B cells, also known as B lymphocytes or lymphoid lineage cells, are a historically understudied cell population with regard to brain-related injuries and diseases. However, an increasing number of publications have begun to elucidate the different phenotypes and roles B cells can undertake during central nervous system (CNS) pathology, including following ischemic and hemorrhagic stroke. B cell phenotype is intrinsically linked to function following stroke, as they may be beneficial or detrimental depending on the subset, timing, and microenvironment. Factors such as age, sex, and presence of co-morbidity also influence the behavior of post-stroke B cells. The following review will briefly describe B cells from origination to senescence, explore B cell function by integrating decades of stroke research, differentiate between the known B cell subtypes and their respective activity, discuss some of the physiological influences on B cells as well as the influence of B cells on certain physiological functions, and highlight the differences between B cells in healthy and disease states with particular emphasis in the context of ischemic stroke.

Background: N-methyl-D-aspartate (NMDARs) play a critical role in neuronal excitotoxicity after stroke. The actions of NMDARs have been shown mostly in obligatory GluN1 subunits on neurons and not GluN2A/B subunits. In B cells, these subunits have not been highly characterized though the presence of NMDARs has been shown. The function of the GluN2A/B subunits can be neuroprotective or pro-death in neurons, respectively. We hypothesized that GluN2A and GluN2B subunit presence on B cells would be affected by exposure to extracellular glutamate. Methods: Splenic B cells were isolated from 3-4mo-old C57BL/6 male mice via magnetic separation and treated with physiologic levels of L-glutamate (glu; 1uM) in the presence or absence of 5ug/mL LPS. B cell cytospins were stained for B220, GluN2A, and GluN2B, imaged using confocal microscopy, and quantified in FIJI. An average of 10.7 B cells were quantified per image at 80-157x magnification. RGB channels of the z-stacks were quantified to identify positive B220 expression. The z-stacks were split into 2D images and quantified plane-by-plane to identify GluN2A/B subunit clusters. Each cluster of subunits was recorded per cell in view across all planes of the original z-stack to yield total subunit count. Groups included 14-43 B cells quantified, and the number of subunits per cell were analyzed via ordinary two-way ANOVA, Sidak post-hoc test (Graphpad Prism). Significance was p<0.05. Results: There was an average of 19.3±7.2 GluN2A subunits and 19.0±5.0 GluN2B subunits per cell for unstimulated, untreated B cells. Neither glu treatment (p=0.23) nor LPS stimulation (p= 0.10) impacted the number of GluN2A subunits per B cell. LPS decreased GluN2B subunits when compared to unstimulated B cells (11.1±5.1 subunits; p=0.02). Glu treatment normalized GluN2B subunits per B cell near untreated baseline levels (18.2±11 subunits per cell; p=0.01), resulting in an interaction between LPS stimulation and glu treatment in B cells (F (1, 86) =6.180, P=0.015). Conclusions: Our data suggests activated B cells downregulate GluN2B-containing NMDARs following LPS stimulation. This downregulation mimics that of NMDAR activity on neurons upon excitoxicity (PMID: 24361499) but future studies should confirm GluN2B internalization.

Background: Stroke injury following a middle cerebral artery occlusion (MCAo) induces a rapid migration of leukocytes into the injured brain that lasts for weeks. The current study focuses on whether a focal cortical stroke using the distal MCAo (dMCAo) model induces similar long-term immune cell diapedesis into the brain parenchyma as seen following transient (t)MCAo stroke. Methods: Cells were isolated from spleens and brain hemispheres of adult 1-year old male C57BL/6J (B6; Jackson Labs) mice 30 days after a dMCAo (n=10). Sham animals (n=5) received a craniotomy without the distal MCA ligation. Peripheral migration was assessed in the spleen and brain using flow cytometry (FACSymphony) to identify viable (Ghost dye 780) CD3, CD4, CD8b, CD11b, Ly6C/Ly6G, CD19, CD45, and NK1.1 leukocytes. Populations were analyzed with FlowJo and assessed via repeated measures two-way ANOVA, Sidak post-hoc test (Graphpad Prism). Significance was p<0.05. Results: CD45 + leukocytes were elevated in the ipsilesional (ipsi) hemisphere compared to the contralesional hemisphere (p=0.01) after dMCAo, though a group hemispheric effect (F(1,13)=5.4; p=0.04) suggests long-term inflammation in sham-treated mice. Hemispheric effects also occurred for CD8 + T cells (p=0.046), B cells (p=0.03), monocytes (p=0.01), and macrophages (p=0.06), with elevations in both dMCAo and sham-treated mice in the ipsi vs. contralesional hemispheres. Only monocyte populations were significantly elevated (p=0.03) in dMCAo vs. sham mice. Conclusions: Our study shows that immune cells remain elevated in the injured hemisphere at 30 days after a focal stroke confined to the neocortex, but inflammation occurred in both sham and dMCAo-treated animals. Only monocytes were differentially affected by dMCAo, and infiltrating cell numbers are not as robust as after tMCAo. This demonstrates a long-term injury response from the craniotomy in the dMCAo model that should be considered for long-term studies using this model.