OVA-challenged Rats Research Articles

Jinkui Shenqi Pills Ameliorate Asthma with "Kidney Yang Deficiency" by Enhancing the Function of the Hypothalamic-Pituitary-Adrenal Axis to Regulate T Helper 1/2 Imbalance.

The aim of the study was to investigate the effects and underlying mechanism of JKSQP in a rat model of asthma with kidney-yang deficiency (KYD). Materials and Methods. Hydrocortisone (HYD) was used to establish the rat model of KYD; rats were then sensitized and challenged with ovalbumin (OVA). JKSQP was administered to OVA-challenged rats, and the changes in signs and symptoms of KYD were observed. The leukocyte number and subpopulations in bronchoalveolar lavage fluid (BALF) were counted and the cells were stained with Wright–Giemsa dye. Serum adrenocorticotropic hormone (ACTH), corticosterone (CORT), corticotropin-releasing hormone (CRH), total immunoglobulin E (IgE), and OVA-specific IgE levels were determined using relevant enzyme-linked immunosorbent assays (ELISA) kits. Results. JKSQP not only reversed the phenomenon of KYD but also significantly inhibited the number of leukocyte and eosinophils in the BALF, increasing the level of interferon (IFN)-γ and decreasing the levels of interleukin-4 (IL-4) and IgE in the serum compared with the OVA-challenged groups. Conclusions. Taken together, the antiasthma effects of JKSQP were likely mediated by the enhancement of the function of the hypothalamic-pituitary-adrenal axis and the reversal of T helper 1/2 imbalance.

Maternal exposure to environmental DEHP exacerbated OVA-induced asthmatic responses in rat offspring

Di (ethylhexyl) phthalate (DEHP) is a commonly used phthalates (PAEs) compound as plasticizer and becomes a severe environmental pollutant worldwide. Studies show that DEHP, as an environmental endocrine disruptor, has potential adverse effects on human. Epidemiologic studies indicate that DEHP is positively correlated to allergic diseases. Maternal exposure to DEHP may contribute to the increasing incidence of allergic diseases in offspring. However, the role of DEHP and its detailed mechanism in allergic disease of the offspring are still unclear. The aim of our study is to investigate whether DEHP maternal exposure could aggravate the allergic responses in offspring and its mechanism. Pregnant Wistar rats were randomly divided into three groups and exposed to different doses of DEHP. Half of the offspring were challenged with OVA after birth. All the pups of each group were sacrificed at postnatal day (PND)14, PND21 and PND28. The number of inflammatory cells in bronchoalveolar lavage was counted, lung pathological changes were observed, Th2 type cytokines expressions were checked, and the expression of TSLP signaling pathway were examined. Our results showed that maternal exposure to DEHP during pregnancy and lactation aggravated the eosinophils accumulation and the pathological inflammatory changes in pups' lung after OVA challenge. And maternal exposure to DEHP during pregnancy and lactation also elevated the levels of typical Th2 cytokines in OVA-challenged rats. What's more, maternal exposure to DEHP during pregnancy and lactation increased the levels of TSLP, TSLPR and IL-7R in the offspring after OVA challenge. Our study suggested that DEHP maternal exposure could aggravate the OVA-induced asthmatic responses in offspring. And this adjuvant effect of DEHP was related with the TSLP/TSLPR/IL-7R and its downstream signal pathways.

Inhibitory effects of a standardized extract of Justicia pectoralis in an experimental rat model of airway hyper-responsiveness

Justicia pectoralis is a plant useful for the treatment of respiratory diseases. Here, we studied the antiasthmatic properties of a standardized extract of J. pectoralis (Jp). Ovalbumin (OVA)-sensitized rats were actively challenged with saline or OVA to study airway hyper-responsiveness after oral treatment with saline or Jp. The ability of Jp to inhibit hyper-reactivity was evaluated in isolated trachea mounted in isolated organ bath chamber. Using KCl or carbachol as contractile agents, tracheal rings of OVA-challenged rats contracted with higher magnitude than trachea of rats challenged with saline. Such hyper-responsive phenotype of OVA-challenged tissues decreased with Jp administration. In Ca+ -free medium, Jp or its major constituent coumarin inhibited preferentially the contractions induced by Ca2+ addition in tissues of OVA-challenged rats stimulated with KCl or acetylcholine. In tissues depleted of their internal Ca+ stores in the presence of thapsigargin, Jp inhibited the contraction induced by capacitative Ca2+ entry. By gavage, Jp abolished the increase caused by challenge with OVA on the levels of IL-1β and TNF-α in the bronchoalveolar fluid and also impaired the changes in gene expression of canonical transient receptor proteins. Jp has antiasthmatic properties in an experimental model that reproduces tracheal hyper-reactivity.

