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To investigate the role of oxidative stress in itch-indicative scratching behavior in mice, and furthermore, to define the cellular and molecular mechanisms underlying oxidative stress-mediated itch.
Scratching behavior was induced by intradermal injection of oxidants, including hydrogen peroxide (H2O2) and tert-butylhydroperoxide (tBHP) into the nape of the neck in mice and observed for 30 min.
Intradermal H2O2 (0.03-1%) or tert-butylhydroperoxide (tBHP, 1-30 μmol) elicited robust scratching behavior, displaying an inverted-U-shaped dose-related curve. Naloxone, an opioid receptor antagonist, but not morphine, largely suppressed the oxidants-induced scratching. Chlorpheniramine, a histamine H1 receptor antagonist, blocked histamine but not oxidants-induced scratching, indicating the involvement of histamine-independent mechanism in oxidants-evoked itch. Further, resiniferatoxin (RTX) treatment abolished oxidants-induced scratching, suggesting an essential role of C-fibers. Notably, blockade of transient receptor potential subtype ankyryn 1 (TRPA1) by selective TRPA1 antagonist HC-030031, or genetic deletion of Trpa1 but not Trpv1 resulted in a profound reduction in H2O2-evoked scratching. Finally, systemic administration of the antioxidants N-acetyl-L-cysteine (NAC) or trolox (a water-soluble vitamin E analogue) attenuated scratching induced by the oxidants.
Oxidative stress by different oxidants can induce profound scratching behavior, which is largely histamine and TRPV1-independent but TRPA1-dependent. Antioxidants and TRPA1 antagonists may be used to treat human itch conditions associated with oxidative stress.
Itch (pruritus) is defined as an unpleasant sensation that elicits the desire or reflex to scratch. Acute itch severs as a warning and self-protective mechanism to protect us from potentially harmful irritations. However, chronic itch is a common clinical problem that is associated with skin diseases (e.g., atopic dermatitis and psoriasis)[2,3], systemic diseases (e.g., chronic renal failure and cholestasis)[4,5], and metabolism disorders (e.g., diabetes). Although itch sensation can be transiently relieved by scratching, itch-scratch cycles often exacerbate cutaneous problems and lead to further injury. Histamine is one of the best studied itch mediators, and antihistamines serve as the first choice for treating clinical itch[9,10]. However, most types of chronic itch are resistant to antihistamine treatment . Chronic itch substantially reduces the quality of life that is inflicted[12,13]. Thus, there is urgency to identify novel mediators and the signaling pathways involved in the pathogenesis of itch, which may provide new targets for anti-pruritic treatment.
Oxidative stress is chemically associated with over-production of reactive oxygen species (ROS), or reduction in the capability of antioxidant defense. In human, oxidative stress has long been proposed to contribute to pathogenesis of systemic and metabolism diseases, cardiovascular diseases and neurodegenerative diseases[14-17]. Oxidative stress and ROS have been strongly implicated in the pathogenesis of pain, such as inflammatory pain and neuropathic pain[18,19]. Of interest, recent studies proposed different mechanisms for pain and itch[11,20-23]. However, the relationship between oxidative stress and itch has not been investigated. Strikingly, chronic itch is commonly accompanied with many of oxidative stress-mediated diseases, such as atopic dermatitis, psoriasis, chronic renal failure, cholestasis, and diabetes[24-26].
The aim of the present study was to test whether oxidative challenges can induce itch-associated behaviors and further elucidate the underlying molecular and cellular mechanisms. In animal studies, itch can be quantitatively evaluated by measuring the scratching behavior elicited by itch-evoking agents[20,27-30]. We investigated the scratching responses in mice induced by intradermal (i.d.) injection of hydrogen peroxide (H2O2) or tert-butylhydroperoxide (tBHP), two commonly used oxidants for producing oxidative injury. Using both pharmacological and genetic manipulations, we found that oxidant could elicit robust itch via TRPA1 activation.
