Challenge-induced Increases Research Articles

SGK1 mediates hypotonic challenge-induced proliferation in basilar artery smooth muscle cells via promoting CREB signaling pathway

Hypotonic stimulus enlarges cell volume and increased cell proliferation with the exact mechanisms unknown. Glucocorticoid-induced kinase-1 (SGK1) is a serine/threonine kinase that can be regulated by osmotic pressure. We have revealed that SGK1 was activated by hypotonic solution-induced lowering of intracellular Cl− concentration. Therefore, we further examined whether SGK1 mediated hypotonic solution-induced proliferation and the internal mechanisms in basilar smooth muscle cells (BASMCs). In the present study, BrdU incorporation assay, flow cytometry, western blotting were performed to evaluate cell viability, cell cycle transition, and the expression of cell cycle regulators and other related proteins. We found that silence of SGK1 largely blunted hypotonic challenge-induced increase in cell viability and cell cycle transition from G0/G1 phase to S phase, whereas overexpression of SGK1 showed the opposite effects. The effect of SGK1 on proliferation was related to the upregulation of cyclin D1 and cyclin E1, and the downregulation of p27 and p21, which is mediated by the interaction between SGK1 and cAMP responsive element-binding protein (CREB). Moreover, we overexpressed ClC-3 Cl− channel to further verify the role of SGK1 in low Cl− environment-induced proliferation. The results revealed that overexpression of ClC-3 further enhanced hypotonic solution-induced cell viability, cell cycle transition, and CREB activation, which were alleviated or potentiated by silencing or overexpression of SGK1. In summary, this study provides compelling evidences that SGK1, as a Cl−-sensitive kinase, is a critical link between low osmotic pressure and proliferation in BASMCs, and shed a new light on the treatment of proliferation-associated cardiovascular diseases.

Neuronal CXCL10/CXCR3 Axis Mediates the Induction of Cerebral Hyperexcitability by Peripheral Viral Challenge.

Peripheral infections can potently exacerbate neuropathological conditions, though the underlying mechanisms are poorly understood. We have previously demonstrated that intraperitoneal (i.p.) injection of a viral mimetic, polyinosinic-polycytidylic acid (PIC) induces a robust generation of CXCL10 chemokine in the hippocampus. The hippocampus also features hyperexcitability of neuronal circuits following PIC challenge. The present study was undertaken to determine the role of CXCL10 in mediating the development of hyperexcitability in response to PIC challenge. Briefly, young female C57BL/6 mice were i.p. injected with PIC, and after 24 h, the brains were analyzed by confocal microscopy. CXCL10 staining of neuronal perikarya and a less intense staining of the neuropil was observed in the hippocampus and cortex. CXCL10 staining was also evident in a subpopulation of astrocytes, whereas microglia were CXCL10 negative. CXCR3, the cognate receptor of CXCL10 was present exclusively on neurons, indicating that the CXCL10/CXCR3 axis operates through an autocrine/paracrine neuronal signaling. Blocking cerebral CXCR3 through intracerebroventricular injection of a specific inhibitor, AMG487, abrogated PIC challenge-induced increase in basal synaptic transmission and long-term potentiation (LTP), as well as the reduction of paired-pulse facilitation (PPF), in the hippocampus. The PIC-mediated abolishment of hippocampal long-term depression (LTD) was also restored after administration of AMG487. Moreover, CXCR3 inhibition attenuated seizure hypersensitivity induced by PIC challenge. The efficacy of AMG487 strongly strengthens the notion that CXCL10/CXCR3 axis mediates the induction of cerebral hyperexcitability by PIC challenge.

In vivo and in vitro protective effect of arginine against intestinal inflammatory response induced by Clostridium perfringens in broiler chickens

