Investigation of conjunctivochalasis histopathology with light and electron microscopy in patients with conjunctivochalasis in different locations.

PURPOSE: To investigate changes in conjunctival tissue of conjunctivochalasis (CCh) patients and to determine the relationship between pathological findings and localization of loose conjunctiva. METHODS: Our study included nineteen eyes of 19 patients who were referred to Cukurova University Ophthalmology Department based on ocular surface symptoms and CCh detected in ocular examination. Amniotic membrane was applied after conjunctival excision as surgical treatment. The control group was formed with five eyes of five patients who are similar in terms of age and gender distribution with our study group. Tissue samples obtained from the study and control groups were investigated with light and electron microscopy. RESULTS: Results of pathological examination of conjunctival tissues revealed increased inflammation in 13 patients (68%), lymphatic ectasia in 12 patients (63%), and loss of goblet cells in 17 patients (89%). Destruction of elastic fibers was detected in all cases by staining with elastic van Gieson. After semiquantitative assessment, varying degrees of light microscopic findings were noted considering the localization of CCh. No statistically significant relationship was observed between light microscopic findings and CCh location (p > 0.05 for all). Electron microscopic investigation revealed increase in intercellular spaces, increased cytoplasmic electron density, and the presence of slight vacuolization in cell cytoplasm, and heterochromatin clumping in nuclei of cells in conjunctival samples. CONCLUSIONS: Mechanical and inflammatory factors induce development of CCh, and signs associated with these factors can be detected with light and electron microscopy of conjunctival tissue. No relationship was observed between CCh localization and pathological changes in tissues examined in our study, and large-scale case series are required to evaluate the possible effect of CCh localization on pathological findings.

Comparison of the ocular surface changes following the use of two different prostaglandin F2α analogues containing benzalkonium chloride or polyquad in rabbit eyes.

OBJECTIVE: The aim of this study is to compare the effect of prostaglandin analogues preserved with either 0.015% or 0.001% benzalkoium chloride (BAK); or 0.001% polyquad (PQ) on the ocular surface of rabbit eyes. METHODS: Forty white rabbits were randomized to receive four-times daily instillation of either 0.0015% tafluprost (TF) preserved with 0.001% BAK (TF-BAK); 0.004% travoprost (TR) with 0.015% BAK (TR-BAK) or 0.001% PQ (TR-PQ); or preservative-free artificial tears in one eye for a 4-week period. Tear samples collected from the 40 rabbits were analyzed by enzyme-linked immunosorbent assays (ELISA) to identify the presence of inflammatory cytokines: interleukin (IL)-1ß and IL-6 on day 14. Subsequently, harvested cornea and bulbar conjunctiva were evaluated using light and transmission electron microscopy (TEM). RESULTS: IL-6 was significantly increased in TF-BAK and TR-BAK groups compared to controls and TR-PQ group (p = 0.005); however, IL-1ß level was not significantly different among four groups (p = 0.360). Rabbits treated with TR-BAK showed decreased goblet cell density of bulbar conjunctiva and increased pyknotic change and vacuolization of corneal epithelial cells on light microscopy; similar change occurred but was less severe in TF-BAK group. The TR-PQ group showed similar results as the controls. The destruction of the microvillar architecture of bulbar conjunctiva and cornea was most prominent in the TR-BAK group. CONCLUSIONS: Preservatives included in the anti-glaucoma eye-drops showed different ocular surface changes according to the concentration and type in the rabbits. Prostaglandin analogues preserved with higher level of BAK may cause more harmful effects on the ocular surface than PQ-preserved medications.

VEGF induces TGF-ß1 expression and myofibroblast transformation after glaucoma surgery.

