Janeil Belle

Effect of unilateral diaphragmatic paralysis on postpneumonectomy lung growth

Respiratory muscle-associated stretch has been implicated in normal lung development (fetal breathing movements) and postpneumonectomy lung growth. To test the hypothesis that mechanical stretch from diaphragmatic contraction contributes to lung growth, we performed left phrenic nerve transections (PNT) in mice with and without ipsilateral pneumonectomy. PNT was demonstrated by asymmetric costal margin excursion and confirmed at autopsy. In mice with two lungs, PNT was associated with a decrease in ipsilateral lung volume (P<0.05) and lung weight (P<0.05). After pneumonectomy, PNT was not associated with a change in activity level, measureable hypoxemia, or altered minute ventilation; however, microCT scanning demonstrated altered displacement and underinflation of the cardiac lobe within the first week after pneumonectomy. Coincident with the altered structural realignment, lung impedance measurements, fitted to the constant-phase model, demonstrated elevated airway resistance (P<0.05), but normal peripheral tissue resistance (P>0.05). Most important, PNT appeared to abrogate compensatory lung growth after pneumonectomy; the weight of the lobes of the right lung was significantly less than pneumonectomy alone (P<0.001) and indistinguishable from nonsurgical controls (P>0.05). We conclude that the cyclic stretch associated with diaphragmatic muscle contraction is a controlling factor in postpneumonectomy compensatory lung growth.

Elastin Cables Define the Axial Connective Tissue System in the Murine Lung.

The axial connective tissue system is a fiber continuum of the lung that maintains alveolar surface area during changes in lung volume. Although the molecular anatomy of the axial system remains undefined, the fiber continuum of the lung is central to contemporary models of lung micromechanics and alveolar regeneration. To provide a detailed molecular structure of the axial connective tissue system, we examined the extracellular matrix of murine lungs. The lungs were decellularized using a 24 hr detergent treatment protocol. Systematic evaluation of the decellularized lungs demonstrated no residual cellular debris; morphometry demonstrated a mean 39 ± 7% reduction in lung dimensions. Scanning electron microscopy (SEM) demonstrated an intact structural hierarchy within the decellularized lung. Light, fluorescence, and SEM of precision‐cut lung slices demonstrated that alveolar duct structure was defined by a cable line element encased in basement membrane. The cable line element arose in the distal airways, passed through septal tips and inserted into neighboring blood vessels and visceral pleura. The ropelike appearance, collagenase resistance and anti‐elastin immunostaining indicated that the cable was an elastin macromolecule. Our results indicate that the helical line element of the axial connective tissue system is composed of an elastin cable that not only defines the structure of the alveolar duct, but also integrates the axial connective tissue system into visceral pleura and peripheral blood vessels. Anat Rec, 298:1960–1968, 2015.

Laser Microdissection of the Alveolar Duct Enables Single-Cell Genomic Analysis

Complex tissues such as the lung are composed of structural hierarchies such as alveoli, alveolar ducts, and lobules. Some structural units, such as the alveolar duct, appear to participate in tissue repair as well as the development of bronchioalveolar carcinoma. Here, we demonstrate an approach to conduct laser microdissection of the lung alveolar duct for single-cell PCR analysis. Our approach involved three steps. (1) The initial preparation used mechanical sectioning of the lung tissue with sufficient thickness to encompass the structure of interest. In the case of the alveolar duct, the precision-cut lung slices were 200 μm thick; the slices were processed using near-physiologic conditions to preserve the state of viable cells. (2) The lung slices were examined by transmission light microscopy to target the alveolar duct. The air-filled lung was sufficiently accessible by light microscopy that counterstains or fluorescent labels were unnecessary to identify the alveolar duct. (3) The enzymatic and microfluidic isolation of single cells allowed for the harvest of as few as several thousand cells for PCR analysis. Microfluidics based arrays were used to measure the expression of selected marker genes in individual cells to characterize different cell populations. Preliminary work suggests the unique value of this approach to understand the intra- and intercellular interactions within the regenerating alveolar duct.

Stretch-induced intussuceptive and sprouting angiogenesis in the chick chorioallantoic membrane.

