Quantitative Magnetization Transfer Detects Renal Fibrosis in Murine Kidneys With Renal Artery Stenosis.

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.

Radiation Induced Metabolic Alterations Associate With Tumor Aggressiveness and Poor Outcome in Glioblastoma.

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.

Distinct Influence of Omega-3 Fatty Acids on the Plasma Metabolome of Healthy Older Adults.

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.

Measurement of murine kidney functional biomarkers using DCE-MRI: A multi-slice TRICKS technique and semi-automated image processing algorithm.

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 (n = 7 for both techniques) by comparing to the validated single-slice technique. Utility of this method was demonstrated on mice after either sham surgery (n = 7) or induction of unilateral renal artery stenosis (RAS, n = 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 1.23 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€¯277.9 mL/100 g/min, P = 0.002), blood flow (1.6 ±â€¯0.4 vs. 0.7 ±â€¯0.7 mL/min, P < 0.001), and GFR (142.9 ±â€¯17.9 vs. 58.0 ±â€¯42.8 µL/min, P < 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.

Three-dimensional NMR microscopy of zebrafish specimens.

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.

Multiparametric MRI detects longitudinal evolution of folic acid-induced nephropathy in mice.

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.

A rapid T1 mapping method for assessment of murine kidney viability using dynamic manganese-enhanced magnetic resonance imaging.

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.

Measurement of Murine Single-Kidney Glomerular Filtration Rate Using Dynamic Contrast-Enhanced MRI.

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.

Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.

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.

Noninvasive Assessment of Renal Fibrosis with Magnetization Transfer MR Imaging: Validation and Evaluation in Murine Renal Artery Stenosis.

Purpose To test the utility of magnetization transfer imaging in detecting and monitoring the progression of renal fibrosis in mice with unilateral renal artery stenosis. Materials and Methods This prospective study was approved by the Institutional Animal Care and Use Committee. Renal artery stenosis surgery (n = 10) or sham surgery (n = 5) was performed, and the stenotic and contralateral kidneys were studied longitudinally in vivo at baseline and 2, 4, and 6 weeks after surgery. After a 16.4-T magnetic resonance imaging examination, magnetization transfer ratio was measured as an index of fibrosis (guided by parameters selected in preliminary phantom studies). In addition, renal volume, perfusion, blood flow, and oxygenation were assessed. Fibrosis was subsequently measured ex vivo by means of histologic analysis and hydroxyproline assay. The Wilcoxon rank sum or signed rank test was used for statistical comparisons between or within groups, and Pearson and Spearman rank correlation was used to compare fibrosis measured in vivo and ex vivo. Results In the stenotic kidney, the median magnetization transfer ratio showed progressive increases from baseline to 6 weeks after surgery (increases of 13.7% [P = .0006] and 21.3% [P = .0005] in cortex and medulla, respectively), which were accompanied by a progressive loss in renal volume, perfusion, blood flow, and oxygenation. The 6-week magnetization transfer ratio map showed good correlation with fibrosis measured ex vivo (Pearson r = 0.9038 and Spearman ρ = 0.8107 [P = .0002 vs trichrome staining]; r = 0.9540 and ρ = 0.8821 [P < .0001 vs Sirius red staining]; and r = 0.8429 and ρ = 0.7607 [P = .001 vs hydroxyproline assay]). Conclusion Magnetization transfer imaging was used successfully to measure and longitudinally monitor the progression of renal fibrosis in mice with unilateral renal artery stenosis. © RSNA, 2016 Online supplemental material is available for this article.

Utilizing magnetization transfer imaging to investigate tissue remodeling in a murine model of autosomal dominant polycystic kidney disease.

PURPOSE: Noninvasive 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. METHODS: C57Bl/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. RESULTS: The 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 R(2) = 0.84 and R(2) = 0.70, respectively. CONCLUSION: MT 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.

Use of Ultra-high Field MRI in Small Rodent Models of Polycystic Kidney Disease for In Vivo Phenotyping and Drug Monitoring.

Several in vivo pre-clinical studies in Polycystic Kidney Disease (PKD) utilize orthologous rodent models to identify and study the genetic and molecular mechanisms responsible for the disease, and are very convenient for rapid drug screening and testing of promising therapies. A limiting factor in these studies is often the lack of efficient non-invasive methods for sequentially analyzing the anatomical and functional changes in the kidney. Magnetic resonance imaging (MRI) is the current gold standard imaging technique to follow autosomal dominant polycystic kidney disease (ADPKD) patients, providing excellent soft tissue contrast and anatomic detail and allowing Total Kidney Volume (TKV) measurements.A major advantage of MRI in rodent models of PKD is the possibility for in vivo imaging allowing for longitudinal studies that use the same animal and therefore reducing the total number of animals required. In this manuscript, we will focus on using Ultra-high field (UHF) MRI to non-invasively acquire in vivo images of rodent models for PKD. The main goal of this work is to introduce the use of MRI as a tool for in vivo phenotypical characterization and drug monitoring in rodent models for PKD.

