A multicenter in vivo proton-MRS study of HIV-associated dementia and its relationship to age

www.elsevier.com/locate/ynimg NeuroImage 23 (2004) 1336 – 1347

A multicenter in vivo proton-MRS study of HIV-associated dementia and its relationship to age L. Chang,a P.L. Lee,b C.T. Yiannoutsos,c T. Ernst,a C.M. Marra,d T. Richards,e D. Kolson,g G. Schifitto,h J.G. Jarvik,e,f E.N. Miller,i R. Lenkinski,j G. Gonzalez,b and B.A. Naviak,* HIV MRS Consortium a

Department of Medicine, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI 96813, USA Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA c Department of Medicine/Biostatistics, School of Medicine, Indiana University, Indianapolis, IN 46202, USA d Department of Neurology, University of Washington, School of Medicine, Seattle, WA 98101, USA e Department of Radiology, University of Washington, School of Medicine, Seattle, WA 98101, USA f Department of Neurosurgery (JGJ), University of Washington, School of Medicine, Seattle, WA 98101, USA g Department of Neurology, University of Pennsylvania, Pennsylvania, PA 19104, USA h Department of Neurology, School of Medicine, University of Rochester, Rochester, NY 14642, USA i Department of Psychiatry, Center for Health Sciences, UCLA, David Geffen School of Medicine, Los Angeles, CA 90095, USA j Department of Radiology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA k Departments of Neurology and Psychiatry, Tufts-New England Medical Center, Boston, MA 02111, USA b

Received 27 February 2004; revised 17 July 2004; accepted 29 July 2004

Objective: Differences in diagnostic criteria and methods have led to mixed results regarding the metabolite pattern of HIV-associated brain injury in relation to neurocognitive impairment. Therefore, a multicenter MRS consortium was formed to evaluate the neurometabolites in HIV patients with or without cognitive impairment. Methods: Proton magnetic resonance spectroscopy (MRS) at shortecho time (30 ms) was assessed in the frontal white matter, basal ganglia, and parietal cortex of 100 HIV patients [61 with AIDS dementia complex (ADC) and 39 neuroasymptomatic (NAS)] and 37 seronegative (SN) controls. Results: Compared to SN, NAS had higher glial marker myoinositol-to-creatine ratio (MI/Cr) in the white matter (multivariate analyses, adjusted P = 0.001), while ADC showed further increased MI/Cr in the white matter and basal ganglia (both P b 0.001), and increased choline compounds (Cho)/Cr in white matter (P = 0.04) and basal ganglia (P b 0.001). Compared to NAS, ADC showed a reduction in the neuronal marker N-acetyl compound (NA)/Cr in the frontal white matter (P = 0.007). CSF, but not plasma, viral load correlated with MI/Cr and Cho/Cr in white matter and NAA/Cr in parietal cortex. HIV infection and aging had additive effects on Cho/Cr and MI/Cr in the basal ganglia and white matter. Conclusions: The results suggest that glial activation occurs during the NAS stages of HIV infection, whereas further inflammatory activity in the basal ganglia and neuronal injury in the white matter

* Corresponding author. Departments of Neurology and Psychiatry, Tufts-New England Medical Center, Boston, MA 02111. E-mail address: [email protected] (B.A. Navia). Available online on ScienceDirect (www.sciencedirect.com.) 1053-8119/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuroimage.2004.07.067

is associated with the development of cognitive impairment. Aging may further exacerbate brain metabolites associated with inflammation in HIV patient and thereby increase the risk for cognitive impairment. D 2004 Elsevier Inc. All rights reserved. Keywords: HIV; Spectroscopy; Inflammation; Dementia; Neuroasymptomatic

Introduction HIV causes a spectrum of neurological abnormalities ranging from subclinical invasion of the brain to cognitive impairment and frank dementia (Glass et al., 1993; Navia et al., 1986a,b). Pathological abnormalities are commonly found in the subcortical regions of the brain, specifically the deep white matter and basal ganglia, including gliosis and inflammatory infiltrates (Glass et al., 1993, 1995; Navia et al., 1986a,b). Additionally, the cortex shows significant neuronal loss as well as damage to the synaptic dendritic tree (Everall et al., 1993; Masliah et al., 1992; Wiley et al., 1991). The extent of pathology in the white matter and basal ganglia generally correlates with the onset and severity of cognitive impairment (Navia et al., 1986a,b). However, the relative regional contributions of inflammation and neuronal injury to the clinical syndrome remain unclear. Furthermore, as the introduction of highly active antiretroviral therapies (HAART) has dramatically

