Cell size and nucleo-cytoplasmic ratios of meibocytes in the anterior acini of the upper eyelid Meibomian glands in rabbits.

OBJECTIVE: To assess the morphological details of the acini of the normal meibomian gland. ANIMALS STUDIED: Six young adult pigmented rabbits. METHODS: The upper eyelid was prepared in extended configuration by glutaraldehyde fixation. Tissue block sections approximately 0.5-1 mm from the eyelid margin were assessed by light microscopy in sagittal sections and transmission electron microscopy (TEM) in coronal sections. TEM images at between 1000× and 2000× magnification were enlarged onto A3-sized prints and cell size and nuclei measured by planimetry. RESULTS: Light microscopy sagittal sections revealed clusters of variable sized acini, sometimes appearing to be slightly overlapping and without any obvious spatial organization of the internal cells (meibocytes). The estimated areas of the acini were close to 6500 sq micron. Coronal sections, as examined by TEM, allowed for visualization of small to large acini (average diameter 82 ± 17 microns, with an estimated area of 5500 sq. microns) containing variable numbers of immature (partly differentiated) or mature (fully differentiated) meibocytes with a distinct spatial organization. The average area of the meibocytes was 158 ± 81 square microns, and they usually appeared to have a single nucleus (with an average sectional area of 29 ± 12 square microns). Within individual acini, peripherally located immature meibocytes tended to be smaller and have higher nucleo-cytoplasmic area ratios, while more centrally mature located meibocytes tended to be slightly larger and had lower or much lower nucleocytoplasmic ratios. CONCLUSIONS: Comparative studies on meibomian glands can be undertaken with objective assessments to assess for normality or abnormality.

Scanning Electron Microscopic Features of the External and Internal Surfaces of Normal Adult Lacrimal Drainage System.

AIM: The aim of this study was to examine the ultrastructural features of the external and internal surfaces of healthy lacrimal drainage systems. METHODS: A prospective interventional study was performed on the healthy adult lacrimal drainage systems obtained from fresh exenterated specimens. Exenteration was performed for malignancies unrelated to lacrimal system where preoperative lacrimal evaluation was normal. A careful and thorough dissection was carried out to isolate the entire lacrimal drainage system from the punctum to the nasolacrimal duct. The analysis was performed using the standard protocols of scanning electron microscopy. RESULTS: Inner punctal surfaces showed a definite and slightly elevated junction between the luminal surfaces of punctum and beginning of the vertical canaliculus. Similar junction could be identified between the lacrimal sac and nasolacrimal duct. The valves of the canaliculi showed broad rugae-like mucosal surfaces, whereas the external surfaces of the canaliculi demonstrated well-defined orbicularis muscle with collagenous attachments. The walls of the lacrimal sac and nasolacrimal duct showed dense vascular plexus which included wide luminal arteries, throttle veins, and large capacitance vessels. CONCLUSIONS: Ultrastructural features of external and internal surfaces of lacrimal drainage system help in better understanding of its anatomy and physiology. The junctional area between the punctum-vertical canaliculus and lacrimal sac-nasolacrimal duct needs further exploration to understand their roles.

Punctal stenosis: histopathology, immunology, and electron microscopic features-a step toward unraveling the mysterious etiopathogenesis.

