Respiratory mechanics evaluation of mice submitted to intravenous methacholine: Bolus vs. continuous infusion.

IMPACT STATEMENT: Respiratory mechanics studies are associated with fundamental research and translational studies; the present work thus investigates this particular matter. Our current research describes differences and similarities between two different ways of administrating a very prevalent bronchoconstrictor (methacholine) in an aging process scenario. The core issue of our work is related with troubles we find with the bolus protocol and the application of the mathematical model used to assess the respiratory mechanics. Our findings reveal the continuous infusion as an alternative to these problems and we hope to provide the proper foundations to a more reliable assessment in the respiratory field.

Methacholine dose response curve and acceptability criteria of respiratory mechanics modeling.

Aim: This study aimed to analyze the Constant Phase Model (CPM) Coefficient of Determination (COD) and an index of harmonic distortion ([Formula: see text]) behavior in intravenous methacholine dose response curve. We studied the COD and [Formula: see text] behavior of Control and Lung Inflammation (OVA) groups of mice and we proposed an alternative for moments when the CPM should not be applied. Methods: 9-week female BALB/c mice were studied, 8 of the control group (23.11 ± 1.27 g) and 11 of the lung inflammation group (OVA) (21.45 ± 2.16 g). The COD values were obtained during the respiratory mechanics assessment via Forced Oscillation Technique (FOT) and the [Formula: see text] was estimated a posteriori. Both control and OVA groups were submitted to 4 doses of Methacholine (MCh) protocol. Results: A strong correlation between COD and [Formula: see text] was present at the last two doses (0.3 mg/kg: r = -0.75, p = 0.0013 and 1 mg/kg: r = -0.91; p < 0.0001) in the OVA group. Differences were found in doses of 0.3 mg/kg between control and OVA for the maximum values of Rn (Newtonian Resistance) and G (tissue viscous); and between groups at PBS and doses of 0.03, 0.1 and 0.3 mg/kg for H (Elastance). A similar behavior was observed for the analysis of Area Under the Curve with the exclusion of the 3 first measurements of each dose. However, in this scenario, the comparison with the maximum value presented a higher discriminatory capacity of the parameters associated with the parenchyma. Conclusions: During severe bronchoconstriction there is a strong negative correlation between model goodness of fit and nonlinearities levels, reinforcing that COD is a robust acceptance criterion, whether still simple and easily obtained from the ventilator. We also pointed out the area under the CPM parameters dose response curve is a useful and can be used as a complementary analysis to peak comparison following bolus injections of methacholine.

Actualización en la prueba de provocación bronquial con metacolina en el niño / Update in the bronchial challenge test with methacholine in children

This document updates the recommendations of the bronchial challenge test with methacholine in children. It is based primarily on the recommendations contained in the guide on the technical standard of the bronchial challenge test for methacholine from the European Society of Respiratory Diseases. The main change is the recommendation to use PD20 (methacholine dose that causes a 20% drop in FEV1) instead of PC20 (methacholine concentration that causes a 20% drop in FEV1), which allows for comparable results when different devices and different protocols are used.

Este documento actualiza las recomendaciones de la prueba de provocación bronquial con metacolina en niños. Se basa fundamentalmente en las recomendaciones contenidas en la guía sobre el estándar técnico de la prueba de provocación bronquial de metacolina de la Sociedad Europea de Enfermedades Respiratorias. El principal cambio es la recomendación de utilizar la PD20 (dosis de metacolina que provoca una caída de 20% del VEF1) en vez de PC20 (concentración de metacolina que provoca una caída del 20% en el VEF1), lo cual permite tener resultados comparables cuando se usan diferentes dispositivos y diferentes protocolos.

Sex-specific lung remodeling and inflammation changes in experimental allergic asthma.

