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%.

possible protective role of curcumin versus garlic in amiodarone-induced keratopathy in rats: light and electron microscopic study

Amiodarone is a highly effective antiarrhythmic drug. However, amiodarone-induced corneal keratopathy is one of the most common side effects in humans. Amiodarone toxicity is attributed to its metabolites, which induce a significant increase in inflammatory mediators and cytokine release as well as free-radical formation. To clarify the possible protective effect of the natural antioxidants curcumin versus garlic on amiodarone-induced keratopathy. This study was carried out on 40 male albino rats weighing 180-200 g. The rats were divided randomly into four main groups [10 rats each]: group I included rats that were used as controls. The rats in group II received amiodarone [30 mg/kg/day] daily by a gavage for 6 weeks. The rats in groups III and IV received curcumin [100 mg/kg/day] and garlic [300 mg/kg/day], respectively, plus amiodarone. After 6 weeks, blood samples were collected for the determination of malondialdehyde and superoxide dismutase serum levels. Then, corneal specimens were rapidly taken from all rats and processed for both light and electron microscopic examination. Light microscopic examination of group II corneal sections showed that the majority of the basal and intermediate epithelial cell layers of the corneal epithelium showed a vacuolated cytoplasm with pyknotic nuclei. The substantia propria became thick and showed abundant collagen bundles, focal masses of disorganized irregularly arranged collagen, and neovascularization. The Descemet's endothelial cell layer appeared thick with focal areas of stratification. In addition, the mean corneal thickness in group II was significantly increased [P<0.05]. Ultrastructural examination showed shrunken irregular basal and intermediate epithelial cell layers with multiple cytoplasmic vacuoles, wide intercellular spaces, and partial loss of the desmosomal junction. The keratocytes and stratified Descemet's endothelial cells showed numerous cytoplasmic vacuoles, lysosomes, and lamellar bodies. Moreover, the mean serum level of malondialdehyde was significantly increased [P<0.05] and the mean serum level of superoxide dismutase was significantly decreased [P<0.05]. In the rats of group IV, garlic significantly attenuated the amiodarone-induced corneal changes and the biochemical alterations compared with curcumin [group III]. Garlic exerts a greater ameliorative effect on amiodarone-induced keratopathy over curcumin and it can be recommended as an adjuvant therapy with amiodarone in patients treated with this drug for a long time

Histological study on the effect of aminoguanidine on the cornea of rat of streptozotocin induced diabetes

Diabetes mellitus is increasing worldwide at an alarming rate. Patient morbidity related to diabetic induced ocular complications has increased year on year proportionate with the worldwide increase in the incidence of diabetes. Diabetic keratopathy is a common ocular complication of diabetes. The present study tried to investigate the effects of experimentally induced diabetes by Streptozotocin [STZ] on the structure of cornea and the role of aminoguanidinc administration to ameliorate these effects.Twenty adult male albino rats were divided into four groups five animals each; Group I [control group]. Group II [diabetic]. Group III [diabetic and aminoguanidine]. Group IV [non diabetic and aminoguanidine]. At the end of experiment, the rats were sacrificed and the corneas of different groups were processed for light and electron microscopic examination. Immunohistochemical study was done using caspase-3 to detect the apoptotic changes. The thickness of corneal layers was measured by image analyzer and statistical analysis was done. Light microscopic examination of group II revealed marked histological alteration in the form of degenerative changes. Immunohistochemical reaction showed increased number of apoptotic cells in most layers of the cornea. Statistical analysis of group II revealed a significant increase in thickness of all corneal layers as compared to all groups. Electron microscopic examination revealed irregularity of the basement membrane of corneal epithelium. The stroma showed focal loss of collagen fibrils. The endothelial coils were enlarged and distorted. Group III showed a more or less restoration of normal histological and morphometric structures of the cornea. Group IV was comparable to control group. Diabetes caused structural alterations in the cornea. However, Aminoguanidine improved structural changes caused by diabetes