We purchased H2O2 solution (30%), tBHP, histamine, RTX, chlorpheniramine, naloxone, N-acetyl-L-cysteine (NAC), and trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) from Sigma-Aldrich (St. Louis, MO, USA). HC-030031 was from Tocris. Morphine sulphate was obtained from Hospira Inc. RTX was dissolved in 10% DMSO. HC-030031 was dissolved in 20% DMSO. Trolox was prepared in 1 M NaHCO3. The pH was adjusted to 7.0 with 1 N HC1, and the solution was diluted with PBS to obtain the desired concentration.
All animal experimental procedures were performed in accordance with the guidelines of the International Association for the Study of Pain and the animal protocol was approved by Harvard Medical School Animal Care. Male adult CD1 mice (8-10 weeks) used in this study were purchased from Charles River and were housed in controlled room temperature (22 ± 2 oC) and humidity (60-80%) under a 12 h/12 h light/dark cycle. Food and water were provided ad libitum except during the experiments. In some experiments, C57BL/6 wild-type and Trpv1 and Trpa1 knockout mice were also used. Wild-type and Trpv1 and Trpa1 knockout mice were obtained from the Jackson Laboratory. All the behavioral tests were done by the observer blinded to the treatment or genotype of the animals.
As described previously, mice were shaved at the nape of the neck the day before experiments. On the day of experiments, mice were individually placed in small plastic chambers (14 × 18 × 12 cm) on an elevated metal mesh floor and allowed 30 min for habituation before examination. Mice were then given an intradermal injection of 50 μl of agents into the nape of the neck under brief anesthesia of isoflurane. Immediately after the injection, mice were returned to their chambers and scratching behavior was quantified by counting the number of scratches for 30 min. A scratch was counted when a mouse lifted its hindpaw to scratch the shaved region and returned the paw to the floor or to the mouth for licking.
To distinguish itch and pain responses simultaneously, we used the cheek model by injection of chemical into the cheek of mice[32,33]. Mice were shaved on cheeks (approx. 5×8 mm area) one day before the experiment. On the day of experiment, mice were intradermally injected of 10 μl of reagent (0.3% H2O2 or 2 μmol tBHP) into the cheek under brief anesthesia with isoflurane. Immediately after the injection, mice were returned to their chambers and the wiping and scratching were quantified by counting the number of wipes and scratches for 30 min. One wipe was counted when mouse unilaterally wipes the injected site with the forelimb, which was not part of grooming behavior. One scratch was defined as a lifting of the hind paw toward the injection site on the cheek and then returning the paw to the floor or to the mouth.
As previously described , tail immersion test was used to assess heat pain sensitivity in mice. Briefly, the terminal 3 cm of a mouse’s tail was immersed in hot water bath at 52 0C and the latency of tail flick was recorded with a cutoff time of 10 seconds to avoid tissue injury.
To test the effects of μ-opioid receptor agonist or antagonist on oxidants-induced scratching, μ-opioid receptor agonist morphine (1 mg/kg) or antagonist naloxone (1 mg/kg) was i.p. injected into mice 20 min before i.d. injection of 0.3% H2O2 or 10 μmol tBHP, the most effective dose to induce scratching in mice.
To assess the involvement of histamine H1 receptor in oxidants-induced scratching behavior, chlorpheniramine (10 mg/kg), the selective histamine H1 receptor antagonist, was i.p. injected 20 min before i.d. injection of 0.3% H2O2 or 10 μmol tBHP in mice.
To examine the role of TRPV1-expressing C-fibers in the oxidants-induced itch, we destroyed the C-fibers by daily treatment with the potent TRPV1 receptor agonist resiniferatoxin (RTX, 30, 70 and 100 μg/kg, subcutaneously for 3 consecutive days), one week before the oxidants injection, as we described previously.
To assess the involvement of TRPA1 in oxidants-induced scratching behavior, HC-030031 (10 or 20 μg in 50 μl vehicle), the selective TRPA1 antagonist, was intradermally co-administrated with 0.3% H2O2. 20% DMSO was used as vehicle to dissolve HC-030031.
All the data were expressed as Mean ± S.E.M. Student’s t test or one-way ANOVA followed by post-hoc Bonferroni test were used to determine statistical significance. The criterion for statistical significance was set as P<0.05.
