Journal of the Neurological Sciences, 104 (1991) 197-202 © 1991 Elsevier Science Publishers B.V. 0022-510X/91/$03.50
197
JNS 03575
Regional differences in the orbicularis oculi muscle: conservation between species Linda Kirschen McLoon and Jonathan D. Wirtschafter Department of Ophthalmology, Universityof Minnesota, Minneapolis, Minnesota (U.S.A.) (Received 28 January, 1991)
(Revised, received 15 March, 1991) (Accepted 19 March, 1991) Key words: Facial muscles; Mimic muscles; Fiber typing; Enzyme histochemistry
Summary The orbicularis oculi muscle is a complex facial muscle involved in eyelid closure. The central parts ofpretarsal and preseptal regions of the palpebral part of the orbicularis oculi muscle in rabbit and cynomolgus monkey lower eyelids were examined histologically and were analyzed for muscle fiber number, muscle fiber cross-sectional area and fiber type composition. Distinct regional differences were seen in the muscle fiber composition in these two regions of the muscle. The pretarsal portion of the muscle, that closest to the eyelid margin, was quite homogeneous and almost completely composed of type 2 fibers. These fibers were the smallest in cross-sectional area. Type 2 fibers also predominated in the preseptal portion of the muscle, but this region contained between 10 and 20% type 1 fibers. There appeared to be a gradient in muscle fiber size, whereby the fiber size increased as a function of the distance from the eyelid margin. The same pattern of regional differences were found in both rabbit and monkey orbicularis oculi. Thus, there is a clear conservation of these regional differences in these two species. While the developmental significance is unknown, the identification of this pattern may facilitate the evaluation of chemomyectomy agents for treatment of eyelid spasms in humans and allow a more accurate analysis of biopsy material from this muscle.
Introduction The determination of pathological changes in injured and regenerated muscle is important to our understanding of the effects of various diseases or surgical and drug treatments on normal muscle fiber structure and function. Histological studies of muscles are often directed at the assessment of fiber size and composition in the affected muscle after a specific drug treatment or disease state. One problem with many of these studies is that the histological analysis is based on biopsy material that is randomly removed from the affected muscles even though it is now recognized that muscles are not homogeneous entities. The extraocular muscles, as an extreme case, show marked differences in muscle fiber types in various regions of the muscles (Spencer and Porter 1988). The masseter muscle shows regional and compartmental specialization in terms of fiber
Correspondence to: Linda Kirschen McLoon, Ph.D., Department of Ophthalmology, University of Minnesota, Box 493, Mayo Memorial Building, Minneapolis, MN 55455, U.S.A. Tel.: (612) 624-0409; Fax: (612) 626-3119.
type content (Bredman et al., 1990). Even the diaphragm has different fiber type and size composition on the thoracic and abdominal surfaces (Kilarski and Sjostrom 1990). Precise knowledge of the fiber sizes and fiber type composition of individual muscle groups would not only aid in our understanding of pathological processes, it would also help in designing treatment protocols where surgical or chemical manipulations of a specific muscle are required. For example, botulinum toxin is injected into the orbicularis oculi muscle to alleviate muscle spasms in patients with blepharospasm and hemifacial spasm and is becoming the current preferred non-surgical treatment (Scott et al. 1985). Knowledge of regional differences in the orbicularis oculi muscle may alter the location of botulinum injections in these patients. Interpretation of supposed histopathologic changes in the botulinum treated muscles may also be obscured by lack of knowledge as to the basic anatomy of this muscle. The anatomy of the facial muscles is unique, due to the fact that these muscles have a complex spatial arrangement and insert directly into the skin. The orbicularis oculi muscle is described as being composed of palpebral and
198 orbital portions. The palpebral portion is in turn described as being composed of pretarsal and preseptal divisions, defined by the border of the meibomian glands and the tarsal plate within the eyelid (Patrinely and Anderson 1988). The palpebral portion of the muscle closes the eyelid in normal blinking, and the orbital portion is involved in forced eyelid closure (Gordon 1951). We have shown that injection of doxorubicin into the eyelids of rabbits and monkeys chemically removes orbicularis oculi muscle fibers (McLoon and Wirtschafter 1988; McLoon et al. 1991). A single set of injections of 2 mg doxorubicin removed up to 70~o of the treated muscle fibers. This muscle loss was selective, however, and the greatest loss was in the preseptal portion of the muscle. This differential susceptibility to muscle injury may be due to the fiber composition in different parts of the muscle. Doxorubicin has also been injected into patients with blepharospasm and hemifacial spasm with major improvement resulting from this treatment (Wirtschafter 1991). Successful reduction of spasms has required several sets of injec-
tions several months apart. A better understanding of orbicularis oculi muscle anatomy may help in choosing better placement for the doxorubicin injections. We have analyzed the muscle fiber numbers, fiber crosssectional areas and fiber type composition in the orbicularis oculi muscle of rabbits and monkeys. We have specifically analyzed the differences between the pretarsal and preseptal portions of the orbicularis oculi muscle fibers for each of these three parameters. This is the first systematic regional examination of fiber type and size in the palpebral portions of the orbicularis oculi muscle in these two species and the first comparison between species using the same methodology.
