Effect Of RTMs On Improving Impairment Of Cognitive Functions Induced By Simulated Microgravity And Its Underlying Mechanisms In Mice

The human beings are accustomed to the gravitational environment on earth during the tens of millions years evolution.However,it is a microgravity environment both in low Earth orbit and deep space.More importantly,thanks to the development of science and technology as well as the efforts of several generations of aerospace researchers,long term stay in low Earth orbit has been achieved.And plans to explore deep space such as exploring Mars have also been put on the agenda.There are significant impact on the actions of astronauts,and even the success of the mission whether the learning cognition ability of astronauts is affected in microgravity environment.Therefore,to investigate the effects of microgravity environment on human body,especially impacts on learning and memory,is necessary.Several researches report that microgravity environment has adverse effects on the learning and memory of astronauts.Due to the restrictions of space,funding,environment and other factors,it is difficult to study the relevant mechanisms in the real space environment.Therefore,the rodent hindlimb unloading model(HU),developed in the 1980's,is now widely used to simulate microgravity.The studies using this model find that simulated microgravity could cause spatial learning and memory impairment in rats and could change the expressions of genes related to learning and memory in the brain of mice.Repetitive transcranial magnetic stimulation(rTMS),developed in recent years,is a non-invasive physical therapy and has been widely used in the treatment of psychiatric disorders and neurological diseases.At the same time,several studies show that rTMS could improve learning and memory.However,whether rTMS could mitigate the cognitive impairment caused by simulated microgravity has not been reported yet.Therefore,we investigate the effect of high frequency(15Hz)rTMS on learning and cognition in simulated microgravity treated mice and further explore the underlying mechanisms.Methods:Adult male C57BL/6J mice were randomly divided into four groups: Control group(Sham Non-HU/Sham NH,n = 8),Magnetic stimulation group(rTMS Non-HU/ rTMS NH,n = 8),Simulated microgravity group(Sham HU,n = 8),and Magnetic stimulation + microgravity group(rTMS HU,n = 8).Mice in Sham HU and rTMS HU group were tail-suspended for 14 days to simulate the effects of microgravity.Mice in Sham NH and rTMS NH group were housed individually in normal cage.After establishment of the HU model,mice in rTMS NH group and rTMS HU group were given a 14-day rTMS(15Hz,90% motor threshold,300 pulses daily).Mice in Sham NH group and Sham HU group were handled similarly to those treated with rTMS while the coil was 100 mm away from the mice's head.After 14 days,the novel object recognition test was conducted to evaluate the learning and memory of mice in each group.Then the in vivo electrophysiological experiments were performed to detect the paired pulse facilitation(PPF)and long term potentiation(LTP)in the hippocampus of mice to reflect the synaptic plasticity in the hippocampi of mice.The right hemisphere of the mice brain were taken for Golgi-Cox staining to investigate the spin density of tertiary dendrites from granulosa cells of hippocampal dentate gyrus(DG)region.Finally,the levels of NR2 B,NR2A and PSD95 in the hippocampus of mice were detected by Western blotting assay.The levels of BDNF,TrkB,and the phosphorylation level of Akt were also detected by Western blotting.Results:Novel object recognition experiment showed that the percent of novel object visits and percent of time spent in exploring novel object were significantly lower in Sham HU group than that in Sham NH group.While rTMS HU mice showed a significant improvement in the percent of novel object visits and percent of time spent in exploring novel object than Sham HU mice.There was no statistical difference in total exploration time of both objects in the test session among the four groups.These results indicated that rTMS could alle viate recognition memory impairment induced by HU.In vivo electrophysiological experiments showed that LTP was impaired in Sham HU mice compared to Sham NH mice.While rTMS HU mice showed a significant improvement in LTP level than that in Sham HU mice.There was no statistical difference in PPF among the four groups.These results suggested that rTMS could mitigate hippocampal LTP impairment induced by HU.However,the effects of rTMS and HU on the presynaptic mechanism of synaptic plasticity may not be remarkable.Golgi-Cox staining experiment showed that the tertiary dendritic spine density in DG region in Sham HU mice was significantly lower than that in Sham NH group.While rTMS HU mice showed a significant enhancement in spine density than that in Sham HU group.These results showed that rTMS could increase the spine density in DG region which was decreased by HU.Western blotting essay showed that the expressions of NR2 B,NR2A,PSD95,BDNF,TrkB,and p-Akt were significantly decreased in Sham HU group than those in Sham NH group.While the levels of these proteins were markedly increased in rTMS HU group than those in Sham HU group.These results indicated that rTMS could notably increase expressions of the postsynaptic function-associated proteins and BDNF/TrkB as well as p-Akt.The increased level of these proteins may be related to the improvement of LTP and recognition memory.Conclusions:High frequency(15 Hz)rTMS could dramatically attenuate learning and recognition ability defects and improve LTP in a mice simulated microgravity model.These alterations may be closely related to the increase of spine density and postsynaptic function-associated proteins,NR2 B,NR2A and PSD95.Furthermore,the activation of BDNF/TrkB signaling and p-Akt may also partly be responsible for the improvement of recognition memory and LTP.