by It must be noted that our study used mature adult mice, and further understanding would necessitate the incorporation of female and aged ( 18 mo) mice. neuroprotection that could be induced by B cells is usually a direct neurotrophic effect of B cells on neurons at risk for cell death (14). Using an in vitro approach to determine a direct role in neuronal protection, mixed cortical cells were subjected to 2 h of oxygen-glucose deprivation (OGD) followed immediately by addition of na?ve B cells to the culture for 4 d (Fig. 2 and 0.001) and loss of arborization ( 0.01; Fig. 2 and 0.05) and loss of dendritic arborization ( 0.05). In the absence of ischemic injury, the addition of na?ve B cells increased the number of surviving MAP2+ neurons ( 0.05; Fig. 2 0.05, 0.1:1.0 ratio; 0.01, 1:1; Fig. 2 0.05; Fig. 2and 0.05), suggesting that IL-10 is a redundant mechanism that supports maintenance of mature neurons with dendrites. In summary, these data confirm the capability of na?ve B cells for direct neuronal protection in vitro by an IL-10 mechanism within the context of neuronal ischemic injury. Open in a separate windows Fig. 2. B cells induce a (S)-(?)-Limonene neurotrophic effect in mixed cortical cultures. (and test was used for untreated non-OGD:OGD comparisons. Data graphed as mean SD, and significance determined by nonparametric one-way ANOVA or Students test (* 0.05, ** 0.01, *** 0.001 vs. untreated control unless C5AR1 otherwise indicated by brackets). B Cells Are Present within the Parenchyma of the Poststroke Brain. At the whole-brain level, B cells are an abundant leukocyte population (S)-(?)-Limonene within the stroke-injured brain 48 h after stroke onset (22, 23). In fact, the cortical and subcortical brain vascular endothelium exhibits an up-regulation of CXCL13, a B cell homing chemokine (22). Recent advances in volumetric whole-brain imaging methods including serial two-photon tomography (STPT) have enabled automated, unbiased methods to visualize and computationally detect signals of interest, including fluorescently labeled cell populations as well as neuronal substructures in the entire brain (24C26). We established a custom pipeline including STPT, supervised machine (S)-(?)-Limonene learning-based pixel classification, and image registration to visualize and quantify adoptively transferred immune cells labeled with the fluorophore e450 throughout the whole mouse brain. We verified this methodology to accurately quantify labeled CD8+ T cell diapedesis into the whole brain after tMCAo in Poinsatte et al., 2019 (27). The output of our machine learning-based pixel classification step is visualized as a probability map of pixels automatically detected by the trained algorithm as B cells (Fig. 3 0.05 vs. respective PBS-treated control). To quantify whole brain neuroinflammation, we used two cohorts of mice: (i) B cell-depleted recipient hCD20+ mice or wild type (WT) hCD20? B cell donor littermate controls, with all recipient mice receiving rituximab prior to tMCAo to target hCD20+ B cells for depletion (29) (Fig. 3 0.05 for both hemispheres vs. PBS; Fig. 3tests (* 0.05, ** 0.01 vs. PBS controls unless indicated by brackets). Table 1. STPT data for e450+ pixels for each brain region = 3)B cells (= 4)valuePBS control (= 3)B cells (= 4)valueLaterality indextest: * 0.05; bolded text, 0.06. B cell diapedesis was elevated in five brain regions associated with motor function (i.e., cerebral cortex,.