Ultrastructure studies of a macular dystrophy cornea-case study

Macular corneal dystrophy [MCD] is an autosomal recessive disease characterised by deposition of glycosaminoglycans in corneal. In the present studies we describe ultrastructura] features of a macular dystrophy cornea. Corneal buttons of A 54 years old female patient who was suffering with Macular dystrophy, removed and fixed in freshly prepared 2.5% glutaraldehyde containing 0.05% cuprolinic blue [BDH Ltd, Dorset, U.K.] using acritical electrolyte concentration mode. The tissue was infiltrated in Spurr resin three times [8 hrs each] and polymerised in Spurr resin at 70°C for Shrs. Ultrathin sections were studied with transmission microscope. Ultrastructural studies showed irregular distribution of variable diameter collagen fibrils in disturbed lamellae. The proteoglycans in the stroma were very large and aggregated in large groups around the keratocytes. Keratocytes were also degenerated and full of large vacuoles. There were numerous electron lucent vacuoles distributed in the stroma. Collagen fibrils irregularly distributed and proteoglycans were abnormally large. The progressive development of the clouding in MCD corneas could be due to alteration in the keratan sulphate proteoglycans such as lumican, kerotocan and mimican which normally regulate the uniform distribution of collagen fibrils

Assessment of the use of cryopreserved x freeze-dried amniotic membrane (AM) for reconstruction of ocular surface in rabbit model / Avaliação do uso da membrana amniótica (MA) criopreservada x liofilizada na reconstrução da superfície ocular em coelhos

PURPOSE: To determine the efficacy of freeze-dried amniotic membrane (AM) for reconstruction of the ocular surface in rabbit eyes. METHODS: The sterilized, freeze-dried amniotic membrane (lyophilized or FD-AM) is a preservative method that uses the drying by freezing process to maintain the AM well preserved for a long time even at room temperature. This paper is an experimental animal interventional study. One eye of each of 15 male New Zealand rabbits (1.5 - 3.0 kg) had the central cornea marked with a 6.0 mm trephine. The marked area was deepithelialized with a No.15 blade. The denuded corneal surface was covered as follows: Group 1: cryopreserved AM (n=6); Group 2: freeze-dried AM (n=6); and Group 3: not covered (control group, n=3). The AM in group 1 and 2 and the periphery of the denuded area in group 3 were secured with continuous 10-0 nylon sutures. The clinical evaluation was made by a blinded observer and graded on a four-point scale (1= minimal, 4= marked) for conjunctival and ciliary hyperemia, eyelid edema, corneal neovascularization, corneal opacity and reepithelialization on postoperative (PO) days 1, 7 and 30 . After PO day 30, the rabbits were euthanized and their corneas were sent for histopathological and ultrastructural analysis to evaluate tissue inflammation, reepithelialization, and basement membrane integrity. RESULTS: Two eyes in group 2 had a corneal infection and were excluded from the analysis. No statistically significant differences among the three groups were found (p>0.05) regarding the clinical evaluation on 1st, 7th and 30th PO days. On transmission electron microscopy, the basement membrane in lyophilized and control groups was more continuous and homogeneous than in the glycerol group. CONCLUSIONS: The freeze-drying method seems to be a good option to preserve human amniotic membrane to be used in ocular surface reconstruction. This preservative method reduces the preservation costs and may enhance...

