Zonula Research Articles

Imaging zonular abnormalities using ultrasound biomicroscopy.

To evaluate the use of ultrasound biomicroscopy (UBM) in detecting ciliary zonular defects and to describe the UBM signs of such defects. Eighteen eyes of 18 patients with clinically suspected zonular abnormalities were evaluated using UBM. Predisposing factors included pseudoexfoliation, congenital spherophakia, surgical procedure, trauma, and the Marfan syndrome. Of 18 eyes, 11 showed UBM evidence of missing zonules and 11 showed evidence of zonular stretch. Four of the 18 eyes had both missing and stretched zonular fibers. All of the eyes examined showed increased lenticular sphericity in the area of zonular disorder. Nine eyes showed ciliary body flattening. Pupillary block was seen in 5 patients, and angle crowding due to direct iridal rotation was noted in 3. Ultrasound biomicroscopy can detect zonular loss and stretching directly. Increased lenticular sphericity and ciliary body flattening are signs of zonular defects. Angle closure mechanisms include pupillary block and direct iridal rotation.

Intracapsular foldable posterior chamber lens implantation in eyes with posterior capsule tears or zonular fiber instability

A surgical technique is described for foldable posterior chamber intraocular lens implantation in the capsular bag in the presence of a posterior capsule tear or weakened zonular fiber support. Haptics are compressed by suturing before endocapsular insertion, minimizing capsular and zonular fiber stress.

Saudi Experience with Classic Homocystinuria

Classic homocystinuria is an autosomal recessive disorder due to cystathionine ss-synthase deficiency. The clinical, radiological and neurophysiological findings of classic homocystinuria diagnosed at King Faisal Specialist Hospital and Research Centre (KFSH&RC) are presented in this report. Twenty-four patients (15 females and 9 males) were referred to KFSH&RC for work-up of mental retardation, seizures, thrombo-embolic episodes and dislocation of the ocular lenses. The common clinical findings included ectopia lentis (20 patients), skeletal system involvement (18 patients), vascular system involvement (9 patients), and mental retardation (all patients to varying degrees). Unusual findings consisted of a patient who developed severe lower gastrointestinal bleeding, a patient with insulin-dependent diabetes mellitus, probably due to vasculopathy, and another having severe bronchiectasis, which may have been due to fibrillin disruption, and required the resection of a lobe of the lung. The parents of 21 patients were first-degree relatives, and 19 patients had one or more family members affected by the same disease. All patients had markedly elevated plasma levels of methionine. Cystathionine synthase activity in the fibroblast was measured in 25% of the patients and was deficient. Only four patients responded to pyridoxine and their methionine level decreased to almost normal range. The aim of this study was to increase the awareness of this disease in the scientific and medical community, in particular in the general pediatrician working in Saudi Arabia who first encounters the clinical manifestations of the disease. Early detection through tandem mass spectrometry of blood spot screening and treatment are important, and may prevent the major complications of this disease.

Resistance of the human posterior lens capsule and zonules to disruption

Purpose: To determine the resistance of the human posterior lens capsule and zonules to disruption during cataract surgery.Setting: Wet lab at Walkergate Hospital, Newcastle upon Tyne, England.Method: Human donor eyes were obtained after removal of a corneoscleral disc. Lens extraction was performed by an extracapsular technique leaving an intact capsular bag. The resistance of the zonules and posterior capsule to disruption by aspiration was measured in each eye, and the resistance of the zonules to damage from direct mechanical stretch was determined.Results: Posterior capsules tolerated a median suction pressure of 200 mm Hg (range 150 to 300 mm Hg) before disruption. Values for female eyes were slightly greater than for male; no significant correlation with age was shown. The zonular fibers tolerated a median suction pressure of 200 mm Hg (range 150 to 300 mm Hg). The median mechanical zonular stretch tolerance was 3.00 mm (range 2.75 to 4.00 mm). No significant correlation was found between zonular suction tolerance or stretch tolerance and sex or between suction tolerance and age, but a negative relationship was noted between maximum tolerated stretch and age.Conclusion: Setting the maximum aspiration pressure during irrigation/aspiration to 150 mm Hg should help prevent capsular or zonular rupture. If more powerful aspiration is required, it should be used with great care so as not to engage the capsule.

