| Objective: The statistics found that every 17 seconds one person will suffer from the cerebrovascular disease,the biggest health problem of the national fitness.At the mean time,it causes a huge burden to the national economy,of which ischemic cerebrovascular weigh a lot.At present,thrombolytic therapy is still limited by the nowadays technic,therefore,protecting-neural on time and effectively is a very important part in the clinical treatment.Currently,secondary brain injury is known as the main pathological mechanism,-including inflammation,oxidative stress,cell apoptosis etc.The inflammation plays an important role in secondary brain injury.The infiltration of inflammatory cells after cerebral ischemia,release big amount of inflammatory factor,causes neuron damage,resulting in nerve function defect.Microglias are recognized that immune cells in the central nervous system,which can gobble up the cell debris and participate in a variety of pathological reaction.The clodronate liposome is currently recognized as macrophage scavenger.After clodronate liposomes consumed by macrophages,they can affect cell energy metabolism leading cell to death.Microglia functions as macrophages in the central nervous system,as thus we speculated that clodronate liposomes injection by cortex methods can inhibit microglial cells,and reduce its quantity.When the central nervous system is damaged,the microglia can be activated and release inflammatory factors etc.,which will participate in the inflammatory reactions.By reducing the number of the microglia,inhibiting its function,the function of the microglia in inflammatory reactions can be further inwestigated.The main goal of this experiment is to study whether the cortexclodronate liposomes injection can be an effective method of inhibiting microglial cells or not;at the same time,how the microglia plays a role in inflammatory response after cerebral infarction will be studied,which is assumed to find new targets for clinical treatment.Methods: Male C57BL/6 MICE(age 8 to 10 weeks,weight 25 to 30 g)were subjected to MCAO,as described previously.Clodronate liposomes or the vehicle were injected to the right cerebral cortex at day 1 before and after stroke.In the first set of studies: part one,the observation of the microglia after clodronate liposomes is injected in the cerebral cortex will be studied.Male C57BL/6 mice were randomly assigned into 3 groups: Clodronate ?PBS and NS group.And corresponding chemicals will be injected.The number of the microglia will be recorded at day 1 after the treatment.Part two,Male C57BL/6 mice were randomly assigned to 3 groups: Clodronate?MCAO and Sham groups.Clodronate liposomes or the vehicle were injected to the right cerebral cortex at day 1 before and after stroke.The number of the microglia will be recorded at day 1 and 3 after stroke.In the second set of studies: observe neurologic after the clodronate lipopsomes injected to the cerebral cortex.Male C57BL/6 mice were randomly assigned to 3 groups: Clodronate ? MCAO and Sham groups.Clodronate liposomes or the vehicle were injected to the right cerebral cortex at 1 day before and after stroke.Functional test was examined at day 3,7,14 and 28 after stroke.In the third set of studies: investigate the influence of clodronate liposomes-inhibited microglia on acute inflammatory reaction.Male C57BL/6mice were randomly assigned to 3 groups: Clodronate ? MCAO and Sham groups.Clodronate liposomes or the vehicle were injected to the right cerebral cortex at day 1 before and after stroke.Infarct wolume and the percentage water content of brain will be examined at day 1 and 3 after stroke.Besides,the mice were used for Western blot to analyze inflammatory factors.Results:1 Injected clodronate liposomes in the cortex reduces the number of the microglia,and weakens the activity of the microglia.According to the results of confocal,the Iba1+ cells in the right cerebral hemisphere was significantly reduced in Clodronate group vs.PBS and NS group after day 1,the clodronate liposomes injected(P <0.05).The depletion rate was 31.6%.At the meanwhile,the fluorescence intensity of Iba1 had decreased.The number of Iba1+ cells had no significant difference in PBS group vs.NS group(P >0.05)(Fig.1,Fig.2).The number of NeuN+ cells was not changed between Clodronate and MCAO group,while the number of Iba1+ cells was reduced obviously(P >0.05)(Fig.3).The number of Iba1+ cells was reduced in Clodronate group,and the fluorescence intensity had decreased,but beyond all that,the micromorphological features of microglias had changed.The Iba1+ cells in the right cerebral hemisphere was significantly reduced in Clodronate group vs.MCAO and Sham group at day 1 and 3 after stroke(P <0.05)(Fig.4,Fig.5).These changes were not discovered in the contralateral cortex(Fig.6,Fig.7).The TLR4+ cells in the right cerebral hemisphere was significantly reduced in Clodronate group vs.MCAO and Sham group at day 1 and 3 after stroke(P<0.05)(Fig.8,Fig.9).The CD206+ cells in the right cerebral hemisphere was significantly reduced in Clodronate group vs.MCAO and Sham group at day 1and 3 after stroke(P <0.05)(Fig.10,Fig.11).And we did not find the similar changes in the contralateral cortexs(P >0.05).2 Injected clodronate liposomes in the cortex reduces the number of microglias,but does not affect the number of neutrophilsFCM showed the Iba1+ cells in the right cerebral hemisphere was significantly reduced in Clodronate group vs.MCAO group at day 1 and 3after stroke(P <0.05)(Fig.12,Fig.13).And the Ly-6G+ cells in the left cerebral hemisphere had no significant difference in Clodronate group vs.MCAO and Sham group at day 1 and 3 after stroke(P >0.05)(Fig.14,Fig.15).3 Clodronate liposomes treatment significantly inproves functional recoveryNeurological deficit was examined and scored by the Rotarod test evaluation weekly.The mice in Sham group did not show obvious neurologic deficits.In the ischemic mice,treatment with clodronate liposomes starting day 3 after stroke showed significant functional improvent from day 7 to day28 after stroke onset compared with vehicle-treated mice(P <0.05)(Fig.16).The therapeutic efficacy peaked at day 7 to day 14.4 Injected clodronate liposomes in the cortex reduces the infarct volumeThe percentage hemisphere lesion volume was significantly reduced in Clodronate group vs.MCAO group at day 1 and 3 after stroke(P <0.05)(Fig.17,Table.1).The decrease of infarct volume was more obviously at day 3after stroke.5 Injected clodronate liposomes in the cortex has no effect on cerebral edemaThe experimental data showed there was no obvious difference in the percentage of brain water content between the two groups(P >0.05).The water content of mice brain in Clodronate group was lower,but there was no pronounced difference(Table.2).6 Injected clodronate liposomes in the cortex reduces the expression of TLR4?TNF-??MyD88?IL-1?,and down-regulates inflammatory responsesAccording to the results of western blot,the protein levels of inflammatory factor was significantly reduced among the group of Clodronate,MCAO and Sham at day 1 and 3 after stroke in ipsilateral cortex(P <0.05)(Fig.18,Fig.19).There was no significantly differences in protein levels of inflammatory factor in the contralateral cortex.The reduced of the protein levels of inflammatory factor led to the down-regulation of inflammatory response.Conclusions: Injected clodronate liposomes in the cortex is an effective method to inhibit the microglia,not only it can reduce the number of the microglia and weaken the activity of the microglia,but also,the clodronate liposomes have no negative effect on the neuron.Inhibited the microglia can reduce the protein levels of inflammatory factor,reduce the infarct volume,appropriately alleviate cerebral edema,and eventually improve nerve function.This is a novel way to protect neuron.Injected clodronate liposomes in the cortex can reduce the expression of IL-1?,TNF-?,MyD88 and TLR4,and finally reduce the inflammatory response.The mechanism might be associated with TLR4 signaling pathway. |
PDF Full Text Request