The overall goal of this procedure is to non-invasively deliver aqueous reagents to the mouse mammary gland.Introductory. This is accomplished by first anesthetizing the mouse and removing the hair surrounding the nipples. The second step is to remove dead skin covering the tip of the nipples using micro tweezers.
Next, an aqueous solution is injected into the nipple using a 33 gauge needle. The final step is to assure that the injection was successful by observing the injection site for potential swelling, which would suggest a mammary fat pad injection instead of an injection into the mammary gland. Ultimately, success of the procedure can be determined by visualization of the ductile tree following Evans blue injection.
This method can help answer key questions in the breast cancer field, such as the contribution of specific genes during various stages of memory tumorgenesis. To begin, prepare 160 microliters of 0.2%Evans blue dye in sterile phosphate buffered saline for each mouse to be injected prior to injection. Weigh each mouse and record its body weight.
Then anesthetize the mouse one at a time, using an isof fluorine chamber and apply eye lubricant. Once sedated, place an isof fluorine nose and inject meloxicam at five to 10 milligrams per kilogram subcutaneously prior to the procedure As an analgesic during the procedure, continuously monitor the mouse for changes in respiratory rate and adjust the level of isof fluorine accordingly, especially if the respiratory rate of the mouse indicates that the level of anesthesia needs to be reduced. Next, prepare the nipple area for injection by applying an over-the-counter hair removal cream.
Wait five minutes and then gently remove the loose hair with the cotton tip applicator. Using a circular motion, remove any residual cream using damp paper towels wedded with warm water. Then secure the mouse under the stereoscope by gently taping down the extremities and clean the injection sites with alcohol swabs.
To start locate the nipples to be injected under the stereoscope. Then use fine micro dissecting tweezers to remove any dead skin that covers the nipple opening. It is not necessary to cut the nipple for the injection.
Next load 11 microliters of injection solution into a 50 microliter syringe equipped with a 33 gauge metal hub needle affixed to it. One microliter of the injection fluid often leaks following injection, resulting in the final goal of 10 microliters of solutions successfully injected. Hold the nipple gently with the fine tweezers and lift it slightly to position it for injection.
Then inject 11 microliters into each gland in the first and last pairs of mammary glands as they require lower volume than the middle pairs to fully fill the ductile tree of the gland. Next, inject 21 microliters into each gland in the second through fourth pairs of mammary glands. The injection rate should be maintained at approximately 40 microliters per minute in order to minimize any potential damage caused by rapidly moving fluid within the ductal lumens.
Following injection. Observe the injection site. There should be no signs of trauma to the nipple region or surrounding tissue.
Swelling in the area surrounding the nipple. Likely indicates a mammary fat pad injection rather than a successful in ductal injection. Then remove the animal from the nose cone and move it to a separate cage for recovery.
Place the cage under a heat lamp to prevent hypothermia and assist in recovery. House the animal singly and monitor them closely until they regain consciousness and mobility. Upon completion of the study, euthanize the mice by cervical dislocation following CO2 compressed gas in an isolated chamber.
Then excise the mammary glands and continue with the proper sample handling for the analysis of interest. Shown here is the mammary gland of a mouse before and after it was injected through the nipple with Evan's Blue Dye. The nipple region shows no swelling or tissue damage, which would be caused by injection of the dye into the fat pad surrounding the ducts.
The diffuse blue dye seen in the right image is a good indicator of a correct injection. This can be validated by looking at the entire gland or a cross section following excision of the gland. In the whole mount analysis shown here, the Evans blue injected gland shows the entire gland filled with dye without damaging the native duck structures.
This is confirmed with Carmen Dye, which also highlights the ductile structures. It is also possible to utilize this method for the delivery of therapeutic molecules such as irna to ductal cells. In the image shown here, a fluorescently tagged irna that targets sickle fillin, a non-essential gene was injected and then the tissue was imaged 48 hours later to show localization of the molecules.
Following this procedure. Other methods like SRNA knockdown can be performed in order to answer additional questions relating to the role of a particular gene in memory development or tumorgenesis.