BackgroundNecrotic enteritis is a widespread disease in poultry caused by Clostridium perfringens. We previously reported that dietary arginine supplementation protected the intestinal mucosa of broiler chickens with necrotic enteritis, but the related protective mechanisms remain unclear. The in vivo trial was designed as a 2 × 2 factorial arrangement to evaluated the effects of arginine supplementation on inflammatory responses, arginine transporters, arginine catabolism and JAK-STAT signalling pathway in broiler chickens challenged with C. perfringens or without C. perfringens. Furthermore, we validated the in vivo results using intestinal epithelial cells of chicken embryos.ResultsC. perfringens infection markedly increased gut gross pathological and histopathological lesion scores, promoted liver C. perfringens invasion, reduced serum arginine levels, and elevated jejunal mucosal lysozyme activities (P < 0.05), but these effects were significantly reversed by arginine supplementation in vivo (P < 0.05). The challenge significantly increased serum procalcitonin levels, jejunal mucosal iNOS activities and jejunal IL-6, TGF-β3, cationic amino acid transporter (CAT)-1, and CAT-3 mRNA expression (P < 0.05), whereas arginine supplementation significantly reduced jejunal IFN-γ, IL-1β, IL-6, IL-10, TGF-β3, and CAT-3 mRNA expression (P < 0.05). Arginine supplementation significantly attenuated the C. perfringens challenge-induced increases in jejunal iNOS, arginase 2, arginine decarboxylase, arginine:glycine amidinotransferase, JAK1, JAK3, STAT1, and STAT6 mRNA expression (P < 0.05). The in vitro experiment showed that C. perfringens challenge markedly increased cellular cytotoxicity and the mRNA expression of IL-1β, IL-8, IL-10, CAT-1 and CAT-3 (P < 0.05), which were significantly reversed by 50 μmol/L and/or 400 μmol/L arginine pre-treatment (P < 0.05).ConclusionsArginine prevented C. perfringens challenge-induced circulated arginine deficiency, normalized intestinal arginine transport and catabolism, down-regulated JAK-STAT signalling pathway and attenuated the inflammatory response, which exerted protective effects on the intestine of broiler chickens.

Dietary leonurine hydrochloride supplementation attenuates lipopolysaccharide challenge-induced intestinal inflammation and barrier dysfunction by inhibiting the NF-κB/MAPK signaling pathway in broilers.

This study was performed to evaluate the beneficial effects of dietary leonurine hydrochloride (LH) supplementation on intestinal morphology and barrier integrity and further illuminate its underlying antioxidant and immunomodulatory mechanisms in lipopolysaccharide (LPS)-treated broilers. A total of 120 1-d-old male broilers (Ross 308) were assigned to 4 treatment groups with 6 replicates of 5 birds per cage. The experiment was designed in a 2 × 2 factorial arrangement with LH (0 or 120 mg/kg) and LPS (injection of saline or 1.5 mg/kg body weight) as treatments. On days 14, 16, 18, and 20 of the trial, broilers were intraperitoneally injected with LPS or physiological saline. Compared with the control group, LPS-challenged broilers showed impaired growth performance (P < 0.05) from day 15 to day 21 of the trial, increased serum diamine oxidase (DAO) and D-lactic acid (D-LA) levels coupled with reduced glutathione (GSH) content and total superoxide dismutase (T-SOD) activity (duodenal and jejunal mucosa), reduced malondialdehyde (MDA) content (duodenal, jejunal, and ileal mucosa), and compromised morphological structure of the duodenum and jejunum. Additionally, LPS challenge increased (P < 0.05) the mRNA expression of proinflammatory cytokine genes and reduced tight junction (TJ) protein expression in the jejunum. However, dietary LH prevented LPS-induced reductions in average daily gain (ADG) and average daily feed intake (ADFI) in broilers. It also alleviated LPS challenge-induced increases in serum DAO levels, MDA content (duodenal and jejunal mucosa), and jejunal crypt depth (P < 0.05) but reduced villus height, GSH content (jejunal mucosa), and T-SOD activity (duodenal and jejunal mucosa) (P < 0.05). Additionally, LH supplementation significantly downregulated the mRNA expression of nuclear factor (NF)-κB, cyclooxygenase-2 (COX-2), and proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and upregulated the mRNA expression of zonula occludens-1 (ZO-1) and Occludin in the jejunal mucosa induced by LPS (P < 0.05). On the other hand, LH administration prevented LPS-induced activation of the p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) and attenuated IkB alpha (IκBα) phosphorylation and nuclear translocation of NF-κB (p65) in the jejunal mucosa. In conclusion, dietary LH supplementation attenuates intestinal mucosal disruption mainly by accelerating the expression of TJ proteins and inhibiting activation of the NF-κB/MAPK signaling pathway.

