Ykl 40

Formalin-fixed and paraffin-embedded brain sections of frontal cortex were dewaxed and pretreated with Tris/EDTA buffer pH 9 at high temperature. The following primary antibodies were incubated overnight at 4 °C: polyclonal goat anti-YKL-40 (R&D Systems, AF2599, dilution 1:200), rabbit anti-GFAP (Sigma, G9269, dilution 1:500), phosphorylated tau clone AT8 (Thermo Scientific, MN1020, dilution 1:1000), monoclonal mouse anti-MAP2 (Sigma, M4403, dilution 1:500) and rabbit anti-Iba-1 (Wako Chemicals, 019-19741, 1:500). For IHC, the endogenous peroxidase activity was blocked, sections were HRP-labelled (Dako, Glostrup, Denmark, dilution 1:200) and the reaction was visualized by the EnVision+ system peroxidase procedure (DAKO, Glostrup, Denmark). For IF, sections were incubated for 1 h with Alexa Fluor 488, 555 or 647 (Invitrogen, Carlsbad, CA, USA, dilution 1:1000) secondary antibodies and stained with Sudan black B (Merck, Whitehouse Station, NJ, USA) to mask tissue autofluorescence. Nuclei were stained with Hoechst 33258 (Life Technologies, Carlsbad, CA, USA, dilution 1:1000), and coverslips were added with Immu-Mount (Fisher Scientific, Rockford, USA) mounting medium.

Plasma samples were obtained by venipuncture after an overnight fast. Glucose was analyzed based on enzymatic spectrophotometric reactions by an automated analyzer (Hitachi Modular P800, Roche, Basel, Switzerland). Serum concentrations of triacylglycerol and free fatty acids (FFA) were measured by enzymatic methods using commercially available kits (Infinity^™, Thermo Electron Corporation, Melbourne, Australia) [26 (link)]. High sensitivity C-reactive protein (CRP) and fibrinogen concentrations were determined as previously reported [27 (link)]. The carcinoembryonic antigen (CEA) was analyzed based on electrochemiluminescence (ECL) reactions by an automated analyzer (Cobas 8000, Roche, Basel, Switzerland). White blood cell (WBC) count was measured using an automated cell counter (Beckman Coulter, Inc., Fullerton, CA). Commercially available ELISA kits were used to assess circulating levels of OPN (R & D systems, Minneapolis, MN), YKL-40 (R & D systems), TNC (IBL International GMBH, Hamburg, Germany) and LCN-2 (R & D systems) according to the manufacturer’s instructions. The intra- and interassay coefficients of variation were: 3.2 and 5.9% for OPN; 4.6% and 6.0% for YKL-40; 4.9% and 5.4% for TNC and 3.7% and 6.5% for LCN-2, respectively.

Glial fibrillary acidic protein (GFAP) and S100 calcium binding protein B (S100β), previously used to indicate neurodegeneration (Petzold et al., 2003 (link); Yardan et al., 2011 (link)), and PMS (Avsar et al., 2012 (link); Michetti et al., 2012 (link)) were measured in all samples with sufficient vCSF (27 PMS and 15 controls, 51% of samples) using western blot, with protein levels normalised to total protein level measured with BLOT-FastStain™. Chitinase 3 like 1 (CHI3L1), used to differentiate active and inactive PMS (Sellebjerg et al., 2017 (link)), and Chitinase 3 like 2 (CHI3L2), used to differentiate PMS and RRMS (Hinsinger et al., 2015 ), were measured by ELISA (Cusabio, YKL-40 and R&D systems DC3 L10, respectively) in the same samples as above (i.e. 27 PMS and 15 controls) as per manufacturers guidelines.