The overall goal of this procedure is to establish an experimental infective endocarditis model in rats. This is accomplished by first culturing aureus and preparing surgery catheters for infection. Next, the surgical area is prepared and the animal is anesthetized.
Once the animal is anesthetized, a catheter is inserted through the right carotid artery toward the heart. The final step is to infect animals with aureus by tail vein injection. Ultimately, the target tissues are dissected and prepared for quantitative culture.
The main advantage of this technique over existing methods, like a rapid endocarditis model, is that the red endocarditis model can be used with the in vivo imaging system.Ivus. This method can help answer key question in step oral pathogenesis field, such as virulence and efficacy of antimicrobial agent. Generally, Individuals new to this technique will struggle because the surgical procedure is challenging.
First inoculate a loop full of M SSA culture from a frozen stock to a sheep blood trip case. Soy auger plate incubate the culture overnight at 37 degrees Celsius after making sure there is no contamination. Pick one colony from the plate and use it to inoculate five milliliters of try toay soy broth in a 15 milliliter snap cap tube.
Incubate the inoculum at 37 degrees Celsius overnight in a shaking incubator. Set at 200 RPM to prepare catheters first. Cut polyethylene tubing to 10 centimeter lengths and sterilize the catheters in cex disinfecting solution for 30 minutes.
Next, melt one end of a sterilized tube by pressing the tip with heated sterile forceps to create a catheter that will pass from the right carotid artery into the left ventricle and cause damage to the endocardium when the heart beats. Then anesthetize the rat by placing it into a chamber containing a one-to-one isof fluorine oxygen mixture until the anesthetic takes effect. Confirm anesthesia by checking whether muscles are relaxed and pedal reflexes are absent.
And maintain an anesthetized state during the surgery by using a nose cone which is connected with the gas mixture. Clean the surgical area with Betadine and 70%ethanol using sterile technique throughout the surgery. Place the rat in a dorsal position and make an incision vertically through the skin layer of the neck above the sternum using blunt dissection.
Separate the fascia to expose the right carotid artery. The right carotid artery is located just slightly right of midline above the clavicles. Carefully pull the artery up out of the neck cavity and place two 10 centimeter lengths of silk suture under the artery to tie it off at the exposed cephalic end.
Then place a clip on the artery to prevent bleeding. Make a small hole in the top of the artery using a catheter introducer using forceps. Insert the unsealed end of the catheter through the hole in the artery.
Remove the clip and push the catheter down toward the heart. Until resistance is met inserted properly, the catheter should extend four to five centimeters into the artery and should pulse with the rat's heartbeat. Once the catheter is in place, tie loose suture around the coddle end of the artery and secure the catheter in place.
With silk suture, cut off the excess suture and catheter ends, and then tuck the ends under the skin of the neck. Close the skin with skin clips. Place the rat in a clean cage and keep it in a warm place until recovered from the anesthesia.
Provide food and water and check the rat frequently, both during and after recovery. Infection can be performed between one and seven days post-surgery, but the time should remain consistent Within experiments. Clean the rat's tail with 70%ethanol and inject 0.5 milliliters of the MRSA culture at the desired cell.
Count into the tail vein. Using a 27 gauge half inch needle, apply pressure to the injection site until any bleeding has stopped before returning the rat to its cage. After sacrificing the rat, wipe the chest with 70%ethanol and then make a V-shaped incision in the chest below the sternum.
Cut the cartilage of the ribs on each side of the sternum. To expose the heart, gently pull the heart up and clip through the tissue close to the aorta and dissect upwards To free the heart. Place the heart on a sterile petri dish lined with four by four inch gauze, and then make a cut through the left ventricular inner wall and open the left-sided chamber.
Check to confirm correct catheter placement, and then remove it. Examine and remove vegetations from the valve with scissors and forceps. Also remove the kidneys and spleen, and after weighing and homogenizing the tissues and vegetations make serial dilutions in PBS for quantitative culture.
Shown here is a correctly positioned catheter as indicated by the leftmost arrow. It extends across the aortic valve and into the left side of the heart chamber, and numerous vegetations are visible around the aortic valves as indicated by the middle and right arrows. This table provides an example of the virulence of an ssus strain in the rat endocarditis model.
All animals challenged with inocular of 10 to the fifth and 10 to the sixth. CFU developed endocarditis with high ssus densities in cardiac vegetations as well as in the kidneys and spleen Once mastered. This surgical procedure can be done within 10 minutes if it's performed Properly.
While attempting this procedure, it's important to remember to maintain the animal in an assessed state during the surgery Following this procedure. Not methods like ivus can be performed to answer additional questions like real time monitoring the efficacy of anti-microbial agents And the in vivo gene expression after its development. This technique be the way for researcher in the field of step orus and fiction to explore its pathogenic virulence factor in the endovascular infection.
After watching this video, you should have a good understanding of how to establish a experimental endocarditis Model in rats. Don't forget that working with MRA can be extremely hazard and precautions such as wearing lab, coat and glove should be taken while performing this procedure.
