Lung Functions under the Microscope
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 the study of the response 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. 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
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 water bath 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).
Example of an Application
As an example Figure 1 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.