Ocular surface findings in impression cytology after interferon a2b or mitomycin C in rabbits / Achados em citologia de impressão da superfície ocular após uso de interferon alfa-2b ou mitomicina C: estudo em coelho

ABSTRACT Objective: To describe ocular surface findings in impression cytology obtained from healthy rabbit conjunctiva treated with interferon alpha-2b eyedrop, and compare them to findings after use of mitomycin C 0.02%. Methods: An experimental study using a rabbit model was performed between September 2013 and October 2014 at the Faculdade de Medicina de Marília, Universidade Federal de São Paulo, Clínica de Olhos Moacir Cunha. Thirty New Zealand white rabbits were divided into 6 groups and received interferon alpha-2b or mitomycin C 0.02%. Impression cytology (IC) was performed prior to topical applications and at15, 30 and 60 days of use. The following variables were analyzed in impression cytology: goblet cells, cellularity, cell-to-cell adhesion, nucleus/cytoplasm ratio, nuclear chromatin, inflammatory cells keratinization, and cytomegaly. Results: The major findings in impression cytology after us of interferon alpha-2b included loss of goblet cells (50.8%), reduced cell-to-cell adhesion (26.2%), abnormal nucleus/cytoplasm ratio (20%) and reduced cellularity (15.4%). After use of mitomycin C 0.02%, the most common changes included loss of goblet cells (46.2%), abnormal nucleus/cytoplasm ratio (25.6%), less cell-to-cell adhesion (23.1%), and reduced cellularity (20.5%). There were no significant differences in any variable when comparing impression cytology after interferon alpha-2b and after mitomycin C 0.02%. Goblet cell loss was more pronounced at days 30 and 60, as compared to impression cytology at day 15 for both drugs. Conclusion: The loss of goblet cells, reduced cell-to-cell adhesion and cellularity, along with abnormal nucleus/cytoplasm ratio were the most common findings in impression cytology after use of interferon alpha-2b. These findings are similar to those described for use of mitomycin C 0.02%. ..

RESUMO Objetivo: Descrever os achados em citologia de impressão de conjuntiva sadia de coelho submetida ao uso de colírio de interferon alfa-2b e compará-los ao que foi encontrado após uso da mitomicina C 0,02%. Métodos: Estudo experimental realizado em modelo animal no período entre setembro de 2013 e outubro de 2014 nas dependências da Faculdade de Medicina de Marília, da Universidade Federal de São Paulo e da Clínica de Olhos Moacir Cunha. Trinta coelhos albinos da raça Nova Zelândia foram divididos em seis grupos e receberam interferon alfa-2b ou mitomicina C. A citologia de impressão foi realizada antes do início dos colírios e após 15, 30, 60 dias de seu uso. As seguintes variáveis foram analisadas na citologia de impressão: células caliciformes, celularidade, adesão intercelular, razão núcleo/citoplasma, cromatina, células inflamatórias, queratinização e citomegalia. Resultados: Os principais achados na citologia de impressão após o uso do interferon alfa-2b foram a redução de células caliciformes (50,8%), a diminuição da adesão intercelular (26,2%), a alteração da razão N/C (20%) e a redução da celularidade (15,4%). Após o uso da mitomicina C 0,02%, foram mais frequentes a redução das células caliciformes (46,2%), a alteração da razão N/C (25,6%), a adesão intercelular (23,1%) e a redução da celularidade (20,5%). Não houve diferença estatisticamente significante para nenhuma das variáveis estudas quando se compararam as citologias de impressão após interferon alfa-2b com as citologias de impressão após mitomicina C 0,02%. Independentemente da substância utilizada, as citologias colhidas 30 e 60 dias após início das drogas apresentaram maior redução de células caliciformes quando comparadas com as citologias de impressão colhidas após 15 dias. Conclusão: A redução das células caliciformes, a diminuição da adesão intercelular, a alteração da razão N/C e a diminuição da celularidade foram as alterações mais frequentes na citologia de impressão colhida após o uso de interferon alfa-2b. Os achados em citologias de impressão após o uso de interferon alfa-2b são semelhantes àqueles encontrados após o uso da mitomicina C 0,02%.

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.

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.

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.

Ocular Permeation and Sustained Anti-inflammatory Activity of Dexamethasone from Kaolin Nanodispersion Hydrogel System.

