Schwann cell TRPA1 elicits reserpine-induced fibromyalgia pain in mice.

BACKGROUND AND PURPOSE: Fibromyalgia is a complex clinical disorder with an unknown aetiology, characterized by generalized pain and co-morbid symptoms such as anxiety and depression. An imbalance of oxidants and antioxidants is proposed to play a pivotal role in the pathogenesis of fibromyalgia symptoms. However, the precise mechanisms by which oxidative stress contributes to fibromyalgia-induced pain remain unclear. The transient receptor potential ankyrin 1 (TRPA1) channel, known as both a pain sensor and an oxidative stress sensor, has been implicated in various painful conditions. EXPERIMENTAL APPROACH: The feed-forward mechanism that implicates reactive oxygen species (ROS) driven by TRPA1 was investigated in a reserpine-induced fibromyalgia model in C57BL/6J mice employing pharmacological interventions and genetic approaches. KEY RESULTS: Reserpine-treated mice developed pain-like behaviours (mechanical/cold hypersensitivity) and early anxiety-depressive-like disorders, accompanied by increased levels of oxidative stress markers in the sciatic nerve tissues. These effects were not observed upon pharmacological blockade or global genetic deletion of the TRPA1 channel and macrophage depletion. Furthermore, we demonstrated that selective silencing of TRPA1 in Schwann cells reduced reserpine-induced neuroinflammation (NADPH oxidase 1-dependent ROS generation and macrophage increase in the sciatic nerve) and attenuated fibromyalgia-like behaviours. CONCLUSION AND IMPLICATIONS: Activated Schwann cells expressing TRPA1 promote an intracellular pathway culminating in the release of ROS and recruitment of macrophages in the mouse sciatic nerve. These cellular and molecular events sustain mechanical and cold hypersensitivity in the reserpine-evoked fibromyalgia model. Targeting TRPA1 channels on Schwann cells could offer a novel therapeutic approach for managing fibromyalgia-related behaviours.

Stephalagine, an aporphinic alkaloid with therapeutic effects in acute gout arthritis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Gout is an inflammatory disease characterized by the accumulation of monosodium urate crystals (MSU) in the joints, leading to severe pain and inflammation. Stephalagine is a Brazilian Savanna aporphine alkaloid isolated from Annona crassiflora Mart. Fruit peel, that has been popularly used to treat rheumatism and have been described with antinociceptive properties. However, no studies evaluated the possible therapeutic properties of stephalagine in arthritic pain. AIM OF THE STUDY: To evaluate the possible antinociceptive and anti-inflammatory effects of stephalagine in an acute gout attack in mice. MATERIALS AND METHODS: Adult male wild type C57BL/6/J/UFU mice (20-25 g) were used (process number 018/17). The treated group received stephalagine (1 mg/kg, by gavage) and the vehicle group received saline (10 mL/kg, by gavage), both 1 h before the MSU crystals (100 µg/ankle joint) administration. All groups were analyzed for mechanical allodynia, thermal hyperalgesia, overt pain-like behaviors, and edema development at 2, 4, 6 and 24 h after injections. Synovial fluid and the ankle articulation from the injected joint were collected 4 h after administrations for myeloperoxidase enzyme activity, IL-1ß measurement, and histological analysis. RESULTS: Stephalagine had a significant antinociceptive effect on mechanical allodynia, when compared to vehicle group at 2-24 h after intra-articular injection of MSU and 2 h for spontaneous and cold thermal sensitivity. Stephalagine was also able to significantly reduce the articular edema (45 ± 1%), the activity of the myeloperoxidase enzyme (37 ± 6%), and IL-1ß levels (43 ± 3%). The histological analysis confirms that stephalagine dramatically reduced the number of infiltrating inflammatory cells (75 ± 6%) in MSU injected animals. Also, stephalagine treatment did not alter the uric acid levels, xanthine oxidase activity, AST and ALT activities, urea and creatinine levels, neither cause any macroscopic changes in the mice's weight, deformations, changes in the coat, or feces. CONCLUSION: Stephalagine may be an alternative for the management of gout, once it was able to induce antinociceptive and anti-inflammatory effects without causing adverse effects on the evaluated parameters.

CdSe magic-sized quantum dots attenuate reactive oxygen species generated by neutrophils and macrophages with implications in experimental arthritis.