Selaginella uncinata flavonoids ameliorated ovalbumin-induced airway inflammation in a rat model of asthma

Ethnopharmacological relevanceSelaginella uncinata (Desv.) Spring, known as “Cuiyuncao”, is a perennial herb widely distributed in the Southeast Asian countries. In the folk medicine, the local minority commonly use it to treat cough and asthma for centuries. Aim of the studyThis study was carried out to investigate the protective mechanisms of total flavonoids from S. uncinata (SUF) on airway hyperresponsiveness, cytokine release and bitter taste receptors (T2Rs) signaling with emphasis on inflammatory responses in a rat model of ovalbumin (OVA)-induced asthma. Materials and methodsRats were sensitized and challenged with OVA to induce typical asthmatic reactions. Pathological changes of lung tissue were examined by HE staining. The serum levels of T cell-associated cytokines (IFN-γ, IL-4, IL-5 and IL-13), total IgE and OVA-specific IgE were determined by enzyme-linked immunosorbent assay (ELISA). Gene expressions of T2R10, IP3R1 and Orai1 in lung tissue were assayed by fluorescence quantitative real-time polymerase chain reaction (FQ-PCR) while protein expressions of NFAT1 and c-Myc were assayed by western blot analysis. The activation of SUF was investigated on tansgentic T2R10-GFP HEK293 cells. ResultsSUF treatment attenuated airway hyperresponsiveness and goblet cell hyperplasia compared with OVA-challenged asthmatic rats. The serum levels of IL-4, IL-5 and IL-13 as well as total and OVA-specific IgE were decreased while serum IFN-γ was increased in SUF-treated rats. SUF treatment significantly up-regulated T2R10 gene expression, down-regulated IP3R1 and Orai1 gene expression. SUF further suppressed eotaxin, NFAT1 and c-Myc protein expression in lung tissues of OVA-challenged rats. ConclusionsThese results imply that SUF exerts anti-inflammatory function through the T2R10/IP3R1/NFAT1 dependent signaling pathway, and may warrant further evaluation as a possible agent for the treatment of asthma.

Phytochemical profiles and inhibitory effects of Tiger Milk mushroom (Lignosus rhinocerus) extract on ovalbumin-induced airway inflammation in a rodent model of asthma.

BackgroundLignosus rhinocerus (L. rhinocerus), which is known locally as Tiger Milk mushroom, is traditionally used in the treatment of asthma by indigenous communities in Malaysia. However, to date, its efficacy on asthma has not been confirmed by scientific studies and there is also sparse information available on its active constituents. In this study, the volatile constituent of L. rhinocerus hot water extract was investigated using gas chromatography mass spectrometry (GC-MS). The potential effects of L. rhinocerus extract for anti-asthmatic activity was further investigated on ovalbumin (OVA)-sensitized asthmatic Sprague Dawley rats.MethodsSequential extraction using five solvents (petroleum ether, diethyl ether, hexane, ethyl acetate and methanol) was conducted prior to GC-MS analysis. Male Sprague Dawley rats were divided into the following four groups of five animals each: 1) normal rats, 2) sensitization plus OVA-challenged rats 3) sensitization plus OVA-challenged with L. rhinocerus treatment and 4) sensitization plus OVA-challenged with dexamethasone treatment. The levels of immunoglobulin E (IgE) in the serum and T-helper 2 cytokines, including interleukin (IL)-4, IL-5 and IL-13, in bronchoalveolar lavage fluid (BALF), as well as eosinophil infiltration in the lungs, were investigated.ResultsGC-MS analysis revealed the presence of five main groups (alkane, fatty acids, benzene, phenol and dicarboxylic acid) with a total of 18 constituents. Linoleic acid (21.35 %), octadecane (11.82 %) and 2,3-dihydroxypropyl elaidate (10.47 %) were present in high amounts. The extract significantly ameliorated the increase in total IgE in serum and IL-4, IL-5 and IL-13 levels in BALF and also effectively suppressed eosinophils numbers in BALF while attenuating eosinophil infiltrations in the lungs.ConclusionL. rhinocerus hot water extract has the potential to be used as an alternative for the treatment of acute asthma.

