Therapeutic Effect of Alpha-Pinene on In Vitro and In Vivo Models of Mild Traumatic Brain Injury

We aimed to investigate the therapeutic effects of alpha-pinene (α-pinene), a major component of phytoncide, in in vitro and in vivo models of mild traumatic brain injury (mTBI), raising the possibility of treating mTBI, a condition currently lacking adequate therapies. An in vitro model was established using SH-SY5Y cells and a cell injury controller, and was treated with α-pinene (0.5 g/mL). An in vivo model was induced by a stereotaxic impactor in male C57BL/6J mice and treated with α-pinene intravenously (50 mg/kg and 100 mg/kg) for 3 days post-injury. Histopathological and immunohistochemical comparisons were conducted alongside cognitive function tests to evaluate -pinene treatment. In vitro analysis showed that alpha-pinene treatment significantly increased TUNEL-positive cells. Elevated NOX4 and p22phox mRNA expressions and a high Bax/Bcl-2 protein expression ratio were noted following alpha-pinene treatment. mTBI mice treated with alpha-pinene exhibited a notable decrease in brain water content with fewer FJB-positive neurons and lower protein expression of Bax and Bcl-2 compared to untreated mTBI mice. Immunofluorescence staining for NOX4 and GFAP-positive or Iba-1-positive cells demonstrated that the increased oxidative stress and astrogliosis or activated microglia triggered by mTBI were alleviated after alpha-pinene treatment. Cognitive function testing revealed a general improvement in mTBI mice treated with alpha-pinene, with statistical significance observed in the NOR test. Alpha-pinene appears to be beneficial for neuroprotection and enhancing cognitive function in the early phases of mTBI.