A pharmacokinetic model for hyperpolarized 13C-pyruvate MRI when using metabolite-specific bSSFP sequences.

PURPOSE: Metabolite-specific balanced SSFP (MS-bSSFP) sequences are increasingly used in hyperpolarized [1-13C]Pyruvate (HP 13C) MRI studies as they improve SNR by refocusing the magnetization each TR. Currently, pharmacokinetic models used to fit conversion rate constants, kPL and kPB, and rate constant maps do not account for differences in the signal evolution of MS-bSSFP acquisitions. METHODS: In this work, a flexible MS-bSSFP model was built that can be used to fit conversion rate constants for these experiments. The model was validated in vivo using paired animal (healthy rat kidneys n = 8, transgenic adenocarcinoma of the mouse prostate n = 3) and human renal cell carcinoma (n = 3) datasets. Gradient echo (GRE) acquisitions were used with a previous GRE model to compare to the results of the proposed GRE-bSSFP model. RESULTS: Within simulations, the proposed GRE-bSSFP model fits the simulated data well, whereas a GRE model shows bias because of model mismatch. For the in vivo datasets, the estimated conversion rate constants using the proposed GRE-bSSFP model are consistent with a previous GRE model. Jointly fitting the lactate T2 with kPL resulted in less precise kPL estimates. CONCLUSION: The proposed GRE-bSSFP model provides a method to estimate conversion rate constants, kPL and kPB, for MS-bSSFP HP 13C experiments. This model may also be modified and used for other applications, for example, estimating rate constants with other hyperpolarized reagents or multi-echo bSSFP.

A Simulation of the Effects of Diffusion on Hyperpolarized [1-13C]-Pyruvate Signal Evolution.

OBJECTIVE: Hyperpolarized [1-13C]-pyruvate magnetic resonance imaging is an emerging metabolic imaging method that offers unprecedented spatiotemporal resolution for monitoring tumor metabolism in vivo. To establish robust imaging biomarkers of metabolism, we must characterize phenomena that may modulate the apparent pyruvate-to-lactate conversion rate (kPL). Here, we investigate the potential effect of diffusion on pyruvate-to-lactate conversion, as failure to account for diffusion in pharmacokinetic analysis may obscure true intracellular chemical conversion rates. METHODS: Changes in hyperpolarized pyruvate and lactate signal were calculated using a finite-difference time domain simulation of a two-dimensional tissue model. Signal evolution curves with intracellular kPL values from 0.02 to 1.00 s-1 were analyzed using spatially invariant one-compartment and two-compartment pharmacokinetic models. A second spatially variant simulation incorporating compartmental instantaneous mixing was fit with the same one-compartment model. RESULTS: When fitting with the one-compartment model, apparent kPL underestimated intracellular kPL by approximately 50% at an intracellular kPL of 0.02 s-1. This underestimation increased for larger kPL values. However, fitting the instantaneous mixing curves showed that diffusion accounted for only a small part of this underestimation. Fitting with the two-compartment model yielded more accurate intracellular kPL values. SIGNIFICANCE: This work suggests diffusion is not a significant rate-limiting factor in pyruvate-to-lactate conversion given that our model assumptions hold true. In higher order models, diffusion effects may be accounted for by a term characterizing metabolite transport. Pharmacokinetic models used to analyze hyperpolarized pyruvate signal evolution should focus on carefully selecting the analytical model for fitting rather than accounting for diffusion effects.

A sensitive LC-MS/MS method for the study of exogenously administered 13 C-oleoylethanolamide in rat plasma and brain tissue.

