NIH Public Access

Author Manuscript
J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.
Published in final edited form as: J Magn Reson Imaging. 2010 November ; 32(5): 10451053. doi:10.1002/jmri.22366.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Lower Brain Glutamate Is Associated With Cognitive Deficits in HIV Patients: A New Mechanism for HIV-Associated Neurocognitive Disorder
Thomas Ernst, PhD1, Caroline S. Jiang, MS1, Helenna Nakama, MD2, Steven Buchthal, PhD1, and Linda Chang, MD1 1 Department of Medicine, John A. Burns School of Medicine, University of Hawaii, and Queens Medical Center, Honolulu, HI
2

Department of Psychiatry, John A. Burns School of Medicine, University of Hawaii, and Queens Medical Center, Honolulu, HI

Abstract
PurposeTo determine whether subjects with HIV-associated neurocognitive disorder (HAND) show altered concentrations of brain glutamate (GLU), and whether lower GLU levels correlate with cognitive deficits. Materials and MethodsGLU concentrations were measured in the basal ganglia, frontal gray and white matter, and parietal gray matter of 45 HIV-positive and 46 age-and-education-matched HIV-negative subjects, using echo-time averaged proton magnetic resonance spectroscopy (1H MRS). ResultsCompared to controls, HIV subjects with cognitive deficits had lower GLU in the parietal gray matter, while those without cognitive deficits tended to show higher basal ganglia GLU. Lower parietal and frontal gray matter GLU were associated with a greater number of nucleoside reverse transcriptase inhibitors, and were predictive of poorer cognitive performance. Correlations between GLU and cognitive performance, but not the other findings, remained significant after correction for multiple comparisons. ConclusionParietal gray matter GLU is lower in HIV subjects with cognitive deficits. This reduction might result from reduced astrocytic reuptake of GLU, secondary excitotoxicity, and mitochondrial toxicity from antiretroviral treatments. The glutamatergic system may play an important role in the pathophysiology of HAND, and brain GLU on 1H MRS may provide an early surrogate marker for monitoring disease severity and treatment effects. Keywords Glutamate; brain; magnetic resonance spectroscopy (MRS); HIV; cognition

INTRODUCTION
Neurocognitive deficits continue to occur in 2040% of patients infected with the human immunodeficiency virus (HIV), despite effective systemic suppression of viral replication with combined antiretroviral medications. With the recently updated research definition of HIV-associated neurocognitive disorder (HAND), which requires cognitive impairment in at

Corresponding Author: Thomas Ernst, PhD, Department of Medicine, 1356 Lusitana Street, 7th Floor, Honolulu, HI 96813, Tel.: (808) 585 5153, Fax: (808) 585 5160, tmernst@hawaii.edu.

Ernst et al.

Page 2

least two of seven ability domains evaluated (1), up to half of HIV-infected individuals may have HAND. Common symptoms and signs of HAND include decreased ability to attend and concentrate, and reduced motor and cognitive processing speed. Neuropathology often shows leukoencephalopathy and axonal damage, with variable neuronal loss (2). HIV patients also demonstrate microglial and glial activation (2), which in turn may lead to the release of neurotoxic substances that may contribute to neuronal apoptosis or neuronal dysfunction. Furthermore, several recent studies indicate that glutamate (GLU)-mediated excitotoxicity may contribute to HIV brain injury. GLU is an amino acid and neurotransmitter with important roles in normal cell function and neurotransmission. However, excessive concentrations of extracellular GLU may be neurotoxic and induce cell death (3). Increases in extracellular GLU may be caused by attenuation of astrocytic reuptake of GLU due to HIV infection (4), as well as by increased GLU production by HIVinfected macrophages (5). In turn, decreased re-uptake of GLU by activated astrocytes would lead to incomplete recycling of GLU, a net loss of GLU into the extracellular space, and eventually decreased intracellular concentrations of GLU. Therefore, the goal of this study was to evaluate whether in vivo brain GLU levels are abnormal in HIV-positive subjects. Proton magnetic resonance (1H-MRS) can measure the concentrations of several neurochemicals, but conventional in vivo 1H-MR spectra show the GLU signal as a broad, complex pattern that is obscured by overlapping glutamine peaks and macromolecule signals, commonly named GLX (Figure 1C). A technique called timeto-echo-averaged point-resolved spectroscopy, or TE-averaged PRESS, can unambiguously resolve GLU (6), by averaging multiple, brief spectra at different echo time values (Figure 1A). The averaging process yields a distinct pseudo-singlet for the GLU resonance at 2.35ppm, with markedly attenuated signals from glutamine and macromolecules and minimal baseline distortions in the GLU region (Figure 1D). Since HIV infection leads to activated astrocytes, which would be associated with incomplete recycling and a net loss of GLU, we hypothesized that HIV positive individuals would show lower brain GLU than seronegative control subjects as assessed by TEaveraged PRESS. We further hypothesized that those with lower brain GLU would show greater cognitive deficits on neuropsychological tests.

