Migration of CD11b+ accessory cells during murine lung regeneration

In many mammalian species, the removal of one lung leads to growth of the remaining lung to near-baseline levels. In studying post-pneumonectomy mice, we used morphometric measures to demonstrate neoalveolarization within 21 days of pneumonectomy. Of note, the detailed histology during this period demonstrated no significant pulmonary inflammation. To identify occult blood-borne cells, we used a parabiotic model (wild-type/GFP) of post-pneumonectomy lung growth. Flow cytometry of post-pneumonectomy lung digests demonstrated a rapid increase in the number of cells expressing the hematopoietic membrane molecule CD11b; 64.5% of the entire GFP(+) population were CD11b(+). Fluorescence microscop…

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 venti…

Regulatory Network Of Angiogenesis Gene Expression During Post-Pneumonectomy Compensatory Growth

Mechanostructural adaptations preceding postpneumonectomy lung growth

In many species, pneumonectomy results in compensatory growth in the remaining lung. Although the late mechanical consequences of murine pneumonectomy are known, little is known about the anatomic adaptations and respiratory mechanics during compensatory lung growth. To investigate the structural and mechanical changes during compensatory growth, mice were studied for 21 days after left pneumonectomy using microCT and respiratory system impedance (FlexiVent). Anatomic changes after left pneumonectomy included minimal mediastinal shift or chestwall remodeling, but significant displacement of the heart and cardiac lobe. Mean displacement of the cardiac lobe centroid was 5.2 ± 0.8 mm. Lung imp…

Sprouting and intussusceptive angiogenesis in postpneumonectomy lung growth: mechanisms of alveolar neovascularization

In most rodents and some other mammals, the removal of one lung results in compensatory growth associated with dramatic angiogenesis and complete restoration of lung capacity. One pivotal mechanism in neoalveolarization is neovascularization, because without angiogenesis new alveoli can not be formed. The aim of this study is to image and analyze three-dimensionally the different patterns of neovascularization seen following pneumonectomy in mice on a sub-micron-scale. C57/BL6 mice underwent a left-sided pneumonectomy. Lungs were harvested at various timepoints after pneumonectomy. Volume analysis by microCT revealed a striking increase of 143 percent in the cardiac lobe 14 days after pneum…

Alveolar Epithelial Dynamics in Postpneumonectomy Lung Growth

The intimate anatomic and functional relationship between epithelial cells and endothelial cells within the alveolus suggests the likelihood of a coordinated response during postpneumonectomy lung growth. To define the population dynamics and potential contribution of alveolar epithelial cells to alveolar angiogenesis, we studied alveolar Type II and I cells during the 21 days after pneumonectomy. Alveolar Type II cells were defined and isolated by flow cytometry using a CD45(-) , MHC class II(+) , phosphine(+) phenotype. These phenotypically defined alveolar Type II cells demonstrated an increase in cell number after pneumonectomy; the increase in cell number preceded the increase in Type …

Alveolar macrophage dynamics in murine lung regeneration

In most mammalian species, the removal of one lung results in dramatic compensatory growth of the remaining lung. To investigate the contribution of alveolar macrophages (AMs) to murine post-pneumonectomy lung growth, we studied bronchoalveolar lavage (BAL)-derived AM on 3, 7, 14 and 21 days after left pneumonectomy. BAL demonstrated a 3.0-fold increase in AM (CD45(+), CD11b(-), CD11c(+), F4/80(+), Gr-1(-)) by 14 days after pneumonectomy. Cell cycle flow cytometry of the BAL-derived cells demonstrated an increase in S + G2 phase cells on days 3 (11.3 ± 2.7%) and 7 (12.1 ± 1.8%) after pneumonectomy. Correspondingly, AM demonstrated increased expression of VEGFR1 and MHC class II between days…

Pulmonary Mechanics Suggest Mechanical Forces Trigger Neoalveolarization In A Murine Model Of Compensatory Lung Growth

Mapping cyclic stretch in the postpneumonectomy murine lung

In many mammalian species, the removal of one lung [pneumonectomy (PNX)] is associated with the compensatory growth of the remaining lung. To investigate the hypothesis that parenchymal deformation may trigger lung regeneration, we used respiratory-gated micro-computed tomography scanning to create three-dimensional finite-element geometric models of the murine cardiac lobe with cyclic breathing. Models were constructed of respiratory-gated micro-computed tomography scans pre-PNX and 24 h post-PNX. The computational models demonstrated that the maximum stretch ratio map was patchy and heterogeneous, particularly in subpleural, juxta-diaphragmatic, and cephalad regions of the lobe. In these…

Dendritic Cell Migration To The Post Pneumonectomy Lung

Selective Laser Photocoagulation Manipulates Blood Flow Dynamics in Microcirculation

Sequence of vascular patterning and gene transcription in the chick chorioallantoic membrane (15.1)

Introduction: The chick chorioallantoic membrane (CAM) is a well-established model of both vasculogenesis and angiogenesis; however, little is known about the genetic control of vascular patterning in the CAM. Methods: Using recent advances in chicken genomics, we investigated the relative expression of 84 angiogenesis genes during the growth and remodeling of the CAM microcirculatory network. Chick embryos, cultured ex ovo, were studied during embryonic development days (EDD) 8-14. UV laser microdissection was used to harvest capillary plexus and 1st, 2nd, and 3rd order conducting vessels for qRT-PCR analysis. Results: Two transcription peaks were observed between EDD 8 and 14. The first p…

Post‐pneumonectomy Lung Deformation is Associated with Alveolar Type II Cell Apoptosis and Altered Parenchymal Mechanics

Regulation Of Post-Pneumonectomy Angiogenesis By Type II Pneumocytes

Molecular Imaging in the Regenerating Post Pneumonectomy Lung

Intravascular pillars and pruning in the extraembryonic vessels of chick embryos.

