in-vivo-measurement-of-the-mouse-pulmonary-endothelial-surface-layer

<em>In vivo</em> Measurement of the Mouse Pulmonary Endothelial Surface Layer

The endothelial glycocalyx is a layer of proteoglycans and associated glycosaminoglycans lining the vascular lumen. In vivo, the glycocalyx is highly ...

direct-intrabronchial-administration-to-improve-selective-agent

Direct Intrabronchial Administration to Improve the Selective Agent Deposition Within the Mouse Lung

Intratracheal (IT) administration of experimental agents is an essential technique in murine models of diffuse lung diseases, such as bleomycin-induced ...

Fowl (males more than females) show maturation-dependent rises in blood pressure (BP) and formation of neointimal plaques (NPs), resembling balloon catheter injury-induced neointima, in the abdominal aorta (AbA) just above the bifurcation. The plaque comprises neointimal cells containing abundant endoplasmic reticulum and extracellular matrix. Hence, we investigated whether rapid incremental BP increases in male chicks trigger NP formation, possibly via endothelial injury in hemodynamically selective areas. In 6-wk-old chicks (n = 8) treated 4 wk with solvent (Sv; minipump) or arginine supplement (Arg; 0.3% in drinking water), BP increased from 140 +/- 5 to 159 +/- 4 (Sv) and from 138 +/- 4 to 157 +/- 3 (Arg) mmHg, whereas propranolol treatment (Prop, 8 mg.kg(-1).day(-1); minipump) prevented the rise. Arg and Prop groups had, respectively, 73% and 77% smaller (P < 0.05) NP areas and 19% and 25% less (P < 0.01) AbA medial thickness than Sv controls. In 16-wk-old cockerels, established BP remained high after Sv and Arg treatments. In the Prop group, BP decreased, but neither NP area nor medial thickness was lower than in the Sv group, whereas the Arg group showed greater NP area and medial thickness. Pulse pressure, determined by intravascular transducer, increased as the pulse wave descended the aorta. The results suggest that maturation-dependent rises in BP in chicks may trigger NP formation in the lower segment of the AbA, which was prevented by inhibition of BP increase, or via a possible increase in nitric oxide availability. BP reduction exerts no effect once BP reaches a plateau. Involvement of endothelial injury leading to NP formation and hemodynamic forces selective for the lesion-prone area remain to be determined.

Do incremental increases in blood pressure elicit neointimal plaques through endothelial injury?

Gefl&uuml;gel (M&auml;nner mehr als Frauen) Karte Reifung-abh&auml;ngige entspringt im Blutdruck (BP) und die Bildung von Neointimal Plaques (NPs), &auml;hnlich Ballon Katheter Sch&auml;digung induziert Neointima in die Abdominal-Aorta (AbA) oberhalb der Bifurkation. Die Plakette besteht aus Neointimal Zellen, die reichlich Endoplasmatisches Reticulum und extrazellul&auml;ren Matrix. Daher untersucht wir, ob schnelle inkrementelle BP Anstieg m&auml;nnliche K&uuml;ken NP-Bildung, m&ouml;glicherweise &uuml;ber endotheliale Sch&auml;digung in hemodynamically selektive Bereichen ausl&ouml;sen. In 6-wk-alte K&uuml;ken (n = 8) behandelt 4 wk mit L&ouml;sungsmittel (Sv; Minipumpe) oder Arginin Erg&auml;nzung (Arg; 0,3 % im Trinkwasser), BP erh&ouml;ht von 140 +/-5 bis 159 +/-4 (Sv) und von 138 +/-4 bis 157 +/-3 (Arg) MmHg, w&auml;hrend die Propranolol-Behandlung (Prop, 8 mg.kg(-1).day(-1); Minipumpe) verhindert den Aufstieg. Arg und Prop Gruppen hatten, bzw. 73 % und 77 % kleiner (P &lt; 0,05) NP Bereiche und 19 % und 25 % weniger (P &lt; 0,01) AbA medialen Dicke als Sv-Steuerelemente. In 16 wk alte H&auml;hne etablierten BP hoch geblieben nach Sv und Arg Behandlungen. Klicken Sie in der Gruppe Prop BP verringert, aber weder die NP-Bereich als auch die mediale Dicke war niedriger als in der Sv-Gruppe, w&auml;hrend die Arg-Gruppe NP Gro&szlig;raum und medialen Dicke zeigte. Pulsdruck, bestimmt durch intravaskul&auml;re Messumformer, erh&ouml;ht die Impuls-Welle die Aorta abstammen. Die Ergebnisse legen nahe, dass Reifung-abh&auml;ngige entspringt im BP in K&uuml;ken NP Bildung im unteren Segment der ABA ausl&ouml;sen k&ouml;nnten, die durch Hemmung der BP erh&ouml;hen oder &uuml;ber eine m&ouml;gliche Erh&ouml;hung in Stickstoffmonoxid Verf&uuml;gbarkeit verhindert wurde. BP Reduktion &uuml;bt keine Wirkung, wenn BP ein Plateau erreicht. Einbeziehung der endotheliale Sch&auml;digung f&uuml;hrt zur Bildung von NP und selektiv f&uuml;r den L&auml;sion-gef&auml;hrdeten Bereich h&auml;modynamischen Kr&auml;fte m&uuml;ssen noch bestimmt werden.

