Orbicularis Oculi Muscle Fibers Are Relatively Short and
Heterogeneous in Length
Timothy Lander, Jonathan D. Wirtschafter, and Linda Kirschen McLoon
Purpose. The anatomy of individual myofibers within the orbicularis oculi muscle was examined
to determine individual myofiber lengths in the different regions of the muscle. A wide
variety of eyelid conditions require eyelid surgery or drug injections direcdy into the eyelid.
Knowledge of regional myofiber anatomy and physiology is important for accurate treatment
of these conditions.
Methods. Eyelid specimens from rabbits were treated with collagenase, fixed, and stained for
neuromuscular junction location. Individual myofibers were dissected from these muscle
specimens and were measured to determine individual myofiber length and neuromuscular
junction position. Additional eyelid specimens of rabbits and humans were stained en bloc
to visualize neuromuscular junction location in the pretarsal and preseptal regions of the
orbicularis oculi muscle.
Results. The myofibers showed variable lengths, shorter in the pretarsal region of the muscle
and longer in the preseptal region. The average individual myofiber length in the pretarsal
region was 36% as long as the entire length of the pretarsal muscle region. In the preseptal
region, the myofibers were slighdy longer, covering 54% of the entire length of this region
of the muscle. In both the pretarsal and preseptal regions of the rabbit and human orbicularis
oculi muscle, there were many clusters of neuromuscular junctions throughout the medial
to lateral length of the muscle, with the majority of the neuromuscular junctions in the medial
and lateral canthal regions of the preseptal portion of the lid. This indicates that the muscle
is composed of relatively short, overlapping myofibers, and that the shortest myofibers reside
in the medial and lateral canthal regions of the eyelid. Multiple innervation of one rabbit
myofiber was observed as a rare occurrence.
Conclusions. Individual myofibers of the orbicularis oculi muscle are relatively short, end
intrafascicularly, and are of heterogeneous lengths varying regionally within the muscle. Thus,
for drug injections into the eyelid, optimal drug effectiveness may require treatment of the
entire lid from medial to lateral canthus to overcome the tissue barriers to diffusion. The
existence of muscle fibers of heterogeneous lengths suggests that the complex organization
of muscle fibers may play previously unappreciated but important roles in normal function,
pathophysiology, and age-related changes in the eyelid. Invest Ophthalmol Vis Sci.
1996;37:1732-1739.
1 he orbicularis oculi muscle, and facial muscles in general, have a complex anatomy. This is related in part to
the complicated spatial arrangement of the myofibers
From the Department of Ophthalmology, University of Minnesota, Minneapolis.
Presented in part at the annual meeting of tlie Association for Research in Vision
and Ophthalmology, Sarasota, Florida, May 1994.
Supported try National Eye Institute, grant EY07935 (LKM, fDW), the Minnesota
Lions and Lionesses, and an unrestricted grant to the Department of Ophthalmology
from Research to Prevent Blindness.
Submitted for publication January 16, 1996; revised March 27, 1996; accepted
April 15, 1996.
Profnietaty interest calgegory: N.
Reprint requests: Linda Kirschen Mcljxm, Department of Ophthalmology,
University of Minnesota, 2001 6th Street SE, Room 374 IJiB, Minneapolis, MN
55455.
1732
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and their insertion directly into the facial skin. The orbicularis oculi muscle is divided into several regions
based on location in the eyelid and on the face. The
pretarsal region extends from the lid margin to the end
of the tarsal glands and tarsal plate within the eyelid.
