Detection of Murine Post-Pneumonectomy Lung Regeneration by 18FDG PET Imaging

Background: An intriguing biologic process in most adult mammals is post-pneumonectomy lung regeneration, that is, the removal of one lung (pneumonectomy) results in the rapid compensatory growth of the remaining lung. The spatial dependence and metabolic activity of the rodent lung during compensatory lung regeneration is largely unknown. Methods: To determine if murine lung regeneration could be detected in vivo, we studied inbred mice 3, 7, 14, and 21 days after left pneumonectomy. The remaining lung was imaged using microCT as well as the glucose tracer 2-deoxy-2-[18 F]fluoro-d-glucose (18FDG) and positron-emission tomography (PET). Because of the compliance of the murine chest wall, reproducible imaging required orotracheal intubation and pressure-controlled ventilation during scanning. Results: After left pneumonectomy, the right lung progressively enlarged over the first 3 weeks. The cardiac lobe demonstrated the greatest percentage increase in size. Dry weights of the individual lobes largely mirrored the increase in lung volume. PET/CT imaging was used to identify enhanced metabolic activity within the individual lobes. In the cardiac lobe, 18FDG uptake was significantly increased in the day 14 cardiac lobe relative to preoperative values (p < .05). In contrast, the 18FDG uptake in the other three lobes was not statistically significant at any time point. Conclusions: We conclude that the cardiac lobe is the dominant contributor to compensatory growth after murine pneumonectomy. Further, PET/CT scanning can detect both the volumetric increase and the metabolic changes associated with the regenerative growth in the murine cardiac lobe

Brain angioarchitecture and intussusceptive microvascular growth in a murine model of Krabbe disease

Abstract Defects of the angiogenic process occur in the brain of twitcher mouse, an authentic model of human Krabbe disease caused by genetic deficiency of lysosomal b-galactosylceramidase (GALC), leading to lethal neurological dysfunctions and accumulation of neurotoxic psychosine in the central nervous system. Here, quantitative computational analysis was used to explore the alterations of brain angioarchitecture in twitcher mice. To this aim, customized ImageJ routines were used to assess calibers, amounts, lengths and spatial dispersion of CD31? vessels in 3D volumes from the postnatal frontal cortex of twitcher animals. The results showed a decrease in CD31 immunoreactivity in twitcher brain with a marked reduction in total vessel lengths coupled with increased vessel fragmentation. No significant changes were instead observed for the spatial dispersion of brain vessels throughout volumes or in vascular calibers. Notably, no CD31? vessel changes were detected in twitcher kidneys in which psychosine accumulates at very low levels, thus confirming the specificity of the effect. Microvascular corrosion casting followed by scanning electron microscopy morphometry confirmed the presence of significant alterations of the functional angioarchitecture of the brain cortex of twitcher mice with reduction in microvascular density, vascular branch remodeling and intussusceptive angiogenesis. Intussusceptive microvascular growth, con- firmed by histological analysis, was paralleled by alterations of the expression of intussusception-related genes in twitcher brain. Our data support the hypothesis that a marked decrease in vascular development concurs to the onset of neuropathological lesions in twitcher brain and suggest that neuroinflammation-driven intussusceptive responses may represent an attempt to compensate impaired sprouting angiogenesis

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

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 pneumonectomy. Analysis of microvascular corrosion casting demonstrated spatially heterogenous vascular densitities which were in line with the perivascular and subpleural compensatory growth pattern observed in anti-PCNA-stained lung sections. Within these regions an expansion of the vascular plexus with increased pillar formations and sprouting angiogenesis, originating both from pre-existing bronchial and pulmonary vessels was observed. Also, type II pneumocytes and alveolar macrophages were seen to participate actively in alveolar neo-angiogenesis after pneumonectomy. 3D-visualizations obtained by high-resolution synchrotron radiation X-ray tomographic microscopy showed the appearance of double-layered vessels and bud-like alveolar baskets as have already been described in normal lung development. Scanning electron microscopy data of microvascular architecture also revealed a replication of perialveolar vessel networks through septum formation as already seen in developmental alveolarization. In addition, the appearance of pillar formations and duplications on alveolar entrance ring vessels in mature alveoli are indicative of vascular remodeling. These findings indicate that sprouting and intussusceptive angiogenesis are pivotal mechanisms in adult lung alveolarization after pneumonectomy. Various forms of developmental neoalveolarization may also be considered to contribute in compensatory lung regeneration. Electronic supplementary material The online version of this article (doi:10.1007/s10456-013-9399-9) contains supplementary material, which is available to authorized users

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 3 days of stretch, Sholl analysis of the CAM demonstrated a 7-fold increase in conducting vessel intersections within the stretch field (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.National Institutes of Health (U.S.) (NIH grant HL95678

Resection of the mesopancreas (RMP): a new surgical classification of a known anatomical space

BACKGROUND: Prognosis after surgical therapy for pancreatic cancer is poor and has been attributed to early lymph node involvement as well as to a strong tendency of cancer cells to infiltrate into the retropancreatic tissue and to spread along the peripancreatic neural plexuses. The objective of our study was to classify the anatomical-surgical layer of the mesopancreas and to describe the surgical principles relevant for resection of the mesopancreas (RMP). Immunohistochemical investigation of the mesopancreatic-perineural lymphogenic structures was carried out with the purpose of identifying possible routes of metastatic spread. METHODS: Resection of the mesopancreas (RMP) was performed in fresh corpses. Pancreas and mesopancreas were separated from each other and the mesopancreas was immunohistochemically investigated. RESULTS: The mesopancreas strains itself dorsally of the mesenteric vessels as a whitish-firm, fatty tissue-like layer. Macroscopically, in the dissected en-bloc specimens of pancreas and mesopancreas nerve plexuses were found running from the dorsal site of the pancreatic head to the mesopancreas to establish a perineural plane. Immunohistochemical examinations revealed the lymphatic vessels localized in direct vicinity of the neuronal plexuses between pancreas and mesopancreas. CONCLUSION: The mesopancreas as a perineural lymphatic layer located dorsally to the pancreas and reaching beyond the mesenteric vessels has not been classified in the anatomical or surgical literature before. The aim to ensure the greatest possible distance from the retropancreatic lymphatic tissue which drains the carcinomatous focus can be achieved in patients with pancreatic cancer only by complete resection of the mesopancreas (RMP)