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improved survival rates among HIV-infected persons, the impact of such treatment on brain and cognitive function will require further study (Palella et al., 1998). The current model for HIV-related neuronal damage postulates that neurotoxic substances, such as viral envelope proteins, cytokines, and chemokines, which are released by immuneactivated and HIV-infected macrophages and microglia contribute to the pathogenesis of HIV-associated brain injury (Kaul et al., 2001). In fact, activated rather than infected macrophages have been shown to be a better marker of ADC (Glass et al., 1995). Therefore, it remains unclear to what extent viral load within the brain compartment contributes to brain injury and whether it reflects a separate pool from the systemic compartment. Further studies are therefore needed to define the course of CNS injury in HIV-infected persons in relationship to their cognitive and virological status and to identify therapies that may protect against the damaging effects of the virus on cerebral function. Proton magnetic resonance spectroscopy (1H-MRS) provides a reliable, noninvasive method to study the HIV-infected brain (Barker et al., 1995; Chang, 1995; Chang et al., 1999a,b; Chong et al., 1993; Jarvik et al., 1993; Laubenberger et al., 1996; McConnell et al., 1994; Meyerhoff et al., 1993; Tracey et al., 1996). Reduced levels of the N-acetyl methyl group (NA), which contains primarily the neuronal marker N-acetylaspartate (NAA) and N-acetyl-aspartylglutamate (NAAG), or the more commonly used ratio of NA to total creatine (NA/Cr), are seen in patients with moderate to severe AIDS dementia complex (ADC) (Barker et al., 1995; Chang et al., 1999a,b; Chong et al., 1994; Laubenberger et al., 1996; Lopez-Villegas et al., 1997; Meyerhoff et al., 1994; Moller et al., 1999; Paley et al., 1995, 1996; Tracey et al., 1996). NA and NA/Cr of neuroasymptomatic (NAS) patients and those with mild ADC are usually within normal limits (Chang et al., 1999a,b; Jarvik et al., 1996; Lopez-Villegas et al., 1997; Meyerhoff et al., 1999; Moller et al., 1999; Tracey et al., 1996), although decreased NA/Cr in cognitively asymptomatic HIV patients has been reported (Suwanwelaa et al., 2000; Wilkinson et al., 1997a,b). More recent studies have shown that decreased NA/Cr may be reversible in response to treatment with AZT (Pavlakis et al., 1998; Salvan et al., 1997; Wilkinson et al., 1997a,b) or highly active antiretroviral therapy (HAART) (Chang et al., 1999a,b; Stankoff et al., 2001). Significant elevations in the cell membrane markers of choline compounds (Cho) and Cho/Cr, and in the glial markers of myoinositol (MI) (Brand et al., 1993) and MI/Cr, have also been reported in HIV patients (Chang et al., 1999a,b; Laubenberger et al., 1996; Lopez-Villegas et al., 1997; Meyerhoff et al., 1999; Salvan et al., 1997; Stankoff et al., 2001; Tracey et al., 1996; von Giesen et al., 2001). The majority of these studies, however, were performed at a single institution and, with few exceptions, the metabolite data were measured in a single brain region, often the parietal-occipital area (Chong et al., 1993; Jarvik et al., 1996; Paley et al., 1996; Salvan et al., 1997; Tracey et al., 1996; Wilkinson et al., 1997a,b). Furthermore, most studies were done at long echo times (e.g., 135–270 ms) (Barker et al., 1995; Chong et al., 1993; Meyerhoff et al., 1994; Moller et al., 1999; Paley et al., 1996; Salvan et al., 1997; Wilkinson et al., 1997a,b), thus preempting measurement of the MI peak, a potentially important marker of HIV-associated cognitive impairment (Chang et al., 1999a,b; Laubenberger et al., 1996; Lopez-Villegas et al., 1997; von Giesen et al., 2001). Nevertheless, the collective findings

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demonstrate that 1H-MRS may be useful for monitoring the progression of neurocognitive impairment and response to treatment. However, differences in data acquisition and processing, as well as in criteria for neurological classification of patients and treatment regimens, make it difficult to build a cohesive description of how brain metabolite changes relate to cognitive performance over the course of HIV infection. Potentially confounding these relationships is the introduction of potent antiretroviral therapies, which have significantly prolonged survival rates among HIV-infected subjects (Palella et al., 1998). Because normal aging is also associated with elevated Cho/Cr and MI/Cr, the effects of aging in HIV patients might further exacerbate changes in these brain metabolites. To address these issues, an MRS consortium was formed with the goal of characterizing the regional metabolite patterns of the HIV-infected brain in relation to neurocognitive function and viral burden using a uniform protocol for patient enrollment, imaging, and spectroscopy. Metabolite data were obtained at a short echo time (35 ms) from three brain regions: the frontal white matter, basal ganglia, and the parietal cortex in ADC, HIV-positive NAS, and HIV-seronegative (SN) controls. Distinct metabolite patterns emerged among the three groups, and aging was found to compound the effects of HIV infection. These results represent the largest multicenter 1H-MRS study of HIV brain injury undertaken in the United States.

Methods Participating subjects Subjects with ADC were recruited as part of the AIDS Clinical Trials Group (ACTG) protocol 301, a phase II study of memantine for the treatment of ADC and peripheral neuropathy. ADC subjects participating in the MRS study were enrolled into ACTG 700, a substudy of ACTG 301, along with HIV seropositive NAS and SN controls, who were recruited in parallel from the local communities during the study. All subjects were enrolled between December 1997 and December 1999; only the baseline assessments, before memantine, are reported here. HIV-positive subjects fulfilled the following inclusion criteria: (1) documented HIV positivity; (2) ADC stages 1, 2, or 3 (ADC group) or ADC stage 0 (NAS group). ADC staging was diagnosed based on the scale established by Price and Brew (1988) and by decreased performance on a neuropsychological battery of eight tests previously shown to be sensitive to the effects of HIV infection on cognitive function (Miller et al., 1990); (3) stable antiretroviral therapy for at least eight consecutive weeks before study entry; (4) estimated premorbid intelligence quotients of z70, or the ability to read at the 6th grade level; (5) laboratory test results within the defined acceptable ranges specified in ACTG 301; (6) age z18 years; (7) Karnofsky score (Karnofsky and Burchenal, 1949) z40; (8) ability to provide written consent. SN subjects had to fulfill inclusion criteria 4–8 above. All subjects were excluded if they had (1) neoplasms that required systemic chemotherapy; (2) confounding psychiatric illnesses (e.g., schizophrenia, bipolar disorder, or current active depression requiring pharmacotherapy); (3) active symptomatic AIDS-defining opportunistic infection; (4) confounding neurological disorders (e.g., multiple sclerosis, chronic seizure disorders, or significant head injuries); (5) central nervous system

