A total of 15 skeletally mature female New Zealand White rabbits (3-months-old, weighing 2–2.5 kg; Chongqing Kangda Juxin Rabbit Co., Ltd., Qingdao, China) were randomly divided into three groups: Group A, punctured blank control group (degeneration and placebo injection, n=5); group B, punctured empty vector control group (degeneration and an empty LV injection, n=5); and group C, treatment group (degeneration and LV-survivin injection, n=5). LV-survivin and empty LV were purchased from Genechem Co., Ltd. (Shanghai, China). The titers of LV-survivin and empty LV were 2×10⁸ and 1×10⁹ TU/ml, respectively. The Animal Care and Use Committee of the Affiliated Hospital of Qingdao University (Shandong, China), approved all animal experimental protocols, which followed the principles expressed in the National Institute of Health Guide. The rabbits were maintained separately at room temperature with 16:8 h light:dark cycle, and were fed with rabbit feed (Qingdao Kangda Foodstuffs Co., Ltd., Qingdao, China).

Computed tomography (CT)-guided percutaneous needle puncture technology was used to establish this model (18). Magnetic resonance imaging (MRI) and histology highlighted alterations that resembled the hallmarks of human disc degeneration. In brief, rabbits were anesthetized with Su Mian Xin II (0.15 ml/kg intramuscularly; Kangda Medical Products Co., Ltd., Shanghai, China) and atropine sulfate (0.05 mg/kg intramuscularly; Jiangsu Lianshui Pharmaceutical Co., Ltd., Lianshui, China), the position of the target discs (L3-L4 and L4-/L5) were identified using CT, and then a 16-gauge needle was inserted toward the center of the disc, with CT guidance. The needle pinpoint was confirmed to be positioned in the disc center by CT (Fig. 1). Postoperatively, the rabbits were housed in individual cages and injected intramuscularly with penicillin at 80×10⁵ U to prevent infection.

At 3 weeks post-puncture surgery, groups A, B and C were injected with equivalent phosphate-buffered saline (PBS; 50 µl), empty LV (5×10⁶/50 µl; Genechem Co., Ltd., Shanghai, China), and LV-survivin (5×10⁶/50 µl; Genechem Co., Ltd.), respectively, using CT-guided percutaneous needle injection technology. This time point was selected due to the fact that it is the earliest that MRI could identify changes of degeneration with this model (18). An 100 µl microinjector (Hamilton Bonaduz AG, Bonaduz, Switzerland) was used for therapeutic injections into the center of the NP.

MRIs were obtained at time-point 0 (prior to puncture), 3 weeks post-puncture (prior to injection), and 12 weeks post-puncture (prior to sacrifice) for all rabbits. A 3-T standard human knee coil was used to obtain T2-weighted images (repetition time=2120 ms, echo time=113 ms, slice thickness=0.6 mm). The rabbits were anesthetized and placed in the knee coil in the supine position.

All rabbits were sacrificed by air injection into the ear vein 12 weeks subsequent to the initial puncture surgery, following the final MRI scan. Immediately after sacrifice, spines were dissected out en bloc. The L3-L4 discs (n=5 per group) were prepared for histology. Two discs from each group were fixed and then dehydrated using a histology tissue processor and embedded in paraffin. Next, the discs were sectioned at a thickness of 5 µm in the coronal plane. The sections were stained with hematoxylin and eosin (H&E; Beijing Leagene Biotechnology Co., Ltd., Beijing, China). The other discs were embedded in Tissue-Tek™ (Sakura, CA, USA) and then sectioned at a thickness of 5 µm in the coronal plane in a freezing microtome (Leica CM1950; Leica Microsystems GmbH, Wetzlar, Germany). The sections were stained for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL; Nanjing KeyGEN Biotech Co., Ltd., Nanjing, China) and were imaged using an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan).

A total of 3 L4-L5 discs from each group (n=5 per group) were prepared for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. mRNA was extracted from NP tissue using TRIzol™ (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and the absorbance at was measured at 260 and 280 nm for quantification and quality control. A total of 1 µg mRNA was reverse transcribed to cDNA using PrimeScript™ RT reagent kit (cat no. DRR037A; Takara Bio, Inc., Otsu, Japan), and the reaction product was treated with RNAse-free DNase I. PCR was conducted using the cycling conditions (LightCycler 480 Instrument II) as stated by the manufacturer's instructions. Primers were designed and purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China) (Table I). Another specific primer pair for human GAPDH was used as an internal control. In each experiment, samples were analyzed in duplicate. The normalized target gene expression was determined through the comparative Cq method (ΔΔCq method) (19).

