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

实验得到了 Marga 和基金会, Kerpen, 德国的支持;项目 210-02-16 (MAAA), 项目 210-03-15 (MAAA) 和由德国研究基金会 (DFG;AL1632-02;MAAA), 波恩, 德国;分子医学的中心科隆 (CMMC;大学医院科隆;职业发展计划;MAAA), 科隆财富 (医学学院, 科隆大学;KD)。

所有动物程序都是按照地方政府当局批准的议定书进行的 (土地北威州, 亚利桑那州: 2012. A424), 并符合德国动物福利法和关于动物福利的条例, 用于试验或为其他科学目的。由于肺功能分析可能会影响肺结构, 因此随后的组织学分析, 应在不同的动物中进行 Cdyn 的测定和肺形态计量学的制备和固定。然而, ipGTT 后的 Cdyn 的测量是可能的。由于 ipGTT 期间的压力可能会干扰肺功能测试所需的麻醉, 因此建?...

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本报告为三种不同方法分析肥胖对葡萄糖代谢和肺部结局的影响提供三项议定书。首先, 葡萄糖耐受性试验提供了分析细胞内葡萄糖吸收的机会, 并可以表明胰岛素抵抗。第二, 全身 plethysmography 是一种测量肺功能的技术, 从而有助于测试新疗法的疗效。第三, 标准化的固定协议是定量形态学分析的关键, 以评估肥胖对结构变化的影响。
饮食诱发肥胖在动物研究中的作用
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根据世界卫生组织 (卫生组织) 的数据, 在 2008年, 超过14亿个年龄在20岁以上的成年人超重, 身体质量指数 (BMI) 大于或等于 25;此外, 2亿多名男子和近3亿名妇女肥胖 (BMI≥30)1。肥胖和代谢综合征是多种疾病的主要危险因素。肥胖和随之增加的白脂肪组织质量与2型糖尿病有密切关系2,3, 心血管疾病包括冠心病 (CHD), 心力衰竭 (HF), 心房颤动4和骨关节炎5, 它们在呼吸道疾病发病机制中的功能作用仍然不甚了解。然而, 流行病学研究表明, 肥胖与慢性呼吸道疾病密切相关, 包括劳力呼吸困难、阻塞性睡眠呼吸暂停综合征 (OSAS)、肥胖低通气综合征 (职业职业治疗)、慢性阻塞性肺病 (COPD), 肺栓塞, 吸入肺炎和支气管哮喘6,7,8,9。将肥胖和摄动代谢、例如、胰岛素抵抗和 II 型糖尿病与慢性肺病发病机制联系起来的潜在机制, 不仅包括对通气的体重增加的机械和物理后果, 而且还诱发慢性亚急性炎症状态10,11。过去十年中肥胖和肺部疾病的增加, 加上缺乏有效的预防策略和治疗方法, 突显出需要调查分子机制, 以确定管理肥胖相关肺的新途径。疾病.
在这里, 我们描述了三标准测试, 这是研究肥胖及其对小鼠肺结构和功能的影响的重要基础知识: (1) 腹腔葡萄糖耐受 (ipGTT) (2) 气道阻力 (Res) 和呼吸的测量系统遵从性 (Cdyn);(3) 肺的制备和固定, 供随后的定量组织学评估。ipGTT 是一个强有力的筛选测试, 以测量葡萄糖摄取量, 从而影响肥胖对新陈代谢。这种方法的简单性使得标准化很好, 因此实验室之间的结果可比性。更复杂的方法, 如高血糖钳或研究孤立胰岛, 可用于详细分析代谢表型12。在这里, 我们评估葡萄糖耐受性, 以确定肥胖相关的系统性和代谢紊乱的状态, 作为进一步研究肺部结局的基础。为了评估肥胖和代谢紊乱对肺功能的影响, 我们测量了气道阻力 (Res) 和呼吸系统依从性 (Cdyn)。为描述肺部疾病, 有无节制的和克制的方法评估肺功能。无拘无束的 plethysmography 在自由运动的动物模仿自然状态, 反映呼吸模式;与此相反, 侵入性方法, 如输入阻抗测量的 cDyn 在深麻醉小鼠评估动态肺力学, 是更准确的13。由于慢性呼吸道疾病是由肺组织的组织学改变反映出来的, 因此适当的肺部固定术是迫在眉睫的。组织固定和制备方法的选择取决于肺的隔间, 例如传导气管或肺实质14。在这里, 我们描述了一种方法, 允许定性和定量评估传导气道, 以研究肥胖对哮喘发展的影响。

<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

肥胖和呼吸系统疾病是主要的健康问题。肥胖正在成为一个新兴的流行病, 预计到2030年世界各地有超过10亿的肥胖人口, 这就代表了社会经济负担的增加。同时, 肥胖相关的并发症, 包括糖尿病以及心脏和慢性肺病, 都在不断上升。虽然肥胖与哮喘加重的风险、呼吸道症状恶化和控制不力有关, 但肥胖和摄动代谢在慢性肺病发病中的功能作用往往被低估,而潜在的分子机制仍然难以捉摸。本文旨在探讨肥胖对代谢的影响以及肺结构和功能的评估方法。在这里, 我们描述了三技术的老鼠研究: (1) 评估腹腔葡萄糖耐受 (ipGTT), 以分析肥胖对葡萄糖代谢的影响;(2) 测量气道阻力 (Res) 和呼吸系统依从性 (Cdyn), 分析肥胖对肺功能的影响;(3) 肺的制备和固定, 供随后的定量组织学评估。肥胖相关的肺部疾病可能是多因素的, 其产生于全身炎症和代谢失调, 可能对肺功能和治疗反应产生不利影响。因此, 研究分子机制和新疗法效果的标准化方法是必不可少的。

腹腔葡萄糖耐受试验 (ipGTT) (图 4)、肺功能测试 (图 5) 的代表性结果, 以及说明苏木精和红红染色肺部的代表性图像 (图 6)。
ipGTT 7 周高脂饮食 (风) 后, 在肥胖小鼠 (蓝色) 中进行。标准膳食喂养的老鼠作为控制 (黑色)。肥胖小鼠在腹腔葡萄糖注射液后显示血糖水...

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

肥胖的发病率在上升, 并增加了慢性肺病的风险。为了建立基本机制和预防战略, 需要有明确定义的动物模型。在这里, 我们提供三种方法 (葡萄糖耐受性测试, 身体 plethysmography 和肺固定), 以研究肥胖对肺结局的影响小鼠。

腹腔葡萄糖耐受试验、肺功能测定和肺固定研究肥胖和代谢障碍对肺部结局的影响

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.

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