Spherophakia associated with molybdenum cofactor deficiency

Molybdenum cofactor deficiency is an autosomal recessive disorder characterized by lack of activity of the enzymes sulfite oxidase, aldehyde oxidase, and xanthine dehydrogenase or oxidase. The clinical manifestations are indistinguishable from those of isolated sulfite oxidase deficiency: craniofacial alterations, intractable neonatal convulsions, very severe mental retardation, lens dislocation, and death in the first decade of life. Lens dislocation is found in nearly all patients after neonatal age. In the present case it developed late (at the age of 8 years) and was preceded by bilateral spherophakia. We hypothesize that an abnormal relaxation of the zonular fibers is the cause of spherophakia in this disease; this causes lens dislocation eventually, after days, months, or years.

HNK-1 epitope in the lens-ciliary zonular region in normal and pseudoexfoliative eyes. Immunohistochemistry and ultrastructure.

To determine the specific binding sites for the HNK-1 adhesion molecule epitope in the lens-ciliary zonular region in normal and pseudoexfoliative (PSX) eyes. Normal and PSX eyes and adjacent tissues from individuals aged 10 months to 89 years were investigated for the HNK-1 epitope by immunohistochemistry and immunogold electron microscopy. Concentration of HNK-1 epitope was high in the anterior central lens capsule into old age, but it tapered earlier and progressively in the inner capsule and elsewhere. On zonular fibers, label was strongest at their lens and ciliary attachments and intense on related elastic microfibrils in adjacent ciliary stroma. Label was also strong on PSX fibers and above normal on PSX central capsule. In contrast, HNK-1 was absent on PSX aggregates in conjunctiva and skin and on normal elastic microfibrils in these tissues. The prominent HNK-1 epitope in lens capsule and zonule has age- and site-specific variations that favor adhesive roles in each. The high density of epitope on ocular PSX material suggests an aberrant increase in expression that may contribute to adhesiveness of these aggregates, HNK-1 negativity on extraocular elastic microfibrils and PSX aggregates indicates a unique difference in composition of intraocular and extraocular elastic tissue, of potential significance for ocular function in aging and lens-dislocating diseases.

Hyaluronan and chondroitin sulfate proteoglycans are colocalized to the ciliary zonule of the rat eye: a histochemical and immunocytochemical study.

In previous studies, chondroitin sulfate proteoglycans have been localized to the periphery of the zonular fibers and the individual zonular fibrils (or microfibrils) after Cuprolinic blue staining in conjunction with chondroitinase digestions and immunogold labelling with 2-B-6 antibody. In the present study, we wished to determine if these proteoglycans are linked to hyaluronan to form a large multimolecular aggregate. To accomplish this, we localized the hyaluronan using a biotinylated hyaluronan-binding protein fragment of chondroitin sulfate proteoglycan, containing also the link protein, purified from bovine nasal cartilage. The results showed that the ciliary zonule of the rat eye was reactive with the biotinylated hyaluronan-binding probe as demonstrated by streptavidin-peroxidase-diaminobenzidine staining and streptavidin-gold labelling. Hyaluronan-gold labelling showed that the gold particles were mostly localized on the periphery of the zonular fibers, which was similar to the localization pattern of the zonule associated-proteoglycans. This hyaluronan-binding probe also strongly labelled the sites of zonule insertion over the basement membrane of the inner ciliary epithelium at the pars plana and the lens capsule at the equatorial region, which suggests its probable role in the attachment of ciliary zonule to the basement membranes. To demonstrate whether these two molecules are linked to one another, ultrastructural colocalization of both hyaluronan and chondroitin sulfate proteoglycans was performed on the same sections by double-gold labelling, and combined Cuprolinic blue staining and hyaluronan-gold labelling. Gold particles of 15 and 10 nm in sizes labelling both hyaluronan and chondroitin 4-sulfate, were colocalized to the surface of the zonular fibers. The combined Cuprolinic blue staining and hyaluronan-gold labelling showed that the gold particles were localized towards the ends of the Cuprolinic blue-stained rodlets, which strongly suggests that these chondroitin sulfate proteoglycans are linked to the hyaluronan chain to form a large aggregate surrounding the periphery of the zonular fibers. These ciliary zonule-associated proteoglycan-hyaluronan aggregates may play a role in organizing the individual zonular fibrils (microfibrils) into bundles of zonular fibers.

