Download Orbicularis oculi muscle in children. Histologic and

Investigative Ophthalmology & Visual Science, Vol. 32, No. 3, March 1991
Copyright © Association for Research in Vision and Ophthalmology
Orbicularis Oculi Muscle in Children
Hisro/ogic and Hisrochemical Characteristics
Christine C. Nelson* and Mila Blaivasf
This is the first study devoted to the histologic and histocheniical characteristics of the orbicularis
oculi muscle in children to the authors' knowledge. The orbicularis muscle was compared with extraocular, facial, and limb striated muscle. Light microscopy showed the orbicularis oculi muscle to be
much smaller and more loosely packed than skeletal limb muscles. It further showed these muscle
fibers to have greater variation in fiber size and shape and more endomysial and perimysial connective
tissue. Finally, analysis of the histochemical reactions showed the orbicularis oculi had a higher
percentage of fast-contracting fibers (Type II). This study establishes the histologic and histochemical
standard characteristics for the orbicularis oculi muscle in children. It was found that orbicularis oculi
muscles have some histologic and histochemical features in common with other facial muscles and
other features in common with extraocular muscles. Invest Ophthalmol Vis Sci 32:646-654,1991
We describe the histologic and histochemical characteristics of the orbicularis oculi muscle (OOM).
This muscle has received little attention in the pathology or ophthalmology literature.' A human light and
electron microscopic study was reported in 1975,2
and the innervation and ultrastructure of monkey
OOM was reported in 1989.3
The OOM is a complex muscle which forcibly
closes the eyelids and takes part in facial expressions.
This lid protractor muscle is a major cause of blepharospasm, a chronic, unremitting, bilateral, variably
progressive disease that may render the affected individual functionally blind or occupationally disabled.
To provide morphologic control for any therapeutic effort, the normal histology of the muscle must be
evaluated. There is an abundance of histologic literature on other striated muscles, and the morphologic
and histochemical characteristics are well known for
many. The OOM differs from both limb and extraocular muscles (EOM) in its histology and histochemistry. Therefore, it is essential to establish the normal
characteristics of this important protractor muscle
before studying the effects of aging, disease, and
treatment.
Materials and Methods
Biopsy specimens of OOM from 20 children's eyelids were studied (Table 1). Thirteen were from boys,
and seven were from girls. The age range was from 9
months to 16 yr. Informed consent was obtained before the procedure. The samples of the entire thickness of the muscle were obtained from the pretarsal
OOM tissue normally excised during surgery for
ptosis or epiblepharon repair. None of these patients
had any reported systemic neufomuscular disease
and no apparent OOM abnormality. In two patients
with associated trauma, the trauma did not involve
the area of the OOM biopsy site. Nineteen muscie
biopsy specimens were obtained from the central
pretarsal portion of the upper eyelid and one from the
medial pretarsal portion of the lower lid. Local anesthetic injection in the area of the muscle biopsy was
used only for five of the six adolescent patients. They
received 2% lidocaine hydrochloride with 1:1000 epinephrine mixed equally with 0.5% bupivacaine. The
muscle specimens were obtained within 20 min of the
anesthetic injection.
After excision, each fresh undamped specimen was
inspected by gross examination, oriented, and
stretched to resting length to avoid contraction artifact. Samples were then snap frozen in isopentane
cooled with liquid nitrogen. Eight-micron thick cross
sections were cut from the frozen blocks in a cryostat
at —20°C and allowed to thaw and air dry on glass
slides at room temperature for approximately 30
min. Sections were stained with hematoxylin and
eosin (H & E), modified Gomori trichrome, periodic
From the Departments of "Ophthalmology and f Pathology, The
University of Michigan Medical Center, Ann Arbor, Michigan.
Supported in part by research grant #E605715 from the National
Institute of Health.
Submitted for publication: January 20, 1989; accepted March
23, 1990.
