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

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.