In this procedure, solutes can be directly delivered into the lungs. Fire the trachea first. Remove the fur from the small rodent and make a small incision near the anterior aspect of the neck.
Expose the trachea. Use a one milliliter subcutaneous needle to instill three microliters of solution per gram of body weight via the trachea. Remove the syringe and suture the wound.
Hi, I'm Dr.Ma Helms from the Department of Physiology at Emory University. Hi, I am Dr.Mauricio Rojas from the Department of Medicine here at Emory University. Today we'll show you a procedure to directly instill solutes into the distal portions of the lung.
We routinely use this procedure in my laboratory to study lung fluid clearance in response to several various compounds instilled, And we use this technique in my lab to deliver bli. Creating a mouse male of lung injury. Direct installation of solutes into small rodent lungs may be useful in your studies.
So let's get started. In preparation for surgery, the mouse is initially anesthetized with isof fluorine and then further sedated with an intraperitoneal injection of a mixture of xylazine and ketamine. Perform a toe pinch to confirm that the animal is fully anesthetized.
Then use an electric razor to shave the fur from the surgical area. To remove remaining hair, apply a thick layer of hair removal cream to the surgical area. Leave the hair removal cream on for three minutes, and then wipe away the fur and cream with a damp towel.
Wipe the surgical area with 70%alcohol, followed with Betadine surgical scrub. To aseptically prepare the animal. The animal is now ready for surgery.
To begin the procedure for an intratracheal installation position the anesthetized animal onto a sloped surgical board. Make a small incision near the anterior aspect of the neck or throat region. Use forceps to push back the platysma and then make an incision on the anterior tracheal muscles.
This will enable you to visualize and access the tracheal rings. Once the trachea is exposed, administer the appropriate amount of installate using a one milliliter subcutaneous needle and syringe. Hold the syringe bevel side up and parallel with the trachea and inject the full volume of installate into the trachea.
A typical intra tracheal injection volume is three microliters per gram of body weight. Remove the syringe from the trachea. The animal may respond by gasping, following the installation of solu into the lungs.
Introducing air through the syringe and observing whether the lungs expand will also confirm that the intubation was successful. Close the incision with four oh nylon sutures. Grasp the needle swage the thickest portion of the needle where the suture material is attached with a needle holder penetrate the skin with the tip of the needle at a 90 degree angle, approximately one to three millimeters away from the edge of the wound.
Depending on the thickness of the skin, grasp the exiting needle after one pass through the tissue. Simple stitches, individually nodded, will efficiently close the wound shown here are some representative results of intra tracheal installations. This first image shows a successful installation of a fluorescent compound sci-fi 0.5 conjugated to dextrin in a black six mouse lung.
The co-registration of the SCI 5.5 signal with the x-ray image confirms that the fluorescent dye is primarily targeted to the lung and is evenly distributed immediately following installation. In contrast, this second image shows the outcome in a poorly instilled mouse. The fluorescent signal in the gastrointestinal tract immediately following the procedure indicates that the esophagus was inadvertently intubated during the procedure.
We've just shown you how to directly instill solutes into the distal portions of the lung. When you're doing this or any other procedure, it's important to follow the guidelines of your own institution for the use of animals in research. So that's it.
Thanks for watching and good luck with your experiments.
