The subjective symptoms were assessed using a questionnaire befor

The subjective symptoms were assessed using a questionnaire before and after the 2-week treatment. The questionnaire was composed of 2 parts: a general impression (Fig. 3) and a visual analogue scale (VAS) (Table 2). Following the completion of the 2-week treatment, a substantial improvement was reported by the treatment group (Fig. 3). The post-treatment VAS value was significantly decreased compared with that of the pre-treatment period in sensation of oral dryness, thirstiness, and burning sensation of the tongue in the treatment group (Table 2). A hypothesis was proposed that unconscious LY294002 episodes of rhythmic masticatory muscle activity play an important role in lubricating the oral

cavity [26] and [27] and that the night guard could help to regulate appropriate rhythmic muscle activity. A second possible mechanism for nocturnal xerostomia improvement with the night guard is maintenance

of saliva volume in the oral cavity. It was suggested that patients recognized the sensation of dryness when there was not enough saliva to cover the various oral surfaces, especially the palate [28]. A third possible factor could be that the night guard decreases the vaporization of saliva from the oral cavity, which is dependent upon how long the mouth is open. The night guard possibly prevents mouth opening, which occurs due to malocclusion, nasal congestion, and enlarged adenoids. We concluded that the application of a bite guard this website is therefore a useful and simple method for the management of nocturnal xerostomia. Furthermore, the device can be used not only during the

night, but also during the daytime. Dry mouth patients can use this device to maintain the presence of artificial gel type lubricants in the mouth (Fig. 2B). As a result, 3-oxoacyl-(acyl-carrier-protein) reductase such a bite guard could also be useful for Sjögren’s syndrome patients. Immunological management is one of the options for patients who do not respond to secretagogues or moisturizing saliva substitutes with or without the bite guard. However, no trials concerning the immunological treatment of primary Sjögren’s syndrome, such as interferon-alpha [29] and [30] and anti CD antibodies [31], [32] and [33] have been conducted so far in Japan, although interferon-alpha and anti CD20 antibody were recognized to be an effective and safe treatment strategy for patients with primary Sjögren’s syndrome in double-blind placebo-controlled clinical trials [30], [33] and [34]. Furthermore, no clinicians in Japan have accepted even systemic administration of corticosteroids for the primary Sjögren’s syndrome patients, and thus, the corticosteroid irrigation of the parotid gland is the only currently available immunological treatment option [35]. Acute stress, such as anger and anxiety induce dry mouth even for healthy people. Chronic psychological and psychiatric factors including anxiety and depression are related to the occurrence of dry mouth sensation [36], [37] and [38].

GC/MS analyses were performed on a Shimadzu QP5050A GC/MS system

GC/MS analyses were performed on a Shimadzu QP5050A GC/MS system equipped with an AOC-20i auto-injector. A J&W Scientific DB-5MS fused capillary column (30 m × 0.25 mm × 0.25 μm film thickness; Agilent, Santa Clara, CA) was used as the stationary phase. MS data were taken at 70 eV with a scan interval of 0.5 s from m/z 40 to 500. All other conditions were similar to the GC analysis. Essential oil components were identified by comparing the retention times of the GC peaks with standard compounds run under identical conditions and by comparison of retention indices (Van Den Dool & Kratz, 1963) and mass spectra (Adams, 2007) with those

found in the literature, and by comparison of mass spectra with those stored in the NIST 107 LGK974 and NIST 21, and Wiley 229 libraries. Tumour cell growth was determined by the ability of living cells to reduce the yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to a purple formazan product, as described by Mossman (1983). For all experiments, cells were seeded in 96-well plates (0.7 × 105 cells/ml for adherent cells or 0.3 × 106 cells/ml for suspended cells in 100 μl of medium). After 24 h, the drugs (0.78–50 μg/ml) were dissolved in pure DMSO and added to IDH inhibitor each well using HTS – High-Throughput Screening (Biomek 3000, Beckman Coulter Inc., Fullerton, CA). Then, the

cells were incubated for 72 h. Doxorubicin (purity > 98%; Sigma Chemical Co., St. Louis, MO) was used as positive control. At the end of incubation, the plates were centrifuged, and the medium was replaced by fresh medium (150 μl) containing 0.5 mg/ml MTT. Three hours later, the formazan product was dissolved

