Lupus (2014) 23,

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1 (2014) 23, PAPER In vivo evaluation of brain damage in the course of systemic lupus erythematosus using magnetic resonance spectroscopy, perfusion-weighted and diffusion-tensor imaging A Zimny 1, M Szmyrka-Kaczmarek 2, P Szewczyk 1, J Bladowska 1, A Pokryszko-Dragan 3, E Gruszka 3, P Wiland 2 and M Sasiadek 1 1 Department of General and Interventional Radiology and Neuroradiology; 2 Department of Rheumatology and Internal Diseases; and 3 Department of Neurology, Wroclaw Medical University, Poland Twenty-two neuropsychiatric (NPSLE) and 13 systemic lupus erythematosus (SLE) patients with a normal appearing brain on plain magnetic resonance (MR) as well as 20 age-matched healthy controls underwent MR spectroscopy (MRS), perfusion-weighted (PWI) and diffusion-tensor imaging (DTI). In MRS NAA/Cr, Cho/Cr and mi/cr ratios were calculated from the posterior cingulate cortex and left parietal white matter. In PWI, values of cerebral blood volume (CBV) were assessed from 14 regions, including gray and white matter. In DTI fractional anisotropy (FA) values were obtained from 14 white matter tracts including projection, commissural and association fibers. All MR measurements were correlated with clinical data. SLE and NPSLE patients showed significantly (p < 0.05) lower NAA/Cr ratios within both evaluated regions and FA values within the cingulum, as well as a tendency to cortical hypoperfusion. Compared to SLE, NPSLE subjects revealed lower FA values within a wide range of association fibers and corpus callosum. Advanced MR techniques are capable of in vivo detection of complex microstructural brain damage in SLE and NPSLE subjects regarding neuronal loss, mild hypoperfusion and white matter disintegrity. MRS and DTI seem to show the highest usefulness in depicting early changes in normal appearing gray and white matter in SLE patients. (2014) 23, Key words: Systemic lupus erythematosus; neuropsychiatric systemic lupus erythematosus; magnetic resonance spectroscopy; perfusion-weighted imaging; diffusion-tensor imaging; cerebral perfusion; cerebral metabolism Introduction The nervous system is affected in up to 75% patients with systemic lupus erythematosus (SLE) and its involvement is associated with greater morbidity and mortality. 1 Neuropsychiatric SLE (NPSLE) includes 19 syndromes affecting the central and peripheral nervous system of both a neurologic and psychiatric nature. 2 The underlying pathophysiology in NPSLE is still not fully understood. The pathogenic etiologies are likely to be diverse and complex and may involve autoantibody production, intrathecal production of proinflammatory cytokines, as well Correspondence to: Magdalena Szmyrka-Kaczmarek, Department of Rheumatology and Internal Diseases, Wroclaw Medical University, ul. Borowska 213, Wroclaw, Poland. magda_szmyrka@yahoo.com Received 6 February 2013; accepted 11 October 2013 as cerebral microangiopathy with atherosclerosis, blood-brain barrier disintegrity, chronic diffuse ischemia or thromboembolic disease associated with antiphospholipid antibody syndrome. 3 Neuropsychiatric SLE is difficult to diagnose and manage, as there are neither identified specific biomarkers nor typical lesions within the central nervous system detected by imaging methods. Magnetic resonance imaging (MRI) is a standard imaging method in the evaluation of cerebral lesions in patients with both SLE and NPSLE. The MR findings in these groups are very often nonspecific and in the majority of cases (40% to 80%) consist of small focal lesions concentrating in the periventricular and subcortical white matter, larger infarcts as well as cortical atrophy and ventricular dilation. 4 On the other hand, it has to be stressed that even in patients with neuropsychiatric manifestations, conventional MRI of! The Author(s), Reprints and permissions: /

