| BackgroundStroke (ischemic stroke especially) is still one of the main killers in threatening ournational health. In China, stroke has characteristics of high incidence, high prevalence,high mortality and morbidity. Meanwhile, stroke brings a heavy social economic burdenand huge financial outlays to our country. Currently, the main clinical treatment istissue-type plasminogen activator (tPA), However, due to the narrow therapeutic window,this only effective treatment has been limited extremely in application. So, to understandthe mechanism of stroke and develop new and effective targeted interventions is urgent.Blood brain barrier (BBB) is a physical barrier between the blood and central nervoussystem, its main function is to regulate substances′transport and protect the centralnervous system from damage by a variety of internal and external factors. So, BBB playsan important role in maintaining homeostasis in the brain environment. However, bloodbrain barrier damage will result in neuroinflammatory reaction, neurodegeneration andcentral nervous dysfunction. There are many factors in causing blood-brain barrier damage,including matrixmetallop roteinases (MMPs), oxidative stress, angiogenic factors,inflammatory cytokines, autoantibodies, leukocyte adhesion molecules, immune cellinfiltration and a variety of pathogens, Their common feature is causing reduction of tightjunction protein expression in vascular endothelial cells, degradation of the basementmembrane and increased blood-brain barrier permeability, and thus aggravate brain injury. MMPs, having a capable of degrading and remodeling the extracellular matrix, are agroup of Zn2dependent endopeptidases. Various exogenous factors or brain injuryischemia-reperfusion injury would cause an increase in inflammatory response and releaseof oxygen radicals, these harmful factor activated MMP9, MMP3and other matrixmetalloproteinases resulted in degradation of BBB basement membrane and extracellularmatrix components, thereby caused damage to blood brain barrier, the destroyedblood-brain barrier could increase the degree of brain injury in turn, leading to vasogeniccerebral edema and a series of brain damage. The gelatinases-MMP9related closely withthe degree of cerebral infarction among MMPs; the upregulation of matrixmetalloproteinase-9(MMP-9) after cerebral ischemia/reperfusion injury could damageblood-brain barrier and lead to vasogenic cerebral edema or a series of brain damage bydegrading the basement membrane. Therefore, to protect the integrity of blood brainbarrier through inhibiting the expression of MMP-9after cerebral ischemia may be animportant measure in reducing brain damage.Discoid domain receptor1(DDR1) belongs to a disk-like domains of receptortyrosine kinases (DDRs); its main function is the regulation of collagen synthesis anddegradation, regulating the activity of enzymes and monitoring the formation ofextracellular matrix (ECM). Previous studies[7]have shown that the destruction of ECMplays a key role in invasion and metastasis of tumor. Many studies in tumors also havedemonstrated that DDR1could mediate the expression and activity of MMP9by bindingwith ligand (collagen IV type), and play a crucial role in transferring and proliferation oftumor cells[8,9]. But the study about DDR1and MMP9in the central nervous system isless, and their relationship after cerebral ischemic injury has not been reported. So webuild MCAO model of rat to observe the change of DDR1mRNA in ischemia and itseffect on MMP9, and then provide a new and effective treatment measure for ischemicbrain injury after the destruction of blood brain barrier by exploring the furthermechanisms.Experiment1The expression of DDR1mRNA and its phosphorylation after cerebral ischemic injuryObjective: Using MCAO model of rat to observe the expression of DDR1mRNA and itsphosphorylation level after cerebral ischemic injuryMethods:55adult male SD rats, weighing280~320g, were randomly divided into shamgroup（sham）, ischemia-reperfusion group(MCAO), DDR1-siRNA group(siRNA) andDDR1scramble-siRNA group(siRNA-c). Cerebral artery was not occluded with suture insham group and was only separated from the blood vessels. In MCAO group, aftercerebral ischemia-reperfusion injury, cerebral artery were occluded for two hours, thenwere sacrificed at3h,6h,12h,24h after reperfusion under1.5times of normal dose ofanesthesia(n=5in each time); siRNA group were transfected with DDR1-siRNA in lateralventricle three days before MCAO, then rats were sacrificed after ischemia for2h andreperfusion for24h; siRNA-c group were transfected with DDR1-siRNA-c in lateralventricle three days before MCAO, then rats were sacrificed after ischemia for2h andreperfusion