The overall goal of this procedure is to show how to dissect and prepare a ma gland hole mount and analyze its morphology to predict memory cancer risk. This is accomplished by first dissecting a rat abdominal memory gland, and then preparing a memory gland hole mount from the dissected gland. The next step is to analyze the memory gland.
Morphology made visible in the hole mounts. The final step of the procedure is to palpate a rat for detection of memory tumor, and to measure the tumor volume in millimeters. Using a caliper, ultimately results can be obtained that show changes in memory gland morphology through identifying the structures in the memory, home mounts and counting or scoring their amounts under a microscope.
This method can help answer key questions in early life exposures and breast cancer field questions such as how dietary or environmental exposures acting during development can affect MA lung differentiation in this breast cancer risk later in life. The implication of this technique extent also to about breast cancer prevention. We can study, for instance, environmental and dietary exposes during the development and see the changes in the mammary gland, whether they are related to increased or decreased breast cancer risk.
Demonstrating the tumor palpation procedure will be Idli Cruz, an animal technician and manager for animal shared resources. To begin the procedure for removing the memory gland, first, use needles to pin the euthanized animal onto the surface board on its back from its legs. Then wipe the skin wet with ethanol.
Lift the skin a little with forceps and with sharp scissors. Make a midline incision into the skin, starting between the pair number five nipples and cut toward the neck. Then make an inverted Y incision from the midline toward the hind legs.
Dissect the skin open with scissors on one side of the inverted Y incision to expose the abdominal number four memory gland, which is attached to the skin. Pin the skin onto the surface board with needles to completely expose the gland. Next, use sharp scissors to dissect the memory gland free from the skin.
Start from the proximal area close to the nipple, and work toward the distal end of the gland toward the spine of the animal. Once the memory gland is detached, immediately spread it onto a glass slide for preparation of a whole mount. The glass slide must be bigger than the gland and appropriately labeled Using a permanent marker and or a diamond pen, spread the gland carefully corresponding to its original size and shape in C two.
After spreading the gland onto the slide, let it sit on the table for about 30 minutes so that the gland sticks onto the slide, but to not let it dry. Next, put the slide into a jar containing car noise fixative, and leave it at room temperature in the fume hood for two days or longer after at least two days. Wash the slide in 70%ethanol for one hour at room temperature.
Rinse the slide and distilled water for 30 minutes at room temperature. Next, stain the gland in Carmine alum stain for two days or longer. Staining is done when you check the back of the slide and see that the lymph nodes have stained through.
Wash the slide in a series of increasing ethanol concentrations, each for one hour. Start with 70%ethanol, followed by 95%ethanol and thirdly in 100%ethanol. To obtain mam gland whole mounts that are technically good for morphological analysis, two steps are essential dissection of the whole gland and the lip liquidation of the mammary fat bed.
The gland is placed in xylene to remove the lipids and to increase transparency. This clearing will take place two days or longer depending on the size of the gland and how much fat there is. After the last wash in absolute ethanol, put the slide in.Xylene.
Allow the gland to remain in xylene at room temperature for at least two days. Once dilapidation is complete, mount the slide with a cover slip. Using mounting media such as per mount, let the slide dry for several days in the fume hood before observing it under a stereo microscope, the morphology of the memory gland hole mount is analyzed according to three end points, epithelial elongation, number of terminal end buds and differentiation.
The results are then correlated with memory cancer risk. We will first demonstrate the analysis of a prepubertal age memory gland hole mount to access epithelial elongation. Use a ruler to measure in millimeters the distance from the nipple to the end of the epithelial tree.
The second endpoint number of terminal end buds or T Bs reflects the potential from malignant transformation. Under a light microscope count the number of tbs, which are the largest pulpous structures, located only at the distal end of the memory epithelial tree for the third end point, which is differentiation. The whole mount is also examined under the light microscope.
Differentiation is measured by scoring the alveolar buds, which are spread across the epithelium. The two alveolar bud types designated AB one and AB two are each given a score from zero to five based on their abundance in the gland. The score values for AB one and AB two are then added for a final differentiation score.
The morphological analysis of a post pubertal H memory gland home mount is similar to that of a pre-pubertal H memory gland home mount with the following differences. To examine epithelial elongation, first, use a ruler to measure the distance in millimeters from the lymph node to the end of the epithelial tree. Second, measure the distance from the tip of the epithelial tree to the end of the fat pad.
Also in millimeters for analysis of differentiation. In a post pubertal H memory gland hole mount, in addition to alveolar buds, lobules are scored on a scale of zero to five under a light microscope. The score reflects the abundance of lobules in the gland.
Differentiation is then assessed in two ways. First, the score values from AB one, AB two, and LOEs are added for a final differentiation score. Second, the ratio of the LOEs score to the AB one plus AB two score is calculated.
Since ABS differentiate two LOEs, a more differentiated gland will have a higher ratio of LOEs to abs. To begin this procedure, hold the rat by grasping the whole body with the palm over the back with forefinger behind the head and the thumb and second finger under the opposite axilla. Turn the rat so it is lying on its posterior and palpate to detect any memory tumors, palpable tumors should feel like a lump.
Next, use a caliper to measure the width, length and height of the tumor. Calculate the tumor volume using the ellipsoid formula. Volume equals one sixth pi, A, B, C, where A equals width, B equals length and C equals height.
Manually record the location and size of the tumor in a notebook on a weekly basis. This data will be transferred to a spreadsheet later when dissected and processed correctly. The memory gland hole mount should have all its components clearly visible as shown in this example.
In contrast, this is an example of a poorly prepared mammary gland hole mount. The gland has not been properly dissected because the distal portion is missing. In addition, the gland was not properly stretched and dilapidation has not occurred.
Completely shown. Here are representative results of how changes in memory gland morphology can predict memory cancer risk. In rats that were exposed to the carcinogen DMBA when the number of terminal N buds or TBS was counted, the treatment group had a higher number of TBS at 21 days compared to the control group.
These changes in ma gland morphology were associated with a higher proportion of memory tumors in rats of the treatment group compared to the control group. As shown in this graph. While attempting this procedure is important to remember to dissect the whole MA gland and to remove all lipids to ensure good visualization of the mam gland structures.
After watching this video, you should have a good understanding how to dissect and prepare a mammary hole mount and how to analyze its morphology to predict breast cancer risk.
