Mechanical Hypersensitivity In Mice Research Articles

Quantification of stimulus-evoked tactile allodynia in free moving mice by the chainmail sensitivity test.

Chronic pain occurs at epidemic levels throughout the population. Hypersensitivity to touch, is a cardinal symptom of chronic pain. Despite dedicated research for over a century, quantifying this hypersensitivity has remained impossible at scale. To address these issues, we developed the Chainmail Sensitivity Test (CST). Our results show that control mice spend significantly more time on the chainmail portion of the device than mice subject to neuropathy. Treatment with gabapentin abolishes this difference. CST-derived data correlate well with von Frey measurements and quantify hypersensitivity due to inflammation. Our study demonstrates the potential of the CST as a standardized tool for assessing mechanical hypersensitivity in mice with minimal operator input.

4E-BP1-dependent translation in nociceptors controls mechanical hypersensitivity via TRIM32/type I interferon signaling.

Activation of the mechanistic target of rapamycin complex 1 (mTORC1) contributes to the development of chronic pain. However, the specific mechanisms by which mTORC1 causes hypersensitivity remain elusive. The eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) is a key mTORC1 downstream effector that represses translation initiation. Here, we show that nociceptor-specific deletion of 4E-BP1, mimicking activation of mTORC1-dependent translation, is sufficient to cause mechanical hypersensitivity. Using translating ribosome affinity purification in nociceptors lacking 4E-BP1, we identified a pronounced translational up-regulation of tripartite motif-containing protein 32 (TRIM32), an E3 ubiquitin ligase that promotes interferon signaling. Down-regulation of TRIM32 in nociceptors or blocking type I interferon signaling reversed the mechanical hypersensitivity in mice lacking 4E-BP1. Furthermore, nociceptor-specific ablation of TRIM32 alleviated mechanical hypersensitivity caused by tissue inflammation. These results show that mTORC1 in nociceptors promotes hypersensitivity via 4E-BP1-dependent up-regulation of TRIM32/interferon signaling and identify TRIM32 as a therapeutic target in inflammatory pain.

SAFit2 ameliorates paclitaxel-induced neuropathic pain by reducing spinal gliosis and elevating pro-resolving lipid mediators

BackgroundChemotherapy-induced neuropathic pain (CIPN) describes a pathological pain state that occurs dose-dependently as a side effect and can limit or even impede an effective cancer therapy. Unfortunately, current treatment possibilities for CIPN are remarkably confined and mostly inadequate as CIPN therapeutics themselves consist of low effectiveness and may induce severe side effects, pointing out CIPN as pathological entity with an emerging need for novel treatment targets. Here, we investigated whether the novel and highly specific FKBP51 inhibitor SAFit2 reduces paclitaxel-induced neuropathic pain.MethodsIn this study, we used a well-established multiple low-dose paclitaxel model to investigate analgesic and anti-inflammatory properties of SAFit2. For this purpose, the behavior of the mice was recorded over 14 days and the mouse tissue was then analyzed using biochemical methods.ResultsHere, we show that SAFit2 is capable to reduce paclitaxel-induced mechanical hypersensitivity in mice. In addition, we detected that SAFit2 shifts lipid levels in nervous tissue toward an anti-inflammatory and pro-resolving lipid profile that counteracts peripheral sensitization after paclitaxel treatment. Furthermore, SAFit2 reduced the activation of astrocytes and microglia in the spinal cord as well as the levels of pain-mediating chemokines. Its treatment also increased anti-inflammatory cytokines levels in neuronal tissues, ultimately leading to a resolution of neuroinflammation.ConclusionsIn summary, SAFit2 shows antihyperalgesic properties as it ameliorates paclitaxel-induced neuropathic pain by reducing peripheral sensitization and resolving neuroinflammation. Therefore, we consider SAFit2 as a potential novel drug candidate for the treatment of paclitaxel-induced neuropathic pain.

Morphine-induced mechanical hypersensitivity in mice requires δ receptors, β-arrestin2, and c-Src activity.