Materials and methods Six New Zealand white rabbits and 4 cynomolgus monkeys were obtained through the University of Minnesota Research Animal Resources. All research con-
Fig. 1. Photomicrographsof the (A,C) pretarsal portion of the orbicularisoculi,the area of the muscle closest to the eyelidmargin, and (B,D) preseptal portion of the orbicularis oculi muscle of the rabbit (A,B) and monkey(C,D) histochemicallyprocessed for alkaline myosinATPase differentiationof muscle fiber types. Arrowheads indicate type 2 muscle fibers. Arrows indicate type 1 muscle fibers. Bar = 50 #m.
199 formed to the guidelines set up by the National Institutes of Health on the use of animals in research. Prior to biopsy which immediately preceded sacrifice, the animals were deeply anesthetized with intramuscular injections of either ketamine HC1 (Ketalar HC1, 100 mg/ml, at a dose of 10 mg/kg) for the monkeys or a 1 : 1 mixture of ketamine HC1 and Rompun (20 mg/ml, at a dose of 2 mg/kg) for the rabbits. Proparacaine HC1 was placed in the conjunctival cul-de-sac. The central part of the lower eyelids was removed and frozen in methylbutane chilled on liquid nitrogen. The tissue was immediately sectioned at 12 #m in a cryostat and processed for alkaline ATPase histochemistry for differentiation of type 1 and type 2 muscle fiber types (Brooke and Kaiser 1970). Rabbit eyelid sections were stained at pH 10.5 and monkey eyelid sections were stained at pH 9.4 to provide for adequate differentiation between the muscle fiber types of the two species. The border between the pretarsal and preseptal portions of the muscle was identified histologically. The lower border of the pretarsal portion was defined by the position of the tarsal glands in the section. The border of preseptal and orbital regions was defined by the end of the conjunctival surface. Since it was hard to define the peripheral margin of the orbital region of the muscle, morphometric analysis of this region was not performed. The individual muscle fibers were traced and quantified using the Bioquant digitizing morphometry program (R and M Biometrics, Inc., TN) to determine the number of muscle fibers in each eyelid section and the individual muscle fiber cross-sectional areas in #m 2 for the pretarsal and preseptal parts of the palpebral part of the orbicularis oculi muscle. The standard error of the means was determined for each set of measured parameters. Differential counts were made of the number of cross-sectional areas of the type 1 muscle fibers as compared to the rest of the muscle. The results were analyzed statistically using a paired Student t-test.
There were approximately 600 muscle fibers in the pretarsal region of the rabbit and approximately 380 in the pretarsal region of the monkey orbicularis oculi (Figs. 2,3). In the preseptal regions, there were 1,652 fibers and 1,318 fibers in the rabbit and monkey, respectively. The cross-sectional areas of the individual fibers differed significantly in the pretarsal and preseptal regions of the muscle. The muscle cross-sectional areas were distinctly and significantly smaller in the pretarsal regions of the muscle in both rabbit and monkey (Figs. 4,5). The most marked difference between the two regions lay in their fiber type composition (Figs. 6,7). In both rabbit and monkey, the pretarsal portion of the muscle was almost totally composed of type 2 fibers. Only 3.5% of the fibers were type 1 in rabbit and only 2.7% were type 1 in monkey orbicularis oculi. The preseptal region of the muscle had
Muscle Fiber T y p e s in the Rabbit ©rbicularis Oculi type 1
~
type 2
1800 1500 1200 900 600 300
~m
m
0
pretarsal
preseptal
total
Fig. 2. Number of muscle fibers and fiber types in the pretarsal and preseptal regions of the orbicularis oculi muscle of the rabbit.
Muscle Fiber T y p e s in the Monkey Orbicularis Oculi Results
The two areas of the orbicularis oculi muscle appeared to be heterogeneous upon histological examination (Fig. 1). The pretarsal region of the muscle was very distinct in appearance. It appeared to be fairly homogeneous in both fiber type composition and individual fiber cross-sectional areas. The pretarsal region was composed largely of type 2 muscle fibers of relatively small cross-sectional area. The preseptal portion of the muscle was more heterogeneous in its composition. It was composed of a mixture of type 1 and type 2 fiber types and the fibers appeared to be larger in cross-sectional area. The differences between the pretarsal and preseptal regions of the orbicularis oculi muscle were quantified.
