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The antipruritic activity of gabapentin, an anticonvulsant, was studied in a mouse model of allergic itch. In mice sensitized by an extract of the salivary glands of the mosquito (ESGM), an intradermal injection of ESGM elicited scratching and increased peripheral nerve firing. Oral or intradermal administration of gabapentin at the ESGM injection site inhibited ESGM-induced scratching and peripheral nerve firing. However, gabapentin did not affect histamine-induced scratching. The distributions of immunoreactivity to the voltage-dependent calcium channel α 2 δ-1 subunit, a site of gabapentin action, and the histamine H 1 receptor differed in the mouse dorsal root ganglia. The α 2 δ-1 subunit was mainly found in neurons that were 15-20 µm in diameter, whereas the H 1 receptor was mainly in 20-30 µm neurons. In addition, α 2 δ-1 subunit immunoreactivity co-localized with that of transient receptor potential vanilloid 1 (TRPV1). These results suggest that gabapentin regulates allergic itch by acting on the calcium channel α 2 δ-1 subunit in peripheral TRPV1-positive neurons., (Copyright © 2018 Elsevier B.V. All rights reserved.)

An itch is experientially well known that the scratching response of conditions such as atopic dermatitis is enhanced under psychological stress. Morphine is typical narcotic drug that induces a scratching response upon local application as an adverse drug reaction. Although long-term treatment with morphine will cause tolerance and dependence, morphine withdrawal can cause psychologically and physiologically stressful changes in humans. In this study, we evaluated the effects of morphine withdrawal on histamine-induced scratching behavior in mice. Administration of morphine with progressively increasing doses (10-50 mg/kg, i.p.) was performed for 5 consecutive days. At 3, 24, 48, and 72 hr after spontaneous withdrawal from the final morphine dose, histamine was intradermally injected into the rostral part of the back and then the number of bouts of scratching in 60 min was recorded and summed. We found that at 24 hr after morphine withdrawal there was a significant increase in histamine-induced scratching behavior. The spinal c-Fos positive cells were also significantly increased. The relative adrenal weight increased and the relative thymus weight decreased, both significantly. Moreover, the plasma corticosterone levels changed in parallel with the number of scratching bouts. These results suggest that morphine withdrawal induces a stressed state and enhances in histamine-induced scratching behavior. Increased reaction against histamine in the cervical vertebrae will participate in this stress-induced itch enhancement.

The present study was conducted to determine whether cutaneous itch involves mu-opioid receptors in either of the spinal cord or lower brainstem or in both regions in mice. An intraplantar injection of serotonin hydrochloride (100 nmol/site) induced biting, an itch-related behavior. The behavior was inhibited by subcutaneous (0.3-1 mg/kg) and intracisternal (1--10 nmol/site), but not intrathecal (1--10 nmol/site), injections of naloxone hydrochloride. An intradermal injection of serotonin (100 nmol/site) to the rostral back induced scratching, an itch-related behavior, which was inhibited by subcutaneous (1 mg/kg) and intracisternal (10 nmol/site) injections of naloxone. These results suggest that mu-opioid receptor in the lower brainstem, but not spinal cord, is a site of central pruritogenic action of opioids and is involved in the facilitatory regulation of itch signaling.

The common adverse effect of centrally-injected mu-opioid receptor (mu-OR) agonists is pruritus. This study was conducted using mice to examine whether different subtypes of mu-OR would be responsible for pruritus and analgesia. Intracisternal injections of morphine and morphine-6beta-glucronide (M6G), but not M3G, produced an antinociceptive effect. Morphine, but neither M6G nor M3G, induced facial scratching, a pruritus-related response. Facial scratching following morphine was not affected by the mu(1)-OR antagonist naloxonazine at doses that inhibited the antinociceptive effects. The results suggest that different subtype and/or splice variants of mu-OR are separately involved in pruritus and antinociception of opioids.

Although mosquito allergy induces the release of histamine, the itch-related response, scratching, is not effectively suppressed by blockade of H1 histamine receptors. To address this question, we examined the effects of neonatal capsaicin treatment on allergic reactions and H1 histamine receptor-expressing sensory neurones in mice. Neonatal capsaicin treatment almost completely abolished allergy-associated scratching, without effects on plasma extravasation or increase in serum concentrations of immunoglobulins E and G1. An injection of edema contents from an animal exhibiting allergic reaction elicited scratching in naive animals, suggesting the production of pruritogen(s) by allergic reaction; this production was not suppressed by neonatal capsaicin treatment. This treatment markedly decreased the number of sensory neurones immunoreactive for TRPV1 capsaicin receptor, with little effect on sensory neurones immunoreactive for neurofilament 200, a marker of myelinated A-fibre neurones. In addition, there was a trend towards a reduction in numbers of sensory neurones immunoreactive for H1 histamine receptor. The results suggest that capsaicin-sensitive sensory neurones that lack H1 histamine receptors play a key role in signalling of allergic itch.