Morphine diminishes pain, but its long-term use is compromised by tolerance and hyperalgesia. Studies implicate δ receptors, β-arrestin2 and Src kinase in tolerance. We examined whether these proteins are also involved in morphine-induced hypersensitivity (MIH). A common pathway for tolerance and hypersensitivity may provide a single target to guide improved analgesic approaches. We examined mechanical sensitivity using automated von Frey in wild-type (WT) and transgenic male and female C57Bl/6mice before and after hind paw inflammation by complete Freund's adjuvant (CFA). CFA-evoked hypersensitivity ceased on day 7 in WT but persisted for the 15-day testing period in μ-/- . Recovery was delayed until day 13 in δ-/- . We explored the expression of opioid genes in the spinal cord using quantitative RT-PCR. Restoration to basal sensitivity in WT occurred with increased δ expression. By contrast, κ expression was reduced, while μ remained unchanged. Daily morphine reduced hypersensitivity in WT on day 3 compared to controls; however, hypersensitivity recurred on day 9 and beyond. By contrast, WT had no recurrence of hypersensitivity in the absence of daily morphine. We used β-arrestin2-/- , δ-/- and Src inhibition by dasatinib in WT to establish whether these approaches, which diminish tolerance, also attenuate MIH. While none of these approaches affected CFA-evoked inflammation or acute hypersensitivity, all caused sustained morphine anti-hypersensitivity, abolishing MIH. Like morphine tolerance, MIH in this model requires δ receptors, β-arrestin2 and Src activity. Our findings suggest that MIH is caused by a tolerance-induced reduction in endogenous opioid signalling. KEY POINTS: Morphine is effective for treating severe acute pain, but tolerance and hypersensitivity often develop during its use in treating chronic pain. It is unclear whether these detrimental effects share similar mechanisms; if so, it might be possible to develop a single approach to minimise both phenomena. Mice deficient in μ receptors, δ receptors or β-arrestin2 and wild type mice treated with the Src inhibitor dasatinib exhibit negligible morphine tolerance. We show that these same approaches also prevent the development of morphine-induced hypersensitivity during persistent inflammation. This knowledge identifies strategies, such as the use of Src inhibitors, which may mitigate tolerance and morphine induced hyperalgesia.

Blockade of receptor for advanced glycation end-products with azeliragon ameliorates streptozotocin-induced diabetic neuropathy.

Treatment options for diabetic neuropathy are suboptimal, so development of a new therapeutic strategy is urgent. We focused on the role of receptor for advanced glycation end-products (RAGE) in diabetic neuropathy. We elaborated the effects of azeliragon (orally available small-molecule antagonist of RAGE) on streptozotocin (STZ)-induced mechanical hypersensitivity in mice. A reduction in mechanical nociceptive threshold observed 28 days after STZ treatment was improved by single administration of azeliragon (10 and 30mg/kg) at 3h, but this effect disappeared at 24h. Conversely, repeat administration (three times; days 28, 30, and 32) of azeliragon (30mg/kg) enhanced the antinociceptive effect significantly compared with that obtained upon single administration, and this effect persisted at least up to 24h. The antinociceptive effect of azeliragon (30mg/kg) was almost comparable with that of pregabalin (30mg/kg). These drug treatments had no effect on blood glucose levels. Our findings suggest that RAGE might be an effective target for diabetic neuropathy treatment.

(2R,6R)-hydroxynorketamine (HNK) reverses mechanical hypersensitivity in a model of localized inflammatory pain

The ketamine metabolite (2R,6R)-hydroxynorketamine, or (2R,6R)-HNK, was recently reported to evoke antinociception in response to a noxious thermal stimulus in healthy mice and reverse mechanical hypersensitivity in a murine model of neuropathic pain. This study reports the behavioral effects of (2R,6R)-HNK in male and female C57BL/6J mice exposed to a localized inflammatory pain condition and the broad pharmacological mechanism underlying this effect. Hind paw intraplantar injection of λ-carrageenan (CARR) caused inflammation and mechanical hypersensitivity in mice within 2 h, lasting at least 48 h. Administration of (2R,6R)-HNK (10–30 mg/kg i.p.) 2 h following CARR injection significantly reversed mechanical hypersensitivity within 1 h in male and female mice, and the effect persisted for 24 h following a single dose. The magnitude and timing of the analgesic effect of (2R,6R)-HNK were comparable to the non-steroidal anti-inflammatory drug carprofen. The reversal of hypersensitivity by (2R,6R)-HNK was blocked at 4 and 24 h after administration by pretreatment with the AMPA receptor antagonist NBQX and was not accompanied by changes in locomotor activity. These findings reinforce the growing evidence supporting (2R,6R)-HNK as a novel analgesic in multiple preclinical pain models and further support an AMPAR-dependent mechanism of action. SignificanceThe ketamine metabolite (2R,6R)-HNK reversed mechanical hypersensitivity associated with localized inflammation with onset less than 1 h and duration greater than 24 h, an effect comparable to the NSAID carprofen. Reversal of mechanical hypersensitivity by (2R,6R)-HNK is AMPAR-dependent.

