Ethical Approval and Animal Grouping
The experimental protocols were reviewed and approved by the Ethics Committee on Animal Studies of Shanghai East Hospital, Tongji University School of Medicine. Animal welfare was assessed and guided strictly following the guideline in Research Animal welfare of Shanghai East Hospital. Thirty three-months-old male Sprague-Dawley rats (SD) were used in this study (SLAC Laboratory Animal Co., Ltd, Shanghai, China). All rats were group-housed in standard plastic cages by 3 per cage and raised in the specific pathogen-free (SPF) laboratory of Shanghai east hospital. A total of twenty seven rats were randomly assigned into three groups: the operational Group A applied with compressive device (n = 9), the Sham Group B carrying devices without compression (n = 9), and the control Group C (n = 9), respectively. Three rats were randomly chosen for sacrifice in each group at time frames (4, 8 and 12 weeks).
Establishment Of The Compression Device
The custom-made device consists of three main parts (Fig. 1). 1) For the compressive unit, one geared motor equipped with screw rod were penetrated through three aluminum rings (with four drilled holes); one pressure sensor was inserted in between two aluminum rings (Fig. 1a, b). 2) The control box that connects the compressive unit through wire provides one screen which shows the compressive force, one signal receiving unit and one power bank that connects to the above two units for electrical energy (inside of the box), as shown in Fig. 1c. 3) An infrared remote controller was provided for increasing or decreasing compressive force, as shown in Fig. 1d.
Surgical Procedures
As shown in Fig. 2, the model was both installed in the tail of SD rats in operation group A and the sham group B. Animals were anesthetized with the injection of 4% chloral hydrate (0.1 ml/kg body wt, Sangon Biotech, Shanghai, China) by means of intra-peritoneal local anesthesia (Fig. 2a). ). The prone position was taken for the operation. First, the compressive unit was penetrated through the tail. Four Kirschner wires (0.9 mm) were respectively inserted into the center of 8th and 9th coccygeal vertebrae (Co 8/9) through two aluminum rings. Then an instruction of increasing force was taken by pressing the button on the infrared remote controller in order to press the disc between Co 8 and 9. As previous studies described [10, 13], 1.3 MPa compressive stress was installed to the targeted disc. The diameters and area of Co 8/9 disc was measured and calculated using a Vernier caliper tightened to the skin of Co 8/9 disc (Fig. 2b). Then the force value that needed to be conducted was calculated according to pressure-force-area formula. The force value in Newton (N) can be displayed in real time on the screen of the control box, therefore an increased compression command was continuously given to the control box until the pre-determined force value was reached (Fig. 2c). The loading duration was set as 8-hours loading and 16-hours free loads in one single day [14]. A protection of shield was then installed around the compressive unit in case of rat-biting damage to the connecting wires. For rats in sham operation group B, force compression command was not performed after the puncture of Kirschner wires. Amoxicillin trihydrate subcutaneous injection (7 mg/kg body wt, Yuanye Bio-Technology Co., Ltd, Shanghai, China) was used to prevent infection within one week after the surgery. Pain killer analgesic ibuprofen granules (5 mg/kg body wt, HPGC, Harbin, China) for one week were used by intragastric administration.
Mri Examinations
After 4, 8 and 12 weeks of the operation, the Co 8/9 disc of each 3 rats in Group A, B and C were respectively sacrificed for magnetic resonance imaging (MRI) examination. A MRI scanner (Achieva 3.0T; Philips Medical Systems, Best, the Netherlands) was used with the following parameters: T1-weighted imaging: TR = 300 ms, TE = 20 ms, FOV = 60mm × 60 mm, data matrix = 148 × 150, and slice thickness = 1 mm. T2-weighted imaging: TR = 5000 ms, TE = 50 ms, FOV = 60 mm × 60 mm, data matrix = 200 × 200, and slice thickness = 1 mm. Two senior radiologists independently read and assessed the MRI image results. The degree of IVD was assessed using the Pfirrmann grading classification system [15].
Formalin-fixed Paraffin-embedded Tissue Preparation
After 4, 8 and 12 weeks of axial compression, the rats were sacrificed by fixation procedure using 4% paraformaldehyde perfused via the circulation system [16]. The targeted disc unit (Co8/9) was extracted and fixed in 4% paraformaldehyde, decalcified in 10% 0.5M ethylenediaminetetraacetic acid (EDTA) (Servicebio Co., Ltd, Wuhan, China) and then embedded in EM-400 embedding medium paraffin. Disc tissues were sectioned at 5 µm thickness using a microtome (Leica RM2235, Biosystems, Wetzlar, Germany).
Histological Evaluations
The disc sections were stained with hematoxylin and eosin (HE) and Safranin-O Fast Green (SOFG) and analyzed qualitatively to observe the morphological changes of degenerated cartilage endplates using a microscope (Leica DM6000B, Microsystems, Wetzlar, Germany). A histological classification system was used to assess the characteristics of degeneration [17].
Immunohistochemistrical Staining
Matrix collagen II and collagen II were performed as markers of endplate and IVD degeneration. The immunohistochemical staining was performed using the following primary antibodies: mouse monoclonal collagen II antibody (1:150, ab185430, Abcam, Cambridge, UK); mouse monoclonal collagen II antibody (1:150 dilution, ab3773, Abcam, Cambridge, UK). For detection, 3, 30-diaminobenzidine was used; positive cells were stained brown. Nuclei were counter-stained blue with hematoxylin. All stained preparations were photographed under a microscope (Leica DM6000B, Microsystems, Wetzlar, Germany). Then the stained sections were semi-quantitatively analyzed by means of Image-Pro Plus 7.0 software (Media Cybernetics, Inc., Rockville, USA). The average optical density was measured on the images at 400 × magnification.
Statistical analysis
Two-tailed Student t-test was performed using SPSS 20.0 version (IBM Inc., Chicago, IL, USA). The experiment data were presented as mean ± standard deviation (SD). The level of statistical significance was set at P < 0.05.