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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, expensive, and access to equipment and expertise is limiting. Robotic ultrasound (US) imaging has lower spatial resolution but is faster, more cost effective, and accessible. Similarly, Picrosirius red (PSR) staining and brightfield microscopy is 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 (TKV) measurements using US and MR. We additionally compared PSR imaging and quantification using standard brightfield with that by circularly polarized light with hue analysis, and fluorescence imaging analyzed using CT-FIRE software for automatic detection of individual collagen fibers. RESULTS: Increased TKV was detected by US in Pkd1RC/RC vs wild type (WT) at timepoints spanning early to established disease. US inter-observer variability was greater but allowed scanning in 2-5 minutes/mouse while MR required 20-30 minutes/mouse. While no change in fibrotic index was detected in this cohort of relatively mild disease using brightfield, polarized light showed fibers skewed thinner in Pkd1RC/RC vs WT. Fluorescence imaging showed a higher density of collagen fibers in Pkd1RC/RC vs WT, and fibers were thinner and curvier with no change in length. Additionally, fiber density was higher in both glomeruli and tubules in Pkd1RC/RC, and glomeruli had a higher fiber density than tubules in Pkd1RC/RC, and trended higher in WT. CONCLUSIONS: These studies show robotic ultrasound is a rigorous imaging tool for pre-clinical PKD research. Additionally, they demonstrate the increased sensitivity of polarized and fluorescence analysis of PSR-stained collagen.
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BACKGROUND: The urine metabolites and chemistries that contribute to kidney stone formation are not fully understood. This study examined differences between the urine metabolic and chemistries profiles of first-time stone formers and controls. METHODS: High-resolution 1H-nuclear magnetic resonance (NMR) spectroscopy-based metabolomic analysis was performed in 24-hour urine samples from a prospective cohort of 418 first-time symptomatic kidney stone formers and 440 controls. In total, 48 NMR-quantified metabolites in addition to 12 standard urine chemistries were assayed. Analysis of covariance was used to determine the association of stone former status with urine metabolites or chemistries after adjusting for age and sex and correcting for the false discovery rate. Gradient-boosted machine methods with nested cross-validation were applied to predict stone former status. RESULTS: Among the standard urine chemistries, stone formers had lower urine oxalate and potassium and higher urine calcium, phosphate, and creatinine. Among NMR urine metabolites, stone formers had lower hippuric acid, trigonelline, 2-furoylglycine, imidazole, and citrate and higher creatine and alanine. A cross-validated model using urine chemistries, age, and sex yielded a mean AUC of 0.76 (95% CI, 0.73 to 0.79). A cross-validated model using urine chemistries, NMR-quantified metabolites, age, and sex did not meaningfully improve the discrimination (mean AUC, 0.78; 95% CI, 0.75 to 0.81). In this combined model, among the top ten discriminating features, four were urine chemistries and six NMR-quantified metabolites. CONCLUSIONS: Although NMR-quantified metabolites did not improve discrimination, several urine metabolic profiles were identified that may improve understanding of kidney stone pathogenesis.
