Immunohistochemistry

Under sevoflurane deep anesthesia, the rats were perfused through the heart with paraformaldehyde, and the L3–L5 segments of the lumbar spinal cord were quickly removed and embedded in paraffin. The paraffin-embedded tissues were cut transversely and mounted on glass slides. Sections were washed with phosphate-buffered saline (PBS) and incubated overnight at 4°C with a primary antibody targeting the microglia-specific marker ionized calcium binding adaptor molecule 1 (IBA-1; 1:2000; Abcam). Hematoxylin and eosin staining was used to counterstain the sections. Microglial activation of the spinal cord was quantified using ImageJ software by counting IBA-1-positive cells. ²²