2 the brain may show no detectable lesions. 5,6 Since brain pathology in SLE patients seems to be multifactorial and complex and neurometabolic impairment, neurochemistry as well as perfusion abnormalities may precede anatomic lesions, the need for a multimodality approach, with the use of multiple new neuroimaging techniques, has been recently emphasized by other authors. 7 In our study we applied three advanced MR methods such as spectroscopy (MRS), perfusionweighted (PWI) and diffusion-tensor imaging (DTI), which are capable of evaluating subtle metabolic, hemodynamic and microstructural changes within the brain that are not detectable with standard MRI. Magnetic resonance spectroscopy (MRS) is a widely used method for assessing cerebral biochemical structure, by evaluation of different metabolite ratios reflecting molecular and cellular processes. N-acetylaspartate (NAA) levels correlate with neuronal and axonal density, myoinositol (mi) is thought to be a marker of gliosis, choline (Cho) represents myelin content and reflects membrane turnover while creatine (Cr) is used as a reference metabolite. 8 The majority of the previous studies have revealed decreased levels of NAA both in SLE and NPSLE patients with or without the elevation of peaks of Cho and mi. These changes were found not only within focal brain lesions but also in normal appearing white matter PWI is an MR technique that enables measurements of hemodynamics of cerebral microcirculation with the use of the cerebral blood volume (CBV) parameter. The value of CBV correlates with vessel density and is found to be lower in hypoperfused brain areas. 12 It has been proved that PWI provides information similar to that obtained in positron-emission tomography (PET) and single-photon emission computed tomography (SPECT) studies but with several advantages, such as no ionizing radiation, high spatial resolution and relatively low cost. 13,14 MR perfusion studies in SLE patients have brought conflicting results, ranging from decreased to increased CBV values and single studies reporting no detectable changes in perfusion parameters DTI is a noninvasive MRI technique for in vivo mapping of brain microstructure, particularly used for assessing white matter integrity. It is a quantitative technique that measures the movement of water within the different tissue compartments. Because of the linear architecture of fiber bundles and the presence of myelin sheaths, the mobility of water within normal white matter is restricted and preferentially oriented to the directions of the fibers, which is called anisotropic diffusion, in contrast to isotropic diffusion characteristic of fluid spaces in which the mobility of water is unrestricted. DTI uses parameters of fractional anisotropy (FA), which is a marker of white matter fiber integrity and its decrease is consistent with degradation or impairment of white matter coherence due to microstructural pathology. 19,20 The majority of DTI studies have revealed decreased FA values within white matter that is more pronounced in NPSLE compared to SLE subjects Though neuroimaging studies using new functional methods have already been performed in patients with SLE and NPSLE, our study still brings novelty to the topic. To our knowledge this is the first study that evaluates together three advanced MR techniques: MRS, PWI and DTI performed in groups of well-matched NPSLE and non-npsle patients with a normal appearing brain in a standard MR examination. The majority of previous studies reporting the results of advanced MR techniques in SLE patients have been conducted using one or two imaging methods and included patients with more advanced brain damage with visible focal brain lesions that may have influenced the final results. The use of a novel multimodality approach enabled us to assess three major aims of the study: 1) to evaluate early metabolic, perfusion and microstructural changes within normal appearing gray and white matter measured using MRS, PWI and DTI methods in the same groups of subjects, 2) to search for correlations between different aspects of brain damage and clinical data and 3) to compare different advanced neuro-imaging techniques in order to find the most clinically useful combination of imaging methods that are able to visualize early brain pathology. Material and methods Material The study group consisted of 35 patients diagnosed with SLE according to the 1997 revised American College of Rheumatology (ACR) criteria (mean age: 34.4 years, range years, 86% female) and 20 age-matched healthy controls (mean age 36.8, range years, 82% female). Healthy controls did not suffer from any neurologic, psychiatric or systemic illness known to influence cerebral function, such as serious vascular disease, head trauma, tumor, current depression, alcoholism, epilepsy and did not have a history of psychoactive 11

