Targeting TRPV4 Channels for Cancer Pain Relief.

Despite the unique and complex nature of cancer pain, the activation of different ion channels can be related to the initiation and maintenance of pain. The transient receptor potential vanilloid 4 (TRPV4) is a cation channel broadly expressed in sensory afferent neurons. This channel is activated by multiple stimuli to mediate pain perception associated with inflammatory and neuropathic pain. Here, we focused on summarizing the role of TRPV4 in cancer etiology and cancer-induced pain mechanisms. Many studies revealed that the administration of a TRPV4 antagonist and TRPV4 knockdown diminishes nociception in chemotherapy-induced peripheral neuropathy (CIPN). Although the evidence on TRPV4 channels' involvement in cancer pain is scarce, the expression of these receptors was reportedly enhanced in cancer-induced bone pain (CIBP), perineural, and orofacial cancer models following the inoculation of tumor cells to the bone marrow cavity, sciatic nerve, and tongue, respectively. Effective pain management is a continuous problem for patients diagnosed with cancer, and current guidelines fail to address a mechanism-based treatment. Therefore, examining new molecules with potential antinociceptive properties targeting TRPV4 modulation would be interesting. Identifying such agents could lead to the development of treatment strategies with improved pain-relieving effects and fewer adverse effects than the currently available analgesics.

Topical application of a TRPA1 antagonist reduced nociception and inflammation in a model of traumatic muscle injury in rats.

Musculoskeletal pain is a widely experienced public healthcare issue, especially after traumatic muscle injury. Besides, it is a common cause of disability, but this pain remains poorly managed. However, the pathophysiology of traumatic muscle injury-associated pain and inflammation has not been fully elucidated. In this regard, the transient receptor potential ankyrin 1 (TRPA1) has been studied in inflammatory and painful conditions. Thus, this study aimed to evaluate the antinociceptive and anti-inflammatory effect of the topical application of a TRPA1 antagonist in a model of traumatic muscle injury in rats. The mechanical trauma model was developed by a single blunt trauma impact on the right gastrocnemius muscle of Wistar male rats (250-350 g). The animals were divided into four groups (Sham/Vehicle; Sham/HC-030031 0.05%; Injury/Vehicle, and Injury/HC-030031 0.05%) and topically treated with a Lanette® N cream base containing a TRPA1 antagonist (HC-030031, 0.05%; 200 mg/muscle) or vehicle (Lanette® N cream base; 200 mg/muscle), which was applied at 2, 6, 12, 24, and 46 h after muscle injury. Furthermore, we evaluated the contribution of the TRPA1 channel on nociceptive, inflammatory, and oxidative parameters. The topical application of TRPA1 antagonist reduced biomarkers of muscle injury (lactate/glucose ratio), spontaneous nociception (rat grimace scale), inflammatory (inflammatory cell infiltration, cytokine levels, myeloperoxidase, and N-acetyl-ß-D-glucosaminidase activities) and oxidative (nitrite levels and dichlorofluorescein fluorescence) parameters, and mRNA Trpa1 levels in the muscle tissue. Thus, these results demonstrate that TRPA1 may be a promising anti-inflammatory and antinociceptive target in treating muscle pain after traumatic muscle injury.

Bipartite Activation of Sensory Neurons by a TRPA1 Agonist Allyl Isothiocyanate Is Reflected by Complex Ca2+ Influx and CGRP Release Patterns: Enhancement by NGF and Inhibition with VAMP and SNAP-25 Cleaving Botulinum Neurotoxins.