Effects of dipeptidyl peptidase-4 inhibition in an animal model of experimental asthma: a matter of dose, route, and time

The CD26-associated enzymatic activity of dipeptidyl peptidase-4 (DPP4) as well as the recruitment of CD26+ T cells increase under allergic airway inflammation. Furthermore, genetic deficiency of CD26/DPP4 exerts protective effects in experimental asthma. Therefore, CD26/DPP4 might represent a novel therapeutic target in asthma. To study the effects of pharmacological inhibition of DPP4 on allergic airway inflammation the DPP4-inhibitor isoleucine thiazolidide was tested using different doses at different time points (at sensitization, immediately before and simultaneously with the allergen challenge, as well as continuously via drinking water), and different routes (intraperitoneal, oral, and by inhalation). Allergic-like airway inflammation was induced in Fischer 344 rats (Charles River) sensitized against ovalbumin (OVA) using OVA aerosols. Intraperitoneal application of the DPP4 inhibitor showed effects neither at sensitization nor at challenge, whereas a continuous application via drinking water using high doses of the inhibitor led to an aggravation of the histomorphological signs of airway inflammation. In contrast, aerosolization of the DPP4 inhibitor simultaneously with the allergen significantly reduced airway hyperresponsiveness and ameliorated histopathological signs compared to controls. In addition, this treatment resulted in increased mRNA levels of surfactant proteins, suggesting an involvement of DPP4 inhibitors in surfactant metabolism in OVA-challenged rats. Continuous systemic inhibition of DPP4 via the oral route aggravates allergic airway inflammation. In contrast, topical inhibition of DPP4 exerts potential protective effects, and further research in humans is needed.

Chronic Intermittent Hypoxia Induces Airflow Limitation in a Rodent Model of Allergen‐Induced Lower Airway Inflammation

RationaleObstructive sleep apnea (OSA) aggravates asthma, but the mechanisms are unclear. Chronic intermittent hypoxia (CIH) is one hallmark feature of OSA. We tested the effects of CIH on lower airways in an ovalbumin (OVA) rodent model.MethodsFour groups (n=5–6/group) of Brown‐Norway rats underwent CIH (10% FiO2, 30 episodes/h, 10h/day for 30 days) vs. normoxia (NORM) and OVA vs. vehicle (VEH). Lung physiology and bronchoalveolar lavage (BAL) WBC differentials were compared between groups, 2 days after last challenge.ResultsOn lung physiology: 1) CIH reduced the Forced Expiratory Volume in 0.1 second (FEV0.1) to Forced Vital Capacity (FVC) ratio (FEV0.1/FVC) (p=0.008) and peak expiratory flow (p=0.03); OVA did not affect these measures. 2) FVC was increased by CIH (p=0.0009), but reduced by OVA (p <0.0001). In the BAL: 1) CIH did not change total cell counts (p = 0.25); 2) CIH induced a 2.3‐fold reduction in % eosinophils (p = 0.001); 3) In OVA‐challenged rats, CIH increased neutrophil fraction among infiltrating granulocytes (p = 0.03, Figure).ConclusionsCIH induces airflow limitation even in the absence of inhaled allergen. CIH induces a more neutrophil‐predominant response to allergen. These findings highlight the potential of OSA to aggravate lung dysfunction in patients with pre‐existent airway inflammation.Supported by UW‐Madison Department of Medicine Pilot Funding.

Zinc Suppressed the Airway Inflammation in Asthmatic Rats: Effects of Zinc on Generation of Eotaxin, MCP-1, IL-8, IL-4, and IFN-γ

