Cloning of the rat MKP1 gene
Spinal cord tissue from Sprague Dawley rats (Harlan, Indianapolis, IN, 250 g) was harvested and stored at –80°C. Frozen tissues were homogenized and total RNA was extracted using RNA easy columns (Qiagen, Basel, Switzerland), following the manufacturer’s protocol. Total RNA was stored at –20°C until further use. Single strand cDNA was synthesized from 1 μg of rat spinal cord total RNA using the Quantitect reverse transcription kit (Qiagen, Basel, Switzerland), following the manufacturer’s protocol. For MKP-1 polymerase chain reaction (PCR), forward (5’CGACAGACTATTTCTGGAGAGCTGC3’) and reverse (5’CGGTGGCACGTGAAACTCCACA3’) primers were designed to be homologous to the rat dual-specificity phosphatase 1 gene (Genbank accession number NM_053769). These primers were used to generate 1.198 kb PCR fragment from spinal cord cDNA samples. PCR was performed as follows: 95°C for 5 min followed by 30 cycles at 95°C for 15 sec, 61°C for 1 min, 72°C for 1 min 15 sec, and 72°C for 10 min. The PCR product was gel-purified and cloned into pcDNA3.1 expression vector (Invitrogen, San Diego, CA). The MKP-1 gene was sequenced on both strands using the Big Dye terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster city, CA) and confirmed the expected rat MKP-1.
Cell culture, transfection, and lipopolysaccharide (LPS) treatment
The immortalized mouse microglial cell line BV-2 (a generous gift of Dr. Weihua Zhao, Methodist Hospital, Houston, TX) was cultured in DMEM (Mediatech, Manassas, VA) supplemented with 10% charcoal-stripped FBS (Hyclone), 1.1% GlutaMax (Invitrogen, San Diego, CA), and 1% Penicillin-Streptomycin (100 U/ml penicillin, 100 μg/ml streptomycin, Mediatech) and maintained in 5% CO2 at 37°C. BV-2 cell viability was assessed by trypan blue staining (Sigma, St. Louis, MO) and hemocytometer. A cell line stably expressing MKP-1 was created by transfection of rat MKP-1 (5 μg) cDNA into 1x106 BV-2 by electroporation (Lonza Biologics, Basel, Switzerland) following manufacturer’s instructions. After 48 h, cells were selected in growth medium containing neomycin (G418 sulfate, 800 μg/ml) for about 2 weeks. Stable transformants were then split at different dilutions in plates containing growth medium and neomycin selection drug. Cells were challenged with 1 μg/mL of LPS (0111:B4 serotype, Sigma, St. Louis, MO) in complete media for 1, 2, 6, and 12 h. Following LPS treatment, supernatants were removed and stored at –80oC until nitric oxide (NO) and cytokine measurement. Cells were used for quantitative RT-PCR and Western blot analyses.
The stealth siRNA oligonucleotides were synthesized by Invitrogen (Invitrogen, San Diego, CA) with sequence complementary to rat MKP-1 5’UCGUGAAGCAGAGGCGGAGUAUUAU3’ and 5’AUAAUACUCCGCCUCUGCUUCACGA3’. The stealth siRNA negative control duplex was used as control oligonucleotide (5’UUCCUCUCCACGCGCAGUACAUUUA3’ and 5’UAAAUGUACUGCGCGUGGAGAGGAA3’). Transfection efficiency (~85%) was monitored using BLOCK-iT Alexa Fluor red fluorescent oligo (Invitrogen, San Diego, CA). Stealth MKP-1 siRNAs (0.1, 1, and 10 nM) were transfected for 24 hr in BV-2 stably expressing MKP-1 using RNAi max transfection reagent (Invitrogen, San Diego, CA), following manufacturer’s instructions.