Small Synthetic Hyaluronan Disaccharide BIS014 Mitigates Neuropathic Pain in Mice

Neuropathic pain (NP) is a challenging condition to treat, as the need for new drugs to treat NP is an unmet goal. We investigated the analgesic potential of a new sulfated disaccharide compound, named BIS014. Oral administration (p.o.) of this compound induced ameliorative effects in formalin-induced nociception and capsaicin-induced secondary mechanical hypersensitivity in mice, but also after partial sciatic nerve transection (spared nerve injury), chemotherapy (paclitaxel)-induced NP, and diabetic neuropathy induced by streptozotocin. Importantly, BIS014, at doses active on neuropathic hypersensitivity (60 mg/kg/p.o.), did not alter exploratory activity or motor coordination (in the rotarod test), unlike a standard dose of gabapentin (40 mg/kg/p.o.) which although inducing antiallodynic effects on the NP models, it also markedly decreased exploration and motor coordination. In docking and molecular dynamic simulation studies, BIS014 interacted with TRPV1, a receptor involved in pain transmission where it behaved as a partial agonist. Additionally, similar to capsaicin, BIS014 increased cytosolic Ca2+ concentration ([Ca2+]c) in neuroblastoma cells expressing TRPV1 receptors; these elevations were blocked by ruthenium red. BIS014 did not block capsaicin-elicited [Ca2+]c transients, but inhibited the increase in the firing rate of action potentials in bradykinin-sensitized dorsal root ganglion neurons stimulated with capsaicin. PerspectiveWe report that the oral administration of a new sulfated disaccharide compound, named BIS014, decreases neuropathic pain from diverse etiology in mice. Unlike the comparator gabapentin, BIS014 does not induce sedation. Thus, BIS014 has the potential to become a new efficacious non-sedative oral medication for the treatment of neuropathic pain.

Vertebrate Lonesome Kinase Phosphorylates EphB2 Receptors on an Extracellular Residue to Drive NMDA-Dependent Pain

EphB/ephrinB signaling plays a key role in synaptic function and is linked to several neuropathological conditions, including neuropathic pain. The EphB2 receptor tyrosine kinase is known to interact with N-Methyl-D-aspartate glutamate receptors (NMDAR), resulting in increased surface retention of NMDARs at the synapse and an enhancement of postsynaptic currents. As changes in NMDAR signaling have been heavily implicated in the development of both acute and chronic pain, targeting this receptor interaction may produce clinically relevant therapeutics. We have recently demonstrated that the phosphorylation of a tyrosine residue (Y504) on the extracellular domain of the EphB2 receptor is necessary and sufficient to induce its interaction with NMDARs and cause mechanical pain hypersensitivity. The kinase responsible for this phosphorylation is unknown. We hypothesized that Vertebrate Lonesome Kinase (VLK) may be the key effector. Since the phosphorylation site of interest is located on the extracellular face of the EphB2 receptor, a secreted tyrosine kinase like VLK is an ideal candidate. Our results provide strong support for our hypothesis that VLK drives pain via extracellular phosphorylation of EphBs. We show that intrathecal injection of VLK resulted in long-lasting mechanical hypersensitivity in mice. Additionally, the effects observed for VLK were dependent on kinase activity and entirely dependent upon NMDARs because they were blocked by AP-5. We also show evidence of VLK protein and mRNA expression in dorsal root ganglion and spinal cord neurons of both mice and humans. Our results provide evidence for the involvement of VLK in pain behaviors and spinal cord NMDAR signaling, and identify a completely unique extracellular kinase target for the alleviation of pain. Grant support from NIH grants NS111976 & NS115441.