Subconjunctival fibrosis at the surgical site determines the outcome of glaucoma surgery. Myofibroblast transformation has a significant role in fibrosis, and vascular endothelial growth factor (VEGF) is reported to trigger myofibroblast transformation by inducing transforming growth factor (TGF)-ß1. In the present study, we used IHC, Western blot analysis, enzyme-linked immunosorbent assay, and electron microscopy to determine the contribution of VEGF to myofibroblast transformation in subconjunctival fibrosis after glaucoma surgery. A rabbit trabeculectomy model was generated, and VEGF stimulation or VEGF inhibition was performed during surgery. VEGF stimulation induced TGF-ß1 expression in a dose-dependent manner. Down-regulation of epithelial markers (E-cadherin and ß-catenin) and up-regulation of mesenchymal marker (α-smooth muscle actin) were observed in the subconjunctival layers after trabeculectomy with VEGF stimulation. Up-regulations of Smad and Snail, which play a central role in myofibroblast transformation, were observed in the conjunctival and subconjunctival layers at the site of trabeculectomy. Electron microscopy revealed changes of the conjunctival epithelial cells, especially the presence of myofilaments and increased rough endoplasmic reticulum in the cytoplasm. Myofibroblast transformation was activated by VEGF stimulation and decreased by VEGF inhibition. These findings suggest that VEGF potentially affected the TGF-ß1/Smad/Snail pathway, thereby triggering myofibroblast transformation. Therapeutic approaches modulating VEGF may control myofibroblast transformation and reduce subconjunctival fibrosis after glaucoma surgery.

Effects of serum-free storage on morphology, phenotype, and viability of ex vivo cultured human conjunctival epithelium.

The use of amniotic membrane (AM) represents one of the major developments in ocular surface reconstruction. However, in a study on patients with primary pterygium, transplantation of AM with ex vivo expanded human conjunctival epithelial cells (HCjE) promoted earlier epithelialization than AM alone. We previously showed that cultured human limbal epithelial cells maintain their morphology, phenotype, and viability for one week when stored at 23°C. The current study investigates the feasibility of storing HCjE in HEPES-MEM and Optisol-GS at 23°C for 4 and 7 days, respectively. The five experimental groups were analyzed by light microscopy, immunohistochemistry, transmission electron microscopy, and a viability assay. The ultrastructural integrity of cultured HCjE was well preserved following 4 days of storage, however, 7 days of storage resulted in some loss of cell-cell contacts and epithelial detachment from the amniotic membrane. The number of microvilli in cultured HCjE not subjected to storage was 2.03±0.38 microvilli/µm. In comparison, after 4 and 7 days of HEPES-MEM storage this number was 1.69±0.54 microvilli/µm; P=0.98 and 0.89±1.0 microvilli/µm; P=0.28, respectively. After Optisol-GS storage for 4 and 7 days, the mean number of microvilli was 1.07±1.0 microvilli/µm; P=0.47 and 0.07±0.07 microvilli/µm; P=0.03, respectively. The number of cell layers in cultured HCjE not subjected to storage was 4.4±0.3 cell layers, as opposed to 4.0±0.9 cell layers; P=0.89 after 4 days of HEPES-MEM storage and 2.8±0.6 cell layers; P=0.01 after 7 days of storage in HEPES-MEM. The number of cell layers after 4 and 7 days of storage in Optisol-GS was 3.7±0.2 cell layers; P=0.46 and 3.4±0.4 cell layers; P=0.18, respectively. The expression of markers for undifferentiated cells (ΔNp63α, ABCG2 and p63), proliferating cells (Ki67 and PCNA), goblet cells (Ck7 and MUC5AC), stratified squamous epithelial cells (Ck4), and apoptotic cells (caspase-3) in cultured HCjE appeared to be unchanged after 4 and 7 days of HEPES-MEM and Optisol-GS storage. The percentage of viable cells in cultured HCjE not subjected to storage (91.4%±3.2%) was sustained after 4 and 7 days of storage in HEPES-MEM (94.1%±4.5%; P=0.99 and 85.1%±13.7%; P=0.87, respectively) as well as after 4 and 7 days of storage in Optisol-GS (87.7%±15.2%; P=0.97 and 79.8%±15.7%; P=0.48, respectively). We conclude that cultured HCjE may be stored for at least 4 days in serum-free conditions at 23°C while maintaining the phenotype and viability. HEPES-MEM appears to be comparable to Optisol-GS for serum-free storage with preservation of the ultrastructure for at least 4 days.