Reprint requests: Christine C. Nelson, MD, Kellogg Eye Center,
1000 Wall Street, Ann Arbor, MI 48105.
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Table 1. Clinical data
Patient
#
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Race
Age in years
Sex
Diagnosis
white
oriental
white
white
white
white
white
white
white
white
white
white
white
black
white
0.75
1.5
3
3
3
3
4
4
4
4
4.5
5.5
6
9
13
F
F
M
M
M
M
M
F
M
M
M
F
white
white
white
white
white
14
14
15
16
16
Traumatic Ptosis
Epiblepharon Lower Lid
Congenital Ptosis
Congenital Ptosis
Congenital Ptosis
Congenital Ptosis
Congenital Ptosis
Congenital Ptosis
Congenital Ptosis
Congenital Ptosis
Congenital III Palsy
Congenital Ptosis
Congenital Ptosis
Congenital Ptosis
Congenital vs
Traumatic Ptosis
Congenital Ptosis
Congenital Ptosis
Congenital Ptosis
Ptosis
Congenital Ptosis
acid-Schiff(PAS) with and without diastase digestion,
Oil Red O, alizarin, and a panel of histochemical
reactions which included nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR), myofibrillar adenosine triphosphatase (ATPase) at pH 9.4,
4.6, and 4.2, myophosphorylase, acid phosphatase,
alkaline phosphatase, myoadenylate deaminase
(MADA), cholinesterase, and nonspecific esterase
(ANAE).
In each muscle sample, areas were selected at random, and at least 100 fibers were counted, classified,
and measured. Two small specimens had fewer than
75 muscle fibers, and these were, therefore, not included in the morphometry results. They were included in the histologic evaluation. The muscle fibers
were counted under a microscope with a mounted
video camera which projected the image on a monitor.
Assessment of the muscle samples with regard to
fiber type proportions was made on serial sections
with ATPase reaction preincubated at pH 9.4, 4.6,
and 4.2 on which each fiber was classified as Type I,
IIA, or IIB. The fiber areas were measured directly
from the muscle cross sections incubated for ATPase
activity at pH 4.6. The cross-sectional outlines of
each individual fiber were magnified to 1860X and
then traced with an electronic digitizer.
The quantitative muscle fiber data from each patient were pooled and statistically analyzed, including
measurements of central tendency (mean and median) and measures of variability (standard error and
M
M
F
M
F
M
F
M
Type of
anesthesia
General
General
General
General
General
General
General
General
General
General
General
General
General
General
Local
Local
Local
Local
Local
General
range). Mean fiber area and the percentage of each
type of fiber (percentage composition) were calculated. The differences between the means were evaluated using parametric analysis of variance and nonparametric (Kruskal-Wallis test) procedures. Statistical significance was established only if the application
of the appropriate statistical test resulted in a P- value
less than 0.05.
Frozen sections cause the least amount of shrinkage and distortion of the cross-sectional fibers. The
fiber cross-sectional area was used for measurements
because it offers the greatest accuracy of measurement. Since area increases as the square of the radius,
the area is a more sensitive parameter of fiber alteration than diameter, and it is less susceptible to the
effects of oblique sectioning. Additional calculations
to convert the areas to the diameters were done by the
computer to make our data comparable with the literature that uses measurements of fiber diameter as
the standard size comparison.
Results
The gross examination of muscle biopsy specimens
revealed little useful information. It was at times difficult to distinguish muscle from connective tissue.
Microscopic examination was therefore the principal
level of evaluation in this study.
Histologic Results
H & E: Cross sections of the entire thickness of the
OOM stained with H & E were used to evaluate mus-
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INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / March 1991
clefibersfor size, shape, and structural abnormalities
such as internal nuclei, ring or split fibers, clusters of
pyknotic nuclei, or degenerating-regenerating fibers.
The muscle fibers from the OOM specimens were
arranged in parallel elongated, oval, or fusiform fascicles of variable thickness divided by wide bands of
collagenous connective tissue (Fig. 1). An increased
amount of endomysial and, in particular, perimysial
collagen was noted compared with limb muscles (Fig.
2). In a given fascicle, the fibers were oriented in the
same direction. The muscle fibers were significantly
smaller than those found in limb muscles. Individual
OOM fibers showed a generally rounded shape and
marked variation in fiber size (Fig. 1) which differed
in degree from one fascicle to another. Some fascicles
were composed of almost monotonous groups of medium to large-sized fibers; others contained many
very small fibers. There was no particular repetitive
pattern such as zonal distribution of the fibers described in EOM. Each fiber contained multiple nuclei
oriented parallel to the long axis of the fiber in longitudinal sections and at the periphery near the sarcolemma, in cross sections. Rare internal nuclei were
noted in fibers along the periphery of the fascicle.
