UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE MEDICINA Departamento de Oftalmología y Otorrinolaringología TESIS DOCTORAL Estudio de la coroides de la población sana mediante tomografía de coherencia óptica swept-source Study of the choroid of healthy population using swept-source optical coherence tomography MEMORIA PARA OPTAR AL GRADO DE DOCTOR PRESENTADA POR Jorge Ruiz Medrano Directores José María Ruiz Moreno Julián García Feijóo Francine Behar-Cohen Madrid, 2018 © Jorge Ruiz Medrano, 2017 UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE MEDICINA PROGRAMA DE DOCTORADO EN CIENCIAS DE LA VISIÓN DEPARTAMENTO DE OFTALMOLOGÍA Y OTORRINOLARINGOLOGÍA ESTUDIO DE LA COROIDES DE LA POBLACIÓN SANA MEDIANTE TOMOGRAFÍA DE COHERENCIA ÓPTICA SWEPT-SOURCE STUDY OF THE CHOROID OF HEALTHY POPULATION USING SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY JORGE RUIZ MEDRANO MADRID, 2017 2 3 UNIVERSIDAD COMPLUTENSE DE MADRID FACULTAD DE MEDICINA PROGRAMA DE DOCTORADO EN CIENCIAS DE LA VISIÓN DEPARTAMENTO DE OFTALMOLOGÍA Y OTORRINOLARINGOLOGÍA ESTUDIO DE LA COROIDES DE LA POBLACIÓN SANA MEDIANTE TOMOGRAFÍA DE COHERENCIA ÓPTICA SWEPT-SOURCE STUDY OF THE CHOROID OF HEALTHY POPULATION USING SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY JORGE RUIZ MEDRANO DIRECTORES: JOSÉ MARÍA RUIZ MORENO JULIÁN GARCÍA FEIJÓO FRANCINE BEHAR-COHEN MADRID, 2017 4 5 6 7 8 9 10 11 12 13 COPYRIGHT El copyright de las publicaciones empleadas para el desarrollo de este proyecto de Tesis Doctoral y que se enumeran a continuación pertenece a ARVO Journals (1, 2), American Journal of Ophthalmology-Elsevier (3) y a RETINA-Wolters Kluwer Health, Inc (4-6), respectivamente. 1. Ruiz-Moreno JM, Flores-Moreno I, Lugo F, Ruiz-Medrano J, Montero JA, Akiba M. Macular choroidal thickness in normal pediatric population measured by swept-source optical coherence tomography. Invest Ophthalmol Vis Sci. 2013;54:353-9. doi: 10.1167/iovs.12-10863. 2. Ruiz-Medrano J, Flores-Moreno I, Peña-García P, Montero JA, Duker JS, Ruiz- Moreno JM. Macular choroidal thickness profile in a healthy population measured by Swept-Source Optical Coherence Tomography. Invest Ophthalmol Vis Sci. 2014;55:3532-42. doi: 10.1167/iovs.14-13868. 3. Ruiz-Medrano J, Flores-Moreno I, Montero JA, Duker JS, Ruiz-Moreno JM. Morphological features of the choroidoscleral interface in a healthy population using Swept-source Optical Coherence Tomography. Am J Ophthalmol 2015;160:596-601. doi: 10.1016/j.ajo.2015.05.027. 4. Ruiz-Medrano J, Flores-Moreno I, Peña-García P, Montero JA, Duker JS, Ruiz- Moreno JM. Asymmetry in macular choroidal thickness profile between both eyes in a healthy populations measured by Swept-Source optical coherence tomography. Retina 2015;35:2067-73. doi: 10.1097/IAE.0000000000000590. 5. Ruiz-Medrano J, Flores-Moreno I, Peña-García P, Montero JA, García-Feijóo J, Duker JS, Ruiz-Moreno JM Analysis of age-related Choroidal Layers thinning in Healthy Eyes Using Swept-Source Optical Coherence Tomography. Retina 2016 (epub ahead of print). 6. Ruiz-Medrano J, Ruiz-Moreno JM, Goud A, Vupparaboina KK, Jana S, Chhablani J. Age-related changes in choroidal vascular density of healthy subjects based on image binarization of Swept-Source Optical Coherence Tomography. Retina 2017 (accepted). 14 A mi familia, 15 AGRADECIMIENTOS Me gustaría aprovechar estas líneas para poder dar las gracias a todas las personas que han colaborado en este trabajo y me han ayudado de una manera directa o indirecta a poder hacerlo posible. Mi decisión de realizar un proyecto de tesis doctoral viene de muy lejos, motivada por el hecho de haber crecido con dos doctores en medicina en casa, que me han servido siempre de ejemplo y motivación para tratar de ser mejor en todos los aspectos. Como me han dicho siempre: “las cosas, si se hacen, se hacen bien”. Este trabajo comenzó justo antes de empezar la residencia, hace ya cinco años, cuando tuve la oportunidad de colaborar en el primer trabajo de esta línea de investigación y me enseñaron a medir la primera coroides. Gracias de corazón al Dr. Ignacio Flores Moreno por la ayuda durante esas largas tardes de mediciones, por todas las vitrectomías, las ganas que tiene de enseñar y los ratos fuera del trabajo. Gracias a Pablo Peña García por todo el trabajo estadístico que ha llevado a cabo durante estos años y por las largas explicaciones telefónicas para tratar de hacerme ver todo lo que se podía sacar de las tablas y gráficas que nos proporcionó. A mis compañeros de residencia, muchos de los cuales han llegado a ser para mí no sólo compañeros de trabajo, sino grandes amigos. A mis compañeros de promoción, en especial a los Dres. Jorge Peraza y Pilar Cifuentes, porque sinceramente creo que hemos hecho un gran equipo y de verdad espero que volvamos a tener la suerte de trabajar juntos en el futuro. 16 A mis amigos los Dres. José Fernández-Vigo y Gabriel Arcos, por todas esas horas de guardias que pasamos juntos, y que gracias a ellos no fueron tan largas; los congresos, los partidos de fútbol, el Tour de Francia y los largos en la piscina. Al Dr. Jay S Duker por sus valiosas revisiones y comentarios que tanto han ayudado a la hora de conseguir estas publicaciones. Considero fundamental dar las gracias a mis directores de tesis: Gracias al Profesor Julián García Feijóo, mi Jefe de Servicio durante la residencia, por toda la ayuda durante estos años y las facilidades a la hora de ampliar fronteras con la formación, que me han permitido ver cómo se trabaja en Milán, Barcelona, Albacete, Manchester y Lausana. A la Profesora Francine Behar-Cohen por darme la gran oportunidad de la que ahora estoy disfrutando, con este puesto de Fellow de Retina y Oncología en el Hospital Oftálmico Jules Gonin de Lausana y por sus ganas de transmitir a todo el mundo su pasión por la investigación. Al Dr. Luis Arias Barquet y a su equipo, por los dos meses increíbles que pasé en la sección de retina del Hospital Universitario de Bellvitge, por la confianza que mostró en mí todos los miércoles en quirófano permitiéndome operar sin importarle la dificultad del caso, por su calma y por todo lo que me enseñó en tan poco tiempo. A mis hermanos, Guillermo y Magali, porque a pesar de que no puede haber tres hermanos tan diferentes, tampoco los puede haber mejores. Un agradecimiento muy especial para Rosa, por estos cinco años fantásticos y los que nos quedan por delante, por todos los ratos robados para llevar a cabo este trabajo y por estar SIEMPRE ahí para mí. Gracias. 17 A mi madre. Por ser la persona más fuerte que conozco y enseñarme cosas mucho más importantes que las que te puedan enseñar en una Facultad de Medicina. Gracias. Y principalmente a mi padre, el Profesor José María Ruiz Moreno, director de este proyecto de tesis, sin el cual esto no habría sido posible. Gracias por haberme transmitido el amor por esta profesión desde niño. Por su esfuerzo constante, por su dedicación, por su entrega, su increíble capacidad de trabajo, su apoyo constante y su ayuda inestimable. Por su comprensión y sobre todo por su ejemplo. MUCHAS GRACIAS. 18 LISTADO DE ABREVIATURAS AFG: Angiografía fluoresceínica AVI: Angiografía con verde de indocianina CSC: Coriorretinopatía serosa central CT: Choroidal thickness D: Dioptrías / Diopters DMAE: Degeneración macular asociada a la edad EDI-OCT: Enhanced depth imaging optical coherence tomography EE: Equivalente esférico EPR: Epitelio pigmentario de la retina ICGA: Indocyanine Green Angiography MCT: Macular choroidal thickness MM: Miopía magna/miope magno NVC: Neovascularización coroidea OA-OCT: Óptica adaptativa OCT OCT: Tomografía de coherencia óptica OCTa: Angiografía OCT PIO: Presión intraocular RPE: Retinal pigment epithelium SD-OCT: Tomografía de coherencia óptica de dominio espectral SE: Spherical equivalent SS-OCT: Tomografía de coherencia óptica Swept-Source TD-OCT: Tomografía de coherencia óptica de dominio tiempo 19 US: Ultrasonidos VEGF: Factor de crecimiento vascular endotelial 20 ÍNDICE: 1. Introducción …………………………………………………………………………………………………………..23 1.1. Definición de coroides ………………………………………………………………………………..24 1.2. Embriología e histología ……………………………………………………………………………..25 1.3. Funciones ……………………………………………………………………………………………………29 1.4. Métodos de estudio ……………………………………………………………………………………31 1.4.1. Ultrasonografía ……………………………………………………………………………….31 1.4.2. Angiografía ……………………………………………………………………………………..32 1.4.3. Tomografía de coherencia óptica ……………………………………………………34 1.4.4. OCT “en face” ………………………………………………………………………………….38 1.4.5. Angiografía OCT ………………………………………………………………………………39 2. Justificación ……………………………………………………………………………………………………………41 3. Hipótesis y objetivos ………………………………………………………………………………………………44 4. Publicaciones …………………………………………………………………………………………………………47 4.1. Macular choroidal thickness in normal pediatric population measured by Swept-Source optical coherence tomography. Invest Ophthalmol Vis Sci 2013;54:353-359 ……………………………………………………………………………………………………48 4.2. Macular choroidal thickness profile in a healthy population measured by swept-source optical coherence tomography. Invest Ophthalmol Vis Sci 2014;55:3532-42 ……………………………………………………………………………………………………56 4.3. Morphologic features of the choroidoscleral interface in a healthy population using swept-source optical coherence tomography. Am J Ophthalmol 2015;160:596- 601 …………………………………………………………………………………………………………………………67 21 4.4. Asymmetry in macular choroid thickness profile between both eyes in a healthy population measured by Swept-Source optical coherence tomography. Retina 2015;35:2067–2073 …………………………………………………………………………………….75 4.5. Analysis of age-related choroidal layers thinning in healthy eyes using Swept- Source optical coherence tomography. Retina 2017 (epub ahead of print) ……………83 4.6. Age-related changes in choroidal vascular density of healthy subjects based on image binarization of Swept-Source Optical Coherence Tomography. Retina 2017 (accepted) ………………………………………………………………………………………………………………93 5. Discusión / Discussion …………………………………………………………………………………..111/131 6. Conclusiones por objetivos / Conclusions ……………………………………………………..147/150 7. Futuro ………………………………………………………………………………………………………………….152 7.1. Estudio de la coriocapilar mediante OCTa ……………………………………………….153 7.2. Óptica adaptativa OCT …………………………………………………………………………….154 7.3. OCT de campo amplio ……………………………………………………………………………..155 7.4. Angiografía con verde de indocianina de campo ultra-amplio …………………155 8. Resumen / Summary …………………………………………………………………………………….157/163 9. Bibliografía …………………………………………………………………………………………………………..168 22 23 1. INTRODUCCIÓN 24 1. INTRODUCCIÓN 1.1 DEFINICIÓN La coroides es un tejido pigmentado y densamente vascularizado que forma parte de la úvea, la capa media del ojo y que se sitúa entre la esclerótica y la retina. Alcanza desde el nervio óptico hasta la ora serrata, continuándose en su parte más anterior con el cuerpo ciliar y la raíz del iris. Entre sus funciones encontramos el aporte de oxígeno y nutrientes a la retina externa, así como la absorción de luz gracias a su alta densidad de pigmento, imprescindible para la visión. Mide 0,22 mm en el polo posterior y entre 0,1 y 0,15 mm en la zona más anterior en estudios anatomopatológicos post-mortem,1 aunque alcanzada la vejez puede reducirse hasta entorno a las 80 μm.2 Está compuesta de vasos derivados de la anastomosis de las arterias ciliares posteriores, que proceden a su vez de la arteria oftálmica. Éstas atraviesan la esclerótica posteriormente en un número que se sitúa entorno a 15-20 y a una distancia de 5-6 mm del nervio óptico. Las arterias se ramifican en arteriolas que terminan en los lóbulos coriocapilares. Éstos drenan su contenido hacia las vénulas, que convergen y salen del ojo a nivel del ecuador en forma de 4-5 venas vorticosas.3 Ocasionalmente es posible encontrar una vena vorticosa que denominamos ciliovaginal, que está localizada en el polo posterior y que sale del globo ocular cerca del nervio óptico o a través del mismo. Las venas vorticosas atraviesan la esclerótica en un trayecto oblicuo para acabar uniendo a las venas oftálmicas superior o inferior. 25 La coroides es un tejido derivado de la cresta neural y el mesénquima periocular. La coriocapilar empieza su desarrollo entorno a la 4ª-5ª semana de gestación. Su endotelio se reduce a un espesor de una sola célula y da lugar a sus típicas fenestraciones alrededor de la 9ª semana. Una vez alcanzado el final del primer trimestre la coriocapilar es prácticamente idéntica a la forma adulta. El resto de capas comienzan su desarrollo en la semana 15, momento en que arteriolas y vénulas empiezan a formarse, siento identificables a las 22 semanas.4 1.2 EMBRIOLOGÍA E HISTOLOGÍA La vesícula óptica se forma a partir de una protrusión del neuroectodermo y se invagina sobre sí misma para formar una copa óptica de doble pared. La más externa dará lugar al EPR mientras que la interna está destinada a convertirse en el resto de capas de la retina. La parte más inferior muestra dos bordes donde se une el tejido que es la fisura coroidea y es por ahí por donde la arteria hialoidea penetra en el ojo. La úvea se forma a partir del mesodermo y del tejido neuro-ectodérmico que rodean a la copa óptica. Las células mesodérmicas que la rodean se diferencian en vasos al mismo tiempo que se desarrolla el EPR. La capa coriocapilar comienza a formarse entorno a la 5ª o 6ª semana de desarrollo embrionario y es entorno a la semana 6 que la membrana de Bruch empieza a ser reconocible.4,5 La coriocapilar se organiza con anterioridad al desarrollo del resto de la coroides. Las arterias ciliares posteriores penetran en el tejido coroideo entorno a la 8ª semana de gestación y no maduran hasta la semana 22. 26 Los precursores de los melanocitos migran hacia la úvea en formación desde la cresta neural a finales de la 4ª semana y empiezan el proceso de diferenciación hacia el séptimo mes. La pigmentación de la coroides empieza de atrás hacia delante, desde el nervio óptico hasta la ora serrata y se completa entorno al mes 9.6 Histológicamente la coroides puede dividirse en 5 capas diferentes: La membrana de Bruch; la coriocapilar; la capa de medianos vasos o capa de Sattler; la capa de grandes vasos o capa de Haller; y el espacio supracoroideo.7 La membrana de Bruch es una matriz extracelular localizada en el límite entre la retina y la coroides. Se extiende anteriormente hasta la ora serrata y se continúa por un tejido que se le asemeja hasta el cuerpo ciliar. Cumple funciones importantes como sustrato del EPR por un lado y de las paredes vasculares de la coriocapilar por otro. Está compuesta en su mayoría de fibras elásticas y fibras de colágeno y tiene un espesor de entre 2 y 4 μm y puede ser dividida a su vez en 5 capas: La membrana basal del EPR, capa colágena interna, capa de tejido elástico, capa colágena externa y membrana basal del endotelio coriocapilar. La membrana basal del EPR es un entramado de fibras de colágeno tipo IV, semejante al resto de membranas basales del cuerpo. Contiene el mismo tipo de colágeno IV que las membranas basales del glomérulo renal, con funciones de filtro y transporte.8 La capa colágena interna contiene fibras de colágeno de tipo I, III y V que se cruzan en un plano paralelo a la membrana de Bruch y que se relacionan con condroitin sulfato y dermatan sulfato mayoritariamente.8 La capa de tejido elástico está compuesta por colágeno tipo VI, fibronectina y una serie de capas de 0,8 μm apiladas de fibras elásticas entrecruzadas, que dejan espacios de aproximadamente 27 1 μm.9 Tiene un papel mecánico y como función de barrera antiangiogénica. La capa colágena externa, cuyo grosor oscila entre 1 y 5 μm, muestra una composición similar a la capa colágena interna, pero a diferencia de ésta, presenta una serie de protrusiones en los espacios intervasculares de la coriocapilar llamadas columnas intercapilares.2 Por último la membrana basal del endotelio de la coriocapilar es una membrana de 0,07 μm cuya función teórica sería inhibir la migración celular endotelial hacia la membrana de Bruch al igual que hacen las láminas basales asociadas a los capilares retinianos. La coriocapilar es la más interna de las capas vasculares de la coroides. Está compuesta por un entramado de capilares fenestrados que cumplen la importante función del aporte de nutrientes y oxígeno para el EPR, la capa de fotorreceptores, la capa plexiforme externa y la parte más externa de la capa nuclear interna. La fenestración de esta red vascular es la que permite la libre circulación e intercambio entre coroides y retina, mayoritariamente moléculas pequeñas y solutos que son capaces de atravesar los poros de entorno a 60-70 nm que se sitúan en sus paredes. Asimismo, la mayoría de las fenestraciones así como los tres tipos de receptores del factor de crecimiento vascular endotelial (VEGF) se sitúan de cara a la retina principalmente, mientras que la densidad de pericitos es mayor del lado más escleral.10 Según algunos autores, esta red es más densa en la zona situada bajo la fóvea, alcanzando un espesor teórico de unas 10 μm, en comparación con las 7 μm de zonas más periféricas.11 La capa de medianos vasos o capa de Sattler está formada por las pequeñas arteriolas que suministran a la coriocapilar, formando una red capilar de distribución 28 lobular semejante a los glomérulos renales. Estos lóbulos varían en forma, tamaño y densidad vascular dependiendo de dónde se sitúen y de la localización relativa de las arteriolas nutricias