3 12 medication use. In all subjects the main radiological inclusion criterion was a normal appearance of the brain in the standard MR examination. Each MR examination of the brain was carefully evaluated independently by three experienced neuroradiologists (AZ, JB, PS). Only the patients without any focal brain lesions and without evident cortical or subcortical atrophy during visual inspection of the images were enrolled in the study. In 22 subjects NPSLE was diagnosed based on ACR nomenclature and case definitions for neuropsychiatric lupus syndromes. 24 Thirteen patients suffered from SLE, but without any previous or current neuropsychiatric symptoms. All SLE patients underwent detailed physical examination by experienced rheumatologists and neurologists and a wide range of laboratory tests including assessment of antinuclear antibodies (ANA) and antiphospholipid antibodies (apl) using commercially available tests (EUROIMMUN, Germany). Positive apl antibodies included anticardiolipin (acl), antibeta 2 glycoprotein 1 (antib2gp1) immunoglobulin G (IgG) and IgM antibodies in medium and high titers or positive lupus anticoagulant (LA). In the NPSLE group there were 11/22 (50%) apl-positive patients (LA: seven, acl: five, antib2gp1: three) while in SLE six of 13 (46%) apl-positive patients (LA: two: acl: two, antib2gp1: two). The SLE Disease Activity Index (SLEDAI) was used to assess current disease activity, and was calculated both including and without taking into account NP symptoms 25 while the Systemic International Collaborative Clinics (SLICC) Damage Index was used to assess accumulated damage to various organs. 26 Subjects demographic information and clinical data are listed in Table 1. Among the patients with NPSLE there were 14 subjects with active and eight patients with inactive, previous clinical NP disease. Mean duration of NP involvement was 3.2 years. The majority of NPSLE patients presented with more than one NP syndrome, which included: mood disorders (n ¼ 13), severe headache (n ¼ 13), cognitive decline (n ¼ 12), psychosis (n ¼ 5), seizures (n ¼ 4), anxiety (n ¼ 2), peripheral polineuropathy (n ¼ 3), cranial neuropathy (n ¼ 1) and acute confusional state (n ¼ 1). Additionally, NP syndromes present in our subjects were divided into major (n ¼ 9), which comprised seizures, psychosis, acute confusional state, cranial neuropathy and peripheral neuropathy, and minor (n ¼ 13), which included mood disorders, mild cognitive dysfunction, headaches and anxiety. The study was conducted in accordance with the Declaration of Helsinki and was approved by the Table 1 Demographic and clinical characteristics of patients with SLE Characteristic NPSLE (n ¼ 22) SLE (n ¼ 13) Age in years Gender (F/M) Disease duration (years) SLEDAI score without NP SLEDAI score with NP SLICC/ACR damage index apl positive (%) n ¼ 11 (50%) n ¼ 6 (46 %) Prednisone average daily dose (mg) Antimalarials (% of patients) Azathioprine (% of patients) 27 8 Cyclophosphamide (% of patients) Anticoagulants (% of patients) Except as indicated otherwise values are the mean SD. NPSLE: neuropsychiatric systemic lupus erythematosus; SLE: systemic lupus erythematosus; SLEDAI: Systemic Erythematosus Disease Activity Index; SLICC/ACR: Systemic Erythematosus International Collaborating Clinics/American College of Rheumatology; apl: antiphospholipid antibodies; NP: neuropsychiatric symptoms; F: female; M: male. Wroclaw Medical University Bioethics Committee for conducting research involving humans. All subjects were enrolled after giving their informed consent to participate in the study. Methods Data acquisition: All subjects underwent standard MR studies followed by MRS, DTI and PWI on a 1.5 T MR scanner (SignaHdx, GE Medical Systems) using a 16-channel head-neck-spine (HNS) coil. All axial images were parallel to the anterior commisure-posterior commisure (AC-PC) line. MRS H 1 MRS was performed with a single voxel technique using point-resolved spectroscopy sequence (PRESS) with the following parameters: echo time (TE) ¼35 ms, repetition time (TR) ¼1500 ms, 128 acquisitions, number of excitations (NEX) ¼eight. In each subject two 8 cm 3 (2 2 2 cm) voxels of interest (VOIs) were used: the first one prescribed in the cortex of the posterior cingulate gyrus (PCG) above the parieto-occipital sulcus (Figure 1 (a)), the second one located within white matter of the left parietal lobe. The total acquisition time was 3 min 43 s for each VOI. The data were postprocessed using algorithms provided by the manufacturer (GE, ADW 4.4). Each spectrum was

