Efeito da riboflavina tópica exposta à irradiação ultravioleta A e inserção de segmentos de anéis corneanos intraestromais para ceratocone

ADIMARA DA CANDELARIA RENESTO

EFEITO DA RIBOFLAVINA TÓPICA EXPOSTA À IRRADIAÇÃO

ULTRAVIOLETA A E INSERÇÃO DE SEGMENTOS DE ANÉIS

CORNEANOS INTRAESTROMAIS PARA CERATOCONE

Tese apresentada à Universidade Federal de São Paulo – Escola Paulista de Medicina, para obtenção do Título de Doutor em Ciências pelo Programa de Pós-Graduação em Oftalmologia.

EFEITO DA RIBOFLAVINA TÓPICA EXPOSTA À IRRADIAÇÃO

ULTRAVIOLETA A E INSERÇÃO DE SEGMENTOS DE ANÉIS

CORNEANOS INTRAESTROMAIS PARA CERATOCONE

Tese apresentada à Universidade Federal de São Paulo – Escola Paulista de Medicina, para obtenção do Título de Doutor em Ciências pelo Programa de Pós-Graduação em Oftalmologia.

Orientador:

Prof. Dr. Mauro Silveira de Queiroz Campos

Coorientadora:

Profa. Dra. Marta de Filippi Sartori

Renesto, Adimara da Candelaria

Efeito da riboflavina tópica exposta à irradiação ultravioleta A e inserção de segmentos de anéis corneanos intraestromais para ceratocone / Adimara da Candelaria Renesto. – São Paulo, 2012.

xi, 77f.

Tese (Doutorado) – Universidade Federal de São Paulo. Escola Paulista de Medicina. Programa de Pós-Graduação em Oftalmologia.

Título em inglês: Effect of topical riboflavin exposure to ultraviolet A radiation and insertion of intrastromal corneal ring segments for

keratoconus.

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UNIVERSIDADE FEDERAL DE SÃO PAULO

ESCOLA PAULISTA DE MEDICINA

PROGRAMA DE PÓS-GRADUAÇÃO EM OFTALMOLOGIA

Chefe do Departamento:

Profa. Dra. Denise de Freitas

Coordenador do Curso de Pós-graduação:

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ADIMARA DA CANDELARIA RENESTO

EFEITO DA RIBOFLAVINA TÓPICA EXPOSTA À IRRADIAÇÃO

ULTRAVIOLETA A E INSERÇÃO DE SEGMENTOS DE ANÉIS

CORNEANOS INTRAESTROMAIS PARA CERATOCONE

Presidente da banca:

Prof. Dr. Mauro Silveira de Queiroz Campos

BANCA EXAMINADORA

Prof (a). Dr (a). Seiji Hayashi

Prof (a). Dr (a). Walton Nosé

Prof (a). Dr (a). Vera Lucia Degaspare Monte Mascaro

Prof (a). Dr (a). Belquiz Rodrigues do Amaral Nassaralla

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Dedicatória

Aos meus pais, Adilson e Maria Angela, a quem devo toda minha formação e pelo apoio a esta tese.

À minha irmã, Acássia, parte também desta minha conquista.

À minha avó, Myrthes, pelo carinho e apoio.

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Agradecimentos

Aos pacientes voluntários que participaram deste estudo, pela esperança e confiança depositados em nós.

Ao meu orientador, Prof. Dr. Mauro Silveira de Queiroz Campos, responsável pelo meu aprimoramento profissional, pelo apoio, paciência e incentivo constantes na realização deste trabalho.

À Profa. Dra. Marta de Filippi Sartori, coorientadora, pelos ensinamentos neste projeto.

Ao Prof. Dr. Walton Nosé, pelas sugestões na banca prévia e ensinamentos à realização desta tese.

Aos Profs. Drs. Ricardo Uras, César Lipener e Neusa Vidal, que permitiram meu acesso ao Departamento de Oftalmologia da Universidade Federal de São Paulo – Escola Paulista de Medicina, através do Setor de Lente de Contato.

Ao tecnólogo e amigo Jeison de Nadai Barros, pela ajuda na realização do exame de citologia de impressão e na publicação do artigo original.

Aos tecnólogos Débora, Ricardo, Maira, Janaina, Joyce, Andréia, Daniel, Paulo, Iara e Denise e ao fotógrafo Laércio, pela ajuda no aprendizado e realização dos exames e fotodocumentação dos pacientes desta tese.

Aos colegas oftalmologistas do setor de Cirurgia Refrativa do Departamento de Oftalmologia da Universidade Federal de São Paulo, em especial, à Dra. Ester Sakae Yamazaki, Dra. Danielle Miranda e Dr. André Luiz Alves Salame, pela amizade e apoio à realização deste trabalho.

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À instituição de fomento deste estudo, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), processo número 1915/07, pelo apoio financeiro à realização deste trabalho.

Ao colega Dr. Luiz Alberto Soares Melo Jr., por sua colaboração na análise estatística deste estudo.

