A midline cervical incision is performed to expose the trachea and a small hole is cut into the trachea for insertion of an endotracheal tube. The mouse is turned on its right side and a left sided thoracotomy is performed between the third and fourth ribs. The heart is exposed and the LAD is ligated with a single stitch.
After placement of a chest tube and closing of the incision, the thorax is drained by aspirating the syringe carefully. Finally, the mouse is turned on. Its back.
The tracheal tube is removed and the trachea and cervical incision are closed. Hi, I am Sonya Refa. I'm professor in Immunology and cardiac surgery in the Department of Cardiovascular Surgery at the University Heart Center in Hamburg.
My laboratory is focusing on stem cell immunobiology and transplant immunology. Today we'll introduce you to the LID ligation model. We use this model in our lab to study the effect of stem cell therapy to generate the heart after myocardial infarction.
And I'm KA Wan, a student at Stanford University and a summer fellow in Drer's TSI Laboratory. And I am Mandy Cooch from the TSI Laboratory of the University Heart Center Hamburg. I will show you how to perform in a lady ligation and later we will show you how to verify the success of a myocardial infarction using different methods.
So let's get started. Shave the neck area and the left side of the rib cage of the anesthetized mouse. Place the mouse on its back and place a face mask over its nose and mouth to maintain anesthesia with 2%isof fluorine disinfect.
Using beta isod before starting the surgery, pinch the tail and hind feet of the mouse to confirm that it is sufficiently anesthetized. Under microscopic view, perform a midline cervical incision separating the skin muscle and tissue covering the trachea. Once the trachea is exposed, cut a small hole into the tissue between two cartridge rings below the glottis to insert the endotracheal tube.
Insert the endotracheal tube while holding the cranial part of the trachea. Using microsurgical forceps, check that thoracic movement to make sure that both lungs are well ventilated. The respiration rate should be approximately 110 per minute with an inspiratory pressure of 17 to 18 centimeters.H2O.
Turn the mouse carefully and lay it on its right side while you face its left side. Perform a left sided thoracotomy between the third and the fourth rib and dissect the tissue and muscle. Carefully use a coer to prevent bleeding.
Open the thorax carefully. Once the thorax is opened, find the heart without touching the lung with any sharp objects. Next, remove the part of the pericardial sac that is covering the heart.
The LAD is located between the pulmonary artery and the left Oracle ligate. The proximal LAD with one single eight oh proline suture. Place an endotracheal tube 28 gauge ven catheter between the fourth and the fifth rib.
Close the thoracic incision in layers using six oh proline running sutures to adapt the ribs and four oh proline running sutures. To close the skin, drain the thorax carefully with the help of a two milliliter syringe. After draining the thorax, place the mouse on its back.
Place the face mask on the mouse and take the endotracheal tube out. Adapt the tracheal cartridge rings with one single stitch using seven oh prolene sutures and close the skin using four oh prolene running sutures. We have shown that the LAD ligation mouse model is an elegant and reliable technique to create a myocardial infarction.
There are different ways to confirm a successful myocardial infarction. One method is the troponin test in which 150 microliters of blood from the mouse is applied to a customized troponin test kit. A positive result where two lines appear after a few minutes indicates the release of troponin T from the heart due to tissue injury.
A second approach to confirm a successful myocardial infarction is disdain cross-sections of the heart with 2 3 5 trien tetra oleum chloride or TTC to measure tissue viability and evaluate the infarct size. The heart is covered with TTC and incubated at 37 degrees Celsius for 20 minutes. The viable tissue will appear red while the infarcted area will appear white.
Finally, the heart of the mouse can be paraffin, embedded and sectioned for histology. Sections are stained with tri chrome to visualize fibrotic tissue. I have just shown you how to perform an LID ligation in mice afterwards.
We have shown you different methods how to confirm the successful myocardial infarction either hours after LID ligation or weeks later in performing in an LID ligation. It is important to stay away from the lung tissue to avoid lung injury. So that's it.
Thank you very much for watching the video and good luck with your experiments.