4 13 Figure 1 Examples of measurements within the posterior cingulate region using different MR techniques: MRS (a), PWI (b) and DTI (c, d). (a) Sagittal view showing voxel of interest within the posterior cingulate region. (b) Axial view showing region of interest on perfusion map of cerebral blood volume (CBV). (C), (d): axial images showing two small regions of interest within cingulum fibers on a color-coded map (c) and FA map (d). MR: magnetic resonance; MRS: magnetic resonance spectroscopy; PWI: perfusion-weighted imaging; DTI: diffusion-tensor imaging; FA: fractional anisotropy. automatically fitted to four peaks corresponding to levels of NAA (2.02 ppm), total creatine (3.03 ppm), Cho-containing compounds (3.23 ppm) and mi (3.56 ppm). Ratios of NAA, Cho and mi to creatine (NAA/Cr, Cho/Cr, mi/cr, respectively) were calculated and analyzed. PWI PWI was performed with dynamic susceptibility contrast enhanced method (DSC) using a fastecho planar gradient T2*-weighted sequence with the following parameters: TR ¼ ms, TE ¼ 80 ms, field of view (FOV) ¼ 30 cm, matrix ¼ , slice thickness ¼ 8 mm without spacing, NEX ¼1.0. Ten seconds after the start of image acquisition, a bolus of a 1.0 mol/l gadobutrol formula (Gadovist, Schering, Berlin, Germany) in a dose of 0.2 ml/kg of body weight was injected via a 20-gauge catheter placed in the antecubital vein. Contrast administration was performed using an automatic injector (Medrad) at a rate of 5 ml/s and was followed by a saline bolus (20 ml at 5 ml/s). The whole perfusion acquisition lasted 1 min 26 s in which sets of images from 13 axial slices were obtained before, during and after contrast injection. The dynamic images were post-processed using Functool software (GE, ADW 4.4). Maps of CBV were computed on a pixel-wise basis from the first-pass data as described by Belliveau et al. 27 We used circular (size 525 mm 2 ) regions of interest (ROIs) placed manually within the posterior cingulate gyrus (PCG) (Figure 1 (b)) and bilaterally within fronto-parietal white matter at the convexity as well as hand-drawn irregular ROIs (size mm 2 ) placed in the temporal cortices (at the level of midbrain), occipital cortices (at the level of the basal ganglia), frontal and parietal cortices (at the level of the body of the lateral ventricles) and

5 14 basal ganglia (irregular hand-drawn ROIs outlining the putamen, globus pallidus and caudate nucleus, size mm 2 ). The cortical ROIs were placed carefully by experienced neuroradiologists (AZ, JB, PS) to outline the cortex, avoiding large vessels. All regional CBV values were normalized to the mean values in the cerebellar cortex. DTI DTI studies were performed using a single-shot spin echo (SE) planar imaging (EPI) sequence in 25 encoding directions with the following parameters: TE ¼ 8500 ms, TR ¼ 100 ms, matrix ¼ , FOV ¼ cm, NEX ¼ two, b values 0 and 1000 s/mm 2. Axial 5 mm thick slices with no spacing were obtained. The total acquisition time was 7 min 29 s. DTI data were postprocessed on commercial workstations (GE, ADW 4.4) using Functool software to generate color-coded and parametric maps of FA. Measurements of FA were obtained using small, fixed-in-size (30 40 mm 2 ), elliptical ROIs drawn manually within the white matter tracts under the control of color-coded FA maps. The evaluated fibers were recognized using available anatomy atlases and publications. 28 To avoid a partial volume effect, ROIs were placed carefully by experienced neuroradiologists (AZ, JB, PS) in the middle of each fiber bundle to maximally encompass only the white matter fibers and to avoid other structures such as cerebrospinal fluid or gray matter. FA measurements were performed on axial slices within 14 white matter bundles including association, commissural and projection fiber tracts. The association fibers included inferior longitudinal fasciculi (ILFs) at the level of midbrain, inferior fronto-occipital fasciculi (IFOFs) at the level of inferior aspects of thalami laterally to the occipital horns of lateral ventricles, superior longitudinal fasciculi (SLFs) at the level of superior aspects of lateral ventricles and bilateral posterior cingulum fibers (Figure 1 (c) and (d)). The examined commissural tracts included genu (GCC) and splenium (SCC) of the corpus callosum (CC) at the level of basal ganglia, while projection tracts included posterior limbs of internal capsules (PLICs) and middle cerebellar peduncles (MCPs). Statistical analysis A group comparison of demographics was performed using analysis of variance (ANOVA) for continuous variables (age) and Pearson chi 2 for categorical variables (gender). Differences in MRS ratios, FA and rcbv values among NPSLE, SLE and control subgroups were evaluated using ANOVA followed by the Least Significant Difference (LSD) test as a post hoc test. Correlations among MRS, PWI and DTI measurements as well as between MRS, PWI, DTI measurements and the clinical data, such as disease duration and the presence of ANA, were estimated using Pearson s correlation coefficients. A student t-test was used to assess differences in MRS, PWI and DTI measurements between apl-positive and apl-negative SLE and NPSLE patients as well as between active and inactive NPSLE subjects. Statistical computations were performed using the Statistica PL software package version 4.0, and p < 0.05 was set as a significant level. Additionally, Bonferroni correction for multiple comparisons was used with a new p value established as p < (p < 0.05 divided by 33 variables: six in MRS, 13 in PWI and 14 in DTI). Results There were no significant differences in age (p ¼ 0.8) and sex distribution (chi 2 ¼ 0.37, degree of freedom (df) ¼ 2, p ¼ 0.82) among the three groups of subjects (Table 1). Comparing SLE and NPSLE patients, there were no differences in the disease duration (p ¼ 0.38), measures of disease activity (SLEDAI with NP p ¼ 0.08, SLEDAI without NP p > 0.05), accumulated organ damage measured by SLICC (p ¼ 0.46), levels of ANA (p ¼ 0.1) and presence of apl (p ¼ 0.89). There were no significant differences in the treatment between the NPSLE and SLE subjects. Results of MRS, PWI and DTI measurements in SLE and NPSLE patients Compared to the control group, SLE and NPSLE patients showed significantly lower NAA/Cr values in PCG and parietal white matter with no significant differences in Cho/Cr and mi/cr ratios in those locations, though a slight tendency to higher mi and Cho levels in the two groups of patients was observed within parietal white matter. There were no significant differences in MRS ratios between SLE and NPSLE subjects (Table 2). After Bonferroni correction in SLE and NPSLE subjects, only NAA/Cr values from the PCG showed significantly lower levels compared to controls (Table 2).

6 Table 2 Mean values of spectroscopic measurements in NPSLE, SLE and the control group with the results of analysis of variance (ANOVA) and the post hoc LSD test 15 Mean value (SD) LSD test, p values Parameters evaluated NPSLE SLE CG ANOVA p values NPSLE vs SLE NPSLE vs CG SLE vs CG Posterior cingulate gyrus (PCG) NAA/Cr 1.57 (0.09) 1.56 (0.09) 1.67 (0.08) < < a,b a,b Cho/Cr 0.56 (0.03) 0.55 (0.06) 0.56 (0.06) 0.89 >0.05 >0.05 >0.05 mi/cr 0.57 (0.05) 0.57 (0.05) 0.56 (0.05) 0.9 >0.05 >0.05 >0.05 Parietal white matter (PWM) NAA/Cr 1.8 (0.17) 1.78 (0.09) 1.88 (0.14) a 0.02 a Cho/Cr 1.01 (0.14) 1.01 (0.19) 0.98 (0.13) 0.52 >0.05 >0.05 >0.05 mi/cr 0.66 (0.05) 0.69 (0.06) 0.64 (0.08) 0.13 >0.05 >0.05 >0.05 NPSLE: Neuropsychiatric systemic lupus erythematosus; SLE: Systemic lupus erythematosus; CG: control group; SD: standard deviation; ANOVA: analysis of variance; NAA: N-acetylaspartate; Cr: creatine; Cho: choline; mi: myoinositol. a statistically significant result at p value < 0.05; b statistically significant result after Bonferroni correction at p value < Table 3 Mean values of perfusion measurements in NPSLE, SLE and the control group. No statistically significant differences were found among the evaluated groups Mean rcbv values (SD) Mean rcbv values (SD) Brain region NPSLE SLE CG Brain region NPSLE SLE CG Right frontal cortex 1.09 (0.1) 1.1 (0.18) 1.18 (0.11) Left frontal cortex 1.08 (0.11) 1.09 (0.2) 1.16 (0.08) Right temporal cortex 1.12 (0.1) 1.11 (0.12) 1.16 (0.08) Left temporal cortex 1.13 (0.1) 1.12 (0.13) 1.17 (0.1) Right occipital cortex 1.15 (0.1) 1.13 (0.14) 1.24 (0.16) Left occipital cortex 1.13 (0.13) 1.15 (0.16) 1.23 (0.18) Right parietal cortex 1.13 (0.09) 1.13 (0.16) 1.18 (0.07) Left parietal cortex 1.16 (0.11) 1.16 (0.17) 1.19 (0.09) Right basal ganglia 1.11 (0.08) 1.12 (0.1) 1.12 (0.05) Left basal ganglia 1.09 (0.08) 1.11 (0.11) 1.1 (0.04) Right parietal white matter 0.52 (0.05) 0.54 (0.07) 0.51 (0.05) Left parietal white matter 0.53 (0.06) 0.54 (0.06) 0.50 (0.06) Posterior CG 1.31 (0.13) 1.34 (0.15) 1.33 (0.11) NPSLE: neuropsychiatric systemic lupus erythematosus; SLE: systemic