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Cálculos Renais , Humanos , Estudos Prospectivos , Cálculos Renais/etiologia , Ácido Cítrico , Citratos/urina , Espectroscopia de Ressonância Magnética/efeitos adversosRESUMO
SUMMARY: 1H nuclear magnetic resonance (NMR) spectroscopy is an established bioanalytical technology for metabolic profiling of biofluids in both clinical and large-scale population screening applications. Recently, urinary protein quantification has been demonstrated using the same 1D 1H NMR experimental data captured for metabolic profiling. Here, we introduce NMRpQuant, a freely available platform that builds on these findings with both novel and further optimized computational NMR approaches for rigorous, automated protein urine quantification. The results are validated by interlaboratory comparisons, demonstrating agreement with clinical/biochemical methodologies, pointing at a ready-to-use tool for routine protein urinalyses. AVAILABILITY AND IMPLEMENTATION: NMRpQuant was developed on MATLAB programming environment. Source code and Windows/macOS compiled applications are available at https://github.com/pantakis/NMRpQuant, and working examples are available at https://doi.org/10.6084/m9.figshare.18737189.v1. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Imageamento por Ressonância Magnética , Software , Espectroscopia de Prótons por Ressonância Magnética , Espectroscopia de Ressonância Magnética/métodos , Metabolômica/métodosRESUMO
Objectives: To examine the degree of agreement between MRI and histologically generated volumetric measurements of residual injection laryngoplasty material. Methods: Following left recurrent laryngeal nerve transection, rabbit vocal cords were injected with jellyfish collagen, Cymetra®, or Restylane®. Laryngeal tissue was harvested 4 or 12 weeks post injection followed by MRI imaging and histologic cross-sectioning. Two raters estimated the volume of remaining injection material in specimens within MRI and histologic axial cross sections. Wilcoxon signed rank tests were employed to detect gross differences between inter-rater measurements and between imaging modalities across time. Agreement between rater measurements and imaging (histology and MRI) was assessed using intra-class correlation coefficients. Results: Data was available from 16 rabbits sacrificed at 4 weeks (n = 8) and 12 weeks (n = 8). Inter-rater testing of MRI imaging revealed no significant differences (p > .05) between rater measurements across time points, and excellent agreement (0.93; 95% confidence interval 0.80-0.98) while histologically estimated volumes demonstrated a significant difference at 4 weeks (p < .05) and overall good agreement (0.89; 95% confidence interval 0.59-0.97). Comparison of MRI and histologically estimated volume measurements revealed significant differences at the 4-week time point (p < .05) but not at 12 weeks (p > .05). Overall, there is only moderate agreement between MRI and histology estimates (0.72; 95% confidence interval 0.22-0.90). Conclusions: MRI imaging demonstrates good reliability and similar estimates of volume to histologically estimated measurements of residual injection laryngoplasty material at time points clinically relevant for future injection laryngoplasty experiments. Level of Evidence: NA.
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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 ( T 2 â ∆ x 2 , ∆ x 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.
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Microscopia , Peixe-Zebra , Animais , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Coelhos , Temperatura , ÁguaRESUMO
We described several postprocessing methods to measure protein concentrations in human urine from existing 1H nuclear magnetic resonance (NMR) metabolomic spectra: (1) direct spectral integration, (2) integration of NCD spectra (NCD = 1D NOESY-CPMG), (3) integration of SMolESY-filtered 1D NOESY spectra (SMolESY = Small Molecule Enhancement SpectroscopY), (4) matching protein patterns, and (5) TSP line integral and TSP linewidth. Postprocessing consists of (a) removal of the metabolite signals (demetabolization) and (b) extraction of the protein integral from the demetabolized spectra. For demetabolization, we tested subtraction of the spin-echo 1D spectrum (CPMG) from the regular 1D spectrum and low-pass filtering of 1D NOESY by its derivatives (c-SMolESY). Because of imperfections in the demetabolization, in addition to direct integration, we extracted protein integrals by the piecewise comparison of demetabolized spectra with the reference spectrum of albumin. We analyzed 42 urine samples with protein content known from the bicinchoninic acid (BCA) assay. We found excellent correlation between the BCA assay and the demetabolized NMR integrals. We have provided conversion factors for calculating protein concentrations in mg/mL from spectral integrals in mM. Additionally, we found the trimethylsilyl propionate (TSP, NMR standard) spectral linewidth and the TSP integral to be good indicators of protein concentration. The described methods increase the information content of urine NMR metabolomics spectra by informing clinical studies of protein concentration.
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Metabolômica , Humanos , Espectroscopia de Ressonância MagnéticaRESUMO
OBJECTIVES/HYPOTHESIS: Test a new jellyfish collagen biomaterial aimed to increase duration of injection medialization laryngoplasty (IL) against two products in clinical practice. STUDY DESIGN: Animal model. METHODS: Left recurrent laryngeal nerve sectioning and IL were performed in New Zealand White rabbits (N = 6/group). Group 1 received micronized cross-linked jellyfish collagen (MX-JC) and adipose derived stem cells (ADSCs), Group 2, MX-JC alone, Group 3, cross-linked hyaluronic acid (X-HA), and Group 4, micronized acellular dermis (MACD). Animals were sacrificed at 4 and 12 weeks. Major outcomes were MRI tissue volumes and histopathology. RESULTS: After 100 µL IL MRI volumes (means ± STD) at 4 and 12 weeks were: Group 1: 27.2 ± 15.6 and 13.1 ± 5.2 µL, Group 2: 60.8 ± 18 and 27.8 ± 2.47 µL, Group 3: 27.4 ± 12 and 10.6 ± 8 µL, and Group 4: 37.5 ± 11 and 9.85 ± 1 µL. Group 2 volumes were largest and Group 3 were smallest in all comparisons (P < .05). Histologically, low grade inflammatory responses were observed in Group 1, mild histiocytic infiltration in Group 2, widespread muscle fiber loss in Group 3, and plasmocytic infiltration in Group 4. CONCLUSIONS: MX-JC showed the least resorption at 4 and 12 weeks among all groups. T cell inflammatory responses were observed with MX-JC but were reduced by 12 weeks while B cell immune responses, indicative of antibody priming, were predominantly noted with MACD. MX-JC + ADSC showed low grade immunity while the XHA showed greater myocyte loss compared to the other groups. LEVEL OF EVIDENCE: NA Laryngoscope, 131:E2452-E2460, 2021.