Airway epithelium is rich in labile zinc (Zn), which may have an important protective role in the airway epithelium. The aim of this study is to investigate the effects of Zn on the airway inflammation and the generation of eotaxin, monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-4 (IL-4), and interferon-γ (IFN-γ) in rat models of ovalbumin (OVA)-induced allergic airway inflammation. For this purpose, animal model of asthma was established by OVA challenge and zinc-deficient and zinc-supplemented diets were given. Thirty-two Sprague-Dawley rats were divided into four groups: zinc-deficient diet with OVA treatment group, zinc-supplemented diet with OVA treatment group, zinc-normal diet with OVA treatment group, and zinc-normal diet with saline treatment group. Twenty-four hours after asthma was induced, lung histomorphological changes, cells in bronchoalveolar lavage fluid (BALF), contents of eotaxin, MCP-1, and IL-8 in BALF, and the expression of IFN-γ and IL-4 mRNAs were observed. Compared with the group of zinc-normal diet with OVA challenge rats, the group of zinc-deficient rats had higher numbers of eosinophils, neutrophils, and monocytes in BALF, as well as higher contents of eotaxin and MCP-1 in BALF and lower expression of lung IFN-γ mRNA. Conversely, Zn supplementation would decrease the numbers of eosinophils, neutrophils, and monocytes in BALF; suppress eotaxin and MCP-1 protein secretion; and increase lung IFN-γ mRNA expression. No significant difference was observed in IL-8 and IL-4 among OVA-challenged rats with different zinc diets. These studies suggested that Zn may be an important anti-inflammatory mediator of airway inflammation.

Treatment with a sphingosine-1-phosphate analog inhibits airway remodeling following repeated allergen exposure

Sphingosine-1-phosphate (S1P) is an immunomodulatory lipid mediator that plays an important role in lymphocyte trafficking. Elevated levels of S1P are found in bronchoalveolar lavage (BAL) fluid of patients with asthma; however, its role in disease is not known. FTY720, a synthetic analog of S1P, has been shown to abrogate allergic inflammation and airway hyperresponsiveness following acute allergen challenge. However, its effects on asthmatic airway remodeling induced by repeated allergen exposure are unknown. Ovalbumin (OVA)-sensitized rats were challenged on days 14, 19, and 24 after sensitization. FTY720 or vehicle (PBS) therapy was administered 1 h prior to each challenge. BAL fluid and quantitative histological analysis were performed 48 h after the last challenge. FTY720 inhibited OVA-induced features of airway remodeling including increased airway smooth muscle mass and bronchial neovascularization, without affecting lymphocyte numbers in secondary lymphoid organs. Furthermore, CD3+ cells adjacent to airway smooth muscle bundles were increased in OVA-challenged rats but the increase was inhibited by FTY720. There was an expansion of bronchus-associated lymphoid tissue following FTY720 treatment of OVA-challenged animals. Real-time quantitative PCR revealed that Th2-associated transcription factors were inhibited following FTY720 therapy. Airway remodeling is a cardinal feature of severe asthma. These results demonstrate that allergen-driven airway remodeling can be inhibited by FTY720, offering potential new therapies for the treatment of severe asthma.

Allergen-Induced Airway Remodeling in Brown Norway Rats

Increased proteoglycan (PG) deposition is a feature of airway remodeling in asthma. Glycosaminoglycans (GAGs) mediate many of the biological and mechanical properties of PGs by providing docking sites through their carbohydrate chains to bioactive ligands; therefore, it is imperative to define structural and metabolic changes of GAGs in asthma. Using a Brown Norway (BN) ovalbumin (OVA)-sensitized and -challenged rat model to induce airway remodeling, we found excessive deposition of chondroitin/dermatan (CS/DS)-, heparan (HS), and keratan (KS) sulfate GAGs in the airways and bronchoalveolar lavage cells of OVA-challenged rats. Disaccharide composition of CS/DS of OVA-challenged rats was significantly different compared with saline-treated (SAL) control rats, with increased levels of 0-, 6-, and 4-sulfated disaccharides. Increases in the amount and a change in the proportion of CS/DS versus HS GAGs were noted in OVA-challenged rats. The higher content and sulfation of CS/DS disaccharides was reflected by the increased expression of xylosyltransferase-I, β1,3-glucuronosyltransferase-I, chondroitin-4, and chondroitin-6 sulfotransferase genes and protein expression of xylosyltransferase-I and β1,3-glucuronosyltransferase-I in OVA-challenged rats. Genes encoding the core proteins of the CS/DS and KS-containing PGs, such as versican, biglycan, decorin, and lumican, were overexpressed in OVA-challenged rats. Our results suggest that GAG biosynthetic enzymes may be involved in the altered expression of GAGs in the airways and are potential targets for inhibiting excess PG-GAG deposition and the airway remodeling process in asthma.

