High-quality lung fixation by controlled closed loop perfusion for stereological analysis in a large animal model

Abstract

Background: Stereology is an essential method for quantitative analysis of lung structure. Adequate fixation is a prerequisite for stereological analysis to avoid bias in pulmonary tissue, dimensions and structural details. We present a technique for in situ fixation of large animal lungs for stereological analysis, based on closed loop perfusion fixation.

Materials and methods: Twenty anesthetized ventilated pigs (30 ± 3 kg) underwent cannulation of the pulmonary artery and ligation of the right hilus. Following circulatory arrest a continuous positive pressure of 12 mbar was applied to the airways and lung perfusion started with the fixative solution (1.5% paraformaldehyde; 1.5% glutaraldehyde in 0.15 M HEPES). In five animals, a single-pass perfusion technique was performed, in 15 subsequent animals, the closed-loop technique was applied. Afterwards, lungs were removed, externally postfixed in the recycled fixative solution, and stored at 4 °C. Fifteen lung specimens underwent stereological analysis with volume estimation and subsequent systematic uniform random sampling for light and electron microscopic analysis.

Results: Singlepass perfusion did not result in satisfactory fixation. Left lung closed loop perfusion rate was 0.5-0.7 L/min with total median [min-max] perfusion time of 15 min (11-19). Perfusion pressure was 15 mm Hg (9-33). Subsequent lung analysis revealed well-preserved cell and tissue ultrastructure.

Conclusion: The closed loop perfusion technique represents a valuable and reproducible fixation method in large animal models. Pressure controlled fixation perfusion results in high-quality preservation of in situ parenchymal architecture of lungs with or without injury, which is ideally suited for quantitative assessment of lung structure by stereology.