PURPOSE: Kaolin can adhere to the mucosa and protect it by absorbing toxins, bacteria, and viruses. Ocular delivery and anti-inflammatory activity of dexamethasone hydrogel system could be advantageous after kaolin incorporation. METHODS: Hydroxypropyl methylcellulose (HPMC) films of dexamethasone have been prepared without and with kaolin by solvent casting method. Differential scanning calorimetry (DSC), X-ray diffractometry (XRD), and scanning electron microscopy (SEM) were utilized for evaluating thermal property, crystallinity, and morphology of the film preparations respectively. In vitro drug release and corneal permeation ex vivo were carried out in phosphate buffer saline of pH 7.4 (PBS) at 34 ± 0.5°C for 6 h. Anti inflammatory effect of the prepared film was evaluated using carrageenan induced rabbit eye. RESULTS: Disappearance of melting endotherm in the DSC thermogram is the indication of almost complete amorphization of drug in all the films. High-intensity reflections with characteristic peaks of pure drug crystal have resulted extensively reduced ordering of the crystal lattice in the X-ray pattern of all the films. Photomicrographs revealed that the plate-shaped geometry of the drug crystal has almost been lost in absence and presence of the nano-kaolin particles in the films. Kaolin incorporation controlled the drug release up to 6 h. Ocular permeation was diffusion controlled and extended for 6 h or more without exhibiting significant "Burst effect". Adsorption of drug onto the surface of nano-kaolin prolonged the permeation due to cation exchange and hydrogen bonding. Signs of inflammation of the carrageenan induced rabbit eye have been disappeared almost completely after 2 h of film application. CONCLUSIONS: Local controlled delivery sustained anti-inflammatory activity of dexamethasone has been achieved using kaolin incorporated HPMC film.

Decellularised conjunctiva for ocular surface reconstruction.

Conjunctival reconstruction is an integral component of ocular surface restoration. Decellularised tissues are frequently used clinically for tissue engineering. This study identifies porcine decellularised conjunctiva (PDC) and human decellularised conjunctiva (HDC) as promising substitutes for conjunctival reconstruction. PDC and HDC were nearly DNA-free, structurally intact and showed no cytotoxic effects in vitro, which was confirmed by DNA quantification, histology, transmission electron microscopy, collagen quantification and cytotoxicity assay. Comparing the biomechanical properties to amniotic membrane (AM), the most frequently applied matrix for ocular surface reconstruction today, the decellularised conjunctiva was more extensible and elastic but exhibited less tensile strength. The in vivo application in a rabbit model proofed significantly enhanced transplant stability and less suture losses comparing PDC and HDC to AM while none of the matrices induced considerable inflammation. Ten days after implantation, all PDC, 4 of 6 HDC but none of the AM transplants were completely integrated into the recipient conjunctiva with a partially multi-layered epithelium. Altogether, decellularised conjunctivas of porcine and human origin were superior to AM for conjunctival reconstruction after xenogeneic application in vivo. STATEMENT OF SIGNIFICANCE: Conjunctival integrity is essential for a healthy ocular surface and clear vision. Its reconstruction is required in case of immunological diseases, after trauma, chemical or thermal burns or surgery involving the conjunctiva. Due to limitations of currently used substitute tissues such as amniotic membrane, there is a need for the development of new matrices for conjunctival reconstruction. Decellularised tissues are frequently applied clinically for tissue engineering. The present study identifies porcine and human decellularised conjunctiva as biocompatible and well tolerated scaffolds with superior integration into the recipient conjunctiva compared to amniotic membrane. Decellularised conjunctiva depicts a promising substitute for conjunctival reconstruction in ophthalmology.

The Corneal Epithelial Barrier and Its Developmental Role in Isolating Corneal Epithelial and Conjunctival Cells From One Another.

Purpose: During development, the corneal epithelium (CE) and the conjunctiva are derived from the surface ectoderm. Here we have examined how, during development, the cells of these two issues become isolated from each other. Methods: Epithelia from the anterior eyes of chicken embryos were labeled with the fluorescent, lipophilic dye, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). DiI was placed on the epithelial surface of the developing anterior eye and its diffusion was monitored by fluorescence microscopy. Concomitant morphologic changes in the surface cells of these epithelial were examined by scanning electron microscopy. Immunofluorescence was used to analyze the expression of cytokeratin K3, ZO-1, N-cadherin and Connexin-43 and the function of gap junctions was analyzed using a cut-loading with the fluorescent dye rhodamine-dextran. Results: Prior to embryonic day 8 (E8), DiI placed on the surface of the CE spreads throughout all the epithelial cells of the anterior eye. When older eyes were similarly labeled, dye diffusion was restricted to the CE. Similarly, diffusion of DiI placed on the conjunctival surface after E8 was restricted to the conjunctiva. Scanning electron microscopy showed that developmentally (1) physical separations progressively form between the cells of the CE and those of the conjunctiva, and (2) by E8 these separations form a ring that completely encompasses the cornea. The functional restriction of gap junctions between these tissues did not occur until E14. Conclusions: During ocular development, a barrier to the diffusion of DiI forms between the contiguous CE and conjunctiva prior to the differential expression of gap junctions within these tissues.

