprevention of AMS, acetazolamide could promote hypoxia adaptation and relievesymptoms of AMS. Recently, it has been reported that the extract of ginkgo biloba,EGb-761, could protect brain from impairment through its regulative effects on cerebralblood flow during hypoxia exposure. Though may have some beneficial effects, theprotective effects of the two drugs mentioned above remain to be clarified during cold andhypoxia exposure. We hypothesize that acetazolamide and EGb-761might protect peoplefrom impairment of learning and memory during acute cold and hypoxia exposure.Objective.The objectives of our study are to determine the impairment effects of acute cold andhypoxia exposure on learning and memory, and assess the protective effects of acetazolamide and EGb-761in human field tests. In animal experiments, we will furtherexplore the protective and regulative effects of Ginkgolide B (GKB), monomer of ginkgobiloba extract, on prevention of learning and memory impairment during acute cold andhypoxia exposure.Methods.1. Subjects and experimental procedure.Male volunteers aging17-20were recruited. And they were randomized into twogroups that ascent to Lhasa in summer and winter separately. And subjects in each groupwere treated with acetazolamide (125mg BID), EGb-761(120mg BID) and placeborandomly. The medication was started3days before ascent and lasted for4days. All thesubjects were lifted from Xianyang (396m) to Lhasa (3658m) where the cognitive testswere performed. The ambient temperature in Lhasa was18-21°C in summer and0-3°Cin winter。2. Incidence of acute mountain sicknessLake Louise scoring system was used to assess the incidence of AMS, which isconsisted of the self-reported score (S-LLS) and clinical assessment score (C-LLS).3. Assessment of learning and memory capacityNeurocognitive test battery was used to assess the effects of acute altitude exposureon learning and memory in summer and winter. The test battery was consists of immediaterecall (immediate word recall/immediate figure recall), PVSAT (paced visual serialaddition task), DSST (digit symbol substitution test) and delayed recall (delayed wordrecall/delayed figure recall).4. Cold and hypoxia exposure modelMale C57J mice weighing18-24g was used and exposed to5000m/4°C for2hours.Animals were randomized into control group (Norm), GKB treated control group (Norm+GKB), cold and hypoxia exposure group (Hypo) and GKB treated cold and hypoxiaexposure group (Hypo+GKB). GKB (40mg/kg body weight) was injectedintraperitoneally (i.p.)1hour before the onset of exposure. 5. Behavior testsNovel object recognition test and step-down inhibitory avoidance test were used toassess the impact of cold and hypoxia exposure on learning and memory.6. Molecular biological studyThe expression and activation level of NMDA, GluR1, Akt, GSK-3β and PKC wasdetected by western blot.Results.1. Effects of acetazolamide and EGb-761on learning and memory during acutehypoxia exposure(1) Incidence of AMS among groups during acute hypoxia exposureDuring the stay in Lhasa, all the drug treatment groups were reported to have AMS,but the mean score of S-LLS in acetazolamide group (1.5) was significantly lower thanthat in the placebo (3.0) and EGb-761(2.8) group (p<0.05).(2) Effects of acute hypoxia exposure on learning and memoryThe capacity of learning and memory was severely impaired8h after exposure andgot to be recovered in the following time.(3) Effects of acetazolamide on learning and memory during hypoxia exposureDuring the stay in Lhasa, the subjects in acetazolamide group performed significantl yworse in the delayed recall, DSST and PVSAT tests when compared with placebo group(p<0.05). And further analysis showed that the subjects in acetazolamide group performedsignificantly worse in the immediate recall and PVSAT tests after8h of exposure whencompared with placebo group (p<0.05).