Inhibition of phosphodiesterase-4 in the spinal dorsal horn ameliorates neuropathic pain via cAMP-cytokine-Cx43 signaling in mice.

BackgroundThe spinal phosphodiesterase‐4 (PDE4) plays an important role in chronic pain. Inhibition of PDE4, an enzyme catalyzing the hydrolysis of cyclic adenosine monophosphate AMP (cAMP), produces potent antinociceptive activity. However, the antinociceptive mechanism remains largely unknown. Connexin43 (Cx43), a gap junction protein, has been shown to be involved in controlling pain transduction at the spinal level; restoration of Cx43 expression in spinal astrocytes to the normal levels reduces nerve injury‐induced pain. Here, we evaluate the novel mechanisms involving spinal cAMP‐Cx43 signaling by which PDE4 inhibitors produce antinociceptive activity.MethodsFirst, we determined the effect of PDE4 inhibitors rolipram and roflumilast on partial sciatic nerve ligation (PSNL)‐induced mechanical hypersensitivity. Next, we observed the role of cAMP‐Cx43 signaling in the effect of PDE4 inhibitors on PSNL‐induced mechanical hypersensitivity.ResultsSingle or repeated, intraperitoneal or intrathecal administration of rolipram or roflumilast significantly reduced mechanical hypersensitivity in mice following PSNL. In addition, repeated intrathecal treatment with either of PDE4 inhibitors reduced PSNL‐induced downregulation of cAMP and Cx43, and upregulation of proinflammatory cytokines tumor necrosis factor‐α (TNF‐α) and interleukin‐1β. Furthermore, the antinociceptive effects of PDE4 inhibitors were attenuated by the protein kinase A (PKA) inhibitor H89, TNF‐α, or Cx43 antagonist carbenoxolone. Finally, PSNL‐induced upregulation of PDE4B and PDE4D, especially the PDE4B subtype, was reduced by treatment with either of the PDE4 inhibitors.ConclusionsThe results suggest that the antinociceptive effect of PDE4 inhibitors is contributed by increasing Cx43 expression via cAMP‐PKA‐cytokine signaling in the spinal dorsal horn.

Evaluation of calcium-sensitive adenylyl cyclase AC1 and AC8 mRNA expression in the anterior cingulate cortex of mice with spared nerve injury neuropathy

The anterior cingulate cortex (ACC) is a critical region of the brain for the emotional and affective components of pain in rodents and humans. Hyperactivity in this region has been observed in neuropathic pain states in both patients and animal models and ablation of this region from cingulotomy, or inhibition with genetics or pharmacology can diminish pain and anxiety. Two adenylyl cyclases (AC), AC1 and AC8 play an important role in regulating nociception and anxiety-like behaviors through an action in the ACC, as genetic and pharmacological targeting of these enzymes reduces mechanical hypersensitivity and anxiety-like behavior, respectively. However, the distribution of these ACs in the ACC has not been studied in the context of neuropathic pain. To address this gap in knowledge, we conducted RNAscope in situ hybridization to assess AC1 and AC8 mRNA distribution in mice with spared nerve injury (SNI). Given the key role of AC1 in nociception in neuropathic, inflammatory and visceral pain animal models, we hypothesized that AC1 would be upregulated in the ACC of mice following nerve injury. This hypothesis was also founded on data showing increased AC1 expression in the ACC of mice with zymosan-induced visceral inflammation. We found that AC1 and AC8 are widely expressed in many regions of the mouse brain including the hippocampus, ACC, medial prefrontal cortex and midbrain regions, but AC1 is more highly expressed. Contrary to our hypothesis, SNI causes an increase in AC8 mRNA expression in NMDAR-2B (Nr2b) positive neurons in the contralateral ACC but does not affect AC1 mRNA expression. Our findings show that changes in Adcy1 mRNA expression in the ACC are insufficient to explain the important role of this AC in mechanical hypersensitivity in mice following nerve injury and suggest a potential unappreciated role of AC8 in regulation of ACC synaptic changes after nerve injury.