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.

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.

Evaluation of Oxidative Stress Markers in Human Conjunctival Epithelial Cells Exposed to Diesel Exhaust Particles (DEP).

PURPOSE: The aim of this study was to evaluate oxidative stress markers in human conjunctival epithelial cells (IOBA-NHC) exposed to diesel exhaust particles (DEP). METHODS: Reactive oxygen (ROS) and nitrogen (RNS) species production; hydrogen peroxide (H2O2) levels; protein oxidation; antioxidant enzymes activities (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx], glutathione S-transferase [GST], and glutathione reductase [GR]); total reactive antioxidant potential (TRAP); reduced (GSH) and oxidized glutathione (GSSG) were evaluated. Transmission electron microscopy was performed to evaluate DEP uptake. RESULTS: Diesel exhaust particles were entrapped by membrane protrusions developed by IOBA-NHC. Cells exposed to DEP 50 and 100 µg/mL showed a significant increase in ROS, RNS, H2O2 levels, SOD, GPx, and GST compared with the control group. A significant decay in GR was observed in both groups, meanwhile CAT levels remained unchanged. The group exposed to DEP 100 µg/mL displayed a significant increase in protein oxidation. In both groups, TRAP was significantly reduced as well as the GSH/GSSG ratio. CONCLUSIONS: The decrease in nonenzymatic antioxidants and the compensatory increase of SOD, GPX, and GST activities are consequence of the increase in ROS and RNS production due to DEP exposure and its accumulation inside the cells. The decay in GR activity leads to the decrease in GSH/GSSG recycling. These results suggest that oxidative stress could play an important role in the development of DEP effects on human conjunctival epithelial cells.

Immunomodulatory effects of galectin-1 on an IgE-mediated allergic conjunctivitis model.

PURPOSE: Galectin (Gal)-1, a lectin found at sites of immune privilege with critical role in the inflammation, has been poorly investigated in the ocular inflammatory diseases. Here, we evaluated the therapeutic potential of Gal-1 in ocular allergy using a model of ovalbumin (OVA)-induced AC. METHODS: OVA-immunized BALB/c male mice were challenged with eye drops containing OVA on days 14 through 16 with a subset of animals pretreated intraperitoneally with recombinant Gal-1 (rGal-1) or dexamethasone (Dex). RESULTS: Recombinant Gal-1 and Dex administration on days 14 through 16 was effective in reducing the clinical signs of allergic conjunctivitis (AC), plasma anti-OVA IgE levels, Th2 (IL-4 and IL-13), and eotaxin/RANTES levels in the lymph nodes. Four hours after the last OVA challenge, rGal-1 markedly increased Gal-1 endogenous levels in the conjunctiva, and provoked eosinophilia, which persisted at 24 hours. Recombinant Gal-1 had no effect on eosinophil activation, as evidenced by the similar pattern of peroxidase eosinophil expression between cells of rGal-1-treated and untreated AC groups. Conjunctival migrated eosinophils and neutrophils exhibited high levels of Gal-1 and ß2-integrin, with points of colocalization, in the rGal-1-treated groups. These different effects observed for rGal-1 were correlated with elevated levels of activated ERK and p38 at 4 hours, and diminished levels of activated JNK and p38 at 24 hours in the eyes. CONCLUSIONS: Gal-1 has an important role in ocular allergic inflammation and represents a potential target for the development of new therapeutic strategies in eye diseases.

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.

Functional morphology of mucosal goblet cells based on spatial separation of orifice openings to the surface--application to the rabbit bulbar conjunctiva.

