In vivo phenotyping of the ob/ob mouse by magnetic resonance imaging and 1H-magnetic resonance spectroscopy

Objective: We studied ob/ob and wild‐type (WT) mice to characterize the adipose tissues depots and other visceral organs and to establish an experimental paradigm for in vivo phenotyping. Research Methods and Procedures: An in vivo evaluation was conducted using magnetic resonance imaging and ¹H‐magnetic resonance spectroscopy (¹H‐MRS). We used T1‐weighted images and three‐dimensional spin echo T1‐weighted images for the morphological analysis and ¹H‐MRS spectra on all body mass, as well as ¹H‐MRS spectra focalized on specific lipid depots [triglyceride (TG) depots] for a molecular analysis. Results: In ob/ob mice, three‐dimensional evaluation of the trunk revealed that ～64% of the volume consists of white adipose tissue, which is 72% subcutaneous and 28% visceral. In vivo ¹H‐MRS showed that 20.00 ± 6.92% in the WT group and 58.67 ± 6.65% in the ob/ob group of the total proton content is composed of TG protons. In in vivo‐localized spectra of ob/ob mice, we found a polyunsaturation degree of 0.5247 in subcutaneous depots. In the liver, we observed that 48.7% of the proton signal is due to water, whereas in the WT group, the water signal amounted to 82.8% of the total proton signal. With the sequences used, the TG amount was not detectable in the brain or kidneys. Discussion: The present study shows that several parameters can be obtained by in vivo examination of ob/ob mice by magnetic resonance imaging and ¹H‐MRS and that the accumulated white adipose tissue displays low polyunsaturation degree and low hydrolipidic ratio. Relevant anatomical alterations observed in urinary and digestive apparatuses should be considered when ob/ob mice are used in experimental paradigms.     

Cerebral creatine kinase deficiency influences metabolite levels and morphology in the mouse brain: a quantitative in vivo 1H and 31P magnetic resonance study

Creatine kinase (CK)-catalysed ATP-phosphocreatine (PCr) exchange is considered to play a key role in energy homeostasis of the brain. This study assessed the metabolic and anatomical consequences of partial or complete depletion of this system in transgenic mice without cytosolic B-CK (B-CK-/-), mitochondrial ubiquitous CK (UbCKmit-/-), or both isoenzymes (CK -/-), using non-invasive quantitative magnetic resonance (MR) imaging and spectroscopy. MR imaging revealed an increase in ventricle size in a subset of B-CK-/- mice, but not in animals with UbCKmit or compound CK mutations. Mice lacking single CK isoenzymes had normal levels of high-energy metabolites and tissue pH. In the brains of CK double knockouts pH and ATP and Pi levels were also normal, even though PCr had become completely undetectable. Moreover, a 20-30% decrease was observed in the level of total creatine and a similar increase in the level of neuronal N-acetyl-aspartate compounds. Although CKs themselves are not evenly distributed throughout the CNS, these alterations were uniform and concordant across different brain regions. Changes in myo-inositol and glutamate peaks did appear to be mutation type and brain area specific. Our results challenge current models for the biological significance of the PCr-CK energy system and suggest a multifaceted role for creatine in the brain.     

High-field (9.4 T) 1H magnetic resonance microscopy of mouse brain

The FLASH and STEAM pulse sequences were used to perform the microimaging and localized spectroscopy of brain of living and dead mice, respectively. The phase-shift presaturation approach was used to sup-press water NMR signal. The experimental results show that the differences in localized spectra and MR images of brain between live and dead mice can be observed by means of magnetic resonance microscopy.     

Metabolic changes detected by proton magnetic resonance spectroscopy in vivo and in vitro in a murin model of Parkinson's disease, the MPTP-intoxicated mouse