This study investigated endogenous mediators involved in mosquito allergy-associated itching in mice. An intradermal injection of an extract of mosquito salivary gland elicited marked scratching in sensitized mice. The 5-lipoxygenase inhibitor zileuton (100 mg/kg), the 5-lipoxygenase activating peptide inhibitor MK-886 (10 mg/kg), and the glucocorticoid betamethasone 17-valerate (3 mg/kg) inhibited the scratching. The scratching was not affected by the cyclooxygenase inhibitors indomethacin and ketoprofen, the TP prostanoid receptor antagonist SQ-29548, the leukotriene B(4) antagonist ONO-4057, the cysteinyl leukotriene antagonist pranlucast, the leukotriene D(4) antagonist MK-571, the platelet-activating factor antagonist CV-3988, the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester, the H(2) histamine-receptor antagonist cimetidine, the H(1) histamine-receptor antagonist terfenadine plus cimetidine, and cypoheptadine that blocks the 5-HT(1/2) serotonin receptors. Zileuton (100 mg/kg) inhibited the increased activity of the cutaneous nerve branch induced by an intradermal injection of the extract, suggesting the peripheral action. Zileuton and MK-886 (10 and 100 microM) did not affect high K(+)-induced increase in intracellular Ca(2+) concentration in cultured dorsal root ganglion neurons. The results suggest that 5-lipoxygenase metabolite(s) other than leukotriene B(4) and cysteinyl leukotrienes are involved in mosquito allergy-associated itching.

Thromboxane A2 (TXA2), a metabolite of arachidonic acid produced by cyclooxygenase and thromboxane synthase, is thought to participate in chronic dermatitis. This study investigated the involvement of TXA2 in cutaneous itch. An intradermal injection of U-46619, a stable analogue of TXA2, elicited scratching, an itch-associated response, in mice. Dose-response curve was bell shaped with a maximum effect at 10 nmol per site. The action of U-46619 was inhibited by a coinjection of the TP antagonist ONO-3708 and was abolished by TP receptor deficiency. TP receptor was mainly expressed in nerve fiber in the skin and keratinocytes. Thromboxane synthase was also expressed in keratinocytes. U-46619 increased intracellular Ca2+ ion concentration in primary cultures of dorsal root ganglion neurons and keratinocytes. The results suggest that TXA2 synthesized by keratinocytes acts as an itch mediator. It may elicit itch through the activation of TP receptors on primary afferents and keratinocytes; keratinocytes may produce itch mediators including TXA2. Thus, thromboxane synthase inhibitor and TP receptor antagonists will be candidates for antipruritic medicines.

JTE-907, N-(benzo[1,3]dioxol-5-ylmethyl)-7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxamide, is a selective cannabinoid CB2 receptor antagonist/inverse agonist. The anti-pruritic activity of JTE-907 was studied in NC mice with chronic dermatitis, a model of atopic dermatitis. The oral dose of JTE-907 (1 and 10 mg/kg/day), an immunosuppressant agent tacrolimus (1 mg/kg/day) and a glucocorticoid betamethasone 17-valerate (1 mg/kg/day) for 20 days suppressed the spontaneous scratching and cutaneous nerve activity of NC mice. JTE-907 (10, but not 1, mg/kg) and tacrolimus, but not betamethasone, tended to alleviate the dermatitis. Betamethasone inhibited the body weight gain. These results suggest that JTE-907 suppresses spontaneous itch-associated responses of NC mice without adverse effects such as weight loss.

The pruritogenic potency of tryptase and its involvement in anti-pruritic effect of intravenous nafamostat mesilate (NFM) were studied in mice. An intradermal injection of tryptase (0.05-1 ng/site) elicited scratching in ICR mice, while chymase was without effects at doses of 0.05-50 ng/site. The dose-response curve of tryptase action was bell-shaped and the effect peaked at 0.1 ng/site (approximately 0.7 fmol/site). NFM (10 mg/kg) inhibited scratching induced by tryptase but not by histamine and serotonin. NFM (1-10 mg/kg) produced the dose-dependent inhibition of scratching induced by intradermal compound 48/80 (10 microg/site). The inhibition by NFM (10 mg/kg) was abolished in mast cell-deficient (WBB6F1 W/W(V)) mice, but not in wild-type (WBB6F1 +/+) mice. NFM (10 mg/kg) suppressed tryptase activity in the mouse skin. Proteinase-activated receptor-2 (PAR-2) neutralizing antibody (0.1 and 1 microg/site) and the PAR-2 antagonist FSLLRY (10 and 100 microg/site) inhibited scratching induced by tryptase (0.1 ng/site) and compound 48/80 (10 microg/site). These results suggest that mast cell tryptase elicits itch through PAR-2 receptor and that NFM inhibits itch-associated responses mainly through the inhibition of mast cell tryptase.

