A C T A O P H T H A L M O L O G I C A V O L . 5 3 1 9 7 5

University Eye Department (Head: Professor Thore Lie Thomassen), and the Institute o f Pathology, Electron Microscopic Laboratory,

(Head: Torstein Hovig, M.D.) , Rikshospitalet, Oslo, Norway

THE SUSPENSORY APPARATUS OF THE LENS. THE SURFACE OF THE CILIARY BODY

A scanning electron microscopic study

BY

MARTIN DAVANGER

Normal anterior segments from five human eyes were prepared for scan- ning electron microscopy by fixation in glutaraldehyde and 0 ~ 0 4 , careful dissection, drying and coating. The lens equatorial region, the zonules and the ciliary body were examined from behind and front. The zonules were found to insert at circular lines immediately in front of and behind the equator of the lens, and some directly on to the equator. In the ciliary body they inserted along the ridges of the processes. Their insertions by ramifi- cation are studied in detail. No zonules were found to insert into or pass through the ciliary valleys, nor did they insert on pars plana or ora serrata. Short zunule-like strands connected the posterior end of the processes with pars plana, and a mat consisting of confluent zonule-like fibers, running in a meridional direction, covered the surface of pars plana. The zonules inserting behind the lens equator seemed to adhere to the anterior face of the vitreous, but they did not merge into it.

Key words: zonules of Zinn - lens - ciliary body - vitreous - accommoda- dation - scanning electron microscopy.

Details of the gross anatomy of the zonules and their relation to neighboring structures are difficult to establish. This part of the eye has a very complicated macroscopic architecture. The tissues are delicate and of varying texture. It is difficult to reconstruct the three-dimensional anatomy of this region from micro- scopic sections. The value of an examination with a dissecting microscope is limited because of the relative invisibility of the vitreous and the zonules, and

Received January 3, 1975.

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Martin Davanger

also because of the low magnification and poor depth resolution inherent in this method.

The insertion and course of the zonules, and also their relations to neighboring structures, is therefore still in dispute. Inconsistent information is obtained from the literature.

Most authors (McCulloch 1954, Vail 1957, Rohen & Rentsch 1969, Hogan et al. 1971) assert that the majority of zonular fibers arise from the region of ora serrata or from pars plana ciliaris (Raviola 1971). It is stated that the zonules course forward from ora serrata, hugging the concavity of pars plana, being pushed into place by the vitreous. When reaching the posterior margin of the ciliary processes they separate into groups which run forward in the valleys be- tween the processes, and supporting fibers merging with the main system of zonules arise in the valleys. It is firmly asserted that the zonules do not insert on the crest of the ciliary processes (for ref., see Vail 1957).

On the other hand, Kaczurowski (1964) found that the posterior zonules emerge from the ciliary processes, also from their apices, while the anterior zonules come from the bottom of the ciliary valleys and at times from pars plana.

It was thought that scanning electron microscopy might be a valuable method for the examination of this region. The scanning electron micrographs have the advantage of giving information on the three-dimensional architecture of con- volute surfaces. The depth resolution is increased about 200 times as compared with incident-light micrographs of the same magnification. The magnification may be varied within a wide range without changing the position of the speci- men, whereby pictures taken with high magnification are easily localized on the specimen.

The purpose of this work is to study the detailed gross anatomy of the zonules and the surfaces of their neighboring structures by the help of the scanning electron microscope.

Material and Methods

Five eyes from adult humans were examined. Three of these were enucleated because of a choroidal melanoma affecting the posterior segment only, while two were donor eyes taken from the same person. The former were processed immediately after enucleation, while the latter were enucleated some hours post mortem and kept in a moist chamber at 4 O C a few hours before processing. The eyes were carefully cut along equator. The anterior halves were prefixed for 2 hours in 2.5 O/o glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. After rinsing in isotonic buffer, the specimens were postfixed for 2 hours in 1 O/O Os04 in Millonig's phosphate buffer, pH 7.4, and dehydrated in graded ethanol solutions.

20

The Suspensory Apfiaratus of the Lens

At this stage, the vitreous of three eyes was carefully removed from behind under a dissecting microscope, while the eyes were submerged in ethanol. The vitreous was firmly adherent to the region of ora serrata, but after being torn at this site, the anterior face of the vitreous was only loosely adherent to pars plana, the posterior zonules and the lens, and could be lifted off in most cases in a con- tinuous membrane (as was later confirmed by a scanning electron microscopy of the anterior face of the vitreous; to be published). The careful removal of the vitreous did not cause any visible damage to the zonules.