PURPOSE: To study the histologic, immunohistochemical, and electron microscopic features of puncta and proximal vertical canaliculi to understand the etiopathogenesis of punctal stenosis. METHODS: Prospective study of 26 stenosed punctae that were collected following a punctoplasty. Sixteen were from lower eyelid and 10 from upper eyelid. Histopathological examination was performed on 20 punctae using hematoxylin-eosin, periodic acid-Schiff, and Masson trichrome staining. Immunohistochemical patterns were analyzed after staining with leukocyte common antigen or CD45, CD3, CD5, CD10, CD20, CD138, and smooth muscle actin. Six punctae (3 upper, 3 lower) were separately processed for electron microscopic studies as per standard protocols. RESULTS: All punctae showed evidence of subepithelial and subconjunctival fibrosis. Thirty percent (6/20) showed extensive fibrosis. Inflammation was noted in 80% (16/20) of the samples; however, 20% (4/20) showed severe inflammation. Strong immunoreactivity was noted, with CD45 and CD3 in 80% (16/20) with predominance in the subepithelial areas. Focal immunoreactivity was noted for CD10, CD20, and CD138. Immunoreactivity was negative for CD5. Electron microscopic features include blunted epithelial microvilli, numerous fibroblasts, extensive and irregularly arranged collagen bundles, mononuclear infiltration in the vicinity of fibroblasts or in between collagen bundles, and inter- and intracellular edema in areas of inflammation. CONCLUSIONS: Chronic inflammation and subsequent fibrosis appear to be the basic ultrastructural response to various noxious stimuli. Mononuclear inflammatory infiltration in the vicinity of fibroblasts could possibly reflect a close cellular interaction between these 2 cells.

Microscopic and ultrastructural changes of Müller's muscle in patients with simple congenital ptosis.

PURPOSE: To study microscopic and ultrastructural changes of Müller's muscle in patients with isolated congenital ptosis. METHODS: In this prospective, observational case-control study, Müller's muscle specimens were collected during ptosis surgical correction for 18 consecutive patients. Each specimen was divided into 2 parts. One part was embedded in formalin for light microscopy, and the other one was fixed in 3% glutaraldehyde for electron microscopy. A neuropathologist, serving as a masked evaluator, blindly reviewed all the different features for every case and counted the number of myocytes showing distinct myofilaments in the whole grid for every case. Statistical analysis using compare means and correlation tests was conducted to investigate potential associations and/or differences within and across groups. RESULTS: Twelve Müller's muscle specimens from patients with simple congenital ptosis of various severities and 6 specimens from patients with aponeurotic ptosis (controls) were collected and studied. Under light microscopy, congenital ptosis slides showed a small number of dispersed myocytes in a fibrotic background, whereas acquired ptosis slides showed a greater number of well-defined myocytes. Under electron microscopy, all congenital ptosis specimens had only a very small number of myocytes with clear, distinct myofilaments. Most myocytes in the aponeurotic ptosis group showed clear, distinct myofilaments, indicating a well-preserved muscle. No relationship existed between the number of clear, distinct myofilaments observed in the congenital ptosis group by transmission electron microscopy and patient age or ptosis severity. CONCLUSION: Substantial Müller's muscle atrophy was observed in patients with different severities of isolated congenital ptosis.

Damage from periorbital ageing to the multilayered structures and resilience of the skin in Chinese population.

Ageing dynamically disrupts the multilayered supporting components of the skin that are held together by cell adhesion molecules (CAMs). Skin specimens from 33 female Chinese patients undergoing lower blepharoplasty were divided into three age groups and examined by haematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and Elastica-van Gieson (EVG) stains, western blotting, surface electron microscopy (SEM) and biomechanical tension analysis. The SEM density (skin surface topology) showed a negative linear relationship with age. The triangular pattern of the skin surface in the younger group gradually broke down into quadrangular and irregular patterns in the older group. Collagens and elastic fibres in the dermis showed anisotropy and decreased density in the older groups compared with the younger group, especially in the papillary dermis. Anisotropy means that physical properties differ according to the direction of measurement. E-cadherin and integrin αv (whose functions are to bind epidermal and dermal elements respectively) increased and decreased, respectively, in the oldest group. Skin resilience decreased significantly in this group under repetitive stress. In conclusion, a loss of skin surface textures, integrin αv expressions, epidermal-dermal connections and dermal compactness led to the multilayered structure of the skin becoming separated. This in turn decreased resilience during ageing. These findings may therefore explain why aged skins cannot tolerate repetitive facial expressions, and why this action produces further dynamic wrinkles.