OBJETIVO: Avaliar a eficácia da liofilização da membrana amniótica (MA) para a reconstrução da superfície ocular em coelhos. MÉTODOS: A liofilização é processo de preservação que mantém a MA estável durante longo tempo mesmo em temperatura ambiente. A córnea de um olho de cada coelho macho da raça Nova Zelândia foi marcada e desepitelizada. Essa área desepitelizada foi coberta com: Grupo 1: MA criopreservada (n=6); Grupo 2: MA liofilizada (n=6) e Grupo 3: Não coberta (n=3). A MA nos grupos 1 e 2 e a periferia da córnea no grupo 3 foram suturadas com nylon 10-0. A avaliação clínica foi realizada por um observador cego em relação à hiperemia, neovascularização e edema de córnea e reepitelização nos dia 1, 7 e 30 pós-operatórios. Após o dia 30 os ratos foram eutanizados e suas córneas enviadas para análise histopatológica e ultra-estrutural. RESULTADOS: Dois olhos no grupo 2 foram excluídos da análise devido à infecção. Não foi encontrada diferença estatisticamente significante entre os grupos em relação à avaliação clínica. Na microscopia eletrônica de transmissão, a membrana basal nos grupos de MA liofilizada e controle foi mais contínua e homogênea em relação ao grupo da MA criopreservada. CONCLUSÕES: O processo de liofilização parece ser boa opção para a preservação da membrana amniótica humana para utilização na reconstrução da superfície ocular.

Dose-dependent ultrastructural changes in rat cornea after oral methylphenidate administration

To investigate dose-dependent ultrastructural changes in rat cornea after oral methylphenidate [Ritalin] administration. This study was conducted in the Department of Anatomy, Gazi University Faculty of Medicine, Ankara, Turkey between March and May 2005, with a total of 27 female prepubertal Wistar albino rats, divided into 3 different dose groups [5mg/kg, 10 mg/ kg, 20 mg/kg], and their control groups. They were treated orally with methylphenidate, and eye tissue was removed to process for electron microscopic studies. We observed that all cells, and prominently basal cells of the corneal epithelium show dose-dependent degenerative changes such as apoptotic bodies, chromatin condensation, and ondulation in their nuclei and crystolysis of the mitochondrion. In the stroma, the most evident finding was the increase of the collagen fiber. In addition to dose-dependent changes related to the apoptotic process, which is chromatin condensation in their nuclei, electron dense material accumulation, and pericellular edema in the cytoplasm were also seen. In the endothelial cell lines, disruption of the junctional complexes, vacuolization in the cell cytoplasms, and crystolysis of the mitochondrion's with rough endoplasmic reticulum cisternae activity were observed. Ritalin is inducing an evident degeneration, especially in epithelium cells with increasing doses. Ultrastructural cell organelle composition degeneration with stromal fibrosis has a negative effect on cornea dehydration. In light of these findings, we believe that the Ritalin treatment doses need to be kept to a minimum to maintain healthy cornea ultrastructure and related physiology

Light and electron microscopic study on the protective role of corneal epithelium against the effect of "UVB" radiation on rabbit cornea

The human eye might be subjected to repeated exposure to ultraviolet radiation [UVR] either during a single day or over a longer period of time. It is known that the cortical epithelium strongly absorbs UVR. The aim of this study was to evaluate the protective role of corneal epithelium against the effect of UVB radiation on corneas of albino rabbits in comparison with that on de-epithelialized corneas. Sixteen adult female New Zealand albino rabbits [3-3.5 Kg] were divided into 4 groups, 4 rabbits each. Group I served as control. Group II served also as control, but their corneas underwent manual de-. In group III, corneas were exposed to UVB irradiation, peaked at 3l2nm as a single dose [3.12J/cm2] for 30 minutes. In group IV corneas underwent manual de-epithelialization. 5 minutes prior to UVB exposure. All animals were sacrificed after 48 hours, and the corneas were removed and processed for histological and transmission electron microscopic study. The histological results of group III revealed that the corneas had slight edema and the keratocytes in the outer stroma appeared to be affected. Ultrastructural evaluation of this group showed affected epithelial cells and the keratocytes revealed early apoptotic changes like chromatin condensation. Results of group IV showed that the stromal damage was deeper and more extensive. The keratocytes had been markedly affected in the entire thickness and there was thinning out of Descemet's membrane. Ultrastructurally, the keratocytes showed chromatin condensation, fragmentation and cell shrinkage. Disorganized collagen lamellae were also seen. It was concluded that the removal of the corneal epithelium makes the underlying ocular structures-more susceptible to damage by UVB in the 312nm range. Further studies are needed to investigate whether the corneal epithelium has protective properties against UVB at other wavelengths

