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Col1a1+ perivascular cells in the brain are a source of retinoic acid following stroke
Kathleen K. Kelly1,
Amber M. MacPherson1,
Himmat Grewal2,
Frank Strnad2,
Jace W. Jones3,
Jianshi Yu3,
Keely Pierzchalski3,
Maureen A. Kane3,
Paco S. Herson2,4,5 &
Julie A. Siegenthaler1
BMC Neuroscience volume 17, Article number: 49 (2016) Cite this article
Perivascular stromal cells (PSCs) are a recently identified cell type that comprises a small percentage of the platelet derived growth factor receptor-β+ cells within the CNS perivascular space. PSCs are activated following injury to the brain or spinal cord, expand in number and contribute to fibrotic scar formation within the injury site. Beyond fibrosis, their high density in the lesion core makes them a potential significant source of signals that act on neural cells adjacent to the lesion site.
Our developmental analysis of PSCs, defined by expression of Collagen1a1 in the maturing brain, revealed that PSCs first appear postnatally and may originate from the meninges. PSCs express many of the same markers as meningeal fibroblasts, including expression of the retinoic acid (RA) synthesis proteins Raldh1 and Raldh2. Using a focal brain ischemia injury model to induce PSC activation and expansion, we show a substantial increase in Raldh1+/Raldh2+ PSCs and Raldh1+ activated macrophages in the lesion core. We find that RA levels are significantly elevated in the ischemic hemisphere and induce signaling in astrocytes and neurons in the peri-infarct region.
This study highlights a dual role for activated, non-neural cells where PSCs deposit fibrotic ECM proteins and, along with macrophages, act as a potentially important source of RA, a potent signaling molecule that could influence recovery events in a neuroprotective fashion following brain injury.
Following CNS injury, the initial, local cellular response is protective, confining the injury and preventing extensive inflammation and neurodegeneration [7, 41], but large amounts of fibrosis and extracellular matrix (ECM) protein deposition can impede neuronal repair [8, 46]. Pericyte-like cells termed perivascular stromal cells (PSCs) or type "A" pericytes have been identified as a major source of ECM proteins that generate the fibrotic scar, separate from the well-characterized astroglial scar, within the injured CNS tissue following both spinal cord and stroke injury [9, 11, 31, 48]. Beyond their role in fibrotic scar formation, however, PSCs in the lesion core are uniquely positioned to interact with a variety of resident (glia, neurons, microglia) and infiltrating inflammatory cell types in the days and weeks post-injury.
Despite the profound response of PSCs to brain injury and their role in fibrotic scar formation, much remains unknown about this cell type. A few unique and defining characteristics of PSCs have been reported. These include (1) expression of platelet derived growth factor receptor-β (PDGFrβ), but not the pericyte marker desmin and (2) they are labeled in GLAST-CreErt [11] and Collagen1a1-GFP (Col1a1-GFP) [48] transgenic mouse lines. It is not clear, however, if the cells labeled by the two transgenic mouse lines are the exact same cell population. That said, both lines label a subset of perivascular cells that are PDGFrβ+/desmin-negative and cells labeled by the two lines increase in number and fill the lesion core following spinal cord injury in a similar manner. In addition to lineage ambiguity, developmental characterization of PSCs is lacking and other, non-fibrosis functions for PSCs post-injury have not been identified.
Activity of the Col1a1 promoter (Col1a1-GFP) has previously been shown to identify PSCs in the spinal cord [48]. Here we use protein expression of Col1a1 to identify PSCs and characterize the temporal and spatial distribution of Col1a1+ PSCs in the maturing brain. We observe that Col1a1+ PSCs are largely absent in the early postnatal brain, but increase in number over the first three postnatal weeks. Initially, Col1a1+ cells are associated with vessels near the meningeal surface, but as their numbers increase so does their depth within brain regions. This information, along with the observation that Col1a1+ PSCs express many of the same markers as meningeal fibroblasts, suggests that Col1a1+ PSCs may originate from the meninges during postnatal brain development. Meningeal fibroblasts express enzymes Raldh1 and Raldh2 required for synthesis of retinoic acid (RA), a hormone with diverse functions in the CNS including neurogenesis and cell survival. We find Col1a1+ PSCs in the un-injured brain express both Raldh1 and Raldh2. This sets up the possibility that expansion of PSCs capable of RA synthesis within a brain injury site creates a source of RA that can act on cells within and adjacent to the injury. We tested this idea using a focal brain ischemia model in mice (middle cerebral artery occlusion or MCAO) which has previously been shown to lead to a substantial increase in PSCs within the lesion site [9]. Analysis of Raldh1 and Raldh2 expressing cells at day 7 post-injury revealed that in addition to numerous Raldh1 and 2-expressing PSCs, Raldh1 was expressed by macrophages within the lesion core. Consistent with an increase in Raldh1 and Raldh2 expressing cells, RA levels were significantly elevated in the ischemic hemispheres as was RA signaling in astrocytes and neurons in the peri-infarct region.
Wild type [postnatal day 0 (P0), P7, P14, P21 and 6 week (wk)] C57Bl/6J, were used throughout this study unless otherwise noted. RA signaling was assessed using the retinoic acid response element or RARE transgenic mouse line (RARE-hsp68-LacZ) [43] purchased from Jackson Laboratories. Strict adherence to The Recommended Guide for Care and Use of Laboratory Animals issued by the National Institutes of Health was performed using animal protocols approved by the Institutional Animal Care and Use Committee at The University of Colorado, Anschutz Medical Campus. All surgery was performed under anesthesia and suffering was minimized.
Tissue collection, immunohistochemistry and imaging
Animals were anesthetized with cryoanesthesia (P0) or 60 mg/kg pentobarbital (≥P6) followed by transcardiac perfusion initially with PBS to clear red blood cells then 4 % paraformaldehyde. Whole brain tissue was isolated and incubated in 4 % paraformaldehyde for 2 h, 20 % sucrose overnight and mounted in Tissue Tek for cryosecting. Sections at 12 µm were generated from fixed brain tissue and immunohistochemistry was performed as described in Zarbalis et al. [53] using the following antibodies: mouse anti-Col1a1 1:100 (Sigma); rabbit anti-Col1 1:100 (Abcam); rabbit anti-PDGFrβ 1:200 (Cell Signaling Technology); rat anti-PDGFrβ 1:200 (Novus Biologicals); rat anti-PDGFrα 1:200 (BD Bioscience); mouse anti-CoupTF2 1:300 (R&D); rabbit anti-Raldh1 1:200 (Abcam); rabbit anti-Raldh2 1:400 (Sigma-Aldrich); mouse anti-CRABP1 1:100 (Abcam); rabbit anti-CRABP2 1:100 (Proteintech); mouse CD68 1:500 (Dako); rabbit anti-β-galactosidase 1:500 (MP Biomed); chicken anti-β-galactosidase 1:500 (Abcam); mouse anti-NeuN 1:500 (Millipore); rabbit anti-glial fibrillary acidic protein (GFAP) 1:500 (Sigma-Aldrich). Following incubation with primary antibodies, sections were incubated with appropriate Alexafluor-conjugated secondary antibodies (Invitrogen) at 1:500, Alexafluor 633-conjugated isolectin-B4 (Invitrogen) at 1:500 to label blood vessels and microglia [3], and DAPI (4′,6-diamidino-2-phenylindole) to label nuclei (Invitrogen). The immunostained tissue sections were imaged using confocal imaging (Zeiss 780 LSM).
Analysis of PSCs in postnatal and adult brain
Sagittal sections of P0, P7, P14, P21 and adult (6–8 week old mice) brains were produced as described above and immunostained with Col1a1, IB4 and DAPI. Tile scan, 10× confocal images of the entire sagittal section of the brain at each time point were collected using a Zeiss 780 LSM and associated Zen software. Brain regions were analyzed using the Zen Blue software for (1) the number of vessels (defined by labeling with IB4) with Col1a1+ cells (as defined by DAPI+ nuclei) per area of a brain region (area: cm2) and (2) average distance of vessels surrounded by Col1a1+ cells from the pial surface (distance: µm). Regions measured at each time point were the cerebral cortex, hippocampus, striatum, hypothalamus, thalamus, midbrain, hindbrain/cerebellum. We used the Allen Mouse Brain Atlas to define regional boundaries at each post-natal time point. For quantification of Raldh1 and Raldh2 expressing cells in stroke brains at 7 days post-MCAO, 10, 10× fields per brain at the level of the ischemic core were analyzed for the number of Raldh1+ or Raldh2+ cells. Individual cells with positive staining were defined by presence of DAPI+ nuclei. The ischemic core was defined by high density of IB4+ macrophages with amoeboid morphology. The number of positive cells was divided by the area analyzed (mm2) to generate a cell density for each field and averaged across all images to generate a mean for each brain. For comparison, we analyzed the non-ischemic cerebral cortex in the opposite hemisphere for Raldh1+/DAPI+ and Raldh2+/DAPI+ cell density in the same manner. To analyze the number of GFAP+ astrocytes and NeuN+ neurons with RARE-LacZ transgene activity (defined by expression of β-galactosidase via antibody) in the non-ischemic cerebral cortex and peri-infarct region, we collected 20×, 4-by-4 tile scan confocal images of the peri-infarct region and a corresponding region in the cerebral cortex of the opposite hemisphere. For each brain, 4 tile scan images were collected in each hemisphere and analyzed for the number of β-gal+/GFAP+ and β-gal+/NeuN+ per area (mm2). The peri-infarct region was defined by activated, GFAP+ astrocytes. For each time point and type of analysis, a minimum of 3 animals were used for analysis (n ≥ 3).
