Chemicals and Reagents
Primary antibodies against Aggrecan (ab36861), Adamt5 (ab41037), Calcitonin gene-related peptide (Cgrp, ab81887) were purchased from Abcam (Cambridge, UK), Type II collagen (Col2,MAB1330) was purchased from Millipore (Billerica, MA, USA), IL-1β (bs-6319R) was purchased from Bioss (Woburn, MA, USA), Nod-like receptor protein-3 (Nlrp3, 19771-1-AP), Caspase-1 (22915-1-AP), and Lef1 (14972-1-AP) were purchased from Proteintech (Wuhan, China), Mmp3 (RLT4465) and β-catenin (RLM3403) were purchased from Ruiying Biological Co. (Jiangsu, China). Unless otherwise mentioned, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Experimental Animals And Treatments
A total of 72 C57BL/6J male mice (8-week-old, 22 ± 2 g) were purchased from Shanghai Laboratory Animal Center and housed at Animal Care Facility of Chinese Medical University according to the institutional guidelines for laboratory animals. All the animal procedures were approved by the Ethical Committee of Zhejiang Chinese Medical University. All mice were randomly divided into LSI surgery group (IVDD group), WBV group and SHAM group (n = 24 per group).
After anesthetized with pentobarbital sodium, mice in the IVDD group and the WBV group were operated by resecting the lumbar 3rd − 5th (L3–L5) spinous processes along with the supraspinous and interspinous ligaments to induce instability of lumbar spine [21, 22] (Fig. 1a). Three days post-surgery, mice in the WBV group was further exposed to a commercial WBV platform (Jin Ri Li Co., Shenzhen, China) with a fixed frequency, amplitude, and duration of exposure (3 HZ with peak acceleration at 0.4 g, 1 h per day, 5 days per week) (Fig. 1b). Mice in the SHAM group were only treated with the detachment of the posterior paravertebral muscles from the L3–L5 vertebrae. L3–L5 vertebrae of mice were harvested at 0, 1, 2, 4 and 8 weeks after the LSI surgery for further analysis (n = 6 per group at the indicated time) (Fig. 1c).
MRI Evaluation
Mice underwent T2-weighted lumbar MRI scan using a 3.0T MR scanning system (Philips, Holland) 8 weeks after LSI surgery. T2-weighted sagittal sections were rendered using a fast spin-echo sequence with time to a repetition of 500 ms and a time to echo of 8 ms.
Micro Computed Tomography (μCT)
The lower thoracic and whole lumbar spine from mice were dissected, fixed in 4% buffered paraformaldehyde for 72 h, transferred into phosphate-buffered saline, and then examined by high-resolution µCT (Skyscan 1176; Bruker µCT, Kontich, Belgium). The ribs on the lower thoracic were included for the identification of L4-L5 IVD localization. Images were reconstructed and analyzed using NRecon v1.6 and CTAn v1.9 (Skyscan company, San Jose, CA, USA), respectively. Three-dimension model visualization software, CTVol v2.0 (Skyscan company, San Jose, CA, USA), was used to analyze parameters of the L4-L5 IVD with the half-height of the L4 and L5 vertebrae. The scanner was set at a voltage of 90 kV, a current of 300 µA, and a resolution of 9 µm per pixel to measure the IVD. A resolution 9 µm per pixel was set for the whole L5 vertebral body measurement. Coronal images of the L4-L5 IVD were used to perform three-dimension histo-morphometric analyses of IVD. IVD volume was defined by the region of interest to cover the whole invisible space between L4 and L5 vertebrae.
Histology, Immunohistochemistry (IHC), And Immunofluorescent (IF)
After fixed in 4% buffered Paraformaldehyde for 72 h, the tissues were decalcified in 14% EDTA (pH 7.4) for 21 days at room temperature, dehydrated and then embedded in paraffin. Five-micrometer-thick coronal-oriented sections of the L4–L5 IVD, were processed for Safranin O/Fast green staining. A histological grading scale established by Norcross et al was developed and tested for the degree of IVDD in a blind fashion, with some modifications (Table 1) [23]. Immunostaining was performed using a standard protocol. Sections were incubated with primary antibodies to Aggrecan (1:300), Col2 (1:1000), Mmp3 (1:300), Adamt5 (1:300), Nlrp3 (1:800), Casepase1 (1:300), IL-1β (1:800), Cgrp (1:200), β-catenin (1:500) and Lef1 (1:300) at 4℃ overnight. For IHC staining, a horseradish peroxidase streptavidin detection system (ZSGB-BIO, Beijing, China) was subsequently used to detect the immunoactivity, followed by counterstaining with hematoxylin. For IF assay, the slides were incubated with secondary antibodies conjugated with fluorescence at room temperature for 1 h. DAPI staining was used to estimate the total cell number. The images were captured using a microscope (Olympus, Tokyo, Japan).
