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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. 646 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017 ORDICULARIS OCULI MUSCLE IN CHILDREN / Nelson and Blaivas No. 3 647 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- Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017 648 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 Vol. 32 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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017 No. 0 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. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017 649 650 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. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017 Vol. 32 No. 3 651 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) Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017 652 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / March 1991 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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017 No. 3 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 Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017 654 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 References 1. Polgar J, Johnson MA, Weightman D, and Appleton D: Data on fibre size in thirty-six human muscles: An autopsy study. J NeurolSci 19:307, 1973. Vol. 32 2. Kuwabara T, Cogan DB, and Johnson CC: Structure of the muscles of the upper eyelid. Arch Ophthalmol 93:1189, 1975. 3. Porter JD, Burns LA, and May PJ: Morphological substrate for eyelid movements: Innervation and structure of primate levator palpebrae superioris and orbicularis oculi muscles. J Comp Neurol 287:64, 1989. 4. Brooke MH and Engel WK: The histographic analysis of human muscle biopsies with regard to fiber types: IV. Children's biopsies. Neurology 19:591, 1969. 5. Pachter BR and Colbjornsen C: Rat extraocular muscle: II. Histochemical fibre types. J Anat 137:161, 1983. 6. Spencer RF and Porter JD: Structural organization of the extraocular muscles. Biittner-Enniver JA, editor. In Neuroanatomy of the Oculomotor System. New York, Elsevier, 1988, pp. 33-79. 7. Ringel SP, Wilson WB, Barden MT, and Kaiser KK: Histochemistry of human extraocular muscle. Arch Ophthalmol 96:1067, 1978. 8. Hoogenroad TU, Jennekens FGI, and Tan KEWP: Histochemical fiber types in human extraocular muscle: An investigation of inferior oblique muscle. Acta Neuropathol (Bed) 45:73, 1979. 9. Schwarting S, Schroder M, Stennert E, and Goebel MM: Enzyme histochemical and histographic data on normal human facial muscles. ORL J Otorhinolaryngol Relat Spec 44:51, 1982. 10. Dubowitz V: Muscle Biopsy: A Practical Approach, 2nd ed. London, Bailliere-Tindall, 1985, pp. 82-128, 208-219. 11. Kakulas BA and Adams RA: Disease of Muscle: Pathologic Foundations of Clinical Myology, 4th ed. Philadelphia, Harper and Row, 1985, pp. 254-305. 12. Engel AG and Banker BQ: Myology, Basic and Clinical, Vols 1 and 2. New York, McGraw-Hill, 1986, pp. 845-902. 13. OertelG: Morphometric analysis of normal skeletal muscles in infancy, childhood, and adolescence: An autopsy study. J NeurolSci 88:303, 1988. 14. Martinez AJ, Albert WB, and Hiles DA: Structural features of extraocular muscles of children with strabismus. Arch Ophthalmol 98:533, 1980. Downloaded From: http://iovs.arvojournals.org/pdfaccess.ashx?url=/data/journals/iovs/933161/ on 04/28/2017