364 Streeten est. Ophthalmol. Visual Set. April 1977

21. Jones, R. E., and Moscona, A. A.: Effects 24. Hogan, M. J., Alvarado, J. A., and Weddell, of cytosine arabinoside on differential gene J. E.: Histology of the Human . An Atlas expression in embryonic neural . I. Ac- and Textbook, Philadelphia, 1971, W. B. cumulation of glutamine synthetase with Saunders Company. suppression of macromolecular synthesis, J. 25. Hughes, W. F., and Coogan, P. S.: Pathology CellBiol. 61: 688, 1974. of the pigment epithelium and retina in rab- 22. Fine, B. S., and Yanoff, M.: Ocular Histol- bits poisoned with lead, Am. J. Pathol. 77: ogy. A Text and Atlas, Hagerstown, Md., 237, 1974. 1972, Harper & Row, Publishers. 26. Kuwabara, T., and Weidman, T. A.: De- 23. Rasmussen, K.-E.: A morphometric study of velopment of the prenatal rat retina, INVEST. the Miiller cell cytoplasm in the rat retina, OPHTHALMOL. 13: 725, 1974. J. Ultrastruct. Res. 39: 413, 1972.

The zonular insertion: a scanning electron microscopic study

Barbara W. Streeten

The zonular insertion on the human capsule has been studied with scanning electron microscopy. The whole insertional area is covered by a variety of 35 to 55 nm. fibrils, includ- ing those of the major inserting zonules, the meridional zonules, capsuhr surface fibrils, and a nonoriented fibrillar layer over the zonules. All appear to be zonular in type. The meridional zonules span the equator and are a form of insertion shared by the three levels of major zonules, probably helping to distribute their lateral putt. Surface rippling over the zonular insertion appears to result from greater contraction of the oriented fibers than of the non- oriented fibrils and superficial capsule. Surface meshwork fibrils attach to the capsule by becoming embedded in a granular capsular matrix 0.5 to 1 n thick.

Key words: zonule of the lens, scanning electron microscopy, zonular insertion, , zonular lamella.

-Lhe mechanism of zonular insertion on sporadic attention in the modern era, al- the human lens capsule has received only though it is essential to an understanding of both normal accommodative function and pathology involving these structures. From the Departments of Ophthalmology and An enduring concept of zonular attach- Pathology, State University of New York ment has been that of a "zonular lamella" Upstate Medical Center, Syracuse, N. Y. or special layer on the lens surface into This study was supported by Research Grant EY which the zonules insert, as described by 01602 from the National Eye Institute. Berger1 in 1882. By transmission electron Reprint requests: Dr. Streeten, Eye Pathology microscopy (TEM) the zonular lamella of Laboratory, State University of New York 2 Upstate Medical Center, Syracuse, N. Y. 13210. light microscopy resolves into two layers:

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Fig. 1. Equator of lens. Zonular insertional area appears light against dark lens capsule back- ground. Three levels of zonular insertion are seen: anterior (A), equatorial (E), and posterior (P). Tearing away of anterior hyaloid membrane has left posterior zonular insertion ragged. (Scanning electron micrograph, xG4.)

(1) the loose superficial capsule surround- lems of zonular fiber distribution, since the ing the lens and (2) an outer layer of in- zonule is essentially a surface tissue. How- serting zonules in the equatorial region.1' * ever, reports of the zonular insertion by It is not clear whether the zonular portion SEM have been fragmentary in both is a continuous layer or intermittent, ap- man51- and animals.13"15 Technical prob- pearing only where the zonules directly lems with preservation of the fragile zonule attach. Since the number of zonules is said and diying of the dense lens material, to be considerably reduced in aging,2 with coupled with low resolution, decrease the those at the equator being especially sparse value of some of the early descriptions. or absent, an intermittent covering might In only a few studies has resolution been be expected in the adult. sufficiently high to visualize the individual 12 14> ls Scanning electron microscopy (SEM) fibrils of the normal zonule. - The should be ideal for studying these prob- presence of a zonular lamella into which

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Fig. 2. A, Anterior zonules terminating on lens capsule. Zonular surfaces show contraction furrows. (Scanning electron micrograph, x470.) B, Fibrillar structure of zonules near their terminus. Some fibrils blending into meshwork on capsular surface. (Scanning electron micro- graph, x6,750.)