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infection or neoplasms; (6) use of investigational agent within past 30 days; (7) pregnant females; (8) active alcohol or drug abuse that might confound the evaluations. A group of 161 subjects, 121 HIV-positive subjects, and 40 SN controls, fulfilled the study criteria and were recruited for the study. Twenty-four subjects (15 ADC, 6 NAS, and 3 SN) were excluded from the current analyses because their data did not fulfill the quality assurance criteria (see MRS methods below), were incomplete, or were lost during transfer. The final study cohort comprised 37 SN controls and 100 HIV-positive subjects, 61 were diagnosed with ADC (stage 1: N = 50, stage 2: N = 10, and stage 3: N = 1) and 39 were NAS controls. All subjects underwent neurological and neuropsychological assessments at entry, and had the MRI/MRS within 3 weeks of these clinical assessments. HIVpositive study participants (ADC and NAS) additionally had CD4 count and plasma and CSF viral load measurements, using the Roche ultrasensitive assay (detection limit 50 copies/ml). All subjects signed consent forms approved by the institutional review board at each institution as well as by the ACTG. Proton magnetic resonance spectroscopy (1H-MRS) Data were obtained at 10 imaging centers: Massachusetts General Hospital (MGH; the central site), Boston Medical Center, Harbor-UCLA Medical Center, University of Washington, University of Pennsylvania, University of Rochester, Mt. Sinai Medical Center, University of Texas, Galveston, San Francisco General Hospital, and University of Nebraska Medical Center. All MRS/MRI scans were performed on Signa 1.5-T MR scanners (GE Medical Systems, Milwaukee, WI) with operating systems 5.6 to 5.8, and using the standard GE quadrature head coil. The MRI and MRS study protocol, detailing all parameter settings under operator control, was prepared and distributed by the central site to ensure that data were acquired uniformly at all imaging centers. The MRS/MRI examination included high-resolution T1weighted sagittal images, and high-resolution proton-density and T2-weighted axial double spin-echo images (TR/TE1/TE2 = 2500/30/80 ms). MRS data were obtained with the GE pulse sequence PROBE-P, which is a point resolved spectroscopy (PRESS) sequence with water suppression. The gradient order of the pulse sequence was optimized to suppress susceptibility-induced

artifacts (Ernst and Chang, 1996). PROBE-P includes automated shimming and water suppression, thus minimizing differences in operator experience. MRS parameters were TE = 35 ms, TR = 3000 ms, voxel size = 6 cm3 (20  20  15 mm3), 128 acquisitions, spectral width = 2500 Hz, and 2 K data points. Using the proton density images, spectra were localized in three regions (Fig. 1) that correspond to different tissue composition: midline posterior parietal cortex (predominantly gray matter), frontal white matter, and basal ganglia (primarily deep gray matter with some white matter). Images and raw spectroscopic data were transferred electronically to the MGH for processing. All raw data sets were analyzed in an identical manner using the commercial software package Sage IDL (GE Medical Systems) without knowledge of the subject’s disease status. Metabolite ratios NA/Cr, Cho/Cr, and MI/Cr were determined as described (Lee et al., 2003). Films showing voxel placement were also forwarded to the central site for verification of the correct anatomic locations. Structural MRIs were visually inspected and the presence of brain atrophy (1—yes, 2—no), the degree of atrophy (1—mild, 2—moderate or marked, 3—none), and the types of white matter lesions (1—diffuse, 2— patchy, 3—both, 4—none, 5—other) were also documented. The combination of uniform data acquisition and uniform data processing eliminated many of the systematic differences between sites. A multicenter validation study, using a standardized phantom and subject data accrued from the first third of the current study, was first performed to establish intersite reliability (Lee et al., 2003); the major metabolite ratios in control subjects generally had a standard deviation below 15% (Lee et al., 2003). Based on the MRS data from phantoms and normal volunteers, quality assurance (QA) thresholds were determined for magnetic field homogeneity, sensitivity, and suppression of the water signal (Lee et al., 2003). Within the ACTG 301/700 study, MRS data from 37 SN and 100 HIV patients (39 NAS and 61 ADC) met the QA criteria and were included in the analyses. Preliminary data from a subset of ADC subjects and HIV-negative controls has been reported previously (Lee et al., 2003). Statistical procedures An initial univariate analysis of demographic and clinical differences with respect to interval data such as age, CD4 count

Fig. 1. Axial proton density-weighted MRI showing the three voxel locations. Left image: voxels for centrum semiovale (or frontal white matter) and parietal gray matter (midline); right image: voxel for basal ganglia, including putamen and globus pallidus. Voxels in the frontal white matter and the basal ganglia alternated between the left and right side for each consecutive subject.