Immunoelectron microscopic localization of the HNK-1 carbohydrate epitope in the anterior segment of pseudoexfoliation and normal eyes

Purpose. To study the presence of the cell-adhesion related HNK-1 carbohydrate epitope in the anterior segment of pseu-doexfoliation and normal eyes by immunoelectron microscopy. Methods. Anterior segment tissue of 6 autopsy eyes with pseu-doexfoliation (PEX) syndrome (5 eyes without glaucoma and 1 with glaucoma), and 6 normal autopsy eyes without PEX syn-drome were studied by an electron microscopic immunogold technique using a monoclonal antibody to the HNK-1 epitope. Results. In both normal and PEX eyes, the HNK-1 epitope could be immunolocalized to the basement membranes of both ciliary epithelia and posterior iris pigmented epithelium, to the lens capsule and zonular lamella, and to the pigmented epithe-lial cells of iris and ciliary body. Within the inner connective tissue layer of the ciliary body, the gold label was mainly asso-ciated with the periphery of elastic fibers and microfibrillar bundles. PEX material on the surfaces of posterior iris, ciliary body, anterior lens capsule, zonular fibers, and uveal part of the trabecular meshwork reacted strongly with the HNK-1 anti-body. In contrast, PEX material accumulations within the iris stroma or the juxtacanalicular tissue of the trabecular mesh-work showed only weak immunoreactivity, while PEX mate-rial in the conjunctiva was totally negative. Conclusions. The wide distribution of the HNK-1 epitope in anterior segment tissues and its association with a variety of extracellular and cellular structures was ultrastructurally dem-onstrated. In PEX syndrome, the varying labelling density of PEX fibers indicates a deviating carbohydrate composition in different locations of the eye. The HNK-1 epitope might be involved in the adhesiveness of PEX deposits on intraocular surfaces.

Fibrillin-1 and Fibulin-2 Interact and Are Colocalized in Some Tissues

Microfibrils 10-12 nm in diameter are found in elastic and non-elastic tissues with fibrillin as a major component. Little is known about the supramolecular structure of these microfibrils and the protein interactions it is based on. To identify protein binding ligands of fibrillin-1, we tested binding of recombinant fibrillin-1 peptides to different extracellular matrix proteins in solid phase assays. Among the proteins tested, only fibulin-2 showed significant binding to rF11, the N-terminal half of fibrillin-1, in a calcium-dependent manner. Surface plasmon resonance demonstrated high affinity binding with a Kd = 56 nM. With overlapping recombinant fibrillin-1 peptides, the binding site for fibulin-2 was narrowed down to the N terminus of fibrillin-1 (amino acid positions 45-450). Immunofluorescence in tissues demonstrated colocalization of fibrillin and fibulin-2 in skin, perichondrium, elastic intima of blood vessels, and kidney glomerulus. Fibulin-2 was not present in ocular ciliary zonules, tendon, and the connective tissue around kidney tubules and lung alveoli, which all contain fibrillin. Immunogold labeling of fibulin-2 on microfibrils in skin was found preferentially at the interface between microfibrils and the amorphous elastin core, suggesting that in vivo the interaction between fibrillin-1 and fibulin-2 is regulated by cellular expression and deposition as well as by protein-protein interactions.

Scanning electron microscopy of the human zonule of the lens (Zonula ciliaris).