There is evidence that sex and sex hormones influence the severity of asthma. Airway and lung parenchyma remodeling and the relationship of ultrastructural changes to airway responsiveness and inflammation in male, female, and oophorectomized mice (OVX) were analyzed in experimental chronic allergic asthma. Seventy-two BALB/c mice were randomly divided into three groups (n=24/each): male, female, and OVX mice, whose ovaries were removed 7 days before the start of sensitization. Each group was further randomized to be sensitized and challenged with ovalbumin (OVA) or saline. Twenty-four hours after the last challenge, collagen fiber content in airways and lung parenchyma, the volume proportion of smooth muscle-specific actin in alveolar ducts and terminal bronchiole, the amount of matrix metalloproteinase (MMP)-2 and MMP-9, and the number of eosinophils and interleukin (IL)-4, IL-5, and transforming growth factor (TGF)-ß levels in bronchoalveolar lavage fluid were higher in female than male OVA mice. The response of OVX mice was similar to that of males, except that IL-5 remained higher. Nevertheless, after OVA provocation, airway responsiveness to methacholine was higher in males compared with females and OVX mice. In conclusion, sex influenced the remodeling process, but the mechanisms responsible for airway hyperresponsiveness seemed to differ from those related to remodeling.

Role of the endocannabinoid system in ethanol-induced inhibition of salivary secretion.

AIM: The aim of the present study was to determine whether the endocannabinoid system could be involved in the ethanol-induced inhibition of salivation in adult male Wistar rats. METHODS: Salivary secretion induced by different concentrations of methacholine, a cholinergic agonist, and the endocannabinoid arachidonoyl ethanolamide (anandamide, AEA) production in the submandibular gland (SMG) were determined in rats after ethanol (3 g/kg) administration by gastric gavage. To study the participation of cannabinod receptors in ethanol action, we evaluated methacholine-induced salivary secretion after ethanol administration when CB1 or CB2 receptors were blocked by intra-SMG injections of their selective antagonists AM251 and AM630, respectively. Additionally, we evaluated the in vitro effect of ethanol (0.1 M) on SMG production of cAMP, alone or combined with AM251 or AM630. RESULTS: Acute ethanol administration increased AEA production in SMG and also inhibited the methacholine-induced saliva secretion that was partially restored by intraglandular injection of AM251 or AM630. In addition, ethanol significantly reduced the forskolin-induced increase in cAMP content in SMG in vitro while treatment with AM251 blocked this response. CONCLUSION: We conclude that the inhibitory effect produced by ethanol on submandibular gland salivary secretion is mediated, at least in part, by the endocannabinoid system.

Lymphatic-borne IL-1beta and the inducible isoform of nitric oxide synthase trigger the bronchial hyporesponsiveness after intestinal ischema/reperfusion in rats.

Intestinal I/R (i-I/R) is an insult associated to further adult respiratory distress syndrome and multiple organ failure. This study was designed to evaluate the repercussions of i-I/R on bronchial reactivity to the cholinergic agent methacholine. Anesthetized rats were subjected to superior mesenteric artery occlusion (45 min) and killed after clamp release and defined intestinal reperfusion periods (30 min, 2, 4, or 24 h). Intestinal I/R caused a progressive bronchial hyporesponsiveness (BHR) that was maximal upon 2 h but reverted within 24 h of intestinal reperfusion. The BHR observed at 2-h i-I/R was prevented by NOS inhibitors (N-L-nitroarginine methyl ester and aminoguanidine) or the KATP channel blocker glibenclamide. Moreover, 2-h i-I/R increased the pulmonary iNOS mRNA expression, a fact prevented by lymphatic thoracic duct ligation. The methacholine reactivity of 2-h i-I/R bronchial segments incubated with NOS inhibitors or glibenclamide was similar to that of naive tissues. In vivo blockade of IL-1beta receptors or lymphatic duct ligation before 2-h i-I/R both abolished BHR. Incubation of naive bronchial segments with lymph collected from 2-h i-I/R rats determined BHR, an effect fully preventable by ex vivo blockade of IL-1beta receptors. Incubation of naive bronchial segments with IL-1beta, but not with IL-10 or TNF-alpha, significantly induced BHR that was prevented by N-L-nitroarginine methyl ester. Our data suggest that a gut ischemic insult generates IL-1beta that, upon reperfusion, travels through the lymph into the lungs. In this tissue, IL-1beta would stimulate the generation of NO that orchestrates the ensuing BHR for which the opening of KATP channels seems to play a pivotal role.

Untreated asthma, final height and sitting height/leg length ratio in Chile.