The trafficking of transient receptor potential (TRP) channels to the plasma membrane and the release of calcitonin gene-related peptide (CGRP) from trigeminal ganglion neurons (TGNs) are implicated in some aspects of chronic migraines. These exocytotic processes are inhibited by cleavage of SNAREs with botulinum neurotoxins (BoNTs); moreover, type A toxin (/A) clinically reduces the frequency and severity of migraine attacks but not in all patients for unknown reasons. Herein, neonatal rat TGNs were stimulated with allyl isothiocyanate (AITC), a TRPA1 agonist, and dose relationships were established to link the resultant exocytosis of CGRP with Ca2+ influx. The CGRP release, quantified by ELISA, was best fit by a two-site model (EC50 of 6 and 93 µM) that correlates with elevations in intracellular Ca2+ [Ca2+]i revealed by time-lapse confocal microscopy of fluo-4-acetoxymethyl ester (Fluo-4 AM) loaded cells. These signals were all blocked by two TRPA1 antagonists, HC-030031 and A967079. At low [AITC], [Ca2+]i was limited because of desensitisation to the agonist but rose for concentrations > 0.1 mM due to a deduced non-desensitising second phase of Ca2+ influx. A recombinant BoNT chimera (/DA), which cleaves VAMP1/2/3, inhibited AITC-elicited CGRP release to a greater extent than SNAP-25-cleaving BoNT/A. /DA also proved more efficacious against CGRP efflux evoked by a TRPV1 agonist, capsaicin. Nerve growth factor (NGF), a pain-inducing sensitiser of TGNs, enhanced the CGRP exocytosis induced by low [AITC] only. Both toxins blocked NGF-induced neuropeptide secretion and its enhancement of the response to AITC. In conclusion, NGF sensitisation of sensory neurons involves TRPA1, elevated Ca2+ influx, and CGRP exocytosis, mediated by VAMP1/2/3 and SNAP-25 which can be attenuated by the BoNTs.

Botulinum Neurotoxin Chimeras Suppress Stimulation by Capsaicin of Rat Trigeminal Sensory Neurons In Vivo and In Vitro.

Chimeras of botulinum neurotoxin (BoNT) serotype A (/A) combined with /E protease might possess improved analgesic properties relative to either parent, due to inheriting the sensory neurotropism of the former with more extensive disabling of SNAP-25 from the latter. Hence, fusions of /E protease light chain (LC) to whole BoNT/A (LC/E-BoNT/A), and of the LC plus translocation domain (HN) of /E with the neuronal acceptor binding moiety (HC) of /A (BoNT/EA), created previously by gene recombination and expression in E. coli., were used. LC/E-BoNT/A (75 units/kg) injected into the whisker pad of rats seemed devoid of systemic toxicity, as reflected by an absence of weight loss, but inhibited the nocifensive behavior (grooming, freezing, and reduced mobility) induced by activating TRPV1 with capsaicin, injected at various days thereafter. No sex-related differences were observed. c-Fos expression was increased five-fold in the trigeminal nucleus caudalis ipsi-lateral to capsaicin injection, relative to the contra-lateral side and vehicle-treated controls, and this increase was virtually prevented by LC/E-BoNT/A. In vitro, LC/E-BoNT/A or /EA diminished CGRP exocytosis from rat neonate trigeminal ganglionic neurons stimulated with up to 1 µM capsaicin, whereas BoNT/A only substantially reduced the release in response to 0.1 µM or less of the stimulant, in accordance with the /E protease being known to prevent fusion of exocytotic vesicles.

NGF Enhances CGRP Release Evoked by Capsaicin from Rat Trigeminal Neurons: Differential Inhibition by SNAP-25-Cleaving Proteases.

Nerve growth factor (NGF) is known to intensify pain in various ways, so perturbing pertinent effects without negating its essential influences on neuronal functions could help the search for much-needed analgesics. Towards this goal, cultured neurons from neonatal rat trigeminal ganglia-a locus for craniofacial sensory nerves-were used to examine how NGF affects the Ca2+-dependent release of a pain mediator, calcitonin gene-related peptide (CGRP), that is triggered by activating a key signal transducer, transient receptor potential vanilloid 1 (TRPV1) with capsaicin (CAP). Measurements utilised neurons fed with or deprived of NGF for 2 days. Acute re-introduction of NGF induced Ca2+-dependent CGRP exocytosis that was inhibited by botulinum neurotoxin type A (BoNT/A) or a chimera of/E and/A (/EA), which truncated SNAP-25 (synaptosomal-associated protein with Mr = 25 k) at distinct sites. NGF additionally caused a Ca2+-independent enhancement of the neuropeptide release evoked by low concentrations (<100 nM) of CAP, but only marginally increased the peak response to ≥100 nM. Notably, BoNT/A inhibited CGRP exocytosis evoked by low but not high CAP concentrations, whereas/EA effectively reduced responses up to 1 µM CAP and inhibited to a greater extent its enhancement by NGF. In addition to establishing that sensitisation of sensory neurons to CAP by NGF is dependent on SNARE-mediated membrane fusion, insights were gleaned into the differential ability of two regions in the C-terminus of SNAP-25 (181-197 and 198-206) to support CAP-evoked Ca2+-dependent exocytosis at different intensities of stimulation.

