THE ANATOMICAL RECORD 218:14-19 (1987)

Extraocular Muscles in the Microphthalmic Rat KOICHI TANAKA, KATSUMI OTANI, AND SHOE1 SUGITA

The Third Department of Anatomy, School of Medicine, Chiba University, 1-8-1 Inohana, Chiba 280 Japan

ABSTRACT The extraocular muscles in a mutant microphthalmic strain of rat were studied. The eyeball of this strain of rat is reduced to about a third in diameter of that of the normal rat. Nevertheless, in the orbit of the mutant rat, every one of the extraocular muscles was identified; their origins and courses were the same as in the normal rat, but differences existed in the insertions. These insertions could be classified into three groups: Group A (retractor bulbi): like normal insertion into the eyeball. Group B (superior rectus and superior oblique): attachment of tendonlike insertions to each other; these muscles come from opposite directions and form a loop. Group C (lateral, medial, and inferior rectus and inferior oblique): insertion into connective tissue surrounding the reduced eyeball.

The volume of each muscle of the mutant rat was smaller than that of the normal rat; moreover, significant differences existed in the degree of reduction in the volume of each muscle group classified according to the change of insertion. In the group A muscle the volume was only 33% of the normal volume, whereas group B was 74% and group C was about half of normal.

Congenital abnormalities of the eye have been re- Kobayashi and Otani (1981) studied the embryogene- ported in many species of vertebrates. Malformation of sis of abnormal eyeball and retina in the microph- the eye during the embryonic stage generally occurs as thalmic rat, but they did not mention the extraocular a result of physical (Coulombre, 1956; Brent and Frank- muscles. lin, 19601, chemical (Wilson, 1955; Tuchman-Duplessis We studied serial celloidin sections of the eye region of and Mercier-Parot, 1960; Kreibig et al., 1976), genetic the adult microphthalmic rat, the same strain used by (Bourne et al., 1938; Tansley, 1951; Gorthy and Abdel- Kobayshi and Otani (1981), Sugita (1982), and Sugita baki, 1974; Yates et al., 1974; Wyse and Hollenberg, and Otani (19831, to determine whether or not extraocu- 1977), and other factors (Yudkin, 1927; Kalter and War- lar muscles were present, and, if they were, to ascertain kany, 1959). Several reports note the morphogenesis of their origin, course, and volume. At the same time, genetic anophthalmia or microphthalmia in the mouse using Epon sections, we counted the number of muscle (Chase and Chase, 1941; Muller, 1951; Truslove, 1962; fibers of each muscle.

MATERIALS AND METHODS Packer, 1967; Konyukhov and Vakhrusheva, 1969) and the rat (Browman and Ramsey, 1943; Browman, 1961; Otani et al., 1978; Kobayashi and Otani, 1981). Owing Adult Donryu albino rats weighing 250-300 gm, in- to these studies, the morphogenesis of the abnormal eye cluding five microphthalmic and five normal rats, were in two strains has been studied in detail. One strain is used. The microphthalmic rats had eyeballs which the anophthalmic mouse (ZRDCT-An), which has been lacked the optic nerve and were reduced bilaterally to studied by Chase and Chase (1941); the other is the about a third of the normal diameter. The normal ones microphthalmic rat, the object of Kobayashi and Otani serving as controls had eyes of normal diameter. All rats (1981). In both those mutant strains the optic cup re- were anesthetized with intraperitoneal pentobarbital so- gresses early in embryogenesis, with a failure of eye dium (60mgkg) and perfused through the left ventricle formation. with 300 ml of 10% formalin.

The extraocular muscles of the ZRDCT-An mouse have To identify each extraocular muscle and to measure been studied by Chase (1945) and Paterson and Kaiser- the volume, the heads of these rats were decalcified with man-Abramof (1981). Chase concluded that the ano- Plank and Rychlo (1952) solution by means of aluminum phthalmic adult mouse had no extraocular muscles, but chloride and formic acid, and embedded in celloidin. the levator muscles of the upper eyelid were present. On Frontal serial sections 50pm thick were cut through the the other hand, Paterson and Kaiserman-Abramof (1981) orbit. These sections were stained with hematoxylin and stated that in the orbit of the anophthalmic mouse sev- eosin. The actual area of each extraocular muscle was era1 bundles of striated muscles occupied a location com- outlined at x 50 magnification using the X-Y panto- parable to that of the extraocular muscles in the normal graph charting system and was subsequently measured mouse. As stated above, there is disagreement regard- by a MUTOH image analyzer. From this area and the ing the existence of the extraocular muscles. However, although the extraocular muscles do, in fact, seem to exist, detailed information about them is still not available. Received July 25, 1986; accepted December 30, 1986.

