Abstract Background Methotrexate (MTX), a widely used antifolate agent in oncology and autoimmune disorders, has been increasingly associated with neurotoxic effects in the central nervous system. Hyperbaric oxygen (HBO) therapy has emerged as a potential neuroprotective approach through modulation of oxidative stress and cellular stress-response pathways, including autophagy and apoptosis. However, the region-specific effects of MTX and HBO on the autophagy–apoptosis axis within the hippocampus remain incompletely understood. Materials and Methods Adult male Sprague Dawley rats were randomly assigned to four groups: SALINE, HBO, MTX, and MTX + HBO. Hippocampal dentate gyrus (DG) and cornu ammonis (CA) subregions were analyzed. Immunohistochemistry was used to assess autophagy- and apoptosis-related markers, including LC3, Beclin-1, p62/SQSTM1, and cleaved Caspase-3 (Asp175). DNA fragmentation was evaluated using the TUNEL assay. Quantitative analyses were performed using animal-based mean values, and statistical comparisons were conducted using one-way ANOVA followed by Tukey post hoc testing ( p < 0.05). Results MTX administration was associated with increased LC3 and Beclin-1 immunoreactivity, indicating activation of early-phase autophagy-related signaling, accompanied by p62 accumulation and elevated Caspase-3 immunoreactivity, a pattern consistent with disrupted downstream autophagic processing and enhanced apoptotic signaling. HBO treatment alone induced a more limited increase in LC3 and Beclin-1 while reducing basal p62 and Caspase-3 levels, suggesting a modulatory and adaptive cellular response rather than overt stress activation. In the MTX + HBO group, p62 and Caspase-3 levels were reduced relative to MTX alone, indicating partial attenuation of MTX-associated cellular stress. TUNEL labeling was negative in all groups, suggesting that apoptotic processes were captured at an early or pre-fragmentation stage. Conclusions The present findings indicate that MTX exposure is associated with preserved autophagy initiation signals but impaired downstream processing and increased apoptotic signaling in the hippocampus. HBO treatment appears to modulate this imbalance by attenuating autophagy-associated stress and apoptotic signaling in a region-dependent manner, without providing definitive evidence of fully restored autophagic flux. These results support the biological plausibility of HBO as an adjunctive strategy to mitigate MTX-associated hippocampal stress while underscoring the need for future studies incorporating direct flux assessments, sex-specific analyses, and functional outcomes.
Cabir et al. (Thu,) studied this question.