Hervorrufen inkrementelle Erh&ouml;hungen des Blutdrucks Neointimal Plaketten durch endotheliale Sch&auml;digung?

Avian kidneys have loopless and looped nephrons; a countercurrent multiplier mechanism operates in the latter by NaCl recycling. We identified an aquaporin-2 (AQP2) homolog in apical/subapical regions of cortical and medullary collecting duct (CD) cells in kidneys of Japanese quail (q), Coturnix japonica. We investigated whether undernutrition during the embryonic/maturation period retards kidney and AQP2 development in quail and programs impaired volume regulation in adults. Protocols included 1) time course and 2) effects of 5-10% egg white withdrawal (EwW) or 48-h post-hatch food deprivation (FD) on nephron growth and qAQP2 mRNA expression, and 3) effects of EwW and FD on qAQP2 mRNA responses to 72-h water deprivation in adults. In metanephric kidneys, qAQP2 mRNA is expressed in medullary CDs at embryonic day 10; distribution and intensity increase during maturation. The number and size of glomeruli continue to increase after birth, whereas nephrogenic zones decrease. In EwW embryos, qAQP2 mRNA expression is initially delayed, then restored; birth weight and hatching rate are lower than in controls. Adults from EwW embryos and FD chicks have fewer (P < 0.01) glomeruli. Water deprivation reduces body weight more in EwW birds than in controls. The results suggest that qAQP2 evolved in metanephric kidneys and that undernutrition may retard nephrogenesis, leading to impaired adult water homeostasis.

Aquaporin-2 water channel in developing quail kidney: possible role in programming adult fluid homeostasis.

Vogelgrippe Nieren haben loopless und geschlungene Nephronen; ein Gegenstrom Multiplikator-Mechanismus arbeitet im letzteren durch das recycling von NaCl. Wir haben ein Aquaporin-2 (AQP2) Homolog in apikale/retroflexer Regionen kortikale und medull&auml;re sammeln Duct (CD) Zellen in Nieren der japanischen Wachtel (Q), Coturnix Japonica identifiziert. Wir untersucht, ob die Unterern&auml;hrung w&auml;hrend der embryonalen/Reifezeit verz&ouml;gert, Nieren- und AQP2-Entwicklung in Wachteln und Programme beeintr&auml;chtigt Volumenregulation bei Erwachsenen. Protokolle werden auf Nephron Wachstum und qAQP2 mRNA Ausdruck sowie 3) Auswirkungen der EwW und FD auf qAQP2 mRNA Antworten um 72-h-Wasser Deprivation bei Erwachsenen 1) zeitlichen Verlauf und 2) Auswirkungen von 5 bis 10 % Eiwei&szlig; R&uuml;ckzug (EwW) oder 48-h der Lebensmittel Mangelerscheinungen (FD) enthalten. Metanephric Nieren ist qAQP2-mRNA in medull&auml;re CDs bei embryonalen 10.Tag ausgedr&uuml;ckt; Verteilung und Intensit&auml;t erh&ouml;hen w&auml;hrend der Reifung. Die Anzahl und Gr&ouml;&szlig;e der Glomerula weiterhin nach der Geburt erh&ouml;hen w&auml;hrend Nephrogene Zonen verringern. EwW Embryonen qAQP2 mRNA Ausdruck zun&auml;chst verz&ouml;gert dann wiederhergestellt; Geburtsgewicht und Schraffur-Preise sind niedriger als in Steuerelementen. Erwachsene ab EwW Embryonen und FD-K&uuml;ken haben weniger (P &lt; 0,01) Glomerula. Wasser-Entzug verringert K&ouml;rpergewicht mehr im EwW V&ouml;gel als in Steuerelementen. Die Ergebnisse legen nahe, dass qAQP2 in metanephric Nieren entwickelt und die Unterern&auml;hrung Nephrogenesis, f&uuml;hrt zu gest&ouml;rter Erwachsenen Wasser Hom&ouml;ostase verz&ouml;gern kann.