Increased expression of CysLT2 receptors in the lung of asthmatic mice and role in allergic responses

Compared with CysLT1 receptors, the functional role of CysLT2 receptors in asthma has not been clarified. The purpose of this study was to determine 1) whether CysLT2 receptors are expressed in the lung of mice and if expression increases in asthmatic mice, and 2) whether CysLT2 receptors are involved in allergic leukocyte infiltration into the lung and in the development of airway remodeling in asthmatic mice. BALB/c mice were sensitized with ovalbumin (OVA) + Al(OH)3, and intratracheally challenged with OVA 4 times. Lung tissue was isolated before and after the 4th OVA challenge for detection of CysLT2 receptors by immunohistochemistry and flow cytometry. The effect of a CysLT2 receptor antagonist BayCysLT2RA on multiple antigen challenge-induced leukocyte infiltration into the lung and the development of airway remodeling was evaluated. Even in non-challenged mice, CysLT2 receptors were expressed in bronchial smooth muscle. After multiple challenges, expression was also observed in leukocytes infiltrating into alveolar spaces. CysLT2R+ leukocytes included alveolar macrophages, conventional dendritic cells, and eosinophils. BayCysLT2RA significantly inhibited multiple antigen challenge-induced increases in eosinophils and mononuclear cells in the lung. The development of airway remodeling was tended to be suppressed by CysLT2 receptor antagonist. In conclusion, CysLT2 receptors were constitutively expressed in the lung, and expression was strengthened in asthmatic mice. Activation of CysLT2 receptors was functionally involved in allergic leukocyte infiltration into the lung. The CysLT2 receptor can be a molecular target for the development of new pharmacotherapies for asthma.

Behavioral changes after nicotine challenge are associated with \u03b17 nicotinic acetylcholine receptor-stimulated glutamate release in the rat dorsal striatum

Neurochemical alterations associated with behavioral responses induced by re-exposure to nicotine have not been sufficiently characterized in the dorsal striatum. Herein, we report on changes in glutamate concentrations in the rat dorsal striatum associated with behavioral alterations after nicotine challenge. Nicotine challenge (0.4 mg/kg/day, subcutaneous) significantly increased extracellular glutamate concentrations up to the level observed with repeated nicotine administration. This increase occurred in parallel with an increase in behavioral changes in locomotor and rearing activities. In contrast, acute nicotine administration and nicotine withdrawal on days 1 and 6 did not alter glutamate levels or behavioral changes. Blockade of α7 nicotinic acetylcholine receptors (nAChRs) significantly decreased the nicotine challenge-induced increases in extracellular glutamate concentrations and locomotor and rearing activities. These findings suggest that behavioral changes in locomotor and rearing activities after re-exposure to nicotine are closely associated with hyperactivation of the glutamate response by stimulating α7 nAChRs in the rat dorsal striatum.

Improved Efficacy of Oral Immunotherapy Using Non-Digestible Oligosaccharides in a Murine Cow's Milk Allergy Model: A Potential Role for Foxp3+ Regulatory T Cells.

Oral immunotherapy (OIT) is a promising therapeutic approach to treat food allergic patients. However, there are some concerns regarding its safety and long-term efficacy. The use of non-digestible oligosaccharides might improve OIT efficacy since they are known to directly modulate intestinal epithelial and immune cells in addition to acting as prebiotics. To investigate whether a diet supplemented with plant-derived fructo-oligosaccharides (FOS) supports the efficacy of OIT in a murine cow's milk allergy model and to elucidate the potential mechanisms involved. After oral sensitization to the cow's milk protein whey, female C3H/HeOuJ mice were fed either a control diet or a diet supplemented with FOS (1% w/w) and received OIT (10 mg whey) 5 days a week for 3 weeks by gavage. Intradermal (i.d.) and intragastric (i.g.) challenges were performed to measure acute allergic symptoms and mast cell degranulation. Blood and organs were collected to measure antibody levels and T cell and dendritic cell populations. Spleen-derived T cell fractions (whole spleen- and CD25-depleted) were transferred to naïve recipient mice to confirm the involvement of regulatory T cells (Tregs) in allergy protection induced by OIT + FOS. OIT + FOS decreased acute allergic symptoms and mast cell degranulation upon challenge and prevented the challenge-induced increase in whey-specific IgE as observed in sensitized mice. Early induction of Tregs in the mesenteric lymph nodes (MLN) of OIT + FOS mice coincided with reduced T cell responsiveness in splenocyte cultures. CD25 depletion in OIT + FOS-derived splenocyte suspensions prior to transfer abolished protection against signs of anaphylaxis in recipients. OIT + FOS increased serum galectin-9 levels. No differences in short-chain fatty acid (SCFA) levels in the cecum were observed between the treatment groups. Concisely, FOS supplementation significantly improved OIT in the acute allergic skin response, %Foxp3+ Tregs and %LAP+ Th3 cells in MLN, and serum galectin-9 levels. FOS supplementation improved the efficacy of OIT in cow's milk allergic mice. Increased levels of Tregs in the MLN and abolished protection against signs of anaphylaxis upon transfer of CD25-depleted cell fractions, suggest a role for Foxp3+ Tregs in the protective effect of OIT + FOS.

