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Cerebral metabolite changes prior to and after antiretroviral therapy in primary HIV infection.......
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"presumed natural history of HIV neuropathogenesis is progressive inflammation in the CNS leading to clinical neurologic dysfunction......ART-naive PHI cohort revealed increases in metabolite levels suggestive of progressive inflammation.......ART initiation during the first year of infection attenuated the increase of these inflammatory cerebral markers, but it did not appear to reverse them within the follow-up period.........early diagnosis and adoption of ART is associated with low prevalence of neurocognitive impairment, providing further support for early intervention"
"Our observations consistently show longitudinal increases of the inflammatory brain metabolite markers Cho/Cr and MI/Cr that suggest worsening inflammation
during early untreated HIV. ART initiation during the first year of infection attenuated the increase of these inflammatory cerebral markers, but it did not appear to reverse them within the follow-up period. It is unknown whether after longer-term treatment and follow-up, ART initiated during early infection might reduce or eliminate inflammation, which has been observed in the setting of chronic, treated HIV infection. However, early diagnosis and adoption of ART is associated with low prevalence of neurocognitive impairment, providing further support for early intervention.39 Our MRS-based observations suggest that early ART initiation may be advisable to prevent neurologic dysfunction by slowing and possibly halting HIV-associated neuropathology."
"......Although ART appears to limit Glu excess, mitochondrial toxicity may play a role and must be considered in early adoption of ART....."
"Fifty-three participants with PHI were followed longitudinally with a total of 154 scans over a median 6.0 months (IQR 0-26.4). ART was initiated independently
from the study by 23 of the 53 PHI participants at a median of 6.4 months (IQR 3.2-16.8) post HIV transmission......
.......The effect of ART in PHI was assessed with longitudinal mixed linear models, revealing attenuation of increasing inflammatory cerebral metabolite ratios Cho/Cr and MI/Cr with initiation of therapy (figure 2). While effective in preventing the increase of inflammatory cerebral metabolites, no significant reduction in inflammation markers was detected after ART initiation. A recent acute infection study has identified elevations of Cho/Cr in the basal ganglia at 14 days postexposure and its subsequent reduction to control levels with 6 months of ART.29 The reduction of basal ganglia Cho/Cr in that study may be related to the earlier initiation of ART than in our study. It is also possible that this is a region-specific observation, as the study also noted a Cho/Cr elevation in the occipital gray matter at baseline that was not reduced upon ART initiation. Our results suggest attenuation but not reversibility of Cho/Cr and MI/Cr in frontal white matter and parietal gray matter. Reversibility of Cho/Cr and MI/Cr may occur beyond follow-up, but this is unlikely as persistent neuroinflammation has been reported in chronic infection on stable ART.9"
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Neurology
Published online before print September 26, 2014
Andrew C. Young, Constantin T., Yiannoutsos, PhD, Manu Hegde, MD, PhD, Evelyn Lee, Julia Peterson, Rudy Walter, Richard W. Price, MD, Dieter J. Meyerhoff, PhD, Serena Spudich, MD
"The presumed natural history of HIV neuropathogenesis is progressive inflammation in the CNS leading to clinical neurologic dysfunction. Our findings suggest that inflammation and gliosis, the presumed substrates of later neurologic injury, initially manifest during the first year of HIV infection and worsen during early infection in the absence of treatment. In addition, our findings suggest that initiation of ART can alter this trajectory such that markers of inflammatory changes are no longer increasing, possibly limiting the extent of neurologic injury."
"Following the prospective ART-naive PHI cohort revealed increases in metabolite levels suggestive of progressive inflammation: Cho/Cr and MI/Cr increased in the frontal white matter and MI/Cr increased in the parietal gray matter. The effect of ART in PHI was assessed with longitudinal mixed linear models, revealing attenuation of increasing inflammatory cerebral metabolite ratios Cho/Cr and MI/Cr with initiation of therapy (figure 2). While effective in preventing the increase of inflammatory cerebral metabolites, no significant reduction in inflammation markers was detected after ART initiation."
