Zonula Research Articles

Morphological and biochemical studies of the structure of the vitreous and the zonular fibres

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Johann Gottfried Zinn - ein fränkischer Anatom und Botaniker

Johann Gottfried Zinn (1727-1759), a franconian anatomist and botanist made important contribution to the anatomy of the eye during his short life span. In his main book "Descriptio anatomica oculi humani" he gave the first detailed and comprehensive description of the anatomy of the human eye. During his professorship at the university of Göttingen the emphasis of his scientific work was in the field of botany. His memory will be kept by the anatomical terms "zonula ciliaris Zinnii" and the circulus arteriosus sclerae = Zinn-Haller's arterial circle. As a botanist Zinn was honoured by the fact that a flower (Zinnie) was named after him.

Extraction of extendable beaded structures and their identification as fibrillin-containing extracellular matrix microfibrils.

High molecular weight aggregates were extracted from human amnion using buffers containing 6 M guanidine hydrochloride. Rotary shadowed preparations and negatively stained samples examined by electron microscopy showed that each aggregate appeared to be a string of globular structures joined by fine filaments, giving the appearance of beads on a string. The periodicity of the beads was variable. A mouse monoclonal antibody directed against a previously characterized pepsin fragment of fibrillin was used with gold-conjugated secondary antibody and immunoelectron microscopy to show that the aggregates contained fibrillin. Similar structures were found in non-denaturing homogenates of skin, tongue, ligament, ciliary zonule, cartilage, and vitreous humor. When immunogold-labeled beaded structures were prepared for electron microscopy in the same manner as tissue, the beaded structures could no longer be seen. Instead, gold-labeled microfibrils were found which appeared to be the same as the fibrillin-containing matrix microfibrils observed in connective tissues and often associated with elastin. Thus, standard TEM protocols including fixation, dehydration, and embedding alter the ultrastructural appearance of microfibrils as compared with negative stain or rotary shadowing techniques. When skin was stretched and prepared for electron microscopy while still under tension, beaded filaments were seen in the tissue sections, but were not visible in non-stretched controls. In addition, when stretched ligament was immunolabeled with antibody directed against fibrillin while still under tension, the periodicity of antibodies along the microfibrils increased compared with non-stretched controls. We propose that microfibrils contain globular structures connected by fine filaments composed at lease in part of highly ordered, periodically distributed fibrillin molecules, whose periodicity is subject to change dependent on the tensional forces applied to the tissue in which they are contained.

The .ALPHA.1 Chain of Type VIII Collagen is Associated with Many butNot All Microfibrils of Elastic Fiber System.

The antigen of monoclonal antibodies which had labeled the hexagonal lattice of Descemet's membrane in a specific manner was shown to be the alpha 1 chain of type VIII collagen by immunoblotting followed by amino acid sequence analysis. With this antibody, the localization of alpha 1 (VIII) in various tissues was studied by several immunocytochemical methods. Under light microscopy, the alpha 1 (VIII) was found in a fine fibrillar form in various capsular tissues such as capsules of the liver, kidneys, adrenals, lungs and so on. It was also present in dense connective tissues such as the Achilles tendon, and periodontal and perivertebral ligaments. When some dense connective tissues which had been negative to the label including the intima of aorta, perimysium and Glisson's sheath of the liver, were subjected to pepsin digestion, epitopes were revealed which showed a specific immunofluorescence pattern. In many locations the pattern of localization coincided with that of elastic fiber components, and full or partial colocalization with tropoelastin or costaining with resorcin-fuchsin staining was observed. In immunoelectron microscopy, the antigen (alpha 1 (VIII)) was localized on the surface of, but not inside, elastic fibers. However, some tissues which are rich in elastic fibers or microfibrils remained unlabeled. These included elastic fibers of the aortic media and ligamentum nuchae as well as ciliary zonules. Therefore it is suggested that alpha 1 (VIII) is a collagen associated with microfibrils of some elastic fiber systems, but is not an intrinsic component of either elastic fibers or of microfibrils.

