Susceptibilidad de las mucosas oculares al SARS COV-2: bioseguridad sanitaria / Susceptibility of eye mucosa to SARS CoV-2: sanitary biosecurity

RESUMEN: El SARS CoV-2, agente causal de la enfermedad llamada Covid-19, infecta las mucosas digestivas y respiratorias, afectando las células epiteliales. El virus ingresa a través del receptor de membrana ACE2 provocando la disrupción de la homeostasis celular. Frecuentes reportes indican la presencia de conjuntivitis ocular en pacientes diagnosticadas con Covid-19, lo cual ha alertado a los científicos sobre el potencial foco de infección viral de las secresiones lagrimales.Los epitelios de la conjuntiva ocular sub-palpebral y corneal, se caracterizan por presentar el receptor de la enzima convertidora de angiotensina 2 (ACE2) y proteasa transmembrana asociada serina 2 (TMPRSS2), cuya interacción activa los mecanismos de liberación de citoquinas, capaces de instalar un proceso de conjuntivitis infecciosa por SARS CoV-2, pero no necesariamente hacer extensiva la infección hacia los sistemas digestivo y respiratorio.Aunque este proceso inflamatorio es más frecuente como una expresión de la infección general y más grave. Sin embargo, cualquiera sea la vía de infección o ingreso del virus SARS CoV-2 es importante considerar el riesgo de infectividad de las lágrimas y las secresiones conjuntivales en los pacientes. Este estudio pretende llamar la atención sobre las medidas de cuidados y control sanitario, incorporando mejores normas de protección personal y bioseguridad, especialmente en el áreas de oftalmología, asumiendo que la mucosa ocular puede ser una vía de entrada del virus y a la vez una fuente de contagio.También considerar la potenciación de la infección viral con las enfermedades de base asociadas, como glaucoma y diabetes.Se sugiere además incorporar estudios histológicos de la mucosa ocular para diferenciar epitelios sanos e infectados.

SUMMARY: SARS CoV-2, the causal agent of the Covid- 19 disease, infects the digestive and respiratory mucosa, affecting epithelial cells. The virus enters through the ACE2 membrane receptor causing the disruption of cell homeostasis. Frequent reports indicate the presence of ocular conjunctivitis in patients diagnosed with Covid-19, which has alerted scientists to the potential source of viral infection from lacrimal secretions. The epithelia of the sub-palpebral and corneal ocular conjunctiva are characterized by presenting the receptor for angiotensin-converting enzyme 2 (ACE2) and associated transmembrane protease serine 2 (TMPRSS2), whose interaction activates cytokine release mechanisms, with the ability to start the infectious conjunctivitis process by SARS CoV-2, but not necessarily extend the infection to the digestive and respiratory systems. Although this inflammatory process is more frequent as an expression of the general and more serious infection. However, whatever the route of infection or entry of the SARS CoV-2 virus, it is important to consider the risk of infection of tears and conjunctival secretions in patients. This study aims to draw attention to health care and control measures, incorporating better standards of personal protection and biosafety, especially in the areas of ophthalmology, assuming that the ocular mucosa can be a route of entry for the virus, and at the same time a source of contagion. A further consideration is the potential of viral infection with associated underlying diseases, such as glaucoma and diabetes. It is also suggested to incorporate histological studies of the ocular mucosa to differentiate healthy and infected epithelia.

Tratamento de crescimento epitelial persistente pós-Lasik com debridamento mecânico, uso de álcool a 20% e cola de fibrina / Treatment of persistent epithelial ingrowth after Lasik with mechanical debridement, use of 20% alcohol and fibrin glue

RESUMO O Lasik é a técnica de cirurgia refrativa mais utilizada no mundo. Apesar de segura e efetiva, ela pode levar a algumas complicações. O crescimento epitelial pós-Lasik é uma complicação pós-operatória incomum, com prevalência maior em casos de retratamento. Geralmente, é um achado não progressivo e assintomático, que não requer tratamento, mas, em uma minoria de pacientes, os sintomas podem ser clinicamente significantes e variados. O tratamento é feito com debridamento mecânico do crescimento epitelial, mas alguns recursos adjuvantes também podem ser utilizados. O presente estudo consiste em um relato de caso de paciente com crescimento epitelial pós-Lasik que apresentou quatro recidivas após intervenções de debridamento epitelial, sutura de lamela corneana e ablação a laser. No quinto procedimento, o paciente foi finalmente tratado com combinação de debridamento epitelial, uso de álcool a 20% e cola de fibrina. Entretanto, a regressão do crescimento epitelial e a melhora da acuidade visual só ocorreram ao longo dos meses após a intervenção, o que mostra a importância de esperar um tempo para que ocorra a melhora da visão no pós-operatório, evitando-se reintervenções.

ABSTRACT Lasik is the most often performed laser refractive surgery worldwide. Despite its efficacy and safety, some complications may occur. Epithelial ingrowth is a rare postoperative complication of Lasik, with an increased prevalence in cases of retreatment. Epithelial ingrowth is usually a nonprogressive and asymptomatic finding, which requires no treatment; however, in a minority of cases, symptoms may be clinically significant and diverse. Treatment is done with mechanical debridement of the affected interface, and additional interventions may be required. This study reported a case of recalcitrant epithelial ingrowth after Lasik, whichrelapsed four times after mechanical debridement, flap lift and laser ablation. In the fifth intervention, the patient was finally treated with a combined scraping/use of 20% alcohol and fibrin glue. However, regression of epithelial ingrowth and better visual acuity were only observed some months after the intervention, which shows the importance of waiting for better vision in the postoperative period, thus avoiding new reinterventions.

