Histochemical Mapping and Fiber Size Analysis of Mimic Muscles Gerhard Freilinger, M.D., Wolfgang Happak, M.D., Georg Burggasser, and Helmut Gruber, M.D. Vienna, Austria Fourteen functionally relevant mimic muscles of nine human bodies were analyzed with respect to their muscle fiber sizesand their histochemical fiber type composition In cryostat sections stained for actomyosin ATPase, type and type 2 fibers were evaluated separately by means of computer-assisted image analysis. The fiber diameters varied between 20.24 and 41.45 ym. According to the proportions of the fiber types, the mimic muscles could be classified into three groups: (1) phasic muscles, with 14 10 15 percent type 1 fibers, (2) intermediate muscles, h 28 to 37 percent type | fibers, and (3) tonic muscles, containing 41 to 67 percent type I fibers. Itis concluded that one has to consider this diversity of mimic muscles when planning the surgical reconstruction of facial pa- alysis. For dynamic reanimation of irreversible facial paralysis, free-muscle transplantation is becom- ig the method of choice throughout the world.' Donor muscles are selected with respect to their superficial position, ease of surgical access, and well-defined neurovascular pedicles. Further im- provement in the functional results will be pos- sible only if more appropriate donor muscles are used in facial reanimation. The muscle or mus- cles to be grafted should be more similar to mimic muscles with respect to their dimension, fiber sizes, and fiber type composition. In order to identify suitable donor muscles, however, one primarily needs more information on the re ent side, namely, the mimic muscles themselves. ‘The appreciation of the fiber type composition of mimic muscles, which varies dramatically from muscle to muscle," also should shed new light on the subject of this delicate muscle system with its myriads of shades of expression and conse- quently may help to stimulate our efforts toward better restoration The aim of the present study was to add new aspects to the spare knowledge of this distinct mimic muscle system.** Fourteen mimic mus- cles, which are of special interest for facial expression, were analyzed with respect to their fiber sizes, and the results in terms of their con- tent of tonic type 1 fibersand phasic type 2 fibers were laid down in a histochemical mapping. MATERIALS AND METHODS Fourteen mimic muscles of 9 human bodies were dissected within 1 to 6 days after death.’ ‘The average age was 63, ranging from 24 to 79 years, All subjects died acutely and did not suffer froma chronic disease, which could have affected the histologic and histochemical characteristics of the mimic muscles.'®"' The causes of death were acute ventricular failure (5 cases), apoplexia (2 cases), and pulmonary embolia (2 cases). The whole muscles were excised and stretched by means of two forceps to the original length and frozen in 2-methylbutane, cooled by dry ice. Then 12-um-thick cryostat sections were stained for actomyosin ATPase after alkaline (pH 10.4) and acid (pH 4.3) preincubation." For the sub- sequent image analysis, the sections stained after acid preincubation were used. Of the complete cross sections, representative fields were trans- mitted by means of TV camera from the micro- scope (Zeiss Axiomat) to a personal computer (IBM PS 2/80). From each tissue section at least 300 muscle fibers were evaluated. Type 1 and type 2 muscle fibers were analyzed separately. For each muscle fiber, the diameter was calcu- lated from a circle of the same area. Counts and mean fiber diameter of each fiber type and the From the Department of Pstic Surgery at che Second Suryical Cini and the ‘Third Department af Anatomy of the University of Vienna Reveised for publication March 13, 1989: revived August 1, 1989, 42Vol. 86, No. 3 / MAPPING OF MIMIC MUSCLES fiber-type_composition—expressed by the per- centage of type I fibers—were printed out. The following 14 muscles (see also Fig.1) were excised, prepared for histochemical staining, and analyzed: M. occipitofron- talis (venter fron- talis and occipi- talis) M. corrugator su- M. procerus percilii M. nasalis M. orbicularis oculi M. 2ygomaticus M. levator labi major superior M. levator anguli M. orbicularis oris oris M. buccinator M. depressor an- guli oris, M. mentalis M. depressor labii inferioris Platysma Fig. 1. Schematic drawing and list of the 14 mi phumbers of specimens and of fibers eval of type | and type 2 muscle fibers based on the histochemical