Intrascleral outflow after deep sclerectomy with absorbable and non-absorbable implants in the rabbit eye.

BACKGROUND: The purpose of the study is an analysis of intrascleral drainage vessels formed in rabbits' eyes after non-penetrating deep sclerectomy (NPDS) with absorbable and non-absorbable implants, and comparison to eyes in which surgery was performed without implanted material. MATERIAL/METHODS: NPDS was carried out in 12 rabbits, with implantation of non-absorbable methacrylic hydrogel (N=10 eyes) or absorbable cross-linked sodium hyaluronate (N=6 eyes), or without any implant (N=8 eyes). All the animals were euthanized 1 year after surgery. Twenty-one eyeballs were prepared for light microscopy and 3 were prepared for transmission electron microscope (TEM) analysis. Aqueous humour pathways were stained with ferritin in 6 eyeballs. RESULTS: By light microscopy, small vessels adjacent to the areas of scarring were the most common abnormality. Vessel density was significantly higher in operated sclera compared to normal, healthy tissue, regardless of the type of implant used. The average vessel densities were 2.18±1.48 vessels/mm2 in non-implanted sclera, 2.34±1.69 vessels/mm2 in eyes with absorbable implants, and 3.64±1.78 vessels/mm2 in eyes with non-absorbable implants. Analysis of iron distribution in ferritin-injected eyes showed a positive reaction inside new aqueous draining vessels in all groups. TEM analysis showed that the ultrastructure of new vessels matched the features of the small veins. CONCLUSIONS: Aqueous outflow after NPDS can be achieved through the newly formed network of small intrascleral veins. Use of non-absorbable implants significantly increases vessel density in the sclera adjacent to implanted material compared to eyes in which absorbable implants or no implants were used.

Goblet cells of the normal human bulbar conjunctiva and their assessment by impression cytology sampling.

Goblet cells of the conjunctiva are the main source of mucus for the ocular surface. The objectives of this review are to consider the goblet cells as assessed by various histological, cytological and electron microscopy methods, and to assess the consistency of published reports (over more than 25 years) of goblet cell density (GCD) from impression cytology specimens from nominally healthy human subjects. Reported GCD values have been notably variable, with a range from 24 to 2226 cells/mm² for average values. Data analysis suggests that a high density of goblet cells should be expected for the healthy human conjunctiva, with a tendency toward higher values in samples taken from normally covered locations (inferior and superior bulbar conjunctiva) of the open eye (at 973 +/- 789 cells/ mm²) than in samples taken from exposed (interpalpebral) locations (at 427 +/- 376 cells/mm²). No obvious change in GCD was found with respect to age, perhaps because the variability of the data did not allow detection of any age-related decline in GCD. Analyses of published data from 33 other sources indicated a trend for GCD to be lower than normal across a spectrum of ocular surface diseases.

Effect of hypothyroidism on postnatal conjunctival development in rats.

