Martin C, Uhlig S, Ullrich V
University of Konstanz, Germany.
Eur Respir J. 1996 Dec;9(12):2479-87. doi: 10.1183/09031936.96.09122479.
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 microns were prepared from Wistar 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) microM; n = 64) contraction of single airways. In the presence of hydrocortisone, the EC50 was about six times greater, i.e. 3.7 +/- 0.9 microM (n = 7), and the effect of the steroid was largely abolished by propanolol (EC50 = 1.1 +/- 0.1 microM; n = 7). Airways with an area smaller than 35,000 microns2 were nearly nine times more sensitive to methacholine (EC50 = 0.1 +/- 0.03 microM; n = 20) than larger ones (EC50 = 87 +/- 0.27 microM; 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.
可通过视频显微镜在活的肺切片中研究不同大小气道的收缩情况。然而，目前该技术的应用受到所获得的异质性反应的限制。我们研究了使用精密切割肺切片来检测单个气道的收缩。从Wistar大鼠制备250±20微米的肺切片，并在无血清基本培养基（MEM）中于滚轴培养箱中培养。在这些条件下，如乳酸脱氢酶漏入上清液、胸腺嘧啶核苷掺入和纤毛摆动所示，切片至少存活70小时。将切片置于新开发的孵育室中，并用固定在铂丝上的尼龙线固定。整个孵育室放置在显微镜载物台上，在与CCD相机相连的显微镜下观察单个气道的收缩。气道面积的减小被视为支气管收缩的指标，并通过计算机程序确定。加入乙酰甲胆碱导致单个气道出现浓度依赖性收缩（产生最大效应一半时的浓度（EC50）=0.64±0.08（平均值±标准差）微摩尔；n=64）。在存在氢化可的松的情况下，EC50约大6倍，即3.7±0.9微摩尔（n=7），并且该类固醇的作用在很大程度上被普萘洛尔消除（EC50=1.1±0.1微摩尔；n=7）。面积小于35000平方微米的气道对乙酰甲胆碱的敏感性（EC50=0.1±0.03微摩尔；n=20）几乎是较大气道（EC50=87±0.27微摩尔；n=22）的9倍。我们得出结论，培养的精密切割肺切片是用于常规研究各种大小单个气道收缩的有用模型。测量精确且可重复，表明较小气道对乙酰甲胆碱比较大气道更敏感。