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and brain metabolite ratios was performed between subject groups (ADC, NAS controls, and SN controls, subsequently called stratification groups) using the nonparametric Kruskal–Wallis test. Cuzick’s trend test (Cuzick, 1985), an extension of the Kruskal– Wallis test, was used to determine whether the mean MRS metabolite ratios among the three stratification groups followed either a positive or negative linear trend depending on disease state (from SN, to NAS, to ADC). To account for the impact of age and other demographic and disease-related predictive factors on brain metabolite ratios, an additional multivariate analysis of covariance was performed on each of the three metabolite-to-creatine ratios in the three brain regions (Table 3). Age was dichotomized as below 40 years or above 40 years (the median age for the study). Because MRSmeasured metabolite levels did not follow a normal distribution, a Box–Cox transformation (Box and Cox, 1964) was performed before fitting each to a multivariate analysis of variance (ANOVA) model. To determine the factors, in addition to stratification (SN, NAS or ADC), that were significantly associated with each metabolite ratio, a backward elimination procedure was performed. Predictors that were significant at the 20% alpha level were allowed to remain in the model. This conservative alpha level was chosen to ensure that significant factors were not excluded from the model. In the final model, if the effect of stratification was statistically significant, pairwise post hoc comparisons between stratification group means were carried out, and were adjusted by the Tukey–Kramer method for multiple comparisons (Kramer, 1956). Trends in metabolite levels among these three groups were assessed via linear contrasts and tested by the F test. MRS levels between the two age groupings were tested via the Kruskal–Wallis test in the univariate case and the t test in the multivariate case; this comparison adjusted for the inclusion of the stratification group into the statistical model. The Spearman correlation was used to explore the correlation between metabolite ratios and CD4 count or viral load. Associations between categorical measures were assessed by the Fisher’s exact test. All statistical tests were carried out at the 5% alpha level. Multiple comparisons were adjusted for by the Bonferroni procedure (except for the exploratory Spearman correlations) to ensure that the overall significance level of the tests was at least 95%. A power calculation was performed to determine the sample size before the study. Based on our preliminary data, we expected differences of 8–17% for NA/Cr and 13–20% for Cho/Cr and MI/Cr between HIV patients and control subjects, similar to the interindividual standard deviations (Lee et al., 2003), i.e., effect sizes of approximately 1. To maintain an overall alpha level of 5% for 27 pairwise comparisons (three pairwise comparisons groups, involving three metabolites in three brain regions), each comparison was to be carried out at the 0.0019 level of significance (=0.05/27). We projected that a total of 160 subjects (40 in each control group and 80 in the ADC group) provided sufficient power (80% or higher) to address all these simultaneous comparisons. Due to the inherently conservative nature of the Bonferroni procedure, we also considered two additional multiple-comparison adjustments: The hierarchical approach suggested by Holm (1979) and the graphical procedure discussed by Turkheimer et al. (2000). However, due to a sharp difference between the P values of statistically significant and nonsignificant results, multiple-comparison adjustment methods produced identical results. For this reason, the simpler Bonferroni adjustment has been maintained throughout the subsequent presentation.

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Results Subject characteristics Subject demographic data are summarized in Table 1. The median age of ADC subjects (44 years) was significantly higher (P b 0.001) than that of SN controls (34 years) and NAS controls (37 years). Accordingly, the percentages of subjects younger than 40 years were significantly different among the three subject subgroups (SN: 68%; NAS: 69%; and ADC: 28%; P b 0.001). Compared to the subjects in the SN control group, there were more African-Americans (P b 0.034) and males (P b 0.001) in the HIV subject group. A small proportion (12%) of study participants reported previous experience with intravenous drugs, which was higher in the ADC cohort (18%) and the NAS controls (13%) compared to the SN controls (3%). Educational levels were slightly different among the three subgroups (P = 0.043), with ADC subjects having lower education. NAS controls had higher plasma viral load (P = 0.009) compared to ADC subjects, although CD4 counts and CSF viral load were comparable. Compared to the NAS group, ADC subjects were more likely to be treated with protease inhibitors and less likely to be ART-naRve, which might account for higher rates of undetectable plasma viral loads in the ADC group (39%) compared to the NAS group (7.7%). In contrast, undetectable CSF viral loads were no more common in the NAS group (46%) compared to the ADC group (64%). 1 H-MRS differences among subject groups: relationship to ADC (univariate analyses)

Mean values for the MRS regional metabolite ratios are presented in Table 2. Metabolite ratios for the NAS group were comparable to those of the SN controls, or fell between those of the SN and ADC groups. Compared to SN controls, the NAS group had higher MI/Cr ratio in the white matter (+8.2%), a small nonsignificant increase in Cho/Cr in the parietal cortex, and no difference in NA/Cr. Compared to the NAS group, ADC subjects had higher Cho/Cr (+12%) and MI/Cr (+17%) in the basal ganglia, as well as significantly lower NA/Cr ( 8%) in the white matter. Compared to the SN group, however, ADC subjects exhibited significantly higher Cho/Cr (+16% in basal ganglia, +12% in white matter) and MI/Cr (+27% in basal ganglia; +16% in white matter). Thus, the comparison of ADC with SN subjects identified further increases in the inflammatory markers in the basal ganglia and white matter but no change in NA/Cr. Trend analysis revealed that advancing cognitive impairment was associated with significantly increasing MI/Cr and Cho/Cr in the basal ganglia (P b 0.001 in both cases) and in the white matter (P b 0.001 and P = 0.003), and with decreasing NA/Cr in the white matter (P b 0.001) (Table 2, middle column Fig. 3). Effect of age on metabolite ratios: univariate analysis To assess the effect of age on brain metabolites, regional metabolite ratios were further examined in subjects below and above 40 years old (the overall median age). Independent of HIV stratification, subjects older than 40 years of age had higher Cho/Cr and MI/Cr in the basal ganglia (P b 0.001), higher Cho/Cr (P = 0.004) and lower NA/Cr in the white matter (P b 0.001) (Fig. 2).