(4) Effects of EGb-761on learning and memory during hypoxia exposureDuring the stay in Lhasa, the subjects in EGb-761group performed significantl ybetter in delayed recall, PVSAT and DSST tests when compared with placebo group(p<0.05). And further analysis showed that the subjects in EGb-761group performedsignificantly better in delayed recall test after8h of exposure when compared withplacebo group (p<0.05). (5) Effects of AMS on learning and memory during hypoxia exposureThere were no significant differences between the subjects with and without AMSwhen compared of their scores in all the neurocognitive tests, no matter what drug theywere treated with.2. Effects of acetazolamide and EGb-761on learning and memory during acutecold and hypoxia exposure(1) Incidence of AMS among groups during acute cold and hypoxia exposureDuring the stay in Lhasa, all the groups were reported to have AMS, but the meanscore of S-LLS in acetazolamide group (2.2) was significantly lower than that in theplacebo (3.9) and group EGb-761(3.3)(p<0.05).(2) Effects of acute cold and hypoxia exposure on learning and memoryThe capacity of learning and memory was severely impaired8h after exposure andgot to be recovered as the exposure time prolonged in all the groups. After8hours ofhypoxia exposure, subjects in each group performed significantly worse in theimmediate/delayed recall, DSST and PVSAT tests (p<0.05).(3) Effects of acetazolamide on learning and memory during cold and hypoxiaexposureDuring the stay in Lhasa, the subjects in acetazolamide group performed significantl yworse in PVSAT test when compared with placebo group (p<0.05). And further analysisshowed that the subjects in acetazolamide group performed significantly worse in theDSST and PVSAT tests after8h of exposure when compared with placebo group(p<0.05).(4) Effects of EGb-761on learning and memory during cold and hypoxia exposureDuring the stay in Lhasa, the subjects in EGb-761group performed significantl ybetter in PVSAT and delayed recall tests when compared with placebo group (p<0.05).And further analysis showed that subjects in EGb-761group performed significantlybetter in delayed recall test when compared with placebo group (p<0.05).(5) Effects of AMS on learning and memory during hypoxia exposureThere were no significant differences between the subjects with and without AMS when compared of their scores in all the neurocognitive tests, no matter what drug theywere treated with.(6) Effects of cold temperature on the impairment effects of hypoxia exposure onlearning and memoryThe cognitive performances of the subjects in placebo group in the summer trail andwinter trial were compared. And we found that the performance of subjects in the wintertrail was significantly worse than those in the summer trail (p<0.05).3. Effects of GKB on learning and memory performance of mouse after coldand hypoxia exposure(1) Effects of acute cold and hypoxia exposure on learning and memory performanceThe time spent in exploring of the novel object as well as the discrimination indexwere significantly reduced in the novel object recognition test (p<0.05), and the latency ofstep down of the platform was significantly shorter in the step-down inhibitory avoidancetask (p<0.05) after2h of cold and hypoxia exposure at5000m.(2) Effects of GKB on learning and memory performance after cold and hypoxiaexposureGKB significantly increased the time spent in exploring of the novel object as well asthe discrimination index (p<0.05), and it also significantly increased the latency of stepdown of the platform (p<0.05) when compared with those treated with saline.(3) Effects of cold and hypoxia exposure and GKB on the expression andphosphorylation level of proteins relative with learning and memoryThe expression level of NMDA and PKCα/β and the phosphorylation level of Akt,GSK-3β and Erk were not affected by cold and hypoxia exposure. However, thephosphorylation level of GluR1at ser831/845and the activity of PKA decreasedsignificantly after2h of cold and hypoxia exposure at5000m. This change could bereversed by GKB treatment1h before exposure.Conclusion.1. Acute hypoxia exposure could impair learning and memory, and cold temperature could aggravate the impairment effects.2. Acetazolamide impaired learning and memory during hypoxia as well as cold andhypoxia exposure. However, it could prevent people from AMS.3. EGb-761could potently protect people from impairment of learning and memory.But it has no protective effects on AMS.4. GKB might be the key component of EGb-761in prevention of learning andmemory impairment by regulating the phosphorylation of GluR1during cold andhypoxia exposure. |
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