X-Ray Phase-Contrast Tomography of Renal Ischemia-Reperfusion Damage

Purpose: The aim of the study was to investigate microstructural changes occurring in unilateral renal ischemia-reperfusion injury in a murine animal model using synchrotron radiation. Material and Methods: The effects of renal ischemia-reperfusion were investigated in a murine animal model of unilateral ischemia. Kidney samples were harvested on day 18. Grating-Based Phase-Contrast Imaging (GB-PCI) of the paraffin-embedded kidney samples was performed at a Synchrotron Radiation Facility (beam energy of 19 keV). To obtain phase information, a two-grating Talbot interferometer was used applying the phase stepping technique. The imaging system provided an effective pixel size of 7.5 mu m. The resulting attenuation and differential phase projections were tomographically reconstructed using filtered back-projection. Semi-automated segmentation and volumetry and correlation to histopathology were performed. Results: GB-PCI provided good discrimination of the cortex, outer and inner medulla in non-ischemic control kidneys. Post-ischemic kidneys showed a reduced compartmental differentiation, particularly of the outer stripe of the outer medulla, which could not be differentiated from the inner stripe. Compared to the contralateral kidney, after ischemia a volume loss was detected, while the inner medulla mainly retained its volume (ratio 0.94). Post-ischemic kidneys exhibited severe tissue damage as evidenced by tubular atrophy and dilatation, moderate inflammatory infiltration, loss of brush borders and tubular protein cylinders. Conclusion: In conclusion GB-PCI with synchrotron radiation allows for non-destructive microstructural assessment of parenchymal kidney disease and vessel architecture. If translation to lab-based approaches generates sufficient density resolution, and with a time-optimized image analysis protocol, GB-PCI may ultimately serve as a non-invasive, non-enhanced alternative for imaging of pathological changes of the kidney

Contralateral Control Kidney and Ischemic Kidney in three Different Contrast Modalities.

<p>(a), (d) Phase contrast. (b), (e) Dark-field contrast. (c), (f) Attenuation contrast. All images are scaled individually for best visual appearance. (a) Phase-contrast images of the contralateral kidney allow to differentiate the functional compartments of the kidney. (d) Phase-contrast images of I/R kidneys show considerably less variation in gray value between the different compartments. Stripes of the outer medulla cannot be distinguished. The number of visible tubular structure or voids is highly reduced. In dark-field contrast, (b) control kidneys show scattering at interfaces, which is reduced in (e) I/R kidneys. Conventional absorption-based images show considerably lower contrast and relatively higher noise in both (c) control and (f) ischemic kidney. CO – cortex; OM – outer medulla; OSOM – outer stripe outer medulla; ISOM – inner stripe outer medulla; IM – inner medulla.</p

Comparison of Histological Slices of Clamped Kidney.

<p>(a) PAS histological slice of the clamped kidney. (b) HE-stained histological slice of the clamped kidney, with a similar slice of the phase-contrast volume (c). For both histology slices a respective zoom view of the inner medulla is given. Scarring and beginning tubular atrophy as well proteinaceous casts could be detected correlating with increasing medullary density in PCI.</p

Photograph of the Interferometer Setup at Beamline ID19, ESRF, Grenoble, France.

<p>Monochromatic, coherent X-rays travel through the sample and introduce distortions in the interference pattern created by the phase grating. Those are evaluated by a lateral movement of the phase grating located behind the sample. Photograph adapted from I. Zanette, <i>Interférometrie X à réseaux pour l'imagerie et l'analyse de front d'ondes au synchrotron</i>, PhD thesis, University of Grenoble, 2011.</p

Mean Gray Values of Kidney Compartments.

<p>Mean value and standard deviations of phase gray values of the respective compartment. Note that OSOM and cortex were assessed together in healthy kidneys and OSOM and ISOM together in I/R kidneys. OSOM – outer stripe outer medulla ISOM – inner stripe outer medulla.</p

Comparison of a HE-Stained Histological Slice of Control Kidney.

<p>Phase contrast images provide similar morphological information as a HE-stained slice. The different functional compartments (IM, ISOM, OSOM/CO) can be readily differentiated. CO – cortex; OSOM – outer stripe outer medulla; ISOM – inner stripe outer medulla; IM – inner medulla.</p

Volumetric data from three-dimensional segmentation of the functional compartments of the kidneys.

<p>The voxel size is equal to 4.22·10⁻⁷ mm³.</p><p>Volumetric data from three-dimensional segmentation of the functional compartments of the kidneys.</p