Parkinson's disease is a neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta, which project to the striatum. The aim of this study was to analyze in vivo and in vitro consequences of dopamine depletion on amount of metabolites in a mouse model of Parkinson's disease using proton (1)H magnetic resonance spectroscopy (MRS). The study was performed on control mice (n = 7) and MPTP-intoxicated mice (n = 7). All the experiments were performed at 9.4 T. For in vivo MRS acquisitions, mice were anesthetized and carefully placed on an animal handling system with the head centered in birdcage coil used for both excitation and signal reception. Spectra were acquired in a voxel (8 microL) centered in the striatum, applying a point-resolved spectroscopy sequence (TR = 4000 ms, TE = 8.8 ms). After in vivo MRS acquisitions, mice were killed; successful lesion verified by tyrosine hydroxylase immunolabeling on the substantia nigra pars compacta and in vitro MRS acquisitions performed on perchloric extracts of anterior part of mice brains. In vitro spectra were acquired using a standard one-pulse experiment. The absolute concentrations of metabolites were determined using jmrui (Lyon, France) from (1)H spectra obtained in vivo on striatum and in vitro on perchloric extracts. Glutamate (Glu), glutamine (Gln), and GABA concentrations obtained in vivo were significantly increased in striatum of MPTP-lesioned mice (Glu: 15.5 +/- 2.5 vs. 12.9 +/- 1.0 mmol/L, p < 0.05; Gln: 2.3 +/- 0.9 vs. 1.8 +/- 0.6 mmol/L, p < 0.05; GABA: 2.3 +/- 0.9 vs. 1.3 +/- 0.6 mmol/L, p < 0.05). The in vitro results confirmed these results, Glu (10.9 +/- 2.5 vs. 7.9 +/- 1.7 micromol/g, p < 0.05), Gln (6.8 +/- 2.9 vs. 4.3 +/- 1.0 micromol/g, p < 0.05), and GABA (2.9 +/- 0.9 vs. 1.5 +/- 0.4 micromol/g, p < 0.01). The present study strongly supports a hyperactivity of the glutamatergic cortico-striatal pathway hypothesis after dopaminergic denervation in association with an increase of striatal GABA levels. It further shows an increased of striatal Gln concentrations, perhaps as a strategy to protect neurons from Glu excitotoxic injury after striatal dopamine depletion.     

In vivo hepatic lipid quantification using MRS at 7 Tesla in a mouse model of glycogen storage disease type 1a

The assessment of liver lipid content and composition is needed in preclinical research to investigate steatosis and steatosis-related disorders. The purpose of this study was to quantify in vivo hepatic fatty acid content and composition using a method based on short echo time proton magnetic resonance spectroscopy (MRS) at 7 Tesla. A mouse model of glycogen storage disease type 1a with inducible liver-specific deletion of the glucose-6-phosphatase gene (L-G6pc^−/−) mice and control mice were fed a standard diet or a high-fat/high-sucrose (HF/HS) diet for 9 months. In control mice, hepatic lipid content was found significantly higher with the HF/HS diet than with the standard diet. As expected, hepatic lipid content was already elevated in L-G6pc^−/− mice fed a standard diet compared with control mice. L-G6pc^−/− mice rapidly developed steatosis which was not modified by the HF/HS diet. On the standard diet, estimated amplitudes from olefinic protons were found significantly higher in L-G6pc^−/− mice compared with that in control mice. L-G6pc^−/− mice showed no noticeable polyunsaturation from diallylic protons. Total unsaturated fatty acid indexes measured by gas chromatography were in agreement with MRS measurements. These results showed the great potential of high magnetic field MRS to follow the diet impact and lipid alterations in mouse liver.     

Neurochemical profile of the developing mouse cortex determined by in vivo 1H NMR spectroscopy at 14.1 T and the effect of recurrent anaesthesia

The neurochemical profile of the cortex develops in a region and time specific manner, which can be distorted by psychiatric and other neurological pathologies. Pre-clinical studies often involve experimental mouse models. In this study, we determined the neurochemical profile of C57BL/6 mice in a longitudinal study design to provide a reference frame for the normal developing mouse cortex. Using in vivo proton NMR spectroscopy at 14 T, we measured the concentrations of 18 metabolites in the anterior and posterior cortex on postnatal days (P) 10, 20, 30, 60 and 90. Cortical development was marked by alterations of highly concentrated metabolites, such as N-acetylaspartate, glutamate, taurine and creatine. Regional specificity was represented by early variations in the concentration of glutamine, aspartate and choline. In adult animals, regional concentration differences were found for N-acetylaspartate, creatine and myo-inositol. In this study, animals were exposed to recurrent isoflurane anaesthesia. Additional experiments showed that the latter was devoid of major effects on behaviour or cortical neurochemical profile. In conclusion, the high sensitivity and reproducibility of the measurements achieved at 14 T allowed us to identify developmental variations of cortical areas within the mouse cortex.     

