Monitoring setup, ECG and breathing detection.

<p>Rodents were placed on a temperature monitored heating pad and connected to ECG electrodes. A thermal probe was used to control the body temperature of the animal. Data of heart and breathing rates were collected with analog digital converters (ADC, <b>A</b>). Recordings from an awake P7 mouse showing a raw data trace of breathing movements including breathing cycle duration (<b>B, trace 1</b>). Heart rate recordings in awake animals (raw data: <b>B, </b><b>trace 2</b>) were filtered at 10–200 Hz band pass which allowed identification of QRS complexes (<b>B, </b><b>trace 3</b>). Effect of urethane on breathing and heart rate in representative recordings of the same animal 30–60 minutes after urethane administration (<b>C</b>). Note the different pattern of the PZT signal (<b>C, trace 1</b>) in comparison with the awake state in B trace 1. After filtering the ECG raw data (<b>C, trace 2</b>) in some recordings beside QRS complexes P and T waves could be identified (<b>C, trace 3 inset</b>).</p

A Novel <i>In Vitro</i> Model to Study Pericytes in the Neurovascular Unit of the Developing Cortex

<div><p>Cortical function is impaired in various disorders of the central nervous system including Alzheimer’s disease, autism and schizophrenia. Some of these disorders are speculated to be associated with insults in early brain development. Pericytes have been shown to regulate neurovascular integrity in development, health and disease. Hence, precisely controlled mechanisms must have evolved in evolution to operate pericyte proliferation, repair and cell fate within the neurovascular unit (NVU). It is well established that pericyte deficiency leads to NVU injury resulting in cognitive decline and neuroinflammation in cortical layers. However, little is known about the role of pericytes in pathophysiological processes of the developing cortex. Here we introduce an in vitro model that enables to precisely study pericytes in the immature cortex and show that moderate inflammation and hypoxia result in caspase-3 mediated pericyte loss. Using heterozygous EYFP-NG2 mouse mutants we performed live imaging of pericytes for several days in vitro. In addition we show that pericytes maintain their capacity to proliferate which may allow cell-based therapies like reprogramming of pericytes into induced neuronal cells in the presented approach. </p> </div

Pericyte impairment in the developing cortex during pathologic conditions.

<p>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.</p

Differences of urethane metabolism in mice and rats within the first postnatal week.

<p>In P0/1 mice ethanol concentrations in blood plasma 60 minutes after urethane administration were significantly higher than in P0/1 rats. Ethanol in plasma from P6/7 rodents was not detected. Box and whisker plots (displaying 75th percentile, median and 25th percentile) are shown, whiskers indicate minimum and maximum values. ***P<0.001.</p

Effect of urethane on blood gases in P6/7 mice.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062628#pone-0062628-t001" target="_blank">Table 1:</a> n = 3–4 animals, body weight [g] = 4.3±0.3, Values are mean ± SD, arterial/venous. blood.</p

The neurovascular unit in COSC.

<p>Immunohistochemical stainings show a preserved morphology of astrocytes and pericytes that cover cortical microvessels in COSC (<b>A</b>). Stainings with the neuronal marker NeuN demonstrated a preserved neuronal morphology within the neurovascular unit of COSC (<b>B</b>).</p

Developmental changes in respiratory and heart rate during the first postnatal week.

<p>In rats and mice breathing frequency significantly increased during the first postnatal week (<b>A</b>). We did not observe differences in RPM at corresponding ages between mice and rats. Heart rate increased from P0/1 to P6/7 in both species (<b>B</b>). Note that mice have significantly faster heart frequencies than rats at the same age. Box and whisker plots (displaying 75th percentile, median and 25th percentile) are shown, whiskers indicate minimum and maximum values. *P<0.05, **P<0.01, ***P<0.001.</p

Pericytes in COSC are capable of cell division.

<p>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).</p

Live cell imaging of pericytes in COSC from EYFP-NG2 mice.

<p>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>B</b>). Co-stainings with PDGFR beta (<b>C</b>, <b>D</b>), Cl-5 and NG2 demonstrate that perivascular EYFP-NG2 expressing cells are indeed pericytes (E, insets).</p

Basement membrane in the neurovascular unit of COSC.

<p>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 #).</p