the inserting zonules "disappear" has gen- fixed immediately in 2.5 percent glutaraldehyde. erally been accepted but not defined.5'Si 15 Twenty-four lenses were obtained from Eye Bank , 4 to 12 hours after death. These lenses The ultimate attachment of the zonules were prepared in a variety of ways, including in to the lens is known to involve penetration situ fixation and extraction while unfixed, with of zonular fibers into the superficial lens the use of a capsule forceps. Most lenses were capsule,3'll! but may also include some type removed before fixation by cutting the zonules and of physicochemical binding commonly re- anterior vitreous close to the under the dissecting microscope, to preserve the ferred to as "glue." With one exception the maximum number of zonules on the lens capsule. SEM studies have not thrown light on this Eight lenses were fixed in 2.5 percent glutaralde- problem. Recently Farnsworth and co-work- hyde and the remainder in 10 per cent neutral ers1- have described a fibrillar ridging of buffered formalin. Most of the specimens were the anterior lens surface which they sug- postfixed in osmium, dehydrated in graded gest may serve as zonular attachment sites acetones, and dried in a critical-point drier. They were then coated with carbon and gold-palladium of the Velcro type. The surface fibrils on and examined in an ETEC or a Coates and Welter these ridges were considered by the authors scanning electron microscope. Eight lenses were to -be capsular in origin, although they postfixed in osmium-thiocarbohydrazide (TCH) were of the same 50 to 70 nm. diameter and coated only with gold-palladium. as the fibrils of the zonule. Results The present study will describe all areas of the zonular insertion as seen by SEM, When a lens is extracted with its at- with special consideration of the zonular tached zonules the entire width of the lamella and capsular attachments. zonular insertion can be viewed in the scanning electron microscope (Fig. 1). Methods and materials This insertional region appears as a well- Thirty human lenses were examined with SEM. demarcated light area against a darker Six were lenses removed at cataract surgery and lens capsule background. The attached an-

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Fig. 3. Final termination of smallest anterior zonular bundles (Z). Fibrils partially obscured by interfibrillar material as they join surface fibrillar meshwork. (Scanning electron micro- graph, x21,000.)

Fig. 4. Anterior lens capsule. A, 1 mm. in front of zonular insertion showing mixture of ran- dom fibrils and pebbled surface. (Scanning electron micrograph, xl6,800.) B, Center of lens with pebbled texture. (Scanning electron micrograph, xl6,800.)

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Fig. 5A. Abrasion of flbrillar surface layer in anterior insertion. Note variable rippling of the remaining layer. (Scanning electron micrograph, x645.)

terior, equatorial, and posterior zonules 2, A). (Linear measurements are not accu- join this band at fairly regular intervals, rate due to a 20 percent shrinkage of the often in double rows. lens in processing.) An interfibrillar ma- The anterior and posterior zonules in- terial often obscures the fibrils of the larger sert as bundles of 25 to 60 p. diameter in zonules, but where visible they are 35 to their contracted state. The equatorial bun- 55 nm. in diameter. Loose peripheral fibrils dles can be as large (Fig. 1) but are often blend into a flbrillar meshwork on the small, from 10 to 15 \x. in diameter, seen capsular surface (Fig. 2, B). This mesh- at all ages into the ninth decade. As the work is thrown into consistent but irregular zonules spread out on the capsule the ridges in the inserting area. The anterior larger ones rapidly separate into 5 to 10 zonules end as 0.3 to 0.4 /± fanning bundles, /J. bundles. The anterior zonules continue blending into the surface layer in basket- to intermingle and decrease in size over a weave fashion (Fig. 3). 0.3 to 0.4 mm. flat insertional area (Fig. The fibrils on the capsular surface are

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Fig. 5B. Edge of abraded area shown in box in Fig. 5A. Fibrils of surface layer adherent to deep meshwork. Bare surface is granular with evidence of interfibrillar matrix. (Scanning electron micrograph, xlO,75O.)

also 35 to 55 nm. in diameter. They con- Where the base of the fibrillar layer is tinue for 0.2 to 0.3 mm. beyond the ap- exposed the attachment of its fibrils to a parent ends of the zonules. Random fibrils deep meshwork of fibrils can be appre- may be seen for another 0.4 to 0.6 mm. ciated (Fig. 5B). An admixture of inter- centrally (Fig. 4, A). The typical pebbly fibrillar matrix and capsular granules covers surface shown by basement membranes is them. Some radially oriented longer fibers increasingly evident among these surface of 0.3 to 0.7 p. width apparently travel fibrils (Fig. 3). In the central 3 to 4 mm. in this layer also (Fig. 6). of capsule pebbly granules form the only Between the anterior and posterior zonu- textural element (Fig. 4, B). They range lar insertions the fibrillar layer is thickened in size from 40 to 160 nm., with the larger by the addition of large numbers of merid- ones more common near the zonules. This ional fibers of 3 to 7 JX in width, causing characteristic granularity of bare lens cap- striations longitudinally across the whole sule is useful in identifying areas of zonular equator (Fig. 7). These are contributed loss or admixture of zonule and capsular to by vertical arms from all three levels material. of zonules. Horizontal rippling across this Patches in which the fibrillar layer has whole layer of zonules can be marked been abraded are helpful in elucidating (Fig. 7, B). Over the surface here as in the nature of this layer (Fig. 5). In the most other areas there is a loose mesh- anterior insertional area it is about 0.5 work of fibrils the same size as the zonular to 1 ix thick, with a tendency to contract, fibrils, bridging the spaces between them roll outward, and be thrown into ripples. (Fig. 7, D). When abraded parallel to the

