Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices

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.