NIH-PA Author Manuscript NIH-PA Author Manuscript

Subjects

MATERIALS AND METHODS

45 HIV-positive subjects and 46 HIV-seronegative (SN) healthy control subjects 18 years or older were recruited from the local community. After signing a written consent form approved by our institution, participants completed detailed clinical assessments, including a physical and neuropsychiatric examination by a board-certified Neurologist or a Psychiatrist, a detailed medical and drug use history and a battery of neuropsychological tests (7). Additional assessments included the Center for Epidemiological Studies Depression (CESD) scale and the Symptom Checklist (SCL-90) for mood, and the New Adult Reading Test Revised (NART-R) to estimate premorbid intellectual functioning. HIV-1 seronegativity in controls was verified by ELISA blood test. HIV-positive subjects also had to be either on no antiretroviral medications or stable on antiretrovirals for at least 8 weeks, and had a nadir CD4 count<500/mm3. Subjects in either group were excluded if they had any of the following: 1) chronic medical or neuropsychiatric illnesses that might confound the study; 2) severe hepatic or renal dysfunction; 3) history of drug dependence according to Diagnostic Statistical Manual-IV criteria or positive urine toxicology screen (for stimulants, benzodiazepines or opiates); 4) contraindications for MRI; 5) pregnancy (for females); 6) less than 8th grade level English reading skill.

NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 3

Neuropsychological Testing Each participant performed a battery of neuropsychological tests sensitive for detecting cognitive deficits in patients with HIV infection (7). The tests evaluated the following seven ability domains: 1) Memory: Rey Osterrieth Complex Figure Delayed Recall and Rey Auditory Verbal Learning Test Trial 7; 2) Learning/Perceptual: Rey Auditory Verbal Learning Test Trial 5 and Rey Osterrieth Complex Figure Immediate Recall; 3) Attention/ working memory: Wechsler Adult Intelligence Scale (WAIS)-III Digit Span Subtest Full, Arithmetic and Letter-Number Sequencing, and Paced Auditory Serial Addition Test 1; 4) Executive function: Stroop interference and Trail Making Test B; 5) Verbal/Language: Ruff Figural Fluency and Verbal Fluency (FAS); 6) Motor skills: Grooved Pegboard Dominant & Non-Dominant hands; and 7) Speed of information processing: Symbol Digit, Trail Making Test A, Stroop color, and California Computerized Assessment Package (CalCAP) Simple Reaction Time. Z-scores adjusted for age and education based on our normative database of 273 subjects were calculated for each cognitive domain and across all domains (global z-score). HIVpositive subjects were classified according to the Updated Research Nosology for HAND (1), in combination with self reports of cognitive symptoms, neurological evaluations, the Functional Activities Questionnaire (8) and Karnofsky score (9). Eighteen HIV subjects had cognitive deficits (HIV-CD); six of those met the criteria for asymptomatic neurocognitve impairment [ANI, impairment in 2 ability domains by 1 standard deviation (SD)], 10 had minor neurocognitive disorder or (MND = ANI plus interference with daily functioning), and two had HAD [impairment in 2 cognitive domains by 2 SDs, and functional deficits]. The remaining 27 HIV subjects had normal neuropsychological test performance, and were defined as having normal cognition (HIV-NC). Imaging Studies MR studies were performed on a Siemens 3 Tesla Trio scanner, using an 8-channel head coil. Following T1 and T2-weighted structural imaging, TE-averaged PRESS (6) was performed in 4 brain regions (each approximately 8cc): basal ganglia, parietal gray matter, frontal white matter, and anterior cingulate gray matter (Figure 2, left). Data were acquired at TE values between 35 and 190 ms (32 5 ms increments; TR=2s; 8 water-suppressed and 1 unsuppressed spectra each); consequently, the resulting signal intensities are dependent on the metabolite T2 values. Additionally, the T2-decay of the water signal in each voxel was measured by acquiring the unsuppressed, fully relaxed, water signal at 7 different TE-values (TE=30ms to 1.5 seconds; TR=10 seconds). Data were processed in IDL and LCModel (10) to determine concentrations of major brain metabolites, including those of N-acetyl aspartate (NAA), Choline (Cho), total creatine (tCr), glutamate (GLU), and myo-inositol (MI). Each spectrum met the following criteria from the LCModel fit: 1) full width at half minimum <0.07 ppm (<0.1ppm for basal ganglia), 2) signal-to-noise ratio >7, and 3) Cramer-Rao bounds 25%. Spectra that did not meet these criteria were repeated. Spectra were corrected for eddy currents and the percentage of %CSF in each voxel (11), averaged over the 32 TE values, and averaged across receiver channels using the relative unsuppressed water signal intensity as a weighting factor. Also, the percentage of gray matter and white matter in each voxel was determined by segmenting high-resolution MP-RAGE scans using FMRIBs Automated Segmentation Tool (FAST, version 4.1) (12) for cortical voxels, and FMRIBs Integrated Registration and Segmentation Tool (FIRST, version 1.2) (13) for subcortical structures. Statistical Analyses Statistical analyses were performed using SAS version 9.1 (SAS Institute Inc., Cary, NC). One-way analysis of covariance (ANCOVA) was performed to compare brain metabolite
J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Ernst et al.