"Our observations consistently show longitudinal increases of the inflammatory brain metabolite markers Cho/Cr and MI/Cr that suggest worsening inflammation during early untreated HIV. ART initiation during the first year of infection attenuated the increase of these inflammatory cerebral markers, but it did not appear to reverse them within the follow-up period. It is unknown whether after longer-term treatment and follow-up, ART initiated during early infection might reduce or eliminate inflammation, which has been observed in the setting of chronic, treated HIV infection. However, early diagnosis and adoption of ART is associated with low prevalence of neurocognitive impairment, providing further support for early intervention.39 Our MRS-based observations suggest that early ART initiation may be advisable to prevent neurologic dysfunction by slowing and possibly halting HIV-associated neuropathology."
Abstract
Objective: We examined the longitudinal effects of primary HIV infection (PHI) and responses to early antiretroviral therapy (ART) on the brain using high-field magnetic resonance spectroscopy (MRS).
Methods: Cerebral metabolites were measured longitudinally with 4T proton MRS and assessed for ART effects in participants with PHI. Levels of glutamate (Glu), N-acetylaspartate (NAA), myo-inositol (MI), and choline-containing metabolites (Cho) were measured relative to creatine + phosphocreatine (Cr) in anterior cingulate, basal ganglia, frontal white matter, and parietal gray matter.
Results: Fifty-three participants recruited at median 3.7 months post HIV transmission were followed a median 6.0 months. A total of 23 participants initiated ART during follow-up. Prior to ART, increases per month were observed in Cho/Cr (slope = 0.0012, p = 0.005) and MI/Cr (slope = 0.0041, p = 0.005) in frontal white matter as well as increases in MI/Cr (slope = 0.0041, p < 0.001) and NAA/Cr (slope = 0.0024, p = 0.030) in parietal gray matter. After initiation of ART, prior positive slopes were no longer significantly different from zero, while Glu/Cr in basal ganglia decreased (slope = -0.0038, p = 0.031).
Conclusions: Early in HIV infection, increases of Cho/Cr and MI/Cr in treatment-naive participants suggest progressive inflammation and gliosis in the frontal white matter and parietal gray matter, which is attenuated after initiation of ART. Elevated baseline Glu/Cr in basal ganglia may signal excitotoxicity; its subsequent stabilization and downward trajectory with ART may lend further support for early ART initiation.
GLOSSARY
ART = antiretroviral therapy; CHI= chronic HIV infection; Cho = choline-containing metabolites; Cr = creatine-containing
metabolites; Glu = glutamate; 1H-MRS = proton magnetic resonance spectroscopy; IQR = interquartile range; MI = myoinositol;
MPRAGE = magnetization-prepared rapid gradient echo; MR = magnetic resonance; NAA = N-acetylaspartate;
PHI = primary HIV infection; TCA = tricarboxylic acid; TE = echo time; VOI = volume of interest.
Proton magnetic resonance spectroscopy (1H-MRS) can detect dynamic cerebral metabolite changes indicative of inflammation and neuronal injury in individuals with HIV.1-5 Biochemical cerebral metabolite evidence found in chronic HIV infection (CHI) are low N-acetylaspartate (NAA, a putative marker of neuronal health), high choline-containing metabolites (Cho, a marker of macrophage infiltration and inflammation), and high myo-inositol (MI, an astrocyte marker associated with astrocytosis, gliosis, and inflammation).1,4,5 Low glutamate (Glu) is also reported in CHI with cognitive impairment and is thought to reflect neuronal and glial cell dysfunction.6 Antiretroviral therapy (ART) offers limited reversibility7,8 even for CHI on stable long-term therapy,9 suggesting that early HIV invasion of the CNS10,11 and accrued neuronal damage12 may contribute to the substantial prevalence of HIV-associated neurocognitive disorders in the era of ART.13,14 1
H-MRS studies during primary HIV infection (PHI, the first year after HIV transmission) describes inflammation with trends of elevated Cho/creatine-containing metabolites (Cr) and MI/Cr with potentially neurotoxic levels of Glx (combined glutamate and glutamine peak)10,15; this corresponds with neuropathologic immune activation previously reported in PHI.16-20
To date, no 1H-MRS study has examined the longitudinal effects of untreated early HIV in the brain, assessing for potentially deleterious effects that may be impacted by intervention with ART. Utilizing a 4T magnetic resonance (MR) scanner, we studied ART-naive PHI participants prospectively, hypothesizing increases in Cho/Cr and MI/Cr. In participants who initiated ART during follow-up, we predicted reduction in markers of inflammation and excitoxicity after treatment.