Middle cerebral artery occlusion (MCAO)
MCAO animals were generated as described in Kelley et al. [20]. Briefly, adult male, wildtype C57Bl/6J or RARE-hsp26-LacZ/+ maintained on the ICR background (8–12 weeks old) mice were anesthetized and following baseline measurements of blood flow, unilateral MCAO was performed by inserting a 6.0 nylon monofilament into the internal carotid artery through a midline ventral neck incision. The filament was positioned for occlusion at 6 mm from the internal carotid/pterygopalatine artery bifurcation for 60 min. After the occlusion period animals were removed from anesthesia and no pain relieving drugs were administered to prevent confounding of outcomes. Brain tissue was collected 7 days post MCAO as described for histology or analysis of retinoid and RA levels.
RA quantification
Non-injury or MCAO brains were hemisected and the cortex/striatum from the right and left hemispheres were harvested in low light conditions to prevent RA degradation, frozen separately and kept at −80 °C until assayed. Tissue was homogenized and retinoids were extracted with a liquid–liquid extraction method for retinoids that has been described in detail previously [17]. RA was quantified by high performance liquid chromatography (HPLC)-multistage tandem mass spectrometry using a Fast HPLC-multiple reaction monitoring cubed (LC-MRM3) method [14]. Retinol and total retinyl ester were quantified by high performance liquid chromatography–ultraviolet detection (HPLC–UV) [16]. Results are given as mole retinoid per gram tissue and expressed as mean ± SEM with n = 2 for each control brain hemisphere (right and left) and n = 3 for each stroke brain hemisphere (right and left). Statistical significance was assessed using a two-tailed, unpaired Student's t test (GraphPad).
One-Way ANOVA and Student–Newman–Keuls post hoc analysis were used to assess statistically significant differences for PSC counts and distances. To address correlation between cell counts and distance, Pearson Correlation was performed. To detect statistically significant differences in mean values between two groups, Student t-tests were used. The standard error of the mean (SEM) is reported on all graphs.
Temporal and spatial distribution of Col1a1+ PSCs in the early postnatal mouse brain
The Col1a1-GFP transgenic line has been used to identify a subset of perivascular cells that dramatically expand in number in response to a spinal cord injury [48] that have the same characteristics of PSCs described in stroke injury [9]. Therefore, Col1a1 expression can be used to identify PSCs in the uninjured brain. Using an antibody to Col1a1, we observe Col1a1+ cells in the meninges surrounding the mouse brain (Fig. 1A, B, carets). Within the brain parenchyma at P7 and P21, we observe some large diameter vessels with Col1a1+ cells surrounding them (Fig. 1A, B, arrows; arrows in A′, B′ insets indicate Col1a1+/DAPI+ cells surrounding IB4+ blood vessel). We next characterized the spatial and temporal distribution of vessels with associated Col1a1+ PSCs from P0 to adulthood. We did not observe Col1a1+ PSCs at E16.5 in the developing embryonic brain (data not shown). At P0, vessels with Col1a1+ cells were observed in the hippocampus, but very infrequently in all other brain regions analyzed (cortex, striatum, hypothalamus, thalamus, midbrain and hindbrain/cerebellum, Fig. 1C). PSC numbers in the hippocampus were reduced to the infrequent levels found in other brain regions at 1 week (P7, 1C). As the postnatal vasculature and neural tissue continued to mature, there was an increase in Col1a1+ cell-associated vessels at 2 weeks in all brain regions (P14, Fig. 1C) followed by a fairly stable PSC cell population during the remainder of postnatal development and into adulthood (P21 and Adult, Fig. 1C). The increase in number of vessels with Col1a1+ PSCs was significant for the cortex and striatum from P0 to adult (Fig. 1C, *p < 0.05) and for the hippocampus and hypothalamus from P14 (Fig. 1C, #p < 0.05).
PSCs appear postnatally and increase in abundance and distance from meninges between P0 and adulthood. A, B Sagittal sections of P7 and P21 brain depicting Collagen-1a1 (Col1a1, green) PSCs (arrows) in the midbrain and in the meninges (carets). A′, B′ Higher magnification insets of large diameter, IB4+ vessels (caret indicates DAPI+ nuclei in the inner endothelium of the vessel) with Col1a1+ PSCs surrounding them (arrows). Open-arrow in B′ indicates small diameter IB4+ vessel, likely a capillary. C Graph depicting analysis of density of blood vessels with Col1a1+ PSCs for each brain area (cortex, hippocampus, striatum, hypothalamus, thalamus, midbrain and hindbrain/cerebellum) in P0, P7, P14, P21 and adult brain. Asterisks (*) mark significant differences between P0 and adult and # symbols mark significant differences between P7 and adult. D Graph depicting scoring for the average distance from the pial surface of Col1a1+ PSC associated vessels per brain region (cortex, hippocampus, striatum, hypothalamus, thalamus, midbrain and hindbrain/cerebellum). Significant differences between P0 and adult are marked (*). E Table of significant Pearson Correlation Coefficients (r) and associated p values (p) for PSC cell count and distance measurements shown in C and D. In C and D, 3 animals were scored at each time point with standard error bars. Scale bars 2 mm (A, B) and 50 µm (A′, B′)
The majority of vessels with Col1a1+ cells at P7 were located near the meningeal surface of the brain (Fig. 1A, arrows), but as the Col1a1+ cell population expanded there appeared to be a corresponding increase in distance from the meningeal surface (Fig. 1B, arrows). To quantify this we measured the distance of Col1a1+ PSC-associated vessels from the meningeal surface in all brain regions at P0, P7, P14, P21 and 6 weeks (Fig. 1D). We observed a relationship between developmental time progression and PSC distance from the pial surface in that the average distance from the pial surface increased over time in all brain regions (Fig. 1D). The correlation of cell number and distance from the pial surface was statistically significant for four of the seven brain regions scored (Fig. 1E). An increase in Col1a1+ PSC distance from the pial surface might be the result of increasing CNS tissue around the PSCs due to brain growth or, possibly, Col1a1+ cells originate in the meninges and "enter" the perivascular space via arterioles and venules that penetrate the surface of the brain from the meningeal space. Consistent with this, Col1a1-GFP+ PSCs are associated with large diameter arterioles and venules, structures which are physically linked to the meningeal surface, but not small diameter capillaries [48]. This analysis also reveals new distinctions between pericytes and vascular smooth muscle cells (vSMCs) and Col1a1+ PSCs in that the postnatal spatial and temporal expansion of Col1a1+ cells contrasts with that of pericytes/vSMCs that undergo expansion and spatial distribution during prenatal neurovascular development [2].
PSCs dynamically express both pericyte and meningeal cell markers
The positional analysis of Col1a1+ cell distribution suggested that PSCs may originate from the meninges, thus we examined expression of meningeal cell markers within this population at P0 when PSCs are first observed in the mouse brain and in PSCs in the more mature brain at P21. We also examined expression of these markers in "activated" PSCs at 7 days post ischemic injury induced by 60 min MCAO.