y de las vénulas de drenaje. La capa de Haller, más externa, está formada por vasos de un calibre mayor y no guarda un patrón claro. El estroma en que están embebidos estos vasos está formado en su mayoría por fibroblastos, células musculares lisas, mastocitos, linfocitos, macrófagos, melanocitos y células nerviosas. Las arterias y las venas coroideas no siguen un curso estrictamente paralelo de forma que una sección que recibe aporte sanguíneo de una arteria no tiene por qué corresponder exactamente con la sección que drena a una vena. Siempre hay cierta superposición entre segmentos arteriales adyacentes.5 El espacio supracoroideo constituye la capa más externa de la coroides, siendo una capa de transición entre ésta y la esclerótica.7 Este espacio no es visible utilizando las técnicas de imagen habituales, que después veremos en mayor detalle, en el caso de pacientes normales, pero sí en el caso de determinadas patologías. Por el contrario, en el caso de las muestras histológicas aparece artefactada, viéndose mucho más grande tras el procesado de las muestras Los vasos que constituyen la coroides están rodeados por un plexo nervioso que recibe inervación de los sistemas simpático y parasimpático. Asimismo, se han hallado células ganglionares en la proximidad de los vasos coroideos de mayor calibre. La función de esta inervación coroidea no está clara hoy en día, pero dado que estas fibras muestran una tinción positiva para óxido nítrico sintasa y péptido intestinal vasoactivo, los hay que defienden la teoría de que cumplen una función reguladora del flujo sanguíneo.11,12 29 1.3 FUNCIONES La retina es uno de los tejidos con mayor actividad metabólica del cuerpo humano y recibe oxígeno de dos vías diferentes: la circulación retiniana y la coroides, siendo el aporte del mismo su función principal. Los dos tercios más internos de la retina dependen de la circulación retiniana, cuyo origen se encuentra en la arteria central de la retina. Es una red vascular con capacidad de autorregulación ya que es capaz de responder a las variaciones en los niveles de oxígeno y de esta forma mantener unos niveles relativamente constantes en el tejido retiniano. La retina externa y el epitelio pigmentario, por otra parte, dependen en exclusiva del aporte de oxígeno por parte de la circulación coroidea. Asimismo, la coroides peripapilar juega un papel importante en lo que se refiere al aporte sanguíneo de la parte más anterior del nervio óptico.13 La creencia generalizada es que a diferencia de la vascularización retiniana, los vasos de la coroides no poseen capacidad de autorregulación, de manera que los niveles de oxígeno de estas estructuras dependen directamente de los niveles de oxígeno sistémico.14 Los hay, sin embargo, que creen que la coroides podría tener cierta capacidad de autorregulación, basándose en la ya descrita presencia de células ganglionares y de mastocitos en proximidad a los grandes vasos coroideos.11,12 Hayreh explica en sus trabajos como el flujo coriocapilar está en función de diferentes gradientes de presión, lo que permite que las regiones que hayan sufrido un descenso de aporte sanguíneo por problemas en su arteria nutricia reciban lo necesario. La práctica totalidad de los tejidos corporales muestran algún tipo de autorregulación, pero como acabamos de explicar, el caso de la coroides es 30 controvertido. Mientras que algunos defienden que la coroides no puede autorregularse cuando la perfusión se reduce a causa de un aumento de presión intraocular (PIO),15 otros han demostrado que el flujo coroideo varía con la PIO,16 la producción endógena de óxido nítrico y la acción de las células ganglionares coroideas.12 Otra de las explicaciones propuestas para justificar que la autorregulación de la coroides no es completa es el hecho de que a pesar de que el flujo coroideo es mucho mayor que el de otros tejidos, la extracción de oxígeno se mantiene baja. La autorregulación debería ajustar el flujo sanguíneo a las necesidades del tejido concreto. En el caso que nos ocupa, la elevada presión parcial de oxígeno y la baja extracción del mismo implican que el flujo coroideo se mantiene a un nivel mucho mayor de lo que en teoría dictarían las necesidades de la coroides o el EPR. Además, se hace difícil pensar que pueda existir una comunicación o una autorregulación directa entre los elipsoides de los fotorreceptores, donde se encuentran las mitocondrias que consumen la gran mayoría del oxígeno empleado, y la capa coriocapilar, ya que existen enfermedades que afectan directamente a la retina externa y no se asocian con una reducción del espesor coroideo.17 Otra de las funciones clásicas atribuidas a la coroides es la de mejorar la función óptica. Al ser un tejido altamente pigmentado puede la absorción de la luz dispersada, mientras que protege indirectamente del estrés oxidativo. Hay autores que defienden que el hecho de vivir en un medio con una presión parcial de O2 elevada junto con la exposición permanente a la luz pueden ser factores de riesgo para la transformación maligna en melanomas. Hay autores que han propuesto otras 31 teorías para el alto flujo de sangre que recibe la coroides. Entre ellas se incluye una función de refrigeración del calor que se genera por el gran rendimiento metabólico de la retina externa, pero estas hipótesis están en duda, ya que la teórica vasodilatación que debería tener lugar en condiciones de aumento de temperatura no se ha demostrado en ojos normales.5 1.4 MÉTODOS DE ESTUDIO El estudio de la coroides se ha disparado en la pasada década gracias a varios descubrimientos en técnicas de imagen que han permitido una visualización y medición más rápidas y precisas. 1.4.1 ULTRASONOGRAFÍA En 1956 las técnicas ultrasónicas (US) fueron utilizadas por primera vez para el diagnóstico de enfermedades oculares.18 Algunos utilizaron las llamadas técnicas de modulación de intensidad (modo B) que requerían la inmersión de los ojos en agua, mientras se obtenía una tomografía acústica del ojo gracias a un movimiento de barrido de un cristal frente a la estructura a examinar. Otros usaban métodos de amplitud-tiempo (modo A), en los que el eje x de la pantalla conforma el eje tiempo y el eje y conforma el eje de amplitud del eco.19 A pesar de que la coroides normal no podía ser medida, y algunos autores afirmaron que lesiones de menos de 4,0 mm de altura no podían ser completamente evaluadas,20 los melanomas coroideos y desprendimientos de retina podían encontrarse en casos en los que penetraban en la cavidad vítrea en al menos entre 32 1,5 y 2,0 mm. La sangre sub-retiniana coagulada suponía un difícil diagnóstico diferencial en aquel momento. Las sondas ultrasónicas de contacto fueron introducidas en los años setenta, con evoluciones continuas y mejoras en la sensibilidad. A lo largo de las dos últimas décadas la revolución de la tecnología en formato digital ha traído cambios en las técnicas de exploración y almacenamiento de datos, junto con grandes mejoras en la calidad de imagen.21 El melanoma coroideo es el tumor intraocular maligno más común, y a pesar de la aparición de nuevas técnicas de imagen, la ecografía es todavía de gran utilidad. Antes de su aparición, los melanomas tan sólo se sospechaban cuando una masa visible podía ser apreciada a través de medios transparentes, e incluso en casos en los que una masa es visible, el diagnóstico puede que no sea tan evidente.22 Pero es en casos de medios opacos cuando el US es de mayor utilidad. Los patrones ultrasónicos de masas coroideas son aún cruciales para establecer un buen diagnóstico diferencial.22 1.4.2 ANGIOGRAFÍA La angiografía con fluoresceína (AFG) y la angiografía con verde indocianina (AVI) se han practicado durante décadas para obtener información clínica útil sobre la retina y la coroides.23,24 La AFG se desarrolló en los años 60 para el estudio de tumores coroideos,25,26 y se utilizó fundamentalmente para estudiar la vascularización retiniana, por lo que algunos de los primeros autores en utilizar este contraste estudiaron la circulación 33 coroidea a lo largo de las primeras fases del angiograma o a través de áreas de atrofia retiniana, que hacían más visibles y más fáciles de distinguir a los vasos coroideos.24,25 Mientras tanto, la AVI fue el primer colorante utilizado en la industria fotográfica y fue aplicado por primera vez con fines clínicos en 1972 cuando Flower intentó describir y captar en imagen la vascularización coroidea.23 La indocianina es una sustancia lipofílica e hidrofílica con una alta capacidad de unión a proteínas plasmáticas (hasta un 98%).27 Éstas muestran un mayor peso molecular que la albúmina, lo que otorga a la indocianina una menor permeabilidad vascular y penetración en tejidos. Esto la