Exposure to alcohol during adolescence exerts long-term effects on stress response and the adult brain stress circuits

The hypothalamic–pituitary–adrenal (HPA) axis undergoes critical developments during adolescence. Therefore, stressors experienced during this period potentially have long-term effects on adult HPA axis function. We hypothesized that adolescent intermittent ethanol (AIE) exposure would affect adult HPA axis function, resulting in altered responses to an alcohol challenge in young adults or adults. To test these hypotheses, male rats were exposed to alcohol vapor for 6h per day from post-natal day (PND) 28–42, then acutely challenged with alcohol intragastrically (3.2–4.5g/kg) in young adults (PND 70) or adults (PND 90). Overall, we observed blunted HPA axis responses to an alcohol challenge due to AIE exposure. Specifically, AIE tended to inhibit the alcohol challenge-induced increase in plasma corticosterone (CORT) concentrations in young adult and adult rats. As well, AIE significantly blunted the alcohol challenge-induced arginine vasopressin (Avp) mRNA expression in the paraventricular nucleus (PVN) of the hypothalamus of adult rats. Results of the present study are similar to what we have previously shown, that these changes in PVN responsiveness may result from AIE-induced alterations in adrenergic neurons in brain stem regions C1–C3 known to project to the PVN. AIE elevated the number of colocalized c-fos/phenylethanolamine N-methyltransferase (PNMT)-positive cell bodies in the C1 region of adult rats. Together, these data suggest that AIE exposure produces alterations in male HPA axis responsiveness to administration of an acute alcohol challenge that may be long-lasting.

Cocaine challenge increases the expression of immunoglobulin heavy chain binding protein in the rat nucleus accumbens

Repeated cocaine administration increases the endoplasmic reticulum (ER) stress response in the dorsal striatum. This study was performed to investigate that cocaine challenge after cocaine abstinence is associated with the ER stress response in the nucleus accumbens (NAc). The results demonstrated that a renewed cocaine challenge (20mg/kg) after six days of withdrawal following seven daily systemic injections of cocaine (20mg/kg), significantly increased the expression of the ER stress response protein, immunoglobulin heavy chain binding protein (BiP). The elevation of BiP expression was decreased by blocking N-methyl-d-aspartate (NMDA) receptors and ryanodine-sensitive Ca2+ channels. The inhibition of c-Jun N-terminal kinase (JNK) also attenuated the renewed cocaine challenge-induced increase in BiP expression. These findings suggest that JNK phosphorylation, via stimulation of NMDA receptors and ryanodine-sensitive Ca2+ channels evoked by cocaine challenge after cocaine abstinence, is necessary for the induction of the ER stress response in the NAc.

Roles of 5-lipoxygenase and cysteinyl-leukotriene type 1 receptors in the hematological response to allergen challenge and its prevention by diethylcarbamazine in a murine model of asthma.

