Human brain glucose metabolism may evolve during activation: Findings from a modified FDG PET paradigm

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Human brain glucose metabolism may evolve during activation: Findings from a modified FDG PET paradigm Andrei G. Vlassenko, Melissa M. Rundle, and Mark A. Mintun⁎ Mallinckrodt Institute of Radiology, Box 8225, Washington University School of Medicine, 510 South Kingshighway Blvd. St,. Louis, MO 63110, USA Received 21 February 2006; revised 12 June 2006; accepted 20 June 2006 Available online 10 October 2006 In human brain, short-term physiological stimulation results in dramatic and proportional increase in blood flow and metabolic rate of glucose but minimal change in oxygen utilization, however, with continuing stimulation, we have observed that blood flow response diminishes and oxygen utilization increases. Given the temporal limitation of conventional methods to measure glucose metabolism in the human brain, we modified [18F]fluorodeoxyglucose (FDG) PET paradigm to evaluate the short-term and long-term effects of visual stimulation on human brain glucose metabolism. In the present study, seven healthy volunteers each underwent three dynamic FDG PET studies: at rest and after 1 min and 15 min of visual stimulation (using reversing black–white checkerboard) which continued for only 5 min after FDG injection. We found that increase in FDG uptake in the visual cortex was attenuated by 28% when preceded by 15 min of continuous visual stimulation (p < 0.001). This decline in metabolism occurred in the absence of any behavior changes in task performance. The similarity in behavior of blood flow and glucose metabolism over time supports the hypothesis that, in activated brain, blood flow is modulated by changes in cytosolic free NADH/NAD+ ratio related to increased glycolysis. Furthermore, the observed decline in glucose metabolism may reflect a shift from glycolytic to oxidative glucose metabolism with continued activation. © 2006 Elsevier Inc. All rights reserved.

Introduction It is well known that in humans, physiological stimulation results in dramatic and proportional increase in cerebral blood flow (CBF) and glucose consumption (CMRGlc) but much less increase in oxygen utilization (Fox and Raichle, 1986; Fox et al., 1988). Moreover, it has been demonstrated in human subjects that the CBF response to physiological stimulation is not altered significantly by either stepped hypoglycemia (Powers et al., 1996) or hypoxia (Mintun et al., 2001). These results suggest that increased CBF during physiological brain stimulation does not occur to prevent shortage of these metabolic substrates. ⁎ Corresponding author. Fax: +1 314 362 7599. E-mail address: [email protected] (M.A. Mintun). Available online on ScienceDirect (www.sciencedirect.com). 1053-8119/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2006.06.065

Based on our recent observations, we suggest that during physiological activation blood flow is regulated by glycolytically evoked changes in cytosolic free NADH/NAD+ ratio (Mintun et al., 2004; Vlassenko et al., 2006). This hypothesis predicts that CBF response to physiological brain stimulation should parallel that of glucose utilization response. We previously demonstrated that blood flow and oxygen metabolism change over the time during continuous visual stimulation (Mintun et al., 2002). Regional CBF increased dramatically (40.7%) 1 min after the onset stimulation, but then this increase attenuated to 26.3% (a decrease of 35.3% compared to 1 min response) 25 min after the onset of stimulation (Mintun et al., 2002). Regional oxygen metabolism increased only 4.7% initially, however, after 25 min of continuous stimulation it was 15%, having tripled from that measured at 1 min (Mintun et al., 2002). In the current study, we hypothesize that glucose utilization should increase initially in response to visual stimulation; however, this increase should attenuate substantially after prolonged continuous stimulation. Our suggestions are indirectly supported by the findings obtained with other neuroimaging techniques. The blood oxygen level dependent (BOLD) signal is dependent on a drop in paramagnetic deoxyhemoglobin in cerebral draining veins, and for more than a decade, it serves as a routine functional MRI confirmation of the fact that blood flow changes are in excess of oxygen consumption changes during acute alteration in brain functional activity. It is of note that MR signal has been shown to decline significantly after continuous (Hathout et al., 1994) or repetitive (Silva et al., 1999) stimulation; although this decline was not always observed and even when observed it was imputed to represent neuronal habituation (Bandettini et al., 1997). MR spectroscopy has demonstrated an initial decrease in glucose content with subsequent turn toward the baseline level after 5 min (Frahm et al., 1996) or 15 min (Chen et al., 1993) of continuous visual stimulation. Assessment of short-term and long-term effects of brain activation using FDG PET is a challenge because, in standard PET approaches, the measurement of cerebral metabolic rate for glucose (CMRGlc) involves ~ 40 min of a “steady state” application of the activation (Fox et al., 1988; Sokoloff et al., 1977). Unfortunately, this time constraint is clearly too long to