The preseptal region extends from the end of the tarsal
glands to the orbital rim. The pretarsal and preseptal
regions are designated as the palpebral portion. The
rest of the muscle is described as the orbital portion and
extends up into the eyebrow region and down onto the
cheek. Each of these regions plays a different role in
general facial expression, eyelid closure, eyeblink, and
lacrimal system function.1
Investigative Ophthalmology & Visual Science, August 1996, Vol. 37, No. 9
Copyright © Association for Research in Vision and Ophthalmology
Orbicularis Oculi Fiber Length
The myofibers within the orbicularis oculi muscle
have the smallest diameter of any skeletal muscle, including other facial muscles.23 In addition, there are
distinct regional differences in myofiber cross-sectional area and fiber type composition in the orbicularis oculi muscle.4 The pretarsal region has the smallest myofiber cross-sectional areas, and this region is
composed almost completely of type 2 myofibers. As
one moves peripherally away from the eyelid margin,
there is a slow progression to slightly larger cross-sectional areas and an increase in the number of type 1
fibers in the muscle, although the muscle still contains
80% to 90% type 2 fibers.45
The anatomy of the orbicularis oculi muscle is
important in treating a number of conditions that require corrective eyelid surgery, such as entropion and
blepharospasm. It is also important in the physiology
of blinking, corneal wetting, and lacrimal excretion
through the lacrimal pump. Individual myofiber
length and neuromuscular junction location become
important issues for optimal placement of injections
into the eyelid for the treatment of muscle spasm diseases. Descriptions of muscle anatomy in the orbicularis oculi have not distinguished muscle bundle
length from myofiber length and have created the
impression that the individual myofibers ran from medial to lateral canthal tendons. Regional myotoxicity
as a result of local injection of bupivacaine into the
eyelid,'1 coupled with the regionality of the response
of the muscle to injections of doxorubicin for treatment of muscle spasm disease,7"9 suggested that the
myofibers in the orbicularis oculi muscle were shorter
than the classical understanding had implied.
The length of individual myofibers in the orbicularis oculi muscle was assessed using two approaches.
Rabbit eyelid specimens were treated with an in vitro
digestion technique.10" This was followed by the dissection of individual myofibers from the pretarsal and
preseptal regions of the muscle, each of which was
measured using a computerized morphometry program. In a second approach, the entire muscle was
dissected en bloc and stained for the presence of acetylcholinesterase to determine neuromuscular junction location, a standard approach for the determination of intrafascicular myofiber termination.12 Rabbit
and human orbicularis oculi muscles were examined
by this method. Staining of these dissected muscles
confirmed that the neuromuscular junctions were
generally in the middle third of the individual myofibers, thus permitting assumptions concerning minimum muscle fiber length based on the distance from
one end of the myofiber to the neuromuscular junction.
MATERIALS AND METHODS
Eyelid specimens were obtained from New Zealand
white rabbits obtained from Birchwood Valley Farm
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1733
circumference- n(margln)
circumference -
lateral tendon r
cut edge of
dissected eyelid
Zv(arc)
medial tendon
isolated preseptal
myofiber
FIGURE l.
Procedure for estimating total length of myofiber
bundles from which preseptalfibershad been isolated. The
lengths of the two dotted half-circles or pi/2 (arc + margin/
2) are averaged.
(Red Wing, MN) and housed with Research Animal
Resources at the University of Minnesota. Normal
adult human eyelid specimens were obtained as surgical waste tissue. All animal research conformed with
the guidelines set up by the National Institutes of
Health and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Tenets of
the Declaration of Helsinki were followed.
Four normal adult rabbits were killed. Before eyelid removal, the length of the eyelid margin was determined for each rabbit, measured from the medial to
lateral angle. Eyelid height was measured from the lid
margin to the orbital rim. Four human orbicularis
oculi specimens were obtained for neuromuscular
junction staining. Human specimens were from orbital exenterations, with skin, muscle, and conjunctiva
present. A suture was placed in the muscle specimen
for subsequent orientation purposes. Eyelid specimens from normal rabbits and humans were stained
en bloc for visualization of neuromuscular junctions.
The conjunctiva was removed, and eyelid specimens
were pinned to their approximate in vivo length,
based on measurements taken at the time of tissue
removal, and fixed overnight in 4% paraformaldehyde
in phosphate buffer, pH 7.3. The specimens were
rinsed in buffer, incubated in a solution of acetylthiocholine iodide, rinsed in buffer, and developed in
10% ammonium sulfide.13 This identified the locations of neuromuscular junctions throughout the intact orbicularis oculi muscle of these specimens. Measurements from the lateral raphe to the first neuromuscular junctions were obtained from photographs
of four stained specimens and were averaged to determine the minimum distance to the first neuromuscular junctions at the lateral angle of the eyelid.
