New Imaging Techniques Reveal Metabolism of the Aging Brain
New Imaging Techniques Reveal Metabolism of the Aging Brain
PET is currently the most common approach to study brain metabolism. It uses radioactive tracers to produce a 3D image of functional processes (e.g., metabolism) in the body. The system detects pairs of γ-rays emitted indirectly by a positron-emitting tracer, which is introduced into the body on a biologically active molecule. 3D images of tracer concentration are then constructed by computer analysis. PET has several useful attributes for human diagnostic or metabolic studies: the positron-emitting radioisotopes of choice are short lived, rather simplistic, safe within the radioactivity ranges permitted; they require only very low amounts of tracer on the order of 10–10 M, a tolerable risk for organs; and, dynamic tissue uptake and washout of the tracer are measurable in real time (more technical details can be found in).
Glucose Metabolism.FDG is the tracer for studying CMRglc. It simulates a combination of both glucose transport and subsequent phosphorylation. FDG is transported into tissues, including the brain, at almost the same rate as glucose itself. Like glucose, FDG can be phosphorylated by the first glycolytic enzyme (hexokinase). Unlike glucose, FDG cannot be further metabolized to fructose-6-phosphate by glucose-phosphate-isomerase, so FDG remains trapped in the tissue as FDG 6-phosphate. Hence, FDG uptake represents glucose uptake but without subsequent metabolism toward CO2.
Aβ Plaques Aβ plaques are one of the hallmarks of AD. Several PET tracers have been developed and introduced into clinical practice recently for Aβ plaques imaging. The best known tracers are C-PIB (also known as N-methyl-[C]2-[4′methylaminophenyl]-6-hydroxybenzothiazole), 4-N-(C-methyl)amino-4′-hydroxystilbene and 2-(1-[6-([2-F-fluoroethyl][methyl]amino)-2-naph] ethyldene) malono nitrile. Among these tracers, PIB is the most widely utilized and best characterized in terms of tracer kinetics, modeling and analytic methods. PIB binds to fibrillar Aβ plaques with high affinity.
Figure 1 shows representative images of FDG-PET and PIB-PET in an AD patient and an age-matched control.
(Enlarge Image)
Figure 1.
Fluorine-18-labeled 2-fluoro-2-deoxy-D-glucose-PET and Pittsburgh compound-B PET in Alzheimer's disease and normal control individuals. Compared with the controls, FDG (represents brain glucose metabolism) dramatically reduced, while PIB (represents Aβ deposition) significantly increased in AD patients.
AD: Alzheimer's disease; FDG: Fluorine-18-labeled 2-fluoro-2-deoxy-D-glucose; PIB: Pittsburgh compound-B.
Reproduced with permission from [13].
PET scans require short-lived radiopharmaceuticals, which are produced by cyclotrons and synthesized at specially adapted on-site chemical laboratories. High costs of cyclotrons and of maintaining the extensive radiochemistry infrastructure have been the major limitations for PET to be used widespread. This limitation restricts clinical PET primarily to the use of tracers labeled with F. F has a 109.4-min half-life, so that a single large production should permit multiple scans to be performed at multiple PET sites, as is currently the case with F-FDG imaging. By contrast, C has a half-life of 20.4 min. Each patient dose requires an on-site cyclotron run and radiosynthesis of C-PIB immediately before the scan. In addition, PET is limited by its lack of anatomic detail. This usually needs to be complemented by other imaging modalities, such as computed tomography or MRI.
An MRS spectrum is a plot (intensity vs frequency) of the number of nuclei in different local magnetic field environments. The local magnetic field environment results from an intrinsic chemical shift based on the electronic structure of the molecule in which the nuclei resides and the effects of any interactions with other nuclei. The signal recorded from the object volume of interest (Figure 2A) is the sum of all the component signals at the different frequencies. After the spectra acquisition, Fourier transform is used to convert the measured time-varying (time-domain) signal into a list of component frequency (frequency-domain) 'peaks'. Each peak is associated with a specific proton, carbon, or phosphorous position of a biochemical compound such as glucose or glutamate.
(Enlarge Image)
Figure 2.