Oleoylethanolamide is an endogenous molecule with neuroprotective effects. It has been reported that exogenous oleoylethanolamide can be administered therapeutically, but the confounding presence of the endogenous molecule has led to conflicting reports regarding the mechanisms of the effects and highlights a need for an adequate methodology to differentiate them. We have developed a liquid chromatography-tandem mass spectrometry method to study oleoylethanolamide in rat plasma and brain using a 13 C-labeled isotope, 13 C-oleoylethanolamide. 13 C-oleoylethanolamide was extracted using a liquid-liquid extraction employing acetonitrile and tert-butyl methyl ether (1:4). Analysis was performed using a gradient with a total run time of 12 min. 13 C-oleoylethanolamide, d4 -oleoylethanolamide (internal standard), and 12 C-oleoylethanolamide (endogenous background) eluted simultaneously at 1.64 min. The method was validated for specificity, sensitivity, accuracy, and precision and found to be capable of quantification within acceptable limits of ±15% over the calibration range of 0.39-25 ng/mL for the plasma and 1.17-75 ng/g for the brain. It was then applied to quantify 13 C-oleoylethanolamide over 90 min after intravenous administration of a solution (1 mg/kg) in rats. Results suggest that 13 C-oleoylethanolamide does not reach therapeutic concentrations in the brain, despite a relatively prolonged plasma circulation, suggesting that rapid degradation in the brain remains an obstacle to its clinical application to neurological disease.

How the molecular weight affects the in vivo fate of exogenous hyaluronan delivered intravenously: A stable-isotope labelling strategy.

There is inconsistent information regarding the size effects of exogenously given hyaluronan on its in vivo fate. The data are often biased by the poor quality of hyaluronan and non-ideal labelling strategies used for resolving exogenous/endogenous hyaluronan, which only monitor the label and not hyaluronan itself. To overcome these drawbacks and establish the pharmacokinetics of intravenous hyaluronan in relation to its Mw, 13C-labelled HA of five Mws from 13.6-1562 kDa was prepared and administered to mice at doses 25-50 mg kg-1. The elimination efficiency increased with decreasing Mw. Low Mw hyaluronan was rapidly eliminated as small hyaluronan fragments in urine, while high Mw hyaluronan exhibited saturable kinetics and complete metabolization within 48 h. All tested Mws exhibited a similar uptake by liver cells and metabolization into activated sugars. 13C-labelling combined with LC-MS provides an excellent approach to elucidating in vivo fate and biological activities of hyaluronan.

Kinetic Modeling of Hyperpolarized Carbon-13 Pyruvate Metabolism in the Human Brain.

Kinetic modeling of the in vivo pyruvate-to-lactate conversion is crucial to investigating aberrant cancer metabolism that demonstrates Warburg effect modifications. Non-invasive detection of alterations to metabolic flux might offer prognostic value and improve the monitoring of response to treatment. In this clinical research project, hyperpolarized [1-13C] pyruvate was intravenously injected in a total of 10 brain tumor patients to measure its rate of conversion to lactate ( kPL ) and bicarbonate ( kPB ) via echo-planar imaging. Our aim was to investigate new methods to provide kPL and kPB maps with whole-brain coverage. The approach was data-driven and addressed two main issues: selecting the optimal model for fitting our data and determining an appropriate goodness-of-fit metric. The statistical analysis suggested that an input-less model had the best agreement with the data. It was also found that selecting voxels based on post-fitting error criteria provided improved precision and wider spatial coverage compared to using signal-to-noise cutoffs alone.

Diverse Roads Taken by 13C-Glucose-Derived Metabolites in Breast Cancer Cells Exposed to Limiting Glucose and Glutamine Conditions.

In cancers, tumor cells are exposed to fluctuating nutrient microenvironments with limiting supplies of glucose and glutamine. While the metabolic program has been related to the expression of oncogenes, only fractional information is available on how variable precarious nutrient concentrations modulate the cellular levels of metabolites and their metabolic pathways. We thus sought to obtain an overview of the metabolic routes taken by 13C-glucose-derived metabolites in breast cancer MCF-7 cells growing in combinations of limiting glucose and glutamine concentrations. Isotopologue profiles of key metabolites were obtained by gas chromatography/mass spectrometry (GC/MS). They revealed that in limiting and standard saturating medium conditions, the same metabolic routes were engaged, including glycolysis, gluconeogenesis, as well as the TCA cycle with glutamine and pyruvate anaplerosis. However, the cellular levels of 13C-metabolites, for example, serine, alanine, glutamate, malate, and aspartate, were highly sensitive to the available concentrations and the ratios of glucose and glutamine. Notably, intracellular lactate concentrations did not reflect the Warburg effect. Also, isotopologue profiles of 13C-serine as well as 13C-alanine show that the same glucose-derived metabolites are involved in gluconeogenesis and pyruvate replenishment. Thus, anaplerosis and the bidirectional flow of central metabolic pathways ensure metabolic plasticity for adjusting to precarious nutrient conditions.