The biological applicability of nanomaterials has been limited due to cytotoxicity. Studies have described the effects of nanomaterials on different tissues and cell types, but their actions on immune cells are less elucidated. This study describes unprecedented in vitro and in vivo antioxidant activities of cadmium selenide magic-sized quantum dots (CdSe MSQDs) with implications on rheumatoid arthritis. While the generation of ROS induced by nanomaterials is linked to cytotoxicity, we found that CdSe MSQDs reduced ROS production by neutrophils and macrophages following opsonized-zymosan stimuli, and we did not find cytotoxic effects. Interestingly, inherent antioxidant properties of CdSe MSQDs were confirmed through DPPH, FRAP, and ORAC assays. Furthermore, CdSe MSQDs reduced ROS levels generated by infiltrating leukocytes into joints in experimental model of rheumatoid arthritis. Briefly, we describe a novel application of CdSe MSQDs in modulating the inflammatory response in experimental rheumatoid arthritis through an unexpected antioxidant activity.

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.

Inhibitors of angiotensin I converting enzyme potentiate fibromyalgia-like pain symptoms via kinin receptors in mice.

Fibromyalgia is a potentially disabling chronic disease, characterized by widespread pain and a range of comorbidities such as hypertension. Among the mechanisms involved in fibromyalgia-like pain symptoms are kinins and their B1 and B2 receptors. Moreover, angiotensin I converting enzyme (ACE) inhibitors, commonly used as antihypertensive drugs, can enhance pain by blocking the degradation of peptides such as substance P and bradykinin, besides enhancing kinin receptors signalling. We investigated the effect of ACE inhibitors on reserpine-induced fibromyalgia-like pain symptoms and the involvement of kinins in this effect in mice. Nociceptive parameters (mechanical and cold allodynia and overt nociception) were evaluated after ACE inhibitors administration in mice previously treated with reserpine. The role of kinin B1 and B2 receptors was investigated using pharmacological antagonism. Additionally, bradykinin levels, as well as the activity of ACE and kininase I, were measured in the sciatic nerve, spinal cord and cerebral cortex of the mice. The ACE inhibitors enalapril and captopril enhanced reserpine-induced mechanical allodynia, and this increase was prevented by kinin B1 and B2 receptor antagonists. Substance P and bradykinin caused overt nociception and increased mechanical allodynia in animals treated with reserpine. Reserpine plus ACE inhibitors increased bradykinin-related peptide levels and inhibited ACE activity in pain modulation structures. Since hypertension is a frequent comorbidity affecting fibromyalgia patients, hypertension treatment with ACE inhibitors in these patients should be reviewed once this could enhance fibromyalgia-like pain symptoms. Thus, the treatment of hypertensive patients with fibromyalgia could include other classes of antihypertensive drugs, different from ACE inhibitors.

Protective effects of a polyphenol-enriched fraction of the fruit peel of Annona crassiflora Mart. on acute and persistent inflammatory pain.

Different parts of Annona crassiflora Mart., a native species from Brazilian savanna, were traditionally used for the treatment of a wide variety of ailments including arthritis. Thus, this study aimed to investigate the possible antinociceptive and anti-inflammatory properties of a polyphenol-enriched fraction of the fruit peel of A. crassiflora, named here as EtOAc, in mice. Pro-inflammatory cytokines and nitric oxide (NO) production were evaluated in LPS-activated macrophages. Then, EtOAc fraction was administered by oral route in male C57BL/6/J mice, and the animals were submitted to glutamate-induced nociception and complete Freund's adjuvant (CFA)-induced monoarthritis tests to assess nociception (mechanical, spontaneous and cold pain) and inflammation (edema and neutrophil infiltration), and to the open-field and rotarod tests for motor performance analysis. EtOAc fraction inhibited the production of IL-6 and NO in the LPS-induced macrophages, and reduced spontaneous nociception induced by glutamate, without altering the animals' locomotor activity. In addition, the polyphenol-enriched fraction was able to revert the early and late hyperalgesia induced by CFA, as well as edema at the acute phase. Reduction of myeloperoxidase activity and inflammatory cell infiltration was observed in the paw tissue of mice injected with CFA and treated with EtOAc fraction. Together, our results support the antinociceptive and anti-inflammatory effects of the polyphenol-enriched fraction of A. crassiflora fruit peel and suggest that these effects are triggered, at least in part, by suppressing pro-inflammatory cytokines and neutrophils infiltration.

