Repurposing Leucovorin for Mild Traumatic Brain Injury: Evidence from Biochemical and Behavioral Outcomes in Rats

Background/Objectives: Mild traumatic brain injury (mTBI) is common and may result in persistent cognitive and affective disturbances driven, at least in part, by delayed secondary injury mechanisms, including oxidative stress, neuroinflammation, apoptosis-related signaling, and impaired neuroplasticity. Pharmacological strategies targeting these interconnected processes remain limited. The present study investigated leucovorin, also known as folinic acid, a clinically approved reduced folate, as a potential repurposing candidate in an experimental model of mTBI. Methods: Male Wistar rats were subjected to mild diffuse brain injury using a modified weight-drop model and received a single intraperitoneal dose of leucovorin (20 mg/kg). Behavioral performance was evaluated using the open field, elevated plus maze, forced swim, and novel object recognition tests. Oxidative stress markers, including total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI), as well as inflammatory mediators tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2), caspase-3, brain-derived neurotrophic factor (BDNF), and acetylcholinesterase (AChE), were measured in hippocampal tissue and plasma. Histopathological and immunohistochemical evaluations were also performed in cortical and hippocampal regions. Results: Experimental mTBI was associated with anxiety-like and depressive-like behaviors and impaired recognition memory, whereas basal locomotor activity was not significantly altered. Trauma was also associated with increased oxidative stress, elevated inflammatory and apoptosis-related markers, reduced BDNF levels, altered AChE activity, and histopathological abnormalities. Compared with untreated mTBI animals, leucovorin-treated animals showed attenuation of biochemical and tissue alterations, accompanied by improved behavioral outcomes. Immunohistochemical findings were consistent with reduced inflammatory labeling and relative preservation of tissue architecture following leucovorin treatment. Conclusions: Leucovorin attenuated behavioral, biochemical, histopathological, and immunohistochemical alterations associated with experimental mTBI. These findings suggest that leucovorin may have neuroprotective potential in this setting; however, further studies are needed to clarify the underlying mechanisms, optimal treatment paradigms, and translational relevance.