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Table 1 Baseline demographics and clinical variables

Gender (% male) Age (years; min–max) Race/ethnicity White Black Hispanic Other IV drug use (% used) Education level High school Some college College Graduate study CD4 count (cells/ml) Log CSF viral loadd Undetectable (%) Log plasma viral loade Undetectable (%) Classes of Anti-HIV drugs Antiretroviral therapy (ART) naRve ART without protease inhibitor ART with protease inhibitor a b c d e

Total N = 137

HIV-negative controls (SN) N = 37

HIV-positive controls (NAS) N = 39

ADC subjects (ADC) N = 61

P value

106 (77%) 40 (19–63)

18 (49%) 34 (19–58)

34 (87%) 37 (19–51)

54 (89%) 44 (31–63)

0.001a 0.001a 0.003a

93 (68%) 21 (15%) 18 (13%) 5 (4%) 17 (12%)

25 (68%) 2 (5%) 6 (16%) 4 (11%) 1 (3%)

21 (54%) 9 (23%) 9 (23%) 0 (0%) 5 (13%)

47 (77%) 10 (16%) 3 (5%) 1 (2%) 11 (18%)

25 (18%) 52 (38%) 28 (20%) 32 (23%) N/A 1.70 (1.7–4.8) 57 (57%) 2.56 (1.7–4.8) 27 (27%)

2 (5.4%) 14 (37.8%) 9 (24.3%) 12 (32.4%) N/A N/A N/A N/A

8 (20.5%) 18 (46%) 8 (20.5%) 5 (13%) 316 F 224 1.70 (1.7–4.8) 18 (46%) 3.98 (1.7–4.8) 3 (7.7%)

15 (24.5%) 20 (33%) 11 (18%) 15 (24.5%) 343 F 212 1.70 (1.7–4.8) 39 (64%) 2.20 (1.7–4.8) 24 (39%)

N/A N/A N/A

6 (15%) 2 (5%) 30 (77%)

1 (2%) 1 (3%) 58 (95%)

0.072a,b 0.043c

0.152c 0.734c 0.811a 0.009c 0.054a 0.006a

Fisher’s exact test for R  C tables. Current and previous IV drug use combined versus no use. Kruskal–Wallis test. HIV RNA levels in the CSF were available on 86 subjects (NA-28, ADC-58). HIV RNA levels in the plasma were available in 77 subjects (NA-19, ADC-58).

observed in the univariate analysis were attenuated with age, metabolite ratios of the NAS group remained generally intermediate between the SN and ADC groups (Fig. 2). Specifically, Cho/Cr in the younger NAS group was similar to that in the older SN

To adjust for the effect of age on metabolite levels among the three subgroups, the same univariate comparisons were performed separately for subjects less than 40 years of age and subjects over 40 years of age. Although some of the differences between groups

Table 2 MRS ratios by subject group and P values (univariate and multivariate analyses) P value (univariate)a

Stratification group

P value (multivariate)b

NAS (N = 39) Mean (SD)

ADC (N = 61) Mean (SD)

SN vs. NAS

NAS vs. ADC

SN vs. ADC

Trend

SN vs. NAS

NAS vs. ADC

SN vs. ADC

Trendd

Basal ganglia NA/Cr 1.42 (0.15) Cho/Cr 0.73 (0.10) MI/Cr 0.48 (0.10)

1.39 (0.16) 0.76 (0.08) 0.52 (0.10)

1.42 (0.21) 0.85 (0.15) 0.61 (0.13)

0.266 0.094 0.033

0.832 0.002 0.001

0.401 b0.001 b0.001

0.418 b0.001 b0.001

0.733 0.281 0.129

0.648 0.071 0.090

0.986 b0.001 b0.001

0.874 b0.001 b0.001

Frontal white matter NA/Cr 1.82 (0.17) Cho/Cr 1.02 (0.15) MI/Cr 0.61 (0.07)

1.82 (0.15) 1.08 (0.14) 0.66 (0.05)

1.67 (0.20) 1.14 (0.19) 0.71 (0.13)

0.913 0.093 0.001

b0.001 0.163 0.056

0.004 b0.001 b0.001

b0.001 0.003 b0.001

0.774 0.265 0.001

0.007 0.699 0.344

0.055 0.044 b0.001

N/Ae 0.017 b0.001

Parietal cortex NA/Cr 1.55 (0.09) Cho/Cr 0.60 (0.06) MI/Cr 0.60 (0.05)

1.56 (0.10) 0.62 (0.06) 0.62 (0.06)

1.51 (0.12) 0.65 (0.08) 0.63 (0.07)

0.734 0.044 0.238

0.043 0.460 0.390

0.135 0.007 0.042

0.085 0.007 0.039

0.896 0.261 0.618

0.232 0.931 0.809

0.475 0.116 0.251

0.245 0.048 0.113

SN (N = 37) Mean (SD)

a

c

Kruskal–Wallis test. After Bonferroni corrections, only P values 0.002 or less (0.05/27) are considered significant. Pairwise t test comparison adjusted for age; multiple comparisons were adjusted by the Tukey–Kramer procedure (adjustment was made within each model but not among models). P V 0.05 is considered significant. c Cuzick’s nonparametric test for linear trend. After Bonferroni adjustment, only P values 0.005 or less (0.05/9) are considered significant. d Linear contrast applied to the MRS means; tested via an F test. P V 0.05 is considered significant. e Not applicable due to the presence of age  group interaction. b

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Fig. 2. MRS metabolite ratios according to the three subject groups and the two age categories.

group, but significantly higher compared to the younger SN group (P = 0.002). Similar results were observed for the MI/Cr ratio in the basal ganglia. By contrast, MI/Cr in the frontal white matter changed little with age in the SN group, was elevated in both the ADC and NAS groups, and increased further in the older ADC group (Fig. 2). Trend analysis further illustrated these changes, because inflammatory markers in the basal ganglia significantly increased with neurological disease progression in both age groups (Figs. 3 and 4). In the white matter, an increase was observed only in the MI/Cr in both age groups, whereas a trend toward a decrease in the NAA/Cr was found only in subjects b40 years. 1

H-MRS metabolites among subject groups (multivariate analyses)

