Animal Studies

Spinal high mobility group box 1 protein (HMGB1) plays crucial roles in arthritis-induced pain, however the involvement of peripheral HMGB1 has not been examined previously. In this study, we addressed the role of peripheral HMGB1 and explored if sex contributes differentially to nociception in arthritis. We found Hmgb1 expression to be elevated in the ankle joints of male and female mice subjected to collagen antibody-induced arthritis (CAIA). Blocking the action of peripheral HMGB1, however, only reversed CAIA-induced hypersensitivity in males. Intra-articular injection of the toll-like receptor (TLR)4-activating, partially reduced disulfide, but not the fully reduced all-thiol, HMGB1 evoked mechanical hypersensitivity in both sexes. A sex-dependent temporal profile in expression of inflammatory factors in the ankle joint was observed in response to intra-articular injection of disulfide HMGB1, with male mice showing a delayed, yet longer lasting increase in mRNA levels for several of the investigated factors. Intra-articular HMGB1 did not induce cellular infiltration in the ankle joint suggesting its action on tissue resident cells. To further explore possible sex differences in cellular involvement, we used the macrophage inhibitor, minocycline, and mice with specific TLR4 depletion in myeloid cells or nociceptors. We found that inhibition of resident macrophages attenuated HMGB1-induced pain-like behavior only in male mice. Interestingly, while the contribution of TLR4 on myeloid cells to nociception was minimal in females compared to males, TLR4 on nociceptors are important for HMGB1-induced pain in both sexes. Collectively, our work highlights sex- and cellular location-dependent roles of HMGB1 and TLR4 in peripheral pain mechanisms.

When skin afferents are activated, the sensory signals are transmitted to the spinal cord and eventually reach the primary somatosensory cortex (SI), initiating the encoding of the sensory percept in the brain. While subsets of primary afferents mediate specific somatosensory information from an early age, the subcortical pathways that transmit this information undergo striking changes over the first weeks of life, reflected in the gradual emergence of specific sensory behaviours. We therefore hypothesised that this period is associated with differential changes in the encoding of incoming afferent volleys in SI. To test this, we compared SI responses to A fibre skin afferent stimulation and A+C skin afferent fibre stimulation in lightly anaesthetised male rats at postnatal day (P) 7, 14, 21 and 30. Differences in SI activity following A and A+C fibre stimulation changed dramatically over this period. At P30, A+C fibre stimulation evoked significantly larger gamma, beta and alpha energy increases compared to A fibre stimulation alone. At younger ages, the changes in S1 oscillatory activity evoked by the two afferent volleys were not significantly different. Silencing TRPV1+ C fibres with QX-314 significantly reduced the gamma and beta SI oscillatory energy increases evoked by A+C fibres, at P30 and P21, but not at younger ages. Thus, C fibres differentially modulate SI oscillatory activity only from the third postnatal week, well after the functional maturation of the somatosensory cortex. This age-related change in afferent evoked S1 oscillatory activity may underpin the maturation of sensory discrimination in the developing brain. Behavioural responses to sensory stimulation of the skin undergo major developmental changes over the first postnatal weeks. Here we show that this is accompanied by a shift in the differential frequency encoding of sensory A fibre and C fibre afferent inputs into the developing rat somatosensory cortex. The results demonstrate major postnatal changes in the ability of the cortex to differentiate between afferent sensory inputs arriving in the mammalian brain.