Profundidad de la línea de demarcación del estroma corneal después de la reticulación del colágeno corneal usando epitelial corneal personalizado versus convencional desbridamiento / Corneal stromal demarcation line depth after corneal collagen cross-linking using customized versus conventional corneal epithelial debridement

Aim of the Study: Compare the corneal demarcation line (DL) depth after corneal collagen cross-linking (CXL) with subtotal versus customized corneal epithelium debridement using anterior segment optical coherence tomography (AS-OCT). Design: Prospective case-control study. Materials and Methods: The study enrolled 18 patients with bilateral progressive keratoconus. Both eyes treated by CXL using 3 mW/cm2 / 30 minutes setting. One eye with subtotal epithelial debridement (about 9 mm diameter) and the contralateral eye with customized debridement (approximately 1 mm single horizontal central scratch). One month postoperatively, patients had AS-OCT imaging to detect and measure the depth ofDL. Results: Patients' age mean was 25.17 years +/- 4.81 SD. Epithelial healing completed with in 3-7 days in conventional treatment group and demarcation line was evident in 16 eyes (89%) with a mean depth of 290.31 µm while in customized debridement group, the epithelial healing lasted less than 24 hours and DL was detectable in 10 patients (55.5 %) with a mean DL depth of 221 µm with a statistically significant difference (p < 0.05). Subjective postoperative pain graded as "moderate to severe" in about 77% of eyes underwent 9 mm epi-off CXL compared with 55% of those with customized debridement group. Conclusions: Although the DL if found in majority cases, but the shallow location in customized corneal debridement cases questioned the efficacy of this technique despite the quick re-epithelialization and less postoperative pain that accompanied it

Severe corneal burn due to the accidental application of salicylic acid packed in a plastic dropper bottle / Quemadura corneal grave debida a la aplicación accidental de ácido salicílico envasado en un frasco gotero plástico

Eye burns due to the accidental application of pharmacological or nonpharmacological substances packaged in plastic dropper bottles have been described for more than three decades and continue to occur. These burns can cause potentially serious corneal injuries. We report the case of a patient who mistakenly applied salicylic acid to the right eye after confusing it with an eye lubricant, which caused him a severe corneal burn. Fortunately, after aggressive medical and surgical management (including oxygen therapy and amniotic membrane grafting), the visual results were good. We suggest conducting educational campaigns and taking legislative measures in our country to avoid packaging corrosive substances in this type of dropper bottle to reduce the risk of accidental burns.

Las quemaduras oculares por aplicación accidental de sustancias farmacológicas o no farmacológicas envasadas en frascos goteros plásticos, se han descrito desde hace más de tres décadas y siguen ocurriendo. Estas quemaduras pueden causar lesiones potencialmente graves de la córnea. Se presenta el caso de un paciente que se aplicó ácido salicílico en el ojo derecho al confundir el envase con el de un lubricante ocular, lo que le causó una grave quemadura corneal. Afortunadamente, tras un manejo médico y quirúrgico agresivo (incluida oxigenoterapia e injerto de membrana amniótica) los resultados visuales fueron buenos. Se sugiere hacer campañas educativas y adoptar normas en el país para evitar el envase de sustancias corrosivas en este tipo de frascos goteros, con el fin de disminuir el riesgo de quemaduras accidentales.

Análise da eficácia do crosslinking transepitelial em pacientes portadores de ceratocone / Analysis of the effectiveness of transepithelial crosslinking in patients with keratoconus

Resumo Objetivo: Avaliar os resultados clínicos do Crosslinking Transepitelial (CXL) analisando sua eficácia em pacientes portadores de ceratocone progressivo. Métodos: Estudo transversal retrospectivo com 49 olhos e 37 pacientes com idades entre 10 e 50 anos, submetidos à técnica de CXL em 2017 no Instituto Panamericano da Visão, em Goiânia, Goiás, Brasil. Foi utilizado o sistema KXL Avedro programado no modo pulsado com intervalo (1/1 segundo), usando 45 mW/cm² com 7,2 J e solução de riboflavina a 0,25% da Avedro com córneas irradiadas por 8 minutos. Foram coletados os dados: sexo, idade, acuidade visual sem correção (AVSC), acuidade visual com correção (AVCC), astigmatismo, paquimetria no ponto mais fino e astigmatismo ceratométrico no pré-operatório e pós-operatório com 1, 6 e 12 meses. Foram utilizados os testes Kolmogorov-Smirnov, Qui-quadrado de Pearson, Friedman, Dunnett à posteriori e a correlação de Spearman. Resultados: Vinte e três pacientes (62,2%) eram do sexo feminino e 14 (37,8%) do sexo masculino. A média de idade foi de 27,89 (±10,89) anos. A AVSC e AVCC melhoraram significativamente no pré-operatório em relação a 1 mês (p=0,01) e (p<0,001), 6 meses (p<0,001 ambas) e 12 meses (p<0.001 ambas). O astigmatismo reduziu significativamente no pré-operatório em relação a 6 meses (p=0,02) e 12 meses (p=0,02). A paquimetria no ponto mais fino manteve-se constante no período (p=0,95). A diferença entre k2 e k1 (astigmatismo ceratométrico) mostrou redução significativa no pré-operatório em relação a 1 mês (p=0,01). Conclusão: A técnica de CXL foi segura e eficaz no tratamento e estagnação da doença em pacientes com ceratocone progressivo.

