W. Mitzner

Disruption of Nrf2 enhances susceptibility to severe airway inflammation and asthma in mice

Oxidative stress has been postulated to play an important role in the pathogenesis of asthma; although a defect in antioxidant responses has been speculated to exacerbate asthma severity, this has been difficult to demonstrate with certainty. Nuclear erythroid 2 p45-related factor 2 (Nrf2) is a redox-sensitive basic leucine zipper transcription factor that is involved in the transcriptional regulation of many antioxidant genes. We show that disruption of the Nrf2 gene leads to severe allergen-driven airway inflammation and hyperresponsiveness in mice. Enhanced asthmatic response as a result of ovalbumin sensitization and challenge in Nrf2-disrupted mice was associated with more pronounced mucus cell hyperplasia and infiltration of eosinophils into the lungs than seen in wild-type littermates. Nrf2 disruption resulted in an increased expression of the T helper type 2 cytokines interleukin (IL)-4 and IL-13 in bronchoalveolar lavage fluid and in splenocytes after allergen challenge. The enhanced severity of the asthmatic response from disruption of the Nrf2 pathway was a result of a lowered antioxidant status of the lungs caused by lower basal expression, as well as marked attenuation, of the transcriptional induction of multiple antioxidant genes. Our studies suggest that the responsiveness of Nrf2-directed antioxidant pathways may act as a major determinant of susceptibility to allergen-mediated asthma.

In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry

The mouse is the species of choice for creating genetically engineered models of human disease. To study detailed systolic and diastolic left ventricular (LV) chamber mechanics in mice in vivo, we developed a miniaturized conductance-manometer system. alpha-Chloralose-urethan-anesthetized animals were instrumented with a two-electrode pressure-volume catheter advanced via the LV apex to the aortic root. Custom electronics provided time-varying conductances related to cavity volume. Baseline hemodynamics were similar to values in conscious animals: 634 +/- 14 beats/min, 112 +/- 4 mmHg, 5.3 +/- 0.8 mmHg, and 11,777 +/- 732 mmHg/s for heart rate, end-systolic and end-diastolic pressures, and maximum first derivative of ventricular pressure with respect to time (dP/dtmax), respectively. Catheter stroke volume during preload reduction by inferior vena caval occlusion correlated with that by ultrasound aortic flow probe (r2 = 0.98). This maneuver yielded end-systolic elastances of 79 +/- 21 mmHg/microliter, preload-recruitable stroke work of 82 +/- 5.6 mmHg, and slope of dP/dtmax-end-diastolic volume relation of 699 +/- 100 mmHg.s-1.microliter-1, and these relations varied predictably with acute inotropic interventions. The control normalized time-varying elastance curve was similar to human data, further supporting comparable chamber mechanics between species. This novel approach should greatly help assess cardiovascular function in the blood-perfused murine heart.The mouse is the species of choice for creating genetically engineered models of human disease. To study detailed systolic and diastolic left ventricular (LV) chamber mechanics in mice in vivo, we developed a miniaturized conductance-manometer system. α-Chloralose-urethan-anesthetized animals were instrumented with a two-electrode pressure-volume catheter advanced via the LV apex to the aortic root. Custom electronics provided time-varying conductances related to cavity volume. Baseline hemodynamics were similar to values in conscious animals: 634 ± 14 beats/min, 112 ± 4 mmHg, 5.3 ± 0.8 mmHg, and 11,777 ± 732 mmHg/s for heart rate, end-systolic and end-diastolic pressures, and maximum first derivative of ventricular pressure with respect to time (dP/d t max), respectively. Catheter stroke volume during preload reduction by inferior vena caval occlusion correlated with that by ultrasound aortic flow probe ( r 2 = 0.98). This maneuver yielded end-systolic elastances of 79 ± 21 mmHg/μl, preload-recruitable stroke work of 82 ± 5.6 mmHg, and slope of dP/d t max-end-diastolic volume relation of 699 ± 100 mmHg ⋅ s-1 ⋅ μl-1, and these relations varied predictably with acute inotropic interventions. The control normalized time-varying elastance curve was similar to human data, further supporting comparable chamber mechanics between species. This novel approach should greatly help assess cardiovascular function in the blood-perfused murine heart.

Effect of thoracic blood volume changes on steady state cardiac output.

