Challenges of microtome-based serial block-face scanning electron microscopy in neuroscience.

Serial block-face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape-collecting ultramicrotome, focused ion-beam scanning electron microscopy and SBEM (microtome serial block-face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines.

Physical activity and the prevention, reduction, and treatment of alcohol and other drug use across the lifespan (The PHASE review): A systematic review.

The aim of this review is to systematically describe and quantify the effects of PA interventions on alcohol and other drug use outcomes, and to identify any apparent effect of PA dose and type, possible mechanisms of effect, and any other aspect of intervention delivery (e.g. key behaviour change processes), within a framework to inform the design and evaluation of future interventions. Systematic searches were designed to identify published and grey literature on the role of PA for reducing the risk of progression to alcohol and other drug use (PREVENTION), supporting individuals to reduce alcohol and other drug use for harm reduction (REDUCTION), and promote abstinence and relapse prevention during and after treatment of alcohol and other drug use (TREATMENT). Searches identified 49,518 records, with 49,342 excluded on title and abstract. We screened 176 full text articles from which we included 32 studies in 32 papers with quantitative results of relevance to this review. Meta-analysis of two studies showed a significant effect of PA on prevention of alcohol initiation (risk ratio [RR]: 0.72, 95%CI: 0.61 to 0.85). Meta-analysis of four studies showed no clear evidence for an effect of PA on alcohol consumption (Standardised Mean Difference [SMD]: 0.19, 95%, Confidence Interval -0.57 to 0.18). We were unable to quantitatively examine the effects of PA interventions on other drug use alone, or in combination with alcohol use, for prevention, reduction or treatment. Among the 19 treatment studies with an alcohol and other drug use outcome, there was a trend for promising short-term effect but with limited information about intervention fidelity and exercise dose, there was a moderate to high risk of bias. We identified no studies reporting the cost-effectiveness of interventions. More rigorous and well-designed research is needed. Our novel approach to the review provides a clearer guide to achieve this in future research questions addressed to inform policy and practice for different populations and settings.

Failure of hypoxic pulmonary vasoconstriction in the canine asthma model. Effect of prostaglandin inhibitors.

Measurements of respiratory mechanics, arterial blood gases, and pulmonary vascular resistance were made before and 15 min after inhalation challenge with Ascaris suum extract in dogs with natural sensitivity to this antigen. 25 of 47 dogs were treated before inhalation challenge with a prostaglandin inhibitor (90 mg/kg of aspirin or 2 mg/kg of indomethacin by intravenous infusion). In response to the challenge, bronchospasm developed in approximately half (responders) of each group reflected by decreases in mean specific respiratory system conductance and arterial oxygen tension. While the dogs were breathing room air, pulmonary vascular resistance remained unchanged after antigen challenge in the responders not given aspirin or indomethacin, but increased significantly and was associated with a lesser degree of arterial hypoxemia in the responders pretreated with either of the prostaglandin inhibitors. Prevention of arterial hypoxemia by oxygen breathing blocked an increase in pulmonary vascular resistance in four pretreated responders. No changes in respiratory mechanics, pulmonary hemodynamics, or arterial blood gases were noted in the 21 dogs who did not develop bronchospasm regardless of whether or not they were pretreated. 12 additional dogs in whom arterial hypoxemia was produced by 10% oxygen breathing, showed an increase in pulmonary vascular resistance that was not potentiated by pretreatment with aspirin in 6. We conclude that in acute experimental canine asthma, vasodilator prostaglandins appear to blunt the hypoxic pulmonary vasoconstrictor response, thereby further compromising gas exchange but preventing the development of pulmonary hypertension.

Relationship between frequency dependence of lung compliance and distribution of ventilation.