Periorbital Nociception in a Progressive Multiple Sclerosis Mouse Model Is Dependent on TRPA1 Channel Activation.

Headaches are frequently described in progressive multiple sclerosis (PMS) patients, but their mechanism remains unknown. Transient receptor potential ankyrin 1 (TRPA1) was involved in neuropathic nociception in a model of PMS induced by experimental autoimmune encephalomyelitis (PMS-EAE), and TRPA1 activation causes periorbital and facial nociception. Thus, our purpose was to observe the development of periorbital mechanical allodynia (PMA) in a PMS-EAE model and evaluate the role of TRPA1 in periorbital nociception. Female PMS-EAE mice elicited PMA from day 7 to 14 days after induction. The antimigraine agents olcegepant and sumatriptan were able to reduce PMA. The PMA was diminished by the TRPA1 antagonists HC-030031, A-967079, metamizole and propyphenazone and was absent in TRPA1-deficient mice. Enhanced levels of TRPA1 endogenous agonists and NADPH oxidase activity were detected in the trigeminal ganglion of PMS-EAE mice. The administration of the anti-oxidants apocynin (an NADPH oxidase inhibitor) or alpha-lipoic acid (a sequestrant of reactive oxygen species), resulted in PMA reduction. These results suggest that generation of TRPA1 endogenous agonists in the PMS-EAE mouse model may sensitise TRPA1 in trigeminal nociceptors to elicit PMA. Thus, this ion channel could be a potential therapeutic target for the treatment of headache in PMS patients.

TRPA1 involvement in depression- and anxiety-like behaviors in a progressive multiple sclerosis model in mice.

Progressive multiple sclerosis (PMS) is a neurological disease associated with the development of depression and anxiety, but treatments available are unsatisfactory. The transient receptor potential ankyrin 1 (TRPA1) is a cationic channel activated by reactive compounds, and the blockage of this receptor can reduce depression- and anxiety-like behaviors in naive mice. Thus, we investigated the role of TRPA1 in depression- and anxiety-like behaviors in a PMS model in mice. PMS model was induced in C57BL/6 female mice by the experimental autoimmune encephalomyelitis (EAE). Nine days after the PMS-EAE induction, behavioral tests (tail suspension and elevated plus maze tests) were performed to verify the effects of sertraline (positive control), selective TRPA1 antagonist (A-967,079), and antioxidants (α-lipoic acid and apocynin). The prefrontal cortex and hippocampus were collected to evaluate biochemical and inflammatory markers. PMS-EAE induction did not cause locomotor changes but triggered depression- and anxiety-like behaviors, which were reversed by sertraline, A-967,079, α-lipoic acid, or apocynin treatments. The neuroinflammatory markers (AIF1, GFAP, IL-1ß, IL-17, and TNF-α) were increased in mice's hippocampus. Moreover, this model did not alter TRPA1 RNA expression levels in the hippocampus but decrease TRPA1 levels in the prefrontal cortex. Moreover, PMS-EAE induced an increase in NADPH oxidase and superoxide dismutase activities and TRPA1 endogenous agonist levels (hydrogen peroxide and 4-hydroxynonenal). TRPA1 plays a fundamental role in depression- and anxiety-like behaviors in a PMS-EAE model; thus, it could be a possible pharmacological target for treating these symptoms in PMS.

Role of TRPA1 expressed in bone tissue and the antinociceptive effect of the TRPA1 antagonist repeated administration in a breast cancer pain model.