staining fo less than 82 am, and muscles 8 to 14 are built up by sd for each muscle and the mean dian 423 For some mimic muscles it was not possible to get specimens from each of the nine cadavers. In some cases, the procerus and the corrugator su- percilii muscles could not be clearly delineated from their neighboring muscles. In some other cases, the nasalis muscle was not developed. Only five mentalis muscles could be used in this study because of technical problems. ResuLTs, Muscle Fiber Diameters The average fiber diameters of the 14 mimic muscles examined varied between 20.24 and 41.45 um (Figs. | and 2). The smallest fiber sizes were observed in the nasalis muscle and the largest in the occipitofrontalis muscle. All mus- cles around the eye (M. nasalis, M. procerus, M corrugator supercilii), the two circular muscles (orbicularis oculi and orbicularis oris muscle), and the two muscles below the lower lip (depres- sor labii inferioris and mentalis) showed very small muscle fiber diameters ranging from 20.24 to 31.84 zm (Fig. 1). In contrast, the muscles Muscle Number Counts Mean diameter ofspee aypel Type Mt nasalis 1793 20.704 20202 1987 23.205. 1 2M. procerus, 3M. orbiculars ons 23.9102 25.8003 28.1:04 1546 28.1.0 31.5:04 4M depressor lab. int 5M. orbielaris cout! 6M mentalis g 7M. corrugator sup. 8 Mduccnawr «75852 93.002 38.0008 8 Mrygomatcusma. 8 6618 «97.9404 39.1002 10 Mevatorlab. sup. 9 5634 3850S 32.702 L Mdepress.ang ors 9 6167 39.6:02 97.502 12 Platysma 8 4551 99.404 395105 18 Mooccipitoftontals 9 56453980 96.4208 14 Mlevtorangulions 9 5454 41.503 39.8:02 ‘The attached table indicates the rl error of the mean, sof more than $2 ya,424 Fic. 2, Photomicrographs of muscles representative for the three groups of mimic muscles. (2) M. orbicularis ocul, orbital part, containing only 15 percent type 1 fibers. (6) M. levator labii superioris, built up by 82 percent type 1 fibers. (c) M ‘occipitofrontalis, containing 57 percent type 1 fibers. Actomyosin ATPase staining after preincubation at pH 4.3. Type 1 fibers are darkly stained (Bars = 250 ym). inserting at the oral angle (M. buccinator, M. zygomaticus major, M. levator anguli oris, M. depressor anguli oris), the levator labii superioris muscle, and the occipitofrontal muscle, as well as the platysma, showed larger fiber diameters vary- ig between 32.73 and 41.45 am (Fig. 1). Muscle Fiber-Type Composition The distribution of type 1 and type 2 muscle fibers differed greatly from one mimic muscle to the other. The percentage of slow-contracting type 1 fibers varied between 14 and 67 percent, (Figs. 2 and 3). Based on their amount of type 1 bers, three groups of mimic muscles may be distinguished: 1. Group 1 consists of muscles that have less than 20 percent of type 1 fibers and i represented by the M. procerus, M. or! cularis oculi, and M. nasalis. 2. Group 2 is composed of muscles containing 20 to 40 percent of type I fibers, as is the case in the Platysma, M. zygomaticus major, M. levator labii superioris, M. mentalis, M orbicularis oris, M. levator anguli oris, and M. depressor anguli oris. 3. Group 3 consists of mimic muscles built up by more than 40 percent of type 1 fibers M. corrugator supercilii, M. depressor labii inferioris, M. occipitofrontalis, and M. bue- cinator. Figure 2 contains the photomicrographs of ATPase-stained sections representative of each of the three groups of mimic muscles. Homogeneity of the Fiber-Type Distribution Most of the muscles examined showed homo- geneous distribution of fiber types throughout the whole cross section. However, in three mus- cles, clearly different zones were observed throughout: In the orbicularis oculi muscle, the palpebral and orbital parts differed with respect to mean muscle fiber size and fiber-type composition. The orbital part showed larger fiber diameters than the palpebral part. The difference was 5 um for type 1 and 7 um for type 2 fibers. In addition, the orbital part contained 2 percent more type 1 fibers than the palpebral part.Vol. 86, No. 3 / MAPPING OF MIMIC MUSCLES In the orbicularis oris muscle, three to five muscle fiber bundles immediately adjacent to the edges of the lips (Fig. 2, center) consist of 100 percent type 1 fibers. The remaining muscle is mixed, containing 30 percent of type I fibers. In the same muscle, the fiber diameters of the lower lip were 5 um smaller than in the upper lip for both fiber types. In the buccinator muscle, the medial (oral) part, also consists predominantly of type 1 fibers (77 percent) in contrast to the lateral (buccal) part (55 percent). Discussion The skeletal muscles of the human body— more than 600 in number—and of most labora- tory animals have been very well investigated morphologically, histochemically, biochemically, and functionally (for review see Peachey"). A great number of publications have focused on the choice of donor muscles and the functional recovery of muscle transplants with and without microvascular anastomoses.'