The previously demonstrated empirical association between frequency dependence of lung compliance and distribution of ventilation, the latter determined by the N(2) washout technique, was confirmed by establishing a mathematical link between the two tests. By assuming a two-compartment system with known compliances and making corrections for Pendelluft and common dead space mixing effects, the ratio of dynamic to static compliance (C(dyn)/C(st)) for any respiratory frequency can be calculated from the compartmental analysis of the N(2) washout at a single respiratory frequency. By using these equations, a good correlation was found between calculated and measured C(dyn)/C(st) in dogs with artificially induced bronchial obstruction and in young smokers or young nonsmokers after carbachol inhalation. A two-compartment N(2) washout was demonstrated in 10 young healthy smokers at one or two respiratory frequencies whereas all 10 normal controls showed a single exponential curve. These findings indicate that the non-invasive N(2) washout test is capable of predicting C(dyn)/C(st) and at the same time gives a direct measure of gas distribution. Further, it appears to be a highly sensitive method for the detection of "small airway disease."

Integrated Exposure Assessment Survey (INES) exposure to persistent and bioaccumulative chemicals in Bavaria, Germany.

The Integrated Exposure Assessment Survey (INES) was started in the year 2005. Altogether 50 healthy adults living in Bavaria, Germany, were included into the study. Monitoring was conducted in accordance with relevant routes of human exposure (inhalation, ingestion) and integrated different pathways (indoor air, food, house dust). This approach consisted of a combination of external measurements of contaminants with the determination of these substances or their metabolites in body fluids. The target substances were phthalates, perfluorinated compounds (PFC), polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs), and polybrominated diphenylethers (PBDEs). This paper gives a brief description of the objectives and the concept of INES as well as methods of sampling and analyses of target compounds. Some preliminary results of biomonitoring data for PFC and phthalates as well as of the dietary intake of DEHP will be discussed.

Exclusion of an extracolonic disease modifier locus on chromosome 1p33-36 in a large Swiss familial adenomatous polyposis kindred.

Familial adenomatous polyposis (FAP), an autosomal dominantly inherited colorectal cancer predisposition syndrome, displays considerable inter- and intrafamilial phenotypic heterogeneity, which represents a major problem in genetic counselling of APC mutation carriers. The Min mouse model indicated a putative disease modifier locus on chromosome 4, which is syntenic to human chromosome 1p35-36. This finding was subsequently supported by parametric and nonparametric linkage analyses in FAP families, however, without identifying functional variants in candidate genes. Recently, germline mutations in the base-excision repair gene MYH (1p33-34) have been described in patients with multiple adenomas, pointing to a possible role as disease modifier in FAP. Here, we present critical reassessment of one of the largest FAP kindreds published, which was previously used in linkage mapping of 1p35-36. In this family, all affected members harbour the same APC germline mutation (5945delA), but display marked phenotypic variability, in particular regarding the occurrence of extracolonic disease that segregates in several branches of the family tree. Using updated clinical information, additional mutation carriers and polymorphic markers, fine mapping of the critical region as well as mutation analysis of the MYH gene were performed. These investigations allowed us to significantly exclude (i) the 1p33-36 region as a modifier locus and (ii) MYH as a modifier gene for extracolonic disease in this FAP kindred. Our results do not eliminate 1p33-36 from suspicion in other families, but clearly indicate that in our family linkage analysis of further putative candidate regions is necessary to identify a disease modifier locus in FAP.

The role of mucociliary dysfunction in bronchial asthma.

Abnormalities of mucociliary function in the airways of patients with bronchial asthma are suggested by the clinical observation of excessive tracheobronchial secretions which are difficult to expectorate and may contribute to bronchial obstruction. Pathologic and functional studies in animals and patients have demonstrated an impairment of mucociliary transport mechanisms, but the pathogenesis of this abnormality is still poorly understood. In patients with allergic asthma, the elaboration of chemical mediators in the lung seems to depress mucociliary function. Although pharmacologic agents which increase mucous transport rates have been identified, more potent stimulators will probably be needed to produce a clinical improvement in patients with bronchial asthma.