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.

Kinins and their B1 and B2 receptors are involved in fibromyalgia-like pain symptoms in mice.

Fibromyalgia is a disease characterised as generalised chronic primary pain that causes functional disability and a reduction in patients' quality of life, without specific pathophysiology or appropriate treatment. Previous studies have shown that kinins and their B1 and B2 receptors contribute to chronic painful conditions. Thus, we investigated the involvement of kinins and their B1 and B2 receptors in a fibromyalgia-like pain model induced by reserpine in mice. Nociceptive parameters (mechanical allodynia, cold sensitivity and overt nociception) and behaviours of burrowing, thigmotaxis, and forced swimming were evaluated after reserpine administration in mice. The role of kinin B1 and B2 receptors was investigated using knockout mice or pharmacological antagonism. The protein expression of kinin B1 and B2 receptors and the levels of bradykinin and monoamines were measured in the sciatic nerve, spinal cord and cerebral cortex of the animals. Knockout mice for the kinin B1 and B2 receptor reduced reserpine-induced mechanical allodynia. Antagonism of B1 and B2 receptors also reduced mechanical allodynia, cold sensitivity and overt nociception reserpine-induced. Reserpine altered thigmotaxis, forced swimming and burrowing behaviour in the animals; with the latter being reversed by antagonism of kinin B1 receptor. Moreover, reserpine increased the protein expression of kinin B1 and B2 receptors and levels of kinin, as well as reduced the levels of monoamines in peripheral and central structures. Kinins and its B1 and B2 receptors are involved in fibromyalgia-like pain symptoms. B1 or B2 receptors might represent a potential target for the relief of fibromyalgia-like pain symptoms.

Transient receptor potential ankyrin 1 (TRPA1) plays a critical role in a mouse model of cancer pain.

There is a major, unmet need for the treatment of cancer pain, and new targets and medicines are required. The transient receptor potential ankyrin 1 (TRPA1), a cation channel expressed by nociceptors, is activated by oxidizing substances to mediate pain-like responses in models of inflammatory and neuropathic pain. As cancer is known to increase oxidative stress, the role of TRPA1 was evaluated in a mouse model of cancer pain. Fourteen days after injection of B16-F10 murine melanoma cells into the plantar region of the right hind paw, C57BL/6 mice exhibited mechanical and thermal allodynia and thigmotaxis behavior. While heat allodynia was partially reduced in TRP vanilloid 1 (TRPV1)-deficient mice, thigmotaxis behavior and mechanical and cold allodynia were absent in TRPA1-deficient mice. Deletion of TRPA1 or TRPV1 did not affect cancer growth. Intrathecal TRPA1 antisense oligonucleotides and two different TRPA1 antagonists (HC-030031 or A967079) transiently attenuated thigmotaxis behavior and mechanical and cold allodynia. A TRPV1 antagonist (capsazepine) attenuated solely heat allodynia. NADPH oxidase activity and hydrogen peroxide levels were increased in hind paw skin 14 days after cancer cell inoculation. The antioxidant, α-lipoic acid, attenuated mechanical and cold allodynia and thigmotaxis behavior, but not heat allodynia. Whereas TRPV1, via an oxidative stress-independent pathway, contributes partially to heat hypersensitivity, oxidative stress-dependent activation of TRPA1 plays a key role in mediating thigmotaxis behavior and mechanical and cold allodynia in a cancer pain model. TRPA1 antagonists might be beneficial in the treatment of cancer pain.

Potentiation of Paclitaxel-Induced Pain Syndrome in Mice by Angiotensin I Converting Enzyme Inhibition and Involvement of Kinins.