PSCs co-express the population defining marker Col1a1 and PDGFrβ at both P0 (Fig. 2A, arrows), P21 (Fig. 2B, arrows) and in the lesion core (Fig. 2C, arrows). Transcription factor CoupTF2 (Fig. 2D–F, nuclear) was expressed by the Col1a1+ meninges (Fig. 2D, carets) and by Col1a1+ PSCs at P0 (Fig. 2D, arrows), P21 (Fig. 2E, arrows) and in the stroke lesion by PDGFrβ+ PSCs (Fig. 2F, arrows) but not by Ib4+ macrophages (Fig. 2F, carets). We assayed for PDGFrα expression, previously shown to be expressed by inactive and active PSCs [9, 11, 48], and found that Col1a1+ cells had weak PDGFrα expression at P0 (Fig. 2G, open-arrows), but it was strongly expressed at P21 and in lesion PSCs (Fig. 2H, I, arrows). PDGFrα expression is noteworthy because this receptor is also expressed by some meningeal fibroblasts (see Fig. 2G, H, carets), again pointing to a possible meningeal origin of PSCs.
Expression of perictye and meningeal cell markers changes over maturation and post-injury activation of PSCs. Confocal images on sections from hippocampus (P0), thalamus or midbrain (P21) and cerebral cortex or striatum (MCAO) with antibodies that label pericytes and/or meningeal cells to characterize Col1a1+ PSC marker expression. Col1a1+ PSCs (green, A–C) express PDGFrβ (red, A–C) at P0 and P21 and in the post-MCAO lesion (arrows, A–C). At P0 and P21, PDGFrβ is also expressed by perivascular pericytes that do not have Col1a1 expression (carets, A, B). CoupTF2 (red, D–F) is expressed by Col1a1+ (green, D, E) PSCs surrounding IB4+ blood vessels at P0 and P21 (arrows, D, E) and by PDGFβ+ PSCs (green, F) in the stroke lesion (arrows, F). Carets in D indicate CoupTF2 +/Col1a1+ cells in the meninges. IB4 in F also labels macrophages (blue, caret). PDGFrα is expressed in the meninges (red, carets, G, H), is weakly expressed by Col1+ PSCs at P0 (green, open-arrows, G) but is strongly expressed by these cells at P21 (arrows, H) and in the stroke lesion (arrows, I). IB4 (blue) labels blood vessels and microglia/macrophages in A, D–H and DAPI (blue) marks nuclei in I. Scale bars 100 μm
PSCs express RA synthesis proteins and, along with macrophages, are a source of lesion-derived RA
RA is a hormone with a variety of functions in the developing and adult brain [30]. Subsets of meningeal fibroblasts are one source of RA for the brain as they express enzymes required for RA synthesis [26, 45]. Our evidence points to a possible meningeal origin for Col1a1+ PSCs, thus we looked at expression of RA synthesis enzymes Raldh1 and Raldh2 in Col1a1+ PSCs during postnatal development and in the 7 day post MCAO ischemic core. Raldh1 was expressed by some neurons in the postnatal brain as reported (Fig. 3A, arrows) [50] and by some Col1a1+ meningeal cells (Fig. 3A, carets) but not by Col1a1+ PSCs at P0 (Fig. 3A, open-arrows). At P21, however, we observed Raldh1 expression in Col1a1+ PSCs (Fig. 3B, arrows). Within the ischemic core, CoupTF2+ PSCs expressed Raldh1+ however we observed CoupTF2+ PSCs that did not express Raldh1 (Fig. 2C, arrows, open arrows respectively). We observed that IB4+ cells with ameboid morphology, typical of macrophages/microglia that are present in large numbers in the lesion core at this time point [12, 47, 49], also expressed Raldh1 (Fig. 3C, carets). Raldh2, strongly expressed by the meninges, was also expressed by Col1a1+ PSCs at both P0 and P21, albeit very weakly at P0 (Fig. 3D, E, open-arrows and arrows mark PSCs and carets indicate meninges). Many, but not all, CoupTF2+ PSCs in the lesion core were also Raldh2+ (Fig. 3F, arrows mark some Raldh2+/CoupTF2+ cells and open-arrows mark the CoupTF1+/Raldh2-negative cells). We did not assay for Raldh3 expression as it is not reported to be expressed by the meninges [40].
PSCs express RA pathway proteins. RA biosynthesis protein Raldh1 (red) is not expressed by Col1a1+ PSCs at P0 (green, open-arrows in A) but is expressed by some cells in the meninges (carets in A) and some neurons in the brain (arrows in A). At P21, Raldh1 (red, B) is expressed by Col1a1+ PSCs (green, B) surrounding blood vessels (arrows in B). In the stroke lesion, Raldh1 (red, C) is expressed in CoupTF2+ PSCs (green, arrows in C) and IB4+ macrophages (magenta, carets in C). Raldh2 (red, D, E) is expressed by Col1a1+ meningeal cells (green; carets in D, E), is only very weakly expressed by Col1a1+ PSCs surrounding IB4+ vessels at P0 (open-arrows, D), but Raldh2 signal is strong in Col1a1+ PSCs at P21 (arrows in E). Following stroke, Raldh2 (red, F) is expressed by CoupTF2+ PSCs (green, arrows in F). Some CoupTF2+ PSCs in the lesion do not express Raldh1 or Raldh2 (open-arrows in C, F). CRABP1 (red, G) is not expressed by Col1a1+ (green, G) meninges or PSCs at P0 (carets and open-arrows, respectively in G) though is expressed by Col1a1+ PSCs (green, H) surrounding IB4+ vessels at P21 (arrows in H). PDGFrα+ PSCs (red, I) in the stroke lesion express CRABP1 (green, arrows in I) though there are other CRABP1-expressing cells in the lesion (caret in I). CRABP2 (green, J, K) is expressed by PDGFrα meninges (red, carets in J, K) but not by PDGFrα+ PSCs (red open-arrows in J, K) at P0 or P21. CRABP2 (green, L) is expressed by some PDGFrα+ PSCs (red, arrows in L) in the stroke lesion. Open-arrows in L indicate PDGFrα+/CRABP2-negative cells. IB4 labels blood vessels and microglia/macrophages in A, C, D, G, H, J and L; DAPI (blue) marks nuclei in B, C, E, F, I, and K. Scale bars 100 μm
Cellular Retinoic Acid Binding Proteins-1 and 2 (CRABP1 and CRABP2) play a key part in RA signaling within a cell in that they bind RA and shuttle it to its nuclear hormone receptors [39]. CRAPB1 was not extensively expressed by Col1a1+ meninges or PSCs at P0 (Fig. 3G, carets and open-arrows respectively) though we did observe CRABP1 in Col1a1+ PSCs at P21 (Fig. 3H, arrows). CRABP1 was broadly expressed in the lesion core, co-localizing with PDGFrα+ PSCs and other, unidentified cell types (Fig. 3I, arrows and carets, respectively). CRABP2 was expressed in the meninges (Fig. 3J, K, carets) at P0 and P21 but not in PDGFrα+ PSCs (Fig. 3J, K, open-arrows). In the lesion, a subset of PDGFrα+ PSCs co-expressed CRABP2 (Fig. 3L, arrows indicate PDGFrα+/CRABP2+ PSCs and open-arrows indicate PDGFrα+/CRABP2-negative PSCs).
Expression of RA synthesis enzymes (Raldh1 and Raldh2) indicates that cells in the ischemic lesion are a source of RA. We characterized this further by (1) co-labeling sections from ischemic brains (60 min MCAO, 7 days post-injury) with antibodies to Raldh1 and Raldh2 (Fig. 4A–C) and analyzing the density of Raldh1+ and Raldh2+ cells in the non-ischemic hemisphere versus the lesion core (Fig. 4E, F) and (2) quantifying RA levels in uninjured, non-ischemic and ischemic hemisphere at 7 days post 60 min MCAO (Fig. 4G). In non-ischemic cerebral cortex, Raldh1/Raldh2 double positive PSCs were occasionally found in the perivascular space (Fig. 4A, carets). Within the ischemic core at 7 days post 60 min MCAO, the number of Raldh1+ and Raldh2+ cells was dramatically increased (Fig. 4B). Some Raldh1+ cells co-localized with Raldh2 (Fig. 4C, arrows) and these likely represent activated PSCs in the lesion core that express both Raldhs. Other Raldh1+ cells did not co-localize with Raldh2 and, as shown in Fig. 3C, these are macrophages. We further confirmed Raldh1 expression in macrophages by showing that Raldh1+ cells with ameboid morphology express the macrophage marker CD68 (Fig. 4D, carets) [36]. Raldh1+ (Fig. 4E) and Raldh2+ (Fig. 4F) cell density was significantly increased in the lesion core as compared to the contralateral, non-ischemic cerebral cortex (p < 0.05). We next tested whether the ischemia-induced increases in Raldh expressing PSCs and macrophages elevated levels of RA in the ischemic hemisphere. RA levels were not significantly different between hemispheres from a non-injured brain and the non-injured left hemisphere (Fig. 4G) whereas the ischemic right hemisphere had significantly elevated levels of RA (Fig. 4E) (p < 0.05). Additionally, retinol (vitamin A, a precursor to RA) as well as total RE (a storage form of vitamin A) were unchanged (data not shown), further supporting a change in production of RA due to elevated number of Raldh+ cells in the lesion capable of synthesizing RA rather than an increase in available vitamin A. Collectively this data suggests that focal ischemia leads to elevated RA levels through injury-induced expansion of RA-synthesizing PSCs and macrophages.
Elevated RA synthesis and signaling in the ischemic hemisphere. Compared to the uninjured hemisphere (A), the number of cells expressing RA synthesizing enzymes Raldh1 (red) and Raldh2 (green) increases dramatically 7 days following 60 min MCAO (B). Carets in A and A' insets mark Raldh1+/Raldh2+ PSCs around vessels in non-ischemic cortex. Arrows in C depict Raldh1+/Raldh2+ PSCs in the lesion. Carets in D indicate CD68+/IB4+ macrophages (green/magenta) express Raldh1 (red) in the lesion core. Graphs depicting quantification of cells expressing Raldh1 (E) and Raldh2 (F) in injured and un-injured hemispheres. Fast LC-MRM3 quantification of RA (G) from left and right hemispheres of uninjured and 60 min MCAO (right hemisphere) animals at 7 days post-injury. A minimum of 3 separate animals were analyzed (n ≥ 3) and error bars represent SEM. Asterisks indicate statistically significant difference (p < 0.05) from uninjured/non-ischemic hemisphere to ischemic hemisphere. DAPI marks nuclei in blue. Scale bars 20 µm (C), 50 µm (A′–A‴), and 100 μm (A, B, D)