Evolution of cardiac and renal impairment detected by high-field cardiovascular magnetic resonance in mice with renal artery stenosis.

BACKGROUND: Renal artery stenosis (RAS) promotes hypertension and cardiac dysfunction. The 2-kidney, 1-clip mouse model in many ways resembles RAS in humans and is amenable for genetic manipulation, but difficult to evaluate noninvasively. We hypothesized that cardiovascular magnetic resonance (CMR) is capable of detecting progressive cardiac and renal dysfunction in mice with RAS and monitoring the progression of the disease longitudinally. METHODS: RAS was induced at baseline in eighteen mice by constricting the renal artery. Nine additional animals served as normal controls. CMR scans (16.4 T) were performed in all mice one week before and 2 and 4 weeks after baseline. Renal volumes and hemodynamics were assessed using 3D fast imaging with steady-state precession and arterial spin labelling, and cardiac function using CMR cine. Renal hypoxia was investigated using blood oxygen-level dependent (BOLD) MR. RESULTS: Two weeks after surgery, mean arterial pressure was elevated in RAS mice. The stenotic kidney (STK) showed atrophy, while the contra-lateral kidney (CLK) showed hypertrophy. Renal blood flow (RBF) and cortical oxygenation level declined in the STK but remained unchanged in CLK. Moreover, cardiac end-diastolic and stroke volumes decreased and myocardial mass increased. At 4 weeks, STK RBF remained declined and the STK cortex and medulla showed development of hypoxia. Additionally, BOLD detected a mild hypoxia in CLK cortex. Cardiac end-diastolic and stroke volumes remained reduced and left ventricular hypertrophy worsened. Left ventricular filling velocities (E/A) indicated progression of cardiac dysfunction towards restrictive filling. CONCLUSIONS: CMR detected longitudinal progression of cardiac and renal dysfunction in 2K, 1C mice. These observations support the use of high-field CMR to obtain useful information regarding chronic cardiac and renal dysfunction in small animals.

Micro-computed tomography and nuclear magnetic resonance imaging for noninvasive, live-mouse cholangiography.

The cholangiopathies are a diverse group of biliary tract disorders, many of which lack effective treatment. Murine models are an important tool for studying their pathogenesis, but existing noninvasive methods for assessing biliary disease in vivo are not optimal. Here we report our experience with using micro-computed tomography (microCT) and nuclear magnetic resonance (MR) imaging to develop a technique for live-mouse cholangiography. Using mdr2 knockout (mdr2KO, a model for primary sclerosing cholangitis (PSC)), bile duct-ligated (BDL), and normal mice, we performed in vivo: (1) microCT on a Siemens Inveon PET/CT scanner and (2) MR on a Bruker Avance 16.4 T spectrometer, using Turbo Rapid Acquisition with Relaxation Enhancement, IntraGate Fast Low Angle Shot, and Half-Fourier Acquisition Single-shot Turbo Spin Echo methods. Anesthesia was with 1.5-2.5% isoflurane. Scans were performed with and without contrast agents (iodipamide meglumine (microCT), gadoxetate disodium (MR)). Dissection and liver histology were performed for validation. With microCT, only the gallbladder and extrahepatic bile ducts were visualized despite attempts to optimize timing, route, and dose of contrast. With MR, the gallbladder, extra-, and intrahepatic bile ducts were well-visualized in mdr2KO mice; the cholangiographic appearance was similar to that of PSC (eg, multifocal strictures) and could be improved with contrast administration. In BDL mice, MR revealed cholangiographically distinct progressive dilation of the biliary tree without ductal irregularity. In normal mice, MR allowed visualization of the gallbladder and extrahepatic ducts, but only marginal visualization of the diminutive intrahepatic ducts. One mouse died during microCT and MR imaging, respectively. Both microCT and MR scans could be obtained in ≤20 min. We, therefore, demonstrate that MR cholangiography can be a useful tool for longitudinal studies of the biliary tree in live mice, whereas microCT yields suboptimal duct visualization despite requiring contrast administration. These findings support further development and application of MR cholangiography to the study of mouse models of PSC and other cholangiopathies.

Preclinical (1)H-MRS neurochemical profiling in neurological and psychiatric disorders.

