Nemo-like Kinase(NLK) Involves In Neuronal Apoptosis In The Hippocampus After Cerebral Ischemia/Reperfusion

Background: Ischemia stroke is the major cause of mortality and morbidity andinduces a significiant burden throughout the world. The sharply reduce of cerebralblood flow and oxygen levels can not meet the normal brain function need duringcerebral ischemia, eventually leading to damgage to brain. Cerebral ischemia leadsto different degrees of cellular damage relying on the vulnerability of the cells toischemia. Hippocampus is the most sensitive area to the change of oxygen content.Therefore, Hippocampus, especially CA1region, is easier to be injuried than others.Lots of mechanisms, such as inflammatory reaction, autophagy, apoptosis andoxygen radicals reaction, are involved in neuronal apoptosis of CA1region causedby cerebral ischemia/reperfusion(I/R). During this process, apoptosis plays animportant role. Activated Caspase-3is the key molecule of apoptosis. Hippocampalfunction includes hearing and memory. Once hippocampus is damaged, the normalliving of patients is under adverse impact. Therefore, it was especially important tostudy the injury mechanisms of neuron leaded by cerebral ischemia/reperfusion.NLK is a neurotrophic factor, which plays roles in neurogenesis, maintenance andtraumatic brain injury. Although NLK expressed in nerve tissue, yet the mechanismand action of NLK during cerebral ischemia/reperfusion are still unclear.Objective: 1. To investigate the expression of NLK in Hippocampus of rat following cerebralischemia/reperfusion, we established MCAO in our experiments.2. To detect cellular localization of NLK.3.To investigate the relationship between NLK and neuronal apoptosis.Methods:We used MACO as our experimental model. Sixty adult male Sprague-Dawleyrats were randomly assigned to two groups:sham-operated group （S） andischemia/reperfusion(I/R) groupe.Based on the different time points after surgery,I/R group was divided into six subgroups:1h,4h,8h,1d,3d and7d. Western blot wasused to detect the change of NLK and activated Caspase-3in Hippocampus of ratfollowing cerebral ischemia/reperfusion. Immunohistochemistry and Immunofluore-cence staining were used to investgate its cellular location and relationship betweenNLK and neuronal apoptosis. In vitro, Immunofluorescence staining was used forPC12cells to certify nuclear transfer of NLK.Results:We performed Western blot to detect the level of NLK. We found that the levelof NLK is relatively lower in the hippocampus of sham control group, whichunderwent a gradual increase post I/R, and peaked at8h (p<0.05), then graduallydecreased to the lowest level at3d, finally it gradually increased. Caspase-3progressively increased and reached peak at3d, then decreased (p<0.05).Immunohistochemistry staining showed NLK immunoreactivity was principallyfound in neurons, especially in pyramidal neurons of CA1region in thehippocampus. Immunofluorescence staining suggested that the colocalizationbetween NLK and Caspase-3was found. Meanwhile, cell expermient displayed:NLK mainly localized in cytoplasm of neuron before stimulus, and graduallytranfered to nuclues after stimulus. Therefore, We initially concluded that nuclear transfer phenomena of NLK might happen in our study.Conclusion:Firstly, The remarkable change of NLK after I/R suggested that NLK might beinvolved in this process. Secondly, the colocalization between NLK and neuron ofCA1region showed that NLK might Play roles in neuron of CA1region. Nucleartransfer phenomena of NLK happened. The level of activated Caspase-3wasgradually increased with the apoptosis of neurons. Finally, the colocalizationbetween NLK and activated Caspase-3suggested that NLK might be involved inneuronal apoptosis of CA1region after cerebral ischemia/reperfusion.