Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices
Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices
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DOI：
10.1183/09031936.96.09122479
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发表时间：
1996-12-01
影响因子：
24.3
通讯作者：
Ullrich, V
中科院分区：
文献类型：
--
作者：
Martin, C;Uhlig, S;Ullrich, V
视频显微镜可以在活肺切片上研究不同大小气道的收缩，但目前该技术的应用受到所获得的异质性反应的限制。我们研究了使用精确切割的肺切片来检查单个气道的收缩。取Wister大鼠肺切片（250+/-20 μ m），在无血清最低必需培养基（MEM）的滚柱培养箱中培养。在这些条件下，切片至少可以存活70小时，如乳酸脱氢酶渗漏到上清，胸苷结合和纤毛加热。切片放置在新开发的孵育室中，用尼龙线固定在铂丝上，整个室放置在显微镜台上，在连接到ccd相机的显微镜下观察单个气道的收缩。以气道面积缩小作为支气管收缩的指标，由计算机程序确定。甲胆碱的加入导致浓度依赖(产生一半最大效应的浓度(EC50)=0.64+/-0.08（平均值+/-SD） mu M；在氢化可的松存在的情况下，EC50约为6倍，即3.7+/-0.9 μ M (n=7)，并且类固醇的作用在很大程度上被丙丙醇所消除（EC50=1/1+/-0.1 μ M; n=7）。面积小于35000亩m(2)的气道对乙酰胆碱的敏感性（EC50=0.1+/-0.03亩m， n=20）是较大气道（EC50=87+/-0.27亩m， n=22）的近9倍。我们的结论是，培养的精确切割肺片是常规研究不同大小单个气道收缩的有用模型。测量是精确的和可重复的，并表明较小的气道比较大的气道对甲胆碱更敏感。
Contraction of airways of different size can be studied in viable lung slices by videomicroscopy, However, at present, application of this technique is limited by the heterogeneous responses obtained. We investigated the use of precision-cut lung slices to examine contraction of individual airways.Lung slices of 250+/-20 mu m were prepared from Wister rats and cultured in a roller incubator in serum-free minimum essential medium (MEM). Under these conditions, the slices were viable for at least 70 h, as indicated by leakage of lactate dehydrogenase into the supernatant, thymidine incorporation and ciliary beating, The slices were placed in a newly developed incubation chamber and mounted by a nylon thread that was fixed to a platinum wire, The whole chamber was positioned on a microscope stage, and contraction of single airways was followed under a microscope that was coupled to a CCD-camera. Reduction in airway area was taken as an index of bronchoconstriction and was determined by a computer program.Addition of methacholine resulted in a concentration-dependent (concentration producing half the maximal effect (EC50)=0.64+/-0.08 (mean+/-SD) mu M; n=64) contraction of single airways, In the presence of hydrocortisone, the EC50 was about six times greater, i.e. 3.7+/-0.9 mu M (n=7), and the effect of tbe steroid was largely abolished by propanolol (EC50=1/1+/-0.1 mu M; n=7). Airways with an area smaller than 35,000 mu m(2) were nearly nine times more sensitive to methacholine (EC50=0.1+/-0.03 mu M; n=20) than larger ones (EC50=87+/-0.27 mu M; n=22).We conclude that cultured precision-cut lung slices are a useful model for routine study of contraction of individual airways of various sizes. The measurements were precise and reproducible and showed that smaller airways are more sensitive to methacholine than larger ones.