in 150 μl DMSO, and absorbance was measured using a multiplate reader (DTX 880 Multimode Detector, Beckman Coulter Inc.). The drug effects were expressed as the percentage of control absorbance of reduced dye at 595 nm. The in vivo antitumour effect Cyclin-dependent kinase 3 was evaluated using Sarcoma 180 ascites tumour cells, following protocols previously described ( Bezerra et al., 2006, Bezerra et al., 2008 and Britto et al., 2012). Ten-day-old Sarcoma 180 ascites tumour cells (2 × 106 cells per 500 μl) were implanted subcutaneously into the left hind groin of mice. The essential oil was dissolved in 5% DMSO and given to mice intraperitoneally once a day for 7 consecutive days. At the beginning of the experiment, the mice were divided into four groups of 8–15 animals as follows: Group 1: animals treated by injection of vehicle 5% DMSO (n = 15); Group 2: animals treated by injection of 5-fluorouracil (5-FU, purity > 99%; Sigma Chemical Co.) (25 mg/kg/day) (n = 9); Group 3: animals treated by injection of the essential oil (50 mg/kg/day) (n = 8); Group 4: animals treated by injection of the essential oil (100 mg/kg/day) (n = 8). The treatments were started one day after tumour injection. The dosages were determined based on previous articles.

For initial characterisation of the assay systems (DGGR and olive

For initial characterisation of the assay systems (DGGR and olive oil) three replicates were used. The analysis between the level of inhibition by alginates from two seaweed sources was tested with a one way ANOVA with Tukey post test. All subsequent measurements used six replicates. The number of replicates is shown in each figure legend. Using DGGR as a substrate, the activity of lipase could be measured by the increase in absorbance

(Panteghini et al., 2001). As expected, there was a marked change in the absorbance over time for the negative control (lipase selleck screening library plus substrate) illustrating the maximum rate of reaction (Fig. 1), whereas, for the inhibition control (Orlistat (0.025 mg/ml)) there was minimal change in absorbance over time. Fig. 1 also shows that alginate could inhibit the activity of lipase. To compare the inhibition of a range of alginates, the absorbance at 12 min reaction time was chosen. This time point was used because the reaction was still close to the linear phase. Fig. 2A shows that there was a significant difference in the level of inhibition depending

Dinaciclib chemical structure on the seaweed source of the alginate. The alginates extracted from Laminaria hyperborea seaweed inhibited pancreatic lipase to a significantly higher degree (two way ANOVA, p = 0.0015) than the alginates extracted from Lessonia nigrescens. A dose dependent inhibition was seen for both sets of seaweed alginates. Fig. 2B shows that

for Laminaria hyperborea alginate the percentage of lipase inhibition increased with increasing concentration. For LFR5/60, there was a 75% relative increase in inhibition when the alginate concentration was increased fourfold from 0.21 mg/ml to 0.86 mg/ml, and a 56% increase from 0.86 mg/ml to 3.43 mg/ml. Similarly, the increases for alginates SF120 and SF/LF were 90% and 122%, respectively when the alginate concentration was increased from 0.21 to 0.86 mg/ml, and again 64% and 47%, respectively increased to 3.43 mg/ml. The alginate SF200 level of inhibition increased 44% and 46% when the alginate concentration increased from 0.21 to 0.86 and then 3.43 mg/ml. MTMR9 As seen in Fig. 2, not all alginates inhibited lipase to the same extent, even those from the same genus of seaweed. To understand why the levels differed, the compositional characteristics of the various alginates were correlated with the level of lipase inhibition (Table 2). Statistical significant positive correlations were found between levels of inhibition and increasing guluronate content (F(G)), the fraction of guluronate dimers (F(GG)), the fraction of guluronate trimers (F(GGG)) and the number of guluronate blocks greater than one in the alginate polymer (N(G > 1)). Surprisingly, the reciprocal correlations with mannuronate levels were not always significant. Only F(M) and F(MG) were statistically significant negative correlations.