diferencia de la fluoresceína, y nos permite realizar un mejor estudio de la vascularización coroidea. Se metaboliza por el hígado y se excreta vía biliar. Se inyecta vía intravenosa utilizado concentraciones de 5 mg/ml y para capturar su circulación son necesarios filtros de excitación y de barrera con picos de 805 y 835 nm respectivamente. Las imágenes se toman normalmente desde 8-10 hasta 40 minutos después de la inyección del colorante.28 Estudios posteriores sugirieron que estadios más tempranos de la AVI podían ser útiles para localizar los vasos nutricios de los complejos de neovascularización coroidea (NVC) y así ayudar a guiar tratamientos focales de los mismos.29 Fue de hecho la degeneración macular asociada a la edad (DMAE) la que centró la gran mayoría de los estudios realizados usando AVI, pero también ha resultado de ayuda para los investigadores en la fisiología de ciertas alteraciones inflamatorias coriorretinianas, así como patologías del segmento anterior.27 34 La mejor forma de visualizar la vascularización coroidea es mediante la AVI, y no está limitado a casos en los que puedan encontrarse zonas de atrofia del EPR y la coriocapilar como en el caso de AFG.30 Teniendo en cuenta que la coroides es un tejido tridimensional y que las imágenes que se obtienen son reproducidas en dos dimensiones, es importante tener un apropiado conocimiento de la anatomía de la coroides para poder levar a cabo una interpretación precisa de la angiografía. Variaciones anatómicas normales del drenaje sanguíneo, entre otras cosas, muestran un patrón asimétrico en hasta el 50% de los pacientes, con preferencia por una de las venas vorticosas, que puede llevar a interpretaciones erróneas.31 La AVI resulta ser especialmente útil para la detección de NVC recurrente en casos difíciles, como desprendimientos del epitelio pigmentario (DEP) o áreas adyacentes a cicatrices de láser.32,33 Es más precisa que el AFG para la localización de NVC debajo de hemorragias subretinianas o sub-EPR debido a la mayor capacidad de penetración que otorga la luz infrarroja.34 La AVI se ha convertido en la herramienta más útil para la detección de pólipos maculares, extramaculares o peripapilares.11 También es utilizada para localizar los puntos de fuga en la coroides o los vasos coroideos dilatados en pacientes con coroidopatía serosa central (CSC).35,36 1.4.3 TOMOGRAFÍA DE COHERENCIA ÓPTICA Desde su desarrollo original hace más de veinte años, la tomografía de coherencia óptica (OCT) se ha convertido en una herramienta casi indispensable en el día a día del estudio del paciente con patología retiniana, ya que permite el estudio del 35 paciente de una manera rápida, “in vivo” y no invasiva. Esta tecnología comenzó su desarrollo en los años 80, siendo las primeras imágenes similares a las ecografías en modo A.37 En 1992, fueron desarrolladas las imágenes transversales en dos dimensiones, con los primeros escáneres de retina para humanos “in vivo” viendo la luz en 1993.38 La OCT se basa en el principio de la interferometría de baja coherencia. Se dirige un haz de luz de baja coherencia al tejido objetivo (habitualmente el área macular o el nervio óptico) y la luz que se refleja en el tejido de manera dispersa es combinada con un haz de luz de referencia produciendo un patrón de interferencia. Éste es utilizado para construir un A-scan axial que no es sino la representación de las propiedades dispersivas de los tejidos que han sido alcanzado por la luz. De la combinación de diferentes A-scans con distintos puntos de incidencia podemos generar un B-scan, que es la representación en dos dimensiones de un corte transversal de la zona estudiada.38 Los primeros modelos de OCT fueros denominados OCT de dominio tiempo (TD-OCT). Esta tecnología utilizaba un único detector y los A-scans eran generados a base de mover un espejo para modificar el haz de referencia y así estudiar el tejido en diferentes planos de profundidad. El principal inconveniente era la velocidad de exploración, que junto con la falta de resolución y de penetrancia en los tejidos hicieron que esta tecnología quedara rápidamente obsoleta.38,39 A continuación en el año 2006 apareció el OCT de dominio espectral o de dominio Fourier que utiliza múltiples detectores en lugar de múltiples rayos de referencia generado a partir de un espejo en movimiento para obtener los A-scans. 36 Su velocidad es aproximadamente de unos 30000 A-scans por segundo, llegando a alcanzar los 100000. La resolución axial de estos aparatos se sitúa entorno a las 5 μm y la resolución lateral, limitada por la difracción de la pupila es de unas 20 μm. La velocidad de el SD-OCT nos ha permitido también obtener imágenes de una mayor calidad al reducir el ruido en las mismas, que es el responsable del aspecto poco definido de algunos B-scans. Al adquirir, guardar y combinar múltiples tomas de un mismo B-scan situado en una misma posición conseguimos una imagen promedio, con mínimo ruido y una definición mucho más alta.39 Poco después fue desarrollado el software para el “eye-tracking” ocular, que nos ayudó a asegurarnos de que las imágenes se obtienen en la misma localización exacta en diferentes exploraciones. Sin embargo, a pesar de las obvias ventajas sobre el TD-OCT, las distorsiones y atenuaciones en la señal debidas a la presencia de tejidos pigmentados u opacidades de medios hacían aún difícil la obtención de imágenes de la coroides con buena calidad en la mayoría de los ojos. Más tarde, Spaide publicó los primeros artículos utilizando la nueva técnica “enhanced-depth imaging” OCT (EDI-OCT), que proporcionaba una visualización consistente de la coroides en la mayoría de los ojos y que permitió mediciones de espesor reproducibles y precisas.5,40,41 Una de las consecuencias de utilizar una transformación Fourier para descodificar la señal de interferometría es la aparición de dos imágenes conjugadas. Sin embargo, sólo una de ellas es utilizada o mostrada en la mayoría de aparatos utilizados en la práctica clínica habitual, habitualmente la que muestra la retina hacia la parte superior de la pantalla y la coroides debajo de ésta. Si modificamos el punto de foco para ubicarlo en la esclerótica interna, las 37 estructuras profundas como la coroides pueden verse con mucha mayor definición. De esta forma la imagen conjugada e invertida aparece en pantalla, mientras que la imagen con orientación clásica aparece en blanco debido a la falta de información. De esta forma ganamos unas 500-800 μm de penetrancia en los tejidos oculares, que en ciertos pacientes con coroides más delgadas como puedan ser los miopes magnos (MM) o los pacientes con DMAE, puede permitirnos visualizar la práctica totalidad de la esclerótica e incluso la grasa orbitaria. Pero con todas estas ventajas también surge un inconveniente, y es el hecho de que al obtener la máxima sensibilidad de imagen en la coroides, perdemos calidad en la visualización del vítreo. Las técnicas más recientes para imágenes de OCT consisten en una OCT con una longitud de onda más larga y alta penetración: el OCT Swept-Source (SS-OCT).42– 44 El SS-OCT utiliza un laser sintonizable que funciona a 100000 Hz con una longitud de onda entorno a 1 mm. Estos aparatos pueden realizar un promedio de imágenes de hasta 96 B-scans en la misma posición, y en caso de utilizar el modo de exploración de una línea sencilla genera imágenes que llegan a contener un promedio de hasta 1024 A-scans y una longitud de 12 mm.45 De acuerdo con algunas publicaciones, el espesor coroideo puede ser medido de forma fiable con este dispositivo hasta en el 100% de los pacientes.46,47 Los vasos de la retina tienen una apariencia hiper-reflectiva en las imágenes de OCT, al tiempo que los vasos de la coroides son hipo-reflectivos. Esta diferencia parece estar relacionada con la velocidad de las células sanguíneas que van a través de los vasos de cada tejido. La velocidad con la que fluye la sangre dentro de los 38 vasos de la coroides es mucho más alta que la velocidad con la que fluye en los vasos de la retina. Como ha sido explicado con anterioridad, las producción de imágenes de OCT está basada en la interferometría, donde un margen de interferencia es detectado para generar la intensidad de la señal. La alta velocidad de las células sanguíneas hace que el margen de interferencia se desvanezca, por lo que no se observa ninguna señal dentro de los vasos de la coroides. Por otro lado, la velocidad de las células sanguíneas es relativamente baja en el caso de estructuras vasculares retinianas, lo que contribuye a la señal de interferencia como una señal hipereflectiva.48 1.4.4 OCT “EN FACE” Las últimas mejoras en técnicas de OCT permiten la adquisición de múltiples B-scans generando imágenes de alta resolución. La OCT en modo “en face” apareció recientemente como una nueva técnica de imagen que proporciona una nueva forma de visualización del polo posterior. Mientras que los nuevos “C-scans” nos proporcionan un plano coronal del polo posterior, la tecnología en face general un aplanamiento artificial de los tejidos del polo posterior usando como referencia la capa seleccionada (habitualmente el EPR) y permitiéndonos navegar a través de distintas capas al mismo tiempo que se modifica la profundidad de la imagen a través de estas capturas en tres dimensiones.49 De esta manera, la OCT en face permite una nueva visión de la coriocapilar, la capa de Sattler y la de Haller sin necesidad de contraste de indocianina, llegando a diagnosticar incluso el vaso nutricio del algún complejo neovascular. 