Clinical and pathologic eyelid alterations in a patient with Darier disease.

Darier disease (DD) is a rare autosomal dominant dermatosis that has infrequent ocular manifestations, especially those involving the eyelids. The authors describe a patient with long-standing DD who presented with both classic and unique clinical findings. Eyelid biopsy samples studied with electron microscopy demonstrated histopathological changes consistent with DD. The authors postulate how clinical findings not previously reported as "classic" to DD may be associated. To the authors' knowledge, electron micrographs detailing changes associated with DD have not been published for eyelid tissue.

ROCK-I regulates closure of the eyelids and ventral body wall by inducing assembly of actomyosin bundles.

Rho-associated kinase (ROCK) I mediates signaling from Rho to the actin cytoskeleton. To investigate the in vivo functions of ROCK-I, we generated ROCK-I-deficient mice. Loss of ROCK-I resulted in failure of eyelid closure and closure of the ventral body wall, which gave rise to the eyes open at birth and omphalocele phenotypes in neonates. Most ROCK-I(-/-) mice died soon after birth as a result of cannibalization of the omphalocele by the mother. Actin cables that encircle the eye in the epithelial cells of the eyelid were disorganized and accumulation of filamentous actin at the umbilical ring was impaired, with loss of phosphorylation of the myosin regulatory light chain (MLC) at both sites, in ROCK-I(-/-) embryos. Stress fiber formation and MLC phosphorylation induced by EGF were also attenuated in primary keratinocytes from ROCK-I(-/-) mice. These results suggest that ROCK-I regulates closure of the eyelids and ventral body wall through organization of actomyosin bundles.

c-Jun regulates eyelid closure and skin tumor development through EGFR signaling.

To investigate the function of c-Jun during skin development and skin tumor formation, we conditionally inactivated c-jun in the epidermis. Mice lacking c-jun in keratinocytes (c-jun(Deltaep)) develop normal skin but express reduced levels of EGFR in the eyelids, leading to open eyes at birth, as observed in EGFR null mice. Primary keratinocytes from c-jun(Deltaep) mice proliferate poorly, show increased differentiation, and form prominent cortical actin bundles, most likely because of decreased expression of EGFR and its ligand HB-EGF. In the absence of c-Jun, tumor-prone K5-SOS-F transgenic mice develop smaller papillomas, with reduced expression of EGFR in basal keratinocytes. Thus, using three experimental systems, we show that EGFR and HB-EGF are regulated by c-Jun, which controls eyelid development, keratinocyte proliferation, and skin tumor formation.

The structure and possible functions of the milkfish Chanos chanos adipose eyelid.

Basic histological sections (with different staining methods) and scanning electron microscopy (SEM) examinations showed that there were three distinctive layers in the adipose eyelid of milkfish Chanos chanos, which is found in the cephalie region and covers the entire eye. The outer and inner layers were epithelial tissues and the middle layer was composed of connective tissue formed by type I collagen fibrils. No adipose tissue was found in any of the three layers of the so-called adipose eyelid. Examination by transmission spectrophotometer showed that the adipose tissue could filter out ambient light with a wavelength shorter than 305 nm. A photoretinoscope was used to investigate whether the adipose eyelid influenced the mechanism of eye focusing. Eye diopter values did not differ before or after eyelid removal, which indicated that the adipose eyelid did not play a role in eye focusing. In light of these findings, it is suggested that the adipose eyelid serves to block exposure of harmful ultraviolet light into eyes and may also to offer some protection against impact to the eye in the aquatic environment.

Light and electron microscopic study of the eyelids, conjunctiva-associated lymphoid tissue and lacrimal gland in Bilgorajska Goose (Anser anser).