The extract of propolis obtained with canola oil and dried (ODEP)

The extract of propolis obtained with canola oil and dried (ODEP) displayed moderate cytotoxicity against leukemia (HL-60), melanoma (MDA-MB-435) and glioblastoma (SF-295) cancer cells, a better result than the ethanolic extract (EEP70). When analysing cytotoxicity from ODEP fractions, it was evident that the fractions were less active than the propolis extract (ODEP) in the cell lines evaluated, whereas OLSx4 and OLSx5 showed moderate cytotoxicity against leukemia (HL-60) and colon (HCT-8). To check if the cytotoxic effects observed in vitro also occured in vivo, we used the

Sarcoma 180 (S-180) model which is a mouse-originated Ipilimumab concentration tumour frequently used in in vivo antitumour related research ( Gonzaga et al., 2009). Fig. 2 shows the effects of the propolis extracts on mice transplanted with S-180 tumour. There was a significant DAPT price reduction of the tumour weight in all extracts tested. The differences between experimental groups were compared by ANOVA followed by Student Newman Keuls or Bonferroni tests (p < 0.05). On the 8th day, the average tumour weight of the control mice inoculated with sarcoma

180 was 2.05 ± 0.22 g. In the presence of EEP70, the sarcoma 180 weight was reduced to 0.94 ± 0.35 and 0.90 ± 0.22 g at doses of 50 and 80 mg/kg, respectively. These reductions are equivalent to inhibition ratios of 53.94% and 56.29%. In the presence of ODEP, the sarcoma 180 weight was reduced to 0.92 ± 0.14 and 0.96 ± 0.24 g at doses of 50 and 80 mg/kg, respectively. These reductions are equivalent to inhibition ratios of 54.94% and 53.35%. At 25 mg/kg, 5-FU reduced tumour weight by 51.56% within the same period. These results showed that the inhibition ratio of the ethanolic extract was the same as that of oil extract and no differences were observed when the extracts were tested at doses of Megestrol Acetate 50 and 80 mg/kg. It was demonstrated that both extracts of propolis inhibited the Sarcoma 180 tumour growth in mice. After killing the animals, the organs were weighed. No significant changes in the organ weights were

seen in any of the extract-treated animals (Fig. 2). After treatment with 5-FU, however, the spleen weights were significantly reduced when compared with the control group (p < 0.05). No significant gain in body weight was seen among the groups (p > 0.05) (data not shown). No significant changes in the renal (urea and creatinine levels) or liver [enzymatic activity of transaminases aspartate aminotransferase (AST) and alanine aminotransferase (ALT)] parameters were seen in the animals treated with propolis extracts (data compared by ANOVA followed by Student Newman Keuls or Bonferroni tests p < 0.05) in mice transplanted with Sarcoma 180 tumour ( Fig. 3). The animals treated with 5-FU have alteration on renal and liver parameters.

However, sampling season should be regarded as a possible source

However, sampling season should be regarded as a possible source of variation in exposure data. Taken together the results from this study indicate that the wild mink is a species suitable for sensitive and cost efficient environmental monitoring of PFAA exposure. This work was funded by the Environmental Monitoring Program at the Swedish University of Agricultural MAPK Inhibitor Library high throughput Sciences. The mink hunters B. Almberg, G. Anttila, Å. Degermark, B. Engström, M. Eriksson, W. Eriksson,

T Hall, J. Karlsson, S. Karlsson, S-G. Lunneryd, M. Nilsson, B. Nyberg, A. Olofsson, E. Olsson, S. Sundin and S-A. Ängwald are thanked for their work of providing mink carcasses for this study. “
“On 26 and 27 November 2009, 29 experts met in Brussels to discuss current opinions on the effects of dietary exposure to endocrine-active pesticide residues. Representatives from academia, industry, government and non-profit organisations participated in a workshop combining expert lectures and

breakout sessions. The workshop was organised such that 8 presentations by experts with specialties in different aspects of endocrine disruption were followed by a breakout group session. Each of the 4 breakout groups discussed 2 questions posed to them by the workshop organisers and then gave a short presentation on their responses to Selleck JQ1 the questions, including whether or not they reached consensus on those responses. In the pages that follow, the 8 expert presentations and the presentations from the breakout sessions are summarised in detail. Interestingly, only one of the breakout groups was able to say that they had no significant disagreements, and for 3 of the questions 2 groups indicated that they could not reach any significant agreement at all (‘Is endocrine disruption a mechanism essentially different from other toxicological mechanisms?’, ‘Should [endocrine disrupting chemicals] Dipeptidyl peptidase therefore be regulated using different criteria?’

and ‘Is the effort currently dedicated to this subject [of pesticides with potentially endocrine disrupting properties] proportional to the real health risk?’). In each of these cases, the reason for the lack of consensus was attributed to a lack of adequate knowledge: Either on how to measure endocrine effects or on what the real health risks of these effects might be. These responses clearly underline the need for more research and more discussion on defining endocrine disrupting properties and then regulating potentially endocrine active substances. The November 2009 workshop in Brussels was the first in a series to be organised by the SAFE consortium with the aim of bringing scientific clarity to this discussion. The paper that follows expresses solely the opinions and recollections of the author. Each expert presentation summarised below was offered to the presenter for review and comment; those presentations with an * next to the author’s name were indeed reviewed and approved by the presenter.

