The authors wish to acknowledge Yerkes National Primate Research Center for access to chimpanzee and rhesus macaque specimens, and Chris Vinyard (Northeast Ohio Medical University) for access to common marmoset specimens. We thank Marissa Boettcher, Kaitlyn Leonard, and Antonia Meza at the University of North Carolina for assistance with the scanning process. This work was performed in part at the Duke University Shared Materials Instrumentation Facility (SMIF), a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), which is supported by the National Science Foundation (Grant ECCS-1542015) as part of the National Nanotechnology Coordinated Infrastructure (NNCI). This is Duke Lemur Center publication number 1405.

Because these procedures use animals that died from natural causes at zoos or were sacrificed in research labs where they were part of unrelated studies, these protocols do not require IACUC approvals.
1. Reverse Dissection
Note: The protocol for reverse dissection is effective for very small mammals, such as laboratory mice, all the way to large land mammals, such as the domestic horse. The mimetic muscles are often best preserved and best visualized...

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	<li>Gregory, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Our face from fish to man. , (1929).</li><li>Burrows, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+facial+expression+musculature+in+primates+and+its+evolutionary+significance.">The facial expression musculature in primates and its evolutionary significance.</a> BioEssays. 30, 212-215 (2008).</li><li>Santana, S. E., Dobson, S. D., Diogo, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plain+faces+are+more+expressive:+comparative+study+of+facial+colour,+mobility+and+in+primates.">Plain faces are more expressive: comparative study of facial colour, mobility and in primates.</a> Biology Letters. , (2014).</li><li>Burrows, A. 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Following the steps for the &#34;reverse dissection&#34; protocol typically produces a facial mask that can be slowly and methodically dissected to reveal mimetic musculature, regardless of the size of the head. It is especially important to move slowly and continuously assess whether the muscles have been completely cut through inadvertently, especially in smaller bodied species.
In order to determine where the musculature is located, it is especially critical to allow the developing mask to ...

Mimetic musculature, or facial expression musculature, is skeletal muscle and is found throughout Mammalia1. While most mammalian skeletal muscle attaches to discrete bony landmarks, mimetic musculature is unique in its attachments primarily into the skin of the face, scalp, and the ventral aspect of the neck1,2,3,4. Mimetic musculature contraction deforms the &#34;facial mask&#34; into expressions or facial displays of social and emotional intent, changes the size and shape of the sphincters of the eye, nasal cavity, and oral cavity used in feeding, respiration, and in vocalizations, and is part of the overall close-proximity visual communication mechanism found among most mammals2,3,4,5. Across Mammalia, the facial displays generated by mimetic muscles assist in regulating and maintaining territorial boundaries, social bonds, and the social group by cuing conspecifics on the emotional and behavioral intentions of the sender2,5.
Among mammals, primates are characterized in part as employing a high level of social behavior throughout the life cycle with all species living in a social group2,5. While some taxa, such as the nocturnal galagos and lorises, may live in groups consisting only of a mother and offspring, other taxa, such as the diurnal macaques and baboons, may live in groups of over 100 individuals6. No matter the size of the social group, primates often use stereotyped social behaviors associated with rank and territoriality and these behaviors typically include a facial display component. Facial displays are part of the process of maintaining bonds among member of social groups, dominance hierarchies, reproduction, and the communication that is part of daily life, especially in diurnal species2,5,7. While it has been clear for some time that facial musculature is used to create these facial displays, it has only recently become apparent that facial musculature form and physiology are associated with the functional demands of social variables2,8. Previous studies on phylogenetically and behaviorally diverse ranges of primates have shown that diurnal species living in large, complex social groups tend to have a high number of discrete facial displays that focus on movement of the lips, eyebrows, and eyelids with a high number of facial muscles clustered around the lips and orbital region9. In contrast, there have been few studies on nocturnal species living in small groups, but these species have a high number of discrete facial muscles with attachments around the external ear and lips, which may be associated with movements of the ear and lips (which have been documented in some nocturnal species in agonistic encounters with conspecifics and locating sounds)2,9,10,11. In addition, humans have a relatively higher percentage of slow-twitch myosin fibers in mimetic musculature than either rhesus macaques or chimpanzees, which may be related to the &#34;slowing down&#34; in contraction of human mimetic musculature around the lips used during production of speech sounds or to general fatigue-resistance capability of the muscles8.
Humans are, arguably, the most social of all primates, and have developed language as one component of social communication. Still, though, humans use facial expression as a means of visual communication and have the greatest known facial display repertoire among primates. In an effort to more completely understand the variables surrounding the evolution of human and general primate social behavior, an increased understanding of the morphology and physiology of primate mimetic musculature is highly desirable. Because mimetic musculature is attached to the skin itself and may, in some species, be exceptionally thin and difficult to visualize, we have developed a unique method of visualizing this musculature for both the processes of recording gross presence/absence and attachments as well as sampling for microanatomical processing.
&#34;Reverse dissection&#34; is a method for preserving the mimetic musculature by removing the entire facial mask from the head and increasing visibility of even small muscles. Because reverse dissection is a destructive process, rare and valuable specimens may not always be available for this methodology. DiceCT is an effective method that can visualize many of the mimetic muscles in even tiny species12,13,14. This method can be used in concert with reverse dissection or in cases where rare, valuable specimens may not be dissected and can provide much information without having to remove the facial mask in &#34;reverse dissection&#34;12,13,14. The present protocol describes a set of methods for combining reverse dissection with DiceCT in order to examine primate mimetic musculature.

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			<td height="21" style="height:21px;width:216px;">Nikon XTH 225 ST</td>
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			<td height="42" style="height:42px;width:216px;">10% buffered formalin</td>
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			<td height="63" style="height:63px;width:216px;">Iodine, ACS Grade</td>
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reverse dissection and dicect reveal otherwise hidden data in the evolution of the primate face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

This section presents examples of results on facial musculature form that can be achieved by using &#34;reverse dissection&#34; in concert with DiceCT scanning. By using &#34;reverse dissection&#34; to create a facial mask, a fuller representation of mimetic (facial) muscle can sometimes be seen than in traditional dissection methodology. This method works on a range of body sizes from the tiny, small-bodied primates, for example the common marmoset Callithrix jacchus (<strong cl...

Watch this Scientific Journal Video about Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face at JoVE.com

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

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

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.

reverse-dissection-dicect-reveal-otherwise-hidden-data-evolution

Research

JoVE Journal

Biology

6.8K visualizações.  Duquesne University. Esse método pode ajudar a responder a questões-chave na biologia evolutiva, como a evolução da comunicação social. As principais vantagens dessa técnica são que ela fornece evidência direta para forma e presença de musculatura mimética. Elimina adivinhação no processo de dissecção, e preserva camadas musculares.Combinado com o DiceCT, a dissecção reversa é o novo padrão-ouro para a investigação muscular mimética. Para começar, siga o protocolo de texto que acompa...