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ix Sumário

Dedicatória ... v 

Agradecimentos ... vi 

Resumo ... x 

1. INTRODUÇÃO ... 1 

1.1 Objetivos ... 4 

2. ARTIGOS ... 5 

2.1 Cross-linking e segmento de anel corneano intraestromal ... 8 

2.2 Impression cytologic analysis after corneal collagen cross-linking using riboflavin and Ultraviolet-A light in the treatment of keratoconus ... 16 

2.3 Sequential topical riboflavin with or without Ultraviolet A radiation with delayed intracorneal ring segment insertion for keratoconus ... 22 

2.4 Impression cytologic analysis after corneal cross-linking and insertion of corneal ring segments for keratoconus: two-year results ... 37 

2.5 Conclusões ... 60 

3. ANEXOS ... 61 

Anexo 1 – Carta de aprovação do Comitê de Ética em Pesquisa da Universidade Federal de São Paulo/Hospital São Paulo ... 62 

Anexo 2 – Termo de consentimento livre e esclarecido aprovado pelo Comitê de Ética em Pesquisa da Universidade Federal de São Paulo/Hospital São Paulo ... 63 

Anexo 3 – Quadro de exames nos momentos de avaliação ... 66 

Anexo 4 – Descrição cirúrgica intraoperatória ... 67 

4. REFERÊNCIAS ... 68 

Abstract 

x Resumo

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O ceratocone é uma doença ectásica da córnea, geralmente bilateral e assimétrica, que afeta aproximadamente 1 em 2000 na população geral. Inicia-se na puberdade e devido à sua progressão, 20% dos pacientes serão submetidos ao transplante de córnea conforme descrito por Rabinowitz* em 1998 (citado por Wollensak et al., 2003a). A etiologia desta doença é desconhecida, provavelmente multifatorial com uma variedade de diferentes processos patológicos. Em uma córnea com ceratocone, haveria predisposição genética para uma maior sensibilidade a mediadores apoptóticos pelos ceratócitos (Edwards et al., 2001). A forma mais comum de apresentação da doença é como uma desordem esporádica, mas há também um histórico familiar caracterizado por uma herança autossômica dominante com expressão fenotípica variável (Edwards et al., 2001).

O quadro clínico do ceratocone se caracteriza pela presença de afinamento e abaulamento corneano, levando à diminuição da acuidade visual secundária ao astigmatismo irregular causado pelas alterações corneanas. A córnea também pode apresentar áreas com cicatriz e depósito de hemossiderina, formando o anel de Fleischer conforme descrito por Krachmer** em 1984 (citado por Miranda et al., 2003). O tratamento inicial da doença é clínico, com o uso de óculos e/ou lentes de contato.

Caso o paciente não tolere o uso das lentes de contato, há algumas alternativas cirúrgicas para o tratamento. Em adição aos transplantes lamelares e penetrantes, a introdução dos segmentos de anéis corneanos intraestromais caracteriza-se como outra opção de tratamento conforme descrito por Barraquer*** em 1966 (citado por Miranda et al., 2003) (Tan et al., 2006). Os segmentos de anéis corneanos intraestromais criam um efeito de encurtamento da lamela corneana, aplanando a área central da córnea, diminuindo a irregularidade da superfície anterior, deslocando o

*

Rabinowitz YS. Keratoconus. Surv Ophthalmol. 1998;42(4):297-319 apud Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen crosslinking for the treatment of keratoconus. Am J

Ophthalmol. 2003a;135(5):620-7.

**

Krachmer JH, Feder RS, Belin MW. Keratoconus and related noninflammatory corneal thinning

disorders. Surv Ophthalmol. 1984;28(4):293-322 apud Miranda D, Sartori M, Francesconi C, Allemann N, Ferrara P, Campos M. Ferrara intrastromal corneal ring segments for severe keratoconus. J Refract Surg. 2003;19(6):645-53.

***

I n t r o d u ç ã o | 3

ápice em direção ao centro e preservando a asfericidade positiva da córnea (Assil et al., 1995).

Inicialmente indicados para baixa miopia (Nosé et al., 1993; Assil et al., 1995), a sua principal indicação atualmente é a pacientes com ceratocone. Outras indicações incluem astigmatismo irregular (ex.: transplante de córnea, ceratotomia radial, trauma corneano), ectasia corneana após cirurgia refrativa, degeneração marginal pelúcida (Ferrara, Bicalho, 2006) e, recentemente, associado à técnica do cross-linking (CXL) do colágeno corneano (Coskunseven et al., 2009a).

O cross-linking do colágeno corneano pelo fotossintetizador riboflavina (vitamina B2) e irradiação ultravioleta A (UVA) aumenta a rigidez corneana e é descrito como um método efetivo para a estabilização da córnea em pacientes com ceratocone progressivo (Wollensak, 2006). A ideia do uso do CXL para aumentar a rigidez corneana foi desenvolvida na Alemanha nos anos 90 (Wollensak et al., 2003a).

A técnica do CXL é similar à fotopolimerização de polímeros e estabiliza a biomecânica da córnea pelo aumento da formação de ligações covalentes intrafibrilares e interfibrilares do colágeno corneano por uma reação fotoquímica oxidativa (Spoerl et al., 2007). Em casos em que o astigmatismo irregular é progressivo (ex.: ceratocone, degeneração marginal pelúcida, ectasia após cirurgia refrativa), o estroma corneano encontra-se enfraquecido e, em alguns casos, podem piorar ao longo do tempo (Coskunseven et al., 2009a).

1.1 Objetivos

Este estudo tem como objetivos:

1. Avaliar se o cross-linking do colágeno corneano antes da inserção de segmentos de anéis corneanos intraestromais altera o tratamento do ceratocone;

4.1 – Cross-linking e segmento de anel corneano intraestromal/ Cross-linking and intrastromal corneal ring segment

Adimara da Candelaria Renesto, MD, Marta Sartori, MD, Mauro Campos, MD.

