The innate immune system is increasingly appreciated to play an important role in the mediation of chronic pain, and one molecule implicated in this process is the Toll-like receptor 4 (TLR4). Here using pharmacological and genetic manipulations we found that activating TLR4 in the spinal cord, with the agonist lipopolysaccharide (LPS), causes robust mechanical allodynia but only in male mice. Spinal LPS had no pain-producing effect in female mice. TLR4 also has a sex-specific role in inflammatory (complete Freund’s adjuvant) and neuropathic (spared nerve injury) pain: pain behaviors were TLR4-dependent in males but TLR4-independent in females. The sex differences appear to be specific to the spinal cord, as LPS administered to the brain or the hindpaw produces equivalent allodynia in both sexes, and specific to pain, as intrathecal LPS produces equivalent hypothermia in both sexes. The involvement of TLR4 in pain behaviors in male mice is dependent on testosterone, as shown by gonadectomy and hormone replacement. We found no sex differences in spinal Tlr4 expression at baseline or after LPS, suggesting the existence of parallel spinal pain processing circuitry in female mice not involving TLR4s.

Chronic pain is often associated with sexual dysfunction, suggesting that pain can reduce libido. We find that inflammatory pain reduces sexual motivation, measured via mounting behavior and/or proximity in a paced mating paradigm, in female but not male laboratory mice. Pain was produced by injection of inflammogens zymosan A (0.5 mg/ml) or -carrageenan (2%) into genital or nongenital (hind paw, tail, cheek) regions. Sexual behavior was significantly reduced in female mice experiencing pain (in all combinations); male mice similarly treated displayed unimpeded sexual motivation. Pain-induced reductions in female sexual behavior were observed in the absence of sex differences in pain-related behavior, and could be rescued by the analgesic, pregabalin, and the libido-enhancing drugs, apomorphine and melanotan-II. These findings suggest that the well known context sensitivity of the human female libido can be explained by evolutionary rather than sociocultural factors, as female mice can be similarly affected.

Supplemental Digital Content is Available in the Text. DNA methylation undergoes rapid and large changes in mouse prefrontal cortex at multiple time points postinjury, implicating hundreds of genes in a time-dependent manner.

Chronic pain is associated with persistent structural and functional changes throughout the neuroaxis, including in the prefrontal cortex (PFC). The PFC is important in the integration of sensory, cognitive and emotional information and in conditioned pain modulation. We previously reported wide-spread epigenetic reprogramming in the PFC many months following nerve injury in rodents. Epigenetic modifications, including DNA methylation, can drive changes in gene expression without modifying DNA sequences.To date, little is known about epigenetic dysregulation at the onset of acute pain or how it progresses as pain transitions from acute to chronic. We hypothesize that acute pain following injury results in rapid and persistent epigenetic remodelling in the PFC that evolves as pain becomes chronic. We further propose that understanding epigenetic remodelling will provide insights into the mechanisms driving pain-related changes in the brain. Epigenome-wide analysis was performed in the mouse PFC 1 day, 2 weeks, 6 months, and 1 year following peripheral injury using the spared nerve injury (SNI) in mice.SNI resulted in rapid and persistent changes in DNA methylation, with robust differential methylation observed between SNI and sham-operated control mice at all time points.Hundreds of differentially methylated genes were identified, including many with known function in pain. Pathway analysis revealed enrichment in genes related to stimulus response at early time points, immune function at later time points and actin and cytoskeletal regulation throughout the time course. Increased attention to pain chronicity as a factor is recommended for both pain research and management.

Objective: Determine if chronic low back pain (LBP) is associated with DNA methylation signatures in human T cells that will reveal novel mechanisms and potential therapeutic targets and explore the feasibility of epigenetic diagnostic markers for pain-related pathophysiology. Methods: Genome-wide DNA methylation analysis of 850,000 CpG sites in women and men with chronic LBP and pain-free controls was performed. T cells were isolated (discovery cohort, n = 32) and used to identify differentially methylated CpG sites, and gene ontologies and molecular pathways were identified. A polygenic DNA methylation score for LBP was generated in both women and men. Validation was performed in an independent cohort (validation cohort, n = 63) of chronic LBP and healthy controls. Results: Analysis with the discovery cohort revealed a total of 2,496 and 419 differentially methylated CpGs in women and men, respectively. In women, most of these sites were hypomethylated and enriched in genes with functions in the extracellular matrix, in the immune system (ie, cytokines), or in epigenetic processes. In men, a unique chronic LBP DNA methylation signature was identified characterized by significant enrichment for genes from the major histocompatibility complex. Sex-specific polygenic DNA methylation scores were generated to estimate the pain status of each individual and confirmed in the validation cohort using pyrosequencing. Conclusion: This study reveals sex-specific DNA methylation signatures in human T cells that discriminates chronic LBP participants from healthy controls.