ThoracicA method for evaluating the murine pulmonary vasculature using micro-computed tomography
Introduction
The lung is a complex organ that is composed of airways, blood vessels, and parenchyma. Acquired diseases such as emphysema and bronchopulmonary dysplasia, and congenital diseases such as congenital diaphragmatic hernia, congenital heart disease, and primary pulmonary hypertension may have alterations in the pulmonary vasculature from vessel wall remodeling and reduction in vessel numbers due to vessel rarefaction or failed angiogenesis.1, 2, 3, 4, 5, 6 Pulmonary hypertension is a source of significant morbidity and mortality. The mechanisms are not fully understood, and treatments to prevent or reverse it are lacking.
Animal models have been used to investigate the histology, physiology, and molecular mechanisms of pulmonary hypertension and pulmonary vascular disease.7, 8, 9 A major limitation in studying pulmonary hypertension and other pulmonary vascular disease is the lack of appropriate quantitative morphometric techniques to evaluate the entire intact pulmonary vascular tree. Current analysis of the pulmonary vasculature is restricted to histologic or stereologic techniques that require the use of random or systematic sampling from two-dimensional tissue sections to draw conclusions about three-dimensional (3D) structure.10, 11, 12, 13, 14, 15 Verified techniques such as point discrimination, line intercept count, and transection count use standardized methods such as a uniform grid to overlay on tissue section images and thus limit the amount of error associated with probing the structures of interest.10, 14, 16, 17, 18 Such methods are limited by the assumption that each analyzed sample is sufficiently random yet simultaneously representative of the entire lung.10 Hence, these methods do not account for the full complexity of the pulmonary vasculature. Furthermore, they do not allow whole-lung evaluation of branch-patterning, vessel generation, length, diameter, or volume of the pulmonary vasculature. Moreover, tissue sections alone may miss the true density of blood vessels or changes in vessel density and structure from one lobe to another.15
In recent years, 3D techniques to evaluate the pulmonary vasculature in small animal models have evolved to avoid the above limitations. Multidetector computed tomography (CT), magnetic resonance imaging, and micro-CT (μCT) have been used.14, 19, 20, 21, 22 Micro-CT has a number of advantages. Images can be acquired in vivo or on postmortem tissue samples. Analysis may be combined with unbiased sampling procedures and can encompass the entire pulmonary vascular tree.14, 20, 22 μCT can acquire images with resolution that is comparable to microscopy.14 Recently, μCT was combined with arterial casting to evaluate pulmonary vascular development in the rat. Data were obtained and analyzed with proprietary software to provide automated measurements.22 Besides the limitation of using noncommercially available software, few studies have applied these techniques to mice. Mouse models of pulmonary vascular disease are important for understanding the basis of pulmonary vascular disease. Thus, the purpose of this study was to develop a method for quantitative analysis of the total murine lung vascular tree using current techniques and commercially available software.
Section snippets
Animals
Male C57BL/6-SVJ/129 hybrid mice from our existing colony were sacrificed at 2 weeks, 4 weeks, and 3 months (adult). Animal care and procedures were approved by the University of North Carolina Institutional Animal Care and Use Committee.
Lung perfusion and casting technique
Perfusion cannulas were constructed by inserting polyethylene-10 (PE-10) into polyethylene-50 (PE-50) tubing (Solomon Scientific, San Antonio, TX) in which a wire mandrel was inserted to maintain luminal patency during heating over a soldering iron tip to seal
Results
Cleared lungs from three different mice from each age group showed qualitative increases in vascular complexity and density (Fig. 3A and C). The tortuosity and dilated nature of the main pulmonary artery declined with adulthood (Fig. 3E). Inspection of the lung periphery at high magnification revealed that more of the “side” branches of high-generation vessels are present in the periphery of the lung at 2 weeks (Fig. 3B). By 4 weeks, there were high-generation side branches that sprouted off of
Discussion
This study combined high-resolution CT, vascular casting of cleared whole-lung, histologic analysis, and automated quantification of vessels with commercially available software to evaluate the murine pulmonary vasculature. Generation number, vessel lengths, diameters, and densities were determined with the methods developed for this study. This study is important because it uses a readily available method for automated quantification of the entire mouse pulmonary vasculature with high
Conclusions
In conclusion, we describe methods for automated quantitative morphometry of the pulmonary vasculature of the mouse. This limits the possibility for bias that can occur when using sampling or supervised stereologic techniques. We believe that these methods will aid lung imaging in mouse models of pulmonary vascular disease.
Acknowledgment
The authors thank Maria Gambarian for her expert care of the mice and technical support. They thank Ted Hobgood for assistance with figures and cover art. This work was supported by the Robert Wood Johnson Foundation Harold Amos Faculty Development Program (67069) (SEM), UNC Department of Surgery, and the National Institutes of Health grant-GM008450 (MRP).
Author's contributions: M.R.P. and S.E.M. did conception, design, experimentation, analysis and interpretation, writing article, and revision
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