The Mechanism And Rehabilitation Effect Of TDCS On Motor Function Deficit After Ischemic Stroke

BackgroundStroke,resulting from a lack of blood flow to the brain,carries a high rate of mortality,incidence and morbidity and is one of the most common diseases of the central nervous system.As the social aging intensifies,stroke has become the leading cause of death in people above the age of 60 worldwide.Among stroke-related deaths,ischemic events account for up to 80%of the stroke events.It is reported that more than two-thirds of the stroke survivors develop post-stroke sequelae including motor functions deficit,learning memory impairment and aphasia,which has severely reduced patients'quality of life and put great economic burden on society and family.Currently,intravenous recombinant tissue plasminogen activator and endovascular intervention have showed good curative effects in ischemic stroke treatment.However,not all patients can receive above therapy since it must be administered within 4.5 hs after ischemic stroke onset.Other therapeutic options,such as brain hypothermia protection,cerebral edema dehydration treatment,neurotrophic factor therapy and microcirculation improvement,are symptomatic support therapy,which could reduce secondary injury of brain,but has limited curative effect on formed brain injury and neural function deficit.Stem cell transplantation has been suggested as an effective therapy for tissue repairation.However,it presents several outstanding risks,including surgical complications,immunological rejection and the potential capacity of oncogenicity,which restrains its clinical transformation and application.It was found that the proliferation of endogenous neural stem cells(NSCs)was activated after brain injury,but the proliferation level was limited,which could not exert the effects of nerve tissue regeneration and functional reconstruction.Therefore,how to promote those endogenous NSCs to proliferation,migration and differentiation after brain injury has become a focus of nerve regeneration and brain damage repairation.Transcranial direct current stimulation(tDCS)is a noninvasive neural control technology by using low intensity direct current(?2 mA)to regulate cerebral cortex neuron activity.This techniche has some unique advantages such as easy-operation,higher safety and noninvasive.Recently,it has been reported that tDCS could promote motor function deficit rehabilitation after stroke,but the mechanism is unclear,which restrains its clinical transformation.In this study,we investigated the effects of tDCS on motor function deficit recovery in rats after ischemic stroke and explored the underlying mechanism from the aspect of neurogenesis and neuroprotection.ObjectiveThis study aims to confirm the treatment efficacy of tDCS on motor function recovery in rat after ischemic stroke and clarify the underlying mechanism.Part ?:A Biological Safety Study of tDCS in RatMaterials and Methods1.Animals and groups:24 male Sprague-Dawley rats(260-280 g)were randomly divided into two groups:control group(Control,n=12)and Cathodal tDCS group(tDCS,n=12).2.Stimulating electrode implantation and tDCS administration:2.1 Stimulating electrode implantation was carried out in rats under 1%pentobarbital sodium anesthesia(45 mg/kg i.p.).After removal of the scalp and underlying tissues,a homemade cup-shaped stimulating electrode with contact area of 3.5 mm~2 was mounted on the intact skull at the coordinates bregma AP+2.0 mm,ML+2.0 mm using glass ionomer dental cement.2.2 Prior to the tDCS,the stimulating electrode was filled with saline solution(0.9%NaCl)to reduce the skull impedance,and a counter electrode made from a large conventional rubber plate electrode(7 cm~2)was placed onto the thorax of the unrestrained animal by a corset.Animals in tDCS group were received 5 consecutive days of cathodal tDCS(current intensity of 500?A,15 min,once per day)from POD 2,then followed by a2-day tDCS-free interval and then 5 days of tDCS again.Animals in Control group were connected to the stimulator for 15 min as tDCS group but without current output.Notably,animals were maintained in a waking state during tDCS.3.Methods:Computational 3D rat model was adopted to calculate the current density distributions in brain during tDCS administration.Essential brain functions were evaluated by open field test,rotarod test and morris water maze.Commonly used indexes for evaluating animals'health such as hematology,serum biochemical markers and the morphology in vital organs were examined.Additionally,to estimate the neurotoxicity of tDCS,the neurotransmitter levels and cerebral temperature were investigated.Results1.Currents