In a second set of experiments, rabbit eyelids were
removed completely, and the specimens were treated
with a modification of a collagenase digestion technique. 10 " The conjunctiva was dissected carefully
from the overlying tissues, and the specimens were
placed in a low-calcium saline solution (pH 7.4) con-
1734
Investigative Ophthalmology & Visual Science, August 1996, Vol. 37, No. 9
FIGURE 2. Photomicrograph of a montage of a dissected pretarsal myofiber from rabbit orbicularis oculi muscle stained
for the presence of neuromuscular junctions. The length of
thisfiberwas 8.8 mm. Curved arrows indicate neuromuscular junctions. This fiber was the only one seen with two
neuromuscular junctions.
V
taining 0.2 mM calcium, 2 mg/ml collagenase (Sigma,
St. Louis, MO) and 1 U/ml hyaluronidase (Wyeth,
Philadelphia, PA) for between '/2 to 4 hours at 37°C
with constant oxygenation using carbogen. After enzymatic digestion, the lids were pinned flat and fixed
in 4% paraformaldehyde in phosphate buffer before
further dissection. The fixed specimens were stained
en bloc for acetylcholinesterase as described in die
preceding paragraph,13 This identified the location of
neuromuscular junctions on individual myofibers and
helped differentiate the myofibers from the surrounding connective tissue elements in the eyelid
specimens. Individual muscle fibers were teased from
pretarsal and preseptal regions within the treated orbicularis oculi muscle, examined under a light microscope, and measured using a computerized morphometry program (Bioquant; R and M Biometrics, Nashville, TN). The pretarsal region extends from die
eyelid margin to the end of die tarsal glands, which
were clearly visible in the treated specimens. The preseptal region was defined histologically as extending
from the end of the tarsal glands to die end of die
conjunctiva1 because removal from the orbit was nec-
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essary for tissue processing; thus, bony landmarks
could not be used.
The preseptal fibers proved to be especially difficult to tease free of their connections into the skin.
Although all pretarsal fibers were dissected by the
mediod described in the preceding paragraph, only
two preseptal fibers could be dissected with that
method. To ensure a better representation of actual
myofiber length in the preseptal region of the orbicularis oculi, another method was devised for these fibers. Myofibers were dissected carefully from the muscle mass if a tapered end of a single myofiber could be
isolated. This fiber was followed toward the tendinous
insertion as far as possible, at which point the fiber
was broken deliberately and measured from the broken end to the tapered end. The remaining distance
from the site of breakage to the tendinous insertion
was then measured using a caliper under the dissection microscope. The two measured lengths were
added to give the total myofiber length. The position
of the neuromuscular junctions on individual myofibers also was determined. These were clearly visible
under the microscope as brown spots on the myofibers
1735
Orbicularis Oculi Fiber Length
FIGURE 3. Higher magnification photomicrograph of each
end of the myofiber from Figure 2. Note that one end is
tapered A), whereas the other
end is slightly rounded. (B).
The muscle scriations are
clearly visible widiin the fiber
up to the ends.
(see Fig. 2). The distance from the fiber end to the
neuromuscular junction was determined using the
Bioquant analysis program.
Once individual fiber lengths were determined,
the percent of each individual myofiber length, compared to the total eyelid length, was determined for
engi
E
each fiber. For the pretarsal fibers, this was a simple
calculation based on the length of the eyelid margin
as measured with calipers before removal of the eyelid
specimen from the animal. The preseptal fibers arc
across the eyelid (Fig. 1). To estimate the medial tendon to lateral tendon arc length of each myofiber, the
following formula was used: arc length = pi/2 (arc +
margin/2), where arc is the radial distance from the
center of the eyelid margin to the isolated myofiber
and margin is the length of the eyelid margin from
medial to lateral angle (Fig. 1).