~
type 1
~77~] type 2
1800
i _e
1500 12 O0 900
I
600 3OO
0
m
pretarsal
preseptal
total
Fig. 3. Number of muscle fibers and fiber types in the pretarsal and preseptal regions of the orbicularis oculi muscle of the monkey.
200 C r o s s - S e c t i o n a l A r e a s o f Individual F i b e r s in R a b b i t E y e l i d
Type
1 F i b e r s in O r b i c u l a r i s Oculi in R a b b i t and M o n k e y
rabbit
~ 600
3O
~ zoo
25
°4°°[
"~
200
0
~
20
10
F ~
monkey
o
5 -
-
pretarsal
-
total
preseptal
0
Fig. 4. Cross-sectional areas of the fiber types in the pretarsal and preseptal portions of the orbicularis oculi muscle in rabbit. Asterisk indicates statistically significant differences in the cross-sectional areas of the muscle fibers in the two regions.
|
-
-
total muscle
C r o s s - S e c t i o n a l A r e a s o f Individual M u s c l e F i b e r s in R a b b i t E y e l i d
7OO
d
-
preseptal
Fig. 6. Percentage of type 1 fibers in the pretarsal and preseptal regions of the orbicularis oculi muscle of rabbit and monkey. Asterisk indicates statistically significant differences in the percentage of type 1 fibers in the pretarsal and preseptal regions in the rabbit and the open circle represents significant differences in the monkey.
C r o s s - S e c t i o n a l A r e a s o f Individual Fibers in M o n k e y E y e l i d
Cq
-
pretarsal
60O
:oor
5OO
4o0 d
t,j~2
type I
500 t
t ~oo b
3O0
lOO
2OO
o
pretarsal
total
Fig. 5. Cross-sectional areas of the fiber types in the pretarsal portion of the orbicularis oculi muscle in monkey compared with the cross-sectional areas of the fibers in the entire palpebral part of the muscle. Asterisk indicates statistically significant differences in the cross-sectional areas of the muscle fibers pretarsally compared with the muscle as a whole.
more type 1 fibers, but it was still largely a type 2 muscle. In rabbit, 23.5?/0 of the fibers in the preseptal region were type 1, while only 9.9% of the fibers in the preseptal region of the monkey muscle were type 1. The overall percentages within the entire palpebral portion of the orbicularis oculi were 15.2% and 8.3% type 1 fibers for rabbit and monkey, respectively. Note that these figures mask the marked regional differences in fiber types within this muscle. Interestingly, the cross-sectional areas of the type 1 and type 2 fibers in the pretarsal region of the rabbit orbicularis were relatively similar, approaching 400/~m 2. The same relation held true for type 1 and type 2 muscle fibers in the preseptal region, where they were significantly larger than the pre-
b
IO0 0
- -
pres~tal
Fig. 7. Cross-sectional areas of type 1 and type 2 fiber types in the pretarsal and preseptal regions of the orbicularis oculi muscle of the rabbit. Asterisk indicates a statistically significant difference between the type 1 fibers in the pretarsal compared with the preseptal regions and the open circle indicates a significant difference between the type 2 fiber crosssectional areas in the pretarsal and preseptal regions.
tarsal fibers, but relatively similar in size to each other. This would indicate that the size of the fibers is region-specific not type-specific.
Discussion These results clearly demonstrate the heterogeneity within the pretarsal and preseptal regions of the palpebral portion of the orbicularis oculi muscle. The pretarsal region
201 is anatomically distinct from the rest of the orbicularis oculi muscle. If these results can be extended to human orbicularis oculi muscle, then care must be given as to the area from which a pathological sample of orbicularis oculi muscle is taken during a biopsy. A number of reports in the literature have described the fiber size in the facial musculature of humans. In general the facial muscles were smaller in cross-sectional diameter than limb muscles (Happak etal. 1988; Polar etal. 1973; Stennert et al. 1985). The orbicularis oculi muscle had the smallest fibers of any of the facial muscles examined. In one study, fiber cross-sectional diameters of the orbital and palpebral regions of the orbicularis oculi were examined (Happak et al. 1988). Although the pretarsal and preseptal portions of the palpebral region of the muscle were not analyzed separately, the cross-sectional diameters of the palpebral fibers as a group were smaller than those of the orbital fibers examined. The measurement of fiber crosssectional area has been described as a more accurate means of muscle measurement than fiber diameter (Bennington and Krupp 1984). Our results confirm and extend these studies showing that there are regional variations in fiber size in the pretarsal and preseptal