Glucose transporter 2 mediates the hypoglycemia-induced increase in cerebral blood flow.

Glucose transporter 2 (Glut2)-positive cells are sparsely distributed in brain and play an important role in the stimulation of glucagon secretion in response to hypoglycemia. We aimed to determine if Glut2-positive cells can influence another response to hypoglycemia, i.e. increased cerebral blood flow (CBF). CBF of adult male mice devoid of Glut2, either globally (ripglut1:glut2 <sup>-</sup> <sup>/</sup> <sup>-</sup> ) or in the nervous system only (NG2KO), and their respective controls were studied under basal glycemia and insulin-induced hypoglycemia using quantitative perfusion magnetic resonance imaging at 9.4 T. The effect on CBF of optogenetic activation of hypoglycemia responsive Glut2-positive neurons of the paraventricular thalamic area was measured in mice expressing channelrhodopsin2 under the control of the Glut2 promoter. We found that in both ripglut1:glut2 <sup>-</sup> <sup>/</sup> <sup>-</sup> mice and NG2KO mice, CBF in basal conditions was higher than in their respective controls and not further activated by hypoglycemia, as measured in the hippocampus, hypothalamus and whole brain. Conversely, optogenetic activation of Glut2-positive cells in the paraventricular thalamic nucleus induced a local increase in CBF similar to that induced by hypoglycemia. Thus, Glut2 expression in the nervous system is required for the control of CBF in response to changes in blood glucose concentrations

Novel Calibrated Short TR Recovery (CaSTRR) Method for Brain-Blood Partition Coefficient Correction Enhances Gray-White Matter Contrast in Blood Flow Measurements in Mice

The goal of the study was to develop a novel, rapid Calibrated Short TR Recovery (CaSTRR) method to measure the brain-blood partition coefficient (BBPC) in mice. The BBPC is necessary for quantifying cerebral blood flow (CBF) using tracer-based techniques like arterial spin labeling (ASL), but previous techniques required prohibitively long acquisition times so a constant BBPC equal to 0.9 mL/g is typically used regardless of studied species, condition, or disease. An accelerated method of BBPC correction could improve regional specificity in CBF maps particularly in white matter. Male C57Bl/6N mice (n = 8) were scanned at 7T using CaSTRR to measure BBPC determine regional variability. This technique employs phase-spoiled gradient echo acquisitions with varying repetition times (TRs) to estimate proton density in the brain and a blood sample. Proton density weighted images are then calibrated to a series of phantoms with known concentrations of deuterium to determine BBPC. Pseudo-continuous ASL was also acquired to quantify CBF with and without empirical BBPC correction. Using the CaSTRR technique we demonstrate that, in mice, white matter has a significantly lower BBPC (BBPCwhite = 0.93 ± 0.05 mL/g) than cortical gray matter (BBPCgray = 0.99 ± 0.04 mL/g, p = 0.03), and that when voxel-wise BBPC correction is performed on CBF maps the observed difference in perfusion between gray and white matter is improved by as much as 14%. Our results suggest that BBPC correction is feasible and could be particularly important in future studies of perfusion in white matter pathologies

Novel Calibrated Short TR Recovery (CaSTRR) Method for Brain-Blood Partition Coefficient Correction Enhances Gray-White Matter Contrast in Blood Flow Measurements in Mice

The goal of the study was to develop a novel, rapid Calibrated Short TR Recovery (CaSTRR) method to measure the brain-blood partition coefficient (BBPC) in mice. The BBPC is necessary for quantifying cerebral blood flow (CBF) using tracer-based techniques like arterial spin labeling (ASL), but previous techniques required prohibitively long acquisition times so a constant BBPC equal to 0.9 mL/g is typically used regardless of studied species, condition, or disease. An accelerated method of BBPC correction could improve regional specificity in CBF maps particularly in white matter. Male C57Bl/6N mice (n = 8) were scanned at 7T using CaSTRR to measure BBPC determine regional variability. This technique employs phase-spoiled gradient echo acquisitions with varying repetition times (TRs) to estimate proton density in the brain and a blood sample. Proton density weighted images are then calibrated to a series of phantoms with known concentrations of deuterium to determine BBPC. Pseudo-continuous ASL was also acquired to quantify CBF with and without empirical BBPC correction. Using the CaSTRR technique we demonstrate that, in mice, white matter has a significantly lower BBPC (BBPCwhite = 0.93 ± 0.05 mL/g) than cortical gray matter (BBPCgray = 0.99 ± 0.04 mL/g, p = 0.03), and that when voxel-wise BBPC correction is performed on CBF maps the observed difference in perfusion between gray and white matter is improved by as much as 14%. Our results suggest that BBPC correction is feasible and could be particularly important in future studies of perfusion in white matter pathologies

