A history of the renewal of the corneal epithelium : a 3D animation

Background: To compare the different schemes that have been proposed during the last thirteen years to explain the renewal of the corneal epithelium. Material and Methods:We analyzed all the data present in the literature to explain the renewal of the corneal epithelium in mammals. According to the schemes proposed in the literature we developed a 3D animation to facilitate the understanding of the different concepts. Results:Three different schemes have been proposed to explain the renewal of the corneal epithelium in mammals during the last thirteen years. 1950-1981: the corneal epithelium was thought being renewed by mitosis of cells located in the basal layer. At this time scientist were not talking about stem cells. 1981-1986 was the period of the "XYZ hypothesis" or the transdifferentiation paradigm. At this time the conjunctival epithelium renewed the corneal epithelium in a centripetal migration. 1986-2008: the limbal stem cell paradigm, there were no stem cells in the corneal epithelium, all the corneal stem cells were located in the limbus and renewed the central cornea after a migration of 6 to 7 mm of transient amplifying cells toward the centre of the cornea. 2008, epithelial stem cells were found in the central cornea in mammals (Nature, Majo et al. November 2008). Discussion:We thought that the renewal of the corneal epithelium was completely defined. According to the last results we published in Nature, the current paradigm will be revisited. The experiments we made were on animals and the final demonstration on human has still to be done. If we find the same results in human, a new paradigm will be define and will change the way we consider ocular surface therapy and reconstruction.

A novel cyclosporin a aqueous formulation for dry eye treatment: in vitro and in vivo evaluation.

PURPOSE: The aim of the present study was the in vitro and in vivo evaluation of a novel aqueous formulation based on polymeric micelles for the topical delivery of cyclosporine A for dry eye treatment. METHODS: In vitro experiments were carried out on primary rabbit corneal cells, which were characterized by immunocytochemistry using fluorescein-labeled lectin I/isolectin B4 for the endothelial cells and mouse monoclonal antibody to cytokeratin 3+12 for the epithelial ones. Living cells were incubated for 1 hour or 24 hours with a fluorescently labeled micelle formulation and analyzed by fluorescence microscopy. In vivo evaluations were done by Schirmer test, osmolarity measurement, CyA kinetics in tears, and CyA ocular distribution after topical instillation. A 0.05% CyA micelle formulation was compared to a marketed emulsion (Restasis). RESULTS: The in vitro experiments showed the internalization of micelles in the living cells. The Schirmer test and osmolarity measurements demonstrated that micelles did not alter the ocular surface properties. The evaluation of the tear fluid gave similar CyA kinetics values: AUC = 2339 ± 1032 min*μg/mL and 2321 ± 881.63; Cmax = 478 ± 111 μg/mL and 451 ± 74; half-life = 36 ± 9 min and 28 ± 9 for the micelle formulation and Restasis, respectively. The ocular distribution investigation revealed that the novel formulation delivered 1540 ± 400 ng CyA/g tissue to the cornea. CONCLUSIONS: The micelle formulation delivered active CyA into the cornea without evident negative influence on the ocular surface properties. This formulation could be applied for immune-related ocular surface diseases.

Jumping Jack Flash.

A 38-year-old woman presented with blurred vision and "jumping" of the right eye for 7 months. Magnetic resonance imaging of the head was normal. Intermittent intorsion of the right eye was noted on examination, consistent with superior oblique myokymia. She was initially treated with carbamazepine but stopped after becoming light-headed. The diagnosis and treatment of superior oblique myokymia are discussed.