Development of actin polygonal arrays in rabbit lens epithelial cells

In searching for a clue to the role of actin filament bundles organized into polygonal arrays, or geodomes, in lens epithelial cells, we examined several physical events occurring in the young rabbit lens which may initiate their formation. We used NZW rabbits between the ages of 24 days gestation and 50 days postnatal. Data were obtained from TEM, SEM and fluorescence microscopy. Parameters measured were lens weights, apical surface areas of cells in epithelial whole mounts, epithelial cell thickness, and timing of eyelid opening, breakdown of the tunica vasculosa lentis (TVL) and formation of the ciliary zonules; these findings were correlated with the first signs of development of the arrays. Polygonal arrays formed slowly beginning at one to two days after birth, and with advancing time these thickened and made more numerous connections with the lateral plasma membranes. Development of the arrays was not correlated with onset of vision or disappearance of the TVL or a sudden increase in cell area, since these events occur postnatally at about 9-10 days, nor with the development of zonular fibers since these are already in place at 24 days of gestation. Only lens weights showed a dramatic increase between 24 days gestation and birth. It is surmised that the expanding lens mass may be involved in some way in signaling the organization of actin filaments into geodomes.

Lens Luxation in the Dog and Cat

Dislocation, or luxation, of the lens arises as a result of rupture of the zonular fibers, which suspend the lens from the ciliary body. In the dog, lens luxation is most frequently encountered as a primary, heritable condition in which there appears to be an inherent weakness of the zonule. The condition is limited mainly to the Terrier breeds but has also been described in the Tibetan Terrier and Border Collie. The disease is essentially bilateral but seldom becomes apparent clinically before 3 or after 7 years of age. In most primary luxations, the lens passes into the anterior chamber and such cases must be regarded as emergencies on account of the likely development of secondary glaucoma; posterior luxations are usually less troublesome. Other causes of lens luxation include cataract formation, glaucoma, and uveitis. Congenital dislocations and those attributable to trauma alone are rare. In the cat, lens luxations are usually secondary and arise in later life.

Ultrastructural immuno-localization of tropoelastin in the chick eye

Immuno-gold labeling at the electron-microscopy level was used to investigate the distribution of tropoelastin in the chick eye. Intense staining was found in the amorphous part of mature elastic fibers in different regions of the organ. In elaunin fibers, both the amorphous core and the surrounding microfibrils were clearly labeled. In addition, reactive sites were detected in the oxitalan fibers of the stroma- of the cornea and in Descemet's membrane, which showed a gradient of reactive sites increasing from the center toward the periphery. Oxitalan fibers of the stroma often fused with Descemet's membrane; the pattern of immunological staining suggested a continuity between the two structures. In the ciliary zonule, labeling for tropoelastin was observed in discrete areas on the bundles of microfibrils. The results show a complex structural organization of elastic tissue; this may be important in endowing the various parts of the eye with different mechanical properties.

Posterior Capsule Tears Using Phaco-emulsification Causes, Prevention and Management

A 2-year study of posterior capsule tears during cataract extraction and lens implantation was carried out at the Gimbel Eye Centre. 7174 consecutive cases of cataract extraction were analyzed in a prospective study. 7159 cases or 99.8% were accomplished using phaco-emulsification. Eight or 0.1% were by extracapsular extraction (ECCE), 2 of which were planned and 6 which were converted from phaco-emulsification because the nucleus was too dense. Seven or 0.1% were by intracapsular extraction (ICCE), 3 of which were planned and 4 which were converted from phaco-emulsification, all because the zonular fibres were too loose to support the capsule. Of the 7169 cases attempted by phaco-emulsification, an intraoperative posterior capsule (PC) tear occurred in 36 cases or an incidence of 0.5%. Of these 36 cases, 35 involved planned intraocular lens implantation, all of which were achieved. The causes of PC tears, their management, and outcomes were examined. The contribution of anterior Continuous Circular Capsulorhexis (CCC) in the prevention of posterior capsule tears is discussed and the application of this same principle to posterior continuous circular capsulorhexis (PCCC) as developed by the author for management of some posterior capsule tears is presented.

In Vivo Videography of the Rhesus Monkey Accommodative Apparatus

Fourteen rhesus monkeys, aged 1 to 24 years, underwent permanent implantation of a bipolar stimulating electrode into the Edinger-Westphal nucleus and complete unilateral or bilateral iridectomy. Slit-lamp Scheimpflug videography of the lens and slit-lamp goniovideography of the lens equator, zonule, and ciliary body allowed direct real-time observation and video recording of the movements of these structures during centrally stimulated accommodation and during disaccommodation. Scalloping of the lens capsule at the zonular insertion sites was clearly visible during disaccommodation and even during accommodation when the zonules were folded. During accommodation, the lens became axially thicker, the ciliary ring narrowed, and, at high levels of accommodation, the zonular fibers slackened and even folded and the lens moved downward. With increasing age and concomitantly decreasing accommodative amplitude, these excursions all diminished, so that in the oldest animals, they were very minimal or absent. Maximum centrally stimulated accommodative amplitude declined with age on a time scale similar to that for cholinomimetic drug-induced accommodation in the rhesus monkey and voluntary accommodation in the human.

