We recently described four distinct types of plexiform lesions in human idiopathic and familial pulmonary arterial hypertension (PAH) [1], visualising the three-dimensional lesion structure using synchrotron-based phase-contrast micro-computed tomography (SPµCT). Two types, 1 and 2, are shunt-type lesions that connect pulmonary arteries to the bronchial circulation: type 1 to the vasa vasorum, and type 2 to peribronchial vessels. Type 3 lesions are found peripherally in the lung as spherical structures abruptly terminating the distal pulmonary artery/arteriole, and type 4 lesions are characterised by recanalisation of an occluded artery/arteriole. Our observation of type 1 and type 2 lesions in PAH supports previous work that demonstrated intrapulmonary bronchopulmonary anastomoses (IBAs) connected to plexiform lesions in human PAH, suggesting that shunting of blood can occur within lesions in the setting of supra-systemic pulmonary arterial pressure [2]. Further haemodynamic studies of distinct subtypes of plexiform lesions have been hampered by the lack of available animal models with plexiform lesions representative of the full range of lesion types found in human disease. Plexiform lesions have previously been described in the Sugen5416/hypoxia rat model of pulmonary hypertension when time until sacrifice following hypoxia is extended to 13-14 weeks. Initially plexiform lesions were identified within the pulmonary artery, as well as in the form of aneurysm-like lesions projecting outside the vessel lumen [3], and recently the latter type was shown to form in supernumerary arteries [4]. However, neither study observed plexiform lesions communicating with the bronchial circulation, possibly because of methodological limitations of the histological analysis.Here, we set out to further characterise the range of plexiform lesion types in the prolonged Sugen5416/ hypoxia rat model using SPµCT combined with injections of a radiopaque dye. As previously suggested by our group and others [5], SPµCT grants superior three-dimensional imaging of biological structures with low and homogenous attenuation and is an emerging tool in the fields of pulmonary vascular physiology and digital pathology.The well-established Sugen5416/hypoxia model, combining vascular endothelial growth factor receptor 2 inhibitor injections with a second hit of hypoxia, was used as initially described [6], but sacrifice was delayed until 15 weeks post injections. No animals died during the study period. Following removal of the heart-lung block, the main pulmonary artery was injected with a radiopaque green dye (CDI's Tissue Marking Dye; Cancer Diagnostics, Durham, NC, USA), diluted with an equal volume of tap water to ensure low viscosity. Super-resolution x-ray radiographies of paraffin-embedded lobes were acquired using a laboratory setup as previously described [7], confirming the presence of contrast agent (radiopaque green dye) and thus enabling tracing of corresponding areas between individuals for subsequent high-resolution SPµCT imaging, ...