distributions in computational rat model:The current density varied from different rat tissues,and the current density inside the brain was less than 20 A/m~2 and skull was 40-60 A/m~2 in computational model.2.The results of behavioral analysis:Compared with Control group,the distance traveled in open field and retention time on the ratarod showed no difference in tDCS group at each testing timepoint.The results of morris water maze test showed that the escape latency to the hidden platform and duration in the target quadrant did not differ between Control and tDCS group.The results indicated that tDCS had no significant effect on motor function and learning and memory ability of healthy rats under this experimental condition.3.The results of morphology,hematology and serum biochemical markers:HE staining results showed that the morphology of main organs(i.e.,heart,lung,kidney,liver and spleen)did not obviously change after tDCS administration compared with the Control group.Hematology analysis showed that no significant differences were found between the tDCS and Control groups.The results of serum biochemical markers for liver and kidney showed no difference between Control and tDCS groups.Those above indicated that tDCS had no significant effect on morphology of main organs in healthy rats,as well as the function of hematopoiesis,liver and kidney under this experimental condition.4.The results of brain morphology,neurotransmitters and brain temperature:HE staining and Nissl staining results revealed that the morphology of nerve cells and the shape and density of intraneural Nissl bodies in cortex and hippocampus did not change compared with Control group.The level of neurotransmitters in hippocampus and cortex was assessed using LC-MS.These indexes showed no difference between Control and tDCS group.Compared with Control group,the cerebral temperatures exhibited no obvious change during tDCS.These results indicated that tDCS did not induce neurotoxicity in healthy rats under this experimental condition.Part ?:tDCS Accelerates Rehabilitation of Motor Function Deficit in rats afterIschemic StrokeMaterials and Methods1.Stimulating electrode implantation:175 Male Sprague-Dawley rats were anesthetized under 1%pentobarbital sodium(45 mg/kg i.p.).After removal of the scalp and underlying tissues,a homemade cup-shaped stimulating electrode with contact area of 3.5 mm~2 was mounted on the intact skull at the coordinates bregma AP+2.0 mm,ML+2.0 mm using glass ionomer dental cement.2.Ischemic stroke model establishment and groups:Animals with implanted electrode were anesthetized,and then underwent a transient focal ischemia surgery by temporary occlusion of the right middle cerebral artery(MCAO)with a silicone-coated nylon suture.Following 2 h of MCAO,reperfusion was initiated by withdrawing the filament.62 animals with moderately neurologic deficits were selected by zea longa evaluation from 117 rats underwent MCAO operation.And then animals were randomly divided into two experimental groups:tDCS group(MCAO+tDCS,n=32)and sham tDCS group(MCAO+Sham,n=30).The other 58 animals that underwent a sham MCAO operation were randomly divided into two control groups:Control+tDCS(n=29)and Control+Sham(n=29).3.tDCS administration:Animals in two tDCS groups were received tDCS administration as mentioned in part I from 2-day post MCAO operation(POD 2).4.Methods:Open field test,rotarod test and foot print analysis were adopted to evaluate the motor functions deficit rehabilitation of MCAO animals during and after tDCS administration.The baseline of motor functions was recorded before MCAO surgery,and then motor functions were evaluated every two days.Results1.Effects of tDCS on locomotor activity recovery in rats after ischemic stroke:Compared with two Control groups,the total distance traveled in open field was significantly decreased in MCAO groups(P<0.05).Compared with MCAO+Sham group,this index was significantly increased in MCAO+tDCS group(P<0.05).There was no significant difference in total distance between Control+Sham and Control+tDCS group.2.Effects of tDCS on athletic endurance recovery in rats after ischemic stroke:Compared with two Control groups,the retention time on rotarod was significantly decreased in MCAO groups(P<0.01).After 5 days tDCS administration,this index was significantly increased in MCAO+tDCS group,compared with MCAO+Sham group(P<0.01).There was no significant difference in retention time between Control+Sham and Control+tDCS group.3.Effects of tDCS on limping gait recovery in rats after ischemic stroke:Compared with two Control groups,the contact area and stride of left forepaw significantly