parts of the orbicularis oculi. There would appear to be a gradient of muscle fiber size, whereby the fiber size increases in size as you move away from the eyelid margin. Several studies have examined the distribution of muscle fiber types in human facial muscles and specifically in the orbicularis oculi (Happak et al. 1988; Johnson et al. 1973; Stennert et al. 1985). In all these studies the orbicularis oculi had significantly more type 2 fibers than other facial muscles. The human orbicularis oculi muscle was described as containing 15~o type 1 fibers. Our studies indicate that these numbers can be misleading. The total percentage of type 1 fibers was 14.5~o in rabbits but only 8.4?/0 in the monkey. This concurs with the work of Porter, showing that 10~o of the fibers in the orbicularis oculi were type 1 (Porter et al., 1989). In our study the pretarsal and preseptal regions of the muscle were analyzed separately, and it was clear that the majority of the type 1 fibers were in the preseptal and orbital regions of the muscle, where the percentages in rabbit and monkey were 19~o and 10~o, respectively. The pretarsal part of the muscle, however, was almost exclusively type 2 fibers. Only 3 - 4 ~o of the fibers were type 1. We can speculate on the functional implications of these results as applied to our understanding of lower eyelid physiology. Type 1 fibers are mitochondria-rich and have a highly oxidative metabolism, whereas type 2 fibers have few mitochondria and have a glycolytic metabolism. Physiologically type 2 fibers are phasic and should take part in short duration contractions during eyelid closure. Type 2 fibers are suited to brief movements, since they are fast contracting, fatigable fibers (Ranatunga and Thomas 1990). It may
be that when relaxed the muscle closest to the eyelid margin functions to provide static eyelid tone and support to the eyelid. When contracting they may provide a rapid, but weak initiating phase in concert with a slower onset but more sustained and forceful eyelid contraction produced by the contraction of the larger diameter type 1 preseptal and orbital fibers. Even when the orbicularis oculi muscle is totally paralyzed and perhaps made temporarily atrophic by botulinum injections, blink induced lid-lowering kinematics are significantly decreased, whereas saccadic lid-lowering is relatively unchanged (Manning et al. 1990). These findings may indicate that the orbicularis oculi controls both actively, due to contraction, and passively, due to stretch, in normal eyelid function. The homogeneity of the pretarsal portion of the orbicularis oculi muscle is particularly interesting in light of our previous observations that this area of the orbicularis oculi muscle is the most resistant to myotoxic injury and degeneration (McLoon and Wirtschafter 1988; McLoon et al. 1991). This differential sensitivity to injury was also seen when eyelids were injected with bupivacaine (McLoon, unpublished), a local anesthetic known to have myotoxic effects (Hall-Craggs 1974). While there are a number of possible explanations for the recalcitrance of the pretarsal portion of the orbicularis oculi to toxic injury, it lends support for the hypothesis that the pretarsal region is a unique muscle with specific, identifiable characteristics distinct from the rest of the orbicularis oculi muscle. These differences are conserved between species. Acknowledgements Supported by NEI grant EY07935, the Benign Essential Blepharospasm Foundation, the Donald and Louise Gabbert Neuroophthalmology Research Fund, Minnesota Lions and Lionesses and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc.
References
Bennington, J.L. and M. Krupp (1984) Morphometricanalysis of muscle. In: Muscle Pathology, Churchill Livingston, New York, pp. 43-71. Bredman, J.J., W.A. Weijs, A.F.M. Moorman and P. Brugman (1990) Histochemical and functional fiber typing of the rabbit masseter muscle. J. Anat., 168: 31-47. Brooke, M.H. and K.K. Kaiser (1970) Muscle fiber types. How many and what kind? Arch. Neurol., 23: 369-379. Gordon, G. (1951) Observations upon the movementsof the eyelids. Br. J. Ophthalmol., 35: 339-351. Hall-Craggs, E.C.B. (1974) Rapid degeneration and regeneration of a whole skeletal muscle following treatment with bupivacaine. Exp. Neurol., 43: 349-358. Happak, W., G. Burggasserand H. Gruber (1988) Histochemicalcharacteristics of human mimic muscles. J, Neurol. Sci., 83: 25-35. Johnson, M.A., J. Polgar, D. Weightman and D. Appleton (1973) Data on the distribution of fibre types in thirty-six human muscles. An autopsy study. J. Neurol. Sci., 18:111-129.