Diethylcarbamazine (DEC), which blocks leukotriene production, abolishes the challenge-induced increase in eosinopoiesis in bone-marrow from ovalbumin- (OVA-) sensitized mice, suggesting that 5-lipoxygenase (5-LO) products contribute to the hematological responses in experimental asthma models. We explored the relationship between 5-LO, central and peripheral eosinophilia, and effectiveness of DEC, using PAS or BALB/c mice and 5-LO-deficient mutants. We quantified eosinophil numbers in freshly harvested or cultured bone-marrow, peritoneal lavage fluid, and spleen, with or without administration of leukotriene generation inhibitors (DEC and MK886) and cisteinyl-leukotriene type I receptor antagonist (montelukast). The increase in eosinophil numbers in bone-marrow, observed in sensitized/challenged wild-type mice, was abolished by MK886 and DEC pretreatment. In ALOX mutants, by contrast, there was no increase in bone-marrow eosinophil counts, nor in eosinophil production in culture, in response to sensitization/challenge. In sensitized/challenged ALOX mice, challenge-induced migration of eosinophils to the peritoneal cavity was significantly reduced relative to the wild-type PAS controls. DEC was ineffective in ALOX mice, as expected from a mechanism of action dependent on 5-LO. In BALB/c mice, challenge significantly increased spleen eosinophil numbers and DEC treatment prevented this increase. Overall, 5-LO appears as indispensable to the systemic hematological response to allergen challenge, as well as to the effectiveness of DEC.

Essential roles of endogenous glucocorticoids and TNF/TNFR1 in promoting bone-marrow eosinopoiesis in ovalbumin-sensitized, airway-challenged mice

AimsStress mechanisms paradoxically contribute to allergic episodes in humans and mice. Glucocorticoids (GC) and interleukin (IL)-5 synergically upregulate murine bone-marrow eosinophil production. Here we explored the role of endogenous GC in allergen-stimulated bone-marrow eosinophil production in ovalbumin-sensitized/challenged mice. Main methodsIn BALB/c or C57BL/6 mice, sensitized and intranasally challenged with ovalbumin, we monitored eosinophil numbers in freshly harvested or cultured bone-marrow, and plasma corticosterone levels. Metyrapone (MET) was used to inhibit GC synthesis, and RU486 to block GC actions. In sensitized mice challenged intraperitoneally, we examined the relationship between eosinophilia of bone-marrow and peritoneal cavity, in the absence or presence of RU486. In experiments involving in vivo neutralization of tumor necrosis factor-α (TNF) by specific antibodies, or using mice which lack functional type I TNF receptors (TNFRI), we evaluated the relationship between TNF blockade, corticosterone levels, RU486 or MET treatment and challenge-induced bone-marrow eosinophilia. Key findingsRU486 or MET pretreatments abolished challenge-induced increases in eosinophil numbers in bone-marrow (in vivo and ex vivo), and in the peritoneal cavity. MET, but not RU486, prevented the challenge-induced increase in corticosterone levels. Challenge-induced bone-marrow eosinophilia and corticosterone surge were abolished in TNFRI-deficient mice. Anti-TNF-treatment very effectively prevented challenge-induced bone-marrow eosinophilia, in the absence of RU486 or MET, but had no independent effect in the presence of either drug. SignificanceEndogenous GC was essential for allergen challenge-induced increases in eosinophil numbers inside bone-marrow. This effect required TNF and TNFRI, which suggests an immunoendocrine mechanism.

Sustained Suppression of IL-13 by a Vaccine Attenuates Airway Inflammation and Remodeling in Mice

We previously reported that a recombinant IL-13 peptide-based virus-like particle vaccine significantly suppressed murine acute airway allergic inflammatory responses. The impact of this strategy on the development of chronic airway inflammation and remodeling has not been investigated. We evaluated whether the vaccine-mediated sustained suppression of IL-13 attenuates features of chronic airway inflammation and remodeling in mice repeatedly challenged with allergen. BALB/c mice received two intraperitoneal sensitizing injections of ovalbumin (OVA) and alum, followed by six consecutive 2-day intranasal OVA challenges at 12-day intervals and then a 4-week recovery period. Anti-IL-13 antibodies were induced with a vaccine before (preventive experiments) or after (interventional experiments) the OVA challenge commenced. Respiratory mechanics were assessed using low-frequency forced oscillation with a small animal ventilator. Cytokine concentrations in bronchoalveolar lavage fluid (BALF) and lung histology were also assessed. In the preventive experiments, vaccination significantly suppressed IL-13 concentrations, the accumulation of inflammatory cells in BALF, lung mucus production, and collagen deposition. Furthermore, vaccination significantly attenuated OVA challenge-induced increases in airway resistance, tissue resistance, and tissue elastance, both acutely and after a 4-week recovery from allergen challenges. In the interventional experiments, vaccination decreased IL-13, TGF-β1, and IL-12p40 concentrations in BALF, as well as mucus production and collagen deposition. Chronic inflammation and sustained airway hyperresponsiveness were not significantly reversed. The persistent suppression of IL-13 with a vaccine inhibits chronic airway inflammation and the development of several key components of airway remodeling, and this intervention is more effective at early stages than later during chronic inflammation.