Association of fibronectin with the microfibrils of connective tissue

The association of fibronectin with the microfibrils of connective tissue was examined in the zonular fibers of the mouse eye by immunohistochemical methods at the light and electron microscopic level. Mouse eyes fixed in formaldehyde were embedded either in paraffin for immunostaining by the peroxidase-antiperoxidase (PAP) method or in Lowicryl for immunolabeling by antirabbit globulin antibodies bound to 5 or 15 nm gold particles. Ultrastructural studies were also carried out after glutaraldehyde perfusion. Both the PAP and immunogold procedures demonstrated the association of fibronectin with microfibrils. After immunolabeling with 5 nm gold particles, examination at high magnification localized fibronectin to fine filaments that appeared to be attached to the surface of microfibrils. The filaments extended outward singly or formed loose aggregates. Their diameter ranged from 1.2 to 3 nm, with a mean of 1.5 nm. Because of their similarity to the fibronectin molecules previously described after rotary shadowing, the filaments were likely to be fibronectin molecules themselves. Since fibronectin is known to have high affinity for the amyloid P component, a model is presented in which fibronectin filaments are bound to the amyloid P component making up the tubular core of microfibrils in mice. Evidence is presented that fibronectin filaments may link microfibrils to one another and thus insure the continuity and strength of zonular fibers. More generally, it is likely that connective-tissue microfibrils, whether or not inserted into elastic fibers, are bonded through fibronectin to surrounding cells, collagen fibrils, or proteoglycans, and thus insure cohesion among connective tissue elements.

Glycoconjugates in exfoliation syndrome. A lectin histochemical study of the ciliary body and lens.

The binding of ten biotin-coupled lectins with different carbohydrate specificities to exfoliative material and neighbouring tissues was studied in 16 formalin-fixed and paraffin-embedded human eyes. Eight of the eyes were exfoliation positive while the rest were exfoliation negative. Exfoliative material reacted intensely with Lens culinaris (LCA), Canavalia ensiformis (ConA) and Ricinus communis (RCA-I) agglutinins. Positive reaction was also seen with wheat germ (WGA), peanut (PNA) and soybean (SBA) agglutinins. The superficial zonular lamella, zonular fibers and the non-pigmented epithelium of the ciliary body had a rather similar lectin-binding profile to exfoliative material. The lens capsule was essentially unreactive with all the lectins used. The lens epithelium reacted faintly with ConA, LCA, WGA and RCA-I. Pre-treatment with neuraminidase to remove sialic acid resulted in increased binding of PNA and SBA to exfoliative material, zonular fibers and the zonular lamella, and in decreased binding of WGA to the non-pigmented epithelium of the ciliary body, zonular fibers and the zonular lamella. The results indicate that alpha-mannosyl, beta-galactosyl, N-acetyl-D-glucosaminyl and N-acetylneuraminic acid residues are present in glycoconjugates of exfoliative material and neighbouring tissues.

Functional morphology of accommodation in the raccoon

The raccoon ( Procyon lotor) is a small carnivore which eats in the upright position, using hand- and finger-like front paws and digits to wash, hold and examine its food at close range. These anatomic and behavioral characteristics prompted structural and functional studies of the accommodative capability of this species. By light and electron microscopy, we observed a prominent ciliary smooth muscle and zonular apparatus. When stimulated by carbachol or pilocarpine, the muscle and zonular apparatus exhibited a shift from longitudinal to reticular or circular orientation of some ciliary muscle bundles, anterior movement of the muscle as a whole, and more oblique crossing of the zonular fiber bundles in the zonular plexus. Maximum carbachol-induced accommodative amplitude measured by coincidence refractometry ranged from 3 to 19 diopters in these 1 to 9 yr old animals, with no definite age-accommodation relationship. A-scan ultrasonographic biometry showed that during accommodation the lens thickened very little, if at all, but moved anteriorly, while the apparent cornea to retina distance increased slightly. The raccoon thus exhibits the greatest accommodative capability of any non-primate terrestrial mammal so far studied.

