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

The experiments were supported by the Marga and Walter Boll-Stiftung, Kerpen, Germany; Project 210-02-16 (MAAA), Project 210-03-15 (MAAA) and by the German Research Foundation (DFG; AL1632-02; MAAA), Bonn, Germany; Center of Molecular Medicine Cologne (CMMC; University Hospital Cologne; Career Advancement Program; MAAA), K&#246;ln Fortune (Faculty of Medicine, University of Cologne; KD).

All animal procedures were conducted in compliance with protocols approved by local government authorities (Land NRW, AZ: 2012.A424), and were in accordance with the German animal welfare law and the regulations on the welfare of animals used for experiments or for other scientific purposes. Since lung function analysis may affect lung structure and therefore subsequent histological analyzes, the measurement of Res and Cdyn and the preparation and fixation of the lung for histomorphometry should be performed in different...

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This report provides three protocols for three different methods to analyze the impact of obesity on glucose metabolism and pulmonary outcomes. First, the glucose tolerance test offers the opportunity to analyze intracellular glucose uptake and can be indicative of insulin resistance. Second, whole body plethysmography is a technique to measure lung function and is thereby helpful to test the efficacy of novel treatments. Third, a standardized fixation protocol is essential for quantitative morphometric analysis to asses...

According to the World Health Organization (WHO) in 2008, more than 1.4 billion adults, aged 20 and older, were overweight with a body mass index (BMI) greater than or equal to 25; further, over 200 million men and nearly 300 million women were obese (BMI&#8805;30)1. Obesity and metabolic syndrome are major risk factors for a multitude of diseases. While obesity and concomitant increased white adipose tissue mass has been intimately linked to type 2 diabetes2,3, cardio-vascular diseases including coronary heart disease (CHD), heart failure (HF), atrial fibrillation4 and osteoarthritis5, their functional roles in the pathogenesis of respiratory disorders remain poorly understood. However, epidemiological studies have demonstrated that obesity is strongly associated with chronic respiratory conditions, including exertional dyspnea, obstructive sleep apnea syndrome (OSAS), obesity hypoventilation syndrome (OHS), chronic obstructive pulmonary disease (COPD), pulmonary embolism, aspiration pneumonia and bronchial asthma6,7,8,9. Potential mechanisms linking obesity and perturbed metabolism, e.g., insulin resistance and type II diabetes, to the pathogenesis of chronic lung disease not only comprise mechanical and physical consequences of weight gain on ventilation but also induce a chronic subacute inflammatory state10,11. The rise of obesity and lung diseases during the last decade, coupled with the lack of effective preventive strategies and therapeutic approaches, highlights the need to investigate the molecular mechanisms to define new avenues to manage obesity-related lung diseases.
Here, we describe three standard tests, which are important basics to investigate obesity and its impact on lung structure and function in mouse models: (1) intraperitoneal glucose tolerance (ipGTT) (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn); and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. The ipGTT is a robust screening test to measure glucose uptake, and thus the effect of obesity on metabolism. The simplicity of the method allows good standardization, and therefore the comparability of results between laboratories. More sophisticated methods, such as hyperglycemic clamps or studies on isolated islets, can be used for detailed analysis of the metabolic phenotype12. Here we assess glucose tolerance to define an obesity-associated state of systemic and metabolic disorder as the basis for further studies on a pulmonary outcome. To assess the effect of obesity and metabolic disorder on lung function, we measured airway resistance (Res) and respiratory system compliance (Cdyn). To characterize lung disease, unrestrained as well as restrained methods for assessment of lung function are available. Unrestrained plethysmography in freely moving animals mimics a natural state, reflecting breathing patterns; in contrast, invasive methods, such as input impedance measurement of Res and cDyn in deeply anesthetized mice to assess dynamic lung mechanics, are more accurate13. Since chronic respiratory conditions are reflected by histologic alterations of the lung tissue, proper lung fixation for further analysis is imminent. The choice of the method of tissue fixation and preparation depends on the compartment of the lung which will be studied, for example, conducting airways or lung parenchyma14. Here, we describe a method that allows qualitative and quantitative assessment of the conducting airways to study the effect of obesity on asthma development.

<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>
		</tr>
		<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>
		</tr>
		<tr>
			<td>Blunt scissors</td>
			<td>FST, British Columbia, Canada</td>
			<td>14105-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissors</td>
			<td>FST, British Columbia, Canada</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Perma-Hand 4-0</td>
			<td>Ethicon, Puerto Rico, USA</td>
			<td>736H</td>
			<td>Surgical suture</td>
		</tr>
		<tr>
			<td>Roti-Histofix 4%</td>
			<td>Roth</td>
			<td>P087.1</td>
			<td>4% Paraformaldehyd</td>
		</tr>
		<tr>
			<td>Ketaset</td>
			<td>Zoetis, Berlin, Germany</td>
			<td>10013389</td>
			<td>Ketamine</td>
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		<tr>
			<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

Obesity and respiratory disorders are major health problems. Obesity is becoming an emerging epidemic with an expected number of over 1 billion obese individuals worldwide by 2030, thus representing a growing socioeconomic burden. Simultaneously, obesity-related comorbidities, including diabetes as well as heart and chronic lung diseases, are continuously on the rise. Although obesity has been associated with increased risk for asthma exacerbations, worsening of respiratory symptoms, and poor control, the functional role of obesity and perturbed metabolism in the pathogenesis of chronic lung disease is often underestimated, and underlying molecular mechanisms remain elusive. This article aims to present methods to assess the effect of obesity on metabolism, as well as lung structure and function. Here, we describe three techniques for mice studies: (1) assessment of intraperitoneal glucose tolerance (ipGTT) to analyze the effect of obesity on glucose metabolism; (2) measurement of airway resistance (Res) and respiratory system compliance (Cdyn) to analyze the effect of obesity on lung function; and (3) preparation and fixation of the lung for subsequent quantitative histological assessment. Obesity-related lung diseases are probably multifactorial, stemming from systemic inflammatory and metabolic dysregulation that potentially adversely influence lung function and the response to therapy. Therefore, a standardized methodology to study molecular mechanisms and the effect of novel treatments is essential.

Representative results of intraperitoneal glucose tolerance test (ipGTT) (Figure 4), lung function test (Figure 5), and representative images illustrating hematoxylin and eosin stained lungs (Figure 6).
The ipGTT was performed in obese mice (blue) after 7 weeks of high-fat-diet (HFD). Standard diet-fed mice served as controls (black). Obese...

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

The incidence of obesity is rising and increases the risk of chronic lung diseases. To establish the underlying mechanisms and preventive strategies, well-defined animal models are needed. Here, we provide three methods (glucose-tolerance-test, body plethysmography, and lung fixation) to study the effect of obesity on pulmonary outcomes in mice.

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

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