Subcutaneous adipose tissue free fatty acid uptake measured using positron emission tomography and adipose biopsies in humans.

Positron emission tomography (PET) radiopharmaceuticals can noninvasively measure free fatty acid (FFA) uptake into adipose tissue. We studied 29 volunteers to test whether abdominal and femoral subcutaneous adipose tissue FFA uptake measured using [1-11C]palmitate PET agrees with FFA storage rates measured using an intravenous bolus of [1-14C]palmitate and adipose biopsies. The dynamic left ventricular cavity PET images combined with blood sample radioactivity corrected for the 11CO2 content were used to create the blood time activity curve (TAC), and the constant (Ki) was determined using Patlak analysis of the TACs generated for regions of interest in abdominal subcutaneous fat. These data were used to calculate palmitate uptake rates in abdominal subcutaneous adipose tissue (µmol·kg-1·min-1). Immediately after the dynamic imaging, a static image of the thigh was taken to measure the standardized uptake value (SUV) in thigh adipose tissue, which was scaled to each participant's abdominal adipose tissue SUV to calculate thigh adipose palmitate uptake rates. Abdominal adipose palmitate uptake using PET [1-11C]palmitate was correlated with, but significantly (P < 0.001) greater than, FFA storage measured using [1-14C]palmitate and adipose biopsy. Thigh adipose palmitate measured using PET calculation was positively correlated (R2 = 0.44, P < 0.0001) with and not different from the biopsy approach. The relative differences between PET measured abdominal subcutaneous adipose tissue palmitate uptake and biopsy-measured palmitate storage were positively correlated (P = 0.03) with abdominal subcutaneous fat. We conclude that abdominal adipose tissue FFA uptake measured using PET does not equate to adipose FFA storage measured using biopsy techniques.

xCT knockdown in human breast cancer cells delays onset of cancer-induced bone pain.

Cancers in the bone produce a number of severe symptoms including pain that compromises patient functional status, quality of life, and survival. The source of this pain is multifaceted and includes factors secreted from tumor cells. Malignant cells release the neurotransmitter and cell-signaling molecule glutamate via the oxidative stress-related cystine/glutamate antiporter, system xC-, which reciprocally imports cystine for synthesis of glutathione and the cystine/cysteine redox cycle. Pharmacological inhibition of system xC- has shown success in reducing and delaying the onset of cancer pain-related behavior in mouse models. This investigation describes the development of a stable siRNA-induced knockdown of the functional trans-membrane system xC- subunit xCT ( SLC7A11) in the human breast cancer cell line MDA-MB-231. Clones were verified for xCT knockdown at the transcript, protein, and functional levels. RNAseq was performed on a representative clone to comprehensively examine the transcriptional cellular signature in response to xCT knockdown, identifying multiple differentially regulated factors relevant to cancer pain including nerve growth factor, interleukin-1, and colony-stimulating factor-1. Mice were inoculated intrafemorally and recordings of pain-related behaviors including weight bearing, mechanical withdrawal, and limb use were performed. Animals implanted with xCT knockdown cancer cells displayed a delay until the onset of nociceptive behaviors relative to control cells. These results add to the body of evidence suggesting that a reduction in glutamate release from cancers in bone by inhibition of the system xC- transporter may decrease the severe and intractable pain associated with bone metastases.

Microglial glutamate release evoked by α-synuclein aggregates is prevented by dopamine.