A texture analysis approach to quantify ventilation changes in hyperpolarised 3He MRI of the rat lung in an asthma model

In preclinical research, allergic asthma is investigated in rats sensitised with the antigen ovalbumin (OVA), followed by a challenge with aerosolised OVA to induce an inflammatory reaction of the lower airways. This causes diffuse, nonfocal ventilation defects that lead to heterogeneously distributed signal intensities in hyperpolarised (HP) (3)He MR images, which are difficult to assess directly by diagnostic grading or volumetry. Texture analysis can characterise these changes and does not require segmentation of the lung structures prior to the analysis. The aim of this work was to evaluate a texture analysis approach to quantify changes in lung ventilation in HP (3)He MRI of OVA-challenged rats. OVA-challenged animals were treated with two different compound doses to evaluate the sensitivity of the texture analysis. Four groups were investigated using HP (3)He MRI at 4.7 T: controls, vehicle-treated, and low- and high-dose budesonide-treated rats. In addition, broncho-alveolar lavage was performed and the eosinophil cell count was used as a biological reference marker. First-order texture, geometrical features and features based on second-order statistics using run-length and grey-level co-occurrence matrices were calculated. In addition, wavelet transforms were applied to compute first-order statistics on multiple scales. The texture analysis was able to show significant differences between the control and untreated vehicle groups as well as between the vehicle and treatment groups. This is in agreement with the findings of the eosinophil cell counts, which were used as a marker for the severity of inflammation. However, not all features used in the different texture analysis methods could differentiate between the treatment groups. In conclusion, texture analysis can be used to quantify changes in lung ventilation as measured with HP (3)He MRI after therapeutic intervention with budesonide.

Enhancement of the pulmonary allergic granulocyte recruitment in rats exposed to DMTI-II, a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds

DMTI-II (23-kDa trypsin inhibitor purified from Dimorphandra mollis seeds) promotes acute inflammation accompanied by an early infiltration of eosinophils, a critical cell type involved in allergic diseases. We have evaluated here the capacity of DMTI-II to enhance the allergic pulmonary inflammation, looking over time to the leukocyte trafficking from bone marrow to peripheral blood, and their recruitment into the allergic airways. Male Wistar rats were sensitized and challenged with ovalbumin (OVA). At 2 to 16 h prior to OVA challenge, animals were exposed to DMTI-II (10 μg). Bronchoalveolar lavage fluid (BAL), circulating blood and bone marrow were examined at 24 h post-OVA challenge. Challenge with OVA significantly increased the influx of total inflammatory cells, neutrophils and eosinophils in BAL and lung tissue. Pre-exposure to DMTI-II potentiated total inflammatory cell and neutrophil recruitment ( p < 0.05). Neutropoiesis and neutrophilia accompanied pulmonary cell influx. Pre-exposure to DMTI-II also significantly increased eosinophil recruitment to BAL, an effect starting at 4 h, remaining markedly elevated at 16 h ( p < 0.05). Eosinopoiesis and eosinophilia (seen within 2 to 4 h) were also observed. Exposure to DMTI-II alone increased the IL-4 levels, and further increased the IL-4 levels in OVA-challenged rats. The levels of IgE, LTB 4 and eotaxin in OVA-challenged rats were greater compared with non-sensitized rats, but DMTI-II exposure failed to further enhance such levels. In summary, our study shows that DMTI-II itself presents granulocytopoietic activity, and enhances allergen-induced neutrophil and eosinophil mobilization from bone marrow to lung tissues that is accompanied by enhanced IL-4 production.

Differential effects of formaldehyde exposure on the cell influx and vascular permeability in a rat model of allergic lung inflammation

Exposure to air pollutants such as formaldehyde (FA) leads to inflammation, oxidative stress and immune-modulation in the airways and is associated with airway inflammatory disorders such as asthma. The purpose of our study was to investigate the effects of exposure to FA on the allergic lung inflammation. The hypothesized link between reactive oxygen species and the effects of FA was also studied. To do so, male Wistar rats were exposed to FA inhalation (1%, 90 min daily) for 3 days, and subsequently sensitized with ovalbumin (OVA)-alum by subcutaneous route. One week later the rats received another OVA-alum injection by the same route (booster). Two weeks later the rats were challenged with aerosolized OVA. The OVA challenge of rats upon FA exposure induced an elevated release of LTB 4, TXB 2, IL-1β, IL-6 and VEGF in lung cells, increased phagocytosis and lung vascular permeability, whereas the cell recruitment into lung was reduced. FA inhalation induced the oxidative burst and the nitration of proteins in the lung. Vitamins C, E and apocynin reduced the levels of LTB 4 in BAL-cultured cells of the FA and FA/OVA groups, but increased the cell influx into the lung of the FA/OVA rats. In OVA-challenged rats, the exposure to FA was associated to a reduced lung endothelial cells expression of intercellular cell adhesion molecule 1 (ICAM-1). In conclusion, our findings suggest that FA down regulate the cellular migration into the lungs after an allergic challenge and increase the ability of resident lung cells likely macrophages to generate inflammatory mediators, explaining the increased lung vascular permeability. Our data are indicative that the actions of FA involve mechanisms related to endothelium–leukocyte interactions and oxidative stress, as far as the deleterious effects of this air pollutant on airways are concerned.