Oeil et environnement

Résumé L'oeil est exposé à de multiples facteurs environnementaux qui influent sur le développement visuel, le confort et la santé visuelle. Une exposition excessive à la lumière solaire peut causer des pathologies de la surface oculaire et de la rétine, mais une exposition insuffisante à la lumière du jour pourrait être incriminée dans l'apparition ou la progression de la myopie. Les lumières artificielles, LED en particulier, comportent un risque accru de phototoxicité rétinienne. L'augmentation importante de la prévalence des allergies oculaires pourrait être liée à la multiplication des polluants environnementaux, comme la fumée de tabac, les dérivés de combustion de carburants ou les phtalates. Enfin, certains compléments alimentaires peuvent jouer un rôle protecteur dans certains types d'atteintes oculaire, rétinienne en particulier. Abstract The eyes are exposed to multiple environmental factors, which affect visual development, comfort, and visual health. While overexposure to sunlight can cause ocular surface and retinal pathologies, insufficient exposure to daylight could significantly contribute to myopia progression. New artificial lights, namely LED, have a higher risk of retinal phototoxicity, and could alter ocular circadian rhythm. The significant increase of prevalence of ocular allergies could be caused by the proliferation of environmental polluting substances, like tobacco smoke, fuel combustion by-products, or phtalates, which are found in many types of plastics. Finally, some dietary supplements could play a protective role in certain types of ocular pathologies, namely retinal pathologies.

The health effects of sea buckthorn berries and oil

Sea buckthorn (Hippophaë) berries are ingredients of the Chinese traditional medicine. In addition to China, they are nowadays cultivated for food in several European countries, Russia, Canada, the USA, and Japan. Sea buckthorn berries are a rich source of flavonoids, mainly flavonol glycosides and proanthocyanidins. Depending on the genetic background, growth conditions, and ripeness of the berries, vitamin C concentrations up to over 1 g/100 ml juice, have been reported. Sea buckthorn berries contain inositols and methyl inositols, components of messenger molecules in humans. Sea buckthorn seed oil is rich in essential aplha-linolenic and linoleic acids, whereas the most abundant fatty acids in the berry oil are palmitoleic, palmitic and oleic acids. Other potentially beneficial lipophilic compounds of sea buckthorn seeds and berries include carotenoids, phytosterols, tocopherols and tocotrienols. The effects of sea buckthorn fractions on inflammation, platelet aggregation, oxidation injuries, the liver, skin and mucosa, among others, have been reported. The aim of the thesis work was to investigate the health effects of sea buckthorn berries and oil in humans. The physiological effects of sea buckthorn berries, berry components, and oil have mostly been studied in vitro and in animal models, leaving a demand for more clinical trials. In the first randomized, placebo-controlled trial of this thesis healthy adults consumed 28 g/day of sea buckthorn berries for three months. The main objective was to investigate the effects on the common cold. In addition, effects on other infections, inflammation and circulating lipid markers associated with cardiovascular disease risk were studied. In the second randomized, placebocontrolled trial participants reporting dry eye symptoms consumed 2 g/day of sea buckthorn oil from the seeds and berries for three months. The effects on symptoms and clinical signs of dry eye were monitored. In addition, the effects on circulating markers of inflammation and liver functions were analyzed. Sea buckthorn berries did not affect the common cold or other infections in healthy adults. However, a decrease in serum C-reactive protein was detected, indicating effects on inflammation. Fasting concentrations of serum flavonols, typical to sea buckthorn berry, increased without affecting the circulating total, HDL, LDL cholesterol, or triacylglycerol concentrations. Tear film hyperosmolarity and activation of inflammation at the ocular surface are among the core mechanisms of dry eye. Combined sea buckthorn berry and seed oil attenuated the rise in tear film osmolarity taking place during the cold season. It also positively affected some of the dry eye symptoms. Based on the tear film fatty acid analysis, the effects were not mediated through direct incorporation of sea buckthorn oil fatty acids to tear film lipids. It is likely that the fatty acids, carotenoids, tocopherols and tocotrienols of sea buckthorn oil affected the inflammation of the ocular surface, lacrimal and/or meibomian glands. The effects on the differentiation of meibomian gland cells are also possible. Sea buckthorn oil did not affect the serum concentrations of inflammation markers or liver enzymes investigated. In conclusion, this thesis work suggests positive effects of sea buckthorn berries and oil on inflammation and dry eye, respectively, in humans.