We have investigated the extent to which shifts of blood volume out of or into the thoracic region influence the steady state cardiac output. The systemic circulation of anesthetized dogs was replaced with an artificial circuit which simulated the pertinent mechanical characteristics of an intact circulation. As in the normal animal, the steady state venous return was proportional to the pressure gradient for venous return (i.e., mean systemic minus right atrial pressure). Cardiac function was altered either by administration of epinephrine or by changes in left ventricular afterload. At a constant mean aortic pressure of 100 mm Hg, epinephrine administration increased the steady state cardiac output by 55%. Half of this increase resulted from the lowered mean right atrial pressure (caused by improved cardiac function); the remainder resulted from an increased mean systemic pressure (caused by the volume shift to the systemic circulation). Increases in afterload transferred sufficient volume to the heart-lung compartment to reduce significantly the mean systemic pressure and, hence, the steady state venous return. Our results indicate that the heart-lung compartment contains a significant volume which is under cardiac control. In addition to being able to alter the right atrial pressure, the heart can modulate the steady state cardiac output by adjusting the mean systemic pressure. To this degree the heart can adjust its own venous return.

Quantitative 3D reconstruction of airway and pulmonary vascular trees using HRCT

Accurate quantitative measurements of airway and vascular dimensions are essential to evaluate function in the normal and diseased lung. In this report, a novel method is described for three-dimensional extraction and analysis of pulmonary tree structures using data from High Resolution Computed Tomography (HRCT). Serially scanned two-dimensional slices of the lower left lobe of isolated dog lungs were stacked to create a volume of data. Airway and vascular trees were three-dimensionally extracted using a three dimensional seeded region growing algorithm based on difference in CT number between wall and lumen. To obtain quantitative data, we reduced each tree to its central axis. From the central axis, branch length is measured as the distance between two successive branch points, branch angle is measured as the angle produced by two daughter branches, and cross sectional area is measured from a plane perpendicular to the central axis point. Data derived from these methods can be used to localize and quantify structural differences both during changing physiologic conditions and in pathologic lungs.

Interaction between high frequency jet ventilation and cardiovascular function

We have studied the interaction of high frequency jet ventilation with cardiovascular pressures and flows. Results in dogs show that the amplitude of all intrathoracic pressures and flows fluctuate with a frequency equal to the difference between the heart rate and ventilator rate. The magnitude of this amplitude variation may be sufficient to obliterate periodically the pulsations in pulmonary artery and right atrial pressures. It is also shown that these cardiovascular beats can occur when the ventilator rate is close to integral multiples of the heart rate. Direct measurement of pleural pressure and the observation that the beats are markedly reduced when the chest is open support the hypothesis that the primary mechanism responsible for these beats is the interaction of the respiratory fluctuations in pleural pressure with the cardiacgenerated pressure pulsations.

Assessment of air space size and surface area

to the editor: Dr. Knudsen and colleagues (1) are to be congratulated for a very important paper, comparing two different approaches for quantifying air space chord lengths in fixed lungs. The ability to make these measurements in an unbiased manner is very important in assessing pathological changes in emphysema models. The paper also highlights the relation between mean air space chord length (Lm; with either method) and lung volume, which is often overlooked by many investigators. However, with regard to this latter point, there is an issue in the data that is not entirely clear. In the rabbit model, the lung is inflated such that the lung air volume at 80% of total lung capacity (D80) is increased by a factor of 2 compared with that at 40% of total lung capacity (D40) (Table 1, 72 vs. 36 ml). Such a doubling of volume would be consistent with a ≈25% increase in any linear dimension of similar structures. However, the reported measurements of Lm show increases of 80% with the direct method and 64% with the indirect method. Thinking in the other direction, an 80% increase in linear dimension would be consistent with over a fivefold increase in air volume, but this is not even close to what was measured. It is not clear what accounts for this large discrepancy, but it clearly will also lead to a substantial underestimate of the calculated internal surface area. Since the surface area calculation assumes a similar shape, perhaps the shifting volume between alveoli and ducts observed contributes to an error of this magnitude. However, a similar error is present in the treated and untreated surfactant protein-D null mice, which show a smaller change in duct/alveoli partitioning. We hope that the authors may be able to clarify why there is such a discrepancy between the Lm and lung volume measurements, and when it is appropriate to use Eq. 1 [Lm = 4·(V/S), where V/S is volume-to-surface ratio] to calculate surface area in other experimental models with pathophysiological changes in the lung parenchyma. If the surface area calculation is so exquisitely sensitive to the duct/alveoli partitioning, then is it still useful to use it as a reliable index of pathological changes in structure?

Effect of Bronchial Thermoplasty on Airway Closure

Background Bronchial Thermoplasty, a procedure that applies thermal energy to the airway wall has been shown to impair the ability of airway to contract in response to methacholine chloride (Mch). The technique has been advocated as an alternative treatment for asthma that may permanently limit airway narrowing. In previous experimental studies in dogs and humans, it was shown that those airways treated with bronchial thermoplasty had significant impairment of Mch responsiveness. Methods In the present study, we investigated the ability of canine airways to close completely with very high concentrations of Mch after bronchial thermoplasty. Bronchial thermoplasty was performed on dogs using the Alair System, comprising a low power RF controller and a basket catheter with four electrodes. A local atomization of Mch agonist was delivered directly to the epithelium of the same airway locations with repeated challenges. Airway size was measured with computed tomography, and closure was considered to occur in any airway where the lumen fell below the resolution of the scanner (< 1 mm). Results Our results show that, while treated airways still have the capacity to close at very high doses of Mch, this ability is seriously impaired after treatment, requiring much higher doses. Conclusions Bronchial thermoplasty as currently applied seems to simply shift the entire dose response curve toward increasing airway size. Thus, this procedure simply serves to minimize the ability of airways to narrow under any level of stimulation.