An HDAC6 inhibitor reverses chemotherapy-induced mechanical hypersensitivity via an IL-10 and macrophage dependent pathway

Chemotherapy-induced peripheral neuropathy (CIPN) impacts a growing number of cancer survivors and treatment options are limited. Histone deacetylase 6 (HDAC6) inhibitors are attractive candidates because they reverse established CIPN and may enhance anti-tumor effects of chemotherapy. Before considering clinical application of HDAC6 inhibitors, the mechanisms underlying reversal of CIPN need to be identified. We showed previously that deletion of Hdac6 from sensory neurons did not prevent cisplatin-induced mechanical hypersensitivity, while global deletion of Hdac6 was protective, indicating involvement of HDAC6 in other cell types. Here we show that local depletion of MRC1 (CD206)-positive macrophages without affecting microglia by intrathecal administration of mannosylated clodronate liposomes reduced the capacity of an HDAC6 inhibitor to reverse cisplatin-induced mechanical hypersensitivity. The HDAC6 inhibitor increased spinal cord Il10 mRNA and this was M2-macrophage dependent. Intrathecal administration of anti-IL-10 antibody or genetic deletion of Il10 prevented resolution of mechanical hypersensitivity. Genetic deletion of the IL-10 receptor from Advillin+ neurons prevented resolution of mechanical hypersensitivity in mice treated with the HDAC6 inhibitor. These findings indicate that treatment with an HDAC6 inhibitor increases macrophage-derived IL-10 signaling to IL-10 receptors on Advillin+ sensory neurons to resolve mechanical hypersensitivity. Cisplatin decreases mitochondrial function in sensory axons, and HDAC6 inhibition can promote axonal transport of healthy mitochondria. Indeed, the HDAC6 inhibitor normalized cisplatin-induced tibial nerve mitochondrial deficits. However, this was independent of macrophages and IL-10 signaling. In conclusion, our findings indicate that administration of an HDAC6 inhibitor reverses cisplatin-induced mechanical hypersensitivity through two complementary pathways: macrophage HDAC6 inhibition to promote IL-10 production and IL-10 signaling to DRG neurons, and neuronal HDAC6 inhibition to restore axonal mitochondrial health.

Distinct synaptic mechanisms underlying the analgesic effects of γ-aminobutyric acid transporter subtypes 1 and 3 inhibitors in the spinal dorsal horn.

Normalization of the excitatory and inhibitory balance by increasing the levels of endogenous inhibitory neurotransmitters by blocking their reuptake is a promising therapeutic strategy for relieving chronic pain. Pharmacological blockade of spinal γ-aminobutyric acid (GABA) transporter subtypes 1 and 3 (GAT1 and GAT3) has been reported to generate analgesic effects in animal models of neuropathic pain. Here, we explored the synaptic mechanisms underlying their analgesic effects in the spinal dorsal horn. Whole-cell recordings were made from dorsal horn neurons in spinal slices with attached dorsal roots from adult mice, and the effects of GAT inhibitors on miniature and evoked postsynaptic currents were examined. Behaviorally, GAT inhibitors were intrathecally applied to assess their effects on mechanical hypersensitivity in mice developing neuropathic pain after partial sciatic nerve ligation. The GAT1 inhibitor NNC-711 reduced the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of C-fiber-mediated EPSCs, and the GAT3 inhibitor SNAP-5114 reduced the amplitude of A-fiber-mediated and C-fiber-mediated EPSCs. These effects were antagonized by the GABAB receptor antagonist CGP55845. Consistently, the analgesic effect of intrathecally injected NNC-711 and SNAP-5114 in mice developing mechanical hypersensitivity after partial sciatic nerve ligation was abolished by CGP55845. Thus, GAT1 and GAT3 inhibitors exert distinct GABAB receptor-mediated inhibitory effects on excitatory synaptic transmission in the spinal dorsal horn, which most likely contributes to their analgesic effects.

Action of mefloquine/amitriptyline THN101 combination on neuropathic mechanical hypersensitivity in mice.