*“** There is no doubt that very little research of any sort has been done on the recipient side, ie., on the muscles innervated by the facial nerve, as May mentions in his book, The Facial Nerve.** In many aspects, the mimic muscle system is distinct from the rest of the body muscles, in that it represents flat, straplike muscle sheets close to the skin with muscle fibers interdigitating or ending in the skin. Mimic muscles have very short tendons, if any, they are not enveloped by a fascia, and typical muscle bellies are never ob- served. There is enough knowledge on func- tional and clinical anatomy,**>** a and we do have extensive information on gross and microana- tomy, excursion measurement, surface anatomy, density of innervation, and data concerning weight and length relationship.'*” There also exist detailed descriptions on the embryology of the facial musculature.?** Unlike the limb muscles, which are mainly responsible for force development, joint stabili- zation, and erection, the delicate mimic muscles control the movement of the face with its myriads of shades known as facial or mimic expression, which even is present immediately in newborn babies.®* The great variability in the fiber com- position of this specific muscular system is re- flected in such highly differentiated functions as facial tonus, emotional expression, phasic reflec- 425 tory protection, and the more forceful activity during mastication and the integrated synergism during phonati The number of histologic and histochemical studies of the facial muscles is rather limited and confined to few muscles.*”**** Primary work on skeletal muscle by Polgar et al.°° and earlier by Brooke and Engel" has shown that the diameters of type 2 fibers are generally larger than those of type I fibers. This is not true for the mimic muscles examined (Fig. 1). A wide variation in fiber-type proportions in as many as 36 different human muscles taken from many parts of the body also has been demonstrated. It is well established that predominantly tonic muscles show a high percentage of type 1 fibers and predominantly phasic muscles contain high per- centages of type 2 fibers.°”°* Most of the muscles, however, are known to fulfill both tonic and phasic functions and show no striking predomi- nance of either type. It is remarkable that the range of variation concerning fiber-type propor- tions in mimic muscles is even larger than in skeletal muscles. The results of the present study show that the fastest muscles of the human body are the phasic mimic muscles arranged around the eye and nose (Fig. 3). On the other hand, the high percentages of slow type 1 fibers typical of postural muscles of the body are observed also in some tonic mi muscles, such as the buccinator and the occipi- tofrontal muscles. Our data do not agree with the view discussed by Rayment et al.”® suggesting that all mimic muscles are of the tonic type. We were surprised to find that the buccinator muscle is one of the slowest in the whole muscular system of the body, inhomogeneous in itself, showing a discrepancy of type 1 fibers in the medial and lateral parts in a magnitude of 22 percent. This muscle is known to be markedly and constantly active during chewing, swallow- ing, blowing, and sucking, as well as during var- ious lip and mandibular movements. Basmajian and White write: *... in a relaxed study we showed the marvellous control man has over various parts of the buccinator and further: with minimal feedback training a series of subjects rapidly learned to consciously control contrac- tions of different parts of the muscle while re- maining silent in the remainder and they could do this while performing on the clarinet.” It was of further interest that the orbicularis oculi mus- cle, which is one of the quickest acting, perform- ing the blinking reflex, is as inhomogeneous as426 PLASTIC AND RECONSTRUCTIVE SURGERY, Seplember 1990 Muscle ype! mn 1M, procerus| “ M. orbiewaris ocull 1s Mnasalis 15 ‘M.zygomaticus major 28 M.orbicularis ons 29 2M mentalis 30 