To investigate the protective effect of edaravone on the blood-spinal cord barrier using compression spinal cord injury in rats and human brain microvascular endothelial cells (HBMECs) injury

The protective effect produced by the application of edaravone after spinal cord injury was firstly explored by in vivo experiments, including the BBB functional score, footprinting experiments, and examination of HE, Nissl and LFB pathology sections. The blood-spinal cord barrier disruption was analyzed by tail vein injection of Evans blue, including Evans blue extravasation, spinal cord edema, spinal cord vasculature and cellular connectivity proteins in spinal cord blood vessels. The mechanism of edaravone protection against spinal cord injury, including the effects on autophagy and necroptosis, was analyzed by western blotting and immunofluorescence co-localization experiments. Using the HBMECs injury model induced by rapamycin and necrosis activator, respectively, we analyzed the effects of edaravone on the properties of endothelial cells in vitro, such as permeability, migration, and tube-forming, and analyzed the changes of endothelial cell connectivity and autophagy by western blotting and immunofluorescence co-localization experiments.

The results of this study indicate that edaravone treatment promotes functional recovery, improves vascular injury, and upregulates the expression of blood-spinal cord barrier-related proteins after spinal cord injury in rats. In in vitro experiments, edaravone increased BBB scores after spinal cord injury and there was a significant difference in the recovery of rats in the edaravone group compared with the SCI group on day 7 after injury. Spinal cord injury resulted in pathological changes including decreased tissue densification, vacuolization, and decreased neuron numbers, which were ameliorated by edaravone treatment. The application of edaravone reduced the permeability of the blood-spinal cord barrier and tissue edema after spinal cord injury. In addition, edaravone increased cellular connectivity in the spinal cord vasculature, increased autophagy in spinal cord tissues, and attenuated the expression of proteins associated with necroptosis. In vitro experiments, edaravone increased the viability of HBMECs, reduced endothelial cell permeability, restored intercellular connections, and promoted cellular angiogenic activity. Notably, the autophagic process was activated after spinal cord injury, and the phosphorylation of RIP1/RIP3/MLKL was significantly upregulated. However, edaravone treatment attenuated this trend in vivo and in vitro. Moreover, the application of rapamycin (Rapa) exacerbated the expression of RIP1/RIP3/MLKL phosphorylation while at the same time diminishing the protective effect of edaravone, including decreased expression of endothelial cell junction proteins, increased cell permeability, and reduced migration and tube-forming capacity. In the model of HBMECs induced by the necrosis-activating reagent TSZ, the expression of autophagy was increased, the expression of connexin in endothelial cells was decreased, the permeability was increased, and the migratory and tube-forming abilities of the cells were decreased, whereas the application of edaravone inhibited TSZ-induced autophagy and the phosphorylation of RIP1/RIP3/MLKL, and it was capable of improving the permeability, migratory, and tube-forming ability of endothelial cells.

In general, our results suggested that edaravone is capable of reducing the destruction of blood-spinal cord barrier and promoting functional recovery after spinal cord injury. The possible underlying mechanism is that edaravone is capable of protecting angiogenic activity and reducing autophagy and the phosphorylation of RIP1/RIP3/MLKL, as well as their mutual deterioration. Accordingly, edaravone can be a favorable option for the treatment of spinal cord injury.