Tricyclic antidepressants that inhibit serotonin and noradrenaline reuptake, such as amitriptyline, are among the first-line treatments for neuropathic pain, which is caused by a lesion or disease affecting the somatosensory nervous system. These treatments are, however, partially efficient to alleviate neuropathic pain symptoms, and better treatments are still highly required. Interactions between neurons and glial cells participate in neuropathic pain processes, and importantly, connexins-transmembrane proteins involved in cell-cell communication-contribute to these interactions. In a neuropathic pain model in rats, mefloquine, a connexin inhibitor, has been shown to potentiate the antihyperalgesic effect of amitriptyline, a widely used antidepressant. In this study, we further investigated this improvement of amitriptyline action by mefloquine, using the cuff model of neuropathic pain in mice. We first observed that oral mefloquine co-treatment prolonged the effect of amitriptyline on mechanical hypersensitivity by 12 hours after administration. In addition, we showed that this potentiation was not due to pharmacokinetic interactions between the 2 drugs. Besides, lesional and pharmacological approaches showed that the prolonged effect was induced through noradrenergic descending pathways and the recruitment of α2 adrenoceptors. Another connexin blocker, carbenoxolone, also improved amitriptyline action. Additional in vitro studies suggested that mefloquine may also directly act on serotonin transporters and on adenosine A1 and A2A receptors, but drugs acting on these other targets failed to amplify amitriptyline action. Together, our data indicate that pharmacological blockade of connexins potentiates the therapeutic effect of amitriptyline in neuropathic pain.

N-acylethanolamine-hydrolysing acid amidase: A new potential target to treat paclitaxel-induced neuropathy.

Although paclitaxel is an effective chemotherapeutic agent used to treat multiple types of cancer (e.g. breast, ovarian, neck and lung), it also elicits paclitaxel-induced peripheral neuropathy (PIPN), which represents a major dose-limiting side effect of this drug. As the endogenously produced N-acylethanolamine, palmitoylethanolamide (PEA), reverses paclitaxel-induced mechanical hypersensitivity in mice, the main goals of this study were to examine if paclitaxel affects levels of endogenous PEA in the spinal cord of mice and whether exogenous administration of PEA provides protection from the occurrence of paclitaxel-induced mechanical hypersensitivity. We further examined whether inhibition of N-acylethanolamine-hydrolysing acid amidase (NAAA), a hydrolytic PEA enzyme, would offer protection in mouse model of PIPN. Paclitaxel reduced PEA levels in the spinal cord, suggesting that dysregulation of this lipid signalling system may contribute to PIPN. Consistent with this idea, repeated administration of PEA partially prevented the paclitaxel-induced mechanical hypersensitivity. We next evaluated whether the selective NAAA inhibitor, AM9053, would prevent paclitaxel-induced mechanical hypersensitivity in mice. Acute administration of AM9053 dose-dependently reversed mechanical hypersensitivity through a PPAR-α mechanism, whereas repeated administration of AM9053 fully prevented the development of PIPN, without any evidence of tolerance. Moreover, AM9053 produced a conditioned place preference in paclitaxel-treated mice, but not in control mice. This pattern of findings suggests a lack of intrinsic rewarding effects, but a reduction in the pain aversiveness induced by paclitaxel. Finally, AM9053 did not alter paclitaxel-induced cytotoxicity in lung tumour cells. Collectively, these studies suggest that NAAA represents a promising target to treat and prevent PIPN. The present study demonstrates that the chemotherapeutic paclitaxel alters PEA levels in the spinal cord, whereas repeated exogenous PEA administration moderately alleviates PIPN in mice. Additionally, targeting NAAA, PEA's hydrolysing enzyme with a selective compound AM9053 reverses and prevents the PIPN via the PPAR-α mechanism. Overall, the data suggest that selective NAAA inhibitors denote promising future therapeutics to mitigate and prevent PIPN.

