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Reduced cerebrospinal fluid concentrations of oxysterols in response to natalizumab treatment of relapsing remitting multiple sclerosis
Journal of the Neurological Sciences, Volume 358, Issue 1-2, November 2015, Pages 201 - 206
Natalizumab therapy reduces inflammation and degeneration of the CNS in relapsing-remitting multiple sclerosis (RRMS). In cerebrospinal fluid (CSF) the concentration of 24S-hydroxycholesterol (24OHC) reflect neurodegeneration, whereas 27-hydroxycholesterol (27OHC) is dependent on the integrity of the blood-brain barrier (BBB).
To measure the impact from natalizumab treatment on 24OHC and 27OHC concentrations in serum and CSF of RRMS.
In serum and CSF obtained from 31 patients before and following 12 months of natalizumab treatment, 24OHC and 27OHC were analyzed by isotope-dilution mass spectrometry.
Natalizumab treatment reduced CSF-24OHC concentrations (p = 0.002), CSF-27OHC concentrations (p = 0.01) and serum-24OHC concentrations (p = 0.029). There was no significant effect of the treatment on serum-27OHC concentrations. Serum concentrations of 24OHC correlated with Symbol Digit Modalities Test scores before (r = 0.5, p = 0.007) and after natalizumab treatment (r = 0.403, p = 0.033).
We showed for the first time that natalizumab treatment of RRMS reduced the concentrations of 24- and 27OHC in CSF, indicating reduced neurodegeneration and improved integrity of the BBB, respectively. Our results imply a role for serum 24OHC as a biomarker of cognition (visuo-spatial ability and processing speed) in RRMS.
Keywords: Multiple sclerosis, Natalizumab, Biomarkers, Neurofilament, Cerebrosterol, 24S-hydroxycholesterol, 27-hydroxycholesterol, SDMT, Cerebrospinal fluid, Neurodegeneration, Blood-brain barrier.
24S-hydroxycholesterol (24OHC, cerebrosterol) is a brain-specific cholesterol hydroxylation product that passes freely over the blood-brain barrier  . Almost all 24OHC in blood circulation originates from the brain , , and  and in healthy individuals the concentrations seem constant throughout adult life  . It has been shown in several studies that serum concentrations of 24OHC reflect CNS cholesterol turnover , , and  and is suggested as a potential biomarker for neurodegeneration  and  reflecting the amount of metabolically active neurons. Less than 1% of 24OHC is excreted into the cerebrospinal fluid (CSF)  , but this fraction may increase as a consequence of neuronal damage  . In contrast, almost all cells of the body can synthesize 27-hydroxycholesterol (27OHC), which is another side-chain oxidized oxysterol. The level of this oxysterol in CSF correlate with the corresponding levels in the circulation  and the flux into the brain is to some extent dependent upon the integrity of the blood-brain barrier (BBB)  . Because the metabolism of 27OHC in the brain is dependent upon CYP7B1, an enzyme located in neuronal cells, neuronal loss may lead to increased concentrations of 27OHC in CSF  .
In active lesions of multiple sclerosis (MS) the neuronal tissue integrity is damaged and axonal loss occurs  . This is the culprit of cerebral atrophy and development of neurological disability in MS. It has been suggested that increased serum concentrations of 24OHC in MS patients may reflect enhanced neuronal damage  and they also correlate to cerebral atrophy in relapsing-remitting (RR) MS  . In neurodegenerative diseases such as Alzheimer disease, Huntington disease and also MS, decreased concentrations of 24OHC was found in the peripheral circulation, indicating reduced flux from the brain and/or increased neuronal loss , , , , and .
Neurofilament light (NFL) is a cytoskeletal protein of myelinated axons and is regarded as a CSF biomarker of axonal damage. Increased CSF-NFL concentrations are found during all stages of MS and peak during acute relapses or during the appearance of contrast enhancing lesions on MRI  and . Treatment of MS with natalizumab (NZ), a monoclonal antibody that inhibit leucocyte migration over the BBB, effectively reduce cerebral inflammation  and . NZ therapy of RRMS also reduce NFL concentrations, comparable to concentrations, found in healthy controls (HCs)  . Thus, effective immunomodulation of MS seems also to reduce the rate of neurodegeneration.