AIM: To study the conjunctival development in hypothyroid genetically epilepsy-prone rats (GEPRs) with serum T3 and T4 significantly lower than in normal rats. METHODS: A structural, ultrastructural and histochemical study on the conjunctival epithelium of GEPRs and of control Sprague-Dawley (SD) rats before and after eyelid opening, with particular regard to goblet cell differentiation. RESULTS: From birth to day 12, no goblet cells were demonstrated on the conjunctival surface of both strains, so that the epithelium was formed only by a cuboidal basal layer and by a superficial layer of roundish or flattened cells. On day 16, after the eyelid opening, Alcian blue (AB)-positive goblet cells filled with homogeneous granules were demonstrated isolated, in GEPRs, or clustered, in SD rats, in both the fornices and palpebral conjunctiva. The epithelium showed a basal layer and many layers of flattened cells and was taller in SD rats (8-10 layers) than in GEPRs (6-7 layers). At 3 months, the epithelium in SD rats was higher with generally clustered goblet cells, whilst in GEPRs goblet cells were both isolated or clustered. In both strains, the goblet cells showed a marked AB/periodic acid-Schiff positivity all over the conjunctival surface and were filled with granules of different density. In both strains, goblet cells were absent at birth and their appearance, as AB-positive cells, was concomitant with eyelid opening. CONCLUSIONS: Hypothyroid rats showed a conjunctival development different than that of normothyroid rats for both epithelial and goblet cells. It appears that thyroid hormone imbalance may influence conjunctival development.

Characterization of membranous (M) cells in normal feline conjunctiva-associated lymphoid tissue (CALT).

OBJECTIVE: To characterize conjunctival lymphoid nodules obtained from the nictitans of healthy cats to determine if the follicle-associated epithelium (FAE) of conjunctiva-associated lymphoid tissue (CALT) in this species contains membranous (M)-cells analogous to those described in other regions of mucosa-associated lymphoid tissue (MALT). METHODS: Lymphoid follicles from nictitan bulbar surfaces of 10 healthy cats (20 eyes total) were examined. Nictitans from five cats were harvested immediately post-mortem and a minimum of 12 lymphoid nodules from each third eyelid were isolated using a Zeiss operating microscope. At least three lymphoid follicles from each eye were examined using light microscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) using standard fixation and embedding protocols. Nictitan-lymphoid follicles from another five healthy cats were processed for immunohistochemistry to characterize the distribution of T- and B-lymphocytes present beneath the FAE. RESULTS: The FAE overlying CALT from 10 healthy cats demonstrated morphology characteristic of M-cells including attenuated apical cell surface with blunted microvilli and microfolds, invaginated basolateral membrane forming a cytoplasmic pocket, and diminished distance between the apical and pocket membrane. Immunohistochemistry of lymphoid tissue subtending the FAE demonstrated B-cell dependent regions in the germinal centers surrounded by T-cell dependent interfollicular zones. CONCLUSIONS: Healthy feline CALT contains morphologic features analogous to those described in other regions of MALT. Documentation of feline conjunctival M-cells is of clinical relevance in the study of primary infectious, allergic, and autoimmune ocular diseases, as well as a potential means of vaccination or drug delivery.

The presence and distribution of elastin in the posterior and retrobulbar regions of the mouse eye.

The Presence and distribution of elastin in the posterior and retrobulbar regions of the mouse eye was investigated. Mice of two strains (C57/BL6 and DBA/2J) were studied at 2 months and 8-12 months of age. Light, confocal, and transmission electron microscopy were used to identify elastin, using immunohistochemical techniques and ultrastructural evaluation. Elastin was found in the following ocular structures: conjunctiva, muscle tendons, sclera, choroid, and meninges. The elastin in the sclera was most dense in a ring surrounding the peripapillary optic nerve head, with its presence in the inner sclera declining with greater distance from the nerve head. Elastin fibers were oriented in the sclera along what would be expected to be the principal stress directions generated from the intraocular pressure, though actual biomechanical measurements have not yet been made in the mouse sclera. Elastin comprises a portion of the mouse sclera and its distribution in the peripapillary area is similar to that in human eyes.

Histological and immunohistochemical characterization of the porcine ocular surface.