When activated, microglial cells have the potential not only to secrete typical proinflammatory mediators but also to release the neurotransmitter glutamate in amounts that may promote excitotoxicity. Here, we wished to determine the potential of the Parkinson's disease (PD) protein α-Synuclein (αS) to stimulate glutamate release using cultures of purified microglial cells. We established that glutamate release was robustly increased when microglial cultures were treated with fibrillary aggregates of αS but not with the native monomeric protein. Promotion of microglial glutamate release by αS aggregates (αSa) required concomitant engagement of TLR2 and P2X7 receptors. Downstream to cell surface receptors, the release process was mediated by activation of a signaling cascade sequentially involving phosphoinositide 3-kinase (PI3K) and NADPH oxidase, a superoxide-producing enzyme. Inhibition of the Xc- antiporter, a plasma membrane exchange system that imports extracellular l-cystine and exports intracellular glutamate, prevented the release of glutamate induced by αSa, indicating that system Xc- was the final effector element in the release process downstream to NADPH oxidase activation. Of interest, the stimulation of glutamate release by αSa was abrogated by dopamine through an antioxidant effect requiring D1 dopamine receptor activation and PI3K inhibition. Altogether, present data suggest that the activation of microglial cells by αSa may possibly result in a toxic build-up of extracellular glutamate contributing to excitotoxic stress in PD. The deficit in dopamine that characterizes this disorder may further aggravate this process in a vicious circle mechanism.

Influence of parameter accuracy on pharmacokinetic analysis of hyperpolarized pyruvate.

PURPOSE: To explore the effects of noise and error on kinetic analyses of tumor metabolism using hyperpolarized [1-13 C] pyruvate. METHODS: Numerical simulations were performed to systematically investigate the effects of noise, the number of unknowns, and error in kinetic parameter estimates on kinetic analysis of the apparent rate of chemical conversion from hyperpolarized pyruvate to lactate (kPL ). A pharmacokinetic model with two physical and two chemical pools of hyperpolarized spins was used to generate and analyze the synthetic data. RESULTS: The reproducibility of kPL estimates worsened quickly when peak signal-to-noise ratio for hyperpolarized pyruvate was below approximately 20. The accuracy of kPL estimates was most sensitive to errors in high excitation angles, the vascular blood volume fraction (vb ), and the rate of pyruvate extravasation (kve ), and was least sensitive to errors in the T1 of pyruvate. When vb and/or kve were fit as additional unknowns, the accuracy of kPL estimates suffered, and when the vascular input function of pyruvate was also fit, the reproducibility of kPL estimates worsened. CONCLUSIONS: The accuracy and precision of kPL estimates improve substantially for peak signal-to-noise ratio above approximately 20. Accurate estimates of perfusion parameters (combinations of vb , kve , and the pyruvate vascular input function) and transmit calibration at high excitation angles have the greatest effect on the accuracy of kinetic analyses. Magn Reson Med 79:3239-3248, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Measurement of 13 C turnover into glutamate and glutamine pools in brain tumor patients.

Malignant brain tumors are known to utilize acetate as an alternate carbon source in the citric acid cycle for their bioenergetics. 13 C NMR-based isotopomer analysis has been used to measure turnover of 13 C-acetate carbons into glutamate and glutamine pools in tumors. Plasma from the patients infused with [1,2-13 C]acetate further revealed the presence of 13 C isotopomers of glutamine, glucose, and lactate in the circulation that were generated due to metabolism of [1,2-13 C]acetate by peripheral organs. In the tumor cells, [4-13 C] and [3,4-13 C]glutamate and glutamine isotopomers were generated from blood-borne 13 C-labeled glucose and lactate which were formed due to [1,2-13 C[acetate metabolism of peripheral tissues. [4,5-13 C] and [3,4,5-13 C]glutamate and glutamine isotopomers were produced from [1,2-13 C]acetyl-CoA that was derived from direct oxidation of [1,2-13 C] acetate in the tumor. Major portion of C4 13 C fractional enrichment of glutamate (93.3 ± 0.02%) and glutamine (90.9 ± 0.03%) were derived from [1,2-13 C]acetate-derived acetyl-CoA.

Hyperpolarized 13C MR metabolic imaging can detect neuroinflammation in vivo in a multiple sclerosis murine model.