H magnetic resonance spectroscopy study design and spectrum. (A) A mid-sagittal MRI view of the voxel placement for MRS experiments. (B) A representative spectrum of proton MRS.
Cho: Choline; Cr: Creatine; Glx: Glutamate and glutamine; mI: Myo-Inositol; MRS: Magnetic resonance spectroscopy; NAA: N-acetyl aspartate; ppm: Parts per million.
H- MRS studies have become popular due to the high natural abundance of protons. However, other nuclei, such as C and P, have also been introduced to study the dynamic properties of brain metabolism. These MRS techniques have become very useful in vivo investigative tools for driving knowledge about the physiological processes of normal aging and the pathophysiological progression of neurodegenerative disorders.
H-MRS: Neurochemical Profile.H-MRS permits visualization of a variety of markers of cellular integrity and function, including those of living neurons (N-acetyl aspartate [NAA]), glia (myo-Inositol [mI]), high-energy metabolic products (creatine), cell membrane synthesis or degradation (choline), and some less well resolved amino acids, such as glutamate and glutamine (Figure 2B). Characterization of NAA, mI, Creatine and choline may provide a diagnostic tool, a monitor of disease progression and insight into mechanism of treatment response.
C-MRS: Neuroenergetics & Neurotransmitter Cycling.H[C] or proton-observed carbon-edited MRS can provide noninvasive measurements of neuroenergetics and neurotransmitter cycling in the human brain. With the infusion of C labeled glucose (or acetate), the mitochondrial glucose oxidation rates in neurons and glia can be measured in vivo. Figure 3 shows a diagram of neuronal and astrocyte (a type of glial cell) cell metabolism, and the interplay of neuronal and astrocyte metabolism via the glutamate/glutamine cycle. Both neurons and astrocytes can take up glucose and oxidize it in their mitochondria via the tricarboxylic acid (TCA) cycle. Excitatory glutamatergic neurons, which account for over 80% of the neurons and synapses in the cerebral cortex, release glutamate as a neurotransmitter, most of which is taken up by astrocytes and converted to glutamine or oxidized. Neurons lack the enzymes required for the de novo synthesis of glutamate, and therefore depend on astrocytes to provide substrates for the synthesis of glutamate lost during neurotransmission. The neuron then converts glutamine to glutamate via phosphate-activated glutaminase. The complete series of steps from neuronal glutamate release to the resynthesis of glutamate from glutamine is called the 'glutamate/glutamine cycle'. During the process, the rates of neuronal glucose oxidation (CMRglc(ox), N; μmol/g/min), astrocytic glucose oxidation (CMRglc(ox), A; μmol/g/min) and glutamate/glutamine (neurotransmitter) cycling (Vcyc; μmol/g/min) can be detected (for a recent review see). In addition to the glutamate/glutamine cycle C and H[C] MRS can be used to measure GABA synthesis and GABA neurotransmitter cycling. However because of the lower concentration of GABA these measurements need to be performed in larger voxel sizes or at higher magnetic fields.
(Enlarge Image)
Figure 3.
The glutamate/glutamine cycle and C spectra. (A) The diagram shows the metabolic pathways within glutamatergic neurons and surrounding astroglial cells. Glucose and lactate enter both the glial (VTCAa) and neuronal (VTCAn) TCA cycles via pyruvate dehydrogenase (VPDH), β-HB is directly incorporated into the neuronal and astroglial TCA cycles, and acetate is near-exclusively incorporated into the glial TCA cycle. Neuronal Glu that is released via neurotransmission is taken up by astroglial cells and converted by Gln synthetase to Gln at a rate proportional to the Glu/Gln cycle. The synthesis of Gln is believed to be exclusively within astroglia and other glial cells. In addition to neurotransmitter cycling, Gln may be synthesized de novo starting with the pyruvate carboxylase reaction (VPC). Gln synthesized via pyruvate carboxylase can replace neurotransmitter Glu oxidized in the astrocyte or elsewhere (and be recycled back to the neuron) or leave the brain (Vefflux) to remove ammonia and maintain the nitrogen balance. To measure the rates of these pathways, C-labeled substrates are used and the flow of C isotope into Glu and Gln is measured using C MRS. For detailed descriptions of how these pathways are tracked using C-MRS, and how isotopically labeled substrates and rates are calculated by metabolic modeling, see [16,17]. (B) A C spectrum with of [1C] glucose infusion.