Development of immunoreagents to ciliary zonules that react with protein components of elastic fiber microfibrils and with elastin-producing cells

We describe the generation of a monoclonal antibody library to ocular zonule components and the characterization of three monoclonal antibodies: 1) one specific for microfibrillar associated glycoprotein (MAGP), a component of both ocular zonules and microfibrils of elastin fibers, 2) an antibody to an as yet unidentified 70,000 dalton antigen that is present in abundance in the extracellular matrix (ECM) of elastin-producing cells, and 3) an antibody reacting with the 67000 dalton subunit of the elastin receptor. The presence of antigenic determinants common to the ocular zonule and elastic fiber microfibrils suggests that zonules, which can be obtained in relatively pure form, can provide a valuable resource for characterizing proteins common to both microfibrillar structures.

Histochemical study on glycosaminoglycans in the developing mouse vitreous.

The distribution of polyanionic glycosaminoglycans (GAGs) in the developing mouse vitreous was studied histologically by P.A.S. reaction, metachromatic staining by toluidin blue at various pH's, alcian blue at pH 0.5 and alcian blue at various pH's, alcian blue at pH 0.5 and alcian blue C.E.C. stainings, modified Hale's method with colloidal iron, and enzymatically with bovine testicular hyaluronidase. A subdivision of the vitreous developmental period into four phases and an early distinction between, the posterior and equatorial vitreous portions are suggested on basis of the results. The early vitreous, during the first developmental phase, exhibits a high content in GAGs. This property gradually vanishes in the posterior part during the second phase of development, while acid GAGs including possibly hyaluronate are present in the equatorial zone. During this second phase, the lens capsule present a strong P.A.S.-reactivity, especially positive in it's posterior part. During the third phase, sulphated GAGs reappear in the posterior vitreous while non-sulphated material remains present in the equatorial zone. During the first two postnatal weeks (fourth developmental phase), acid GAG's disappear in the equatorial part of the vitreous but the maturing zonular fibres display the properties of sulphated GAGs. It is suggested that the histochemical maturation of the secondary vitreous starts around the 16th or 17th fetal day, i.e. much earlier than its morphological differentiation.

Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils.

A new connective tissue protein, which we call fibrillin, has been isolated from the medium of human fibroblast cell cultures. Electrophoresis of the disulfide bond-reduced protein gave a single band with an estimated molecular mass of 350,000 D. This 350-kD protein appeared to possess intrachain disulfide bonds. It could be stained with periodic acid-Schiff reagent, and after metabolic labeling, it contained [3H]glucosamine. It could not be labeled with [35S]sulfate. It was resistant to digestion by bacterial collagenase. Using mAbs specific for fibrillin, we demonstrated its widespread distribution in the connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule. Electron microscopic immunolocalization with colloidal gold conjugates specified its location to a class of extracellular structural elements described as microfibrils. These microfibrils possessed a characteristic appearance and averaged 10 nm in diameter. Microfibrils around the amorphous cores of the elastic fiber system as well as bundles of microfibrils without elastin cores were labeled equally well with antibody. Immunolocalization suggested that fibrillin is arrayed periodically along the individual microfibril and that individual microfibrils may be aligned within bundles. The periodicity of the epitope appeared to match the interstitial collagen band periodicity. In contrast, type VI collagen, which has been proposed as a possible microfibrillar component, was immunolocalized with a specific mAb to small diameter microfilaments that interweave among the large, banded collagen fibers; it was not associated with the system of microfibrils identified by the presence of fibrillin.

The microfibrils of connective tissue: II. Immunohistochemical detection of the amyloid P component.

Immunohistochemical methods were used for the detection of the amyloid P component in the microfibrils of two regions: the zonule of the eye and the connective tissue of the foot pad in 20- to 50-gm mice. Following fixation by immersion in 4% formaldehyde, the eyes and foot pads were embedded in paraffin, and sections were immunostained for light microscopy by using antiamyloid P component antiserum followed by peroxidase-antiperoxidase procedure. For electron microscopy, formaldehyde-fixed tissues were immunostained for the amyloid P component with protein A-gold by using either thin Lowicryl sections or frozen sections which were then embedded in Epon for thin sectioning. In the zonule of the eye, the light microscope showed that zonular fibers were strongly immunostained for the amyloid P component; there was also weak staining of the nonpigmented ciliary epithelium at the distal end of the fibers and of the zonular lamella at their proximal end. The electron microscope revealed clear-cut immunolabeling of the microfibrils making up zonular fibers as well as of individual microfibrils. In the foot pad, the light microscope detected a weak diffuse staining of connective tissue, whereas the electron microscope showed immunolabeling restricted to microfibrils. It was concluded that the amyloid P component was present in, or associated with, microfibrils. Purified amyloid P component was prepared and examined in the electron microscope after either negative staining or routine processing. After negative staining, it appeared as flat pentagonal units, frequently associated into columns. After routine processing, the units looked like cross sections of microfibrillar tubules. The dimensions of the units matched those of the hypothetical segments of the tubules. It was concluded that this tubule consisted of a column of amyloid P units. The cohesion of the units within the column was likely to be reinforced by the bands present at the surface of microfibrils.

