To address this question COSC were treated with the selective and irreversible caspase-3 inhibitor Z-DEVD-fmk [17] during hypoxia and inflammation and pericyte numbers in confocal z-stacks were compared with solvent treated normoxic and non-inflammatory control COSC

Groups that did not pass the examination for normalization were analyzed with a two sided MannWhitney U test. Variations had been regarded to be significant at alpha < 0.05.Immunohistochemical quadruple stainings show that in the presented in vitro approach almost all of the cell types of the neurovascular unit are present and display a preserved morphology in COSC after 3 DIV. Using high power imaging of cortical microvessels, pericytes and astrocytes we found that the endothelium is enwrapped by pericytes that are further in close contact with astrocytes (Figure 2 A, Movie S3). GSK 3203591NeuN immunoreactivity revealed the presence of neurons with an intact cellular morphology within the COSC preparation (Figure 2 B). It has been reported that the basement membrane (BM), which consists of laminins, proteoglykans, nidogens as well as collagen IV [23], is of high relevance for neurovascular integrity [24,25]. Therefore we were interested in the question if a BM is present in the cortical neurovascular in vitro model. We found that cortical microvessels and pericytes in COSC possess a BM as demonstrated by pan-Laminin stainings (antibody that binds to Laminin chains alpha-1, beta-1, alpha-2 and gamma-1, and thereby many laminin isoforms that contain at least one of these chain types) in COSC [24,26]. Laminins are heterotrimers composed of an alpha, beta and gamma chain [23]. They are a hallmark for a proper formation of a BM and therefore are a marker of the BM in microvessels in the CNS [25,27]. We observed a preservation of the BM in the neurovascular unit of our in vitro preparation after 4 DIV (representative images from more than 10 COSC preparations,In the CNS the PDGFR beta receptor is exclusively expressed by pericytes and therefore represents a sensitive pericyte marker [4,20]. To identify perivascular PDGFR beta positive cells in COSC we performed immunohistochemical analyses with different vessel markers (CD105, Claudin 5), PDGFR beta and DAPI. Our COSC preparations displayed a well preserved cytoarchitecture of cortical layers after 5 days in vitro (DIV) as demonstrated by DAPI staining (Figure 1 A). The transmembraneous tight junctional protein Claudin 5 (Cl-5) is expressed by brain endothelial cells [17] in COSC and is Figure 1. Pericytes in cortical organotypic slice cultures. Microvessels and pericytes in COSC were immunolabelled with different vascular markers (CD105, Cl-5), the pericyte marker PDGFR beta and DAPI. COSC preparations displayed a well preserved cytoarchitecture of cortical layers after 5 DIV (DAPI, epifluorescent image, A). A high number of microvessels covered by pericytes remained in COSC (epifluorescent image, B). PDGFR beta (green) partly co-localized (yellow) with the proteoglykan NG2 (red) in COSC which is another pericyte marker (arrowhead, confocal Z-stack with orthogonal section views, C). Similar to the perivascular localization of PDGFR beta positive cells in newborn mice at the age of postnatal day 3 (D) we observed PDGFR beta positive cells to be in close contact with microvessels in COSC after 5 DIV (E). Note the DAPI positive pericyte cell soma in which the nucleus is mostly spared by PDGFR beta staining (marked by asterisks) and its processes that follow the microvessel (D, E insets). Further the pericyte marker Desmin was found to be expressed by perivascular PDGFR beta positive cells in COSC (Panel F) as well as in native P3 mice (Panel G). Pericyte coverage in cortical microvessels was not significantly different in native P3 mice compared with COSC (H).Figure 2. The neurovascular unit in COSC. Immunohistochemical stainings show a preserved morphology of astrocytes and pericytes that cover cortical microvessels in COSC (A). Stainings with the neuronal marker NeuN demonstrated a preserved neuronal morphology within the neurovascular unit of COSC (B).High power confocal images revealed that cortical microvessels and pericytes are embedded in the BM (Figure 3 B, Movie S4). Animated three-dimensional reconstructions with laser scanning confocal imaging confirmed these findings on an intact NVU (Movies S3, S4).To address the question whether the in vitro model can be used for live cell imaging of pericytes, we labeled cortical microvessels of P4 heterozygous EYFP-NG2 knockin mouse mutants with tomato lectin. This mouse lineage carries an EYFP label inserted in exon 1 of the NG2 gene [28]. Here, in line with our immunohistochemical NG2 analyses, the EYFPNG2 signal was partly present next to cortical microvessels in living COSC (Figure 4 A). However other NG2 cells were not associated with microvessels indicating that they are not pericytes. Pericytes could be identified through the perivascular EYFP-NG2 