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Precision-cut lung slices Lung Functions under the Microscope

A novel incubation chamber for characterization of airway and vascular responses in precision-cut lung slices   Precision-cut lung slices (PCLS) offer a novel and unique way to assess lung functions under cell culture conditions. They can be prepared from nearly any species including murine, rat and human lungs. The method allows to study the responses of airways of different size (down to the terminal bronchioles) and to relate these changes in lung functions to gene expression and mediator release. Slices are viable for at least three days. They can be placed under an inverted microscope, where digital image techniques allow to quantify not only the responsiveness of single airways, but also of single vessels. In addition, it is possible to analyze the ciliary beating frequency. More than 20 slices can be obtained from one lung, thus this method is very economical in terms of experimental costs and animal use. This method obtained in 1997 the Annual European Prize for the substitution of the animal experimentation form the FISEA and in 1998 the Tierschutzpreis Schleswig-Holstein. Tissue cores are prepared from the lungs filled with agarose solution, after cooling to 4°C. From the cores, slices (220 +/- 20µm) are cut using a Krumdieck tissue slicer (For detailed preparation technique see: Martin, C., Uhlig, S. & Ullrich, V. Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices. Eur. Respir. J., 9, 2479-2487; 1996)   Video images of a slice showing a bronchus (B); a bronchiolus (b) and a pulmonary vein (PV)

The incubation chamber was developed to allow incubation and observation of slices by an inverted microscope. The chamber is made of Polycarbonate. It is connected to a waterbath to maintain constant temperature conditions. Two incubation cells are positioned in the center of the chamber. The bottom of the cells is sealed by glass, the cover is made of acrylic glass. The slices are fixed in the incubation cells by positioning them under nylon strings fixed to a bent platinum wire. The incubation cells can be filled with buffer, medium or drug solutions through the filling pipe. Buffer solution can be removed from the cells over a vacuum pipe. In addition, it is possible to gas the incubation cells in order to use bicarbonate buffered media. The incubation chamber is placed on the stage of an inverted microscope and warmed to 37°C. The slices are screened for airways and transferred to the incubation chamber. Lung slices are selected for study using predefined criteria (Martin et al. 1996). Airways and vessels are focused, and finally the images are analyzed by image analysis software (e.g. Optimas or Metamorph ).

View of a lung slice in position in the chambre

Response of a single airway (B) and a vessel (V) by increasing concentrations of U46619 (a-f)

Exposure of a PCLS to increasing concentrations of endothelin-1. Shown is one lung slice containing a small airway (B), a pulmonary artery (PA) and a pulmonary vein (PV). The slice was imaged before (C) and after exposure to increasing concentrations of endothelin-1, ranging from 10-10 M to 10-6 M.

As an example the figure shows exposure of a slice to increasing concentrations of endothelin-1. Shown is a lung slice containing an airway (B), a pulmonary artery (PA) and a pulmonary vein (PV). The pulmonary artery and the airway contracted almost completely, while the pulmonary vein area decreased to only 50% of its initial area. These responses are now easily quantified by digital imaging technique. It is a distinct advantage of this technique that in many ways precision-cut slices can be treated like a cell culture. Thus, the slices can be incubated under various conditions and gene as well as protein expression or mediator release be determined. In contrast to cell culture models, in slices the anatomical structure of the lung is largely maintained, so that the functional consequences of gene expression and mediator release can be evaluated.

The following table compares various in vivo and in vitro techniques that have been used to assess lung functions. Thus as a method, precision-cut lung slices possess all the advantages of an in vitro technique, but still maintain many functions of the intact organ.

Parameters	PCLS	In vivo	Perfused Lung	Tracheal rings	Lung cells
viable for	>70h		8h	24h	>100h
Experiments/day	>10	>10	<3	>10	>10
Experiments/animal	>20	1	1	8-10	>10
Technical requirements	medium	high	high	low	low
Human tissue	yes	yes	difficult	yes	yes
Accessible functions/ parameters
Bronchoconstriction	yes	yes	yes	yes	no
Vasoconstriction	yes	yes	yes	no	no
Relate vessel size to function	yes	difficult	yes	no	no
Relate airway size to function	yes	difficult	no	no	no
Edema	no	yes	yes	no	yes
Ciliary beat frequency	yes	no	no	no	yes
Gas exchange	no	yes	yes	no	no

References:

Brendel K, Gandolfi AJ, Krumdieck CL, Smith PF (1987). Tissue slicing and culturing revisited. Trends Pharmacol. Sci. 8,11-15.

Krumdieck CL, dos Santos JE, Ho KJ (1980). A new instrument for the rapid preparation of tissue slices. Anal. Biochem. 104, 118-123.

Martin C, Uhlig S, Ullrich V (1996). Videomicroscopy of methacholine-induced contraction of individual airways in precision-cut lung slices. Eur. Respir. J. 9, 2479-2487.

Martin C (1997). Study of pharmacology and inflammatory responses in precision-cut lung slices. Stõhle Druck und Verlag, Germany

Held HD, Martin C, Uhlig S (1999) Characterization of airway and vascular responses in murine lungs. Brit. J. of Pharm. 126, 1191-1199.

Martin C, Held HD, Uhlig S (2000) Differential effects of the mixed ETA/ETB-receptor antagonist bosentan on endothelin-induced bronchoconstriction, vasoconstriction and prostacyclin release. Naunyn-Schmiedeberg's Arch Pharmacol (2000) 362: 128-136

Martin C, Ullrich V, Uhlig S (2000) Effects of the thromboxane receptor agonist U46619 and endothelin-1 on large and small airways. Eur Respir J 2000; 16: 316-323.

Martin C, Uhlig S, Ullrich V (2001) Cytokine-induced bronchoconstriction in precision-cut lung slices is dependent upon cyclooxygenase-2 and thromboxane receptor activation. Am. J. Respir. Cell Mol. Biol. 2001 24:139-145.

Wohlsen A, Uhlig S, Martin C (2001). The early allergic response in small airways. Am. J. Respir. Crit. Care Med., in press.