The ocular surface of the white domestic pig (Sus scrofa domestica) is used as a helpful model of the human ocular surface; however, a complete histological description has yet to be published. In this work, we studied porcine eyeballs with intact eyelids to describe and characterize the different structures that form the ocular surface, including the cornea and conjunctiva that covers the bulbar sclera, tarsi, and the nictitating membrane. We determined the distribution of goblet cells of different types over the conjunctiva and analyzed the conjunctival-associated lymphoid tissue (CALT). Porcine eyeballs were obtained from a local slaughterhouse, fixed, processed, and embedded in paraffin blocks. Tissue sections (4 µm) were stained with hematoxylin/eosin, Alcian blue/Periodic Acid Schiff, and Giemsa. Slides were also stained with lectins from Arachis hypogaea (PNA) and Helix pomatia (HPA) agglutinins and immunostained with rabbit anti-CD3. We found that the porcine cornea was composed of 6-8 epithelial cell layers, stroma, Descemet's membrane, and an endothelial monolayer. The total corneal thickness was 1131.0±87.5 µm (mean±standard error of the mean) in the center and increased to 1496.9±138.2 µm at the limbus. The goblet cell density was 71.25±12.29 cells/mm, ranging from the highest density (113.04±37.21 cells/mm) in the lower palpebral conjunctiva to the lowest density (12.69±4.29 cells/mm) in the bulbar conjunctiva. The CALT was distributed in the form of intraepithelial lymphocytes and subepithelial diffuse lymphoid tissue. Lenticular-shaped lymphoid follicles, about 8 per histological section, were also present within the conjunctival areas. In conclusion, we demonstrated that the analyzed porcine ocular structures are similar to those of humans, confirming the potential usefulness of pig eyes to study ocular surface physiology and pathophysiology.

Ocular surface repair using decellularized porcine conjunctiva.

The primary functions of the conjunctiva embody ocular surface protection and the maintenance of the tear film equilibrium. Severe conjunctival defects such as symblepharon may impair the integrity of ocular surface and cause loss of visual functions. Here we report the use of a decellularized porcine conjunctiva (DPC) for conjunctival reconstruction in rabbit models and in clinic. Our results show that the major xenoantigens are efficiently removed, while abundant matrix components and integrated microstructures are well preserved in the DPC. These characteristics provide mechanical support and favorable histocompatibility for repairing damaged conjunctiva. The DPC application has demonstrated enhanced transplant stability and improved epithelial regeneration in severe ocular surface damage comparing to those of amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction nowadays. In order to test the DPC performance in clinic, three patients with pterygium and one patient with symblepharon underwent transplant with DPC. The grafts in all cases were completely re-epithelized and no graft melt or fibroplasia were observed. These results suggest that the strategy we developed is feasible and effective for conjunctival reconstruction and ocular surface repair. STATEMENT OF SIGNIFICANCE: In this study, we adopted an innovative approach to prepare decellularized porcine conjunctiva (DPC). The intricate conjunctiva-specific structures and abundant matrix components were preserved in DPC, which offers favorable mechanical properties for graft. DPC has shown positive effects in ocular surface repair, which has been proven particularly in a rabbit model with severe symblepharon. Reconstructed conjunctiva by DPC exhibited epithelial heterogeneity, extremely resembling that of native conjunctiva. In addition, results from clinical studies were encouraging for pterygium and symblepharon and clinical application of DPC is promising.

Calcium-binding S100 protein expression in pterygium.