Selective blockade of transient receptor potential vanilloid 4 reduces cyclophosphamide-induced bladder pain in mice

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel activated by various physical stimuli such as cell swelling and shear stress. TRPV4 is expressed in bladder sensory nerves and epithelium, and its activation produces urinary dysfunction in rodents. However, there have been few reports regarding its involvement in bladder pain. Therefore, we investigated whether TRPV4 is involved in bladder pain in mouse cystitis model. Intraperitoneal injection of cyclophosphamide (CYP; 300 mg/kg) produced mechanical hypersensitivity in the lower abdomen associated with a severe inflammatory bladder in mice. The mechanical threshold was reversed significantly in Trpv4-knockout (KO) mice. Repeated injections of CYP (150 mg/kg) daily for 4 days provoked mild bladder inflammation and persistent mechanical hypersensitivity in mice. Trpv4-KO mice prevented a reduction of the mechanical threshold without an alteration in bladder inflammation. A selective TRPV4 antagonist also reversed the mechanical threshold in chronic cystitis mice. Although expression of Trpv4 was unchanged in the bladders of chronic cystitis mice, the level of phosphorylated TRPV4 was increased significantly. These results suggest involvement of TRPV4 in bladder pain of cystitis mice. A TRPV4 antagonist might be useful for patients with irritable bladder pain such as those with interstitial cystitis/painful bladder syndrome.

Cilostazol is an effective causal therapy for preventing paclitaxel-induced peripheral neuropathy by suppression of Schwann cell dedifferentiation

Chemotherapy-induced peripheral neuropathy (CIPN) can lead to discontinuation of chemotherapy and is consequently a serious impediment to effective cancer treatment. Due to our limited understanding of mechanisms underlying the pathogenesis of CIPN, no causal therapy has been approved for relief of this condition. We previously demonstrated that taxanes (paclitaxel and docetaxel) induce Schwann cell dedifferentiation, characterized by increased expression of p75 and galectin-3, ultimately leading to demyelination. These changes appear to be responsible for CIPN pathogenesis. This study was designed to identify a novel candidate therapeutic for CIPN with the ability to suppress paclitaxel-induced Schwann cell dedifferentiation. Given that elevation of cyclic adenosine monophosphate (cAMP) signaling participates in Schwann cell differentiation, we performed immunocytochemical screening of phosphodiesterase (PDE) inhibitors. We found that the PDE3 inhibitor cilostazol strongly promoted differentiation of primary cultures of rat Schwann cells via a mechanism involving cAMP/exchange protein directly activated by cAMP (Epac) signaling. Co-treatment with cilostazol prevented paclitaxel-induced dedifferentiation of Schwann cell cultures and demyelination in a mixed culture of Schwann cells and dorsal root ganglia neurons. Notably, continuous oral administration of cilostazol suppressed Schwann cell dedifferentiation within the sciatic nerve and the development of mechanical hypersensitivity in a mouse model of paclitaxel-related CIPN. Importantly, cilostazol potentiated, rather than inhibited, the anti-cancer effect of paclitaxel on the human breast cancer cell line MDA-MB-231. These findings highlight the potential utility of cilostazol as a causal therapeutic that avoids the development of paclitaxel-related CIPN without compromising anti-cancer properties.

HDAC6-selective inhibitors decrease nerve-injury and inflammation-associated mechanical hypersensitivity in mice.

HDAC6 is a class IIB histone deacetylase expressed at many levels of the nociceptive pathway. This study tested the ability of novel and selective HDAC6 inhibitors to alleviate sensory hypersensitivity behaviors in mouse models of peripheral nerve injury and peripheral inflammation. We utilized the murine spared nerve injury (SNI) model for peripheral nerve injury and the Complete Freund's Adjuvant (CFA) model of peripheral inflammation. We applied the Von Frey assay to monitor mechanical allodynia. Using the SNI model, we demonstrate that daily administration of the brain-penetrant HDAC6 inhibitor, ACY-738, abolishes mechanical allodynia in male and in female mice. Importantly, there is no tolerance to the antiallodynic actions of these compounds as they produce a consistent increase in Von Frey thresholds for several weeks. We observed a similar antiallodynic effect when utilizing the HDAC6 inhibitor, ACY-257, which shows limited brain expression when administered systemically. We also demonstrate that ACY-738 and ACY-257 attenuate mechanical allodynia in the CFA model of peripheral inflammation. Overall, our findings suggest that inhibition of HDAC6 provides a promising therapeutic avenue for the alleviation of mechanical allodynia associated with peripheral nerve injury and peripheral inflammation.

Bartonella henselae infection induces a persistent mechanical hypersensitivity in mice.

