In experimental traumatic brain injury the neuroprotective effectiveness of melatoni caspase-dependent apoptotic examination of signal pathways

Objective: In this study, the neuroprotective effect of melatonin in rats with traumatic brain injury (TBI) was investigated in terms of caspase-dependent apoptotic signaling pathways. Materials and Methods: 21 mice obtained from Dicle University Health Sciences Application and Research Center were divided into 3 groups (Control, Trauma, Trauma+Melatonin). The animals in the control group were not subjected to any application during the experiment and were sacrificed at the end of the experiment and their brain tissues were collected. Trauma and Trauma + melatonin groups were placed under general anesthesia, and an experimental TBI model was created by free fall to the frontal lobes of animals with a height of 80 cm and a weight of 30 g, by means of the prepared burette stand and pipe assembly. Intraperitoneal injection of 10 mg/kg melatonin was administered to the animals in which trauma model was created 30 minutes later, and 1 hour after the injection, the animals were sacrificed by decapitation and their blood and brain tissues were taken. Collected brain samples were fixed in 10% buffered formalin solution and subjected to routine histological follow-up. 5 µm thick sections taken from paraffin-embedded tissues were stained with Hematoxylin & Eosin (H&E), Luxol Fast Blue for routine histopathological evaluation. In addition, the expression levels of apaoptotic proteins were examined by applying Bax, Bcl-2, Caspase-3 and Caspase-9 immunohistochemistry to serial section samples taken from brain tissues. The Kruskal Wallis Median Test was used for statistical analysis. Mann-Whitney U test was used for pairwise comparisons. Results: In the control group, it was observed that cerebral neurons, neuroglia and nerve fibers, most of which were myelinated, were in normal appearance. In the trauma and trauma + melatonin groups, brain tissue loss was clearly observed in the cortex region due to trauma. In the myelin stained samples, it was observed that myelinated nerve fibers occupied a large area in the white matter in the control group, while the paler stained state in the trauma group showed demyalination. In the trauma + melatonin group, the presence of both pale and dark staining areas with myelinated nerve fibers was detected, and demyelination was found to be decreased compared to the Trauma group. In the samples with immunohistochemistry staining, the Bax distribution ratio was 27.12±1.83% in the control group, this increased significantly in the Trauma group and constituted a rate of 47.79±8.86%. It was determined that it had a ratio of ±6.20 and was somewhere between both groups. As a result of Bcl-2 immunohistochemistry analysis, the presence of 4.00±0.18% of the tissues in the control sections was observed. This rate increased to 6.50±.13% in the Trauma group and increased to 7.91±2.17% in the Trauma+melatonin group, which was significantly different in both groups. As a result of our caspase-3 immunohistochemistry analyzes, the presence of immunopositivity was observed in 1.60±0.08% of the tissues in the control group. In the trauma group, this rate increased significantly to 3.51±0.74%, while in the trauma + melatonin group, a rate of 1.60±0.08% was obtained and the presence of immunopositivity similar to the control group was detected. In the analyzes in which we evaluated caspase-9 immunopositivity, a rate of 1.50±0.09% was observed in the control group. On the other hand, it was determined that immunopositivity increased significantly to 3.12%±0.49% in the trauma group, and this immunopositivity rate was 1.60±0.06% in the trauma + melatonin group, which was similar to the control group Conclusion: As a result of our study, it was determined that traumatic brain injury triggered neurodegeneration by causing an increase in the expression level of both internal and external apoptosis-related proteins. It was concluded that the 10 mg/kg melatonin dose we applied alleviated TBI-induced apoptotic protein expression levels and demyelination, and also triggered anti-apoptotic protein expression in the planned experimental animal model. Key words: Traumatic brain injury, Melatonin, Apoptosis, Neurodegeneration, Mice