Arquivos Brasileiros de Oftalmologia. 2011;74(1):67-74.

4.2 – Impression cytologic analysis after corneal collagen cross-linking using riboflavin and Ultraviolet-A light in the treatment of keratoconus

Adimara da Candelaria Renesto, MD, Jeison de Nadai Barros, PhD, and Mauro Campos, MD.

Cornea. 2010;29(10):1139-1144.

4.3 – Sequential topical riboflavin with or without Ultraviolet A radiation with delayed intracorneal ring segment insertion for keratoconus

Adimara da Candelaria Renesto, MD, Luiz Alberto S. Melo Jr, MD, Marta de Filippi Sartori, MD, and Mauro Campos, MD.

American Journal of Ophthalmology [periódico na Internet]. 2012 [citado 2012

Jan 20]; [cerca de 15 p.]. Disponível em: http://www.ajo.com/inPress

4.4 – Impression cytologic analysis after corneal cross-linking and insertion of corneal ring segments for keratoconus: two-year results

Adimara da Candelaria Renesto, MD, Jeison de Nadai Barros, PhD, and Mauro Campos, MD.

Arquivos Brasileiros de Oftalmologia [artigo aceito para publicação em

A r t i g o s | 7

- Trabalho apresentado como pôster na American Academy of Ophthalmology Annual Meeting, San Francisco, EUA, outubro de 2009;

(Intraocular Pressure and Corneal Biomechanical Properties of Keratoconic Eyes After

Cross-Linking. Mauro Campos, MD, Adimara da Candelaria Renesto, MD, Rossen

Hazarbassanov, MD, and Marta Sartori, MD).

- Contemplado com o prêmio melhor trabalho de pós-graduação em oftalmologia no Annual Research Days Meeting da Universidade Federal de São Paulo - Escola Paulista de Medicina, São Paulo, Brasil, dezembro de 2011;

(Topical Riboflavin Exposure To Ultraviolet A Radiation And Corneal Ring Segment

Insertion For Keratoconus: Two-Year Results. Adimara da Candelaria Renesto, MD,

2.2 Impression cytologic analysis after corneal collagen cross-linking

2.3 Sequential topical riboflavin with or without Ultraviolet A radiation with

A r t i g o s | 37

2.4 Impression cytologic analysis after corneal cross-linking and insertion

of corneal ring segments for keratoconus: two-year results

Artigo aceito para publicação nos Arquivos Brasileiros de Oftalmologia:

1. PÁGINA DE ROSTO

a) Título em inglês: Impression Cytologic Analysis after Corneal Cross-Linking and Insertion of Corneal Ring Segments for Keratoconus:Two-Year Results

b) Título em português: Citologia de Impressão após Cross-Linking Corneano e Inserção dos Segmentos de Anéis Corneanos para Ceratocone:Dois Anos de Seguimento

c) Título resumido para cabeçalho: Citologia de impressão após tratamento para ceratocone

d) Nome dos autores:

1. Adimara da Candelaria Renesto1 (1ª autora ) 2. Jeison de Nadai Barros2

3. Mauro Campos1

e) Titulação dos autores:

1=Médicos oftalmologistas, Departamento de Oftalmologia, Setor de Cirurgia Refrativa, Universidade Federal de São Paulo-UNIFESP- São Paulo-SP, Brasil

2= Doutor em Ciências Visuais, Departamento de Oftalmologia, Universidade Federal de São Paulo-UNIFESP- São Paulo-SP, Brasil

f) Número do projeto e instituição responsável pelo parecer do Comitê de Ética:

-Projeto Nº 1915/07; Comitê de Ética em Pesquisa da Universidade Federal de São Paulo/Escola Paulista de Medicina

g) Os autores não possuem nenhum conflito de interesses.

2. ABSTRACT

Purpose: To report the impression cytologic results after corneal cross-linking and insertion of intrastromal corneal ring segments for keratoconus. Methods: Thirty-nine eyes were distributed into two groups: 1) cross-linking group (patients underwent corneal cross-linking procedure), and 2) riboflavin eyedrops group (patients received riboflavin 0.1% (w/v) eyedrops in 20% dextran solution for 1 month). After 3 months, all patients underwent insertion of intrastromal corneal ring segments. Impression cytologic specimens were obtained from all eyes at baseline, at 1 month and 3 months after cross-linking or riboflavin eyedrops, and again at 6 months, 1 year, and 2 years after intrastromal corneal ring segment insertion. Results: Patients in the cross-linking group demonstrated improvement in the cell-to-cell contact of epithelial cells and the nucleus-to-cytoplasm ratio on the temporal conjunctiva after treatment (P = 0.008 and P = 0.047), respectively. On the superior conjunctiva, increases in goblet cell density (P = 0.037) and level of organization of nuclear chromatin (P = 0.010) after treatment were noted. Patients in the riboflavin eyedrops group demonstrated improvement in the cell-to-cell contact of epithelial cells on the superior conjunctiva after treatment (P = 0.021). On the temporal conjunctiva, an improvement in the cell-to-cell contact of epithelial cells (P < 0.001) and increases in the nucleus-to-cytoplasm ratio (P < 0.001), goblet cell density (P = 0.001), and less keratinization (P = 0.011) were noted. No changes were identified on the cornea for either group. Fisher’s exact test comparison of the impression cytologic total scores after treatment revealed no difference between groups. Conclusion: Despite changes in some conjunctival parameters (e.g., cell-to-cell contact of epithelial cell-to-cells, nucleus-to-cytoplasm ratio, level of organization of nuclear chromatin , goblet cell density, and keratinization), comparison of the total impression cytologic scores revealed no difference between groups.