We first tested whether i.d. injection of H2O2 and tBHP, two commonly used oxidants for producing oxidative injury, would induce scratching behavior in CD1 mice. As shown in Figure 1, injection of H2O2 (i.d., 0.03%-1% in 50 μl saline) into the nape of mouse neck produced very striking scratching behavior in CD-1 mice, in a dose-dependent manner. H2O2 began to elicit scratching at the concentration of 0.03% and reached a maximum (230 scratches in 30 min) at the concentration of 0.3%. However, the highest concentration of H2O2 (1%) produced less scratches than that of 0.3% H2O2 (P<0.05), indicating an invert-U-shaped dose-response curve (Figure 1A), as shown for other pruritogens such as imiquimod and chloroquine. The concentration of 0.3% H2O2 was chosen for the following experiments, as this is the most effective one. Figure 1B shows the time course (0-30 min) of scratching behavior after i.d. injection of 0.3 % H2O2. H2O2-induced scratching occurred rapidly in the first 5 min, peaked after 15 min, and declined after 30 min. We did not observe any abnormal behaviors of mice after i.d. administration of H2O2.
The oxidant, tBHP (1-30 μmol in 50 μl saline, i.d.) also induced marked scratching response in CD-1 mice (Figure 1C). Scratching began to be induced at the dose of 1 μmol and reached a maximum at the dose of 10 μmol tBHP. However, a higher dose (30 μmol) of tBHP evoked less scratches compared to 10 μmol tBHP (P<0.05, Figure 1C), suggesting that tBHP also induces a bell-shaped dose-response curve as H2O2. The most effective dose of tBHP (10 μmol) was chosen for the following experiments. Time course analysis showed that tBHP-elicited scratching was significant within 5 min, reached to the peak at 10 min and maintained at 30 min (Fig. 1D). We also did not observe abnormal behaviors in the tBHP-treated mice.
In order to distinguish itch and pain responses simultaneously, we used the cheek model by i.d. injection of oxidants into cheek rather than the neck of mice. In this model injection of pain-inducing agents, such as capsaicin, can only elicit wiping behavior by forelimb, while injection of itch-inducing agents, such as histamine, can only elicit scratching behavior by hindlimb. As shown in Figure 2, i.d. injection of H2O2 (0.3% in 10 μl saline) or tBHP (2 μmol in 10 μl saline) into the cheek of mice elicited both wiping and scratching behavior, indicating these oxidants can induce mixed pain and itch sensations at a given dose. Notably, both H2O2 and tBHP induced much more scratching than wiping behavior, suggesting that itch may be one of the major sensory modalities induced by oxidants.
We tested whether oxidant-induced scratching could be modulated by μ-opioid receptor, which has been implicated in itch in animals and humans. Morphine (1 mg/kg, i.p.), a μ-opioid receptor agonist, increased the latency of tail-flick in hot water immersion test, indicating the analgesic effect of morphine (Figure 3A). In contrast, morphine (1 mg/kg, i.p.) did not reduce H2O2 or tBHP-induced scratching behavior (Figure 3B), consistent with clinical observations that morphine can only reduce pain but not itch. The result also suggests that H2O2 or tBHP-induced scratching could be itch-associated behavior, rather than pain-like behavior in mice. The reason we chose low-dose morphine (1 mg/kg) is because high doses of morphine (e.g., 5-10 mg/kg) could induce psychoactive behaviors which may interfere with scratching behavior . Of note, naloxone (1 mg/kg, i.p.), an opioid receptor antagonist, significantly attenuated scratching induced by H2O2 or tBHP (Figure 3B; P<0.05; Student’s t test). It is suggested that endogenous opioids may be involved in H2O2 or tBHP-induced itch in mice.
We tested chlorpheniramine, a histamine H1 receptor antagonist to assess the involvement of histamine, one of the best-known itch mediator that was stocked and released from skin mast cells, in oxidants-induced itch. As expected, chlorpheniramine (10 mg/kg, i.p.) blocked scratching induced by i.d. injection of histamine in mice (Figure 4). Nonetheless, chlorpheniramine at the same dose was not able to inhibit the scratching response induced by H2O2 or tBHP in mice (Figure 4). Thus, oxidative challenges-elicited scratching behavior is histamine-independent.
