Presentation Authors: Kait Al*, Danny Poinapen, Joanna Konopka, Chris Norley, Jaques Milner, David Holdsworth, Hassan Razvi, Jeremy Burton, London, Canada
Introduction: The incidence of nephrolithiasis in North America is currently around 10%, with rates rising. One of the most well-studied models of nephrolithiasis utilizes Drosophila melanogaster (DM). Here we introduce a new tool to assay the stone burden in DM. Previous studies have utilized X-ray micro-computed tomography (uCT) to evaluate stones in narcotized, wax-embedded flies. We designed and implemented a novel method for live imaging of DM using uCT. We utilized this tool in a longitudinal, repeated-scanning protocol to visualize and evaluate in 3D the potential of an oxalate-degrading bacterium (Bacillus subtilis strain 168, BS168) to decrease stone burden.
Methods: One-week old DM adults were reared under standard conditions. DM were administered 10^8 CFU BS168 on day 0, and transferred to 1.0% sodium oxalate food on day 1. The first uCT scan was performed on day 3, and the second was performed on day 7. During scanning, the DM were immobilized under constant CO2 gas flow in a custom designed acrylic apparatus, and scanned for 3 hours using uCT (90 kVp; 70 uA; 0.3 degree incremental angle over 360 degrees; GE Locus MS scanner). A threshold of 700 Hounsfield Units (HU) was applied during analysis, and particles >10 voxels in size were retained for stone quantification.
Results: DM tolerated 3 hours of constant CO2 anesthesia in combination with X-ray radiation exposure throughout the uCT acquisition, during which the optimal scanning protocol achieved DM survival and minimal image-noise. With this method, successful 3D reconstruction and visualization of DM adults were reproduced at 5.72 um isotropic voxel spacing, with stones clearly present (Fig.1). Analysis of the impact of BS168 on stone formation is ongoing.
Conclusions: We have demonstrated that using CO2 gas during uCT imaging of live DM induces safe, repeatable, temporary full-body immobilization without motion artefacts, for the purpose of visualizing stones. DM could fully recover after scans and we demonstrated the capability of this tool in time-course studies, as the same individuals were repeatedly scanned throughout the experiment. Currently this technique is being applied to characterize the effect of stone-modulating treatments such as probiotic bacteria with future potential in evaluating pharmaceuticals.
Source of Funding: W. Garfield Weston FoundationNatural Sciences and Engineering Research Council of Canada