Platelet adhesion and intracellular calcium levels in antigen-challenged rats

There is considerable evidence that platelet activation occurs in allergic airways diseases. In this study we aimed to investigate platelet adhesion to immobilized fibrinogen and intracellular calcium levels in a rat model of allergic inflammation. Male Wistar rats were challenged with ovalbumin (OVA). At 30 min to 24 h after OVA-challenge, assays of platelet adhesion to immobilized fibrinogen and intracellular calcium levels using fura 2-AM loaded platelets were performed. The serum levels of IgE were approximately 5-fold greater in OVA-sensitized rats. A marked eosinophil influx in bronchoalveolar lavage (BAL) fluid of OVA-challenged rats at 24 h after OVA-challenge was also seen. OVA-challenge resulted in a marked thrombocytopenia, as observed within 12 h after OVA-challenge. The agonists ADP (0.5–50 μM) and thrombin (30–100 mU/ml) concentration-dependently increased platelet adhesion to immobilized fibrinogen. At an early time after OVA-challenge (30 min), platelets exhibited greater platelet adhesion compared with the non-sensitized group, whereas at a late time (24 h) they exhibited lower platelet adhesion to both agonists. Moreover, at 30 min after OVA-challenge, intracellular calcium levels to ADP (20 μM) and thrombin (100 mU/ml)-activated platelets were greater compared with non-challenged rats. As opposed, at 24 h after OVA challenge, a lower intracellular calcium level to ADP- and thrombin-activated platelets was observed. In conclusion, OVA-challenge in rats promotes a biphasic response in platelet adhesion consisting of an increased adhesion and intracellular calcium levels at an early phase (30 min), which progress to a reduction in adhesion and intracellular calcium levels at a late time (24 h) after antigen challenge.

Pre-exposure to Staphylococcal enterotoxin A exacerbates the pulmonary allergic eosinophil recruitment in rats

Gram-positive Staphylococcus aureus releases classical enterotoxins which aggravates allergic airway diseases. However, little is known about the mechanisms underlying the cell influx exacerbation in asthmatic individuals under exposure to Staphylococcal enterotoxins. We therefore aimed to investigate the effects of airways exposure to Staphylococcal enterotoxin A (SEA) to pulmonary leukocyte recruitment in rats sensitized and challenged with ovalbumin (OVA). Rats were exposed to SEA at 4 h prior to OVA challenge or at 4 h post-OVA challenge. Bronchoalveolar lavage (BAL) fluid, bone marrow and lung tissue were obtained at 24 h after OVA challenge. Pre-exposure to SEA markedly enhanced the eosinophil counts in both BAL fluid and pulmonary tissue in OVA-challenged rats, whereas neutrophil and mononuclear cell counts remained unchanged. In bone marrow, pre-exposure to SEA alone significantly increased the number of eosinophils, and that was further increased in OVA-challenged rats. Exposure to SEA post-OVA challenge did not affect the number of eosinophils, neutrophils and mononuclear cells in BAL fluid. Pre-exposure to the endotoxin lipopolyssacharide (LPS) in OVA-challenged animals rather enhanced the neutrophil number in BAL fluid. In rats pre-exposed to SEA and OVA-challenged, a marked elevation in the levels of TNF-α and eotaxin (but not of IL-10) in BAL fluid was observed. The eotaxin levels increased by about of 3-fold in alveolar macrophages treated with SEA in vitro. In conclusion, airways pre-exposure to SEA causes a selective increase in eosinophil number in BAL fluid and bone marrow of OVA-challenged rats by mechanisms involving enhancement of TNF-α and eotaxin synthesis.