In the present study we followed both serum and CSF concentrations of 24OHC and 27OHC in parallel with CSF concentrations of NFL before and after NZ treatment of RRMS patients. The aim was to explore oxysterols as potential biomarkers of the immunopathogenesis of MS and their usefulness as markers of disease activity, disease progression and therapeutic efficacy.
2. Materials and methods
2.1. Patients and healthy controls
The study was approved by the regional ethical board of the University of Gothenburg, Sweden. Informed consent was obtained from patients and HCs upon their recruitment to the study. The study cohort consisted of 31 RRMS patients and 16 HCs. Patients had been diagnosed with MS according to the revised McDonald criteria  . They were recruited prospectively at the MS Center, Department of Neurology, Sahlgrenska University Hospital, Gothenburg, Sweden, and constituted a sub-group of a larger population that had been described previously  . Disease duration was estimated from onset of the first demyelinating symptoms. Patients were treated with NZ 300 mg intravenously once monthly. Three patients had no previous treatment, 8 were previously treated with mitoxantrone and 20 were previously treated with interferon beta or glatiramer acetate. Patients treated with mitoxantrone terminated their treatment on average 6 months (range 3–12) prior to natalizumab treatment. Ten patients had relapse within 3 months before assessment and 21 were in remission. Of those ten patients who had relapse, 8 had relapse prior and 2 had relapse during treatment with NZ.
2.2. Clinical assessment and specimen sampling
Neurological disability was scored using the Expanded Disability Status Scale (EDSS)  and cognitive function (visuo-spatial ability and processing speed) was tested with the Symbol Digit Modalities Test (SDMT) , , , and . None of the HCs had a history of neurological disease and all had normal clinical neurological examination. SDMT was not tested in HCs. Serum and CSF were sampled once in HCs and at baseline and after 12 months of NZ treatment in patients. Lumbar puncture was done according to the procedures recommended in the consensus protocol of the BioMS-EU network for CSF biomarker research in MS  . CSF was transported on ice and the first 12 mL of CSF was carefully mixed; after centrifugation, fractions were snap-frozen within 2 h in 0.5 mL aliquots and stored at − 80 °C until analysis.
2.3. Determination of albumin ratio and serum cholesterol
Albumin concentration in serum and CSF was measured by immunonephelometry on a Beckman Immage Immunochemistry system (Beckman Instruments, Beckman Coulter, Brea, CA, USA). CSF/serum albumin ratio, as a function of the permeability of the BBB, was calculated as CSF albumin (mg/L) divided by serum albumin (g/L). Serum (S) cholesterol concentration was determined using an enzymatic colorimetric method on the c501 module of the Roche 6000 analyzer according to instructions from the manufacturer (Roche, Penzberg, Germany).
2.4. NFL enzyme-linked immunosorbent assay
CSF NFL protein was measured with a sensitive sandwich ELISA method (NF-light® ELISA kit, UmanDiagnostics AB, Umeå, Sweden) by board-certified laboratory technicians at the Clinical Neurochemistry Laboratory, the Sahlgrenska University Hospital, according to protocols approved by the Swedish Board for Accreditation and Conformity Assessment. The lower limit of quantification of the assay was 31 ng/L. Intra- and inter-assay coefficients of variation were below 10%.
2.5. 24OHC and 27OHC spectrometry
24OHC and 27OHC in serum and CSF were analyzed by isotope-dilution mass spectrometry using deuterium-labeled internal standards, as described elsewhere  . In brief, serum and CSF samples were subjected to saponification to hydrolyze oxysterol esters. The hydrolysis was performed at 22 °C for 2 h with concentration 0.35 M KOH. In order to prevent cholesterol autoxidation during sample preparation and handling, cholesterol was separated from oxysterols by solid-phase extraction using silica columns. A deuterium-labeled internal standard were added for both oxysterols analyzed. 27OHC and 24OHC are the most abundant cholesterol oxidation products (mean serum values 154 and 64 ng/ml respectively in a healthy population).
