To explore the protective effect of in-taking high concentration hydrogen gas on neurons and dendritic spines in hippocampus CA1 region of rats after globe cerebral ischemia/reperfusion (I/R) injury and its mechanism.

Four-vessel occlusion (4VO) was used to establish the models of global cerebral I/R injury in rats. One hundred and twenty healthy male Sprague-Dawley rats were randomly divided into 3 groups using a random number table: sham-operated group (inhaled 67% N₂ and 67% O₂, n=40), model group (inhaled 67% N₂ and 67% O₂ during reperfusion, n=40), and treatment group (inhaled 67% H₂ and 67% O₂ during reperfusion, n=40). After 72 h, 5 and 9 d reperfusion, neuron-specific nuclear (NeuN) protein expression in the pyramidal neurons of the hippocampal CA1 region was detected with immumohistochemical staining and the positive cells were counted. And the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) in serum were tested with colorimetry. Water maze test was used to measure the spatial orientation and memory function and Golgi staining to detect the number of dendritic spines in neurons 9 d after reperfusion.

(1) Immunohistochemical staining of NeuN results showed that as compared with those in the model group, the neurons of hippocampus CA1 region were significantly closer to normal with relatively intact structure, and the number of positive neurons was significantly increased in the treatment group 72 h, 5 d, and 9 d after reperfusion (P<0.05). With the reperfusion time being prolonged, the number of NeuN stained positive neurons at different time points of reperfusion in model group was gradually decreased (P<0.05), and the numeric of the NeuN stained positive neurons at different time points of reperfusion in treatment group was slightly declined without significant difference (P>0.05). (2) The serum SOD activity in the treatment group was significantly higher than that in the model group and sham- operated group (P<0.05), while the MDA content in the treatment group was significantly lower than that in model group 72 h, 5 d, and 9 d after reperfusion (P<0.05). And with the reperfusion time being prolonged, the SOD activity at different time points of reperfusion showed no significant difference among the difference groups (P>0.05). But the MDA content at different time points of reperfusion between model group and treatment group was significantly different (P<0.05); with the reperfusion time being prolonged, the MDA content was gradually decreased in both groups. (3) Nine d after reperfusion, water maze test found that the incubation period of treatment group was significantly shorter than that of model group (P<0.05); the IV quadrant swimming time of space exploration in the treatment group was significantly longer than that in the model group (P<0.05). (4) Golgi staining showed that the complexity of the neuronal dendrites branch and the number of dendritic spines of neurons in the hippocampal CA1 region of treatment group were increased than those in the model group; high-power oil microscopy indicated that the density of dendritic spines in the treatment group was significantly higher than that in the model group (P<0.05).

In-taking of high concentrations hydrogen gas during reperfusion can definitely protect neurons in hippocampal CA1 region after globe cerebral I/R injury, and improve learning and memory function, whose mechanism may be related to hydrogen protecting the structure and function of neurons and dendritic spines, and inhibiting oxidative stress to reduce oxidative damage.

Hydrogen; Ischemia-reperfusion injury; Neuron; Dendritic spine; Oxidative stress