Expression Of G-protein-coupled Receptor Kinase6(GRK6)after Acute Spinal Cord Injury In Adult Rat

Objective1. To investigate the expression profiles of GRK6on the acute spinal cord injury (SCI) model in adult rats and study its biological functions in the process of spinal cord injury and repair.2. To study the expressions and potential role of GRK6during the activation of microglia in vitroMethods:1. Male Sprague Dawley rats (n=72) with an average body weight of250g (220-275g) were used in this study. These included animals used for western blot analysis (n=48), immunohistochemistry and immunofluorescence (n=24). With improved Allen’s method, contusions were induced at T9using the NYU impactor. After SCI, we observed the expression, tissue distribution and cellular localization of GRK6. Western blot analysis, immunohischemistry and double immunofluorescent staining were performed to identify potential role of GRK6in microglia2. We applied LPS to induce HAPI microglia activation in vitro. Western blot analysis was used to observe the changes in protein expression of GRK6and F4/80in microglia. Results1. Western blot analysis showed the expression of GRK6was upregulated significantly at protein level in spinal cord after SCI.2. Immunohistochemistry revealed wide expression of GRK6in the normal spinal cord. After injury, the GRK6stainings were increased in both neurons and glial cells. Quantitative analysis showed the number of cells expressing GRK6was increased significantly in the white matter at3day after injury.3. Based on the double immunofluorescent staining, the colocalization of GRK6/neurons and GRK6/microglia was detected in the injured spinal cords. Quantitative analysis showed the changes of GRK6expression after SCI were prominentin in microglia (p<0.05), but not in neurons4. We found that F4/80increased remarkably at3day (p<0.05) after SCI and the co-expression of GRK6and F4/80was detected following the spinal cord injury via Western blot analysis and double immunofluorescent staining. Moreover, we discovered GRK6expression was increased significantly in CD68-positive microglia at3day after SCI.5. Western blot analysis demonstrated up-regulation in GRK6protein expression after LPS stimulation was time-and dose-dependent and that up-regulation in F4/80expression was concomitant with GRK6.ConclusionsGRK6might be implicated in microglia activation after SCI.