High mobility group box 1 protein (HMGB1) is increasingly regarded as an important player in the spinal regulation of chronic pain. While it has been reported that HMGB1 induces spinal glial activation in a Toll-like receptor (TLR)4-dependent fashion, the aspect of sexual dimorphisms has not been thoroughly addressed. Here, we examined whether the action of TLR4-activating, partially reduced disulfide HMGB1 on microglia induces nociceptive behaviors in a sex-dependent manner. We found disulfide HMGB1 to equally increase microglial Iba-1 immunoreactivity in lumbar spinal dorsal horn in male and female mice, but evoke higher cytokine and chemokine expression in primary microglial culture derived from males compared to females. Interestingly, TLR4 ablation in myeloid-derived cells, which include microglia, only protected male mice from developing HMGB1-induced mechanical hypersensitivity. Spinal administration of the glial inhibitor, minocycline, with disulfide HMGB1 also prevented pain-like behavior in male mice. To further explore sex difference, we examined the global spinal protein expression using liquid chromatography-mass spectrometry (LC-MS/MS) and found several antinociceptive and anti-inflammatory proteins to be upregulated in only male mice subjected to minocycline. One of the proteins elevated, alpha-1-antitrypsin, partially protected males but not females from developing HMGB1-induced pain. Targeting downstream proteins of alpha-1-antitrypsin failed to produce robust sex differences in pain-like behavior, suggesting that several proteins identified by LC-MS/MS are required to modulate the effects. Taken together, the current study highlights the importance of mapping sex dimorphisms in pain mechanisms and point to processes potentially involved in the spinal antinociceptive effect of microglial inhibition in male mice.

The present study aimed to investigate the effects of high-mobility group box-1 (HMGB-1) on mechanical pain hypersensitivity and the underlying mechanism. Mouse primary astrocytes were isolated and treated as specified. A CCK-8 assay was used to determine cytotoxicity and a gap junctional communication assay was performed. Ethidium bromide (EtBr) uptake was used to evaluate the hemichannel activity of primary astrocytes. A mouse model of neuropathic pain was developed and paw withdrawal threshold was used to evaluate hind paw sensitivity. RT-qPCR and Western blot were used to determine mRNA and protein expression of genes, respectively. ELISA was used to measure the release of inflammatory cytokines. Treatment with HMGB-1 increased the expression of both toll-like receptor-4 (TLR-4) and connexin 43 (Cx43)in mouse primary astrocytes. HMGB-1 also promoted gap junctional intercellular communication and hemichannel function. Our results also demonstrated that HMGB-1 regulated Cx43 through the JNK signaling pathway, and Cx43 was involved in HMGB-1-mediated inflammation in astrocytes. In vivo analysis supported the idea that HMGB-1-induced mechanical hypersensitivity was associated with Cx43. We therefore conclude that HMGB-1-induced mechanical pain hypersensitivity occurs through modulating astrocytic Cx43 via the TLR-4/JNK signaling pathway.

Patients with neuropathic pain often experience exaggerated pain and anxiety. Central sensitization has been linked with the maintenance of neuropathic pain and may become an autonomous pain generator. Conversely, emerging evidence accumulated that central sensitization is initiated and maintained by ongoing nociceptive primary afferent inputs. However, it remains elusive what mechanisms underlie this phenomenon and which peripheral candidate contributes to central sensitization that accounts for pain hypersensitivity and pain-related anxiety. Previous studies have implicated peripherally-localized cGMP-dependent protein kinase I (PKG-I) in plasticity of nociceptors and spinal synaptic transmission as well as inflammatory hyperalgesia. However, whether peripheral PKG-I contributes to cortical plasticity and hence maintains nerve injury-induced pain hypersensitivity and anxiety is unknown. Here we demonstrated significant upregulation of PKG-I in ipsilateral L3 DRG, no change in L4 DRG and downregulation in L5 DRG upon spared nerve injury (SNI). Genetic ablation of PKG-I specifically in nociceptors or posttreatment with intervertebral foramen injection of PKG-I antagonist, KT5823 attenuated the development and maintenance of SNI-induced bilateral pain hypersensitivity and anxiety. Mechanistic analysis revealed that activation of PKG-I in nociceptors is responsible for synaptic potentiation in ACC upon peripheral neuropathy via presynaptic mechanisms involving BDNF signaling. Our results revealed that PKG-I expressed in nociceptors is a key determinant for cingulate synaptic plasticity after nerve injury, which contributes to the maintenance of pain hypersensitivity and anxiety. Thereby, this study presents a strong basis for opening up a novel therapeutic target, PKG-I in nociceptors for treatment of comorbidity of neuropathic pain and anxiety with least side effects.

