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Acid-sensing ion channels (ASICs) are important acid sensors involved in neural modulation in the central nervous system and pain-associated tissue acidosis in the peripheral system. Among ASIC subtypes, ASIC1b is the most selectively expressed in peripheral sensory neurons. However, the role of ASIC1b is still elusive in terms of its functions and expression profile. In this study, we probed the role of ASIC1b in acid-induced muscle pain in -knockout () and transgenic ( ) mice. We tested the effect of ASIC1b knockout in a mouse model of fibromyalgia induced by dual intramuscular acid injections. In this model, a unilateral acid injection to the gastrocnemius muscle induced transient bilateral hyperalgesia in wild-type () but not mice; a second acid injection, spaced 1 or 5 days apart, to the same muscle induced chronic hyperalgesia lasting for 4 weeks in mice, but the duration of hyperalgesia was significantly shortened in mice. Mambalgin-1, an ASIC1b-containing channel inhibitor that was mixed with acid saline at the first injection, dose-dependently blocked the acid-induced transient and chronic hyperalgesia in mice. In contrast, psalmotoxin 1 (PcTx1), an ASIC1a-selective antagonist, had no effect on acid-induced transient or chronic hyperalgesia. We used whole-cell patch clamp recording to study the properties of acid-induced currents in ASIC1b-expressing dorsal root ganglia (DRG) neurons from -TdTomato reporter mice. Medium- to large-sized ASIC1b-expressing DRG neurons mainly exhibited an amiloride-sensitive ASIC-like biphasic current () in response to acid stimulation, whereas small- to medium-sized ASIC1b-expressing DRG neurons predominantly exhibited an amiloride-insensitive sustained current. Specifically, mambalgin-1 selectively inhibited the in most ASIC1b-expressing DRG neurons. However, PcTx1 or APETx2 (an ASIC3-selective antagonist) had only a mild inhibitory effect on in about half of the ASIC1b-expressing DRG neurons. hybridization revealed that ASIC1b-positive DRG neurons co-expressed highly with ASIC1a and ASIC2a mRNA and partially with ASIC3 and ASIC2b. Thus, ASIC1b might form a wide variety of heteromeric channels. ASIC1b-containing heteromeric channels might be promising targets for the therapeutic treatment of acid-induced chronic muscle pain.
17β-estradiol plays a role in pain sensitivity, analgesic drug efficacy, and neuropathic pain prevalence, but the underlying mechanisms remain unclear. Here, we investigated whether voltage-gated chloride channel-3 (ClC-3) impacts the effects of 17β-estradiol (E2) on spared nerve injury (SNI)-induced neuropathic pain in ovariectomized (OVX) female Sprague Dawley rats that were divided into OVX, OVX + SNI, OVX + SNI + E2, OVX + SNI + E2 + DMSO (vehicle, dimethyl sulfoxide), or OVX + SNI + E2+Cltx (ClC-3-blocker chlorotoxin) groups. Changes in ClC-3 protein expression were monitored by western blot analysis. Behavioral testing used the paw withdrawal threshold to acetone irritation and paw withdrawal thermal latency (PWTL) to thermal stimulation. Immunofluorescence indicated the localization and protein expression levels of ClC-3. OVX + SNI + E2 rats were subcutaneously injected with 17β-estradiol once daily for 7 days; a sheathed tube was implanted, and chlorotoxin was injected for 4 days. Intrathecal Cltx to OVX and OVX + SNI rats was administered for 4 consecutive days (days 7-10 after SNI) to further determine the contribution of ClC-3 to neuropathic pain. Patch clamp technology in current clamp mode was used to measure the current threshold (rheobase) dorsal root ganglion (DRG) neurons and the minimal current that evoked action potentials (APs) as excitability parameters. The mean number of APs at double-strength rheobase verified neuronal excitability. There was no difference in behaviors and ClC-3 expression after OVX. Compared with OVX + SNI rats, OVX + SNI + E2 rats showed a lower paw withdrawal threshold to the acetone stimulus, but the PWTL was not significantly different, indicating increased sensitivity to cold but not to thermal pain. Co-immunofluorescent data revealed that ClC-3 was mainly distributed in A- and C-type nociceptive neurons, especially in medium/small-sized neurons. 17β-estradiol administration was associated with increased expression of ClC-3. 17β-estradiol-induced increase in ClC-3 expression was blocked by co-administration of Cltx. Cltx causes hyperalgesia and decreased expression of ClC-3 in OVX rats. Patch clamp results suggested that 17β-estradiol attenuated the excitability of neurons induced by SNI by up-regulating the expression of ClC-3 in the DRG of OVX rats. 17β-estradiol administration significantly improved cold allodynia thresholds in OVX rats with SNI. The mechanism for this decreased sensitivity may be related to the upregulation of ClC-3 expression in the DRG.