It is well established that TRPV1-expressing C-fibers mediate itch sensation induced by various pruritogens[39-41]. To determine the role of TRPV1-expressing C-fibers in oxidants-induced itch, we employed systemic pretreatment of resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, to destroy TRPV1-expressing C-fibers in mice. Our previous work showed that pretreatment with RTX resulted in depletion of TRPV1-expressing C-fibers in mice. As shown in Figure 5A, RTX-treated mice were insensitive to noxious heat (52 0C hot water bath), demonstrating functional loss of TRPV1-expressing C-fibers in mice after RTX treatment. Notably, scratching responses induced by H2O2 or tBHP were almost abolished in RTX-treated mice, compared with control mice (Figure 5B), indicating that TRPV1-expressing C-fibers mediate H2O2 or tBHP-induced itch.
To further define the involvement of TRPV1 in oxidants-induced itch, we tested H2O2 or tBHP-induced scratching behavior in Trpv1 knockout mice (Trpv1−/−). Notably, H2O2 or tBHP-induced scratching behavior was comparable in wild-type (WT) control mice and Trpv1−/− mice (Fig. 5C). Together, these results suggest that TPRV1-expressing C-fibers, but not TRPV1 per se, were required for oxidants-induced scratching behavior in mice.
Recent work suggested that TRPA1 is essential for histamine-independent, Mas-related G protein-couple receptor (Mrgpr)-mediated itch in mice. Given the fact that H2O2 is able to activate TRPA1[43,44], we subsequently examined the role of TRPA1 in oxidants-induced scratching behavior, by means of both pharmacological and genetic manipulations. Co-administration of H2O2 and HC-030031, a selective TRPA1 antagonist, dose-dependently attenuated H2O2-induced scratching behavior in mice (Figure 6A). Consistently, H2O2-induced scratching behavior was also substantially reduced in Trpa1−/− mice relative to WT control mice (Figure 6B). Together, these data suggest that TRPA1 plays a critical role in oxidants-induced pruritus.
We tested whether two common antioxidants N-acetyl-L-cysteine (NAC) and trolox (a water-soluble vitamin E analogue) are able to attenuate oxidants-induced scratching behavior. Administration of NAC (200 mg/kg, i.p.) or trolox (100 mg/kg), 20 min prior to the injection pruritogens, significantly reduced H2O2 or tBHP-induced scratching behavior in mice (Figure 7A and B). Thus, oxidants-induced scratching behavior can be attenuated by antioxidants.
Itch is a major somatic sensation and can be acute (e.g., mosquito bite) and chronic (e.g., atopic dermatitis). Although acute itch is self-protective and served as a warning system, chronic itch represents a common clinical problem, as there is still a lack of therapeutics that can consistently and effectively alleviate itch[45,46]. Antihistamines serve as a standard treatment for clinical itch; however, most types of chronic itch are resistant to antihistamines. Recent studies have identified several novel itch mediators that are largely independent of histamine[47-49]. To our knowledge, this is the first study to demonstrate that oxidative challenges can induce itch-indicative scratching behavior. We showed that TRPA1, but not TRPV1, mediated oxidants-elicited itch in mice. We further demonstrated that antioxidants substantially attenuated oxidants-induced itch responses. Thus, we offered novel mouse itch models elicited by oxidative stress and these models should be useful for testing new anti-pruritic drugs. These animal models should also be helpful to investigate the molecular and cellular mechanisms of oxidative stress-induced itch that are associated with chronic itch conditions in humans.
Great progress has been made in recent years that reveals distinct molecular basis for itch sensation[11,20,21,49-51]. Primary sensory neurons located in trigeminal ganglion and dorsal root ganglion (DRG) are responsible for transducing itch stimuli to the central nervous system[1,40,45]. TRPV1-expressing C-fibers are required for both histamine-dependent and independent itch[39,52]. Histamine, released form mast cells, binds H1 and H4 receptors on nerve terminals in skin to elicit itch via activation PLCbeta3 and TRPV1. Of interest, histamine-independent itch induced by chloroquine (an anti-malaria drug and an agonist of sensory neuron-specific G-protein-coupled receptor (GPCR) MrgprA3) and BAM8-22, an endogenous agonist of MrgprC11), requires TRPA1 but not TRPV1[11,42]. In spinal cord, gastrin releasing peptide (GRP) released from primary TRPV1-expressing C-fibers, may activate GRP receptor (GRPR)-expressing neurons in superficial laminas of dorsal horn to elicit itching sensation[20,21].