Page 4

concentrations between HIV-NC, HIV-CD and SN control subjects, with age included as a covariate (14). P-values corrected for the percentages of gray or white matter were also calculated since brain metabolite levels may vary with voxel composition (15,16). Linear regression was used to examine the relationships between neuropsychological test performance and brain glutamate levels with significant group differences, using Pearson correlations for post-hoc testing. In all analyses, a p-value <0.05 without a correction for multiple comparisons was considered significant.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

RESULTS
Clinical Characteristics of Research Participants Table 1 shows that the three groups were similar in age, education, gender distribution, viral load, Karnofsky score, and duration of HIV infection. As expected, the HIV-CD subjects tended to have lower scores on the HIV dementia scale compared to HIV-NC subjects, as well as lower scores on all 7 ability domains. None of the participants had vascular or abnormal focal brain lesions on the MP-RAGE or FLAIR images. MR Findings In the parietal gray matter, the GLU concentration was highest in the SN controls, intermediate in the HIV-NC subjects, and lowest in the HIV-CD subjects (10.2% compared to SN, p=0.02; Figure 2 right); these findings remained significant when values were adjusted for gray-matter differences (p=0.04). A similar stepwise pattern was observed in the frontal gray matter, although the HIV-CD subjects only showed a trend to be lower than the SN controls (7.1%, p=0.08) Basal ganglia GLU was higher in the HIV-NC group compared to control subjects (+10.0%, p=0.04 and p=0.07, without and with correction for %gray-matter). None of the metabolite group differences remained significant when pvalues were adjusted for multiple comparisons. The gray matter content was lower in the HIV compared to the SN group in the parietal gray matter voxel, but not in the other three voxels (Table 2). The parietal GLU concentration declined with subjects age (7.5%/decade; r=0.46, p<0.0001; both groups combined; no interaction; Figure 3). Therefore, HIV subjects on average have a parietal gray matter GLU level equivalent to that in SN subjects who are 10years older. Age-related decreases of GLU were also seen in the basal ganglia (r=0.37, p=0.004) and frontal gray matter (r=0.23, p=0.03; no interactions). HIV-positive subjects also had other alterations. The Cho concentration of HIV-CD subjects was lower in the basal ganglia compared to control (13.7%, p=0.01) and HIV-NC subjects (14.7%, p=0.02), and in the parietal gray matter compared to controls after correction for gray-matter percentage (12.5%, p=0.01). HIV-CD subjects also tended to show lower NAA concentrations in frontal gray matter compared to control (5.8%, p=0.08) and HIVNC subjects (7.3%, p=0.04), as well as in parietal gray matter compared to the HIV-NC subjects (9.0%, p=0.08). Additionally, the MI concentration was higher in the frontal white matter of HIV-CD subjects compared to controls (+14.0%, p=0.04), and tended to be higher in HIV-NC subjects compared to controls in the basal ganglia and parietal gray matter. Finally, the total Cr levels were slightly lower in the basal ganglia and parietal gray matter of the HIV-CD group compared to SN controls (approximately 7% for both voxels), as well as in parietal gray matter of HIV-CD subjects compared to controls (10.2%, p=0.08). Relationships Between Glutamate And Other Variables On linear regressions, lower parietal gray matter GLU concentrations across all subjects were associated with poorer performance on the Grooved Pegboard tasks (Non-Dominant

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 5

hand: r=0.25, p=0.04; Dominant hand: r=0.23, p=0.065), Trail Making Test A (r=0.37, p=0.002), and on the AVLT Delayed Recall test (r=0.26, p=0.04); see Figure 4. Many of these correlations remained significant within individual groups. Likewise, lower frontal gray matter GLU was associated with poorer performance on the CalCAP Reaction Time for Degraded Words (r=0.24, p=0.03) and Word Reversal (r=0.30, p=0.006), and on the Wechsler Adult Intelligence Scale (WAIS) Backward test (r=0.32, p=0.004) across all subjects. Furthermore, lower basal ganglia GLU concentration correlated with poorer performance on the CalCAP Reaction Time for Degraded Words (r=0.27, p=0.05). No interactions between HIV status and GLU levels were present for any of these tasks, and none of the other correlations between GLU and clinical variables or neuropsychological test scores were significant. Likewise, no correlations were observed between GLU levels in any of the brain regions and CD4 count, nadir CD4 count or viral load. Because nucleoside reverse transcriptase inhibitors (NRTIs) can exhibit mitochondrial toxicity (17,18), which in turn might affect de novo synthesis of GLU via the Krebs cycle, we studied the association between the number of NRTIs in each subjects antiretroviral regimen and GLU concentrations. Of the 45 HIV subjects, 6 did not receive any NRTIs, 5 received one NRTI, 31 received two, and 3 received 3 NRTIs as part of their HIV regimen. A significant inverse correlation was observed between parietal GLU and the number of NRTIs (r=0.40, p=0.04, Spearman correlation), with a drop in the parietal GLU concentration of approximately 9% for each additional NRTI drug in the treatment regimen (using linear regression). The number of NRTIs also correlated inversely with frontal gray matter GLU (r=0.36, p=0.02).