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investigate the early temporal changes in CMRGlc after the onset of activation. We now introduce a modified paradigm, designed to evaluate changes in human brain glucose metabolism from shortterm and long-term visual stimulation. This approach limits our abilities for precise quantitative evaluation of local CMRGlc values, but it is very well suited for assessment of relative changes in cerebral glucose metabolism during continuous physiological stimulation. Materials and methods Seven healthy, right-handed subjects, two females and five males (ages 22–32 years; mean age ± SD was 24.7 ± 3.7 years), were recruited from the Washington University community. The Humans Studies Committee and the Radioactive Drug Research Committee of our institution approved the protocol of this study. Written informed consent was obtained. PET imaging All seven subjects underwent three FDG PET scans on three different days. One FDG PET study was done at rest state (eyes closed starting 15 min prior to PET scan). In another FDG PET study, visual stimulation (6 min total) started 1 min prior to FDG injection and continued for 5 min into uptake. In a third FDG study, visual stimulation (20 min total) started 15 min prior to FDG injection and continued for 5 min into the uptake. After the end of visual stimulation, subjects had their eyes closed until the completion of the scan. The order of the three FDG scans was randomized. Studies were done using a Siemens/CTI ECAT EXACT HR 47 tomograph (Wienhard et al., 1994). This scanner collects 47 simultaneous slices with 3.125 mm spacing encompassing an axial field of view of 15 cm. Transaxial and axial spatial resolution is approximately 4.3 mm full-width half-maximum (FWHM) at slice center. Studies were done in the 2-D acquisition mode (interslice septa extended). Subjects had an intravenous catheter in the antecubital vein for FDG injection and sampling for blood glucose measurements. Each FDG study consisted of a 60 min dynamic scan (rebinned to 12 × 5-minute frames) initiated with ~ 20 s bolus intravenous injection of 5 mCi of FDG. Visual stimulation paradigm The visual stimulus has been presented using a computer monitor showing a reversing black–white checkerboard that extends radially from the center to subtend a diameter of 8 visual degrees. The checkerboard reversed at 8 Hz, which yields a robust neural activation response (Fox and Raichle, 1984). We ensured attention to the visual stimulus during the PET scanning. The checkerboard had a small white cross at the center. The subjects were told to fixate and attend to this white cross. Periodically, the cross briefly dimmed. The subjects were instructed to respond by pushing a button (PsyScope button box, Carnegie Mellon University, Pittsburgh, PA) under their forefinger when they detect the dimming. The inter-stimulus intervals were random with mean interval of 10 s with a uniform distribution over a range of 5 to 15 s. At the end of visual stimulation, data about the number of stimuli and number of correct and false responses were recorded and analyzed using in-house software.