RESULTS
Study participant characteristics. A total of 53 men with PHI, 16 men and 2 women with CHI, and 19 HIV-uninfected men were studied cross-sectionally. Laboratory and demographic characteristics of the 3 groups are shown in table 1. At baseline, PHI was scanned at median 3.7 months post infection (interquartile range [IQR] 2.1-6.2) while CHI was scanned at median 93 months post diagnosis (IQR 62.9-216.1). Of the 53 PHI participants enrolled in the
study, only 5 had a history of ART, of median duration 22 days prior to scanning. In the CHI group, 13 participants were ART-naive, 4 had less than a 2-week history of ART, and 1 had a 4-month proximal history of ART. Significant differences between groups in CD41 T-cell count, CSF HIV RNA, and CSF
leukocytes were identified in the baseline measures (table 1). As compared to HIV-uninfected participants, participants with PHI had lower median
values in CD41 T-cell count, measurable CSF HIV RNA, and higher CSF leukocytes.
Fifty-three participants with PHI were followed longitudinally with a total of 154 scans over a median 6.0 months (IQR 0-26.4). ART was initiated independently
from the study by 23 of the 53 PHI participants at a median of 6.4 months (IQR 3.2-16.8) post HIV transmission. ART regimens consisted of 21 nucleoside reverse transcriptase inhibitors, 8 non-nucleoside reverse transcriptase inhibitors, 14 protease inhibitors, 4 integrate inhibitors, and 1 CCR5 entry inhibitor. Participants received ART for a median of 12.9 months (IQR 3.1-29.3) by the conclusion of follow-up.
Cross-sectional baseline cerebral metabolites.
Group differences between PHI and HIV-uninfected participants were noted in parietal gray matter where PHI had lower Cho/Cr (p = 0.007) and MI/Cr (p 5 0.030). PHI participants had higher Glu/Cr in the basal ganglia relative to HIV-uninfected participants (p = 0.027). Comparisons between PHI and CHI revealed higher NAA/Cr in frontal white matter (p = 0.003) as well as higher NAA/Cr (p50.014) andMI/Cr (p=0.045) in anterior cingulate in PHI. Finally, CHI compared to
HIV-uninfected had lower NAA/Cr in frontal white matter (p = 0.037) and parietal gray matter (p = 0.014) as well as lower Glu/Cr in parietal gray matter
(p= 0.033) (table 2 and figure 1). Kruskal-Wallis posttest results were not adjusted for multiple comparisons.
Cerebral metabolite changes prior to ART in PHI.
Cho/Cr andMI/Cr in the frontal white matter and parietal gray matter increased prior to ART initiation. In frontal white matter, the trajectory of ART-naive Cho/Cr and MI/Cr increased at 0.0012/month (p = 0.005) and 0.0041/month (p = 0.005), respectively (figure 2, A and B). Similarly, in the parietal gray matter, ART-naïve MI/Cr increased by 0.0041/month (p < 0.001) (figure 2C). In parietal gray matter, NAA/Cr increased by 0.0024/month (p = 0.030) (figure 2D).
Effects of ART on cerebral metabolite changes in PHI.
Treatment with ART was associated with an apparent elimination of prior positive slopes for Cho/Cr and MI/Cr (i.e., slopes became insignificantly different from 0). In frontal white matter, the ART-initiated Cho/Cr slope decreased to 0.00017/month (p =0.654) and ART-initiated MI/Cr slope decreased to 0.000797/month (p = 0.578) (figure 2, A and B). In parietal gray matter, the ART-initiated MI/Cr slope decreased to 20.0011/month (p = 0.219) (figure 2C).