M. tevator tab. sup, a1 Playsma a -M. levator angull ora 37 1M. depressor ang. ons 37 a 46 . a or Fic. 3, Histochemical mapping of the 14 mimic muscles studied. Muscles of the “fast” group 1 with 14 to 15 percent type | fibers appear in yellow, the “intermediate” group 2 with 28 to 37 percent type 1 fibers are in blue, and the “slow” group 3 containing 41 to 67 percent type I fibers appear in red. The percentage of type I fibers of each muscle is listed on the left side of the figure. The size of the samples is identical to that of Figure 1 the orbicularis oris when considering the inner part as opposed to the outer. According to the Nomina Anatomica," the sys- tem of mimic muscles in total comprises 23 mus- cles. Fight of them were excluded in our study: ‘The three muscles around the ear (M. auricularis anterior, superior, and posterior) were not eval- uated because they have no direct effect on facial expression and are of minor clinical importance. Further, five other muscles (M. depressor septi, M. depressor supercilii, M. transversus menti, M. risorius, M. zygomaticus minor) were excluded because they are inconstant and often consist of only few muscle fibers. The M. levator labii superioris alaeque nasi was evaluated together with the M. levator labii superioris, which showed the same fiber composition We are convinced that no single muscle can ever take over the difficult tasks of the mimic muscle system; therefore, we have to graft more muscles into the area to be reinnervated by dif- ferent branches of the facial nerve. Mapping of the mimic muscles with respect to phasic, inter- mediate, and tonic properties implies that the motoneurons of the facial nerve are firing with different impulse patterns and that their nerve fibers are running in functionally different branches of the facial nerve. Following this concept of mapping the mimic muscles allows us to differentiate between nerve branches to slow- (e.g-, M. buccinator) and quick- acting muscles (e.g., M. orbicularis oculi), respec- tively, and use them accordingly. Conctusions Histochemical examination has revealed that the mimic muscle system is not uniform with respect to fiber diameters and fiber-type com- position. On the contrary, the analyzed muscles show a remarkable variation from the quickest (M. procerus) to one of the slowest (M. buccina- tor) in the whole body. These findings might be of interest when rean- imation of facial paralyses is planned. SpecificVol. 86, No. 3 / MAPPING OF MIMIC MUSCLES branches supplying tonic muscles (e.g., the buc- cinator) should therefore be used for restoration of such tonic functions as elevation of the oral angle. On the other hand, for achieving phasic and protective functions, parts of the facial nerve that normally innervate phasic mimic muscles should be anastomosed. We are convinced that the better understanding of the recipient side presented in this paper also will influence the choice of more appropriate donor muscles in addition to the selection of specific branches of the facial nerve. Gerhard Freilinger, M.D. Department of Plastic Surgery 2nd Surgical University Clinic Spitalgasse 23 1090 Vienna, Austria ACKNOWLEDGMENTS: The authors wish co express their thanks to Ms. G. Schaden and Mrs. M. Pensch for their excellent histologie and histochemical assistance. REFERENCES: 1, Manktelow, R.'T. Microvascular Reconstruction: Anat ‘omy, Applications, and Surgical Technique. Berlin: Springer-Verlag, 1986. 2. Schwarting, S., Schroeder, M., Stennert, E., and Goe- bel, HH. “Enzyme histochemical and histographic data on normal human facial muscles. Otorkinalaryn= (gol. Relat. Spec, ORL J. 44: 51, 1982. 3. Happak, W., Burggasser, G., and Gruber, H. Histo- chemical characteristics of human mimic muscles. J Neurol. Sri. 83: 25, 1988, 4. Ruge, G. Zur Eintheilung der Gesichtsmuskulacur, speciell des Muskel orbicularis oculi, Morphol. Jahr uch 13: 184, 1888, 5, Virchow, H. Gesichismuskeln und Gesichtsausdruck Arch, Anat, Physiol. 21: 1908. 6. Lightoller, G. H.S. Facial muscles. The modiolus and ‘muscles surrounding the rima oris with some remarks about the panniculus adiposus. J. Anat. 60: 1, 1995. 7. Schwarting, S., Schroeder, M., Siennert, E., and Goe bel, H. H. Morphology of denervated human facial muscles. ORL J. Otorkinolaryngol. Relat. Spec. 46: 248, 1984, 8. Bardosi, A., Goebel, H. H., and Stennert, E. The ultrastructure of normal and denervated human fa- cial muscle, Plast, Reconstr. Surg. 79: 171, 1987. 