Growing evidence indicates cannabinoid receptors as potential therapeutic targets for chronic pain. Consequently, there is an increasing interest in developing cannabinoid receptor agonists for treating human and veterinary pain. To better understand the actions of a drug, it is of paramount importance to know the cellular distribution of its specific receptor(s). The distribution of canonical and putative cannabinoid receptors in the peripheral and central nervous system of dogs is still in its infancy. In order to help fill this anatomical gap, the present study has been designed to identify the cellular sites of cannabinoid and cannabinoid-related receptors in canine spinal ganglia. In particular, the cellular distribution of the cannabinoid receptors type 1 and 2 (CB and CB) and putative cannabinoid receptors G protein-coupled receptor 55 (GPR55), nuclear peroxisome proliferator-activated receptor alpha (PPARα), and transient receptor potential vanilloid type 1 (TRPV1) have been immunohistochemically investigated in the C6-C8 cervical ganglia of dogs. About 50% of the neuronal population displayed weak to moderate CB receptor and TRPV1 immunoreactivity, while all of them were CB-positive and nearly 40% also expressed GPR55 immunolabeling. Schwann cells, blood vessel smooth muscle cells, and pericyte-like cells all expressed CB receptor immunoreactivity, endothelial cell being also PPARα-positive. All the satellite glial cells (SGCs) displayed bright GPR55 receptor immunoreactivity. In half of the study dogs, SGCs were also PPARα-positive, and limited to older dogs displayed TRPV1 immunoreactivity. The present study may represent a morphological substrate to consider in order to develop therapeutic strategies against chronic pain.
Traumatic spinal cord injury (SCI) has devastating implications for patients, including a high predisposition for developing chronic pain distal to the site of injury. Chronic pain develops weeks to months after injury, consequently, patients are treated after irreparable changes have occurred. Nociceptors are central to chronic pain; however, the diversity of this cellular population presents challenges to understanding mechanisms and attributing pain modalities to specific cell types. To begin to address how peripheral sensory neurons below the injury level may contribute to the below-level pain reported by SCI patients, we examined SCI-induced changes in gene expression in lumbar dorsal root ganglia (DRG) below the site of injury. SCI was performed at the T10 vertebral level, with injury produced by a vessel clip with a closing pressure of 15 for 1 min. Alterations in gene expression produce long-term sensory changes, therefore, we were interested in studying SCI-induced transcripts before the onset of chronic pain, which may trigger changes in downstream signaling pathways and ultimately facilitate the transmission of pain. To examine changes in the nociceptor subpopulation in DRG distal to the site of injury, we retrograde labeled sensory neurons projecting to the hairy hindpaw skin with fluorescent dye and collected the corresponding lumbar (L2-L6) DRG 4 days post-injury. Following dissociation, labeled neurons were purified by fluorescence-activated cell sorting (FACS). RNA was extracted from sorted sensory neurons of naïve, sham, or SCI mice and sequenced. Transcript abundances validated that the desired population of nociceptors were isolated. Cross-comparisons to data sets from similar studies confirmed, we were able to isolate our cells of interest and identify a unique pattern of gene expression within a subpopulation of neurons projecting to the hairy hindpaw skin. Differential gene expression analysis showed high expression levels and significant transcript changes 4 days post-injury in SCI cell populations relevant to the onset of chronic pain. Regulatory interrelationships predicted by pathway analysis implicated changes within the synaptogenesis signaling pathway as well as networks related to inflammatory signaling mechanisms, suggesting a role for synaptic plasticity and a correlation with pro-inflammatory signaling in the transition from acute to chronic pain.
Despite being routinely used for pain management, opioid use is limited due to adverse effects such as development of tolerance and paradoxical pain, including thermal hyperalgesia and mechanical allodynia. Evidence indicates that continued morphine administration causes increased expression of proinflammatory mediators such as tumor necrosis factor-alpha (TNF-α). The objectives of the present study were to determine the effects of B1 (-[(1-benzimidazol-2-yl)methyl]-4-methoxyaniline) and B8 (-{4-[(1-benzimidazol-2-yl)methoxy]phenyl}acetamide), benzimidazole derivatives, on thermal nociception and mechanical allodynia during repeated morphine (intraperitoneal; 5 mg/kg twice daily for 6 days)-induced paradoxical pain and TNF-α expression in the spinal cord in mice. Our data indicate that administration of benzimidazole derivatives attenuated morphine-induced thermal hyperalgesia and mechanical allodynia. Benzimidazole derivatives also reduced TNF-α expression in mice. Taken together, these results suggest that benzimidazole derivatives might be useful for the treatment of neuroinflammatory consequences of continued morphine administration and could be potential drug candidates for the management of opioid-induced paradoxical pain.
Acupuncture therapy is effective for relieving postoperative pain. Our previous study showed that electroacupuncture (EA) at Futu (LI18) and Hegu (LI4)-Neiguan (PC6) could alleviate incisional neck pain, which was related with its effect in upregulating γ-aminobutyric acid (GABA) expression in cervical (C3-6) dorsal root ganglions (DRGs); but whether its receptor subsets GABAα2R and GABAR1 in C3-6 DRGs are involved in EA analgesia or not, it remains unknown.
