Background: With the first breath, the newborn’s circulatory system must quickly adapt to obtaining oxygen through respiration rather than mother’s circulation. The fetal circulation is designed to allow oxygenated blood from the placenta to bypass the developing lungs and be directed to the systemic circulation. This is accomplished by a tonic level of vasoconstriction of the pulmonary artery (PA) in utero, as well as the wide patency of a large fetal artery, the ductus arteriosus (DA), connecting PA and aorta to bypass the unventilated fetal lung [1]. At birth, the elevated levels of oxygen rapidly induce the constriction of DA and force blood to travel through the newly ventilated lungs and eliminate the fetal right to left blood shunting. This miracle of birth occurs flawlessly in most term infants but often fails in prematurity, promoting congenital heart diseases like persistent DA (PDA) and persistent pulmonary hypertension of the newborn (PPHN) [2,3]. Testing the novel therapeutics to constrict the DA and relax the PAs in PDA and PPHN patients, hinges on the ability to image the DA and PAs in preclinical animal models. 

State-of-the-art in vivo and ex vivo contrast-enhanced micro-computed tomography (micro-CT) imaging is being developed to visualize and quantitively measure the effects of increased oxygen tension (from fetal PO₂ of 40mmHg to neonatal PO₂ of 100mmHg) on DA and PA dimensions. In vivo X-ray CT imaging of cardiopulmonary system in small-animal models is challenging mostly because of heart’s fast motion and small caliber of blood vessels (DA diameter <0.5mm and small PAs <0.1mm). The use of intravascular contrast agents overcomes the micro-CT’s poor contrast sensitivity for soft tissues [4]. This study entails optimization of the choice of contrast agents and perfusion techniques used to image both the DA and small PAs in the same intact rabbit kit [5]. We image changes in DA and PA diameters in hypoxic fetal kits before the first breath (hypoxia) and after increased oxygen tension to fetal PO₂ (normoxia), with the goal of imaging of vascular reactivity resulting from changes in oxygen tension in situ evaluation. 

Experimental Methods: At 29 days of gestation, rabbit kits were delivered via the caesarian section and exposed to either hypoxia (being anesthetized before the first breath) or normoxia (breathing 100% O₂ prior to be anesthetized). They were perfused with either Omnipaque 300 mgI/mL or 25% barium sulfate (8 mL, 2 mL/min) via injection into the beating left ventricle, and scanned using VECTor⁴CT (MILabs B.V., Utrecht, Netherlands).

Results and Conclusion: Figure 1(a-b) shows micro-CT images taken after in vivo heart perfusion with Omnipaque following delivery, indicating an open DA (linking PA to aorta) in hypoxic rabbit kit compared to the complete absence (closure) of DA in normoxic kit [6]. To study the effects of therapies on both DA and PAs, we modified the protocol to use lower viscosity contrast agents, like barium sulfate [7]. As shown in Figure 1(c-d) barium solution achieved superior visualization of the entire lung vasculature network, including small distal arteries, without barotrauma.

Oxygen has opposing effects on the DA vs PA, simultaneously constricting the DA while vasodilating the pulmonary circulation. These physiologic changes can be detected and quantified in a single micro-CT scan by optimizing contrast agents. Optimized perfusion techniques enable us for the first time to visualize the transition from fetus to air-breathing neonate using micro-CT through perfusion of the newly ventilated lung vasculature and the DA closure in fetal rabbit kits exposed to hypoxia vs normoxia. In the future we hope that these robust micro-CT imaging methodologies will be valuable to evaluate therapeutic efficacy of novel drugs in small-animal models of human diseases. 

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Image/Figure Caption:

Figure 1. Maximum intensity projection (MIP) micro-CT images taken after perfusion with contrast agent Omnipaque (mixed with 3% gelatin). (a) The DA is shown connecting to the pulmonary artery (PA) and descending thoracic aorta (Ao) in rabbit kits perfused immediately following cesarean delivery (hypoxia); (b) only the beak of the DA remains at its connection to the aorta, for rabbit kits exposed to 20 minutes of 100% O₂ (normoxia) prior to perfusion. (c-d) MIP micro-CT ex vivo images taken after perfusion with 25% barium sulfate solution (mixed with 3% gelatin) indicating lungs vasculature in rabbit kits exposed to hypoxia (c) and normoxia (d).

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