The sclera and cornea were removed in one piece by careful dissection with a spatula inserted between the sclera and the ciliary body by a procedure similar to that used in cyclodialysis. In two eyes, in which the vitreous remained, the iris was removed by tearing along the iris root. These two eyes were viewed from the front in the scanning electron microscope. One of the two donor eyes was seen from the front, while its fellow eye was examined from behind.

The two first processed eyes were dryed in air at this stage. It was found, however, that because of an uneven shrinkage of the different tissues, the ciliary body and the zonules became irregularly deformed during the drying process. Therefore, only parts of the specimens could be examined, and a general view could not be obtained. T o avoid this deformation, the following procedure was applied to the remaining three eyes: The specimens were suspended in propylene oxide, thereafter in Epon 812, and kept in room temperature for 2 days. They were then washed in propylene oxide under a dissecting microscope with the help of a pipette until the Epon solution seemed to be completely washed away from the surfaces. It is thought that some partly polymerized Epon, forming a denser skeleton, remained within the tissue. The specimens thus treated were less de- formed during the following air drying process. Some details of the surface, as seen in high magnification, may be influenced by this procedure, but it is felt that serious artifacts are not introduced in the gross anatomy of the specimens.

The specimens were air dryed in room temperature for 3 days. The first part of the drying process was followed by the help of a dissecting microscope. The specimens were mounted on specimen holders, and coated in a vacuum with a thin layer of carbon and gold-palladium. Scanning electron microscopy was per- formed with a Jeol JSM-SO A scanning electron microscope.

Results

Fig. 1 gives a general view of part of a specimen seen from behind. The zonules bridge the space between the lens equator and the ciliary processes. They tend to be collected in groups which are directed towards the processes. The zonules

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Martin Davanger

insert along the ridge and slope of the processes. They do not pass through or insert in the valleys as most earlier authors have described (Vail 1957, Rohen & Rentsch 1969, Hogan et al. 1971). At the site of insertion the zonules split up in a number of fibrils (Figs. 1, 2 and 3 ) , which can be followed as distinct entities along the surface of a considerable part of the processes. The fibrils gradually

Fig. 1. General view of a part of the posterior equatorial region of the lens (L), the zonules, the iris (i), the ciliary processes (CP), and pars plana ciliaris (PP). The zonules are partly collected in irregular bundles directed towards the processes, where the zonules insert mainly along their ridges. Short zonule-like strands (arrows) connect the posterior end of the processes with the anterior part of pars plana. Zonula-like strands insert on the posterior iris surface (double arrows). Areas indicated by right angles are seen in

higher magnification in Figs. 2 and 3. x 49. Bar = 0.5 mm.

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The Suspensory Ap/xzrutiis of the Lens

Fig. 2. Area indicated in Fig. 1 (left), in higher magnification. The insertion of groups of zonules along the ridge of two ciliary processes. The zonules ramify along the epithelial surface. In thc valley (V) between the main processes, thin strands are extended between

small irregular processes. x 190. Bar = 0.1 mm.

thin out and disappear in the posterior part of the processes (Fig. 1). Some strands of the fan may bridge over to neighboring processes, or to small, irregu- lar processes situated in the valleys between two main processes (Fig. 3). These small processes are often connected by thin strands, which do not originate from the main or true zonules (Fig. 2).

In the region of the ciliary processes and the valleys the surface is very convolute.

Zonule-like short fibers bridge between the posterior end of the processes and pars plana (Figs. 1 and 4). These fibers are not continuous with the zonules which attach to the lens. Anteriorly, they split up in a fan and attach to the posterior part of the processes in much the same way as was described for the zonules coming from the lens. Posteriorly, these fibers merge into the surface of pars plana. This surface is covered by a continuous mat of confluent long fibers running in a meridional direction (Fig. 5 ) . A few strands attached to the surface

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Martin Davanger

of this mat cross the main fiber direction. They may end rather abruptly, ramify- ing into a short fan (Fig. 5, arrow and inset).

Turning towards the attachment of the zonules to the lens (Fig. 6), the zonules are seen to split up into a few thinner units a short distance above the lens surface. Here, the zonules ramify further into thinner and thinner fibrils (Fig. 7 ) . The fans lie flat on the lens surface and broaden until they merge with neigh- boring fans and form a continuous mat of thin fibrils directed towards the lens pole (Figs. 8 and 9). As seen from the surface, the mat of fibrils terminates by the fibrils gradually becoming thinner (Fig. 10).

