Ical tools [24,25,26,27], with few reports of changes in DNA methylation in chronic pain conditions [28,29,30,31]. In this study, a mouse model of neuropathic pain following peripheral nerve injury was used to test the hypothesis that ongoing, chronic painful neuropathy induces changes in global DNA methylation in the brain. Our data show decreases in global DNA methylation in the PFC and amygdala six months following a peripheral nerve injury in the hindlimb. This is consistent with many of the comorbidities that develop when pain has transitioned from being acute to chronic, such as chronic-pain associated depression [32]. Furthermore, these global changes were region-specific; similar effects were not observed in the thalamus or the visual cortex, even though the former receives direct input from nociceptive neurons. It is important to note that regions that did not show global changes may still undergo changes in 1326631 DNA methylation at the individual gene level that are not detectable by a global methylation assay such as the LUMA. However, the fact that alterations were observed in the PFC and amygdala shows a strong link between nerve injury-induced hypersensitivity and changes in DNA methylation in the 
brain and provides a potential link between injury, chronic pain and co-morbidities such as cognitive dysfunction, depression and anxiety. The magnitude of the nerve injury-associated changes in global methylation in the PFC from 60 to 48 suggests that the changes are broad and affect wide parts of the genome. It is estimated that the mouse genome contains ,20 million CpG sites, the targets for DNA methylation. The LUMA assay used in this study is sensitive to ,1.5 million of these sites. Therefore, a decrease of 12 in this assay corresponds to a minimum estimated demethylation of ,180,000 CpG sites following nerve injury, a number predicted to alter the expression of hundreds of individual genes [33].Global methylation is an indicator of the overall state of the DNA methylation machinery and has long-range consequences on genome function and organization [34,35,36]. Recent data suggests that the landscape 
of altered DNA methylation in other pathologies such as cancer spans thousands of genes [37] and intergenic regions [38]. Programming of DNA methylation encompasses both global changes in genome methylation and gene-specific changes that target discreet regulatory regions, thus affecting gene expression. Changes in global DNA methylation state affect high-level organization of genome function [39]. These changes produce lasting effects on the regulation of the transcriptome and higher order chromatin folding [40] and are 11089-65-9 capable of affecting many aspects of cell function. The population of methylated CpG sites in gene promoters and 1326631 known regulatory regions constitutes only a small fraction of global DNA methylation. Given the magnitude of the pathological changes in DNA methylation observed in this study, they must therefore also involve regions in the genome beyond individual gene promoters and gene regulatory sequences. Indeed, demethylation/methylation of all known promoters and regulatory regions in the genome would in itself not have a significant impact on global DNA methylation.Dynamic Mechanisms Mediating Chronic PainIn this study, the epigenetic changes were Rubusoside site attenuated by a behavioral intervention. Environmental enrichment reversed nerve injury-related reductions in global DNA methylation in the PFC and reduced hypersensi.Ical tools [24,25,26,27], with few reports of changes in DNA methylation in chronic pain conditions [28,29,30,31]. In this study, a mouse model of neuropathic pain following peripheral nerve injury was used to test the hypothesis that ongoing, chronic painful neuropathy induces changes in global DNA methylation in the brain. Our data show decreases in global DNA methylation in the PFC and amygdala six months following a peripheral nerve injury in the hindlimb. This is consistent with many of the comorbidities that develop when pain has transitioned from being acute to chronic, such as chronic-pain associated depression [32]. Furthermore, these global changes were region-specific; similar effects were not observed in the thalamus or the visual cortex, even though the former receives direct input from nociceptive neurons. It is important to note that regions that did not show global changes may still undergo changes in 1326631 DNA methylation at the individual gene level that are not detectable by a global methylation assay such as the LUMA. However, the fact that alterations were observed in the PFC and amygdala shows a strong link between nerve injury-induced hypersensitivity and changes in DNA methylation in the brain and provides a potential link between injury, chronic pain and co-morbidities such as cognitive dysfunction, depression and anxiety. The magnitude of the nerve injury-associated changes in global methylation in the PFC from 60 to 48 suggests that the changes are broad and affect wide parts of the genome. It is estimated that the mouse genome contains ,20 million CpG sites, the targets for DNA methylation. The LUMA assay used in this study is sensitive to ,1.5 million of these sites. Therefore, a decrease of 12 in this assay corresponds to a minimum estimated demethylation of ,180,000 CpG sites following nerve injury, a number predicted to alter the expression of hundreds of individual genes [33].Global methylation is an indicator of the overall state of the DNA methylation machinery and has long-range consequences on genome function and organization [34,35,36]. Recent data suggests that the landscape of altered DNA methylation in other pathologies such as cancer spans thousands of genes [37] and intergenic regions [38]. Programming of DNA methylation encompasses both global changes in genome methylation and gene-specific changes that target discreet regulatory regions, thus affecting gene expression. Changes in global DNA methylation state affect high-level organization of genome function [39]. These changes produce lasting effects on the regulation of the transcriptome and higher order chromatin folding [40] and are capable of affecting many aspects of cell function. The population of methylated CpG sites in gene promoters and 1326631 known regulatory regions constitutes only a small fraction of global DNA methylation. Given the magnitude of the pathological changes in DNA methylation observed in this study, they must therefore also involve regions in the genome beyond individual gene promoters and gene regulatory sequences. Indeed, demethylation/methylation of all known promoters and regulatory regions in the genome would in itself not have a significant impact on global DNA methylation.Dynamic Mechanisms Mediating Chronic PainIn this study, the epigenetic changes were attenuated by a behavioral intervention. Environmental enrichment reversed nerve injury-related reductions in global DNA methylation in the PFC and reduced hypersensi.