The purpose of the study was to assess spatial separation of goblet cell orifices observed at the surface of the rabbit bulbar conjunctiva by scanning electron microscopy (SEM) specimens of the bulbar conjunctiva from 8 healthy pigmented rabbits were obtained using a special preparation technique by which the tissue was carefully stretched out during glutaraldehyde fixation. On high magnification prints of SEM images of the conjunctival surface, the locations of goblet cell openings (orifices) to the apical surface were marked and the centre-to-centre spacing between all such orifices measured. Across the regions of interest (ROI), with averaged dimensions of 322 µm × 230 µm (adjusted for tissue shrinkage), the averaged value for the distances between all orifices was 196 µm (range 141-241 µm), with the calculated density of orifices being 412 mm(-2). A sequential order-based analysis of the spatial separation between orifices indicated a predictable value of 6 µm, a separation that showed a nearly linear inter-dependence over distances of at least 200 µm. The openings of goblet cells to the surface of unstimulated bulbar conjunctiva have a organized spatial distribution that is consistent with there being an organized control of goblet cell secretion.

Estudio del epitelio conjuntival de las ampollas filtrantes mediante citología de impresión / Evaluation of conjunctival epithelium of filtering blebs by impression cytology

OBJETIVO: Estudiar la superficie conjuntival de ampollas filtrantes mediante citología de impresión y comparar los resultados con el área de conjuntiva circundante. MÉTODO: Se incluyeron 12 ampollas de 8 pacientes; 4 tras trabeculectomía sin mitomicina C (MMC), 6 tras trabeculectomía con MMC y 2 tras esclerectomía profunda no perforante sin MMC. Se realizaron citologías de impresión conjuntival de las ampollas filtrantes y del área circundante. Se usó una clasificación de 0 a 3 para describir el grado de cohesión de las células epiteliales y la densidad de células caliciformes, siendo el grado 0 indicador de células epiteliales en placas y abundantes células caliciformes y el grado 3, de pérdida de cohesión del epitelio y ausencia de células caliciformes. RESULTADOS: El grado medio de la citología de impresión en las ampollas de filtración fue de 2,4 ± 0,9 y en el área circundante de 0,8 ± 0,3 (p < 0,001). Estas diferencias fueron independientes del uso de MMC (p = 0,48). El 83% de las ampollas presentaban células epiteliales cilíndricas dehiscentes y ausencia de células caliciformes. En la conjuntiva circundante, el 100% de los casos presentaban células epiteliales en placas con distintos grados de células caliciformes. CONCLUSIÓN: El epitelio de las ampollas filtrantes presentó cambios significativos que consistían en aumento de los espacios intercelulares y pérdida de células caliciformes. Estos espacios intercelulares aumentados podrían explicar un paso transcelular del humor acuoso. La pérdida de mucina secundaria a la pérdida de células caliciformes contribuiría al riesgo de infección de las ampollas filtrantes

PURPOSE: To study the ocular surface in filtering blebs using impression cytology, comparing the bleb side and areas outside the bleb edges. METHODS: Twelve filtering blebs of 8 patients were included: 4 cases of trabeculectomy without mitomycin C (MMC), 6 cases of trabeculectomy with MMC, and 2 cases of non-penetrating glaucoma surgery. Impression cytology specimens were taken from filtering blebs as well as outside the bleb area. A classification scale from 0 to 3 was used to describe the distribution of epithelial cells and the density of goblet cells. Grade 0 indicated cohesive epithelial cells and abundant goblet cells; and the grade 3 indicated loss of epithelial cohesion and absence of goblet cells. RESULTS: The mean grade of cytology in filtering blebs was 2.4 ± 0.9, and in the outside bleb area of 0.8 ± 0.3 (P<0.001). These differences were independent of the use of MMC (P=0.48). The large majority (83%) of filtering blebs showed a decrease in epithelial cohesion and absence of goblet cells. Outside the bleb area, 100% of the cases had cohesive epithelial cells with different grades of goblet cells. CONCLUSION: The conjunctival epithelium overlying the filtering blebs showed significant changes that consisted of increased intercellular spaces and loss of goblet cells. These increased intercellular spaces could explain the trans-epithelial pathway of aqueous humor. The least amount of mucin due to loss of goblet cells could contribute to increase the risk of infection in filtering blebs

Phototoxic effects of an operating microscope on the ocular surface and tear film.