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3. RESUMO

Objetivo: Relatar os resultados de citologia de impressão após o cross-linking da córnea e inserção dos segmentos de anéis corneanos intraestromais para ceratocone.

Métodos: Trinta e nove olhos foram distribuídos em dois grupos: pacientes no grupo cross-linking foram submetidos ao procedimento do cross-linking corneano e pacientes no grupo colírio de riboflavina receberam o colírio de riboflavina 0,1% (w/v) -20% dextran para uso tópico por 1 mês. Após três meses, todos os pacientes foram submetidos à inserção dos segmentos de anéis corneanos intraestromais. Citologia de impressão foi realizada em todos os olhos no pré-operatório, 1 mês e 3 meses após o cross-linking ou colírio de riboflavina, e novamente com 6 meses, 1 ano e 2 anos após a inserção dos segmentos de anéis. Resultados: Pacientes do grupo cross-linking apresentaram melhora na adesividade das células epiteliais e proporção núcleo:citoplasma na conjuntiva temporal após o tratamento (P=0,008 e P=0,047), respectivamente. Na conjuntiva superior, houve um aumento na densidade das células caliciformes (P=0,037) e nível de organização da cromatina nuclear (P=0,010) após o tratamento. Pacientes do grupo colírio de riboflavina apresentaram melhora na adesividade das células epiteliais na conjuntiva superior após o tratamento (P=0,021). Na conjuntiva temporal, houve melhora na adesividade das células epiteliais (P<0,001), aumento na proporção núcleo:citoplasma (P<0,001), células caliciformes (P=0,001) e menor queratinização (P=0,011). Ambos os grupos não apresentaram alterações corneanas. O Teste exato de Fisher para comparação de citologia de impressão no escore total após o tratamento não revelou diferença entre os grupos. Conclusão:

Apesar de mudanças em alguns parâmetros conjuntivais ( adesividade das células epiteliais, proporção núcleo:citoplasma, nível de organização da cromatina nuclear, células caliciformes e queratinização), a comparação do escore total na citologia de impressão não apresentou diferença entre os grupos.

4. MAIN TEXT

INTRODUCTION

Keratoconus is a noninflammatory cone-like ectasia of the cornea that can be managed by glasses, contact lenses, keratoplasty, cross-linking (CXL),(1) and intrastromal corneal ring segment (ICRS)(2-3) insertion, based on the stage of the disease and the patient’s symptoms. Despite being an evolutive disease, CXL has recently been utilized as a therapeutic option for stabilizing the cornea(1).

An optical microscopic study(4) demonstrated that the morphology grade of the most superficial epithelial cells obtained by impression cytology (IC) from the cornea and bulbar conjunctiva did not change after CXL using riboflavin and ultraviolet-A (UVA) light in the treatment of keratoconus.

Some patients have been pretreated with CXL, and ICRS can be performed in conjunction to produce flattening of the central corneal curvature, which decreases corneal irregularity and improves visual acuity(5). To the authors’ knowledge, no report on the cytological features of the ocular surface epithelium after CXL and ICRS insertion in the treatment of keratoconus currently exists in the literature.

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METHODS

An interventional prospective randomized clinical trial was conducted between March 2008 and December 2010 at the Vision Institute, Department of Ophthalmology, Federal University of São Paulo, São Paulo, Brazil. This study was approved by the Institutional Ethics Committee and conducted according to the tenets of the Declaration of Helsinki. All patients provided informed consent.

Patients diagnosed with documented keratoconus with a corneal thickness of at least 400 µm and a history of intolerance to contact lens fitting were recruited for the study. Exclusion criteria included previous ocular surgery; only one functional eye; corneal opacity on the axis; corneal curvature >65 diopters; history of herpetic keratitis; moderate dry eye; autoimmune disease; and pregnancy. The criteria for the diagnosis of dry eye included tear film, punctacte keratitis, and patient complaint.

A total of 39 eyes of 31 patients were included. All eyes were graded according to the Amsler-Krumeich classification(6), based on patient’s refraction, mean central K-reading, corneal signs, and corneal thickness. All treated eyes were graded stage II or III. Using a randomized table, patients were distributed into two study groups according to the treatment: CXL group (19 eyes underwent CXL procedure using riboflavin and UVA light) and riboflavin eyedrops (RE) group (20 eyes received riboflavin 0.1% (wt/vol) eyedrops [10 mg riboflavin-5-phosphate in 20% (wt/vol) dextran-T-500; Ophthalmos, São Paulo, Brazil] 4 times per day for 1 month.

In eight patients, both eyes were included. In these eight cases, the right eye received only RE, and the left eye underwent the CXL procedure. After 3 months of this first step (CXL or RE), all patients then underwent insertion of ICRS (Keraring, Mediphacos, Belo Horizonte, Brazil), which comprised the second step.

treatment and at 1 month and 3 months after CXL or RE, and again at 6 months, 1 year, and 2 years after ICRS insertion.

