Transverse relaxation in fixed tissue: Influence of temperature and resolution on image contrast in magnetic resonance microscopy.

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 ∝ ∆ 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.

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.

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.

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.

MR microscopy of formalin fixed paraffin embedded histology specimens.

PURPOSE: Archived 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. METHODS: A 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. RESULTS: Using 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. CONCLUSION: MRM 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.

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.

Decoded calreticulin-deficient embryonic stem cell transcriptome resolves latent cardiophenotype.

Genomic perturbations that challenge normal signaling at the pluripotent stage may trigger unforeseen ontogenic aberrancies. Anticipatory systems biology identification of transcriptome landscapes that underlie latent phenotypes would offer molecular diagnosis before the onset of symptoms. The purpose of this study was to assess the impact of calreticulin-deficient embryonic stem cell transcriptomes on molecular functions and physiological systems. Bioinformatic surveillance of calreticulin-null stem cells, a monogenic insult model, diagnosed a disruption in transcriptome dynamics, which re-prioritized essential cellular functions. Calreticulin-calibrated signaling axes were uncovered, and network-wide cartography of undifferentiated stem cell transcripts suggested cardiac manifestations. Calreticulin-deficient stem cell-derived cardiac cells verified disorganized sarcomerogenesis, mitochondrial paucity, and cytoarchitectural aberrations to validate calreticulin-dependent network forecasts. Furthermore, magnetic resonance imaging and histopathology detected a ventricular septal defect, revealing organogenic manifestation of calreticulin deletion. Thus, bioinformatic deciphering of a primordial calreticulin-deficient transcriptome decoded at the pluripotent stem cell stage a reconfigured multifunctional molecular registry to anticipate predifferentiation susceptibility toward abnormal cardiophenotype.

Brainstem 1H nuclear magnetic resonance (NMR) spectroscopy: marker of demyelination and repair in spinal cord.

Measuring in vivo spinal cord injury and repair remains elusive. Using magnetic resonance spectroscopy (MRS) we examined brainstem N-acetyl-aspartate (NAA) as a surrogate for spinal cord injury in two mouse strains with different reparative phenotypes following virus-induced demyelination. Swiss Jim Lambert (SJL) and Friend Virus B (FVB) mice progressively demyelinate with axonal loss. FVB mice demyelinate similarly but eventually remyelinate coincident with functional recovery. Brainstem NAA levels drop in both but recover in FVB mice. Chronically infected SJL mice lost 30.5% of spinal cord axons compared to FVB mice (7.3%). In remyelination-enhancing or axon-preserving clinical trials, brainstem MRS may be a viable endpoint to represent overall spinal cord dysfunction.

Influence of water based embedding media composition on the relaxation properties of fixed tissue.

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 7 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.

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.

Effect of hydrogel porosity on marrow stromal cell phenotypic expression.

This study describes investigation of porous photocrosslinked oligo[(polyethylene glycol) fumarate] (OPF) hydrogels as potential matrix for osteoblastic differentiation of marrow stromal cells (MSCs). The porosity and interconnectivity of porous hydrogels were assessed using magnetic resonance microscopy (MRM) as a noninvasive investigative tool that could image the water construct inside the hydrogels at a high-spatial resolution. MSCs were cultured onto the porous hydrogels and cell number was assessed using PicoGreen DNA assay. Our results showed 10% of cells initially attached to the surface of scaffolds. However, cells did not show significant proliferation over a time period of 14 days. MSCs cultured on porous hydrogels had increased alkaline phosphatase activity as well as deposition of calcium, suggesting successful differentiation and maturation to the osteoblastic phenotype. Moreover, continued expression of type I collagen and osteonectin over 14 days confirmed osteoblastic differentiation of MSCs. MRM was also applied to monitor osteogenesis of MSCs on porous hydrogels. MRM images showed porous scaffolds became consolidated with osteogenic progression of cell differentiation. These findings indicate that porous OPF scaffolds enhanced MSC differentiation leading to development of bone-like mineralized tissue.

Use of a vaccine strain of measles virus genetically engineered to produce carcinoembryonic antigen as a novel therapeutic agent against glioblastoma multiforme.