Sigma-1 (σ) receptor antagonists are promising tools for neuropathic pain treatment, but it is unknown whether σ receptor inhibition ameliorates the neuropathic signs induced by nerve transection, in which the pathophysiological mechanisms and response to drug treatment differ from other neuropathic pain models. In addition, σ antagonism ameliorates inflammatory pain through modulation of the endogenous opioid system, but it is unknown whether this occurs during neuropathic pain. We investigated the effect of σ inhibition on the painful hypersensitivity associated with the spared nerve injury (SNI) model in mice. Wild-type (WT) mice developed prominent cold (acetone test), mechanical (von Frey test), and heat hypersensitivity (Hargreaves test) after SNI. σ receptor knockout (ခσ-KO) mice did not develop cold allodynia and showed significantly less mechanical allodynia, although they developed heat hyperalgesia after SNI. The systemic acute administration of the selective σ receptor antagonist S1RA attenuated all three types of SNI-induced hypersensitivity in WT mice. These ameliorative effects of S1RA were reversed by the administration of the σ agonist PRE-084, and were absent in σ-KO mice, indicating the selectivity of S1RA-induced effects. The opioid antagonist naloxone and its peripherally restricted analog naloxone methiodide prevented S1RA-induced effects in mechanical and heat hypersensitivity, but not in cold allodynia, indicating that opioid-dependent and -independent mechanisms are involved in the effects of this σ antagonist. The repeated administration of S1RA twice a day during 10 days reduced SNI-induced cold, mechanical, and heat hypersensitivity without inducing analgesic tolerance during treatment. These effects were observed up to 12 h after the last administration, when S1RA was undetectable in plasma or brain, indicating long-lasting pharmacodynamic effects. These data suggest that σ antagonism may have therapeutic value for the treatment of neuropathic pain induced by the transection of peripheral nerves.
GNF-2 is an allosteric inhibitor of Bcr-Abl. It was developed as a new class of anti-cancer drug to treat resistant chronic myelogenous leukemia. Recent studies suggest that c-Abl inhibition would provide a neuroprotective effect in animal models of Parkinson's disease as well as in clinical trials. However, the role of c-Abl and effects of GNF-2 in glia-mediated neuroinflammation or pain hypersensitivity has not been investigated. Thus, in the present study, we tested the hypothesis that c-Abl inhibition by GNF-2 may attenuate the inflammatory activation of glia and the ensuing pain behaviors in animal models. Our results show that GNF-2 reduced lipopolysaccharide (LPS)-induced nitric oxide and pro-inflammatory cytokine production in cultured glial cells in a c-Abl-dependent manner. The small interfering ribonucleic acid (siRNA)-mediated knockdown of c-Abl attenuated LPS-induced nuclear factor kappa light chain enhancer of activated B cell (NF-κB) activation and the production of pro-inflammatory mediators in glial cell cultures. Moreover, GNF-2 administration significantly attenuated mechanical and thermal hypersensitivities in experimental models of diabetic and inflammatory pain. Together, our findings suggest the involvement of c-Abl in neuroinflammation and pain pathogenesis and that GNF-2 can be used for the management of chronic pain.
Peripheral mechanisms of primary headaches such as a migraine remain unclear. Meningeal afferents surrounded by multiple mast cells have been suggested as a major source of migraine pain. Extracellular ATP released during migraine attacks is a likely candidate for activating meningeal afferents via neuronal P2X receptors. Recently, we showed that ATP also increased degranulation of resident meningeal mast cells (Nurkhametova et al., 2019). However, the contribution of ATP-induced mast cell degranulation in aggravating the migraine pain remains unknown. Here we explored the role of meningeal mast cells in the pro-nociceptive effects of extracellular ATP. The impact of mast cells on ATP mediated activation of peripheral branches of trigeminal nerves was measured electrophysiologically in the dura mater of adult wild type (WT) or mast cell deficient mice. We found that a spontaneous spiking activity in the meningeal afferents, at baseline level, did not differ in two groups. However, in WT mice, meningeal application of ATP dramatically (24.6-fold) increased nociceptive firing, peaking at frequencies around 10 Hz. In contrast, in mast cell deficient animals, ATP-induced excitation was significantly weaker (3.5-fold). Application of serotonin to meninges in WT induced strong spiking. Moreover, in WT mice, the 5-HT3 antagonist MDL-7222 inhibited not only serotonin but also the ATP induced nociceptive firing. Our data suggest that extracellular ATP activates nociceptive firing in meningeal trigeminal afferents via amplified degranulation of resident mast cells in addition to direct excitatory action on the nerve terminals. This highlights the importance of mast cell degranulation via extracellular ATP, in aggravating the migraine pain.