Several fibers with larger nuclei close to the sarcolemma were seen in the specimen from the youngest
patient, age 9 months. Some of her fibers also contained central basophilic material which stained with
PAS, NADH-TR, and MADA.
Trichrome: The modified Gomori trichromestained sections (Fig. 3) were evaluated for the presence of the cytoplasmic inclusions, nemaline rods,
and ragged red fibers. The latter are commonly seen
in the mitochondrial myopathies, including the
ophthalmoplegic group of neuromuscular disorders.
Although no true ragged red fibers were observed,
several fibers had some of their features as confirmed
by mitochondrial enzymatic reactions (Fig. 4). This is
apparently due to the aggregates of mitochondria
found in the typical ragged red fiber. To prove this
point, further study at the electron-microscopic level
is needed. The number, size, and distribution of the
intramuscular nerves and the connective-tissue content of the OOM were also increased compared with
limb muscle and were similar to these features in
EOM and facial muscles. In skeletal limb muscle,
cross sections of nerves are uncommon. Both the
number and size of intramuscular nerves were relatively increased in the OOM. Multiple cross sections
of the nerves were seen in all but the smallest biopsy
specimens. In one average-sized specimen, 22 cross
sections of intramuscular nerves were counted. The
smaller nerve branches, 6-50 j^m in diameter, were
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scattered haphazardly in the endomysium, but the
larger branches, up to 200 ^rn in diameter, were located in the perimysium or in bands of endomysial
connective tissue (Fig. 3).
Oil Red O: Oil red O stains lipid droplets in muscle
fibers. The dye has an affinity for fatty acids, triglycerides, and neutral fats, and it stains these from red to
orange. As a rule, there were no stainable lipids in the
OOM fibers. In young children the amount of lipids
in limb muscle fibers is also usually low; adults have
variable amounts of lipid droplets in their fibers.
PAS: The PAS stain revealed the distribution of the
diastase-digestible glycogen in the OOM fibers to be
similar to limb muscles. The major difference was the
relative coarseness of the stainable network and absence of sarcolemmal enhancement that is often
prominent in the Type II fibers of limb muscles.
Alizarin: Alizarin red S, an anthraquinone derivative, stains calcium deposits in tissue sections orangered. The reaction is not very specific, but interference
by other elements is usually negligible. Rare calcium
granules were noted along the periphery of OOM
fibers bordering the wide connective tissue bands and
in the perimysium. Only one of the teenagers had
three fibers containing calcium as a single row of
granules along the perimeter of the fibers. These
fibers did not appear to be degenerated or otherwise
abnormal. No calcium was seen in the blood vessels
of these specimens apparently due to the young age of
the patients.
Histochemical Results
ATPase: The myofibrillar ATPase in the biopsy
specimens showed excellent contrast between fiber
types and provided further identification of fiber II
subtypes with selective inhibition. Serial sections
demonstrated the reversal of ATPase in the same
fibers at pH 9.4, 4.6, and 4.2. Small Type I fibers
stained lightly at pH 9.4 (Fig. 5) but darkly at pH 4.2
(Fig. 6). Fiber II subtypes A and B were clearly differentiated at pH 4.6 (Fig. 7). Type IICfiberswere identified as rare intermediately stained fibers at pH 4.2
(Fig. 6). This corresponds to the fiber-type differentiation seen in limb muscle (Fig. 8). In all examined
OOM, Type II fibers were the predominant type and
the Type Ifiberswere smaller in size and number. In
limb skeletal muscles the staining results in a checkerboard pattern because of an almost equal ratio of
Type I to the subtypes of Type II fibers. There are
exceptions, however, such as deltoid and soleus
which have 60-80% Type I predominance and triceps
which has Type II fiber predominance.4 The mosaic
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ORBICULARIS OCULI MUSCLE IN CHILDREN / Nelson ond Dloivos
%»A
'Mi
Fig. 1. Orbicularis oculi muscle, Hematoxylin and eosin stain, 235X, 6 yr female.