Preventive and therapeutic effects of rapamycin, a mammalian target of rapamycin inhibitor, on food allergy in mice

Because few curative treatments are available for food allergy, we investigated the therapeutic potential of rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, on mouse food allergy. The preventive and therapeutic effects of oral rapamycin on anaphylactic symptoms induced by oral ovalbumin (OVA) challenge in food allergy mice were investigated. Mast cell functions in response to rapamycin were also measured in the passive systemic anaphylaxis model and bone marrow-derived mast cells (BMMCs). Daily rapamycin from the first challenge (preventive protocol) attenuated food allergy symptoms including diarrhea, anaphylactic reactions, and hypothermia in mice. The treatment decreased the challenge-induced increases in mouse mast cell protease-1 in serum and mast cell numbers in the intestine. Notably, the mice that already showed food allergy symptoms by previous challenges recovered from the disease with daily administration of rapamycin (therapeutic protocol). Anti-OVA IgG1 and IgE levels in serum, as well as IFN-γ, IL-4, IL-13, IL-9, IL-10, and IL-17 secretion from splenocytes, were decreased by the treatments. In contrast, a single dose of rapamycin failed to affect passive systemic anaphylaxis. Spontaneous and IL-9-dependent survival and IgE-induced IL-13 secretion, but not degranulation, of BMMCs were reduced by rapamycin. Our data show that mouse food allergy was attenuated by rapamycin through an immunosuppressive effect and inhibition of intestinal mast cell hyperplasia. Inhibition of the IL-9 production-mast cell survival axis is one of the mechanisms of the therapeutic effect of rapamycin. Rapamycin and other mTOR inhibitors might be good candidates for therapeutic drugs for food allergy.

The Protective and Recovery Effects of Fish Oil Supplementation on Cedar Pollen-Induced Allergic Reactions in Mice

Obesity is associated with the production of pro-inflammatory cytokines and therefore can lead to worsening of allergic reactions. Thus, there is cross-talk between obesity and allergic reactions. In this study, we investigated whether the anti-obesity action of fish oil supplementation is involved in the anti-allergic action also induced by fish oil. We observed attenuation of cedar pollen-induced IgE serum increases in two experimental protocols: 15% fish oil supplementation for 8 weeks, which attenuated body weight increases compared with 15% soybean supplementation, and 4% fish oil supplementation for 2 weeks, which did not affect body weight increases compared with 4% soybean or 4% lard supplementation. The former but not the latter protocol attenuated sneezing after pollen challenge. Gene expression of TNF-α and IL-13, a Th2 cytokine, was moderately reduced in the trachea and nasal mucosa in mice fed fish oil supple- ments. In a third protocol, fish oil was administered for up to 15 weeks after a cedar pollen sensitization and challenge-induced increase in IgE levels. These levels decreased following fish oil supplementation but were almost unaffected by soybean oil supplementation. Surprisingly, the IL-13 gene expression level in the nasal mucosa and trachea was higher in the fish oil group than in the soybean oil group. These results suggest that fish oil supplementation before, during, or after antigen sensitization and challenge in mice helped to reduce cedar pollen-induced allergic reactions in- dependent of its anti-obesity action. Fish oil supplementation can also exhibit pro- or anti-inflammatory action against IL-13 expression depending on the experimental protocol.

