Six Mice from each group without undergoing behavior test were anesthetized with chloral hydrate and then sacrificed. To evaluate histological damage, mice were perfused with saline and paraformaldehyde. Their brains were removed, fixed in paraformaldehyde for 24 h, dehydrated in alcohol, and embedded in wax. The wax was trimmed and sectioned into 4 μm slices for staining with HE and Nissl. Then, the sections were dewaxed and dehydrated using xylene and ethanol solutions before being rinsed with tap water. For HE staining, they were stained with hematoxylin solution (Servicebio, G1003) and treated with differentiation and bluing solutions before being fixed with ethanol and stained with Eosin dye. The sections were then dehydrated and placed in xylene before being sealed with neutral gum. For Nissl Staining, they were stained with Nissl dye (Servicebio, G1036) and treated with a differentiation solution before being rinsed and sealed with neutral gum. The pathological changes in the hippocampus were observed under a light microscope. The Nissl-stained positive neurons in the hippocampal dentate gyrus (DG) region were counted under a light microscope. Besides, each section were visually counted in a blinded manner. The results show the different number of surviving neurons in CIH group compared to the CON group in same regions (Chu et al., 2019 (link); Ke et al., 2020 (link)).
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