PURPOSE: We evaluated light exposure-induced dry eye syndrome by investigating the phototoxic effects of an operating microscope on the ocular surface and tear film in rabbits. METHODS: Sixty eyes of 30 rabbits were divided into 3 groups based on the intensity of light exposure received from an operating microscope: Control group, no exposure to light; group A, 40,000-lx intensity for 30 minutes; and group B, 100,000-lx intensity for 30 minutes. To evaluate the potential damage to the ocular surface and tear film, Schirmer tests, rose bengal staining, and conjunctival impression cytology were performed before the light exposure and at 1, 3, and 5 days afterward. In addition, the expression of interleukin 1-beta was analyzed in tear samples. The expression of mucin 5AC was evaluated using immunofluorescence staining, and periodic acid-Schiff staining was conducted on conjunctival tissues. Corneal and conjunctival tissues were observed by means of electron microscopy. RESULTS: Potential damage to the ocular surface and tear film was found in the light-exposed groups as evidenced by decreased aqueous tear production, devitalized corneal and conjunctival epithelial cells, squamous metaplasia of conjunctival epithelial cells, decreased conjunctival goblet cell density, decreased expression of mucin 5AC, ultrastructural cellular damage to corneal and conjunctival tissues, and increased interleukin 1-beta expression in tears. This damage was more noticeable in group B than in group A (P < 0.05). CONCLUSIONS: Light exposure from an operating microscope had phototoxic effects on the ocular surface and tear film in this in vivo experiment. These changes seemed to intensify as the intensity of the light increased. Therefore, excessive light exposure during ophthalmic procedures could be a pathogenic factor in dry eye syndrome after a surgery is performed.

Development, alteration and real time dynamics of conjunctiva-associated lymphoid tissue.

PURPOSE: Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model. METHODS: Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy. RESULTS: Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min. CONCLUSIONS: CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.

Scanning electron microscopy applied to impression cytology for conjunctival damage from glaucoma therapy.

PURPOSE: To apply scanning electron microscopy to impression cytology (IC) to evaluate conjunctival damage in patients undergoing topical glaucoma therapy. METHODS: All patients undergoing glaucoma therapy and without ocular surface disorders between September 2012 and January 2013 were enrolled. An age- and gender-matched group without glaucoma served as the control group. Conjunctival epithelium was evaluated with the ferning test (FT), impression cytology with light optic microscopy (ICOM), and impression cytology with scanning electron microscopy (ICSEM). RESULTS: Twenty patients (40 eyes; 11 men and 9 women, mean age 59.9 ± 11 years) constituted the treated group. The mean duration of glaucoma therapy was 25.5 ± 13.8 months (range, 6-48 months). The mean FT, ICOM, and ICSEM grades were 2.52 ± 0.5, 2.52 ± 0.6, and 2.55 ± 0.7, respectively. Treatment duration was not significantly correlated with FT/IC grade (P = 0.1), whereas it was significantly correlated with microvilli count at ICSEM (P = 0.01). The mean FT, ICOM, and ICSEM grades were significantly lower in the control group (40 eyes; 11 men and 9 women, mean age 61.1 ± 7.12 years) than in the treated group (1.22 ± 0.4, 1.25 ± 0.4 and 1.15 ± 0.3, respectively, P < 0.001). CONCLUSIONS: The FT, ICOM, and ICSEM grades were lower in eyes undergoing glaucoma therapy than in control eyes. Treatment duration was significantly associated with a reduced microvilli count at ICSEM, but not with FT or ICOM grades. Reduction of microvilli could be the first sign of cellular damage during chronic glaucoma therapy.

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.

The ocular surface: from physiology to the ocular allergic diseases.

Allergic conjunctivitis (AC) is an inflammation of the conjunctiva secondary to an immune response to exogenous antigens, usually called allergens. In fact, AC is a syndrome that involves the entire ocular surface, including conjunctiva, lids, cornea, and tear film. The signs and symptoms of AC have a meaningful effect on comfort and patient health, and could be influenced by environment, genetics and immune regulation mechanisms, all of which work together in a complex immunological homeostasis. Dysregulation in such immune responses could turn into a variety of ocular allergic diseases (OAD). This review describes some of the current understanding of cellular and molecular pathways involved in different OAD.

La conjuntivitis alérgica es la inflamación de la conjuntiva secundaria a una respuesta inmunitaria contra antígenos exógenos, usualmente llamados alergenos. De hecho, la conjuntivitis alérgica es un síndrome que involucra la totalidad de la superficie ocular, incluyendo la conjuntiva, los párpados, la córnea y la película lagrimal. Los signos y síntomas de la conjuntivitis alérgica tienen un efecto significativo en el bienestar y salud del paciente y pueden ser influidos por el ambiente, la genética y mecanismos de regulación inmunológicos, todos los cuales trabajan en conjunto en una compleja homeostasia inmunológica. La disregulación de estos mecanismos puede desembocar en una gran variedad de enfermedades alérgicas oculares. Esta revisión describe algunos de los conocimientos celulares y moleculares actuales, involucrados en las diferentes enfermedades alérgicas oculares.