Mishra, Prasanna K.

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.

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.

BackgroundRenal fibrosis is a common pathway in tubulointerstitial injury and a major determinant of renal insufficiency. Collagen deposition, a key feature of renal fibrosis, may serve as an imaging biomarker to differentiate scarred from healthy kidneys.PurposeTo test the feasibility of using quantitative magnetization transfer (qMT), which assesses tissue macromolecule content, to measure renal fibrosis.Study TypeProspective.Animal ModelFifteen 129S1 mice were studied 4 weeks after either sham (n = 7) or unilateral renal artery stenosis (RAS, n = 8) surgeries.Field Strength/SequenceMagnetization transfer (MT)‐weighted images were acquired at 16.4T using an MT‐prepared fast‐low‐angle‐shot sequence. Renal B0, B1, and T1 maps were also acquired, using a dual‐echo gradient echo, an actual flip angle, and inversion recovery method, respectively.AssessmentA two‐pool model was used to estimate the bound water fraction (f) and other tissue imaging biomarkers. Masson's trichrome staining was subsequently performed ex vivo to evaluate renal fibrosis.Statistical TestsComparisons of renal parameters between sham and RAS were performed using independent samples t‐tests. Pearson's correlation was conducted to investigate the relationship between renal fibrosis by histology and the qMT‐derived bound pool fraction f.ResultsThe two‐pool model provided accurate fittings of measured MT signal. The qMT‐derived f of RAS kidneys was significantly increased compared to sham in all kidney zones (renal cortex [CO], 7.6 ± 2.4% vs. 4.6 ± 0.6%; outer medulla [OM], 8.2 ± 4.2% vs. 4.2 ± 0.9%; inner medulla [IM] + P, 5.8 ± 1.6% vs. 2.9 ± 0.6%, all P < 0.05). Measured f correlated well with histological fibrosis in all kidney zones (CO, Pearson's correlation coefficient r = 0.95; OM, r = 0.93; IM + P, r = 0.94, all P < 0.05).Data ConclusionThe bound pool fraction f can be quantified using qMT at 16.4T in murine kidneys, increases significantly in fibrotic RAS kidneys, and correlates well with fibrosis by histology. Therefore, qMT may constitute a valuable tool for measuring renal fibrosis in RAS.Level of Evidence1Technical Efficacy Stage3

While zebrafish embryos in the first five days after fertilization are clear and amenable to optical analysis, older juveniles and adults are not, due to pigmentation development and tissue growth. Thus other imaging methods are needed to image adult specimens. NMR is a versatile tool for studies of biological systems and has been successfully used for in vivo zebrafish microscopy. In this work we use NMR microscopy (MRM) for assessment of zebrafish specimens, which includes imaging of formalin fixed (FF), formalin fixed and paraffin embedded (FFPE), fresh (unfixed), and FF gadolinium doped specimens. To delineate the size and shape of various organs we concentrated on 3D MRM. We have shown that at 7 T a 3D NMR image can be obtained with isotropic resolution of 50 μm/pxl within 10 min and 25 μm/pxl within 4 h. Also, we have analyzed sources of contrast and have found that in FF specimens the best contrast is obtained by T 1 weighting (3D FLASH, 3D FISP), whereas in FFPE specimens T 2 weighting (3D RARE) is the best. We highlight an approach to perform segmentation of the organs in order to study morphological changes associated with mutations. The broader implication of this work is development of NMR methodology for high contrast and high resolution serial imaging and automated analysis of morphology of various zebrafish mutants.

The rodent model of folic acid (FA)-induced acute kidney injury (AKI) provides a useful model for studying human AKI, but little is known about longitudinal changes in renal hemodynamics and evolution of renal fibrosis in vivo. In this work, we aimed to longitudinally assess renal structural and functional changes using multiparametric magnetic resonance imaging (MRI). Ten adult mice were injected with FA, after which a multiparametric MRI was used to measure kidney volume, hypoxia index R2*, magnetization transfer ratio (MTR), perfusion, T1, and glomerular filtration rate (GFR) at 2 wk posttreatment. Then five mice were euthanized for histology, and the other five underwent MRI again at 4 wk, followed by histology. Control mice ( n = 5) were injected with vehicle and studied with MRI at 2 wk. Trichrome and hematoxylin-eosin staining were performed to assess FA-induced tissue injuries. Whereas kidney size and oxygenation showed progressive deterioration, a transient impairment in renal perfusion and normalized GFR slightly improved by 4 wk. Kidney fluid content, as reflected by T1, was prominent at 2 wk and tended to regress at 4 wk, consistent with observed tubular dilation. Trichrome staining revealed patchy necrosis and mild interstitial fibrosis at 2 wk, which exacerbated at 4 wk. MTR detected increased fibrosis at 4 wk. In conclusion, multiparametric MRI captured the longitudinal progression in kidney damage evolving within the first month after treatment with folic acid and may provide a useful tool for assessment of therapeutic strategies.