39 Los B-scans de OCT y imágenes en face muestran los vasos coroideos de forma hipo-reflectiva en comparación con lo que les rodea.50 Pero no todas las imágenes oscuras son en realidad vasos coroideos, ya que sombras de los vasos retinianos pueden provocar imágenes que induzcan a error. Se registran imágenes del fondo al mismo tiempo en la mayoría de los aparatos actuales, lo que fácilmente ayuda a distinguir cuál es cuál.51 Esta tecnología se ha utilizado en patologías como la CSC, en la que ha permitido demostrar la presencia de vasos engrosados a lo largo de todas las capas de la coroides.50 Otros estudios han permitido también la visualización de más del 90% de los pólipos diagnosticables mediante AVI o las ramificaciones vasculares de las MNV bajo la membrana de Bruch y bajo estos pópipos.52 También ha sido utilizada para confirmar hallazgos anteriores, como la atrofia coriocapilar que encontramos en los pacientes afectados de DMAE.53 1.4.5 ANGIOGRAFÍA OCT Mientras que la OCT en face ayuda a visualizar una vascularización coroidea normal, la angiografía OCT (OCTa), el último desarrollo en el campo de OCT, proporciona buenas imágenes de los vasos retinianos y complejos neovasculares sin necesidad de utilizar ningún contraste.54,55 Esta nueva herramienta identifica los vasos en base a variaciones entre múltiples B-scans consecutivos. El tejido retiniano es estático pero la sangre está en constante movimiento, y el procesamiento informático de estas imágenes ayuda a eliminar todo el tejido estático, mostrando una imagen del contenido de los vasos retinianos, dibujando de esta manera el perfil de los mismos, 40 con el inconveniente de que no permite apreciar la fuga o exudación vascular.54 Esto limita el uso de esta técnica de imagen en lo que se refiere a las alteraciones de permeabilidad vascular. De la misma manera, y como detecta el flujo sanguíneo, las MNV inactivas pueden ser casi idénticas a las activas, lo cual puede llevar a una malinterpretación. La imagen de OCTa se genera con la presentación de esta señal en una escala de blanco y negro. El escáner de alta densidad de áreas en dos dimensiones general un esquema volumétrico del flujo sanguíneo que nos permite visualizar de manera directa vasos retinianos y coroideos, tanto normales como alterados. De esta manera, esta tecnología tiene una aplicación importante para captura de complejos neovasculares que se hallen tanto por encima como por debajo del EPR. Jia56 mejoró esta tecnología introduciendo la angiografía Split-spectrum amplitude-decorrelation (SSADA). Esta herramienta permite la mejora del índice de señal/ruido para la detección de flujo mostrando la red microvascular y eliminando automáticamente errores de movimiento. Dado que la OCTa no precisa de la utilización de contrastes externos por vía intravenosa, esta técnica se puede realizar de manera repetida y sin el riesgo de sufrir efectos adversos. 41 2. JUSTIFICACIÓN 42 2. JUSTIFICACIÓN Como se ha comentado con anterioridad, la retina, y en particular la capa de fotorreceptores presentan una demanda muy alta de oxígeno que se suple de manera casi exclusiva gracias al aporte aporte de nutrientes y de la oxigenación que le proporciona la coroides, una de las estructuras de mayor flujo sanguíneo por gramo del cuerpo humano, y ha quedado de sobra demostrado a través de numerosas publicaciones el importante papel que juega la coroides en multitud de patologías como puedan ser la CSC,27,57–59 la DMAE y la VCP,27,59–62 la miopía magna,63–66 las uveítis posteriores27,58,59,67,68 y los tumores coroideos27,69–71 entre otras. La suposición de que alteraciones vasculares de la coroides pudieran causar problemas de visión no es nueva. Autores como Spaide han observado envainamientos y oclusiones de los canales vasculares coroideos con la edad, en lo que se ha llamado esclerosis coroidea senil. Estudios histopatológicos de pacientes ancianos ha mostrado una importante atrofia del tejido coroideo con una marcada pérdida de los vasos de calibre pequeño y mediano, llegando al punto en que la membrana de Bruch aparece en ocasiones en una situación contigua a la esclerótica, haciendo que los vasos de mayor calibre ocupen la práctica totalidad del espesor coroideo. Si las modificaciones de la coroides efectivamente juegan un papel o participan en la génesis de algunas enfermedades de la retina, el perfil normal de grosor coroideo y su morfología deben ser conocidos con el mayor detalle posible, para poder diferenciar las variaciones de la normalidad de los hallazgos anómalos y 43 poder señalar y cuantificar los cambios patológicos en el momento preciso de su aparición lo más precozmente posible. 44 3. HIPÓTESIS Y OBJETIVOS 45 3. HIPÓTESIS Y OBJETIVOS El objetivo principal de este trabajo se centra en el estudio del grosor y del perfil de la coroides del sujeto sano, planteando la hipótesis de que: - No existen diferencias entre hombres y mujeres. - No existen diferencias entre ojos derechos e izquierdos. - Existe una reducción progresiva del grosor coroideo con la edad conservando un mismo perfil. - No existen diferencias en el perfil coroideo entre la población infantil y la adulta. - La densidad vascular de la coroides disminuye con la edad. 3.1. OBJETIVO 1 Evaluar el grosor coroideo de una población pediátrica sana utilizando tecnología de coherencia óptica Swept-Source. 3.2. OBJETIVO 2 Determinar el perfil de grosor coroideo macular de una población sana utilizando tecnología de coherencia óptica Swept-Source. 3.3. OBJETIVO 3 Analizar las características morfológicas de la interfaz esclero-coroidea de una población sana utilizando tecnología de coherencia óptica Swept-Source. 46 3.4. OBJETIVO 4 Determinar si existen diferencias en el perfil de grosor coroideo macular entre los dos ojos en una población sana utilizando tecnología de coherencia óptica Swept- Source. 3.5. OBJETIVO 5 Estudiar los cambios que la edad induce en el grosor de las diferentes capas de la coroides de una población sana utilizando tecnología de coherencia óptica Swept- Source. 3.6. OBJETIVO 6 Analizar la densidad vascular de la coroides de la población sana utilizando tomografía de coherencia óptica Swept-Source. 47 4. PUBLICACIONES 48 4.1 Macular Choroidal Thickness in Normal Pediatric Population Measured by Swept-Source Optical Coherence Tomography 49 T Retina Macular Choroidal Thickness in Normal Pediatric Population Measured by Swept-Source Optical Coherence Tomography José M. Ruiz-Moreno,1,2 Iñaki Flores-Moreno,1 Francisco Lugo,2 Jorge Ruiz-Medrano,2 Javier A. Montero,3 and Masahiro Akiba4 PURPOSE. To evaluate choroidal thickness in healthy pediatric population by swept-source longer-wavelength optical coher- ence tomography (SS-OCT). METHODS. This was a cross-sectional comparative, noninterven- tional study. The macular area of 83 eyes from 43 pediatric patients (<18 years) was studied with an SS-OCT prototype system. Macular choroidal thickness was manually determined at 750-lm intervals by measuring the perpendicular distance from the posterior edge of the RPE to the choroid/sclera junction, along a horizontal 4500-lm line centered in the fovea. Three observers independently determined choroidal thick- ness. Pediatric choroidal thickness was compared with choroidal thickness from 75 eyes from 50 normal healthy adult volunteers (18 years or older). RESULTS. Mean age was 10 6 3 years (3–17) in the pediatric population versus 53 6 16 (25–85) in the adult population (P < 0.001). Mean spherical equivalent was not different (P ¼ 0.06) between both groups. Mean subfoveal choroidal thick- ness was 312.9 6 65.3 lm in the pediatric versus 305.6 6 102.6 lm in the adult population (P ¼ 0.19). Mean macular choroidal thickness was 285.2 6 56.7 lm in the pediatric versus 275.2 6 92.7 lm in the adult population (P ¼ 0.08). The distribution of choroidal thickness along the horizontal line was different for both populations; the temporal choroid was thicker