ABSTRACT Bartonella spp. are re-emerging and neglected bacterial pathogens. The natural reservoirs for several species of this genus are domestic animals such as cats and dogs, the most common pets in the USA and Brazil. Some cat studies suggest that the infection is more prevalent in tropical and poverty-stricken areas. These bacteria were associated with a wide spectrum of clinical manifestations: fever of unknown origin, endocarditis, angiomatosis, chronic lymphadenopathy, hepatitis, fatigue, paresthesia and pain. Our group has already demonstrated that B. henselae -infected sickle cell disease mice present with hyperalgesia. We hypothesized that even immunocompetent mice infected by B. henselae would show an increased and persistent mechanical sensitivity. Five ten-week old male BALB/c mice were intraperitoneally inoculated with a 30 µL of suspension containing 10 4 CFU/mL of B. henselae, while five others were inoculated with an equal volume of saline solution. Four days after bacterial inoculation, the mechanical paw withdrawal threshold was measured using von Frey filaments in all animals, for five consecutive days. The infected animals showed hypersensitivity to mechanical stimuli for five consecutive days. The present study has demonstrated that B. henselae infection induces persistent mechanical hypersensitivity, a signal consistent with pain.

Identification and characterization of saikosaponins as antagonists of transient receptor potential A1 channel.

Neuropathic pain is associated with an increased sensitivity to painful stimuli or abnormal sensitivity to otherwise innocuous stimuli. However, in addition to adverse effects, currently available drugs have shown limited response in patients with neuropathic pain, which provides a rationale to explore new drug classes acting on novel targets and with better efficacy and safety profiles. Here, we found that saikosaponins potently inhibit agonist-induced activation of the transient receptor potential A1 (TRPA1) channel, which has been reported to mediate neuropathic pain by sensing a variety of chemical irritants. Molecular docking and site-directed mutagenesis analyses suggested that saikosaponins bind to the hydrophobic pocket in TRPA1 near the Asn855 residue, which, when mutated to Ser, was previously associated with enhanced pain perception in humans. In support of these findings, saikosaponin D significantly attenuated agonist-induced nociceptive responses and vincristine-induced mechanical hypersensitivity in mice. These results indicate that saikosaponins are TRPA1 antagonists and provide a basis for further elaboration of saikosaponin derivatives for the development of new therapeutics for neuropathic pain.

Skin/Muscle Incision and Retraction Induces Evoked and Spontaneous Pain in Mice.

Background Surgery is a frequent cause of persistent pain. Unrelieved chronic postsurgical pain causes unnecessary patient suffering and discomfort and usually leads to psychological complications. The rat model of skin/muscle incision and retraction (SMIR) with decreased paw withdrawal thresholds developed by Flatters was usually used to investigate the underlying mechanism of chronic postsurgical pain. Objectives The aim of our study was to develop a new mice model of SMIR for further investigation with transgenic mice and so on and to evaluate the analgesic effects of clonidine and gabapentin on pain behavior with this new mice model. Methods Male C57BL/6 mice were anesthetized, and a 1.0–1.3 cm incision was made in the skin of the medial thigh approximately 3 mm medial to the saphenous vein to reveal the muscle of the thigh. The paw withdrawal threshold (PWT) to mechanical stimuli and the paw withdrawal latency to heat stimuli were measured before and after SMIR. Furthermore, the PWT to mechanical stimuli and conditioned place preference (CPP) was measured before and after the systemic injection of clonidine and gabapentin. Results SMIR-evoked mechanical hypersensitivity in mice began on day 1 after the procedure, prominent between days 1 and 10 after the procedure, persisted at least until day 14, and disappeared on day 18 after the procedure. However, the mice model of SMIR did not evoke significant heat hypersensitivity. Systemic injection of clonidine and gabapentin raised the PWT in the SMIR mice dose-dependently. Compared with the mice that underwent the sham operation, mice of SMIR spent a longer time in the clonidine-paired chamber than those of NS, while the gabapentin-paired chamber has no difference with that of NS in the CPP paradigm. Conclusion These data suggested that the mice model of SMIR demonstrated a persistent pain syndrome, including evoked pain and spontaneous pain. Clonidine and gabapentin could relieve mechanical hypersensitivity dose-dependently simultaneously. However, clonidine but not gabapentin could alleviate the spontaneous pain of SMIR in the mice model.