Quantitative real time (RT)-PCR
For mRNA quantification, total RNA isolated from BV-2 was reverse transcribed into single-strand cDNA as described above. Quantitative RT-PCR was performed using a 96-well plate with the Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems, Foster city, CA) according to the following conditions: 1 cycle of 50°C for 2 min, 1 cycle of 95°C for 10 min, then 40 cycles at 95°C for 15 sec, 60°C for 1 min. All samples were run in duplicate using Taqman gene expression assays (Applied Biosystems, Foster City, CA) with rat MKP-1 (Rn00678341_g1) predesigned and preformulated primer and probe. Expression of MKP-1 was normalized to a predesigned and preformulated rat (Rn01775763_g1) or mouse (Mm99999915_g1) control gene GAPDH (Applied Biosystems, Foster City, CA). The difference in MKP-1 mRNA expression between treatments was analyzed using the comparative CT method. The threshold cycle (CT) is defined as the cycle at which the amount of amplified PCR product from the target cDNA reaches a fixed threshold. In each treatment, ΔCT = MKP1CT – CT for the endogenous reference, GADPH. ΔΔCT = ΔCT
treatment – ΔCT
control. The N-fold of differential expression of MKP-1 transcript in the treated group compared to that in the control group is expressed as 2- ∆∆CT. This number is converted to percent of control, where control is set at 100. Total RNA for each sample was also included during each run as a negative control. Predesigned and preformulated primers and probes for rat and mouse cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β mRNAs—i.e., for rat, Rn 99999011_m1, Rn 99999017_m1, and Rn 00580432_m1 respectively, and for mouse, Mm 99999064_m1, Mm 00443258_m1 and Mm 01336189_m1 respectively—were used for quantitative RT-PCR as described above.
Western blot analyses
BV-2 cell protein or hemi-sectioned spinal cord (ipsilateral to injury) was collected in 100 μL of 1x Laemmli buffer (Bio-Rad, Hercules, CA) containing either 5% 2-β Mercaptoethanol (Sigma, St. Louis, MO). Protein (40–50 μg) or standard protein markers were subjected to SDS polyacrylamide gel electrophoresis (10% or 18% gels, Bio-Rad, Hercules, CA) and transferred to polyvinylidene difluoride (PVDF, Bio-Rad, Hercules, CA) membranes. Membranes were blocked with 5% BSA in TBS-Tween 20 (0.05%, Sigma, St. Louis, MO), then incubated overnight at 4°C with mouse anti rat MKP-1 monoclonal antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-phospho-ERK 44/42 (Phospho-MAP Kinase 1:500, Cell Signaling, Danvers, MA), mouse anti-phospho-p38 (1:500, Cell Signaling, Danvers, MA), or mouse anti-phospho-JNK (1:500, Cell Signaling, Danvers, MA). Blots were incubated with goat HRP-conjugated secondary antibody (1:3000, Pierce, Rockford, IL), treated with SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Rockford, IL), and imaged with Syngene G-box (Synoptics, Frederick, MD). Membranes were stripped and re-probed with rabbit anti-ERK 44/42 (Total MAP Kinase 1:500, Cell Signaling, Danvers, MA), rabbit anti-p38 (total MAP kinase 1:500, Cell Signaling, Danvers, MA), rabbit anti-JNK (1:500, Cell Signaling, Danvers, MA), or mouse anti-β-actin antibody (1:3000, Abcam, Cambridge, MA). Band density was quantified in relation to loading controls using Syngene Tools software (Synoptics, Frederick, MD). MAPKs were evaluated at 1 and 2 hr following LPS stimulation in BV-2 cells, since these early activated molecules are upstream cytokines, chemokines and nitric oxide production (which were measured at later time points, 6 and 12 hr after LPS stimulation in BV-2 cells).
Griess assay (Nitric oxide production) and ELISA assays
Supernatants collected from LPS-treated cells or total proteins (20 μg) extracted from L5 spinal cord were assayed for (1) nitric oxide (NO), measured as nitrite, using the Griess Assay (Promega, Madison, WI), (2) MCP-1 using the BD OptEIA™ ELISA kits for mouse and rat MCP-1 (BD Biosciences, San Diego, CA), and (3) IL-1β, IL-6, and TNF-α using the respective R&D Systems DuoSet ELISA (R&D Systems, Minneapolis, MN) kits following the manufacturer’s protocol.