Given the significant age differences between subgroups and the possible age-by-subject-group interaction, multivariate analyses were performed to isolate the effect of ADC on metabolite levels. As age, group status, and other factors such as race, gender, and intravenous drug exposure were significantly different between groups, a backward elimination procedure was performed to determine possible associations with each metabolite ratio as described in Methods (Table 3). Intravenous drug exposure reached statistical significance at the 5% level with respect to Cho/Cr levels in the white matter. Race/ethnicity was included in some models but never reached statistical significance at the 5% level, which suggests at best a minor effect after adjustment for the remaining predictive factors. Both age and dementia severity contributed significantly to increases in Cho/Cr and MI/Cr in the basal ganglia,

Cho/Cr in the frontal white matter, and decreases in NA/Cr in the frontal white matter (Table 3). In contrast, the increase in MI/Cr in the frontal white matter and the Cho/Cr in the parietal cortex were driven by factors related to the presence of ADC. The results of adjusted pairwise comparisons between subject groups were similar to those from the univariate analyses (Table 2; right columns). The only significant difference between the NAS and SN subjects was higher MI/Cr in the white matter of HIVpositive subjects (P = 0.001; Table 2, right column). Compared to SN, ADC subjects showed increased Cho/Cr and MI/Cr in the basal ganglia (P b 0.001) and the white matter (P = 0.044 and P b 0.001, respectively), as well as a trend for decreased NA/Cr in the frontal white matter (P = 0.055). ADC subjects also had lower white matter NA/Cr than the NAS controls (P = 0.007). The tests of trend (with disease severity) remained statistically significant with respect to Cho/Cr in all three regions and MI/Cr in the basal ganglia and white matter (Table 2, last column; Fig. 3). Trend analysis over subject groups with respect to NA/Cr in the white matter was not applicable, as there was a significant age-by-group interaction (see below). Interaction between HIV status and age on metabolite ratios As the effects of normal aging are similar to those of ADC (i.e., elevated Cho/Cr and MI/Cr) (Chang et al., 1996; Schuff et al., 1999), the possible interaction between HIV and age was evaluated as part of the multivariate analysis model (Fig. 2). A significant interaction between HIV and age was observed for NA/Cr in the white matter ( P = 0.045), in that the younger (b40

L. Chang et al. / NeuroImage 23 (2004) 1336–1347 Fig. 3. Bar graphs of adjusted mean metabolite ratios (FSE) measured in the basal ganglia (left graph), the white matter (middle graph), and the parietal cortex (right graph). Means were estimated in the multivariate analyses and have been adjusted for all statistically significant factors (see Table 3). Standard errors were calculated by the delta method. Statistically significant trends as well as their direction are shown along with adjusted P values produced by the F test. P values correspond to the multivariate trend test in Table 2. Black bars: HIV-negative controls; light gray bars: NAS controls; dark gray bars: ADC subjects.

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Table 3 Factors included in the optimal statistical modela Metabolite Transformation Factors includeda Comment level Basal ganglia NA/Cr y = 1/x 3

Cho/Cr

y = 1/x

Race/ethnicity IV drug experience Group Age

MI/Cr

y = 1/x

Group Age Group-by-age interaction

Frontal white matter NA/Cr y = x3

Group Age

Cho/Cr

None required

Group Age IV drug experience

MI/Cr

Y = 1/x

Group

ADC had higher Cho/Cr levels than SN subjects Younger subjects had lower Cho/Cr levels ADC had higher MI/Cr levels than SN subjects Younger subjects had lower MI/Cr levels NA/Cr was not reduced among NAS subjects

NAS had lower NA/Cr levels than SN subjects Younger subjects had higher NA/Cr levels ADC had higher Cho/Cr levels than SN subjects Young subjects had lower Cho/Cr levels Non-users had lower Cho/Cr than current/previous users ADC had higher MI/Cr levels than SN subjects

IV drug experience Parietal cortex NA/Cr None required Cho/Cr y = log(x)

MI/Cr

None required

Age Group IV drug experience Education IV drug experience

ADC had higher Cho/Cr levels than SN subjects Nonusers had lower Cho/Cr levels No evident trend

a

Subject subgroup membership (SN, NAS, ADC) is a factor always included in the model.

years), but not older, HIV subjects had lower NA/Cr than SN controls (Fig. 4). Significant age effects were present on Cho/Cr (basal ganglia: P b 0.001; frontal white matter: P = 0.004) and MI/Cr (basal ganglia: P b 0.001; frontal white matter: P = 0.015) that paralleled the HIV effects (as described above); however, no significant interaction between HIV and age were observed on these metabolite ratios. This implies that HIV infection and aging have an additive effect on these metabolite ratios. Similar results were obtained when age was used as a continuous variable in an analysis of covariance. Association between 1H MRS metabolites and clinical parameters The log10 CSF viral load correlated with MI/Cr in the white matter (r = 0.359, P = 0.002), while weaker correlations were found with white matter Cho/Cr (r = 0.3, P = 0.009) and parietal

L. Chang et al. / NeuroImage 23 (2004) 1336–1347

Fig. 4. MRS metabolite ratios (mean F SE) in the three brain regions in the younger (upper row) and older subjects (lower row).

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NA/Cr (r = 0.26, P = 0.029). However, no differences in MRS metabolite levels were observed between subjects with detectable and those with undetectable viral load in either plasma or CSF. Metabolite ratios also did not correlate with log10 HIV plasma viral load (range of detection: 50–750,000 copies/ml) or CD4 count, except for the basal ganglia NA/Cr, which showed a trend for correlation with CD4 (Spearman correlation coefficient 0.23, unadjusted P value 0.025).

