Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

This method can help answer key questions in evolutionary biology, such as the evolution of social communication. The main advantages of this technique are that it provides direct evidence for form and presence of mimetic musculature. It eliminates guesswork in the dissection process, and it preserves muscle layers.

Combined with DiceCT, reverse dissection is the new gold standard for mimetic muscle investigation. To begin, follow the accompanying text protocol for specimen fixation and preparation. Then, transfer the specimen to the fume hood.

Then, rinse the specimen for several hours in running water. Once the specimen has been rinsed, pat it dry with paper towels, and transfer it to a work station. With the brain and overlying calvaria removed, make one incision near the glabella region of the skull, and another along the caudal aspect of the skull, where the cranial bone is intact.

Palpate for the external occipital protuberance at the caudal edge of the skull. This marks the area where the skull meets the spinal column. Then use a scalpel and make a midline incision, beginning at the external occipital protuberance.

Cut the specimen rostrally over the parietal and frontal region of the skull. Continue and pass over the orbital region in between the eyes, down the nasal region, all the way through the external nasal area between the nares. Next, make a cut on the mandible between the lower and central incisors, and move the scalpel caudally toward the clavicle.

At this point, choose one side of the face mask, and release it from the skull, leaving the other side in place. Starting in the occipital region, use the scalpel to cut the facial mask, including mimetic musculature, away from the occipital bone of the skull, pulling the mask rostrally and laterally. Cut the occipitalis muscle away from the skull, leaving a small amount of the muscle behind on the skull, so that its attachment can still be visualized, once the facial mask has been removed.

By leaving some portion of the muscle behind on the bony and cartilaginous skull, attachments can be preserved and recorded at a later time. Once the external ear is hit, locate the auricular muscles. Cut through these muscles, so that a small portion of each muscle remains with the skull.

Then, cut through the elastic cartilage attaching the external ear to the skull. Continue to pull the mask rostrally, and release each mimetic muscle from the skull, leaving a small portion of each muscle behind on the skull. Once there is an intact facial mask from one side of the skull, allow it to sit out with the musculature exposed to the air, for one to three hours, depending on the size of the specimen.

This will desiccate the connective tissue on the face mask, and increase the color contrast between muscle and connective tissue. With the contrast between muscle and connective tissue now enhanced, use the number three scalpel, forceps, and micro scissors to remove sufficient connective tissue, in order to visualize the musculature. The superficial layer of mimetic musculature in this reverse dissection, gently lift and separate the deep layer of musculature from the superficial layer, and remove the connective tissues surrounding the superficial musculature.

When finished, return the resulting face mask back into formalin. The procedure may be paused here, before moving on to further dissection, or before continuing on to staining. At each intervening step, allow sufficient drying time of the facial mask, so that the connective tissue can be easily discerned from the muscle tissue.

In order for the mimetic musculature to be visualized using CT scanning, stain the masks using an iodine solution by placing the mask into Lugol's staining solution, as described in the accompanying text protocol. The staining process generally takes about two weeks, in 1.75%Lugol's solution. However, this timing varies, depending on several factors.

Intermittent scanning with accompanied staining adjustment is recommended. For CT scanning, first prepare face mask by using wooden toothpicks to mount the face mask onto a low density material, such as floral foam. This will eliminate any wrinkles in the mask, and limit its movement as it dries out slightly during the scan.

Next, place the specimen in a low density container, and secure it into place. Once secure, place the specimen into the scanner, close the door, and turn on the X-rays. Then, set up the CT scan parameters for a high resolution X-ray computed tomography scan.

Using inter-slice spacing and inter pixel distance of around 0.05 millimeters for these scans, as the small fascicles will be obscured at lower resolution. If fascicles are unable to be fully visualized, due to overstaining, place face mask into a de-stain solution such as 10%formalin, or 5%sodium thiosulfate to remove some of the stain. After adjusting the staining, scan the face mask again, and continue to make the necessary adjustments, until all fascicles are able to be fully visualized.

By using reverse dissection to create a facial mask, a fuller representation of mimetic muscle can sometimes be seen, than in traditional dissection methodology. This method works on a range of body sizes, from the tiny, small body of the Marmoset, to large-bodied primates such as the chimpanzee. In small-bodied primates that have gracil facial muscles, some of the facial musculature may be indistinguishable from the surrounding connective tissue, and may be lost during dissection.

An iodine stain, such as Lugol's, may improve the contrast and help to resolve both individual mimetic muscles, as well as individual muscle fascicles, and for the first time, obtain whole muscle volumes of these gracil muscles. As shown here, some of the very small muscles, associated with the external ear, are clearly visible in the DiceCT scans. It is not uncommon for these muscles to be missed in some reverse dissection procedures, perhaps due to their small size.

This three dimensional volume rendering allows for basic volume thresholding and color mapping, to visualize the musculature more easily, than with cross-examination alone. Here you can see that the individual muscle fascicles are clearly visible in cross section, making digital dissection feasible. While attempting this procedure, it's important to keep in mind, that you should move slowly, cautiously, and with deliberation, as this method is destructive in nature.

Following this procedure, other methods, such as histological processing, may be used to answer additional questions, such as fiber diameter, or myosin fiber typing. After its development, this technique paved the way for researchers in the field of evolutionary biology, to explore the evolution of facial expression and visual communication in non-human mammals, and in humans. Don't forget, that working with formalin, and a loaded scalpel can be extremely hazardous.

Precautions, such as rinsing your specimen, and knowing where your scalpel is at all times, should be taken while performing this procedure.