Mouse Cheek Research Articles

Acupuncture attenuates comorbid anxiety- and depressive-like behaviors of atopic dermatitis through modulating neuroadaptation in the brain reward circuit in mice

Atopic dermatitis (AD) is highly comorbid with negative emotions such as anxiety and depression. Although acupuncture has demonstrated efficacy in AD, its influence on comorbid anxiety and depression remains unclear. We sought to explore the impact and mechanisms of action of acupuncture on comorbid anxiety and depression of AD. AD-like skin lesions were induced by the topical application of MC903 to the mouse cheek. Acupuncture was performed at Gok-Ji (LI11) acupoints. AD-like phenotypes were quantified by lesion scores, scratching behavior, and histopathological changes. The effects of acupuncture on comorbid anxiety and depression-like behaviors were assessed using the elevated plus-maze (EPM), open-field tests (OFT), and tail-suspension test (TST). In addition, biochemical changes in the brain reward regions were investigated by immunoblotting for the expression of tyrosine hydroxylase (TH), dopamine D1 receptor (D1R), phospho-dopamine and cAMP-regulated phosphoprotein-32 kDa (pDARPP-32), phospho-cAMP response element binding protein (pCREB), ΔFosB, and brain-derived neurotrophic factor (BDNF) in the nucleus accumbens, dorsolateral striatum, and ventral tegmental area. Acupuncture effectively improved the chronic itching and robust AD-like skin lesions with epidermal thickening. Additionally, it considerably reduced comorbid anxiety- and depression-like symptoms, as indicated by more time spent in the open arms of the EPM and in the center of the open field and less time spent immobile in the TST. Higher pCREB, ΔFosB, BDNF, and pDARPP-32 levels, and reduced TH and D1R protein expression in the brain reward regions of AD mice were reversed by acupuncture treatment. The beneficial effects of acupuncture on clinical symptoms (scratching behavior) and comorbid psychological distress in AD strongly correlated with dorsal striatal ΔFosB levels. Collectively, these data indicate that acupuncture had a significant, positive impact on comorbid anxiety- and depression-like behaviors by modulating neuroadaptation in the brain reward circuit in mice with AD, providing a novel perspective for the non-pharmacological management of psychiatric comorbidities of AD.

Innate immune regulates cutaneous sensory IL-13 receptor alpha 2 to promote atopic dermatitis

The clinical significance and regulators of IL-13Rα2 in itch and atopic dermatitis (AD) remain unclear. To identify disease-driven regulatory circuits of IL-13Rα2, transcriptomic/pathological analysis was performed in skin from patients with AD, psoriasis, healthy subjects, and murine AD model. Functionality was investigated in sensory neurons, keratinocytes and animal model, by using knockdown (KD), calcium imaging, RNA-seq, cytokine arrays, pharmacological assays, and behavioural investigations. In our study, an upregulated IL-13Rα2 expression was revealed in skin of AD patients, but not psoriasis, in a disease activity-dependent manner. In cultured human keratinocytes, IL-13 increased IL-13Rα2 transcription levels, and this were downregulated by IL-13Rα1KD. IL-13Rα2KD reduced transcription levels of EDNRA, CCL20, CCL26. In contrast, sensory neuron-derived IL-13Rα2 was upregulated by TLR2 heterodimer agonists, Pam3CSK4 and FSL-1. In a mouse cheek model, pre-administration of Pam3CSK4 and FSL-1 enhanced IL-13-elicited scratching behaviour. Consistently, in cultured sensory neurons Pam3CSK4 enhanced IL-13-elicted calcium transients, increased number of responders, and orchestrated chemerin, CCL17 and CCL22 release. These release was inhibited by IL-13Rα2KD. Collectively, IL-13 regulates keratinocyte-derived IL-13Rα2 and TLR2 to modulate neuronal IL-13Rα2, thereby promoting neurogenic inflammation and exacerbating AD and itch. Thus, the cutaneous IL-13-IL-13Rα2 and neuronal TLR2-IL-13Rα2 pathway represent important targets to treat AD and itch.

