Electroacupuncture Improves Cognition And Neuroethology Following Brain Ischemia Via Upregulation Of Neurogenin2Expression And Promoting Neurogenesis

BackgroundWith the progress of medical means, the survival rate after stroke has been greatlyincreased. But the stroke sequela is still a problem needed to be solved.It is now acceptedthat the reason why nervous system dysfunction after stroke is that there is not so muchnew neurons to replace the dead ones. The present study reveals that there are numerousneural stem cells in the subventricular zone (SVZ) and hippocampal dentate gyrus (DG) inadult mammalian central nervous system. Under certain conditions，these stem cells canproliferate and differentiate into neurons and glial cells, improving nerve functional restoration process，this phenomenon is called adult neurogenesis.Acupuncture has a remarkable curative effect in treating stroke. The effects ofelectroacupuncture (EA) is the integration of acupuncture and biological stimulation,which can play the same role as acupuncture. EA stimulation is painless, safe and reliable,and now has been widely used in clinical treatment after stroke. But the specificmechanism remains to be disclosed.Neurogenin2(Neurog2) is a key transcription factor which regulate the neurogenesisand neuronal radial migration in embryonic brain. In different regions of the nervoussystem, it is involved in the neuronal differentiation and subtype of decision. Theexpression of Neurog2in the adult mammalian brain after ischemia remains not clear.Our research used the global cerebral ischemia model to observe the effect of EAtreatment on motor and cognitive dysfunction after ischemia and to investigate thepossible mechanism. Also we observed the Neurog2expression after ischemia and explorewhether early EA treatment could improve cognitive and motor dysfunction after ischemiais associated with promoting Neurog2expression and boosting neurogenesis. This studywould provide relevant new experimental evidence for EA therapy for stroke and injectionneurog2protein to treat stroke.Experiment IThe effect of EA treatment on neurologic and cognitivedysfunction after brain ischemiaObjectiveTo investigate the effectiveness of early EA stimulation on neurological and cognitionoutcome after cerebral ischemia.MethodsThe animals were provided by Experiment Animal Center of the Fourth MilitaryMedical University. C57BL/6J male mouse, weight more than20g, were randomly dividedinto four groups: sham group (n=6), EA+sham group (n=6), the global cerebral ischemia group (I/R)(n=6), and EA+I/R (n=6). The ischemia model was established bybilateral comman cervical artery ligation for20minutes. The sham groups received thesame operation but no ligation. The two EA groups received the same EA stimulation inthe next five days. EA treatment was performed at the acupoint “Baihui (GV20)” with anintensity of1mA and frequency of15HZ for four weeks (30min/day,5day/week). Theneurological behavior scores of four groups were evaluated by a person who was unawareabout the grouping. Then the four groups received the water Morris test.Results1)Neurobehavioral score: Compared with the sham group, the EA treatment shamgroup showed a little better, which was not statistically significant. In the two I/R groups,the EA treatment group showed much better. The neurobehavioral performances in I/R andEA+I/R groups were damaged more severely than that of sham and EA+sham groups.2).Morris water maze: Water maze test showed that the spatial learning and memory functionwere significantly improved in EA+I/R group, with the extension of target quadrant timeand the shorten of escape latency in comparison with the I/R group (P<0.05). At the sametime, compared with sham group, a slight improvement of spatial learning and memoryfunction were also detected in EA+sham group，but with no statistical significance.Theneurobehavioral performances in I/R and EA+I/R groups were more severely damagedthan that of sham and EA+sham groups.ConclusionEarly EA stimulation could remarkably improve motor and cognition function aftercerebral ischemia, thus EA have a better therapeutic effect on stroke. Experiment2EA treatment inhibits neuron apoptosis and promote neurogenesis.ObjectiveTo study whether the effect of EA treatment against brain ischemia is associatedwith