The Effects And Mechanisms Of Low Frequency Pulsed Electromagnetic Fields Via Intrinsic Neural Stem Cells In Recovery Of Intracerebral Hemorrhage

Stroke is a major threat to human health that always results in permanent injuries and causes a series of complications in patients.Intracerebral hemorrhage（ICH）with evident high morbidity and mortality comprises approximately one-third of stroke cases,although its pathophysiologic mechanism remains unclear and effective treatments for stroke are lacking.A clinical trial of extrinsic drugs did not obtain satisfactory results.Intrinsic neural stem cells（NSCs）,discovered in 1992,provide a direct way to repair the brain.Activation of proliferation,differentiation,and migration has been reported as the possible major intrinsic protective mechanism after ICH.However,accompanied by limited restoration,a method of influencing the intrinsic and natural repair system in the body demonstrates a promising means of ICH rehabilitation.Recent research has shown the existence of an intrinsic biological electromagnetic field during the period of development and repair after neuronal damage.Different properties of electromagnetic fields with spatial and temporal differences may exert various influences on NSCs,especially on their proliferation,differentiation,and migration.Repetitive transcranial magnetic stimulation（rTMS）,which is widely used in neural diseases,is a physical method that can noninvasively penetrate the scalp to exert a focal electromagnetic field on neural cells.Though effects of this noninvasive treatment are substantial,little research has focused on the influence of rTMS on NSCs in ICH since its invention in 1985.In recent reports,researchers showed that rTMS could promote the proliferation of NSCs in ischemic rats.Nevertheless,no research has focused on the effects and potential underlying mechanism of rTMS on ICH.This study was designed to examine the influence of rTMS on neurological function,cerebral edema,glial aggregation,and the migration,proliferation,and neuronal differentiation of NSCs in a mouse model of experimental ICH.In addition,the potential mechanisms underlying the alterations in NSCs triggered in vitro by the treatment of rTMS were investigated and discussed in this study.The main results and conclusions of this study are summarized as following:1.Biological characteristics of intrinsic neural stem cells in mice after intracerebral hemorrhage（1）Brain water content and neurological function after ICHTo testify if ICH animal model was up to standard,we first assessed neurological function deficits.The single-blind method was employed for assessments at 0 h,24 h,72 h,and 120 h after the surgery.Compared with the sham group,the group that underwent ICH suffered a worse neurobehavioral deficit at all time points（p<0.05）.Because brain edema is considered a main pathological feature for evaluating the severity of brain damage and inflammation,we also used the determination of brain water content to measure the percent change in brain water content in various parts of the brain at24 h,72 h,and 120 h after the surgery.In accordance with previous study,only the ipsilateral basal ganglia（ips-BG）and cortex brain（ips-CX）water content was increased in the ICH group compared with those in the sham group（p<0.05）.These results showed that ICH increased brain water content and resulted in neurological function deficit.（2）NSCs around lesion after ICHThe Ki-67 protein（also known as MKI67）is a cellular marker for cell proliferation.During interphase,the Ki-67 antigen can be specifically detected in the cell nucleus;hence,Ki-67 immunofluorescence labeling was used to evaluate the proliferation of NSCs.In accordance with previous study,Immunofluorescence staining demonstrated that the percentage of Ki-67-positive cells in Nestin-positive cells in the perihematomal region（basal ganglia）significantly increased at 72 h after ICH,suggesting NSCs were activated by ICH to proliferate and migrate for repairing which shows a natural protection and recovery mechanism after ICH.