NAD is an essential metabolite that exists in NAD + or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD + /NADH redox state and modulating cellular signaling processes through the activity of the NAD + -dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD + and NADH contents and the NAD + /NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD + , total NAD contents, and NAD + /NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.redox state | NAD | in vivo 31 P MR spectroscopy | human brain | aging N AD, a multifunctional metabolite found in all living cells, has been the interest of many scientific investigations since its discovery in the early 20th century (1). NAD is known to convert between its oxidized NAD + and reduced NADH forms during the breakdown of nutrients; hence, the intracellular NAD + /NADH redox state reflects the metabolic balance of the cell in generating ATP energy through oxidative phosphorylation in mitochondria and/or glycolysis in cytosol (2). More recently, after several protein families associated with cell survival were found to use NAD + as their main substrate with activities also regulated by the availability of the NAD + , the full extent of the NAD's function as a metabolic regulator began to unfold (3-5). A growing number of studies have indicated that NAD + can modulate metabolic signaling pathways and mediate important cellular processes, including calcium homeostasis, gene expression, aging, degeneration, and cell death; therefore, the cellular NAD could serve as a therapeutic target for treating various metabolic or age-related diseases and promoting longevity (6-12).Despite the critical relevance of the intracellular NAD metabolism to human health and diseases, assessment of NAD contents and NAD + /NADH redox state is extremely challenging. Only a few invasive techniques based on biochemical assays or autofluorescence methods have been used to analyze tissue samples or cell extracts (13,14). However, during the preparation of such ex vivo sample, the NAD + and NADH contents are likely altered, beca...

A comprehensive technique was developed for using threedimensional 17 O magnetic resonance spectroscopic imaging at 9.4T for rapidly imaging the cerebral metabolic rate of oxygen consumption (CMRO2) in the rat brain during a two-min inhalation of 17 O2. The CMRO2 value (2.19 ؎ 0.14 mol͞g͞min, n ‫؍‬ 7) was determined in the rat anesthetized with ␣-chloralose by independent and concurrent 17 O NMR measurements of cerebral H2 17 O content, arterial input function, and cerebral perfusion. CMRO2 values obtained were consistent with the literature results for similar conditions. Our results reveal that, because of its superior sensitivity at ultra-high fields, the 17 O magnetic resonance spectroscopic imaging approach is capable of detecting small dynamic changes of metabolic H2 17 O during a short inhalation of 17 O2 gas, and ultimately, for imaging CMRO2 in the small rat brain. This study provides a crucial step toward the goal of developing a robust and noninvasive 17 O NMR approach for imaging CMRO2 in animal and human brains that can be used for studying the central role of oxidative metabolism in brain function under normal and diseased conditions, as well as for understanding the mechanisms underlying functional MRI.

Radiofrequency (RF) field wave behavior and associated nonuniform image intensity at high magnetic field strengths are examined experimentally and numerically. The RF field produced by a 10-cm-diameter surface coil at 300 MHz is evaluated in a 16-cm-diameter spherical phantom with variable salinity, and in the human head. Temporal progression of the RF field indicates that the standing wave and associated dielectric resonance occurring in a pure water phantom near 300 MHz is greatly dampened in the human head due to the strong decay of the electromagnetic wave. The characteristic image intensity distribution in the human head is the result of spatial phase distribution and amplitude modulation by the interference of the RF traveling waves determined by a given sample-coil configuration. Enhancements in signal-to-noise ratio (SNR) and T* 2 contrast arising from high static magnetic field strengths are desirable for in vivo MR applications. Thus, the number of high-field human MRI systems has increased rapidly in recent years (1-10). The advent of high-field human imaging systems introduces new challenges in radiofrequency (RF) engineering (11,12). Because at high frequencies the wavelength of the RF field is comparable to or less than that of the dimension of the human body, the RF magnetic field (B 1 ) inside a sample exhibits prominent wave behavior (13-16). Additionally, the homogeneity of the B 1 field and source currents in the RF coil are strongly perturbed by sample loading (17-19). The B 1 field distribution inside a sample is important for both specific absorption rate (SAR) assessment and RF coil engineering at high frequency. However, mathematical treatment of the RF field in such systems can be extremely complicated because 1) the quasi-static approximations are no longer valid, and Maxwell's wave equation must be employed; and 2) the geometry of the human body is irregular, and electromagnetic properties of tissues are heterogeneous. Thus, computer numerical calculation becomes an effective and indispensable tool for studying interactions of the RF field with the human body at high field (20 -24). Associated with the RF field wave behavior, the distributions of the B 1 field and its circularly polarized components B ϩ and B -, which are directly responsible for the MR image intensity distribution, become distinctively different from one another. Consequently, the relationship of RF field polarization to coil configuration and sample electric properties needs to be analyzed in order to understand the resultant image intensity distribution. Computer modeling provides an effective way to study this problem, and may provide insight into complex RF field wave behavior and its dependence on the electrical properties of the sample. In this report, we present a study specifically devised to analyze high-frequency wave behavior of the RF field with the aid of numerical calculation and parallel experimental measurements. METHODSThe study was carried out using water and saline phantoms with a 10-cm-diameter sur...