202 Kilarski, W. and M. Sjostrom (1990) Systemic-distribution of muscle fibre types in the rat and rabbit diaphragm: a morphometric and ultrastructural analysis. J. Anat., 168: 13-30. Manning, K.A., C. Evinger and P.A. Sibony (1990) Eyelid movements before and after botulinum therapy in patients with lid spasm. Ann. Neurol., 28: 653-660. McLoon, L.K. and J. Wirtschaffer (1988) Doxorubicin chemomyectomy: injection of monkey orbicularis oculi results in selective muscle loss. Invest. Ophthal. Vis. Sci., 29: 1854-1859. McLoon, L.K., G. Bauer and J. Wirtschafter (1991) Quantification of muscle loss in the doxorubicin treated orbicularis oculi of the monkey. Invest. Ophthal. Vis. Sci., 32: 1667-1673. Patrinely, J.R. and R.L. Anderson (1988) Anatomy of the orbicularis oculi and other facial muscles. Adv. Neurol., 49: 15-23. Polgar, J., M.A. Johnson, D. Weightman and D. Appleton (1973) Data on fibre size in thirty six human muscles. An autopsy study. J. Neurol. Sci., 19: 307-318. Porter, J.D., L.A. Burns and P.J. May (1989) Morphological substratc for
eyelid movements: innervation and structure of primate levator palpebrae superioris and orbicularis oculi muscle. J. Comp. Neurol., 287: 64-81. Ranatunga, K.W. and P.E. Thomas (I 990) Correlation between shortening velocity, force-velocity relation and histochemical fibre-type composition in rat muscles. J. Muscle Res. Cell Motil., 11: 240-250. Scott, A.B., R.A. Kennedy and H.A. Stubbs (1985) Botulinum A toxin injection as a treatment for blepharospasm. Arch. Ophthalmol., 103: 347-350. Spencer, R.F. and J.D. Porter (1988) Structural organization of the extraocular muscles. In: Buttner-Ennever (Ed.), Neuroanatomy of the Oculomotor System, Elsevier, Amsterdam, pp. 33-79. Stennert, E., H.H. Goebel, S. Schwarting, M. Schroder (1985) Morphology of human mimic musculature. In: Portmann, M. (Ed.), Symposium on the Facial Nerve, Masson, Paris, pp. 177-180. Wirtschafter, J.D. (1991) Clinical doxorubicin chemomyectomy. An experimental treatment for benign essential blepharospasm and hemifacial spasm. Ophthalmology, 98: in press.
197
JNS 03575
Regional differences in the orbicularis oculi muscle: conservation between species Linda Kirschen McLoon and Jonathan D. Wirtschafter Department of Ophthalmology, Universityof Minnesota, Minneapolis, Minnesota (U.S.A.) (Received 28 January, 1991)
(Revised, received 15 March, 1991) (Accepted 19 March, 1991) Key words: Facial muscles; Mimic muscles; Fiber typing; Enzyme histochemistry
Summary The orbicularis oculi muscle is a complex facial muscle involved in eyelid closure. The central parts ofpretarsal and preseptal regions of the palpebral part of the orbicularis oculi muscle in rabbit and cynomolgus monkey lower eyelids were examined histologically and were analyzed for muscle fiber number, muscle fiber cross-sectional area and fiber type composition. Distinct regional differences were seen in the muscle fiber composition in these two regions of the muscle. The pretarsal portion of the muscle, that closest to the eyelid margin, was quite homogeneous and almost completely composed of type 2 fibers. These fibers were the smallest in cross-sectional area. Type 2 fibers also predominated in the preseptal portion of the muscle, but this region contained between 10 and 20% type 1 fibers. There appeared to be a gradient in muscle fiber size, whereby the fiber size increased as a function of the distance from the eyelid margin. The same pattern of regional differences were found in both rabbit and monkey orbicularis oculi. Thus, there is a clear conservation of these regional differences in these two species. While the developmental significance is unknown, the identification of this pattern may facilitate the evaluation of chemomyectomy agents for treatment of eyelid spasms in humans and allow a more accurate analysis of biopsy material from this muscle.
Introduction The determination of pathological changes in injured and regenerated muscle is important to our understanding of the effects of various diseases or surgical and drug treatments on normal muscle fiber structure and function. Histological studies of muscles are often directed at the assessment of fiber size and composition in the affected muscle after a specific drug treatment or disease state. One problem with many of these studies is that the histological analysis is based on biopsy material that is randomly removed from the affected muscles even though it is now recognized that muscles are not homogeneous entities. The extraocular muscles, as an extreme case, show marked differences in muscle fiber types in various regions of the muscles (Spencer and Porter 1988). The masseter muscle shows regional and compartmental specialization in terms of fiber
Correspondence to: Linda Kirschen McLoon, Ph.D., Department of Ophthalmology, University of Minnesota, Box 493, Mayo Memorial Building, Minneapolis, MN 55455, U.S.A. Tel.: (612) 624-0409; Fax: (612) 626-3119.