Animals, vector delivery, surgeries, and behavior testing to assess allodynia
Sprague-Dawley rats weighing 250–300 g (Harlan) at the start of the study were housed individually and maintained in 12:12 h light-dark cycle with ad libitum access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee at Dartmouth College (Dartmouth Medical School, Hanover, New Hampshire) and in accordance with the Guidelines for Animal Experimentation of the International Association for the study of Pain. Plasmid DNA expressing MKP-1 (pcDNA-MKP-1) or empty plasmid DNA (pcDNA3.1) used in this study was purified using an endotoxin-free Qiagen plasmid purification kit and resuspended in sterile water. After determining the concentration of pcDNA-MKP-1 using 260 nm adsorption, aliquots of 8 μg were stored at –20°C. The purity of pcDNA-MKP-1 was determined by 260:280 nm adsorption and ranged between 1.9 to 2.0.
We used the in vivo non-viral nanoparticle preparation polyethylenimine (PEI), in vivo-JetPEI (PEI, Polyplus Transfection, New York, NY) for our in vivo gene transfection and induction. Endotoxin-free PEI is a cationic polymer that protects nucleic acids from degradation when administered in vivo [33, 34]. The positively charged PEI-nucleic acid complex interacts with the cellular membrane anionic proteoglycans, facilitating entry of the molecule into cells by endocytosis . Genes complexed with PEI have shown efficient induction into the brain and spinal cord parenchyma (glial and neuronal) of mice and rats when administered intrathecally or intracerebrally [36–39]. This CNS tissue penetration is not achieved with microparticles limited to the meningeal tissue following intrathecal administration . CNS administration of PEI nanoparticles with DNA plasmids has shown minimal toxicity, no necrosis , no inflammatory response , and no overt side effects in CNS animal transfections . Additionally, PEI transfection does not alter the electrophysiological properties of neurons , which makes this nanoparticle transfection agent a suitable tool for pain research.
On the day of intrathecal (i.t.) injection, aliquot of pcDNA-MKP-1 or empty pcDNA3.1 was thawed on ice and complexed with in vivo-JetPEI as described by the manufacturer: 8 μg of pcDNA plus in vivo-JetPEI in 5% glucose in a final ratio of nitrogen residues of in vivo-JetPEI per nucleic acid phosphate of 15 (N/P = 15) and a total volume of 20 ul. The preparation was incubated at room temperature for 15 min before the injections. The ready complex PEI + pcDNA-MKP1 (PEI + MKP-1, n = 9 animals), PEI + pcDNA alone (empty cDNA, n = 4 animals), or PEI alone (n = 9 animals) was administered by lumbar-puncture i.t. injection using a Hamilton syringe and a 25-gauge 5/8 hypodermic needle, as we have described before , under brief inhalational anesthesia (2–3% isoflurane in oxygen). The needle was inserted intrathecally, on the midline between the fourth and fifth lumbar vertebrae. Injection site was confirmed by stimulation of nerves in the cauda equina when the needle penetrated the dura and manifested with a brief and obvious movement of the tail and/or the hind paws. The animals regained consciousness 2–3 min after discontinuation of anesthesia.
Animals underwent L5 nerve transection (L5NT) procedure as previously described . Briefly, rats were anesthetized with 2% isoflurane in oxygen and a small incision was made to the skin overlying L5–S1, followed by retraction of the paravertebral musculature. The L6 transverse process was then removed and the L5 spinal nerve identified, lifted slightly, and transected. The wound was sutured. Sham surgeries were performed similarly, except for nerve manipulation or transection. Intrathecal injection of PEI + cDNAs or PEI only was performed immediately after surgery.
Behavioral testing was performed to assess the development of mechanical allodynia before injection or surgery and on postoperative days 1–4 . The withdrawal threshold to mechanical stimuli was assessed ipsilaterally to surgery using calibrated von Frey filaments (Stoelting, Wood Dale, IL) and an up-down statistical method . The investigator was blinded to treatment in all behavioral tests.