Proinflammatory mononuclear phagocytes (MPs) play a crucial role in the progression of multiple sclerosis (MS) and other neurodegenerative diseases. Despite advances in neuroimaging, there are currently limited available methods enabling noninvasive detection of MPs in vivo. Interestingly, upon activation and subsequent differentiation toward a proinflammatory phenotype MPs undergo metabolic reprogramming that results in increased glycolysis and production of lactate. Hyperpolarized (HP) 13C magnetic resonance spectroscopic imaging (MRSI) is a clinically translatable imaging method that allows noninvasive monitoring of metabolic pathways in real time. This method has proven highly useful to monitor the Warburg effect in cancer, through MR detection of increased HP [1-13C]pyruvate-to-lactate conversion. However, to date, this method has never been applied to the study of neuroinflammation. Here, we questioned the potential of 13C MRSI of HP [1-13C]pyruvate to monitor the presence of neuroinflammatory lesions in vivo in the cuprizone mouse model of MS. First, we demonstrated that 13C MRSI could detect a significant increase in HP [1-13C]pyruvate-to-lactate conversion, which was associated with a high density of proinflammatory MPs. We further demonstrated that the increase in HP [1-13C]lactate was likely mediated by pyruvate dehydrogenase kinase 1 up-regulation in activated MPs, resulting in regional pyruvate dehydrogenase inhibition. Altogether, our results demonstrate a potential for 13C MRSI of HP [1-13C]pyruvate as a neuroimaging method for assessment of inflammatory lesions. This approach could prove useful not only in MS but also in other neurological diseases presenting inflammatory components.

The potential of hyperpolarized 13C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents.

BACKGROUND: Targeting tumor vasculature with vascular disrupting agents (VDAs) results in substantial cell death that precede tumor shrinkage. Here, we investigate the potential of hyperpolarized magnetic resonance spectroscopy (HPMRS) to monitor early metabolic changes associated with VDA treatment. METHODS: Mice bearing C3H mammary carcinomas were treated with the VDAs combretastatin-A4-phosphate (CA4P) or the analog OXi4503, and HPMRS was performed following [1-13C]pyruvate administration. Similarly, treated mice were positron emission tomography (PET) scanned following administration of the glucose analog FDG. Finally, metabolic imaging parameters were compared to tumor regrowth delay and measures of vascular damage, derived from dynamic contrast-agent enhanced magnetic resonance imaging (DCE-MRI) and histology. RESULTS: VDA-treatment impaired tumor perfusion (histology and DCE-MRI), reduced FDG uptake, increased necrosis, and slowed tumor growth. HPMRS, revealed that the [1-13C]pyruvate-to-[1-13C]lactate conversion remained unaltered, whereas [1-13C]lactate-to-[13C]bicarbonate (originating from respiratory CO2) ratios increased significantly following treatment. CONCLUSIONS: DCE-MRI and FDG-PET revealed loss of vessel functionality, impaired glucose delivery and reduced metabolic activity prior to cell death. [1-13C]lactate-to-[13C]bicarbonate ratios increased significantly during treatment, indicating a decline in respiratory activity driven by the onset of hypoxia. HPMRS is promising for early detection of metabolic stress inflicted by VDAs, which cannot easily be inferred based on blood flow measurements.

Effects of hepatic ischemia-reperfusion injury on the blood-brain barrier permeability to [14C] and [13C]sucrose.

Hepatic encephalopathy that is associated with severe liver failure may compromise the blood-brain barrier (BBB) integrity. However, the effects of less severe liver diseases, in the absence of overt encephalopathy, on the BBB are not well understood. The goal of the current study was to investigate the effects of hepatic ischemia-reperfusion (IR) injury on the BBB tight junction permeability to small, hydrophilic molecules using the widely used [14C]sucrose and recently-proposed alternative [13C]sucrose as markers. Rats were subjected to 20 min of hepatic ischemia or sham surgery, followed by 8 h of reperfusion before administration of a single bolus dose of [14C] or [13C]sucrose and collection of serial (0-30 min) blood and plasma and terminal brain samples. The concentrations of [14C] and [13C]sucrose in the samples were determined by measurement of total radioactivity (nonspecific) and LC-MS/MS (specific), respectively. IR injury significantly increased the blood, plasma, and brain concentrations of both [14C] and [13C]sucrose. However, when the brain concentrations were corrected for their respective area under the blood concentration-time curve, only [14C]sucrose showed significantly higher (30%) BBB permeability values in the IR animals. Because [13C]sucrose is a more specific BBB permeability marker, these data indicate that our animal model of hepatic IR injury does not affect the BBB tight junction permeability to small, hydrophilic molecules. Methodological differences among studies of the effects of liver diseases on the BBB permeability may confound the conclusions of such studies.