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

DISCUSSION
This study found that HIV subjects, and in particular those with cognitive deficits, have lower brain GLU levels than seronegative control subjects in the parietal and the frontal cortex; however, these findings were no longer significant after correction for multiple comparisons. The tendency for low GLU in the parietal region is in agreement with a recent smaller pilot study (19). In contrast, we did not find the marked (43%) reduction in glutamate levels in the white matter of HIV subjects observed previously (19); in fact, our HIV subjects showed non-significantly and mildly elevated white matter GLU levels. Interestingly, the parietal lobe also showed the most pronounced decline of GLU with normal aging amongst several brain regions, both in the current and a prior study (14). Consequently, HIV subjects across the age span demonstrate parietal GLU levels that are equivalent to control subjects 10 years older. Additionally, since lower GLU was consistently associated with poorer performance on several neuropsychological tests in both subject groups, lower GLU may contribute to cognitive deficits or increase the risk to develop dementia in HIV-positive individuals. Glutamate is synthesized de novo from -ketoglutarate via the tricarboxylic acid (TCA) cycle. Glutamate in neurons has the dual roles of an amino-acid and that of the most abundant excitatory neurotransmitter. Glutamate released during synaptic transmission into the extracellular space (ECS) is taken up by astrocytes, via excitatory amino acid transporters (EAATs), and converted into glutamine. Astrocytes then release glutamine into the extracellular space, which is taken up by neurons and converted back into glutamate. The flux through this glutamate-glutamine shuttle represents 4050% of the total flux from the TCA cycle (20,21). Of note, since the extracellular concentration of GLU is much lower than its concentration in neurons, essentially all of the GLU signal detected by 1H MRS can be attributed to the intracellular compartment.

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 6

Our results are in agreement with our initial hypothesis that incomplete recycling of glutamate by the glutamate-glutamine shuttle would lead to a net output of GLU into the vasculature or ECS, and thereby reduce the total amount of glutamate in neurons. HIV infection causes activation of glial cells, specifically microglia and astrocytes (2,22), which may lead to inhibition of glutamate reuptake (23) and glutamine synthesis (24) and ultimately in increased extracellular GLU and decreased intracellular (neuronal) GLU. Specifically, exposure of astrocytes to HIV-1 or the envelope glycoprotein gp120 reduces their ability to transport GLU (4). Elevated extracellular GLU concentrations can lead to excitotoxicity, which has been implicated in HIV-induced damage to neurons (3). Clinical studies support this notion; for instance, glutamate concentrations in the cerebrospinal fluid (CSF), which likely reflect extracellular GLU levels, were elevated in HIV patients (25). GLU-induced excitotoxicity may be further exacerbated by HIV-infected macrophages, which often migrate to the brain and contribute to the neuroinflammatory cascade, and could dramatically increase GLU production (5). Alternative explanations also might account for lower GLU concentrations. First, lower GLU might result from increased demand for GLU as an amino-acid precursor for synthetic pathways, for instance, for repair of HIV-induced damage to cell membranes. This process would reduce the GLU signal since protons in large molecules generally become MR invisible. However, the normal parietal Cho levels found suggest relatively normal cell membrane metabolism. Second, lower gray matter GLU in the HIV-positive individuals may reflect a loss of gray matter and/or glutamatergic neurons, since the GLU concentration is substantially lower in normal white matter than in gray matter. However, this possibility is less likely since the neuronal marker NAA was only marginally different amongst the three groups, and since findings remained significant even after correcting for differences in gray-white matter voxel composition. Additionally, neuropathology studies find that only HIV patients with dementia show neuronal loss (2,22). Third, lower levels of GLU may result from reduced GLU synthesis via the TCA cycle, due to inhibition of the mitochondrial function as a result of oxidative stress. For instance, oxidized proteins in the CSF from HIV subjects with dementia can attenuate mitochondrial activity (26), as can many NRTIs (17,18). Accordingly, subjects treated with a greater number of NRTIs showed lower GLU concentrations. A down-regulation of the TCA cycle is also suggested by nuclear imaging techniques which demonstrate reductions in cerebral perfusion and glucose consumption (which are closely coupled to the TCA cycle) in the cortex (2734) and specifically in the parietal gray matter (35) of HIV-positive compared to seronegative subjects. Lower glutamate plus glutamine (GLX) in HIV patients was also observed in a prior study (36), although the parietal lobe was not evaluated. Since the parietal cortex appears to show the greatest age-related decline in GLU (14), the current finding of persistently lower GLU in the parietal region across the age span of HIV subjects suggests they have an older than normal brain in terms of glutamate reserve. The associations of lower gray matter GLU concentrations with poorer performance on several cognitive tests suggest an important role of parietal GLU for cognitive functioning. Of note, all of these tasks involve an attentional neural network that includes the parietal lobe, which showed altered activation in HIVpositive subjects (37). Therefore, the combined effect of declining GLU levels with age and HIV infection may reduce cognitive reserve in HIV-infected individuals, and make them more susceptible to developing cognitive deficits.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 7

