Study design and patient population
The procedure for CSF collection was approved by our Institutional Ethics Board and with written informed consent was obtained from each patient (DSRB Ref No. 2011/01710). Patients (both the pain group and the group without pain) were prospectively recruited by the attending anaesthesiologists prior to surgery. All patients recruited in the pain group satisfied the following inclusion criteria: (a) Confirmed OA of knees from history and x-ray of the knees by orthopaedic surgeons, (b) Numeric pain rating scale of more than 3 (0 being no pain; 10 being worst possible imaginable pain), (c) Knee pain of more than six months duration, (d) Scheduled for total knee replacement surgery under spinal anaesthesia, (e) Age of twenty-one years and above, (f) No other concurrent chronic pain conditions. Patients without pain and undergoing surgery with spinal anaesthesia served as the control group. The inclusion criteria for no pain group include: (a) No history of chronic pain over the past one year; (b) Patient consented for surgery below the umbilicus under a spinal anaesthesia, (c) Age of twenty-one years and above. Pregnant patients or patients with severe end-organ function impairment (e.g. heart, lungs, liver and kidneys) were excluded.
CSF was obtained during the conduct of spinal anaesthesia, where two-ml of CSF was withdrawn after the dura was punctured, before injecting local anaesthetic into the subarachnoid space to effect the spinal anaesthesia. A total of 15 chronic knee OA pain patients and 15 no pain patients were recruited. CSF samples were then stored at –80°C. Patients’ demographics such as gender and age were also recorded.
Measurement of pro-inflammatory cytokine release in cell culture
Human glioblastoma cell line T98G (ATCC-CRL1690, American Type Culture Collection, Manassas, VA) was cultured as previously described . In brief, Eagle’s Minimum Essential Medium (Gibco, Grand Island, New York) (EMEM) containing 10% fetal bovine serum (FBS) (HyClone, Utah, USA) and 1X antibiotic-anti-mycotic cocktail (HyClone, Utah, USA) was used.
T98G cells were grown to approximately 90% confluency in 12-well plates. CSF from patients were filtered using 0.22 micron filter prior addition into cultured T98G cells. The culture medium was replaced with FBS-free EMEM medium before addition of 50μl CSF along with 32 μg/ml lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma Aldrich, St Louis MO, USA) to the T98G cells. Cells were induced for 48 hours at 37°C and 5% CO2 environment. LPS was used to enhance cytokine release from the cells [9,10]. After 48 hours, the medium containing “cytokine release” was collected and amount of IL–6 in this supernatant was measured in duplicates using enzyme-linked immunosorbent assay (ELISA) according to manufacturer’s instructions (DY206, R&D Systems, Minneapolis, USA). The detection range for the IL–6 standard used was between 9.38 pg/ml to 600 pg/ml.
Using supernatant from the above 48 hour CSF-triggered T98G protocol, TNFα and IL–1β levels were also determined with ELISA assays- Human TNFα Duoset ELISA (DY210) and Human IL–1β/IL–1F2 Duoset (DY201) respectively (R&D Systems, Minneapolis, USA). Detection range of standards for TNFα was 15.6 pg/ml to 1000 pg/ml; and IL–1β was 3.91 pg/ml to 250 pg/ml.
Nuclear factor kappa light chain enhancer of activated B cells (NFκB) phospho antibody array (PNK215, Fullmoon Biosystems, CA, USA) was used to screen for possible protein activity and protein expression profiling in our samples. This array applied an ELISA-based technique where samples were biotinylated before adding to the array slide containing affixed antibodies. Biotin on samples upon interaction with dye-labelled streptavidin on array would generate the fluorescence signal. Patient CSF-triggered T98G cells were harvested and lysed with CelLytic MT Cell Lysis Reagent (C3228; Sigma Aldrich). Total protein from T98G cell lysate were quantified using bicinchoninic acid (BCA) assay (Pierce BCA Protein Assay Kit, Thermo Scientific, USA). Pooled OA pain sample and no-pain sample (n = 6) was added to each antibody array slide and assay was performed according to manufacturer’s instructions. Array image was captured using array scanner (GenePix 4000B; Molecular Probes, CA, USA). Analysis of array data was done using Genescan software. Comparison of signals between OA pain pooled sample and no pain pooled sample was done after normalization with glyceraldehyde–3-phosphate dehydrogenase (GAPDH).
Western Blot analysis
To validate the fold increase as observed in the antibody array for OA triggered T98G cells, all individual OA and no pain-CSF triggered T98G cell lysates (n = 15 each) were checked for protein expression level of targets with Western Blotting. Ten micrograms of each CSF-triggered T98G cell lysates were loaded per lane of 10% Tris-glycine gels and run using Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on gel were transferred onto Polyvinylidene difluoride (PVDF) membrane. 0.1% Tween–20 added into Tris-buffered saline (TBS-T) was used to wash membranes and prepare antibodies. Non-specific binding sites on the membranes were blocked in 5% w/v Bovine serum albumin (BSA)-TBS-T or milk-TBS-T. BSA was used when checking for spleen tyrosine kinase (Syk); while milk was used for STE20-related kinase adaptor protein (STRAD) and GAPDH. Primary antibodies used in this study are: STRAD (G–8) (Santa Cruz Biotechnology, CA, USA); Syk (D3Z1E) (Cell Signaling Technology Inc, MA, USA); and GAPDH (MAB374) (EMD Millipore, Darmstadt, Germany). GAPDH, with a molecular weight of 37kDa, was used as the loading control for normalization. Membranes were imaged with ChemiDoc XRS+ system. Densitometry quantification was done with ImageLab software.
All data were analysed using GraphPad Prism v5.0 (GraphPad, La Jolla, CA, USA) and SPSS v24.0 (IBM SPSS, Armonk NY, USA). Cytokine levels was checked for normality and deemed not normally distributed, thus non-parametric Mann Whitney-U test was used. Mann-Whitney U test was also used to analyse STRAD and Syk protein levels from western blotting between groups. Simple and multivariate linear regression model analyses were used to study the relationship of pain score and cytokine level. A p-value of less than 0.05 was considered statistically significant.