γ-Tocopherol Attenuates Ozone-induced Exacerbation of Allergic Rhinosinusitis in Rats

Compared to healthy subjects, individuals with allergic airway disease (e.g., asthma, allergic rhinitis) have enhanced inflammatory responses to inhaled ozone. We created a rodent model of ozone-enhanced allergic nasal responses in Brown Norway rats to test the therapeutic effects of the dietary supplement gamma-tocopherol (gammaT). Ovalbumin (OVA)-sensitized rats were intranasally challenged with 0% or 0.5% OVA (in saline) on Days 1 and 2, and then exposed to 0 or 1 ppm ozone (eight hours/day) on Days 4 and 5. Rats were also given 0 or 100 mg/kg gammaT (p.o., in corn oil) on days 2 through 5, beginning twelve hours after the last OVA challenge. On Day 6, nasal tissues were collected for histological evaluation and morphometric analyses of intraepithelial mucosubstances (IM) and eosinophilic inflammation. Nasal septal tissue was microdissected and analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) for mucin glycoprotein 5AC (MUC5AC) expression levels. Histological analysis revealed mild to moderate eosinophil influx in the mucosa lining the nasal airways and maxillary sinus of OVA-challenged rats (eosinophilic rhinosinusitis). Ozone exposure of allergic rats further increased eosinophils in the maxillary sinus (400%), nasolacrimal duct (250%), and proximal midseptum (150%). Storage of intraepithelial mucosubstances (IM) was not significantly affected by OVA challenge in filtered air-exposed rats, but it was increased by ozone in the septum (45%) and maxillary sinus (55%) of allergic compared to control rats. Treatment with gammaT attenuated the ozone/ OVA-induced synergistic increases in IM and mucosal eosinophils in both nasal and paranasal airways. gamma-Tocopherol also blocked OVA and ozone-induced MUC5AC gene expression. Together, these data describe a unique model of ozone enhancement of allergic rhinosinusitis and the novel therapeutic efficacy of a common supplement, gammaT, to inhibit ozone exacerbation of allergic airway responses.

Effect of valsartan on the expression of angiotensin II receptors in the lung of chronic antigen exposure rats

Many studies have suggested that angiotensin II (Ang II) and its receptors may be involved in the development of asthma. However, the expression of angiotensin II receptors (AGTR) is not clear in the lung tissue of chronic asthmatics. This study was designed to determine the relationship between airway remodeling, dysfunction and the expression of AGTRs in a rat model of asthma. Rats were sensitized with ovalbumin (OVA) for 2 weeks. Sixty minutes before an inhalation challenge, the rats were pretreated either with valsartan (15, 30, 50 mg x kg(-1) x d(-1)) or saline intragastrically. Then the rats received an OVA challenge for 30 alternative days. Acetylcholine (Ach)-induced bronchoconstriction was measured after the final antigen challenge. White cell counts in bronchoalveolar lavage fluid (BALF) and morphological changes in the airways were then assessed. The levels of transforming growth factor-beta 1 (TGF-beta(1)) and platelet-derived growth factor (PDGF) in BALF were detected by ELISA. The levels of AGTR1 and AGTR2 mRNA and protein in lung tissues were measured by RT-PCR and Western blotting. AGTR1 mRNA and protein levels in repeatedly OVA-challenged rats were significantly increased as compared with negative controls. The AGTR1 mRNA expression versus white cell counts of BALF and airway wall thickness (mainly in small airways) in lungs of chronic antigen-exposed rats were positively correlated. Valsartan decreased the level of AGTR1 in repeatedly OVA-challenged rats. However, AGTR2 mRNA and protein levels in the OVA-challenged rats and high-dose valsartan-treated rats (50 mg x kg(-1) x d(-1)) were also increased. Valsartan significantly decreased inflammatory cell accumulation and attenuated Ach-evoked bronchoconstriction in repeatedly antigen-challenged rats. Valsartan also decreased allergen-induced structural changes in rat airway (including total airway wall thickness and smooth muscle area) and the levels of TGF-beta(1) and PDGF in BALF. AGTR1 expression is potentially associated with airway remodeling and dysfunction in asthma. Ang II and AGTR1 may participate in airway inflammation and airway remodeling of chronic antigen-exposed rats. Valsartan, a AGTR1 antagonist, could inhibit AGTR1 expression and partially inhibits structural airway changes as well as airway inflammation in chronic OVA-exposed rats.