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Colágeno/farmacologia , Ácido Hialurônico/análogos & derivados , Laringoplastia/métodos , Imageamento por Ressonância Magnética/métodos , Paralisia das Pregas Vocais/terapia , Derme Acelular/efeitos adversos , Animais , Linfócitos B/imunologia , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/farmacologia , Cadáver , Colágeno/administração & dosagem , Modelos Animais de Doenças , Feminino , Ácido Hialurônico/administração & dosagem , Ácido Hialurônico/farmacologia , Imunidade/imunologia , Inflamação/patologia , Imageamento por Ressonância Magnética/estatística & dados numéricos , Células-Tronco Mesenquimais/patologia , Plasmócitos/imunologia , Padrões de Prática Médica , Coelhos , Traumatismos do Nervo Laríngeo Recorrente/complicações , Traumatismos do Nervo Laríngeo Recorrente/patologia , Linfócitos T/patologia , Paralisia das Pregas Vocais/diagnóstico , Paralisia das Pregas Vocais/etiologiaRESUMO
Alzheimer's Disease (AD) is a devastating neurodegenerative disorder without a cure. Here we show that mitochondrial respiratory chain complex I is an important small molecule druggable target in AD. Partial inhibition of complex I triggers the AMP-activated protein kinase-dependent signaling network leading to neuroprotection in symptomatic APP/PS1 female mice, a translational model of AD. Treatment of symptomatic APP/PS1 mice with complex I inhibitor improved energy homeostasis, synaptic activity, long-term potentiation, dendritic spine maturation, cognitive function and proteostasis, and reduced oxidative stress and inflammation in brain and periphery, ultimately blocking the ongoing neurodegeneration. Therapeutic efficacy in vivo was monitored using translational biomarkers FDG-PET, 31P NMR, and metabolomics. Cross-validation of the mouse and the human transcriptomic data from the NIH Accelerating Medicines Partnership-AD database demonstrated that pathways improved by the treatment in APP/PS1 mice, including the immune system response and neurotransmission, represent mechanisms essential for therapeutic efficacy in AD patients.
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Doença de Alzheimer/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Cognição/efeitos dos fármacos , Complexo I de Transporte de Elétrons/antagonistas & inibidores , Pironas/uso terapêutico , Doença de Alzheimer/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/ultraestrutura , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuroproteção , Estudo de Prova de Conceito , Pironas/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Renal 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. PURPOSE: To test the feasibility of using quantitative magnetization transfer (qMT), which assesses tissue macromolecule content, to measure renal fibrosis. STUDY TYPE: Prospective. ANIMAL MODEL: Fifteen 129S1 mice were studied 4 weeks after either sham (n = 7) or unilateral renal artery stenosis (RAS, n = 8) surgeries. FIELD STRENGTH/SEQUENCE: Magnetization 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. ASSESSMENT: A 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 TESTS: Comparisons 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. RESULTS: The 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 CONCLUSION: The 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 EVIDENCE: 1 TECHNICAL EFFICACY STAGE: 3.