Chronic methamphetamine exposure suppresses the striatal expression of members of multiple families of immediate early genes (IEGs) in the rat: normalization by an acute methamphetamine injection

Repeated injections of cocaine cause blunted responses to acute cocaine challenge-induced increases in the expression of immediate early genes (IEGs). The aim of this study was to test if chronic methamphetamine (METH) exposure might cause similar blunting of acute METH-induced increases in IEG expression. Repeated saline or METH injections were given to rats over 14 days. After 1 day of withdrawal, they received a single injection of saline or METH (5 mg/kg). Acute injection of METH increased c-fos, fosB, fra2, junB, Egr1-3, Nr4a1 (Nur77), and Nr4a3 (Nor-1) mRNA levels in the striatum of saline-pretreated rats. Chronic METH treatment alone reduced the expression of AP1, Erg1-3, and Nr4a1 transcription factors below control levels. Acute METH challenge normalized these values in METH-pretreated rats. Unexpectedly, acute METH challenge to METH-pretreated animals caused further decreases in Nr4a2 (Nurr1) mRNA levels. In contrast, the METH challenge caused significant but blunted increases in Nr4a3 and Arc expression in METH-pretreated rats. There were also chronic METH-associated decreases in the expression of cAMP responsive element binding protein (CREB) which modulates IEG expression via activation of the cAMP/PKA/CREB signal transduction pathway. Chronic METH exposure also caused significant decreases in preprotachykinin, but not in prodynorphin, mRNA levels. These results support the accumulated evidence that chronic administration of psychostimulants is associated with blunting of their acute stimulatory effects on IEG expression. The METH-induced renormalization of the expression of several IEGs in rats chronically exposed to METH hints to a potential molecular explanation for the recurrent self-administration of the drug by human addicts.

Availability of serum corticosterone level for quantitative evaluation of morphine withdrawal in mice

Physical dependence on morphine is evidenced by the withdrawal syndromes, including body weight loss, which are induced by the discontinuation of morphine exposure or by the treatment with naloxone, an opioid receptor antagonist. The present study was designed to examine whether the elevation of serum corticosterone (SCS) level induced by naloxone-precipitated morphine withdrawal was a useful index to quantify the physical dependence on morphine in mice, which was compared with body weight loss induced by naloxone-precipitated morphine withdrawal. The SCS level was dependent on the dosage and the number of dosing of morphine and challenging dosage of naloxone. Intraplantar injection of formalin, potentially producing inflammatory pain, inhibited both body weight loss and SCS increase induced by naloxone challenge in mice receiving repeated exposure of morphine, indicating that formalin-induced pain attenuated the development of physical dependence on morphine. The magnitude of body weight loss in morphine withdrawal was significantly correlated with the magnitude of naloxone challenge-induced SCS increase. These results suggest that the naloxoneinduced increase in SCS level is a quantitative index of the magnitude of physical dependence on morphine in mice.

Ritanserin reverses repeated methamphetamine‐induced behavioral and neurochemical sensitization in mice

Chronic administration of methamphetamine (METH) elicits progressive enhancement of locomotor activity known as behavioral sensitization. We have recently shown that chronic METH enhanced METH challenge-induced increase in 5-HT levels in the prefrontal cortex and that 5-HT(1A) receptor activation attenuated this neurochemical sensitization as well as behavioral sensitization. This study examined whether the nonselective 5-HT(2) receptor antagonist, ritanserin affects METH-induced behavioral and neurochemical sensitization in mice. Ritanserin at doses of 1 and 3 mg/kg inhibited the development and expression of METH-induced behavioral sensitization in a dose-dependent manner. Furthermore, chronic administration of ritanserin for a week attenuated the maintenance of behavioral sensitization, indicating the improvement of established behavioral sensitization. Microdialysis analysis showed that chronic ritanserin inhibited the neurochemical sensitization that chronic METH enhanced METH challenge-induced increase in extracellular 5-HT levels in the prefrontal cortex. Furthermore, acute ritanserin inhibited METH challenge-induced increase in extracellular 5-HT but not DA levels in the prefrontal cortex. These results suggest that 5-HT(2) receptors are involved in METH-induced hyperactivity and behavioral sensitization in mice.

