GSM2689977: Spinal cord def diet 04; Rattus norvegicus; OTHER
spinal nerve ligation protocol: Spinal nerve ligation (SNL) in male Sprague-Dawley rats (8-9 weeks old, ± 220 g; Harlan Sprague-Dawley, Indianapolis, IN) was used for this study as the neuropathic pain model. Animals were anesthetized with 2-3% isoflurane, and the left L5 and L6 spinal nerves were isolated and ligated tightly with 4.0-silk suture under a surgical microscope. Sham surgery (without nerve ligation) was performed on the contralateral side as the control. chemotherapy-induced neuropathy: Four intraperitoneal injections of the chemotherapeutic agent paclitaxel (2 mg/kg, every two days) were performed, and vehicle (cremophor ethanol 1:1)-treated rats were used as the control group. intrathecal cathetrization: Under anesthesia with 2% isoflurane, an intrathecal catheter (a PE-10 tubing) was surgically inserted through an incision made in the cisternal membrane. The catheter is directed to the lumbar region of the spinal cord, and the external opening was sealed. Rats displaying signs of the motor or neurological dysfunction were excluded and terminated immediately with an overdose of phenobarbital (200 mg/kg, i.p.). The animals were allowed to recover for five days before intrathecal injections. Ten micrograms of N-phthaloyl-L-tryptophan (RG108, Tocris Bioscience, Bristol, UK) or vehicle (10% DMSO; saline) were injected intrathecally in a 5 µl volume followed by 10 µl saline flush.(Chestnut et al. 2011). Animals received intrathecal injections for four consecutive days. neuronal enrichment: To prepare a neuronal nuclear enrichment, fresh whole DRG samples were cut and mashed onto an ice-cold petri dish. Mashed tissue from 3 DRGs was pooled in one 1.5 ml tube. The tissue was resuspended in buffer NAME (20 mM Tris pH8, 20 mM KCl, 1.5 mM MgCl2) with a cocktail of protease and phosphatase inhibitors cocktails (Pierce), 0.5 mM DTT and 1 mM PMSF (Sigma-Aldrich, MO) to obtain a single cell suspension. Cells were incubated on ice for 5 min before to be spincentrifuged at 3,000 rpm at 4 °C for 2 min. The pPellet was resuspended in buffer NAME containing 0.15% NP-40. Cells were incubated on a rotator overnight to isolate nuclei. Neuronal nuclei were immunoprecipitated using anti-Fox3 (neuronal nuclear marker, Abcam #104225) in PBS. Then, classical and genomic DNA extraction was performed. Neuronal nuclei were lysed with lysis buffer (0.1 M Tris pH 8, 5 mM EDTA, 0.2% SDS, 0.2 M NaCl and, proteinase K) overnight and precipitated with isopropanol. Genomic DNA pellet was washed with 75% ethanol, dried and re-suspended in TE buffer (Tris 10 mM pH8, 1 mM EDTA). The isolation purity was validated by western blot analysis. Nuclear proteins were extracted from one batch. Cytoplasm/nucleus separation was validated by western blot using anti-histone H3 (Cell Signaling Technology#9715; 1/4000) and neuronal enrichment was validated by western blot using the neuronal marker anti-Fox3 (Abcam #104225) and the satelite/glia markers anti-g Glutamine sSynthetase (Millipore #MAB302; 1/200) and anti-GFAP (Abcam #7260; 1/4000). Genomic DNA extracted from tissue samples was spiked with methylation standards and sequentially digested with two restriction endonucleases recognizing CCCGGG sites in DNA. SmaI cleaved only unmethylated sites resulting in blunt ended fragments starting with 5’-GGG. Next, XmaI cleaved the remaining methylated sites creating 5’-CmeCGG overhang sequences. Exonuclease-deficient Klenow fragment DNA polymerase was then used to fill in the 3’ recesses left by XmaI and add 3’-dA overhangs to the ends of all fragments. Thus, specific signatures 5’-GGG and 5’-CCGGG were created at unmethylated and methylated CpG sites, respectively. Illumina sequencing adapters were then ligated to the enzyme-treated DNA; sequencing libraries were generated according to Illumina protocols and run on an Illumina HiSeq 2500 instruments at Fox Chase Cancer Center Genomics facility. Sequencing data were aligned to the rat genome (rn4), and the reads with unmethylated and methylated signatures were counted at individual CCCGGG sites. Methylation levels were calculated as the ratio of reads with the methylated signature to all reads mapping to each respective site. DNA methylation values at individual CpG sites were adjusted based on spiked in standards and filtered for the minimum sequencing depth of 100 reads.