α-KG: α-ketoglutarate; β-HB: β-hydroxybutyrate; AcCoA: Acetyl-CoA; Asp: Aspartate; Gln: Glutamine; Glu: Glutamate; Glut1: Glucose transporter 1; Lac: Lactate; MCT1: Monocarboxylate transporter 1; OAA: Oxaloacetate; ppm: Parts per million; Pyr: Pyruvate; TCA: Tricarboxylic acid; Vefflux: Efflux rate; VTCAα: Tricarboxylic acid flux rate of astrocytes; VTCAn: Tricarboxylic acid flux rate of neurons.
Reproduced with permission from [18].
P-MRS: Energy Production & Redox State.P-MRS enables the noninvasive evaluation of relation concentrations of different intracellular phosphorus metabolites. Substrates of energy metabolism, such as ATP, anorganic phosphate, as well as metabolites of the cell membrane metabolism like phosphomonoesters (PME) and phosphodiesters (PDE), pH level can be estimated. In addition to static measurements, dynamic property, such as ATP production rate, can also be measured using P-MRS to perform a method known as saturation transfer.
Recently, researchers have extended the P-MRS method to determine the redox state in the brain by measuring intracellular NAD/NADH ratio (Figure 4). The basic function of NAD for humans is to release energy from nutrients in our diet through conversion between NADH (reduced form) and NAD (oxidized form) in various redox reactions to form ATP for supporting all cellular activities and functions. Besides regulating bioenergetics and maintaining mitochondrial function, accumulating evidence has suggested that NAD and NADH are also involved in other biological activities, such as calcium homeostasis, antioxidation and oxidative stress, gene expression, immunological functions, aging and cell death. The significantly higher intracellular level of NAD under physiological conditions makes NAD the dominant component in regulating intracellular redox state. Therefore, the concentration ratio of NAD and NADH (defined as RX = NAD/NADH) is widely accepted as a major representation of the intracellular redox state; and its changes have been linked to alterations in energy metabolism and transcription factors under various physiopathological conditions, including aging, diabetes, stroke, cancer and epilepsy.
(Enlarge Image)
Figure 4.
Representative surface-coil localized in vivoP magnetic resonance spectra of normal cat brains at 16.4 T. Full spectrum is shown at the bottom. Enlarged spectral region (chemical shift from -9 to -11.5 ppm) with phase and baseline correction is shown in the bottom inset (gray lines), superimposed by the model predicted spectrum (red line). The model decomposed individual signals of NAD (black line), NADH (green line) and α-ATP (blue line) are shown in the middle inset, and the residue of model fittings to the original P spectra is plotted in the top of inset.
PCr: Phosphocreatine; PDE: Phosphodiester; Pi: Inorganic phosphate; PME: Phosphomonoester; ppm: Parts per million.
Reproduced with permission from [26].
One of the limitations of MRS is that it provides the metabolic composition of a given voxel, which may include more than one type of tissue. In addition, the measurable metabolites have heterogeneous distribution in the brain. Therefore, results would be different if the volume of interest is placed in different brain regions. Collectively, the variation can be found between tissue types, as well as within a tissue type at different locations.
There are other limitations specific to C MRS. One is the heating that results from the applied radiofrequency (RF) energy to the protons bound to C. Although advances in RF coil design have allowed human brain C studies to be performed safely even up to 4 Tesla, concerns remain about RF heating of the eyes, which may be more vulnerable than the brain because of areas of restricted circulation. Another limitation is the requirement for a continuous infusion of the isotopically labeled substrate with venous sampling for fractional enrichment determination. This infusion must occur over a time period (typically 2 h) to capture sufficient kinetic information from spectral time courses for absolute rate estimation using metabolic modeling. However, several studies have shown that it is possible to obtain considerable information on metabolism using simplified infusion schemes or oral ingestion. Given that the majority of information on absolute and relative rates is derived from the early and steady-state portions of the time course, a significant reduction in the time required for a subject to be in the scanner may be possible.