A threshold neurotoxic amphetamine exposure inhibits parietal cortex expression of synaptic plasticity-related genes

Compulsive drug abuse has been conceptualized as a behavioral state where behavioral stimuli override normal decision making. Clinical studies of methamphetamine users have detailed decision making changes and imaging studies have found altered metabolism and activation in the parietal cortex. To examine the molecular effects of amphetamine (AMPH) on the parietal cortex, gene expression responses to amphetamine challenge (7.5 mg/kg) were examined in the parietal cortex of rats pretreated for nine days with either saline, non-neurotoxic amphetamine, or neurotoxic AMPH dosing regimens. The neurotoxic AMPH exposure [three doses of 7.5 mg/kg/day AMPH (6 h between doses), for nine days] produced histological signs of neurotoxicity in the parietal cortex while a non-neurotoxic dosing regimen (2.0 mg/kg/day×3) did not. Neurotoxic AMPH pretreatment resulted in significantly diminished AMPH challenge-induced mRNA increases of activity-regulated cytoskeletal protein (ARC), nerve growth-factor inducible protein A (NGFI-A), and nerve growth-factor inducible protein B (NGFI-B) in the parietal cortex while neither saline pretreatment nor non-neurotoxic AMPH pretreatment did. This effect was specific to these genes as tissue plasminogen activator (t-PA), neuropeptide Y (NPY) and c-jun expression in response to AMPH challenge was unaltered or enhanced by amphetamine pretreatments. In the striatum, there were no differences between saline, neurotoxic AMPH, and non-neurotoxic AMPH pretreatments on ARC, NGFI-A or NGFI-B expression elicited by the AMPH challenge. These data indicate that the responsiveness of synaptic plasticity-related genes is sensitive to disruption specifically in the parietal cortex by threshold neurotoxic AMPH exposures.

Transgenic smooth muscle expression of the human CysLT1 receptor induces enhanced responsiveness of murine airways to leukotriene D4.

Cysteinyl leukotrienes (CysLTs) exert potent proinflammatory actions and contribute to many of the symptoms of asthma. Using a model of allergic sensitization and airway challenge with Aspergillus fumigatus (Af), we have found that Th2-type inflammation and airway hyperresponsiveness (AHR) to methacholine (MCh) were associated with increased LTD(4) responsiveness in mice. To explore the importance of increased CysLT signaling in airway smooth muscle function, we generated transgenic mice that overexpress the human CysLT1 receptor (hCysLT(1)R) via the alpha-actin promoter. These receptors were expressed abundantly and induced intracellular calcium mobilization in airway smooth muscle cells from transgenic mice. Force generation in tracheal ring preparations ex vivo and airway reactivity in vivo in response to LTD(4) were greatly amplified in hCysLT(1)R-overexpressing mice, indicating that the enhanced signaling induces coordinated functional changes of the intact airway smooth muscle. The increase of AHR imposed by overexpression of the hCysLT(1)R was greater in transgenic BALB/c mice than in transgenic B6 x SJL mice. In addition, sensitization- and challenge-induced increases in airway responsiveness were significantly greater in transgenic mice than that of nontransgenic mice compared with their respective nonsensitized controls. The amplified AHR in sensitized transgenic mice was not due to an enhanced airway inflammation and was not associated with similar enhancement in MCh responsiveness. These results indicate that a selective hCysLT(1)R-induced contractile mechanism synergizes with allergic AHR. We speculate that hCysLT(1)R signaling contributes to a hypercontractile state of the airway smooth muscle.

Suplatast tosilate inhibits late response and airway inflammation in sensitized guinea pigs.

The effect of suplatast tosilate, which has been proven to inhibit T-cell synthesis of IL-4 and IL-5, on the response to antigen inhalation challenge was investigated in sensitized guinea pigs. The animals were given an oral dose of 30 or 100 mg/kg of suplatast or vehicle (distilled water) daily for 1 wk before antigen challenge. Measurement of pulmonary resistance for 6 h was followed by bronchoalveolar lavage and lung fixation. After antigen challenge, all guinea pigs in the vehicle group displayed dual-phase airway obstruction and accumulation of eosinophils and lymphocytes in the airways. After 1 wk of treatment with the high dose of suplatast, the late asthmatic response and the recruitment of eosinophils and lymphocytes into the airways were significantly inhibited, but the early asthmatic response was not affected. In situ hybridization revealed that challenge-induced increases in IL-5 mRNA-positive cells in lung tissue were significantly inhibited after treatment. Thus, suplatast inhibited airway obstruction in the late phase by specifically inhibiting the inflammatory process after mast cell degranulation.