lupus erythematosus; rcbv: cerebral blood volume; CG: cingulate gyrus. In PWI there were no significant differences in rcbv values in any of the evaluated locations between SLE and NPSLE patients. Compared to the control subjects, SLE and NPSLE subjects showed a tendency to lower rcbv values in the bilateral temporal, occipital, frontal and parietal cortices as well as a tendency to elevated rcbv values bilaterally in parietal white matter but the results did not reach a significant level (Table 3). DTI SLE patients, compared with the control group, showed lower FA values only in the right cingulum fibers. Compared to the control subjects, NPSLE patients revealed significantly lower FA values in the genu of the CC and within a wide range of association fibers such as both cingulum fibers, both ILFs as well as in the left IFOF and SLF. Compared to SLE, NPSLE subjects showed significantly lower FA values in the left ILF and IFOF (Table 4). After Bonferroni correction only in NPSLE subjects FA values from the right cingulum fibers showed significantly lower values compared to the control group (Table 4). Correlations among MRS, PWI and DTI measurements as well as between MRI results and clinical data In NPSLE, NAA/Cr levels from parietal white matter positively correlated with NAA/Cr levels in PCG (r ¼ 0.59, p < 0.000), as well as rcbv values from both frontal (right: r ¼ 0.56, p ¼ 0.006, left: r ¼ 0.58, p ¼ 0.004) and occipital (right: r ¼ 0.46, p ¼ 0.028, left: r ¼ 0.45, p ¼ 0.03) cortices as well as the right temporal cortex (r ¼ 0.57, p ¼ 0.005) and the left parietal cortex (r ¼ 0.45, p ¼ 0.03). No other significant correlations were found between MR results in the group of NPSLE subjects. In SLE subjects there were no significant associations between MRS, PWI and DTI results. In SLE as well as NPSLE patients there were no significant associations between MRS, PWI and DTI measurements and the duration of the disease. NPSLE and SLE patients positive for apl, compared to NPSLE and SLE subjects without

7 16 Table 4 Mean values of DTI measurements in NPSLE, SLE and the control group with the results of analysis of variance (ANOVA) and the post hoc LSD test Mean FA values (SD) LSD test, p values Brain region NPSLE SLE CG ANOVA p values NPSLE vs SLE NPSLE vs CG SLE vs CG Right ILF (0.03) (0.05) (0.05) a 0.89 Left ILF (0.03) (0.04) (0.04) a a 0.7 Right IFOF (0.03) (0.04) (0.02) 0.42 >0.05 >0.05 >0.05 Left IFOF (0.04) (0.03) (0.03) a a 0.05 Right SLF (0.04) (0.06) (0.03) 0.3 >0.05 >0.05 >0.05 Left SLF (0.04) (0.06) (0.03) a 0.26 Right CG (0.04) (0.04) (0.03) < < a,b a Left CG (0.05) (0.05) (0.04) a 0.1 Genu of CC (0.04) (0.04) (0.02) a 0.05 Splenium of CC (0.04) (0.04) (0.03) 0.54 >0.05 >0.05 >0.05 Right MCP (0.03) (0.03) (0.02) 0.07 >0.05 >0.05 >0.05 Left MCP (0.02) (0.02) (0.03) 0.57 >0.05 >0.05 >0.05 Right PLIC (0.03) (0.02) (0.01) 0.58 >0.05 >0.05 >0.05 Left PLIC (0.04) (0.02) (0.02) 0.59 >0.05 >0.05 >0.05 FA: fractional anisotropy; NPSLE: Neuropsychiatric systemic lupus erythematosus; SLE: Systemic lupus erythematosus; CG: control group; SD: standard deviation; ANOVA: analysis of variance; ILF: inferior longitudinal fasciculus; IFOF: inferior fronto-occipital fasciculus; SLF: superior longitudinal fasciculus; MCP: middle cerebellar peduncle; PLIC: posterior limb of internal capsule; CG: cingulum; CC: corpus callosum. a statistically significant result at p value < 0.05; b statistically significant result after Bonferroni correction at p value < detected apl, showed significantly (p ¼ 0.01) lower FA values within the splenium of the CC (0.841 and 0.807, respectively). There were no other significant (p < 0.05) differences in DTI, PWI and MRS measurements between apl-positive and apl-negative SLE and NPSLE subjects. There were no significant differences in MRS, PWI and DTI results between the NPSLE subjects with major and minor symptoms or between active and inactive NPSLE subjects. Discussion The first aim of the study was to assess the pattern of early gray and white matter damage in SLE and NPSLE patients with a normal appearing brain on conventional MR sequences. To assess metabolic disturbances within the brain, we applied MRS. Similarly to previous reports, we found lower NAA levels within white matter both in SLE and NPSLE subjects However, unlike the majority of previous reports we also evaluated cortical regions and found a similar decrease in NAA levels in the cortex of PCG both in SLE and NPSLE. Since low NAA values are most closely associated with reduced neuronal-axonal density, our results indicate significant neuronal damage both in gray and white matter of these patients. In our study we found only a tendency to higher levels of Cho or mi, though the significant increase of these two metabolites was reported before. 