AIMS: Breast cancer-induced chronic pain is usually treated with opioids, but these compounds cause various adverse effects. Transient receptor potential ankyrin 1 (TRPA1) is involved in cancer pain; also, endogenous TRPA1 agonists are associated with cancer pain development. The aim of this study was to observe the antinociceptive effect of a repeated-dose TRPA1 antagonist administration and the production of endogenous TRPA1 agonists and TRPA1 expression in bone tissue in a model of breast cancer pain in mice. Second, we used a sequence reading archive (SRA) strategy to observe the presence of this channel in the mouse bone and in mouse bone cell lines. MAIN METHODS: We used BALB/c mice for experiments. The animals were subjected to the tumor cell inoculation (4 T1 strain). HC-030031 (a TRPA1 antagonist) treatment was done from day 11 to day 20 after tumor inoculation. TRPA1 expression and biochemical tests of oxidative stress were performed in the bone of mice (femur). SRA strategy was used to detect the TRPA1 presence. KEY FINDINGS: Repeated treatment with the TRPA1 antagonist produced an antinociceptive effect. There was an increase in hydrogen peroxide levels, NADPH oxidase and superoxide dismutase activities, but the expression of TRPA1 in the bone tissue was not altered. SRA did not show TRPA1 residual transcription in the osteoblast and osteoclast cell lines, as well as for mice cranial tissue and in mouse osteoclast precursors. SIGNIFICANCE: The TRPA1 receptor is a potential target for the development of new painkillers for the treatment of bone cancer pain.

Trans fat intake during pregnancy or lactation increases anxiety-like behavior and alters proinflammatory cytokines and glucocorticoid receptor levels in the hippocampus of adult offspring.

Changes in dietary habits, including the increased consumption of processed foods, rich in trans fatty acids (TFA), have profound effects on offspring health in later life. Thus, this study aimed to assess the influence of maternal trans fat intake during pregnancy or lactation on anxiety behavior, as well as markers of inflammation, oxidative stress, and expression of glucocorticoid receptors (GR) of adult male offspring. Female Wistar rats were supplemented daily with soybean oil/fish oil (SO/FO) or hydrogenated vegetable fat (HVF) by oral gavage (3.0 g/kg body weight) during pregnancy or lactation. After weaning, male offspring received only standard diet. On the postnatal day 60, anxiety-like symptoms were assessed, the plasma was collected for the quantification of cytokines levels and the hippocampus removed for biochemical and molecular analysis. Our findings have evidenced that offspring from HVF-supplemented dams during pregnancy or lactation showed significantly greater levels of anxiety behavior. HVF supplementation increased plasma levels of proinflammatory cytokines and these levels were higher in the lactation period. In contrast, HVF supplementation decreased plasma levels of IL-10 in relation to SO/FO in both periods. Biochemical evaluations showed higher reactive species generation, protein carbonyl levels and catalase activity in offspring from HVF-supplemented dams during lactation. In addition, offspring from HVF-supplemented dams showed decreased GR expression in both supplemented periods. Together, these data indicate that consumption of TFA in different periods of development may increase anxiety-like behavior at least in part via alterations in proinflammatory and anti-inflammatory cytokine levels and GR expression in limbic brain regions.

Neonatal Tactile Stimulation Alters Behaviors in Heterozygous Serotonin Transporter Male Rats: Role of the Amygdala.

The serotonin transporter (SERT) gene, especially the short allele of the human serotonin transporter linked polymorphic region (5-HTTLPR), has been associated with the development of stress-related neuropsychiatric disorders. In line, exposure to early life stress in SERT knockout animals contributes to anxiety- and depression-like behavior. However, there is a lack of investigation of how early-life exposure to beneficial stimuli, such as tactile stimulation (TS), affects later life behavior in these animals. In this study, we investigated the effect of TS on social, anxiety, and anhedonic behavior in heterozygous SERT knockouts rats and wild-type controls and its impact on gene expression in the basolateral amygdala. Heterozygous SERT+/- rats were submitted to TS during postnatal days 8-14, for 10 min per day. In adulthood, rats were assessed for social and affective behavior. Besides, brain-derived neurotrophic factor (Bdnf) gene expression and its isoforms, components of glutamatergic and GABAergic systems as well as glucocorticoid-responsive genes were measured in the basolateral amygdala. We found that exposure to neonatal TS improved social and affective behavior in SERT+/- animals compared to naïve SERT+/- animals and was normalized to the level of naïve SERT+/+ animals. At the molecular level, we observed that TS per se affected Bdnf, the glucocorticoid-responsive genes Nr4a1, Gadd45ß, the co-chaperone Fkbp5 as well as glutamatergic and GABAergic gene expression markers including the enzyme Gad67, the vesicular GABA transporter, and the vesicular glutamate transporter genes. Our results suggest that exposure of SERT+/- rats to neonatal TS can normalize their phenotype in adulthood and that TS per se alters the expression of plasticity and stress-related genes in the basolateral amygdala. These findings demonstrate the potential effect of a supportive stimulus in SERT rodents, which are more susceptible to develop psychiatric disorders.