In the present study eight human eyeballs were specifically prepared for scanning-electron-microscopic observation of the zonule. The zonule consisted of two main layers of radial fibres, an anterior and a posterior one, that inserted on the anterior and the posterior lens capsules, respectively. Some fibres inserted on the equator of the lens. Posterior zonular fibres originated at the pars plana, entered the dorsal part of the ciliary valleys and then changed their direction towards the posterior face of the lens. Posterior fibres inserted on the posterior capsule of the lens by branched endings 1 mm behind the equator of the lens. Anterior zonular fibres originated mainly at the pars plana and occasionally at the ciliary valleys. After running completely through the ciliary valleys in close contact with the lateral walls of the ciliary processes, they changed their direction at the anterior endings of the pars plicata and reached the anterior lens capsule. Anterior zonular insertions were achieved by webbed endings that diffused into the anterior capsule 2 mm in front of the lens equator. The extraordinary distension capacity of the zonular fibres was demonstrated by pulling the anterior lens capsule after hydrodissection. As a consequence, the anterior fibres were stretched up to four times their original length without breaking or disinserting.

Fibrillines et fibrillinopathies

Microfibrils contain a variety of proteins, the most prominent of which are the two fibrillins. Fibrillins are large glycoproteins (320 kDa) ubiquitously distributed in connective tissues. Together with amorphous elastin, fibrillin-containing microfibrils form the elastic fibers. Fibrillins are also found in microfibrils that are apparently unendowed with amorphous elastin, such as those forming the ciliary zonule. Fibrillins are encoded by two different genes: FBN1 on human chromosome 15q21 encodes type 1 fibrillin and FBN2 on human chromosome 5q23 encodes type 2 fibrillin. The most notable structural feature of these proteins is the presence of numerous calcium-binding epidermal growth factor like motifs interspersed with a few motifs homologous to those found in the binding protein for transforming growth factor b. The study of the developmental expression of the fibrillin genes has revealed different patterns: FBN1 is mainly expressed during late morphogenesis while FBN2 expression coincides with early morphogenesis and notably the beginning of elatogenesis. Therefore, fibrillins could contribute to the structural and functionnal heterogeneity of microfibrils. This heterogeneity is reflected in the large phenotypic spectrum associated with mutations in the fibrillin genes. These mutations are found in a new groupe of inherited connective-tissue disorders: the fibrillinopathies. To date, 76 mutations have been identified in FBN1 gene. These mutations are associated not only with the Marfan syndrome (MFS) but also with a spectrum of conditions phenotypically related to MFS including dominantly inherited ectopia lentis, severe neonatal Marfan syndrome and isolated typical features of Marfan syndrome. Conversely, only a few mutations have been reported in the FBN2 gene and observed in congenital contractural arachnodactyly. Important issues that will now have to be addressed concern the elucidation of the exact composition of the microfibrils and their spatial organization into tissue-specific macro-aggregates. Les fibrillines sont les constituants majeurs du reseau microfibrillaire. Cette structure du tissu conjonctif est soit etroitement associee aux fibres d'elastine soit presente en agregats isoles dans les tissus depourvus d'elastine. Les travaux de biologie cellulaire et moleculaire ont permis d'elucider la structure des fibrillines et de commencer a apprehender leurs relations avec les autres constituants de la matrice extra-cellulaire. Mais, c'est la description de leur alteration dans differentes maladies du tissu conjonctif, dont le syndrome de Marfan, qui a revele leur importance.

Proteoglycans associated with the ciliary zonule of the rat eye: a histochemical and immunocytochemical study

The structural organization of integral and associated components of the ciliary zonule is still not fully understood. The present study is to localize and characterize the proteoglycans associated with the ciliary zonule of the rat eye by Cuprolinic blue (CB) staining and immunocytochemistry. After CB staining, the proteoglycans appeared as electron dense elongated rodlets and were localized with the zonular fibers. They were seen lying on the periphery of the zonular fibers or along the length of the individual fibrils. Most of the CB rodlets had a size of 60-170 nm long (average 130 nm) and 25 nm wide. Smaller CB rodlets measuring 25-60 nm long (average 45 nm) and 12 nm wide were sometimes found associated with the individual zonular fibrils. The CB rodlets were removed after chondroitinase ABC or chondroitinase AC treatment, but were resistant to heparitinase, nitrous acid, keratanase or Streptomyces hyaluronidase digestions. The ciliary zonule was also immunostained with three monoclonal antibodies: 2-B-6 specific for chondroitin 4-sulfate, 3-B-3 for chondroitin 6-sulfate and 1-B-5 for unsulfated chondroitin, using indirect immunoperoxidase or immuno-colloidal gold methods. The zonular fibers were immunoperoxidase stained and immunogold labeled by 2-B-6, but were not reactive to 3-B-3 and 1-B-5. The results demonstrate that chondroitin sulfate proteoglycan is associated with the ciliary zonule of the rat eye.