in the pediatric population (320, 322, and 324 lm; P ¼ 0.002, 0.001, and 0.06, respectively), followed by the subfoveal (312 lm) and nasal choroid (281, 239, and 195 lm). CONCLUSIONS. Macular choroidal thickness in the pediatric population is not significantly thicker than that of healthy adults. Differences are more remarkable in the temporal side of the fovea. (Invest Ophthalmol Vis Sci. 2013;54:353–359) DOI: 10.1167/iovs.12-10863 From the 1Department of Ophthalmology, Castilla La Mancha University, Albacete, Spain; the 2Alicante Institute of Ophthalmol- ogy, VISSUM, Vitreo-Retinal Unit, Alicante, Spain; the 3Ṕıo del Rı́o Hortega University Hospital, Ophthalmology Unit, Valladolid, Spain; and the 4Topcon Corporation, Tokyo, Japan. Supported in part by a grant of the Spanish Ministry of Health, Instituto de Salud Carlos III, Red Temática de Investigación Cooperativa en Salud ‘‘Patoloǵıa ocular del envejecimiento, calidad visual y calidad de vida’’ (RD07/0062/0019). Submitted for publication August 29, 2012; revised November 5 and December 9, 2012; accepted December 9, 2012. Disclosure: J.M. Ruiz-Moreno, None; I. Flores-Moreno, None; F. Lugo, None; J. Ruiz-Medrano, None; J.A. Montero, None; M. Akiba, TOPCON (E) Corresponding author: Jose M. Ruiz-Moreno, Departamento de Ciencias Médicas, Facultad de Medicina, Avda. de Almansa, 14. 02006. Albacete, Spain; josemaria.ruiz@uclm.es. echnological advances and new information about the role of the choroid in ophthalmic pathology have promoted new research on choroidal anatomy and physiology.1 Choroidal changes are associated with some conditions such as central serous chorioretinopathy,2–5 age-related macu- lar degeneration,4–11 polypoidal choroidal vasculopathy,4–7,10 myopic maculopathy,12–16 posterior uveitis,4,5,17–21 and cho- roidal tumors.4,22,23 Even though indocyanine green angiogra- phy (ICGA) and optical coherence tomography (OCT) have aided in the study of the choroid, adequate morphologic examination using spectral domain OCT (SD-OCT) has not been possible until recently due to the presence of pigment cells that attenuate the incident light, and the limited depth of penetration inherent to the design of SD-OCT instruments.1 It has been previously reported that choroidal thickness (as determined by SD-OCT) decreases with age in healthy eyes.24–29 However, pediatric choroidal thickness has not been previously determined. High penetration, swept-source longer-wavelength OCT (SS- OCT) has an innovative 1-lm band light source,30–33 longer than that of conventional machines, that provides higher penetration through the RPE, enabling deep choroidal imaging. There are no commercially available SS-OCT machines and prototypes are mainly used for research. The aim of this study is to determine choroidal thickness in the pediatric population using a prototype SS-OCT. PATIENTS AND METHODS A cross-sectional comparative, noninterventional study was performed at VISSUM Alicante, Spain. All examinations were obtained in the afternoon to avoid diurnal variations.34,35 The institutional review board of VISSUM Alicante approved the use of the prototype SS-OCT and data collection. This study followed the tenets of the Declaration of Helsinki. The macular area of a healthy pediatric population (<18 years) was studied with an SS-OCT prototype system (Topcon Corporation, Tokyo, Japan), after their parents provided informed consent. The SS-OCT prototype used to image the full-thickness choroid and sclera is based on SS-OCT technology,36 which uses a tunable laser as a light source operated at a 100,000-Hz A-scan repetition rate in the 1-lm wavelength region. The reference mirror is placed at the deepest position of the retina to increase sensitivity at the choroidal level in macular imaging. An OCT image contains 1024 axial scans and up to 96 images are considered for image averaging. Lateral resolution is 20 lm while axial resolution is 8 lm in the retina.37 Lateral and axial resolution are independent. Acquisition time was 1 second. Choroidal thickness was manually calculated as the perpendicular distance from the external surface of the RPE (hyperreflective line) to the internal surface of the sclera. Investigative Ophthalmology & Visual Science, January 2013, Vol. 54, No. 1 Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. 353 50 FIGURE 1. Choroidal thickness determinations (in lm). (A) Pediatric eyes. (B) Adult eyes. Choroidal thickness was determined under the fovea (subfoveal choroidal thickness); three further determinations were performed every 750 lm temporal (T1, T2, and T3) and nasal (N1, N2, and N3) to the fovea (Fig. 1). Average macular horizontal choroidal thickness was calculated as the average of these seven determinations. An average macular profile was calculated as a line formed by the mean values of each point (T3, T2, T1, subfoveal, N1, N2, and N3) in the pediatric and adult groups. Pediatric choroidal thickness was compared with that of normal healthy adult volunteers (18 years or older). Eyes with spherical equivalent (SE) beyond 66 diopters (D) or ocular conditions were excluded from both groups. An experienced technician determined refractive errors using an auto-refractometer (Nidek, Gamagohri, Japan) that was later checked by a certified optometrist. Three observers determined choroidal thickness independently and the final thickness was calculated as the arithmetic mean of the 51 IOVS, January 2013, Vol. 54, No. 1 SS-OCT and Choroidal Thickness in Pediatric Population 355 calculations of the three observers. The interobserver reproducibility was evaluated using intraclass correlation coefficient and Bland Altman plots. Pearson’s correlation was calculated for choroidal thickness and age and SE; P value < 0.05 was considered statistically significant. Statistical analysis was performed using licensed statistical software (SPSS version 14.0; SPSS Inc., Chicago, IL). RESULTS The macular area of 83 eyes from 43 healthy pediatric individuals (<18 years) was studied with an SS-OCT prototype system and compared with 75 eyes from 50 normal healthy adult volunteers (18 years or older). SS-OCT allowed visualization of choroidal thickness in all the cases (100%) in both groups (Fig. 1). Mean age in the pediatric population was 10 6 3 years (3–17) versus 53 6 16 years (25–85) in the adult group (P < 0.001; Student’s t-test). Mean SE was similar in both groups (0.3 6 2.0 D, range þ3.75 to -5.25 in children versus 0.16 6 1.4 D, range þ3.25 to -5.0 in adults; P ¼ 0.06; Student’s t-test). Mean subfoveal choroidal thickness was 312.9 6 65.3 lm (158–469) in children versus 305.6 6 102.6 lm (152–624) in adults (P ¼ 0.19; Mann- Whitney U test). Average macular horizontal choroidal thickness was 285.2 6 56.7 lm (153–399) in children versus 275.2 6 92.7 lm (132–551) in adults (P ¼ 0.08; Mann-Whitney U test; Table 1). Pediatric choroidal thickness was highest in the temporal side (320, 322, and 324 lm for T3, T2, and T1, respectively; confidence intervals 13.2, 12.9, and 13.0 lm, respectively); then in the fovea (312 lm; confidence interval 14.1 lm); and thinnest in the nasal side (281, 239, and 195 lm for N1, N2, and N3 respectively; confidence intervals 14.1, 13.3, and 12.5 lm, respectively). Adult choroidal thickness was highest in the fovea (305 lm; confidence interval 23.3 lm); followed by the temporal (281, 290, 299 lm for T3, T2, and T1, respectively; confidence intervals 18.5, 20.3, and 21.6 lm, respectively); and the nasal side (290, 253, 205 lm for N1, N2, and N3 respectively; confidence intervals 23.8, 23.4, and 22.5 lm, respectively; Fig. 2). Differences in choroidal thickness between both groups were statistically significant at T3 and T2 (P ¼ 0.002 and P ¼ 0.01, respectively, Student’s t-test) and near significance in T1 (P ¼ 0.06, Student’s t-test). Differences in subfoveal and nasal choroidal thickness were not statistically significant. The average temporal choroidal thickness within the pediatric group was lower in the group formed by children 10 to 17 years (n ¼ 35 eyes) than among children aged 3 to 9 years (n ¼ 48 eyes); but the differences between both groups were less marked in the nasal sectors (Fig. 2B). Correlation between macular horizontal choroidal thickness and age or SE and between subfoveal choroidal thickness and SE in the pediatric group was r ¼-0.25 (P ¼ 0.02); r ¼ 0.37 (P ¼ 0.001); and r ¼ 0.41 (P ¼ 0.000), respectively. Correlation between choroidal thickness and age in the whole population was weak or not significant at N3, N2, N1, and fovea, and significant at T1 (r ¼ -0.22, P ¼ 0.004); T2 (r ¼ -0.29, P ¼ 0.000); and T3 (r ¼ -0.33, P ¼ 0.000; Fig. 3). The intraclass correlation coefficient for choroidal thickness for the