The ongoing development of animal models of neurological and psychiatric disorders in combination with the development of advanced nuclear magnetic resonance (NMR) techniques and instrumentation has led to increased use of in vivo proton NMR spectroscopy ((1)H-MRS) for neurochemical analyses. (1)H-MRS is one of only a few analytical methods that can assay in vivo and longitudinal neurochemical changes associated with neurological and psychiatric diseases, with the added advantage of being a technique that can be utilized in both preclinical and clinical studies. In this review, recent progress in the use of (1)H-MRS to investigate animal models of neurological and psychiatric disorders is summarized with examples from the literature and our own work.

Ethanol withdrawal-induced brain metabolites and the pharmacological effects of acamprosate in mice lacking ENT1.

Acamprosate is clinically used to treat alcohol-dependent patients. While the molecular and pharmacological mechanisms of acamprosate remain unclear, it has been shown to regulate γ-aminobutyric acid (GABA) or glutamate levels in the cortex and striatum. To investigate the effect of acamprosate on brain metabolites in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc), we employed in vivo 16.4 T proton magnetic resonance spectroscopy. We utilized type 1 equilibrative nucleoside transporter (ENT1) null mice since acamprosate attenuates ethanol drinking in these mice. Our findings demonstrated that ethanol withdrawal reduced GABA levels and increased phosphorylated choline compounds in the mPFC of both wild-type and ENT1 null mice. Notably, acamprosate normalized these withdrawal-induced changes only in ENT1 null mice. In the NAc, ethanol withdrawal increased glutamate and glutamine (Glx) levels only in wild-type mice. Interestingly, acamprosate reduced Glx levels in the NAc compared to the withdrawal state in both genotypes. These results provide a molecular basis for the pharmacological effect of acamprosate in the cortical-striatal circuit.

Contrasting roles for CD4 vs. CD8 T-cells in a murine model of virally induced "T1 black hole" formation.

MRI is sensitive to tissue pathology in multiple sclerosis (MS); however, most lesional MRI findings have limited correlation with disability. Chronic T1 hypointense lesions or "T1 black holes" (T1BH), observed in a subset of MS patients and thought to represent axonal damage, show moderate to strong correlation with disability. The pathogenesis of T1BH remains unclear. We previously reported the first and as of yet only model of T1BH formation in the Theiler's murine encephalitis virus induced model of acute CNS neuroinflammation induced injury, where CD8 T-cells are critical mediators of axonal damage and related T1BH formation. The purpose of this study was to further analyze the role of CD8 and CD4 T-cells through adoptive transfer experiments and to determine if the relevant CD8 T-cells are classic epitope specific lymphocytes or different subsets. C57BL/6 mice were used as donors and RAG-1 deficient mice as hosts in our adoptive transfer experiments. In vivo 3-dimensional MRI images were acquired using a 7 Tesla small animal MRI system. For image analysis, we used semi-automated methods in Analyze 9.1; transfer efficiency was monitored using FACS of brain infiltrating lymphocytes. Using a peptide depletion method, we demonstrated that the majority of CD8 T-cells are classic epitope specific cytotoxic cells. CD8 T-cell transfer successfully restored the immune system's capability to mediate T1BH formation in animals that lack adaptive immune system, whereas CD4 T-cell transfer results in an attenuated phenotype with significantly less T1BH formation. These findings demonstrate contrasting roles for these cell types, with additional evidence for a direct pathogenic role of CD8 T-cells in our model of T1 black hole formation.

Dynamic phosphometabolomic profiling of human tissues and transgenic models by 18O-assisted ³¹P NMR and mass spectrometry.

