Study On The Effects Of Repetitive Transcranial Magnetic Stimulation (rTMS) Preconditioning On The Local Ischemic Reperfusion Injury Model Of SD Rats

Background and purpose: In 1990 Kitagawa found that transient ischemia of whole brain not only didn't lead to obvious injury on neurons but also conspicuously prevent from subsequent lethal ischemic damage.This phenomenon is called as ischemic tolerance(IT) and the first sublethal transient ischemia is named as ischemic preconditioning(IP) . The phenomenon of ischemic tolerance is of considerable interest because understanding its mechanism could lead to new treatments for preventing ischemic neuronal damage. Data from subsequent studies have revealed that other manipulations besides sublethal ischemia also induce ischemic tolerance, including hypo-oxygen or hyperbaric oxygen, hypothermia or hyperthermia, chemical drugs, cortical spreading depression (CSD) .Although several manipulations may induce tolerance, most are invasive or have the potential for causing direct neuron injury or initiating molecular processes that are also engaged following injury. In terms of potential clinical applications, it is considerably important to develop techniques for inducing ischemic tolerance without causing injury or dysfunction and ideally using noninvasive manipulations.At the basis of the principle of the electromagnetic induction, the transcranial magnetic stimulation (TMS) has been a kind of important electricalphysiological examine methods clinically. As a new noninvasive and good tolerance technique, the volunteers and the patients can accept TMS easily. Repetitive transcranial magnetic stimulation (rTMS) emerged in the last decade which is the development of single pulse TMS and can produce trains of magnetic stimulation with certain intensity and frequency,then affect the functional state of the local stimulus and remote sites. Owning to its specific biophysiology characteristic and the perfect merit of no pain, noninvasive, safe and good tolerance, the technique of rTMS has been applied gradually to therapeutic fields .Besides the ascertained improvement effects on major depression or other dyskinesia diseases, rTMS has been recently noticed to be worthy in improving ischemic vascular disorders including limb movement founctions , language capabilities, visual and spatial sensory, studying and memoring damage. The past studies of our laboratory have also showed that rTMS could affect the pathophysiological conditions of ischemic stroke and could protect the ischemic neurons.So we had consideration for applying the safe and useful rTMS technique to exploring the phenomenon of ischemic tolerance. Altough studies in Japanese have been noticed that rTMS induces ischemic against DND of hippocampal neurons following an otherwise lethal ischemic insult, the mechanism of rTMS wasn't be explored.Morever that study used gerbils as experimental model, which haven't Willis circles intracalvarium .In contrast, SD rats have intact Willis and similar structure with human' cerebral blood vessel.So the present study demonstrate the impact of rTMS pretreatment on the infarct volume and neurofunction at the acute stage of middle cerebral artery occlusion and reperfusion injury using SD rats as focal cerebral ischemic model and mimicking the stroke course in humans.Methods: SD rats were used as research model. At 48h after rTMS were applied to them, the rats were introduced to middle cerebral artery occlusion for 90min and then reperfusion; The control group was set with sham rTMS treatment; other experimental conditions were just the same as the rTMS group. At 24 h, 72 h after the reperfusion, the extent of modified neurological severity scores(NSS) were evaluated. At 72h after reperfusion,the rats were decapitated rapidly and infarct volumes were measured. Changes in these parameters and effects of rTMS preconditioning on these parameters were analyzed. Cerebral blood flow(CBF) effected by rTMS was also measured using Laser Dopplar Flowery(LDF) in present study.Results: The results showed that ischemic reperfusion injury produced distinct focal infarction and neurological deficits, which presented with the growing of neurofunction scores, and deteriorated along with the prolongation of reperfusion time. And rTMS preconditioning could reduce neurological scores as well as infarct volume, and improve neurofunction. Especially at 72 h after reperfusion, the neurological scores of rats receiving rTMS were statistically better than the control group. The change of CBF effected by rTMS was little and not obvious.Conclusions: These results suggested that rTMS preconditioning may provide important neuroprotective role in acute stage of cerebral ischemic reperfusion injury, and should be explored further for its corresponding mechanisms in order to find new assistant therapeutic strategy for ischemic stroke. On the other hand, as the animal sample in this study is limited, and some methods should be perfected further, the conclusion should be validated further.Innovation: It is the first time that we investigate the