2.7. Hematoxylin-Eosin (H&E) Stain

After the behavioral test at each time point, 5 mice in each subgroup were randomly selected for hippocampal tissue preparation. 10% chloral hydrate (0.4 mL/100 g) was intraperitoneally injected. After successful anesthesia, the mice were placed on the wooden board in the supine position. The skin was cut through the sternum, and then, the xiphoid was lifted and the chest cavity was cut. The perfusion needle was then inserted into the left ventricle from the apex while the right atrial appendage was cut. Precooled PBS solution was pushed, and the color of the perfusion to the lungs and liver becomes grayish white, and reperfusion of 4% paraformaldehyde was made until the limbs were stiff. The head of the mouse was cut off, the skull was cut to expose the skull, the skull was separated, and the brain was removed gently. The brain tissue was then fixed in neutral formalin solution for 24 h, then dehydrated by alcohol gradient, and finally paraffin-embedded for H&E staining. Paraffin-embedded hippocampal tissues were cut into 3 μm thick and stained with H&E. The pathological changes in the hippocampus were observed under a light microscope. The number and volume of hippocampus cells were measured by using ImageJ software (NIH, Bethesda, USA).