NP tissues were washed with ice-cold PBS, weighed and then ground using a pestle and mortar. Lysis was performed on ice for 45 min using 200 µl radioimmunoprecipitation assay buffer with 2 µl phenylmethanesulfonylfluoride for every 100 µg of tissue. Subsequently, the lysis solution was centrifuged at 5,700 × g at 4°C for 20 min. For the western blot analysis of survivin, GAPDH was used as an internal control, and proteins were resolved through sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 10% gel, prior to transferring onto Immobilon P membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% fat-free dried milk and probed with a monoclonal rabbit anti-survivin antibody (1:1,000; ab76424; Abcam, Cambridge, MA) and anti-GAPDH antibody (1:2,000; ab9485; Abcam) for 8 h at 4°C. Subsequent to incubation with horseradish peroxidase-conjugated secondary antibodies (1:2,000; CW0103; Beijing Kangwei Century Biotechnology Co., Ltd., Beijing, China) for 4 h at room temperature, the positive bands were visualized using chemiluminescence (Pierce Biotechnology, Inc., Rockford, IL, USA).

The remaining L4-L5 discs were prepared for measuring caspase-3 activity. NP cells were cultured as described in a previous study (20). In brief, the NP tissue was cut into small pieces (~1 mm²) and then digested with 0.25% trypsinase (HyClone™; GE Healthcare Life Sciences, Logan, UT, USA) at 37°C under gentle agitation for 20 min. Subsequently, 0.5% collagenase type II (MP Biomedicals, LLC, Santa Ana, CA, USA) was used at 37°C for approximately 4 h. The cells were transferred to a 12.5 cm² culture flask at a density of 10⁵ cells/cm². The cells were cultured in a CO₂ incubator (SANYO Electric Co., Ltd., Osaka, Japan) at 37°C with humidity, and grown in Dulbecco's modified Eagle's medium (DMEM)-F12 containing 15% fetal calf serum. The experiments were analyzed in duplicate. For ischemic conditions, NP cells were cultured in DMEM culture medium (glucose deprivation) in a CO₂ incubator at 37°C with 1% oxygen and 95% humidity. These cells were subsequently used for analyzing caspase-3 activity. The caspase-3 activity was measured using the Caspase-3 Colorimetric Assay kit (BioVision, Inc., Milpitas, CA, USA). The NP cells were counted and pelleted at 1.5×10⁶ cells. The cells were then re-suspended in cell lysis buffer, and 50 µl 2X reaction buffer (containing 10 mM DTT) and 5 µl of DEVE-pNA were added. The samples were incubated for 90 min at 37°C and their optical densities were read at 405 nm using a microtiter plate reader (Sunrise™; Tecan Group, Ltd., Männedorf, Switzerland).

All values were presented as the mean ± standard error. Student's t-test and one-way analysis of variance with a post hoc Fisher's least significant difference test were applied to measure the statistical significance of the differences. Statistical analyses were performed using SPSS software for Windows (version 19; SPSS, Inc., Chicago, IL, USA).

The T2-weighted midsagittal MRI scans of groups A and B were darkened and reduced in area from 0–12 weeks, consistent with the degeneration. Group C demonstrated reduced qualitative evidence of degeneration when compared with groups A and B; treated NPs did not darken and reduce in area as much as the punctured discs (Fig. 2). The morphology of normal discs indicated an abundance of water and ECM. Discs from groups A and B were reduced in the quantity of both water and ECM, and exhibited fibrotic morphology, consistent with the degree of degeneration. However, the quantity of water and ECM in group C were between that of the normal group (group A) and the punctured group (group B) (Fig. 3).

The gene and protein content of each group were analyzed by RT-qPCR and western blot analysis. Gene expression and protein content of survivin in group C demonstrated a significant increase compared with groups A and B (P<0.05). No significant difference was observed between groups A and B (Fig. 4).

The NP cells were attached to the culture dish after 3–5 days of culture. The cell morphology gradually elongated and became triangular or polygonal, and the cytoplasm became plump and equally distributed, and the attached cells exponentially increased. Subsequently, 10–15 days later, 90% of the cells formed colonies. No significant differences of cell morphology were observed among the three groups (Fig. 5).

The caspase-3 activity of NP cells was assayed. Under the normal culture conditions, group C did not exhibit significantly altered caspase-3 activity compared with groups A and B (P=0.082 and P=0.539, respectively). When exposed to ischemia in vitro (1% oxygen, glucose deprivation), the caspase-3 activity of all groups increased compared with the normal culture conditions (P=0.00). However, caspase-3 activity remained significantly reduced overall for group C (group C + in vitro ischemia vs. group A + in vitro ischemia and group B + in vitro ischemia: P=0.00 and P=0.01, respectively) (Fig. 6).

The histological analysis of NP tissues was performed using H&E staining. Under the highest magnification, the number of NP cells in group C was significantly increased compared with groups A and B (Fig. 7).

Apotosis analysis of NP tissues was performed using TUNEL staining. The TUNEL-positive NP cells (brown cells) in group C were markedly reduced compared with groups A and B (Fig. 7).