ATP metabolism is controlled mainly by ATP synthase (ATP ase ) and creatine kinase (CK) reactions that regulate cerebral ATP production, transportation, and utilization. These coupled reactions constitute a chemical exchange metabolic network of PCr7ATP7Pi characterized by two forward and two reverse reaction fluxes, which can be studied noninvasively by in vivo 31 P MRS combined with magnetization transfer (MT). However, it is still debated whether current MT approaches can precisely determine all of these fluxes. We developed and tested a modified in vivo 31 P MT approach based on a multiple single-site saturation (MSS) technique to study the entire PCr7ATP7Pi network in human occipital lobe at 7T. Our results reveal that 1) the MSS MT approach can explicitly determine all four reaction fluxes with a minimal number of The primary functions of brain cells are excitation and conduction, which are reflected by constant electrophysiological activity in the brain. The cerebral bioenergetics that support sustained electrophysiological activity are ultimately driven by a variety of biochemical processes that maintain the normal function and structural integrity of the brain (1). Of these processes, the most fundamental for supporting various cellular activities is adenosine triphosphate (ATP) metabolism in living cells (2). The majority of ATP is formed from adenosine diphosphate (ADP) and inorganic phosphate (Pi) in the mitochondria through oxidative phosphorylation catalyzed by the ATP synthase (ATP ase ) enzyme, as illustrated by Fig. 1a (3,4). The highly demanding biochemical processes involving ATP production and utilization in the brain cause rapid chemical cycling among ATP, ADP, and Pi (see Fig. 1a). These processes are also accompanied by another important chemical reaction involving phosphocreatine (PCr) and creatine kinase (CK). PCr acts as an ATP reservoir and carrier, and transfers energy from the mitochondria to sites of ATP utilization in the cytosol through reversible CK reactions, ultimately maintaining a stable cellular ATP level (4,5). These two chemical exchange reactions (i.e., PCr7ATP and Pi7ATP) play central roles in regulating ATP metabolism and maintaining normal ATP functionality, both of which are crucial for cerebral bioenergetics and brain function in the healthy brain as well as in neurodegenerative diseases. Moreover, the ATP ase and CK reactions are tightly coupled together, leading to a complex three-31 P-spin chemical exchange kinetic network (i.e., PCr7ATP7Pi) as depicted in Fig. 1b. Thus, it is essential to develop a noninvasive, reliable technique that is capable of assessing the entire kinetic network of PCr7ATP7Pi and associated ATP metabolic fluxes in situ, particularly in the human brain.Measuring all kinetic parameters involved in the PCr7ATP7Pi network requires extensive information, including three steady-state phosphate metabolite concentrations (i.e., [ATP], [PCr], and [Pi]) and four pseudo-firstorder chemical reaction rate constants (forward and reverse rate constants fo...

Spontaneous hemodynamic signals fluctuate coherently within many resting-brain functional networks not only in awake humans and lightly anesthetized primates but also in animals under deep anesthesia characterized by burst-suppression electroencephalogram (EEG) activity and unconsciousness. To understand the neural origin of spontaneous hemodynamic fluctuations under such a deep anesthesia state, epidural EEG and cerebral blood flow (CBF) were simultaneously recorded from the bilateral somatosensory cortical regions of rats with isoflurane-induced burst-suppression EEG activity. Strong neurovascular coupling was observed between spontaneous EEG "bursts" and CBF "bumps," both of which were also highly synchronized across the 2 hemispheres. Functional magnetic resonance imaging (fMRI) was used to image spontaneous blood oxygen level-dependent (BOLD) signals under the same anesthesia conditions and showed similar BOLD "bumps" and dependence on anesthesia depth as the CBF signals. The spatiotemporal BOLD correlations indicate a strong but less-specific coherent network covering a wide range of cortical regions. The overall findings reveal that the spontaneous CBF/BOLD fluctuations under unconscious burst-suppression anesthesia conditions originate mainly from underlying neural activity. They provide insights into the neurophysiological basis for the use of BOLD- and CBF-based fMRI signals for noninvasively imaging spontaneous and synchronous brain activity under various brain states.

