Name: intraperitoneal glucose tolerance test, measurement of lung function, and fixation of the lung to study the impact of obesity and impaired metabolism on pulmonary outcomes
Uploaded: 2018-03-15T13:00:00
Description: Watch this Scientific Journal Video about Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmo

Die Experimente wurden durch die Marga und Walter Boll-Stiftung, Kerpen, Deutschland gestützt; Projekt-210-02-16 (MAAA) Projekt 210-03-15 (MAAA) und von der Deutschen Forschungsgemeinschaft (DFG; AL1632-02; MAAA), Bonn, Deutschland; Zentrum für Molekulare Medizin Köln (CMMC; Universitätsklinikum Köln; Karriere-Förderung-Programm; MAAA), Köln Fortune (medizinische Fakultät, Universität zu Köln; KD).

Alle tierische Verfahren wurden durchgeführt in Übereinstimmung mit den Protokollen, die von lokalen Behörden genehmigt (Land NRW, AZ: 2012.A424), und wurden im Einklang mit dem deutschen Tierschutzgesetz und die Vorschriften über das Wohlergehen der Tiere für Experimente oder für andere wissenschaftliche Zwecke. Da Lunge Funktionsanalyse Lunge Struktur beeinflussen kann und deshalb nachfolgende histologische analysiert, sollte die Messung der Res Cdyn und die Vorbereitung und Fixierung der Lunge für Histomorphome...

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Dieser Bericht enthält drei Protokolle für drei verschiedene Methoden, um die Auswirkungen der Fettleibigkeit auf Glukose-Stoffwechsel und pulmonale Ergebnisse zu analysieren. Erstens kann die Glukose-Toleranz-Test bietet die Möglichkeit, intrazelluläre Glukoseaufnahme analysieren und Hinweis auf Insulin-Resistenz. Zweitens: Ganzkörper-Plethysmographie ist eine Technik zur Messung der Lungenfunktion und ist dabei hilfreich, um die Wirksamkeit von neuen Behandlungsmethoden zu testen. Drittens ist eine standardisierte...

Laut der Weltgesundheitsorganisation (WHO) im Jahr 2008, waren mehr als 1,4 Milliarden Erwachsene, im Alter von 20 und älter, Übergewicht mit einem Body mass Index (BMI) größer als oder gleich 25; Weitere, mehr als 200 Millionen Männer und fast 300 Millionen Frauen waren übergewichtig (BMI≥30)1. Adipositas und metabolisches Syndrom sind Hauptrisikofaktoren für eine Vielzahl von Krankheiten. Bei der Adipositas und damit einhergehenden erhöhten weißen Fettgewebe Masse eng verknüpft wurde um 2 Diabetes2,3, Herz-Kreislauf Erkrankungen, wie koronare Herzkrankheit (KHK), Herzinsuffizienz (HF), Vorhofflimmern4 geben und Arthrose5, ihre funktionelle Rolle in der Pathogenese der Erkrankungen der Atemwege bleiben schlecht verstanden. Epidemiologische Studien haben jedoch gezeigt, dass Übergewicht stark verbunden mit chronischen Atemwegserkrankungen ist, einschließlich exertional Dyspnoe, obstruktive Schlafapnoe-Syndrom (OSAS), Adipositas Hypoventilation Syndrom (OHS), chronische obstruktive Lungenerkrankung (COPD), Lungenembolie, Aspirationspneumonie und Asthma bronchiale6,7,8,9. Mögliche Mechanismen Verknüpfung von Fettleibigkeit und gestörten Stoffwechsel, z.B., Insulinresistenz und Typ-II-Diabetes, zur Pathogenese der chronischen Lungenerkrankungen umfassen nicht nur mechanische und physikalische Konsequenzen des Gewichts zu gewinnen sondern auch über be-und Entlüftung induzieren Sie eine chronische subakute entzündliche Zustand10,11. Der Anstieg von Adipositas und Lungenerkrankungen während des letzten Jahrzehnts, gekoppelt mit dem Mangel an wirksame Präventionsstrategien und Therapieansätze, betont die Notwendigkeit, untersuchen die molekularen Mechanismen zu definieren, neue Wege zur Verwaltung von Adipositas Lunge Krankheiten.
Hier beschreiben wir drei standard-Tests, die wichtige Grundlagen, Adipositas und ihre Auswirkungen auf Lunge Struktur und Funktion in Mausmodellen zu untersuchen sind: (1) intraperitoneal Glukose Toleranz (IpGTT) (2) Messung des Atemwegswiderstandes (Res) und der Atemwege System-Compliance (Cdyn); und (3) Vorbereitung und Fixierung der Lunge für anschließende quantitative histologische Bewertung. Die IpGTT ist eine robuste Screening-Test Maßnahme Glukoseaufnahme, und damit die Wirkung von Adipositas auf den Stoffwechsel. Die Einfachheit der Methode ermöglicht gute Standardisierung und damit die Vergleichbarkeit der Ergebnisse zwischen den Labors. Ausgefeiltere Methoden, z. B. hyperglykämischen Klemmen oder Studien auf isolierte Inseln können für eine detaillierte Analyse der metabolischen Phänotyp12verwendet werden. Hier bewerten wir Glukosetoleranz um eine Adipositas-assoziierten Staat der systemischen und metabolische Störung als Grundlage für weitere Studien auf eine pulmonale Ergebnis zu definieren. Um die Auswirkungen von Übergewicht und Stoffwechselstörungen auf die Lungenfunktion zu beurteilen, haben wir Atemwegswiderstand (Res) und Atemwege Compliance (Cdyn) gemessen. Zur Charakterisierung von Lungenerkrankungen stehen hemmungslose sowie zurückhaltende Methoden zur Bewertung der Lungenfunktion. Hemmungslose Plethysmographie im frei beweglichen Tieren ahmt einen natürlichen Zustand, Atemmuster widerspiegelt; im Gegensatz dazu sind invasive Methoden, wie Eingangs-Impedanz-Messung von Res und cDyn in tief narkotisierten Mäuse dynamische Lungenmechanik bewerten genauer13. Da chronische Atemwegserkrankungen durch histologischen Veränderungen des Lungengewebes übernommen werden, droht richtige Lunge Fixierung zur weiteren Analyse. Die Wahl der Methode der Gewebe Fixierung und Vorbereitung hängt das Fach der Lunge die, beispielsweise untersucht werden Durchführung der Atemwege oder Lunge Parenchym14. Hier beschreiben wir eine Methode, die qualitative und quantitative Bewertung der Durchführung von Fluglinien, die Auswirkungen der Fettleibigkeit auf Asthma Entwicklung zu studieren zu können.