Normal structure of the accessory organs of the eye is essential for normal eye physiology. Among the most important accessory organs of the eye are the eyelids, the conjunctiva-associated lymphoid tissue (CALT) and the lacrimal gland (LG). The aim of this study was to demonstrate the histological structure of the eyelids and LG by histochemical and ultrastructural analysis. The study was performed on 13 adult female Bilgorajska geese. Eyelid samples were stained with the Alcian blue (AB pH 2.5) and periodic acid-Schiff (PAS) methods. Staining methods used for LG were AB pH 2.5, aldehyde fuchsin (AF), PAS and Hale's dialysed iron (HDI). Within the connective tissue of the eyelids, well-developed, diffuse, CALT follicles were observed, mostly under the conjunctival epithelium. Numerous lymphocytes were present within loose connective tissue. Staining of the eyelids with the PAS method demonstrated the presence of goblet cells of a mucous nature, and AB pH 2.5 staining indicated the presence of sulfated acid mucopolysaccharides. PAS staining of LG revealed the presence of secretory cells containing weakly PAS-positive granules. All epithelial cells of the corpus glandulae and the duct systems reacted positively to AB pH 2.5. HDI staining detected the presence of carboxylated acid mucopolysaccharides. Transmission electron microscopy investigations revealed two types of secretory epithelial cells in LG. Both types of LG cells contained drop-like secretory vesicles of different sizes with low or high electron density in cytoplasm, as well as small and large lipid vacuoles, and numerous small primary lysosomes.

[Comparative characteristics of the lymphatic bed of organ of vision in animals].

This investigation was aimed at the study of peculiarities of of lymphatic bed of organ of vision in herbivorous (cattle) and predatory (dog) animals, which have some anatomical differences in the eye structure. The methods of preparation, morphometry, light and electron microscopy were used. It was found that in the cattle, lymphatic capillaries of the sclera have lower density of distribution than those in dogs. In the cattle, extraorgan lymphatic vessels of lower and upper eyelids could be subdivided into a lateral and medial groups. The former group drains into parotid lymph node, while the latter group is connected to a mandibular one. In dogs an additional pathway of lymph transport was found that carried the lymph through the extraorgan lymphatic vessels into the facial lymph node.

Disruption of eyelid and cornea development by targeted overexpression of the glucocorticoid receptor.

Glucocorticoid hormones act through the glucocorticoid receptor (GR) and they affect almost all physiological systems in the organism. We have previously reported that transgenic mice overexpressing GR under the control of the keratin k5 promoter (K5-GR mice) display severe phenotypic alterations in the epidermis and other ectoderm derivatives (Perez et al., 2001). In this work, we aimed to characterize the pathological consequences of GR targeted overexpression in the eyelid and cornea at late developmental stages. Despite glucocorticoids being widely prescribed as a topical treatment in ophthalmology, their potential role during ocular development in the embryo is not well understood. As shown by scanning electron microscopy analysis as well as by our histopathological and immunohistochemical data, long-term and newborn transgenic embryos showed unfused eyelids, along with proptosis of the globe and exposure of the anterior surface. In addition, epithelial defects were evident at the cornea. Our results indicate that GR overexpression affected the proliferation rate of targeted epithelia of the cornea and eyelid, thus demonstrating that GR was responsible for the arrest of epithelial proliferation of the developing eyelid edges, as well as for their destruction. We conclude that constitutive targeted overexpression of GR in the eyelid and corneal epithelium dramatically impairs ocular function in these transgenic mice.

Histological and Ultrastructural Studies on the Conjunctiva of the Barred Owl (Strix varia).

