Fig. 2: Inflammatory cytokines in brain tissue are detected before the arrival of peripheral immune cells.

a Diagram outlining the regions of the brain, relative to infarct location: contralateral (CL), ipsilateral (IL), peri-infarct tissue and core-infarct tissue. Image created with BioRender.com. b–f Mice were subjected to photothrombotic (PT) stroke for 3 or 24 h, and perfused brains were homogenized, and supernatant was analyzed using a LEGENDplex cytokine panel. b Analytes taken from infarct core homogenates were measured for cytokines and presented as log₂ fold-change over sham control. Concentration (pg/g of brain tissue) of c IL-1α d IL-1β, e TNF-α and f IL-6 in regions of the brain at 3 and 24 h post-stroke is shown (sham n = 10, 3 h PT n = 9, 24 h PT n = 10). g–l. Mice were subjected to PT Stroke for 3 or 24 h, perfused brains were dissociated, and cells were stained for analysis by spectral flow cytometry. g Gating strategy of single, viable cells (see also Fig. S2) to identify neutrophils, monocytes/monocyte-derived macrophages and microglia. Total count of h neutrophils (CD45^hiCD11b⁺Ly6G⁺), i monocytes (CD45^hiCD11b⁺Ly6G⁻F4/80^loLy6C⁺), j monocyte-derived macrophages (CD45^hiCD11b⁺Ly6G⁻F4/80^midLy6C⁺) and k microglia (CD45^midCD11b⁺Ly6G⁻F4/80^hiLy6C⁻) in contralateral (CL) or ipsilateral (IL) regions of the brain (Sham n = 4, PT n = 6). l Representative fluorescent image of a 30 µm section of a Catchup^IVM-red (neutrophil reporter; red) perfused mouse brain at 24 h post-stroke, with DAPI (blue) and anti-GPIbβ (platelets; cyan), imaged using an Olympus VS120 slide scanner. Arrows point to extravasated neutrophils, and the dotted line indicates the infarct core region (n = 3). Graphs are presented as mean ± SD. A one-way ANOVA was performed for the analysis of multiple groups. *p  <  0.05, **p  <  0.01, ***p  <  0.001, ****p  <  0.0001, ns no significance.