Next-generation screening of disease-related metabolomic phenotypes requires monitoring of both metabolite levels and turnover rates. Stable isotope (18)O-assisted (31)P nuclear magnetic resonance (NMR) and mass spectrometry uniquely allows simultaneous measurement of phosphometabolite levels and turnover rates in tissue and blood samples. The (18)O labeling procedure is based on the incorporation of one (18)O into P(i) from [(18)O]H(2)O with each act of ATP hydrolysis and the distribution of (18)O-labeled phosphoryls among phosphate-carrying molecules. This enables simultaneous recording of ATP synthesis and utilization, phosphotransfer fluxes through adenylate kinase, creatine kinase, and glycolytic pathways, as well as mitochondrial substrate shuttle, urea and Krebs cycle activity, glycogen turnover, and intracellular energetic communication. Application of expanded (18)O-labeling procedures has revealed significant differences in the dynamics of G-6-P[(18)O] (glycolysis), G-3-P[(18)O] (substrate shuttle), and G-1-P[(18)O] (glycogenolysis) between human and rat atrial myocardium. In human atria, the turnover of G-3-P[(18)O], which defects are associated with the sudden death syndrome, was significantly higher indicating a greater importance of substrate shuttling to mitochondria. Phosphometabolomic profiling of transgenic hearts deficient in adenylate kinase (AK1-/-), which altered levels and mutations are associated to human diseases, revealed a stress-induced shift in metabolomic profile with increased CrP[(18)O] and decreased G-1-P[(18)O] metabolic dynamics. The metabolomic profile of creatine kinase M-CK/ScCKmit-/--deficient hearts is characterized by a higher G-6-[(18)O]P turnover rate, G-6-P levels, glycolytic capacity, γ/ß-phosphoryl of GTP[(18)O] turnover, as well as ß-[(18)O]ATP and ß-[(18)O]ADP turnover, indicating altered glycolytic, guanine nucleotide, and adenylate kinase metabolic flux. Thus, (18)O-assisted gas chromatography-mass spectrometry and (31)P NMR provide a suitable platform for dynamic phosphometabolomic profiling of the cellular energetic system enabling prediction and diagnosis of metabolic diseases states.

A single dose of neuron-binding human monoclonal antibody improves spontaneous activity in a murine model of demyelination.

Our laboratory demonstrated that a natural human serum antibody, sHIgM12, binds to neurons in vitro and promotes neurite outgrowth. We generated a recombinant form, rHIgM12, with identical properties. Intracerebral infection with Theiler's Murine Encephalomyelitis Virus (TMEV) of susceptible mouse strains results in chronic demyelinating disease with progressive axonal loss and neurologic dysfunction similar to progressive forms of multiple sclerosis. To study the effects of rHIgM12 on the motor function of TMEV-infected mice, we monitored spontaneous nocturnal activity over many weeks. Nocturnal behavior is a sensitive measure of rodent neurologic function because maximal activity changes are expected to occur during the normally active night time monitoring period. Mice were placed in activity boxes eight days prior to treatment to collect baseline spontaneous activity. After treatment, activity in each group was continuously recorded over 8 weeks. We chose a long 8-week monitoring period for two reasons: (1) we previously demonstrated that IgM induced remyelination is present by 5 weeks post treatment, and (2) TMEV-induced demyelinating disease in this strain progresses very slowly. Due to the long observation periods and large data sets, differences among treatment groups may be difficult to appreciate studying the original unfiltered recordings. To clearly delineate changes in the highly fluctuating original data we applied three different methods: (1) binning, (2) application of Gaussian low-pass filters (GF) and (3) polynomial fitting. Using each of the three methods we showed that compared to control IgM and saline, early treatment with rHIgM12 induced improvement in both horizontal and vertical motor function, whereas later treatment improved only horizontal activity. rHIgM12 did not alter activity of normal, uninfected mice. This study supports the hypothesis that treatment with a neuron-binding IgM not only protects neurons in vitro, but also influences functional motor improvement.

MRI in rodent models of brain disorders.

Magnetic resonance imaging (MRI) is a well-established tool in clinical practice and research on human neurological disorders. Translational MRI research utilizing rodent models of central nervous system (CNS) diseases is becoming popular with the increased availability of dedicated small animal MRI systems. Projects utilizing this technology typically fall into one of two categories: 1) true "pre-clinical" studies involving the use of MRI as a noninvasive disease monitoring tool which serves as a biomarker for selected aspects of the disease and 2) studies investigating the pathomechanism of known human MRI findings in CNS disease models. Most small animal MRI systems operate at 4.7-11.7 Tesla field strengths. Although the higher field strength clearly results in a higher signal-to-noise ratio, which enables higher resolution acquisition, a variety of artifacts and limitations related to the specific absorption rate represent significant challenges in these experiments. In addition to standard T1-, T2-, and T2*-weighted MRI methods, all of the currently available advanced MRI techniques have been utilized in experimental animals, including diffusion, perfusion, and susceptibility weighted imaging, functional magnetic resonance imaging, chemical shift imaging, heteronuclear imaging, and (1)H or (31)P MR spectroscopy. Selected MRI techniques are also exclusively utilized in experimental research, including manganese-enhanced MRI, and cell-specific/molecular imaging techniques utilizing negative contrast materials. In this review, we describe technical and practical aspects of small animal MRI and provide examples of different MRI techniques in anatomical imaging and tract tracing as well as several models of neurological disorders, including inflammatory, neurodegenerative, vascular, and traumatic brain and spinal cord injury models, and neoplastic diseases.