This report is the first characterization of the histology and ultrastructure of the barred owl conjunctiva. The inferior eyelid was dominated by a large disk-shaped plate covered by a non-keratinized stratified squamous or cuboidal epithelium of variable thickness. The apical surface of the plate epithelium varied from flat to long microvilli or even short cytoplasmic extensions similar to those seen in the third eyelid. All specimens had a few goblet cells filled with mucous secretory granules in the plate region. The underlying connective tissue was a dense fibroelastic stroma. Eosinophils were surprisingly common in the epithelial layer and underlying connective tissue in the plate and more distal orbital mucosal region. The orbital mucosa contained goblet cells with heterogeneous glycosylation patterns. The leading edge and marginal plait of the third eyelid are designed to collect fluid and particulate matter as they sweep across the surface of the eye. The palpebral conjunctival surface of the third eyelid was covered by an approximately five-cell-deep stratified squamous epithelium without goblet cells. The bulbar surface of the third eyelid was a bilayer of epithelial cells whose superficial cells have elaborate cytoplasmic tapering extensions reaching out 25 µm. Narrow cytofilia radiated outwards up to an additional 15-20 µm from the cytoplasmic extensions. Lectin labeling demonstrated heterogeneous glycosylation of the apical membrane specializations but only small amounts of glycoprotein-filled secretory granules in the third eyelid.

Meibomian gland changes in the rhino (hrrhhrrh) mouse.

The rhino mouse, a single gene recessive mutation, is characterized by abnormal epidermal differentiation and maturation leading to the loss of hair at 1 month of age as well as follicular and epidermal hyperkeratoses. We evaluated the lids and corneas of nine rhino mice and their normal litter mates at various ages from 3 months to 1 year. Tissue specimens were studied by light microscopy, scanning and transmission electron microscopy as well as immunoperoxidase using a polyclonal rabbit anti-keratin antibody. At 3 months of age there was a thickening and hyperkeratinization of the palpebral epidermis which extended into and included the meibomian gland central duct. Whereas in the skin, hyperkeratinization is followed by follicular hyperkeratosis and dermal cyst formation, in the meibomian gland, ductal hyperkeratinization appeared to lead to loss of well developed acini followed by atrophy of the gland at 1 year as confirmed by immunostaining for keratin proteins. Scanning electron microscopy revealed marked plugging of the meibomian gland orifice with keratinized cells or debris in contrast to the patent orifice of the normal lid. Ocular surface changes included the presence of a whitish exudate covering the surface of the eye and increased numbers of preexfoliative corneal epithelial cells. These findings suggest that the rhino mouse may represents the first naturally occurring disorder of the meibomian gland.

Lectin electron microscopic histochemistry of the pseudoexfoliative material in the skin.

PURPOSE: To investigate the hypothesis that pseudoexfoliation (PSX) syndrome is a systemic disorder, the authors studied the composition of glycoconjugates in the intraocular and extraocular PSX material at the electron microscopic level, using a panel of lectins as cytochemical probes. METHODS: The authors examined 8 lid skins, 11 trabecular tissues, and 3 cataractous lenses from human eyes with PSX syndrome. Tissues were processed for electron microscopical histochemistry and stained with PNA, RCA120, DBA, SBA, ConA, WGA, UEA-I, and Lotus, with an indirect lectin-colloidal gold technique. RESULTS: Both the intraocular and extraocular PSX materials manifested almost identical reactivity to lectins, which indicated that glycoconjugates in the PSX material contained with sugar residues of galactose (PNA, RCA120), alpha-mannose (ConA), and N-acetyl-D-glucosamine (WGA). On the other hand, it was indicated that sugar residues of N-acetyl-D-galactosamine (DBA, SBA) and fucose (UEA-I, Lotus) were absent. Granular inclusions and microfibrils in the capsule and ocular zonules were stained similarly and weakly. CONCLUSIONS: The intraocular and extraocular PSX materials contained the same sugar residues of glycoconjugates, which suggested that those materials had the same nature. This study, the first documentation of lectin-binding sites on the extraocular PSX material, supported the hypothesis of PSX syndrome as a systemic disorder.

Increased extracellular deposition of fibrillin-containing fibrils in pseudoexfoliation syndrome.