Fig. 3. Area indicated in Fig. 1 (center), in higher magnification. Detail of the insertion of

zonules on the convolute surface of ciliary processes. x 465. Bar = 50 .urn.

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The Suspensory Apparatus of the Lens

Fig. 4. The posterior parts of the ciliary processes and the anterior part of pars plana ciliaris (PP). The posterior end of the processes are connected with short zonule-like strands to

the anterior part of pars plana. x 120. Bar = 0.1 mm.

In the eyes examined from the front, the anterior zonules are seen to attach to the anterior equatorial region of the lens along a circular line in much the same way as the posterior zonules (Fig. 9). The anterior zonules are directed towards the anterior end of the ciliary processes, where they attach to the ante- rior part of their ridges. In Fig. 9, the anterior face of the vitreous can be seen in the background. The zonules from the posterior lens surface are seen to lie against the vitreous face, to which they seem to adhere, but they do not seem to merge into it.

In addition to the zonules which insert along a circular line on the anterior and posterior equatorial region of the lens, some zonules insert directly on the

25

M a r t i n D a v a n g e r

F i g . 5.

F i g . 6 .

26

The Suspensory Afi@nratirs of the Lcns

Fig. 7. Area indicated in Fig. 6, in higher magnification. Ramification of the zonules at their insertion on the posterior lens surface. Meridional striation of the lens equatorial region.

x 130. Bar = 0.1 mm.

Fig. 5. Pars plana ciliaris. The surface is covered by a continuous mat consisting of confluent zonule-like fibers running in a meridional direction. A few superficial fibers cross the main fiber direction. Inset demonstrates the ramification of the end 0 1 one of the cross-

ing fibers (at the arrow). x 130, inset x 690. Bar = 0.1 mm, in inset Bar = 20 pm.

Fig. 6. The insertion of the zonules on the equatorial region of the lens. Oblique view from behind. This area is seen directly from behind in Fig. 1 (lower part, central and right). Most zonules insert along a equator. Area indicated by

line parallel to the lens equator, some insert directly on the right angles is seen in higher magnification in Fig. 7 . x 7 7

Bar = 0.2 mm.

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Martin Davanger

Fig. 8. Detail of brush-like ramification of a zonule inserting on the posterior lens surface.

x 1150. Bar= 10pm.

equator (Fig. 6). The whole equatorial region of the lens is striated in a meridio- nal direction (Fig. 7 ) .

In Fig. 1 it is seen that a few zonule-like strands attach to the posterior surface of the iris. These strands are irregularly distributed along the corona ciliaris, and their number is about half that of the ciliary processes. Thin strands run- ning in an oblique or circular direction may connect neighboring zonules (Figs. 1 and 6). In high magnification, a cobweb-like veil can be seen in the ramifica- tions of zonules or at their insertions on the lens. By this method of preparation, no matrix enveloping the zonules can be recognized.

The main zonules do not form a cross when bridging between the lens and the ciliary body (Fig. 6).

In some specimens, all the fibrils running along the posterior lens surface end abruptly along a sharp continuous circular line, obviously by being torn (Fig. 11). This line is probably Egger’s line, to which the vitreous has been attached. When

28

The Suspensory A/?/xiratiis of !lie Lens

the edge of the sheet seen in Fig. 1 1 is examined in high magnification, it can not be seen that fibrils enter into the lens capsule.

The diameter of the zonules is found to vary up to 40-50 pm in these speci- mens. Their cross-section seems to be circular, and their surface is usually smooth and even, also in high magnification. However, the zonules may have longitudinal stripes or grooves, consistent with their being composed of thinner subunits. These subunits split apart in the ramification near the insertion of the zonules, and they can also split from one zonule to merge with a neighboring zonule.

Fig. 9. The anterior equatorial region of the lens, the zonules, the anterior face of the vitreous (V), and the anterior end of the ciliary processes. The posterior zonules lie upon and seem to adhere to the anterior surface of the vitreous (arrow). The anterior zonules insert

near the anterior end of the ciliary processes. x 67. Bar = 0.2 mm.

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Martin Davanger

Fig . 10. The central termination of the zonular insertion on the anterior lens surface. The brushes on the lens surface seen in Fig. 9 terminate in the gradual thinning out of the bundles

of fibrils. Highlight on a fold of the lens surface. x 1680. Bar = 10 pm.

Discussion

It is felt that this method is appropriate for the study of the gross, detailed anatomy of the region to be examined. It is presumed that the scanning electron micrographs give a representative impression of the conditions in vivo. The zonules seem to remain intact and their insertions and course appear undisturbed after the processing of the specimen as described above.