Discussion HIV infection causes cellular changes in the brain that follow a predominantly subcortical distribution as well as significant cortical neuronal injury and loss. In this large, multicenter in vivo MRS study, statistically significant differences in MRS metabolite ratios were detected among ADC patients, neuroasymptomatic HIV subjects, and HIV-negative controls. These differences remained significant even after adjustment for multiple comparisons and age. One of the main findings of this study is that the metabolite ratios in the basal ganglia and frontal white matter show increasing abnormalities from the seronegative to the neuroasymptomatic and cognitive impairment stages (see Table 2 and Fig. 3). Notably, these differences and trends are observed although most subjects in the ADC group had only mild dementia. These findings clarified some of the controversial results from prior reports with smaller sample sizes (Barker et al., 1995; Chang et al., 1999a,b; Marcus et al., 1998; Meyerhoff et al., 1999; von Giesen et al., 2001) and suggest that the neurological impact of HIV infection results in a continuum of metabolite changes that can be evident even during its neuroasymptomatic stages. The pathological process begins with an inflammatory response localized in the white matter and basal ganglia, and progresses to neuronal injury and eventual cognitive impairment. The significance of the increase in the MI/Cr, a marker that may reflect glial or inflammatory activity, has remained controversial, since several prior single-site studies of HIV-infected patients who were either neuroasymptomatic or had minor-cognitive motor disorder have yielded conflicting results (Chang et al., 1999a,b; Lopez-Villegas et al., 1997; von Giesen et al., 2001). Our study confirms that the MI/Cr in white matter is significantly elevated in NAS compared to SN controls, even after adjustments for potentially confounding factors such as age. The specificity of this marker is further observed in the white matter of the ADC group as MI/Cr ratios continue to increase with disease severity compared to those in the SN and NAS groups. An increase in MI/Cr or MI with disease progression or severity also has been observed in other brain disorders, including Alzheimer’s disease (Ernst et al., 1997), the pre-Alzheimer stage of Down syndrome (Huang et al., 1999), and myotonic dystrophy (Chang et al., 1998). Therefore, our study clarifies that elevated MI/Cr in the white matter reflects early HIV brain disease before the onset of cognitive impairment. Whether NAS subjects with this particular metabolite abnormality are at higher risk for further neurological injury remains to be studied. In contrast to several prior single-site reports of reduced NA/Cr in the gray matter of ADC patients (Chang et al., 1999a,b; Meyerhoff et al., 1994; Paley et al., 1995; Salvan et al., 1997; Stankoff et al., 2001; Suwanwelaa et al., 2000), our HIV patients had relatively normal NA/Cr in the gray matter, which is surprising

given the reports of significant neuronal loss and inflammation in the cortex of patients with AIDS (Everall et al., 1993; Masliah et al., 1992; Wiley et al., 1991). This result may reflect the lesser degree of cognitive impairment in our subjects (81% with mild dementia or ADC stage 1) compared to those previously reported, the positive effects of combined antiretroviral therapies, or regional differences in metabolite ratio abnormalities, as other groups have reported reduced NA/Cr in the frontal lobe (Chang et al., 1999a,b; LopezVillegas et al., 1997; Marcus et al., 1998). Our study, however, found lower NA/Cr ratio in the white matter of the ADC group, as well as in the younger NAS subjects (less than 40 years of age), suggesting that the process may be more evident in younger neuroasymptomatic subjects (see below). Of interest, significant correlations have been observed between dendritic or synaptic damage and levels of gp41 in the white matter during early stages of cognitive impairment (Masliah et al., 1992). These cellular changes might be reflected in a decrease of the NA/Cr ratio in this region; similar findings also have been observed in macaques infected with simian immunodeficiency virus (Gonzalez et al., 2000). One of the primary goals of this multicenter study was to identify an MRS-derived metabolic signature of ADC distinct from that of neuroasymptomatic HIV-positive subjects and seronegative controls. ADC subjects showed the following patterns: (1) elevated Cho/Cr and MI/Cr in the basal ganglia and white matter relative to the NAS group; (2) further and significant increases in these metabolite ratios, reflecting inflammatory processes compared to the SN group; and (3) significantly decreased NA/Cr in the white matter compared to NAS subjects. In the only prior multicenter MRS study, Salvan et al. (1997) identified a choline pattern (elevated Cho/Cr) in NAS subjects and an NA pattern (decreased NA/Cr) or a pattern with both decreased NA/Cr and increased Cho/Cr in subjects with cognitive impairment. Metabolite ratios, however, were measured solely from the parietal region using a long echo time protocol, thus precluding the detection and measurement of MI and detailed regional assessment of the effects of HIV infection on brain metabolites. Together, the results from our study suggest that neurological progression from asymptomatic to symptomatic disease results from further inflammatory activity in the basal ganglia and white matter accompanied by neuronal injury in the white matter. Previous MRS studies (Angelie et al., 2001; Chang et al., 1996; Saunders et al., 1999; Schuff et al., 2001), as well as our results, show that Cho/Cr and MI/Cr increases and NA/Cr decreases with normal aging in some brain regions. Thus, when the differences in age were adjusted in the multivariate analyses, the highly significant HIV effect on the metabolite ratios in the basal ganglia and the white matter is attenuated to some degree. However, even after correcting for aging effects, notable changes in these regions persisted and distinguished one group from the other. The age-like effect of HIV-disease progression is evident in the multivariate analysis as a significant age effect along with a parallel significant group effect, as well as from the results in the NAS group younger than 40 years of age (Fig. 3). Specifically, the Cho/Cr and MI/Cr in the basal ganglia and Cho/Cr in the frontal white matter are elevated with HIV as well as aging, while NA/Cr in the frontal white matter is decreased. These findings agree with those from previous reports on MRS of normal aging (Angelie et al., 2001; Chang et al., 1996; Saunders et al., 1999; Schuff et al., 2001), and suggest that aging may compound the effects of HIV infection on brain function, potentially leading to greater cerebral injury and increasing the risk for cognitive impairment.