Muscle-associated triglyceride measured by computed tomography and magnetic resonance spectroscopy

Objective: Muscle triglyceride can be assessed in vivo using computed tomography (CT) and ¹H magnetic resonance spectroscopy (MRS), two techniques that are based on entirely different biophysical principles. Little is known, however, about the cross‐correlation between these techniques and their test—retest reliability. Research Methods and Procedures: We compared mean muscle attenuation (MA) in soleus and tibialis anterior (TA) muscles measured by CT with intra‐ and extramyocellular lipids (IMCL and EMCL, respectively) measured by MRS in 51 volunteers (26 to 72 years of age, BMI = 25.5 to 39.3 kg/m²). MA of midthighs was also measured in a subset (n = 19). Test—retest measurements were performed by CT (n = 6) and MRS (n = 10) in separate sets of volunteers. Results: MA of soleus was significantly associated with IMCL (r = ?0.64) and EMCL, which by multiple regression analysis was explained mostly by IMCL (p < 0.001) rather than EMCL (β = ?0.010, p = 0.94). Muscle triglyc‐eride was lower in TA than in soleus, and MA of TA was significantly correlated with EMCL (r = ?0.40) but not IMCL (r = ?0.16). By CT, MA of midthighs was correlated with MA in soleus (r = 0.40, p = 0.07) and whole calf (r = 0.62, p < 0.05). Finally, both MA and IMCL were highly reliable in soleus (coefficient of variation = <2% and 6.7%, respectively) and less reliable in TA (4% and 10%, respectively). Discussion: These results support the use of both CT and MRS as reliable methods for assessing skeletal muscle lipid.     

Region-selective alterations of glucose oxidation and amino acid synthesis in the thiamine-deficient rat brain: a re-evaluation using 1H/13C nuclear magnetic resonance spectroscopy

Thiamine deficiency provides an effective model of selective neuronal cell death. ¹H and ¹³C-NMR was used to investigate the effects of thiamine deficiency on the synthesis of amino acids derived from [1-¹³C]glucose in vulnerable (medial thalamus; MT) compared to non-vulnerable (frontal cortex; FC) brain regions. Following 11 days of thiamine deficiency, a time-point associated with the absence of significant neuronal cell death, regional concentrations of glutamate, glutamine and GABA remained unaffected in FC and MT; however, decreased levels of aspartate in MT at this time-point were a predictor of regional vulnerability. De novo synthesis of glutamate and GABA were unaffected at 11 days of thiamine deficiency, while synthesis of [2-¹³C]aspartate was significantly impaired. Glucose loading, which has been shown to exacerbate symptoms in patients with thiamine deficiency, resulted in further decreases of TCA cycle flux and reduced de novo synthesis of glutamate, aspartate and GABA in thiamine-deficient (TD) rats. Isotopomer analysis revealed that impaired TCA cycle flux and decreased aspartate synthesis due to thiamine deficiency occurred principally in neurons. Glucose loading deteriorated TD-related decreases in TCA cycle flux, and concomitantly reduced synthesis of aspartate and glutamate in MT.     

In vivo quantification of neuro‐glial metabolism and glial glutamate concentration using 1H‐[13C] MRS at 14.1T

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo ¹³C NMR spectroscopy coupled with the infusion labeled glial‐specific substrate, such as acetate. In this study, we infused alpha‐chloralose anesthetized rats with [2‐¹³C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using ¹H‐[¹³C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two‐compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (V_g) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (V_NT) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (V_PC) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (V_x^g and V_xⁿ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations.

     

Intrauterine hyperexposure to dexamethasone of the common marmoset monkey revealed normal cerebral metabolite concentrations in adulthood as assessed by quantitative proton magnetic resonance spectroscopy in vivo

Background  Animal models of human brain disorders often have to rely on non-human primates because of their immunological, physiological, and cognitive similarities to humans.
Methods  Localized proton magnetic resonance spectroscopy was performed to assess cerebral metabolite profiles of male common marmoset monkeys in vivo and to determine putative alterations of adult brain metabolism in response to intrauterine hyperexposure to the synthetic glucocorticoid hormone dexamethasone.
Results  Excellent spectral quality allowed for absolute quantification of the concentrations of major metabolites in predominantly white matter, gray matter, and thalamus. Marmoset monkeys intrauterinely hyperexposed to dexamethasone revealed normal neurochemical profiles at adulthood.
Conclusions  Prenatally applied dexamethasone does not lead to persistent metabolic alterations affecting adult brain integrity.     

