Animal experiments were approved by the Institutional Animal Care and Use Committee. Wistar rats (250 to 300 g; Charles River Laboratories International, Inc., Wilmington, MA, USA) were euthanized with CO₂ and the spinal columns were removed en bloc under aseptic conditions. Lumbar IVDs comprising the vertebral endplates, AF and NP were harvested (six discs per animal). Soft tissues and posterior elements were removed, and discs were rinsed in saline solution before being placed in 48-well culture plates. All experimental specimens were cultured in an hypoxia workstation (Invivo₂; Ruskinn Technology Ltd, Pencoed, UK) in 1% O₂, 5% CO₂, and 93% N₂ in Dulbecco's modified Eagle's medium (DMEM) containing 1% fetal bovine serum (FBS), tumor necrosis factor-alpha (TNF-α) 100 ng/mL, interleukin-1beta (IL-1β) 10 ng/mL (R&D Systems, Inc., Minneapolis, MN, USA), 50 μg/mL L-ascorbate, 40 mM NaCl, antibiotics, and antimycotics (Cellgro; Mediatech, Inc., Manassas, VA, USA). Concentration of cytokines was based on the literature 2829. Importantly, slightly higher doses of cytokines were chosen to compensate for the lack of systemic feedback present from immune and neuronal cells during degeneration. Control groups were cultured in DMEM containing 20% FBS, 50 μg/mL L-ascorbate, 40 mM NaCl, and antibiotics. The discs were maintained for 3 and 10 days. The complete medium was replaced every 2 days for all groups.

To measure the diffusion in the organ-cultured discs, 5 to 10 μg/mL of QuantiLum Firefly luciferase (61 kDa) (Promega Corporation, Madison, WI, USA) and 50 to 100 μg/mL of Renilla luciferase (38 kDa) (Prolume Ltd., Pinetop, AZ, USA) were added to the medium. After 24 hours in culture, discs were immersed in Hank's balanced salt solution supplemented with 40 mM NaCl. An incision was made through the middle of the AF with #15 scalpel blades, and the two halves of the disc were opened. The NP was then scooped out with a Meyhoefer Curette (World Precision Instruments, Sarasota, FL, USA) (Figure 1A). Tissue was harvested, and a Dual-Luciferase Reporter Assay System (Promega Corporation) was used for measurements of Firefly and Renilla luciferase. Quantification was carried out with a luminometer (TD-20/20; Turner Designs, Sunnyvale, CA, USA). All analysis was carried out in triplicate.

For in situ identification of apoptotic cells, a TACS 2 TdT-Fluor In Situ Apoptosis Detection Kit was used on histological sections in accordance with the instructions of the manufacturer (Trevigen Inc., Gaithersburg, MD, USA). In brief, 8-μm tissue sections from paraffin-embedded discs were deparaffinized in xylene, rehydrated, and pretreated with proteinase K solution for 15 minutes at 37°C. The slides were rinsed twice in deionized water and incubated with 50 μL of labeling reaction mixture for 1 hour at 37°C in a humidified chamber. After washing with phosphate-buffered saline (PBS), 50 μL of Strep-Fluor solution was applied, and the slides were incubated for 20 minutes. The slides were washed three times in PBS and mounted under glass coverslips using fluorescence mounting media (Vector Laboratories, Burlingame, CA, USA) and analyzed under a fluorescence microscope using a 495-nm filter (Nikon eclipse TE 2000-U; Nikon, Tokyo, Japan).

Whole disc units, including the endplates, were fixed in 10% formaldehyde in PBS for routine histological and immunohistochemical evaluation. After fixation, disc units were serially dehydrated in alcohol. Decalcification was accomplished with 12.5% ethylenediaminetetraacetic acid (EDTA) (pH 7.4) followed by paraffin embedding. The tissue blocks were sectioned parallel to the endplate (6 to 8 μm in thickness), and sections were deparaffinized in xylene and rehydrated through graded ethanol and stained with Alcian blue, eosin, and hematoxylin. For localizing matrix metalloproteinase 3 (MMP-3), sections were incubated with rabbit monoclonal MMP-3 antibody (EP1186Y; Abcam, Cambridge, MA, USA) in 2% bovine serum albumin in PBS at a dilution of 1:250 at 4°C overnight. After washing of the sections, the bound primary antibody was incubated with Alexa fluor-488 conjugated anti-rabbit secondary antibody (Invitrogen Corporation, Carlsbad, CA, USA) at a dilution of 1:100 for 1 hour at room temperature. Sections were mounted in mounting media containing DAPI (4',6' diamino-2-phenylindole) and visualized with a fluorescence microscope (Olympus, Tokyo, Japan).