Acupuncture ameliorates not only atopic dermatitis-like skin inflammation but also acute and chronic serotonergic itch possibly through blockade of 5-HT2 and 5-HT7 receptors in mice

Acupuncture has been known to be effective for atopic dermatitis, especially ameliorating itch; however, its mechanisms are still unclear. The aim of this study was to test the anti-itch effects of acupuncture and to investigate its possible mechanisms. Acupuncture was performed at Gok-Ji (LI11) acupoints just before the injection of pruritogens in the mouse cheek model of acute itch and of MC903-induced atopic dermatitis displaying serotonergic chronic itch. Acupuncture significantly reduced acute itch triggered by compound 48/80, chloroquine, or especially serotonin. It also markedly reduced scratching behaviors evoked by the serotonin 5-HT2 receptor agonist α-methylserotonin and selective 5-HT7 receptor agonist LP 44. In addition, acupuncture treatment at LI11 had the preventive and therapeutic effects on persistent itch as well as the robust skin inflammation with epidermal thickening in mice with MC903-induced atopic dermatitis. It also considerably reduced the increased expression of 5-HT2A, 5-HT2B and 5-HT7 receptors in atopic dermatitis-like skin lesions in mice treated with MC903. Taken together, these findings highlight that acupuncture significantly ameliorates not only skin inflammation, but also acute and chronic serotonergic itch, possibly through blockade of serotonin 5-HT2 and 5-HT7 receptors.

マウスおいて硫化物の頬皮内投与により誘起される痒みと痛み：Ca<sub>v</sub>3.2 T型カルシウムチャネル遺伝子欠失の影響

マウスおいて硫化物の頬皮内投与により誘起される痒みと痛み：Ca<sub>v</sub>3.2 T型カルシウムチャネル遺伝子欠失の影響

Extracellular vesicles released from irradiated neonatal mouse cheek tissue increased cell survival after radiation.

Alopecia is one of the common symptoms after high-dose radiation exposure. In our experiments, neonatal mice that received 7 Gy X-ray exhibited defects in overall hair growth, except for their cheeks. This phenomenon might suggest that some substances were secreted and prevented hair follicle loss in the infant tissues around their cheeks after radiation damage. In this study, we focused on exosome-like vesicles (ELV) secreted from cheek skin tissues and back skin tissues, as control, and examined their radiation protective effects on mouse fibroblast cell lines. We observed that ELV from irradiated cheek skin showed protective effects from radiation. Our results suggest that ELV from radiation-exposed cheek skin tissue is one of the secreted factors that prevent hair follicle loss after high-dose radiation.

Atopic dermatitis induces anxiety- and depressive-like behaviors with concomitant neuronal adaptations in brain reward circuits in mice

Clinically, it has been reported that atopic dermatitis (AD) has been linked with negative emotional problems such as depression and anxiety, thereby reducing the quality of life, but little is known about the molecular mechanism that underlies AD-associated emotional impairments. We sought to determine whether AD could induce anxiety- and depressive-like symptoms in mice and to identify pertinent signaling changes in brain reward circuitry. AD-like lesions were induced by the repeated intradermal application of MC903 into the cheek of the mouse. We assessed dermatitis severity with scratching behavior, histopathological changes, anxiety- and depressive-like behaviors using the elevated plus maze, open field and tail suspension tests, and serum corticosterone levels. In the nucleus accumbens (NAc), dorsal striatum (DS) and ventral tegmental area (VTA), protein levels of dopamine- and plasticity-related signaling molecules were determined by Western immunoblotting assay. Intradermal administration of MC903 into mouse cheek provoked a strong hind limb scratching behavior as well as the robust skin inflammation with epidermal thickening. MC903-treated mice also displayed markedly increased anxiety- and depressive-like behaviors, along with elevated serum corticosterone levels. Under these conditions, enhanced cAMP response element binding protein (CREB) and dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP32) phosphorylation, significantly higher brain-derived neurotrophic factor (BDNF) and ΔFosB, but reduced tyrosine hydroxylase (TH) and dopamine D1 receptor (D1R) protein expression were found in the NAc, DS and VTA. Striatal BDNF, phospho-DARPP32 and phospho-CREB levels were significantly associated with the levels of depressive-like behavior in these mice. Taken together, these findings demonstrate that AD-like skin lesion elicits anxiety- and depressive-like phenotypes that are associated with neuroplasticity-related changes in reward circuitry, providing a better understanding of AD-associated emotional impairments.

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors.