@ 1987 ALAN R. LISS. INC.

EXTRAOCULAR MUSCLE IN THE MICROPHTHALMIC RAT 15

section thickness, the volume occupied by these extra- struction of the muscles allow the classification of these ocular muscles was calculated for each side. Sets of abnormal insertions into three groups. Group A: (RB) as sections of the extraocular muscles of the two strains of in the normal rat, it inserts into the eyeball, which is rats, one of each, were reconstructed at a 50:l scale by reduced to about one-third of the diameter of the normal means of a styrene board. Gross dissections of the orbits eye. Group B: (SRI and SO) the insertions of these mus- of additional microphthalmic rats after fixation supple- cles attach together after coming from opposite direc- mented the study. tions, thereby making a loop. Group C: (LR, MR, IR, and

To count the number of muscle fibers, four rats of each 10) they insert into dense connective tissue surrounding strain were used. After perfusion with 2.5% glutaralde- the reduced eyeball. hyde in 0.1M phosphate buffer (pH 7.41, the mid-portion of each muscle was osmicated in 2% osmium tetroxide The Volume of the Extraocular Muscles in 0.1 M phosphate buffer (pH 7.4) for 90 minutes, dehy- Table 1 presents the volume of the extraocular mus- drated in graded alcohol, and embedded in Epon 8.12. cles of five normal (ten sides) and also five micro- Transverse semithin sections 0.5 pm thick were pre- phthalmic (ten sides) rats. Shrinkage of the muscles pared and stained with a toluidine blue solution. These after fixation and dehydration was not corrected. For all sections were photographed at ~ 5 0 0 magnification. The extraocular muscles the mean value for the volume in number of muscle fibers was counted from these the mutant rats is always smaller than that in the photographs. normal rats. Probability values derived from one-way

analysis of variance indicate that these differences be- tween normal and microphthalmic rats are statistically RESULTS

The Origin, Course, and Insertion of Each Extraocular Muscle significant (p < 0.01). Moreover, a significant difference exists in the degree of the reduction in volume of each

Normal rat muscle group classified according to the change of inser- The funnel-shaped, nondivisible, retractor bulbi mus- tion. In the group A muscle (RB), volume in the micro-

cle (RB) originates from the lateral aspect of the anterior Phthalmic rats is only 33% Of normal, whereas the group part of the sphenoid body, ventral to the canalis opticus. (SR and so), which make a lOOP7 have 74% of It is penetrated dorsally by the optic nerve. It inserts and group MR7 IR, and into the sclera immediately caudal to the rectus mus- 10) are about half of normal. cles. The four rectus muscles (superior = SR, medialis The Numbers of Muscle Fiber = MR, inferior = IR, and lateralis = LR) arise from the ventral edge of the optic canal and insert with short Table 2 shows the number of muscle fibers contained tendons into the equator of the eyeball. The superior in each extraocular muscle of normal (four rats, four oblique muscle (SO) originates from the orbital part of sides) and microphthalmic (four rats, four sides) rats. As the frontal bone and runs nasally and dorsally along the with the volume of muscles, the number of fibers was osseous wall of the orbit. Near the temporal margin of always smaller in the microphthalmic rat, and the de- the lacrimal bone i t is deviated by the trochlea and then gree of reduction in number was different in the groups passes to the dorsal aspect of the eyeball. The S o is classified according to their change of insertion. But the attached to the eyeball near or over the position where difference was not as prominent as the difference in the tendon of SR is inserted. The inferior obliaue muscle volume. (10) originates from the orbital part of the f&ntal bone close to the margin of the lacrimal bone. It crosses the nasoventral edge of the Harderian gland and inserts into the ventral temporal quadrant of the eyeball.

