Ozone Treatment Attenuates Neuroinflammation and Alters miRNA Expression in a Rat Model of Post-Traumatic Epilepsy

The aim of this study was to investigate the effects of intraperitoneal ozone therapy in a post-traumatic epilepsy (PTE) model. An in vivo PTE model was established in male Sprague-Dawley rats, which were randomised to control (n = 8), PTE (n = 10), and PTE + Ozone (n = 10) groups. 0.7 mg/kg ozone was administered intraperitoneally for 3 consecutive days. Seizure activity was video recorded for 120 min and evaluated for latency, frequency, duration, and severity. Behavioral assessments of locomotor activity, anxiety, and spatial memory were conducted using open field, elevated plus, and radial arm maze tests on days 4-6 after the first ozone application. Blood and brain tissues were collected for biochemical assays (SUR1, TRPM4, IL-1β, IL-6, TNF-α, TAS, TOS, OSI, thiol-disulfide homeostasis), histological analyses (H&E, Cresyl Violet, and 8-OHdG immunostaining), and qRT-PCR of epilepsy-related miRNAs. Significant differences were observed among the groups for all serum and brain biomarkers (p < 0.001). The PTE group showed marked increases in SUR1, TRPM4, IL-1β, IL-6, TNF-α, TOS, OSI, TT, NT, and DIS levels, accompanied by a decrease in TAS. Ozone treatment partially reversed these changes by reducing cytokine and oxidative stress markers, improving thiol-disulfide balance, and restoring TAS levels. Behavioural testing revealed beneficial effects of ozone, including reduced immobility, fewer errors in the radial arm maze, and increased open-arm exploration. Although seizure severity, latency, and duration were not significantly altered, seizure frequency showed a decreasing trend (p = 0.067). Immunofluorescence for 8-OHdG revealed increased hippocampal oxidative DNA damage in the PTE group, which was attenuated following ozone treatment. Analysis of miRNA expression revealed downregulation in the PTE group, whereas ozone treatment resulted in overall upregulation. There was no statistically significant difference between miRNA expression results and the PTE + Ozone group (p = 0.056-0.076). Ozone therapy mitigated oxidative stress and inflammation, improved redox homeostasis, enhanced cognitive and locomotor performance, and reduced hippocampal DNA damage in the PTE model. Furthermore, the observed upregulation of specific miRNAs following ozone treatment highlights a potential molecular mechanism contributing to its neuroprotective effects.