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Interactions between age and disease status were observed on NA/Cr in the frontal white matter, which was lower in HIVpositive subjects younger than 40 years of age, but not in those 40 years or older, compared to SN controls. Therefore, HIV appears to have a more profound effect on NA/Cr in the white matter of younger but not the older subjects. One possible explanation is that a more robust immune status in the younger patients compared to that of the older patients may lead to a greater inflammatory response, which in turn may cause greater neuronal injury. Furthermore, elevated Cr with normal aging in the older SN subjects but decreased Cr in older HIV subjects, as had been observed previously (Ernst and Chang, 2004), could also lead to such interaction effects. Combined potent antiretroviral therapies have significantly altered the natural history of HIV infection (Palella et al., 1998) and may have confounded some of the relationships between metabolite ratios, plasma viral load, and CD4 counts in this study. While there are previous reports of such correlations (Chang et al., 1999a,b; Lopez-Villegas et al., 1997; Tracey et al., 1996; Wilkinson et al., 1997a,b), other groups found no effect (Jarvik et al., 1996; Meyerhoff et al., 1996; Paley et al., 1996; Salvan et al., 1997; Suwanwelaa et al., 2000). A recent study of antiretroviralnaive HIV patients found that MI correlates positively with plasma viral load and negatively with CD4 (Chang et al., 2002). The lack of correlation in our study may simply reflect the timing of therapy, as antiretroviral treatments can rapidly suppress viral replication and lead to a dramatic rebound of the CD4 count (Connors et al., 1997). Alternatively, it may suggest that neurological damage may occur through indirect mechanisms mediated by host and viral products (Kaul et al., 2001) that may take place in the CNS even when systemic viral replication has been suppressed and stabilized. The current study indeed showed a significant correlation between CSF viral load and MI/Cr in the white matter, despite the use of combined antiretroviral therapies. Combined regimens have also been reported to improve cognitive impairment, although the majority of protease inhibitors display poor CNS penetration. In fact, emerging evidence suggests an increase in the incidence of HIV-associated cognitive impairment (Sacktor et al., 1999, 2000). The results from this study further support this observation and suggest that even in the setting of such therapy, CNS injury may persist and correlate more strongly with virologic burden in the CSF than in the peripheral compartment. This finding is consistent with previous studies supporting a similar relationship between CSF HIV RNA concentration and cognitive performance (Ellis et al., 1997; McArthur et al., 1997). It also suggests that at a certain point during HIV infection or in a subgroup of patients, immunological and virological events in the CNS compartment may become independent of those in the periphery and thereby contribute to cerebral injury in the setting of chronic disease and treatment. We have recently shown in a study of memantine for HIV-associated dementia that detectable HIV RNA concentration in the CSF at baseline is a significant predictor of worsening cognitive function among subjects with ADC (Navia and Yiannoutsos, unpublished observations). The metabolic findings from the present study confirm and extend those previously reported from other forms of brain imaging such as positron emission tomography and functional MR studies, which have shown a predilection for the subcortical regions with disease progression (Rottenberg et al., 1987, 1996; Tracey et al., 1998; von Giesen et al., 2000). In the basal ganglia,

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NA/Cr appeared to be unchanged with disease progression; however, this constant level of NA/Cr could reflect either lack of changes in both NA and Cr, or more likely, concomitant decreases in NA and Cr in the basal ganglia (Chang et al., 2002). Decreased Cr in this region suggest decreased energy metabolism, which is consistent with prior PET studies that found decreased glucose metabolism in the basal ganglia, especially in more advanced HIV dementia patients (Rottenberg et al., 1996). HIV patients with mild or early dementia, however, typically show increases in glucose metabolism (Rottenberg et al., 1987) and cerebral blood volume (Tracey et al., 1998) in the basal ganglia. These earlier changes in the basal ganglia are consistent with immunohistopathological data, which have shown that increased host inflammatory response and viral burden in the basal ganglia and frontal white matter are significant predictors of worsening cognitive impairment in HIV infected persons (Rostasy et al., 1999). We have shown that regional analysis of MRS-derived metabolites acquired from multiple sites can clarify and extend the results of smaller single site studies and provide further insight into the course of cerebral injury in the HIV-infected brain. The findings reported here provide a regional assessment of injury in the HIV-infected brain that is consistent with the known subcortical pathology of the disorder. It further illustrates that HIV and aging have similar effects with respect to certain metabolites (Cho, Cr, and MI) and suggests that cognitive impairment in the older HIV population may reflect, to some degree, the compounding effects of age and HIV infection. As HIV infection evolves into a chronic disease, neurocognitive impairment is likely to remain an important source of morbidity in HIV-infected subjects, including those who are stable on HAART. With the advent of newer antiretroviral therapies that have prolonged survival in a substantial number of subjects, the impact of such treatments and the effects of chronic infection on CNS function can now be evaluated noninvasively by 1 H-MRS. Future longitudinal studies will address the dynamic relationship of these metabolic alterations with the onset of neurological disease and its response to changes in viral burden and antiretroviral therapy.

Acknowledgments This work was supported by the AIDS Clinical Trials Group and grants from NINDS (R01 NS36524), NIAID (R01 AI38855), NIMH (R01 MH64409), NIDA (K24 DA16170; K02 DA16991), and NCRR (MO1 RR00425; RR13213). We especially thank Diane Rausch from NIMH for her guidance and generous support for this study. We also thank Linda Millar, Sharon Shriver, and Dodi Colquhoun for their dedicated work on the AACTG operations and data management. We appreciate and acknowledge the exceptional efforts from all personnel and members of the HIV MRS Consortium who were involved with subject recruitment, evaluation, and data acquisition.

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