PURPOSE: To localize the distribution of fibrillin-containing microfibrils in normal human anterior segment tissues and to characterize the role of fibrillin in the pathogenesis of pseudoexfoliation syndrome. METHODS: Anterior segment tissues were obtained from 10 eyes with pseudoexfoliation syndrome and 10 normal eyes and investigated by indirect immunofluorescence and electron microscopic immunogold labeling using a monoclonal antibody to fibrillin-1. RESULTS: In addition to labeling of zonular fibers, fibrillin-immunoreactive microfibrillar bundles generally were found in the corneal stroma; the stromal connective tissues of conjunctiva, ciliary body, and iris, especially in the iris root area; the periphery of Schlemm's canal, the scleral spur, and the most anterior portion of the trabecular meshwork; the ciliary muscle, and the dilator and sphincter muscles of the iris; the basement membranes of peripheral corneal epithelium, conjunctival epithelium, ciliary pigmented epithelium, and the lens capsule. The microfibrillar bundles were found to be isolated or in association with elastic fibers and cellular basement membranes. In pseudoexfoliation eyes, an additional strong immunoreaction was localized to pseudoexfoliation fibers and their microfibrillar subunits in close proximity to surfaces of cells involved in pseudoexfoliation fiber production. CONCLUSIONS: The fibrillin-containing microfibrillar system in normal ocular tissues is suggested to have a substantial role in the maintenance of tissue integrity by providing tensile strength and flexibility to mechanically strained tissues. The findings further provide evidence for fibrillin as an intrinsic component of pseudoexfoliation fibers, suggesting the possibility that enhanced expression of fibrillin or abnormal aggregation of fibrillin-containing microfibrils may be involved in the pathogenesis of pseudoexfoliation syndrome.

Leishmaniasis affecting the eyelids.

Leishman-Donovan bodies were recognized in the smear of a biopsy specimen from an eyelid ulcer. The infecting organisms were identified serologically as Leishmania braziliensis panamensis. The ulcer responded to pentavalent antimony. Ultrastructurally, the organisms had double-unit membranes, beneath which lay a palisade of microtubules. At one end of the organism, there was a rudimentary flagellum; at the other, the nucleus. A kinetoplast basal complex separated the two.

Structure of the muscles of the upper eyelid.

The human and monkey orbicularis muscle has fibers that are more uniform in size and structure than those of rectus muscles. They have distinct myofibrils, a moderate number of mitochondria, and a well-developed transverse T-tube system. The levator muscle also has relatively uniform fibers, but the myofibrils are less distinct than those of the orbicularis. Especially noteworthy is the unusual arrangement whereby Muller muscle arises directly from the undersurface of the levator muscle, causing an intimate intermingling of smooth and striated fibers. Muller muscle then inserts on the tarsus, whereas the levator muscle extends by an aponeurosis into the septa of the orbicularis muscle. In surgical specimens from patients with ptosis, the levator fibers show varying degrees of abnormality, whereas Muller fibers are normal.

The ultrastructural defect in conjunctiva from a case of recessive dystrophic epidermolysis bullosa.

The predominant feature in the several forms of epidermolysis bullosa is the formation of cutaneous bullous lesions arising after minimal mechanical trauma. Ocular involvement has been noted as a complication. To our knowledge to date, only four investigators have correlated clinical eye disease with light microscopic findings. Ultrastructure of the ocular lesions has not been described previously. We present four cases of recessive dystrophic epidermolysis bullosa emphasizing their associated ocular complications. Diagnosis was confirmed by skin biopsy specimen and in one patient by demonstrating light and electron microscopic findings in eyelid skin. This tissue exhibited ultrastructural recessive cutaneous lesions; namely, bullous separation occurring below the basal lamina and absence of anchoring fibrils in both bullous and nonbullous areas. By electron microscopy, the conjunctiva in this patient exhibited an absence of clear anchoring fibrils that were numerous in control tissue. This defect may increase the susceptibility of the conjunctiva to minor mechanical trauma, resulting in the bullous and cicatricial changes seen clinically.