To test whether elevated RA levels in the stroke hemisphere activate RA signaling following stroke, we used the RA activity reporter mouse line RARE-hsp68-LacZ [43] to look for sites of active RA signaling at 7 days post 60 min MCAO. Using an antibody to β-galactosidase (β-gal) to detect activation of the RARE-LacZ reporter gene, we observed expression of the reporter in the hippocampus in both hemispheres (Fig. 5A; Hp) as has been described previously [10]. In the lesion hemisphere there was elevated reporter activity in the peri-infarct region (Fig. 5A–A″, infarct region outlined by dotted lines in 5A). Robust transgene activation was specific to the ischemic hemisphere as only low levels of transgene activity were observed in the contralateral hemisphere (Fig. 5A). To identify which cell types had RARE-hsp68-LacZ transgene activity following injury, we assayed for specific cell markers within the cerebral cortex. In the non-injury hemisphere, neuronal marker NeuN co-localized with some weakly positive β-gal expressing cells (Fig. 5B, carets), but we did not observe overlap between β-gal and the astrocyte marker GFAP (Fig. 5C). In the peri-infarct areas in the ischemic hemisphere, strongly labeled β-galactosidase+ cells co-localized with both NeuN (Fig. 5D, carets) and with astrocyte marker GFAP (Fig. 5E, carets). Consistent with analysis of RA levels, the number of NeuN+/β-gal+ (Fig. 5F) and GFAP+/β-gal+ (Fig. 5G) was significantly increased in peri-infarct region as compared to a similar region in the non-ischemic hemisphere (p < 0.05). We used PDGFrα (Fig. 5H) and PDGFrβ (Fig. 5I) to look for RARE-hsp68-LacZ transgene activity PSCs in the non-lesion and lesion tissue, respectively. Cells in the granule cell layer of the hippocampal dentate gyrus had transgene activity however PDGFrα+ PSCs did not co-localize with β-galactosidase (Fig. 5H, arrows). In the lesion core, PDGFrβ+ PSCs did not have overlapping β-gal expression (Fig. 5I, carets) but some had closely adjacent expression that appeared to be processes (Fig. 5I, I′ open-arrows) possibly β-galactosidase+ astrocyte or neuronal processes as these cells were present in the same area (Fig. 5I, arrows). Collectively these data show that neurons and astrocytes in the peri-infarct region are responding to RA synthesized by macrophages and PSCs within the lesion core.
Assessment of in vivo RA signaling activity. Spatial RA activity assayed using RARE-hsp68-LacZ mouse 7 days post 60 min MCAO injury. Confocal, tile-scan image of β-galactosidase immunolabeling (β-gal, green, A) with IB4 (magenta, A) at the level of the lesion (outlined with dashed line). A′ and A″ indicate magnified images in A. Tile stitched confocal images of RA activity in non-ischemic hemispheres (β-gal, green, B, C) and ischemic (β-gal, green, D–E) with markers for neurons (NeuN, red, carets, B, D), astrocytes (GFAP, red, carets, C, E). Insets are magnified areas with dotted lines in D and E. Graphs depict quantification of NeuN+/β-gal+ neurons (F) and GFAP+/β-gal+ astrocytes (G) from non-ischemic and ischemic hemispheres 7 days following a 60 min MCAO (n ≥ 3 and bars represent SEM). Asterisks indicate statistically significant difference (p < 0.05) from uninjured/non-ischemic hemisphere and the ischemic hemisphere. PDGFrα + PSCs (red) in the non-ischemic cortex did not co-localize with β-gal (arrows, H) though β-gal+ cells are present in the granule cell layer of the dentate gyrus (DG) (carets, H). β-gal+ processes (green, I) appear to contact PDGFrβ+ PSCs (red) in the lesion core (open-arrows in I and I′) but PDGFrβ+ PSCs do not appear to co-label with β-gal (caret, I). β-gal+/PDGFrβ-negative cell bodies are adjacent to PDGFrβ+ PSCs (arrows, I). DAPI marks nuclei in blue. Scale bars 2 mm (A), 200 μm (B–E, I), 100 µm (H, I′)
PSCs, aka Type "A" pericytes, represent a subset of PDGFrβ-expressing perivascular cells that are activated in the days following a CNS injury, expand in number and contribute significantly to fibrotic scar deposition [9, 11, 31, 48]. The Col1a1-GFP mouse line labels a subset of perivascular cells that fit the primary characteristics of a PSC, most notable of which is expansion and ECM protein deposition following CNS injury. In this study, we first use the unique expression of Col1a1 by PSCs to delineate this population from pericytes and vSMCs in the perivascular space and gain knowledge about this cell type during CNS development. This analysis led to the observation that PSCs, like meningeal fibroblasts, express RA synthesis enzymes Raldh1 and Raldh2. We show that following a focal ischemic injury, increased numbers of Raldh-expressing PSCs and macrophages elevates RA levels that activates RA signaling in peri-infarct neurons and astrocytes. These findings support a novel function for PSCs and macrophages within the infarct core and raise new questions regarding the role of endogenous RA synthesis and signaling following focal brain ischemia.
Our observation that an expanded population of PSCs are, along with macrophages, a source of RA following brain injury resulted from our interest in identifying when and from where Col1a1+ PSCs originate. We find that Col1a1+ PSCs are only found in the postnatal brain and we have evidence that they may originate from the meninges. The latter is based on shared expression of meningeal fibroblast markers (e.g. Col1a1, PDGFrα, Raldh2, CoupTF2 and PDGFrβ) and the expanding distance of PSCs from the meningeal surface during postnatal development. Postnatal appearance of Col1a1+ PSCs contrasts with pericytes and vSMCs that enter the CNS along with blood vessels during prenatal CNS angiogenesis [2] and is another characteristic defining this unique perivascular population. It is also consistent with reports that there is almost no fibrotic scar deposition in the lesion site at P7 following experimental brain injury in mice or rats [19, 33], a day point when we observe that Col1a1+ PSCs are infrequent. That Col1a1+ PSCs may originate in the meninges and "move" into the perivascular space along arterioles and venules that penetrate the surface of the brain is consistent with the anatomy of the interface between the meninges and perivascular gaps called Virchow-Robin spaces (VRs) (Fig. 6A). VRs start at the point where a vessel in the pial layer of the meninges penetrates the brain surface and runs the length of penetrating arterioles and venules [4]. In this way, VRs are largely continuous with the pial space. VRs may play an important role in interstitial flow, essentially helping to flush cellular debris, notably β-amyloid, out of the brain parenchyma [4, 38]. Possibly, VRs also represent an entry point for Col1a1+ meningeal cells from the pial layer of the meninges to enter the perivascular space. Our model is supported by electron microscopy analysis at both the meninges-VRs interface and arterioles within the brain where researchers have characterized pial fibroblasts entering VRs from the meninges [25, 32] and surrounding the vSMC layer of arterioles within the brain parenchyma [54].
Model of PSC development in the post-natal brain and as a source of RA following stroke injury. Potential model of how during post-natal brain development Col1a1+ cells (putative PSCs) could 'move' from the meningeal space to the perivascular space around large diameter blood vessels via Virchow-Robin Spaces (A). Following injury Raldh1+ macrophages/microglia and Raldh1/2+ PSCs within the ischemic core produce RA capable of activating RA signaling in adjacent peri-infarct astrocytes and neurons (B)