Microphthalmic rat In the mutant rat, the eyeball is reduced to about a

third of the normal diameter, and the retina and the optic nerve could not be observed (Fig. 1C). The funnel- shaped, although very slender, RB originates from the same position as in the normal rat. It runs directly toward the eyeball, where it is attached. The MR, IR, and LR arise from the ventral edge of the optic canal, as in the normal rat, but do not insert into the eyeball. They become more and more slender and insert into dense connective tissue (Figs. 1C,D,2). The SR arises with other rectus muscles and runs toward the point of the insertion of the normal muscle where it attaches to the insertion of SO. The SO also arises and follows the same course as in the normal one, but after the trochlea it becomes attached to SR (Figs. lC, D,2). The I0 origi- nates from the same part of the frontal bone and ends in the connective tissue like many of the rectus muscles. As a whole, the origins and courses of all extraocular muscles are the same as in the normal rat, but differ- ences exist in the insertions. Gross dissection and recon-

The Semithin Sections of the Mid-Portion of the Extraocular Muscles

The extraocular muscles of the normal rat are com- posed of two rather distinct layers. One layer looks to- ward the eyeball (=global layer), the other surrounds the first layer on three sides, has a C-shaped appearance in cross section, and looks toward the walls of the orbit (=orbital layer), just as Mayr (1971) described. A connec- tive tissue layer of perimysium internum often sepa- rates the two muscle layers very distinctly. The orbital layer is composed of small-diameter muscle fibers, which have numerous capillaries and connective tissue ele- ments located between them. This layer organization is more distinct in the rectus muscles (Fig. 3C) than in the oblique muscles (Fig. 3A), where the orbital layer may enclose the global one completely. No layer organization is found in RB.

In the microphthalmic rat the layer organizations are observed as being the same as in the normal rat (Fig. 3A, B). Moreover, the tendency €or the orbital layer to be composed of smaller muscle fibers than the global layer is also observed in the microphthalmic rat. When comparing the same layers, the microphthalmic rat ap- pears to have the same size of muscle fibers as the normal rat.

Fig. 1. Frontal sections through the centers of the eyeball of a normal (A, B) and a microphthalmic (C,D) rat. In the microphthalmic rat, the eyeball locates on a more caudal level than that in the normal rat. The SR, LR, and MR run to a more distal point than the eyeball. Celloidin section 50pm thick, hematoxylin-eosin stain. x 18. Scale bar=2 mm.

EXTRAOCULAR MUSCLE IN THE MICROPHTHALMIC RAT 17

Fig. 2. Ventrolateral view of the extraocular muscles of a microphthalmic rat, left side. The I 0 has been removed. The IR and LR do not attach to the eyeball. The tendons of SO and SR are attached, making a loop. x 10. Scale bar= 2 mm.

TABLE 1. Volume of the extraocular muscles per side in normal and microphthalmic rats (mm3)

Microphthalmic (b) L) x loo(%) Muscle Normal (a)

Group A 33 M. retractor bulbi 1.55 k 0.08 0.51 f 0.09 33

Group B M. rectus sup. M. obliquus sup.

1.13 & 0.07 1.86 k 0.13

0.84 f 0.06 1.38 & 0.17

74 74 74

Group C 51 M. rectus lat. 1.36 & 0.10 0.72 & 0.07 53 M. rectus med. 1.28 & 0.10 0.69 & 0.06 54

M. obliquus inf. 1.15 & 0.10 0.56 f 0.09 49 M. rectus inf. 2.05 k 0.13 1.02 k 0.14 50

TABLE 2. Number of muscle fibers per side in normal and microphthalmic rats

x 100 (%I Muscle Normal (a) Microphthalmic 03) a

Group A

Group B M. retractor bulbi

M. rectus sup. M. obliquus sup.

M. rectus lat. M. rectus med. M. rectus inf. M. obliauus inf.

Group C

33

65 1,945 k 173 1,169 k 110 60 1,634 k 42 1,162 k 169 71

52 2,455 f 387 1,124 82 46

1,632 & 41 534 f 58 33

2,336 & 261 1,248 & 154 53 2,260 k 339 1,190 k 231 53 1,132 + 255 662 f 65 58

DISCUSSION

As for extraocular muscles of the anophthalmic mouse, Chase (1945) stated that such an adult mouse had none. However, Paterson and Kaiserman-Abramof (1981) found that in the orbit of the anophthalmic mouse, sev- eral bundles of striated muscles occupied a location com- parable to that of extraocular muscles in the normal

mouse. In our specimens, using not the anophthalmic mouse but the microphthalmic rat with complete lack of the optic nerve, we could identify all of the extraocular muscles. Their origin and course are identical with those of normal rats; however, differences exist in the inser- tions. Owing to the reduction of the eyeball to about one- third the normal diameter, neither all the rectus mus- cles nor the two oblique muscles are attached to the

18 K. TANAKA, K. OTANI, AND S. SUGITA

Fig. 3. Transverse section of SR of a normal (A), and a microphthalmic (B) rat; and SO of a normal (C), and a mutant (D) rat. x200. Scale bar=O.Z mm. OL=orbital layer; GL=global layer.

