The overall goal of the following experiment is to demonstrate the microsurgical methods for performing temporary or permanent coronary artery ligation, as well as intra myocardial injection in the mouse heart. This is achieved by first prepping and disinfecting the surgical area. The mouse is then intubated with PE 90 tubing to provide positive pressure ventilation during the thoracotomy.
Once isolated, the coronary artery can be either temporarily or permanently occluded. In addition, a substance can be injected into the heart. Results are obtained that show the degree of injury imposed on the heart based on histological and morphometric endpoints, tissue and serum protein markers, as well as functional indices.
My name is TKA Rag and I'm in the Department of Physiology at Brody School of Medicine at East Carolina University. The purpose of this video is to demonstrate the microsurgical procedures used for temporary and permanent coronary artery ligation, as well as intra myocardial injection into a mouse heart. To begin place sterile surgical instruments and three inch cotton tipped applicators onto a sterile underpad.
Next turn on a bead sterilizer so that it is ready to sterilize instruments between individual surgeries. To prepare the animal for surgery, apply a sterile lubricant to the eyes of an anesthetized mouse. Next, remove the fur from the left side of the chest with the depilatory agent, such as nare.
Thoroughly wipe off the depilatory cream to avoid chemical burns or dermatitis. Place the mouse onto a delta phase isothermal pad fixed to a plexiglass table. Immobilize each limb with tape and position a thick thread under the top teeth to hold the upper jaw in place.
Wipe the surgical site with Betadine and then alcohol a total of three times. Use a fiber optic light to illuminate the neck region to help visualize the trachea during insertion of the PE tubing. Once illuminated, carefully insert the tubing through the mouth.
Connect the tubing to a ventilator to administer positive pressure ventilation. Once ventilation is confirmed by synchronous chest movements, fix the connection to the pad with tape to keep it in place. During the surgery, use toothed forceps to pull the skin up and away from the chest and to make a 1.5 centimeter incision in the skin parallel to the sternum.
Next, use curved Vana micro scissors to cut the pectoralis muscles and to make a small hole in the intercostal muscle. After retracting the muscle, use straight blunt micro scissors to cut through three ribs. Use a speculum to retract the rib cage.
Gently open the pericardial sack to visualize the heart using a TRO Viejo needle holder and a tapered point needle thread, a polyethylene suture underneath the left anterior descending coronary artery for a temporary ligature that can be removed For timed reperfusion, cut a 0.5 to one centimeter piece of PE 90 and place it on the heart. Parallel to the coronary artery, tie the suture that is looped under the coronary artery to the tubing. After a set time period, loosen the ligature to allow reperfusion to perform a permanent occlusion.
The ligature laced under the coronary artery is tied. Blanching and dyskinesia should be apparent. Cut away the long end of the suture for intra myocardial injections, insert a 30 gauge sterile needle into the base of the heart above the area of injury to the right of the ligature.
Advance the needle to the area of injury and withdraw slightly so that the bevel can be seen approximately at the border zone. Inject a small amount of the solution and hold the needle in place. Withdraw the syringe, another one to three millimeters and inject the rest of the solution.
Hold the syringe in place until the blood that is formed by the solution dissipates. Once dissipated, remove the needle. If bleeding occurs, gently press a cotton tipped applicator onto the site until the bleeding stops.
Once the myocardial manipulations are complete, remove the rib retractors and close the thoracic cavity with two to three mattress sutures. Next, close the pectoralis muscles with two to three mattress sutures. Apply one to three drops of 0.25%marking in sterile saline to the muscle, and use an additional two to three mattress sutures to close the skin.
Remove the mouse from the ventilator and ensure rhythmic rapid shallow breathing. Inject 0.5 milliliters of warm sterile saline into the dorsal subcutaneous space and place the mouse in a cage containing a heating pad until it regains.Mobility. For survival experiments, the mice are placed back into their home cages and returned to the vivarium until the time of sacrifice.
Wipe the surgical instruments clean with ethanol and insert them into the bead sterilizer To prepare for the next surgery to collect samples for analysis, isolate the heart and puncture the posterior basal region of the ventricle with a needle containing cold potassium chloride, or two three butane Dion Monoxide solution. Inject the solution slowly into the chamber until the heart is arrested into diastole. Remove the heart and perfuse with PBS to remove any remaining blood.
Once perfused immerse the heart in fixative, the heart is then processed and embedded according to routine procedures. The acute injury caused by temporary ligation can be visualized through staining. In this image, the blue dye is distributed to the occluded regions of the heart and TTC staining marks metabolically viable tissue.
In red, necrotic areas do not stain and so remain pale. Imaging software can be used to calculate the area at risk as a function of the left ventricle and infarct area as a function of the area at risk. In this example, an injured heart is cut transversely through the infarct region, four days post mi and stained with hemat, hematin, and eoin.
The star denotes tissue necrosis while the arrows point to granulation tissue collagen staining with prose. Serious red, fast green can be used to measure interstitial fibrosis, which correlates to functional indices of wall stiffening. This image of a mouse heart four weeks post mi shows the cytoplasm in green and collagen fibers in red.
This series of images represent the distribution of six microliters of Evan's blue dye solution injected into the border zone of the heart following permanent artery ligation. Notice that the dye proceeds in the direction of the injury as well as toward the base, and also transmural Visualizing the coronary artery and reliably occluding. It is very challenging and and takes a lot of practice to become proficient.
It's very important to remember during the procedure to avoid puncturing the lungs and additionally to avoid extubation of the animal during the surgical manipulations.I.