（3）Glia in the lesion area after ICHAggregation of glial cells,which is treated as a process of inflammatory response in the central nervous system,occurs after brain damage.GFAP is an intermediate filament protein that is expressed by numerous cell types of the central nervous system,especially glial cells.GFAP immunofluorescence labeling was used to detect inflammatory glial cells after ICH.In accordance with previous study,results showed that the number of GFAP-positive cells significantly increased around the hematoma at 72 h after ICH（p<0.05）,suggesting more substitute gila from differentiation of NSCs and inflammatory glia aggregated after ICH.2.Effect of rTMS on the repair of intracerebral Hemorrhage Injury by intrisinc neural stem cells（1）Brain Water Content and Neurological FunctionTo learn about how rTMS plays an important role in recovery from ICH,we assessed neurological function deficits.The single-blind method was employed for assessments at 0h,24 h,72 h,and 120 h after the surgery.rTMS treatment improved the neural function of ICH mice after one,two,and three treatments（24 h:ICH group,7.47±0.35 vs.ICH/rTMS group,9.00±0.45,p=0.012,F factor=232.8;72 h:ICH group,3.67±0.40 vs.ICH/rTMS group,6.67±0.53,p=0.0001,F factor=325.0;120 h:ICH group,12.07±0.51 vs.ICH/rTMS group,13.87±0.62,p=0.034,F factor=31.35）,and mice exhibited the most improvement on the third day after ICH.Determination of brain water content was used to measure the percent change in brain water content in various parts of the brain at 24 h,72 h,and 120 h after the surgery.The rTMS treatment relieved the swelling after one,two,and three treatments（ips-BG:24 h,ICH group,80.44±0.26%vs.ICH/rTMS group,79.08±0.42%,p=0.010,F factor=16.38;72 h:ICH group,81.87±0.22%vs.ICH/rTMS group,79.82±0.35%,p<0.0001,F factor=63.04;120 h:ICH group,80.96±0.20 vs.ICH/rTMS group,79.61±0.79%,p=0.0023,F factor=40.06;ipsCX:24 h,ICH group,80.39±0.25%vs.ICH/rTMS group,79.34±0.27%,p=0.0083,F factor=19.34;72 h:ICH group,80.48±0.23%vs.ICH/rTMS group,79.71±0.29%,p=0.044,F factor=22.73;120 h:ICH group,80.23±0.18%vs.ICH/rTMS group,79.43±0.31%,p=0.034,F factor=13.75）.In accordance with the neurological evaluation,mice had a better recover on the third day after the surgery.It is suggested that rTMS Reduced Brain Water Content and Neurological Function Deficit After ICH.（2）Proliferation of Intrinsic NSCsKi-67 immunofluorescence labeling was used to evaluate the proliferation of NSCs.The results demonstrated that the percentage in the ICH/rTMS group increased almost 46%compared with that in the ICH group after the 72 h rTMS treatment.（53.8±6.9 vs.78.8±7.6,p=0.041）,suggesting that rTMS enhanced the proliferation of intrinsic NSCs（3）Neuronal differentiattion of intrinsic NSCsThe prognosis of ICH is mainly dependent on the renewal of functional neurons,which reconstruct the serviceable connections among cells in the central nervous system.Glial scars,which consist of glial cells,are the regular pathological course that results in a poor outcome.Whether rTMS plays an important role in recovery from ICH primarily lies in the direction of terminal differentiation of intrinsic NSCs.Doublecortin is a microtubule-associated protein expressed by neuronal precursor cells and immature neurons in embryonic and adult cortical structures.Neuronal precursor cells begin to express DCX while actively dividing,and their neuronal daughter cells continue to express DCX for 2–3weeks as the cells mature into neurons.DCX immunofluorescence labeling was used in the experiment to determine whether rTMS treatment can promote neuronal repair after ICH.The results showed that a few DCX-positive cells emerged after ICH and increased significantly by 72%in the ICH/TMS group compared with those in the ICH group（176.4±21.8 vs.304.2±39.4,p=0.022）,suggesting that rTMS guided intrinsic NSCs to differentiate into neurons（reconstruct the neural function）after ICH（4）Aggregation of glial cells after ICHAggregation of glial cells,which is treated as a process of inflammatory response in the central nervous system,occurs after brain damage.GFAP is an intermediate filament protein that is expressed by numerous cell types of the central nervous system,especially glial cells.GFAP immunofluorescence labeling was used to detect inflammatory glial cells after ICH.The results showed that after the 72 h rTMS treatment,GFAP-positive cells showed a marked decrease of approximately 41%（267.6±26.5 vs.156.2±20.3,p=0.010）,suggesting that rTMS reduced the aggregation of glial cells after ICH3.Study on the biological behavior of neural stem cells regulated by rTMS in vitro（1）Proliferation of NSCs In vitroAfter rTMS treatment,the neurospheres were examined under a light microscope,which showed a notable increase of 83%in the diameter of neurospheres after rTMS exposure（67.6±18.3 vs.124.2±11.5,p=0.031）.SRY-box 2,also known as SOX2,is a transcription factor that is essential for maintaining self-renewal,or pluripotency of undifferentiated embryonic stem cells.Sox2 plays a critical role in the maintenance of embryonic and neural stem cells.Sox2 and Ki-67 immunofluorescence labeling was used in this experiment to examine the proliferation of NSCs in vitro.The result demonstrated a significant increase in the number of Sox2-positive cells（117%,283.0±69.6 vs.615.4±98.0,p=0.024）and in the percentage of Ki-67-positive cells per neurosphere compared with those in the control group.