Skeletal myofiber vascular endothelial growth factor is required for the exercise training-induced increase in dentate gyrus neuronal precursor cells

Exercise signals neurogenesis in the dentate gyrus of the hippocampus. This phenomenon requires vascular endothelial growth factor (VEGF) originating from outside the blood–brain barrier, but no cellular source has been identified. Thus, we hypothesized that VEGF produced by skeletal myofibers plays a role in regulating hippocampal neuronal precursor cell proliferation following exercise training. This was tested in adult conditional skeletal myofiber‐specific VEGF gene‐ablated mice (VEGFHSA−/−) by providing VEGFHSA−/− and non‐ablated (VEGFf/f) littermates with running wheels for 14 days. Following this training period, hippocampal cerebral blood flow (CBF) was measured by functional magnetic resonance imaging (fMRI), and neuronal precursor cells (BrdU+/Nestin+) were detected by immunofluorescence. The VEGFf/f trained group showed improvements in both speed and endurance capacity in acute treadmill running tests (P < 0.05). The VEGFHSA−/− group did not. The number of proliferating neuronal precursor cells was increased with training in VEGFf/f (P < 0.05) but not in VEGFHSA−/− mice. Endothelial cell (CD31+) number did not change in this region with exercise training or skeletal myofiber VEGF gene deletion. However, resting blood flow through the hippocampal region was lower in VEGFHSA−/− mice, both untrained and trained, than untrained VEGFf/f mice (P < 0.05). An acute hypoxic challenge decreased CBF (P < 0.05) in untrained VEGFf/f, untrained VEGFHSA−/− and trained VEGFHSA−/− mice, but not trained VEGFf/f mice. VEGFf/f, but not VEGFHSA−/−, mice were able to acutely run on a treadmill at an intensity sufficient to increase hippocampus VEGF levels. These data suggest that VEGF expressed by skeletal myofibers may directly or indirectly regulate both hippocampal blood flow and neurogenisis

CALIBRATED SHORT TR RECOVERY MRI FOR RAPID MEASUREMENT OF BRAIN-BLOOD PARTITION COEFFICIENT AND CORRECTION OF QUANTITATIVE CEREBRAL BLOOD FLOW

The high prevalence and mortality of cerebrovascular disease has led to the development of several methods to measure cerebral blood flow (CBF) in vivo. One of these, arterial spin labeling (ASL), is a quantitative magnetic resonance imaging (MRI) technique with the advantage that it is completely non-invasive. The quantification of CBF using ASL requires correction for a tissue specific parameter called the brain-blood partition coefficient (BBPC). Despite regional and inter-subject variability in BBPC, the current recommended implementation of ASL uses a constant assumed value of 0.9 mL/g for all regions of the brain, all subjects, and even all species. The purpose of this dissertation is 1) to apply ASL to a novel population to answer an important clinical question in the setting of Down syndrome, 2) to demonstrate proof of concept of a rapid technique to measure BBPC in mice to improve CBF quantification, and 3) to translate the correction method by applying it to a population of healthy canines using equipment and parameters suitable for use with humans. Chapter 2 reports the results of an ASL study of adults with Down syndrome (DS). This population is unique for their extremely high prevalence of Alzheimer’s disease (AD) and very low prevalence of systemic cardiovascular risk factors like atherosclerosis and hypertension. This prompted the hypothesis that AD pathology would lead to the development of perfusion deficits in people with DS despite their healthy cardiovascular profile. The results demonstrate that perfusion is not compromised in DS participants until the middle of the 6th decade of life after which measured global CBF was reduced by 31% (p=0.029). There was also significantly higher prevalence of residual arterial signal in older participants with DS (60%) than younger DS participants (7%, p = 0.005) or non-DS controls (0%, p \u3c 0.001). This delayed pattern of perfusion deficits in people with DS differs from observations in studies of sporadic AD suggesting that adults with DS benefit from an improved cardiovascular risk profile early in life. Chapter 3 introduces calibrated short TR recovery (CaSTRR) imaging as a rapid method to measure BBPC and its development in mice. This was prompted by the inability to account for potential changes in BBPC due to age, brain atrophy, or the accumulation of hydrophobic A-β plaques in the ASL study of people with DS in Chapter 2. The CaSTRR method reduces acquisition time of BBPC maps by 87% and measures a significantly higher BBPC in cortical gray matter (0.99±0.04 mL/g,) than white matter in the corpus callosum (0.93±0.05 mL/g, p=0.03). Furthermore, when CBF maps are corrected for BBPC, the contrast between gray and white matter regions of interest is improved by 14%. This demonstrates proof of concept for the CaSTRR technique. Chapter 4 describes the application of CaSTRR on healthy canines (age 5-8 years) using a 3T human MRI scanner. This represents a translation of the technique to a setting suitable for use with a human subject. Both CaSTRR and pCASL acquisitions were performed and further optimization brought the acquisition time of CaSTRR down to 4 minutes which is comparable to pCASL. Results again show higher BBPC in gray matter (0.83 ± 0.05 mL/g) than white matter (0.78 ± 0.04 mL/g, p = 0.007) with both values unaffected by age over the range studied. Also, gray matter CBF is negatively correlated with age (p = 0.003) and BBPC correction improved the contrast to noise ratio by 3.6% (95% confidence interval = 0.6 – 6.5%). In summary, the quantification of ASL can be improved using BBPC maps derived from the novel, rapid CaSTRR technique

