Characterization of the Kallikrein-Kinin system post chemical and traumatic brain injury : an in vivo and In vitro biochemical approach

Abstract

Background: Traumatic Brain Injury (TBI) is the result of a mechanical impact on the brain that can provoke mild to moderate to severe symptoms. It is acknowledged that TBI leads to apoptotic and necrotic cell death; however, the exact mechanism by which brain trauma leads to neuronal injury is not fully elucidated yet. Noteworthy, the Kallikrein Kinin System (KKS) represents the first inflammatory pathway activated following tissue injury. Thus, the KKS’s contributing role represents a good target to assess in the area of TBI.Aims: The aim of this study is to investigate the expression and the role of the KKS’s main players: Bradykinin and its receptors, in mediating neuronal injury. This will be evaluated under TBI (in vivo) and chemical (in vitro) neurotoxicity paradigms. The neuronal cell line PC12 will be treated with the apoptotic drug Staurosporine (STS) to simulate the cell activity after TBI to a certain degree.Methods: The effect of STS on the viability and proliferation of pre-treated PC12 cells was investigated using MTT, ROS and LDH assays. DAPI nuclear staining was performed to assess for apoptotic bodies. MitoPT JC-1 and Annexin-V staining were presented as a proof of apoptosis. Intracellular calcium release was evaluated by Fluo 4-AM staining. Immunofluorescence (IF) was also performed on KKS markers, notably bradykinin 1 and 2 receptors (B1R and B2R). Western blotting (WB) was performed on both apoptotic and KKS markers. Real-time PCR, IF and WB were conducted to study the effect of STS on the transcriptional, translational and cellular localization of KKS markers. The same methods were employed on primary neurocortical cells and post-CCI ipsilateral cortical tissues. Finally, proteomics was used to find relevant proteins associated to STS and KKS in PC12 cells.Results: STS inhibited the proliferation of pre-treated PC12 cells in a time-dependent manner. DAPI, WB and IF confirmed that apoptosis is the mechanism that STS is inducing and that it is mainly the B2R that is being altered