Sweden is an ideal case for such an analysis since the retention

Sweden is an ideal case for such an analysis since the retention approach has been practiced in this country for more than two decades (Eckerberg, 1988, Götmark et al., 2009 and Gustafsson and Perhans, 2010), and an extensive and high-quality National Forest Inventory data-base exists that can be used for detailed analysis. Due to a long history of industrial forestry in North Europe, and especially in Sweden and Finland, production forests have become more even-aged and much less structurally diverse than intact forests. Amounts of dead wood, old trees and other properties of importance to biodiversity are much lower

compared with natural forest landscapes (Fridman GSI-IX and Walheim, 2000, Peterken, 2001 and Josefsson and Östlund, 2011). The importance of incorporating old-growth elements in managed forests is increasingly being recognized

(e.g. Bauhus et al., 2009), and dead trees and old living trees are known to be of large importance to biodiversity, not the least to threatened species (e.g. Bernes, AZD5363 research buy 2011). A multiscale model for forest conservation is applied in Sweden, implying that conservation actions are taken at different scale-levels from individual trees to areas embracing hundreds or thousands of ha. The highest level, up to 1000 ha or more, includes formally protected areas such as national parks and nature reserves. At the next, intermediate level (ca. 1–50 ha) there are both formally protected and voluntary set-asides through certification, many to of which are so called woodland key habitats (Timonen et al., 2010). Retention approaches represent the lowest scale level, implying that trees of importance to biodiversity and ecosystem function

are left unlogged, mainly at final felling operations, but also during thinning. Single living trees are retained, and tree patches may be left as ‘islands’ in felled areas or adjacent to non-felled stands, often as buffer strips along lakes, rivers, wetlands and near settlements. Standing and lying dead trees are also retained, and according to instructions they should not be harmed during logging operations. Dead wood is created, in Sweden primarily through artificially creating snags by cutting trees at a height of 3–4 m, but also by retaining living trees of which some or many will eventually become windthrown. The state is responsible for establishing nature reserves, while both the state and the forest owners protect also smaller areas. Retention requirements have been part of Swedish forest legislation since the 1970s, and were made well-known to landowners through the “Richer Forest” campaign by the Swedish Forest Agency in the beginning of the 1990s. They were further consolidated with the launch of a new forest policy in the mid 1990s in which environmental and production goals were assigned equal value (Bush, 2010).

, 2012, Hansen et al , 2012 and Schwartz et al , 2007) While fie

, 2012, Hansen et al., 2012 and Schwartz et al., 2007). While field trials continue to be expensive and time consuming, the costs of genetic marker studies are decreasing. With increasing ability to handle large amounts of data and combine available information from genetic studies R428 with other geographically based information, it now seems possible to suggest indicators of genetic diversity that are both relevant and not prohibitively costly. The purpose of this paper is to provide a framework and a typology for the application

of such indicators of tree genetic diversity commensurate with the current international scheme provided by the Strategic Plan for Biodiversity 2011–2020 and the BIP. To do so, we first describe the Strategic Plan and the work of BIP to identify indicators within the established framework that are relevant for tree genetic diversity (Section 2). Next, a review of past attempts to define

and report on possible tree genetic diversity indicators is provided, in order to reveal why they have not been widely applied (Section 3). We then move on to suggest Veliparib cost what we consider meaningful and realistic indicators of genetic diversity of trees that can be embedded within the Strategic Plan and BIP, and constitute a framework and typology for management of trees within, as well as outside, forests (Section 4). Finally, conclusions