Fig. 2. Normal limb muscle, Hematoxylin and eosin stain 235X, 2 yr female.
Fig. 3. Orbicularis oculi muscle, Trichrome stain 752x, 3 yr male.
Fig. 4. Orbicularis oculi muscle, NADH-TR 752X, 5 yr male.
Fig. 5. Orbicularis oculi muscle, ATPase, pH 9.4 470X, 6 yr female.
Fig. 6. Orbicularis oculi muscle, ATPase, pH 4,2 470X, 6 yr female.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1991
Fig. 7. Orbicularis oculi muscle, ATPase, 4.6 470X, 6 yr female.
Fig. 8. Limb muscle, ATPase, 9.4 296x dystrophy, adult.
Fig. 9. Limb muscle, Hematoxylin and Eosin 118X dystrophy, adult.
Fig. 10. Orbicularis oculi muscle, Cholinesterase 47OX, 6 yr female.
Fig. 11. Limb muscle dystrophy, NADH-TR 235X, young adult female.
Fig. 12. Orbicularis oculi muscle, Phosphorylase 470X, 4.5 yr male.
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ORBICULARIS OCULI MUSCLE IN CHILDREN / Nelson and Blaivas
pattern in normal OOM is obscured by the paucity
and smallness of Type I fibers and prevalence of
larger Type II fibers. These features along with increased variation of muscle-fiber size, rounding of
muscle fibers, and increased amount of endomysial
connective tissue are usually interpreted as myopathic or dystrophic in the limb muscles in ATPase
and other enzymatic reactions and histologic stains
(Figs. 8, 9).
Esterases: Cholinesterase and nonspecific esterase
were used to identify the number and size of the endplates. They also provided additional information on
variation in fiber size, state of innervation, fiber typing, and the presence of fiber degeneration or denervation atrophy. The nonspecific esterase reaction was
similar to that found in limb muscle biopsy specimens because the identification of the neuromuscular
junctions was not as reliable as in the cholinesterase
reaction. No multiinnervated fibers were seen
(Fig. 10).
Phosphatases: Muscle sections were stained for
both acid and alkaline phosphatases activity. Acid
phosphatase is located in lysosomes and can be found
in increased activity in diseases causing myofiber necrosis, in storage disorders, and in atrophied muscle
fibers. Alkaline phosphatase is usually enhanced in
regenerating muscle fibers, vessel walls, and in the
endomysium in inflammatory myopathies. Both of
these enzymes were found to have minimal activity in the OOM as would be expected in a normal
muscle.
MADA: The MADA is known to catalyze the deamination of 5' adenosine monophosphate to inosine
monophosphate with the production of ammonia.
The ammonia provides a source for synthesis of
amino acids. The reaction is also associated with the
regulation of the level of high-energy phosphate compounds in the cell. The absence of this enzyme is seen
in exercise exertion and in some patients with muscle
cramps. All of the OOM specimens had MAE>A. The
enzyme did not usually allow the fiber-type recognition, although in some of our specimens the pattern
was similar to that of NADH-TR.
NADH-TR: The NADH-TR reaction snowed that
the larger fibers were weak in oxidative enzymatic
activity, with a preponderance of pale and intermediate-type fibers (Fig. 4). The smaller fibers were rich
in oxidative enzyme activity and stained darkly. The
staining characteristics of the muscle fibers was odd
due to the coarse network of the sarcoplasm and frequent thickened dark rim of sarcolemma. The OOM
of the teenagers and one 4-year-old had varying numbers of lobulated, ring-like ("partial" ring) fibers and
fibers with thick dark contours and pale sarcoplasm
(Fig. 4). This further emphasizes the similarity of
OOM histochemical features with those of diseased
limb muscle (Fig. 11). The reliability of NADH-TR
infiber-typedifferentiation was not as good as that of
ATPase.
Phosphorylase: The phosphorylase reaction was
used along with PAS stain to identify the glycolytic
pathway. It was positive in all specimens (Fig. 12).
Local anesthetics in conjunction with epinephrine
are known to exhaust the phosphorylase activity and
deplete glycogen. Other causes of depleted glycogen
include strenuous exercise before biopsy, recent denervation, and degeneration or necrosis offibers.The
local injection of anesthetic agent with epinephrine
used in five specimens did not alter the histochemical
characteristics of the fibers compared with those obtained under general anesthesia.