Table 1
Histological grading scale criteria based on Norcross et al.
Scores | Symptoms |
Nucleus pulposus (NP) |
5 Large, bulging central cavity with abundant NP material; >2/3 (IVD) height; smooth borders with minimal disruption |
4 Slightly reduced central cavity size with some NP material present; >1/3 IVD height and < 2/3 IVD height; minimal border disruption may be present |
3 Markedly reduced and disrupted cavity with minimal NP material and compartmentalization; total cavity > 1/3 IVD height and < 2/3 IVD height |
2 Severe disruption of NP with minimal cavity; total cavity < 1/3 IVD height but > 0; consists only of a few small pockets lined by NP-like cells |
1 Complete obliteration of cavity with no NP-lined pockets |
Annulus fibrosus (AF) |
5 Discrete, well-opposed lamellae bulging outward with no infolding; minimal preparation defect with “simple radial clefting” |
4 Discrete lamellae, less well-opposed; minimal infolding may be present; fibers remain well-organized, but with ‘‘complex radial clefting’’ |
3 Moderate to severe infolding of discrete, relatively well-opposed lamellae; moderate fragmentation of lamellae; AF fibers remain well organized |
2 Severe infolding and distortion of poorly opposed lamellae; severe fragmentation of lamellae; small regions of disorganized fibrous material replacing central lamellae |
1 Severe infolding, distortion, and fragmentation of lamellae; extensive amount of disorganized fibrous material replacing central lamellae |
Cartilage endplate (EP) |
5 Neatly arranged chondrocytes without any lesion or ectopic bone formation |
4 Hyaline cartilage cells were replaced by the spindle-shaped chondrocytes, ectopic bone formation only located at the “junction”. |
3 Ectopic bone formation located at superior cartilage endplate with smooth cartilage surface |
2 Ectopic bone formation located at whole cartilage endplate with smooth cartilage surface |
1 Ectopic bone formation located at whole cartilage endplate with flawed cartilage surface |
This scale mainly scores the disruption of nucleus pulposus central cavity, cellularity and collagen fiber orientation of annulus fibrosus and the degree of ectopia ossification of cartilage endplate. Simple radial clefting = the presence of radial gaps between AF lamellae with minimal fragmentation; complex radial clefting = the presence of radial, transverse, and/or oblique gaps in the lamellae with significant fragmentation; junction = the triangle junction between the cartilage endplate and the annulus fibrosus. |
TUNEL Assay
The TUNEL assay for detecting DNA breaks in IVD tissues was performed with TUNEL BrightGreen Apoptosis Detection Kit according to the manufacturer’s instruction (Vazyme Biotech, Nanjing, China). Negative controls were incubated in a TdT free-enzyme solution. The number of positive cells was quantified in three randomly selected fields of view using three sections from each sample. DAPI staining was used to estimate the total cell number.
Quantitative Real-time PCR
Total RNA was extracted from L4 - L5 IVD using TRIzol reagent (Invitrogen, CA, USA) following the manufacturer’s protocol. The yield and purity of RNA were quantified using nanodrop 2000C spectrophotometer (Thermo, Massachusetts, USA) and 2 µg of RNA were reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit protocol (Takara Bio, Dalian, China). Real-time PCR amplification was performed using murine gene-specific primers (Supplementary Table 1) and TB Green Premix Ex Taq II (Takara) to quantify the mRNA expression levels of Aggrecan, β-Catenin and Lef1. Target mRNA expression levels were normalized against β-actin. The relative expression levels were calculated using the 2-ΔΔCT method [24].
Statistics Analysis
All the data were expressed as mean ± s.d., as indicated in figure legends. All data analyses were performed using SPSS 15.0 analysis software (SPSS Inc, Illinois, USA). Statistical differences between groups were determined using one-way analysis of variance (ANOVA) or Student’s t-test. and the level of significance was defined as P < 0.05.