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).
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).
Figure 5.
Pericytes in COSC are capable of cell division.
Confocal analyses revealed that BrdU (3 hours exposition, 10µmol/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).
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 [50µmol/l, 1 h pre-incubation] was not able to block caspase-3 cleavage under moderate hypoxic conditions. *P<0.05, ***P<0.001.
Figure 8.
Pericyte impairment in the developing cortex during pathologic conditions.
Hypoxia and inflammation result in caspase-3 dependent loss of pericytes in cortical layers. Pericyte loss may be of relevance for perturbances in neurovascular integrity in the developing brain and may be involved in a variety of pathologic sequelae e.g. cognitive decline in ageing or neuroinflammation.