The aim of this procedure is to execute a double thoracotomy on rats in order to perform coronary ligation and epicardial application. This is accomplished by first performing a thoracotomy at the fourth intercostal space. Next, the left anterior descending coronary artery or LAD is ligated.
Then a second thoracotomy is performed two weeks later at the fifth intercostal space. Finally, the cells and matrix are applied to the surface of the infarcted beating heart. Ultimately, echocardiography is performed to assess heart function and histology is carried out by infarct size measurements.
The main advantage of this technique over existing methods like sternotomy is that it is a minimally invasive approach demonstrating the procedure will be and ine Lopez sent collaborators from our laboratory. All animals were treated in compliance with the recommendations of the F-E-L-A-S-A and the Swiss Law on animal protection. After exsanguinating a rat and isolating and cleaning the femur bones according to the text protocol, use scissors to cut both tips of the bones.
Then to flush the bone marrow, inject sterile PBS into the core of the bone, and collect the marrow into a 15 milliliter tube. Centrifuge the collected bone marrow for seven minutes at 300 times G.Remove the supinate with three milliliters of red blood cell lysis buffer. Suspend the pellet, incubate the suspension for one minute at room temperature before spinning again for seven minutes at 300 times G after resus.
Suspending the pellet in sterile culture medium. Add additional cell culture medium in a 150 milliliter culture flask and seed the cells on the second day of incubation. Change the medium to remove non-adherence cells and continue to replace the medium every second day for two to three weeks until the desired cell density is achieved.
To carry out the first T thoracotomy, begin by weighing the rat and set up the ventilator parameters accordingly. The ventilation outflow is automatically defined. Then shave the left part of the thorax, turn on a heating pad and set it to 37 degrees Celsius.
After anesthetizing the rat according to the text protocol, perform a toe or tail pinch before placing the rat on the heating pad in a supine position using a 14 gauge IV catheter, intubate the animal, then connect the intubation catheter to a rodent ventilator. Programmed for 2.5 liters per minute of oxygen, 2.5%isof fluorine, a tidal volume of two milliliters, and a breathing frequency of 90 breaths per minute. After preparing a syringe with buprenorphine 0.1 milligrams per kilogram subcutaneously, inject one third of the solution, then use 1%Betadine solution to disinfect the area.
Next, incise the skin perpendicular to the sternum of the fourth intercostal space and separate the three layers of thoracic muscle. Then open the fourth intercostal space between ribs four and five. Use a small retractor to spread the ribs and expose the heart.
Then carefully open the pericardium and locate the left anterior descending coronary artery or LAD, and use a 7.0 suture to ligate it four millimeters below the atrium. Then control the color change of the ischemic area with three zero sutures positioned approximately and distally from the sternum. Make two stitches to be used to close the intercostal space.
Tighten the distal suture. Clamp the ventilator exhaust tubing for two seconds in order to inflate the chest and avoid pneumothorax. Then tighten the second suture.
Position the muscle layers back in place. Then use five zero suture to close the skin with 1%Betadine solution. Disinfect the suture and inject the remaining buprenorphine solution.
Turn off the anesthesia system and remove the intubation catheter. Keep the rat in a cage under a warm lamp for one to two hours before returning it to an IVC unit. Two weeks after the first thoracotomy, healing is complete and a scar is barely visible.
Following the procedure outlined earlier in this video for preparing the rat for surgery, incise the skin perpendicular to the sternum at the fifth intercostal space. Separate the three layers of muscle and open the fifth intercostal space between ribs five and six. Use a small retractor to spread the ribs and expose the heart.
If there is some adherence, use fine forceps to carefully relieve it. Next, locate the infarct area that appears as a pale area below the ligature. If necessary, insert a 10 centimeter piece of seven zero suture at the apex and pull on it gently to better visualize the left ventricle.
Then to apply cells and matrix to the surface of the ischemic area of the heart. After pelleting, four times 10 to the six cells as outlined in the text protocol, deposit the palette onto the heart before using a fibrin sealant to cover it. The layer of glue fixes the cells to the surface of the heart.
After removing the piece of suture from the apex position, the muscle layers back in place suture and disinfect the animal. As demonstrated earlier in the video as shown here. After surgical intervention, animals lost weight and recovered quickly before gaining weight independently of the treatment applied As demonstrated in this Kaplan graph, survival rate is high after the first T thoracotomy, animals treated with fibrin and or cells demonstrated lower survival rates than other groups as seen in this graph.
Nevertheless, the overall survival rates stayed high after a second. Thoracotomy and treatment heart function was evaluated by echocardiography. These representative M mode images for healthy and infarcted animals illustrate that LV wall contractility is abolished two weeks Post LAD ligation fractional shortening was recorded two weeks post LAD lation on a total of 104 animals.
The frequency distribution shows reasonably reproducible methods with a coefficient of variation of 17%This figure depicts graphical representation of heart function by the measured ejection fraction two and six weeks post LAD lation Heart function continues to decrease during this time period. Heart remodeling also occurs during this period and is represented here by an increase in LV volumes recorded in both s sisterly and diastole. Finally, golden staining on heart sections revealed that fibrotic tissue and eventually a transmural scar developed by six weeks post LAD ligation Once mastered, this technique can be done in 30 to 40 minutes if it is performed properly.
After watching this video, you should have a good understanding of how to perform a minimally invasive double thoracotomy.
