Protective Effect Of Electric Stimulation On Cerebral Ischemia Injury In Rats

Background and objective：Stroke is a medical emergency and can cause permanent neurological damage,complications, or even death. In china, there are2million stroke patients growing and anestimated1.5million stroke patients die every year, of which60%～70%are ischemicstroke. At present，reperfusion is the best way to protect the brain against ischemic braindamage. But the narrow therapeutic window is the bottleneck of the thrombolytic therapy.While tissue-type plasminogen activator (tPA) remains the only FDA-approved treatmentfor ischemic stroke, clinical use of tPA has been constrained to roughly3%-4%of allpatients. So the research on the treatment of patients who suffer from permanent middlecerebral artery occlusion is very important.Electrical stimulation has been used in the clinical treatment for various disease of central nervous system, including dystonia, essentialtremor, epilepsy, particularly inParkinson’s disease. For cerebral ischemia, electric stimulation to the affected cortex,spinal cord, epidural space of the motor cortex has been shown to exert functionalrecovery. In these studies, electric stimulation was thought to participate in the increasingATP content, enhancing of synaptic plasticity, and facilitating the expression ofneurotrophic factors. Thus it can be seen that electric stimulation has been widely used inclinical disease. But does the electric stimulation technology also have protective effect onpermanent middle cerebral artery occlusion? And the range of frequencies, therapeutictime window and the mechanisms of therapeutic effect were unknown. Therefore, thisstudy tries to treat the stroke with the electric stimulation in different frequency and time,in order to study the detailed mechanisms of electric stimulation and develop new ideasfor clinical treatment.Methods：In this study, our therapeutic strategy was tested in the rat model of permanent middlecerebral artery occlusion (pMCAO). Electric stimulation was performed at differentfrequency and different model. The primary aim of this study was to investigate whetherthere exist a potential protective effect of electric stimulation in the acute stage of pMCAOrat model. The second aim was to determine optimum parameters by hemispheric infractratio, behavioral test and TUNEL staining. And then the underlying neuroprotectivemechanisms were elucidated by western-blot and Evans blue staining.Results:Part Ⅰ: Neuroprotective effect of DBS on cerebral ischemia injury in rats(1) We used the bipolar deep brain stimulation. Rats were randomly divided intotwo groups: control group and experiment group. Then the experiment group were furtherdivided into four sub-groups:2Hz group,20Hz group,100Hz group and100/2Hz group.Stroke animals were conducted to behavioral test at5/24hours after pMCAO. Comparedwith other stimulation groups, the function recovery is the best in2Hz stimulation group.(2) The hemispheric infarct ratio was detected at24hours after pMCAO. The study found that the2Hz(40.36±6.27%) group exhibited significantly decreased in thehemispheric infract ratio compared with other groups[control group（62.51±5.84%）,20Hz（56.42±4.17%)、100Hz(56.79±6.37%)、100/2Hz（50.61±3.67%)](3) Basing on the hemispheric infarct ratio and behavioral test, we assumed thatelectrical stimulation at2Hz was the optimal therapeutic parameter for exertingneuroprotection in ischemic stroke which would be used in subsequent experiment.Western-blot analysis of BDNF/VEGF were carried out to see whether it is up or downregulated by electric stimulation. We found that the expressions of VEGF weresignificantly down-regulated in the electrical stimulation group (51.4%±2.6%) thancontrol group (88.9%±7%).However, the expressions of BDNF were not significantlydifferent between two groups (control:59.1%±7.9%stimulation:57.6%±4.7%)Part Ⅱ: Neuroprotective effect ofcortical electric stimulation on cerebralischemia injury in rats(1) We used the unipolar cortical electric stimulation(CS). Rats were randomlydivided into four groups: sham group, vehicle group, anode group and cathode group. Theanode group and cathode group [12(10～14)and11(9～13)] significantly promote thebehavioral test score compared with vehicle group [9(7～13)]. The therapeutic effects ofanode group and cathode group did not differ, although anode group displayed a trend ofmore potential behavioral benefits than cathode group.(2) The hemispheric infarct ratio was detected at24hours after pMCAO. The studyfound that the anode group and cathode group (39.83%±3.93%and49.22%±5.94%)significantly decreased the hemispheric infarct ratio compared with vehicle(62.51%±5.84%) group. The anode group exhibited significantly decreased in thehemispheric infract ratio compared with cathode group.(3) Western-blot analysis of BDNF/VEGF were carried out to see whether it is up ordown regulated by electric stimulation. The expressions of BDNF/VEGF were markedlyup-regulated in vehicle group in comparison with sham group (P<0.05). The results ofwestern-blot showed that the expressions of VEGF were significantly down-regulated inthe anode group and cathode group (23.46%±1.50%and29.46%±1.46%) than vehicle group (42.7%±3.06%). The expressions of VEGF were significantly down-regulated inanode group in comparison with cathode group. However, the expressions of BDNF werenot significantly different between CS groups and vehicle group.(4) To measure the effect of CS on blood-brain barrier integrity after pMCAO, wedetected tissue contents of Evans blue dye. The contents of Evans blue dye weresignificantly increased in vehicle group in comparison with sham group (P<0.01). In CSgroup, the contents of Evans blue were markedly decreased. The anode group exhibitedsignificantly decreased the contents of Evans blue dye compared with cathode group.(5) TUNEL staining was used to assess the typical DNA laddering pattern of neurons.We observed a significant increase in the number of TUNEL-positive cells in the ischemicarea at24h in pMCAO rats compared with shams. TUNEL staining revealed significantlyfew TUNEL-positive cells in the ischemic cortex of rats receiving CS compared withthose of vehicle rats. The anode group exhibited significantly decreased the number ofTUNEL-labeled apoptotic cells compared with the cathode group.Conclusion:(1) Electric stimulation exerted greater neuroprotective effects in the acute stage ofpMCAO stroke rats.2Hz was the optimal therapeutic parameter in deep brain stimulation.And the anodal group exerted greater neuroprotective effects than the cathodal group incortical electric stimulation.(2) The expressions of BDNF/VEGF were markedly up-regulated in vehicle groupin comparison with sham group. Inhibition of VEGF is thought to be neuroprotective inthe acute stage of injury. The down-regulation of VEGF expression is positively correlatedwith neuroprotective effects. And the neuroprotective effects have no significantcorrelation with the expression of BDNF.(3) Reduction the expression of VEGF in acute stage of pMCAO model couldpreserve the functional and structural integrity of the Blood Brain Barrier (BBB). Thismay be one of the mechanisms of neuroprotective effects.