Paclitaxel is a chemotherapeutic agent used to treat solid tumours. However, it causes an acute and neuropathic pain syndrome that limits its use. Among the mechanisms involved in neuropathic pain caused by paclitaxel is activation of kinin receptors. Angiotensin converting enzyme (ACE) inhibitors can enhance kinin receptor signalling. The goal of this study was to evaluate the role of kinins on paclitaxel-associated acute pain syndromes (P-APS) and the effect of ACE inhibition on P-APS and paclitaxel-associated chronic peripheral neuropathy (P-CPN) in mice. Herein, we show that paclitaxel caused mechanical allodynia and spontaneous nociceptive behaviour that was reduced by antagonists of kinin receptors B1 (DALBk and SSR240612) and B2 (Hoe140 and FR173657). Moreover, enalapril (an ACE inhibitor) enhanced the mechanical allodynia induced by a low dose of paclitaxel. Likewise, paclitaxel injection inhibited ACE activity and increased the expressions of B1 and B2 receptors and bradykinin-related peptides levels in peripheral tissue. Together, our data support the involvement of kinin receptors in the P-APS and suggest kinin receptor antagonists to treat this syndrome. Because hypertension is the most frequent comorbidity affecting cancer patients, treatment of hypertension with ACE inhibitors in patients undergoing paclitaxel chemotherapy should be reviewed, since this could enhance the P-APS and P-CPN.

Nanoencapsulation of rice bran oil increases its protective effects against UVB radiation-induced skin injury in mice.

Excessive UV-B radiation by sunlight produces inflammatory and oxidative damage of skin, which can lead to sunburn, photoaging, and cancer. This study evaluated whether nanoencapsulation improves the protective effects of rice bran oil against UVB radiation-induced skin damage in mice. Lipid-core nanocapsules containing rice bran oil were prepared, and had mean size around 200 nm, negative zeta potential (∼-9 mV), and low polydispersity index (<0.20). In order to allow application on the skin, a hydrogel containing the nanoencapsulated rice bran oil was prepared. This formulation was able to prevent ear edema induced by UVB irradiation by 60 ± 9%, when compared with a hydrogel containing LNC prepared with a mixture of medium chain triglycerides instead of rice bran oil. Protein carbonylation levels (biomarker of oxidative stress) and NF-κB nuclear translocation (biomarker of pro-inflammatory and carcinogenesis response) were reduced (81% and 87%, respectively) in animals treated with the hydrogel containing the nanoencapsulated rice bran oil. These in vivo results demonstrate the beneficial effects of nanoencapsulation to improve the protective properties of rice bran oil on skin damage caused by UVB exposure.

Antinociceptive effect of 3-(4-fluorophenyl)-5-trifluoromethyl-1H-1-tosylpyrazole. A Celecoxib structural analog in models of pathological pain.

Pain is the most common complaint in the medical field and the identification of novel compounds that can effectively treat painful states without causing side effects remains a major challenge in biomedical research. The aim of the present study is to investigate the antinociceptive effect of 3-(4-fluorophenyl)-5-trifluoromethyl-1H-1-tosylpyrazole (FTosPz) in models of pathological pain in mice and compare these effects with those produced by Celecoxib. FTosPz (100-500 µmol/kg) or Celecoxib (26-260 µmol/kg) was administrated orally. The administration of either FTosPz or Celecoxib reduced the hyperalgesia but not the edema or leukocyte infiltration that was caused by Complete Freund's Adjuvant (CFA), used as an arthritis model. Oral administration of both FTosPz and Celecoxib also attenuated the postoperative hyperalgesia as well as the hyperalgesia caused by partial sciatic nerve ligation, used as a neuropathic pain model. FTosPz and Celecoxib produced antinociceptive effects without altering the locomotor activity of animals. Furthermore, FTosPz neither altered AST/ALT enzyme activity nor the urea/creatinine levels. Still, the FTosPz did not alter the COX-1 and COX-2 enzyme activities. Thus, FTosPz is an interesting prototype for the development of novel analgesic drugs.

Anti-inflammatory and antioxidant effects of Aloe saponaria Haw in a model of UVB-induced paw sunburn in rats.

Ultraviolet B (UVB) irradiation mainly affects biological tissues by inducing an increase in reactive oxygen species (ROS) production which leads to deleterious outcomes for the skin, including pain and inflammation. As a protective strategy, many studies have focused on the use of natural products. The aim of this study was to investigate the effects of Aloe saponaria on nociceptive, inflammatory, and oxidative parameters in a model of UVB-induced sunburn in adult male Wistar rats. Sunburned animals were topically treated with vehicle (base cream), 1% silver sulfadiazine (positive control) or A. saponaria (10%) once a day for 6days. UVB-induced nociception (allodynia and hyperalgesia), inflammation (edema and leukocyte infiltration) and oxidative stress (increases in H2O2, protein carbonyl levels and lipid peroxidation and a decrease in non protein thiol content) were reduced by both A. saponaria and sulfadiazine topical treatment. Furthermore, A. saponaria or its constituents aloin and rutin reduced the oxidative stress induced by H2O2 in skin homogenates in vitro. Our results demonstrate that topical A. saponaria treatment displayed anti-nociceptive and anti-inflammatory effects in a UVB-induced sunburn model, and these effects seem to be related to its antioxidant components.