EXTRAOCULAR MUSCLE IN THE MICROPHTHALMIC RAT 19

eyeball. Among these, LR, MR, IR, and I0 are inserted into connective tissue around the eyeball. The SO and SR are attached together and make a loop.

These changes of insertions are strongly related to the degree of reduction in volume of the muscles. The SO and SR which make a loop, have volumes of about three- quarters of normal, whereas LR, MR, IR, and 10, which are inserted into connective tissue, have only half of the normal volume. This correlation between the changes of insertion and the degree of reduction in volume seems to depend on the contraction ability of each muscle. The SO and SR are attached from opposite directions, so each of them can be stretched by the other; and therefore they can contract even in the mutant animal. But in the case of LR, MR, IR, and 10, once they contract, there is nothing to stretch them again.

There are reports mentioning that the weight of cat muscles which are immobilized by a plaster cast for 14 weeks decreases to about half of control value (Cooper, 1972). The situation of LR, MR, IR, and I 0 in the microph- thalmic rat seems to resemble this case of immobiliza- tion. He demonstrated that immobilization initiates muscle fiber disintegration. Immobilized muscle fibers undergo a more-or-less well-defined sequence of degen- erative changes. During these changes many fibers re- main simply as sarcotubes, enclosed by basement membrane and containing only fluid, precipitated pro- tein, and fragments of the sarcolemma. But in the pres- ent study of transverse Epon sections of muscle fibers by light microscopy, the extraocular muscles of the mi- crophthalmic rat show no degeneration. Moreover, the mutant rat appears to have the same size of muscle fibers as the normal rat.

The number of muscle fibers in each extraocular mus- cle of the mutant rat was always smaller than that of the normal one. Moreover, the degree of reduction in the number of fibers was about the same as that in the volume of muscles. This indicates that the volume of muscles in the microphthalmic rat decreased because of the reduction of the number of muscle fibers rather than because of any narrowing.

The question of the developmental relationship of the eye to the oculomotor system was raised by the experi- ments of Bronson and Hedley-Whyte (1977). These au- thors reported that removal of the eye 5 days postnatally in the rat results in only slight changes in the extraocu- lar muscles after varying survival times; the muscles became thinner and shorter, and interestingly, they were inserted into dense connnective tissue, as in our case. Their study suggests that the absence of the eyeball exerts only slight influence on the postnatal develop- ment of the extraocular muscles. Then, how is it in the embryonic stage? In the rat embryo with a normal optic cup, the anlagen of extraocular muscles can be recog- nized by 14 days of gestation (Kobayashi and Otani, 1981). At this time, the muscles appear in the micro- phthalmic embryo also, although by then the malfor- mation of the eye has already started (Kobayashi and Otani, 1981). It is noteworthy that at the time when the muscles first arise the optic cup has regressed in the mutant embryos. These facts indicate a greater degree of independence in the development of the eyeball and the extraocular muscles than was previously supposed.

ACKNOWLEDGMENTS The authors wish to thank Mr. K. Miyama, Mrs. F.

Saito, and Mrs. T. Saito for their expert technical assis-

tance. We are also grateful to Dr. A. Gerz for his critical reading of the manuscript. This work was supported by grant 60570023 from the Japanese Ministry of Educa- tion, Science and Culture.

LITERATURE CITED Bourne, M.C., D.A. Cambell, and K. Tansley (1938) Hereditary degen-

eration of the rat retina. Br. J. Ophthalmol., 22:613-623. Brent, R.L., and J.B. Franklin (1960) Uterine vascular clamping: New

procedure for the study of congenital malformations. Science,

Bronson, R.T., and E.T. Hedley-Whyte (1977) Morphometric analysis of the effects of exenteration and enucleation on the development of third and sixth cranial nerves in the rat. J. Comp. Neurol., 176:315- 330.

Browman, L.G. (1961) Microphthalmia and optic blood supply in the rat. J. Morphol., 109:37-55.