PSCs that contribute to fibrotic scar formation were, for a long time, mistaken for meningeal fibroblasts that move into the injured CNS after injury-induced damage to the pial basement membrane [1, 51]. This was based, in part, on the observation that fibrotic cells in the scar were continuous with the meningeal space and expressed markers also expressed by meningeal fibroblasts [5]. Recent lineage tracing using the GLAST-CreErt and Col1a1-GFP lines, however, provide compelling evidence that the fibrotic scar is generated by the resident PSC population and not meningeal fibroblasts moving into injured tissue [13, 48]. Our data provides new insight into why PSCs were erroneously identified as meningeal fibroblasts in that we show that PSCs express many of the same proteins. It is not clear, however, how or why cells might move from the meninges into the perivascular space of the early postnatal brain. It is possible that PSCs in the brain are an artifact of blood vessel development during postnatal brain growth. Conceivable, pial meningeal fibroblasts are "pulled" into the developing brain along the large blood vessel tracts and brain growth around the vessels displaces PSCs deeper into brain structures. It cannot be an entirely passive process, however, since we observe that PSCs are proliferating in the early postnatal brain (data not shown). Also, Col1a1+ PSCs increase expression of certain markers like Raldh1, Raldh2, PDGFrα, and CRABP1 over time, suggesting that they undergo differentiation in the perivascular space and thus are likely a unique cell type from meningeal fibroblasts. Our analysis also indicates there is heterogeneity in PSCs within the injury site. We observe that only some PSCs in the lesion express Raldh1, Raldh2 and CRABP2, suggesting they may be more than one type of PSC. More studies are needed to define different subtypes of PSCs, their origin and function following brain injury.
The meninges are a source of RA for the developing and adult brain [26, 45] and we find that Col1a1+ PSCs express RA biosynthesis and signaling proteins. The dramatic expansion of this population, along with infiltration of large numbers of Raldh1-expressing macrophages, following brain ischemia creates a concentrated pool of RA synthesizing cells within the lesion core. Consistent with this, we show that the presence of numerous Raldh-expressing cells correlates with a significant increase in RA levels in the stroke hemisphere at 7 days post-injury. Significantly, when we use the RA signaling reporter, RARE-hsp68-LacZ, we show that elevated RA synthesis in the core stimulates RA signaling activity in peri-infarct neurons and astrocytes at the same time-point. Our data are consistent with transcription profiling of stroke tissue from rat showing up-regulation of both Raldh2 and CRABP2 in the ischemic hemisphere following injury [15]. Following spinal cord injury, increased numbers of Raldh2+ cells have been reported, along with elevated RA levels and signaling [21, 34, 35]. Considering our characterization of Raldh2+ cells in stroke injury, Raldh2+ cells in spinal cord lesions described in these studies are very likely PSCs. If that is the case, RA synthesis by PSCs may be a common function of this injury-activated cell type following CNS injury.
Our identification of cells in the stroke lesion as a source of RA has implications for how bioactive ligands emanating from the lesion may play a previously unknown role in recovery from ischemic injury. Reports in the stroke literature regarding the effect of exogenous treatment of RA on focal ischemic injury generally conclude that RA is beneficial though different stroke models and exogenous RA treatment regimens were used. Exogenous RA results in a smaller lesion [6, 24], improved blood brain barrier integrity [24], reduced neuroinflammatory response accompanied by decreased hippocampal cell death and improved behavioral recovery [22], and increased neurogenesis and improved neurobehavioral outcomes when coupled with environmental enrichment [42]. Possibly, endogenous RA synthesis mediated by PSCs and macrophages that we describe here has similar effects as reported for exogenous RA in the post-stroke environment. In this way, injury-induced production of RA in the lesion may create a dynamic signaling environment for neurons and glia that could aid in recovery following brain ischemia. Future studies aimed at pharmacologically inhibiting endogenous RA synthesis following stroke injury will provide important insight into the function of endogenous, lesion-derived RA in stroke recovery.
Work thus far on PSCs has focused on their pro-fibrotic function with the idea that therapies to inhibit PSC population expansion would block fibrotic scar deposition and promote neural recovery. Inhibition or reduction of collagen IV and CSPGs (chondroitin sulfate proteoglycans) can attenuate CNS fibrosis [44, 52], however the extent to which that promotes neuronal repair in vivo following injury is not clear [18]. Blocking the TGFβ pathway following CNS injury decreases ECM deposition and size of the fibrotic scar, but there is not a corresponding recovery of axonal growth [23, 27–29, 37]. These results suggest that blocking fibrotic scar formation is not sufficient for full, functional recovery of the tissue. Importantly, though PSCs contribute to fibrotic scar formation, our work outlined here suggests they may also have a protective function via secretion of bioactive ligands that promote recovery, namely RA. In this way, PSCs may play an unexpected, dual role in regeneration and fibrosis within the injured brain tissue and therapeutic strategies aimed purely at preventing PSC expansion are potentially not ideal.
Our research supports a dual role for post-injury, activated PSCs where in addition to contributing to fibrosis, PSCs secrete signaling molecule RA. In this way, activated PSCs are potentially capable of influencing of recovery following brain injury.
β-gal:
β-galactosidase
Col1a1:
Collagen1a1
CoupTF2:
COUP transcription factor 2
CRABP1:
Cellular Retinoic Acid Binding Protien 1
CSPG:
chondroitin sulfate proteoglycan
DAPI:
4′,6-diamidino-2-phenylindole
E16.5:
embryonic day 16.5
ECM:
extra-cellular matrix
GFAP:
glial fibrillary acidic protein
GFP:
green fluorescent protein
GLAST:
glutamate aspartate transporter
HPLC:
IB4:
isolectin B4
MCAO:
middle cerebral artery occlusion
NeuN:
neuronal nuclear antigen
PSC:
perivascular stromal cell
P0:
postnatal day 0
P14:
postnatal day 14
PDGFrα:
platelet derived growth factor receptor-α
PDGFrβ:
platelet derived growth factor receptor-β
RA:
retinoic acid
Raldh1:
retinaldehyde dehydrogenase 1
SEM:
standard error of the mean
TGFβ:
transforming growth factor-β
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KKK participated in the study design, data collection, analysis, and drafting the manuscript. AMM participated in data collection and analysis. HG and FS generated stroke animals. JWJ performed the HPLC RA analysis, JY performed the HPLC RA analysis, KP performed the HPLC RA analysis, MAK designed the RA analysis and participated in drafting the manuscript. PH designed the stroke model and participated in drafting the manuscript. JAS conceived and designed the study and participated in drafting the manuscript. All authors read and approved the final manuscript.
This work was supported funding from the AHA (14BGIA18440006), NIH/NINDS (R00 NS070920) and Children's Hospital Colorado Research Institute to JAS, NIH/NINDS (R01 NS080851) to PSH, and NIH/NICHD (R01 HD077260), NIH/NIAID (HHSN272202000046C), and the University of Maryland Mass Spectrometry Center (SOP1841-IQB2014) to MK.
Animal studies were performed with strict adherence to The Recommended Guide for Care and Use of Laboratory Animals issued by the National Institutes of Health and all procedures were approved by the Institutional Animal Care and Use Committee at The University of Colorado, Denver-Anschutz Medical Campus [JAS: Animal protocol number: B-99915(07)1D; PSH: B-83311(10) 1E]. All surgery was performed under anesthesia and suffering was minimized.
Mouse lines and reagents ('materials') used in this study are commercially available as listed in the "Methods" sections.
Department of Pediatrics, Section of Developmental Biology, University of Colorado Denver-Anschutz Medical Campus, 12800 E. 19th Ave MS-8313, Aurora, CO, 80045, USA
Kathleen K. Kelly, Amber M. MacPherson & Julie A. Siegenthaler
Department of Anesthesiology, University of Colorado Denver-Anschutz Medical Campus, Aurora, CO, 80045, USA
Himmat Grewal, Frank Strnad & Paco S. Herson
Department of Pharmaceutical Sciences, University of Maryland, Baltimore School of Pharmacy, Baltimore, MD, 21201, USA
Jace W. Jones, Jianshi Yu, Keely Pierzchalski & Maureen A. Kane
Department of Pharmacology, University of Colorado Denver-Anschutz Medical Campus, Aurora, CO, 80045, USA
Paco S. Herson
Neuronal Injury Program, University of Colorado Denver-Anschutz Medical Campus, Aurora, USA
Kathleen K. Kelly
Amber M. MacPherson
Himmat Grewal
Frank Strnad
Jace W. Jones
Jianshi Yu
Keely Pierzchalski
Maureen A. Kane
Julie A. Siegenthaler
Correspondence to Julie A. Siegenthaler.
Kelly, K.K., MacPherson, A.M., Grewal, H. et al. Col1a1+ perivascular cells in the brain are a source of retinoic acid following stroke. BMC Neurosci 17, 49 (2016). https://0-doi-org.brum.beds.ac.uk/10.1186/s12868-016-0284-5
DOI: https://0-doi-org.brum.beds.ac.uk/10.1186/s12868-016-0284-5
Pericyte
Brain fibrosis
Meninges
Anatomy, development, plasticity and repair
Submission enquiries: bmcneuroscience@biomedcentral.com


