The overall goal of this procedure is to induce lung injury in mice using a ventilator. This is accomplished by performing a tracheotomy and administering high pressure mechanical ventilation. Next, a carotid artery catheter is inserted to measure hemodynamic parameters.
After the animal has been on the ventilator long enough to induce lung injury, arterial blood samples and bronchoalveolar lavage fluid are collected. The final step of the procedure is to harvest lung tissue for further analysis. Ultimately, results can be obtained that correlate changes in the samples collected with the duration of mechanical ventilation.
The main advantage of this technique over existing methods like LPS inhalation is that it induces an acute lung injury, which resembles all major characteristics of acute respiratory distress syndrome found in humans. To begin place an anesthetized mouse in a supine position onto a temperature controlled heated table loop suture material around each extremity, and then tape the sutures to the table. Next, secure the head in place by running a suture underneath the top teeth.
Administer a saline bolus of 500 microliters and insert a rectal probe attached to a thermal feedback controller to ensure the body temperature remains at 37 degrees Celsius. Once prepared, the mouse can be covered with mineral oil to reduce exposure to allergens from the hair. Lastly, cover the animal in commercially available food wrap to help maintain body temperature.
To expose the trachea, make an incision along the neck and blunt, dissect the surrounding muscle and connective tissue. Place two 10 centimeter long surgical sutures underneath the trachea about one centimeter apart. Use mc first and Vanna scissors to carefully incise the trachea between two rings of cartilage, about three to four millimeters below the larynx.
To complete the intubation, insert the tip of a blunt polyethylene cannula at an 85 degree angle into the trachea. Next, tilt the cannula so that it is in line with the tracheal lumen. Slowly advance the tube until the tip has disappeared into the thorax aperture.
Fix the tube in position with the two surgical sutures that were placed under the trachea earlier. Lastly, connect the tube to the ventilator. Lung injury is induced with a servo 900 C from Siemens.
Using a pressure controlled ventilation technique, set the peak in inspiratory pressure to 45 millibars with a frequency of 80 breaths per minute and a positive end expiratory pressure of zero to three millibars with a fractional inspired oxygen equal to one, the inspiration to expiration ratio is one to one. Once the animal is successfully connected to the ventilator, a catheter is inserted into the carotid artery for measuring blood pressure and heart rate. A longer piece of artery will be exposed if the arm is first attached to the side of the body.
Begin by blunt dissecting the para tracheal muscles to expose the carotid artery once isolated, not a suture at the proximal end of the artery, and secure it with a clamp or tape. Place a second suture about one centimeter away from the first and tie, but do not tighten the suture around the artery. Dissect the artery to the very distal end and place a small clamp to prevent blood loss.
Use micro scissors to cut a small diagonal opening into the artery above the second suture. Hold the opening with fine forceps while inserting the catheter and tighten the knot to hold in place. Next, loosen the clamp and advance the catheter.Further.
Advance the catheter past the second suture. Secure the catheter with several knots and tape. Once the catheter is in place, infuse normal saline at a rate of 0.1 milliliter per hour, and join it to the blood pressure measuring device.
Lastly, attach clamps to the extremities to monitor heart rate. After three hours of mechanical ventilation, samples are collected to measure the extent of lung injury. First, collect samples from the carotid artery catheter for blood gas analysis.
After deepening the level of anesthesia, remove the connection from the ventilator and insert a syringe filled with one milliliter of phosphate buffered saline. Plunge the syringe and wait three seconds before collecting the bronchoalveolar lavage fluid back into the syringe. It should be noted that the recovered volume should be significantly less than one milliliter.Flash.
Freeze the bronchoalveolar lavage fluid in liquid nitrogen and store at minus 80 degrees Celsius To isolate the lungs, first, make an incision right below the sternum. Hold the sternum with forceps and extend the incision along the rib cage. Next, cut the diaphragm at the edges and away from the ribs to reveal a clear view into the lower aperture of the thorax.
Lift the sternum up with forceps and open the thorax using long cuts at the right and left side, keeping the cuts as lateral as possible so that the complete anterior thorax wall can be turned upwards. Connect a 27 and a half gauge needle to a syringe filled with five milliliters of PBS. Insert the needle into the right ventricle and flush the ary circulation until the lungs turn completely white.
If this does not happen, make sure that the tip of the needle is correctly placed in the right ventricle and reposition if necessary. Excise the lungs and block by pulling up the heart and cutting the trachea. Use tissues to absorb any blood that obstruct your view.
Pull the heart in the direction of the abdomen and carefully cut along the spine to mobilize all thoracic organs. Cut the aorta and place the thoracic organs onto a clean surgical table. Cut the heart away and clean off any major vessels from the sample.
Make sure that there is no thymus tissue still attached to the lung. Separate the lobes of the lungs place in individual tubes, snap, freeze, and store at minus 80 degrees Celsius. For further analysis.
As seen here, the protein content in the bronchoalveolar lavage fluid increases with the duration of ventilation. The relative change of the protein content is shown normalized to zero hours of ventilation To assess the extent of lung injury. Eliza for albumin and Myla peroxidase are performed and the inflammatory cells in the BL fluid are quantified.