Computer models for computation and verification of bronchial morphology

Accurate physiological measurements of the parameters like branching angles, branch lengths, and diameters of bronchial tree structures help in addressing the mechanistic and diagnostic questions related to obstructive lung disease. In order to facilitate these measurements, bronchial trees are reduced to a central axis tree. The approach we take employs first setting up a theoretical computerized tree structure, and then applying a 3D analysis to obtain the required anatomical data. A stick model was set up in 3D, with segment endpoints and diameters as input parameters to the model generator. By fixing the direction in which the slices are taken, a stack of 2D images of the generated 3D tree model is obtained, thereby simulating bronchial data sets. We design a two pass algorithm to compute the central axis tree and apply it on our models. In the first pass, the topological tree T is obtained by implementing a top-down seeded region growing algorithm of the 3D tree model. In the second pass, T is used to region growth along the axes of the branches. As the 3D tree model is traversed bottom-up, the centroid values of the cross sections of the branches are stored in the corresponding branch of T. At each bifurcation, the branch point and the three direction vectors along the branches are computed, by formulating it as a nonlinear optimization problem that minimizes the sum of least squares error of the centroid points of the corresponding branches. By connecting the branch points with straight lines, we obtain a reconstructed central axis tree which closely corresponds to the input stick model. We also studied the effect of adding external noise to out tree models and evaluating the physiological parameters. We conclude with the results of our algorithm on real airway trees.

Leukocyte Transit Through The Airway Circulation

In pathological conditions such as asthma and COPD, it is likely that the tracheal-bronchial circulations are pivotal in the recruitment of circulating leukocytes. Furthermore, the importance of the airway circulation for the recruitment of inflammatory cells after the inhalation of airway allergens is often implicitly assumed. However, little data exist to confirm and quantify the process of leukocyte recruitment in real time. Airway inflammation requires an orchestrated series of molecular events whereby inflammatory cells leave the airway vasculature and migrate within the airway wall. Leukocytes are initially tethered to the endothelial cell surface of post-capillary venules, then roll, before firmly adhering and subsequently migrating out of the vasculature. Herein we report our methods to study leukocyte recruitment to the airways in real time using intravital microscopy. We report our observations using this experimental approach to visualize and quantify the effects of airways inflammation after challenge with inflammatory (LPS and fMLP) (Lim et al., 2002) and mechanical (PEEP) stimuli (Lim & Wagner, 2003).

Effect of lung orientation on measurement and resolvability of airways using computed tomography

Volumetric data sets in the lung are formed by stacking sequential computed tomography (CT) slices. The resulting volumetric image file, however, is nonuniformly resolved; the in-plane resolution is greater than that in the z-axis (slice thickness) dimension. The purpose of this study was to determine the effect of branch orientation within the image volume on the measurement and resolvability of airway branches. An isolated canine lobe was sequentially scanned at three orientations with a Siemens Somotom Plus S CT Scanner using a 124.4 mm field of view, 137 kVp, 220 mAs, a 2 mm slice thickness, and a 1 mm table feed. A grid size of 2562, resulted in an in- plane pixel dimension of 0.49 mm. The lobe was inflated to an airway pressure of 20 cm H2O with the main bronchus of the lobe aligned approximately perpendicular to the scan plane. The entirety of the lobe was scanned at this orientation. The inflated lobe was then reoriented 90 degree(s) clockwise (as if the lobe was sitting on a clock face) and again the entirety of the lobe was scanned. The lobe was rotated a third time 90 degree(s) in plane from the first lobe alignment and again rescanned. Airway trees were segmented for each orientation and there were significant differences in the number of resolved branches among the three segmented trees. Measurement of airways over four millimeters in diameter was not affected by orientation. Airways smaller than two millimeters in diameter showed surprising similarity in measured diameter, but all airways were not resolved at all orientations. The angle of orientation of the individual airways with respect to the scan axis was therefore calculated to determine the angle at which a branch of given diameter was no longer resolved. There was surprising similarity of measured diameter in these smaller airways with orientation, if the branch was resolved. The orientation at which branches were resolved was quite variable, suggesting a high degree of randomness in the segmented branches at this level of branch resolution.