In contrast to the reduced Glu levels in the parietal cortex of the HIV-CD subjects, the Glu levels in the basal ganglia of HIV-NC subjects were elevated. This may reflect a compensatory response to the ongoing oxidative stress associated with HIV infection. A compensatory response to oxidative stress has been reported in HIV-infected individuals, who were found to have elevated levels of glutathione peroxidase, especially those on antiretroviral treatments (38). Such a compensatory response may necessitate the increased production of intracellular glutamate as a precursor for glutathione. Our HIV subjects with elevated Glu (i.e. those who able to compensate against oxidative stress) had normal cognition and normal NAA level. However, the HIV-CD subjects were unable to compensate against the oxidative stress tended to have lower Glu and NAA level in the frontal and parietal regions, suggesting greater neuronal loss. One of the other interesting findings of the current study is the reduction of Cho-containing compounds in the basal ganglia and parietal gray matter of HIV-CD subjects. Earlier studies of HIV brain disease commonly found increases in Cho concentrations (36,3941), perhaps due to increased cell membrane breakdown, whereas several more recent studies did not report abnormalities in Cho concentrations in subjects with HIV (4245). Lower Cho concentrations in the current study may indicate overall cell loss in these HIV subjects, perhaps due to the prolonged and chronic HIV infection in these subjects (over 10 years). Likewise, the Cr and NAA concentrations tended to be lower in the basal ganglia and parietal gray matter of the HIV-CD subjects in our study. One of the limitations of the current study is that group differences in metabolite concentrations were not significant after correcting for multiple comparisons; therefore, the results require further confirmation. Another limitation is that the intensity of metabolite signals acquired with TE-averaged PRESS is dependent on the transverse (T2) relaxation time. Therefore, interpretation of alterations in measured metabolite concentrations relies on the assumption that metabolite T2-values between groups are unchanged. In conclusion, our findings support a working hypothesis that lower GLU in the parietal region of HIV subjects results from 1) reduced reuptake of GLU by activated astrocytes in HIV-positive individuals; 2) increased usage of GLU for protein synthesis associated with cell injury repair and oxidative stress; and 3) neuronal injury due to excitotoxicity and mitochondrial toxicity from antiretroviral treatments (reduced de novo synthesis via the TCA cycle). The glutamatergic system appears to play an important role in the pathophysiology of HAND, and brain GLU as assessed with 1H MRS may provide an important early or presymptomatic surrogate marker for monitoring disease severity and possibly treatment effects. In vivo GLU levels also might provide a non-invasive means to monitor the effects of excitotoxicity and/or potential neuroprotective treatments (e.g. NMDA-receptor antagonists or antioxidant medications) for patients with HAND.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Acknowledgments
This work was partially supported by the National Institute on Drug Abuse (K24-DA16170 to L.C.; K02-DA16991 to T.E.), and Core resources from the National Center for Research Resources (G12-RR003061, P20-RR011091), National Institute on Neurological Disorders and Strokes (U54-NS56883), and the Office of National Drug Control Policy. We would like to thank the clinical and technical staff in the Neuroscience and MR Research Program for their assistance in data collection and analyses.

References
1. Antinori A, Arendt G, Becker JT, et al. Updated research nosology for HIV-associated neurocognitive disorders. Neurology. 2007; 69:17891799. [PubMed: 17914061]

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 8

2. Gray F, Adle-Biassette H, Chretien F, Lorin de la Grandmaison G, Force G, Keohane C. Neuropathology and neurodegeneration in human immunodeficiency virus infection. Pathogenesis of HIV-induced lesions of the brain, correlations with HIV-associated disorders and modifications according to treatments. Clin Neuropathol. 2001; 20:146155. [PubMed: 11495003] 3. Kaul M, Garden G, Lipton S. Pathways to neuronal injury and apoptosis in HIV-associated dementia. Nature. 2001; 410:988994. [PubMed: 11309629] 4. Wang Z, Trillo-Pazos G, Kim S, et al. Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: potential role in neuropathogenesis. Journal of NeuroVirology. 2004; 10:2532. [PubMed: 14982736] 5. Erdmann N, Zhao J, Lopez AL, et al. Glutamate production by HIV-1 infected human macrophage is blocked by the inhibition of glutaminase. J Neurochem. 2007; 102:539549. [PubMed: 17596215] 6. Hurd R, Sailasuta N, Srinivasan R, Vigneron DB, Pelletier D, Nelson SJ. Measurement of brain glutamate using TE-averaged PRESS at 3T. Magnetic Resonance in Medicine. 2004; 51:435440. [PubMed: 15004781] 7. Chang L, Yakupov R, Nakama H, Stokes B, Ernst T. Antiretroviral treatment is associated with increased attentional load-dependent brain activation in HIV patients. J Neuroimmune Pharmacol. 2008; 3:95104. [PubMed: 18247124] 8. Pfeffer RI, Kurosaki TT, Harrah CH Jr, Chance JM, Filos S. Measurement of functional activities in older adults in the community. J Gerontol. 1982; 37:323329. [PubMed: 7069156] 9. Karnofsky, DA.; Burchenal, JH. The clinical evaluation of chemotherapeutic agents in cancer. MacLeod, C., editor. New York: Columbia University Press; 1949. p. 199-205. 10. Provencher S. Automatic quantitation of localized in vivo 1H spectra with LCModel. NMR Biomed. 2001 Jun; 14(4):260264. [PubMed: 11410943] 11. Ernst T, Kreis R, Ross BD. Absolute quantitation of water and metabolites in the human brain. I: compartments and water. Journal of Magnetic Resonance. 1993; B102:18. 12. Zhang Y, Brady M, Smith S. Segmentation of brain MR images through a hidden Markov random field model and the expectation-maximization algorithm. IEEE Trans Med Imaging. 2001; 20:45 57. [PubMed: 11293691] 13. Woolrich MW, Jbabdi S, Patenaude B, et al. Bayesian analysis of neuroimaging data in FSL. Neuroimage. 2009; 45:S173186. [PubMed: 19059349] 14. Chang L, Jiang CS, Ernst T. Effects of age and sex on brain glutamate and other metabolites. Magn Reson Imaging. 2009; 27:142145. [PubMed: 18687554] 15. Hetherington H, Mason G, Pan J, et al. Evaluation of cerebral gray and white matter metabolites differences by spectroscopic imaging at 4.1T. Magnetic Resonance in Medicine. 1994; 32:565 571. [PubMed: 7808257] 16. Schuff N, Ezekiel F, Gamst AC, et al. Region and tissue differences of metabolites in normally aged brain using multislice 1H magnetic resonance spectroscopic imaging. Magnetic Resonance in Medicine. 2001; 45:899907. [PubMed: 11323817] 17. Dagan T, Sable C, Bray J, Gerschenson M. Mitochondrial dysfunction and antiretroviral nucleoside analog toxicities: what is the evidence? Mitochondrion. 2002; 1:397412. [PubMed: 16120293] 18. Kohler JJ, Lewis W. A brief overview of mechanisms of mitochondrial toxicity from NRTIs. Environ Mol Mutagen. 2007; 48:166172. [PubMed: 16758472] 19. Sailasuta N, Shriner K, Ross B. Evidence of reduced glutamate in the frontal lobe of HIVseropositive patients. NMR Biomed. 2009; 22:326331. [PubMed: 18988228] 20. Morris P, Bachelard H. Reflections on the application of 13C-MRS to research on brain metabolism. NMR Biomed. 2003; 16:303312. [PubMed: 14679497] 21. Shen J, Petersen KF, Behar KL, et al. Determination of the rate of the glutamate/glutamine cycle in the human brain by in vivo 13C NMR. Proc Natl Acad Sci U S A. 1999; 96:82358240. [PubMed: 10393978] 22. Bell J. The neuropathology of adult HIV infection. Revue neurologique. 1998; 154:816829. [PubMed: 9932303]

