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Rivastigmine decreases brain damage in HIV patients with mild cognitive deficits

Abstract Rivastigmine has been shown to improve cognition in HIV+ patients with minor neurocognitive disorders; however, the mechanisms underlying such beneficial effect are currently unknown. To assess whether rivastigmine therapy is associated with decreased brain inflammation and damage, we performed T1/T2* relaxometry and magnetization transfer imaging in 17 aviremic HIV+ patients with minor neurocognitive disorders enrolled on a crossed over randomized rivastigmine trial. Rivastigmine therapy was associated with changes in MRI metrics indicating a decrease in brain water content (i.e., edema reabsorption) and/or reduced demyelination/axonal damage. Furthermore, MRI changes correlated with cognitive improvement on rivastigmine therapy.


Funding Information
This study was supported by unrestricted grants from Abbott and ViiV; the Swiss National Science Foundation under grants PZ00P3_131914/11 and PP00P2-146318; the Swiss MS Society; and the Societ e Acad emique Vaudoise. The funding sources had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.

Introduction
Rivastigmine is a dual acetyl-and butyrylcholinesterase inhibitor (ChEI) that is commonly used to alleviate cognitive deficits in Alzheimer's disease (AD). 1 Recently, we have provided first evidence that rivastigmine improves some cognitive deficits in HIV patients with mild neurocognitive disorder (MND). 2 In AD patients, rivastigmine achieves its positive effects on cognition through multiple mechanisms encompassing enhanced cholinergic neurotransmission and improved clearance of amyloid plaques. 3 The putative beneficial effect of rivastigmine in HIV+ patients may be due to a decrease in brain acetylcholine levels, related to a deficit in choline acetyltransferase. 4 Yet, in HIV patients, rivastigmine may also improve cognitive outcome by reducing brain inflammation and damage. Indeed, rivastigmine has been reported to markedly temper microglia activation, demyelination, and axonal damage in experimental autoimmune encephalomyelitis, leading to improved spatial memory deficits. 5 Also, in a cellular model of HIVassociated dementia, the adjunction of galantamine, another ChEI, resulted in the inhibition of microglial activation induced by HIV-1 proteins. 6 Moreover, the activation of the cholinergic anti-inflammatory system by nicotine was shown to attenuate neuroinflammation via suppression of Th1 and Th17 responses. 7 In this study, we used multiparametric MRI (mpMRI) to investigate the effect of rivastigmine on brain inflammation and damage in MND HIV patients who participated to a blinded randomized crossed over trial to assess rivastigmine efficacy and safety. 2 Multiparametric quantitative MRI represents a sensitive approach to study inflammatory and degenerative brain changes. 8 In fact, quantitative MRI metrics such as T1 and T2* relaxometry and semiquantitative MRI parameters like magnetization transfer ratio (MTR) may provide complementary information regarding the brain tissue structure and its components: T1 relaxation times are sensitive to the tissue organization of micro-and macromolecules, and to a lesser extent to iron content; 9 T2* relaxation times exhibit high sensitivity to quantify paramagnetic substances like iron and provide additional information concerning the presence of free and bound water molecules (i.e., myelin water 9 ). On the other hand, MTR is highly sensitive to the presence and extent of macromolecules such as myelin, cellular membranes components, etc. 9 Therefore, the combination of T1/T2 relaxometry and MTR may help disentangling whether brain structural changes underlie the benefits of rivastigmine in HIV patients.

Subjects' population
We studied 17 patients with HIV infection, undetectable viremia, and MND, who were enrolled in a blinded crossover randomized study to assess efficacy and safety of rivastigmine for the treatment of MND. 2 MND was diagnosed according to Frascati criteria 10 using an extensive neuropsychological and neuropsychiatric battery of tests. 2 Exclusion criteria were previous brain opportunistic infection, any other opportunistic infection in the last year, active drug consumption, and major depression according to DSM-IV criteria. No patient developed psychiatric (e.g., psychosis) or neurological conditions (e.g., stroke), which could have interfered with neuropsychological or MRI evaluation. Patients clinical characteristics are summarized in Table S1.
Patients were randomly assigned to two arms receiving either rivastigmine or placebo for 20 weeks, followed by a 6 weeks washout, and then by 20 weeks where treatments were inversed between the two arms (see Simioni et al. 2 for details).
All patients had undetectable HIV-RNA in the plasma (defined as <20 copies/mL for more than 3 months) and in the cerebrospinal fluid (<200 copies/L) at study entry; at study end, plasmatic HIV-RNA was detected in one patient (147 copies/mL).
Brain MRI scans were performed before the treatment was started (time point 0, TP0), at 20 weeks (TP1), and at 46 weeks (TP2) (Fig. 1). Five patients could not complete the MRI follow-up and were therefore excluded from the analysis.
A neuropsychological evaluation was also performed at TP0, TP1, and TP2 for all subjects. Neuropsychological tests explored cognitive domains which are usually impaired in MND patients. They included reaction time and rapid visual information processing and spatial working memory (strategy and error component) from the Cambridge Neuropsychological Test Automated Battery (CANTAB), Trail Making Test Parts A and B, Wechsler Adult Intelligence Scale-III Symbol Digit Test, CANTAB, Digit Spans Backward/Forward, and Stockings of Cambridge (see 2 for details).