The findings in this study differ in some respects from the descriptions given by most earlier authors. It has been believed that the zonules attached mainly to ora serrata (McCulloch 1954, Vail 1957, Rohen k Rentsch 1969, Hogan et al.

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The Szispensory Apfiaratris of the Lens

1971), to pars plana (Raviola 1971), and to the valleys between the ciliary pro- cesses. According to the findings in the present work, the zonule-like strands found connected with these structures are not true zonules as they do not attach to the lens.

Rohen & Rentsch (1969) based their new theory of the mechanism of accomo- dation on the distinction between two functionally different groups of zonular fibers, the socalled main fibers and the span fibers. Their concept of the archi- tecture of the zonula apparatus is not supported by the present study.

I t has been asserted that "- zonular fibers are but specialized portions of the anterior peripheral vitreous framework, - the hyaloideo-capsular ligament is essentially synonymous with the attachment ring of the posterior zonular fibers." (Fine & Yanoff 1972, Fine & Tousimis 1961). Further, the suspensory apparatus of the lens has been described as a gel-like structure with many of the properties of the vitreous (Hogan et al. 1971), or as a membranous part of the vitreous (Duke-Elder 1930). These concepts may perhaps be explained by the difficulties

F i g . 11. The posterior equatorial region of the lens. The zonules seem to form, or insert in a sheet on the lens surface, this sheet is torn along a distinct line, Egger's line? x 170.

Bar = 0.1 mm.

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Martin Davanger

in distinguishing between true zonules and the hyaloideo-capsular fibrils of the ligament of Wieger inherent in other preparatory techniques, e.g., embedding and sectioning.

According to the findings in the present study, the zonules are distinct, real and independent entities which can not be considered as a part of the vitreous. Their course is direct and simple. The zonules bridge the shortest distance be- tween the lens equatorial region and the ciliary body. Here they insert on the anterior part of the ridges of the ciliary processes. There is no systematic cross- ing of zonules as they span between the lens and the ciliary processes. The zonules do not run continuously from the lens toward ora serrata or pars plana ciliaris. In the valleys between the ciliary processes there are no true zonules. Zonule-like strands which do not insert on the lens run in a meridional direction between the posterior end of the processes and pars plana, and along the surface of pars plana. They seem to reinforce these structures for forces in a meridional direction. The forces involved in the mechanism of accomodation are thought to be transferred to the lens via these strands and the true zonules.

Ac knowledgrnents

Representatives of JEOL are sincerely thanked for instruction in the use of the scanning electron microscope. Thanks are also due to Mrs. Sigrid Lystad, for instruction in the use of the vacuum coating unit.

References

Duke-Elder, W . S. (1930) The nature of the vitreous body. Brit. /. Ophthal., Supfil. 4 . Fine, B. S. & Tousimis, A. J. (1961) The structure of the vitreous body and the suspen-

sory ligaments of the lens. Arch. Ophthal. 65, 95-1 10. Fine, B. S. & Yanoff, M. (1972) Ocular Histology. A Text and Atlas, p. 125. Harper &

Row, New York, Evanston, San Francisco, London. Hogan, M. J., Alvarado, J. A. & Weddell, J. E. (1971) Histology of the Human Eye.

An Atlas and Textbook, pp. 273 and 652. Saunders Co., Philadelphia, London, To- ronto.

Kaczurowski, M. I. (1964) Zonular fibers of the human eye. Amer. /. Ophthal. 58, 1030- 1047.

McCullock, C. (1954) The zonule of Zinn: Its origin, course and insertion, and its rela- tion to neighboring structures. Trans. Amer. Ophthal. Soc. 52, 525-585.

Raviola, Giuseppina (1971) The fine structure of the ciliary zonule and ciliary epithe-

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The Suspensory Apparatus of the Lens

hum. With special regard to the organization and insertion of the zonular fibrils. Invest. Ophthal. 10, 851-8G9.

Rohen, J. W. & Rentsch, F. J. (1969) Der konstruktive Bau des Zonulaapparates beim Menschen und dessen funktionelle Bedeutung. Morphologische Grundlagen fur eine neue Akkomodationstheorie. Albrecht v. Graefes Arch. Ophthal. 178, 1-19.

Vail. D. (1957) The zonule of Zinn and ligament of Wieger. Trans. Ophthal. SOC. U.K. 77, 441-499.

Author’s address: Martin Davanger, M.D., University Eye Department, Rikshospitalet, Oslo, Norway.

33 Acta ophthal. 53 I 3