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MRI To guide anatomical localization, MRI scans were obtained using standard protocols in all subjects. MRI scans were performed on a Magnetom Vision 1.5-T imaging system (Siemens). A magnetization-prepared rapid-gradient echo acquisition was used to acquire anatomic images that consisted of 160 contiguous 1mm-thick sagittal slices. Scanning parameters were repetition time = 1900 ms, echo time = 3.93 ms, inversion time = 1100 ms, flip angle = 15°, matrix = 256 × 256 pixels, voxel size = 1 × 1 × 1. Image reconstruction and registration FDG dynamic data analysis was limited to the frames between 40 and 60 min after FDG injection. These four individual 5minute frames were aligned using rigid body linear affine transformation to correct for any head motion during the scanning and then summed. The summed images collected from the three experimental conditions for a given subject were coregistered and aligned to the subject’s MRI scan which has been coregistered with the laboratory Talairach atlas MRI. All MRI and PET data were resampled into atlas space for viewing and processing (ANALYZE_AVW, Mayo Clinic, Rochester, MN). Each FDG uptake image was scaled to normalize by whole brain FDG uptake values. Volume of interest (VOI) analysis The two activation images for each subject were averaged. The rest image was then subtracted from this average activation image to yield a difference image of FDG uptake increase during activation. For each subject’s difference image, a VOI was created by selecting thresholds that result in visual cortex VOI of ~ 1000 pixels (equal to 8 cm3). In all cases, VOI was confirmed to be centered over the visual cortex by comparing location to coregistered MRI images. This VOI was applied to each subject’s three FDG scans to obtain the qualitative FDG uptake data for the region. Paired two-tailed t-tests were done to examine whether activation states showed increased FDG uptake compared to the “rest” state. Furthermore, a paired two-tailed t-test was used to determine whether the FDG scan begun 1-minute after onset of activation has greater visual cortex FDG uptake than seen in the scan begun 15-minute after onset of activation. A p value of 0.05 was used as a threshold of significance. Results Mean FDG uptake in the visual cortex was increased compared to eyes closed state by 28.5 ± 4.9% (p < 0.0001; two-tailed paired t-test) and 20.5 ± 3.7% (p < 0.00001; two-tailed paired t-test) after 6 and 20 min of visual stimulation, respectively (Fig. 1). The percent attenuation of FDG uptake response from 6 min to 20 min of visual stimulation was calculated as 100 × (FDG6 − FDG20)/FDG6. Thus, after 20-min visual stimulation, CMRGlc response attenuated by 27.9 ±6.7% (p < 0.001; two-tailed paired t-test) compared to that after 6-min stimulation (Fig. 2). Attention to the visual stimulus (percent of correct responses to the dimming of the cross at the center of the checkerboard) did not significantly differ before and during the PET scan, both for 6-min stimulation (96% and 88%, p = 0.22, two-tailed paired t-test) and

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Fig. 1. Percent changes in FDG uptake after short-term (6 min) and prolonged (20 min) visual stimulation. FDG uptake was evaluated in VOI created by selecting threshold that result in visual cortex VOI of ∼ 1000 pixels (equal to 8 cm3).

for 20-min stimulation (82% and 84%, p = 0.65, two-tailed paired t-test). Blood glucose levels before and after FDG scan were 5.1 ± 0.5 and 4.9 ± 0.7 mmol/L, respectively (p = 0.147; two-tailed paired t-test). Discussion Our data demonstrate that during initial period of physiological brain stimulation glucose metabolism increases significantly but after 20 min of continuing stimulation this increase attenuates by ~ 28% (Fig. 2). The observation that cerebral blood flow changes over time parallel changes of glucose utilization indicates a close relationship between these important parameters but does not imply that blood flow increase occurs only to match the delivery of glucose to momentary changes in its utilization. We believe that, in general, the energy requirements of activated brain are increased to a small extent compared to that at rest (baseline) (Raichle and Gusnard, 2002), and the human brain has an oversupply of fuels at almost any level of physiological stimulation (Mintun et al., 2001; Powers et al., 1996). Our recent and previous observations suggest that changes in blood flow in activated brain are related to changes in glycolysis and are aimed to balance the cytosolic NADH/NAD+ ratio. This ratio is increased by glycolysis (due to reduction of NAD+ to NADH), and it should be properly balanced because, otherwise, glycolysis could not proceed and no ATP would be generated. Several ways to regenerate cytosolic NAD+ (and to