three independent observers was between 0.91 and 0.98. The Bland-Altman plots showed small differences and narrow limits of agreement for choroidal thickness for interobserver comparison, suggesting satisfactory agreement between the observers. Most of the data points were tightly clustered around the zero line of the difference between the two choroidal thickness determinations and 95% to 97.5% of the determinations fell within limits of agreement (Fig. 4). FIGURE 2. (A) Choroidal thickness profile in the pediatric versus the adult group. (B) The pediatric group has been split into two subgroups (aged 3–9 and 10–17 years) and the respective profiles are compared with the adult profile. DISCUSSION Choroidal research has always been difficult. ICGA permits visualization of choroidal vessels4,5 and recent advances in OCT technology have added cross-sectional information about the choroid.1 Enhanced-depth imaging provided by SD-OCT has permitted cross-sectional research of the choroid, increas- ing our knowledge on the pathophysiology and etiology of several ocular conditions.2,3,6–23 Long wavelength SS-OCT prototypes (1050–1060 nm) have been used in patients improving image quality. Faster and higher quality software may overcome RPE barrier effect and movement arti- facts.11,26,38 52 FIGURE 3. Scatterplot showing choroidal thickness at T3, T2, and T1 in the whole group. Choroidal thickness and age correlate significantly: T1 (r ¼ -0.22, P ¼ 0.004); T2 (r ¼ -0.29, P ¼ 0.000); and T3 (r ¼ -0.33, P ¼ 0.000). Papers on choroidal thickness report a progressive choroi- dal thinning associated with age.24–29 Margolis described 1.56- lm thinning for each year of life.28 Agawa38 and Li39 reported that such correlation between choroidal thickness and age did not exist in eyes with axial length <25 mm. The effect of age on pediatric choroidal thickness has not been studied previously. In our series, SS-OCT allowed visualization of the choroid in all the cases with high-quality images (Fig. 1), permitting choroidal thickness determination. Our data suggest that the temporal choroid may become thinner with age, even thinner than the subfoveal choroid in the adult population. This finding is reinforced by the significant inverse correlation between choroidal thickness and age in the whole group at T1, T2, and T3 (Fig. 3). The average values of choroidal thickness in our adult group were in agreement with previously reported series with similar age distribution (Table 2).25,27,28 Due to the strong correlation between age and choroidal thickness in adults,28 the age factor should be carefully considered when comparing populations with different age distributions.26,29,38,39 Mean subfoveal choroidal thickness in our adult group (312.9 6 65.3 lm) TABLE 1. Patients’ Demographics and CT Pediatric Population Adult Population P Value n (eyes) 83 75 Mean age; y 9.6 6 3.1; 3–17 53.2 6 15.6; 25–85 P < 0.001; Student’s t-test Mean SE Mean subfoveal CT Mean macular CT Subfoveal CT 95% CI 0.3 6 2.0 D; 3.75 to -5.25 312.9 6 65.3 lm; 158–469 285.2 6 56.7 lm; 153–399 298.7–327.3 lm -0.16 6 1.4 D; 3.25 to -5.0 305.6 6 102.6 lm; 152–624 275.2 6 92.7 lm; 132–551 281.9–329.4 lm P ¼ 0.06; Student’s t-test P ¼ 0.19; Mann-Whitney U test P ¼ 0.08; Mann-Whitney U test Macular CT 95% CI 272.9–297.8 lm 250.0–293.3 lm Definite choroid/sclera junction, % 100 100 CT, choroidal thickness. 53 M !OVS, Ja.rnnry 2013, Vol. 54. No. 1 SS-OCT and Cll.oroidal Thidmess in Pedia!ric Population 357 Bl<11.nd A.ltmann Plot Bland Ahmann Plot c 0 MunU Bland Altmann Plo1 ::: 0 Munll FIlid tines rcprcscnt mc:an dîfl'crcncc 2nddasbed lines show the lowcr 2nd upper95% limîts of 2gr«mcnt.Moot of the d2t2points uc tightly dustcrcduound the zero Une of the dîfl'cR:ncc bctween the two choroi60 y P, ANOVA Mean choroidal thickness 286.0 6 43.5 277.7 6 68.2 264.0 6 61.9 223.4 6 62.2 229.7 6 66.1 <0.001 95% CI 276.04–296.08 257.90–297.54 246.41–281.64 203.54–243.34 213.11–246.42 Subfoveal choroidal thickness 325.6 6 51.1 316.7 6 90.1 313.9 6 80.3 264.6 6 79.3 276.3 6 88.8 <0.001 95% CI 313.92–337.44 290.54–342.91 291.11–336.78 239.11–290.06 253.94–298.67 FIGURE 4. Choroidal thickness profile in different age groups (microns/measurement locations). Mean SFCT of the different study groups (Table 2) was 325.6 6 51.1 lm in subjects 0 to 10 years, 316.7 6 90.1 lm in those 11 to 20 years, 313.9 6 80.3 lm in those 21 to 40 years, 264.6 6 79.3 lm in those 41 to 60 years, and 276.3 6 88.8 lm in those older than 60 years, differences that were statistically significant (P < 0.001; ANOVA test). Mean horizontal MCT was 286.0 6 43.5, 277.7 6 68.2, 264.0 6 61.9, 223.4 6 62.2, and 229.7 6 66.1 lm for subjects 0 to 10, 11 to 20, 21 to 40, 41 to 60, and >60 years, respectively (P < 0.001; ANOVA test). The CT profile was different in each age group (Figs. 4, 5, 6), showing an evident statistically significant difference; CT was thicker as age decreased except in the group older than 60 years, where it was thicker than among those 41 to 60 years (Table 3). However, differences in mean MCT and SFCT were not significant between subjects ages 41 to 60 versus those older than 60 years (P ¼ 0.629 and 0.502; Student’s t-test, respectively). Spherical equivalent was not different between groups except for the group 0 to 10 years (P ¼ 0.016; Kruskal-Wallis test), with 0.51 6 1.49 D in this group. Mean SE of the other groups was not different (P ¼ 0.429; Kruskal-Wallis test) (Table 3). Correlation Analysis and Linear Regression Models There exists a high correlation between CT and age, which is statistically significant (Table 4; Fig. 7). This correlation was found to be stronger the farther the CT was measured 61 SS-OCT and CT in a Healthy Population IOVS j June 2014 j Vol. 55 j No. 6 j 3536 FIGURE 5. Mean choroidal thickness (microns) in different age groups. temporally from the foveal center. Correlations between SE and CT were significant only in subfoveal and nasal measurement points (Table 4). Macular CT multiple regression analysis including age and SE was R ¼ 0.407 (r2 ¼ 0.166), showing that subtle changes appear with the variation of this parameter but with a high significance (P < 0.001). The equation predicted is as follows: MCTðmicronsÞ ¼ 292:484 - 1:006*AgeðyearsÞ þ 5:292*SEðDÞ: ð1Þ Following this model, a mean reduction of 10 lm in MCT per decade (1 lm every year) and of 5.3 lm per SE diopter can be predicted. We evaluated the intereye correlation through bivariate correlations. In all measurements the correlation was strong and statistically significant (P < 0.001; Pearson correla- tion test), with values oscillating from r ¼ 0.680 (T5) to r ¼ 0.830 (N1) and r ¼ 0.864 for MCT. This suggests that similar results should be obtained using one eye (randomly selected) or two eyes in the models developed. In fact, the results predicted are very similar. Evaluating only one eye per patient (taking right or left randomly), the most accurate equation to predict MCT according to age would be the following: MCT in microns ¼ 294:133 - 0:942*AgeðyearsÞ þ3:492*SEðDÞ ðr2 ¼ 0:15Þ: ð2Þ The SE is not statistically significant, so the equation to calculate MCT would then be MCTðmicronsÞ ¼ 291:847 - 0:943*AgeðyearsÞ þ3:492*SEðDÞ ðr2 ¼ 0:15Þ: ð3Þ The decrease in MCT is calculated to be 9.4 lm per decade (versus 10 lm when both eyes are included). FIGURE 6. Mean subfoveolar choroidal thickness (microns) in different age groups. 