In Vivo Neuroimaging for Brain Metabolism Measurement
PET
PET is currently the most common approach to study brain metabolism. It uses radioactive tracers to produce a 3D image of functional processes (e.g., metabolism) in the body. The system detects pairs of γ-rays emitted indirectly by a positron-emitting tracer, which is introduced into the body on a biologically active molecule. 3D images of tracer concentration are then constructed by computer analysis. PET has several useful attributes for human diagnostic or metabolic studies: the positron-emitting radioisotopes of choice are short lived, rather simplistic, safe within the radioactivity ranges permitted; they require only very low amounts of tracer on the order of 10–10 M, a tolerable risk for organs; and, dynamic tissue uptake and washout of the tracer are measurable in real time (more technical details can be found in).
Glucose Metabolism.FDG is the tracer for studying CMRglc. It simulates a combination of both glucose transport and subsequent phosphorylation. FDG is transported into tissues, including the brain, at almost the same rate as glucose itself. Like glucose, FDG can be phosphorylated by the first glycolytic enzyme (hexokinase). Unlike glucose, FDG cannot be further metabolized to fructose-6-phosphate by glucose-phosphate-isomerase, so FDG remains trapped in the tissue as FDG 6-phosphate. Hence, FDG uptake represents glucose uptake but without subsequent metabolism toward CO2.
Aβ Plaques Aβ plaques are one of the hallmarks of AD. Several PET tracers have been developed and introduced into clinical practice recently for Aβ plaques imaging. The best known tracers are C-PIB (also known as N-methyl-[C]2-[4′methylaminophenyl]-6-hydroxybenzothiazole), 4-N-(C-methyl)amino-4′-hydroxystilbene and 2-(1-[6-([2-F-fluoroethyl][methyl]amino)-2-naph] ethyldene) malono nitrile. Among these tracers, PIB is the most widely utilized and best characterized in terms of tracer kinetics, modeling and analytic methods. PIB binds to fibrillar Aβ plaques with high affinity.
Figure 1 shows representative images of FDG-PET and PIB-PET in an AD patient and an age-matched control.
(Enlarge Image)
Figure 1.
Fluorine-18-labeled 2-fluoro-2-deoxy-D-glucose-PET and Pittsburgh compound-B PET in Alzheimer's disease and normal control individuals. Compared with the controls, FDG (represents brain glucose metabolism) dramatically reduced, while PIB (represents Aβ deposition) significantly increased in AD patients.
AD: Alzheimer's disease; FDG: Fluorine-18-labeled 2-fluoro-2-deoxy-D-glucose; PIB: Pittsburgh compound-B.
Reproduced with permission from [13].
Limitations
PET scans require short-lived radiopharmaceuticals, which are produced by cyclotrons and synthesized at specially adapted on-site chemical laboratories. High costs of cyclotrons and of maintaining the extensive radiochemistry infrastructure have been the major limitations for PET to be used widespread. This limitation restricts clinical PET primarily to the use of tracers labeled with F. F has a 109.4-min half-life, so that a single large production should permit multiple scans to be performed at multiple PET sites, as is currently the case with F-FDG imaging. By contrast, C has a half-life of 20.4 min. Each patient dose requires an on-site cyclotron run and radiosynthesis of C-PIB immediately before the scan. In addition, PET is limited by its lack of anatomic detail. This usually needs to be complemented by other imaging modalities, such as computed tomography or MRI.
Magnetic Resonance Spectroscopy
An MRS spectrum is a plot (intensity vs frequency) of the number of nuclei in different local magnetic field environments. The local magnetic field environment results from an intrinsic chemical shift based on the electronic structure of the molecule in which the nuclei resides and the effects of any interactions with other nuclei. The signal recorded from the object volume of interest (Figure 2A) is the sum of all the component signals at the different frequencies. After the spectra acquisition, Fourier transform is used to convert the measured time-varying (time-domain) signal into a list of component frequency (frequency-domain) 'peaks'. Each peak is associated with a specific proton, carbon, or phosphorous position of a biochemical compound such as glucose or glutamate.
(Enlarge Image)
Figure 2.
