Computational protocol: Experimental Cerebral Malaria Pathogenesis—Hemodynamics at the Blood Brain Barrier

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Protocol publication

[…] Multiple time sequences and 3D stacks were recorded for quantification of the number of arrested leukocytes in the vascular lumen. Depending on the experimental conditions, 20–45 postcapillary venules and arterioles were analyzed per mouse. Arrested leukocytes were defined in each vessel segment as cells that did not detach from the endothelial lining within the observation period. CD8+ T cells, CD4+ T cells, neutrophils, monocytes, and macrophages were quantified by counting the number of cells per square millimeter of vessel surface . The relative density of the various leukocyte populations was determined in multiple fields of view per experimental condition and expressed as the mean ± SEM of arrested cells as well as the percentage of the total cell number. Velocities were measured with Imaris Track as described .To quantify endothelial ICAM-1 expression, confocal 3D stacks of postcapillary venules or similarly sized arterioles were acquired from 2 mice each infected with PbA, PyXL, or no parasites. The fluorescence signal intensity across 10 (PbA) or 12 (PyXL and control) vessel volumes was collected from 3D data and quantified in ImageJ. To quantify leukocyte ICAM-1 expression, the relative fluorescence emission from individual leukocytes was measured from mice infected with PbA or PyXL. As for endothelial ICAM-1 expression, confocal 3D stacks were collected and projections were created in AutoDeblur. A total of 6 stacks from 2 mice per experimental condition were analyzed. [...] The cortical microvasculature was imaged with an inverted Leica TCS SP2 AOBS confocal system as described . Time series and 3D data sets were acquired with Leica Confocal Software , , . Imaris 7.4 (Bitplane, Saint Paul, MN), Image-Pro Plus (Media Cybernetics, Bethesda, MD), AutoDeBlur (Media Cybernetics, Bethesda, MD), and NIH ImageJ were used for further image analysis, deconvolution, and 3D reconstruction. […]

Pipeline specifications

Software tools Imaris, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus, Plasmodium yoelii, Homo sapiens, Plasmodium berghei ANKA
Diseases Hypertension, Nervous System Diseases, Wounds and Injuries, Malaria, Cerebral