Abstract Objective: To evaluate the clinical results of Transepithelial Crosslinking (CXL) by analyzing its efficacy in patients with progressive keratoconus. Methods: Retrospective cross-sectional study with 49 eyes and 37 patients aged 10 to 50 years submitted to the CXL technique in 2017 at the Instituto Panamericano da Visão, in Goiânia, Goiás, Brazil. The Avedro KXL system was programmed in pulsed mode with interval (1/1 second), using 45 mW/cm² with 7.2 J and 0.25% riboflavin solution of Avedro with irradiated corneas for 8 minutes. Data were collected: sex, age, uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), astigmatism, pachymetry at the thinnest point and keratometric astigmatism in the preoperative and postoperative periods at 1, 6 and 12 months. The Kolmogorov-Smirnov, Pearson's Chi-square, Friedman, Dunnett, and the Spearman correlation were used. Results: Twenty-three patients (62.2%) were female and 14 (37.8%) male. The mean age was 27.89 ± 10.89 years. The UDVA and CDVA significantly improved in the preoperative period in relation to 1 month (p = 0.01) and (p <0.001), 6 months (p <0.001 both) and 12 months (p <0.001 both). Astigmatism significantly reduced preoperatively in relation to 6 months (p = 0.02) and 12 months (p = 0.02). The pachymetry at the thinnest point remained constant in the period (p = 0.95). The difference between k2 and k1 (keratometric astigmatism) showed a significant reduction in the preoperative period in relation to 1 month (p = 0.01). Conclusion: The CXL technique was safe and effective in the treatment and stagnation of the disease in patients with progressive keratoconus.

Amniotic membrane as a biological scaffold for dental pulp stem cell transplantation in ocular surface reconstruction / Membrana amniótica como substrato biológico para o transplante de células-tronco da polpa do dente de leite na reconstrução da superfície ocular

ABSTRACT Purpose: To evaluate the ability of human immature dental pulp stem cells, which are mesenchymal stem cells of neural crest origin, to differentiate into the corneal epithelium for purposes of corneal transplantation and tissue engineering when cultured on de-epithelized amniotic membranes. Methods: We compared the immunophenotypes (ABCG2, K3/12, and vimentin) of cells grown on amniotic membranes or plastic surfaces under serum-free conditions or in culture media containing serum or serum replacement components. Results: Immature dental pulp stem cells grown on amniotic membranes under basal conditions are able to maintain their undifferentiated state. Our data also suggest that the culture medium used in the present work can modulate the expression of immature dental pulp stem cell markers, thus inducing epithelial differentiation of these cells in vitro. Conclusions: Our results suggest that the amniotic membrane is a good choice for the growth and transplantation of mesenchymal stem cells, particularly immature dental pulp stem cells, in clinical ocular surface reconstruction.

RESUMO Objetivos: Avaliar a capacidade das células-tronco imaturas da polpa do dente de leite que são células-tronco mesenquimais de origem da crista neural, de se diferenciarem no epitélio corneano para fins de transplante de córnea e engenharia de tecidos quando cultivadas em membrana amnióticas desepitelizadas. Métodos: Foram comparamos so imunofenótipo (ABCG2, CK3/12 e vimentina) de células cultivadas em membranas amnióticas ou em superfícies plásticas sob condições livres de soro ou em meios de cultura contendo soro ou componentes de substituição de soro. Resultados: Células-tronco imaturas da polpa do dente de leite cultivadas sobre membrana amniótica em condições basais são capazes de manter seu estado indiferenciado. Nossos dados também sugerem que o meio de cultura utilizado no presente trabalho pode modular a expressão de marcadores de células-tronco imaturas da polpa do dente de leite, induzindo a diferenciação epitelial destas células in vitro. Conclusão: Nossos resultados sugerem que a membrana amniótica é uma boa escolha para o crescimento e transplante de células-tronco mesenquimais, particularmente as células-tronco imaturas da polpa do dente de leite, na reconstrução da superfície ocular.

Biological modulation of corneal epithelial wound healing / Modulação biológica do processo de cicatrização epitelial da córnea

ABSTRACT The transparency and maintenance of corneal epithelial integrity are essential for its optical properties and, to preserve these characteristics, the epithelium undergoes continuous renewal. This renewal depends on the control of cell proliferation and differentiation mediated by mitogenic factors responsible for increasing mitoses and stimulating cellular migration. Cell-cell communication plays a pivotal role in epithelial healing process, and several cytokines and growth factors are involved in this process. Understanding the cross-talk and paracrine effects of these cytokines and growth factors released can help in the search for new therapeutic strategies to treat ocular surface diseases.

RESUMO A transparência e a manutenção da integridade epitelial da córnea são essenciais para suas propriedades ópticas e, para preservar tais características, o epitélio sofre renovação contínua. Essa renovação depende do controle da proliferação e diferenciação celular mediadas por fatores mitogênicos responsáveis pelo aumento das mitoses e estímulo à migração celular. A comunicação célula-célula desempenha um papel fundamental no processo de cicatrização epitelial, e várias citocinas e fatores de crescimento estão envolvidos neste processo. Compreender os efeitos cruzados e paracrinos dessas citocinas e fatores de crescimento liberados pode ajudar na busca de novas estratégias terapêuticas para o tratamento de doenças da superfície ocular.