Complications of Extracapsular Cataract Extraction in Patients with Pseudoexfoliation Syndrome

Objective: To evaluate the complications of extracapsular cataract extraction in patients with pseudoexfoliation (PE) syndrome. Study Design: A retrospective study of patients operated on between 1990 and 1992. Setting: Hacettepe University School of Medicine, Department of Ophthalmology. Patients: Seventy-three patients with PE syndrome. The mean age of the patients was 71.3 ± 8.0 years with ages ranging between 54 and 89. There were 42 male patients with a mean age of 71.8 and 31 female patients with a mean age of 70.6. Results: Pupillary irregularity was the most frequent complication (23.3%) in the study. The other complications were as follows: posterior capsule rupture in 6.8%, posterior capsule rupture with vitreous loss 4.1%, fibrinoid reaction in 4.1%, posterior synechia in 4.1%, intraocular lens (IOL) decentralization in 2.7% and UGH syndrome in 1.4%. Conclusions: Cataract surgery in PE syndrome may be linked to several problems. Eyes with PE may show spontaneous subluxation of the lens. The zonular fibres are known to be weak in PE syndrome and the central posterior lens capsule very thin. However the low rates of complications of ECCE with and without IOL implantation has convinced us that this procedure is relatively safe in patients with PE syndrome.

Marfan syndrome: fibrillin expression and microfibrillar abnormalities in a family with predominant ocular defects.

We have found abnormal fibrillin microfibrils isolated from tissues and cell cultures from two cousins with Marfan syndrome whose major clinical abnormality is bilateral ectopia lentis, but who also have skeletal involvement but no cardiovascular defects. Ultrastructural analysis of ciliary zonules showed the presence of abundant loose microfibril bundles which in many places appeared disorganised. Microfibrils isolated from ciliary zonules and vitreous were highly fragmented when examined by rotary shadowing electron microscopy. Investigation of microfibrils elaborated by patient dermal fibroblasts showed remarkable variations in periodicity and packing. The synthesis and secretion of fibrillin by these cells was confirmed electrophoretically with the identification of metabolically labelled immunoprecipitated fibrillin (M(r) 300,000) in medium and cell layer compartments. These data show that fibrillin expression is normal but that assembled microfibrils are manifestly abnormal both morphologically and functionally. The occurrence of microfibrils with variable periodicities and susceptibility to fragmentation suggests that structural weakness is probably the primary cause of lens dislocation in these patients.

Galactose-containing glycoconjugates of the ciliary body and lens in capsular glaucoma: a lectin histochemical study

This study was carried out in order to obtain information on galactose-containing glycoconjugates in the lens and ciliary body of human eyes with capsular glaucoma and thus on the etiopathogenesis of exfoliation syndrome. Six formalin-fixed, paraffin-embedded human eyes with capsular glaucoma and six control eyes were studied using a panel of 11 biotinylated lectins with extended binding sites to galactose- and N-acetylgalactosamine-containing glycoconjugates. Both pepsin and neuraminidase pretreatments were performed. Gal(beta 1-->3)GalNAc-reactive peanut (PNA) and Bauhinia purpurea alba (BPA) agglutinins and Gal(beta 1-->4)GlcNAc GlcNAc-reactive Ricinus communis (RCA-I) and Phaseolus vulgaris (PHA-E) agglutinins reacted strongly with exfoliation material, while binding of Gal(beta 1-->4)GlcNAc-reactive Erythrina cristagalli and GalNAc-reactive soybean, Caragana arborescens, Vicia villosa, Helix pomatia (HPA) and Dolichos biflorus agglutinins was generally weak. In the nonpigmented ciliary epithelium and on the zonular fibers and lamella, at least moderate reaction was detected with PNA, BPA, RCA-I and PHA-E, and these tissues thus resembled exfoliation material in their reactivity. In contrast, the lens epithelium reacted weakly with PHA-E and HPA only, and the lens capsule was never labeled. No qualitative changes were seen after neuraminidase pretreatment. The findings suggest that galactose- and N-acetylgalactosamine-containing glycoconjugates in exfoliation material are at least partially produced by the nonpigmented ciliary epithelium, rather than by the lens epithelium. Moreover, PNA, PBA, RCA-I and PHA-E seem to be the most suitable of the lectins tested for detection of exfoliation material in histological specimens.