The discs were collected and the AF was separated from the NP. The tissues were stabilized in RNA later in accordance with the instructions of the manufacturer (Ambion, Inc., Austin, TX, USA) and stored at −80°C. Total RNA was extracted with an RNeasy Micro kit in accordance with the instructions of the manufacturer (Qiagen Inc., Valencia, CA, USA). The DNA template from the RNA preparation was removed by using the DNase I digestion. cDNAs were made by using 0.5 μg of total RNA, and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed with SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI 7900 HT sequence detection system. PCR was initiated at 95°C for 10 minutes (to activate the AmpliTaq polymerase) followed by a 40-cycle amplification phase (95°C for 15 seconds, anneal/extend at 60°C for 60 seconds). Melting curve analysis was performed to ensure specific PCR product while excluding primer dimers. The data were normalized to the endogenous control, 18S rRNA message, in each sample and were represented as a relative change to the control. All of the primers used were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA). Sequences for primers are presented in Table 1.

Expression of aggrecan degradation products, MMP-3, MMP-9, and MMP-13 were evaluated by means of Western blot analysis. AF and NP tissue was washed with cold PBS and was extracted with lysis buffer (Cell Signaling Technologies, Danvers, MA, USA) at 4°C for 24 hours. The lysis buffer contains the following: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA (ethylene glycol tetraacetic acid), 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate (Na₃VO₄), 1 μg/mL leupeptin, 1 mM PMSF (phenylmethylsulfonyl fluoride), and 1X complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN, USA). The tissue lysate was centrifuged for 10 minutes at 14,000g to collect the clear tissue extract. Protein concentration in the extract was determined by using a Pierce BCA protein assay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA). To detect aggrecan core protein fragments, lysates were treated with chondroitinase ABC (Sigma-Aldrich, St. Louis, MO, USA) 0.1 U/mL in 50 mM Tris-acetate EDTA buffer at 37°C for 1 hour. Protein extracts (10 μg) were centrifuged at 4°C for 2 minutes at 14,000g and resolved on NuPAGE 4-12% Bis-Tris Gels (Invitrogen Corporation). Proteins were transferred onto Immobilon-P Membrane (Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% nonfat dry milk in PBS with 0.1% Tween-20 and incubated overnight at 4°C in 3% nonfat dry milk in PBS with 0.1% Tween-20 with appropriate antibody (mouse monoclonal [BC-3] to aggrecan ARGxx, rabbit monoclonal [EP1186Y] to MMP-3, rabbit monoclonal [EP1254] to MMP-9, and rabbit polyclonal to MMP-13; all from Abcam). The binding of the secondary antibody was detected by enhanced chemiluminescence (ECL-Plus; GE Healthcare, Little Chalfont, Buckinghamshire, UK). Results were normalized to the content of tubulin detected with monoclonal antibody beta-tubulin (E7; Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, IA, USA).

Data were represented as mean ± standard error from three independent experiments. Statistical analysis was performed with Sigma Plot 11.2 statistical software (Systat Software, Inc., San Jose, CA, USA). Student t test or, where appropriate, Mann-Whitney test was used to measure statistical significance (P <0.05).

To characterize the diffusion of proteins of different molecular-weight proteins in the organ culture disc, we incubated discs with Firefly (61 kDa) and Renilla (38 kDa) luciferase (Figure 1A). After 24 hours of incubation, the NP was dissected and analyzed for the luciferase activity. Both the Firefly and Renilla luciferase show an increase in luminescence in a concentration-dependent manner within the NP (Figure 1B,C).

To ensure that our model reflects cellular and histological changes often seen with early disc degeneration, an assessment of cell viability was performed with the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. Figure 1D shows that TNF-α and IL-1β treatment for 3 days results in an increase in the number of apoptotic cells (Figure 1D-d) both in the NP and AF in comparison with the untreated control discs (Figure 1D-b). We performed a histological analysis of the organ-cultured discs to assess changes in cellular morphology and tissue organization (Figure 1E). Control untreated discs showed lamellar organization of the AF with well-circumscribed NP containing abundant proteoglycan-rich Alcian blue-stained extracellular matrix. NP cells showed well-defined eosinophilic cytosol with prominent cell nuclei. After 3 days of treatment with TNF-α and IL-1β, the discs demonstrated a notable loss of eosinophilic staining of the cytoplasm of the central NP cells. Although tissue architecture was preserved, a decrease in the Alcian blue staining of the extracellular matrix was noted with signs of cell loss and a concomitant increase in vacuole formation. At 10 days after treatment, additional changes of the cellular architecture were observed; the most obvious was a loss of Alcian blue staining of the extracellular matrix. Additionally, the eosinophilic staining of the NP cytosol was nearly gone and lacunae previously occupied by cells were empty. AF architecture, however, was preserved throughout the 10 days of treatment (Figure 1E, bottom row).

Since the disc organ culture is an in vitro system, a direct assessment of pain and inflammation without the involvement of neuronal and immunological components is not feasible. However, to get an indirect assessment of whether the degenerative changes in the discal tissues elicit changes in the expression of genes that activate neuronal and immune components resulting in pain and inflammation changes in expression of marker genes such as nerve growth factor (NGF), TNF-α, and IL-1β were measured. Figures 6A and 6B show that 3 days of treatment results in increased NGF expression by the NP and the AF. This increase in expression is sustained in the NP tissue at 10 days, whereas a small decrease is noticed in the AF. Interestingly, whereas IL-1β demonstrated a strong increase in both tissues (Figure 6C), TNF-α exhibits a small decrease in expression after treatment (Figure 6D).