PURPOSE: Pterygium is an ocular surface disease of unknown etiology associated with epithelial and fibrovascular outgrowth from the conjunctiva onto the cornea. S100 proteins are calcium-activated signaling proteins that interact with other proteins to modulate biological functions such as cell migration, proliferation, and differentiation. The aim of this study was to investigate the presence of various S100 proteins in pterygium compared to normal conjunctiva. METHODS: Immunofluorescent staining using antibodies against S100A4, S100A6, S100A8, S100A9, and S100A11 were conducted to investigate the expression and tissue distribution. S100 protein secretions and expressions were confirmed using western blot and quantitative real-time polymerase chain reaction (RT-PCR), respectively. RESULTS: Immunofluorescent staining demonstrated the presence of S100A4, S100A6, S100A8, S100A8, S100A9, and S100A11 in both conjunctival and pterygial epithelium. No significant difference was found in the localization and expression of S100A4. In both conjunctiva and pterygium, S100A4-positive cells were found in superficial and suprabasal layers. S100A6 expression was strong in the superficial layer of pterygium epithelium but relatively weaker in the suprabasal and superficial cells of normal conjunctiva epithelium. S100A8 and S100A9 were localized in the superficial layer of both pterygium and normal conjunctiva epithelium, with higher levels in pterygium than uninvolved conjunctiva. S100A11 was expressed in the basal cells of conjunctival epithelium but in the suprabasal layers of pterygium epithelium. Western blot and RT-PCR confirmed the presence of S100A4, S100A6, S100A8, S100A9, and S100A11 in pterygium and conjunctiva tissue. CONCLUSIONS: Higher levels of S100A6, S100A8, and S100A9 expressions were detected in the pterygium tissue relative to normal conjunctiva. In addition, a distinct alteration of localization of S100A11 expression was observed in pterygium epithelium compared to the conjunctiva. Therefore, these S100 proteins may be associated with the formation of pterygium.

Alteration of ocular surface mucins in MUC5AC-DTA transgenic mice.

PURPOSE: To investigate the compensation of secretory mucins with membranous mucins in mice with goblet cell deficiency. METHODS: A transgenic mouse model in which conjunctival goblet cells were targeted was generated, and the expression of mucins was evaluated through the toxicity of diphtheria toxin A driven by a human mucin, MUC5AC, promoter. Immunohistochemical staining, in situ hybridization, electronic microscopy, and quantitative reverse transcription polymerase chain reaction (RT-PCR) were used to characterize their phenotypes. RESULTS: The external appearance of the ocular surface was normal, and no corneal pathology was found. The quantity of MUC5AC and the number of conjunctival goblet cells decreased in this mouse as expected. However, the membranous mucin, MUC4, compensates the decrease of MUC5AC in terms of the results of immunohistochemical staining, in situ hybridization, electronic microscopy, and quantitative RT-PCR. CONCLUSIONS: The membranous mucin, MUC4, can compensate for the deficiency of the secretory mucin, MUC5AC, in goblet cell deficient mice. This compensation may explain why the symptoms of mucus threads can be found in some goblet deficiency diseases, and it may provide an alternative defensive mechanism in goblet cell deficiency.

Microplicae: characteristic ridge-like folds of the plasmalemma.

Scanning electron microscopy reveals that the free surfaces of stratified squamous epithelial cells lining the alimentary tract, cornea, and conjunctiva exhibit characteristic ridge-like folds of plasmalemma. These microplicae are approximately 0.1-0.2 micronm in width, of variable height (0.2-0.8 micronm) and length, may followstraight or winding paths, often branch, and exhibit a wide variety of patterns over the surfaces of cells. Transmission electron microscopy reveals that microplicae often have a fine (100-150 A) electron-dense zone subjacent to their plasmalemma and an intracellular matrix characterized by a disorderly arrary of fine filaments (40-60 A in diameter). Microplicae appear to arise from plasmalemmal fold which once provided for intercellular interdigitation and desmosome abhesion between adjacent cells. Ruthenium red staining demonstrates that microplicae and interplical grooves are covered with a polyanionic glycocalyx. Although free surface microplicae may merely represent the renmants of intercellular interdigitations or a modified expression of microvillous-like extensions, it is also possible that they serve another specific function. In this regard it is speculated that microplical and interplical grooves may function to hold a layer of lubricating and cushioning mucin designed to protect the underlying plasmalemma from abrasive abuse.

Expression of specific keratin markers by rabbit corneal, conjunctival, and esophageal epithelia during vitamin A deficiency.