Despite the most aggressive medical and surgical treatments, glioblastoma multiforme remains incurable with a median survival of <1 year. We investigated the antitumor potential of a novel viral agent, an attenuated strain of measles virus (MV), derived from the Edmonston vaccine lineage, genetically engineered to produce carcinoembryonic antigen (CEA). CEA production as the virus replicates can serve as a marker of viral gene expression. Infection of a variety of glioblastoma cell lines including U87, U118, and U251 at MOIs 0.1, 1, and 10 resulted in significant cytopathic effect consisting of excessive syncycial formation and massive cell death at 72-96 h from infection. terminal deoxynucleotidyltransferase-mediated nick end labeling assays demonstrated the mechanism of cell death to be predominantly apoptotic. The efficacy of this approach in vivo was examined in BALB/c nude mice by using both s.c. and intracranial orthotopic U87 tumor models. In the s.c. U87 model, mice with established xenografts were treated with a total dose of 8 x 10(7) plaque forming units of MV-CEA, administered i.v. Mice treated with UV light inactivated MV, and untreated mice with established U87 tumors were used as controls. There was statistically significant regression of s.c. tumors (P < 0.001) and prolongation of survival (P = 0.007) in MV-CEA treated animals compared with the two control groups. In the intracranial orthotopic U87 model, there was significant regression of intracranial U87 tumors treated with intratumoral administration of MV-CEA at a total dose of 1.8 x 10(6) plaque forming units as assessed by magnetic resonance image (P = 0.002), and statistically significant prolongation of survival as compared with mice that received UV-inactivated virus and untreated mice (P = 0.02). Histological examination of brains of MV-CEA-treated animals revealed complete regression of the tumor with the presence of a residual glial scar and reactive changes, mainly presence of hemosiderin-laden macrophages. In addition, CEA levels in the peripheral blood in both the s.c. and orthotopic models increased before tumor regression, indicating viral gene expression, and returned to normal when the tumors regressed. Ifnar(ko) CD46 Ge transgenic mice, susceptible to MV infection, were used to assess central nervous system toxicity of MV-CEA. Intracranial administration of MV-CEA into the caudate nucleus of Ifnar(ko) CD46 Ge did not result in clinical neurotoxicity. Pathologic examination demonstrated limited microglial infiltration surrounding the injection site. In summary, MV-CEA has potent antitumor activity against gliomas in vitro, as well as in both s.c. and orthotopic U87 animal models. Monitoring CEA levels in the serum can serve as a low-risk method of detecting viral gene expression during treatment, and could allow dose optimization and individualization of treatment.

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.

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.

Acamprosate reduces ethanol drinking behaviors and alters the metabolite profile in mice lacking ENT1.

Acamprosate is clinically used to treat alcoholism. However, the precise molecular functionality of acamprosate in the central nervous system remains unclear, although it is known to antagonize glutamate action in the brain. Since elevated glutamate signaling, especially in the nucleus accumbens (NAc), is implicated in several aspects of alcoholism, we utilized mice lacking type 1 equilibrative nucleoside transporter (ENT1), which exhibit increased glutamate levels in the NAc as well as increased ethanol drinking behaviors. We found that acamprosate significantly reduced ethanol drinking of mice lacking ENT1 (ENT1(-/-)) while having no such effect in wild-type littermates. We then analyzed the basal and acamprosate-treated accumbal metabolite profiles of ENT1(-/-) and wild-type mice using in vivo 16.4T proton magnetic resonance spectroscopy (MRS). Our data show that basal glutamate+glutamine (Glx), glutamate, glutamine and N-acetylaspartatic acid (NAA) levels are increased in the nucleus accumbens (NAc) of ENT1(-/-) compared to wild-type mice. We then found that acamprosate treatment significantly reduced Glx and glutamine levels while increasing taurine levels in the NAc of only ENT1(-/-) compared to their saline-treated group while normalizing other metabolite compared to wild-type mice. This study will be useful in the understanding of the molecular basis of acamprosate in the brain.

Rigid fixation of the spinal column improves scaffold alignment and prevents scoliosis in the transected rat spinal cord.

STUDY DESIGN: A controlled study to evaluate a new technique for spinal rod fixation after spinal cord injury in rats. Alignment of implanted tissue-engineered scaffolds was assessed radiographically and by magnetic resonance imaging. OBJECTIVE: To evaluate the stability of implanted scaffolds and the extent of kyphoscoliotic deformities after spinal fixation. SUMMARY OF BACKGROUND DATA: Biodegradable scaffolds provide an excellent platform for the quantitative assessment of cellular and molecular factors that promote regeneration within the transected cord. Successful delivery of scaffolds to the damaged cord can be hampered by malalignment following transplantation, which in turn, hinders the assessment of neural regeneration. METHODS: Radio-opaque barium sulfate-impregnated poly-lactic-co-glycolic acid scaffolds were implanted into spinal transection injuries in adult rats. Spinal fixation was performed in one group of animals using a metal rod fixed to the spinous processes above and below the site of injury, while the control group received no fixation. Radiographic morphometry was performed after 2 and 4 weeks, and 3-dimensional magnetic resonance microscopy analysis 4 weeks after surgery. RESULTS: Over the course of 4 weeks, progressive scoliosis was evident in the unfixed group, where a Cobb angle of 8.13 +/- 2.03 degrees was measured. The fixed group demonstrated significantly less scoliosis, with a Cobb angle measurement of 1.89 +/- 0.75 degrees (P = 0.0004). Similarly, a trend for less kyphosis was evident in the fixed group (7.33 +/- 1.68 degrees ) compared with the unfixed group (10.13 +/- 1.46 degrees ). Quantitative measurements of the degree of malalignment of the scaffolds were also significantly less in the fixed group (5 +/- 1.23 degrees ) compared with the unfixed group (11 +/- 2.82 degrees ) (P = 0.0143). CONCLUSION: Radio-opaque barium sulfate allows for visualization of scaffolds in vivo using radiographic analysis. Spinal fixation was shown to prevent scoliosis, reduce kyphosis, and reduce scaffold malalignment within the transected rat spinal cord. Using a highly optimized model will increase the potential for finding a therapy for restoring function to the injured cord.