Emilin, a component of elastic fibers preferentially located at the elastin-microfibrils interface.

The fine distribution of the extracellular matrix glycoprotein emilin (previously known as glycoprotein gp115) (Bressan, G. M., I. Castellani, A. Colombatti, and D. Volpin. 1983. J. Biol. Chem. 258: 13262-13267) has been studied at the ultrastructural level with specific antibodies. In newborn chick aorta the protein was exclusively found within elastic fibers. In both post- and pre-embedding immunolabeling emilin was mainly associated with regions where elastin and microfibrils are in close contact, such as the periphery of the fibers. This localization of emilin in aorta has been confirmed by quantitative evaluation of the distribution of gold particles within elastic fibers. In other tissues, besides being associated with typical elastic fibers, staining for emilin was found in structures lacking amorphous elastin, but where the presence of tropoelastin has been demonstrated by immunoelectron microscopy. This was particularly evident in the oxitalan fibers of the corneal stroma, in the Descemet's membrane, and in the ciliary zonule. Analysis of embryonic aorta revealed the presence of emilin at early stages of elastogenesis, before the appearance of amorphous elastin. Immunofluorescence studies have shown that emilin produced by chick embryo aorta cells in culture is strictly associated with elastin and that the process of elastin deposition is severely altered by the presence of antiemilin antibodies in the culture medium. The name of the protein was derived from its localization at sites where elastin and microfibrils are in proximity (emilin, elastin microfibril interface located protein).

Differentiation of oxytalan fibres from elastic fibres with reagents for detection of magnesium

Elastic fibres in various human and animal tissues revealed positive results with reagents for the detection of magnesium. By contrast, no Mg could be detected in oxytalan fibres or in zonular fibres of the ciliary body, which are composed of microfibrils not containing elastin. Mg is therefore concluded to be associated with the elastin core of elastic fibres. Its antagonism to calcium is speculated to play a protective role in maintaining the extensibility of elastin.

An immunoelectron microscope study of the aged human lens capsule

The distribution of types I–IV collagen and laminin was studied in seven aged human lens capsules using the immunogold EM technique on LR White embedded tissue. Samples were taken from the anterior, equatorial and posterior regions. Labelling for type II collagen was not observed. Type IV collagen was evenly distributed throughout the thickness of the capsule but was absent from the zonules. However, an unexpected finding was strong labelling for types I and III collagen, again evenly distributed throughout the capsule. The presence of type III collagen makes the lens capsule unique among ocular basement membranes. Laminin was present in linear densities, zonular lamellae and zonular fibres, suggesting that linear densities are an integral part of the zonular apparatus.

Capsulorhexis, Phacoemulsification, and Posterior Chamber Lens Placement Following Iridocyclectomy

Two patients developed cataract following iridocyclectomy for iris melanoma invading the angle. Although several clock hours of zonular fibers had been surgically resected, the use of a continuous, circular capsulorhexis followed by phacoemulsification maintained the integrity of the anterior capsular margin and allowed for stable "in-the-bag" placement of a posterior chamber intraocular lens (PC-IOL). Continuous, circular capsulorhexis is the optimal anterior capsulectomy procedure, minimizing the incidence of radial tears and the risk of subsequent PC-IOL decentration.

Kontusionsbedingte traumatische Aniridie

Total aniridia occurred after a severe blow on the eyeball of a 65 year old man. The entire iris was torn off the ciliary body and escaped out of the eye through a small corneal wound. The zonular fibres were found to be intact and the posterior parts of the eye showed no traumatic changes.