Short predictable stress promotes resistance to anxiety behavior and increases dendritic spines in prefrontal cortex and hippocampus.

PURPOSE: Some stress paradigms have shown protective effects of stress, suggesting increased resilience. Here we investigated whether a short predictable psychological stress, continuous or discontinuous, has a protective or negative long-lasting effect on behavior and cerebral morphology, and if there is difference when stress is experienced at different phases of neurodevelopment, namely in juvenile, adolescent, and adult rats. METHODS: Male and female rats were stressed on an elevated open platform at different ages (P23, P40, P75), and behavior was assessed later around 90 days of age. Behavioral analysis included measures of anxiety-like behavior, motor activity, and contextual memory. We also investigated the cerebral morphology by using dendritic spine analysis in pyramidal cells, in medial prefrontal cortex (Cg3), orbital prefrontal (AID) and hippocampal region CA1 as well as granule cells in the dentate gyrus (DG) of the hippocampus. RESULTS: Our results showed that short predictable psychological stress, continuous or discontinuous for 12 days, altered emotional behavior and neuronal morphology. Both behavioral and morphological results were sexually dimorphic and suggest a habituation and protective effect, as seen in the decrease of the anxiety-like behavior in the elevated plus maze (EPM), and an increase in excitatory synapse number, which is inferred from an increase of dendritic spines in the hippocampus and prefrontal cortex. CONCLUSION: We conclude that the response to short predictable psychological stress induces a protective/positive effect in both sexes and at different developmental ages.

Characterisation of nociception and inflammation observed in a traumatic muscle injury model in rats.

Muscle pain is the most prevalent type of pain in the world, but treatment remains ineffective. Thus, it is relevant to develop trustable animal models to understand the involved pain mechanisms. Therefore, this study characterised the nociception and inflammation in a traumatic muscle injury model in rats. A single blunt trauma impact on the right gastrocnemius muscle of male Wistar rats (250-350 g) was used as model for muscle pain. Animals were divided into four groups (sham/no treatment; sham/diclofenac 1%; injury/no treatment; injury/diclofenac 1%) and the topical treatment with a cream containing 1% monosodium diclofenac (applied at 2, 6, 12, 24, and 46 h after muscle injury; 200 mg/muscle) was used as an anti-inflammatory control. Nociception (mechanical and cold allodynia, or nociceptive score) and locomotor activity were evaluated at 26 and 48 h after injury. Also, inflammatory and oxidative parameters were evaluated in gastrocnemius muscle and the creatine kinase (CK) activity and lactate/glicose levels in rat's serum and plasma, respectively. Muscle injury caused mechanical and cold allodynia, and increased nociceptive scores, without inducing locomotor impairment. This model also increased the inflammatory cells infiltration (seen by myeloperoxidase and N-acetyl-ß-D-glucosaminidase activities and histological procedure), nitric oxide, interleukin (IL)-1ß, IL-6, and dichlorofluorescein fluorescence in muscle samples; and CK activity and lactate/glicose ratio. The treatment with 1% monosodium diclofenac reduced inflammatory cells infiltration, dichlorofluorescein fluorescence and lactate/glicose levels. Thus, we characterised the traumatic muscle injury as a reproducible model of muscle pain, which makes it possible to evaluate promising antinociceptive and anti-inflammatory therapies.

Hydroalcoholic extract of leaf of Arachis hypogaea L. (Fabaceae) did not induce toxic effects in the repeated-dose toxicity study in rats.

Arachis hypogaea L. (peanut) leaf is traditionally used for the treatment of insomnia in Asia. However, studies describing the safety and toxicity profile for this plant preparation are limited. Thus, the goal of this study was to investigate the toxicity of peanut leaf hydroalcoholic extract (PLHE) repeated treatment. The extract was administered orally (100, 300 or 1000 mg/kg) in male and female Wistar rats for 28 days (OECD guideline 407). PLHE treatment did not cause mortality or weight variation in the animals. Also, there was no alteration on locomotor activity (open field test), motor coordination (rotarod test), or anxiety behaviour (elevated plus-maze test). Male rats had a reduction in relative liver weight (100 mg/kg) and an increase in total kidney weight (1000 mg/kg), but there was no change in biochemical and haematological parameters after PLHE treatment. Free extracellular double-stranded DNA (dsDNA) levels was also evaluated, but PLHE treatment did not increase this parameter in rat organs. Also, the dose of 1000 mg/kg of PLHE significantly increased the total thiols in the liver of females compared with the control animals. Thus, PLHE did not induce toxicity after repeated exposure for 28 days in rats.