Despite the essential role of the brain energy generated from ATP hydrolysis in supporting cortical neuronal activity and brain function, it is challenging to noninvasively image and directly quantify the energy expenditure in the human brain. In this study, we applied an advanced in vivo 31P MRS imaging approach to obtain regional cerebral metabolic rates of high-energy phosphate reactions catalyzed by ATPase (CMRATPase) and creatine kinase (CMRCK), and to determine CMRATPase and CMRCK in pure grey mater (GM) and white mater (WM), respectively. It was found that both ATPase and CK rates are three times higher in GM than WM; and CMRCK is seven times higher than CMRATPase in GM and WM. Among the total brain ATP consumption in the human cortical GM and WM, 77% of them are used by GM in which approximately 96% is by neurons. A single cortical neuron utilizes approximately 4.7 billion ATPs per second in a resting human brain. This study demonstrates the unique utility of in vivo 31P MRS imaging modality for direct imaging of brain energy generated from ATP hydrolysis, and provides new insights into the human brain energetics and its role in supporting neuronal activity and brain function.

In this paper, we demonstrate an approach by which some evoked neuronal events can be probed by functional MRI (fMRI) signal with temporal resolution at the time scale of tens of milliseconds. The approach is based on the close relationship between neuronal electrical events and fMRI signal that is experimentally demonstrated in concurrent fMRI and electroencephalographic (EEG) studies conducted in a rat model with forepaw electrical stimulation. We observed a refractory period of neuronal origin in a two-stimuli paradigm: the first stimulation pulse suppressed the evoked activity in both EEG and fMRI signal responding to the subsequent stimulus for a period of several hundred milliseconds. When there was an apparent site-site interaction detected in the evoked EEG signal induced by two stimuli that were primarily targeted to activate two different sites in the brain, fMRI also displayed signal amplitude modulation because of the interactive event. With visual stimulation using two short pulses in the human brain, a similar refractory phenomenon was observed in activated fMRI signals in the primary visual cortex. In addition, for interstimulus intervals shorter than the known latency time of the evoked potential induced by the first stimulus (Ϸ100 ms) in the primary visual cortex of the human brain, the suppression was not present. Thus, by controlling the temporal relation of input tasks, it is possible to study temporal evolution of certain neural events at the time scale of their evoked electrical activity by noninvasive fMRI methodology. Since the introduction of functional magnetic resonance imaging (fMRI) of the human brain in early 1990s, this noninvasive functional neuro-imaging modality has rapidly gained a prominent position in systems level neuroscience research. The most commonly used fMRI approach relies on blood oxygen level dependent (BOLD) contrast (1), which detects changes in regional deoxyhemoglobin content induced by alterations in cerebral blood flow (CBF) and͞or oxygen consumption rate (CMRO 2 ) that accompany modulations in neuronal activity. It is also possible to image regional CBF changes directly by magnetic resonance imaging with a little more elaborate data acquisition schemes and to generate functional maps based on CBF alterations alone (2).The tight coupling between neural activation and changes in CBF and͞or CMRO 2 that forms the basis of fMRI has been shown to be present mostly under steady state conditions (3-6). Brinker et al. (7) have reported the presence of the tight and highly quantitative coupling between the EEG signal and BOLD signal in their rat model experiments under ␣-chloralose anesthesia, where the frequency of forepaw stimulation rate was varied under steady state conditions. In the cerebellum of anesthetized rats, the regional CBF increase was also shown to be proportional to the product of the frequency of stimulation and the strength of the evoked local field potential near a Purkenje cell (8). Based on such coupling, two parameters that characterize the fMRI ...

17 O is the only stable oxygen isotope that can be detected by NMR. The quadrupolar moment of 17 O spin (I ¼ 5/2) can interact with local electric field gradients, resulting in extremely short T 1 and T 2 relaxation times which are in the range of several milliseconds. One unique NMR property of 17 O spin is the independence of 17 O relaxation times on the magnetic field strength, and this makes it possible to achieve a large sensitivity gain for in vivo 17 O NMR applications at high fields.In vivo 17 O NMR has two major applications for studying brain function and cerebral bioenergetics. The first application is to measure the cerebral blood flow (CBF) through monitoring the washout of inert H 2 17 O tracer in the brain tissue following an intravascular bolus injection of the 17 O-labeled water. The second application, perhaps the most important one, is to determine the cerebral metabolic rate of oxygen utilization (CMRO 2 ) through monitoring the dynamic changes of metabolically generated H O is a stable oxygen isotope that has a magnetic moment and can be detected by NMR. Compared with nuclei such as 1 H, 31 P and 13 C that are commonly used for most in vivo MR applications, 17 O has a spin quantum number of greater than ½ (I ¼ 5/2) and possesses an electric quadrupolar moment. The natural abundance of 17 O is only 0.037%, which is almost 30 times lower than that of 13 C and 2700 times lower than that of 1 H. Moreover, the magnetogyric ratio ( ) of 17 O, which is proportional to the Larmor frequency, is 7.4 times lower than that of 1 H.