Modulatory role of the ketogenic diet in glial scar formation after traumatic brain injury: A Fourier transform infrared, Raman, and X-ray fluorescence microscopy study.

Traumatic brain injuries (TBI) represent a significant clinical challenge, causing not only direct damage to neural tissue but also triggering secondary pathophysiological processes, including glial scar formation. While the glial scar serves a protective role, it simultaneously generates profound biochemical and elemental disturbances that may contribute to secondary neurodegeneration and the development of epilepsy. In recent years, the ketogenic diet (KD), recognized for its anticonvulsant properties, has emerged as a potential therapeutic strategy for TBI. However, despite growing interest, its impact on glial scar formation and biomolecular remodeling in the injured brain remains poorly understood. A better understanding of these mechanisms is essential for developing personalized dietary interventions for TBI treatment. To assess the modulatory effects of KD on glial scar development, male and female Wistar rats maintained on ketogenic or standard diets were subjected to controlled cortical injury. Brain samples collected at 2-, 8-, 16-, and 30-days post-injury were analyzed using Fourier transform infrared (FTIR) microspectroscopy, Raman microscopy, and synchrotron radiation-based X-ray fluorescence (SRXRF) microscopy. FTIR and Raman spectroscopy enabled topographic and semi-quantitative assessment of biomolecules accumulation and structure at the lesion site and adjacent cortex, while SRXRF provided detailed elemental mapping of P, S, K, Ca, Fe, Cu, and Zn within the developing glial scar. Topographic chemical maps obtained with FTIR microspectroscopy consistently revealed reduced biomolecule levels at the lesion site, largely independent of diet. Semi-quantitative biomolecular analyses, however, demonstrated some sex- and time-dependent effects of KD. Raman spectroscopy further highlighted cortical modifications, showing increased lipid unsaturation in males on KD. In females treated with high-fat fodder, elevated levels of lipid esterification, cytochrome, and amide III and/or deoxyhemoglobin was additionally observed. SRXRF imaging revealed dynamic elemental changes, including transient Ca release, delayed Fe accumulation, and progressive Cu increase during scar formation, without a pronounced effect of KD. All findings together suggest that KD may modulate biomolecular responses to TBI in a sex-dependent manner, with females displaying greater susceptibility to the observed changes.