Dose-dependent protective effect of anetol in experimental traumatic brain injury is mediated by apopytotic and inflammatory proteins examination of their expression in terms of oxidative stress

OBJECTIVES: Traumatic brain injury (TBI) is one of the leading causes of morbidity and mortality in all ages. More than 50 million people worldwide suffer from traumatic brain injury each year. The clinical features of TBI and the underlying complex pathophysiology increase the value of preclinical studies and phase I/II studies. However, a therapeutic method other than surgical intervention for the treatment of traumatic brain injuries has not yet been developed. The fact that Anetol has antioxidant, anticarcinogenic, anti-inflammatory and antihypernociceptive effects has made this vegetable oil gain an important place in recent studies. Findings on its strong antioxidant potential have provided data suggesting that Anetol may be protective in cardiac ischemia reperfusion injury. In our current study, we plan to investigate the protective effect of Anetol in the TBI model in a dose-dependent manner, thanks to its cheap herbal component and easy availability. In our literature review, there is no study to investigate the dose-dependent protective power of Anetol, which is an antioxidant component, in animals with TBI model. For this reason, this research has the potential to bring a new perspective to the TBI research topic. MATERIALS AND METHODS: For this study, 35 adult male mice weighing 28-30 g were obtained from DÜSAM (Dicle University Health Sciences Application Research Center). These 35 animals were divided into 5 groups in equal numbers. After these groups were formed, the animals in the control group were not given any treatment for 72 hours. The animals in the trauma group were traumatized by weight reduction method and then only analgesia was applied without any application and followed up under optimal conditions and decapitation was performed after sacrification at the end of the experiment.In the post-traumatic dose-dependent groups, the animals were firstly anesthetized with the help of xylazine and ketamine, then moderate traumatic brain injury was induced by free weight reduction method and 3 hours after the trauma was applied and for the following 72 hours on the same day and hour, 40mg/kg anethole for one group, 80mg/kg anethole for the other group and 160mg/kg anethole for the last group were administered by oral gavage.All experimental animals were sacrificed 1 hour after the last treatment doses. The collected brain samples were fixed in 10% buffered formalin solution and subjected to routine histologic follow-up. Paraffin embedded 5 µm thick sections were stained with Hematoxylin & Eosin (H&E) for routine histopathologic evaluation. In addition, the expression levels of apapitotic proteins were examined by immunohistochemistry of TNF-alfa, Bax, Caspase-3 in serial sections taken from brain tissues. Finally, biopsy samples were taken for MDA levels in the experimental study. FINDINGS: In the control group, the brain tissue had normal morphology, was covered with meninges and contained abundant neurons and glial cells, but in the trauma group, intense hemorrhage and vascular congestion were observed. Pyknotic nuclei, karyolysis, increased perineural cavity and edema areas were observed in neuron cell bodies. Post-traumatic administration of 80mg/kg or more of anethole after trauma showed that pyknosis occurred but the pyramidal triangle image of pyramidal neurons was strongly preserved. However, oxidative stress, inflammation, apopitotic induction were affected at a low level and a healthy neuron image was observed. Immunohistochemical results obtained from studies on TNF-alpha, Bax and Caspase-3 levels after trauma showed a significant decrease in the levels of these molecules at high doses. When the results of MDA analysis were analyzed, a significant decrease was found in the anetol-treated groups in the MDA levels that increased after trauma. CONCLUSIONS: In this study, we found that anethole increased the amount of glial cells in the post-traumatic administration of anethole, however, the pyramidal triangle image was strongly preserved in the pyramidal neurons of the brain tissue, and it was affected at a low level by factors such as oxidative stress, inflammation, and apopitotic induction accompanying trauma. In addition, there was a significant decrease in Bax immunopositivity, a significant decrease in Caspase-3 immunointensity and significant decreases in TNF-alpha levels. When the results of MDA analysis were analyzed, a significant decrease was found in the anetol-treated groups in the MDA levels that increased after trauma. Keywords; Traumatic brain injury, anetol, apoptosis