Comparación: injerto libre o cierre primario más lente de contacto, en resección de tumores de conjuntiva limbar

INTRODUCCION: Existe controversia en cuanto a la técnica quirúrgica para el manejo de tumores del limbo conjuntival. El uso de cierre primario con uso de lente de contacto puede ofrecer una mejor cicatrización y tener ventajas adicionales sobre la técnica tradicional con el uso de plastia. OBJETIVOS: Comparar los resultados en cuanto a grado de dolor, picadas, prurito, porcentaje de epitelización y cicatrización, comodidad del paciente, grado de quemosis y tiempo de retorno a actividades diarias en ambas técnicas quirúrgicas. MATERIALES Y METODOS: Experimento clínico controlado aleatorizado en dos grupos: Al primer grupo se le realizó cirugía de resección de la lesión más plastia. Al segundo grupo se le practicó la resección de la lesión cierre primario y lente de contacto. El seguimiento se realizó al primer y cuarto día, y cada semana durante el primer mes de postoperatorio. Se utilizó el SPSS 20.0 ® para análisis estadístico de datos y se utilizó estadística no paramétrica. RESULTADOS: Se conto con 10 pacientes por grupo. El dolor y porcentaje de cicatrización al primer día postoperatorio fueron mayores en el grupo usando lente de contacto (p=0.048). Al cuarto día postquirúrgico se encontró un mayor porcentaje de cicatrización en el grupo usando lente de contacto. (p=0.075). CONCLUSIONES: El cierre por afrontamiento con uso de lente de contacto mostró dolor y picadas mayores al primer y cuarto día postoperatorio. Pero la epitelización y cicatrización fueron tempranas con un retorno corto a actividades cotidianas.

Ex vivo expansion of limbal stem cells is affected by substrate properties

Limbal epithelial stem cells play a key role in the maintenance and regulation of the corneal surface. Damage or destruction of these cells results in vascularisation and corneal opacity. Subsequent limbal stem cell transplantation requires an ex vivo expansion step and preserving cells in an undifferentiated state remains vital. In this report we seek to control the phenotype of limbal epithelial stem cells by the novel application of compressed collagen substrates. We have characterised the mechanical and surface properties of conventional collagen gels using shear rheology and scanning electron microscopy. In doing so, we provide evidence to show that compressive load can improve the stiffness of collagen substrates. In addition Western blotting and immunohistochemistry display increased cytokeratin 3 (CK3) protein expression relating to limbal epithelial cell differentiation on stiff collagen substrates. Such gels with an elastic modulus of 2900 Pa supported a significantly higher number of cells than less stiff collagen gels (3 Pa). These findings have substantial influence in the development of ocular surface constructs or experimental models particularly in the fields of stem cell research, tissue engineering and regenerative medicine.

Towards the use of hydrogels in the treatment of limbal stem cell deficiency

Corneal blindness caused by limbal stem cell deficiency (LSCD) is a prevailing disorder worldwide. Clinical outcomes for LSCD therapy using amniotic membrane (AM) are unpredictable. Hydrogels can eliminate limitations of standard therapy for LSCD, because they present all the advantages of AM (i.e. biocompatibility, inertness and a biodegradable structure) but unlike AM, they are structurally uniform and can be easily manipulated to alter mechanical and physical properties. Hydrogels can be delivered with minimum trauma to the ocular surface and do not require extensive serological screening before clinical application. The hydrogel structure is also amenable to modifications which direct stem cell fate. In this focussed review we highlight hydrogels as biomaterial substrates which may replace and/or complement AM in the treatment of LSCD.