PurposeDynamic manganese‐enhanced MRI (MEMRI) allows assessment of tissue viability by tracing manganese uptake. We aimed to develop a rapid T1 mapping method for dynamic MEMRI to facilitate assessments of murine kidney viability.MethodsA multi‐slice saturation recovery fast spin echo (MSRFSE) was developed, validated, and subsequently applied in dynamic MEMRI at 16.4T on ischemic mouse kidneys after 4 weeks of unilateral renal artery stenosis (RAS). Baseline T1 values and post‐contrast R1 (1/T1) changes were measured in cortex (CO), outer (OSOM), inner (ISOM) strips of outer medulla, and inner medulla (IM).ResultsValidation studies showed strong agreement between MSRFSE and an established saturation recovery Look‐Locker method. Baseline T1 (s) increased in the stenotic kidney CO (2.10 [1.95–2.56] vs. 1.88 [1.81–2.00], P = 0.0317) and OSOM (2.17 [2.05–2.33] vs. 1.96 [1.87–2.00], P = 0.0075) but remained unchanged in ISOM and IM. This method allowed a temporal resolution of 1.43 min in dynamic MEMRI. Mn2+ uptake and retention decreased in stenotic kidneys, particularly in the OSOM (ΔR1: 0.48 [0.38–0.56] vs. 0.64 [0.61–0.69] s−1, P < 0.0001).ConclusionDynamic MEMRI by MSRFSE detected decreased cellular viability and discerned the regional responses to RAS. This technique may provide a valuable tool for noninvasive evaluation of renal viability. Magn Reson Med 80:190–199, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

PurposeTo develop and validate a method for measuring murine single‐kidney glomerular filtration rate (GFR) using dynamic contrast‐enhanced MRI (DCE‐MRI).MethodsThis prospective study was approved by the Institutional Animal Care and Use Committee. A fast longitudinal relaxation time (T1) measurement method was implemented to capture gadolinium dynamics (1 s/scan), and a modified two‐compartment model was developed to quantify GFR as well as renal perfusion using 16.4T MRI in mice 2 weeks after unilateral renal artery stenosis (RAS, n = 6) or sham (n = 8) surgeries. This approach was validated by comparing model‐derived GFR and perfusion to those obtained by fluorescein isothiocyanante (FITC)‐inulin clearance and arterial spin labeling (ASL), respectively, using the Pearson's and Spearman's rank correlations and Bland‐Altman analysis.ResultsThe compartmental model provided a good fitting to measured gadolinium dynamics in both normal and RAS kidneys. The proposed DCE‐MRI method offered assessment of single‐kidney GFR and perfusion, comparable to the FITC‐inulin clearance (Pearson's correlation coefficient r = 0.95 and Spearman's correlation coefficient ρ = 0.94, P < 0.0001, and mean difference −7.0 ± 11.0 μL/min) and ASL (r = 0.92 and ρ = 0.84, P < 0.0001, and mean difference 4.4 ± 66.1 mL/100 g/min) methods.ConclusionThe proposed DCE‐MRI method may be useful for reliable noninvasive measurements of single‐kidney GFR and perfusion in mice. Magn Reson Med 79:2935–2943, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

PurposeNoninvasive imaging techniques that quantify renal tissue composition are needed to more accurately ascertain prognosis and monitor disease progression in polycystic kidney disease (PKD). Given the success of magnetization transfer (MT) imaging to characterize various tissue remodeling pathologies, it was tested on a murine model of autosomal dominant PKD.MethodsC57Bl/6 Pkd1 R3277C mice at 9, 12, and 15 months were imaged with a 16.4T MR imaging system. Images were acquired without and with RF saturation in order to calculate MT ratio (MTR) maps. Following imaging, the mice were euthanized and kidney sections were analyzed for cystic and fibrotic indices, which were compared with statistical parameters of the MTR maps.ResultsThe MTR‐derived mean, median, 25th percentile, skewness, and kurtosis were all closely related to indices of renal pathology, including kidney weight/body weight, cystic index, and percent of remaining parenchyma. The correlation between MTR and histology‐derived cystic and fibrotic changes was R2 = 0.84 and R2 = 0.70, respectively.ConclusionMT imaging provides a new, noninvasive means of measuring tissue remodeling PKD changes and may be better suited for characterizing renal impairment compared with conventional MR techniques. Magn Reson Med 75:1466–1473, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance.

PurposeArchived formalin‐fixed paraffin‐embedded (FFPE) tissue collections represent a valuable informational resource for numerous studies. However, there is no NMR signal from FFPE specimens at room temperature. To obtain MR images and enable comparison of magnetic resonance microscopy (MRM) and histology studies we propose to image FFPE tissue at elevated temperature.MethodsA FFPE tissue block was imaged at 7 Tesla (T) and 16.4T at 70–80°C using T2‐weighting methods. The only difference from standard MR microscopy (MRM) is elevated temperature at which the embedding medium melts.ResultsUsing FFPE murine brain tissue, we were able to demonstrate the feasibility of tissue MRM from paraffin embedded specimens. Histology images taken from the specimen after MRM demonstrate that keeping the FFPE specimen in paraffin melt at 70–80°C for dozens of hours does not affect subsequent histology analysis.ConclusionMRM of FFPE tissue from paraffin melt opens new avenues for analyzing archived histological specimens (biopsies, post mortem, etc.) and for correlating MR images with histology (optical microscopy). In addition, 3D MRM of FFPE specimens could guide histology in search for appropriate regions of interest and subsequently minimize occurrences of false negatives in tissue analysis. Magn Reson Med 71:1989–1994, 2014. © 2014 Wiley Periodicals, Inc.