Relationship of ozone application and time in rats with hypoxic-ischemic brain injury

Background: Despite the important advances in pregnancy and newborn follow-up, hypoxic-ischemic encephalopathy is still one of the prominent causes of newborn mortality and disability worldwide, and there is no sufficiently effective treatment for it yet. This study aimed to investigate whether the ozone injection, administered in a single-dose as a preconditioning agent before the hypoxia and in single and repeated doses on different days following the hypoxia, would affect the spatial memory performance of the rats in the Morris water maze test or on their apoptotic cell numbers. Methods: The study consisted of 102 seven-day-old male Wistar baby rats randomly divided into five groups. Rats in all groups were induced with hypoxic-ischemic brain injury (HIBI) except for the Sham group, and 1.2 mg/kg ozone was administered intraperitoneally. For the apoptosis evaluation, eight rats from each of the first four groups were decapitated by cervical dislocation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used for immunohistochemical quantification of apoptosis in the excised brains. Blood malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured in the blood samples collected through cardiac puncture. Fourteen-week-old rats underwent the Morris water maze test to test their long-term spatial memory. Results: On apoptotic quantification in the right hemisphere using the TUNEL assay, the numbers of apoptotic neurons in the ozone preconditioning group (Group 3) and the group given ozone on the day of hypoxia (Group 4) were found to be significantly higher than the Sham group (Group 1), but significantly lower than the non-treatment group (Group 2) (p <0.001; p <0.001, respectively). Group 3 rats had the highest mean MDA level and SOD activity. Considering the platform finding times in the first four days of the tests, Group 4 had the shortest times after Group 1; and on Day 4, Group 4 found the platforms significantly sooner than Groups 2, 3, and 5 (p <0.001). Comparison of Groups 1 and 4 revealed significantly shorter times for Group 1 for each day except for Day 2. Conclusions: Other studies have shown that controlled application of ozone would result in oxidative preconditioning and reduce the damage induced by reactive oxygen species through enabling adaptation to oxidative stress. Our study obtained remarkable and encouraging findings for ozone administration in HIBI by examining Group 4's performance in the first four days and the difference in its platform finding times between Day 1 and Day 4.