In vivo study of the biocompatibility of a novel compressed collagen hydrogel scaffold for artificial corneas

The experiments were designed to evaluate the biocompatibility of a plastically compressed collagen scaffold (PCCS). The ultrastructure of the PCCS was observed via scanning electron microscopy. Twenty New Zealand white rabbits were randomly divided into experimental and control groups that received corneal pocket transplantation with PCCS and an amniotic membrane, respectively. And the contralateral eye of the implanted rabbit served as the normal group. On the 1st, 7th, 14th, 21st, 30th, 60th, 90th, and 120th postoperative day, the eyes were observed via a slit lamp. On the 120th postoperative day, the rabbit eyes were enucleated to examine the tissue compatibility of the implanted stroma. The PCCS was white and translucent. The scanning electron microscopy results showed that fibers within the PCCS were densely packed and evenly arranged. No edema, inflammation, or neovascularization was observed on ocular surface under a slit lamp and few lymphocytes were observed in the stroma of rabbit cornea after histological study. In conclusion, the PCCS has extremely high biocompatibility and is a promising corneal scaffold for an artificial cornea. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Differential effects of alpha-helical and beta-hairpin antimicrobial peptides against Acanthamoeba castellanii

In this work we evaluated the ability of different types of antimicrobial peptides to promote permeabilization and growth inhibition of Acanthamoeba castellanii trophozoites, which cause eye keratitis. We used cationic alpha-helical peptides P5 and a beta-hairpin amphipathic molecule (gomesin), of the spider Acanthoscurria gomesiana haemocytes. A. castellanii permeabilization was obtained after 1 h incubation with micromolar concentrations of both types of peptides. While permeabilization induced by gomesin increased with longer incubations, P5 permeabilization did not increase with time and occurred at doses that are more toxic for SIRC cells, P5, however, at doses below the critical dose used to kill rabbit corneal cells was quite effective in promoting growth inhibition. Similarly, P5 was more effective when serine protease inhibitor was added simultaneously to the permeabilization assay. High performance chromatography followed by mass spectrometry analysis confirmed that, in contrast to gomesin, P5 is hydrolysed by A. castellanii culture supernatants. We conclude that the use of antimicrobial peptides to treat A. castellanii infections requires the search of more specific peptides that are resistant to proteolysis.

Conjunctival effects of canine distemper virus-induced keratoconjunctivitis sicca

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estrabismo: detecção em uma amostra populacional e fatores demográficos associados

OBJETIVOS: Identificar a frequência de ocorrência de desvios oculares e as características dos portadores em uma amostra populacional. MÉTODOS: Estudo transversal, observacional e probabilístico, entre os anos de 2004 e 2005, envolvendo 11 cidades da região centro-oeste do estado de São Paulo. Foram examinados 10.994 indivíduos, sendo utilizada para este estudo uma subamostra desta população, identificada pelo diagnóstico de estrabismo. A população foi abordada por uma equipe treinada e padronizada para os procedimentos da pesquisa. Os dados foram analisados estatisticamente por meio de análise descritiva, frequência de ocorrência, análise de contingência e testes de associação (p<0,05). RESULTADOS: A frequência de ocorrência de estrabismo na população estudada foi de 1,4% (148 portadores de estrabismo), sem diferença entre sexos. Portadores de esodesvios (ET) eram 46,3%, 38,2% casos de exodesvio (XT) e 15,4% de desvios verticais associados a horizontais ou síndromes. A análise de contingência mostrou que 3 indivíduos (2,3%) estrábicos apresentavam cegueira e 7 (5,43%) apresentavam baixa visão em um dos olhos. Tanto a ET, quanto a XT estiveram presentes em indivíduos com graus variáveis de miopia (até -5,75 para XT e -2,50 para ET) e de hipermetropia (até +9,00 para XT e +8,00 para ET). A associação entre estrabismo e o equivalente esférico obtido na refração estática não mostrou diferença significativa (p>0,05). CONCLUSÃO: A frequência de ocorrência de estrabismo em uma amostra populacional foi de 1,4%, sem diferença entre sexos ou tipo de desvio ocular. A presença de cegueira e de baixa visão associadas aos desvios oculares reforçam a necessidade de tratamento precoce.