signal for up to 3 days after COSC preparation (Figure 4 B). To verify that the obtained perivascular EYFPNG2 signals were of pericyte origin we performed immunohistochemical co-stainings of EYFP-NG2 cortices with PDGFR beta, NG2 and Cl-5, which demonstrated a colocalization of EYFP-NG2 and PDGFR beta in a perivascular manner (Figure 4 C, D, insets). Co-stainings with NG2 and Cl-5 confirmed this observation (Figure 4 E).vesicular stomatitis glycoprotein-pseudotyped retroviruses to transduce certain differentiation factors e.g. Mash1 and Sox2 [29]. However, this process is crucially dependent on the capacity of pericytes to proliferate because a successful transduction via retroviral vectors (except for lentiviruses) is dependent on the ability of the targeted cells for cell division. We found that BrdU (exposition for 3 hours on DIV 3, 10 ol/l) was incorporated by some pericytes within 24 hours after exposure (Figure 5 A, Movies S5, S6). In addition some pericytes were positive for Ki67 (Figure 5 B), a marker for cell proliferation, after 4 DIV [30]. Ki67 and BrdU stainings were performed in more than 4 COSC preparations.To elucidate if COSC maintain spontaneous network activity during culture conditions (5% CO2, 20% O2, rest N2, humidified atmosphere, 37) multi-electrode array (MEA) recordings were performed as described previously with slight modifications (Heck et al., 2008). Here we observed spontaneous synchronized neuronal network activity (Figure 6 A, representative image from n = 3 independent recordings, DIV 5). Pericytes could be identified in COSC for up to 3 weeks. Figure 6 B shows a pericyte with characteristic perivascular morphology after 14 days in vitro.It has recently been shown that pericytes from human origin can be reprogrammed into induced neuronal cells by the use of Chronic hypoxia and inflammation have been proposed to be key factors leading to preterm brain injury [31]. In addition we Figure 3. Basement membrane in the neurovascular unit of COSC. Co-labeling of cortical microvessels (Cl-5, green), pericytes (PDGFR beta, red) and laminins with a pan-Laminin antibody (white) reveals the presence of a basement membrane (BM) in the neurovascular unit in COSC after 4 DIV (confocal Z-stacks, maximum projection A). High power confocal magnification visualizes the BM (arrowheads, B) that encloses microvessels (Cl-5, green) and pericytes (PDGFR beta, red). Note the DAPI positive cell nuclei of the Cl-5 positive microvessel (asterisk) and the PDGFR beta positive pericyte (marked by ).Figure 4. Live cell imaging of pericytes in COSC from EYFP-NG2 mice. Lectin stained microvessels in COSC appeared as red labeled vascular structures that were surrounded by EYFP-NG2 expressing pericytes (A, insets). Pericytes could be identified for up to 3 DIV 3 (B). Co-stainings with PDGFR beta (C, D), Cl-5 and NG2 demonstrate that perivascular EYFP-NG2 expressing cells are indeed pericytes (E, insets).and others have shown that caspase-3 is involved in neuronal and BBB pathology during ischemia and inflammation [17,32,33]. Therefore we were interested in the question if inflammation induced by interleukin 1 beta (IL1B), a cytokine which has been shown to disrupt proper white matter formation in the developing brain [34], and moderate hypoxia result in caspase-3 activation in pericytes. Thus we subjected COSC towards prolonged moderate hypoxia and different concentrations of IL1B. The cultures were exposed to a medium maintaining 71 + 2 mmHg pO2 for 24 hours. This pO2 Figure 5. Pericytes in COSC are capable of cell division. Confocal analyses revealed that BrdU (3 hours exposition, 10ol/l) was incorporated by pericytes within 24 hours on DIV 4 (arrowheads and asterisks in A mark a pericyte cell nucleus positive for BrdU). Ki-67 is a marker for cell proliferation. Here, a pericyte cell nucleus immunoreactive for Ki-67 on DIV 4 is shown (arrowheads, asterisks in B).Figure 6. Spontaneous neural network activity and longterm persistence of pericytes in COSC. MEA recordings demonstrated that spontaneous synchronized neural network activity is preserved in COSC under culture conditions on DIV 5 (A). In addition pericytes were found to be present in COSC for weeks. Here, a 2 week old pericytes situated next to a Cl-5 positive microvessel is shown (B).level is about 58% of control medium kept in normoxic cell incubators (hypoxia 71 + 2.2 mmHg vs. normoxia 123 + 0.5 mmHg pO2, P < 0.0001, n = 15-48 measurements). IL1B treatment resulted in significantly higher amounts of caspase-3 positive pericytes (representative image of a caspase-3 positive pericyte treated for 24 hours with IL1B 100 ng/ml, Figure 7 A) after 24 hours compared with control (control: 1 + 0.1 vs. IL1B 10 ng/ml 1.42 + 0.15, P = 0.0269 control: 1 + 0.1 vs. IL1B 100 ng/ml 1.98 + 0.19, P < 0.0001, n = 7 - 8 COSC preparations per group [535-668 pericytes], Figure 7 B). We detected significantly more caspase-3 positive pericytes in COSC treated with IL1B 100 