Macrophages and Schwann cell TRPA1 mediate chronic allodynia in a mouse model of complex regional pain syndrome type I.

Complex regional pain syndrome type I (CRPS-I) is characterized by intractable chronic pain. Poor understanding of the underlying mechanisms of CRPS-I accounts for the current unsatisfactory treatment. Antioxidants and antagonists of the oxidative stress-sensitive channel, the transient receptor potential ankyrin 1 (TRPA1), have been found to attenuate acute nociception and delayed allodynia in models of CRPS-I, evoked by ischemia and reperfusion (I/R) of rodent hind limb (chronic post ischemia pain, CPIP). However, it is unknown how I/R may lead to chronic pain mediated by TRPA1. Here, we report that the prolonged (day 1-15) mechanical and cold allodynia in the hind limb of CPIP mice was attenuated permanently in Trpa1-/- mice and transiently after administration of TRPA1 antagonists (A-967079 and HC-030031) or an antioxidant (α-lipoic acid). Indomethacin treatment was, however, ineffective. We also found that I/R increased macrophage (F4/80+ cell) number and oxidative stress markers, including 4-hydroxynonenal (4-HNE), in the injured tibial nerve. Macrophage-deleted MaFIA (Macrophage Fas-Induced Apoptosis) mice did not show I/R-evoked endoneurial cell infiltration, increased 4-HNE and mechanical and cold allodynia. Furthermore, Trpa1-/- mice did not show any increase in macrophage number and 4-HNE in the injured nerve trunk. Notably, in mice with selective deletion of Schwann cell TRPA1 (Plp1-CreERT;Trpa1fl/fl mice), increases in macrophage infiltration, 4-HNE and mechanical and cold allodynia were attenuated. In the present mouse model of CRPS-I, we propose that the initial oxidative stress burst that follows reperfusion activates a feed forward mechanism that entails resident macrophages and Schwann cell TRPA1 of the injured tibial nerve to sustain chronic neuroinflammation and allodynia. Repeated treatment one hour before and for 3 days after I/R with a TRPA1 antagonist permanently protected CPIP mice against neuroinflammation and allodynia, indicating possible novel therapeutic strategies for CRPS-I.

Nociception in a Progressive Multiple Sclerosis Model in Mice Is Dependent on Spinal TRPA1 Channel Activation.

Central neuropathic pain is a common untreated symptom in progressive multiple sclerosis (PMS) and is associated with poor quality of life and interference with patients' daily activities. The neuroinflammation process and mitochondrial dysfunction in the PMS lesions generate reactive species. The transient potential receptor ankyrin 1 (TRPA1) has been identified as one of the major mechanisms that contribute to neuropathic pain signaling and can be activated by reactive compounds. Thus, the goal of our study was to evaluate the role of spinal TRPA1 in the central neuropathic pain observed in a PMS model in mice. We used C57BL/6 female mice (20-30 g), and the PMS model was induced by the experimental autoimmune encephalomyelitis (EAE) using mouse myelin oligodendrocyte glycoprotein (MOG35-55) antigen and CFA (complete Freund's adjuvant). Mice developed progressive clinical score, with motor impairment observed after 15 days of induction. This model induced mechanical and cold allodynia and heat hyperalgesia which were measured up to 14 days after induction. The hypersensitivity observed was reduced by the administration of selective TRPA1 antagonists (HC-030031 and A-967079, via intrathecal and intragastric), antioxidants (α-lipoic acid and apocynin, via intrathecal and intragastric), and TRPA1 antisense oligonucleotide (via intrathecal). We also observed an increase in TRPA1 mRNA levels, NADPH oxidase activity, and 4-hydroxinonenal (a TRPA1 agonist) levels in spinal cord samples of PMS-EAE induced animals. In conclusion, these results support the hypothesis of the TRPA1 receptor involvement in nociception observed in a PMS-EAE model in mice.