Aquaporin-2 Kanal in Entwicklungsl&auml;ndern Wachtel-Niere: m&ouml;gliche Rolle in der Programmierung von Erwachsenen fl&uuml;ssig Hom&ouml;ostase.

V&ouml;gel und S&auml;ugetiere sind die einzigen Wirbeltiere, die Urin konzentrieren k&ouml;nnen. Vogelgrippe Nieren enthalten strukturell primitive loopless Nephronen und auch vorger&uuml;ckteren geschlungene Nephronen, in den Regionen kortikalen und medull&auml;ren bzw.. Wir haben ermittelt, dass die Gensequenz von einer Aquaporin 2 (AQP2)-Homolog-Wasser-Kanal im Sammeln von Leitungen der Nieren von Erwachsenen Wachteln, Coturnix Japonica. Obwohl immunreaktivem Wachtel AQP2 (qAQP2) fand sich in beiden Arten von Nephronen, der Ausdruck wird deutlicher in medull&auml;ren Regionen verbessert, nach Entzug von Wasser. Wir daher die Hypothese Verordnung des qAQP2 Ausdrucks in Wachtel, die Nieren &uuml;ber Antidiuretisches Hormon (ADH) ben&ouml;tigen k&ouml;nnte weiter fortgeschritten Nephron-Struktur. In dieser Studie haben wir den Ausdruck der qAQP2 mRNA im Gewebe isoliert aus den kortikalen und medull&auml;ren Regionen vor und nach dem Entzug von Wasser, von konventionellen Reverse Transkriptase-Polymerase-Kettenreaktion (RT-PCR) und quantitative Echtzeit-PCR festgestellt. In Gruppen sowohl normalerweise hydratisiert und Wasser entzogen, qAQP2-mRNA-Stufen in den medull&auml;ren Regionen waren signifikant h&ouml;her (P &lt; 0,01) als in den kortikalen Regionen. Medull&auml;re Bereiche, qAQP2-mRNA-Stufen (Real-Time PCR normalisiert mit 18S) waren signifikant h&ouml;her (P &lt; 0,01, ANOVA) nach Wasser-Entzug (1.09 +/-0,10) als normalerweise hydratisiert Kontrollen (0.46 +/-0,08). In kortikalen Gebieten lagen qAQP2 mRNA-Stufen auch nach Wasser-Entzug (0.37 +/-0,05) als in Steuerelementen (0.11 +/-0,02). qAQP2-mRNA-Signale, die durch in-situ Hybridisierung von Digoxigenin-markierten Riboprobe bestimmt wurden auch nach Wasser Entbehrung in sowohl kortikale und medull&auml;re sammeln Kan&auml;len erweitert. Die Ergebnisse legen nahe, dass entgegen unserer Hypothese, die endogene Produktion von ADH durch Wasser Deprivation qAQP2 mRNA in sowohl die loopless als auch die geschlungene Nephronen stimuliert.

Kontrolle der Aquaporin 2 Ausdruck in Leitungen, der Wachtel Nieren sammeln.