As a model of dry skin pruritus in mice, one hind paw was treated twice daily with a mixture of acetone/diethylether/water (AEW); controls received water only. A protective collar prevented the animals from accessing the treatment area. At 16 days, the collar was removed and AEW-treated mice exhibited marked biting of the treated paw; the number and cumulative duration of bites was significantly greater than in controls. After 3 additional treatment days (collars intact), animals were perfused for c-fos immunohistochemistry. There was significantly more fos-like immunoreactivity in the ipsilateral lumbar spinal cord of AEW-treated animals, with the majority in superficial laminae. It is proposed that biting of the dry skin reflects pruritus, and that neurons predominantly in superficial laminae of the dorsal horn may signal itch sensation.

Unsei-in inhibits substance P (SP)-induced scratching of mice after repeated administration. The involvement of cutaneous nitric oxide (NO) in the SP-induced scratching led us to investigate the effects of Unsei-in on the cutaneous NO system in mice. Seven-day oral administration of Unsei-in (300, but not 100, mg/kg daily) significantly inhibited scratching and the increase of cutaneous NO after intradermal SP injection. The NO synthase 1 (NOS1) inhibitor 7-nitroindazole (1 nmol/site) decreased SP-induced scratching and NO production. Repeated administration of Unsei-in (300 mg/kg) reduced the cutaneous NOS1 level. The results suggest that the inhibition of cutaneous NOS1 expression and NO production participates in the antipruritic action of Unsei-in.

Using a dry skin model in rats, we assessed spontaneous itch-related scratching behavior and associated c-fos expression in the superficial dorsal horn of the spinal cord. The number of spontaneous bouts of hind limb scratching directed toward the nape of the neck was significantly higher after 5 days of topical application of acetone-diethylether-water (AEW) compared to pre-treatment levels or to control animals treated with water only. In AEW-treated animals, neurons expressing Fos-like immunoreactivity (FLI) were observed in superficial laminae of the dorsal horn at C3-C5 levels. There was a significant, positive correlation between the number of neurons in lamina I expressing FLI and the number and cumulative duration of spontaneous scratching bouts. These results suggest that this model may be useful to assess mechanisms of dry skin pruritus, and that lamina I neurons are activated as a consequence of itching and/or scratching in this model.

We investigated the role of serotonin (5-hydroxytryptamine; 5-HT)2 and 5-HT3 receptor subtypes in acute itch-associated scratching behavior as well as in an allergic pruritus model in rats. Intradermal 5-HT evoked hind limb scratching directed toward the injection site in naïve rats. Scratching behavior was significantly reduced by pretreatment with the 5-HT2 receptor antagonist ketanserin. Intradermal injection of alpha-methylserotonin, a 5-HT2 receptor agonist, also elicited scratching behavior in a dose-dependent manner, indicating that acute 5-HT-induced scratching is mediated via peripheral 5-HT2 receptors. To produce a model of allergic pruritus, skin was sensitized by topical application of 5% dinitrofluorobenzene (DNFB). One month later, repeated challenge of the skin with 0.2% DNFB at weekly intervals elicited scratching as part of the immediate allergic response. Scratching was not affected by ketanserin or by the 5-HT3 receptor antagonist ondansetron, indicating that neither 5-HT2 nor 5-HT3 receptors is involved in itch-associated scratching behavior caused by allergic skin dermatitis in rats.

Hind limb scratching is used increasingly as an itch model in rodents. Scratching is usually quantified as the number of scratching bouts over a 60 min period. Since the antipruritic effect of scratching should depend on the total time of skin contact, then the duration of scratching bouts and within-bout scratching frequency may also be important factors. Therefore, we measured these parameters during episodes of scratching directed toward the site (nape of neck) of intradermal injection of serotonin in Sprague-Dawley rats. Serotonin elicited significantly more scratching bouts than saline. There was a biphasic pattern of scratching over time, with peaks at 10-20 and 40-50 min. Although cumulative bout duration (2-min intervals) had a similar biphasic distribution, the mean individual bout duration (2.1 s) did not change significantly over time. Within-bout scratching frequency remained constant over time at 8 Hz. The number of scratching bouts was suppressed in a dose dependent manner by naltrexone (3 and 5 mg/kg), while the individual bout duration and the within-bout frequency were not significantly different compared with serotonin-evoked scratching without naltrexone. These results validate the total number of scratching bouts as an indicator of the magnitude of itch-related scratching.