Morphometry
Type I fibers were noted to be small and sparsely
scattered throughout the fascicles. The Type II fibers
were always larger, more numerous, and varied more
widely in size (Table 2). These general characteristics
were found in all specimens. The percentage by number of Type I varied from 2.3-30%; of Type IIA
fibers, from 17.9-37.7%; and of Type IIB, from
51.6-67.7%.
Table 2. Fiber area and diameter measurements
of orbicularis oculi muscle in children
Fiber type
/
Patient #
1
2
3
4
5
6
7
8
9
10
11
13
14
16
17
18
19
20
Overall
Mean
IIA
IIB
Area (diameter) Area (diameter) Area (diameter)
in microns
in microns
in microns
193.9(15.71)
197.7(15.83)
195.3(15.76)
153.3 (J3.97)
178.3(15.07)
124.8(12.60)
116.5(12.17)
169.6(14.69)
154.1 (14.00)
125.4(12.88)
181.2(15.18)
183.6(15.28)
123.5(12.53)
148.4(13.74)
118.7 (12.29)
167.3(14.59)
148.0(13.72)
154.8(14.03)
237.0(17.37)
210.0(16.35)
252.3 (17.92)
555.4 (26.59)
187.3(15.44)
208.1 (16.27)
353.6(21.21)
325.6 (20.36)
187.7(15.42)
238.5(17.42)
168.5(14.64)
225.3(16.93)
376.7(21.90)
274.0(18.67)
384.2 (22.11)
337.2 (20.72)
337.0(20.71)
340.0 (20.80)
391.5(22.32)
316.7(20.08)
446.4 (23.84)
714.8(30.16)
494.7 (25.09)
570.1 (26.94)
604.7 (27.74)
606.5 (27.78)
341.9(20.86)
393.6 (22.38)
231.7(17.17)
403.1 (22.65)
534.1 (26.07)
598.1(27.59)
615.8(28.00)
779.4(31.50)
516.9(26.65)
158.4(14.15)
270.8(18.55)
499.4 (24.87)
171.9 (14.79)
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The mean cross-sectional fiber area of each of the
three,fibertypes in boys and girls demonstrated that
Type I was the smallest. The greatest variation in size
in one type occurred in Type IIB, with a standard
error of 51.9. The Kruskal-Wallis test, a nonparametric, multisample comparison procedure, indicated
that the average fiber areas for the three fiber types
differed significantly (P < 0.0001). There were no
significant difference in thesefiber-type-specificareas
between boys and girls.
The overall percentage by area was 6.8% for Type I,
20.8% for Type IIA, and 72.0% for Type IIB. The
overall percentage of Type Ifibersby area (6.8%) was
significantly lower than the percentage by number
(14.2%) since these fibers were small. Type IIB fibers
were large, and therefore, the overall percentage of
area of the muscle of this fiber type was large (72%)
compared with the number offibers(54.8%). Statistical comparisons of the fiber-type means by sex, using
the student t-test, showed no gender effect or consistent pattern of variance.
Although the data pool was small, no significant
difference was found in thefiber-typecomposition by
area between the juveniles and adolescents. Only one
lower lid specimen, patient 2, was examined, and
therefore, no statistical analysis could be made. However, the morphometric data of this lower lid OOM
was similar to that of the upper lid muscle.
Discussion
Mammalian muscles can be classified by fiber
types based on their morphologic and histochemical
characteristics.4 All human muscles with the exception of the EOM and some other small highly specialized muscles are a combination of red (Type I) and
white (Type II) muscles. Type Ifibersare functionally
slow-contracting fibers and capable of long continuous activity. They are metabolically aerobic and high
in oxidative activity but low in glycolytic enzyme activity. Type IIAfibersare fast contracting but are also
capable of sustained activity. These fibers are both
aerobic and anaerobic and contain much glycogen.
As might be expected, myophosphorylase activity is
high. Type IIBfibersare fast contracting, phasic, and
anaerobic. Therefore, they fatigue easily, in contrast
to Type I or Type IIA fibers. Moderate amounts of
glycogen and high levels of myophosphorylase are
present.