Although pain is known to suppress itch in physiological and acute conditions, pain and itch also share remarkable similarities, especially in clinical and pathological conditions[1,45,54]. First, both are unpleasant sensory experiences and consist of multidimensional components, including sensory discriminative, affective and motivational components. Second, both peripheral sensitization (sensitization of primary sensory neurons) and central sensitization (sensitization of spinal cord and other CNS neurons) are implicated in pain and itch hypersensitivity in pathological conditions[54-57]. Third, similar inflammatory mediators and neurotransmitters are involved in both chronic itch and chronic pain, including opioids, proteases, and substance P, as well as their respective receptors such as μ- and κ-opioid receptors, protease-activated receptor PAR-2, and NK-1 receptor. Finally, oxidative stress has been demonstrated to contribute to the genesis of peripheral and central sensitization related to chronic pain, and antioxidants treatment is able to attenuate pain behavior in animal pain models of inflammatory pain and neuropathic pain. In parallel, we demonstrated in this study that oxidative challenges elicited itch behavior in mice and antioxidants attenuated the oxidants-induced itch. Thus, our results support that oxidative stress is positively correlated with both pain and itch.
TRP channels, such as TRPV1, TRPV3 and TRPA1, play a key role in pain and itch signaling transduction in sensory neurons [42,59-62]. Previous reports demonstrated that TRPV1 mediated histamine-induced itch while TRPA1 mediated histamine-independent itch in mice. Our present work showed that TRPA1, but not TRPV1, mediated oxidative challenges-induced itch in mice, which was also independent of histamine. Oxidative stress results from metabolic activity or environmental stimuli, such as ultraviolet radiation, chemotherapeutic agents and hyperthermia, and produces highly reactive chemicals (e.g., H2O2) and oxidizing lipid products (e.g. 4-hydroxynonenal) , which is known to activate TRPA1, but not TRPV1[43,44,63]. Thus, endogenous TRPA1 agonists, such as H2O2 and 4-hydroxynonenal, produced by oxidative stress, activate TRPA1 on sensory neurons to elicit or/and modulate pain and itch signaling. TRPA1 is known to be expressed by a subset of TRPV1-expressing population[40,64,65]. We found that ablation of TRPV1-expressing C-fibers almost abolished oxidative challenges-induced itch, which may be attributed to elimination of TRPA1-expressing neurons within the TRPV1 population. We provided both pharmacological and genetic evidence supporting an essential role of TRPA1 but not TRPV1 in mediating oxidants-induced pruritus. First, the TRPA1 antagonist HC-030031 reduced oxidants-induced itch. Second, H2O2-induced scratching was largely prevented in Trpa1 knockout mice but intact in Trpv1 knockout mice.
In summary, we have demonstrated that oxidative challenges are sufficient to induce scratching behavior in mice via activation TRPA1 in C-fibers (TRPV1+). Oxidative stress is not only present in neurodegenerative conditions[66,67], but also exists in chronic itch conditions associated with atopic dermatitis, psoriasis, chronic renal failure, cholestasis, and diabetes[24-26]. Moreover, our findings showed that oxidative stress-induced itch response is largely independent of histamine, consistent with the clinical observation that chronic itch associated with oxidative stress is resistant to antihistamines treatment. Although further investigation is needed to establish that oxidative stress also drives chronic itch, our findings strongly suggest that oxidative stress is a novel mechanism for pruritus. Targeting oxidative stress by antioxidants or TRPA1 activation by selective TRPA1 antagonists may lead to the development of novel and effective anti-itch therapies.
The work was supported by US National Institutes of Health grants R01-DE17794, R01-NS54362 and R01-NS67686 to RR Ji.
Conflict of interest
The authors state no conflict of interest