9. Eriksson, O., Eriksson, A., Ringqvist, M.,and Thorne, L-E. "The reliability of histochemical fiber typing fof human necropsy muscles. Histochemistry 65: 198, 1980. 10. Engel, A. G., and Banker, B. Q. Myology: Basic and Clinical. Parts Ito 3. New York: McGraw-Hill, 1986. 1, Schroeder, J. M. Pathologie der Muskulatur. Berlin: Springer-Verlag, 1982. 19, Guth, L., and Samaha, F. J. Procedure for the histo chemical demonstration of actomyosin ATPase. Exp. Neurol. 28: 365, 1970. 13, Peachey, L. D. Handbook of Physiology, Section 10: ‘Skeletal Muscle. Bethesda, Md.: American Physiolog- ieal Society, 1983, M4 15, 16. 19, 20. 21 23. 24 25. 26. 27. 28, 29, 30. 31 32. 33. 34 427 “Thompson, N, Autogenous free grafts of skeletal mus- cle. Plast. Reconstr, Surg. 48: 11, 1971 Tamai, S., Komatsu, S., Sakamoto, H., Sano, S., Saschu- chi, N., Hori, ¥., Tatsumi, Y.,and Okuda, H.Free- ‘muscle transplants in dogs, with microneurovascular anastomoses. Plast. Reconstr. Surg. 46: 219, 1970. Yerzis, J. K., Sweet, R. C., Dykes, R. W., and Williams, H. B, Recovery of funetion in free-muscle erans. plants using microneurovascular_ anastomoses. J. Hand Surg. 8: 37, 1978. Studitskii, A. N., and Bosova, N. N. Development of atrophic muscular tissue in conditions of transplan- tation on the site of mechanically injured muscles. Arch, Anat. 39: 18, 1960, Bosova, N.N._ Free autoplasty of whole muscles. Biull. Eksp. Biol. Med. 3: 88, 1962. Carlson, B.M. Regeneration of the rat gastrocnemius ‘muscle from sibling and nonsibling muscle fragments. Am. J. Anat. 128: 21, 1970, ‘Thompson, N. Investigations of autogenous skeletal muscle free grafis in the dog with a report on a successful free graft of skeletal muscle in man, Trans- plantation 12: 853, 1971 Hakelius, L. Transplantation of free autogeneous ‘muscle in the treatment of facial paralysis. Scand. J. Plast. Reconstr. Surg. 8: 220, 1974, Freilinger, G. Dynamic Reanimation for Facial Paral- ysis: A Survey. In G. Freilinger, J. Holle, and B. M. Carlson (Eds.), Muscle Transplantation, Berlin: Sprin- iger-Verlag, 1981. P. 135. Lischka, A. J-, Holle, J., and Freilinger, G. Morpho- logische Verdinderungen von Muskelfasern bei freier autologer Muskeltransplantation, Verh. Anat. Ges. 69: 755, 1975. May, M. The Facial Nerve. New York: Thieme, 1986. Niklison, J. Contribution to the subject of facial paral- ysis, Plast. Reconstr. Surg. 17: 276, 1956. Preilinger, G., Gruber, H., Happak, W., and Pechmann, U. ‘Surgical anatomy of the mimic muscle system and the facial nerve: Importance for reconstructive and aesthetic surgery. Plast. Reconstr. Surg. 80: 686, 1987. ‘Terzis, J. K. New Concepts in Facial Reanimation. In ‘Transactions of the VIII International Congress of Plastic Surgery. 1983, Futamura, R. Uber die Entwicklung der Facialismus- kulatur des Menschen. Anat. Hefie 30: 435, 1906. Ranson, 8. W., and Clark, S. L. The anatomy of the nervous system: Its development and function. Phil adelphia: Saunders, 1961 Vermeij-Keere, C. De facialis musculatwur en trans: formaties in het kopgebied. Doctoral thesis, Leiden, 1967. More, K. L. The Developing Human: Clinically Orien- tated Embryology. Philadelphia: Saunders, 1974, Fernande2 Villoria, J. M.A study of the development of the orbicularis oris muscle. Plast. Reconstr. Surg. 55: 205, 1975, Schiefenhoevel, W., Scheidt, M., and Grammer, K. Mimik und Emotion: Verhaltensbiologische As- pekte. In V. Schubert (Ed), Der Mensch wnd seine Gefuehle. St. Ottilien: Eos-Verlag, 1985. P. 175. Johnson, M. A., Polgar, J, Weightman, D., and Apple: ton, D. Data on the distribution of fiber types in 36 hhuman muscles: An autopsy study. J. Neurol. Sei. 18: 11, 1973,428 Polgar, J. Johnson, M. A., Weightman, D., and Apple- ton, D.” Data on fiber size in 36 human muscles: An autopsy study. J. Newrol. ci 19: 307, 1973, 36. Brooke, M. H., and Engel, W. K. The histographic ‘analysis of human muscle biopsies with regard to fiber types. Newrolagy 19: 221, 1969. 37. Barany,M. ATPase activity of myosin correlated with speed of muscle shortening. J. Gen. Physiol. 50(Suppl 2}: 197, 1967, PLAST! 38, 3a, 40. ND RECONSTRUCTIVE SURGERY, September 1990 Burke, R. E, Motor unit types of cat triceps surae muscle. J. Physiol. (Lond.) 193: 141, 1967 Rayment, R., Poole, M.D.,and Rushworth, G. Cross: facial nerve transplants: Why are spontaneous smiles, not restored. Br. J. Plast. Surg. 40: 592, 1987, Basmajian, J. V., and White, E. R. Neuromuscular control of wrumperer's lip. Nature 241: 70, 1973. Nomina Anatomica, 5th Ed. Baltimore: Williams & Wil. kins, 1983,