TRPA1 activation mediates nociception behaviors in a mouse model of relapsing-remitting experimental autoimmune encephalomyelitis.

Central neuropathic pain is the main symptom caused by spinal cord lesion in relapsing-remitting multiple sclerosis (RRMS), but its management is still not effective. The transient receptor potential ankyrin 1 (TRPA1) is a pain detecting ion channel involved in neuropathic pain development. Thus, the aim of our study was to evaluate the role of TRPA1 in central neuropathic nociception induced by relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE) mouse model. In this model, we observed the development of similar clinical conditions of RRMS in C57BL/6 female mice through RR-EAE using MOG35-55 antigen and Quil A adjuvant. At the thirty-fifth day post-induction, C57BL/6 female mice demonstrated alteration in the RR-EAE score without motor impairment, mechanical and cold allodynia. Also, significative changes in demyelinating (Mog and olig-1) and neuroinflammatory (Iba1, Gfap and Tnfa) markers were observed, but this model did not alter Trpa1 RNA expression levels in the spinal cord. The hydrogen peroxide and 4-hydroxynonenal levels (TRPA1 agonists) were increased in RR-EAE induced mice, as well as the NADPH oxidase activity. The intragastric treatment of RR-EAE induced mice with TRPA1 antagonists (HC-030031 and A-967079) and antioxidant (α-lipoic acid and apocynin) caused an antiallodynic effect. Moreover, the intrathecal administration of TRPA1 antisense oligonucleotide, HC-030031, α-lipoic acid, and apocynin transiently attenuated mechanical and cold allodynia. Thus, TRPA1 plays a key role in the induction of neuropathic pain in this model of RR-EAE and can be a possible target for investigating the development of pain in RRMS patients.

Role of transient receptor potential ankyrin 1 (TRPA1) on nociception caused by a murine model of breast carcinoma.

Breast carcinoma causes severe pain, which decreases the quality of life of patients. Current treatments produce adverse effects and have limited efficacy. Transient potential receptor ankyrin 1 (TRPA1) is related to the onset of cancer and neuropathic pain. The aim of this study was to evaluate the involvement of TRPA1 in a model of breast carcinoma. We injected 4T1 cells in the fourth caudal mammary fat pad of female BALB/c mice, and after 20 days we observed mechanical and cold allodynia and spontaneous nociception behavior (mouse grimace scale detection, MGS). TRPA1 selective antagonist (HC-030031 or A-967079) administration or intrathecal administration of TRPA1 antisense (AS) oligonucleotide was performed. The activity of NADPH oxidase, superoxide dismutase (SOD) and hydrogen peroxide (H2O2) levels were evaluated. The chemical hyperalgesia produced by a TRPA1 agonist (allyl isothiocyanate, AITC) was also detected. The administration of TRPA1 antagonists, TRPA1 AS, or antioxidant, transiently attenuated MGS, or mechanical and cold allodynia. Intraplantar injection of AITC also caused nociception. NADPH oxidase or SOD activity and H2O2 levels were increased in the sciatic nerve and hind paw skin samples. The 4T1 cells did not express TRPA1, and the use of HC-030031 or α-lipoic acid did not reduce the cytotoxic effect of a chemotherapeutic drug (paclitaxel). Thus, TRPA1 could be investigated as a target for breast carcinoma pain treatment.

Dacarbazine alone or associated with melanoma-bearing cancer pain model induces painful hypersensitivity by TRPA1 activation in mice.