decrease NADH/NAD+ ratio) have been proposed including signaling pathways that promote increase in blood flow (Ido et al., 2001, 2004). Experiments testing this hypothesis have been based on the known near-equilibrium between the cytosolic NADH/NAD+ and lactate/pyruvate ratios. Studies in anesthetized rats and in awake humans demonstrated that changes in plasma lactate/pyruvate ratio induced by intravenous injection of lactate or pyruvate evoke corresponding changes in blood flow response to physiological brain stimulation (Ido et al., 2001, 2004; Mintun et al., 2004; Vlassenko et al., 2006). Lactate injection augments the regional cerebral blood flow response to visual stimulation, and pyruvate injection attenuates this response (Ido et al., 2001, 2004; Mintun et al., 2004; Vlassenko et al., 2006). Our observations of matched changes in blood flow and glucose utilization over the time of continuous brain stimulation are well consistent with the hypothesis of glycolytically related blood flow regulation. These observations indicate that a decrease in glycolysis with continuing visual stimulation may be associated with a decrease in blood flow response through NADH/NAD+-related mechanisms. It is likely that close regulatory relationship between glycolysis and brain blood flow occurs not only in physiological conditions but also in some disorders accompanied by increased glycolysis. As an example, epileptic seizures, both experimental in animals and clinical in humans, demonstrate regional increase in glycolysis and corresponding increase in blood flow (Franck et al., 1986; Kuhl et al., 1980; Theodore et al., 1983), which exceeds that of oxygen utilization, and also significant decrease in oxygen extraction fraction (Franck et al., 1986). Of note, these changes in brain blood flow and energy metabolism are associated with significant increase in cytosolic free NADH/NAD+ ratio, and this increase has been demonstrated in electroconvulsive seizures (Merrill and Guynn, 1976) as well as in sustained seizures induced by known experimental convulsants (Ackermann and Lear, 1989; Chapman et al., 1977; Folbergrova et al., 1985; Ingvar et al., 1984; Pinard et al., 1984). The time course and magnitude of changes in blood flow and energy metabolism in activated brain suggest that the energy demands of brain activation are initially met predominantly by increased glycolysis with less increase in oxidative phosphorylation. In astrocytes, this initial increase in glycolysis occurs in support of the uptake of glutamate from the synaptic cleft and its conversion to glutamine (Erecinska and Silver, 1990; Hertz, 1979; Pellerin and Magistretti, 1994). The advantage of glycolysis over oxidative phosphorylation in this situation may be its more rapid delivery of ATP in a setting, where intermittent changes in neu-

Fig. 2. Coregistered sagittal MRI (left) and PET (middle and right) images from a single subject. PET images are subtractions of FDG resting scan from FDG 6min (middle) and 20-min (right) activation scans. The area delineated with a red line is a representative of the selected VOI.

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ronal activity are occurring in tens of milliseconds. Activation increases energy production in neurons as well, however, neurons are better equipped for oxidative phosphorylation than astrocytes: neurons have more mitochondria (Wong-Riley, 1989) and higher level of important carriers for malate-aspartate shuttle of electrons and protons from cytosolic NADH to mitochondrial NAD+ (Ramos et al., 2003). Thus, neurons should have more proportional changes in glycolysis and oxidative phosphorylation in activated state compared to astrocytes. We suggest that ability to increase glycolysis initially in response to brain stimulation and to return efficiently to oxidative utilization of glucose with continuing stimulation is a key factor for maintaining the adequate level of brain functioning. There are reasons to speculate that aging may be associated with alterations in this metabolic control. A number of studies have reported age-related reductions in resting glucose and oxygen metabolism (Kuhl et al., 1982; Leenders et al., 1990; Marchal et al., 1992; Pantano et al., 1984; Yamaguchi et al., 1986), while others (de Leon et al., 1984; Duara et al., 1984) have failed to confirm these findings. Functional MRI studies allowed demonstrating age-related reduction in BOLD response to photic (Ross et al., 1997) or motor–sensory stimulation (Buckner et al., 2000). Activation associated with visual word identification exhibited an age-related decline in the CBF measured with 15O-water PET in visual sensory (striate) cortex (Madden et al., 2002). In patients with Alzheimer disease (AD), the magnitude of the CMRGlc PET responses to audiovisual stimulation (Pietrini et al., 2000) or visual recognition task (Kessler et al., 1991) demonstrated significant decline compared to age-matched healthy volunteers. CBF response to visual activation measured with 15O-water PET is diminished in Alzheimer disease (AD) subjects proportionally to severity of dementia compared to age-matched controls (Mentis et al., 1996, 1998). Of note, a specific set of brain regions was described recently, which presumably comprise a brain “default network” (Raichle et al., 2001). These regions are chronically active in baseline functional state of the brain in order to support some default activities such as monitoring the environment, monitoring one’s internal state and emotions, and various forms of undirected thought (Raichle et al., 2001). Recent fMRI and PET amyloid imaging studies demonstrated that these default areas undergo age-related functional changes and are even more disturbed in patients with AD (Buckner et al., 2005; Lustig et al., 2003). Studies of hemodynamic and metabolic effects of continuous physiological stimulation with involvement of different brain regions in elderly subjects and patients with AD may contribute substantially to our understanding of the role of ageing and neurodegenerative changes in alteration of the regulation of brain energy metabolism and blood flow. In our study, modification of an experimental paradigm was designed to evaluate and compare short-term and long-term effects of visual stimulation with FDG PET scans. The effects of shortterm physiological stimulation were investigated with visual stimulation initiated 1 min before FDG injection and the start of PET scan; and the effects of continuous physiological stimulation were investigated with visual stimulation initiated 15 min before FDG injection and the start of PET scan. Visual stimulation continued for only 5 min of FDG uptake and PET scanning, thus allowing us comparison of short-term (1 min + 5 min = 6 min) and “long-term” (15 min + 5 min = 20 min) stimulation. We chose 5 min of stimulus during FDG uptake as a compromise: we wanted to use a stimulus time shorter than what has been associated with changes in the blood flow response (Mintun et al., 2002), but long enough

