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Mild traumatic brain injury (mTBI) is a low-level injury, which often remains undiagnosed, and in most cases it leads to death and disability as it advances as secondary injury. Therefore, it is important to study the underlying signaling mechanisms of mTBI-associated neurological ailments. While transforming growth factor-beta1 (TGF-β1) has a significant role in inflammation and apoptosis in myriads of other pathophysiological conditions, the precise function of increased TGF-β1 after mTBI is unknown. In this study, our objective is to study the physiological relevance and associated mechanisms of TGF-β1-mediated inflammation and apoptosis in mTBI. Using an in vitro stretch-injury model in rat neuronal cultures and the in vivo fluid percussion injury (FPI) model in rats, we explored the significance of TGF-β1 activation in mTBI. Our study demonstrated that the activation of TGF-β1 in mTBI correlated with the induction of free radical generating enzyme NADPH oxidase 1 (NOX1). Further, using TGF-β type I receptor (TGF-βRI) inhibitor SB431542 and transfection of TGF-β1 siRNA and TGF-β antagonist Smad7, we established the neuroinflammatory and apoptotic role of TGF-β1 in mTBI. Inhibition of TGF-βRI or TGF-β1 diminished TGF-β1-induced inflammation and apoptosis. Further, the enhanced TGF-β1 activation increased the phosphorylation of R-Smads including Smad2 and Smad3 proteins. By immunofluorescence, western blotting, ELISA and TUNEL experiments, we demonstrated the up-regulation of pro-inflammatory cytokines IL-1β and TNF-α and apoptotic cell death in neurons. In conclusion, this study could establish the significance of TGF-β1 in transforming the pathophysiology of mTBI into secondary injury.

Traumatic brain injury (TBI), even at mild levels, can activate matrix metalloproteinases (MMPs) and the induction of neuroinflammation that can result in blood brain barrier breakdown and neurodegeneration. MMP2 has a significant role in neuroinflammation and neurodegeneration by modulating the chemokine CXCL12α (stromal cell derived factor SDF-1α) signaling pathway and the induction of apoptosis. SDF-1α is responsible for cell proliferation and differentiation throughout the nervous system and is also implicated in various neurodegenerative illnesses. We hypothesized that TBI leads to MMP2 activation and cleavage of the N-terminal 4 amino acid residues of CXCL12α with generation of the highly neurotoxic fragment SDF-1(5-67). Using an in vitro stretch-injury model of rat neuronal cultures and the in vivo fluid percussion injury (FPI) model in rats, we found that oxidative stress has a significant role in the activation of MMP2. This is initiated by the induction of free radical generating enzyme NADPH oxidase 1 (NOX1). Induction of NOX1 correlated well with the signatures of oxidative stress marker, 4HNE in the injured neuronal cultures and cerebral cortex of rats. Further, using MMP2 siRNA and pharmacological MMP2 inhibitor, ARP100, we established the neurodegenerative role of MMP2 in cleaving SDF-1α to a neurotoxic fragment SDF-1(5-67). By immunofluorescence, western blotting and TUNEL experiments, we show the cleaved form of SDF leads to apoptotic cell death in neurons. This work identifies a new potential therapeutic target to reduce the complications of brain damage in TBI.