Effects of CAG repeat length, HTT protein length and protein context on cerebral metabolism measured using magnetic resonance spectroscopy in transgenic mouse models of Huntington's disease

Huntington's disease is a neurodegenerative illness caused by expansion of CAG repeats at the N-terminal end of the protein huntingtin. We examined longitudinal changes in brain metabolite levels using in vivo magnetic resonance spectroscopy in five different mouse models. There was a large (>50%) exponential decrease in N-acetyl aspartate (NAA) with time in both striatum and cortex in mice with 150 CAG repeats (R6/2 strain). There was a linear decrease restricted to striatum in N171-82Q mice with 82 CAG repeats. Both the exponential and linear decreases of NAA were paralleled in time by decreases in neuronal area measured histologically. Yeast artificial chromosome transgenic mice with 72 CAG repeats, but low expression levels, had less striatal NAA loss than the N171-82Q mice (15% vs. 43%). We evaluated the effect of gene context in mice with an approximate 146 CAG repeat on the hypoxanthine phosphoribosyltransferase gene (HPRT). HPRT mice developed an obese phenotype in contrast to weight loss in the R6/2 and N171-82Q mice. These mice showed a small striatal NAA loss (21%), and a possible increase in brain lipids detectable by magnetic resonance (MR) spectroscopy and decreased brain water T1. Our results indicate profound metabolic defects that are strongly affected by CAG repeat length, as well as gene expression levels and protein context.     

Lipopolysaccharide‐bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad‐spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram‐negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram‐negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α‐helical structure in a solution containing LPS. For NMR experiments, we expressed ¹⁵N‐labeled and ¹³C‐labeled CP1 in bacterial cells and successfully assigned almost all backbone and side‐chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr‐NOE) experiments in LPS. We performed ¹⁵N‐edited and ¹³C‐edited Tr‐NOE spectroscopy for CP1 bound to LPS. Tr‐NOE peaks were observed at the only C‐terminal region of CP1 in LPS. The results of structure calculation indicated that the C‐terminal region (Lys15–Gly29) formed the well‐defined α‐helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Hepatic 31P‐magnetic resonance spectroscopy identified the impact of melatonin‐pretreated mitochondria in acute liver ischaemia‐reperfusion injury

Acute liver ischaemia‐reperfusion injury (IRI), commonly encountered during liver resection and transplantation surgery, is strongly associated with unfavourable clinical outcome. However, a prompt and accurate diagnosis and the treatment of this entity remain formidable challenges. This study tested the hypothesis that ³¹P‐magnetic resonance spectroscopy (³¹P‐MRS) findings could provide reliable living images to accurately identify the degree of acute liver IRI and melatonin‐pretreated mitochondria was an innovative treatment for protecting the liver from IRI in rat. Adult male SD rats were categorized into group 1 (sham‐operated control), group 2 (IRI only) and group 3 (IRI + melatonin [ie mitochondrial donor rat received intraperitoneal administration of melatonin] pretreated mitochondria [10 mg/per rat by portal vein]). By the end of study period at 72 hours, ³¹P‐MRS showed that, as compared with group 1, the hepatic levels of ATP and NADH were significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1. The liver protein expressions of mitochondrial‐electron‐transport‐chain complexes and mitochondrial integrity exhibited an identical pattern to ³¹P‐MRS finding. The protein expressions of oxidative stress, inflammatory, cellular stress signalling and mitochondrial‐damaged biomarkers displayed an opposite finding of ³¹P‐MRS, whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. Microscopic findings showed that the fibrotic area/liver injury score and inflammatory and DNA‐damaged biomarkers exhibited an identical pattern of cellular stress signalling. Melatonin‐pretreated mitochondria effectively protected liver against IRI and ³¹P‐MRS was a reliable tool for measuring the mitochondrial/ATP consumption in living animals.     