Effects of methylxanthines on airway mucociliary function.

Mucociliary interaction and hence mucus clearance in the airways is governed by ciliary activity and the depth and rheologic properties of periciliary fluid and mucus. Therefore, a defect in one or more of these component functions must be responsible for the impairment of mucociliary clearance in patients with a variety of airway diseases. Methylxanthines stimulate ciliary beat frequency, augment net ion secretion with a substantial increase in water flux toward the lumen, and promote mucus secretion in the lower airways. The net result is an enhancement of mucociliary clearance. This beneficial action of methylxanthines on mucociliary function may complement their effects on bronchoconstriction and respiratory muscle dysfunction in patients with chronic bronchitis, cystic fibrosis, and bronchial asthma.

Effect of ipratropium bromide on airway mucociliary function.

Airway mucociliary dysfunction leading to a depression of mucus transport has been demonstrated in patients with acute and chronic bronchitis, cystic fibrosis, and bronchial asthma; use of bronchodilators that might further impair mucociliary function, therefore, generally has been discouraged. Atropine and ipratropium bromide are cholinergic antagonists that are effective bronchodilators in various clinical settings. Atropine has been shown to block the production of respiratory secretions in response to cholinergic stimulation, but to have no effect on baseline secretions. Atropine has also been clearly demonstrated to depress ciliary beat frequency and to slow airway mucociliary clearance, whereas the short-term and long-term administration of ipratropium bromide at higher than clinically recommended doses seems to lack these effects. No satisfactory explanation has thus far been offered for this difference between the two cholinergic antagonists. Nevertheless, with respect to airway mucociliary function, ipratropium bromide appears to be preferable to atropine in the treatment of obstructive airways disease.

Pilot detection study of alpha(1) antitrypsin deficiency in a targeted population.

Screenings for the genetic disorder alpha(1) antitrypsin deficiency (AAT Deficiency) have been one of two models: large screenings of general populations and small targeted detection programs in high-risk groups. The most appropriate screening and detection methodologies in terms of target populations, subject participation and yield of positive tests, however, have not been well defined. The major objective of this pilot study was to evaluate the effectiveness in terms of participation of two different AAT Deficiency detection programs using a self-administered fingerstick blood test. Individuals ages 30-60 under the care of a pulmonary physician and with a diagnosis of emphysema, COPD, chronic bronchitis, or bronchiectasis were the targeted population. Participants were offered AAT Deficiency testing in the pulmonary physician's office compared with testing offered through mail. Participation (i.e., frequency of subject participation in the detection program) of two different AAT Deficiency detection programs. Non-participation was due to fear of self-administered testing and research studies; women were more likely to participate than men. Eligible subjects were significantly more likely to participate when offered testing by their pulmonary physician in-office (83%) than mail-only (42%) (P < 0.02). Although self-administered genetic testing is available, highest participation in AAT Deficiency detection program was found when offered directly by the physician. This finding may have implications for screening and detection of other genetic diseases. Future studies need to evaluate the yield (i.e., frequency of positive tests) of these detection methodologies in highly targeted populations.

The role of mucus in chronic obstructive pulmonary disease.

Chronic bronchitis is characterized by mucociliary dysfunction resulting from structural and functional defects of cilia and the secretory apparatus. The combination of hypersecretion and ciliary impairment leads to disruption of mucociliary interaction and hence the accumulation of secretions in the lower airways. Cigarette smoke appears to play a critical role in the pathogenesis of chronic bronchitis-associated mucociliary dysfunction. While the excessive lower airway secretions may have only minor effects on the natural course of airflow obstruction, they could transiently compromise airway function during acute exacerbations. In addition, altered aerosol deposition in the airways resulting from excessive airway secretions could influence the airway responses to inhaled irritants and pharmacologic agents. There are currently no direct, non-invasive methods available to assess the quantity and distribution of airway secretions in vivo. Indirect indices such as cough frequency, sputum volume, respiratory function, and mucociliary clearance are nonspecific and subject to misinterpretation. The clinical utility of mucotropic pharmacologic agents and of physical maneuvers directed at removing excessive lower airway secretions is therefore difficult to evaluate objectively.