the inhibiting neuron apoptosis and promoting neurogenesis.MethodsC57BL/6J male mice, weight20-25, were randomly divided into four groups: shamgroup (n=9), EA+sham group (n=9), the global cerebral ischemia group (I/R)(n=9),and EA+I/R (n=9). The two EA group received the same EA stimulation in thefollowing five days. EA treatment was performed at the acupoint “Baihui” with anintensity of1mA and frequency of15HZ for four weeks (30min/day,5day/week).Fourgroups were injected with Brdu (50mg/kg) from day2-7after brain ischemia. Then weprepared mouse hippocampal slice for TUNEL, Nissl’s, and immunofluorescent stainings.Results1) Neurogenesis: Compared to the sham group, brain ischemia could promote Brduincorporation. EA treatment could make more neurogenesis. Brdu and neurog2have thesame localization.2) TUNEL test: The positive cells in the ischemia group received EAtreatment was less than that in the ischemia group, indicating that EA could suppressneuron apoptosis.3) The Nissls staining: The neurons in two sham groups were normal,but two ischemia groups showed disordered arrangement of neurons and Nissl bodydisappeared in the cell. The pathology of cellular structures in EA+I/R groups was betterthan I/R group.ConclusionThe mechanism of therapeutic effect of EA against brain ischemia may beassociagted with inhibiting neuronal apoptosis and promoting neurogenesis. Experiment3The expression of Neurogenin2after brain ischemia and the effect ofelectroacupuncture treatment in adult animalsObjectiveTo explore the expression change of the neural precursor gene Neurog2after brainischemia and the effect of EA treatment in adult animals.MethodsFive-month-old healthy C57BL/6J male mice, weight20-25, were randomly dividedinto four groups: sham group (n=9), EA+sham group (n=9), the global cerebralischemia group(I/R)(n=15), and EA+I/R (n=9). The ischemia model was established bybilateral common cervical artery ligation for20minutes. The sham groups give the sameoperation but no ligation. At3d,5d,7d, the I/R group were executed three mice respectively.The two EA groups received EA stimulation in the following five days. The experimentalparameters based on our laboratory early results:30min, frequency15HZ, the intensity is1-2mA. When the treatment finished, the mouse brain were obtained to extract RNA.Besides, we prepared mouse hippocampal slice for immunofluorescent staining.Results1) The expression of Neurog2after ischemia gradually increased at3d after cerebralischemia, peaked at5d, and at7d began to fall. The P value was below0.01.2) Comparedwith the sham group, the expression of Neurog2increased significantly (P <0.05) in two I/Rgroups. After EA treatment,the mRNA level of Neurog2(P <0.001) also increased.3）Inimmunofluorescence staining,we observed that the Neurog2expression increased aftercerebral ischemia, and electroacupuncture obviously enhanced this expression.ConclusionThe expression of Neurog2in adult is activated after cerebral ischemia reperfusion and presentsa dynamic change, EA treatment can promote the expression of Neurog2. Experiment4The injection of Neurog2to the brain could improves neuroethologyand boost neurogenesisObjective: To study the effect of injection Nueorg2to brain on nervous behavior andneurogenesis in hippocampus after brain ischemia.Methods: Eighteen male C57mice were randomly divided into three groups: shamgroup, ischemia group (I/R group, and ischemia+neurog2injection group（neurog2+I/Rgroup）.The third group were injected with neurog2(10umol/l)4uL before ischemia. Thenthe two ischemia groups received operations to construct global brain ischemia model.Thesham group received similar operation without ligation. In the following7days, weinjected Brdu to the three groups. At last, we observed the neurological behavioralmanifestation and neurogenesis in hippocampus.Results:1）TMS results: Compared to sham group, the two ischemia groups showedworse motor behavior, After injection Neurog2, the third group showed much better thanthe ischemia group in neurological functional deficit scales.2) From immunofluorescentstaining, we noticed that the neurogenesis in the third group was more than other twogroups.Conclusion: Injection Neurog2to the ischemia brain would improve neurologicalimpairment, also administration of Neurog2can increase neurogenesis.