All results were compared for statistical significance
using an unpaired, two-tailed Rest. An F-test indicated
that the variances of the groups were not significantly
different. Statistical tests were run using the Instat Biostatistics Program (Graphpad, San Diego, CA).
RESULTS
A
pretarsal
preseptal
FIGURE 4. Analysis of myofiber lengths in the pretarsal and
preseptal regions of die orbicularis oculi muscle of rabbits.
Pretarsal fibers: N = 18. Preseptal fibers: N = 12.
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Individual rabbit myofibers were dissected from collagenase-treated eyelid specimens (Fig. 2). This is a difficult dissection in facial muscles because of the interweaving of the myofibers in the dense connective tissue of the eyelid and myofiber insertion into the eyelid
skin. Myofibers were considered to be intact if die
ends tapered or ended in a slightly rounded tip at
which muscle Striations were visible within die entire
myofiber (Fig. 3). Myofibers were considered damaged if they showed a clean break with a rectangular
end or obvious signs of breakage and/or contraction
of the end of the muscle fiber with a large, bulbous
formation. In the pretarsal region, the average fiber
length was 6.8 mm with a range in length of 1.5 to
12.2 mm (Fig. 4). This means that an average single
myofiber was 35.7% as long as the entire length of the
1736
Investigative Ophthalmology 8c Visual Science, August 1996, Vol. 37, No. 9
100
Q)
V
+
o
0
O
a
pretarsal
preseptai
FIGURE 5. Analysis of myofiber lengths as a percentage of
measured (pretarsal) or calculated (preseptai) total lid
length in rabbits. These measurements reflect the medial
canthal tendon to lateral canthal tendon distance.
pretarsal region in the medial to lateral direction (Fig.
5). In the preseptai region, the average fiber length
was 17.8 mm, with a range of 8.2 to 31.4 mm (Fig. 4).
This represents an average of 54.2% of the estimated
entire length of the preseptai region (calculated as
arc length) (Fig. 5).
In several of the dissected fibers, the position of
the neuromuscular junction on individual dissected
myofibers was determined. If the entire measured
length of a myofiber was 100%, in the pretarsal region,
neuromuscular junctions were, on average, 40.6%
from one myofiber end (N = 4) and are skewed
slightly from the exact myofiber middle. In the preseptai region, the neuromuscular junctions were placed
slightly more eccentrically and were, on average, 36%
from the end of an individual myofiber (N = 3) and
are skewed even farther from the middle of each individual myofiber. Although the majority of the neuromuscular junctions were in the middle one diird of
individual myofibers, in at least one case, a neuromuscular junction was located at a distance of one quarter
of the length of that myofiber. All dissected individual
myofibers in this study were singly innervated except
one, which had two sites of innervation as demonstrated by acetylcholinesterase staining (Fig. 2).
The acetylcholinesterase-stained orbicularis oculi
specimens from rabbit and human eyelids all showed
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a similar pattern of neuromuscular junction staining.
Clusters of neuromuscular junctions were numerous
and were scattered along the entire length of the pretarsal region of the muscle, from medial to lateral
angles (Fig. 6). This suggests that there are many short
myofibers in this region of the orbicularis oculi muscle.
The neuromuscular junction staining pattern in
the preseptai region of the muscle had a markedly
different pattern (Fig. 7). In rabbit eyelids, the majority of the neuromuscular junctions were clustered
within 1.7 and 12 mm from the medial and lateral
angles of each lid, with few visible in the central part
of the preseptai region. This suggests that the lengths
of individual orbicularis oculi myofibers varies within
the medial to lateral extent of this region of the muscle.
The distance from the musculotendinous junction of the rabbit preseptai muscle region to the
stained neuromuscular junction clusters closest to the
raphe was measured. The first neuromuscular junctions were an average of 3 mm from the raphe. Assuming that the neuromuscular junctions are in the middle third of each fiber, this results in an average fiber
length of 9 mm in the lateral preseptai region. A similar picture is seen in the medial preseptai muscle closest to the medial angle of the eyelid. These measurements indicate that there is a population of shorter
myofibers in the medial and lateral canthal regions of
the orbicularis oculi muscle.