Vascular systems grow and remodel in response to not only metabolic needs, but also mechanical influences as well. Here, we investigated the influence of tissue-level mechanical forces on the patterning and structure of the chick chorioallantoic membrane (CAM) microcirculation. A dipole stretch field was applied to the CAM using custom computer-controlled servomotors. The topography of the stretch field was mapped using finite element models. After 3days of stretch, Sholl analysis of the CAM demonstrated a 7-fold increase in conducting vessel intersections within the stretch field (p<0.01). The morphometric analysis of intravital microscopy and scanning electron microscopy (SEM) images demonstrated that the increase vessel density was a result of an increase in interbranch distance (p<0.01) and a decrease in bifurcation angles (p<0.01); there was no significant increase in conducting vessel number (p>0.05). In contrast, corrosion casting and SEM of the stretch field capillary meshwork demonstrated intense sprouting and intussusceptive angiogenesis. Both planar surface area (p<0.05) and pillar density (p<0.01) were significantly increased relative to control regions of the CAM. We conclude that a uniaxial stretch field stimulates the axial growth and realignment of conducting vessels as well as intussusceptive and sprouting angiogenesis within the gas exchange capillaries of the ex ovo CAM.

Structural and functional evidence for the scaffolding effect of alveolar blood vessels

ABSTRACT A contribution of pulmonary blood distension to alveolar opening was first proposed more than 100 years ago. To investigate the contribution of blood distension to lung mechanics, we studied control mice (normal perfusion), mice after exsanguination (absent perfusion) and mice after varying degrees of parenchymal resection (supra-normal perfusion). On inflation, mean tracheal pressures were higher in the bloodless mouse (4.0 ± 2.5 cm H2O); however, there was minimal difference between conditions on deflation (0.7 ± 0.9 cm H2O). To separate the peripheral and central mechanical effects of blood volume, multi-frequency lung impedance data was fitted to the constant-phase model. The presence or absence of blood had no effect on central airway resistance (p > .05). In contrast, measures of tissue damping (G), tissue elastance (H) and hysteresivity (η) demonstrated a significant increase in bloodless mice relative to control mice (p < .001). After varying amount of surgical resection and associated supra-normal perfusion of the remaining lung, there was an increase in G and H. Although the absolute difference in G and H increased with the amount of parenchymal resection, the proportional contribution of blood was identical in all conditions. The presence of blood in the pulmonary vasculature resulted in a constant 64 ± 5% reduction in tissue damping (G) and a 55 ± 4% reduction in tissue elastance (H). This nearly-constant contribution of blood to lung hysteresivity was only reduced by positive end-expiratory pressure (PEEP). To identify a distinct structural subset of vessels in the lung potentially contributing to these observations, vascular casting and scanning electron microscopy of the lung demonstrated morphologically distinct vascular rings at the alveolar opening. Our results suggest that intravascular blood distension, likely attributable to a subset of vessels in the alveolar entrance ring, contributes a measurable scaffolding effect to the functional recruitment of the peripheral lung.

Extracellular Assembly of the Elastin Cable Line Element in the Developing Lung: ELASTIN LINE ELEMENT IN DEVELOPING LUNG

In the normal lung, a dominant structural element is an elastic “line element” that originates in the central bronchi and inserts into the distal airspaces. Despite its structural importance, the process that leads to development of the cable line element is unknown. To investigate the morphologic events contributing to its development, we used optical clearing methods to examine the postnatal rat lung. An unexpected finding was numerous spheres, with a median diameter of 1–2 µm, within the primary septa of the rat lung. The spheres demonstrated green autofluorescence, selective fluorescent eosin staining, reactivity with carboxyfluorescein succinimidyl ester, and specific labeling with anti‐tropoelastin monoclonal antibody—findings consistent with tropoelastin. The sphere number peaked on rat postnatal day 4 (P4) and were rare by P14. The disappearance of the spheres was coincident with the development of the cable line element in the rat lung. Transmission electron microscopy demonstrated no consistent association between parenchymal cells and sphere alignment. In contrast, the alignment of tropoelastin spheres appeared to be the direct result of interactions of scaffold proteins including collagen fibers and fibrillin microfibrils. We conclude that the spatial organization of the cable line element appears to be independent of tropoelastin deposition, but dependent on crosslinking to scaffold proteins within the primary septa. Anat Rec, 300:1670–1679, 2017.

Transcriptional Regulation of Post-Pneumonectomy Angiogenesis in Peripheral Lung Regeneration

RESULTS: We identified 8,930 lung resection patients; 59.5% of resections were performed for lung cancer and of these, 73% were lobectomy. Overall, 56% of cases were performed using VATS. Morbidity and mortality rates were 9% and 1.2% for VATS vs 15.3% and 2% for THOR (p<0.001). Median length of stay was 4 days for VATS (interquartile range [IQR] 2-6 days), and 5 days (IQR 4-8 days) for THOR (p<0.001). Unplanned readmission occurred within 30 days in 7.4% of patients and was not dependent on surgical approach or preoperative diagnosis. The reason for readmission was more likely related to air leak in the VATS group vs infections in the THOR group. Multivariate predictors of readmission included American Society of Anesthesiologists (ASA) 3, pneumonectomy, and the occurrence of postoperative complications as shown in the table.