Birds and mammals are the only vertebrates that can concentrate urine. Avian kidneys contain structurally primitive loopless nephrons and also more advanced looped nephrons, in the cortical and medullary regions, respectively. We have identified the gene sequence of an aquaporin 2 (AQP2)-homologue water channel in collecting ducts of kidneys from adult quail, Coturnix japonica. Although immunoreactive quail AQP2 (qAQP2) was found in both types of nephrons, the expression is enhanced more clearly in the medullary regions after water deprivation. We therefore hypothesized that regulation of qAQP2 expression in quail kidneys via antidiuretic hormone (ADH) may require more advanced nephron structure. In this study, we determined the expression of qAQP2 mRNA in tissues isolated from the cortical and medullary regions before and after water deprivation, by conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and quantitative real-time PCR. In both normally hydrated and water-deprived groups, qAQP2 mRNA levels in the medullary regions were significantly higher (P<0.01) than in the cortical regions. In medullary areas, qAQP2 mRNA levels (real-time PCR normalized with 18S) were significantly higher (P<0.01, ANOVA) after water deprivation (1.09+/-0.10) than in normally hydrated controls (0.46+/-0.08). In cortical areas, qAQP2 mRNA levels were also higher after water deprivation (0.37+/-0.05) than in controls (0.11+/-0.02). qAQP2 mRNA signals determined by in situ hybridization of digoxigenin-labeled riboprobe were also enhanced after water deprivation in both cortical and medullary collecting ducts. The results suggest that, contrary to our hypothesis, the endogenous production of ADH by water deprivation stimulates qAQP2 mRNA in both loopless and looped nephrons.

Control of aquaporin 2 expression in collecting ducts of quail kidneys.

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-Î± (TNF-Î±)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-Î±-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis.

Sepsis, eine systemische entz&uuml;ndliche Reaktion auf eine Infektion, h&auml;ufig fortschreitet, akute Lungeninsuffizienz (ALI), eine entz&uuml;ndliche Lungenerkrankung mit hohen Morbidit&auml;t. Wir postuliert, dass Sepsis-assoziierten ALI neutrophil festhalten und Entz&uuml;ndungen durch Abbau von der pulmonalen endothelialen Glykokalyx initiiert wird. Mit intravital-Mikroskopie, fanden wir, dass Endotoxin&auml;mie in M&auml;use schnell induzierten pulmonalen mikrovaskul&auml;re Glykokalyx Abbau durch Tumor-Nekrose-Faktor-&alpha; (TNF-&alpha;)-abh&auml;ngige Mechanismen. Glykokalyx Abbau den spezifischen Verlust von Heparansulfat beteiligt und fiel mit Aktivierung der endothelial Heparanase, ein TNF-&alpha;-responsive, Heparan Sulfat-spezifische Glukuronidase. Glykokalyx Abbau erh&ouml;ht die Verf&uuml;gbarkeit von endothelial Oberfl&auml;che Adh&auml;sionsmolek&uuml;le auf zirkulierenden Mikrosph&auml;ren und trug zur neutrophil Adh&auml;sion. Heparanase Hemmung verhindert Endotoxin&auml;mie-assoziierte Glykokalyx-Verlust und Neutrophilen-Adh&auml;sion und dementsprechend abgeschw&auml;cht Sepsis-induzierter ALI und Sterblichkeit in den M&auml;usen. Diese Erkenntnisse sind f&uuml;r Krankheiten des Menschen potenziell relevant, wie Sepsis-assoziierten Atemstillstand beim Menschen h&ouml;here Plasma Heparan Sulfat Abbau Aktivit&auml;t zugeordnet wurde; Dar&uuml;ber hinaus lag Heparanase Inhalt in Lunge Biopsien diffusen alveol&auml;ren Schaden als in normalen menschlichen Lungengewebe zeigen.

Die pulmonale endothelialen Glykokalyx regelt neutrophil Adh&auml;sion und Lungeninsuffizienz w&auml;hrend experimentelle Sepsis.

While reductionist in vitro approaches have allowed for careful interrogation of cellular pathways that underlie innate immune responses, they often fail to capture the complex multicellular interactions characteristic of acute inflammation. Intravital microscopy, by directly observing alveolar cell-cell interactions, provides unique insight into the complex intercellular mechanisms responsible for alveolar inflammation. This review discusses multiple potential approaches to intravital pulmonary imaging, with specific attention to in vivo microscopy of the freely moving mouse lung.

Intravital Microscopy in the Mouse Lung.