Biocompatibilidade e biodegradabilidade de micropartículas de poli-lactato-co-glicolato na ceratite infecciosa em coelhos

Avaliaram-se a biocompatibilidade e a biodegradabilidade do sistema de liberação controlada de poli-lactato-co-glicolato (PLGA) no tratamento com ciprofloxacina das ceratites por Staphylococcus aureus em coelhos. Foram utilizados 20 coelhos, distribuídos em quatro grupos (G). Os animais dos G1, G3 e G4 foram inoculados com 2,5µL da bactéria - 108UFC, no estroma corneano. Os do G2 não receberam a aplicação do inóculo. O tratamento foi realizado com solução salina básica para os animais do G1, micropartículas de PLGA contendo ciprofloxacina nos animais dos G2 e G4 e colírio de ciprofloxacina naqueles do G3. Suabe e biópsia da superfície ocular foram coletados para cultura. Apenas um animal do G1 apresentou cultura positiva para S. aureus. Exame histológico revelou a presença bacteriana em todos os animais do G1 e em dois animais do G3. Também foi constatada reação inflamatória no local da aplicação do sistema de liberação controlada. O tratamento com micropartículas de PLGA foi eficiente no tratamento de ceratites bacterianas, ao eliminar por completo a presença do S. aureus, mas entretanto não foi completamente biocompatível e biodegradável após cinco dias.

Effect of amniotic membrane transplantation on corneal healing and proteoglycan expression in an experimental model of limbal deficiency in rabbits

PURPOSE. Amniotic membrane transplantation (AMT) has been used as a graft or as a dressing in ocular surface reconstruction, facilitating epithelization, maintaining normal epithelial phenotype, and reducing inflammation, vascularization, and scarring. The corneal transparency is due, at least in part, to the arrangement in orthogonal lamellae of collagen fibrils, surrounded by proteoglycans (PGs). These PGs regulate fibrilogenesis, the matrix assembly, and ultimately the corneal transparency. The purpose of the present study was to investigate the effects of AMT upon the corneal PGs after severe limbal injury.METHODS. Experiments were performed on the right corneas of 22 New Zealand female albino rabbits, and their left corneas were used as matched controls. These animals were divided into 3 groups: G1 (n = 10): total peritomy and keratolimbectomy, followed by application of 0.5 M NaOH; G2 (n = 10): submitted to the same trauma as G1, and treated by AMT; G3: no trauma, only AMT (n = 2). The right corneas of G2 and G3 were covered by DMSO 4 cryopreserved human amniotic membrane, fixed by interrupted 9-0 mononylon sutures, with its stromal face toward the ocular surface. After 7 or 30 days, the corneas were removed and PGs were extracted.RESULTS. Normal corneas contained approximately 9 mg of PGs per gram of dry tissue. AMT on intact cornea (G3) did not cause any changes in the concentration of PGs. In contrast, injured corneas contained much less PGs, both on the seventh and on the 30th day posttrauma. The PG concentration was even lower in injured corneas treated by AMT. This decrease was due almost exclusively to dermatan sulfate PGs, and the structure of dermatan sulfate was also modified, indicating changes in the biosynthesis patterns.CONCLUSIONS. Although beneficial effects have been observed on clinical observation and concentration of soluble proteins after AMT, the normal PG composition of cornea was not attained, even 30 days postinjury, indicating that the normal ocular surface reconstruction, if possible, is a long-term process. (Eur J Ophthalmol 2010; 20: 290-9)

Microalterations in the third eyelid gland of dogs with keratoconjunctivits sicca secondary to distemper

Analisaram-se, à microscopia óptica, 28 glândulas da terceira pálpebra de cães, que morreram com cinomose. O diagnóstico de ceratoconjuntivite seca baseou-se nas alterações da superfície ocular e no teste da lágrima de Schirmer. À histologia, visibilizaram-se atrofia tubuloacina, espessamento do tecido conjuntivo e exudação inflamatória de leve a intensa com presença de plasmócitos, de histiócitos e por hipertrofia compensatória de algumas glândulas. O lume das glândulas encontrava-se hipertrofiado contendo células epiteliais descamadas, polimorfonucleares e hemácias, além de muco.