The use of stable isotopes in the study of human pathophysiology.

The growing prevalence of metabolic diseases including fatty liver disease and Type 2 diabetes has increased the emphasis on understanding metabolism at the mechanistic level and how it is perturbed in disease. Metabolomics is a continually expanding field that seeks to measure metabolites in biological systems during a physiological stimulus or a genetic alteration. Typically, metabolomics studies provide total pool sizes of metabolites rather than dynamic flux measurements. More recently there has been a resurgence in approaches that use stable isotopes (e.g. 2H and 13C) for the unambiguous tracking of individual atoms through compartmentalised metabolic networks in humans to determine underlying mechanisms. This is known as metabolic flux analysis and enables the capture of a dynamic picture of the metabolome and its interactions with the genome and proteome. In this review, we describe current approaches using stable isotope labelling in the field of metabolomics and provide examples of studies that led to an improved understanding of glucose, fatty acid and amino acid metabolism in humans, particularly in relation to metabolic disease. Examples include the use of stable isotopes of glucose to study tumour bioenergetics as well as brain metabolism during traumatic brain injury. Lipid tracers have also been used to measure non-esterified fatty acid production whilst amino acid tracers have been used to study the rate of protein digestion on whole body postprandial protein metabolism. In addition, we illustrate the use of stable isotopes for measuring flux in human physiology by providing examples of breath tests to measure insulin resistance and gastric emptying rates.

A ten fold reduction of nicotine yield in tobacco smoke does not spare the central cholinergic system in adolescent mice.

The tobacco industry has gradually decreased nicotine content in cigarette smoke but the impact of this reduction on health is still controversial. Since the central cholinergic system is the primary site of action of nicotine, here, we investigated the effects of exposure of adolescent mice to tobacco smoke containing either high or low levels of nicotine on the central cholinergic system and the effects associated with cessation of exposure. From postnatal day (PN) 30 to 45, male and female Swiss mice were exposed to tobacco smoke (whole body exposure, 8h/day, 7 days/week) generated from 2R1F (HighNic group: 1.74mg nicotine/cigarette) or 4A1 (LowNic group: 0.14mg nicotine/cigarette) research cigarettes, whereas control mice were exposed to ambient air. Cholinergic biomarkers were assessed in the cerebral cortex and midbrain by the end of exposure (PN45), at short- (PN50) and long-term (PN75) deprivation. In the cortex, nicotinic cholinergic receptor upregulation was observed with either type of cigarette. In the midbrain, upregulation was detected only in HighNic mice and remained significant in females at short-term deprivation. The high-affinity choline transporter was reduced in the cortex: of HighNic mice by the end of exposure; of both HighNic and LowNic females at short-term deprivation; of LowNic mice at long-term deprivation. These decrements were separable from effects on choline acetyltransferase and acetylcholinesterase activities, suggesting cholinergic synaptic impairment. Here, we demonstrated central cholinergic alterations in an animal model of tobacco smoke exposure during adolescence. This system was sensitive even to tobacco smoke with very low nicotine content.

Metabolic flux profiling of MDCK cells during growth and canine adenovirus vector production.

Canine adenovirus vector type 2 (CAV2) represents an alternative to human adenovirus vectors for certain gene therapy applications, particularly neurodegenerative diseases. However, more efficient production processes, assisted by a greater understanding of the effect of infection on producer cells, are required. Combining [1,2-(13)C]glucose and [U-(13)C]glutamine, we apply for the first time (13)C-Metabolic flux analysis ((13)C-MFA) to study E1-transformed Madin-Darby Canine Kidney (MDCK) cells metabolism during growth and CAV2 production. MDCK cells displayed a marked glycolytic and ammoniagenic metabolism, and (13)C data revealed a large fraction of glutamine-derived labelling in TCA cycle intermediates, emphasizing the role of glutamine anaplerosis. (13)C-MFA demonstrated the importance of pyruvate cycling in balancing glycolytic and TCA cycle activities, as well as occurrence of reductive alphaketoglutarate (AKG) carboxylation. By turn, CAV2 infection significantly upregulated fluxes through most central metabolism, including glycolysis, pentose-phosphate pathway, glutamine anaplerosis and, more prominently, reductive AKG carboxylation and cytosolic acetyl-coenzyme A formation, suggestive of increased lipogenesis. Based on these results, we suggest culture supplementation strategies to stimulate nucleic acid and lipid biosynthesis for improved canine adenoviral vector production.