Proton diffusion spectroscopy and modeling of brain metabolism at 14.1T

As a field at the CIBM, Nuclear magnetic resonance (NMR)spectroscopy can be applied non-invasively to explore the metabolic fate of energy fuel substrates, as well as the rate at which they are consumed, using 13C and 1H nuclei. The work of this thesis encompasses both nuclei, and focuses on (1) improving the quantification and modeling of glucose-derived metabolites; and (2) characterizing diffusion-related parameters of the purportedly glial-specific energy substrate, acetate. Both aim to quantitatively explore cerebral energy metabolism, at ultra-high magnetic field, in vivo, in the healthy rat. 13C NMR spectroscopy, as a tool, enables measuring the progressive incorporation of 13C-glucose into brain glucose and then NMR detectable amino acids (glutamate and glutamine); this relies on the infusion of the 13C-labeled energy substrate. The experimentally obtained 13C labelling curves are analyzed using suitable mathematical models to provide an estimation of cerebral metabolic rates. Here, a dynamic model of time-courses of 13C multiplets arising from isotopomers was considered. So beyond the two-compartment neuronal-glial model, we took into account additional data on the dynamics of 13C isotopomers, available from the fine structure multiplets in 13C spectra of glutamate and glutamine, measured under prolonged [1-6,13C] glucose infusion. We concluded that the dynamic analyses of 13C multiplet time courses of glutamate and glutamine resulted in a higher precision for estimating the absolute values of most cerebral metabolic rates. Acetate metabolism is challenging because dynamic metabolic modeling requires prior knowledge of the transport and uptake kinetics of infused acetate. We sought this information by determining the apparent concentration and distribution volume (V_d) of cerebral acetate between the intracellular and the extracellular compartments. Experimentally, the diffusion characteristics of cerebral acetate were measured, relative to that of N-Acetylaspartate (NAA, known to be mainly intracellular) using diffusion-weighted 1H NMR spectroscopy at 14.1T, under prolonged acetate infusion. The detection of an acetate and NAA signal at large diffusion weighting provided direct experimental evidence of intracellular cerebral acetate and NAA, although a substantial fraction of acetate was extracellular. To estimate the apparent concentration of in vivo brain acetate, T1 and T2 relaxation times of acetate were measured. The longer T1 relaxation and shorter T2 relaxation times of acetate compared with NAA provided evidence of its small molecular size, and possibly different chemical environment. Our experimentally determined value of V_dled to cerebral metabolic rates of acetate (CMR acetate) of the same order reported for the glial Krebâs cycle rate, an indication that estimates of CMR acetate are highly dependent on V_d. Finally, in order to pursue metabolic mapping of cerebral acetate uptake in the rat, in vivo, at 14.1 T, the design and construction of a combined transmit-birdcage coil and receive-quadrature pair surface coil was considered. Its performance was compared to a single birdcage coil in the transmit/receive mode. So far, the preliminary results of the 2-coil configuration are promising: homogenous excitation and a gain in sensitivity up to a distance of 5 mm are achievable. Improvements are ongoing for NMR spectroscopic and imaging applications at 14.1 T

