| ObjectiveBrain injury has been proposed as the major cause of the poor outcomes associated with intracerebral hemorrhage(ICH).A large number of studies suggest that secondary brain injury(SBI)is one of the main pathological factors of poor prognosis of ICH.Emerging evidence indicates that the nuclear receptor,peroxisome proliferator-activated receptor ?/?(PPAR?/?),plays a crucial role in the pathological process of central nervous impairment.The present study was undertaken to evaluate the protective effects of PPAR-?/? activation using a selective PPAR-?/? agonist,GW0742,against SBI after ICH in a mouse model.Methods1.Induction and evaluation of ICH model:healthy male C57BL/6 mice were randomly divided-into sham-operated group(Sham group),control group(Vehicle group),GW0742 group.The ICH model was generated by injecting the bacterial collagenase ? into the right caudate putamen of mice.And the Bederson score was used to test and confirmed the model.The agonist GW0742 or the same volume of saline was injected intraperitoneally into mice.The neurological score of each group was tested with the Rotarod test,Forelimb placement test and corner test to evaluate the protective effect of activating PPAR ?/? on SBI induced neurological impairment after ICH,and the subsequent research time point was determined according to the results.2.The mice were executed after operation,and the wet and dry weight method was used to examine the changes of cerebral edema in each group.The extravasation of Evans Blue and FITC-dextran was used to detect the permeability change of BBB.The change of hematoma volume of ICH mice was calculated using coronary slices of brain tissue method.3.The detection of neuron damage:Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)and neuronal nuclei protein(NeuN)double fluorescent label staining was used to detect the apoptosis of neuron in perihematomal tissue,Nissl staining was used to assess the survival of neurons in perihematomal tissue.4.The expression level of PPAR?/? was detected by using Immunohistochemical staining and Western Blot.And also,the cell specificity of PPAR?/? in mice brain tissue was examined with double fluorescence double label staining.5.The expression level of inflammation-related protein interleukin-1(IL-1),tumor necrosis factor-?(TNF-?)and apoptosis-related protein B-cell lymphoma-2(Bcl-2),Bcl-2-related X(Bax)in perihematomal tissue after ICH was tested with Western Blot(WB).6.The expression of inflammation-related protein IL-1,TNF-? and apoptosis-related protein Bcl-2,Bax in perihematomal tissue was examined with Immunohistochemical staining.7.The expression level of microglia maker protein ionized calcium-binding adapter molecular-1(Ibal)and astrocyte maker protein glial fibrillary acidic protein(GFAP)in perihematomal tissue was examined with Western Blot to evaluate the microgliosis and astrogliosis.Results1.The mice showed paralysis of the left limb,with spontaneous left turn movement,and slow movement after ICH.The behavioral score of ICH mice on days 1 and 3 showed that the neuro-dysfunction of ICH mice was more severe than those in the Sham group.The neurological function score of ICH mice on day 1 was improved by using GW0742 intervention,but there was no statistical difference in the results compared to the Vehicle group(P>0.05).GW0742 improved the neurological deficit scores of ICH mice on 3d with significant statistical differences(P<0.05).Therefore,we selected the day 3 as the point in follow-up study.2.The BBB permeability and brain water increased significantly after ICH.Treatment with GW0742 significantly reduced the extravasation of EB and FITC-dextran,and alleviated brain water content(P<0.05).GW0742 decreased the volume of hematoma in the brain of ICH mice,but there was no statistical difference in results compared to the Vehicle group(P>0.05).3.TUNEL and NeuN double fluorescent label staining showed a significant increase in the number of apoptosis neurons in the perihematomal tissue in mice after ICH,and the GW0742 intervention significantly reduced the number of apoptotic neurons(P<0.05).Nissl staining showed a significant decrease in the number of surviving neurons in the perihematomal tissue after ICH,and together with an obvious reduction in the number of Nissl bodies.Compared to the Vehicle group,GW0742 significantly increased the number of surviving neurons and Nissl bodies in the perihematomal tissue(P<0.05).4.The results of immohistochemical staining and Western Blot showed that the expression level of PPAR?/? in the perihematomal tissue after ICH increased significantly,mainly located in cytoplasm,and also in the nucleus.GW0742 treatment increased the expression level of PPAR?/? in the brain of mice(P<0.05).The results of immunofluorescent double-label staining showed that PPAR?/? is expressed in neurons,not microglia or astrocytes.5.The results of immunohistochemical staining and Western Blot showed that the expression level of the anti-apoptotic protein Bcl-2 in the perihematomal tissue decreased significantly,while the expression levels of the pro-inflammatory cytokines IL-1?,TNF-?and pro-apoptotic protein Bax increased significantly.Compared to the Vehicle group,the treatment of GW0742 increased the expression level of Bcl-2 and reduced the expression levels of IL-1?,TNF-?,and Bax(P<0.05).6.The results of Western Blot showed that the expression levels of Ibal and GFAP in perihematomal tissue were increased after ICH.GW0742 significantly reduced the protein expression levels of Ibal and GFAP compared to the Vehicle group(P<0.05).Conclucion1.Activation of PPAR?/? with GW0742 alleviated neurological function impairment in ICH mice,improved BBB permeability,and reduced cerebral edema.2.Activation of PPAR?/? with GW0742 mitigated neurological damage,inhibited the microgliosis and astrogliosis and participated in the pathological process of SBI after ICH.3.Activation of PPAR?/? with GW0742 exerted a neuroprotective effect on SBI caused by ICH,possibly through anti-inflammatory and anti-apoptosis pathways. |
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