Because of the small sample size and non-normal distribution of serum 27OHC (Shapiro-Wilk test, p < 0.05), non-parametric statistics was used. Mann–Whitney U-test were used to investigate group differences and Wilcoxon signed rank sum test were used for analysis of matched pair data, i.e. before and after NZ treatment. Correlation coefficients were calculated using Pearson and Spearman two-tailed correlation test. Multiple regression analysis included the following variables: age, gender, disease duration, EDSS, SDMT and relapse within 3 months before assessment. Statistical calculations were performed in SPSS Statistics 22 software and in Microsoft Office Excel 2013.
Clinical characteristic and demographic features of patients and HC are presented in Table 1 . NZ treatment did not influence S-cholesterol concentration. Mean S-cholesterol was 4.68 (SD = 1.0) mmol/l prior to and 4.61 (SD = 0.87) mmol/l after NZ treatment. NZ decreased the concentrations of 24- and 27OHC and NFL in CSF ( Table 2 ). Analyses were performed in all patients (n = 31), those in remission (n = 21), and those with relapse (n = 10) within 3 months at the time of collecting the samples. Stratification of the patients into 3 different subgroups depending on previous treatment (no treatment, treatment with mitoxantrone, treatment with interferon beta or glatiramer acetate) did not influence the results significantly. Individual oxysterol concentrations in serum and CSF before and after NZ treatment for each patient are shown in Fig 1, Fig 2, Fig 3, and Fig 4.
(n = 31)
|Patients in remission (n = 21)||Patients with relapse (n = 10)||HC
(n = 16)
|Gender male/female, no||11/20||8/13||3/7||11/5|
|Mean age, years (range)||36 (13–60)||35 (13–60)||39 (26–53)||41 (27–53)|
|Mean disease duration, years (range)||8.4 (0.5–26)||7.9 (2–26)||9.5 (0.5–21)||NA|
|Median EDSS before treatment (range)||3.5 (0–6.5)||3.0 (0–6.5)||5.0 (2.5–6.5)||NA|
|Median EDSS after treatment (range)||3.5 (0–6.5)||2.5 (0–6.5)||4.5 (0–6.5)||NA|
HC: healthy controls.
|Before treatment||After treatment||HC|
|(n = 31)
|(n = 21)
|(n = 10)
|(n = 31)
|(n = 21)
|(n = 10)
|(n = 16)|
|CSF NFL (ng/L)||2391 ± 5274||1389 ± 1665||4493 ± 8906||485 ± 264a and b||498 ± 307a and b||457 ± 148a and b||308 ± 95|
|CSF 24OHC (ng/mL)||1.9 ± 0.7||2.0 ± 0.8||1.7 ± 0.6||1.7 ± 0.6a and b||1.8 ± 0.6a and b||1.6 ± 0.6||2.3 ± 0.7|
|S 24OHC (ng/mL)||58.9 ± 14.8||57.9 ± 15.7||61.1 ± 13.3||56.6 ± 13.8||55.4 ± 13.9||59.1 ± 13.9||57.3 ± 14.3|
|CSF 27OHC (ng/mL)||1.0 ± 0.4||0.9 ± 0.3||1.2 ± 0.6||0.9 ± 0.3||0.8 ± 0.3||0.9 ± 0.4||1.0 ± 0.4|
|S 27OHC (ng/mL)||133.9 ± 39.1||127.9 ± 39.0||146.7 ± 37.9||134.1 ± 40.0||129.6 ± 37.3||143.6 ± 45.5||162.8 ± 49.7|
a p < 0.05: patients vs. HC.
b p < 0.05: before vs after natalizumab treatment.
RRMS: relapsing-remitting multiple sclerosis; HC: healthy controls; NFL: neurofilament light; 24OHC: 24S-hydroxychelesterol; 27OHC: 27-hydroxycholesterol; CSF: cerebrospinal fluid; S: serum; ±: standard deviation.