Itch was defined as "an unpleasant cutaneous sensation that provokes a desire to scratch"by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

665 Pruritogen activation of human MRGPRX2

Histamine-independent mechanisms mediate the itch associated with the majority of dermatologic and systemic diseases. SLIGRL is a prototypic histamine-independent pruritogen. It is derived from the tethered sequence of the PAR2 receptor. Previous studies have demonstrated that SLIGRL activates mouse MrgprC11 to induce itch. We demonstrate that SLIGRL specifically activates human MRGPRX2, a receptor implicated in itch, allergy, and inflammation. We also evaluated the effect of QWF, an MRGPRX2 antagonist, on SLIGRL-induced activation of Mrgprs and SLIGRL-induced itch in mice. The in vitro interactions of SLIGRL and QWF with Mrgprs were studied using HEK-293 cells transfected with cDNAs encoding mouse MrgprC11, human MRGPRX1, MRGPRX2 MRGPRX3, and MRGPRX4. Intracellular calcium ([Ca2+]) was determined by ratiometric Fura-2 imaging as an indicator of receptor activation. For in vivo experiments, the mouse cheek model, which differentiates between itch and pain, was used to evaluate the effect of QWF on SLIGRL-induced itch in C57BL/6 mice. Consistent with previous studies, SLIGRL activates mouse MrgprC11. We demonstrate that SLIGRL specifically activates human MRGPRX2. The MRGPRX2 antagonist, QWF inhibits activation HEK-293 cells expressing mouse MrgprC11 and human MRGPRX2. SLIGRL-induced itch is significantly decreased (p<0.05), to baseline, with QWF. In conclusion, SLIGRL activates human MRGPRX2, a receptor implicated in itch, allergy, and inflammation. Receptor activation is blocked by QWF. Antagonists of human MRGPRX2 may have therapeutic role in the treatment of itch, allergy and inflammation.

A single amino acid in MRGPRX2 necessary for binding and activation by pruritogens

A single amino acid in MRGPRX2 necessary for binding and activation by pruritogens

Endothelin-1 induces itch and pain in the mouse cheek model

AimsTo date, suggestions that endothelin-1 (ET-1) causes nociception and pruritus are based on results in preclinical models in which responses to pruritic and nociceptive stimuli cannot be distinguished. This study reexamines these sensory effects of ET-1 in the new mouse cheek model, in which pruritogens and algogens evoke distinct behavioral responses. Main methodsMice received intradermal (i.d.) injections of test substances into the left cheek and bouts of hind limb scratches or forepaw wipes, directed to the injection site, were considered indicative of pruritus and nociception, respectively. Key findingsHistamine and capsaicin selectively evoked scratching and wipes, respectively, whereas ET-1 (3–60pmol) promoted dose-dependent bouts of both behaviors. While scratching and wipe responses to ET-1 (30pmol) were potentiated by BQ-788 (an ETB receptor antagonist) and reduced by co-injection of BQ-788 plus BQ-123 (an ETA receptor antagonist), BQ-123 alone inhibited scratching responses only. CTOP (μ-opioid receptor selective antagonist) only augmented scratching responses to ET-1, whereas DAMGO (μ-opioid receptor selective agonist) reduced both behaviors. Loratadine (histamine H1 receptor antagonist) marginally reduced scratching, but markedly suppressed wipes. SignificanceThese results demonstrate that ET-1 evokes pruritic and nociceptive behaviors in the mouse cheek model. Both responses to ET-1 appear to be mediated via ETA receptors and subjected to limitation by simultaneous ETB receptor activation. Local endogenous opioids acting on μ-opioid receptors selectively modulate the pruritic response to ET-1, whereas histamine, possibly derived from mast cells and acting on H1 receptors, contributes importantly to the nociceptive effect of ET-1 in this model.

A New Type of Colored Alumina/Glass Composite Biological Safety Assessment - Oral Mucous Membrane Irritation and Skin Sensitivity Tests

Objective: to describe and demonstrate an oral mucosa irritation and a skin sensitivity test for evaluation of the biological safety of a colored alumina/glass composite (AGC). Methods: colored alumina/glass composite and control materials were sutured to mucine bilateral cheek pouch mucosa, in a position for gross observation and for histological and cytological examination; skin irritation activity of the composite was tested by intradermal injection of a guinea pig test solution, by inducing, enhancing, and stimulating the allergic response observed after local tissue exposure. Results: colored AGC in mouse cheek pouch mucosa stimulated the same reaction as the control, with no significant inflammatory reaction or infiltration of inflammatory cells. Guinea pigs showed no obvious erythema or edema from the irritation test, such that the assessment of colored AGC skin allergy response in mice was 0%. Conclusion: colored AGC was benign towards mouse oral mucosa and elicited no allergic skin inflammation response, indicating that in these aspects this material had good biological safety.

