Infarct volume after MCAO-reperfusion. Mice were sacrificed by PFA perfusion and brains were collected for histology at 6, 24, or 72 hr following MCAO-reperfusion. Representative serial sections collected every 600 μm from bregma −3.7 to 3.5 were stained with cresyl violet for infarct volume determination. This MCAO-reperfusion protocol produced consistent, progressive infarcts of the ipsilateral (right) hemisphere.

The distribution of EYFP+DC accumulation in the ischemic hemisphere over time in response to MCAO-reperfusion. Brain maps were generated from sections processed for DAB-IHC to illustrate the temporal and spatial pattern of EYFP+DC in the ipsilateral ischemic hemisphere. Each mark represents approximately 2 cells; maps are not intended to display exact number. The blue dashed line defines the cerebral infarct.

Radiation chimeras demonstrated the distribution of brain resident versus peripheral DC populations. Representative brain sections from radiation chimeras 72 hr post-MCAO-reperfusion were processed by DAB-IHC for detection of EYFP+ cells (A). In CD11c/EYFP Tg host with WT BM donors, EYFP+DC (considered of brain origin) were abundant in the ipsilateral hemisphere and accumulated in the border regions of the infarct (white arrows). In WT hosts with BM from CD11c/EYFP Tg mice, EYFP+DC (considered of peripheral origin) were confined to the core regions of the cerebral infarct (black arrows). Higher magnification images of DAB-IHC sections from radiation chimeras (B) confirmed the presence of bDC in the border and peripheral DC in the core regions of the cerebral infarct at 72 hr post-MCAO-reperfusion. EYFP+DC from the CD11c/EYFP Tg host mice of brain origin, bDC, displayed ramified morphology and resided in the border region of the infarct. EYFP+DC of the WT host mice of BM origin, peripheral DC, populated the core of the infarct and appeared ovoid in morphology. To verify the location of sampling from core and border regions of the infarct, IFC staining for GFAP was conducted in neighboring serial sections and examined for presence (border) or absence (core) of astrocytes (D). Scale bars A = 400 microns, B = 100 microns (black), 50 microns (white) ; red = IFC for GFAP, green = EYFP+DC

Cells positive for EYFP and mature DC markers were increased in the ischemic hemisphere in response to MCAO-reperfusion. The mean (+/− SEM) percentage of EYFP+DC within the CD11b population was significantly increased at 24 hr and 72 hr post-MCAO-reperfusion (A). The percentage of cells within the CD11b population positive for mature DC markers was also increased in the ipsilateral hemisphere in response to MCAO-reperfusion (B) as represented by means (+/− SEM) for CD11c, MHC II, and the co-stimulatory molecules CD86 and CD80. * = p<0.05, ** = p<0.01, *** = p<0.001 compared to control

T lymphocytes infiltrated the ischemic hemisphere at 72 hr post-MCAO-reperfusion and accumulated in the border region near resident bDC. The neuroanatomical distribution of lymphocytes (arrows) relative to the bDC (asterisks) was examined by IFC for CD3+ T cells and B220+ B cells (A). CD3+ T cells (arrows) were found in high numbers in the infarct border region in close proximity to the resident EYFP+ bDC and their processes (asterisks). FACS analysis of the CD45+CD11b^NegLy6G^Neg cell population demonstrated a mean (+/− SEM) percent increase of lymphocytes at 72 hr post-MCAO-reperfusion (B). The majority of observed lymphocytes were CD3+CD4+ T cells (B, C). Scale bars = 50 microns. red = IFC for CD3 or B220, green = EYFP+DC, blue = DAPI