3.1. 24OHC concentrations in CSF
NZ treatment reduced the CSF concentration of 24OHC in all patients (p = 0.002) and in patients in remission (p = 0.018) but not in patients with relapse (p = 0.091). Prior to NZ treatment the concentrations of CSF-24OHC was significantly lower in all patients and in patients with relapse compared to HCs (p = 0.027 and p = 0.037, respectively), but after 12 months of NZ treatment the CSF-24OHC concentrations became significantly lower in all patient groups compared to HCs, irrespective of clinical activity ( Table 2 ).
3.2. 24OHC concentrations in serum
The serum concentrations of 24OHC in the patient groups were not significantly different from that in HCs but were decreased significantly by NZ treatment in all patients and in patients in remission (p = 0.029 and 0.038, respectively, Table 2 ).
3.3. 27OHC concentrations in CSF
NZ treatment reduced CSF-27OHC concentrations significantly in all patients and in patients in remission (p = 0.01 and p = 0.027, respectively). Although lower CSF-27OHC concentrations were recorded after NZ treatment in patients with relapse, they did not reach statistical significance (p = 0.113). The 27OHC concentrations in CSF were similar in all patient groups compared to HCs before and after NZ treatment ( Table 2 ).
3.4. 27OHC concentrations in serum
NZ treatment did not influence the 27OHC concentrations in serum. Prior to NZ treatment the serum concentrations of 27OHC of all patients and patients in remission had significantly lower serum-27OHC concentrations than HCs (p = 0.044 and p = 0.014, respectively) but patients with relapse showed no difference (p = 0.618). After NZ treatment the serum concentrations of 27OHC were significantly lower in patients in remission (p = 0.04) but no differences were found between serum-27OHC concentrations of all patients or in patients with relapse and HCs (p = 0.059 and p = 0.370, respectively, Table 2 ).
3.5. NFL concentrations in CSF
The concentration of CSF-NFL prior to NZ treatment was significantly higher in all patients, in patients in remission and in patients with relapse compared to HCs (p < 0.001, p < 0.001 and p = 0.003, respectively). NZ treatment significantly reduced the CSF-NFL concentrations to a level similar to that found in HC in all patients, in patients in remission and those with relapse (p < 0.001, p = 0.001 and p = 0.028, respectively, Table 2 ).
3.6. Correlations between 24OHC, 27OHC and NFL
A significant correlation was found between 24OHC and 27OHC in serum before (r = 0.413, p = 0.021) and after NZ treatment (r = 0.552, p < 0.001) as well as in CSF before (r = 0.597, p < 0.001) and after treatment (r = 0.568, p < 0.001). S-cholesterol correlated with 24OHC and 27OHC in serum before (r = 0.595, p < 0.001 and r = 0.538, p = 0.002, respectively) and after NZ treatment (r = 0.641, p < 0.001 and r = 0.557, p = 0.001, respectively) but there was no correlation between S-cholesterol and oxysterol in CSF.
No significant correlations were found between CSF and serum concentrations of 24OHC or between CSF and serum concentrations of 27OHC. Furthermore, no significant correlations were found between NFL and 24OHC or between NFL and 27OHC.
3.7. The influence on NFL, 24OHC and 27OHC concentrations from albumin ratio, age, disease duration, gender, EDSS and SDMT
Albumin ratio as a marker of the integrity of the BBB correlated to CSF-27OHC both prior to and after NZ treatment (r = 0.603, p = 0.001 and r = 0.679, p < 0.001, respectively) but not to CSF 24OHC or NFL.
Following NZ treatment EDSS decreased from 3.6 (SD = 1.9) to 3.3 (SD = 2.1, p = 0.034) and SDMT increased from 47.8 (SD = 10.0) to 61.4 (SD = 17.2, p < 0.001). EDSS was negatively correlated to SDMT before (r = − 0.43, p = 0.024) and after treatment (r = − 0.59, p = 0.001) and this was also true for patients in remission (r = − 0.57, p = 0.011 and r = − 0.63, p = 0.004, respectively). Serum-24OHC correlated to SDMT before (r = 0.5, p = 0.007) and after (r = 0.40, p = 0.033) NZ treatment ( Fig. 5 ). In patients in remission the corresponding correlation coefficients were 0.5 (p = 0.031) and 0.4 (p = 0.091) respectively.