decreased in MCAO groups(P<0.01).After tDCS administration,those indexes were significantly improved in MCAO+tDCS group,compared with MCAO+Sham group(P<0.05).There was no significant difference in footprint analysis between Control+Sham and Control+tDCS group.Part ?:The Effect of tDCS on Neurogenesis and Relative Signal Pathway in Rats after Ischemic StrokeMaterials and Methods1.The animals used for behavioral experiments in part II were adopted in part III.2.Methods:The ischemic damage area in brain was examined by 2,3,5-triphenyltetrazolium chloride(TTC)staining at POD 3.The proliferation and differentiation of NSCs were detected by immunofluorescence staining confocal(IFC)at POD 3 and POD7.Western blot(WB)and transmission electron microscope(TEM)were adopted to measure the number of myelinated nerve fibers and the expression level of myelin related protein at POD 14.The distribution and protein expression of Notch1 signaling molecules were examined by IFC and WB.Results1.Effects of tDCS on NSCs proliferation and migration in subependymal region(SVZ)in MCAO model:TTC staining and IFC results showed that much more Nestin~+(a recognized marker for NSCs)cells appeared in ischemic striatum of MCAO+tDCS group at POD 3,compared with MCAO+Sham group(P<0.01).No Nestin~+cells were observed in striatum of two Control groups.The results of IFC showed that the percentage of proliferating NSCs in SVZ significantly increased after MCAO surgery at POD 3 and POD 7,compared with two Control groups(P<0.01),and tDCS administration significantly promoted this proliferating index at POD 3(P<0.01,vs MCAO+Sham group).The total number of Nestin~+cells in SVZ of MCAO+tDCS group was similar to that of two Control groups,but much less than that of MCAO+Sham(P<0.01).Besides,the number of NSCs in striatum of MCAO+tDCS obviously increased compared with that in MCAO+Sham group(P<0.01).No obvious differences were found in the number of Nestin~+cells and proliferating rate in both SVZ and striatum between Control+Sham and Control+tDCS group.2.Effects of tDCS on differentiation of NSCs in ischemic striatum in MCAO model:The results from IFC,WB and TEM showed that the NSCs in ischemic striatum subsequently differentiated to neuron and oligodendrocyte after MCAO surgery.Compared with MCAO+Sham group,the number of immature neurons,mature neurons and oligodendrocytes in ischemic striatum obviously increased in MCAO+tDCS group(P<0.01).In Control+Sham and Control+tDCS group,NSCs and immature neurons were rarely observed in the striatum.3.Effects of tDCS on the activation of Notch1 signal pathway in ischemic striatum:Compared with the Control+Sham group,the expression of DLL1,Notch1(NICD)and the target gene Hes1 was significantly increased in MCAO+Sham group(P<0.01).Compared with MCAO+Sham group,the expression of DLL1,Jagged1,NICD and Hes1was significantly decreased,and the expression of Notch1 and NUMB was significantly increased in MCAO+tDCS group(P<0.01),which indicated that tDCS inhibited the activation of Notch1 signaling pathway in the ischemic striatum.IFC results showed that the percentage of Nestin~+/Hes1~+double stained cells was significantly decreased in MCAO+tDCS group,compared with MCAO+Sham group(P<0.01),which suggested that tDCS mainly inhibited Notch1 pathway in NSCs.No Nestin~+/Hes1~+cells were observed in Control+Sham and Control+tDCS group.Part ?:The Neuroprotective Effect of tDCS in rats after ischemic stroke Materials and Methods1.Ischemic stroke model establishment and groups:Stimulating electrode implantation and MCAO operation were performd as Part II.55 animals met the experimental design were randomly divided into two experimental groups:MCAO+tDCS(n=27)and MCAO+Sham(n=28).Another 54 animals with implanted electrode underwent sham MCAO operation and were randomly divided into two control groups:Control+tDCS(n=27)and Control+Sham(n=27).2.tDCS administration:Animals in two tDCS groups were received tDCS administration as mentioned in part II from POD 2.3.Methods:Boby weight and modified neurological severity score(mNSS)were adopted to evaluate the effects of tDCS on general health and neurologic deficit improvement in rats after ischemic stroke.The ischemic damage area and edema score of brain was examined by TTC staining;The morphology of the cortex was detected by HE staining and Nissl staining;The level of neuron-specific enolase(NSE),interleukin-6(IL-6),interleukin-1b(IL-1b),interleukin-10(IL-10)and tumor necrosis factor-a(TNF-a)were detected by enzyme-linked immunosorbent assay(ELISA);The activaton of astrocyte and microglia were examined by IFC and WB;The apoptosis level in CIP(Cerebral ischemic penumbra)was