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text:
# FTC Vision Library [![Build Status](https://travis-ci.org/lasarobotics/FTCVision.svg?branch=master)](https://travis-ci.org/lasarobotics/FTCVision) [![Documentation Status](https://img.shields.io/badge/documentation-1.0.0%20(up%20to%20date)-blue.svg)](http://ftcvision.lasarobotics.org)
Computer Vision library for FTC based on OpenCV, featuring **beacon color and position detection**, as well as an easy-to-use `VisionOpMode` format and many additional detection features planned in the future.

## Installing into FIRST Official App *(recommended)*

- Clone FTCVision into a clean directory (outside your robot controller app) using the following command: `git clone --depth=1 https://github.com/lasarobotics/ftcvision`.
- Navigate to the FTCVision directory that you just cloned and copy the `ftc-visionlib` and `opencv-java` folders into your existing robot controller app.
- Open your robot controller app in Android Studio. Make sure you have the `Project` mode selected in the project browser window (so you can see all of the files in your project).
- Find your `settings.gradle` file and append the following two lines:
```
include ':opencv-java'
include ':ftc-visionlib'
```
- Find the `AndroidManifest.xml` file in your app's `FTCRobotController/src/main` folder.
- Insert the following `uses-permission` tag in `AndroidManifest.xml` just below the `<mainfest ...>` tag and just before the `application` tag:
```
<uses-permission android:name="android.permission.CAMERA" android:required="true" />
```
- Find the `FTCRobotController/build.release.gradle` AND `TeamCode/build.release.gradle` files and insert the following lines under `dependencies` **into both files**:
```
compile project(':ftc-visionlib')
compile project(':opencv-java')
```
- Update Gradle configuration by pressing the green "Sync Project with Gradle Files" button in the header (this may take a minute)
- Copy in Vision opmodes (those that end in `VisionSample.java`, located in `[vision-root]/ftc-robotcontroller/src/main/java/com/qualcomm/ftcrobotcontroller/opmodes`) from the FTCVision directory into a folder within the `TeamCode` directory (where your place any other opmode).
- Before running the app for the first time, install the "OpenCV Manager" from the Google Play Store to enable Vision processing.
- Run and test the code! Let us know if you encounter any difficulties. *Note: depending on your version of the FIRST app, you may need to modify the OpModes slightly (such as adding `@Autonomous`) in order to get them to work for your version of the SDK.*
- You can now write your custom `VisionOpMode`!
- *(Optional)* Add Vision testing app (see pictures of it below!) by copying all files from `ftc-cameratest` into the root of your project. Then, add `include ':ftc-cameratest'` to your `settings.gradle` in the root of your project. To run the camera test app, click the green "Sync Project with Gradle Files" button to update your project, then select `ftc-cameratest` from the dropdown next to the run button.

## Installing via Git Submodule *(advanced)*
When installing via Git submodule, **every person cloning your repo will need to run `git submodule init` and `git subomodule update` for every new clone.** However, you also get the advantage of not copying all the files yourself and you can update the project to the latest version easily by navigating inside the `ftc-vision` folder then running `git pull`.

- Inside the root of your project directory, run
```
git submodule init
git submodule add https://github.com/lasarobotics/ftcvision ftc-vision
```
- Follow the guide "Installing into Existing Project" starting from the third bullet point. Please note that since everything will be in the `ftc-vision` folder and thus directories will need to be modified. Once you get to the step that modifies `settings.gradle`, add the following lines:
```
project(':opencv-java').projectDir = new File('ftc-vision/opencv-java')
project(':ftc-visionlib').projectDir = new File('ftc-vision/ftc-visionlib')
project(':ftc-cameratest').projectDir = new File('ftc-vision/ftc-cameratest') <- only if you want to enable the camera testing app
```
- You can now write your custom `VisionOpMode`!

## Installing from Scratch *(for testing only - deprecated)*

1. Clone FTCVision into a clean directory (outside your robot controller app) using the following command: `git clone --depth=1 https://github.com/lasarobotics/ftcvision`.
2. Open the FTCVision project using Android Studio
3. Copy your OpModes from your robot controller directory into the appropriate directory within `ftc-robotcontroller`. Then, modify the `FtcOpModeRegister` appropriately to add your custom OpModes.
4. Before running the app for the first time, install the "OpenCV Manager" from the Google Play Store to enable Vision processing.
5. Run and test the code! Let us know if you encounter any difficulties.
6. You can now write your own `VisionOpMode`!

## Status
This library is complete as of World Championship 2016. If you have any questions or would like to help, send a note to `smo-key` (contact info on profile) or open an issue. Thank you!

## Documentation [![Documentation Status](https://img.shields.io/badge/documentation-1.0.0%20(up%20to%20date)-blue.svg)](http://ftcvision.lasarobotics.org)

Documentation for the stable library is available at http://ftcvision.lasarobotics.org.

## Does it work?

**Yes!** FTCVision can detect a beacon 0.5-4 feet away with 90% accuracy in 0.2 seconds. Here are some pictures. :smiley:

#### Accuracy Test
![Can it detect the beacon?](https://raw.githubusercontent.com/lasarobotics/ftcvision/img/test4.png)

#### Old Accuracy Test
![Can it detect the beacon?](https://raw.githubusercontent.com/lasarobotics/ftcvision/img/test2.png)

#### Distance Test
![A test from 8 feet away](https://raw.githubusercontent.com/lasarobotics/ftcvision/img/test1.png)

#### Basic Analysis Demo
![FAST isn't the greatest](https://raw.githubusercontent.com/lasarobotics/ftcvision/img/analysisdemo.gif)

#### Ambient Analysis (Color and Rotation Detection) Test
![A test from 8 feet away](https://raw.githubusercontent.com/lasarobotics/ftcvision/img/test3.gif)

#### Analysis Methods
![FAST vs. COMPLEX](https://raw.githubusercontent.com/lasarobotics/ftcvision/img/methods.png)

- The **FAST** method analyzes frames at around 5 FPS. It looks for the primary colors in the image and correlates these to locate a beacon.
- The **COMPLEX** method analyzes frames at around 2-4 FPS. It uses statistical analysis to determine the beacon's location.
- Additionally, a **REALTIME** method exists that retrieves frames and analyzes them as fast as possible (up to 15 FPS).

## Goals
- To make it easy for teams to use the power of OpenCV on the Android platform
- Locate the lit target (the thing with two buttons) within the camera viewfield
- Move the robot to the lit target, while identifying the color status of the target
- Locate the button of the target color and activate it

## Progress
- Beacon located successfully with automated environmental and orientation tuning.
- A competition-proof `OpMode` scheme created so that the robot controller does not need to be modified to use the app.
- Now supports nearly every phone since Android 4.2, including both the ZTE Speed and Moto G.

redpajama_set_name: RedPajamaGithub


text:
Home › Call of Duty: Black Ops Cold War - Ultimate Edition Xbox One Game Global Activation Key
Call of Duty: Black Ops Cold War - Ultimate Edition Xbox One Game Global Activation Key
Product Type: game
Product SKU: codCWEDxbx
You will receive an activation key to your email for the game which can be redeemed via Xbox Live.
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If you do not have an Xbox/Microsoft Account you can register FREE at Xbox (or Log in with your existing account).
Check Activation Guide.
Third-person
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Call of Duty: Black Ops Cold War - Ultimate Edition Xbox Key
Call of Duty: Black Ops Cold War (Ultimate Edition) includes:
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Confrontation Weapons Pack;
One Season of Call of Duty: Black Ops Cold War Battle Pass Bundle, including 20 Tier Skips;
The Land/Sea/Air Pack for use in Call of Duty: Black Ops Cold War;
Three Operator Skins;
Three Weapon Blueprints;
Three Vehicle Skins.
Miss the Cold War Call of Duty games? Buy Call of Duty: Black Ops Cold War (Ultimate Edition) Xbox One key and you'll return to the era of political tensions between global superpowers on the edge of a nuclear exchange. With the aid of Raven Software, studio Treyarch is bringing back the COD: Black Ops spin-off series to the scene with a direct sequel to the very first and highly acclaimed Call of Duty: Black Ops game from 2011. Call of Duty Black Ops Cold War price pays-off with a refined FPS experience involving espionage, Cold War-era weapons, spying technology, and never before seen gameplay features.
Will Call of Duty: Black Ops Cold War (Ultimate Edition) have a campaign?
While Call of Duty: Black Ops 4 was a multiplayer-only first-person shooter, the fifth installment in Treyarch's COD subseries will feature a fully-realized single-player component. Buy Call of Duty: Black Ops Cold War (Ultimate Edition) Xbox One key to witness and participate in the events of the Cold War during the 1980s, with some of the segments also focusing on the events of the Vietnam War. Major characters from previous COD Cold War games like Mason, Woods, and Hudson make a comeback, real-life historical figures like 40th US president Ronald Regan make an appearance for the first time, while the main protagonist is fully-customizable for ultimate immersion!
Call of Duty: Black Ops Cold War (Ultimate Edition) multiplayer mode
An engaging and addictive Call of Duty multiplayer component is assured to anyone who chooses to buy Call of Duty: Black Ops Cold War (Ultimate Edition) Xbox One key. Killstreak rewards, callsigns, and a variety of other unlocks are once again at the core of the experience, with all of them being gradually made available through a battle pass system. Progression as well as gameplay are both cross-platform and cross-generational. The fifth Cold War Call of Duty won't have its own battle royale mode but it nevertheless shares content and unlocks with the free-to-play Call of Duty: Warzone. Treyarch also has prepared a few surprises for Zombie mode fans – yet another reason to buy Call of Duty: Black Ops Cold War (Ultimate Edition) Xbox One key!
Call of Duty: Black Ops Cold War (Ultimate Edition) features:
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Multiple endings. Just like Call of Duty: Black Ops 2 the game will feature multiple endings – the conclusion of the single-player campaign depends solely on your choices;
Customizable protagonist. Players will also get to modify the gender and appearance of the main protagonist which results in much higher immersion;
Engaging FPS multiplayer. The game features cross-platform, cross-generational, multiplayer and Zombies modes with content and progression shared with Call of Duty: Warzone.
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redpajama_set_name: RedPajamaCommonCrawl