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 9

23. Zou JY, Crews FT. TNF alpha potentiates glutamate neurotoxicity by inhibiting glutamate uptake in organotypic brain slice cultures: neuroprotection by NF kappa B inhibition. Brain Res. 2005; 1034:1124. [PubMed: 15713255] 24. Muscoli C, Visalli V, Colica C, et al. The effect of inflammatory stimuli on NMDA-related activation of glutamine synthase in human cultured astroglial cells. Neurosci Lett. 2005; 373:184 188. [PubMed: 15619540] 25. Ferrarese C, Aliprandi A, Tremolizzo L, et al. Increased glutamate in CSF and plasma of patients with HIV dementia. Neurology. 2001; 57:671675. [PubMed: 11524477] 26. Turchan J, Sacktor N, Wojna V, Conant K, Nath A. Neuroprotective therapy for HIV dementia. Current HIV Research. 2003; 1:373383. [PubMed: 15049424] 27. Pohl P, Vogl G, Fill H, Rossler H, Zangerle R, Gerstenbrand F. Single photon emission computed tomography in AIDS dementia complex. J Nucl Med. 1988; 29:13821386. [PubMed: 3261331] 28. Masdeu JC, Yudd A, Van Heertun RL, et al. Single photon emission computed tomography in human immunodeficiency virus encephalopathy: a preliminary report. Journal of Nuclear Medicine. 1991; 32:14711475. [PubMed: 1869964] 29. Holman BL, Garada B, Johnson KA, et al. A comparison of brain perfusion SPECT in cocaine abuse and AIDS dementia complex. Journal of Nuclear Medicine. 1992; 33:13121315. [PubMed: 1613571] 30. Harris GJ, Pearlson GD, McArthur JC, Zeger S, LaFrance ND. Altered cortical blood flow in HIVseropositive individuals with and without dementia: a single photon emission computed tomography study. AIDS. 1994; 8:495499. [PubMed: 8011253] 31. Schwartz RB, Komaroff AL, Garada BM, et al. SPECT imaging of the brain: comparison of findings in patients with chronic fatigue syndrome, AIDS dementia complex, and major unipolar depression. American Journal of Roentgenology. 1994; 162:943951. [PubMed: 8141022] 32. Rosci MA, Pignorini F, Bernabei A, et al. Methods for detecting early signs of AIDS dementia complex in asymptomatic subjects: a quantitative tomography study of 18 cases. AIDS. 1996; 6:13091316. [PubMed: 1361744] 33. Rottenberg DA, Moeller JR, Strother SC, et al. The metabolic pathology of the AIDS dementia complex. Annals of Neurology. 1987; 22:700706. [PubMed: 3501695] 34. Rottenberg DA, Sidtis JJ, Strother SC, et al. Abnormal cerebral glucose metabolism in HIV-1 seropositive subjects with and without dementia. Journal of Nuclear Medicine. 1996; 37:1133 1141. [PubMed: 8965184] 35. Chang L, Ernst T, Leonido-Yee M, Speck O. Perfusion MRI Detects rCBF Abnormalities in Early Stages of HIV-Cognitive Motor Complex. Neurology. 2000; 54:389396. [PubMed: 10668700] 36. Chang L, Ernst T, Leonido-Yee M, Walot I, Singer E. Cerebral metabolite abnormalities correlate with clinical severity of HIV-cognitive motor complex. Neurology. 1999; 52:100108. [PubMed: 9921855] 37. Chang L, Tomasi D, Yakupov R, et al. Adaptation of the attention network in human immunodeficiency virus brain injury. Annals of Neurology. 2004; 56:259272. [PubMed: 15293278] 38. Stephensen CB, Marquis GS, Douglas SD, Kruzich LA, Wilson CM. Glutathione, glutathione peroxidase, and selenium status in HIV-positive and HIV-negative adolescents and young adults. Am J Clin Nutr. 2007; 85:173181. [PubMed: 17209194] 39. Jarvik JG, Lenkinski RE, Grossman RI, Gomori JM, Schnall MD, Frank I. Proton MR spectroscopy of HIV-infected patients: Characterization of abnormalities with imaging and clinical correlation. Radiology. 1993; 186:739744. [PubMed: 8430182] 40. Tracey I, Carr CA, Guimaraes AR, Worth JL, Navia BA, Gonzalez RG. Brain choline-containing compounds are elevated in HIV-positive patients before the onset of AIDS dementia complex: A proton magnetic resonance spectroscopic study. Neurology. 1996; 46:783788. [PubMed: 8618683] 41. English C, Kaufman M, Worth J, et al. Elevated frontal lobe cytosolic choline levels in minimal or mild AIDS dementia complex patients: A proton magnetic resonance spectroscopy study. Biological Psychiatry. 1997; 41:500502. [PubMed: 9034546]