<table>
	<tbody>
		<tr>
			<td>GlucoMen LX</td>
			<td>A.Menarini diagnostics, Firneze, Italy</td>
			<td>38969</td>
			<td>blood glucose meter</td>
		</tr>
		<tr>
			<td>GlucoMen LX Sensor</td>
			<td>A.Menarini diagnostics, Firneze, Italy</td>
			<td>39765</td>
			<td>Test stripes</td>
		</tr>
		<tr>
			<td>Glucose 20%</td>
			<td>B. Braun, Melsung, Germany</td>
			<td>2356746</td>
			<td></td>
		</tr>
		<tr>
			<td>FinePointe Software</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1831-002</td>
			<td></td>
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		<tr>
			<td>FinePointe RC Single Site Mouse Table</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1831-001</td>
			<td></td>
		</tr>
		<tr>
			<td>FPRC Controller</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1075-001</td>
			<td></td>
		</tr>
		<tr>
			<td>FPRC Aerosol Block</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-1106-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Aerogen neb head-5.2-4um</td>
			<td>DSI, MC s&#180;Hertogenbosch, Netherlands</td>
			<td>601-2306-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST, British Columbia, Canada</td>
			<td>11065-07</td>
			<td></td>
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			<td>Blunt scissors</td>
			<td>FST, British Columbia, Canada</td>
			<td>14105-12</td>
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			<td>Micro scissors</td>
			<td>FST, British Columbia, Canada</td>
			<td>15000-00</td>
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			<td>Perma-Hand 4-0</td>
			<td>Ethicon, Puerto Rico, USA</td>
			<td>736H</td>
			<td>Surgical suture</td>
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		<tr>
			<td>Roti-Histofix 4%</td>
			<td>Roth</td>
			<td>P087.1</td>
			<td>4% Paraformaldehyd</td>
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		<tr>
			<td>Ketaset</td>
			<td>Zoetis, Berlin, Germany</td>
			<td>10013389</td>
			<td>Ketamine</td>
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			<td>Rompun 2%</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>770081</td>
			<td>Xylazine</td>
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intraperitoneal glucose tolerance test, measurement of lung function, and fixation of the lung to study the impact of obesity and impaired metabolism on pulmonary outcomes