DISCUSSION
We have developed a working model of myofiber
length based on the neuromuscular junction staining
within the orbicularis oculi muscles of rabbits and hu-
FICURE 6. Photomicrograph of the pretarsal region of a human orbicularis oculi muscle stained for the visualization of
neuromuscular junctions (arroxvs). Note that the clusters of
neuromuscular junctions are found along the entire length
of the muscle. Bar = 1 mm.
1737
Orbicularis Oculi Fiber Length
I
IA«MOln«umoiii«ulif«inc>wre
FIGURE 8. Schematic drawing of myofiber length in the preseptal region of the rabbit orbicularis oculi muscle. Shaded
regions indicate the observed areas of the greatest concentrations of neuromuscular junctions in the stained material
(see Fig. 8). The increased concentrations are found in the
medial and lateral portions of the eyelid compared with a
relative paucity of stained neuromuscular junctions in the
central region. This model would predict fibers of greater
length in the central region of the eyelid and shorter fibers
medially and laterally.
J
FIGURE 7. Photomicrograph of the preseptal region of the orbicularis oculi of rabbit eyelid stained for visualization of neuromuscular junctions. In the preseptal region of the muscle, there is a
concentration of neuromuscular junction clusters in the medial
region (A), fewer neuromuscular junction clusters in the central
region (B), and an increased number of neuromuscular junctions in the lateral region (C). Bar = 1 mm.
mans and on single fiber measurements in rabbits
(Figs. 8, 9). Although single fiber measurements were
made only in the rabbit orbicularis oculi muscle, the
similarity of neuromuscular junction staining patterns
in the human and rabbit muscles allowed us to extrap-
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olate that the rabbit data would be applicable in the
human orbicularis oculi as well. Thus, in the pretarsal
region, there appear to be many short, overlapping
myofibers that end intrafascicularly (Fig. 9). In the
preseptal region of the eyelid, again based on the
neuromuscular junction staining patterns and on individual fiber measurements in rabbit muscle, there appear to be a larger number of relatively shorter fibers
in the medial and lateral portions of the eyelid compared to relatively longer myofibers within the central
region of the eyelid (Figs. 8, 9).
An alternative explanation of the pattern of neuromuscular junction staining is that neuromuscular
junctions may not be in the middle third of individual
preseptal myofibers. If the distribution of neuromuscular junctions of individual myofibers was skewed toward the myofiber ends, this might give the same picture seen in stained specimens, with a preponderance
of neuromuscular junction clusters stained in the medial and lateral regions of the muscle. However, based
on our determination of neuromuscular position on
individual teased myofibers, this seems a less likely
possibility.
Another alternative explanation for the large
number of neuromuscular junction clusters throughout the muscle is that there are multiply innervated
myofibers within the orbicularis oculi muscle, such as
seen in the extraocular muscles.1" The existence of
multiple innervation of single myofibers has been described in other skeletal muscles as well.12 In all the
dissected orbicularis oculi myofibers thus far examined, only one was found that had two innervation
sites (Fig. 2). Although possible, the existence of many
multiply innervated myofibers in orbicularis oculi is
unlikely.
The presence of overlapping, relatively short myo-
1738
Investigative Ophthalmology & Visual Science, August 1996, Vol. 37, No. 9
Orbital
Arrow at Lacrinial Punctum
FIGURE 9. Schematic drawing of die postulated myofiber
structure of the palpebral portion of die human eyelid based
on the human and rabbit neuromuscular junction staining
patterns and on single myofiber lengths in rabbit muscle
extrapolated to the human. This model illustrates die heterogeneity of the relatively short, overlapping myofibers in
the pretarsal and preseptal regions of the orbicularis oculi
muscle. The muscle has a greater number of relatively
shorter fibers in the medial and lateral one diird of each
eyelid, with longer fibers in die more central region of the
lid. Also illustrated is the overlapping myofiber configuration; not all myofibers are attached to die canthal tendons,
and most fibers end intrafascicularly.
