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...

<ol>
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The authors have nothing to disclose.

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>
		</tr>
		<tr>
			<td>Rompun 2%</td>
			<td>Bayer, Leverkusen, Germany</td>
			<td>770081</td>
			<td>Xylazine</td>
		</tr>
	</tbody>
</table>

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...

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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

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 ...

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.

intraperitoneal-glucose-tolerance-test-measurement-lung-function

Research

JoVE Journal

Immunology and Infection

13.9K visualizações.  University of Cologne. 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.

Vídeo: 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