29,30 Mioinositol is a marker of gliosis, while Cho is regarded as a marker of myelin turnover and in NPSLE elevated levels of Cho and mi were found to be significantly associated with vascular and reactive changes including gliosis, vasculopathy, inflammatory cell infiltration or edema. 31 The almost normal levels of mi and Cho in our patients could be explained by the main inclusion criterion, which was a normal brain appearance on plain MR images indicating an early stage of cerebral damage without significant gliosis and inflammation. To assess changes in cerebral microcirculation we used an MR technique called dynamic susceptibility contrast-enhanced (DSC) perfusion. To our knowledge there are only a few studies on perfusion changes measured with MR in patients with SLE and they gave contradictory results ranging from hypo- to hyperperfusion These studies were performed using different MR scanners and were conducted on different subject groups including SLE patients with depression and visible brain lesions, which may explain the discrepancy between their and our results. Our results are in accordance with the study by Emmer et al. 15 performed with a 1.5 Tesla scanner. Similarly to that study we also did not find any significant hypoperfusion, within either gray or white matter of patients with NPSLE and SLE, though we noticed a tendency to lower values of rcbv in several cortical regions in both patient subgroups. To investigate white matter pathology we used DTI and measured FA values in several white

8 matter fibers including projection, commissural and association tracts. Comparison of our results with previous studies is difficult because of different methodologies used in almost every study and different characteristics of the study groups. The previous DTI studies found no or minimal FA changes in SLE and more pronounced alterations in NPSLE subjects, either within the bigger areas of white matter or within specific fiber tracts ,32,33 Similarly to previous reports, we found more pronounced and widespread FA abnormalities in NPSLE patients compared to SLE subjects. In SLE patients we showed significantly lower FA values only within right cingulum fibers, while in the NPSLE group damage of the white matter bundles included CC and several association tracts such as both cingulum fibers (part of the limbic system), both ILFs (connecting temporal and parietal association cortices), left IFOF (connecting frontal and occipital cortices) and left SLF (consisting of fibers running from frontal to parietal, occipital and temporal regions). Our study indicates profound damage of commissural and many association tracts to be the major difference between the SLE patients with or without neurological symptoms. Damage to commissural tracts and the wide range of association tracts that are necessary for interand intrahemispheric connections may explain the variety of neurological and behavioral problems in patients with NPSLE manifestations and this may reflect global nervous system dysfunction. The interesting result of our study is the pathology of the posterior cingulate region in SLE and NPSLE patients. In both groups of patients we observed a decrease of NAA/Cr within the posterior cingulate cortex and lower FA values within cingulum fibers, which is typically detected in patients with mild cognitive impairment and Alzheimer s disease. 34 The posterior cingulate cortex and cingulum fibers are parts of the limbic system and are important in the memory and learning processes. Since these structures showed significant changes both in SLE and NPSLE subjects our results may suggest that all SLE patients are prone to cognitive decline because of microstructural damage to the posterior cingulate region that is present even in the early stages of SLE. It is well known that SLE can produce cognitive impairment even without overt NP features, though the pathogenesis of this dysfunction is not well understood. 