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 10

42. Marcus C, Taylor-Robinson S, Sargentoni J, et al. 1H MR spectroscopy of the brain in HIV-1 seropositive subjects evidence for diffuse metabolic abnormalities. Metabolic Brain Disorders. 1998; 13:123136. 43. Suwanwelaa N, Phanuphak P, Phanthumchinda K, et al. Magnetic resonance spectroscopy of the brain in neurologically asymptomatic HIV-infected patients. Magnetic Resonance Imaging. 2000; 18:859865. [PubMed: 11027880] 44. von Giesen H, Wittsack H, Wenserski F, Koller H, Arendt G. Basal ganglia metabolite abnormalities in minor motor disorder associated with human immunodeficiency virus type 1. Archives of Neurology. 2001; 58:12811286. [PubMed: 11493169] 45. Chang L, Ernst T, Witt M, et al. Relationships Among Cerebral Metabolites, Cognitive Function and Viral Loads in Antiretroviral-Nave HIV Patients. NeuroImage. 2002; 17:16381648. [PubMed: 12414302]

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 11

NIH-PA Author Manuscript

Figure 1.

NIH-PA Author Manuscript NIH-PA Author Manuscript

Individual spectra at different echo times, or TEs, using a solution of major brain metabolites (A). In vivo spectra from the parietal gray matter voxel (B) are shown as acquired with regular PRESS at a single TE of 30ms (C), versus a TE-averaged PRESS acquisition (D). Abbreviations: NAA = N-acetyl aspartate compounds, GLU = glutamate, GLX = glutamate plus glutamine, tCr = total creatine, Cho = choline-containing compounds, and MI = myo-inositol.

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 12

NIH-PA Author Manuscript

Figure 2.

Locations of voxels used for TE-averaged PRESS acquisitions (left panel): frontal gray matter, parietal gray matter, frontal white matter, and basal ganglia. The graph (right) shows glutamate concentrations in these four brain regions for control and HIV-positive subjects with normal cognition (HIV-NC) and cognitive deficits (HIV-CD), as measured with TEaveraged PRESS. Abbreviations: [GLU] = glutamate concentration, WM = white matter, GM = gray matter.

NIH-PA Author Manuscript NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 13

NIH-PA Author Manuscript

Figure 3.

Dependence of the GLU concentration in the parietal gray matter on subject age, separately for the two groups. GLU levels in the HIV subjects are equivalent to those in SN subjects who are approximately 10-years older. P-values are not corrected for multiple comparisons.

NIH-PA Author Manuscript NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Ernst et al.

Page 14

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

Figure 4.

Dependence of subject performance on the Trails A and AVLT (after interference) task on the parietal gray matter GLU concentration (top left and right), as well as dependence of the CalCAP reaction time for word reversal and WAIS backward on the frontal gray matter GLU concentration. In both groups and on both tests, lower GLU levels were associated with poorer subject performance. The r- and p-values reflect linear regressions across both groups; there were no significant interactions between GLU levels and HIV status. Regressions in HIV subjects are represented by solid lines and those in control subjects by dashed lines. P-values are not corrected for multiple comparisons.

Table 1

Clinical Characteristics and Neuropsychological Performance of Research Participants (mean standard error)
SN (n=46) 43.33 1.85 37 (80%) 14.54 0.35 388.65 35.29 2.14 0.19 91.67 2.05 14.70 0.30 153.26 16.52 1.48 0.15 0.05 0.13 0.26 0.10 0.08 0.12 0.31 0.15 0.03 0.12 0.04 0.09 0.16 0.10 0.23 0.12 0.15 0.07 0.82 0.12 <0.0001 0.69 0.21 0.0001 0.92 0.22 <0.0001 0.89 0.18 <0.0001 1.09 0.23 <0.0001 <0.0001 0.03 0.12 0.01 0.10 0.21 0.12 0.22 0.12 0.10 0.08 1.00 0.16 0.30 0.13 0.48 0.17 0.0003 ns 0.05 ns ns ns ns ns 0.02 0.18 0.03 ns 0.64 0.28 2.00 0.16 0.01 0.0003 <0.0001 <0.0001 <0.0001 <0.0001 0.0003 <0.0001 124.72 19.22 13.00 0.52 87.78 2.07 2.37 0.30 439.78 73.11 14.74 0.50 14.06 0.51 ns ns ns ns ns ns ns 0.004 ns 0.03 0.05 0.0005 <0.0001 <0.0001 <0.0001 <0.0001 0.0002 <0.0001 25 (96%) 16 (89%) ns 0.08 ns ns 46.79 1.86 44.95 2.53 ns ns ns ns HIV-NC (n=27) HIV-CD (n=18) ANOVA or Fishers exact p-value SN vs. HIV-NC SN vs. HIV-CD HIV-NC vs. HIV-CD

