Correction: Platelets Recognize Brain-Specific Glycolipid Structures, Respond to Neurovascular Damage and Promote Neuroinflammation

[This corrects the article DOI: 10.1371/journal.pone.0058979.].

. Depletion of platelets and white blood cell populations and its effect on the ability of brain lipid rafts to cause an anaphylactic-like reaction 1  Table S2. Analysis of the composition and ability of brain lipid rafts to cause an anaphylactic-like reaction 1
2 100 l of lipid rafts were injected i.v. per mouse. 3 The amount of phospholipids was determined and the concentrations of lipid rafts from various organs were adjusted to the concentration of phospholipids of the lipid rafts from brain (10 g/ml); 100 l/mouse of the lipid rafts (1 g of phospholipids per mouse) were injected i.v.

Receptor/ molecule
Method of blocking 2
Mice were injected i.v. with the brain lipid rafts obtained by the homogenization of brain tissue in PBS with 0.5% Triton X-100 as described in Methods. The anaphylactic-like reaction (see Methods for details) was observed during a ten-minute period and scored as follows: 1) restless behavior; 2) loss of consciousness; 3) dyspnea; 4) death. The anaphylactic score (a) and body temperature (b) with mean  S.E. of the total number of animals from the five experiments with groups of five mice per experiment is shown. Figure S2. Fractionation of brain lipid rafts. Lipid rafts were isolated by the homogenization of brain tissue in PBS with 0.5% Triton X-100, overlayed on a 40%/35%/5% sucrose discontinuous gradient and centrifuged at a high speed as described in Methods. The total of the 11 fractions shown in (a) were analyzed by taking 1 ml portions from the centrifuge tube (fractions are numbered from bottom to top) and by measuring the concentration of phospholipids (b) and cholesterol (d) in each fraction. Four bands were also identified visually in the centrifuge tube as shown in (b), separated, washed with PBS and re-suspended in equal volumes identical to the initial volume of non-separated brain lipid rafts. For each of the four fractions identified in (b), anaphylaxis was tested by the i.v. injection of 100 l/mouse of fractionated lipid rafts (c) and the concentrations of phospholipids and cholesterol were measured (e). The amount of the transferin receptor (TfR) was measured by Western blot for all the 11 fractions (g) and for the four fractions (b) identified visually (h). Lipid rafts were isolated from the astroglial C8-D1A line and treated with proteinase K, saponin, sphyngomyelinase, neuroaminidase, -galactosidase and fucosidase. Non-treated and treated lipid rafts were injected i.v. and the anaphylactic-like reaction was assessed during a ten-minute period. The mean maximum score ± S.E. of the total number of animals of the three separate experiments with groups of 4-5 mice in each experiment is shown.  Abbreviations: BCbrain capillary, BV-blood vessel, SBV-superficial blood vessel.    (a) Mouse platelets were co-incubated with brain lipid rafts or thrombin as in Suppl. Fig. 8 and the concentration of intracellular Ca 2+ was measured using a Fura 2M fluorescent probe and TECAN spectrofluorometer. The arrow indicates the time when the lipid rafts or thrombin were added to the platelets.
(b) Mouse platelets were co-incubated with brain lipid rafts and the concentration of IL-1 in the platelet free supernatant was measured by ELISA as described in Methods.
In (a,b) one representative experiment of the three is shown.    Mice were observed for a ten-minute period. Mean maximal clinical score ± S.E. of a group of 4-5 animals is shown (**, p<0.01; ***, p<0.001; unmanipulated mice vs. mice with EAE). (b) EAE was induced in B6 mice by immunization with MOG/CFA. PTx was injected on day 0 and day 2 post-immunization. Platelet rich plasma (PRP) was obtained from the unmanipulated mice or mice with EAE on day 2, 4 or 6 post-immunization. PRP was stained for CD41 and CD61 as described in Methods and analyzed by two-color flow cytometry. Forward (x-axes) and side scatter (y-axes) parameters and the percentages of events in the "microparticle" gates (subplatelet gates) are shown in the top contour plots, and the percentages of CD41 + microparticles are shown in the bottom histograms. (c) Platelet free plasma was obtained from the unmanipulated mice, or from the injected mice 10 minutes post-i.v. injection of brain lipid rafts, or the mice with EAE (d6-10). The levels of the platelet factor 4 (PF4/CXCL4) expression were assessed as described in Methods.  Figure S16. Immune cells in the CNS and spleen following intravenous administration of brain lipid rafts. Mice were injected i.v. with 75 l of brain lipid rafts and immune cells were analyzed in the CNS and spleen at 10min, 1hr, 24hr and 48hr after the injection.
Euthanized mice were perfused with PBS and mononuclear cells from the CNS were isolated using Percoll gradient as described in Methods. The cells were stained for surface markers CD11b, CD45 and MHC class II and analyzed by three-color flow cytometry. Percentages of activated CD11b + CD45 low MHC class II + microglia (a), CD11b + CD45 hi macrophages (c) and CD11b -CD45 hi lymphocytes (e) are shown.
In a-f, the mean percentage ± S.E. of the total number of animals from the three separate experiments with groups of 4-5 animals is shown.