Adipositas und Erkrankungen der Atemwege sind große gesundheitliche Probleme. Adipositas ist mit einer erwarteten Zahl von mehr als 1 Milliarde übergewichtige Personen weltweit bis 2030, stellvertretend für eine zunehmende sozioökonomische Belastung einer aufstrebenden Epidemie. Gleichzeitig sind Adipositas verbundenen Komorbiditäten, einschließlich Diabetes sowie Herz- und chronische Lungenerkrankungen, kontinuierlich auf dem Vormarsch. Übergewicht mit einem erhöhten Risiko für Asthma-Exazerbationen in Verbindung gebracht hat, zwar Verschlechterung der respiratorischen Symptomen und mangelhafte Kontrollen, die funktionale Rolle der Fettleibigkeit und gestörten Stoffwechsel in der Pathogenese der chronischen Lungenerkrankungen oft unterschätzt, und zugrunde liegenden molekulare Mechanismen bleiben schwer. Dieser Artikel zielt darauf ab, Methoden zur Beurteilung der Wirkung von Fettleibigkeit auf Stoffwechsel, sowie Lunge Struktur und Funktion zu präsentieren. Hier beschreiben wir drei Techniken für Mäuse-Studien: (1) Beurteilung der intraperitonealen Glukosetoleranz (IpGTT) analysieren die Auswirkungen der Fettleibigkeit auf Glukose-Stoffwechsel; (2) Messung des Atemwegswiderstandes (Res) und Atemwege Compliance (Cdyn) zu analysieren, die Wirkung der Fettleibigkeit auf die Lungenfunktion; und (3) Vorbereitung und Fixierung der Lunge für anschließende quantitative histologische Bewertung. Adipositas Lungenerkrankungen sind wahrscheinlich multifaktoriell, aus systemische entzündliche und metabolische Dysregulation, die Lungenfunktion und das Ansprechen auf die Therapie möglicherweise negativ beeinflussen. Daher unbedingt eine standardisierte Methode zur molekularen Mechanismen und die Wirkung von neuartigen Behandlungen zu studieren.

Repräsentative Ergebnisse der intraperitonealen Glucose-Toleranz-Test (IpGTT) (Abbildung 4), gebeizt Funktion Test (Abbildung 5) und Vertreter Lungenaufnahmen Hämatoxylin und Eosin veranschaulichen Lunge (Abbildung 6).
Die IpGTT wurde bei fettleibigen Mäusen (blau) nach 7 Wochen von High-Fat-Diät (HFD) durchgeführt. Standard-Diät gef?...

Watch this Scientific Journal Video about Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmo

Intraperitoneal Glucose Tolerance Test, Measurement of Lung Function, and Fixation of the Lung to Study the Impact of Obesity and Impaired Metabolism on Pulmonary Outcomes

Die Inzidenz der Adipositas steigt und erhöht das Risiko von chronischen Lungenerkrankungen. Die zugrunde liegenden Mechanismen und Präventionsstrategien, wohldefinierte Tier sind Modelle erforderlich. Hier bieten wir drei Methoden (Glukose-Toleranz-Test, Body-Plethysmographie und Lunge Fixierung), um die Wirkung der Fettleibigkeit auf die pulmonale Ergebnisse bei Mäusen untersuchen.

Intraperitoneale Glucose-Toleranz-Test, Messung der Lungenfunktion und Fixierung der Lunge, um die Auswirkungen von Übergewicht und beeinträchtigter Stoffwechsel auf pulmonale Ergebnisse studieren

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese ...