GIXEN Forum - View topic - Great!!!
Posted: Mon Feb 12, 2007 9:25 am Post subject: Great!!!
Leave this message to say that I'm really happy to have used Gixen.
This was my first time in ebay and was buying an article that seemed to be no interesting for anybody.
I had just made a bid, but was 'afraid' of the possibility of someone in the last minute to make a higher bid, so decided to try Gixen.
An it happened, somebody made a higher bid in the last minute, but thanks to Gixen I won!!!!!
By the way, did you use Spanish version of Gixen?
I'd love to hear suggestions or problems (translation, bugs, etc), as very few users use Gixen in Spanish. Feel free to email me about it.
I used the English version, as I don't have problems with that.
Anyway, I you wish me to check the spanish translation for you I will do and send my comments.


A simple, elegant rendition of the symbols of the Tarot drawn and colored by Ferdinando Crippa in 1978 and now published in a limited edition of 700 copies by Meneghello of Milan. The drawings are bold and clear and the line work is wonderful. Subtle, sometimes wry, emotions conveyed from the characters expressions are generated with an economy of line that is the hallmark of the finest illustration. This is artwork of the highest caliber.
Full 78 card deck; Majors and Courts are not numbered or labeled; Minors are numbered pips.
Includes 8 page leaflet in English and Italian with a biographical profile of Ferdinando Crippa written by his brother Giuliano. The front cover of the booklet has the limited edition number. Packaged in the classic Meneghello 2-part telescoping box with wax seal. Destined to be a collector's favorite.
English: 2 3/8" x 3 15/16" x .012"


Now BBM is going to include Voice Calls, Video Calls and a new social and promotional element called BBM Channels.
BBM Channels allows you to create you own personal Channel or subscribe to another users Channel.
In your own Channel you can engage in discussions with your subscribers without having to divulge any personal details or even add them to your contact list.
You can control the interaction of your subscribers by moderating comments before they are published, you can also set chat hours to control the time period where interaction can take place, it's like running your own little chat room.
With BBM's built in notifications you will get instant updates when a subscriber posts a comment on your channel or a channel you are subscribed to.
BBM Channels comes with a built in analytics tool so you can measure engagement with every post you create, like how many followers your Channel has, how many visits, likes, comments and unique subscribers.
Where Channels will undoubtedly come into its own is for brands wishing to promote services, products and promotions, sort of like a mini Twitter, Facebook and messaging app all in one and I can see advertising coming to highly subscribed Channels very soon.
BBM has always been one of the biggest attractions for BlackBerry users, and it seems like BlackBerry have got a cunning plan to finally monetize this app after years of free ad-free messaging.
The beauty for Brands, companies, and anyone trying to promote their business or services through BBM Channels, is that BBM is an 'always on' application, giving them 24 hour access to subscribers devices.
All in all this seems like a smart move for BlackBerry, enhancing a very popular application into a standalone business model that could possibly rival Twitter and Facebook as a promotional marketing tool for anyone with an opinion, to a multinational company and everything in between.
Download BBM for here for Android or for your iPhone from the Apple App Store.


Fans may know Falk best from his work as Arliss Fulton in the CBS series "Reckless," he was also featured in the film "Transcendence" with Johnny Depp and Morgan Freeman. As for "DC'S LEGENDS OF TOMORROW" the midseason drama will follow time-traveling renegade Rip Hunter as he assembles a group of both heroes and villains to confront an unstoppable threat.
DC'S LEGENDS OF TOMORROW stars Victor Garber ("The Flash," "Alias"); Brandon Routh ("Arrow," "Superman Returns"); Arthur Darvill ("Doctor Who"); Caity Lotz ("Arrow"); Ciarra Renee ("Pippin"); Franz Drameh ("Edge of Tomorrow"); with Dominic Purcell ("The Flash," "Prison Break"); and Wentworth Miller ("The Flash," "Prison Break").


text:
You are here: Home Transport
Travellers Vocabulary
Study Spanish in Madrid
Those who travel to Madrid by plane will land at the Madrid Barajas airport, which is about 12 km northeast of the city centre. Barajas is one of three airports of the Spanish capital, the other two airports are scarcely used for scheduled flights though. Cuatro Vientos is open for general aviation, but is also used by the military and Torrejón is a former American Air Base, which is barely utilized due to the existing capacity of Barajas. In 2005, a new and modern terminal (T4) was opened and is connected to the satellite terminal T4S with its own metro due to the distance from the old airport ground. The connections to public transport are excellent from both areas. Both T1 - T3 and T4 have a connection to the metro (line 8). Those who don't want to take the metro can either take a taxi or to take the bus.
For the metro, a supplement charge of 1€ on top of the regular ticket is payable should you not have a monthly ticket (Abono) or a tourist ticket. For a taxi ride there is also an airport supplement payable. The taxi ride to Puerta del Sol costs about 20-25 €. Please note that a further surcharge is payable at night.
Madrid Barajas is utilized not only by the major UK airlines, but also the budget no-frills airlines. British Airways (www.britishairways.com), Iberia (www.iberia.com), Lufthansa (www.lufthansa.com) and KLM (www.klm.com) all offer flights to the Spanish capital from the popular major United Kingdom airports. Other airlines also operate routes from Ireland. Some of these are Air France (www.airfrance.com) and Swiss (www.swiss.com) as well as the no-frills airlines like Easyjet (www.easyjet.com), Aer Lingus (www.aerlingus.com) and Ryanair (www.ryanair.com).
From the airport (T1 - T3 and T4) the ride with the metro is the cheapest and the most convenient. In the metro stations at the airport you can buy a ten-ticket (MetroBus or Diez Viajes) for 9.30 €. This ticket can be shared with other passengers, but remember that each fellow traveller still has to pay their airport supplement fee (1 €).
From the airport, the journey takes approximately 30-40 minutes to the inner city (Note: In the Nuevos Ministerios station you have to change the subway line - line 6 or 10). It will be a little faster by bus though, the airport line 200 (it costs 1 € or use the MetroBus ticket) goes to Avenida de América, where you can then change to the metro.
Aéroport Barajas
12 km northeast from the centre of Madrid
Connections Madrid Airport
Bus - Line 200
Crossing Avenida de la América, Terminal 1 - 4, daily 6 am - 10.30 pm, 1€
Metro - Line 8
Phone 010, daily 6 am -1.30 am, 1€ + supplement
with its own metro
www.template-land.com


I've always wanted to use the soft fuzzy yarn but, was afraid to really mess up the stitches. This pattern and article has given me hope and confidence try it! I'm looking forward to the next yarn sale!!! Thank You!!!
I purchased Pipsqueak to make a baby blanket for my newest niece and frogged 3-4 skeins several times. I searched and searched for a pattern (yup, I used "Pipsqueak" in my search) and tried but just had too much trouble finding the stitches under all the lovely fluff. So it sat. I made a bath mat using cotton for my first C2C piece. That's when I realized C2C is perfect for fuzzy yarn. I even decided to alternate the purple/green varigated Pipsqueak and white just like the pattern above shows. I am sooooo happy with the results.
Good tutorial. I have a graph I would like to use for a c2c but not sure when to start decrease. It is 23 blocks by 30 blocks. Please advise.