Multiple regression analysis showed a significant relationship between serum-24OHC and EDSS after (beta-coefficient 0.527, p = 0.016) but not prior treatment. Dependence between serum-24OHC and disease duration was noted after (beta-coefficient 0.396, p = 0.039) but not prior treatment. Significant relationships were found between serum-24OHC and SDMT before (beta-coefficient 0.603, p = 0.006) and after NZ treatment (beta-coefficient 0.817, p = 0.001). The concentrations of serum-27OHC and CSF-27OHC were both influenced by gender (p < 0.05). There was no gender or age dependence for the other studied biomarkers. There was no significant relationship between the concentrations of CSF-NFL, CSF-24OHC or CSF-24OHC and EDSS.
We showed for the first time that NZ treatment reduced oxysterol concentrations in CSF of RRMS. NZ treatment had no influence on the serum cholesterol concentration. While the reduction of CSF 27OHC concentration probably reflects improved integrity of the BBB  , the mechanism behind the reduction of 24OHC in CSF seems more complex. Besides neuronal damage, the CSF-24OHC concentration is influenced by the number of metabolically active neurons  , presence of the hydroxylation enzyme — CYP46A1 , , and , and the rate of the 24OHC clearance from the CSF  . However, concentrations of CSF-24OHC are fairly constant in adults  , and less than 1% of the total excretion of 24-OHC is via the CSF , , and . Therefore, the reduction of 24OHC we found was probably more influenced by reduced neuronal damage and degeneration than changes in neuronal metabolism  .
Previous investigations of neurodegenerative diseases such as Alzheimer disease, Huntington disease and MS, have shown decreased circulating concentrations of 24OHC , , and . The concentration of 24OHC in plasma correlated with the degree of brain atrophy  and  and in MS also with the T2 lesion load  and . Increased 24OHC plasma concentrations have been shown during relapse  and  and both 24OHC and 27OHC concentrations were higher in the presence of Gd + lesions  and . The 24OHC-forming enzyme CYP46 was shown in macrophage infiltrates in experimental autoimmune encephalomyelitis  . Thus, the concentration of 24OHC in circulation may be influenced by both inflammatory and degenerative processes in MS.
Our study population consisted of RRMS patients with relatively low mean disease duration and low median EDSS score. With one exception (serum 27OHC in patient in remission), oxysterol concentrations in serum were similar in patients and HCs. This was in line with a previous report, which showed normal 24OHC in early cases and reduced 24OHC concentrations only in severe MS or cases with long disease duration  .
In accordance with a previous report  we found a gender dependence of serum 27OHC but we could not confirm that previously reported for serum 24OHC  . Males had significantly higher concentrations of 27OHC than women. Since patients and HC were not gender-matched, this could have influenced the concentrations of 27OHC, and at least partly explained why the mean 27OHC in serum was higher in HC than in patients. This could also potentially influence the 27OHC concentrations in CSF of HC.
There was no correlation between CSF NFL and serum or CSF concentrations of the two oxysterols. This could probably be explained by differences in their origin, metabolism and elimination. 27OHC is essentially produced in extra-hepatic organs, outside the CNS/CSF compartment  , and is to some extent dependent on the integrity of the BBB  . In contrast, both NFL and 24OHC are brain-specific. While 24OHC freely pass the BBB and is possible to determine in serum/plasma, the passage of NFL over the BBB is restricted and cannot be detected in peripheral blood with our immunoassay  . Moreover, CSF concentrations of 24OHC and NFL represent different aspects of the pathology in MS and their turnover and elimination differ  . NFL in CSF is a marker of axonal damage  and , the concentration peaks during relapse  and  or during gadolinium enhancement of lesions  and thereafter return to low levels within 3 months  and . In contrast, 24OHC is affected by a plethora of pathological processes and the half-life is only 10–14 h  .
Almost all 27OHC observed in CSF originates from peripheral blood and the concentration is to some extent depending on the functional state of the BBB  . NZ therapy is known to have profound impact on the amount of new gadolinium enhancing lesions  , and thereby restore the integrity of the BBB  . Therefore, the decrease of CSF 27OHC concentrations following NZ treatment is probably related to an improved integrity of the BBB. An assumption that was supported by a high correlation between CSF 27OHC concentration and albumin ratio.