Magnetic Resonance Imaging
All MRI scans were performed on a 3T Magnetom Trio (a Tim System, Siemens, Erlangen, Germany) equipped with a 32-channel head coil.
The study protocol consisted a high-resolution, T1weighted magnetization prepared rapid gradient echo (MPRAGE) acquisition for anatomical reference; an MP2RAGE acquisition with the same voxel and matrix size to obtain T1 relaxation maps; 11 and a multiple echo Fast Low Angle SHot (FLASH) with and without magnetization transfer preparation. The signals acquired with (MT) and without (M0) magnetization saturation pulse were used to compute magnetization transfer ratio (MTR) maps as reported previously. 8,12 T2 star maps were calculated using a monoexponential fitting as performed previously. 8,12 Brain segmentation into gray and white matter (GM and WM) was performed using Morphobox 13 (see Data S1) and segmentation quality was visually inspected. Mean T1 rt, T2* rt, and MTR were calculated in each ROI (WM and GM) at each time point. The study was approved by the ethics committee of the Centre Hospitalier Universitaire Vaudois (CHUV) in Lausanne (Switzerland), and all subjects gave informed written consent.

Analysis of variance of multiparametric brain MRI data
To measure the effect of rivastigmine on brain microstructural properties, we computed for each subject the difference in T1rt, T2* rt, and MTR between TP1 and TP0 and TP2 and TP1 in WM and GM. Subsequently, we performed a multivariate analysis of variance (MANOVA) for each MR contrast and each brain tissue. Treatment and sequence of treatment (Arm1, Arm2) were considered as fixed effects. Bonferroni correction for multiple comparison was applied (6 MANOVA; 2 regions of interest, that is, WM and GM; and 3 MR parameters, that is, T1 rt, T2*rt, and MTR).
Our null hypothesis was that there is no difference in parametric MRI between patients treated with rivastigmine and patients who received placebo. To investigate the relationship between changes in MRI metrics and changes in cognition during rivastigmine treatment, we used a generalized linear model (GLM) and assessed whether a linear regression of the variation of T2* and MTR in WM and MTR in GM predicted the variation in the Trail Making Test, Part A (TMTA) z-score (calculated using available normative data), which is the score that improved on rivastigmine in the previous pilot trial in MND patients. 2

Results
Mean T2* rt in WM was significantly different between patients treated with rivastigmine and those treated with placebo (P = 0.004, P-corr = 0.02, degrees of freedom: 11, standard error: 0.69; Table S2). Patients treated with rivastigmine showed a decrease in WM T2* rt over time, while patients treated with placebo exhibited an increase in mean T2* rt (Fig. 2).
Mean MTR in WM and GM was significantly different between patients treated with rivastigmine and those treated with placebo (P = 0.006, P-corr = 0.04, degrees of freedom: 11, standard error: 0.004 for both; Table S2). Patients treated with rivastigmine showed an increase in MTR in both WM and GM, while patients treated with placebo exhibited a decrease in MTR (Fig. 2). T1 rt did not show any significant difference in WM and GM between subjects with and without treatment.
The GLM analysis showed that the difference between MTR after rivastigmine treatment and before rivastigmine treatment (delta MTR) significantly correlated with the difference in TMTAZ after and before treatment (delta TMTAZ; adjusted R 2 = 0.4, P = 0.03; Fig. 3).

Discussion
Our study provides evidence that rivastigmine treatment reduces brain damage in MND HIV patients. Also, we show that changes in MRI metrics on rivastigmine were correlated with clinical improvement.
A recent pilot study performed by our group showed that rivastigmine treatment for 20 weeks leads to an improvement in processing speed and executive function in HIV subjects with minor neurocognitive disorder. 2 Still, whether brain structural changes underlay cognitive improvement on rivastigmine in HIV-infected patients is currently unknown.
Rivastigmine has been previously reported to decrease brain inflammation (i.e., microglia activation, 5 also when triggered by HIV proteins 6 ) and brain damage (demyelination and axonal injury 5 ) in animal models and cell cultures. We hypothesized that the cognitive improvement measured in the rivastigmine trial in HIV patients 2 is paralleled by reduced brain inflammation and reversible damage to myelin and axons.
Multiparametric MRI provides sensitive biomarkers of brain inflammation and degeneration in HIV-infected patients. By combining T1 and T2* relaxometry with magnetization transfer imaging, we have shown that aviremic HIV patients with MND exhibit increased and diffuse brain inflammation and degeneration compared to aviremic HIV patients without MND and to healthy subjects. 12 In this pilot study, we applied exactly the same mpMRI approach as described in Granziera et al. 12 to assess the effect of rivastigmine on brain inflammation and damage.
The concomitant decrease in MTR and T2* rt observed in patients treated with rivastigmine strongly suggests an increase in macromolecules (i.e., myelin or axons) and/or a decrease in free water (i.e., edema reabsorption that may induce an increase in MTR).
In fact, MTR parameter quantifies the interaction between free protons and immobilized protons bound to macromolecules and an increase in MTR has been linked to an increase in myelination/axons [14][15][16] or decrease in water content. [17][18][19] On the other hand, T2* relaxation times reflect the loss of transverse magnetization due to T2 relaxation; therefore, a decrease in T2* relaxation times may be provoked by an increase in macromolecular compounds such as myelin. 20,21 Interestingly, changes in brain white matter MTR (i.e., increase in MTR) were significantly related to the improvement in processing speed (Trail Making Test Part A scores) on rivastigmine treatment, suggesting that the improvement in cognitive functionreported in our previous pilot trial on rivastigmine efficacy 2may be mediated by a decrease in brain damage.
Our study suffers from some methodological limitations. The first one is the small sample size of our cohort. The second is that the MRI at TP1 was performed just before the washout period, decreasing the sensitivity to detect changes among time points. However, we tried to take this latter effect into account by including the study arm (i.e., sequence of treatment) as covariable in our multivariate analysis.
In conclusion, we have shown that rivastigmine treatment leads to a reduction in brain damage, alongside to improved cognition in a group of HIV patients with MND. Future studies should confirm and extend our current findings in larger cohorts of patients and combining mpMRI with biological assessments of inflammatory markers in serum and cerebrospinal fluid before and after rivastigmine treatment.