ART-interaction slopes trended toward significance for Cho/Cr and MI/Cr in frontal white matter at 20.0011/month, p = 0.073 and 20.0033/ month, p = 0.077, respectively, and was significant for MI/Cr in parietal gray matter with a slope of 20.0052/month, p < 0.001. Interestingly, the significantly elevated baseline Glu/Cr levels in basal ganglia decreased after ART initiation by 20.0038/month (p = 0.031), though a nonsignificant ART-interaction slope (20.0022/month, p50.399) is noted (figure 2E). Decreases in ART-initiated NAA/Cr slope in parietal gray matter were not significantly different from zero (20.0002/month, p = 0.820) with an ART-interaction slope trending toward significance (20.0026/month, p = 0.074) (figure 2D). Cerebral metabolite ratio slopes for each region can be found in table 3. p Values were not adjusted for multiple comparison.
DISCUSSION
The presumed natural history of HIV neuropathogenesis is progressive inflammation in the CNS leading to clinical neurologic dysfunction. Our findings suggest that inflammation and gliosis, the presumed substrates of later neurologic injury, initially manifest during the first year of HIV infection and worsen during early infection in the absence of treatment. In addition, our findings suggest that initiation of ART can alter this trajectory such that markers of inflammatory changes are no longer increasing, possibly limiting the extent of neurologic injury.
Our cross-sectional findings of lower Cho/Cr and MI/Cr in parietal gray matter in PHI when compared to HIV-uninfected participants may initially be puzzling. However, these results are consistent with our current understanding of the natural history of HIV infection. Longitudinal SIV/macaque studies report an initial elevation of Cho/Cr in acute infection that is subsequently reduced during the transition from acute to primary infection.27,28 Studies with human participants show a similar progression with elevated Cho/Cr at 16-17 days postexposure10 followed by lower Cho,15 consistent with our results of lower Cho/Cr and MI/Cr months after exposure. A plausible biological mechanism for the low Cho/Cr and MI/Cr in PHI may be the result of reduced viral load after the acute phase24 and concomitant reduced membrane turnover and gliosis related to immune stabilization.
Following the prospective ART-naive PHI cohort revealed increases in metabolite levels suggestive of progressive inflammation: Cho/Cr and MI/Cr increased in the frontal white matter and MI/Cr increased in the parietal gray matter. The effect of ART in PHI was assessed with longitudinal mixed linear models, revealing attenuation of increasing inflammatory cerebral metabolite ratios Cho/Cr and MI/Cr with initiation of therapy (figure 2). While effective in preventing the increase of inflammatory cerebral metabolites, no significant reduction in inflammation markers was detected after ART initiation. A recent acute infection study has identified elevations of Cho/Cr in the basal ganglia at 14 days postexposure and its subsequent reduction to control levels with 6 months of ART.29 The reduction of basal ganglia Cho/Cr in that study may be related to the earlier initiation of ART than in our study. It is also possible that this is a region-specific observation, as the study also noted a Cho/Cr elevation in the occipital gray matter at baseline that was not reduced upon ART initiation. Our results suggest attenuation but not reversibility of Cho/Cr and MI/Cr in frontal white matter and parietal gray matter. Reversibility of Cho/Cr and MI/Cr may occur beyond follow-up, but this is unlikely as persistent neuroinflammation has been reported in chronic infection on stable ART.9
An early infection study with 8 individuals scanned within months of initial HIV exposure reported low NAA in the frontal cortical gray matter as compared to seronegative controls. All individuals reported symptoms of acute HIV syndrome and 5 additionally reported severe headaches, numbness in extremities, and difficulty thinking clearly.30 In predominantly less symptomatic acute infection studies, no significant differences in baseline NAA/Cr were identified,10,29 though this may be limited by sample size. Similarly, we report no significant differences in baseline NAA/Cr in PHI as compared to HIV-uninfected controls, but low NAA/Cr in parietal gray matter trended toward significance (p 5 0.064). Interestingly, we found increasing NAA/Cr in this brain region, which may suggest recovery after an initial drop in NAA/Cr that has been reported in acute infection in SIV31 and early infection in humans.30 Evidence for recovery of NAA/Cr is also reported in acute infection studies with increases (greatest in first months post infection) in NAA/Cr in frontal gray matter, frontal white matter, and occipital gray matter despite not detecting significant differences at baseline.29 Furthermore, baseline NAA/Cr in basal ganglia was significantly elevated in Fiebig III/IV when compared to Fiebig I/II,29 which provides additional context for a temporal relationship of recovering NAA/Cr. We detected increases of NAA/Cr in the ART-naive condition, which implies that NAA/Cr recovery in early infection may not be dependent on ART, and hypothesize that the lack of significance in ART-initiated slope can be attributable to the later timeframe rather than the effect of therapy.
