Micrometer-resolution X-ray tomographic full-volume reconstruction of an intact post-mortem juvenile rat lung.
- 作者列表："Borisova E","Lovric G","Miettinen A","Fardin L","Bayat S","Larsson A","Stampanoni M","Schittny JC","Schlepütz CM
:In this article, we present an X-ray tomographic imaging method that is well suited for pulmonary disease studies in animal models to resolve the full pathway from gas intake to gas exchange. Current state-of-the-art synchrotron-based tomographic phase-contrast imaging methods allow for three-dimensional microscopic imaging data to be acquired non-destructively in scan times of the order of seconds with good soft tissue contrast. However, when studying multi-scale hierarchically structured objects, such as the mammalian lung, the overall sample size typically exceeds the field of view illuminated by the X-rays in a single scan and the necessity for achieving a high spatial resolution conflicts with the need to image the whole sample. Several image stitching and calibration techniques to achieve extended high-resolution fields of view have been reported, but those approaches tend to fail when imaging non-stable samples, thus precluding tomographic measurements of large biological samples, which are prone to degradation and motion during extended scan times. In this work, we demonstrate a full-volume three-dimensional reconstruction of an intact rat lung under immediate post-mortem conditions and at an isotropic voxel size of (2.75 µm)3. We present the methodology for collecting multiple local tomographies with 360° extended field of view scans followed by locally non-rigid volumetric stitching. Applied to the lung, it allows to resolve the entire pulmonary structure from the trachea down to the parenchyma in a single dataset. The complete dataset is available online (https://doi.org/10.16907/7eb141d3-11f1-47a6-9d0e-76f8832ed1b2).
: 在本文中，我们提出了一种非常适合动物模型中肺病研究的 X 射线断层成像方法，以解析从气体摄入到气体交换的完整通路。当前最先进的基于同步加速器的层析相衬成像方法允许在扫描时间为秒的情况下无损地采集三维显微成像数据良好的软组织对比。然而，当研究多尺度分层结构的对象时，如哺乳动物肺,总体样本量通常超过单次扫描中 X 射线照射的视野，实现高空间分辨率的必要性与对整个样本进行成像的需要相冲突。已经报道了几种实现扩展高分辨率视场的图像拼接和校准技术，但是当成像非稳定样本时，这些方法往往会失败,因此排除了大型生物样品的断层扫描测量，这些样品在延长扫描时间内容易发生降解和运动。在这项工作中，我们展示了一个完整的大鼠肺在死后即刻条件下的全体积三维重建，各向同性体素大小为 (2.75 µ m)3。我们提出了用 360 ° 扩展视场扫描收集多个局部断层扫描，然后进行局部非刚性容积缝合的方法，应用于肺，它允许在单个数据集中解析从气管到实质的整个肺结构。完整的数据集可以在线获得 ( https://doi.org/10.16907/7eb141d3-11f1-47a6-9d0e-76f8832ed1b2 )。
METHODS:BACKGROUND AND PURPOSE:A critical role for sphingosine kinase/sphingosine-1-phosphate (S1P) pathway in the control of airway function has been demonstrated in respiratory diseases. Here, we address S1P contribution in a mouse model of mild chronic obstructive pulmonary disease (COPD). EXPERIMENTAL APPROACH:C57BL/6J mice have been exposed to room air or cigarette smoke up to 11 months and killed at different time points. Functional and molecular studies have been performed. KEY RESULTS:Cigarette smoke caused emphysematous changes throughout the lung parenchyma coupled to a progressive collagen deposition in both peribronchiolar and peribronchial areas. The high and low airways showed an increased reactivity to cholinergic stimulation and α-smooth muscle actin overexpression. Similarly, an increase in airway reactivity and lung resistances following S1P challenge occurred in smoking mice. A high expression of S1P, Sph-K2 , and S1P receptors (S1P2 and S1P3 ) has been detected in the lung of smoking mice. Sphingosine kinases inhibition reversed the increased cholinergic response in airways of smoking mice. CONCLUSIONS AND IMPLICATIONS:S1P signalling up-regulation follows the disease progression in smoking mice and is involved in the development of airway hyperresponsiveness. Our study defines a therapeutic potential for S1P inhibitors in management of airways hyperresponsiveness associated to emphysema in smokers with both asthma and COPD.
METHODS::The interim results from this 90-day multi-dose, inhalation toxicology study with life-time post-exposure observation has shown an important fundamental difference in persistence and pathological response in the lung between brake dust derived from brake-pads manufactured with chrysotile, TiO2 or chrysotile alone in comparison to the amphiboles, crocidolite and amosite asbestos. In the brake dust exposure groups no significant pathological response was observed at any time. Slight macrophage accumulation of particles was noted. Wagner-scores, were from 1 to 2 (1 = air-control group) and were similar to the TiO2 group. Chrysotile being biodegradable, shows a weakening of its matrix and breaking into short fibers & particles that can be cleared by alveolar macrophages and continued dissolution. In the chrysotile exposure groups, particle laden macrophage accumulation was noted leading to a slight interstitial inflammatory response (Wagner-score 1-3). There was no peribronchiolar inflammation and occasional very slight interstitial fibrosis. The histopathology and the confocal analyses clearly differentiate the pathological response from amphibole asbestos, crocidolite and amosite, compared to that from the brake dust and chrysotile. Both crocidolite and amosite induced persistent inflammation, microgranulomas, and fibrosis (Wagner-scores 4), which persisted through the post exposure period. The confocal microscopy of the lung and snap-frozen chestwalls quantified the extensive inflammatory response and collagen development in the lung and on the visceral and parietal surfaces. The interim results reported here, provide a clear basis for differentiating the effects from brake dust exposure from those following amphibole asbestos exposure. The subsequent results through life-time post-exposure will follow.
METHODS::The respiratory tract is lined by a pseudo-stratified epithelium from the nose to terminal bronchioles. This first line of defense of the lung against external stress includes five main cell types: basal, suprabasal, club, goblet and multiciliated cells, as well as rare cells such as ionocytes, neuroendocrine and tuft/brush cells. At homeostasis, this epithelium self-renews at low rate but is able of fast regeneration upon damage. Airway epithelial cell lineages during regeneration have been investigated in the mouse by genetic labeling, mainly after injuring the epithelium with noxious agents. From these approaches, basal cells have been identified as progenitors of club, goblet and multiciliated cells, but also of ionocytes and neuroendocrine cells. Single-cell RNA sequencing, coupled to lineage inference algorithms, has independently allowed the establishment of comprehensive pictures of cell lineage relationships in both mouse and human. In line with genetic tracing experiments in mouse trachea, studies using single-cell RNA sequencing (RNAseq) have shown that basal cells first differentiate into club cells, which in turn mature into goblet cells or differentiate into multiciliated cells. In the human airway epithelium, single-cell RNAseq has identified novel intermediate populations such as deuterosomal cells, 'hybrid' mucous-multiciliated cells and progenitors of rare cells. Novel differentiation dynamics, such as a transition from goblet to multiciliated cells have also been discovered. The future of cell lineage relationships in the respiratory tract now resides in the combination of genetic labeling approaches with single-cell RNAseq to establish, in a definitive manner, the hallmarks of cellular lineages in normal and pathological situations.