type content (Bredman et al., 1990). Even the diaphragm has different fiber type and size composition on the thoracic and abdominal surfaces (Kilarski and Sjostrom 1990). Precise knowledge of the fiber sizes and fiber type composition of individual muscle groups would not only aid in our understanding of pathological processes, it would also help in designing treatment protocols where surgical or chemical manipulations of a specific muscle are required. For example, botulinum toxin is injected into the orbicularis oculi muscle to alleviate muscle spasms in patients with blepharospasm and hemifacial spasm and is becoming the current preferred non-surgical treatment (Scott et al. 1985). Knowledge of regional differences in the orbicularis oculi muscle may alter the location of botulinum injections in these patients. Interpretation of supposed histopathologic changes in the botulinum treated muscles may also be obscured by lack of knowledge as to the basic anatomy of this muscle. The anatomy of the facial muscles is unique, due to the fact that these muscles have a complex spatial arrangement and insert directly into the skin. The orbicularis oculi muscle is described as being composed of palpebral and
198 orbital portions. The palpebral portion is in turn described as being composed of pretarsal and preseptal divisions, defined by the border of the meibomian glands and the tarsal plate within the eyelid (Patrinely and Anderson 1988). The palpebral portion of the muscle closes the eyelid in normal blinking, and the orbital portion is involved in forced eyelid closure (Gordon 1951). We have shown that injection of doxorubicin into the eyelids of rabbits and monkeys chemically removes orbicularis oculi muscle fibers (McLoon and Wirtschafter 1988; McLoon et al. 1991). A single set of injections of 2 mg doxorubicin removed up to 70~o of the treated muscle fibers. This muscle loss was selective, however, and the greatest loss was in the preseptal portion of the muscle. This differential susceptibility to muscle injury may be due to the fiber composition in different parts of the muscle. Doxorubicin has also been injected into patients with blepharospasm and hemifacial spasm with major improvement resulting from this treatment (Wirtschafter 1991). Successful reduction of spasms has required several sets of injec-
tions several months apart. A better understanding of orbicularis oculi muscle anatomy may help in choosing better placement for the doxorubicin injections. We have analyzed the muscle fiber numbers, fiber crosssectional areas and fiber type composition in the orbicularis oculi muscle of rabbits and monkeys. We have specifically analyzed the differences between the pretarsal and preseptal portions of the orbicularis oculi muscle fibers for each of these three parameters. This is the first systematic regional examination of fiber type and size in the palpebral portions of the orbicularis oculi muscle in these two species and the first comparison between species using the same methodology.
Materials and methods Six New Zealand white rabbits and 4 cynomolgus monkeys were obtained through the University of Minnesota Research Animal Resources. All research con-
Fig. 1. Photomicrographsof the (A,C) pretarsal portion of the orbicularisoculi,the area of the muscle closest to the eyelidmargin, and (B,D) preseptal portion of the orbicularis oculi muscle of the rabbit (A,B) and monkey(C,D) histochemicallyprocessed for alkaline myosinATPase differentiationof muscle fiber types. Arrowheads indicate type 2 muscle fibers. Arrows indicate type 1 muscle fibers. Bar = 50 #m.
199 formed to the guidelines set up by the National Institutes of Health on the use of animals in research. Prior to biopsy which immediately preceded sacrifice, the animals were deeply anesthetized with intramuscular injections of either ketamine HC1 (Ketalar HC1, 100 mg/ml, at a dose of 10 mg/kg) for the monkeys or a 1 : 1 mixture of ketamine HC1 and Rompun (20 mg/ml, at a dose of 2 mg/kg) for the rabbits. Proparacaine HC1 was placed in the conjunctival cul-de-sac. The central part of the lower eyelids was removed and frozen in methylbutane chilled on liquid nitrogen. The tissue was immediately sectioned at 12 #m in a cryostat and processed for alkaline ATPase histochemistry for differentiation of type 1 and type 2 muscle fiber types (Brooke and Kaiser 1970). Rabbit eyelid sections were stained at pH 10.5 and monkey eyelid sections were stained at pH 9.4 to provide for adequate differentiation between the muscle fiber types of the two species. The border between the pretarsal and preseptal portions of the muscle was identified histologically. The lower border of the pretarsal portion was defined by the position of the tarsal glands in the section. The border of preseptal and orbital regions was defined by the end of the conjunctival surface. Since it was hard to define the peripheral margin of the orbital region of the muscle, morphometric analysis of this region was not performed. The individual muscle fibers were traced and quantified using the Bioquant digitizing morphometry program (R and M Biometrics, Inc., TN) to determine the number of muscle fibers in each eyelid section and the individual muscle fiber cross-sectional areas in #m 2 for the pretarsal and preseptal parts of the palpebral part of the orbicularis oculi muscle. The standard error of the means was determined for each set of measured parameters. Differential counts were made of the number of cross-sectional areas of the type 1 muscle fibers as compared to the rest of the muscle. The results were analyzed statistically using a paired Student t-test.
