Background and Purpose
After ischemic brain injury, glial and inflammatory cells migrate into the lesion and vascular function is perturbed in the lesion. These changes can affect the lesion maturation 1 . Small GTPase-Rho plays a crucial role in the cell motility and polarization via Rho kinase activation. Rho/Rho kinase system may work as a key signal to mature the ischemic lesion. In this study, we investigated the change of Rho/Rho kinase system and its effector molecule in the brain ischemia.
Methods
Adult Wister rats were subjected to permanent middle cerebral artery occlusion by intraluminal suture. Rectal temperature was monitored and was maintained at 37.0+/−0.5 degree. Brains were removed 1 or 2 days after ischemia for following analysis. Ischemic and non-ischemic hemispheres of forebrain were homogenated and subjected to Western blot to observe the expression change of RhoA protein. To assess Rho/Rho kinase activation, we performed the immunohistochemistry of phospho-adducin, an effector molecule of Rho/Rho kinase, in the ischemic brain. Zamboni's solution (2% paraformaldehyde and 0.2 % picric acid in phosphate buffer) fixed-brains were frozen after cryo-protection and 10um coronal sections were prepared for immunohistochemistry.
Result
Western blot showed that RhoA protein expression in the sham-operated brain. Twenty-four hours after ischemia, expression of RhoA protein was increased in the ischemic hemisphere. In order to show the evidence of Rho/Rho kinase system activation and reveal the localization of activated Rho/Rho kinase, phospho-adducin immunohistochemistry was carried out. Phospho-adducin signals were expressed mainly in the neuronal cells in the sham-operated brain. In the ischemic hemisphere, phospho-adducin signals were increased in the glial cells or macrophages at the margin of the infarct area. After 2 days, signals were decreased in the infarct core, whereas many cells still expressed phospho-adducin at the margin of the infarct area. In these regions, brain microvessels were also expressed phospho-adducin strongly. Signals were not increased in the microvessels of the contra-lateral hemisphere.
Conclusion
We demonstrated up-regulation and activation of small GTPase-Rho following cerebral ischemia. It may be involved in the maturation of ischemic brain injury via inflammatory cell infiltration and perturbation of vascular function.