effects of rTMS preconditioning on the rats motor cortical excitability and neurofunction and the infarction in acute stage ofthe middle cerebral artery occlusion reperfusion injury. Background and purpose: Up to date,much ischemic tolerance phenomenon has been found, however the certain mechanism hasn't been fully understood.Some studies showed that transient ischemic preconditioning couldn't increase the energy metabolism and blood flow during the subsequent severe ischemia.Protein synthesis inhibitor CHX could surely inhibit the protection iuduced by ischemic pretreatment, which suggested that preconditioning may leading to serial gene transcription and translation by inducing or changing the course of transcription , and finally synthesize protective protein after 24h.Many important molecular and signal paths may involveled in the brain protective fouction induced by preconditioning.It has been noticed that early gene cFos is an sensitive index reflecting nerve cell fountional status which could present instant response to outside stimulation and play an important role in nerve cell defferentiation and nervous system plasticity. The expression of cFos has close relation with brain ischemia.Moreover the high expression of cFos emerges before the change of morph and biochemics of cell apoptosis, and the expression of cFos may be the premonition of apoptosis.Hot shock protein(HSP) is a kind of new proteins which generally reside in prokaryotic and eucaryotic cells and may be induced by stress such as hot, ischemia and hypoxygen, so named as stress protein.HSP may act as molecular chaperones during protein translocation and folding which participate in cytothesis through redoubling of degenerative protein. Of HSP70-KDa family , HSP70 has conservative structure and is the maximum produced after stress.Studies for focal ischemia have showed HSP70 induced in penumbra could promote protein renaturation after motabolism stress status retroconversing and act as endogenous protective fouction. Vascular Endothelial Growth Factor (VEGF) was firstly purificated from the culture fluid of stellate cell in cattle pituitary gland follicle by Ferrara et al which is also named as vascular permeability factor (VPF) .VEGF is a kind of specific mitogens in endothelial cell, which could promote endothelial cell proliferation through integrating with its receptors.Thus it could accelerate the neovascularization and enhance thevasopermeability.Methods: The rats were randomly assigned to the two groups according to different reperfusion time.The rats in experimental groups were given rTMS stimulation with different stimulus parameters for one time or five time.At 48h After the last rTMS were applied to them, the rats were introduced to middle cerebral artery occlusion for 90min and then reperfusion; The control group was set with sham rTMS treatment; other experimental conditions were just the same as the rTMS group. At 3h or 24 h after the reperfusion, the rats were decapitated to determine the protein expression level of cFos or HSP and VEGF respectively using immunohistochemistry staining and western blotting in order to evaluate the effects of rTMS preconditioning on molecular level.Results: The results suggested that different amount of expression of protein cFos , HSP70 and VEGF in ischemic area after ischemic reperfusion injury and microamount in sham operation groups. rTMS preconditioning with different stimulus parameters could produce different effects on the expression of protein above-mentioned. Compared with the intensity, we found that the stimulus frequency could arise much apparent effects, and high frequency (20Hz) especially in contrast to the low frequency (5Hz). High frequency could decrease the expression of cFos positive neurons and opposite in terms of HSP70 and VEGF positive ones. When the frequency is fixed, the effects of high stimulus intensity (2MT) would be more distinct than the low intensity (1.2MT). These impacts would be more evident with rTMS stimulition time.Conclusions: Our experiments primarily suggested that the rTMS preconditioning with high frequency (20Hz) could provide neuroprotection in the subsequent acute stage of cerebral ischemia reperfusion injury; and when the rTMS frequency is limited, we found that the higher the stimulus intensity, the distinct its effects.Morever the effect of rTMS on protein expression probably has time cumulative effect. And the corresponding mechanisms of inducing ischemic torelance by rTMS may involve with protein expression modulating. These conclusions may provide some basic evidence on the influence of rTMS preconditioning on the subsequent acute stage of ischemic stroke.Innovation: It is the first time that we explore the effects of rTMS preconditioning on protein expression after the rats middle cerebral artery occlusion reperfusion injury, and investingate its corresponding mechanisms. We found that the rTMS pretreatment could provide neuroprotection at the early stage of the cerebral ischemia reperfusion injury; the underlying mechanisms may involve the protein expression modulating.