View Abstract

A4371 - Synchrotron-Based Phase Contrast Micro-CT; a Novel Tool for Investigating Disease Distribution in Pulmonary Arterial Hypertension in 3D Space
Author Block: C. Westoo1, C. Norvik1, M. Bech2, N. Peruzzi2, R. Mokso3, G. Lovric4, H. Brunnstrom5, K. Tran-lundmark1; 1Experimental Medical Sciences, Lund University, Lund, Sweden, 2Clinical Sciences, Medical Radiation Physics, Lund University, Lund, Sweden, 3MAX IV Laboratory, Lund University, Lund, Sweden, 4Swiss Light Source, Paul Scherrer Institute, Villigen, Switzerland, 5Clinical Sciences, Pathology, Lund University, Lund, Sweden.
Rationale:
Over the past 15 years, several new drugs have been approved for treatment of pulmonary arterial hypertension (PAH), with positive effects on symptoms and survival. However, these drugs have limited effects on disease progression in the long-term and novel therapies that specifically target vascular remodeling must be developed. To achieve this, an improved understanding of how the vascular lesions are distributed in 3D space is needed. Galambos and co-workers found, in their recent analyses of serial sections of plexiform lesions, that these severely diseased vessels seem to have connections with the systemic arterial circulation (via bronchial arteries) as well as with pulmonary arteries and veins. The clinical importance of these findings is still not fully determined. Synchrotron-based phase contrast micro-CT combined with immunohistochemistry has the potential to become a powerful tool for improving our understanding of cellular and molecular disease distribution in PAH in 3D space.
Methods:
Paraffin-embedded lung tissue from eight patients with idiopathic PAH were selected from the pathology biobank at Skåne University Hospital, Sweden. One section from each block was prepared and stained with hematoxylin and areas with vascular lesions of interest were identified. The corresponding regions were subsequently imaged with the synchrotron beam; small volumes of interest were imaged with high spatial resolution. The paraffin embedded tissue was imaged at the X02DA-TOMCAT beamline of the Swiss Light Source (PSI, Switzerland).
Results:
We found that a 4x magnifying objective coupled to a 20 µm thick scintillating material (LSO:Tb) and a sCMOS detector (pco.Edge 5.5) yielded the best tradeoff between spatial resolution and field-of view, resulting in a field-of view of 4.2 x 3.5 mm2 and a pixel size of 1.63 x 1.63 µm2. By applying a single-shot phase retrieval algorithm, all biological features could be resolved without paraffin interference. Following sectioning, Elastica van Gieson and Alcian blue PAS staining were selected for histology; enabling arteries to be distinguished from veins and demonstrating extracellular matrix distribution. For mapping of cell distribution, a triple staining for smooth muscle alpha actin, von Willebrand factor and CD45 proved useful.
Conclusion:
Synchrotron-based phase contrast micro-CT is a powerful tool for investigating pulmonary vascular pathology, and combined with immunohistochemistry lesions can now be understood by origin and orientation as well as by composition. We believe this method will advance our understanding of PAH pathobiology significantly and that it will become an important tool in the search for new targets for drug development.