Lung tumor in a patient with congenital unilateral hypoplasia of the pulmonary artery.

The occurrence of an undifferentiated carcinoma in the affected lung of a cigarette smoker with asymptomatic congenital hypoplasia of the right pulmonary artery is reported.

Effect of beta-adrenergic agonists aerosolized by freon propellant on tracheal mucous velocity and cardiac output.

The present study was designed to assess the effects of two beta-adrenergic agonists, isoproterenol sulfate and carbuterol hydrochloride, and aerosolized Freon propellant (a mixture of Freon II, Freon 12, and Freon 114) on tracheal mucous velocity and cardiac output in anesthetized dogs. Five groups of ten animals each received the following dosages of aerosols: Freon, 20 puffs; isoproterenol, four puffs; carbuterol, four puffs; isoproterenol, 20 puffs; and carbuterol, 20 puffs. The puff was delivered by a standard metered aerosol; each puff of isoproterenol spray contained 75 mug of isoproterenol sulfate, and each puff of carbuterol spray contained 100 mug of carbuterol hydrochloride. Tracheal mucous velocity was not changed by receiving Freon, but administration of both isoproterenol and carbuterol caused a significant increase in this measurement, with peak increases ranging from 74 to 111 percent above control values. The duration of action for four and 20 puffs of isoproterenol and for four puffs of carbuterol was two hours. Twenty puffs of carbuterol increased tracheal mucous velocity for three hours. Administration of carbuterol effected a slightly larger increase in cardiac output than isoproterenol. The duration of action for the increased cardiac output was shorter than the duration of action for the increased tracheal mucous velocity. These studies indicate that beta-adrenergic agonists may have an important role in improving mucous transport in patients with chronic obstructive pulmonary disease in whom mucociliary clearance is depressed.

Metaproterenol responsiveness after methacholine- and histamine-induced bronchoconstriction.

We investigated whether the bronchodilator response to a beta-adrenergic agonist is influenced by the mechanism of induced bronchoconstriction. Normal subjects and asymptomatic asthmatics inhaled a dry aerosol (mass median aerodynamic diameter, 1.5 microns) with increasing concentrations of methacholine or histamine to produce a 35% decrease in specific airway conductance (SGaw), followed by a single inhalation of a metaproterenol aerosol. By studying normal subjects and asthmatics, we were able to compare metaproterenol responsiveness after widely divergent doses of the bronchoprovocative agents but the same degree of bronchoconstriction. Airway deposition of methacholine, histamine, and metaproterenol was measured using a quinine fluorescence technique. Mean baseline SGaw, metaproterenol responsiveness, and metaproterenol mass deposited were similar in normal subjects and asthmatics. Likewise, mean SGaw after completion of methacholine and histamine challenge, and the subsequently deposited metaproterenol mass were similar in the two groups. After methacholine challenge (mean +/- SD provocative drug mass causing a 35% decrease in SGaw, PM35: 8.94 +/- 5.96 mumol in normal subject and 0.30 +/- 0.29 mumol in asthmatics), metaproterenol increased mean SGaw by 89 +/- 33% in normal subjects and by 190 +/- 55% in asthmatics (p < 0.05, two-way analysis of variance). After histamine challenge (PM35, 2.92 +/- 2.49 mumol in normal subjects and 0.17 +/- 0.29 mumol in asthmatics), metaproterenol increased mean SGaw by 111 +/- 38% in normal subjects and 113 +/- 69% in asthmatics (p = not significant). Thus, for the same degree of bronchoconstriction, metaproterenol responsiveness was influenced by the dose of methacholine but not the dose of histamine. The differential metaproterenol response could be related to a functional antagonism between muscarinic and beta-adrenergic agonists.