OBJECTIVE: There is uncertainty as to whether asthma has an effect on final height. We investigated using subjective and objective assessments whether untreated asthma is associated with final height, leg length and sitting height to leg length ratio in an area of Chile in which almost no one received asthma treatment. METHODS: We collected data on 1232 males and females aged 22-28 years in a semi-rural area of Chile. Information on asthma was collected using the European Community Respiratory Health Survey (ECRHS) questionnaire. We assessed sensitisation to eight allergens and bronchial hyper-responsiveness (BHR) to methacholine as a dichotomous variable and as a log slope. Information on possible confounders in terms of smoking, birth weight, number of siblings and socio-economic factors such as household possessions, car ownership and education was available. RESULTS: Regardless of the asthma assessment used, there was no association between asthma symptoms, diagnosis of asthma, atopy, BHR as log slope, binary or categorical and height, leg length or the ratio of sitting height to leg length. The latter was used as a potentially more appropriate measure to assess a detriment of growth. CONCLUSION: Asthma as assessed in community studies is unrelated to final height or body proportions.

Modulation of allergic and immune complex-induced lung inflammation by bradykinin receptor antagonists.

OBJECTIVE: The effect of bradykinin (B(1) or B(2)) receptor antagonists was studied in allergic and immune-complex-induced lung inflammation. METHODS: Lungs of BALB/c mice were examined 24 h after induction of lung inflammation, either allergic (ovalbumin-sensitized submitted to two aerosol of antigen, one week apart) or immune-complex induced (intratracheal instillation of IgG antibodies followed by intravenous antigen). The bradykinin B(2) receptor antagonist, HOE-140 or bradykinin B(1) receptor antagonist, R-954 were given intraperitoneally (100 microg/kg), 30 min before induction. RESULTS: In allergic inflammation, pre-treatment with R-954 reduced eosinophil infiltration into the lungs, mucus secretion and the airway hyperreactivity to methacholine. Pre-treatment with HOE-140 increased eosinophil infiltration but did not affect the other parameters. In immune-complex inflammation, HOE-140 increased neutrophil infiltration but not their activation nor the hemorrhagic lesions. R-594 pre-treatment did not change the parameters examined. CONCLUSION: These results show important modulatory effects of bradykinin B(1) and B(2) receptor antagonists in both models of lung inflammation.

Supramaximal flow in asthmatic patients.

In normal subjects supramaximal flows (SF) are known to be correlated with flow limitation. To further understand the mechanisms involved in SF this correlation and the influence of salbutamol and methacholine administration on SF have been investigated in asthmatic subjects. Protocol A involved obtaining basal maximal expiratory flow/volume curves and interrupted curves through a fast valve from 36 asthmatic patients. Maximal flow at 50% of forced vital capacity (V'max50) increase (deltaV'max50) was compared between basal curves and envelope curves passing through SF peaks obtained after each interruption. Protocol B involved the study of 33 asthmatic patients after salbutamol administration and 12 asthmatic patients after methacholine challenge. DeltaV'max50 between basal versus interrupted curves were analysed (deltaV'max50B). Similar procedures were performed after salbutamol (deltaV'max50S) and after methacholine administration (deltaV'max50M). A significant negative correlation between forced expiratory volume in one second and deltaV'max50 was observed. SF decreased significantly after salbutamol administration and increased significantly after methacholine. The results of this study suggest that in asthmatic patients participation of "pendelluft" in SF increases as airflow limitation increases.

A simplified cyclic adenosine monophosphate-mediated sweat rate test for quantitative measure of cystic fibrosis transmembrane regulator (CFTR) function.