IC Technique

After the slit-lamp examination, all patients were subjected to IC sampling by the same researcher (A.C.R.). Following the application of topical anesthesia, IC specimens were collected (Millipore HAWP304; Bedford, MA) from the four quadrants of the cornea, from an exposed area of the bulbar conjunctiva (temporal region), and from an unexposed area of the conjunctiva (superior region) adjacent to the corneal limbus. All strips were processed for periodic acid-Schiff and Gill hematoxylin staining. Glass slides mounted with Entellan (Merck, Darmstadt, Germany) were analyzed under light microscopy by an experienced professional (J. N. B.), operating in a masked fashion. For quality control, only IC specimens with at least one-third of the filter surface covered with visible epithelial cells were included.

Conjunctival samples were evaluated according to established techniquesfor the following parameters(7-8-9): cellularity, cell-to-cell contact of epithelial cells, nucleus-to-cytoplasm (N:C) ratio, nuclear chromatin level, goblet cell density, keratinization, and distribution of inflammatory cells. A score of 0 to 3 was assigned to each of these features. Zero represented normal findings. One represented borderline features, and 2 or 3 represented abnormal features. The scores obtained for each parameter were added together to obtain a total score and classified as A or normal (total score 0-3), B or borderline (total score 4-6), and C or abnormal (total score >6). Goblet cell densities were judged as normal when abundant, borderline when slightly or moderately reduced, and abnormal when distinctly reduced (single to no goblet cells). Corneal samples were evaluated and graded according to the squamous metaplasia classification proposed by Murube and Rivas(10).

Cross-Linking Procedure

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the surgeon confirmed the presence of riboflavin in the anterior chamber before UVA irradiation commenced. The cornea was exposed (using a lid speculum) to UV light (UV-X System; Peschke Meditrade GmbH, Hunenberg, Switzerland), which emits light at a wavelength of 370 ± 5 nm at an irradiance of 3 mW/cm2 or 5.4 J/cm2 for 30 minutes (medium spot). During this time, riboflavin solution was applied every 5 minutes. The limbus and conjunctiva were not protected during irradiation. Topical anesthetic was applied as needed during the surgery.

After the treatment, a soft bandage contact lens was applied until reepithelialization was complete. A combination of moxifloxacin 0.5% (wt/vol) and dexamethasone phosphate 0.1% (wt/vol) eyedrops (Alcon Laboratories, Fort Worth, TX) were prescribed 4 times per day for 2 weeks.

Intrastromal Corneal Ring Segment Implantation

Intrastromal corneal ring segment implantation was performed by one of the authors (M.C.) in an operating room under sterile conditions using topical anesthetic drops. The Purkinje reflex was chosen as the central point and marked under a biomicroscope. A 5.0-mm marker was used to locate the exact ring channel. Tunnel depth was set at 80% of the thinnest corneal thickness on the tunnel location.

The arc length and thickness were chosen according to the manufacturer’s nomogram. A 60-kHz femtosecond laser (IntraLase Corp, Irvine, CA) was used to create the ring channels. The channel’s inner diameter was set to 5.0 mm, and the outer diameter was set to 5.9 mm. The entry-cut thickness was 1 µm, and the ring energy for channel creation was 1.70 µJ. The entry-cut energy was 1.10 µJ, and channel creation timing with the femtosecond laser was 15 seconds. The ICRS was implanted immediately after channel creation and before the disappearance of the bubbles, which revealed the exact tunnel location.

Postoperatively, a combination of moxifloxacin 0.5% (wt/vol) and dexamethasone phosphate 0.1% (wt/vol) eyedrops (Alcon Laboratories) were prescribed 4 times per day for 2 weeks. The patients were instructed to avoid rubbing their eyes and to use artificial tears frequently. On the first postoperative day, slit-lamp biomicroscopic examination was performed. Wound healing and segment migration were evaluated.

Statistical Analysis

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RESULTS

Of the 31 patients, 23 were women (74.19%) and 8 were men (25.80%). The mean ages of the patients were 28.30 ± 9.3 years (range, 17-55 years) in the CXL group and 30.40 ± 9.1 years (range, 22-55 years) in the RE group. Both groups were similar before treatment, as assessed by corneal keratometry (flattest-K1 and steepest-K2 meridian) and spherical equivalent measurements (Table 1).

DISCUSSION

In the present study, the ocular surface of two groups of patients who underwent ICRS insertion was evaluated by IC. The IC technique studies the most superficial layers of cells from the corneal and conjunctival surfaces(12). Morphological changes in the bulbar conjunctiva do not always occur in typical combinations. Therefore, a grading system (IC score) that included several morphological parameters (IC subscores) was devised to provide a complete analysis of conjunctival alteration. The parameters were separately judged and totaled to yield the final score, as described elsewhere(8-9).

Recently, an optical microscopic study(4) by our group demonstrated that despite changes in goblet cell density after corneal CXL in the superior conjunctiva and an improvement in the morphology of epithelial cells after the use of RE, the morphology grade of the most superficial epithelial cells obtained by IC from the cornea and bulbar conjunctiva did not change after CXL using riboflavin and UVA light in the treatment of keratoconus. This finding suggests that the CXL is a safe procedure at the 3-month follow-up. The ICRS had not been inserted at this time point.

