一种转化大鼠 Ex vivoLung 灌注实验的方法。
- 作者列表："Ohsumi A","Kanou T","Ali A","Guan Z","Hwang DM","Waddell TK","Juvet S","Liu M","Keshavjee S","Cypel M
:The application of ex vivolung perfusion (EVLP) has significantly increased the successful clinical use of marginal donor lungs. While large animal EVLP models exist to test new strategies to improve organ repair, there is currently no rat EVLP model capable of maintaining long-term lung viability. Here, we describe a new rat EVLP model that addresses this need, while enabling the study of lung injury due to cold ischemic time (CIT). The technique involves perfusing and ventilating male Lewis rat donor lungs for 4h before transplanting the left lung into a recipient rat, then evaluating lung function 2h after reperfusion. To test injury within this model,lungswere divided into groups and exposed to different CITs (ie, 20 min, 6h, 12h, 18h and 24h).Experiments involving the 24h CIT group were prematurely terminated due to the development of severe edema. For the other groups, no differences in the ratio of arterial oxygen partial pressure to fractional inspired oxygen (PaO2/FiO2) were observed during EVLP; however, lung compliance decreased over time in the 18h group (P=0.012) and the PaO2/FiO2of the blood from the left pulmonary vein 2h after transplantation was lower compared to 20 min CIT group (P=0.0062).This new model maintained stable lung function during 4h EVLP and after transplantation when exposed to up to 12h of CIT.
: Ex vivolung 灌注 (EVLP) 的应用显著增加了边缘供体肺的临床成功使用。虽然存在大型动物 EVLP 模型来测试改善器官修复的新策略，但目前还没有能够维持长期肺活力的大鼠 EVLP 模型。在这里，我们描述了一个新的大鼠 EVLP 模型，解决了这一需要，同时使研究由于冷缺血时间 (CIT) 的肺损伤。该技术涉及在将左肺移植到受体大鼠之前对雄性 Lewis 大鼠供体肺灌注通气 4h，然后在再灌注 2h 后评估肺功能。为了测试该模型内的损伤，肺部被分成组并暴露于不同的 CITs (即 20 min 、 6h 、 12h 、 18h 和 24h)。涉及 24h CIT 组的实验由于严重水肿的发展而提前终止。对于其他组，在 EVLP 期间未观察到动脉氧分压与吸入氧分数的比值 (PaO2/FiO2) 的差异; 然而,18h 组肺顺应性随时间下降 (P = 0.012)移植后 2h 左肺静脉血 PaO2/fio2 低于 CIT 组 20 min (P = 0.0062)。当暴露于 CIT 长达 12h 时，这种新模型在 4h EVLP 期间和移植后保持稳定的肺功能。
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.