OBJECTIVE: Sweat production is stimulated by both cholinergic and beta-adrenergic pathways in the sweat gland secretory coil. beta-Adrenergic pathway-mediated sweating is absent in cystic fibrosis (CF) because cyclic adenosine monophosphate (cAMP)-mediated chloride transport through the cystic fibrosis transmembrane regulator (CFTR) is disrupted. We report the development of a rapid, reproducible, macroscopic, and quantitative methodology to test the hypothesis that beta-adrenergic sweat rate discriminates among 3 different CFTR phenotypes-CF, heterozygote CF carriers, and non-CF. STUDY DESIGN: Intradermal injection of a mixture of 50 micromol/L isoproterenol, 5 mmol/L aminophylline (to potentiate the beta-adrenergic stimulation), and 140 micromol/L atropine (to block potential cholinergic stimulation) in lactated Ringer's solution was performed in duplicate on one forearm. A single injection of 0.5 mmol/L methacholine to stimulate sweat production by the cholinergic pathway was performed on the other forearm. Sweat rate was determined as the amount of sweat collected on filter paper over 20 minutes. RESULTS AND CONCLUSIONS: Median cAMP-mediated sweat rates were 1.45 mg/20 min (CF, n = 29), 2.55 mg/20 min (CF heterozygote carriers, n = 30), and 3.65 mg/20 min (non-CF, n = 30) and were significantly different in all 3 groups (P =.0001, Kruskal-Wallis test). Methacholine-stimulated sweat rates were similar for all 3 groups. The cAMP-mediated sweat rate test may be a useful endpoint for studies of new agents to increase the function of CFTR.

Differential distribution of elastic system fibers in control and bronchoconstricted intraparenchymatous airways in the guinea-pig lung.

The elastic system fibers were studied at the light microscopic level by using Weigert's resorcin-fuchsin method after oxidation. This study was designed to describe the distribution of these fibers in intrapulmonary guinea-pig airways and to characterize their conformational changes during bronchoconstriction induced by methacholine aerosol. Airways present a palisade of elastic system fibers just beneath the epithelial basement membrane; these fibers are also present in the adventitial connective tissue. Thin fibers link the fibers located in the palisade among themselves and also connect them to those fibers located in the bronchial adventitial tissue, by traversing the airway smooth muscle. During bronchoconstriction, the fibers located beneath the epithelial basement membrane are divided into two components: one follows the epithelial invaginations towards airway lumen, while the other population remains attached through airway smooth muscle to the fibers located in the adventitial connective tissue. At the ultrastructural level, the findings corroborated those of the light microscopy and in addition, disclosed that typical mature elastic fibers and also elaunin fibers attach directly to the basal lamina, a feature that has not been reported previously in other tissues studied. This configuration is compatible with the idea that fibers of the elastic system restrict the mucosal folding during bronchoconstriction, and may also provide energy to restore airway configuration to its normal status after contraction.

Efectos sobre el valor de PC20 al emplear VEF1 mas alto o mas bajo para su cálculo / Effect on PC20 calculated using the highest or the lowest FEV1 value

Existe poca información en niños acerca de cual valor de VEF1, si el más alto o el más bajo, debe ser considerado para el cálculo de la PC20 de metacolina. Veinte niños asmáticos atópicos, previamente entrenados hasta alcanzar maniobras de capacidad vital forzada reproducibles, fueron estudiados para determinar si había diferencias en las PC20 de metacolina calculadas con los mayores o los menores valores de VEF1. Las diferencias entre las PC20 usando uno u otro valor de VEF1 no fueron estadísticamente significativas (ANOVA). Hubo una correlación significativa entre las PC20 calculadas con ambos métodos (r= 0,93, p< 0,05). El presente estudio demuestra que, en niños asmáticos entrenados hasta lograr maniobras de capacidad vital forzada reproducible, la PC20 puede ser determinada usando el valor más alto o más bajo de VEF1

Urban levels of air pollution increase lung responsiveness in rats.

This study was designed to investigate if animals exposed to urban levels of air pollution develop pulmonary hyperresponsiveness and to test if this change was reversed after moving the animals to a nonpolluted environment. One hundred twenty male Wistar rats were kept in (a) São Paulo (polluted environment) for 3 months (SP3); (b) Atibaia (clean region), for 3 months (A3); (c) São Paulo for 3 months and then Atibaia for a further 3 months (SPA6); (d) Atibaia for 6 months (A6). After the exposure period, the rats were submitted to dose-response curves to inhaled methacholine. Older animals (SPA6 and A6) had lower responses to methacholine in terms of respiratory system resistance when compared to the animals studied after 3 months of experiment (SP3 and A3). However, the response in terms of respiratory system elastance of the SP3 group was significantly (P = 0.0004) greater than those of the other three groups. Our results suggest that the environmental conditions of the large urban centers can induce pulmonary hyperresponsiveness in rats that can be reversed when the animals are removed to a nonpolluted area.