The overall goal of the following three methods is to study the impact of obesity and metabolic disorders on lung structure and function. These methods can help to answer key questions in the respiratory research field about the impact of metabolic disorders on lung structure and lung function. The advantage of these techniques is that they can be combined to assess the functional role of metabolism on the pathogenesis of chronic lung diseases and to test different therapeutical approaches.The implications of these techniques extend beyond phenotyping to what's elucidating the specific functional role of metabolic targets in the pathogenesis of lung diseases. These methods can also be applied to the study of structure and function in both healthy animals with genetic modifications but also in animal models of COPD and asthma. Generally, individuals new to these methods will struggle as insertion of the tracheal tube, the insufflation of the lung and the administration of the deep anesthesia, they all require experience and training.We first had the idea for these methods when we discovered that early onset obesity in mice but also in humans were intimately linked to impact metabolism and to chronic lung diseases beyond infancy. To measure intraperitoneal glucose tolerance, gently restrain a 12-hour fasted adult mouse by the tail and use sterile scissors to incise the tail tip. Immediately apply a free-flowing drop of blood to the test strip of the blood glucose meter to measure the fasted blood glucose level.The test result will appear on the glucometer screen after four seconds. After measuring the glucose for each animal, label them individually with colored markers and weigh the animals. Then use a 27-gauge needle attached to a one-milliliter syringe to administer 100 microliters of glucose per ton grams of body weight to each animal via intraperitoneal injection and measure the blood glucose levels 15, 30, 60 and 120 minutes later with new test strips as just demonstrated.To assess the lung function, place an adult mouse in the supine position on a pad and apply ointment to the animal's eyes. After confirming a lack of response to toe pinch, wet the fur in the thyroid region with 70%ethanol and use forceps to lift the skin at the midline between the jugular notch at the sternum and the tuber symphysis of the mentum. Using blunt scissors, carefully make a one-centimeter skin incision in the tented skin to visualize the underlying subcutaneous adipose tissue and thyroid gland and expose the trachea with careful blunt separation of both thyroid lobes at the isthmus and dissection of the sternothyroid and sternothyroid muscles.Using sharp forceps, pass a 4-0 braided surgical suture between the trachea and the esophagus and carefully incise the trachea close the the larynx between the tracheal cartilages with microscissors. Intubate with a tracheal tube securing the tube with a surgical suture and move the animal to the heated bed of a body chamber. Connect the tracheal tube to the face plate and turn on the ventilator.Confirm movement of the thorax contemporaneously with the ventilation rate to confirm proper placement of the tracheal tube and watch the pressure signal on the computer screen. To control changes in the transpulmonary pressure during ventilation, insert an esophageal tube to the level of the lungs watching the screen to facilitate placement of the tube to where maximal pressure deflection and minimal heart artifacts can be observed. Begin the data acquisition according to the manufacturer's instructions and apply 10 microliters of PBS on the nebulizer.Start nebulization after five minutes of acclamation followed by a response phase of three minutes during which the airway resistance and respiratory system compliance are measured. Allow the animal to recover for three minutes before starting the next nebulization and follow the software instructions for stepwise application of increasing 10-microliter concentrations of methacholine on the ventilator. For quantitative histomorphometric analysis of the lung tissue, use blunt scissors to make a small incision in the diaphragm of an adult mouse and use curved blunt scissors to make a peristernal incision along the entire length of the rib cage to gently open the thorax.Lift the rib cage to expose the pleural cavity and remove the thymus. After removing the heart, use sharp forceps to prepare and expose the trachea in order to pass a 4-0 braided surgical suture between the trachea and the esophagus followed by careful incision of the trachea close to the larynx between the tracheal cartilages. Intubate with a 26-gauge intravenous cannula and use a fixative agent to inflate the lungs by pressure fixation at a constant pressure of 20 centimeters of water.After 30 minutes at room temperature, ligate the trachea and remove the cannula at the same time. Then carefully excise the lungs without damaging the tissue and store the lungs in fresh fixative agent at four degrees Celsius overnight. Obese mice demonstrate an increase in serum glucose levels 15 and 30 minutes after intraperitoneal glucose injection indicating an impaired cellular glucose uptake compared to standard diet fed mice.Invasive lung function analysis shows an up to 1.5-fold increase of airway resistance in obese, high-fat diet fed mice after stimulation with methacholine compared to control animals. Hematoxylin and eosin staining of lung parenchyma sections after intratracheal installation reveals multiple uninflated areas, thick alveolar septa and polygonal-shaped alveoli in lungs instilled with too little pressure. In contrast, too much instillation pressure leads to overinflated areas with destructed alveolar septa.Application of the appropriate amount of pressure during lung fixation however leads to a completely inflated lung with round-shaped alveoli. Once mastered, the intraperitoneal glucose tolerance tests can be completed in two and a half hour. The lung function tests can be completed in one hour, and the lung insufflation can be completed in under an hour.While attempting this procedure, it's important to remember that adapting the mice, training the investigator and working in a quiet and stress-reduced environment all are recommended for optimal data acquisition. After its development, this technique paved the way for researchers in the fields of metabolism and respiratory diseases to explore the mechanisms involved in and potention of treatment strategies of chronic lung disease using small animal models. After watching this video, you should have a good understanding how to investigate the impact of obesity and metabolic disorders on lung structure and function.Don't forget that working with animals or surgical instruments can cause injuries or skin reactions and that precautions such as wearing the appropriate personal protection equipment should always be taken while performing these procedures.

Research

JoVE Journal

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Forschung

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Pseudomonas aeruginosa Induced Lung Injury Model

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Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

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Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

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Precision-cut Mouse Lung Slices to Visualize Live Pulmonary Dendritic Cells

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The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Das WinCF-Modell - Ein preiswerter und tragbarer Mikrokosmos eines Schleims verstopfte Bronchiole zur Untersuchung der Mikrobiologie von Lungeninfektionen

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