Browman, L.G., and F. Ramsy (1943) Embryology of microphthalmos in Rattus noruegicus. Arch. Ophthalmol., 30:338-351.

Chase, H.B. (1945) Studies on an anophthalmic strain of mice. V. associated cranial nerves and brain centers. J. Comp. Neurol., 83: 121-139.

Chase, H.B., and E.B. Chase (1941) Studies on an anophthalmic strain of mice. I. Embryology of the eye region. J. Morphol., 68:249-301.

Cooper, R.R. (1972) Alterations during immobilization and regenera- tion of skeletal muscle in cats. J. Bone Joint Surg [Am], 54-A:919- 953.

Coulombre, A.J. (1956) The role of intraocular pressure in the devel- opment of the chick eye. I. Control of eye size. J. Exp. Zool., 113:211- 225.

Gorthy, W.C., and Y.Z. Abdelbaki (1974) Morphology of a hereditary cataract in the rat. Exp. Eye Res., 19:147-156.

Kalter, H., and J. Warkany (1959) Experimental production of congen- ital malformations in mammals by metabolic procedure. Physiol. Rev., 39:69-115.

Kobayashi, K., and K. Otani (1981) Morphogenesis of the hereditary microphthalmia in a new strain of rat. J. Morphol., 167:265-276.

Konyukhov, B.V., and M.P. Vakhrusheva (1969) Abnormal develop- ment of eyes in mice homozyous for the fidget gene. Teratology, 2:147-158.

Kreibig, von W., H.H. Schlote, and Th. von Kreybig (1976) Ein seltene Augenmissbildung bei einer CD Rattle. A. Versuchstierkd., 18:25- 31.

Mayr, R. (1971) Structure and distribution of fiber types in the external eye muscles of the rat. Tissue Cell, 3:433-462.

Miiller, G. (1951) Eine entwicklungsgeschichtliche Untersuchung uber das erbliche Klobom mit Microphthalmus bei der Hausmaus. Z. Mikrosk. Anat. Forsch., 56:520-558.

Otani, K., K. Kobayashi, and G. Kato (1978) Morphology of the eye and brain in anophthalmic rats. In: Integrative Control Functions of the Brain. Vol I. M. Ito et al., eds. Eldevier Amsterdam; Kodan- s h d Tokyo, pp 100-101.

Packer, S.O. (1967) The eye and skeletal effects of two mutant alleles at the microphthalmia locus of Mus musculus. J. Exp. Zool., 165~21- 46.

Paterson, J.A., and I.R. Kaiserman-Abramof (1981) The oculomotor nucleus and extraocular muscles in a mutant anophthalmic mouse. Anat. Rec., 200:239-251.

Plank, J., and A. Rychlo (1952) Eine Schnellentkalkungsmethode. Zen- tralbi. Allg. Pathol., 89:252-254.

Sugita, S. (1982) Hypoplasia of the lateral geniculate nucleus in the hereditary microphthalmic rat. Chiba Med. J., 58:151-159. (In Japanese.)

Sugita, S., and K. Otani (1983) Quantitative analysis of the lateral geniculate nucleus in the mutant microphthalmic rat. Exp. Neu- rol., 82~413-423.

Tansley, K. (1951) Hereditary degeneration of the mouse retina. Br. J. Ophthalmol., 35:573-582.

Truslove, G.M. (1962) A gene causing ocular retardation in the mouse. J. Embryol. Exp. Morphol., 10~652-662.

Tuchmann-Duplessis, H., and L. Mercier-Parot (1960) Production of congenital eye malformations, particularly in rat fetuses. Anat. Rec., 136:294.

Wilson, J.G. (1955) Teratogenic activity of several aso dyes chemically related to trypan blue. Anat. Rec., 123~313-333.

Wyse, J.P.H., and M.J. Hollenberg (1977) Complicated colobomatous microphthalmos in the BW rat: A new form of inherited retina1 degeneration. Am. J. Anat., 149:377-412.

Yates, C.M., A.J. Dewar, H. Wilson, A.K. Winterburn, and H.W. Read- ing (1974) Histological and biochemical studies on the retina of a new strain of dystrophic rat. Exp. Eye Res., 18~119-133.

Yudkin, A.M. (1927) Congenital anophthalmos in a family of albino rats. Am. J. Ophthalmol., 10:341-345.

132:89-91.