Effect of high-frequency oral airway and chest wall oscillation and conventional chest physical therapy on expectoration in patients with stable cystic fibrosis.

STUDY OBJECTIVE: To compare the effect of high-frequency oral airway oscillation, high-frequency chest wall oscillation, and conventional chest physical therapy (CPT) on weight of expectorated sputum, pulmonary function, and oxygen saturation in outpatients with stable cystic fibrosis (CF). DESIGN: Prospective randomized trial. SETTING: Pediatric pulmonary division of a tertiary care center. PATIENTS: Fourteen outpatients with stable CF recruited from the CF center. INTERVENTIONS: Two modes of oral airway oscillation (1: frequency 8 Hz; inspiratory to expiratory [I:E] ratio 9:1; 2: frequency 14 Hz; I:E ratio 8:1), two modes of chest wall oscillation (1: frequency 3 Hz; I:E ratio 4:1; 2: frequency 16 Hz; I:E ratio 1:1, alternating with frequency 1.5 Hz, I:E ratio 6:1), and CPT (clapping, vibration, postural drainage, and encouraged coughing) were applied during the first 20 min of 4 consecutive hours. MEASUREMENTS AND RESULTS: Sputum was collected on an hourly basis for a total of 6 consecutive hours. During the first and the last hour, patients collected sputum without having any treatment and underwent pulmonary function tests (PFTs). Oxygen saturation was measured at 30-min intervals during hours 1 to 6. For the first 20 min of the second to the fifth hour, patients received one of the treatments. To assess the effect of the intervention, the weight of expectorated sputum during hours 2 to 6 was averaged and expressed as percentage of the weight expectorated during the first hour (baseline). For the five treatment modalities, mean sputum dry and wet weights ranged between 122% and 185% of baseline. There was no statistically significant difference among the treatment modalities. As measured by sputum wet weight, all oscillatory devices tended to be less effective than CPT (p=0.15). As measured by dry weight, oral airway oscillation at 8 Hz with an I:E ratio of 9:1 and CPT tended to be more effective than the other treatment modalities (p=0.57). None of the treatment modalities had an effect on PFTs and oxygen saturation and all were well tolerated. CONCLUSION: In outpatients with stable CF, high-frequency oscillation applied via the airway opening or via the chest wall and CPT have comparable augmenting effects on expectorated sputum weight without changing PFTs or oxygen saturation. In contrast to CPT, high-frequency oral airway and chest wall oscillations are self-administered, thereby containing health-care expenses.

Distribution of ventilation in normal children.

Measurements of closing volume and the distribution fo ventilation by both single-breath (SBN2) and multiple-breath nitrogen washout methods were obtained in 376 healthy boys and girls, ages 6 to 18 years. A closing volume could be demonstrated in 39 percent of the subjects, and closing volume expressed as percentage of vital capacity did not change with height. Closing capacity expressed as percentage of total lung capacity showed a slight decrease with height. The slope of phase III of the SBN2 curve decreased with height. Single compartment N2 washout curves were observed in 72 percent of the subjects, and the incidence of single compartment curves increased with age. In those subjects with two compartment N2 washout curves, the relative compartmental ventilation became more even with increasing height. Our observations suggest that parallel units among peripheral airways grow at different rates.

Energy dissipation and path instabilities in dynamic fracture of silicon single crystals

Brittle fracture usually proceeds at crack driving forces which are larger than those needed to create the new fracture surfaces. This surplus can lead to faster crack propagation or to the onset of additional dissipation mechanisms. Dynamic fracture experiments on silicon single crystals reported here show several distinct transitions between different dissipation mechanisms. Cleavage fracture is followed by the propagation of a faceted crack front, which is finally followed by a path instability and the propagation of multiple cracks. The fracture surface qualitatively corresponds to the mirror, mist, and hackle morphology of amorphous materials. However, the corresponding fracture mechanisms, which remain largely unknown in the amorphous materials, can clearly be identified here.