One patient had high CSF levels of 27OHC (2.4 ng/mL) and NFL (27 310 ng/L) at baseline, and the concentrations dropped after NZ treatment (0.4 ng/mL and 590 ng/L, respectively). A recent relapse prior initiation of NZ treatment probably caused severe axonal damage and disrupted integrity of the BBB. However, on clinical grounds the patient was comparable with other patients of the study group, and not considered as an outlier. The main results were unaffected by removing this patient from the statistical calculation.
In this study the correlation between serum concentrations of 24OHC and SDMT was found at baseline and following 12 months of NZ treatment, which implies 24OHC as a biomarker of cognition (visuo-spatial ability and processing speed). This role of 24OHC has previously been reported in Alzheimer's disease and in mild cognitive impairment  and . It appeared to be the most sensitive biomarker in the evaluation of patients with cognitive impairment disease compared with other available biomarkers in CSF, such as total tau, phospho-tau and amyloid beta 42  . In our study however, the serum concentrations of 24OHC were not significantly different from those of HCs but were influenced by NZ treatment. Improvement of the integrity of the BBB from NZ treatment might have influenced the release of 24OHC from CNS to the peripheral circulation. Interestingly, serum 24OHC correlated with disease duration and EDSS in this study which may reflect the total spatiotemporal burden of MS showed previously  . SDMT is considered a robust cognitive test for visuo-spatial ability and processing speed not affected by age, education, gender and socioeconomic status  and is suggested as part of the Brief Cognitive Assessment in MS (BICAMS)  , and proposed as a single screening cognitive assessment tool in patients with MS  . Although, we cannot exclude that the reason for improvement of SDMT could be a practice effect from repeated testing  , the reliability of the test was supported by its use as a surveillance tool of progressive multifocal leukoencephalopathy in MS during long-term NZ treatment  . Because the patients included in our study were not assessed for the presence of cognitive impairment with any other test and we did not test HC subjects with SDMT, we lack the possibility to validate our results.
In conclusion, 24- and 27OHC concentrations are both affected by NZ treatment. Decreased concentrations of CSF 24OHC and NFL probably reflect reduction of neuro-axonal damage in RRMS and the influence on 27OHC indicate a restored integrity of the BBB. Our results imply serum 24OHC as a potential biomarker of cognition for visuo-spatial ability and processing speed. However, this has to be explored, confirmed in future investigations.
Conflict of interest statement
L. Novakova, H. Zetterberg, I. Björkhem have no conflict of interest.
C. Malmeström has received lecture honoraria from BiogenIdec, Merck Serono and Novartis and unconditional research grants from BigenIdec.
M. Axelsson has received lecture honoraria from BiogenIdec and Merck Serono.
J. Lycke has received honoraria from Bayer Shering Pharma, BiogenIdec, Novartis and Sanofi-Aventis; has served on scientific advisory boards for Almirall, Teva, Biogen Idec and Genzyme/Sanofi-Aventis; serves on the editorial board of the Acta Neurologica Scandinavica, and has received unconditional research grants from BiogenIdec and Novartis.
V.D. Karrenbauer has received honoraria for speaker's fees from Merck-Serono. She has received scholarship from BiogenIdec. Her MS research is funded by the Stockholm County Council.
This study was funded by grants from Research Foundation of the Multiple Sclerosis Society of Gothenburg, Edit Jacobsson's Foundation, Brain Power, the Swedish Science Council, the City Council of Stockholm (ALF), Västra Götaland Regional Council (ALF) and Biogen Idec MS research scholarship.