BCKDHB (Section 5) are provided. According to Sparks et al. (2011) and UNEP/CBD/AHTEG, 2011a and UNEP/CBD/AHTEG, 2011b, indicators should ideally provide answers to, or shed light on, four basic questions (Table 1). In the case of tree genetic diversity, indicators should monitor the adaptive potential of tree species to help identify and prioritize actions, related to its use and conservation. The UN Strategic Plan for Biodiversity 2011–2020 is made of five strategic goals and 20 specific targets to be achieved by 2020, referred to as the Aichi Targets (UNEP/CBD/COP, 2010 and UNEP/CBD/COP, 2011). To monitor progress, an elaborate indicator framework for assessing the Aichi Targets has been developed by the Ad Hoc Technical Expert Group (AHTEG) on indicators for the Strategic Plan (UNEP/CBD/AHTEG, 2011a and UNEP/CBD/AHTEG, 2011b). This indicator framework consists of 12 proposed headline indicators and 97 proposed operational indicators (see Table 2 for examples). A single indicator, used in isolation, is generally considered insufficient to assess overall progress towards a target, thus the necessity to link multiple indicators (Chenery et al., 2013). The global initiative BIP has been established to promote and coordinate development and delivery of biodiversity indicators in support of the CBD and other sectors.

In general, with the number of SNP loci those studies have consid

In general, with the number of SNP loci those studies have considered, di-allelic SNPs do not resolve all possible ambiguities. While clearly the multiallelic microhaps will be better, per locus, than the di-allelic SNPs, it is not clear what the optimal number of microhap loci will be. The answers will depend on the population-specific number of alleles and level of heterozygosity of each microhap in the panel used. Since we expect more and better microhaps will be identified in the near future, and we are not advocating the present set as optimal, such important statistical questions are better

addressed when a better set of loci has been documented. Because large numbers of loci can be multiplexed with the current sequencing technology, many more loci will be added to any final panel. As new microhaps are identified and added to the panel it would be possible to tune the panel toward individual identification or Venetoclax order ancestry, but with enough microhaps that may be moot. The results for these 31 loci suggest that a large enough panel containing this range of locus patterns may provide good ancestry information and sufficiently low match probabilities globally that the variation among populations becomes irrelevant. Microhap loci with three or four SNPs can have higher heterozygosity

and identifying and adding such loci LY294002 may provide sufficient information to meet all purposes: lineage/kinship as well as individual identification and ancestry. It is also desirable to have other research groups replicate the results reported here on additional samples from the sample populations as well as validate results on new populations. Since the 54 populations studied already cover much of the world and many of the as yet unstudied populations share similar genetic and demographic histories, it is reasonable to expect that most new populations studied will also be found to have excellent heterozygosities and genotype resolvabilities. In order to IMP dehydrogenase make the panel more generally useful it might also be desirable to find some additional

unlinked microhaps that might have enhanced heterozygosities for Native American and Pacific Island populations. Fine tuning the panel might also be desirable by replacing some of the loci in the current panel with loci that are found to have more alleles and better average heterozygosities worldwide and also in particular geographical regions. Microhaps comprised of three SNPs are likely to be significantly better than those based on only two SNPs as are the majority of the loci in this initial panel. We have already identified several additional three-SNP and four-SNP loci with four or more alleles and are now working to collect the population data. The high throughput methods now available with the appropriate read lengths for these microhaps have enormous capacity. Additional microhaps are clearly needed and can easily be accommodated.

Flow and pressure signals were then passed through 8-pole Bessel

Flow and pressure signals were then passed through 8-pole Bessel low-pass filters (902LPF, Frequency Devices, Haverhill, MA, USA) with the corner frequency set at 100 Hz, sampled at 200 Hz with a 12-bit analog-to-digital converter (DT2801A, Data Translation, Marlboro, MA, USA), and stored on a microcomputer. All data were collected using LABDAT www.selleckchem.com/products/dabrafenib-gsk2118436.html software (RHT-InfoData Inc., Montreal, QC, Canada). Lung resistive (ΔP1) and viscoelastic/inhomogeneous (ΔP2) pressures, static elastance (Est), and viscoelastic component

of elastance (ΔE) were computed by the end-inflation occlusion method ( Bates et al., 1985 and Bates et al., 1988). Briefly, after end-inspiratory occlusion, there is an initial fast drop in transpulmonary

pressure (ΔP1) from the pre-occlusion value down to an selleck compound inflection point (Pi) followed by a slow pressure decay (ΔP2), until a plateau is reached. This plateau corresponds to the elastic recoil pressure of the lung (Pel). ΔP1 selectively reflects airway resistance in normal animals and humans and ΔP2 reflects stress relaxation, or viscoelastic properties of the lung, together with a small contribution of time constant of alveoli ( Bates et al., 1988 and Saldiva et al., 1992). Lung static and dynamic elastances (Est and Edyn, respectively) were calculated by dividing Pel and Pi by tidal volume, respectively. ΔE was calculated as Est − Edyn, and reflects the viscoelastic component of elastance ( Bates et al., 1985 and Bates et al., 1988). Heparin (1000 IU) was intravenously injected immediately after the determination of pulmonary mechanics. The trachea was clamped at end-expiration and the animals were euthanized by exsanguinations via sectioning of the abdominal aorta and the vena cava. The lungs were removed and weighed. Functional residual capacity (FRC) was determined by volume displacement (Scherle, 1970). Left lungs were then fixed with Millonig formaldehyde (100 ml HCHO, 900 ml H2O, 18.6 g

