The overall goal of this procedure is to study mastitis or inflammation of the mammary gland, and more specifically to visualize and quantify NF Kary activity within an inflamed lactating gland. This is accomplished by first crossing a female NF Kappa b reporter transgenic with a wild type male. Eight days after the birth of the pups, lipopolysaccharide is introduced into the lactating gland of the female via an introductory injection technique, which does not damage all wound the site of injection six hours after injection.
The mouse undergoes bioluminescent imaging to detect luciferase activity within the mammary gland. Ultimately, results can show significant NF KB activity in the LPS treated glands through the use of the Intraductal injection method, followed by bioluminescent imaging. The main advantage of our approach over other methods of intraductal injection is that the site of injection is not damaged in any way.
This is absolutely critical if you plan to study the inflammation associated with the LPS injection as any damage to the site or to the nipple would overlay and confound these analysis. Female mice positive for the NGL Reporter transgene are used for this procedure.One. Females are at least six weeks of age.
M mate them with an FVB wild type male by har and breeding at 15 days post mating. Begin checking the females regularly for signs of pregnancy, namely large protruding girth. Separate pregnant females to their own cages, and when the last female becomes pregnant, remove the male from the breeding cage.
A litter of at least six pups is required for adequate lactation. Eight days after the birth of such a litter. Proceed with the protocol.
The most challenging part of the procedure is the introductory injection itself. Having steady hands and lots of practice will help, but pitfalls can always occur. Be prepared to have multiple mice ready on the day of injection in order to ensure success.
Begin by diluting e coli derived lipopolysaccharide in PBS to a final concentration of 0.1 micrograms per microliter. 50 microliters of this LPS solution will be injected into the mammary gland. Prepare a second tube of PBS alone to be used for control injection.
Next to the stage of the dissecting scope. Secure a nose cone apparatus to deliver anesthesia. Prepare bright illumination of the stage using extra spotlights as needed.
Once the dissection setup is ready, start the isof fluorine machine and airflow. Next load the 31 gauge insulin syringe with 50 microliters of the LPS solution and set the syringe aside for practice injections. Use triam blue dye diluted in PBS to visualize the results.
Next, anesthetize the mouse to be injected once its breathing has slowed. Place it on the stage and provide iso fluorine via the nose cone. Use a to pinch to confirm the fully anesthetized throughout the procedure.
Monitor the mouse's breathing pattern. Continually adjust the isof fluorine to keep it steady. Use a small drop of 70%ethanol to flatten the hair on the animal's ventral abdomen and locate the nipples.
Locate the nipple of the right number four inguinal mammary gland on the lower abdomen with fine forceps in the non-dominant hand. Carefully draw out the nipple away from the skin. The forceps should never be clamped tight.
Only a light hold should be applied to the nipple in the dominant hand. Bring the preloaded syringe into the field of view and hold it so that no repositioning is required to inject its contents. Carefully aim the needle at the small ductile opening at the center of the nipple.
Insert approximately one quarter of the needle into the duct without repositioning. Inject the contents of the syringe over three to five seconds. Then carefully remove the needle from the nipple.
Load a second syringe with 50 micro releases of PBS and repeat the procedure on the left. Number four, inguinal mammary gland nipple using the PBS control. Once both injections are completed, discontinue the anesthesia and move the mouse to a recovery cage.
The female should be moving within five minutes. After an hour, return her to a home cage with her puffs. The mouse should be imaged six hours injection.
During the interim, monitor the mouse if signs of distress are observed. Discontinue the experiment and euthanize the mice at the imaging facility. Prepare to anesthetize the female using isof fluorine.
Ensure that the anesthesia is flowing to both the box and the imaging chamber. Place the mouse under isof fluorine Anesthesia in the clear anesthesia box. Once breathing has slowed, use a to pinch to confirm the mouse is fully anesthetized.
Next retroorbital, inject 100 microliters of Lucifer substrate through a 26 gauge needle. Immediately transfer the mouse to the imaging chamber after the injection and attach the nose cone delivering Isof Lorraine. Place the mouse in a supine position and secure each foot to the stage with black opaque tape.
Input the appropriate settings to the IVIS software to take both a white light photographic image and an image of the bioluminescent activity. The exposure time should be 10 seconds once the images have been acquired. Follow the same recovery protocol as described for post injection.
The photographic and luminescent images are automatically overlaid and displayed on the screen. Analyze the acquired images by placing region of interest or ROI circles of equal size over each side of the mouse's lower abdomen. One over the PBS injected gland and one over the LPS injected gland.
Set the readout to photons. A number should be displayed indicating the luminescence detected within each ROI use this number in statistical analysis to display the optimum image. Further adjust the threshold of the signal displayed.
This will not alter the total luminescence readout, but will allow the removal of the background signal when the PBS injected gland is free of the background signal. The increased signal in the LPS treated gland should be apparent in the image practice Injections were performed on day 21 of lactation when there is less milk production and the success of an injection can be readily visualized by trian blue Staining in successfully injected memory glands only the ductal tree was filled with solution in an unsuccessful injection. There was a buildup of liquid at the nipple or surrounding tissue.
Successful injection of LPS into the mammary ducts at day eight resulted in increased NF KB activity within the gland due to constitutive NF KB activity in the brain and other abdominal organs. There was a consistent baseline signal prior to any manipulation. Luciferase activity was substantially higher in the LPS injected gland than the PBS injected gland from a group of four mice.
A statistically significant increase in luminescence within the LPS treated glands was measured. This method can provide insight into mammary gland inflammation. It can also be applied to other areas of research such as cancer research.
You can inject cancer cells or carcinogens directly into the mammary duct.