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Glioblastoma (GBM) is uniformly fatal with a 1-year median survival, despite best available treatment, including radiotherapy (RT). Impacts of prior RT on tumor recurrence are poorly understood but may increase tumor aggressiveness. Metabolic changes have been investigated in radiation-induced brain injury; however, the tumor-promoting effect following prior radiation is lacking. Since RT is vital to GBM management, we quantified tumor-promoting effects of prior RT on patient-derived intracranial GBM xenografts and characterized metabolic alterations associated with the protumorigenic microenvironment. Human xenografts (GBM143) were implanted into nude mice 24 hrs following 20 Gy cranial radiation vs. sham animals. Tumors in pre-radiated mice were more proliferative and more infiltrative, yielding faster mortality (p < 0.0001). Histologic evaluation of tumor associated macrophage/microglia (TAMs) revealed cells with a more fully activated ameboid morphology in pre-radiated animals. Microdialyzates from radiated brain at the margin of tumor infiltration contralateral to the site of implantation were analyzed by unsupervised liquid chromatography-mass spectrometry (LC-MS). In pre-radiated animals, metabolites known to be associated with tumor progression (i.e., modified nucleotides and polyols) were identified. Whole-tissue metabolomic analysis of pre-radiated brain microenvironment for metabolic alterations in a separate cohort of nude mice using 1H-NMR revealed a significant decrease in levels of antioxidants (glutathione (GSH) and ascorbate (ASC)), NAD+, Tricarboxylic acid cycle (TCA) intermediates, and rise in energy carriers (ATP, GTP). GSH and ASC showed highest Variable Importance on Projection prediction (VIPpred) (1.65) in Orthogonal Partial least square Discriminant Analysis (OPLS-DA); Ascorbate catabolism was identified by GC-MS. To assess longevity of radiation effects, we compared survival with implantation occurring 2 months vs. 24 hrs following radiation, finding worse survival in animals implanted at 2 months. These radiation-induced alterations are consistent with a chronic disease-like microenvironment characterized by reduced levels of antioxidants and NAD+, and elevated extracellular ATP and GTP serving as chemoattractants, promoting cell motility and vesicular secretion with decreased levels of GSH and ASC exacerbating oxidative stress. Taken together, these data suggest IR induces tumor-permissive changes in the microenvironment with metabolomic alterations that may facilitate tumor aggressiveness with important implications for recurrent glioblastoma. Harnessing these metabolomic insights may provide opportunities to attenuate RT-associated aggressiveness of recurrent GBM.
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BACKGROUND: In MRI of formalin-fixed tissue one of the problems is the dependence of tissue relaxation properties on formalin composition and composition of embedding medium (EM) used for scanning. In this study, we investigated molecular mechanisms by which the EM composition affects T2 relaxation directly and T1 relaxation indirectly. OBJECTIVE: To identify principal components of formaldehyde based EM and the mechanism by which they affect relaxation properties of fixed tissue. METHODS: We recorded high resolution 1H NMR spectra of common formalin fixatives at temperatures in the range of 5⯰C to 45⯰C. We also measured T1 and T2 relaxation times of various organs of formalin fixed (FF) zebrafish at 7â¯T at 21⯰C and 31⯰C in several EM with and without fixative or gadolinium contrast agents. RESULTS: We showed that the major source of T2 variability is chemical exchange between protons from EM hydroxyls and water, mediated by the presence of phosphate ions. The exchange rate increases with temperature, formaldehyde concentration in EM and phosphate concentration in EM. Depending on which side of the coalescence the system resides, the temperature increase can lead to either shortening or prolongation of T2, or to no noticeable change at all when very close to the coalescence. Chemical exchange can be minimized by washing out from EM the fixative, the phosphate or both. CONCLUSION: The dependence of T2 in fixed tissue on the fixative origin and composition described in prior literature could be attributed to the phosphate buffer accelerated chemical exchange among the fixative hydroxyls and the tissue water. More consistent results in the relaxation measurements could be obtained by stricter control of the fixative composition or by scanning fixed tissue in PBS without fixative.