Using an in vivo rabbit model system, we have studied the morphological and biochemical changes in corneal, conjunctival, and esophageal epithelia during vitamin A deficiency. Light and electron microscopy showed that the three epithelia undergo different degrees of morphological keratinization. Corneal and conjunctival epithelia became heavily keratinized, forming multiple layers of superficial, anucleated cornified cells. In contrast, esophageal epithelium underwent only minor morphological changes. To correlate morphological alterations with the expression of specific keratin molecules, we have analyzed the keratins from these epithelia by the immunoblot technique using the subfamily-specific AE1 and AE3 monoclonal antikeratin antibodies. The results indicate that during vitamin A deficiency, all three epithelia express an AE1-reactive, acidic 56.5-kd keratin and an AE3-reactive, basic 65-67-kd keratin. Furthermore, the expression of these two keratins correlated roughly with the degree of morphological keratinization. AE2 antibody (specific for the 56.5- and 65-67-kd keratins) stained keratinized corneal epithelial sections suprabasally, as in the epidermis, suggesting that these two keratins are expressed mainly during advanced stages of keratinization. These two keratins have previously been suggested to represent markers for epidermal keratinization. Our present data indicate that they can also be expressed by other stratified epithelia during vitamin A deficiency-induced keratinization, and suggest the possibility that they may play a role in the formation of the densely packed tonofilament bundles in cornified cells of keratinized tissues.

Evaluation of Subconjunctival Remnant Particles after High-frequency Radio-wave Electrosurgery for Conjunctivochalasis.

PURPOSE: To investigate the clinical manifestations and properties of remnant particles in the subconjunctival space after high-frequency radio-wave electrosurgery for conjunctivochalasis. METHODS: We performed a retrospective, observational case series with in vitro experimental imaging in nine eyes from eight patients who presented with small dark-gray lesions during follow-up after high-frequency radio-wave electrosurgery for conjunctivochalasis. General examination including slit-lamp examination and visual acuity testing was performed preoperatively and postoperatively. During follow-up, we evaluated remnant particles and any other complications including granuloma or conjunctival injection with slit-lamp photography and anterior optical coherence tomography. Coagulation tips were investigated with scanning electron microscope and energy dispersive X-ray spectroscopy to analyze the insulating electrode and assess changes to tips after repeated use. RESULTS: None of the patients included in this study experienced any change in visual acuity or major complications postoperatively. Small dark-gray lesions (0.3 to 0.5 mm in size) were observed in the inferior bulbar sub-conjunctival space in the location where high-frequency radio-wave electrosurgery had been performed. Cirrus high-definition optical coherence tomography images revealed focal hyper-reflection with a posterior shadow, suggesting foreign particles. Scanning electron microscopy and energy dispersive X-ray spectroscopy imaging analysis revealed peaks of carbon and fluorine complexes, consistent with the polytetrafluoroethylene coating on the electrode. CONCLUSIONS: There were no instances of inflammatory reaction, particle migration, or major complications due to particles. Physicians should be aware of the possibility of remnant polytetrafluoroethylene particles in subconjunctival tissue when using insulated coagulation tips subjected to repeat sterilization.

A rabbit dry eye model induced by topical medication of a preservative benzalkonium chloride.