Unsei-in, a traditional medicine, is prescribed against pruritic cutaneous diseases, but the mechanisms of antipruritic action are still unknown. In the present study, we examined the antipruritic effects of Unsei-in in mice. Single administration of Unsei-in did not inhibit substance P-induced itch-associated response (scratching) in mice. However, repeated treatment with Unsei-in for 7 d significantly inhibited substance P-induced scratching. The same repeated treatment with Unsei-in suppressed the expression of NK(1) tachykinin receptors in the skin. These results suggest that Unsei-in inhibits substance P-associated itching and that the inhibition is at least partly due to the suppression of the expression of NK(1) tachykinin receptors in the skin.

We examined whether azelastine would inhibit itch-associated responses of mice to mosquito allergy. Repeated injections of mosquito salivary gland extract increased scratching and sensory nerve activity. Azelastine inhibited the increased scratching and nerve activity, while terfenadine was without effects. Dexamethasone did not affect the increased scratching. Azelastine suppressed high K(+)-induced increase in intracellular free Ca(2+) in primary cultures of mouse sensory neurons. Direct inhibition by azelastine of sensory neurons may be at least involved in the anti-pruritic effect of azelastine. Histamine, substance P, and leukotriene B(4) may not play a key role in the itching of mosquito allergy.

The effects of the opioid antagonist naltrexone on spontaneous itch-associated behaviors and cutaneous nerve activities were examined to determine whether it inhibits pruritus through peripheral action in NC mice with chronic dermatitis. Their rostral-back scratching and caudal-back biting were 19 and 3.4 times more, respectively, than those of control mice. The activities of cutaneous nerves innervating the rostral and caudal back were 9.5 and 5.4 times more, respectively, in affected mice than in control mice. Subcutaneous injections of naltrexone significantly inhibited the scratching and biting, without effects on the nerve activities. The results suggest that the peripheral action does not play a central role in inhibiting chronic itch-associated behaviors.

In the present study, we examined whether cholinergic agonists would elicit an itch-associated response in mice. When mice were given an intradermal injection of carbachol (1-10 nmol) or bethanechol (0.3-100 nmol) into the rostral back, they showed the dose-dependent increase of scratching. Nicotine (1-10 nmol) showed no effect. Pretreatment with naloxone, but not with terfenadine, significantly suppressed the carbachol-induced scratching. When intradermally co-injected with carbachol, atropine and 4-DAMP but neither methoctramine nor pancuronium significantly inhibited the carbachol-induced scratching. Muscarinic agonists are suggested to produce itch through activation of M3 muscarinic receptors in the skin.

The present study was conducted to establish a new mouse model of dry skin pruritus. The rostral back was treated daily with cutaneous application of acetone/ether (1:1) mixture (AE), water following AE (AEW), 1% sodium lauryl sulfate (SLS) or tape stripping (TS). On the day after 5-day treatment, although all four treatments significantly decreased stratum corneum (SC) hydration and increased transepidermal water loss (TEWL), only AEW treatment significantly increased spontaneous scratching. An increase in the frequency of TS produced the marked increase of TEWL, without significant effects on SC hydration and spontaneous scratching. In AEW-treated mice, changes in SC hydration and TEWL were marked in the initial 2-day period, while spontaneous scratching increased gradually from 3 days after starting the treatment. The degranulation of cutaneous mast cells was increased by SLS treatment but not by other treatments. There was no apparent difference in AEW-induced spontaneous scratching between mast cell-deficient mice (WBB6F1-W/Wv) and normal littermates (WBB6F1-+/+). Opioid antagonists, naloxone and naltrexone, (1 mg/kg, subcutaneously) significantly suppressed spontaneous scratching in AEW-treated mice. It is suggested that spontaneous scratching of AEW-treated mice is an itch-related response and a useful model for studying the mechanisms of dry skin pruritus.

We previously screened the anti-itching activities of 33 herbal medicines in substance P (SP)-induced itching model mice. One of the most potent antipruritogenic extracts, the methanol extract of fruits of Cnidium monnieri (Cnidii Fructus) was studied further. The chloroform-soluble fraction of the methanol extract markedly inhibited SP-induced scratching. Among 10 subfractions of the chloroform-soluble fraction, the CS-3 fraction had the most potent inhibitory effect on scratching. Each of 3 subfractions of CS-3 showed significant anti-scratching activities. However, inhibitory potencies were not different among the three and weaker than that of CS-3 itself at a same dose. These 3 subfractions of CS-3 mainly contained xanthotoxin, isopimpinellin, bergapten, imperatorin and osthol. Single administration of osthol did not inhibit SP-induced scratching, and imperatorin very weakly subsided scratching. These results suggest that the strong antipruritic action was focused on the CS-3 fraction of the C. monnieri methanol extract, and it might result from the combined effects of these coumarin derivatives, or by undetermined minor compounds.