ALOX15 NaH2PO4, 4.2 g NaOH), routinely prepared for histology, embedded in paraffin, and two 3-μm-thick longitudinal slides from the left lung were cut and stained with hematoxylin–eosin. Morphometric analysis was performed with an integrating eyepiece with a coherent system made of a 100-point and 50-line (1250-μm-long each) grid coupled to a conventional light microscope (Axioplan, Zeiss, Oberkochen, Germany). The fraction areas of collapsed and normal alveoli were determined by the point-counting technique at a magnification of ×200 across 10 random non-coincident microscopic fields per animal. Points falling on normal or collapsed alveoli were expressed as percentage of points hitting those alveoli (Weibel, 1990). Polymorphonuclear (PMN) and pulmonary tissue were evaluated at ×1000 magnification across 10 random non-coincident microscopic fields in each animal.

In the ovalbumin group (OVA), mice were immunized using an adjuva

In the ovalbumin group (OVA), mice were immunized using an adjuvant-free protocol with intraperitoneal injection of ovalbumin (10 μg in 0.1 mL sterile saline) on each of seven alternate days. Forty days after the beginning of sensitization, 20 μg of OVA in 20 μL

sterile saline were intratracheally instilled. This procedure was performed three times at 3-day intervals. The control group (C) received saline using the same protocol. Eighty-four animals were used for analysis of lung mechanics and histology, and a second group of 84 animals was used for analysis of airway responsiveness and bronchoalveolar lavage fluid (BALF). The BCG Moreau vaccine was donated by the Ataulpho de Paiva Foundation, Brazil. Twenty-four hours after the last challenge, mice were sedated (diazepam 1 mg i.p.), anesthetized (thiopental sodium 20 mg/kg i.p.), tracheotomized, paralyzed (vecuronium bromide, 0.005 mg/kg i.v.), and mechanically PF-02341066 in vitro ventilated with the following settings: respiratory frequency 100 breaths/min, tidal volume (VT) 0.2 mL, and fraction of inspired oxygen (FiO2) 0.21. The anterior chest wall was surgically removed and a positive end-expiratory pressure (PEEP) of 2 cmH2O was applied, and the lung mechanics were computed. At the end of the experiment, the lungs were prepared for histology Vorinostat ic50 and molecular biology.

Airflow, volume and tracheal pressure (Ptr) were measured ( Hsia et al., 2010). In an open chest preparation, Ptr reflects transpulmonary pressure (PL). Lung static elastance and airway resistance were computed by the end-inflation ifenprodil occlusion method ( Bates et al., 1985) using the ANADAT data analysis software (RHT-InfoData, Inc., Montreal, Quebec, Canada). Twenty-four hours after the last challenge, airway responsiveness was measured. Increasing doses of methacholine (Sigma Chemical Co., Saint Louis, MI, USA) (100, 300, 1000, 3000, and 10,000 μg/kg) were administered via a silastic catheter placed in the jugular vein. Data were stored at 30 s, 1, 3, and 5 min after agonist injection. Shortly after each intravenous infusion of methacholine, the maximal increase in Ptr was reached, and the respective airflow

was measured at this moment (Antunes et al., 2009). Respiratory system resistance (R) was obtained using the equation of motion of the respiratory system: Ptr(t) = E·V(t) + R·V′(t), where (t) is time. The right lung was removed, fixed in 4% buffered formaldehyde, paraffin-embedded, and cut into 4 μm-thick slices, which were stained with hematoxylin and eosin (Vetec Química Fina, Rio de Janeiro, Brazil). Fraction area of collapsed and normal lung areas were determined by the point-counting technique at a magnification of 200× across 10 random, non-coincident microscopic fields (Hsia et al., 2010). Points falling on collapsed or normal pulmonary areas were counted and divided by the total number of points in each microscopic field.