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Fixadores/farmacologia , Fixação de Tecidos/métodos , Água/química , Animais , Soluções Tampão , Calibragem , Meios de Contraste/farmacologia , Fixadores/química , Formaldeído , Gadolínio/química , Radical Hidroxila/química , Imageamento por Ressonância Magnética , Espectroscopia de Ressonância Magnética , Modelos Estatísticos , Fosfatos/química , Temperatura , Peixe-ZebraRESUMO
Omega-3 polyunsaturated fatty acids (n3-PUFA) are well recognized for their potent triglyceride-lowering effects, but the potential influence of these bioactive lipids on other biological processes, particularly in the context of healthy aging, remains unknown. With the goal of gaining new insight into some less well-characterized biological effects of n3-PUFAs in healthy older adults, we performed metabolomics of fasting peripheral blood plasma collected from 12 young adults and 12 older adults before and after an open-label intervention of n3-PUFA (3.9 g/day, 2.7 g eicosapentaenoic [EPA], 1.2 g docosahexaenoic [DHA]). Proton nuclear magnetic resonance (1H-NMR) based lipoprotein subclass analysis revealed the expected reduction in total triglyceride (TG), but also demonstrated that n3-PUFA supplementation reduced very low-density lipoprotein (VLDL) particle number, modestly increased high-density lipoprotein (HDL) cholesterol, and shifted the composition of HDL subclasses. Further metabolite profiling by 1H-NMR and mass spectrometry revealed pronounced changes in phospholipids, cholesterol esters, diglycerides, and triglycerides following n3-PUFA supplementation. Furthermore, significant changes in hydroxyproline, kynurenine, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) following n3-PUFA supplementation provide further insight into some less well-recognized biological effects of n3-PUFA supplementation, including possible effects on protein metabolism, the kynurenine pathway, and glucose metabolism.
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Ácidos Graxos Ômega-3/administração & dosagem , Metaboloma , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Cinurenina/metabolismo , Lipídeos/sangue , Lipoproteínas/sangue , Masculino , Adulto JovemRESUMO
PURPOSE: To propose a rapid multi-slice T1 measurement method using time-resolved imaging of contrast kinetics (TRICKS) and a semi-automated image processing algorithm for comprehensive assessment murine kidney function using dynamic contrast-enhanced MRI (DCE-MRI). METHODS: A multi-slice TRICKS sampling scheme was implemented in an established rapid T1 measurement method. A semi-automated image-processing scheme employing basic image processing techniques and machine learning was developed to facilitate image analysis. Reliability of the multi-slice technique in measuring renal perfusion and glomerular filtration rate (GFR) was tested in normal mice (nâ¯=â¯7 for both techniques) by comparing to the validated single-slice technique. Utility of this method was demonstrated on mice after either sham surgery (nâ¯=â¯7) or induction of unilateral renal artery stenosis (RAS, nâ¯=â¯8). Renal functional parameters were extracted using a validated bi-compartment model. RESULTS: The TRICKS sampling scheme achieved an acceleration factor of 2.7, allowing imaging of eight axial slices at 1.23â¯s/scan. With the aid of the semi-automated scheme, image analysis required under 15-min for both kidneys per mouse. The multi-slice technique yielded renal perfusion and GFR values comparable to the single-slice technique. Model-fitted renal parameters successfully differentiated control and stenotic mouse kidneys, including renal perfusion (706.5⯱â¯164.0 vs. 375.9⯱â¯277.9â¯mL/100â¯g/min, Pâ¯=â¯0.002), blood flow (1.6⯱â¯0.4 vs. 0.7⯱â¯0.7â¯mL/min, Pâ¯<â¯0.001), and GFR (142.9⯱â¯17.9 vs. 58.0⯱â¯42.8⯵L/min, Pâ¯<â¯0.001). CONCLUSION: The multi-slice TRICKS-based DCE-MRI technique, with a semi-automated image processing scheme, allows rapid and comprehensive measurement of murine kidney function.
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Meios de Contraste/farmacologia , Testes de Função Renal , Rim/diagnóstico por imagem , Imageamento por Ressonância Magnética , Reconhecimento Automatizado de Padrão , Obstrução da Artéria Renal/diagnóstico por imagem , Algoritmos , Animais , Biomarcadores , Taxa de Filtração Glomerular , Interpretação de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador , Cinética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reprodutibilidade dos TestesRESUMO
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 T1 weighting (3D FLASH, 3D FISP), whereas in FFPE specimens T2 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.
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Imageamento Tridimensional , Espectroscopia de Ressonância Magnética , Microscopia/métodos , Peixe-Zebra/fisiologia , Animais , Gadolínio/química , Inclusão em Parafina , Fixação de TecidosRESUMO
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.