ng/ml than in those treated with 10 ng/ml (IL1B 10 ng/ml 1.42 + 0.15 vs. IL1B 100 ng/ml 1.98 + 0.19, P = 0.0237, n = 8 COSC per group [535 668 pericytes], Figure 7 B). Moderate hypoxia for 24 hours resulted in a significantly higher amount of cleaved caspase-3 positive pericytes (control: 1 + 0.05 vs. hypoxia: 2.48 + 0.23, P < 0.0001, n = 11-41 COSC preparations [571 - 2800 pericytes], Figure 7 C). Exposure of combined hypoxia and IL1B 100 ng/ml significantly elevated cleaved caspase-3 levels in pericytes (control: 1 + 0.05 vs. yhpoxia+IL1B: 2.38 + 0.15, P < 0.0001, n = 18-41 COSC preparations [677-2800 pericytes], Figure 7 C). However, a combination of IL1B and 24 h of moderate hypoxia did not increase caspase-3 cleavage compared with 24 h of moderate hypoxia alone (hypoxia: 2.48 + 0.23 vs. hypoxia+IL1B 2.38 + 0.15, P = 0.9763, n = 11 - 18 COSC preparations [571 - 677 pericytes], Figure 7 C).It has been suggested that oxidative stress in form of reactive oxygen species (ROS) plays a major role in immature brain damage [31]. Therefore we were interested in the question if ROS may contribute to the observed enhancement of caspase-3 cleavage in pericytes after 24 h of hypoxia and whether an inhibition of oxidative stress may prevent caspase-3 cleavage. Stainings for nitrotyrosine, a product that arises from the reaction of the ROS peroxynitrite and the amino acid tyrosine [35] did not show any significant levels of nitrotyrosine in pericytes (representative image from at least 3 COSC preparations, Figure 7 D, E). The NADPH oxidase is a major source of reactive oxygen species [36] in cerebral ischemia and diphenyliodonium chloride (DPI) is a widely used inhibitor of the NADPH oxidase [18]. We therefore tested if a pharmacological inhibition of NADPH oxidase by DPI has an effect on blockade of caspase-3 cleavage in pericytes during hypoxia. We found that DPI was not able to block caspase-3 cleavage in pericytes. Despite DPI treatment caspase-3 levels in the hypoxic group were significantly higher than in the normoxic control (hypoxia+DPI: 3.049 + 0.16 vs. normoxic control: 1 + 0.07, P < 0.0001, n = 24 COSC preparations per group [1597-2812 pericytes], Figure 7 F).Because we observed enhanced levels of cleaved caspase-3 in pericytes upon ischemia and inflammation we were Figure 7. IL1B and moderate hypoxia induce caspase-3 cleavage in pericytes independently from peroxynitrite. Exposition of COSC towards pathologic conditions led to an increase of cleaved caspase-3 positive pericytes as shown in a representative confocal image in panel A. Stimulation of COSC with different concentrations of IL1B resulted in significantly elevated levels of cleaved caspase-3 in pericytes (B). Furthermore 24 hours of moderate hypoxia and a combination of IL1B and 24 hours of moderate hypoxia resulted in caspase-3 cleavage in pericytes (C). Of note, combination of IL1B and hypoxia did not significantly increase caspase-3 cleavage compared with probes treated with 24 hours of moderate hypoxia alone. Immunohistochemical stainings for nitrotyrosine did not reveal any significant formation of this ROS reaction product in pericytes (D, E). The NADPH oxidase inhibitor DPI [50ol/l, 1 h pre-incubation] was not able to block caspase-3 cleavage under moderate hypoxic conditions. P<0.05, P<0.001.interested in the question if caspase-3 activation may result in pericyte loss. 6128652To address this question COSC were treated with the selective and irreversible caspase-3 inhibitor Z-DEVD-fmk [17] during hypoxia and inflammation and pericyte numbers in confocal z-stacks were compared with solvent treated normoxic and non-inflammatory control COSC. We found that hypoxia resulted in a significant decrease of pericytes/volume compared with controls (control: 1 + 0.16 vs. hypoxia 0.31 + 0.02, P = 0.0034, n = 5 – 6 RFV from more than 3 COSC). After IL1B (100 ng/ml) treatment pericytes were also significantly reduced in cortical layers II-IV (control: 1 + 0.16 vs. IL1B 0.46 + 0.05, P = 0.0142, n = 5-6 RFV from more than 3 COSC preparations per group). COSC that were treated with Z-DEVDfmk sustained significantly higher pericyte numbers after hypoxic (hypoxia 0.31 + 0.02 vs. hypoxia/Z-DEVD-fmk 0.52 + 0.08, P = 0.0324, n = 5 RFV from more than 3 COSC) and inflammatory conditions compared with solvent treated groups (IL1B 0.46 + 0.05 vs. IL1B/ZDEVD-fmk 0.95 + 0.16, P = 0.0406, n 5 – 8 RFV from more than 3 COSC preparations per group). However Z-DEVD-fmk was not able to completely block pericyte loss under hypoxic conditions (control: 1 + 0.16 vs.hypoxia/Z-DEVD-fmk 0.52 + 0.08, P = 0.0301, n = 5 – 6 RFV from more than 3 COSC).In this report we demonstrate that cortical organotypic slice cultures from neonatal rodents are a useful model to study pericytes within the intact neurovascular unit under various experimental conditions. Further we show that pathological conditions such as moderate hypoxia and inflammation result in caspase-3 mediated pericyte loss.