Now, new results from a 2-year follow-up period showed improvements in the cell-to-cell contact of epithelial cells and the N:C ratio on the temporal conjunctiva in the CXL group. On the superior conjunctiva, an increase in the values of nuclear chromatin and goblet cells were observed at the 2-year follow-up. The conjunctival goblet cell count and the epithelial cell morphology are objective measures of the ocular surface status. The goblet cells are responsible for the production of gel-forming mucin, MUC5AC, whereas corneal and conjunctival epithelial cells synthesize transmembrane mucins, such as MUC1, 2, and 4. The gel-forming mucin constitutes the main component of the mucus layer (innermost component of the film)(13), and it stabilizes the tear film.

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using IC have shown a greater reduction in goblet cells after femtosecond laser treatment than after mechanical microkeratomy in LASIK procedures due to the length of time that the suction ring exerts pressure on the conjunctiva. The destruction of corneal tissue by LASIK procedures may also directly or indirectly affect the population of goblet cells. These findings were observed at the 3-month follow-up. After 6 months, the goblet cells recovered(14), which was similar to our experience. In our study, the femtosecond laser was used to create the channel ring at a depth of 80% of the thinnest corneal thickness on the tunnel location. Thus, the creation of the channel ring by the femtosecond laser did not affect the goblet cell density on the ocular surface in our group ofpatients.

In the present study, the IC results before and after treatment were normal (grade 0) in all corneal specimens. Although epithelial debridement before corneal CXL can cause necrosis and apoptosis(15) of keratocytes at a depth of 50 to 60 μm, any significant cytotoxic effect of CXL seems to be concentrated in the anterior part of the cornea because of the high absorption of UVA light by riboflavin. This behavior prevents the light from penetrating to deeper levels and preserves the endothelium and lens. Previous reports showed neither clinically evident side effects of CXL treatment nor persistent epithelial defects(16), with the exception of transient corneal edema and a foreign body sensation for 24-48 hours, in combination with burning and tearing that are common in photorefractive surgery. Mazzotta et al.(16) used confocal microscopy to demonstrate that the cytotoxic effects of CXL, although significant, were concentrated in the first 350 μm of the stroma and did not affect the adjacent structures.

Riboflavin alone produced no cell damage, which is not surprising because riboflavin is also present in the retina, liver, and heart as an essential element in normal nutrition(17).In a previous experimental study, riboflavin played a role in the development and maintenance of the surface structures of epithelial cells(18).

CONCLUSION

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TABLES

Table 1. Topographic Results (D) and Spherical Equivalent (D)

Group Measure N Mean SD Minimum Median Maximum P

CXL K1 19 48.1 4.21 41.41 47.50 55.87

0.197

RE K1 20 46.5 3.39 41.66 46.29 54.87

CXL K2 19 53.26 5.11 43.10 52.98 61.25

0.454

RE K2 20 52.17 3.89 45.06 51.09 63.92

CXL SE 19 -7.41 3.48 -14.0 -6.50 -1.75

0.090

RE SE 20 -5.45 3.53 -13.5 -4.50 0.75

Table 2. IC Results

Total Score A B C Corneal class N (%) N (%) N (%)

Total Score A B C Superior class N (%) N (%) N (%) P*

CXL= 0.251 RE = 0.042

Total Score A B C Temporal class N (%) N (%) N (%) P*

CXL= 0.359

RE = 0.018

Pretreatment Pretreatment Pretreatment

CXL RE 19(100) 20(100) - -CXL RE 17(94.4) 16(80) 1(5.6) 3(15) - 1(5) CXL RE 11(64.7) 12(60) 5(29.4) 6(30) 1(5.9) 2(10)

1 Month 1 Month 1 Month

CXL RE 19(100) 20(100) - -CXL RE 15(78.9) 18(94.7) 4(21.1) 1(5.3) - - CXL RE 14(73.7) 12(63.2) 5(26.3) 6(31.6) - 1(5.3)

3 Months 3 Months 3 Months

CXL RE 18(100) 20 (100) - -CXL RE 12(70.6) 17(94.4) 5(29.4) 1(5.6) - - CXL RE 14(87.5) 16(84.2) 1(6.3) 3(15.8) 1(6.3) -

6 Months 6 Months 6 Months

CXL RE 16(94.1) 19(100) 1(5.9) -CXL RE 16(94.1) 17(100) 1(5.9) - - - CXL RE 15(93.8) 15(88.2) 1(6.3) 2(11.8) - -

1 Year 1 Year 1 Year

CXL RE 18(100) 19(100) - -CXL RE 18(100) 19(100) - - - - CXL RE 15(88.2) 17(94.4) 2(11.8) 1(5.6) - -

2 Years 2 Years 2 Years

CXL RE 15(93.8) 19(100) 1(6.3) -CXL RE 17(100) 18(90) - 1(5) - 1(5) CXL RE 17(100) 19(95) - 1(5) - -

IC, impression cytologic; N, number of eyes; CXL, cross-linking group; RE, riboflavin eyedrops group; P*, Friedman test was used to compare IC results before treatment, 1, 3, and 6 months, and at 1 and 2 years.

Artigos

|

A r t i g o s | 51

Table 3. Fisher’s Exact Test to Compare Conjunctival Total Scores Between the Two Groups

P

Region Pretreatment 1 Month 3 Months 6 Months 1 Year 2 Years

Superior 0.174 0.147 0.069 0.500 - 0.285

A r t i g o s | 57

Figure Legends

Figure 1A: Cross-linking group: Cell-to-cell contact of epithelial cells- temporal conjunctiva.

