Intervertebral Disc Degeneration Modulation

All animal experiments in this study were approved by the Animal Care and Use Committee of Henan Provincial Orthopedic Institute, and we followed the protocols of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978). Based on a previous study [11 (link)], male rats weighing 250–300 g were anesthetized with 2.5% pentobarbital sodium, and then, the IVD of L4-5 was exposed using a transabdominal median approach. Subsequently, the IVD was penetrated vertically to reach the NP using an 18-gauge needle at a depth of 2 mm, and then, drugs were administered with a microsyringe. After that, the incision was sutured layer by layer with silk thread, and the animals were kept warm until they regained consciousness. For the behavioral study, seven groups were established: the sham-surgery group, puncture + saline group, puncture + NAC (N-acetyl-L-cysteine) group, puncture + H₂O₂ group, puncture + SP 0.1 μg group, puncture + SP 1 μg group, and puncture + aprepitant (an antagonist of the neurokinin 1 receptor, which blocks the effect of SP) group. To avoid bias, analgesic drugs and antibiotics were not used before or after the surgery. H₂O₂ was diluted in deionized water and administered at a concentration of 100 μM per disc after puncture. NAC (CAS No. 616-91-1, MedChemExpress, NJ, US) was administered at a concentration of 1 mM per disc after puncture. Substance P (cat No. 1156/5, R&D Inc., MN, US) was administered at a concentration of 0.1 μg or 1 μg per disc, and aprepitant (CAS No. 170729-80-3, MedChemExpress, NJ, US) was administered at a concentration of 1 mM per disc. For immunohistochemistry (IHC) and Western blot analysis about H₂O₂-indcued SP, the H₂O₂ was inoculated into rodent IVD with a 24-gauge needle, and the tissue was harvested 24 hours later.