Meloche, Ryan

Combined histopathological and metabolomic analysis of the same tissue specimen can reduce biological variability compared to independent analyses of similar samples. However, conventional workflows are incompatible: tissue fixation leads to metabolite loss through dilution and chemical reactions with fixative, while metabolomic extraction typically destroys the specimen. We propose a method that reconciles these conflicting requirements by enabling metabolite recovery from fixative while preserving tissue for histopathology. In this approach, tissue is initially fixed in a small volume of fixative; an aliquot of the fixative is then collected for nuclear magnetic resonance-based metabolomic analysis before additional fixative is added for standard processing. As a proof-of-concept, mouse kidneys from the same subject were analyzed using two protocols: standard perchloric acid extraction (ensuring complete metabolite recovery but destroying tissue) and metabolite extraction from fixative. Several metabolites were fully recoverable from the fixative, others partially, while some were lost due to chemical reactions. Despite some limitations, this strategy may be useful for analyzing precious specimens, such as human biopsies, that must remain intact for further studies. Moreover, using the same specimen for dual analyses reduces the number of animals required and enhances statistical robustness.

Key Points Robotic ultrasound performed favorably compared with magnetic resonance imaging for evaluating total kidney volume. Collagen evaluation by two novel methods of picrosirius red imaging were more informative than the standard method by brightfield imaging. Findings can improve research by increasing speed and access to total kidney volume determination and sensitivity of collagen assessment. Background 3D imaging and histology are critical tools for assessing polycystic kidney disease (PKD) in patients and animal models. Magnetic resonance (MR) imaging provides micron resolution but is time consuming and expensive, and access to equipment and expertise is limited. Robotic ultrasound (US) imaging has lower spatial resolution but is faster, more cost-effective, and accessible. Similarly, picrosirius red (PSR) staining and brightfield microscopy are commonly used to assess fibrosis; however, alternative methods have been shown in non-kidney tissues to provide greater sensitivity and more detailed structural characterization. Methods In this study, we evaluated the utility of robotic US and alternative methods of quantifying PSR staining for PKD research. We compared longitudinal total kidney volume measurements using US and MR imaging. We additionally compared PSR imaging and quantification using standard brightfield microscopy with that by circularly polarized light with hue analysis and fluorescence imaging analyzed using curvelet transform fiber extraction software for automatic detection of individual collagen fibers. Results Increased total kidney volume was detected by US in Pkd1 RC/RC versus wild-type (WT) at time points spanning from early to established disease. US interobserver variability was greater but allowed scanning in 2–5 minutes/mouse, whereas MR imaging required 20–30 minutes/mouse. While no change in fibrotic index was detected in this cohort of relatively mild disease using brightfield microscopy, polarized light showed fibers skewed thinner in Pkd1 RC/RC versus WT. Fluorescence imaging showed a higher density of collagen fibers in Pkd1 RC/RC versus WT, and fibers were thinner and curvier with no change in length. In addition, fiber density was higher in both glomeruli and tubules in Pkd1 RC/RC , and glomeruli had a higher fiber density than tubules in Pkd1 RC/RC and trended higher in WT. Conclusions These studies show robotic US is a rigorous imaging tool for preclinical PKD research. In addition, they demonstrate the increased sensitivity of polarized and fluorescence analysis of PSR-stained collagen.

To describe transverse relaxation of water in fixed tissue, we propose a model of transverse relaxation accelerated by diffusion and exchange (TRADE) that assumes exchange between free (visible) and bound (invisible) water, which relax by the dipole–dipole interaction, chemical exchange, and translation in the field gradient. Depending on the prevailing mechanism, transverse relaxation time (T2) of water in fixed tissue could increase (when dipole–dipole interaction prevails) or decrease with temperature (when diffusion in the field gradient prevails). Chemical exchange can make T2 even temperature independent. Also, variation of resolution from 100 to 15 μm/pxl (or less) affects effective transverse relaxation. T2 steadily decreases with increased resolution ( , is the read direction resolution). TRADE can describe all of these observations (semi)quantitatively. The model has been experimentally verified on water phantoms and on formalin‐fixed zebrafish, mouse brain, and rabbit larynx tissues. TRADE could help predict optimal scanning parameters for high‐resolution MRM from much faster measurements at lower resolution.