Then washed and redissolved in ddH2O.Samples were incubated (37uC, 4 h) and then heat inactivated (80uC, 20 min). Digested genomic DNA (15 ml) was mixed with pyrosequencing annealing buffer (15 ml; Qiagen, Toronto, ON, Canada). Samples were transferred to 24-well pyrosequencing plates for sequencing (PyroMark 24; Biotage, Uppsala, Sweden). The nucleotide dispensation order used was based on Pilsner et al. [21]. Peak heights for C and A PD 168393 chemical information represent the HpaII and MspI cuts (methylation) and EcoRI (input DNA), respectively. The formula to calculate genomic methylation is: 1-[(HpaII (C)/EcoRI (A)/ MspI (C)/EcoRI (A)]6100. All samples were run in triplicate.Statistical Analysis Luminometric Methylation Assay (LUMA)LUMA is a high throughput assay used to determine genomic global DNA methylation. The LUMA method used in our study is a modification described by Karimi et al. [19,20]. LUMA involves the digestion of genomic DNA by methylation sensitive (HPAII) or insensitive (MSPI) restriction enzymes in combination with an internal control restriction enzyme (EcoRI) to normalize the DNA input. Both HpaII and MspI restriction enzymes recognize and cleave 59-CCGG-39 ML 281 site sequences producing 59-CG overhangs, whereas EcoRI recognizes and cleaves 59-GAATTC-39 sequences and produces 59-AATT overhangs. The extent of cleavage is determined by a bioluminetric polymerase extension assay based on a four-step pyrosequencing reaction. All data are expressed as mean 6 
standard error of the mean (SEM). Comparisons between injured and control groups (Figures 1 and 2) or between enriched vs. impoverished (Figures 3A,B) were performed using 2-tailed, unpaired student’s t-test. The data in Figure 3C was analyzed by one-way ANOVA followed by Tukeys test for multiple comparisons. Significance was set at P,0.05. Welch’s correction was used when the assumption of equal variances was not met. The degree of co-variation between mechanical or cold sensitivity and global methylation was determined by calculating Pearson’s coefficient since our data followed a Gaussian distribution. Statistical analysis was undertaken using Prism (GraphPad Software Inc, San Diego, California).Changes in DNA Methylation following Nerve Injurythat injured animals visited the same number of peripheral squares as control animals (Figure 1E), they spent significantly less time in the central square compared to control mice (Figure 1F).Nerve injury was associated with decreased global methylation in the prefrontal cortex and amygdala, but not the thalamus or visual cortex, six months following injuryPeripheral nerve injury resulted in a decrease in global methylation in the prefrontal cortex (Figure 2A) and the amygdala (Figure 2B) that was detected 6 months after injury. No significant changes were 
observed in the thalamus (Figure 2C) or visual cortex (Figure 2D). Since there were no significant differences in global methylation between right and left brain structures, the results reported here are the average of the two sides.Environmental enrichment reverses nerve injury-induced chronic pain and abnormal global methylation in the prefrontal cortexThree months after peripheral nerve injury, a second set of mice were removed from standard housing and placed in either an enriched or an impoverished environment for two additional months. The enriched environment was associated with decreased nerve injury-induced hypersensitivity to mechanical and cold stimuli compared to the impoverished environment.Then washed and redissolved in ddH2O.Samples were incubated (37uC, 4 h) and then heat inactivated (80uC, 20 min). Digested genomic DNA (15 ml) was mixed with pyrosequencing annealing buffer (15 ml; Qiagen, Toronto, ON, Canada). Samples were transferred to 24-well pyrosequencing plates for sequencing (PyroMark 24; Biotage, Uppsala, Sweden). The nucleotide dispensation order used was based on Pilsner et al. [21]. Peak heights for C and A represent the HpaII and MspI cuts (methylation) and EcoRI (input DNA), respectively. The formula to calculate genomic methylation is: 1-[(HpaII (C)/EcoRI (A)/ MspI (C)/EcoRI (A)]6100. All samples were run in triplicate.Statistical Analysis Luminometric Methylation Assay (LUMA)LUMA is a high throughput assay used to determine genomic global DNA methylation. The LUMA method used in our study is a modification described by Karimi et al. [19,20]. LUMA involves the digestion of genomic DNA by methylation sensitive (HPAII) or insensitive (MSPI) restriction enzymes in combination with an internal control restriction enzyme (EcoRI) to normalize the DNA input. Both HpaII and MspI restriction enzymes recognize and cleave 59-CCGG-39 sequences producing 59-CG overhangs, whereas EcoRI recognizes and cleaves 59-GAATTC-39 sequences and produces 59-AATT overhangs. The extent of cleavage is determined by a bioluminetric polymerase extension assay based on a four-step pyrosequencing reaction. All data are expressed as mean 6 standard error of the mean (SEM). Comparisons between injured and control groups (Figures 1 and 2) or between enriched vs. impoverished (Figures 3A,B) were performed using 2-tailed, unpaired student’s t-test. The data in Figure 3C was analyzed by one-way ANOVA followed by Tukeys test for multiple comparisons. Significance was set at P,0.05. Welch’s correction was used when the assumption of equal variances was not met. The degree of co-variation between mechanical or cold sensitivity and global methylation was determined by calculating Pearson’s coefficient since our data followed a Gaussian distribution. Statistical analysis was undertaken using Prism (GraphPad Software Inc, San Diego, California).Changes in DNA Methylation following Nerve Injurythat injured animals visited the same number of peripheral squares as control animals (Figure 1E), they spent significantly less time in the central square compared to control mice (Figure 1F).Nerve injury was associated with decreased global methylation in the prefrontal cortex and amygdala, but not the thalamus or visual cortex, six months following injuryPeripheral nerve injury resulted in a decrease in global methylation in the prefrontal cortex (Figure 2A) and the amygdala (Figure 2B) that was detected 6 months after injury. No significant changes were observed in the thalamus (Figure 2C) or visual cortex (Figure 2D). Since there were no significant differences in global methylation between right and left brain structures, the results reported here are the average of the two sides.Environmental enrichment reverses nerve injury-induced chronic pain and abnormal global methylation in the prefrontal cortexThree months after peripheral nerve injury, a second set of mice were removed from standard housing and placed in either an enriched or an impoverished environment for two additional months. The enriched environment was associated with decreased nerve injury-induced hypersensitivity to mechanical and cold stimuli compared to the impoverished environment.