Ophthalmologic Clinical Features of Facial Nerve Palsy Patients

PURPOSE: To understand the ophthalmic clinical features and outcomes of facial nerve palsy patients who were referred to an ophthalmic clinic for various conditions like Bell's palsy, trauma, and brain tumor. METHODS: A retrospective study was conducted of 34 eyes from 31 facial nerve palsy patients who visited a clinic between August 2007 and July 2017. The clinical signs, management, and prognosis were analyzed. RESULTS: The average disease period was 51.1 ± 20.6 months, and the average follow-up duration was 24.0 ± 37.5 months. The causes of facial palsy were as follows: Bell's palsy, 13 cases; trauma, six cases; brain tumor, five cases; and cerebrovascular disease, four cases. The clinical signs were as follows: lagophthalmos, 24 eyes; corneal epithelial defect, 20 eyes; conjunctival injection, 19 eyes; ptosis, 15 eyes; and tearing, 12 eyes. Paralytic strabismus was found in seven eyes of patients with another cranial nerve palsy (including the third, fifth, or sixth cranial nerve). Conservative treatments (like ophthalmic ointment or eyelid taping) were conducted along with invasive procedures (like levator resection, tarsorrhaphy, or botulinum neurotoxin type A injection) in 17 eyes (50.0%). Over 60% of the patients with symptomatic improvement were treated using invasive treatment. At the time of last following, signs had improved in 70.8% of patients with lagophthalmos, 90% with corneal epithelium defect, 58.3% with tearing, and 72.7% with ptosis. The rate of improvement for all signs was high in patients suffering from facial nerve palsy without combined cranial nerve palsy. CONCLUSIONS: The ophthalmic clinical features of facial nerve palsy were mainly corneal lesion and eyelid malposition, and their clinical course improved after invasive procedures. When palsy of the third, fifth, or sixty cranial nerve was involved, the prognosis and ophthalmic signs were worse than in cases of simple facial palsy. Understanding these differences will help the ophthalmologist take care of patients with facial nerve palsy.

Effect of the bioprotectant agent trehalose on corneal epithelial healing after corneal cross-linking for keratoconus / Efeito do agente bioprotetor trealose na cicatrização epitelial da córnea após reticulação corneana para ceratocone

ABSTRACT Purpose: This study aimed to compare the effectiveness of eye drops containing a combination of sodium hyaluronate and trehalose and sodium hyaluronate for the treatment of corneal cross-linking and epithelial healing. Methods: This study included 46 eyes of 23 keratoconus patients who underwent corneal cross-linking on both eyes. Unpreserved trehalose 30 mg/mL and sodium hyaluronate 1.5 mg/mL (Thealoz Duo®; Thea, France) were applied six times a day on one eye of each patient and unpreserved sodium hyaluronate 0.15% (Eye Still®; Teka, Inc., Istanbul) was applied on the other eye. Patients were examined daily until complete re-epithelialization. Postoperative examinations to assess the measured size of epithelial defect were performed using slit-lamp biomicroscopy. Results: The study included 23 patients (13 females and 10 males) with a mean age of 20.9 ± 10.3 years. Corneal epithelial healing time after cross-linking was 2.3 ± 1.2 days for the trehalose and sodium hyaluronate group and 3.8 ± 2.9 days for the sodium hyaluronate group (p=0.03). Conclusion: The application of eye drops containing the combination of trehalose and sodium hyaluronate resulted in faster corneal re-epithelialization following corneal cross-linking than that of eye drops containing sodium hyaluronate alone.

RESUMO Objetivo: Este estudo teve como objetivo comparar a eficácia de colírios contendo uma combinação de hialurônico de sódio e hialuronato de sódio e trealose para o tratamento da cicatrização epitelial em pacientes com cross-linking corneano. Métodos: Este estudo inclui 46 olhos de 23 pacientes com ceratocone, que foram operados com cross-linking corneano em ambos os olhos. Trealose não preservada 30 mg/mL e hialuronato de sódio 1,5 mg/mL (Thealoz Duo®; Thea, França) foram aplicados seis vezes por dia em um olho de cada paciente e hialuronato de sódio não conservado, 0,15% (Eye Still®; Teka, Inc., Istambul) foi aplicada no outro olho. Os pacientes foram examinados diariamente até a completa reepitelização. Exames pós-operatórios para avaliar o tamanho medido do defeito epitelial foram realizados usando biomicroscopia com lâmpada de fenda. Resultados: O estudo incluiu 23 pacientes (13 mulheres e 10 homens) com idade média de 20,9 ± 10,3. O tempo de cicatrização epitelial da córnea após o cross-linking foi de 2,3 ± 1,2 dias para o grupo trealose e hialuronato de sódio e 3,8 ± 2,9 dias para o grupo hialurônico de sódio (p=0,03). Conclusão: A aplicação de gotas para os olhos contendo combinação de trealose e hialuronato de sódio resultou em uma re-epitelização da córnea mais rápida após o cross-linking corneano do que a de gotas contendo hialurônico de sódio apenas.