Lymphangiogenesis occurring in transplanted corneas / 华中科技大学学报（医学）（英德文版）

To study corneal lymphangiogenesis after corneal transplantation, corneal allogenic transplantation models were established in rats. 8 female Wister rats were used as donors, and 16 Sprague Dawley (SD) rats were used as recipients and 2 SD served as controls. Corneal lymphangiogenesis and hemangiogenesis was examined by electron microscopy 1 and 2 weeks after corneal penetrating transplantation, and the expression of lymphatic vessel endothelial receptor (LYVE-1) was examined 1, 3, 7, 14 days after the transplantation respectively. In addition, 19 allograft failed human corneas were examined by 5'-nase-alkaline phosphatase (5'-NA-ALP) double-enzyme-histochemistry staining to detect corneal lymphangiogenesis and hemangiogenesis. By immunohistochemistry for LYVE-1, it was found that blown lymphatics were localized in the stroma 3 days after the corneal transplantation. With electron microscopy, new lymphatic vessels and blood vessels were found 1 and 2 weeks after the corneal transplantation. By 5'-NA-ALP enzyme-histochemistry, corneal hemangiogenesis was found in all allograft failed human corneas and 5 of 19 (26.3 %) cases had developed corneal lymphangiogenesis. It is concluded that corneal lymphangiogenesis is present after corneal transplantation, which may play an important role in allograft rejection.

ultrastructural alterations in rat corneas with experimentally-induced diabetes mellitus

To examine the ultrastructural changes of rat corneas in streptozotocin [STZ] induced diabetes mellitus and the follow-up insulin treatment. Sprague-Dawley type rats was used for experimental procedures during the period from January to April 2003 at Baskent University, Ankara, Turkey. Rats were studied in 4 groups; group 1: controls, group 2: sham controls [single dose IV sodium citrate], group 3: STZ-induced diabetes mellitus [single dose 45 mg/kg STZ intravenously], group 4: diabetes mellitus + insulin treatment [8 U/day]. We observed degenerative changes in the epithelial layer, stromal keratocytes and endothelial cells in diabetic group. In contrast, the corneal layers have revealed positive alterations in the insulin-treated group. The statistical analyses showed significant narrowing in the epithelial layer in the diabetic group [p=0.002], whereas thickening was observed in the epithelial basement membrane and Descemet's membrane [p=0.002]. It was determined that diabetes mellitus causes degenerative changes in cornea, which are positively influenced by short-term insulin treatment

Hexavalent chromium [VI]] induced toxicity on rat corneal structure: a light and scanning electron microscopic study

Chromium is one of the most studied chemicals linked to occupational hazards and is a proven toxin, mutagen and carcinogin. People are exposed to chromium compound in a wide variety of situations as it is commonly used in industrial chrome plating, welding, paining, metal finishes, steel manufacturing, alloy, cast iron and wood treatment. So, this study aimed to evaluate the cytotxicity of hexavalent chromium on rat corneas using light and scaning electron microscopic examinations. Ten adult albino rats were used as a control group, whereas twenty adult albino rats were treated with 25 and 2.5 mg/kg hexavalent chromium by intraperitoneal [ip] injection for 7 days respectively. Examination of the control rat cornea demonstrated that, it consisted of five layers. The outer surface was stratified squamous non-keratinized eqithelium with polygonal shape and surface microvilli. After seven daily dose of 25-mg/kg hexavalent chromium adminnistration, the examined corneal specimens revealed increased corneal thickness and loss of cellular demarcation of corneal epithelium with cleft formation and few microvilli. There were cytoplasmic vacuolation and nuclear changes. Also there was loss of the normal stromal lamellar architecture and various empty spaces. Moreover, after 90 days of chromium toxicity there was irregular corneal surface with marked patchy epithelial loss and multiple surface ostia. Also, there was morphological alteration of the surface microvilli and intercellular focal disruption. The Bowman's layer was lost. The corneal stroma showed marked loss of lamellar architecture and empty spaces. Corneal vascularization was also seen with inflammatory infiltrates. So, the present study concluded that, hexavalent chromium is very toxic to the cornea especially after prolonged exposure causing visual impairment and loss