-  D. Lutjohann, O. Breuer, G. Ahlborg, et al. Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholesterol from the brain into the circulation. Proc. Natl. Acad. Sci. U. S. A.. 1996;93:9799-9804 Crossref
-  I. Bjorkhem, D. Lutjohann, U. Diczfalusy, L. Stahle, G. Ahlborg, J. Wahren. Cholesterol homeostasis in human brain: turnover of 24S-hydroxycholesterol and evidence for a cerebral origin of most of this oxysterol in the circulation. J. Lipid Res.. 1998;39:1594-1600
-  S. Meaney, M. Hassan, A. Sakinis, et al. Evidence that the major oxysterols in human circulation originate from distinct pools of cholesterol: a stable isotope study. J. Lipid Res.. 2001;42:70-78
-  L. Bretillon, D. Lutjohann, L. Stahle, et al. Plasma levels of 24S-hydroxycholesterol reflect the balance between cerebral production and hepatic metabolism and are inversely related to body surface. J. Lipid Res.. 2000;41:840-845
-  D. Lutjohann, K. von Bergmann. 24S-hydroxycholesterol: a marker of brain cholesterol metabolism. Pharmacopsychiatry. 2003;36(Suppl. 2):S102-S106
-  V. Leoni. Oxysterols as markers of neurological disease—a review. Scand. J. Clin. Lab. Invest.. 2009;69:22-25 Crossref
-  A. Papassotiropoulos, D. Lutjohann, M. Bagli, et al. 24S-hydroxycholesterol in cerebrospinal fluid is elevated in early stages of dementia. J. Psychiatr. Res.. 2002;36:27-32 Crossref
-  V.D. Karrenbauer, V. Leoni, E.T. Lim, et al. Plasma cerebrosterol and magnetic resonance imaging measures in multiple sclerosis. Clin. Neurol. Neurosurg.. 2006;108:456-460 Crossref
-  V. Leoni, T. Masterman, F.S. Mousavi, et al. Diagnostic use of cerebral and extracerebral oxysterols. Clin. Chem. Lab. Med.. 2004;42:186-191
-  I. Bjorkhem, A. Cedazo-Minguez, V. Leoni, S. Meaney. Oxysterols and neurodegenerative diseases. Mol. Asp. Med.. 2009;30:171-179 Crossref
-  B.D. Trapp, J. Peterson, R.M. Ransohoff, R. Rudick, S. Mork, L. Bo. Axonal transection in the lesions of multiple sclerosis. N. Engl. J. Med.. 1998;338:278-285 Crossref
-  V. Leoni, T. Masterman, U. Diczfalusy, G. De Luca, J. Hillert, I. Bjorkhem. Changes in human plasma levels of the brain specific oxysterol 24S-hydroxycholesterol during progression of multiple sclerosis. Neurosci. Lett.. 2002;331:163-166 Crossref
-  C. van de Kraats, J. Killestein, V. Popescu, et al. Oxysterols and cholesterol precursors correlate to magnetic resonance imaging measures of neurodegeneration in multiple sclerosis. Mult. Scler.. 2014 Apr., 20;4:412-417 (Epub 2013 Aug. 19) Crossref
-  C.E. Teunissen, C.D. Dijkstra, C.H. Polman, E.L. Hoogervorst, K. von Bergmann, D. Lutjohann. Decreased levels of the brain specific 24S-hydroxycholesterol and cholesterol precursors in serum of multiple sclerosis patients. Neurosci. Lett.. 2003;347:159-162 Crossref
-  V. Leoni, C. Mariotti, L. Nanetti, et al. Whole body cholesterol metabolism is impaired in Huntington's disease. Neurosci. Lett.. 2011;494:245-249 Crossref
-  V. Leoni, C. Caccia. Potential diagnostic applications of side chain oxysterols analysis in plasma and cerebrospinal fluid. Biochem. Pharmacol.. 2013;86:26-36 Crossref
-  C. Malmestrom, S. Haghighi, L. Rosengren, O. Andersen, J. Lycke. Neurofilament light protein and glial fibrillary acidic protein as biological markers in MS. Neurology. 2003;61:1720-1725 Crossref
-  J.N. Lycke, J.E. Karlsson, O. Andersen, L.E. Rosengren. Neurofilament protein in cerebrospinal fluid: a potential marker of activity in multiple sclerosis. J. Neurol. Neurosurg. Psychiatry. 1998;64:402-404 Crossref
-  C.H. Polman, P.W. O'Connor, E. Havrdova, et al. A randomized, placebo-controlled trial of natalizumab for relapsing multiple sclerosis. N. Engl. J. Med.. 2006;354:899-910 Crossref