Adaptations in brain communication are associated with multiple pain disorders and are hypothesized to promote the transition from acute to chronic pain. Despite known increases in brain synaptic activity, it is unknown if and how changes in pathways and networks contribute to persistent pain. A tunable rat model that induces transient or persistent temporomandibular joint pain was used to characterize brain network and sub-circuit changes when sensitivity is detected in both transient and persistent pain groups and later when sensitivity is present only for the persistent pain group. Brain activity was measured by F-FDG PET imaging and used to construct inter-subject correlation networks; network connectivity distributions, diagnostics, and community structure were assessed. Activation of sub-circuits was tested by structural equation modeling. Findings reveal differences in the brain networks at day 7 between the persistent and transient pain groups, a time when peripheral sensitivity is detected in both groups, but spontaneous pain occurs only in the persistent pain group. At day 7, increased (p≤0.01) clustering, node strength, network segregation, and activation of prefrontal-limbic pathways are observed only in the group that develops persistent pain. Later, increased clustering and node strength are more pronounced with persistent pain, particularly within the limbic system, and decrease when pain resolves. Pre-treatment with intraarticular etanercept to attenuate pain confirms these adaptations are associated with pain onset. Results suggest that early and sustained brain changes can differentiate persistent and transient pain, implying they could be useful as prognostic biomarkers for persistent pain and in identifying therapeutic targets.

Extrasynaptic α -subunit containing GABA (α -GABA ) receptors participate in chronic pain. Previously, we reported a sex difference in the action of α -GABA receptors in dysfunctional pain. However, the underlying mechanisms remain unknown. The aim of this study was to examine this sexual dimorphism in neuropathic rodents and the mechanisms involved. Female and male Wistar rats or ICR mice were subjected to nerve injury followed by α -GABA receptor inverse agonist intrathecal administration, L-655,708. The drug produced an antiallodynic effect in nerve-injured female rats and mice, and a lower effect in males. We hypothesized that changes in α -GABA receptor, probably influenced by hormonal and epigenetic status, might underlie this sex-difference. Thus, we performed qPCR and western blot. Nerve injury increased α -GABA mRNA and protein in female dorsal root ganglia (DRG) and decreased them in DRG and spinal cord of males. To investigate the hormonal influence over α -GABA receptor actions, we performed nerve injury to ovariectomized rats and reconstituted them with 17β-estradiol (E2). Ovariectomy abrogated L-655,708 antiallodynic effect and E2 restored it. Ovariectomy decreased α -GABA receptor and ER α protein in DRG of neuropathic female rats, while E2 enhanced them. Since DNA methylation might contribute to α -GABA receptor down-regulation in males, we examined CpG island DNA methylation of α -GABA receptor coding gene through pyrosequencing. Nerve injury increased methylation in male, but not female rats. Pharmacological inhibition of DNA methyltransferases increased α -GABA receptor and enabled L-655,708 antinociceptive effect in male rats. These results suggest that α -GABA receptor is a suitable target to treat chronic pain in females.

The adaptive significance of acute pain (to withdraw from tissue-damaging or potentially tissue-damaging external stimuli, and to enhance the salience of the stimulus resulting in escape and avoidance learning) and tonic pain (to enforce recuperation by punishing movement) are well-accepted [1]. Pain researchers, however, generally assert that chronic pain has no adaptive significance, representing instead a pathophysiological state. This belief was recently challenged by the observation [2] that nociceptive sensitization caused by a chronic pain-producing injury reduced predation risk in squid (Doryteuthis pealeii). In that study, injury to an arm (removal of the tip with a scalpel) 6 hours prior led to increased targeting by black sea bass, resulting in decreased survival of the squid in a 30-minute trial featuring free interaction between predator and prey. The surprising finding was that anesthesia during surgery, preventing the chronic nociceptor sensitization associated with such injuries, led to even lower probability of survival. That is, the likely presence of pain increased apparent fitness, and the authors concluded that the chronic pain state and its associated nociceptive sensitization represented an adaptive function. Pain-induced defensive behaviors affecting fitness have also been reported in crustaceans (Gammarus fossarum) [3]. It is, however, currently unknown whether this may also be true in any other species, including in Mammalia.