Erished environment (Figures 3A,B). This was accompanied by the recovery of nerve injury-induced reductions in 115103-85-0 web global methylation in the PFC to normal, control levels (Figure 3C).Figure 2. Global Methylation in the CNS Six Months following Nerve Injury. Nerve injured mice show a decrease in global methylation in the PFC (a) and amygdala (b) six months after the induction of peripheral neuropathy. No such changes were observed in the thalamus (c) or visual cortex (d). *** = p,0.0001, n = 8?0/group, error bars indicate S.E.M. doi:10.1371/79831-76-8 journal.pone.0055259.gResults Behavioral signs of neuropathic pain, motor 1531364 impairment and anxiety six months following peripheral nerve injuryChronic, persistent changes in cutaneous sensitivity to both mechanical and cold stimuli were detected six months following nerve injury. Injured mice displayed decreased mechanical thresholds (Figure 1A) and increased acetone-evoked behaviors (Figure 1B). Animals with nerve injury were significantly impaired in motor capacity in the rotarod assay (Figure 1C). Animals with neuropathy exhibited signs of anxiety in the open field assay six months after injury (Figure 1D). Despite the factCorrelation between global methylation in the prefrontal cortex and hypersensitivity to cutaneous stimuliAnalysis of the data from Figure 3 revealed significant correlations between the magnitude of both mechanical (Figure 4A) and cold (Figure 4B) hypersensitivity and global methylation in the PFC in injured, but not in control animals. Specifically, following environmental manipulation, nerve-injured animals with higher global methylation in the PFC had reduced hypersensitivity to mechanical and thermal stimuli.Figure 3. Environmental Enrichment Reverses Nerve Injury-Induced Neuropathic Pain and Pathological Changes in DNA Methylation in the Prefrontal Cortex. Three months following nerve injury and the establishment of chronic neuropathic pain, mice were subjected to an enriched or an impoverished environment for an additional two months. Environmental enrichment resulted in decreases in mechanical (A) and cold sensitivity (B) in nerve-injured animals towards control values. (C) These changes in cutaneous hypersensitivity following environmental enrichment were accompanied by reversal of the nerve injury-induced decreases in global methylation in the impoverished group such that global methylation was no longer different from control values (1-way ANOVA, F(3,21) = 4.545, p = 0.013, tukeys multiple comparison 1662274 test). * = p,0.05, ** = p,0.01,*** = p,0.001; control, enriched vs. impoverished or injured, enriched vs. impoverished; n = 5?/group, error bars indicate S.E.M. doi:10.1371/journal.pone.0055259.gChanges in DNA Methylation following Nerve InjuryFigure 4. Global Methylation in the Prefrontal Cortex Correlates with the Magnitude of Nerve Injury-Induced Hypersensitivity. Correlation analysis was performed on the data from Figure 3. Significant correlations were observed between global methylation and hypersensitivity to mechanical (a) and cold stimuli (b), in neuropathic but not control mice. * = p,0.05, ** = p,0.01. doi:10.1371/journal.pone.0055259.gDiscussion DNA Methylation and Chronic Neuropathic PainEpigenetic mechanisms triggered by injury have been hypothesized to participate in mediating the lasting changes in the CNS associated with chronic pain [22]. To date, most of the work related to chronic pain has focused on the role of histone acetylation/deacetylation [23], main.Erished environment (Figures 3A,B). This was accompanied by the recovery of nerve injury-induced reductions in global methylation in the PFC to normal, control levels (Figure 3C).Figure 2. Global Methylation in the CNS Six Months following Nerve Injury. Nerve injured mice show a decrease in global methylation in the PFC (a) and amygdala (b) six months after the induction of peripheral neuropathy. No such changes were observed in the thalamus (c) or visual cortex (d). *** = p,0.0001, n = 8?0/group, error bars indicate S.E.M. doi:10.1371/journal.pone.0055259.gResults Behavioral signs of neuropathic pain, motor 1531364 impairment and anxiety six months following peripheral nerve injuryChronic, persistent changes in cutaneous sensitivity to both mechanical and cold stimuli were detected six months following nerve injury. Injured mice displayed decreased mechanical thresholds (Figure 1A) and increased acetone-evoked behaviors (Figure 1B). Animals with nerve injury were significantly impaired in motor capacity in the rotarod assay (Figure 1C). Animals with neuropathy exhibited signs of anxiety in the open field assay six months after injury (Figure 1D). Despite the factCorrelation between global methylation in the prefrontal cortex and hypersensitivity to cutaneous stimuliAnalysis of the data from Figure 3 revealed significant correlations between the magnitude of both mechanical (Figure 4A) and cold (Figure 4B) hypersensitivity and global methylation in the PFC in injured, but not in control animals. Specifically, following environmental manipulation, nerve-injured animals with higher global methylation in the PFC had reduced hypersensitivity to mechanical and thermal stimuli.Figure 3. Environmental Enrichment Reverses Nerve Injury-Induced Neuropathic Pain and Pathological Changes in DNA Methylation in the Prefrontal Cortex. Three months following nerve injury and the establishment of chronic neuropathic pain, mice were subjected to an enriched or an impoverished environment for an additional two months. Environmental enrichment resulted in decreases in mechanical (A) and cold sensitivity (B) in nerve-injured animals towards control values. (C) These changes in cutaneous hypersensitivity following environmental enrichment were accompanied by reversal of the nerve injury-induced decreases in global methylation in the impoverished group such that global methylation was no longer different from control values (1-way ANOVA, F(3,21) = 4.545, p = 0.013, tukeys multiple comparison 1662274 test). * = p,0.05, ** = p,0.01,*** = p,0.001; control, enriched vs. impoverished or injured, enriched vs. impoverished; n = 5?/group, error bars indicate S.E.M. doi:10.1371/journal.pone.0055259.gChanges in DNA Methylation following Nerve InjuryFigure 4. Global Methylation in the Prefrontal Cortex Correlates with the Magnitude of Nerve Injury-Induced Hypersensitivity. Correlation analysis was performed on the data from Figure 3. Significant correlations were observed between global methylation and hypersensitivity to mechanical (a) and cold stimuli (b), in neuropathic but not control mice. * = p,0.05, ** = p,0.01. doi:10.1371/journal.pone.0055259.gDiscussion DNA Methylation and Chronic Neuropathic PainEpigenetic mechanisms triggered by injury have been hypothesized to participate in mediating the lasting changes in the CNS associated with chronic pain [22]. To date, most of the work related to chronic pain has focused on the role of histone acetylation/deacetylation [23], main.
