Cortical microinfarcts caused by single penetrating vessel occlusion lead to widespread reorganization across the entire brain in a CX3CR1 dependent manner

Abstract

Mann-Whitney) and deeper (p=0.0007, student's t-test) lesions (Figure 2E,F). Thirty-two percent of all 101 lesions reached the white matter (15 of 46), while most of these were veins (9 of the 15). 102 2.3. Microglia/macrophages migrate along the corpus callosum following penetrating vessel occlusion 103 To further understand the nature of the observed trail, we performed a immuno-histological survey 104 to identify cells that uptake the FITC-dextran. We chose the 14 days post-occlusion time point from the 105 previous experiments for the analysis, as at this time a clear trail was observed along the corpus callosum 106 in most animals. Consistent with previous reports ([28, 29, 30]), we observed microglia/macrophages and 107 astrogliosis surrounding the lesion site. While microglia/macrophages occupied the infarct, astrocytes 108 did not infiltrate the lesion core, forming a glial scar, as previously reported ([31]) (Supplementary Figure 109 S5). Notably, microglia/macrophages in the penumbra were also FITC-positive, while astrocytes were 110 not co-localized with FITC (Supplementary Figure S6). 111Importantly, the vast majority of the FITC-dextran outside the infarct itself was found to be in-112 side cells, specifically microglia/macrophages (83% positive for both Iba1 and FITC). In line with this 113 finding, 51% of the FITC-positive cells were also positive for CD45 which is preferentially expressed by 114 macrophages and activated microglia (as opposed to Iba1 which is constitutively expressed). In contrast, 115FITC was rarely found inside astrocytes (2.6% positive for both GFAP and FITC). Doublecortin (DCX), 116 a marker for newly formed neurons, was also not colocalized with the FITC-dextran (¡2%). Eight per-117 cent of the FITC-positive cells were oligodendrocytes (colocalized with Olig2). This last observation was 118 replicated using transgenic mice expressing TdTomato under the control of the NG2 promoter, labeling 119 pericytes and oligodendrocytes precursor cells [32] (OPCs; 6% positive for both TdTomato and FITC; 120 Figure 5 A+B).121To verify if the trail is not formed due to passive dextran leakage but as a result of actual mi-122 croglia/macrophage spatial reorganization, we performed again single artery occlusion albeit in CX3CR1 GFP/+ 123 mice, without labeling blood plasma with FITC-dextran. In the CX3CR1-GFP mice either one (heterozy-124 6 gous) or two (homozygous) copies of the CX3CR1 allele are replaced with GFP in cells under control of 125 the endogenous CX3CR1 locus (microglia, macrophages, dendritic cells and more). Arteries were labeled 126 with Alexa fluor 633 hydrazide, a specific arterial marker [33] with good spectral separation from the 127 GFP expressed under CX3CR1 [34] (Figure 5C left). A photothrombotic occlusion was induced, result-128 ing in a small infarct (Figure 5C right). In line with our previous experiments, fourteen days following 129 occlusion of a single penetrating artery, a trail of CX3CR1+ cells formed along the corpus callosum. In 130 general, expressio...