Antineoplastic therapy has been associated with pain syndrome development characterized by acute and chronic pain. The chemotherapeutic agent dacarbazine, used mainly to treat metastatic melanoma, is reported to cause painful symptoms, compromising patient quality of life. Evidence has proposed that transient receptor potential ankyrin 1 (TRPA1) plays a critical role in chemotherapy-induced pain syndrome. Here, we investigated whether dacarbazine causes painful hypersensitivity in naive or melanoma-bearing mice and the involvement of TRPA1 in these models. Mouse dorsal root ganglion (DRG) neurons and human TRPA1-transfected HEK293 (hTRPA1-HEK293) cells were used to evaluate the TRPA1-mediated calcium response evoked by dacarbazine. Mechanical and cold allodynia were evaluated after acute or repeated dacarbazine administration in naive mice or after inoculation of B16-F10 melanoma cells in C57BL/6 mice. TRPA1 involvement was investigated by using pharmacological and genetic tools (selective antagonist or antisense oligonucleotide treatment and Trpa1 knockout mice). Dacarbazine directly activated TRPA1 in hTRPA1-HEK293 cells and mouse DRG neurons and appears to sensitize TRPA1 indirectly by generating oxidative stress products. Moreover, dacarbazine caused mechanical and cold allodynia in naive but not Trpa1 knockout mice. Also, dacarbazine-induced nociception was reduced by the pharmacological TRPA1 blockade (antagonism), antioxidants, and by ablation of TRPA1 expression. TRPA1 pharmacological blockade also reduced dacarbazine-induced nociception in a tumor-associated pain model. Thus, dacarbazine causes nociception by TRPA1 activation, indicating that this receptor may represent a pharmacological target for treating chemotherapy-induced pain syndrome in cancer patients submitted to antineoplastic treatment with dacarbazine.

Nociceptive mechanisms involved in the acute and chronic phases of a complex regional pain syndrome type 1 model in mice.

Complex regional pain syndrome I (CRPS-I) is a chronic painful pathology still undertreated. CTK 01512-2 is a recombinant version of the spider peptide Phα1ß, and it functions as a voltage-gated calcium channel blocker and a transient receptor potential ankyrin 1 (TRPA1) antagonist with antinociceptive effect in different pain models. Here, we investigate the mechanisms involved in the acute and chronic nociceptive phases of a model of CPRS-I in mice and assess the antinociceptive effect of CTK 01512-2 using this model. Adult male and female mice C57BL/6 (20-30 g) were used to determine mechanical (von Frey test) or cold (acetone test) allodynia induction. Inflammatory parameters (serum and tibial nerve lactate levels, hind paw temperature and edema, or tissue cell infiltration) were evaluated after chronic post-ischemia pain (CPIP, a model of CPRS-I) induction. Anti-inflammatory and anti-neuropathic drugs or CTK 01512-2 were tested. First, we detected that CPIP-induced mechanical and cold allodynia in male and female mice in a similar way. In the acute phase (1 day after CPIP), an increase in inflammatory parameters were observed, as well as the anti-allodynic effect of anti-inflammatory compounds. In the chronic phase (17 days after CPIP), mice exhibited mechanical and cold allodynia, and anti-neuropathic drugs induced antinociception, while no inflammatory alterations were found. CTK 01512-2 reversed the CPIP allodynic effect in both nociceptive phases. Thus, this CPRS-I model can be used to understand the mechanisms involved in CPRS-I induced pain and inflammation. Besides, we observed that CTK 01512-2 has a valuable antinociceptive effect in this pain model.

Peanut leaf extract has antioxidant and anti-inflammatory activity but no acute toxic effects.

Arachis hypogaea L. (peanut) leaves have been popularly used for the treatment of insomnia and inflammation, but no toxicological study has been performed for this plant preparation. This study aimed to examine the phytochemical composition of peanut leaf hydroalcoholic extract (PLHE) and describe its potential toxic effects and antioxidant and anti-inflammatory properties. The qualitative chemical analysis of PLHE by UHPLC-ESI-HRMS allowed the identification of eight metabolites types (totaling 29 compounds). The 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay revealed that PLHE had strong antioxidant effects; it also exhibited nitric oxide (NO)-scavenging capacity. Human peripheral blood mononuclear cells (PBMCs) exposed to PLHE showed no reduced cell viability or increased free double-stranded DNA, NO, or reactive species production. PLHE reversed the cytotoxicity, pro-inflammatory (release of interleukin-1ß), and pro-oxidant effects of H2O2 on human PBMCs. Acute PLHE toxicity analysis was performed in vivo using the Organization for Economic Co-operation and Development (OECD) 423 guidelines. PLHE single injection (2000 mg/kg, intragastric) did not cause mortality or morbidity or induce changes in hematological or biochemical parameters after 14 days of administration. Thus, PLHE could be a source of bioactive compounds and possesses antioxidant and anti-inflammatory properties without elicitin cytotoxicity or genotoxicity in human PBMCs or acute toxicity in rats.