The overall goal of this experiment is to observe the effects of compressive loading in the mandibular condylar cartilage of mice using Morphometric measurements in radiographs and histological analysis. These metrics can help answer key questions regarding the structural and cellular changes within the mandible condyle of rodents. The main advantage of the technique shown here is that they could be used to analyze other small born or participated animals.
For this protocol, have isolated, dissected mouse mandibles ready for use. On a Petri dish, transfer the mandibles into an x-ray cabinet system. Then, take radiographs at 26 kilovolts for five seconds.
Now, open the images in the analysis software to make Morphometric measurements. First, use the unit button to setup the scale bar. Next, select six anatomical points using marker style 2.
First, select the most posterior points of the mandibular condyle as point one. Next, select the incisored process for point two. Then, select the deepest point at the sigmoid notch for point three.
Next, select the deepest point in the concavity of the mandibular ramus, point four and then, select the most anterior point of the condylar articular surface, point five. Now, select the most posterior point of the condylar articular surface as point six and proceed with taking length measurements using the length tool. Next, use the perpendicular line tool from points three to four to measure the condyle head length, which is the length of the perpendicular, from point one to the line between points three and four.
Then, measure the mandibular length, between points one and two. Lastly, measure the condyle head width, which is between points five and six. To save the data, copy the measurement list on the right side of the screen.
Before embedding the fixed, but not decalcified mandibles, soak them overnight in 30 percent sucrose in PBS. The next day, dissect away any excess soft tissue and carefully cut the mandibular condyle. Now, place the samples in plastic molds, with the medial surface of the condyle against the base of the mold and the sample parallel to the bottom of the mold.
Then, immerse them in embedding resin and cool the resin with a piece of dry ice. Next, top off the molds with more embedding resin. Transfer them into cold two methyl butane, cooled by a freezer or by dry ice.
Once chilled, store the specimens at negative 20 degrees Celsius or at negative 80 degrees Celsius for sectioning. To quantify Col10a1 expression in the MCC of sagittal sections of condyles, open the histological images in an image analysis software package and select the area of interest using the lasso tool. In this case, the MCC region is selected.
Take note of the number of pixels in the area, which is reported in the histogram box. Next, select the Col10a1 red pixels by adjusting the color range. Use the eye dropper tool to select a red pixel from the image and click OK.Then, record the number of red pixels in the area, as reported in the histogram box.
The fraction of red pixels in the area represents the amount of Col10a1 expression. Now, using the same basic technique, quantify the TRAP activity in the MCC and subchondral bone. First, select the cartilage in subchondral bone regions as the areas to analyze and take note of the total pixels.
Then, select the yellow pixels generated by the ELF97 substrate. Take note of the number of positive pixels and calculate the fraction of TRAP positive pixels. Now, quantify the EDU positive cells that are counter-stained in blue by DEPI.
Again, select the area of interest and then quantify the blue pixels within it. Also use this method to determine the number of the EDU positive pixels in the same region. Morphometric measurements of mice, subjected to compressive TMJ loading, showed that the loading significantly increased mandible and condyle head length.
Nevertheless, loading did not result in a significant change in condyle width. Quantification of the Collagen distribution revealed a significant increase in Col10a1 expression and the MCC of the condyles of loaded animals. On the other hand, Col2a1 expression was not much different from control.
TRAP activity increased in the subchondral region of condyles and loaded mice as did sub proliferation. EDU staining denotes number of proliferative cells. Proteoglycan distribution in the MCC was quantified by evaluating the Toluidine blue stained area and the distance map.
There was a significant increase in distance mapping in the MCC of condyles of the loaded group. However, the proteoglycan stained area was not statistically different between groups. After watching this video, you should have a good understanding of how to analyze the cellular and the structural changes in the mandibular condyle of rodents.