（58%,32.2±3.6 vs.51.0±5.6,p=0.022）,indicating that rTMS promoted the proliferation of NSCs in vitro（2）Differentiation of NSCs in vitroNeurons and glial cells are the main types of cells derived from NSCs and are the major cell types in the nervous central system.These cells are involved in the recovery from ICH,during which they play critical roles.In brief,neurons and glia decide the outcome of ICH.Under light microscopy,the rTMS group NSCs seldom exhibited adherence and had fewer branches compared with the control group NSCs.Based on this finding,NSCs were co-stained for their representative markers DCX and GFAP to examine the differentiation of NSCs in vitro.Confocal imaging indicated that the percentage of DCX in every neurosphere in the rTMS group increased significantly compared with that in the control group（360%,18.4±8.9 vs.84.6±7.2,p=0.0004）.In contrast,there was a remarkable reduction in GFAP（82%,14.4±2.7 vs.5.6±1.9,p=0.028）,indicating that rTMS modulated the differentiation of NSCs in vitro.4.Mechanisms of rTMS regulating intrinsic neural stem cell（1）Interaction network analysis of rTMS-related pathwaysUsing the GCBI online platform,microarray data analysis demonstrated transcriptome differences according to a select criterion（p<0.05,Q<0.05,fold change>1.5）.The rTMS give rise to remarkable alterations in genes and cell function（here,we showed the top 20altered cell functions and the top 10 path-net in Supplementary Tables S1 and S2,respectively）,and pathways affected by rTMS were generated.The outcomes demonstrated that regulation of transcription,DNA-dependent（ranked 1）,transcription,DNA-dependent（ranked 2）,cell cycle（ranked 3）and cell division（ranked 5）,phosphorylation（ranked 12）,phosphorylation protein（ranked 13）,nervous system development（ranked 16）and ion transport（ranked 20）were changed after rTMS treatment.Based on KEGG and the pathways affected by rTMS,Path-net analysis revealed significantly altered pathways and their relationships.The MAPK signaling pathway,calcium signaling pathway,TGF-beta signaling pathway,and axon guidance are significantly linked to neurogenesis.Importantly,calcium signaling pathway,TGF-beta signaling pathway,and axon guidance have been suggested to be closely related to neurogenesis via the MAPK signaling pathway.The genes involved in those significantly altered pathways（MAPK signaling pathway,calcium signaling pathway,TGF-beta signaling pathway,axon guidance）.It is suggested that MAPK signaling pathway,calcium signaling pathway,TGF-beta signaling pathway,axon guidance might be the potential mechniasms underlying those effects.（2）Phosphorylation levels of classic MAPK familyAccording to the bioinformatics analysis,we further investigated the classic MAPK family（Erk1/2,JNK/SAPK,p38）and their phosphorylation levels using western blotting analysis to initially explore possible mechanisms underlying the effects of rTMS on neurogenesis after ICH.The results showed that the expression levels of p-Erk1/2 and p-p38 increased with additional rTMS treatments.When the treatment was finished,the expression levels decreased in a timedependent manner,while there were no significant changes in the expression levels of p-JNK/SAPK,JNK/SAPK,Erk1/2,and p38.In addition,SCH772984,a specific inhibitor of Erk1/2,and SB203580,a specific inhibitor of p38,could block the phosphorylation-promoting effect of rTMS on Erk1/2 and p38.It is suggested that MAPK signaling pathway might be the major downstream and hub signaling pathway.（3）Intracellular Ca²⁺levelIn light of the specific relationship between physical treatment and the MAPK signaling pathway,combined with microarray data analysis,Ca²⁺was considered a possible initiating factor.To prove this,the intracellular Ca²⁺level was qualified by calcium imaging.The outcome indicated that the intracellular Ca²⁺level increased when rTMS treatment was applied and that the effect of rTMS could be blocked by a specific voltage-gated Ca²⁺channel inhibitor,nifedipine,suggesting Ca²⁺whIich is directly modulated by electromagnetic filed might be the intiator of the process.In summary,our results confirm that rTMS,a noninvasive,safe,and convenient tool with positive effects,is a potential therapy for experimental ICH by enhancing the neurogenesis of NSCs,guiding them to neuronal differentiation,and suppressing glial differentiation.The novel mechanism might be involved in neurogenesis,the inflammatory response,reprogramming glial cells,and axon guidance via the MAPK signaling pathway.