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to result in a measurable change of FDG uptake (Huang et al., 1981). For visual stimulation, we used black–white checkerboard reversing at 8 Hz. The highest response of CMRGlc to visual stimulation reported, 51%, was achieved by Fox et al. (1988) with large visual field annular reversing red–black checkerboard in a single scanning session for both resting and activated state. Other studies using various visual and behavioral tasks reported 6%–29% changes in regional CMRGlc (Chang et al., 1987; Greenberg et al., 1981; Kushner et al., 1988; Phelps et al., 1981). Thus, although our data represent the mixed contribution from the initial 5 min with visual stimulation and approximately 55 min of eyes closed, the relative metabolic response in our study is comparable to that obtained in other FDG studies when steady state requirements were fulfilled. It is typically assumed that FDG PET studies require a 30 to 40-minute equilibration time to approach steady state and any changes in brain metabolism during FDG uptake will alter any precise quantitation of CMRGlc. However, the accumulation of FDG is also known to be heavily weighted to the time early after injection (e.g., 0–15 min) when circulating FDG levels are highest (Crosby and Sokoloff, 1983; Gjedde, 1987; Sokoloff et al., 1977). In one report, the impact of a transient change in metabolism was specifically calculated using compartmental modeling and computer simulations and presented in tabular form (Huang et al., 1981). For these calculations, a 5-minute increase in metabolism of a given amount that was present at the time of injection would be reduced to only 23%–50% of its original increase at the time of PET imaging. Our robust (~ 28%) increase in FDG uptake when visual stimulation was presented only for the first 5 min of FDG circulation is somewhat higher than would be expected by these calculations (even assuming that this stimulus results in a 51% increase (Fox et al., 1988)). However, the paradigm of our study does not allow us to be precise in the quantitative evaluation of regional CMRGlc during short-term and long-term visual stimulation. In conclusion, we studied the effects of continuous visual stimulation on brain glucose metabolism and found that initial rise in FDG uptake attenuates substantially after 20 min of continuous stimulation. Our results are consistent with the hypothesis that blood flow in activated brain is modulated by glycolytically evoked changes in NADH/NAD+ ratio. Initial response of the brain metabolism to stimulation may be primarily glycolytic and change back to predominantly oxidative utilization of glucose with continuous stimulation. We suggest that this relationship of different parameters of brain energy metabolism may be important for maintaining healthy brain functioning. Acknowledgments We thank Lori Groh and Lisa Votraw for the help with recruiting subjects and organizing the study and Lenis Lich for skilled technical assistance in PET imaging. This study was supported by NINCDS Grants P50 NS-06833 and P30 NS-048056. References Ackermann, R.F., Lear, J.L., 1989. Glycolysis-induced discordance between glucose metabolic rates measured with radiolabeled fluorodeoxyglucose and glucose. J. Cereb. Blood Flow Metab. 9, 774–785. Bandettini, P.A., Kwong, K.K., Davis, T.L., Tootell, R.B., Wong, E.C., Fox,

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