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 t echnique is limited by the heterogeneous responses obtained. We invest igated the use of precision-cut lung slices to examine contraction of individual airways. Lung slices of 250+/-20 mu m were prepared from Wi ster rats and cultured in a roller incubator in serum-free minimum ess ential medium (MEM). Under these conditions, the slices were viable fo r at least 70 h, as indicated by leakage of lactate dehydrogenase into the supernatant, thymidine incorporation and ciliary beating, The sli ces were placed in a newly developed incubation chamber and mounted by a nylon thread that was fixed to a platinum wire, The whole chamber w as positioned on a microscope stage, and contraction of single airways was followed under a microscope that was coupled to a CCD-camera. Red uction in airway area was taken as an index of bronchoconstriction and was determined by a computer program. Addition of methacholine result ed in a concentration-dependent (concentration producing half the maxi mal effect (EC50)=0.64+/-0.08 (mean+/-SD) mu M; n=64) contraction of s ingle airways, In the presence of hydrocortisone, the EC50 was about s ix times greater, i.e. 3.7+/-0.9 mu M (n=7), and the effect of tbe ste roid was largely abolished by propanolol (EC50=1/1+/-0.1 mu M; n=7). A irways 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 larg er ones (EC50=87+/-0.27 mu M; n=22). We conclude that cultured precisi on-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 t o methacholine than larger ones.