There were approximately 600 muscle fibers in the pretarsal region of the rabbit and approximately 380 in the pretarsal region of the monkey orbicularis oculi (Figs. 2,3). In the preseptal regions, there were 1,652 fibers and 1,318 fibers in the rabbit and monkey, respectively. The cross-sectional areas of the individual fibers differed significantly in the pretarsal and preseptal regions of the muscle. The muscle cross-sectional areas were distinctly and significantly smaller in the pretarsal regions of the muscle in both rabbit and monkey (Figs. 4,5). The most marked difference between the two regions lay in their fiber type composition (Figs. 6,7). In both rabbit and monkey, the pretarsal portion of the muscle was almost totally composed of type 2 fibers. Only 3.5% of the fibers were type 1 in rabbit and only 2.7% were type 1 in monkey orbicularis oculi. The preseptal region of the muscle had
Muscle Fiber T y p e s in the Rabbit ©rbicularis Oculi type 1
~
type 2
1800 1500 1200 900 600 300
~m
m
0
pretarsal
preseptal
total
Fig. 2. Number of muscle fibers and fiber types in the pretarsal and preseptal regions of the orbicularis oculi muscle of the rabbit.
Muscle Fiber T y p e s in the Monkey Orbicularis Oculi Results
The two areas of the orbicularis oculi muscle appeared to be heterogeneous upon histological examination (Fig. 1). The pretarsal region of the muscle was very distinct in appearance. It appeared to be fairly homogeneous in both fiber type composition and individual fiber cross-sectional areas. The pretarsal region was composed largely of type 2 muscle fibers of relatively small cross-sectional area. The preseptal portion of the muscle was more heterogeneous in its composition. It was composed of a mixture of type 1 and type 2 fiber types and the fibers appeared to be larger in cross-sectional area. The differences between the pretarsal and preseptal regions of the orbicularis oculi muscle were quantified.
~
type 1
~77~] type 2
1800
i _e
1500 12 O0 900
I
600 3OO
0
m
pretarsal
preseptal
total
Fig. 3. Number of muscle fibers and fiber types in the pretarsal and preseptal regions of the orbicularis oculi muscle of the monkey.
200 C r o s s - S e c t i o n a l A r e a s o f Individual F i b e r s in R a b b i t E y e l i d
Type
1 F i b e r s in O r b i c u l a r i s Oculi in R a b b i t and M o n k e y
rabbit
~ 600
3O
~ zoo
25
°4°°[
"~
200
0
~
20
10
F ~
monkey
o
5 -
-
pretarsal
-
total
preseptal
0
Fig. 4. Cross-sectional areas of the fiber types in the pretarsal and preseptal portions of the orbicularis oculi muscle in rabbit. Asterisk indicates statistically significant differences in the cross-sectional areas of the muscle fibers in the two regions.
|
-
-
total muscle
C r o s s - S e c t i o n a l A r e a s o f Individual M u s c l e F i b e r s in R a b b i t E y e l i d
7OO
d
-
preseptal
Fig. 6. Percentage of type 1 fibers in the pretarsal and preseptal regions of the orbicularis oculi muscle of rabbit and monkey. Asterisk indicates statistically significant differences in the percentage of type 1 fibers in the pretarsal and preseptal regions in the rabbit and the open circle represents significant differences in the monkey.
C r o s s - S e c t i o n a l A r e a s o f Individual Fibers in M o n k e y E y e l i d
Cq
-
pretarsal
60O
:oor
5OO
4o0 d
t,j~2
type I
500 t
t ~oo b
3O0
lOO
2OO
o
pretarsal
total
Fig. 5. Cross-sectional areas of the fiber types in the pretarsal portion of the orbicularis oculi muscle in monkey compared with the cross-sectional areas of the fibers in the entire palpebral part of the muscle. Asterisk indicates statistically significant differences in the cross-sectional areas of the muscle fibers pretarsally compared with the muscle as a whole.
more type 1 fibers, but it was still largely a type 2 muscle. In rabbit, 23.5?/0 of the fibers in the preseptal region were type 1, while only 9.9% of the fibers in the preseptal region of the monkey muscle were type 1. The overall percentages within the entire palpebral portion of the orbicularis oculi were 15.2% and 8.3% type 1 fibers for rabbit and monkey, respectively. Note that these figures mask the marked regional differences in fiber types within this muscle. Interestingly, the cross-sectional areas of the type 1 and type 2 fibers in the pretarsal region of the rabbit orbicularis were relatively similar, approaching 400/~m 2. The same relation held true for type 1 and type 2 muscle fibers in the preseptal region, where they were significantly larger than the pre-
b
IO0 0
- -
pres~tal
Fig. 7. Cross-sectional areas of type 1 and type 2 fiber types in the pretarsal and preseptal regions of the orbicularis oculi muscle of the rabbit. Asterisk indicates a statistically significant difference between the type 1 fibers in the pretarsal compared with the preseptal regions and the open circle indicates a significant difference between the type 2 fiber crosssectional areas in the pretarsal and preseptal regions.
tarsal fibers, but relatively similar in size to each other. This would indicate that the size of the fibers is region-specific not type-specific.