for24h. Ipsilateral ischemic penumbra in each group were taken to detect theexpression of DDR1mRNA and its phosphorylation level by Western blotting (n=5). Inaddition, each group were used for continuous frozen sections after perfusing and fixing,then we use immunofluorescence of double-labeled NeuN and DDR1to observe DDR1expression in neurons (n=5).Results: Compared with the sham group, the expression of DDR1mRNA and itsphosphorylation level after cerebral ischemia and reperfusion injury were significantlyunregulated following each time and peaked at24h after reperfusion; Compared with theMCAO group, the expression of DDR1mRNA and its phosphorylation was significantlydecreased after intracerebroventricular injection of DDR1-siRNA;（P<0.01MCAO vs.sham； P<0.01siRNA vs.MCAO）. In addition, there was no significant expression changefor DDR1in the group injected siRNA-c compared with MCAO group（P＞0.05）.Conclusion: The expression of DDR1mRNA and its phosphorylation level after cerebralischemia-reperfusion injury have varying degrees of increase at different injury time.Experiment2The expression changes of MMP9mRNA and its enzyme activity after downregulating the level of DDR1mRNA in MCAO.Objective: To observe the enzyme activity and mRNA expression level of MMP9byinhibiting the overexpression of DDR1in MCAO of rat.Methods:35adult male SD rats, grouping and treatment method was identical withexperiment1. Ipsilateral ischemic penumbra tissues were taken and separated to detectMMP9activity changes and mRNA expressions by Western blotting and Gelatinzymography assay.Results: The expression and activity of MMP9is very low under normal circumstances,its expression and activity significantly increased after cerebral reperfusion injuryfollowing each time and peaked at24h after reperfusion; however, MMP9expression andactivity was significantly reduced after the expression of DDR1was suppressed;(P<0.01MCAO vs. sham； P<0.01siRNA vs. MCAO). In addition, there was no significantexpression change for MMP9in siRNA-c group compared with MCAO group（P＞0.05）Conclusion: DDR1siRNA can decrease overexpression and activity change of MMP9inischemic penumbra after cerebral ischemic injury.Experiment3The effect of inhibition of DDR1mRNA on blood-brain barrierpermeability after cerebral ischemic injuryObjective: To observe the effect of downregulation of DDR1expression andphosphorylation on blood-brain barrier permeability, infarct volume and neurologicalbehavior in MCAO of rat.Methods:112adult male SD rats, weighing280~320g, grouping and treatment methodwere identical with experiment1. After middle cerebral artery occlusion for2hours, therats were killed at24h after reperfusion. We use Evans blue to assess the BBBpermeability (n=12); wet and dry specific gravity method was used to determine brainwater content (n=8); TTC staining was used to assess infarct volume (n=8) respectively.Results: Compared with sham group, the blood-brain barrier permeability（Evans bluecontent:9.464±0.5342μg/g; brain water content:83.73±0.44%） was increased,neurobehavioral scores(6.250±0.8864) were decreased and infarct volumes (44.75±3.778%) were increased in rats after cerebral ischemia/reperfusion injury.Compared with the MCAO group, the blood-brain barrier permeability(Evans bluecontent:4.614±1.142μg/g; brain water content:79.76±1.05%) was decreased, neurologicalbehavior scores (12.63±1.408) were significantly improved and infarct volumes（22.45±3.018%）were significantly reduced (P<0.05) in siRNAgroup;（P<0.01MCAOvs. sham； P<0.01siRNA vs.MCAO）.Conclusion: Inhibition of DDR1overexpression and its phosphorylation can effectivelyease the destruction of blood-brain barrier permeability, reduce infarct volume andimprove neurological behavior.SummaryOur results show that DDR1and its phosphorylation levels were significantlyupregulated after cerebral ischemia and reperfusion injury, BBB permeability wasimproved, infarct volume was increased and neurobehavioral score was lowered.Inhibition of DDR1overexpression could reduce BBB permeability and ischemic braininjury after MCAO. Further research that DDR1could increase the permeability of BBBand ischemic brain injury by upregulating MMP9expression and activity. This studyprovides us with a new way of thinking that is: inhibitting the overexpression of DDR1specifically may play an effect on protecting blood-brain barrier and reducing theincidence of ischemic stroke. This topic broadens the idea for BBB damage aftercerebral ischemia research, our next step will start from the middle of molecularmechanisms in DDR1regulating MMP9and further reveal the role of DDR1-MMP9pathway in BBB injury after cerebral ischemia. |
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