H magnetic resonance spectroscopy study design and spectrum. (A) A mid-sagittal MRI view of the voxel placement for MRS experiments. (B) A representative spectrum of proton MRS.
Cho: Choline; Cr: Creatine; Glx: Glutamate and glutamine; mI: Myo-Inositol; MRS: Magnetic resonance spectroscopy; NAA: N-acetyl aspartate; ppm: Parts per million.
H- MRS studies have become popular due to the high natural abundance of protons. However, other nuclei, such as C and P, have also been introduced to study the dynamic properties of brain metabolism. These MRS techniques have become very useful in vivo investigative tools for driving knowledge about the physiological processes of normal aging and the pathophysiological progression of neurodegenerative disorders.
H-MRS: Neurochemical Profile.H-MRS permits visualization of a variety of markers of cellular integrity and function, including those of living neurons (N-acetyl aspartate [NAA]), glia (myo-Inositol [mI]), high-energy metabolic products (creatine), cell membrane synthesis or degradation (choline), and some less well resolved amino acids, such as glutamate and glutamine (Figure 2B). Characterization of NAA, mI, Creatine and choline may provide a diagnostic tool, a monitor of disease progression and insight into mechanism of treatment response.
C-MRS: Neuroenergetics & Neurotransmitter Cycling.H[C] or proton-observed carbon-edited MRS can provide noninvasive measurements of neuroenergetics and neurotransmitter cycling in the human brain. With the infusion of C labeled glucose (or acetate), the mitochondrial glucose oxidation rates in neurons and glia can be measured in vivo. Figure 3 shows a diagram of neuronal and astrocyte (a type of glial cell) cell metabolism, and the interplay of neuronal and astrocyte metabolism via the glutamate/glutamine cycle. Both neurons and astrocytes can take up glucose and oxidize it in their mitochondria via the tricarboxylic acid (TCA) cycle. Excitatory glutamatergic neurons, which account for over 80% of the neurons and synapses in the cerebral cortex, release glutamate as a neurotransmitter, most of which is taken up by astrocytes and converted to glutamine or oxidized. Neurons lack the enzymes required for the de novo synthesis of glutamate, and therefore depend on astrocytes to provide substrates for the synthesis of glutamate lost during neurotransmission. The neuron then converts glutamine to glutamate via phosphate-activated glutaminase. The complete series of steps from neuronal glutamate release to the resynthesis of glutamate from glutamine is called the 'glutamate/glutamine cycle'. During the process, the rates of neuronal glucose oxidation (CMRglc(ox), N; μmol/g/min), astrocytic glucose oxidation (CMRglc(ox), A; μmol/g/min) and glutamate/glutamine (neurotransmitter) cycling (Vcyc; μmol/g/min) can be detected (for a recent review see). In addition to the glutamate/glutamine cycle C and H[C] MRS can be used to measure GABA synthesis and GABA neurotransmitter cycling. However because of the lower concentration of GABA these measurements need to be performed in larger voxel sizes or at higher magnetic fields.
(Enlarge Image)
Figure 3.
The glutamate/glutamine cycle and C spectra. (A) The diagram shows the metabolic pathways within glutamatergic neurons and surrounding astroglial cells. Glucose and lactate enter both the glial (VTCAa) and neuronal (VTCAn) TCA cycles via pyruvate dehydrogenase (VPDH), β-HB is directly incorporated into the neuronal and astroglial TCA cycles, and acetate is near-exclusively incorporated into the glial TCA cycle. Neuronal Glu that is released via neurotransmission is taken up by astroglial cells and converted by Gln synthetase to Gln at a rate proportional to the Glu/Gln cycle. The synthesis of Gln is believed to be exclusively within astroglia and other glial cells. In addition to neurotransmitter cycling, Gln may be synthesized de novo starting with the pyruvate carboxylase reaction (VPC). Gln synthesized via pyruvate carboxylase can replace neurotransmitter Glu oxidized in the astrocyte or elsewhere (and be recycled back to the neuron) or leave the brain (Vefflux) to remove ammonia and maintain the nitrogen balance. To measure the rates of these pathways, C-labeled substrates are used and the flow of C isotope into Glu and Gln is measured using C MRS. For detailed descriptions of how these pathways are tracked using C-MRS, and how isotopically labeled substrates and rates are calculated by metabolic modeling, see [16,17]. (B) A C spectrum with of [1C] glucose infusion.