The microfibrils of connective tissue: I. Ultrastructure.

The ultrastructure of connective tissue microfibrils was examined in two sites: the ciliary zonule of the eye and the foot pad, in 20-day-old mice perfused with glutaraldehyde. The microfibrils were classified into two categories, referred to as typical and atypical. Typical microfibrils predominate in both sites; they are unbranched, straight or gently curving, tubular structures of indefinite length with an overall diameter of 12.8 +/- 1.7 nm in the zonule and 13.8 +/- 2.8 nm in the foot pad. They are composed of two parts: tubule proper and surface band. The tubule is 7- to 10-nm wide and characterized in cross section by an approximately pentagonal wall and an electron-lucent lumen containing a 1- to 2-nm bead referred to as a spherule. When longitudinal sections of microfibrils are examined at high magnification, the wall of the tubule does not appear as a continuous line but as a series of successive dots. The interpretation of these findings is that the tubule is composed of successive annular segments with an approximately pentagonal outline. The surface band is a 3-nm-wide, ribbon-like structure wrapped around the tubule. The band has dense borders called tracks. Along the tracks, densely stained, 4.6-nm-long "spikes" are attached at 4.0-nm intervals. The wrapping of the bands is somewhat irregular. They may be in a transverse position across single or several microfibrils, in which case each band might constitute a distinct belt; more frequently, the bands are oblique and appear to form a continuous helix. It is proposed that surface bands play a role in holding together the juxtaposed segments making up a tubule. A model has been constructed to represent the association of tubule and band into a typical microfibril. Atypical microfibrils, which are more common in foot pad than in ciliary zonule, appear wavy, lack a definite tubule, and are characterized by distorted, irregular surface bands. They are attributed to proteolysis of typical microfibrils.

Pseudoexfoliative disease: histochemical evidence of an affinity with zonular fibres.

The histochemical staining properties of the abnormal material deposited in the anterior segment of the eye in the pseudoexfoliation syndrome and the zonular ligaments of the lens are essentially the same. Both present the staining characteristics of oxytalan, the microfibrillar component of elastic tissue. Reasons are advanced for regarding the pseudoexfoliative material as a product of the ciliary and lenticular epithelium.

Pseudoexfoliative material and oxytalan fibers

Pseudoexfoliative material (PSX) was found to show oxytalan staining like the ocular zonular fibers. These two fibrous proteins also shared positive reactions for disulfide and sulhydryl groups and for acidic mucosubstances, both of which have been suggested as reactive groups in the oxytalan staining reaction. The deep striated zone in the pre-equatorial lens capsule and the lens capsular inclusions shared most of these reactions, indicating that they contain some similar components. PSX material showed histochemical differences from the zonule, including evidence of less PAS-positive glycoprotein and more acidic mucosubstance. Variations in staining within the PSX excrescences indicated that they are a composite of more than one substance. PSX material appears to be a product of local basement membrane-secreting cells, representing an abnormal polymerization of glycoconjugates similar to those in elastic microfibrils.

Model of visual focussing involving extraocular muscles and the causes of myofia and glaucoma

The classical model of visual focus has increased curvature of the lens for accommodation and flattening of the lens for far vision, with both determined by ciliary muscle tension. This new model proposes that changes in the focal length is a second modulator of acuity. This model also proposes that the extraocular muscles are the more important neuromuscular entities for both lens and focal length changes. At accommodation there would be relaxation of the recti muscles and contraction of the superior oblique muscle. This increases focal length while allowing the ciliary muscle ring to contract, releasing tension on the lens capsule. Far object focus is due to contraction of the recti muscles. These pull on the sclera, stretching the zonule fibers taut to give flattening tension of the lens capsule. The myopic eye, in addition to a highly curved lens, also has longer focal length and is misaligned in the orbit. Ageing glaucoma is hypothesized as due to: 1. excessive curvature of an ageing lens; 2. chronic contraction of the recti in compensatory effort to flatten the lens and shorten the focal length; 3. the overcontracted recti pull to excess, stretching the inner tissue and distorting the Schlemm canal and its access mesh area to inhibit efflux of aqueous fluid.