Accepted

Though sex differences in chronic pain have been consistently described in the literature, their underlying neural mechanisms are poorly understood. Previous work in humans has demonstrated that men and women differentially invoke distinct brain regions and circuits in coping with subjective pain unpleasantness. The goal of the present work was to elucidate the molecular mechanisms in the basolateral nucleus of the amygdala (BLA) that modulate hyperalgesic priming, a pain plasticity model, in males and females. We used plantar incision as the first, priming stimulus and prostaglandin E (PGE) as the second stimulus. We sought to assess whether hyperalgesic priming can be prevented or reversed by pharmacologically manipulating molecular targets in the BLA of male or female mice. We found that administering ZIP, a cell-permeable inhibitor of aPKC, into the BLA attenuated aspects of hyperalgesic priming induced by plantar incision in males and females. However, incision only upregulated PKCζ/PKMζ immunoreactivity in the BLA of male mice, and deficits in hyperalgesic priming were seen only when we restricted our analysis to male mice. On the other hand, intra-BLA microinjections of pep2m, a peptide that interferes with the trafficking and function of GluA2-containing AMPA receptors, a downstream target of aPKC, reduced mechanical hypersensitivity after plantar incision and disrupted the development of hyperalgesic priming in both male and female mice. In addition, pep2m treatment reduced facial grimacing and restored aberrant behavioral responses in the sucrose splash test in male and female primed mice. Immunofluorescence results demonstrated upregulation of GluA2 expression in the BLA of male and female primed mice, consistent with pep2m findings. We conclude that, in a model of incision-induced hyperalgesic priming, PKCζ/PKMζ in the BLA is critical for the development of hyperalgesic priming in males, while GluA2 in the BLA is crucial for the expression of both reflexive and affective pain-related behaviors in both male and female mice in this model. Our findings add to a growing body of evidence of sex differences in molecular pain mechanisms in the brain.

HIV-associated neuropathic pain (HNP) is a common complication for AIDS patients. The pathological mechanism governing HNP has not been elucidated, and HNP has no effective analgesic treatment. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic factor family related to the plasticity of the central nervous system. BDNF dysregulation is involved in many neurological diseases, including neuropathic pain. However, to the best of our knowledge, the role and mechanism of BDNF in HNP have not been elucidated. In this study, we explored this condition in an HNP mouse model induced by intrathecal injection of gp120. We found that Wnt3a and β-catenin expression levels increased in the spinal cord of HNP mice, consequently regulating the expression of BDNF and affecting hypersensitivity. In addition, the blockade of Wing-Int/β-catenin signaling, BDNF/TrkB or the BDNF/p75NTR pathway alleviated mechanical allodynia. BDNF immunoreactivity was colocalized with spinal microglial cells, which were activated in HNP mice. Inhibition of spinal microglial cell activation by minocycline relieved mechanical allodynia in HNP mice. This study helped to elucidate the role of the Wing-Int/β-catenin/BDNF signaling axis in HNP and may establish a foundation for further research investigating the Wing-Int/β-catenin/BDNF signaling axis as a target for HNP treatment.

Spinal D-serine plays an important role in nociception via an increase in phosphorylation of the NMDA receptor GluN1 subunit (pGluN1). However, the cellular mechanisms underlying this process have not been elucidated. Here we investigate the possible role of neuronal nitric oxide synthase (nNOS) in the D-serine-induced potentiation of NMDA receptor function and the induction of neuropathic pain in a chronic constriction injury (CCI) model. Intrathecal administration of the serine racemase inhibitor, LSOS or the D-serine degrading enzyme, DAAO on post-operative days 0-3 significantly reduced the CCI-induced increase in NO levels and NADPH-diaphorase staining in lumbar dorsal horn neurons, as well as the CCI-induced decrease in phosphorylation (Ser847) of nNOS (pnNOS) on day 3 post-CCI surgery. LSOS or DAAO administration suppressed the CCI-induced development of mechanical allodynia and PKC-dependent (Ser896) phosphorylation of GluN1 on day 3 post-surgery, which were reversed by the co-administration of the NO donor, SIN-1. In naïve mice, exogenouse D-serine increased NO levels via decreases in pnNOS. D-serine-induced increases in mechnical hypersensitivity, NO levels, PKC-dependent pGluN1, and NMDA-induced spontaneous nociception were reduced by pretreatment with the nNOS inhibitor, 7-nitroindazole or with the NMDA receptor antagonists, 7-chlorokynurenic acid and MK-801. Collectively we show that spinal D-serine modulates nNOS activity and concomitant NO production leading to increases in PKC-dependent pGluN1, and ultimately contributing to the induction of mechanical allodynia following peripheral nerve injury.

The formation of neuromas involves expansion of the cellular components of peripheral nerves. The onset of these disorganized tumors involves activation of sensory nerves and neuroinflammation. Particularly problematic in neuroma is arborization of axons leading to extreme, neuropathic pain. The most common sites for neuroma are the ends of transected nerves following injury; however, this rodent model does not reliably result in neuroma formation. In this study, we established a rodent model of neuroma in which the sciatic nerve was loosely ligated with two chromic gut sutures [1]. This model formed neuromas reliably (~95%), presumably through activation of the neural inflammatory cascade. Resulting neuromas had a disorganized structure and a significant number of replicating cells. Quantification of changes in perineurial and Schwann cells showed a significant increase in these populations. Immunohistochemical analysis showed the presence of β-tubulin 3 (TUJ1) in the rapidly expanding nerve and a decrease in neurofilament heavy chain compared to the normal nerve, suggesting the axons forming a disorganized structure. Measurement of the permeability of the blood-nerve barrier (BNB) shows that it opened almost immediately and remained open as long as 10 days. Studies using an antagonist of the 3-adrenergic receptor (ADRβ3) (L-748,337) or cromolyn showed a significant reduction in tumor size and cell expansion as determined by flow cytometry, with an improvement in the animal's gait detected using a Catwalk system. Previous studies in our laboratory have shown that heterotopic ossification (HO) is also a result of the activation of neuroinflammation. Since HO and neuroma often occur together in amputees, they were induced in the same limbs of the study animals. More heterotopic bone was formed in animals with neuromas as compared to those without. These data collectively suggest that perturbation of early neuroinflammation with compounds such as L-748,337 and cromolyn may reduce formation of neuromas.

Fast inactivation of voltage-gated sodium (Nav) channels is essential for electrical signaling but its mechanism remains poorly understood. Here, we determined the structures of a eukaryotic Nav channel alone and in complex with a lethal α-scorpion toxin, AaH2, by electron microscopy, both at 3.5-A resolution. AaH2 wedges into voltage-sensor domain IV (VSD4) to impede fast activation by trapping a deactivated state in which gating charge interactions bridge to the acidic intracellular C-terminal domain. In the absence of AaH2, the S4 helix of VSD4 undergoes a ~13-Å translation to unlatch the intracellular fast inactivation gating machinery. Highlighting the polypharmacology of α-scorpion toxins, AaH2 also targets an unanticipated receptor site on VSD1 and a pore-glycan adjacent to VSD4. Overall, this work provides key insights into fast inactivation, electromechanical coupling, and pathogenic mutations in Nav channels.