Rats were deeply anesthetized with 2–3% isoflurane in oxygen and transcardially perfused with 10 mM phosphate buffer saline (200 mL) followed by 4% formaldehyde (400 mL) at room temperature. The L5 portion of the spinal cord was removed and placed in 30% sucrose for 48–72 h at 4°C. The tissue was then frozen at –80°C in optimal cutting temperature compound (Sakura Finetek, Torrance, CA). Immunohistochemistry was performed on transverse 20 μm L5 spinal cord free-floating sections. The sections were blocked for 1 h in 5% normal goat serum (NGS, Vector Labs, Burlingame, CA) and 0.01 Triton X-100 (Sigma) in PBS. Then the sections were incubated overnight in mouse anti rat monoclonal antibody for MKP-1 (1:100, Cell Signaling, Danvers, MA). The next day, a TSA Signal Amplification Kit (PerkinElmer LifeSciences Inc, Boston, MA) was used, following the manufacturer’s instructions, to enhance the visualization of MKP-1 expression. Sections were washed 2 times for 5 min in PBS then incubated in a biotinylated Goat anti-rabbit secondary antibody for 1 h at 4°C. Sections were then washed, incubated in SA-HRP (1:100) for 1 h at 4°C, washed again and incubated in the TSA fluorophore (1:250) for 10 min at 4°C. Sections were then washed again and incubated overnight in rabbit anti rat antibody for ionized calcium binding adaptor molecule 1 (Iba-1, 1:1000, microglia marker, Wako, Richmond, VA), rabbit anti glial fibrillary acidic protein (GFAP, 1:10,000, astrocyte marker, Dako, Carpinteria, CA), rabbit anti S100 calcium binding protein B (S100B, 1:15,000, astrocyte marker, Fitzgerald, Concord, MA), or rabbit NeuN/Fox3 (NeuN, 1:10,000, neuron nuclear marker, Biosensis, Australia). The next day sections were washed three times, then incubated for 1 h in goat anti-mouse Alexa 488 IgG3 and goat anti-rabbit Alexa 555 IgG secondary antibodies (1:250, Invitrogen). Tissue sections were washed, mounted on glass slides, dehydrated, treated with Vectashield (Vector Labs, Burlingame, CA), and sealed with a coverslip and nail polish. The specificity of each antibody was tested by omitting the primary antibody on 1–3 additional sections. All MAPK and cell marker antibodies are widely used for their specificity. We confirmed the specificity of our MKP-1 antibody by using Western blots in MKP-1 overexpressing BV-2 cells and normal BV-2 cells (see Results section). The following groups were included to test MKP-1 antibody signal amplification using the TSA kit: (1) only the anti-MKP-1 primary antibody (1:100) and the Alexa 555 Goat anti-rabbit secondary antibody, (2) only the anti-MKP-1 primary antibody and the TSA kit, and (3) the TSA kit, the cell marker primary, and the Alexa 555 Goat anti-rabbit secondary antibody, but excluding the anti-MKP-1 primary antibody. All controls confirmed the specificity of the complete co-stain. Sections for single antibody immunofluorescence were imaged with a Q-Fired cooled CCD camera attached to an Olympus microscope. Confocal microscopy of dual antibody immunofluorescence was performed with a Zeiss LSM 510 Meta confocal microscope (Englert Cell Analysis Laboratory of Dartmouth Medical School), and images were prepared with the Zeiss LSM software (Thornwood, NY) and Adobe Photoshop software (San Jose, CA). All images were taken from laminae I–II of dorsal horn spinal cord ipsilateral to nerve injury.
All in vitro experiments were completed at least three time. Data are expressed as mean ± s.e.m. Statistical analyses were completed using GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA). One- or two-way ANOVA and Bonferroni post test were used when appropriate. Unpaired t-test was use for MKP-1 mRNA expression and cytokine release. Significance was determined at a level of P < 0.05 or with exact p value where available.