fibers in skeletal muscles other than the orbicularis
oculi has been observed in the skeletal muscles extending over a wide range of animal species. Individual myofibers that terminate intrafascicularly can be
seen in human leg muscles such as sartorius,15 and in
cat hind limb muscles,"1'17 as well as in many mammalian strap,18 neck19 muscles, and rectus abdominus
muscles.20 Many of the short myofibers were between
25% and 65% of the length of the entire muscle fascicle,15"I7I9-21 with an average myofiber overlap of approximately 40% the fascicle length. Average myofiber
length in the orbicularis oculi muscle is in a range
similar to that found in studies of other skeletal muscles. Interestingly, individual myofiber length compared to overall length of the muscle fascicles is not
related specifically to animal size or to total muscle
length.1518 Average myofiber length determined by
this in vitro method gave longer measurements than
the 1.1 mm average individual myofiber length previously determined by a three-dimensional reconstruction technique.1 Computer three-dimensional reconstruction is a difficult procedure because of the necessity of having uninterrupted and technically perfect
serially cut histologic sections for analysis. This technique would be biased toward the inclusion of the
shortest myofibers because they are the most likely to
be complete over the course of many sections.
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Relatively short myofibers within muscles have
been postulated to play a role in allowing discrete
regions of a muscle to function independently.20'22
This has been supported by electrophysiological measurements that demonstrate regionalization in muscle
activation within muscle compartments in normal
muscles, including the cat hind limb and human orbicularis oculi,23 and in disease.24
The heterogeneity of myofiber length in different
regions of the orbicularis oculi muscle allows for regional functional activation of the muscles, within
frontal and sagittal planes, that could play a role in
the normal physiology of eyelid closure and eyeblink.
For example, recruitment and decruitment could occur from medial to lateral as well as from pretarsal to
orbital and vice versa. In earlier studies, each sagittally
defined region, or subvolume, of the orbicularis oculi
has been postulated to play a different physiological
role. As well as having a mimic function along with
the other facial musculature, the orbital portion is
involved in forceful and sustained eyelid closure.25
The palpebral portion closes the eyelid in blink, and
lid closure velocity is related directly to the amount
of orbicularis oculi motor unit activity.26 The current
hypothesis concerning pretarsal muscle function is
that it helps maintain lid margin tone.1 Preliminary
studies demonstrate sequential recruitment of myofibers in different medial to lateral locations along the
length of the human orbicularis oculi that, besides
playing a role in eyelid closure and eyeblink, also
could be involved in lacrimal pump function (unpublished observations, 1996). The sagittally defined functional units or regions of the orbicularis oculi are manifested by their specific involvement in patients with
blepharospasm; patients can be grouped by which regions of the orbicularis oculi muscles show abnormal
muscle activity.24
The heterogeneity of myofiber length in the orbicularis oculi muscle is important for optimal drug treatment into the eyelid. To reach each myofiber and
overcome the tissue barriers to diffusion effectively,
the entire length of the eyelid must be treated. The
microanatomy correlates well with patterns of regional
resistance of myofibers to doxorubicin8'9'27 and bupivacaine injections6 into rabbit and monkey eyelids. This
myofiber arrangement also suggests that botulinum
toxin treatment for eyelid spasms be injected, or their
diffusion ensured, throughout the length of the eyelid
to reach all the neuromuscular junctions effectively
and to obtain the maximum effect with the minimum
dose.
The existence of relatively short, overlapping individual muscle fibers within the orbicularis oculi muscle and the differences in individual myofiber lengths
in the different divisions of this muscle suggest that
the complex organization of the muscle fibers themselves forms the structural basis for the wide, dynamic
1739
Orbicularis Oculi Fiber Length
range of action observed in normal eyelid closure and
blink. This anatomic arrangement may be of significance in the evaluation of the structural changes associated with aging and other disease processes involving
the eyelids.
KeyWards
eyelid, facial muscles, myofiber, neuromuscular junctions,
skeletal muscle
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