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Injúria Renal Aguda/diagnóstico por imagem , Ácido Fólico , Rim/diagnóstico por imagem , Imageamento por Ressonância Magnética , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Injúria Renal Aguda/fisiopatologia , Animais , Modelos Animais de Doenças , Progressão da Doença , Fibrose , Taxa de Filtração Glomerular , Rim/patologia , Rim/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Necrose , Tamanho do Órgão , Valor Preditivo dos Testes , Circulação Renal , Fatores de TempoRESUMO
PURPOSE: Dynamic 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. METHODS: A 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). RESULTS: Validation 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). CONCLUSION: Dynamic 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.
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Meios de Contraste/química , Córtex Renal/diagnóstico por imagem , Rim/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Manganês/química , Animais , Peso Corporal , Sobrevivência Celular , Constrição Patológica/diagnóstico por imagem , Coração/diagnóstico por imagem , Aumento da Imagem/métodos , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Imagens de Fantasmas , Reprodutibilidade dos TestesRESUMO
PURPOSE: To develop and validate a method for measuring murine single-kidney glomerular filtration rate (GFR) using dynamic contrast-enhanced MRI (DCE-MRI). METHODS: This 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. RESULTS: The 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. CONCLUSION: The 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.
Assuntos
Meios de Contraste/química , Taxa de Filtração Glomerular , Rim/diagnóstico por imagem , Imageamento por Ressonância Magnética , Animais , Artérias/patologia , Peso Corporal , Fluoresceína-5-Isotiocianato/química , Interpretação de Imagem Assistida por Computador/métodos , Processamento de Imagem Assistida por Computador , Inulina/química , Testes de Função Renal/métodos , Camundongos , Perfusão , Estudos Prospectivos , Reprodutibilidade dos Testes , Marcadores de SpinRESUMO
Medulloblastoma arising from the cerebellum is the most common pediatric brain malignancy, with leptomeningeal metastases often present at diagnosis and recurrence associated with poor clinical outcome. In this study, we used mouse medulloblastoma models to explore the relationship of tumor pathophysiology and dysregulated expression of the NOTCH pathway transcription factor ATOH1, which is present in aggressive medulloblastoma subtypes driven by aberrant Sonic Hedgehog/Patched (SHH/PTCH) signaling. In experiments with conditional ATOH1 mouse mutants crossed to Ptch1+/- mice, which develop SHH-driven medulloblastoma, animals with Atoh1 transgene expression developed highly penetrant medulloblastoma at a young age with extensive leptomeningeal disease and metastasis to the spinal cord and brain, resembling xenografts of human SHH medulloblastoma. Metastatic tumors retained abnormal SHH signaling like tumor xenografts. Conversely, ATOH1 expression was detected consistently in recurrent and metastatic SHH medulloblastoma. Chromatin immunoprecipitation sequencing and gene expression profiling identified candidate ATOH1 targets in tumor cells involved in development and tumorigenesis. Among these targets specific to metastatic tumors, there was an enrichment in those implicated in extracellular matrix remodeling activity, cytoskeletal network and interaction with microenvironment, indicating a shift in transcriptomic and epigenomic landscapes during metastasis. Treatment with bone morphogenetic protein or SHH pathway inhibitors decreased tumor cell proliferation and suppressed metastatic tumor growth, respectively. Our work reveals a dynamic ATOH1-driven molecular cascade underlying medulloblastoma metastasis that offers possible therapeutic opportunities. Cancer Res; 77(14); 3766-77. ©2017 AACR.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Neoplasias Cerebelares/metabolismo , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proliferação de Células , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/patologia , Proteínas Hedgehog , Xenoenxertos , Humanos , Meduloblastoma/genética , Camundongos , Camundongos Transgênicos , Metástase Neoplásica , Transdução de SinaisRESUMO
Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill-defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)-infected transgenic FVB mice that express the Db class I molecule. FVB/Db and wild-type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy via T2-weighted MRI and subsequent 3-dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV-infected FVB/Db mice, but not in wild-type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4-mo observation period. Of importance, virus-infected FVB/Db mice elicited a strong CD8 T-cell response toward the immunodominant Db-restricted TMEV-derived peptide, VP2121-130, and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus-infected neurons; therefore, we hypothesize that class I restricted CD8 T-cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long-term neuropathology.-Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson. A. J. Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.