If a mouse failed to reach the platform within s, it was guided to the platform and allowed to stay for s.Swimming escape latency was automatically recorded and analyzed with a videocompute system. An open field test was conducted to explore the presence of anxiety or disinhibition in CCI injured mice at days after TBI.The open field test was performed, as previously described.Before each behavioral session, the apparatus was cleaned by the solution of ethanol.Animals were removed from their home cage and placed in an open field test arena. Behaviors and parameters were recorded and analyzed by an automated behavioral tracking system. Ambulatory activity was recorded for min and analyzed.Briefly, the brains were fixed in paraformaldehyde for h, then cut into serial coronal sections with a cryostat.A random sampling scheme was used to yield five total sections, by estimating every tenth section from rostral to caudal.Transfections were performed as per the manufacturers instructions. Melatonin was then added hours before the scratch injury.Cell viability was assessed using the cell counting kit. Finally, cells were used for protein extraction, cellular reactive oxygen species detection, and BODIRY C staining.The ROS generation by the cells was measured with a cellular ROS detection assay kit. Harvested cells were washed with serumfree culture medium at C for min.GSH plays an important role in ferroptosis, and reduced GSH levels can trigger ferroptosis.Perls blue staining showed that the number of ironpositive cells was significantly increased at days after TBI and continued to increase gradually afterward. Compared with the sham group, the cell bodies of injured neurons exhibited cytoplasmic shrinkage or nuclear pyknosis at days following TBI.To elucidate whether the protective eect of melatonin is dependent on its receptors following TBI, we first. Our results were consistent with the previous report that both MT and MT levels were reduced in a time pointdependent manner, although the expression of MT and MT was evaluated during the acute period postTBI.Furthermore, melatonin significantly ameliorated MT and MT loss in the brains of melatonintreated TBI mice at h. Taken together, the effect of melatonin was blocked by MT and MT receptors antagonists.In the MWM test, animals that find the platform more quickly are regarded as having better learning and memory function.An open field test was then conducted to explore the presence of anxiety or disinhibition on days after TBI.Animals in the vehicle TBI group traveled markedly shorter total distance than that in the sham group.In parallel, significantly shorter distance moved were observed in the vehicletreated TBI group than that in the sham group.CCK assay was used to assess cell survival rate for choosing the time course of the damage and the dose of melatonin.The results showed that the general trend of cell survival rate was decreased and reached the bottom with at hafter injury, then increased gradually afterwards. Given lipid peroxidation is a hallmark of ferroptosis, we assessed the cytosolic ROS using dichlorodihydrofluorescien diacetate and BODIRY C as convenient proxies.Resistance to ferroptosis correlated with suppression of BODIRYC oxidation.Similar to the above ROS results, we found mechanical scratch injury significantly promoted lipid peroxide formation with red fluorescence of the BODIPY shifted to green fluorescence, indicating that scratch cell injury rises to lipid peroxidation.