Increased responsiveness to 5‐hydroxytryptamine after antigenic challenge is inhibited by nifedipine and niflumic acid in rat trachea in vitro*

Antigenic challenge often induces hyperreactivity in asthmatic airway, although the precise mechanism(s) underlying this increased responsiveness is not entirely known. Tracheae obtained from ovalbumin (OVA)-sensitized saline- or OVA-challenged rats were placed in 10 mL bath chambers for isometric recording of 5-hydroxytryptamine (5-HT)-induced contractions. 5-Hydroxytryptamine induced a stronger contraction compared with control in antigen-challenged trachea under normal or Ca2+-free conditions. In tracheae pretreated with the L-type Ca2+ channel blocker nifedipine (10(-6) mol/L) or the Ca2+-activated Cl- channel blocker niflumic acid (10(-4) mol/L), this hyperresponsiveness was not developed in either normal or Ca2+-free medium. The increased contractile response to 5-HT in allergic rat isolated trachea may be related to a greater ionic (Ca2+ and Cl-) channel involvement.

Effects of Amphetamine on Immune-Mediated Lung Inflammatory Response in Rats

Objective: The present study analyzed the effects of acute amphetamine (AMPH) treatment on immune-mediated lung inflammatory response in rats. Methods: There were four experiments. In the first and second experiments, rats were treated with AMPH (1 mg/kg) or 0.9% NaCl, and locomotor activity (experiment 1) and serum AMPH concentrations (experiment 2) were measured 1 or 12 h after treatment. In the third experiment, rats which were immunized with ovalbumin (OVA) were treated 14 days later with 0.9% NaCl or AMPH (1 mg/kg). Twelve hours after these treatments, all animals were submitted to challenge by 1% OVA inhalation being analyzed afterwards for bronchoalveolar lavage fluid (BAL), peripheral blood and bone marrow cellularity. In the fourth and final experiment, rats were treated and studied as for experiment 3, except that half of the animals within each group were previously treated with metyrapone prior to the OVA challenge. Results: In the non- immunized rats, AMPH treatment induced an increase in locomotor activity synchronized to high serum AMPH concentrations 1 h after, but not 12 h after treatment. In OVA-challenged rats, AMPH treatment decreased the total number of inflammatory cells, recovered in both BAL and peripheral blood and increased the total number of bone marrow cells. These effects, observed 1 day after OVA challenge, were abrogated by previous metyrapone treatment. Conclusion: AMPH treatment changed HPA-axis responsiveness to the stress condition imposed by the OVA challenge decreasing lung and blood leukocytes cellularity most probably via corticosterone actions on bone marrow activity.

Development of a Respiratory Allergy Model in Male Brown Norway Rats: Pulmonary Immune and Gene Expression Studies

The aim of these studies is to develop a respiratory allergy model in Brown Norway (BN) rats using ovalbumin (Ova) as the antigenic stimulus. Specifically, we are interested in studying the mechanisms underlying the development of pulmonary T helper type 2 (Th2) responses following Ova sensitization and inhalation challenge. We have previously reported on the development of an active pulmonary inflammatory response characterized by the presence of eosinophils and measurement of serum immunoglobulin E levels in Ova-sensitized but not in alum-treated, Ova-challenged or sham control rats. In the current study, we have used cDNA microarray technology to assess the gene expression in tracheobronchial, mesenteric and submaxillary lymph nodes, as well as in pulmonary cells and tissues of Ova-sensitized, alum-sensitized or sham control BN rats. Preliminary cDNA microarray results of lung tissue from BN rats exposed to ova–ova (Ova-sensitized, Ova-challenged) 4 or 24 h after ova challenge did not show up-regulation (relative to sham controls) of mRNA for interleukin-4 (IL-4) or IL-5 but did show up-regulation of eotaxin (4 h), intercellular adhesion molecules (ICAM) and vascular cell adhesion molecules (VCAMs), which are likely to be prerequisites for eosinophilic pulmonary inflammatory responses measured at 24 and 48 h post Ova inhalation exposure. In addition, reverse transcriptase–polymerase chain reaction analysis of lung tissue from BN rats exposed to ova–ova 4 and 24 h after challenge demonstrated up-regulation of mRNA for IL-10 (Th2 response). Studies are ongoing to identify the time course and anatomic location of Th2 responses related to the development of Ova-induced respiratory allergy responses in BN rats.