Under physiological pressure, one kidney from each pig was perfused with a radio-opaque silicone polymer (Microfil MV122, Flow Tech, Carver, MA), using a saline-filled cannula (PE 190) and a syringe infusion pump (SIP 22; Harvard Apparatus, Holliston, MA) until the polymer drained freely from a segmental vein. Perfused sections were preserved in 10% buffered formalin, and then scanned using a micro-CT scanner, which generates 3D images consisting of up to a billion cubic voxels, each 5μm to 25μm on a side, with isotropic spatial resolution. Images were reconstructed (20μm voxels), and spatial density of cortical and medullary microvessels (diameters 20–500μm) calculated using Analyze™, as previously described (Zhu et al., 2004 (link); Favreau et al., 2010 (link)). Vessel tortuosity was assessed by the “connectivity index” (vessel length divided by the shortest distance between its ends) (Bentley et al., 2002 (link)). In addition, media-to-lumen ratio was calculated in randomly selected intra-renal vessels in α smooth muscle actin (SMA)-stained sections (DakoCytomation A/S, Glostrup, Denmark).
Protocol detail
Quantifying Renal Microvascular Structure
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Actin
Cannula
Cortical
Ct scanner
Cubic
Formalin
Infusion pump
Medullary
Microfil
Microvessels
Physiological
Polymer
Pressure
Radio opaque
Renal
Saline
Silicone
Smooth muscle
Syringe
Vein
Vessels
Corresponding Organization : WinnMed
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Variable analysis
independent variables
- Perfusion of one kidney from each pig with a radio-opaque silicone polymer (Microfil MV122)
- Spatial density of cortical and medullary microvessels (diameters 20–500μm)
- Vessel tortuosity assessed by the 'connectivity index' (vessel length divided by the shortest distance between its ends)
- Media-to-lumen ratio in randomly selected intra-renal vessels
- Saline-filled cannula (PE 190) used for perfusion
- Syringe infusion pump (SIP 22; Harvard Apparatus) used for perfusion
- Perfusion continued until the polymer drained freely from a segmental vein
- Perfused sections preserved in 10% buffered formalin
- Micro-CT scanner used to generate 3D images with voxels 5μm to 25μm on a side and isotropic spatial resolution
- Images reconstructed to 20μm voxels
- α smooth muscle actin (SMA)-stained sections used for media-to-lumen ratio measurements
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