text:
Ceramics surprise with durable dryness
Hardy water-repelling lanthanide oxides tackle extreme engineering challenges.
Bouncing droplet
The ceramic material coating this surface is so hydrophobic that the droplet bounces off, leaving virtually no residue.
GISELE AZIMI & HYUK-MIN KWON
Coatings that repel water are found in myriad applications — they keep car windscreens clear in storms, for example, and ensure that rainswept hikers stay snug and dry. But they quickly wear away in harsh environments, such as the insides of power-station turbines, where condensation reduces efficiency. As a result, engineers are eager to get their hands on tough materials that are intrinsically hydrophobic.
Now they can, following the discovery that a well-known family of durable ceramics can repel water.
That is surprising because most ceramics are hydrophilic. When water meets a ceramic such as aluminium oxide, the water's oxygen atoms share some of their electrons with vacant electron orbitals on the aluminium atoms, and the oxygens in the ceramic share their electrons with hydrogen in the water. This binds the two together.
But what if a ceramic failed to accept electrons from water? Then the ceramic might actually be hydrophobic, reasoned Kripa Varanasi, a materials scientist at the Massachusetts Institute of Technology (MIT) in Cambridge. He looked to the oxides of the lanthanides — the row of metals nestled almost at the bottom of the periodic table, from cerium to lutetium. The lanthanides' empty orbitals are buried beneath shells of other electrons, which should make them much less attractive to water's oxygen, thought Varanasi.
Proving that sometimes the simplest experiments are the best, he and his colleagues tested the idea by making small ceramic discs of the oxides of all the lanthanides except radioactive promethium. The team then dropped water onto the pellets and watched what happened.
Every single one repelled the liquid. When water condensed on to the discs, it did so in neat droplets rather than the films that would form on non-hydrophobic materials such as silicon. And cerium oxide — the cheapest and most widely available of the lanthanide oxides — remained hydrophobic even after a two-hour sauna at 1,000 °C, or a thorough grinding with abrasive silicon carbide. The results are published today in Nature Materials1.
Gisele Azimi and Adam T. Paxson
A thin film made of a water-repelling ceramic material — here a rare-earth oxide — can help you stay dry.
Hidden effect
The water-repelling properties of cerium dioxide had been noticed before2, for example by a student working with Barry Cheung, a materials scientist at the University of Nebraska–Lincoln. But no one had investigated these properties in detail. Indeed, most chemists would have failed to spot the hydrophobicity of the lanthanide oxides, Cheung speculates, if their samples had defects — specifically, missing oxygen atoms — that allowed water to latch on.
Cheung suggests that Varanasi's ceramics were particularly hydrophobic because they had very few oxygen defects - perhaps a consequence of their formation in dry air at high temperatures of around 1,500 °C.
Varanasi says that hydrophobic ceramics could improve the efficiency of energy generation. As steam passes through the turbines of a thermal power station, water condenses onto the blades and forms droplets many micrometres across. The rotating blades lose energy as they smash into these droplets, accounting for up to 30% of the inefficiency of the turbine, he says.
A hydrophobic coating made of tough ceramic would prevent films of water forming on the blades, he says. Another application could be in wind turbines, where films of water on the blades can, if they freeze in cold weather, lead to catastrophic failures.
Varanasi is now working with energy and technology companies partnered with the MIT Energy Initiative, which co-funded his work, to test the ceramics in real-world applications.
Azimi, G., Dhiman, R., Kwon, H.-M., Paxson, A. T. & Varanasi, K. K. Nature Mater. http://dx.doi.org/10.1038/NMAT3545 (2013).
Lawrence, N. J., Jiang, K. & Cheung, C. L. Chem. Commun. 47, 2703–2705 (2011).
Raindrops on roses
Spongy nanocoating makes for fog-free glass
Water repellent also absorbs
The water marbles
Kripa Varanasi
Commenting is currently unavailable.


Curious as to how support (demand for?) 7-zip format is coming; that is, is it still a "trial" feature requiring a special build? I like to use 7-Zip for its compression is always better than .ZIP format. I supposed it is considered a proprietary format, requiring special legal treatment, as opposed to an "open" standard?
P.S. Sorry if this was not posted in correct forum? I'll re-post if you tell me where it should go?
That's ok. This is the traditional Chinese forum, but I can answer your question here since that's what this user was also asking.
We have read support for 7zip. The trial is the trial of BC3, not of the support for 7zip. This way you can test out our fully featured trial and see if the 7zip support meets your needs or if you have any questions before purchase.
There aren't any legal issues with the 7-zip support. The 7-zip program is open source, and I believe the format is reasonably well documented, so there aren't too many issues with using it instead of zip files. We haven't had enough demand to warrant adding support for modifying them, but we certainly could if we wanted to.
FWIW: One of the reasons I don't use 7-zip more often is because utilities like BC3 do not yet fully support it. I, too, would welcome such an enhancement... though items like 3-way folder compares are higher on my list.


text:
Parents on trial for murder, abuse of son aged 5
Couple accused of confining child in cage, scalding him and using pliers to pinch him
Posed photo of a boy being abused. Azlin Arujunah and Ridzuan Mega Abdul Rahman are on trial for subjecting their child to several kinds of abuse.PHOTO: THE NEW PAPER
Charmaine Ng
Nov 13, 2019, 5:00 am SGT
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https://str.sg/Jiw6
In the months leading to his death in October 2016, a five-year-old boy was subjected to all kinds of alleged abuse, including being confined in a cage measuring 91cm long, 58cm wide and 70cm tall meant for the family's pet cat.
The 1.05m-tall boy was also allegedly pinched with pliers on his thighs and buttocks, hit with various objects and scalded with hot water. These acts of physical and psychological abuse were allegedly inflicted by his own parents, from July 2016 until his death on Oct 23 that year.
When the boy finally collapsed from his injuries that included severe burns across 70 per cent of his body, the married couple allegedly delayed sending him to the hospital for six hours.
Azlin Arujunah and Ridzuan Mega Abdul Rahman, both 27, allegedly did this out of fear that they would get into trouble for child abuse.
Deputy Public Prosecutors Tan Wen Hsien, Daphne Lim and Li Yihong said the couple blatantly disregarded the boy's welfare and his life.
"This is a murder as horrific as it is tragic," said the prosecution team in its opening statement yesterday in the High Court, on the first day of the trial.
"Leaving aside the physical abuse he suffered, one cannot imagine the pain and torment the deceased must have endured in the months leading up to his death. He was tortured to his death, and had no one to turn to," the team added.
His parents each face the charge of murder with common intention, which carries the death penalty, and charges relating to the ill-treatment of the child and voluntarily causing hurt by means of a heated substance.
The court heard that the boy was fostered out shortly after his birth and lived with a close friend of Azlin's in a private arrangement from around March 2011 until May 2015, when he was returned to the couple.
From June 2016, he suffered abuse allegedly at the hands of his parents while living with them and his siblings in a one-room flat in Toa Payoh.
Examples of alleged abuse
The boy developed a limp as a result of a misaligned kneecap he suffered after his mother Azlin allegedly hit him repeatedly with a broom over biscuits she found scattered on the kitchen floor.
Azlin pushed the boy hard, causing his head to hit the wall and blood to flow from his head because he refused to answer a question. Ridzuan then punched the boy in the face, causing him to bleed profusely from the nose.
In the week leading up to his death - between Oct 15 and 22 that year - the boy was scalded with hot water on at least four occasions, causing him to suffer severe scald injury that led to his eventual death.
The day before he died, the boy was hit on his head, hands and legs allegedly by Ridzuan, the father, using a broom, because he had refused to take off his shorts to have a shower.
Ridzuan then allegedly threw several cups of hot water at his son's legs and body, causing the child to shout "don't want, don't want" in Malay.
The prosecution team said the boy crouched over as Ridzuan poured hot water over his son's back and calf area, and the child fell forward onto the bathroom floor and stopped moving.
Upon his admission to KK Women's and Children's Hospital, he was found to be in a critical condition with symptoms such as low body temperature and low blood pressure.
He also had second-to third-degree burns, dehydration, acute kidney injury, multiple facial wounds and a broken nose, and demonstrated isolated speech delay.
Woman charged with murder of son, 5
30 per cent spike in child abuse cases last year, with sharp rise in physical and sexual abuses
Mother pleads guilty to slapping 23-month-old son who fell and hit head, resulting in cerebral palsy
Courts & Crime: Read more stories
Associate Professor Loh Tsee Foong, a senior consultant at the hospital's Children's Intensive Care Unit, testified in court yesterday that the boy also had severe anaemia that was "almost life threatening", suggesting significant blood loss over a period of time.
Prof Loh added that while he was told that the child had been playing with a kettle when it fell on him, he found the injuries to be "very suspicious". Despite the hospital's efforts to resuscitate him, the boy died about 14 hours later.
Ridzuan was arrested on Oct 23, while Azlin was arrested on Oct 25.
Psychiatric assessments of the couple during their remand found that they were both not of unsound mind at the time of the offences.
Correction note: This article has been edited for clarity.
A version of this article appeared in the print edition of The Straits Times on November 13, 2019, with the headline 'Parents on trial for murder, abuse of son aged 5'. Print Edition | Subscribe
SINGAPORE COURTS
CRIMES AGAINST CHILDREN/MINORS