In the basal ganglia, Glu/Cr was elevated prior to ART and significantly declined after initiation of therapy (figure 2E). Elevated Glu/Cr prior to ART is concerning for Glu excitotoxicity via excessive NMDA receptor stimulation by increasing Glu production and release from HIV-infected macrophages or microglia32,33 in addition to dysregulation of astrocyte glutamate reuptake.34 Our data suggest that ART may control viral presence and limit Glu excitotoxicity. Alternatively, nucleoside analog reverse transcriptase inhibitors, which 21 of 23 participants received, are known to cause mitochondrial oxidative stress,35 which may result in reduced Glu synthesis from the tricarboxylic acid (TCA) cycle. Diminished resting cerebral blood flow in the lenticular nucleus of 33 predominantly ART-treated participants in early HIV infection may further suggest reduced TCA cycle metabolism.36 Although ART appears to limit Glu excess, mitochondrial toxicity may play a role and must be considered in early adoption of ART. Regardless of the mechanism of Glu reduction, the elevation of Glu/Cr in basal ganglia prior to ART may suggest utility of 1H-MRS as initial screening for neuronal damage prior to NAA/Cr changes.
One limitation of our study is that we assessed metabolite-to-Cr ratios rather than absolute metabolite concentrations. Absolute cerebral metabolite concentration analysis may overcome possible confounds resulting from Cr level alterations in HIV infection or between subject groups.37,38 It is not known whether absolute concentrations of Cr may change during the early stages of infection, and thus characterization of absolute metabolites over time should be considered in future studies. Readers should note that baseline cross-sectional findings may be attributable to HIV and non-HIV influences. For example, despite matching, there is a nonsignificant but notable 4-year difference in median age between the PHI and HIV- cohort and significant age differences with CHI.
Sufficient longitudinal HIV-uninfected control data were not obtained to compare to with PHI data, thus observed PHI trends cannot be conclusively stated to be the result of infection rather than a general process such as aging. Furthermore, due to the large number of statistical tests performed in this study, the evidence provided by this data need to be carefully weighed as p values were not adjusted for multiplicity, inflating the probability of false-positive type 1 errors. If adjusted for multiple comparisons, only our major findings of increasing MI/Cr in parietal gray matter prior to ART and its subsequent attenuation by ART remain significant. Despite these limitations, our study presents novel hypothesisgenerating data for ART-naive and ART-initiated longitudinal changes in 1H-MRS cerebral metabolite markers in a large and well-characterized PHI cohort.
Our observations consistently show longitudinal increases of the inflammatory brain metabolite markers Cho/Cr and MI/Cr that suggest worsening inflammation during early untreated HIV. ART initiation during the first year of infection attenuated the increase of these inflammatory cerebral markers, but it did not appear to reverse them within the follow-up period. It is unknown whether after longer-term treatment and follow-up, ART initiated during early infection might reduce or eliminate inflammation, which has been observed in the setting of chronic, treated HIV infection. However, early diagnosis and adoption of ART is associated with low prevalence of neurocognitive impairment, providing further support for early intervention.39 Our MRS-based observations suggest that early ART initiation may be advisable to prevent neurologic dysfunction by slowing and possibly halting HIV-associated neuropathology.
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