Effects of rapamycin on cerebral oxygen supply and consumption during reperfusion after cerebral ischemia.

Activation of the mammalian target of rapamycin (mTOR) leads to cell growth and survival. We tested the hypothesis that inhibition of mTOR would increase infarct size and decrease microregional O2 supply/consumption balance after cerebral ischemia-reperfusion. This was tested in isoflurane-anesthetized rats with middle cerebral artery blockade for 1h and reperfusion for 2h with and without rapamycin (20mg/kg once daily for two days prior to ischemia). Regional cerebral blood flow was determined using a C(14)-iodoantipyrine autoradiographic technique. Regional small-vessel arterial and venous oxygen saturations were determined microspectrophotometrically. The control ischemic-reperfused cortex had a similar blood flow and O2 consumption to the contralateral cortex. However, microregional O2 supply/consumption balance was significantly reduced in the ischemic-reperfused cortex. Rapamycin significantly increased cerebral O2 consumption and further reduced O2 supply/consumption balance in the reperfused area. This was associated with an increased cortical infarct size (13.5±0.8% control vs. 21.5±0.9% rapamycin). We also found that ischemia-reperfusion increased AKT and S6K1 phosphorylation, while rapamycin decreased this phosphorylation in both the control and ischemic-reperfused cortex. This suggests that mTOR is important for not only cell survival, but also for the control of oxygen balance after cerebral ischemia-reperfusion.

Selective spectroscopic imaging of hyperpolarized pyruvate and its metabolites using a single-echo variable phase advance method in balanced SSFP.

PURPOSE: In balanced steady state free precession (bSSFP), the signal intensity has a well-known dependence on the off-resonance frequency, or, equivalently, the phase advance between successive radiofrequency (RF) pulses. The signal profile can be used to resolve the contributions from the spectrally separated metabolites. This work describes a method based on use of a variable RF phase advance to acquire spatial and spectral data in a time-efficient manner for hyperpolarized 13C MRI. THEORY AND METHODS: The technique relies on the frequency response from a bSSFP acquisition to acquire relatively rapid, high-resolution images that may be reconstructed to separate contributions from different metabolites. The ability to produce images from spectrally separated metabolites was demonstrated in vitro, as well as in vivo following administration of hyperpolarized 1-13C pyruvate in mice with xenograft tumors. RESULTS: In vivo images of pyruvate, alanine, pyruvate hydrate, and lactate were reconstructed from four images acquired in 2 s with an in-plane resolution of 1.25 × 1.25 mm(2) and 5 mm slice thickness. CONCLUSION: The phase advance method allowed acquisition of spectroscopically selective images with high spatial and temporal resolution. This method provides an alternative approach to hyperpolarized 13C spectroscopic MRI that can be combined with other techniques such as multiecho or fluctuating equilibrium bSSFP. Magn Reson Med 76:1102-1115, 2016. © 2015 Wiley Periodicals, Inc.

Magnetic resonance imaging of tumor with a self-traceable polymer conjugated with an antibody fragment.

A (13)C-enriched phosphorylcholine polymer ((13)C-PMPC) as a self-traceable MR (magnetic resonance) tag was conjugated with a fragment (scFv) of Herceptin, a clinical antibody against antigen Her2. When injected in model mice bearing Her2(+) (gastric) and Her2(-) (pancreatic) tumors, the antibody-tag conjugate (13)C-PMPC-scFv selectively accumulated in the Her2(+) tumor with a rapid build-up/decay (accumulation/clearance) profile and, with the use of the (1)H-(13)C double-resonance (heteronuclear correlation) technique, the Her2(+) gastric tumor was clearly MR imaged.