-  N. Putzki, O. Yaldizli, M. Maurer, et al. Efficacy of natalizumab in second line therapy of relapsing-remitting multiple sclerosis: results from a multi-center study in German speaking countries. Eur. J. Neurol.. 2010;17:31-37 Crossref
-  M. Gunnarsson, C. Malmestrom, M. Axelsson, et al. Axonal damage in relapsing multiple sclerosis is markedly reduced by natalizumab. Ann. Neurol.. 2011;69:83-89 Crossref
-  C.H. Polman, S.C. Reingold, B. Banwell, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann. Neurol.. 2011;69:292-302 Crossref
-  J.F. Kurtzke. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology. 1983;33:1444-1452
-  P. Scherer. Cognitive screening in multiple sclerosis. J. Neurol.. 2007;254(Suppl. 2):II26-II29 Crossref
-  S.A. Morrow, P.W. O'Connor, C.H. Polman, et al. Evaluation of the symbol digit modalities test (SDMT) and MS neuropsychological screening questionnaire (MSNQ) in natalizumab-treated MS patients over 48 weeks. Mult. Scler.. 2010;16:1385-1392 Crossref
-  L.K. Sheridan, H.E. Fitzgerald, K.M. Adams, et al. Normative symbol digit modalities test performance in a community-based sample. Arch. Clin. Neuropsychol.. 2006;21:23-28 Crossref
-  R.H. Benedict, J.A. Duquin, S. Jurgensen, et al. Repeated assessment of neuropsychological deficits in multiple sclerosis using the Symbol Digit Modalities Test and the MS Neuropsychological Screening Questionnaire. Mult. Scler.. 2008;14:940-946 Crossref
-  C.E. Teunissen, A. Petzold, J.L. Bennett, et al. A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking. Neurology. 2009;73:1914-1922 Crossref
-  S. Dzeletovic, O. Breuer, E. Lund, U. Diczfalusy. Determination of cholesterol oxidation products in human plasma by isotope dilution-mass spectrometry. Anal. Biochem.. 1995;225:73-80 Crossref
-  I. Bjorkhem, S. Meaney. Brain cholesterol: long secret life behind a barrier. Arterioscler. Thromb. Vasc. Biol.. 2004;24:806-815 Crossref
-  C.E. Teunissen, S. Floris, M. Sonke, C.D. Dijkstra, H.E. De Vries, D. Lutjohann. 24S-hydroxycholesterol in relation to disease manifestations of acute experimental autoimmune encephalomyelitis. J. Neurosci. Res.. 2007;85:1499-1505 Crossref
-  G.L. Vega, M. Weiner, H. Kolsch, et al. The effects of gender and CYP46 and apo E polymorphism on 24S-hydroxycholesterol levels in Alzheimer's patients treated with statins. Curr. Alzheimer Res.. 2004;1:71-77 Crossref
-  M. Axelsson, C. Malmestrom, M. Gunnarsson, et al. Immunosuppressive therapy reduces axonal damage in progressive multiple sclerosis. Mult. Scler.. 2014;20:43-50 Crossref
-  R.M. Ransohoff. Natalizumab for multiple sclerosis. N. Engl. J. Med.. 2007;356:2622-2629 Crossref
-  R.H. Benedict, M.P. Amato, J. Boringa, et al. Brief International Cognitive Assessment for MS (BICAMS): international standards for validation. BMC Neurol.. 2012;12:55
-  J.M. Sonder, J. Burggraaff, D.L. Knol, C.H. Polman, B.M. Uitdehaag. Comparing long-term results of PASAT and SDMT scores in relation to neuropsychological testing in multiple sclerosis. Mult. Scler.. 2014;20:481-488 Crossref
a Department of Clinical Neuroscience and Rehabilitation, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden
b Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, Mölndal, Sweden
c UCL Institute of Neurology, Queen Square, London, UK
d Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden
e Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden
⁎ Corresponding author at: Department of Neurology, Sahlgrenska University Hospital, Blå stråket 7, 413 45 Gothenburg, Sweden.
1 These authors contributed equally.
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