Male C57BL/6 mice (10-12 weeks) were devoted to generate needle puncture-induced intervertebral disc degeneration model. For intervertebral disc degeneration (IVDD) treatment, hesperidin was administratedorally and the dose was calculated in reference to previous studies using themetrological conversion formula between human and mouse. The dosein mice is = 5.5 mg/kg x 70 kg x 0.0026/20g = 9.1 x 5.5 mg/kg = 50.05 mg/kg ~50 mg/kg.

24 male C57BL/6J mice (8 weeks, weighing 20 ± 2 g) were purchased from Charles River (Beijing, China). Mice were anesthetized via an intraperitoneal injection of 3% pentobarbital sodium (40 mg/kg). Afterward, their extraperitoneal space was isolated and the exterior part of IVD was exposed. A 26 G puncture needle (90% of intervertebral space height) was utilized to punch in the C6/C7 IVD at a depth of 2.5 mm. The puncture needle was inserted into the dorsal annulus fibrosus across the center of the nucleus pulposus and partially across the ventral annulus fibrosus and withdrawn 30 s later.

24 male C57BL/6J mice (8 weeks, weighing 20 ± 2 g) were purchased from Charles River (Beijing, China). Mice were anesthetized via an intraperitoneal injection of 3% pentobarbital sodium (40 mg/kg). Afterward, their extraperitoneal space was isolated and the exterior part of IVD was exposed. A 26 G puncture needle (90% of intervertebral space height) was utilized to punch in the C6/C7 IVD at a depth of 2.5 mm. The puncture needle was inserted into the dorsal annulus fibrosus across the center of the nucleus pulposus and partially across the ventral annulus fibrosus and withdrawn 30 s later.

8-10 weeks old male mice were placed in prone position and anesthetized with isoflurane inhalation. The 1.5 cm-long longitudinal incision was made in 2 mm from the posterior midline. The superior and inferior articular processes, supraspinous ligament and interspinous ligament of the L4-L5 lumbar vertebrae were removed to construct LSI, which would induce IVDD. After the operation, the mice were placed in a warm environment.

8-10 weeks old male mice were placed in prone position and anesthetized with isoflurane inhalation. The 1.5 cm-long longitudinal incision was made in 2 mm from the posterior midline. The superior and inferior articular processes, supraspinous ligament and interspinous ligament of the L4-L5 lumbar vertebrae were removed to construct LSI, which would induce IVDD. After the operation, the mice were placed in a warm environment.

Thirty male C57BL/6 mice (8 weeks old, average weight 25 g) were divided into three groups. Blank group: Ten mice were fed with normal diet and water. HHcy group: Ten mice were fed with a high Met diet (normal diet with 2% l-methionine). HHcy + Folic Acidrescue group: Ten mice were fed withhigh Met diet (normal diet with 2% l-methionine), and injected with Folic Acid (1.0 umol/kg/d) twice a week two months later. At the beginning of treatment, mice were anesthetized with anintraperitoneal injectionof 0.3% (w/v)pentobarbital sodium(20 uL/g) before surgery and disc degeneration was induced by stabbing the C5-6 and C6-7 discs with a 32G needle.

Male C57BL/6 mice (10-12 weeks) were devoted to generate needle puncture-induced intervertebral disc degeneration model. For intervertebral disc degeneration (IVDD) treatment, hesperidin was administratedorally and the dose was calculated in reference to previous studies using themetrological conversion formula between human and mouse. The dosein mice is = 5.5 mg/kg x 70 kg x 0.0026/20g = 9.1 x 5.5 mg/kg = 50.05 mg/kg ~50 mg/kg.