Mice were on C57BL/6J genetic background (at least 10 back-crosses) and WT C57BL/6J mice were used as control. For experiments, 7/11-week-old mice were kept in filtered-cages in a P2 animal facility. All animal experimental protocols complied with the French ethical and animal experiments regulations and were approved by the Ethics Committee for Animal Experimentation of CNRS Campus Orleans (N° CLE CCO 2011–028 to V.Q., UMR7355). Plasmodium berghei ANKA (PbA) 15cy1 line constitutively expressing GFP under EF1α-promoter control, was obtained from Dr. A. Waters [23]. Mice were infected intraperitoneally with buy INCB024360 105 parasitized erythrocytes

as described [41]. Alternatively, mice were infected intravenously with 1000 motile sporozoites obtained from salivary gland homogenate of day-21-PbA-infected females Anopheles stephensi. Mice were observed daily and scored for ECM neurological signs, namely ataxia, paralysis, and coma. Parasitaemia was assessed with EGFP-PbA as described [41] and fluorescent cells analyzed by BD CANTO II flow cytometer. Data were acquired by using DIVA software (BD Bioscience, Rungis, France) and analyzed with FlowJO software (Treestar, Ashland, USA). Blood was drawn under Isofluorane anesthesia (CSP, Fontenay-sous-Bois,

France) into tubes containing Ethylenediaminetetraacetic Acalabrutinib (EDTA), (Vacutainer; Becton, Grenoble, France) and hematological parameters determined using 5-part-differential-hematology Carnitine palmitoyltransferase II analyzer MS9.5 (Melet Schloesing Laboratoires, France). Histological analysis was performed as described [41]. Briefly, mice were euthanized and perfused with intracardiac PBS/2 mM-EDTA. Organs were fixed in PBS/3.6%-formaldehyde for 72

h. Brain and lung microvascular obstruction was quantified on H&E stained sections, using a semiquantitative score with increasing severity of changes (0–5) by two independent observers, including a trained pathologist (B.R.). MRI and MRA measurements of cerebral vascular blood flow were performed using a horizontal 7 T/16 Bruker Biospec MR system (Bruker Biospin, Wissembourg, France), as described [8]. A homogeneous coil with inner diameter of 23 mm and length 55 mm was used to achieve uniform excitation and reception. A custom-built stereotaxic head holder was used to fix the animal into the birdcage coil (see below). The mice were anaesthetized with isoflurane (1.5%) and O2 (0.5 L/min) applied with a face mask allowing free breathing. Respiration was monitored using a balloon taped to the abdomen and connected to a pressure transducer (SA Instruments, Inc., Stony Brook, NY, USA). Body temperature was kept at 37 ± 0.5°C throughout the experiment, using warm water circulation. Brain lesions and global changes in tissue structure were accessed by T2 weighted (T2w) MR images using a MSME sequence, in both axial and sagittal planes, with the following parameters: RARE factor = 8, TR/TEeff.