Differential Itch- and Pain-related Behavioral Responses and µ-opoid Modulation in Mice

Intradermal microinjection of the pruritogen histamine, or the algogen capsaicin, in the mouse cheek differentially elicits mainly hindlimb scratching or ipsilateral forelimb wiping, respectively. We investigated the dose-dependency of these responses elicited by various pruritogens and algogens, and µ-opioid modulation. Histamine, 5-hydro-xytryptamine (5-HT) and agonists of protease-activated receptors PAR-2 and PAR-4, all elicited dose-related hindlimb scratching bouts with little forelimb wiping. In contrast, capsaicin, allyl isothiocyanate and bradykinin elicited dose-related forelimb wiping with little scratching. Morphine reduced capsaicin-evoked wiping but not pruritogen-evoked scratching. The µ-antagonist naltrexone decreased pruritogen-evoked scratching but not capsaicin-evoked wiping. A cowhage spicule inserted intradermal elicited equivalent scratching and wiping, while inactivated cowhage spicules loaded with histamine or capsaicin elicited significantly more scratching or wiping, respectively. The mouse cheek injection model appears to be a useful behavioral test that distinguishes between itch and pain.

Lymphocyte changes in peripheral blood, spleen, and liver in DMBA-induced squamous cell carcinoma of mouse cheek skin

The peripheral blood, spleen, and liver lymphocyte subsets of mice with experimental cheek skin carcinoma were determined. The carcinoma was induced by the topical application of 2% (w/v) 9,10-dimethyl-1,2-benzanthracene (DMBA) to cheek skin twice a week for 12 weeks, and it was examined macroscopically and histopathologically. The composition of lymphocyte subsets (T cells, B cells, CD4+ single-positive [SP] T cells, and CD8+SP T cells) in peripheral blood, spleen, and liver was determined by flow cytometry at 3-week intervals for up to 24 weeks. Spleens and livers were assessed by determining their content of natural killer (NK)T cells. The results showed histopathological progression of the skin lesions from papilloma to squamous cell carcinoma at week 12. Body weight was significantly reduced from weeks 15 to 24, and spleen weight was significantly increased at weeks 21 and 24, but liver weight was not significantly different from the control. The lymphocyte subset composition of peripheral blood showed significant elevation of T cells at weeks 6 and 9, followed by reduced levels at weeks 21 and 24, with significant reduction of B cells at weeks 6 and 9, followed by elevation at weeks 21 and 24. CD4+SP T-cell content was elevated at weeks 6, 9, and 12, and reduced at weeks 21 and 24. CD8+SP T-cell content was significantly reduced at weeks 6, 9, and 12, and elevated at weeks 21 and 24. The composition of the lymphocyte subsets in the spleen was similar to their composition in peripheral blood. The composition of both T and B cells in the liver was significantly different from that in the corresponding control group, but no significant differences were found in either CD4+SP or CD8+SP T cells. These findings revealed that the DMBA-induced cheek skin carcinoma in mice affected not only the lymphocyte subsets in peripheral blood, but the cells in the spleen and liver as well.

マウス頬の発生に関する電子顕微鏡的観察

In order to elucidate the pathogenesis of the buccal area deformity, normal development of the mouse cheek, especially the chronological changes of the area between maxillary and mandibular process (AMM) from the 9.0th day to the 14.5th day of gestation (plug day=0), was investigated by light and electron microscopy. On the 9.0-9.5th day of gestation, the first branchial arch connected directly to the fronto-nasal process and its surface epithelium was simple cuboidal and had many short microvilli. On the 10.0 th day, the maxillary process began to swell up from the first branchial arch. The epithelium between and maxillary mandibular process was flattened and its cellular margins were accentuated by the presence of numerous microvilli. There were two kinds of cells, one had numerous microvilli and the other had not. On the 10.3th day, the arrangement of the AMM cell layer was temporally random and the apoptosis was observed characteristically. After the 11.0th day, squamous peridermal cells, of which boundaries were clearly demarcated with microvilli, appeared at surface of the epithelium. Beneath the periderm, cuboidal epithelial cells arranged as one or two cell layers. After the 14.0th day, the epithelium became striated and squamous.Although the epithelial arrangement became temporally random at the 10.3th day, AMM epithelium existed as the compact mass and the mesenchymal cell did not migrate into the epithelium during AMM development. The phenomena which were reported to occur characteristically at the epithelial fusion during the development of the primary or secondary palate were not observed in this study.From these results, the development of the cheek is not from the epithelial fusion between maxillary and mandibular process but from the proliferation and the differentiation of the cells both of maxillary and mandibular process including mesenchymal cells, and the cheek develops through the relative facial development.