Preclinical MRI of the Kidney

This Open Access volume provides readers with an open access protocol collection and wide-ranging recommendations for preclinical renal MRI used in translational research. The chapters in this book are interdisciplinary in nature and bridge the gaps between physics, physiology, and medicine. They are designed to enhance training in renal MRI sciences and improve the reproducibility of renal imaging research. Chapters provide guidance for exploring, using and developing small animal renal MRI in your laboratory as a unique tool for advanced in vivo phenotyping, diagnostic imaging, and research into potential new therapies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Preclinical MRI of the Kidney: Methods and Protocols is a valuable resource and will be of importance to anyone interested in the preclinical aspect of renal and cardiorenal diseases in the fields of physiology, nephrology, radiology, and cardiology. This publication is based upon work from COST Action PARENCHIMA, supported by European Cooperation in Science and Technology (COST). COST (www.cost.eu) is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation. PARENCHIMA (renalmri.org) is a community-driven Action in the COST program of the European Union, which unites more than 200 experts in renal MRI from 30 countries with the aim to improve the reproducibility and standardization of renal MRI biomarkers

Parallel transit methods for arterial spin labelling magnetic resonance imaging

Vessel selective arterial spin labelling (ASL) is a magnetic resonance imaging technique which permits the visualisation and assessment of the perfusion territory of a specific set of feeding arteries. It is of clinical importance in both acute and chronic cerebrovascular disease, and the mapping of blood supplied to tumours. Continuous ASL is capable of providing the highest signal-to-noise (SNR) ratio of the various ASL methods. However on clinical systems it suffers from high hardware demands, and the control of systematic errors decreases perfusion sensitivity. A separate labelling coil avoids these problems, enabling high labelling efficiency and subsequent high SNR, and vessel specificity can be localised to one carotid artery. However this relies on the careful and accurate positioning of the labelling coil over the common carotid arteries in the neck. It is proposed to combine parallel transmission (multiple transmit coils, each transmitting with different amplitudes and phases) to spatially tailor the labelling field, removing the reliance on coil location for optimal labelling efficiency, and enabling robust vessel selective labelling with a high degree of specificity. Presented is the application of parallel transmission methods to continuous ASL, requiring the development of an ASL labelling coil array, and a two channel transmitter system. Coil safety testing was performed using a novel MRI temperature mapping technique to accurately measure small temperature changes on the order of 0.1 ⁰C. A perfusion phantom with distinct vascular territories was constructed for sequence testing and development. Phantom and in-vivo testing of parallel transmit CASL using a 3D-GRASE acquisition showed an improvement of up to 35% in vessel specificity when compared with using a single labelling coil, whilst retaining the high labelling efficiency and associated SNR of separate coil CASL methods

Preclinical MRI of the kidney : methods and protocols

This Open Access volume provides readers with an open access protocol collection and wide-ranging recommendations for preclinical renal MRI used in translational research. The chapters in this book are interdisciplinary in nature and bridge the gaps between physics, physiology, and medicine. They are designed to enhance training in renal MRI sciences and improve the reproducibility of renal imaging research. Chapters provide guidance for exploring, using and developing small animal renal MRI in your laboratory as a unique tool for advanced in vivo phenotyping, diagnostic imaging, and research into potential new therapies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and thorough, Preclinical MRI of the Kidney: Methods and Protocols is a valuable resource and will be of importance to anyone interested in the preclinical aspect of renal and cardiorenal diseases in the fields of physiology, nephrology, radiology, and cardiology. This publication is based upon work from COST Action PARENCHIMA, supported by European Cooperation in Science and Technology (COST). COST (www.cost.eu) is a funding agency for research and innovation networks. COST Actions help connect research initiatives across Europe and enable scientists to grow their ideas by sharing them with their peers. This boosts their research, career and innovation. PARENCHIMA (renalmri.org) is a community-driven Action in the COST program of the European Union, which unites more than 200 experts in renal MRI from 30 countries with the aim to improve the reproducibility and standardization of renal MRI biomarkers

Continuous arterial spin labeling of mouse cerebral blood flow using an actively-detuned two-coil system at 9.4T.

Among numerous magnetic resonance imaging (MRI) techniques, perfusion MRI provides insight into the passage of blood through the brain's vascular network non-invasively. Studying disease models and transgenic mice would intrinsically help understanding the underlying brain functions, cerebrovascular disease and brain disorders. This study evaluates the feasibility of performing continuous arterial spin labeling (CASL) on all cranial arteries for mapping murine cerebral blood flow at 9.4 T. We showed that with an active-detuned two-coil system, a labeling efficiency of 0.82 ± 0.03 was achieved with minimal magnetization transfer residuals in brain. The resulting cerebral blood flow of healthy mouse was 99 ± 26 mL/100g/min, in excellent agreement with other techniques. In conclusion, high magnetic fields deliver high sensitivity and allowing not only CASL but also other MR techniques, i.e. (1)H MRS and diffusion MRI etc, in studying murine brains