Clinical features and corneal optical anisotropies in a rabbit model of limbal stem cell deficiency / Fenótipos clínicos e anisotropias ópticas da córnea em coelhos com deficiência de células tronco limbais

ABSTRACT Purposes: To investigate the intra-laboratory reproducibility of clinical features and to evaluate corneal optical anisotropies in a rabbit model of limbal stem cell deficiency. Methods: Limbal injury was induced in the right eye of 23 adult New Zealand White rabbits using a highly aggressive protocol that combined 360 degrees limbal peritomy, keratolimbectomy, alkaline chemical burn, and mechanical removal of the epithelium. Clinical evaluation of the injured eyes was performed for 28 days and included corneal impression cytology. Corneas with a severe clinical outcome set typical of limbal stem cell deficiency were then collected, subjected to a histopathological examination, and examined for optical anisotropies. Corneas from healthy rabbit eyes were used as controls. Differences in optical path due to stromal collagen birefringence, as well as linear dichroism related to the expression and spatial orientation of glycosaminoglycan chains from proteoglycans, were measured from cross-sections under a quantitative polarized light microscope. Results: One eye showed signs of hypopyon and was excluded. Signs of ocular inflammation were observed in all eyes studied (n=22). Corneal impression cytology did not detect goblet cells. Twelve of the 22 corneas presented a clinical outcome set typical of limbal stem cell deficiency, which is characterized by the presence of epithelial defects, inflammatory cells, moderate-to-severe opacity, and neovascularization. Microscopic studies under polarized light revealed that relative to controls, limbal stem cell deficiency caused a 24.4% increase in corneal optical path differences. Further, corneas with limbal stem cell deficiency were less dichroic than controls. Conclusions: These results suggest that rabbit models of limbal stem cell deficiency must be rigorously screened for use in preclinical studies to ensure experimental homogeneity because protocols used to create limbal stem cell deficiency could be not associated with good intra-laboratory reproducibility of clinical features. Limbal stem cell deficiency, as induced herein, altered the optical anisotropic properties of the corneal stroma. Such alterations are indicative of changes in collagen packing and the spatial orientation of glycosaminoglycan chains from proteoglycans. Knowledge of these changes is important to potentiate strategies aimed at restoring the morphofunctional integrity of the corneal stroma affected by limbal stem cell deficiency.

RESUMO Objetivos: Investigar a reprodutibilidade intra-la­boratorial dos fenótipos clínicos e avaliar anisotropias ópticas em córneas de coelhos com deficiência de células tronco limbais. Métodos: Lesões ao limbo foram feitas no olho direito de 23 coelhos adultos da Nova Zelândia Branco, usando um protocolo altamente agressivo, que envolveu peritomia limbal em 360 graus, ceratolimbectomia, cauterização por álcali, e remoção mecânica de epitélio remanescente. Os olhos foram clinicamente avaliados por 28 dias, inclusive por citologia de impressão corneal. As córneas que manifestaram um conjunto de alterações típicas de deficiência de células tronco limbais foram coletadas e submetidas à estudos em histopatologia e em anisotropias ópticas. Córneas saudáveis foram usadas como controles. Diferenças de caminho óptico de birrefringência relacionada à organização do colágeno estromal, e dicroísmo linear relacionado à expressão e à orientação das cadeias de glicosaminoglicanos dos proteoglicanos estromais, foram quantificados por microscopia de luz polarizada. Resultados: Um olho apresentou hipópio e foi excluído do estudo. Todos os olhos estudados (n=22) apresentaram sinais de inflamação ocular. A citologia de impressão não detectou células caliciformes na superfície corneal. Doze de 22 córneas manifestaram alterações clínicas típicas de deficiência de células tronco limbais, caracterizado por defeitos epiteliais, infiltrados inflamatórios, opacidade de moderada à severa, e neovascularização. Estudos por microscopia de luz polarizada mostraram que a deficiência de células tronco limbais aumentou a diferenças de caminho óptico corneal em 24,4% (versus controles). As cór­neas com deficiência de células tronco limbais foram menos dicroicas do que as córneas controle. Conclusões: Coelhos com deficiência de células tronco limbais, para aplicações em estudos pré-clínicos, devem ser rigorosamente selecionados para assegurar homogeneidade experimental, pois há evidências de que protocolos utilizados para indução de deficiência de células tronco limbais não estão associados com boa reprodutibilidade intra-laboratorial de fenótipos clínicos. A deficiência de células tronco limbais, como induzida aqui, alterou as propriedades ópticas anisotrópicas do estroma corneal. Tais alterações são indicativas de mudanças no empacotamento de colágeno e na orientação das cadeias de glicosaminoglicanos dos proteoglicanos. Conhecimentos nessas alterações são importantes para potencializar estratégias que visam a restabelecer a integridade morfofuncional do estromal corneal acometido pela deficiência de células tronco limbais.

Evaluation of programmed cell death processes on the lens epithelium of older dogs with diabetic and hypermature cataracts / Avaliação dos processos de morte celular programada no epitélio da lente de cães idosos com catarata diabética e hipermadura

It is well known that posterior capsule opacification (PCO), one of the most common late postoperative complications of cataract surgery, is mainly caused by proliferation and differentiation of remaining lens epithelial cells (LECs) on the posterior lens capsule. Many authors suggest that alterations induced by the pathophysiology of cataracts, its metabolism and the use of 0.1% trypan blue (TB) must cause some degree of cellular damage on these cells, wicht would help to prevent and/or reduce the incidence of PCO after cataract surgery in humans. Therefore, the aim of this study was to evaluate the expression of cell death markers on LECs of older dogs with diabetic and hypermature cataracts, after capsulorhexis, both using 0.1% TB. Twenty samples collected from 13 dogs of different breeds, with ages varying from 8 to 12 years-old, with diabetic and hypermature cataracts, which had been subjected to phacoemulsification surgery (Phaco) using 0.1% TB for staining were studied. Animals were classified as dogs with diabetic (DC) and hypermature cataracts (HC), and expression of molecular markers for apoptosis and autophagy (caspase-3 and beclin-1) on LECs were obtained by immunofluorescence technique. The expression of caspase-3 and beclin-1 was observed in every studied sample and did not differ between groups. In conclusion, our findings suggest that apoptosis and autophagy processes occur to LECs in older dogs presenting diabetic and hypermature cataracts after Phaco utilizing 0.1% TB. Our results may be helpful to future studies of PCO in post-phacoemulsification surgery patients.(AU)