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vw Fig. 6. Frayed patch in fibrillar surface layer showing longer aggregated fibrils among the short fibrils of the meshwoik. (Scanning electron micrograph, x21,500.)

oriented zonules these meshwoik fibrils are zonules have a fanning insertion like the shown adhering to the capsule superficially anterior, but more obscured by meshwork (Fig. 8). This perizonular meshwork ap- fibrils (Fig. 10). The insertion is 0.4 to pears to blend freely with the straight 0.5 mm. in width, ending in a jagged border zonular fibrils so that no significant differ- when the anterior hyaloid membrane is ence is seen between them. Deep zonular torn away (Fig. 1), which is the usual penetration is seen occasionally in abraded occurrence in the adult. Occasional seg- areas (Fig. 9). mentally contracted zonules are seen here The equatorial and posterior zonules in- and in the anterior insertion (Fig. 11). sert more vertically than the anterior zon- ules. Their bundles are lost sight of quick- Discussion ly as they disappear into the even thicker By SEM the whole insertions! region is fibrillar layer present here. The posterior covered by either aggregated zonular bun-

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Fig. 7. Meridional zonules (MZ's). A, MZ's producing longitudinal striations on lens equator. Anterior zonules at top, posterior below. (Scanning electron micrograph, xl95.) B, Hori- zontal rippling across elevated MZ's. (Scanning electron micrograph, x2,000.) C, Equatorial zonule joining meridional rows. (Scanning electron micrograph, x470.) D, Fibrillar meshwork over whole meridional zonular layer. (Scanning electron micrograph, x20,500.)

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Fig. 8. Pre-equatorial zonule inserting into meridional layer. Abraded edge shows layer to be 1 to 2 n thick. Fibrils adherent to bare lens capsule, lower right. Zonular fibrils merge with those of fibrillar surface meshwork. Straight fibrils of meridional zonules visible through the meshwork (arrows). (Scanning electron micrograph, x6,300.)

dies or fibrils. Thus the outer or zonular zonular fibrils. Morphologically there is no portion of the zonular lamella appears to significant difference among them with be a continuous layer, although undoubted- SEM, since they all measure 35 to 55 nm. ly very thin in some interzonular areas. The in diameter. This is four to five times the question is whether all of the fibrils seen size of zonular fibrils we and others have on the lens in SEM are zonular, since their measured in TEM,S possibly representing degree of organization is so different. Be- coating thickness, and is within the range sides the obvious inserting and meridional described by other workers with SEM.12 zonular fibrils there are the less aggre- The suggestion that the capsular surface gated capsular fibrils and the loose peri- fibrils are part of the normal capsular

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Fig. 9. Peeling of equatorial zonular complex from capsule reveals deeper small zonules (arrows). (Scanning electron micrograph, x525.)

fibrillar structure'- does not seem likely, been pictured before in SEM7- s> ll) but since, they do not appear in areas of bare their relation to the large inserting zonules or abraded capsule, and are too large. With has not been fully appreciated. While they TEM the fibrils of the normal lens capsule are clearly contributed to by the three (as distinct from aging inclusions)17 in our levels of zonules, there is some suggestion material are 1 to 3 nm. in diameter, that their bulk is greater than can be whereas zonular fibrils average 10 nm. accounted for by the inserting zonules. The possibility that the perizonular fi- Equatorial zonules are indeed sparse and brils might be vitreals is bound up with do not contribute much to this pattern. the question of whether the whole zonule Functionally the orientation of the merid- is a modified vitreous. TEM should demon- ional zonules and their complete coverage strate whether they have the lucent core of the equator would seem designed to of a typical zonular fiber1 but ultimately spread the lateral pull of the major zon- biochemistry must answer the question of ules more smoothly over the equator, and relationship. perhaps to accomplish an equatorial Most of the insertional area is covered squeeze when the major zonules are relaxed by meridional zonular fibers. The striations in accommodation. which these cause on the lens equator have Concentric rippling of the superficial

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Fig. 10. Insertion of posterior zonules on left. Anterior hyaloid membrane (AH) is peeling back on the right. (Scanning electron micrograph, x210.) Fig. 11. Posterior zonules folded back on themselves showing smaller fibers entering fibrillar meshwork. One zonule is segmented, also seen occasionally in other areas. (Scanning electron micrograph, xl,075.)