35 The second major aim of the study was to assess correlations between different pathological processes in order to gain an insight into the mechanisms of brain damage in SLE and NPSLE patients. In NPSLE subjects only we found decreased NAA levels in parietal white matter to be associated with decreased NAA levels in PCG. This may indicate neuronal deficits in NPSLE to be a global process present at the same time both in gray and white matter, even in the early stages of the disease, in contrast to SLE subjects not showing such associations. In NPSLE we also revealed a strong association between the decrease of NAA levels in parietal white matter and hypoperfusion in multiple cortical locations, such as both frontal and occipital cortices as well as right temporal cortex and left parietal cortex. These results may suggest perfusion deficits play an important role in neuronal damage in these patients. Ischemia as a result of endothelial damage, generalized vasculopathy or due to hypercoagulopathy has been implicated as one of the most important pathogenic mechanisms leading to NP symptoms in SLE. 36 Correlation between NAA levels and values of rcbv was not found in SLE subjects, which may indicate other processes than hypoperfusion are responsible for neuronal damage in this group of subjects. Searching for correlations between brain damage and clinical data, interestingly we found that the presence of apl was associated with lower FA values within the splenium of the CC in SLE and NPSLE patients. Our results may suggest that presence of apl could lead to direct damage of white matter bundles. Our results are in accordance with the previous papers reporting apl to show strong associations with neurological involvement in SLE and to account not only for events of a thromboembolic nature, but also headaches, seizures, multiple-sclerosis like syndrome and cognitive dysfunction, where direct effect on the brain cells through immune-mediated mechanisms may play a role. 3,37 Similarly to the previous report by Emmer et al., 11 we did not find any correlations between the presence of apl and brain perfusion measured with MRI. On the other hand, we also did not detect significant correlations between the presence of apl and decreased NAA levels or increased Cho/Cr ratio as reported before. 38 Our results may be explained by the early stage of brain damage with only mild changes in spectroscopic metabolites without significant perfusion alterations. We also compared MRS, PWI and DTI results between NPSLE with major and minor NP symptoms. In previous MRS studies, patients with major symptoms were reported to show decreased NAA levels compared to subjects with minor symptoms 39 or increased mi levels compared to healthy controls. 40 To our knowledge there are no reports on the differences between patients with major and 17

9 18 minor symptoms using PWI and DTI techniques. In our work we found no differences in MR measurements between patients with major and minor symptoms, which may indicate that the rate of anatomical damage that can be detected with advanced MR techniques is similar in these two groups of patients and the different NP symptoms may be due to functional abnormalities that are difficult to detect with MRS, PWI and DTI techniques. The third aim of the study was to compare three neuroimaging methods and find the technique that could be the most useful in a clinical setting. In our study only MRS and DTI techniques enabled visualization of early brain damage in NPSLE and SLE patients. Among multiple measurements the assessment of NAA/Cr ratios from the posterior cingulate cortex and FA values from the right cingulum fibers and several other association fibers including ILF, IFOF and SLF showed the lowest p values, indicating the highest rate of damage compared to normal controls and thus the highest utility of measurements in these locations in clinical practice. Our study proves that in both groups of SLE and NPSLE subjects, although brain morphology is not yet altered, early microscopic damage of gray and white matter is already present and can be detected using multimodality MRI. In our opinion DTI and MRS seem to be the techniques with the highest usefulness in the visualization of cerebral pathology in the early stages of brain damage in SLE and NPSLE. Of all evaluated brain locations, the area of the posterior cingulate region (both cortex and cingulum fibers) seems to show the most pronounced alterations in MRS and DTI in the early stage of brain pathology. 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