Triterpene 3ß, 6ß, 16ß trihidroxilup-20(29)-ene protects against excitability and oxidative damage induced by pentylenetetrazol: the role of Na(+),K(+)-ATPase activity.

Administration of the compound triterpene 3ß, 6ß, 16ß-trihidroxilup-20(29)-ene (TTHL) resulted in antinociceptive activity in several pain models in mice. Because pain and epilepsy have common mechanisms, and several anticonvulsants are clinically used to treat painful disorders, we investigated the anticonvulsant potential of TTHL. Behavioral and electrographic recordings revealed that pretreatment with TTHL (30 mg/kg; i.g.) increased the latencies to the first clonic seizure to the tonic-clonic and reduced the duration of the generalized seizures induced by the GABA(A) receptor antagonist PTZ (80 g; i.p.). The TTHL pretreatment also protected against PTZ-induced deleterious effects, as characterized by protein carbonylation, lipid peroxidation, [(3)H] glutamate uptake and the inhibition of Na(+),K(+)-ATPase (subunits α(1) and α(2)/α(3)). Although TTHL did not exhibit DPPH, ABTS radical scavenging activity per se and does not alter the binding of [(3)H]flunitrazepam to the benzodiazepinic site of the GABA(A) receptor, this compound was effective in preventing behavioral and EEG seizures, as well as the inhibition of Na(+),K(+)-ATPase induced by ouabain. These results suggest that the protection against PTZ-induced seizures elicited by TTHL is due to Na(+),K(+)-ATPase activity maintenance. In fact, experiments in homogenates of the cerebral cortex revealed that PTZ (10 mM) reduced Na(+),K(+)-ATPase activity and that previous incubation with TTHL (10 µM) protected against this inhibition. Collectively, these data indicate that the protection exerted by TTHL in this model of convulsion is not related to antioxidant activity or GABAergic activity. However, these results demonstrated that the effective protection of Na(+),K(+)-ATPase elicited by this compound protects against the damage due to neuronal excitability and oxidation that is induced by PTZ.

Involvement of mast cells in a mouse model of postoperative pain.

Recent studies have indicated that nearly half of all surgical patients still have inadequate pain relief; therefore, it is becoming increasingly more important to understand the mechanisms involved in postoperative pain in order to be better treated. Previous studies have shown that incisions can cause mast cell degranulation. Thus, the aim of this study was to investigate the involvement of mast cells in a model of postoperative pain in mice. The depletion of mast cell mediators produced by pre-treatment with compound 48/80 (intraplantar (i.pl.)) widely (98 ± 23% of inhibition) and extensively (up to 96 h) prevented postoperative nociception and reduced histamine and serotonin levels (88 ± 4% and 68 ± 10%, respectively) in operated tissue. Furthermore, plantar surgery produced immense mast cell degranulation, as assessed by histology and confirmed by the increased levels of serotonin (three-fold higher) and histamine (fifteen-fold higher) in the perfused tissue, 1h after surgery. Accordingly, pre-treatment with the mast cell membrane stabilizer cromoglycate (200 µg/paw, i.pl.) prevented mechanical allodynia (inhibition of 96 ± 21%) and an increase in histamine (44 ± 10% of inhibition) and serotonin (73 ± 5% of inhibition) levels induced by plantar surgery. Finally, local treatment with H(1) (promethazine, 100 µg/paw, i.pl.), 5-HT(3) (ondansetron, 10 µg/paw, i.pl.) or 5-HT(2A) (ketanserin, 5 µg/paw, i.pl.) receptor antagonists partially decreased postoperative nociception in mice, but when co-administered together it completely reversed the mechanical allodynia in operated mice. Thus, mast cell activation mechanisms are interesting targets for the development of novel therapies to treat postoperative pain.