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capsule in the insertional area, as noted by 4. Fine, B. S., and Yanoff, M.: Ocular Histology, Farnsworth and co-workers,12 is a consistent Hagerstown, Md., 1972, Harper & Row, Publishers. feature in several species with different 5. Oberman, E. A.: Scanning electron micros- fixatives and is seen in many pub- copy of the lens and zonular fibers, Am. J. 1 4 lished TEM figures, - so it must repre- Ophthalmol. 72: 604, 1971. sent a basic characteristic. It is most marked 6. Worthen, D. M.: Scanning electron micros- where the major zonular attachments are copy after <*-chymotrypsin perfusion in man, loose or absent, so the Velcro-type adhe- Am. J. Ophthalmol. 73: 673, 1972. 7. Hervouet, F., Goerge, Y., Tusques, J., et a.L: sive function suggested by these au- Structure du crystallin humain et de la thors seems questionable. Velcro consists of zonule de Zinn en microscopie electronique two different opposing surfaces. The loose a balayage, Bull. Assoc. Anat. (Nancy) 58: capsular fibrils bear a superficial semblance 349, 1973. to the loops of one Velcro surface, but 8. Bornfeld, N., Spitznas, M., Breipohl, W., et al.: Scanning electron microscopy of the there is no apparent counterpart to the zonule of Zinn. 1. Human eyes, v. Graefes stiff hooks of the necessary opposing sur- Arch. Klin. Exp. Ophthalmol. 192: 117, 1974. face. 9. Seland, J. H.: The lenticular attachment of The variability of the concentric rippling the zonular apparatus in congenital simple suggests that it is a plastic feature, related , Acta Ophthalmol. 51: 520, 1973. to the malleability of the loose superficial 10. Davanger, M.: The suspensory apparatus of capsule and the surface fibrillar meshwork the lens. The surface of the , when subjected to the contraction or elas- Acta Ophthalmol. 53: 19, 1975. tic tension of the oriented zonules. Small 11. Reich, M. E., Roll, P., Hofmann, H., et al.: hidden zonular bundles probably also con- Rasterelektronenmikroskopische Untersuchun- tribute to this anteroposterior tension. gen des vorderen Augenabschnittes mit besonderer Berucksichtigung des Verlaufs Rippling of the fibrillar meshwork over the der Zonula Zinnii, v. Graefes Arch. Klin. Exp. meridional zonules clearly results from Ophthalmol. 195: 271, 1975. greater contraction of the oriented zonular 12. Farnsworth, P. N., Mauriello, J. A., Burke- fibrils than the unoriented. Gadomski, P., et al.: Surface ultrastructure Not much can be said of the deep zonu- of the human lens capsule and zonular attachments, INVEST. OPHTHALMOL. 15: 36, lar insertions with SEM. The attachments 1976. which were seen consisted of individual 13. Hansson, H. A.: Scanning electron micros- fibrils blending with the surface meshwork copy of the zonular fibers in the rat eye, Z. or capsule. The surface meshwork fibrils Zellforsch. 107: 199, 1970. were embedded to a depth of 0.5 to 1 ^ 14. Anderson, D. R.: Scanning electron micros- in capsular matrix, as judged by the ad- copy of zonulolysis by a-chymotrypsin, Am. J. Ophthalmol. 71: 619, 1971. mixture of pebbly granules and gluelike 15. Erckenbrecht, J. F., and Rohen, J. W.: material. Rasterelektronenmikroskopische Untersuchun- REFERENCES gen iiber den Zonulaapparat hohere Primaten als weiterer Beitrag zu einer neuen Akkom- 1. Berger, B.: Beitrage zur Anatomie der Zonula modationstheorie, v. Graefes Arch. Klin. Exp. Zinni, v. Graefes Arch. Ophthalmol. 28: 28, Ophthalmol. 193: 19, 1975. 1882. 16. Seland, J. H.: Ultrastructural changes in the 2. McCulloch, C: The zonule of Zinn: its normal human lens capsule from birth to origin, course and insertion, and its relation old age, Acta Ophthalmol. 52: 688, 1974. to neighboring structures, Trans. Am. Oph- 17. Dark, A. J., Streeten, B. W., and Jones, D.: thalmol. Soc. 52: 525, 1954. Accumulation of fibrillar protein in the aging 3. Hogan, M. J., Alvarado, J. A., and Weddell, human lens capsule, Arch. Ophthalmol. 82: J. E.: Histology of the , Phila- 815, 1969. delphia, 1971, W. B. Saunders Company.

(End of Symposium—Part I)

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