Abstract
Comparative, functional, developmental, and some morphological studies on animal anatomy require accurate visualization of three-dimensional structures. Nowadays, several widely applicable methods exist for non-destructive whole-mount imaging of animal tissues. The purpose of this study was to optimize specimen preparation and develop a method for quantitative analysis of the total pulmonary vasculature in fetal rats. Tissues were harvested at E21 and fetuses fixed overnight in 4% paraformaldehyde/phosphate buffered saline. They were treated with 25% Lugol solution for 72 hours to ensure perfusion. Four different methods were used for fetal specimen preparation; isolated lung, upper torso, direct right ventricle contrast injection, and whole body with partial thoracic skin excision. The microCT scan was performed, and pulmonary vasculature was segmented. Vessels were analyzed for diameter, length, and branching. Of the four preparation methods, only whole body with partial thoracic skin excision resulted in adequate reconstruction of the pulmonary vasculature. In silico generated 3D images gathered by micro CT showed pulmonary vasculature distributed throughout the lung, which was representative of the shape and structure of the lungs. The mean number of vessels segmented in the pulmonary tree was 900 ± 24 with a mean diameter of 134.13 µm (range 40.72–265.69 µm). While up to the 30th generation of vessels could be segmented, both for arteries and veins, the majority of branching was between the 21st and 30th generations. Passive diffusion of contrast material enables quantitative analysis of the fetal pulmonary vasculature. This technique is a useful tool to analyze the characteristics and quantify the fetal pulmonary vasculature.
Highlights
In the lung, the vascular network leads the way for tissue growth and pulmonary development during the embryonic and fetal periods
Multiple stains have been applied to the micro computerized tomography (CT) technique, the most commonly used including phosphotungstic acid (PTA), iodine potassium iodide (IKI, Lugol’s solution), and 1% iodine in ethanol or methanol[9]
This study provides a foundation to identify and compare normal lung vasculature variation as well as the fetal vasculature in the diseased organ to fully investigate etiology and pathology
Summary
The vascular network leads the way for tissue growth and pulmonary development during the embryonic and fetal periods. Alternatives to histology have previously been attempted, such as corrosion casts, arteriograms and computerized tomography (CT) scans[4,5,6,7] These methods generally evaluate around 0.1% of the vasculature in the lung which were not representative of the lung as a whole[4,5,6,7]. The non-volatile and excellent penetrative properties of these stains allow for detailed imaging of anatomy through micro CT, while still preserving the sample for further experimental[0] manipulation and analysis[8] In light of these developments, the purpose of this study was to develop a new sample preparation method for quantitative analysis of the total fetal pulmonary vasculature in rats, that is easy to reproduce and validate, and has potential to be translated into human studies
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