DLK-Mediated Axonal Injury After Subarachnoid Hemorrhage In Rats And Its Mechanism

Objective:Subarachnoid hemorrhage(SAH)is a fatal cerebrovascular disease,a very common critical illness in neurosurgery,with a high morbidity and fatality rate.Early brain tissue damage after SAH often occurs within 72 hours after SAH,which is called early brain injury(EBI).The acute pathophysiological events occurring at this stage are considered to be the basis for intervention and treatment of SAH patients.White matter accounts for more than half of human brain tissue and is more susceptible to ischemia / hemorrhagic stroke than gray matter.Dual leucine zipper kinase(DLK)belongs to the mixed lineage kinase(Mixed lineage kinase)family in structure,and is a member of MAPKKK(MAP kinase kinase kinase)in function.DLK is a mitogen-activated protein kinase-kinase-kinase that controls axon growth,apoptosis and neuronal degeneration during neural development,as well as neurodegeneration after various damages to the adult nervous system.In this study,Sprague Dawley rats(SD rats)were used as the research object,and the SAH injury model was established by puncturing the internal carotid artery.Intraventricular injection of DLK recombinant protein or DLK adeno-associated virus(AAV)for intervention,through neuroethology,immunofluorescence(IF),transmission electron microscope(TEM),magnetic resonance diffusion DTI(Diffusion tensor imaging,DTI)and other methods to observe the regulation effect of DLK on white matter axon injury after SAH in rats mechanism.Methods:1.Using the modified Garcia score,Beam Balance score,Rotarod test,and dry-wet weight method to detect SAH during the EBI period 3 h,6 h,12 h,24 h and At 72 h,the nerve injury and brain edema in rats were detected by quantitative real-time polymerase chain reaction(qPCR)and Western blot at the above time points.Protein(Beta-amyloid precursor protein,?-APP).2.Intraventricular injection of DLK adeno-associated virus or DLK recombinant protein to down-regulate or up-regulate the expression of DLK,Garcia,Beam Balance and Rotarod test to detect neurological behavioral changes in rats,dry and wet weight method to detect rat brain edema TEM and DTI were used to detect white matter axon injury in rats,and frozen section IF was used to detect the expression of ?-APP and DLK.The DLK / MKK7 / JIP3 / p-JNK / ?-APP was detected by WB to explore the mechanism of DLK regulating axonal injury after SAH.Results:1.After SAH,the expression of DLK and ?-APP increasedand reached a peak at 24 h.Neurobehavioral manifestations showed obvious damage after 12 hours of SAH,and the water content of brain tissue increased significantly after 24 hours of SAH.2.Inhibition of DLK can improve the neurobehavioral performance of rats after SAH and reduce brain edema.The expression of ?-APP is reduced in the fluorescent section.Transmission electron microscopy shows that the damage of axon subcellular structure is greatly reduced.DTI also found that white matter cellulose travels.And the integrity is greatly improved,and overexpression of DLK will reverse the above changes.Compared with the control group,after knocking down DLK,MKK7 expression will be reduced,JIP3 will increase,JNK phosphorylation will be reduced,?-APP expression will be reduced,and overexpression of DLK will make the protein expression results in the pathway and knockdown The result of DLK is reversed.After knocking down MKK7,there was no difference in the expression of DLK upstream,no difference in the expression of JIP3 downstream,the phosphorylation degree of JNK was reduced,and there was no difference in the expression of ?-APP.After knocking down JIP3,there was no difference in upstream DLK expression,while MKK7 expression decreased,downstream JNK phosphorylation decreased,and there was no difference in ?-APP expression.Conclusion:1.During the EBI period after SAH,the expression ofDLK will increase,and axon damage will occur.The accumulation of DLK and axon damage will reach a peak at 24 h.There is nerve damage and brain edema early in EBI.2.After SAH in rats,inhibition of DLK has obvious neuroprotection,brain edema protection and white matter axon protection,while overexpression of DLK will reverse the neuroprotection and blood-brain barrier(BBB)after SAH Protection and protection of white matter axons.The above-mentioned effects are involved in the regulation of white matter axon injury after SAH through DLK / MKK7 /JIP3 / p-JNK and thus participate in the pathological process of EBI.