PURPOSE: To establish a rabbit dry eye model with topical medication of the ocular preparation preservative benzalkonium chloride (BAC). METHODS: Sixteen white rabbits were used. One eye of each rabbit was chosen randomly for topical administration of 0.1% BAC twice daily for 14 days. The other untreated eyes served as controls. Schirmer test, fluorescein, and rose bengal staining were performed before and after BAC treatment on days 3, 5, 7, and 14. Conjunctiva impression cytology specimens were collected on days 0, 7, and 14. The rabbits were killed after day 14. Immunofluorescence staining was performed to detect mucin-5 subtype AC (MUC5AC) on conjunctival cryosections. Cornea and conjunctiva structures were evaluated by light and electron microscopy. RESULTS: Compared with untreated controls, BAC-treated eyes showed significant decreases in Schirmer scores (P = 0.01) and increases in fluorescein scores (P < 0.001) on days 5, 7, and 14. A significant increase in rose bengal scores was noticed as early as day 3 (P = 0.001). Decreases in goblet cell density occurred on days 7 and 14 (P = 0.001). Decreased MUC5AC and histopathologic and ultrastructural disorders of the cornea and conjunctiva were also observed in the BAC group. CONCLUSIONS: These findings demonstrated that an ophthalmic preservative, benzalkonium chloride, induced a dry eye syndrome in rabbits with damage to the cornea and conjunctiva, decreased aqueous tear basal secretion, goblet cell loss, and MUC5AC deficiency. This rabbit model was consistent with human dry eye syndrome in both aqueous tear and mucin deficiency and may be appropriate for studying dry eye syndrome.

Analysis of conjunctival fibroblasts from a proband with Schnyder corneal dystrophy.

PURPOSE: To analyze for the presence of lipids in conjunctival fibroblasts of a patient with Schnyder corneal dystrophy (SCD). METHODS: A proband with SCD was identified, and the pedigree was examined. The proband underwent an automated lamellar therapeutic keratoplasty (ALTK). At the same time, the proband underwent a skin and conjunctival biopsy. Specimens were sent for histological and ultrastructural examination. Conjunctival fibroblasts were cultured from the biopsy specimen and stained with filipin. RESULTS: The proband showed no evidence of recurrence following the ALTK procedure. Histopathological examination showed no evidence of hydrophobic lipids in the conjunctival or dermal fibroblasts. Lipid particles were detected in the cornea. Ultrastructural examination showed no lipid particles in the conjunctival fibroblasts. Cultured fibroblasts showed no evidence of intracellular unesterified cholesterol unless low density lipoprotein (LDL) was added to the culture medium. CONCLUSIONS: There was no evidence of lipid deposition in conjunctival or skin fibroblasts in our patient with SCD. The evidence suggests local factors are responsible for the lipid deposition in the cornea.

Culture and characterization of oral mucosal epithelial cells on human amniotic membrane for ocular surface reconstruction.

PURPOSE: To culture oral mucosal epithelial cells on deepithelialized human amniotic membrane without the use of feeder cells and to compare the characteristics of cultured oral cells with cultured limbal and conjunctival epithelial cells for use in ocular surface reconstruction. METHODS: Oral biopsies were obtained from healthy volunteers after informed consent and were cultured on deepithelialized amniotic membrane for three to four weeks. Confluent cultures of limbal, oral, and conjunctival cells were subjected to characterization of markers of stem cells and of epithelial differentiation by reverse-transcription polymerase chain reaction (RT-PCR) and by immunohistochemistry. Ultrastructural studies were also performed using electron microscopy. RESULTS: A sheet of healthy, stratified oral epithelial cells was obtained within three to four weeks of culture. Electron microscopy demonstrated that the cells formed gap junctions and desmosomes. RT-PCR analysis showed that cultured oral epithelial cells expressed markers of epithelial differentiation such as cytokeratin K3, K4, K13, K15 and connexin 43. The cells also expressed stem cell markers of epithelial cells such as DeltaN isoforms of p63 as well as p75, a marker for stem cells of oral epithelium. The cells did not express cytokeratin K12 or Pax-6, an eye-specific transcription factor. CONCLUSIONS: Oral epithelial cells can be cultured as explants on deepithelialized amniotic membrane without using feeder cells. Characterization showed that these cells maintain the phenotypic characteristics of oral epithelial cells and that the culture is a heterogeneous population of differentiated cells and stem cells. We find the cultured oral epithelial cells usable for ocular surface reconstruction in patients having bilateral ocular surface diseases.

Chlamydiae and polymorphonuclear leukocytes: unlikely allies in the spread of chlamydial infection.

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.