Neuroprotective Effects of Early Brain Injury after Subarachnoid Hemorrhage in Rats by Calcium Channel Mediating Hydrogen Sulfide

Abstract

The present study explored the modulating apoptosis effect of hydrogen sulfide (H₂S) in subarachnoid hemorrhage (SAH) rats and its exact mechanism. A rat SAH model established by intravascular puncturing was used for the present study. After giving NaHS (donor of H₂S), an L-type calcium channel opener (Bay K8644), or a calcium channel agonist (nifedipine), the neurological function of the rats, associated pathological changes, and expression of apoptosis-related proteins (Bcl-2, Bax, and caspase-3) and microtubule-associated protein (MAP-2) were examined. The concentration of H₂S and expression of cystathionine beta synthase in the hippocampus changed upon early brain injury (EBI) after SAH. Compared with the SAH group, the neurological function of the rats and microstructure observed by electron microscopy were better in the SAH + NaHS group and SAH + Bay K8644 group. It was observed that apoptosis was more obvious in the SAH group than in the control group and was alleviated in the SAH + NaHS group. Furthermore, the alleviating effect of NaHS was partially weakened by nifedipine, indicating that the effect of anti-apoptosis in H₂S might be correlated with the calcium channel. The expression of Bax and caspase-3 was elevated, while the expression of Bcl-2 decreased in the SAH group but improved in the SAH + NaHS and SAH + Bay K8644 group. Compared with the SAH + NaHS group, the expression of pro-apoptotic proteins was higher in the SAH + NaHS + nifedipine group. Therefore, upon EBI following SAH, the H₂S system plays an important neurological protective effect by modulating the function of the L-type calcium channel and inhibiting apoptosis.

Keywords: Apoptosis; Calcium channel; Early brain injury; Hydrogen sulfide; Subarachnoid hemorrhage.

Fig. 1

The neurological scores of the rats in each group at 24 h after surgery. The scores were lower in the four SAH model groups compared with the control group. However, there was no statistical significance among the four SAH groups. *Means compared with the control group (normal), P < 0.05

Fig. 2

a–e structure of the hippocampal neurons of the rats in each group under an electron microscope. a control group; b SAH group; c SAH + NaHS group; d SAH + Bay K8644 group; e SAH + NaHS + nifedipine group. f H₂S concentration in the hippocampus of the rats in each group. *Means compared with the control group (normal), P < 0.05; (filled diamond) means compared with the SAH group, P < 0.05

Fig. 3

The expression of CBS, Bax, and Bcl-2 via the western blotting in each group (a) and the statistical results (b). *Means compared with the control group (normal), P < 0.05; (filled diamond) means compared with the SAH group, P < 0.05; (filled star) means compared with the SAH + NaHS group, P < 0.05. c The average optical density of the immunohistochemical staining results of MAP-2 and Caspase-3 in each group. *Means compared with the control group (normal), P < 0.05; (filled diamond) means compared with the SAH group, P < 0.05; (filled star) means compared with the SAH + NaHS group, P < 0.05. d: The detection of hippocampal neuron apoptosis in each group; the sections were labeled with TUNEL (green) to detect apoptotic cells and counterstained with DAPI (blue) to detect the nuclei. Immunohistochemical staining of MAP-2 and Caspase-3 in each group; scale bar = 50 μm