Discussion These results clearly demonstrate the heterogeneity within the pretarsal and preseptal regions of the palpebral portion of the orbicularis oculi muscle. The pretarsal region
201 is anatomically distinct from the rest of the orbicularis oculi muscle. If these results can be extended to human orbicularis oculi muscle, then care must be given as to the area from which a pathological sample of orbicularis oculi muscle is taken during a biopsy. A number of reports in the literature have described the fiber size in the facial musculature of humans. In general the facial muscles were smaller in cross-sectional diameter than limb muscles (Happak etal. 1988; Polar etal. 1973; Stennert et al. 1985). The orbicularis oculi muscle had the smallest fibers of any of the facial muscles examined. In one study, fiber cross-sectional diameters of the orbital and palpebral regions of the orbicularis oculi were examined (Happak et al. 1988). Although the pretarsal and preseptal portions of the palpebral region of the muscle were not analyzed separately, the cross-sectional diameters of the palpebral fibers as a group were smaller than those of the orbital fibers examined. The measurement of fiber crosssectional area has been described as a more accurate means of muscle measurement than fiber diameter (Bennington and Krupp 1984). Our results confirm and extend these studies showing that there are regional variations in fiber size in the pretarsal and preseptal parts of the orbicularis oculi. There would appear to be a gradient of muscle fiber size, whereby the fiber size increases in size as you move away from the eyelid margin. Several studies have examined the distribution of muscle fiber types in human facial muscles and specifically in the orbicularis oculi (Happak et al. 1988; Johnson et al. 1973; Stennert et al. 1985). In all these studies the orbicularis oculi had significantly more type 2 fibers than other facial muscles. The human orbicularis oculi muscle was described as containing 15~o type 1 fibers. Our studies indicate that these numbers can be misleading. The total percentage of type 1 fibers was 14.5~o in rabbits but only 8.4?/0 in the monkey. This concurs with the work of Porter, showing that 10~o of the fibers in the orbicularis oculi were type 1 (Porter et al., 1989). In our study the pretarsal and preseptal regions of the muscle were analyzed separately, and it was clear that the majority of the type 1 fibers were in the preseptal and orbital regions of the muscle, where the percentages in rabbit and monkey were 19~o and 10~o, respectively. The pretarsal part of the muscle, however, was almost exclusively type 2 fibers. Only 3 - 4 ~o of the fibers were type 1. We can speculate on the functional implications of these results as applied to our understanding of lower eyelid physiology. Type 1 fibers are mitochondria-rich and have a highly oxidative metabolism, whereas type 2 fibers have few mitochondria and have a glycolytic metabolism. Physiologically type 2 fibers are phasic and should take part in short duration contractions during eyelid closure. Type 2 fibers are suited to brief movements, since they are fast contracting, fatigable fibers (Ranatunga and Thomas 1990). It may
be that when relaxed the muscle closest to the eyelid margin functions to provide static eyelid tone and support to the eyelid. When contracting they may provide a rapid, but weak initiating phase in concert with a slower onset but more sustained and forceful eyelid contraction produced by the contraction of the larger diameter type 1 preseptal and orbital fibers. Even when the orbicularis oculi muscle is totally paralyzed and perhaps made temporarily atrophic by botulinum injections, blink induced lid-lowering kinematics are significantly decreased, whereas saccadic lid-lowering is relatively unchanged (Manning et al. 1990). These findings may indicate that the orbicularis oculi controls both actively, due to contraction, and passively, due to stretch, in normal eyelid function. The homogeneity of the pretarsal portion of the orbicularis oculi muscle is particularly interesting in light of our previous observations that this area of the orbicularis oculi muscle is the most resistant to myotoxic injury and degeneration (McLoon and Wirtschafter 1988; McLoon et al. 1991). This differential sensitivity to injury was also seen when eyelids were injected with bupivacaine (McLoon, unpublished), a local anesthetic known to have myotoxic effects (Hall-Craggs 1974). While there are a number of possible explanations for the recalcitrance of the pretarsal portion of the orbicularis oculi to toxic injury, it lends support for the hypothesis that the pretarsal region is a unique muscle with specific, identifiable characteristics distinct from the rest of the orbicularis oculi muscle. These differences are conserved between species. Acknowledgements Supported by NEI grant EY07935, the Benign Essential Blepharospasm Foundation, the Donald and Louise Gabbert Neuroophthalmology Research Fund, Minnesota Lions and Lionesses and an unrestricted grant to the Department of Ophthalmology from Research to Prevent Blindness, Inc.
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