EYFP+DC accumulated in the ischemic hemisphere in response to MCAO-reperfusion. Representative brain sections from CD11c/EYFP Tg mice subject to sham surgery or MCAO-reperfusion were processed for DAB-IHC (A). Sham operated animals demonstrated a similar quantity and distribution of EYFP+ cells as in young naïve male mice, with cells observed primarily in the dentate gyrus of the hippocampus (DG), the interstitial nucleus of the posterior limb of the anterior commissure (IPAC), fiber tracts, hypothalamic nuclei (hyp), the piriform (pir ctx) and entorhinal cortices (ent ctx), as indicated by white arrows. At 6 hr post-MCAO reperfusion, no increase in EYFP+ cells was observed in the ipsilateral or contralateral hemispheres. At 24 and 72 hr-post MCAO-reperfusion, EYFP+ cells accumulated in the ischemic hemisphere and formed clusters in regions surrounding the cerebral infract (black arrows). The distribution of EYFP+DC in the ischemic hemisphere was compared to cresyl violet stained sections used for infarct volume determination (B), demonstrating that EYFP+DC were found in high densities in the border regions as compared to the core of the infarct. Higher magnification images demonstrated that at 24 hours post-MCAO-reperfusion, DC were observed in the border region, whereas the core of the infarct was devoid of EYFP+ cells (C). At 72 hr, DC were present in the core and border of the infarct, although cells in the core adopted an ovoid morphology compared to the ramified cells of the infarct border (D). To verify the location of sampling from core and border regions of the infarct, IFC staining for GFAP was conducted in neighboring serial sections and examined for presence (border) or absence (core) of astrocytes (E). Scale bars A, B = 400, C = 200, D = 200 (small), 100 (large), E = 50 microns; red = IFC for GFAP, green = EYFP+DC

Examination of mature DC markers on EYFP+DC within the core versus border region of the ischemic infarct at 72 hr post-MCAO-reperfusion by IFC. EYFP+DC that were immunopositive (white arrows) for CD11c (A) and MHC II (B) were frequently observed in the core of the infarct but were rare in the border region. Scale bars = 50 microns. red = IFC for CD11c or MHC II, green = EYFP+DC, yellow = merge

FACS analysis of CD45+ cell populations in CD11c/EYFP Tg mice following MCAO-reperfusion. The total number of CD45+ cells (mean, +/− SEM) recovered from the hemispheres of controls and mice subject to MCAO-reperfusion (A). The average percentage of the three CD45+ cell populations, CD11b+Ly6G+ neutrophils (black), CD11b+Ly6G^Neg MG/macrophage/DC (white), and CD11b^NegLy6G^Neg other immune cells (gray), for control and ischemic conditions are represented graphically in B, and an example of the gating strategy is depicted by FACS plot in (C). The CD11b+Ly6G^Neg MG/macrophage/DC population (white) was subsequently analyzed for expression of EYFP and phenotyped for activated DC markers; the other immune cell population (gray) was examined for the presence of lymphocytes.

The resident bDC and MG populations were isolated by gating on CD45 fluorescence intensity and examined for the presence of DC markers. The percentage of CD45^High cells increased in response to MCAO-reperfusion (A) as peripheral immune cells invaded the ipsilateral hemisphere. Comparison of mean (+/− SEM) percentage of CB11b+EYFP+ cells that were either CD45^High (black) or CD45^Int (white) showed that the majority of EYFP+DC at 24 hr were of CD45^Int brain resident origin, yet at 72 hr the percentage of CD45^Int and CD45^High cells did not differ (B). Both the CD45^IntCD11b+EYFP+ and CD45^HighCD11b+EYFP+ cell populations at 72 hr were positive for CD11c compared to CD11b+EYFP-cells (C). Examination of the CD45^Int resident cell populations revealed that the bDC upregulated MHC II and co-stimulatory molecules following MCAO-reperfusion (D). Compared to EYFP^Neg MG (white), a higher mean (+/−SEM) percentage of bDC (black) were positive for MHC II, CD80, and CD86. Assessment of MHC II+ versus CD80+ within the bDC at 72 hr (B) demonstrated that ~25% of the bDC were positive for both MHC II and CD80. B: *** = p<0.001 compared to CD45^High, D: ** = p<0.01, *** = p<0.001 compared to MG; ++ = p<0.01, +++ = p<0.001 compared to 24 hr