Septic patients frequently develop cognitive impairment that persists beyond hospital discharge. The impact of sepsis on electrophysiological and molecular determinants of learning is underexplored. We observed that mice that survived sepsis or endotoxemia experienced loss of hippocampal long-term potentiation (LTP), a brain-derived neurotrophic factor-mediated (BDNF-mediated) process responsible for spatial memory formation. Memory impairment occurred despite preserved hippocampal BDNF content and could be reversed by stimulation of BDNF signaling, suggesting the presence of a local BDNF inhibitor. Sepsis is associated with degradation of the endothelial glycocalyx, releasing heparan sulfate fragments (of sufficient size and sulfation to bind BDNF) into the circulation. Heparan sulfate fragments penetrated the hippocampal blood-brain barrier during sepsis and inhibited BDNF-mediated LTP. Glycoarray approaches demonstrated that the avidity of heparan sulfate for BDNF increased with sulfation at the 2-O position of iduronic acid and the N position of glucosamine. Circulating heparan sulfate in endotoxemic mice and septic humans was enriched in 2-O- and N-sulfated disaccharides; furthermore, the presence of these sulfation patterns in the plasma of septic patients at intensive care unit (ICU) admission predicted persistent cognitive impairment 14 days after ICU discharge or at hospital discharge. Our findings indicate that circulating 2-O- and N-sulfated heparan sulfate fragments contribute to septic cognitive impairment.

Circulating heparan sulfate fragments mediate septic cognitive dysfunction.

Sepsis induces heparanase-mediated degradation of the endothelial glycocalyx, a heparan sulfate-enriched endovascular layer critical to vascular homeostasis, releasing highly sulfated domains of heparan sulfate into the circulation. These domains are oligosaccharides rich in heparin-like trisulfated disaccharide repeating units. Using a chemoenzymatic approach, an undecasaccharide containing a uniformly C-labeled internal 2-sulfoiduronic acid residue was synthesized on a -nitrophenylglucuronide acceptor. Selective periodate cleavage afforded a heparin nonasaccharide having a natural structure. This C-labeled nonasaccharide was intravenously administered to septic (induced by cecal ligation and puncture, a model of polymicrobial peritonitis-induced sepsis) and nonseptic (sham) mice. Selected tissues and biological fluids from the mice were harvested at various time points over 4 hours, and the C-labeled nonasaccharide was recovered and digested with heparin lyases. The resulting C-labeled trisulfated disaccharide was quantified, without interference from endogenous mouse heparan sulfate/heparin, using liquid chromatography-mass spectrometry with sensitive and selective multiple reaction monitoring. The C-labeled heparin nonasaccharide appeared immediately in the blood and was rapidly cleared through the urine. Plasma nonasaccharide clearance was only slightly prolonged in septic mice ( ∼ 90 minutes). In septic mice, the nonasaccharide penetrated into the hippocampus but not the cortex of the brain; no hippocampal or cortical brain penetration occurred in sham mice. The results of this study suggest that circulating heparan sulfates are rapidly cleared from the plasma during sepsis and selectively penetrate the hippocampus, where they may have functional consequences.

ArticleTotal : 2	Year
Publication title Cited by 16	2013
Publication title Cited by 5	2019

Article	Year
Do incremental increases in blood pressure elicit neointimal plaques through endothelial injury? American journal of physiology. Regulatory, integrative and comparative physiology\| PubMed ID: 15319217	2004
Aquaporin-2 water channel in developing quail kidney: possible role in programming adult fluid homeostasis. American journal of physiology. Regulatory, integrative and comparative physiology\| PubMed ID: 17699563	2007
Control of aquaporin 2 expression in collecting ducts of quail kidneys. General and comparative endocrinology\| PubMed ID: 19135443	2009
The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis. Nature medicine\| PubMed ID: 22820644	2012
Intravital Microscopy in the Mouse Lung. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29987798	2018
Circulating heparan sulfate fragments mediate septic cognitive dysfunction. The Journal of clinical investigation\| PubMed ID: 30720464	2019
Circulating heparin oligosaccharides rapidly target the hippocampus in sepsis, potentially impacting cognitive functions. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 31010931	2019

Yimu Yang

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