The ratio of Type I to Type IIfibersmay vary from
fascicle to fascicle in a muscle. It is possible to discern
a difference in the staining quality among the Type II
fibers and subclassify them as Type IIA or IIB. Both
ATPase and NADH-TR reactions can determine any
Vol. 32
abnormalfiber-typedistribution or predominance, as
seen in neuromuscular disorders. The Type I and II
classification of the muscle fibers is not applicable to
EOM due to marked variation and discrepancies in
the enzymatic reactions compared with limb muscles. There has been a notable controversy over the
years in typing EOM which, with the implementation
of more sophisticated methods, resulted in a six-fiber
type of classification based on histochemical and ultrastructural properties and on location and innervation of the fibers.5"7
The human skeletal muscle fiber types, as defined
histochemically, differ functionally and metabolically. The functional differences concern fatigability
and velocity of contraction. The polymorphic characteristics of muscle fiber structure are greatly influenced by the functional demands placed on the
individual muscle. These functional differences correlated well with the histochemical characteristics
found for the OOM. In OOM there is a predominance of Type IIBfibers,the fast fibers which are not
able to sustain contraction for long periods of time
due to fatigue and are, therefore, ideally suited for
blinking. Sustained squeezing of the eyelids can occur
due to the Type IIA fibers which are fast but fatigue
resistant. During sleep the OOM is at rest, and the lid
position is determined by the equilibrium between
the state of relaxation of the levator muscles
and OOM.
The OOM can generate extremely fast movements
and does not rest for more than a few seconds during
waking hours. Apparently lipids are rapidly used in
this constantly active muscle since no stainable lipids
were seen. This suggests a metabolic similarity with
the central area of the EOM which was confirmed in
our study by the ATPase reaction.8
The OOM'differs from some other facial muscles
in regard to the ratio of the Type II muscle fiber
subtypes. Schwarting et al.9 reported the paucity of
Type IIB fibers in the levator labii, zygomaticus
major, and OOM. The Type IIB fibers were particularly scarce (virtually absent) in the zygomaticus
major muscle. The platysma, on the other hand,
closely resembled the normal limb muscle in distribution of the three fiber types. Type II fibers were
more numerous in each of the four facial muscles
examined than in the limb muscles.
Histochemical reactions greatly enhance diagnostic
precision by providing an easy means of identifying
muscle fiber types, their distribution, and disturbances of metabolism with standard light microscopy. Although there is a lack of quantitative measure, the advantages of histochemistry more than
compensate by anatomically locating metabolic
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ORDICULARIS OCULI MUSCLE IN CHILDREN / Nelson and Blaivas
products and enzymatic activities in tissue sections.
By identifying specificfiber-typeratios on the basis of
these various enzyme reactions, selective fiber involvement can be shown with certain disease processes such as small Type I musclefibersin congenital
myopathies and myotonic dystrophy or Type II fiber
atrophy in immobilization, corticosteroid toxicity,
and other conditions.10"12 As seen in limb muscles,
there were essentially no gender-related differences in
fiber size or distribution in children before puberty. It
is known that gender-related differences in skeletal
muscle fiber size become apparent after puberty with
the onset of rapid muscle growth.13 Due to the small
adolescent sample size, no such conclusions can be
made in the OOM. However, the other feature peculiar to this eyelid muscle was that the mean fiber area
and the diameter increased little from early infancy
until adulthood (Table 2).
When a neuromuscular disease or degenerative
process is restricted to the periorbital muscles and
EOM, it is essential to be able to distinguish normal
from diseased muscle. The OOM is more similar to
the group of facial muscles and EOM than to limb
muscle, although it does not appear to possess all the
peculiarities of either of these muscle groups. For instance the EOM consist of fibers arranged in three
concentric zones with no reliable fiber-type differentiation in ATP-ase reaction.5"7 There were quantitative morphologic differences between strabismic and
nonstrabismic muscle.14 Further studies are needed
to determine if a biopsy specimen of the OOM in
patients with ophthalmoplegic syndromes and other
myopathies would assist in the diagnosis. Normal
OOM possesses some features which would be considered consistent with a chronic myopathy or dystrophy in limb muscle. These features include
marked variation in fiber size, rounded fiber shape,
structural alterations such as lobulation and irregular
coarseness of stainable sarcoplasmic network, absence of the checkerboard pattern offiber-typedistribution, and an increase in endomysial and perimysial
connective tissue.