We investigated itch-associated responses (scratching) to mosquito bites and the role of histamine and mast cells in mosquito-induced itching in mice. Although the first bites of mosquito Aedes albopictus did not increase scratching, repeated bites increased scratching. The response was not diminished even after an interval of 2 months. Similarly, repeated intradermal (i.d.) injections of salivary gland extract (SGE) from Aedes albopictus increased scratching after SGE injection itself and mosquito bites. The scratching peaked within 10 min and almost subsided by 60 min. The opioid antagonist naloxone (1 mg/kg, s.c.) inhibited scratching following SGE injection. Although the non-sedative H1-histamine-receptor antagonist terfenadine (30 mg/kg, p.o.) significantly suppressed scratching induced by histamine (100 nmol/site, i.d.) in either naive or mosquito-sensitized mice, it did not affect mosquito-induced scratching in mosquito-sensitized mice. Repeated injections of SGE increased scratching in mast cell-deficient (WBB6F1-W/Wv) mice as well as in normal (WBB6F1-+/+) littermates. Repeated exposure to mosquito bites roughly doubled serum concentrations of total IgE and IgG1, but not IgG2a. Repeated injections of SGE markedly increased plasma extravasation induced by mosquito bites and such an increase was almost completely suppressed by terfenadine (30 mg/kg, p.o.). The results show the presence of histamine-mediated and histamine-independent mechanisms in cutaneous itching and suggest that histamine probably released from mast cells does not play an important role in itching in immediate allergic reaction. Our murine model of mosquito itching may be useful for studying the mechanisms of immediate allergic itching.

To clarify the behavioral and pathological features of spontaneous scratching of NC mice with mite-induced chronic dermatitis, we investigated the spontaneous and pruritogen-evoked scratching of NC mice. Although the frequency of scratching of NC mouse did not increase under specific pathogen-free environment, it gradually and markedly increased from 3 to 6 weeks after transfer to conventional environment. The onset of increase in spontaneous scratching was similar to that of dermatitis development and the elevation of plasma concentration of immunoglobulin E. At chronic stage (16 weeks after environment change), the frequency of spontaneous scratching was roughly parallel to the degree of dermatitis, but not to the plasma concentration of immunoglobulin E. The spontaneous scratching of NC mice with dermatitis was inhibited by distraction and the opioid antagonist naltrexone, suggesting that the scratching is itch-associated response. An intradermal injection of serotonin, but not histamine and substance P, elicited scratching of the injected site. Methysergide and cyproheptadine inhibited the serotonin-induced scratching but not spontaneous scratching. The results suggest that marked elevation of plasma immunoglobulin E is not always the cause of spontaneous itch-associated response of NC mice with dermatitis. Serotonin, histamine and substance P may not play an important role in spontaneous itch-scratch response at a chronic stage.

To assess the itch-associated response of primary afferents innervating the murine skin in vivo, dose-response curves and time-courses for itch-scratching and cutaneous nerve firing responses to intradermal injections of pruritogens (histamine and serotonin) were compared in ICR and ddY mice. Serotonin increased the itch-scratch response and cutaneous nerve firing in either ICR or ddY mice. Histamine increased these two responses in ICR, but not ddY, mice. The dose-response curves and time-courses for serotonin- and histamine-induced nerve firing were similar to those for the itch-scratch response. The results suggest that cutaneous nerve firing evoked by peripherally given pruritogens includes the itch-associated response.

NC mice, a model for atopic dermatitis, showed scratching behavior when kept under conventional environment. The scratching behavior of NC mice was suppressed by distraction or by the administration of naltrexone (1 mg/kg, s.c.), an opioid antagonist. These results suggest that such scratching behavior is itch-associated response. The itch-associated response of the NC mice was significantly suppressed by an intravenous injection of nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), but not D-NAME (10 mg/kg) and saline. Intracutaneous NO production in the rostral back, a region which the NC mice mainly scratched, was markedly increased as compared with the caudal back, a non-scratched region. The increased NO production in the rostral back of NC mice was decreased by the intravenous injection of L-NAME (10 mg/kg). These results suggest that NO and NO synthase are new target in the treatment of atopic pruritus.