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Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice.

ABSTRACT: Dysesthesia is an unpleasant abnormal sensation, which is often accompanied by peripheral neuropathy or vascular impairment. Here, we examined the roles of transient receptor potential ankyrin 1 (TRPA1) in dysesthesia-like behaviours elicited by transient hindlimb ischemia (15-60?min) by tightly compressing the hindlimb, and reperfusion by releasing the ligature. The paw-withdrawal responses to tactile stimulation were reduced during ischemia and lasted for a while after reperfusion. Hindlimb ischemia/reperfusion elicited spontaneous licking of the ischemic hindpaw that peaked within 10?min. The licking was inhibited by reactive oxygen species (ROS) scavengers, a TRPA1 antagonist, or TRPA1 deficiency, but not by TRPV1 deficiency. In human TRPA1-expressing cells as well as cultured mouse dorsal root ganglion neurons, the H2O2-evoked TRPA1 response was significantly increased by pretreatment with hypoxia (80?mmHg) for 30?min. This hypoxia-induced TRPA1 sensitisation to H2O2 was inhibited by overexpressing a catalytically-inactive mutant of prolyl hydroxylase (PHD) 2 or in a TRPA1 proline mutant resistant to PHDs. Consistent with these results, a PHD inhibitor increased H2O2-evoked nocifensive behaviours through TRPA1 activation. Our results suggest that transient hindlimb ischemia/reperfusion-evoked spontaneous licking, i.e. painful dysesthesia, is caused by ROS-evoked activation of TRPA1 sensitised by hypoxia through inhibiting PHD-mediated hydroxylation of a proline residue in TRPA1.

SUBMITTER: So K

PROVIDER: S-EPMC4794653 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice.

So Kanako K Tei Yuna Y Zhao Meng M Miyake Takahito T Hiyama Haruka H Shirakawa Hisashi H Imai Satoshi S Mori Yasuo Y Nakagawa Takayuki T Matsubara Kazuo K Kaneko Shuji S

Scientific reports 20160317

Dysesthesia is an unpleasant abnormal sensation, which is often accompanied by peripheral neuropathy or vascular impairment. Here, we examined the roles of transient receptor potential ankyrin 1 (TRPA1) in dysesthesia-like behaviours elicited by transient hindlimb ischemia (15-60 min) by tightly compressing the hindlimb, and reperfusion by releasing the ligature. The paw-withdrawal responses to tactile stimulation were reduced during ischemia and lasted for a while after reperfusion. Hindlimb is ...[more]

PMID: 26983498

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Project description:Myocardial ischemia-reperfusion (I/R) results in the generation of free radicals, accumulation of lipid peroxidation-derived unsaturated aldehydes, variable angina (pain), and infarction. The transient receptor potential ankyrin 1 (TRPA1) mediates pain signaling and is activated by unsaturated aldehydes, including acrolein and 4-hydroxynonenal. The contribution of TRPA1 (a Ca2+-permeable channel) to I/R-induced myocardial injury is unknown. We tested the hypothesis that cardiac TRPA1 confers myocyte sensitivity to aldehyde accumulation and promotes I/R injury. Although basal cardiovascular function in TRPA1-null mice was similar to that in wild-type (WT) mice, infarct size was significantly smaller in TRPA1-null mice than in WT mice (34.1?±?9.3 vs. 14.3?±?9.9% of the risk region, n = 8 and 7, respectively, P < 0.05), despite a similar I/R-induced area at risk (40.3?±8.4% and 42.2?±?11.3% for WT and TRPA1-null mice, respectively) after myocardial I/R (30 min of ischemia followed by 24 h of reperfusion) in situ. Positive TRPA1 immunofluorescence was present in murine and human hearts and was colocalized with connexin43 at intercalated disks in isolated murine cardiomyocytes. Cardiomyocyte TRPA1 was confirmed by quantitative RT-PCR, DNA sequencing, Western blot analysis, and electrophysiology. A role of TRPA1 in cardiomyocyte toxicity was demonstrated in isolated cardiomyocytes exposed to acrolein, an I/R-associated toxin that induces Ca2+ accumulation and hypercontraction, effects significantly blunted by HC-030031, a TRPA1 antagonist. Protection induced by HC-030031 was quantitatively equivalent to that induced by SN-6, a Na+/Ca2+ exchange inhibitor, further supporting a role of Ca2+ overload in acrolein-induced cardiomyocyte toxicity. These data indicate that cardiac TRPA1 activation likely contributes to I/R injury and, thus, that TRPA1 may be a novel therapeutic target for decreasing myocardial I/R injury. NEW & NOTEWORTHY Transient receptor potential ankyrin 1 (TRPA1) activation mediates increased blood flow, edema, and pain reception, yet its role in myocardial ischemia-reperfusion (I/R) injury is unknown. Genetic ablation of TRPA1 significantly decreased myocardial infarction after I/R in mice. Functional TRPA1 in cardiomyocytes was enriched in intercalated disks and contributed to acrolein-induced Ca2+ overload and hypercontraction. These data indicate that I/R activation of TRPA1 worsens myocardial infarction; TRPA1 may be a potential target to mitigate I/R injury.

Project description:BackgroundMuscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration.MethodsA clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration.Results2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI.ConclusionWIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.

Project description:BackgroundHypoxia preconditioning has been proven to be an effective method to enhance the therapeutic action of mesenchymal stem cells (MSCs). However, the beneficial effects of hypoxic MSCs in ischemia/reperfusion (I/R) lung injury have yet to be investigated. In this study, we hypothesized that the administration of hypoxic MSCs would have a positive therapeutic impact on I/R lung injury at molecular, cellular, and functional levels.MethodsI/R lung injury was induced in isolated and perfused rat lungs. Hypoxic MSCs were administered in perfusate at a low (2.5×105 cells) and high (1×106 cells) dose. Rats ventilated with a low tidal volume of 6 ml/kg served as controls. Hemodynamics, lung injury indices, inflammatory responses and activation of apoptotic pathways were determined.ResultsI/R induced permeability pulmonary edema with capillary leakage and increased levels of reactive oxygen species (ROS), pro-inflammatory cytokines, adhesion molecules, cytosolic cytochrome C, and activated MAPK, NF-κB, and apoptotic pathways. The administration of a low dose of hypoxic MSCs effectively attenuated I/R pathologic lung injury score by inhibiting inflammatory responses associated with the generation of ROS and anti-apoptosis effect, however this effect was not observed with a high dose of hypoxic MSCs. Mechanistically, a low dose of hypoxic MSCs down-regulated P38 MAPK and NF-κB signaling but upregulated glutathione, prostaglandin E2, IL-10, mitochondrial cytochrome C and Bcl-2. MSCs infused at a low dose migrated into interstitial and alveolar spaces and bronchial trees, while MSCs infused at a high dose aggregated in the microcirculation and induced pulmonary embolism.ConclusionsHypoxic MSCs can quickly migrate into extravascular lung tissue and adhere to other inflammatory or structure cells and attenuate I/R lung injury through anti-oxidant, anti-inflammatory and anti-apoptotic mechanisms. However, the dose of MSCs needs to be optimized to prevent pulmonary embolism and thrombosis.

Dataset Information

Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice.

Publications

Hypoxia-induced sensitisation of TRPA1 in painful dysesthesia evoked by transient hindlimb ischemia/reperfusion in mice.

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