Brain development:1H magnetic resonance spectroscopy of rat brain extracts compared with chromatographic methods

We compared in vitro¹H magnetic resonance spectroscopy (MRS) measurements of rat brain extracts (rats: 2–56 days old) with chromatographic measurements and in a further step also with results of in vivo MRS. The following substances can be reliably measured in brain extracts by in vitro MRS: N-acetylaspartate (NAA), total creatine (Cr), phosphorylethanoloamine (PE), taurine (Tau), glutamate (Glu), glutamine (Gln), -aminobutyrate (GABA) and alanine (Ala). Two different methods of MRS data evaluation compared with chromatographic data on Cr and NAA are shown. During development of the rat from day 2–56 brain concentrations of PE, Tau and Ala decrease, those of NAA, Cr, Glu and Gln increase, while GABA does not change. The developmental patterns of these substances are the same, whether measured by in vitro MRS or by chromatographic methods. Quantification of NAA, Cr, Tau, GABA and PE leads to the same results with both methods, while Glu, Gln and Ala concentrations determined by in vitro MRS are apparently lower than those measured chemically. The NAA/Cr ratios of 7 to 35-day-old rats were determined by in vivo¹H MRS. These results correlate with chromatographic and in vitro data. Using appropriate methods in the in vivo and in vitro MR-technique, the obtained data compare well with the chromatographic results.     

Neurochemical changes in Huntington R6/2 mouse striatum detected by in vivo 1H NMR spectroscopy

The neurochemical profile of the striatum of R6/2 Huntington's disease mice was examined at different stages of pathogenesis using in vivo(1)H NMR spectroscopy at 9.4 T. Between 8 and 12 weeks, R6/2 mice exhibited distinct changes in a set of 17 quantifiable metabolites compared with littermate controls. Concentrations of creatine, glycerophosphorylcholine, glutamine and glutathione increased and N-acetylaspartate decreased at 8 weeks. By 12 weeks, concentrations of phosphocreatine, taurine, ascorbate, glutamate, and myo-inositol increased and phophorylethanolamine decreased. These metabolic changes probably reflected multiple processes, including compensatory processes to maintain homeostasis, active at different stages in the development of HD. The observed changes in concentrations suggested impairment of neurotransmission, neuronal integrity and energy demand, and increased membrane breakdown, gliosis, and osmotic and oxidative stress. Comparisons between metabolite concentrations from individual animals clearly distinguished HD transgenics from non-diseased littermates and identified possible markers of disease progression. Metabolic changes in R6/2 striata were distinctly different from those observed previously in the quinolinic acid and 3NP models of HD. Longitudinal monitoring of changes in these metabolites may provide quantifiable measures of disease progression and treatment effects in both mouse models of HD and patients.     

In vitro 1H NMR spectroscopy shows an increase in N-acetylaspartylglutamate and glutamine content in the hippocampus of amygdaloid-kindled rats

We examined energy metabolism and amino acid content in the hippocampus of amygdaloid-kindled rats using (1)H NMR spectroscopy. Three weeks after the last stage 5 seizure, kindled rats were killed by microwave irradiation. The hippocampus was dissected out and subjected to MeOH/CHCl(3) extraction. All (1)H spectra were analyzed to quantify absolute concentrations using a non-linear least squares method, combined with a prior knowledge of chemical shifts. Saturation effects were compensated for by the T1 measurement of each component. Levels of energy metabolism-related compounds, phosphocreatine, creatine, glucose and succinate were the same in both kindled rats and sham controls. Lactate concentration had a tendency to increase, although this was not statistically significant. When compared with sham controls, levels of aspartate, glutamate, glycine and glutamine, as well as GABA and inositol, were increased in the ipsilateral but not the contralateral hippocampus. In contrast, levels of taurine, alanine and threonine were unchanged. Finally, N-acetylaspartylglutamate content was elevated, whereas N-acetyl-l-aspartate content was unaltered in the ipsilateral hippocampus of kindled animals. Our results suggest that amygdala kindling may affects amino acid metabolism, but not energy metabolism.     

Probing a CMP-Kdn synthetase by 1H, 31P, and STD NMR spectroscopy

CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and cytidine-5'-triphosphate (CTP) to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP(i). STD NMR experiments of a recombinant nucleotide cytidine-5'-monophosphate-3-deoxy-d-glycero-d-galacto-nonulosonic acid synthetase (CMP-Kdn synthetase) were performed to map the binding epitope of the substrate CTP and the product CMP-Neu5Ac. The STD NMR analysis clearly shows that the anomeric proton of the ribose moiety of both investigated compounds is in close proximity to the protein surface and is likely to play a key role in the binding process. The relative rates of the enzyme reaction, derived from (1)H NMR signal integrals, show that Kdn is activated at a rate 2.5 and 3.1 faster than Neu5Ac and Neu5Gc, respectively. Furthermore, proton-decoupled (31)P NMR spectroscopy was successfully used to follow the enzyme reaction and clearly confirmed the appearance of CMP-Sia and the inorganic pyrophosphate by-product.