A opacificação da cápsula posterior da lente do globo ocular é a complicação mais observada após a remoção da lente. Essa patologia é causada principalmente pela proliferação e diferenciação das células do epitélio anterior da lente em sua cápsula posterior. Muitos autores sugerem que alterações induzidas pelo metabolismo e/ou patofisiologia da catarata e o uso do corante de azul de tripan a 0,1% devam causar algum dano a essas células, o que supostamente ajudaria a prevenir e reduzir a incidência de tal complicação em humanos. Este trabalho avaliou a expressão de marcadores de morte celular no epitélio anterior da lente de cães idosos com catarata diabética e hipermadura, após capsulorrexe realizada com o emprego do azul de tripan a 0,1%. Foram estudadas vinte amostras colhidas de treze cães de diferentes raças, com idades variando de oito a doze anos, que apresentavam catarata diabética ou hipermadura e que foram submetidos à facoemulsificação utilizando corante de azul de tripan a 0,1%. Foram designados dois grupos: com catarata diabética (DC) e com catarata hipermadura (HC). A expressão molecular dos marcadores de morte celular por apoptose a autofagia (caspase-3 e beclina-1) no epitélio anterior da lente foi avaliada pela técnica de imunofluorescência. Observou-se que a expressão de caspase-3 e beclina-1 ocorreu em todas as amostras e não foi diferente entre os grupos. Os achados deste estudo sugerem que o processo de morte celular por apoptose e autofagia ocorre no epitélio anterior da lente de cães idosos com catarata diabética e hipermadura submetidos à facoemulsificação com o corante de azul de tripan a 0,1%. Este resultado pode ser útil para estudos futuros da opacidade da cápsula posterior da lente em cães submetidos à facoemulsificação.(AU)

A supramolecular look at microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny / Uma visão supramolecular sobre a regulação microambiental de células tronco epiteliais limbais e a diferenciação de sua progênie

ABSTRACT Various approaches have been taken to improve our knowledge of the microenvironmental regulation of limbal epithelial stem cells. Researchers have extensively investigated the roles of growth factors, survival factors, cytokines, enzymes, and permeable molecules secreted by the limbal cells. However, recent evidence suggests that stem cell fate (i.e., self-renewal or differentiation) can also be influenced by biophysical and mechanical cues related to the supramolecular organization and the liquid crystalline (mesophase) nature of the stromal extracellular matrix. These cues can be sensed by stem cells and transduced into intracellular biochemical and functional responses, a process known as mechanotransduction. The objective of this review is to offer perspectives on the supramolecular microenvironmental regulation of limbal epithelial stem cells and the differentiation of their progeny.

RESUMO Muitas abordagens têm sido utilizadas para ampliar entendimentos sobre a regulação microambiental das células tronco epiteliais limbais. Neste contexto, pesquisadores têm exaustivamente investigado a participação de fatores de crescimento, fatores de sobrevida, citocinas, enzimas e moléculas permeáveis secretadas pelas células limbais. Entretanto, evidências recentes sugerem que o destino (ie. autorrenovação ou recrutamento para a via de diferenciação) das células tronco também sofre influência de estímulos biofísicos ou mecânicos relacionados à organização supramolecular e à natureza liquido-cristalina (mesofases) da matriz extracelular estromal. Esses estímulos podem ser percebidos e traduzidos pelas células tronco em sinais bioquímicos que geram respostas funcionais, através de um processo designado de mecanotransdução. Objetiva-se, com a presente revisão, oferecer ao leitor perspectivas supramoleculares sobre a regulação microambiental das células tronco epiteliais limbais e a diferenciação de sua progênie.

Change of Corneal Epithelial and Stromal Thickness after Cataract Surgery through Scleral Tunnel Incision

PURPOSE: To compare preoperative and postoperative thickness and to investigate the difference in the thickness change of corneal epithelium and stroma after cataract surgery through scleral tunnel incision. METHODS: Forty eyes of forty patients who were 40 years old or older and underwent small-incision superior scleral tunnel cataract surgery with phacoemulsification were included. Using the RTVue instrument (Optovue Inc., Fremont, CA, USA), corneal epithelial (ET) and stromal thicknesses (ST) of all subjects were measured preoperatively and at 3 days, 1 week, and 1 month postoperatively. Thicknesses were classified into 3 zones according to the distance from the vertex: central zone (within 2 mm), paracentral zone (2-5 mm diameter) and midperipheral zone (5-6 mm diameter). RESULTS: Mean central ST was 486.68 ± 25.15 µm, 535.16 ± 48.13 µm, 515.98 ± 44.07 µm, and 502.28 ± 34.87 µm preoperatively, and at 3 days, 1 week, and 1 month postoperatively, respectively (p < 0.001 for all). ST showed significant thickening in all three zones from 3 days to 1 month postoperatively (p < 0.001 for all). Mean central, paracentral, and midperipheral ET was 52.13 ± 3.41 µm, 50.42 ± 2.97 µm, 49.12 ± 3.05 µm at preoperatively and 51.03 ± 3.63 µm, 48.96 ± 3.62 µm, 47.67 ± 3.81 µm at 1 month postoperatively, respectively (p = 0.061, 0.006, 0.001, respectively), while there were no signficant changes in all three zones at 3 and 7 days postoperatively. Changes in ET and ST were prominent at the superotemporal incision site. CONCLUSIONS: After scleral tunnel cataract surgery, corneal edema was observed in the stroma immediately after surgery. There was no significant change at early times after surgery in the epithelium, and there was a decrease in the peripheral cornea at 1 month postoperatively. The change in ET was considered a compensatory change due to stromal edema and appeared between 1 week to 1 month postoperatively.