Circulation of the airway mucosa.

The major part of tracheobronchial blood flow is distributed to the mucosa. Its microvasculature comprises 10-20% of the subepithelial tissue volume, with blood flow ranging from 30 to 95 ml.min-1.100 g wet tissue-1 in different animal species. Mucosal blood flow is influenced by vascular and airway pressures, inspired air conditions, and autonomic neurotransmitters. Several inflammatory mediators and neuropeptides are capable of enhancing the permeability for macromolecules in postcapillary venules and of augmenting tissue water volume, often with a concomitant increase in perfusion. These microvascular responses of the lower airway mucosa have an important role under various conditions of physiological stress and in airway inflammation.

Effects of induced bronchoconstriction on pulmonary blood flow.

Allergic bronchoconstriction may be associated with hemodynamic alterations due to changes in respiratory mechanics (or the associated changes in arterial blood gas composition) or the cardiovascular effects of chemical mediators. In an attempt to differentiate between these two possible mechanisms, we obtained measurements of hemodynamics, respiratory mechanics, and O2 consumption (VO2) in nine asymptomatic adult ragweed asthmatics before and after inhalation challenge with either ragweed extract or methacholine. We measured specific airway conductance (sGaw) by body plethysmography, pleural pressure with an esophageal balloon catheter, pulmonary blood flow (Q) and VO2 by a rebreathing technique, and heart rate. For a similar degree of bronchoconstriction after the two types of challenge (mean +/- SD sGaw 0.06 +/- 0.03 and 0.05 +/- 0.02 cmH2O-1 . s-1, P = NS), mean Q increased by 29 and 29%, and mean VO2 by 33 and 37% 15-20 min after ragweed and methacholine, respectively. Since heart rate did not change, there was a concomitant increase in mean stroke volume by 25 and 35%, respectively (P less than 0.05). The respiratory pleural pressure swings during quiet breathing and the rebreathing maneuver and the work of breathing during rebreathing also increased to a similar degree after the two types of challenge. These observations suggest that, if chemical mediators are released into the circulation during antigen-induced bronchoconstriction, their blood concentrations are too low for appreciable cardiovascular effects. The increase in rebreathing cardiac output during allergic and nonallergic bronchoconstriction is probably due to increases in intrathoracic pressure swings and in the work of breathing.

Adrenergic airway vascular smooth muscle responsiveness in healthy and asthmatic subjects.

The purpose of the present study was to determine the responsiveness of airway vascular smooth muscle (AVSM) as assessed by airway mucosal blood flow (Qaw) to inhaled methoxamine (alpha(1)-agonist; 0.6-2.3 mg) and albuterol (beta(2)-agonist; 0.2-1.2 mg) in healthy [n = 11; forced expiratory volume in 1 s, 92 +/- 4 (SE) % of predicted] and asthmatic (n = 11, mean forced expiratory volume in 1 s, 81 +/- 5%) adults. Mean baseline values for Qaw were 43.8 +/- 0.7 and 54.3 +/- 0.8 microl. min(-1). ml(-1) of anatomic dead space in healthy and asthmatic subjects, respectively (P < 0.05). After methoxamine inhalation, the maximal mean change in Qaw was -13.5 +/- 1.0 microl. min(-1). ml(-1) in asthmatic and -7.1 +/- 2.1 microl. min(-1). ml(-1) in healthy subjects (P < 0.05). After albuterol, the mean maximal change in Qaw was 3.0 +/- 0.8 microl. min(-1). ml(-1) in asthmatic and 14.0 +/- 1.1 microl. min(-1). ml(-1) in healthy subjects (P < 0.05). These results demonstrate that the contractile response of AVSM to alpha(1)-adrenoceptor activation is enhanced and the dilator response of AVSM to beta(2)-adrenoceptor activation is blunted in asthmatic subjects.