There was a slight but definite difference between
the OOM in young children and the teenagers. The
younger children had a generally more monotonous
or homogeneous pattern of staining than teenagers.
Only rare fibers in the younger age group were finely
lobulated or had darker contours reminiscent of
ragged red fibers. In teenagers the lobulated and
coarse fibers were more conspicuous. There was no
significant increase in number of internal nuclei.
No typical ragged red, split, or fragmented fibers
were noted in H & E or modified Gomori trichrome
stains as would be characteristic of a diseased mus-
653
cle.10 12 Specimens from several children showed occasional "partial rings," when one half or one third of
the peripheral zone fibrils were oriented perpendicular to the sarcolemma. In all cases, ring fibers were
not as well defined as the ones described in diseased
limb muscle or mature EOM. The sarcoplasmic network in trichrome-stained sections was often irregular and, in the older age group, occasionally had
coarse peripheral mottling; however, no nemaline
rods were identified. Only one specimen contained
muscle spindles, and they were not remarkable in any
of the stains or reactions.
Fibers stained with PAS had a coarse sarcoplasmic
network, without enhancement of the sarcolemmal
outlines. They were randomly distributed throughout
the sections. In each specimen, there were virtually
no fibers containing lipid droplets, as defined by oil
red O stain. The lipids are probably used rapidly in
this constantly active muscle. A single fiber in the
specimen from a 16-year-old patient contained minute droplets of fat at the sarcolemma outlining one
half of the fiber perimeter. Cholinesterase revealed
two to four rather large motor end-plates in children
of all ages. Their size and number were more similar
to the ones reported in EOM67 and facial9 muscles
than in limb muscles.
Alizarin stain revealed occasional fibers containing
red granules of calcium and scattered rare granules in
the wide connective tissue bands of perimysium.
Normal limb muscles of young people have no stainable calcium.1011 Accumulation of calcium in muscle
fibers and vessel walls may be seen in old age and
some muscle diseases such as polymyositis and Duchenne's dystrophy.
Local injection of an anesthetic agent did not alter
the histologic arrangement of fibers and vessels compared with the specimens obtained under general anesthesia. Specifically, there was no difference in
phosphorylase activity. This finding may be due to
the biopsy specimens being taken within 20 min of
administering the anesthetic injection. Preliminary
studies on the OOM show that there is alteration of
the phosphorylase activity in biopsy specimens taken
1 hr or more after injection of anesthetics. Since five
of six specimens examined after local anesthetic injection were from adolescents, further studies are required to ensure these two variables, age and anesthetic, did not have opposing effects which, when
taken together, resulted in the observed lack of effect.
Summary
The OOM as a part of facial musculature has histologic similarities to the other facial muscles such as
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / March 1991
smaller fiber size than limb muscles, greater variation
in fiber size, and increased density of intramuscular
nerves. These features are also characteristic of the
EOM. Thus, although the OOM does not appear to
possess many peculiarities of the EOM, it is similar to
them and the facial group of muscles and can be
placed somewhere between them with respect to the
histologic and histochemical parameters.
The characteristics we described substantiate the
conclusion that the OOM muscle is different from
normal limb muscle and has many features of facial
muscles and EOM. The muscle biopsy specimens
used in this study were taken from patients with clinically normal OOM. Although most patients had
congenital ptosis involving the levator muscle, this
would not be expected to affect the OOM. One of the
most important points in our study was that many
characteristics of normal OOM are considered pathologic when they occur in limb skeletal muscle. These
data establish the histologic and histochemical standards for this muscle in children. Further studies in
adults are needed to evaluate the effect of the aging
process on the OOM. Changes occurring in the OOM
with myopathic and neuropathic diseases will require
definitive characterization. Histochemical and histologic testing of muscle biopsy specimens can be valuable in the differential diagnosis of these diseases.
Key words: blepharospasm, facial muscles, histochemistry,
histology, orbicularis oculi muscle
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