A Case of a Corneal Stromal Penetrating Injury via an Ejected Needle during Stromal Hydration

PURPOSE: We report a case of a horizontally penetrating injury of corneal stroma via an ejected irrigating needle during stromal hydration in cataract surgery. CASE SUMMARY: A 79-year-old woman presented with decreased visual acuity in her right eye. Her best corrected visual acuity in the right eye was 0.3. The cataract score of her right eye was nuclear opacity 3, cortical opacity 1−2, and posterior subcapsular opacity 1. She underwent phacoemulsification using a clear corneal approach while under topical anesthesia, and an intraocular lens was successfully implanted in the bag. A plastic syringe with an irrigating needle was used to hydrate the corneal stroma at the clear cornea site. As a result of the increased pressure applied to the connection between the syringe and irrigating needle, the loosened irrigating needle was forcefully ejected toward the corneal endothelium, horizontally penetrating the stroma without injuring the corneal epithelium. On the 1st postoperative day, her right eye exhibited a visual acuity of 0.5, astigmatism of −1.25 diopter (D) cyl., corneal edema with Descemet's folds, and insignificant scarring of the corneal endothelium. Three weeks after surgery, the visual acuity was 0.9, the astigmatism was −0.50 D, and any other abnormalities such as corneal opacity were no longer present. CONCLUSIONS: Despite a penetrating needle injury through the corneal endothelium and stroma, the corneal wound healing process did not result in corneal opacity or worsened astigmatism. To prevent sequela due to an ejected irrigating needle, operators and assistants should be actively aware of tight locking between the needle and syringe.

Effects of Human Serum on Human Corneal Epithelial Cells in Vitro

PURPOSE: To investigate the effect of human serum on corneal epithelial cells. METHODS: Changes of corneal epithelial cells were evaluated after 1, 4, 12, and 24 hours (hrs) of exposure to various concentrations of human serum (3, 5, 8, and 16%). Cellular metabolic activity and the extent of cellular damage were measured. Effect of human serum on cell migration was also examined. Concentration of procollagen type-I COOH-terminal peptide (PIP), epidermal growth factor (EGF), and laminin after exposure to human serum was further observed. RESULTS: In every concentration of human serum, metabolic activity of the corneal epithelial cells temporarily decreased at 4 hrs of exposure and recovered to baseline levels afterward. With the same exposure time, there was no statistically significant difference in metabolic activity between the human serum-exposed group and the control group. Cellular toxicity of human serum exhibited a time- and dose-dependent relationship. Cellular migration was observed after 24 hrs of exposure to 5% concentration of human serum and after 12 hrs of exposure to 8% and 16% concentration of human serum. The PIP, EGF, and laminin titers increased in time- and dose-dependent manners. CONCLUSIONS: Human serum does not decrease the metabolic activity of corneal epithelial cells as the concentration and exposure time increase, but it can induce cytotoxicity. Considering cellular migration, a serum concentration of 5% or higher should be used.

Gintonin, an exogenous ginseng-derived LPA receptor ligand, promotes corneal wound healing

Ginseng gintonin is an exogenous ligand of lysophosphatidic acid (LPA) receptors. Accumulating evidence shows LPA helps in rapid recovery of corneal damage. The aim of this study was to evaluate the therapeutic efficacy of gintonin in a rabbit model of corneal damage. We investigated the signal transduction pathway of gintonin in human corneal epithelium (HCE) cells to elucidate the underlying molecular mechanism. We next evaluated the therapeutic effects of gintonin, using a rabbit model of corneal damage, by undertaking histochemical analysis. Treatment of gintonin to HCE cells induced transient increases of [Ca²⁺](i) in concentration-dependent and reversible manners. Gintonin-mediated mobilization of [Ca²⁺](i) was attenuated by LPA1/3 receptor antagonist Ki16425, phospholipase C inhibitor U73122, inositol 1,4,5-triphosphate receptor antagonist 2-APB, and intracellular Ca²⁺ chelator BAPTA-AM. Gintonin facilitated in vitro wound healing in a concentration-dependent manner. When applied as an eye-drop to rabbits with corneal damage, gintonin rapidly promoted recovery. Histochemical analysis showed gintonin decreased corneal apoptosis and increased corneal cell proliferation. We demonstrated that LPA receptor activation by gintonin is linked to in vitro and in vivo therapeutic effects against corneal damage. Gintonin can be applied as a clinical agent for the rapid healing of corneal damage.

