title: 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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Anesthesiology, University Hospital Berne, University of Berne, Berne, Switzerland. sebastien.trachsel@insel.ch
authors.forename: Sebastien
authors.initials: S
authors.lastname: Trachsel
authors.forename: Kurosch
authors.initials: K
authors.lastname: Purkabiri
authors.forename: Ophelie
authors.initials: O
authors.lastname: Loup
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Jenni
authors.forename: Balthasar
authors.initials: B
authors.lastname: Eberle
authors.forename: Matthias
authors.initials: M
authors.lastname: Ochs
authors.forename: Alexander
authors.initials: A
authors.lastname: Kadner
mesh_terms: D000818:Animals; D005404:Fixatives; D005557:Formaldehyde; D005976:Glutaral; D007260:Infusion Pumps; D008168:Lung / Q000166:cytology* / Q000648:ultrastructure; D008853:Microscopy; D046529:Microscopy, Electron, Transmission; D023421:Models, Animal; D010477:Perfusion / Q000295:instrumentation / Q000379:methods / Q000592:standards; D011108:Polymers; D011651:Pulmonary Artery / Q000166:cytology / Q000648:ultrastructure; D012121:Respiration, Artificial; D013552:Swine; D016707:Tissue Fixation / Q000295:instrumentation / Q000379:methods / Q000592:standards
chemical_list: D005404:Fixatives; D011108:Polymers; D005557:Formaldehyde; D005976:Glutaral; C003043:paraform