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Abstract: PO0313

Second Harmonic Generation and Fluorescence Imaging Reveal Collagen Fibrils and Cell Nuclei in Mature Randall Plaque

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Canela, Victor Hugo, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Bledsoe, Sharon B., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Winfree, Seth, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Sabo, Angela R., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Worcester, Elaine M., University of Chicago, Chicago, Illinois, United States
  • El-Achkar, Tarek M., Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Williams, James C., Indiana University School of Medicine, Indianapolis, Indiana, United States
Background

The formation of calcium oxalate (CaOx) stones on Randall’s plaque (RP) is a common phenomenon (perhaps 25% of CaOx stones), yet this mechanism of stone formation is still poorly understood. The objective of the study was to devise novel techniques to study RP structure.

Methods

Micro CT was used to orient RP stones for decalcification and sectioning. Sections were examined for collagen using Second Harmonic Generation (SHG) signals with multiphoton excitation. Other sections were stained with the DNA marker 7-aminoactinomycin D (7-AAD).

Results

SHG showed collagen fibrils in the plaque but not in the CaOx overgrowth region. Demineralized RP displayed autofluorescence in the far-blue region, as we have previously described in mineralized RP. Staining of plaque sections with the DNA marker, 7-AAD, confirmed the presence of cell nuclei within mature RP.

Conclusion

Our results show that collagen fibrils and cell nuclei are present in RP. The nature of cells and their role in plaque formation are yet to be determined. Our data suggest that these cells contain ordinary nuclear morphology and were well-preserved within the mature plaque. The presence of cell nuclei in the plaque raises critical questions about the role of apoptosis/necrosis and survival in this mineralized environment. Future studies exploring organization of collagen and the nature of cells in plaque will be invaluable in understanding plaque and stone pathogenesis.

Mature Randall’s plaque (RP) contains collagen and cell nuclei. A. Representative image of a decalcified RP stone. The white arrow is pointing to the decalcified plaque area in the far-blue range spectrum. Its second harmonic generation counterpart image (B) shows collagen within the plaque area (white arrow). C. Fluorescent staining with the DNA marker 7-AAD clearly depicts cell nuclei in regions of RP (arrowheads).

Funding

  • NIDDK Support