To investigate the local mechanical forces associated with intravascular pillars and vessel pruning, we studied the conducting vessels in the extraembryonic circulation of the chick embryo. During the development days 13-17, intravascular pillars and blood flow parameters were identified using fluorescent vascular tracers and digital time-series video reconstructions. The geometry of selected vessels was confirmed by corrosion casting and scanning electron microscopy. Computational simulations of pruning vessels suggested that serial pillars form along pre-existing velocity streamlines; blood pressure demonstrated no obvious spatial relationship with the intravascular pillars. Modeling a Re…

Free-Floating Mesothelial Cells in Pleural Fluid After Lung Surgery

Objectives: The mesothelium, the surface layer of the heart, lung, bowel, liver and tunica vaginalis, is a complex tissue implicated in organ-specific diseases and regenerative biology; however, the mechanism of mesothelial repair after surgical injury is unknown. Previous observations indicated seeding of denuded mesothelium by free-floating mesothelial cells may contribute to mesothelial healing. In this study, we investigated the prevalence of mesothelial cells in pleural fluid during the 7 days following pulmonary surgery. Study design: Flow cytometry was employed to study pleural fluid of 45 patients after lung resection or transplantation. We used histologically validated mesothelial …

CD34+ progenitor to endothelial cell transition in post-pneumonectomy angiogenesis.

In many species, pneumonectomy triggers compensatory lung growth that results in an increase not only in lung volume, but also in alveolar number. Whether the associated alveolar angiogenesis involves the contribution of blood-borne progenitor cells is unknown. To identify and characterize blood-borne progenitor cells contributing to lung growth after pneumonectomy in mice, we studied wild-type and wild-type/green fluorescence protein (GFP) parabiotic mice after left pneumonectomy. Within 21 days of pneumonectomy, a 3.2-fold increase occurred in the number of lung endothelial cells. This increase in total endothelial cells was temporally associated with a 7.3-fold increase in the number of …

Structural contribution of intravascular blood distension to lung mechanics

Scaffolding Effect of Vascular Distension on Peripheral Lung Mechanics

Computational analysis of lung deformation after murine pneumonectomy. [corrected].

In many mammalian species, the removal of one lung (pneumonectomy) is associated with the compensatory growth of the remaining lung. To investigate the hypothesis that parenchymal deformation may trigger lung regeneration, we used microCT scanning to create 3-dimensional finite element geometric models of the murine lung pre- and post-pneumonectomy (24 hours). The structural correspondence between models was established using anatomic landmarks and an iterative computational algorithm. When compared with the pre-pneumonectomy lung, the post-pneumonectomy models demonstrated significant translation and rotation of the cardiac lobe into the post-pneumonectomy pleural space. 2-dimensional maps…

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

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 (4.0α2.5 cmH2O); however, there was minimal difference between conditions on deflation (0.7α0.9 cmH2O). 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 abse…

Mechanical Evidence Of Microstructural Remodeling During Post-Pneumonectomy Compensatory Lung Growth

Cross-circulation and Cell Distribution Kinetics in Parabiotic Mice

Blood-borne nucleated cells participate not only in inflammation, but in tissue repair and regeneration. Because progenitor and stem cell populations have a low concentration in the blood, the circulation kinetics and tissue distribution of these cells is largely unknown. An important approach to tracking cell lineage is the use of fluorescent tracers and parabiotic models of cross-circulation. Here, we investigated the cross-circulation and cell distribution kinetics of C57/B6 GFP(+)/wild-type parabionts. Flow cytometry analysis of the peripheral blood after parabiosis demonstrated no evidence for a "parabiotic barrier" based on cell size or surface characterstics; all peripheral blood cel…

Dynamic determination of oxygenation and lung compliance in murine pneumonectomy.

Thoracic surgical procedures in mice have been applied to a wide range of investigations, but little is known about the murine physiologic response to pulmonary surgery. Using continuous arterial oximetry monitoring and the FlexiVent murine ventilator, the authors investigated the effect of anesthesia and pneumonectomy on mouse oxygen saturation and lung mechanics. Sedation resulted in a dose-dependent decline of oxygen saturation that ranged from 55% to 82%. Oxygen saturation was restored by mechanical ventilation with increased rate and tidal volumes. In the mouse strain studied, optimal ventilatory rates were a rate of 200/minute and a tidal volume of 10 mL/kg. Sustained inflation pressu…