The present study was approved by the Institutional Animal Care and Use Committee. Altogether, 60, 3-month-old Sprague Dawley (SD) rats (Vital River Experimental Animal Technical Co., Ltd., Beijing) weighing 216 ± 14 g (mean ± SD) were randomized into two groups, including the sham group (sham surgery, n = 24) and OVX group (bilateral ovariectomy, n = 36). The animals in the sham surgery group were randomly assigned to the sham group (n = 12) and PLF group (n = 12). The animals in the OVX group were randomly assigned to the OVX group (n = 12), OVX+PLF group (n = 12), and OVX+PLF+RAL group (n = 12). PLF surgery was performed at L4–L5 using a previously described procedure [15, 16]. The rats in the OVX+PLF+RAL group were administered 1 mg/kg/d RAL by gavage for 12 weeks. All rats were euthanized at 12 weeks post-PLF to collect the L3–L6 segment.
The rats were maintained at 21 ± 1 °C and 12-h light/dark cycle conditions and were provided food and water freely (HFK Bioscience Co., Ltd., Beijing).
Manual palpation and X-ray analysis
The L3–L6 segment fusion was evaluated in the lateral recumbent position by soft radiography (DR7500 System, Kodak, USA). The disk height index (DHI) was determined by this formula: anterior disk height + posterior disk height/anterior vertebral bone height + posterior vertebral bone height. Manual palpation is considered a gold standard for assessing the success of pseudarthrosis formation and fusion . The fusion scores were evaluated according to the criteria established by O’Loughlin et al. .
For investigating changes in vertebrae, the SkyScan 1176 microcomputed tomography system (80 kV, 313 μA, 18 μm) was used to scan the L5-L6 segments. One inner cylinder with a diameter and length of 1.5 mm and 3 mm, respectively, was chosen to be the region of interest (ROI) in L6 vertebrae at the cephalad level. To evaluate the vertebral trabecular structural parameters, we determined the bone mineral density (BMD), trabecular number (Tb.N), trabecular separation (Tb.Sp), and bone volume fraction (BV/TV).
Histology and immunohistochemistry examinations
Neutral paraformaldehyde (10%) was used to fix the specimens for 48 h at room temperature. Ethylenediaminetetraacetic acid (EDTA)-2Na (10%) was used for further decalcification for 3 months. Thereafter, each specimen was dehydrated, paraffinized, and embedded. Next, each sample was cut into 8-μm sections for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, Van Gieson (VG) staining, and immunohistochemical (IHC) analysis. VG and TUNEL staining were performed as per the protocols provided with the BA408A VG kit and MA0224 one-step TUNEL apoptosis kit, respectively. The L5–L6 IVD degeneration was studied using histological scoring , which was performed by two independent researchers in a blinded manner.
Expression of caspase 3 (1:100; Gene Tex Inc., USA), ADAMTS-4 (1:100; Abcam Inc, USA), matrix metalloproteinase-13 (MMP-13) (1:500; Boster Co., Ltd., Wuhan, China), aggrecan (1:100; Abcam Inc., USA), BCL2-associated X (bax) (1:200; Abcam Inc., USA), and B cell lymphoma-2 (bcl2) (1:300; Abcam Inc., USA) in NP was detected by IHC. Each section was deparaffinized, rehydrated, and immunostained in succession, followed by 30 min of incubation by pancreatin to retrieve the antigens at 37 °C. Afterwards, 3% H2O2 was used to block endogenous peroxidase activity, and primary antibodies were used to incubate overnight under 4 °C. On the next day, each section was rinsed with Tris-buffered saline for 15 min, followed by further incubation using biotin-labeled goat anti-rabbit IgG (ZSGB-BIO, PV-6000). Later, diaminobenzidine (ZSGB-BIO Corp., China), a chromogenic substrate, was used for color development, and hematoxylin was used to counterstain the sections.
The Imaging Pro Plus 6.0 software (Media Cybernetics, Inc., USA) was used to calculate the ROI and to integrate the optical density (IOD). For measuring the mean IOD of certain proteins, we divided the total IOD by ROI, which was reported as IOD/mm2.
SPSS20.0 (SPSS Inc.; Chicago, IL, USA) was applied in statistical analysis. Values were expressed in as mean ± SD. Normal distribution and homogeneity of variances were evaluated by Shapiro–Wilk and Bartlett’s test. One-way analysis of variance (ANOVA) and Fisher’s protected least significant difference (LSD) test were adopted for analyzing significant differences. Kruskal–Wallis test was applied in analyzing lumbar fusion scores. P < 0.05 suggested that a difference was of statistical significance.