Estudo clínico e ultra-estrutural da ceratoplastia lamelar inversa em coelhos / Clinical and ultrastructural study of inverse lamelar keratoplasty in rabbits

Em 1985, Galväo propôs a utilizaçäo do disco corneano formado pelas camadas posteriores da córnea na realizaçäo da Ceratoplastia Lamelar, o que permitiria à córnea doadora fornecer dois enxertos lamelares. Galväo denominou a nova técnica de Ceratoplastia Lamelar Inversa. A autora realizou o estudo clínico e ultra-estrutural dessa técnica em coelhos. Dezessete coelhos foram operados e posteriormente quinze foram submetidos ao exame biomicroscópico periódico e ultra-estrutural. Os exames ultra-estruturais foram realizados imediatamente após a cirurgia e na primeira, segunda, quarta, oitave e décima-segunda semanas de pós-operatório. A epitelizaçäo completa do enxerto ocorreu entre o oitavo e o décimo-quarto dia. A partir do quinto dia de pós-operatório todos os enxertos estavam transparentes. O endotélio do doador degenerou no per-operatório. A membrana de Descemet do doador permaneceu íntegra até a décima-segunda semana de pós-operatório e foi recoberta pelo epitélio corneano do receptor. O epitélio era morfologicamente normal. Hemidesmosomas associados a fragmentos de membrana basal foram evidenciados a partir da quarta semana. Conforme proposto por Galväo, os resultados desta investigaçäo experimental indicam que uma única córnea doadora propicia duas ceratoplastias lamelares - a inversa e a convencional - e sugerem a possibilidade...

Effect of intraocular epinephrine on the cornea of rabbits

Fifty eyes of 25 albino rabbits were included in this study. Intracameral injection of 0.25 ml of epinephrine with different concentrations had been done. Two eyes were used as control, the other 48 eyes were classified into 3 groups according to the concentration of epinephrine, 1/5000, 1/10000 and 1/20000. Specular microscopy and histopathological examination had been done immediately, one week, 2 weeks and 4 weeks after injection. Manifest changes were found with concentration of 1/5000 in the form of loss of hexagonal pattern, widening of the intercellular spaces and appearance of cell free areas, light microscopic examination revealed vaculated endothelial cells with swollen nuclei. Decrease in the thickness of the Descemet's membrane and stromal edema. These changes were less with 1/10000 concentration and were undetectable with 1/20000 concentration

The effect of combined application of argon and Nd-YAG lasers on iridectomy in rabbits

Recently iridectomy using an argon or Nd-YAG laser to treat narrow angle glaucoma has become popular, and is now the procedure of choice over the standard surgical technique. However, the shock wave of the Nd-YAG laser causes hemorrhage in almost all cases and the high energy level of the Nd-YAG laser, which is required for iridectomy, causes injury to the lens and cornea. Furthermore, there is a tendency toward closure of the iridectomy site after argon laser application. We performed iridectomies by a combined application of argon and Nd-YAG lasers in pigmented rabbits to improve iris bleeding, iridectomy patency, and lens and corneal damage. The iridectomy patency and the lens and corneal damage were examined with a scanning electron microscope. The rabbits that underwent laser iridectomies with only the Nd-YAG laser were used as a control group. Based on the results, it can be concluded that laser iridectomy by a combined application of argon and Nd-YAG lasers results in a lower rate of bleeding, a higher rate of patency, and less damage to the lens and cornea as compared with iridectomy performed by Nd-YAG laser only.