Figure 1B: Cross-linking group: Nucleus-to-cytoplasm ratio-temporal conjunctiva.

Figure 2A: Cross-linking group: Goblet cell density-superior conjunctiva.

Figure 2B: Cross-linking group: Nuclear chromatin level-superior conjunctiva.

Figure 3: Riboflavin eyedrops group: Cell-to-cell contact of epithelial cells-superior conjunctiva.

Figure 4A: Riboflavin eyedrops group: Cell-to-cell contact of epithelial cells-temporal conjunctiva.

Figure 4B: Riboflavin eyedrops group: Nucleus-to-cytoplasm ratio-temporal conjunctiva.

Figure 4C: Riboflavin eyedrops group: Goblet cell density-temporal conjunctiva.

REFERENCES

1. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-A-induced collagen

crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135(5):620-27.

2. Tan BU, Purcell TL, Torres LF, Schanzlin DJ. New surgical approaches to the management of keratoconus and post-lasik ectasia.Trans Am Ophthalmol Soc.

2006;104:212-20.

3. Miranda D, Sartori M, Francesconi C, Allemann N, Ferrara P, Campos M. Ferrara intrastromal corneal ring segments for severe keratoconus. J Refract Surg.

2003;19(6):645-53.

4. Renesto AC, Barros JN, Campos M. Impression cytologic analysis after corneal collagen cross-linking using riboflavin and ultraviolet-A light in the treatment of keratoconus. Cornea. 2010;29(10):1139-44.

5. Torquetti L, Berbel RF, Ferrara P. Long-term follow-up of intrastromal corneal ring segments in keratoconus. J Cataract Refract Surg. 2009;35(10):1768-73.

6. Krumeich JH, Daniel J, Knülle A. Live-epikeratophakia for keratoconus. J Cataract Refract Surg. 1998;24(4):456-63.

7. Pinheiro-Jr MN, Santos PM, Santos RCR, Barros JN, Passos LF, Neto JC. Oral flaxseed oil (Linum usitatissimum) in the treatment for dry-eye Sjögren’s syndrome patients. Arq Bras Oftalmol. 2007;70(4):649-55.

8. Fenga C, Aragona P, Cacciola A, Ferreri F, Spatari G, Stilo A, et al. Ocular

discomfort and conjunctival alterations in operating room workers. A single-institution pilot study. Int Arch OccupEnviron Health. 2001;74(2):123-28.

9. Almeida SFF, Sousa LB, Vieira LA, Chiamollera MI, Barros JN. Clinic-cytologic study of conjunctivochalasis and its relation to thyroid autoimmune diseases. Cornea.

A r t i g o s | 59

10. Murube J, Rivas L. Impression cytology on conjunctiva and cornea in dry eye patients establishes a correlation between squamous metaplasia and dry eye clinical severity. Eur J Ophthalmol . 2003;13(2):115-27.

11. Spoerl E, Mrochen M, Sliney D, Trokel S, Seiler T. Safety of UVA-riboflavin cross-linking of the cornea. Cornea. 2007;26(4):385-89.

12. Gadkari SS, Adrianwala SD, Prayag AS, Khilnani P, Mehta NJ, Shaha NA. Conjunctival impression cytology – a study of normal conjunctiva. J Postgrad Med.

1992;38(1):21-3.

13. Watanabe H. Significance of mucin on the ocular surface. Cornea. 2002;21(1):S17-S22.

14. Rodriguez AE, Rodriguez-Prats JL, Hamdi IM, Galal A, Awadalla M, Alio JL. Comparison of goblet cell density after femtosecond laser and mechanical microkeratome in LASIK. Invest Ophthalmol Vis Sci. 2007;48(6):2570-75.

15. Wollensak G, Spoerl E, Wilsch M, Seiler T. Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment. Cornea. 2003;23(1):43-9.

16. Mazzotta C, Balestrazzi A, Traversi C, Baiocchi S, Caporossi T, Tommasi C, et al. Treatment of progressive keratoconus by riboflavin-UVA-induced cross-linking of corneal collagen. Cornea. 2007;26(4):390-97.

17. Wollensak G, Spoerl E, Reber F, Seiler T. Keratocyte cytotoxicity of riboflavin/UVA-treatment in vitro. Eye. 2004;18(7):718-22.

2.5 Conclusões

1. No tratamento do ceratocone, o cross-linking do colágeno corneano antes da inserção de segmentos de anéis corneanos intraestromais não altera o efeito destes em relação à refração, topografia, sensibilidade ao contraste, microscopia especular, paquimetria, tonometria e biomecânica da córnea.

Anexo 1 – Carta de aprovação do Comitê de Ética em Pesquisa da Universidade Federal de São Paulo/Hospital São Paulo

A n e x o s | 63

Anexo 2 – Termo de consentimento livre e esclarecido aprovado pelo Comitê de Ética em Pesquisa da Universidade Federal de São Paulo/Hospital São Paulo

“Avaliação do uso da riboflavina tópica exposta à irradiação ultravioleta A e

implante de anel intraestromal para tratamento de ceratocone e outros

astigmatismos irregulares”

de promover o enrijecimento da mesma, permitindo melhora da acuidade visual corrigida com óculos e /ou com lente de contato.