Age (years)

Male Sex

Education (years)

CD4

(#/mm3)

Log Viral Load (copies/mL)

Karnofsky Score (0100)

HIV Dementia Scale (016)

Duration HIV infection (months)

# of NRTIs

Motor Skills

Verbal/Language

Executive Function

Attention/Working Memory

Speed Information Processing

Learning/Perceptual

Memory

Global

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

NRTI = nucleoside reverse transcriptase inhibitors

NIH-PA Author Manuscript

Ernst et al. Page 15

NIH-PA Author Manuscript

NIH-PA Author Manuscript

Table 2

Metabolite concentrations in 4 brain regions (mean S.E.)*

HIV-NC (n=27) HIV-CD (n=18) ANCOVA p-value SN vs. HIV-NC SN vs. HIV-CD HIV-NC vs. HIV-CD

Ernst et al.

SN (n=46)

Basal ganglia 4.56 0.15 4.21 0.25 3.74 0.27 1.70 0.07 7.55 0.16 59.3% 2.1% 55.8% 2.2% ns 0.06 ns ns 7.34 0.20 ns ns ns ns 1.45 0.08 0.01 (0.03) ns 0.01 (0.02) 0.02 (0.02) 3.66 0.26 ns 0.08 (0.13) ns ns 3.93 0.35 ns 0.04 (0.07) ns ns 4.23 0.17 ns ns 0.07 (0.20) ns

Cr

4.57 0.10

Glu

3.83 0.14

MI

3.30 0.11

Cho

1.68 0.05

NAA

7.49 0.12

%GM

54.9% 1.3%

Frontal gray matter 5.61 0.13 7.25 0.19 5.15 0.17 2.20 0.05 7.93 0.17 78.5% 1.2% 78.1% 1.6% ns ns ns 7.35 0.20 0.09 (0.08) ns 0.08 (0.07) 2.11 0.11 ns ns ns 4.94 0.28 ns ns ns ns ns 0.04 (0.04) ns 6.85 0.25 ns ns 0.08 (0.16) ns 5.61 0.17 ns ns ns ns

Cr

5.78 0.09

Glu

7.37 0.16

MI

5.00 0.15

Cho

2.20 0.04

NAA

7.80 0.13

%GM

80.1% 0.9%

Frontal white matter 4.71 0.13 5.24 0.19 4.18 0.16 2.36 0.07 7.75 0.19 83.6% 1.0% 81.0% 1.7% ns 7.99 0.16 ns 2.33 0.14 ns ns ns ns 4.39 0.27 0.08 (0.14) ns 5.55 0.24 ns ns 4.85 0.15 ns ns ns ns 0.04 (0.09) ns ns 0.06 ns ns ns ns ns ns

Cr

4.81 0.09

Glu

5.21 0.14

MI

3.85 0.12

Cho

2.31 0.05

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.

(n=17) 6.27 0.22 6.86 0.26 5.66 0.33 1.71 0.11 5.24 0.33 1.46 0.11 6.37 0.36 5.63 0.36 (n=10) 0.10 (0.15) 0.07 (0.13) ns 0.09 (0.06) ns ns 0.16 (0.09) ns 0.08 (0.14) 0.02 (0.04) ns 0.12 (0.01) 0.08 (0.11) ns ns ns

NAA

7.75 0.15

%WM

84.0% 0.7%

Parietal gray matter

(n=41)

Cr

6.06 0.10

Glu

7.09 0.19

MI

5.21 0.15

Page 16

Cho

1.58 0.03

NIH-PA Author Manuscript

NIH-PA Author Manuscript

NIH-PA Author Manuscript

SN (n=46) 8.04 0.12 67.5% 0.6% 64.1% 1.4% 62.7% 1.4% 0.002 0.01 0.001 ns 8.47 0.26 7.71 0.53 ns 0.11 (0.10) ns 0.08 (0.07)

HIV-NC (n=27)

HIV-CD (n=18)

ANCOVA p-value

SN vs. HIV-NC

SN vs. HIV-CD

NAA

Ernst et al.

%GM

All means are adjusted for TIM upgrade

**

All p-values are two-sided from ANCOVA with age as a covariate and are not corrected for multiple comparisons. P-values in parentheses are corrected for gray/white matter segmentation.

NIH-PA Author Manuscript

HIV-NC vs. HIV-CD

NIH-PA Author Manuscript

Page 17

NIH-PA Author Manuscript

J Magn Reson Imaging. Author manuscript; available in PMC 2011 November 1.