Assessment of surgical outcomes of limbal transplantation using simple limbal epithelial transplantation technique in patients with total unilateral limbal deficiency / Avaliação dos resultados cirúrgicos do transplante de limbo utilizando a técnica SLET (simple limbal epithelial transplantation), em pacientes com deficiência límbica total unilateral

ABSTRACT This study aimed to evaluate the effectiveness of the novel simple limbal epithelial transplantation (SLET) technique, which reduces the risk of iatrogenic limbal stem cell deficiency in the donor eye. Four patients with total unilateral limbal stem cell deficiency received a limbal graft, measuring 4 mm × 2 mm, from the contralateral healthy eye in a single surgical procedure. The graft was divided into 10-20 pieces and distributed on the corneal surface. At 6-month follow-up, a completely avascular corneal epithelial surface was obtained in two patients, and there was improvement in visual acuity in one patient. The limbal grafts did not adhere to the cornea in one patient. No serious complications related to the surgery were observed in this study.

RESUMO Este trabalho tem como objetivo avaliar a eficácia de uma nova técnica cirúrgica denominada SLET (simple limbal epithelial transplantation), um procedimento promissor que reduz os riscos de indução de deficiência límbica iatrogênica no olho doador. Quatro pacientes com deficiência límbica total unilateral, secundária a queimadura química, receberam um enxerto de células límbicas, medindo 4 mm x 2 mm, do olho contralateral sadio, em apenas um tempo cirúrgico. Este foi divido em 10 a 20 fragmentos e distribuído sobre a superfície da córnea. Após 6 meses de cirurgia, superfície corneana totalmente epitelizada e avascular foi obtida em dois pacientes. Houve melhora da acuidade visual em um paciente. Não houve aderência dos enxertos de limbo na córnea em um paciente. Nenhum paciente apresentou efeitos colaterais graves decorrentes do procedimento cirúrgico.

Ability of Mn(2+) to Permeate the Eye and Availability of Manganese-enhanced Magnetic Resonance Imaging for Visual Pathway Imaging via Topical Administration / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Manganese-enhanced magnetic resonance imaging (MEMRI) for visual pathway imaging via topical administration requires further research. This study investigated the permeability of the corneal epithelium and corneal toxicity after topical administration of Mn2+ to understand the applicability of MEMRI.</p><p><b>METHODS</b>Forty New Zealand rabbits were divided into 0.05 mol/L, 0.10 mol/L, and 0.20 mol/L groups as well as a control group (n = 10 in each group). Each group was further subdivided into epithelium-removed and epithelium-intact subgroups (n = 5 in each subgroup). Rabbits were given 8 drops of MnCl2in 5 min intervals. The Mn2+ concentrations in the aqueous and vitreous humors were analyzed using inductively coupled plasma-mass spectrometry at different time points. MEMRI scanning was carried out to image the visual pathway after 24 h. The corneal toxicity of Mn2+ was evaluated with corneal imaging and pathology slices.</p><p><b>RESULTS</b>Between the aqueous and vitreous humors, there was a 10 h lag for the peak Mn2+ concentration times. The intraocular Mn2+ concentration increased with the concentration gradients of Mn2+ and was higher in the epithelium-removed subgroup than that in the epithelium-intact subgroup. The enhancement of the visual pathway was achieved in the 0.10 mol/L and 0.20 mol/L epithelium-removed subgroups. The corresponding peak concentrations of Mn2+ were 5087 ± 666 ng/ml, 22920 ± 1188 ng/ml in the aqueous humor and 884 ± 78 ng/ml, 2556 ± 492 ng/ml in the vitreous body, respectively. Corneal injury was evident in the epithelium-removed and 0.20 mol/L epithelium-intact subgroups.</p><p><b>CONCLUSIONS</b>The corneal epithelium is a barrier to Mn2+, and the iris and lens septum might be another intraocular barrier to the permeation of Mn2+. An elevated Mn2+ concentration contributes to the increased permeation of Mn2+, higher MEMRI signal, and corneal toxicity. The enhancement of the visual pathway requires an effective Mn2+ concentration in the vitreous body.</p>

Autologous Platelet-rich Plasma Eye Drops in the Treatment of Recurrent Corneal Erosions

PURPOSE: To evaluate the effect of platelet-rich plasma (PRP) eye drops in the treatment of recurrent corneal erosions (RCE). METHODS: A total of 47 eyes were included in this retrospective study. Clinical records of 20 consecutive patients with RCE who had been treated with conventional lubricant eye drops (conventional treatment group) from June 2006 to December 2008 and 27 consecutive patients treated with autologous PRP eye drops in addition to lubricant eye drops (PRP eye drops treated group) from January 2009 to September 2014 were reviewed. Major and minor recurrences were recorded and compared between two groups. RESULTS: This study included 31 men and 16 women. The mean age was 44.5 ± 14.5 years (range, 19 to 86 years), and the mean follow-up duration was 14.9 ± 14.4 months (range, 6 to 64 months). Of the 27 cases in the PRP eye drops treated group, there were seven major recurrences in six eyes (22.2%) and ten minor recurrences in seven eyes (25.9%). In contrast, 16 eyes (80.0%) from the 20 patients in the conventional lubricant eye drops treated group had major recurrences, and all patients in this group reported minor recurrences. The mean frequency of recurrence was 0.06 ± 0.08 per month in the PRP eye drops treated group and 0.39 ± 0.24 per month in the conventional treatment group (p = 0.003). No side effects were noted in any of the patients over the follow-up period. CONCLUSIONS: The use of PRP eye drops for the treatment of RCE was shown to be effective in reducing the recurrence rate without any significant complications.

Efficacy of Korean Multipurpose Contact Lens Disinfecting Solutions against Acanthamoeba castellanii

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.