Explicação do procedimento:

O anel intracorneal é uma técnica cirúrgica liberada pelo Conselho Federal de Medicina para pacientes portadores de ceratocone grau 3 e 4. Consiste na implantação de uma prótese de acrílico na intimidade do tecido da córnea, de modo a alterar a sua curvatura na quantidade necessária para correção que se deseja obter. A cirurgia é realizada sob anestesia local por meio de gotas de colírio anestésico. O tempo de cirurgia é de aproximadamente 15 minutos e após seu término não há necessidade de se ocluir o olho. É uma técnica reversível, que pode ser ajustada em caso de correção inadequada, pela troca da prótese. O anel pode ser removido em qualquer época sem prejuízo para a saúde da córnea. A riboflavina é uma vitamina largamente utilizada na prática médica há muitos anos. O efeito combinado da riboflavina com a irradiação ultravioleta A (UVA) parece enrijecer a córnea, através da produção de ligações entre as camadas da córnea.

Após o implante do anel, serão instiladas gotas de riboflavina durante trinta minutos, com intervalo de cinco minutos. Nos trinta minutos subseqüentes a córnea receberá uma quantidade pré-determinada de irradiação UVA. A porção nobre da córnea será protegida por uma lente de contato. Todo procedimento é indolor.

visão é lenta e poderá demorar até três meses para estabilizar. Entretanto, já no dia seguinte, o paciente poderá reassumir as suas atividades habituais.

O resultado desta cirurgia não é garantido. Em alguns casos podem ocorrer as seguintes complicações: infecção, cicatrizes e opacidades da córnea, dificuldade na técnica cirúrgica, obrigando o cirurgião a interromper o procedimento e troca da prótese em caso de hipo ou hipercorreção.

Este procedimento conjunto tem natureza investigacional e há necessidade de retornos periódicos ao ambulatório por pelo menos dois anos para avaliação de seu resultado. Em caso de quaisquer dúvidas sobre o tratamento a ser realizado, estas serão imediatamente esclarecidas, e o paciente terá a liberdade de retirar o seu consentimento de participar deste estudo sem prejuízo de continuidade do seu tratamento nesta Instituição. Se existirem gastos adicionais estes serão absorvidos pelo orçamento deste estudo.

A principal investigadora é a Dra. Adimara da Candelaria Renesto, que pode ser encontrada à Rua Borges Lagoa 368, Setor de Cirurgia Refrativa, telefone: 50844864. Se você tiver alguma consideração ou dúvida sobre a ética da pesquisa, entre em contato com o Comitê de Ética em Pesquisa (CEP), localizado à Rua Botucatu 572-1°andar-cj 14, 5571-1062, FAX:5539-7162.

E-mail:cepunifesp@epm.br

As informações obtidas serão analisadas em conjunto com outros pacientes, não sendo divulgada a identificação de nenhum paciente. O paciente tem o direito de ser mantido atualizado sobre os resultados parciais das pesquisas, estando compromissado o pesquisador de utilizar os dados e o material coletado somente para esta pesquisa.

O paciente autoriza que a cirurgia seja filmada, fotografada e que os dados obtidos de seu exame possam ser utilizados para finalidades científicas ou de ensino.

Acredito ter sido suficientemente informado a respeito das informações que li ou que foram lidas para mim, sobre o estudo “Avaliação do uso da riboflavina tópica exposta à irradiação UVA e implante de anel intra-estromal para tratamento de ceratocone e outros astigmatismos irregulares”.

A n e x o s | 65

confidencialidade e de esclarecimentos permanentes. Ficou claro também que minha participação é isenta de despesas e que tenho garantia do acesso a tratamento hospitalar quando necessário. Concordo voluntariamente em participar deste estudo e poderei retirar o meu consentimento a qualquer momento, antes ou durante o mesmo, sem penalidades ou prejuízo ou perda de qualquer benefício que eu possa ter adquirido, ou no meu atendimento neste Serviço.

___________________________________________

Assinatura do paciente/representante legal Data ____/____/____

___________________________________________ Assinatura da testemunha Data ____/____/_____

Para casos de pacientes menores de 18 anos, semi-analfabetos ou portadores de deficiência auditiva ou visual.

Declaro que obtive de forma apropriada e voluntária o Consentimento Livre e Esclarecido deste paciente ou representante legal para a participação neste estudo.

__________________________________________

Anexo 3 – Quadro de exames nos momentos de avaliação

Após inserção de SACI

Exames Preop 1M 3M 1M SACI 3M SACI 6M SACI 12M SACI 24M SACI

Acuidade visual sem

correção X X X X X X X X

Acuidade visual com

correção X X X X X X X X

Refração dinâmica* X X X X X X X X

Biomicroscopia X X X X X X X X

Sensibilidade ao

contraste X X X X X X X X

Topografia

EyeSys-2000® X X X X X X X X

Orbscan IIz® X X X X X X X X

Pentacam® X X X X X X X

Tonometria de

aplanação X X X X X X X X

Tonometria de contorno X X X X X X X X

Paquimetria ultrassônica X X X X X X X X

Biomecânica corneana

(ORA)® X X X X X X X

Visante OCT® X X X X X X

Microscopia especular X X X X X X

Citologia de impressão X X X X X X

Mapeamento de retina X X X

A n e x o s | 67

Anexo 4 – Descrição cirúrgica intraoperatória

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Abstract

keratinization [P≥0.001]) revealed statistical difference between groups. Conclusions:

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