In vivo assessment of the pulmonary microcirculation in elastase-induced emphysema using probe-based confocal fluorescence microscopy

Abstract

Introduction: The alveolar capillary bed, which appears essential for the maintenance of alveolar septa, is altered in pulmonary emphysema. Until recently, techniques that allow its analysis in vivo in spontaneously breathing conditions were lacking. Fibered confocal fluorescence microscopy (FCFM) is a new technique that enables distal lung microstructures imaging in vivo. FCFM can be coupled with I.V fluorescein injection to image the pulmonary capillary network. The aim of this study was to assess the lung microcirculation in vivo using FCFM and I.V fluorescein in rats with experimental emphysema. Results: In vivo pulmonary microcirculation imaging was possible in 7/7 elastase animals and in 6/7 controls. Using FCFM, intercapillary distances and alveolar facets diameters were found significantly higher in the elastase group compared with controls (49.5 vs. 41.8 µm p < 0.001, and 118.5 vs. 95.1 µm p < 0.001, respectively). Ex vivo mean interwall distance (MIWD) was correlated with the alveolar facets diameters measured in vivo (rs = 0.65 ; p = 0.016). Methods: 14 Sprague-Dawley rats were assigned to intratracheal instillation of porcine pancreatic elastase (n = 7) or saline (n = 7). The subpleural microcirculation was assessed using FCFM in spontaneously breathing rats, through a 2mm thoracic window using a continuous aspiration system, after I.V. injection of fluorescein-dextran. FCFM sequences were recorded and the image analysis was performed separately by two observers, blindly to the animal group. Fluorescence intensity (FI), maximal intercapillary distances, and alveolar facets diameters measured with FCFM were compared between groups, and to ex vivo lung morphometric measurements (MIWD). Conclusion: FCFM allows the quantitative assessment of the microcirculation alterations due to emphysema in vivo.