Experimental Study Of The Protective Effect And Mechanism Of Hydrogen On Early Brain Injury In Rabbits After SubarachnoidHemorrhage

Backgroud:Subarachnoid hemorrhage (SAH) is a frequently seen devastating disease with high mortality and disability. After decades of study, considerable advances have been made in diagnostic methods and medical therapy. However, the outcome of the patients with SAH remains poor. Vasospasm has been the focus of the majority of research efforts during the past decades on SAH. Unfortunately, it has led to limited success in improving outcome. Increasing evidence has proved that the early brain injury (EBI) period, encompassing 24-72h following SAH, is closely bound up with the poor prognosis. Especially, lots of studies have demonstrated that oxidative stress and brain edema are critical in the development of EBI after SAH. Hydrogen (H2) has been gradually recognized as a selective anti-oxidative and anti-apoptotic agent. And many studies have shown that H2 possesses cytoprotective effects in different cell types and disease models. Therefore, in this study, we used a rabbit SAH model to test the protective effect and mechanism of H2.Objectives:The aims of this study are:1) to establish a stable SAH model in rabbit; 2) to investigate the protective effect of H2 on EBI following SAH; 3) to explore the underlying mechanism of the protective effect of H2.Methods:Adult male New Zealand white rabbits weighing from 2.4 to 2.8 kg were assigned randomly to four groups:(1) control group, (2) SAH group, (3) vehicle group, and (4) hydrogen-rich saline group. In the rabbits of SAH+hydrogen-rich saline group, hydrogen-rich saline was administered immediately after the first blood injection and was continued every 12 hours for 72 hours. Rabbits of vehicle group received equal volumes of saline at corresponding time points. Both hydrogen-rich saline and saline were injected intraperitoneally. Before execution, the neurological evaluation was finished. Six rabbits in each group were killed with the fixation perfusion method. The brains were taken for Nissl staining, immunofluorescence study and TUNEL staining. The other six rabbits in each group were exsanguinated and decollated, with the brains removed and frozen in liquid nitrogen immediately for Western blot, Realtime-PCR, ELISA, electrophoretic mobility shift assay (EMSA) and MDA test. The rest of the six were exsanguinated and decollated, and the brains were used for brain water content measurement.Results:All the models were qualified 72 hours after SAH. It was observed that SAH induced a significant increase of MDA, caspase-12/3 and brain water content. Correspondingly, in the SAH group, obvious brain injury was found by Nissl and TUNEL staining. Similar results were found in the vehicle group. In contrast, in the hydrogen-rich saline group, the levels of MDA, caspase-12/3 and brain water content decreased markedly and H2 activated NF-κB/Bcl-xL pathway significantly. However, H2 did not change the level of the NF-κB related inflammation factors significantly. Correspondingly, the brain injury was substantially alleviated by H2 treatment.Conclusion:H2 treatment alleviated EBI in the rabbits following SAH, and the protective role of H2 is associated with the decreased oxidative stress, suppressed brain edema and activated NF-κB/Bcl-xL pathway. Thus, H2 may serve as a novel therapeutic for SAH.