α-KG: α-ketoglutarate; β-HB: β-hydroxybutyrate; AcCoA: Acetyl-CoA; Asp: Aspartate; Gln: Glutamine; Glu: Glutamate; Glut1: Glucose transporter 1; Lac: Lactate; MCT1: Monocarboxylate transporter 1; OAA: Oxaloacetate; ppm: Parts per million; Pyr: Pyruvate; TCA: Tricarboxylic acid; Vefflux: Efflux rate; VTCAα: Tricarboxylic acid flux rate of astrocytes; VTCAn: Tricarboxylic acid flux rate of neurons.
Reproduced with permission from [18].
P-MRS: Energy Production & Redox State.P-MRS enables the noninvasive evaluation of relation concentrations of different intracellular phosphorus metabolites. Substrates of energy metabolism, such as ATP, anorganic phosphate, as well as metabolites of the cell membrane metabolism like phosphomonoesters (PME) and phosphodiesters (PDE), pH level can be estimated. In addition to static measurements, dynamic property, such as ATP production rate, can also be measured using P-MRS to perform a method known as saturation transfer.
Recently, researchers have extended the P-MRS method to determine the redox state in the brain by measuring intracellular NAD/NADH ratio (Figure 4). The basic function of NAD for humans is to release energy from nutrients in our diet through conversion between NADH (reduced form) and NAD (oxidized form) in various redox reactions to form ATP for supporting all cellular activities and functions. Besides regulating bioenergetics and maintaining mitochondrial function, accumulating evidence has suggested that NAD and NADH are also involved in other biological activities, such as calcium homeostasis, antioxidation and oxidative stress, gene expression, immunological functions, aging and cell death. The significantly higher intracellular level of NAD under physiological conditions makes NAD the dominant component in regulating intracellular redox state. Therefore, the concentration ratio of NAD and NADH (defined as RX = NAD/NADH) is widely accepted as a major representation of the intracellular redox state; and its changes have been linked to alterations in energy metabolism and transcription factors under various physiopathological conditions, including aging, diabetes, stroke, cancer and epilepsy.
(Enlarge Image)
Figure 4.
Representative surface-coil localized in vivoP magnetic resonance spectra of normal cat brains at 16.4 T. Full spectrum is shown at the bottom. Enlarged spectral region (chemical shift from -9 to -11.5 ppm) with phase and baseline correction is shown in the bottom inset (gray lines), superimposed by the model predicted spectrum (red line). The model decomposed individual signals of NAD (black line), NADH (green line) and α-ATP (blue line) are shown in the middle inset, and the residue of model fittings to the original P spectra is plotted in the top of inset.
PCr: Phosphocreatine; PDE: Phosphodiester; Pi: Inorganic phosphate; PME: Phosphomonoester; ppm: Parts per million.
Reproduced with permission from [26].
Limitations
One of the limitations of MRS is that it provides the metabolic composition of a given voxel, which may include more than one type of tissue. In addition, the measurable metabolites have heterogeneous distribution in the brain. Therefore, results would be different if the volume of interest is placed in different brain regions. Collectively, the variation can be found between tissue types, as well as within a tissue type at different locations.
There are other limitations specific to C MRS. One is the heating that results from the applied radiofrequency (RF) energy to the protons bound to C. Although advances in RF coil design have allowed human brain C studies to be performed safely even up to 4 Tesla, concerns remain about RF heating of the eyes, which may be more vulnerable than the brain because of areas of restricted circulation. Another limitation is the requirement for a continuous infusion of the isotopically labeled substrate with venous sampling for fractional enrichment determination. This infusion must occur over a time period (typically 2 h) to capture sufficient kinetic information from spectral time courses for absolute rate estimation using metabolic modeling. However, several studies have shown that it is possible to obtain considerable information on metabolism using simplified infusion schemes or oral ingestion. Given that the majority of information on absolute and relative rates is derived from the early and steady-state portions of the time course, a significant reduction in the time required for a subject to be in the scanner may be possible.
Source...