determined by WB and terminal dexynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)staining at POD 3.Results1.Effects of tDCS on body weight and neurological deficit recovery in ratsafter ischemic stroke:Compared with two Control groups,the body weight of MCAO rats significantly decreased(P<0.05).After 5-day tDCS administration,the body weight of MCAO rats obviously increased,compared with MCAO+Sham group(P<0.05),and this difference maintained until POD 14.mNSS result showed that the above index significantly increased after MCAO operation,compared with two Control groups.After tDCS administration,the above index significantly decreased in MCAO+tDCS group,compared with that of MCAO+Sham group(P<0.05).At POD 14,mNSS in MCAO+tDCS group was still lower than that in MCAO+Sham group.No differences were found in body weight and mNSS between Control+Sham and Control+tDCS group.2.Effects of tDCS on brain damage in rats after ischemic stroke:TTC staining results showed that compared with MCAO+Sham group,the brain infarction area and cerebral edema score significantly decreased in MCAO+tDCS group(P<0.01)at POD 3.ELISA results showed that compared with MCAO+Sham group,the level of NSE in serum significantly decreased(P<0.05)at POD 3.No differences in above index were found between Control+Sham and Control+tDCS group.3.Effects of tDCS on cortex morphology in rats after ischemic stroke:No differences in cortex morphology were found between two Control groups.Compared with two Control groups,the morphologyof rat cortex in MCAO group was severely damaged by ischemic stroke.It was found that the number of neuron and Nissl body decreased obviously,and the arrangement of cells were disordered accompanying with degeneration and necrosis at the early stage of ischemic stroke(POD 3)(P<0.01).Compared with MCAO+Sham group,the cortex morphology was significantly improved,in addition,the number of Nissl body increased distinctly in MCAO+tDCS group(P<0.01),which indicated that tDCS could exert protective effect on rat brain morphology after ischemic stroke at the early stage.4.Effects of tDCS on glial cell activation in CIP of rats after ischemic stroke:IFC and WB results showed that the number of IBA-1~+(Ionized calcium binding adaptor molecule)and GFAP~+(glial fibrillary acidic protein)cells and the level of IBA-1 and GFAP protein significantly increased in CIP after MCAO,compared with two Control groups(P<0.05);Compared with MCAO+Sham group,the number of IBA-1~+and GFAP~+cells significantly decreased in CIP of MCAO+tDCS group(P<0.01)at POD 3.No difference was found in morphology and number of IBA-1~+and GFAP~+cells as well as their protein levels between Control+Sham and Control+tDCS group.5.Effects of tDCS on the level of inflammatory factors in CIP of rats after ischemic stroke:ELISA results showed that compared with two Control groups,the level of inflammatory factors(i.e.IL-6,IL-1b,TNF-aand IL-10)significantly increased in CIP after MCAO operation(P<0.01).Compared with MCAO+Sham group,the level of proinflammatory factors(i.e.IL-6,IL-1band TNF-a)significantly decreased and anti-inflammatory factors(IL-10)significantly increased in CIP of MCAO+tDCS group(P<0.05)at POD 3.No differences were found in the level of inflammatory factors between Control+Sham and Control+tDCS group.6.Effects of tDCS on cell apoptosis in CIP of rats after ischemic stroke:TUNEL staining and WB results showed that compared with Control+Sham group,the percentage of apoptotic cells and the level of apoptosis related protein Caspase 3 significantly increased in CIP of MCAO+Sham group(P<0.05),and the above index significantly decreased in MCAO+tDCS group,compared with MCAO+Sham group(P<0.01)at POD 3.Conclusions1.tDCS had no effects on vital organs'(i.e.,brain,heart,lung,kidney,liver and spleen)morphology and function under this experimental condition.The data suggested that tDCS performed in the present study(current intensity 500?A,15 min)was safe for rodents.2.tDCS could accelerate motor function deficit recovery in rats after ischemic stroke under this experimental condition.3.tDCS could promote neurogenesis by modulating the proliferation,migration and differentiation of endogenous NSCs and the inhibition of Notch1 pathway activation by tDCS might be involved in the processes of NSCs differentiation after ischemic stroke.4.tDCS could significantly decrease the damage of ischemic stroke in the early stage and promote neurological deficit recovery by inhibiting nerve inflammation and apoptsis.5.Neurogenesis and neuroprotection might be involved in the mechanism of tDCS promoted motor function deficit recovery in rats after ischemic stroke.