62 SS-OCT and CT in a H ealthy P opulation IOVS j June 2014 j Vol. 55 j No. 6 j 3537 TABLE 3. Choroidal Thickness Values According to Age Group in All Measurement Locations Age N3 N2 N1 SF T1 T2 T3 T4 T5 SE, D 0–10: Mean 161.06 221.63 280.65 325.68 330.51 326.75 320.81 313.57 293.8 0.51 SD 45.02 49.1 54.33 51.13 54.33 56.12 59.72 63.7 60.61 1.49 95% CI 150.80–171.52 210.34–232.93 268.15–293.15 313.92–337.44 318.01–343.01 313.84–339.87 307.07–334.55 298.91–328.22 279.85–307.75 0.13–0.81 11–20: Mean 159.21 219.79 279.14 316.73 319.23 312.14 300.49 295.02 297.74 -0.21 SD 61.58 80.94 90.6 90.18 80.54 72.16 69.56 80.11 76.06 1.76 95% CI 141.33–177.09 196.29–243.30 252.83–305.44 290.54–342.91 295.84–342.62 291.18–333.09 280.29–320.69 271.76–318.28 275.65–319.82 -0.71 to 0.33 21–40: Mean 156.37 218.03 277.86 313.94 313.12 298.25 283.55 264.92 250.17 -0.15 SD 54.56 65.16 73.41 80.35 81.79 78.89 77.33 73.84 71.11 1.04 95% CI 140.87–171.88 199.51–236.55 257.00–298.72 291.11–336.77 289.88–336.36 275.83–320.67 261.57–305.53 243.94–285.90 229.96–70.38 -0.50 to 0.10 41–60: Mean 123.99 177.08 230.56 264.69 258.71 252.91 241.79 234.89 226.33 0.03 SD 58.35 84.35 82.25 79.34 80.8 69.42 65.11 64.75 71.46 0.94 95% CI 105.33–142.65 150.10–204.05 204.26–256.87 239.31–290.06 232.87–284.55 230.71–275.11 220.97–262.61 214.18–255.60 203.47–249.18 -0.27 to 0.33 ‡60: Mean 144.79 201.48 253.04 276.3 265.39 250.77 236.98 228.89 210.25 0.2 SD 63.83 81.51 93.72 88.81 85.47 79.29 69.53 70.76 70.31 1.26 95% CI 128.71–160.86 180.95–222.00 229.44–276.44 253.93–298.67 243.86–286.92 230.80–270.74 219.47–254.49 211.07–246.71 192.54–227.95 -0.13 to 0.51 P test 0.001 Kruskal-Wallis 0.002 ANOVA 0.001 ANOVA <0.001 ANOVA <0.001 ANOVA <0.001 ANOVA <0.001 ANOVA <0.001 ANOVA <0.001 ANOVA 0.016 Kruskal-Wallis TABLE 4. Correlations of CT With Age and Spherical Equivalent N3 N2 N1 SF T1 T2 T3 T4 T5 MCT Age, Spearman’s Rho -0.182, -0.180, -0.207, -0.287, -0.344, -0.421, -0.454, -0.447, -0.432, -0.385, P ¼ 0.002 P ¼ 0.003 P ¼ 0.001 P ¼ 10-6 P ¼ 4 3 10-9 P ¼ 3 3 10-13 P ¼ 2 3 10-15 P ¼ 6 3 10-15 P ¼ 6 3 10-14 P ¼ 3 3 10-11 Spherical equivalent, 0.178, 0.174, 0.173, 0.141, 0.104, 0.105, 0.082, 0.084, 0.064, 0.147, Spearman’s Rho P ¼ 0.003 P ¼ 0.004 P ¼ 0.004 P ¼ 0.020 P ¼ 0.086 P ¼ 0.082 P ¼ 0.178 P ¼ 0.166 P ¼ 0.294 P ¼ 0.015 63 ICC 95% Confidence Interval N3 0.973 0.966, 0.978 N2 0.981 0.976, 0.985 N1 0.981 0.976, 0.985 SF 0.987 0.984, 0.990 T1 0.986 0.982, 0.989 T2 0.981 0.977, 0.985 T3 0.984 0.979, 0.987 T4 0.982 0.978, 0.986 SS-OCT and CT in a Healthy Population IOVS j June 2014 j Vol. 55 j No. 6 j 3538 FIGURE 7. Correlation between age and mean choroidal thickness (microns, top). Correlation between age and mean subfoveolar choroidal thickness (microns, bottom). Correlation between mean SFCT and age was R ¼ 0.275, r2 ¼ 0.076. As before, the dependence found is subtle but highly significant (P < 0.001). SFCTðmicronsÞ ¼ 331:61 - 0:887*AgeðyearsÞ: ð4Þ This model predicts a loss of 8.87 lm of SFCT per decade. If only one eye is evaluated per patient (taking right or left randomly), a more accurate calculation yields a SFCT in microns of 333.27 - 0.852*Age (years); R ¼ 0.266, r2 ¼ 0.071. The estimated decrease in SFCT is 8.5 lm per decade (versus 8.87 lm when both eyes are included). Intraclass correlation coefficient in CT for the two independent observers varied between 0.973 and 0.987 (Table 5). Bland-Altman plots show this good interobserver correla- tion (see Fig. 8). There were no statistically significant differences between the variation coefficients obtained by TABLE 5. ICC in Different Measurement Locations T5 0.981 0.976, 0.985 the two observers (Table 6; P > 0.05, Wilcoxon test). 64 SS-OCT and CT in a Healthy Population IOVS j June 2014 j Vol. 55 j No. 6 j 3540 TABLE 6. Coefficients of Variation of Each Observer at Each Measurement Point and Each Age Group Studied Coefficient of Variation Observer 1 Observer 2 N3 0.379 0.386 N2 0.345 0.351 N1 0.302 0.303 SF 0.266 0.268 T1 0.271 0.270 T2 0.264 0.269 T3 0.269 0.273 T4 0.290 0.289 T5 0.304 0.301 MCT >60 y 0.287 0.289 41–60 y 0.277 0.280 21–40 y 0.237 0.234 11–20 y 0.248 0.244 0–10 y 0.152 0.153 SFCT >60 y 0.320 0.324 41–60 y 0.299 0.302 21–40 y 0.258 0.256 11–20 y 0.286 0.285 0–10 y 0.158 0.158 There were no statistically significant differences between the variation coefficients obtained by each observer (P > 0.05; Wilcoxon test). DISCUSSION To our knowledge, there is no previously published large study across all age groups (including pediatric patients) elucidating the normal age-related choroidal thinning that occurs in healthy eyes. Age-dependent thinning is a key factor to establish the role of the choroid in retinal pathology, something that has already been shown in several studies.4,14–33 Although Ikuno et al.36 performed a similar study in which they described the CT in healthy Japanese subjects, their study did not include a wide variation of ages. Furthermore, the loss of SFCT predicted by our study is 0.852 lm per year, far from the 4.32 lm per year predicted by previous papers, leading to different conclusions. This is the largest study to date of CT measured with SS- OCT, and it confirms again the accuracy with which SS-OCT can measure the choroid. Swept-source OCT was chosen to perform this study because its characteristics allow the analysis of the choroid with more precision than previous OCT technology from a theoretical point of view. This accuracy has been shown in papers by Copete et al.12 and Ruiz-Moreno et al.13 using the same device. Full CT was successfully measured in 100% of the patients in those studies. Using SS- OCT we successfully measured the CT of 100% of the eyes in this study as well. Wei et al.45 successfully measured 93.2% of 3468 patients (largest study reported to date) using Heidelberg Spectralis OCT on the EDI setting. The choice of Manjunath et al.37 was Cirrus HD-OCT without EDI, which was able to successfully measure the choroid in only 74% of the eyes. Yamashita et al.46 and Shin et al.47 used Topcon SD-OCT and were able to image 63.3% and 90.7%, respectively. Copete et al.12 compared SD-OCT and SS-OCT, obtaining good measure- ments in 70.4% and 100%, respectively. Margolis et al.38 studied a group of 54 eyes with a mean SE of -1.3 D, finding a mean SFCT of 287 lm, similar to the results of Flores-Moreno et al.48 and those of Manjunath et al.,37 who published a mean SFCT of 272 lm in 34 eyes of 51.1-year-old patients. Xu et al.49 compared diabetic with nondiabetic patients, finding a mean SFCT of 266 lm in the control group (1795 subjects). The choroid has also been studied in pediatric population using SS-OCT. Ruiz-Moreno et al.13 compared both pediatric (83 eyes, 10-year-old patients with mean SE of 0.3 D) and adult (75 eyes, 53-year-old patients with mean SE of 0.16 D) healthy patients, finding a mean SFCT of 312 and 302 lm, respectively. Park and Oh,50 in a recent study with pediatric subjects, found a mean SFCT of 348.4 lm in the 48 eyes studied. In the present study, the mean SFCT was found to be 301.89 lm. This figure is approximately 10 lm greater than that found by other authors using SD-OCT.37–39,45–49 It is likely that the introduction of pediatric eyes into the study group resulted in a slight increase in the mean SFCT. Interestingly, these results match those of Ruiz-Moreno et al.,13 who with the same device found almost the same CT in their patient group consisting of adults only. Swept-source OCT may result in a CT measurement slightly greater than that seen with SD-OCT. This may be explained by the higher quality of the images and by the fact that the measures were taken from the posterior edge of the RPE to the outer aspect of the lamina fusca, rather than the outer limit of the choroidal vessels. Ruiz-Moreno et al.13 with SS-OCT and Flores-Moreno et al.48 with SD-OCT provide data about mean MCT. Ruiz-Moreno found it to be 275 lm in adults compared to 285 lm in pediatric subjects. Flores-Moreno obtained a result of 257 lm in healthy patients and 115 lm in highly myopic patients. We found that mean MCT was 258.69 lm, similar to these authors’ results. Nevertheless, the present study is the first employing a subfoveal 8-mm line of the macula compared to the 6 mm studied by other authors. This was possible due to SS-OCT technology, which provides a more robust imaging of the temporal choroid, as this 2-mm increase was performed on the temporal sector. This may prove to be a clinically important measure, as it is in the temporal sector that greater variations of CT with age take place. We found no statistically significant difference when comparing men to women. Subjects of both sexes showed almost identical choroidal profiles with a minimal difference in the temporal sector (Fig. 3), in which the females usually had slightly thinner choroids. Mean SFCT was 303.9 lm in men versus 296.4 lm in women. These slight, nonstatistically significant differences can be explained by the age differences of the two groups, 30.39 years for men and 37.48 years for women. Choroidal thickness was approximately 7 lm greater in men on average, but the men in this study were, on average, 7 years younger. According to the correlation formulas established by this database, CT in a sex- and age-matched group would likely have been almost identical. A few studies have been previously performed in adults using high-penetration OCT36,43,51 but without comparing different age groups. We analyzed CT comparing several ranges of age with a 20-year gap. The first two age groups spanned from age 0 to 10 and age 10 to 20 years. These ranges were selected because eye growth undergoes two phases, the first up to age 6 years, reaching emmetropization, and the second from 7 to 19 years, when global expansion of the eye takes place.52 Park and Oh50 (mean age of subjects 86.4 months, from 52 to 131 months) and Ruiz-Moreno et al.13 studied CT in pediatric subjects, with only the latter comparing CT simultaneously with adults. The CT profile changed in every group (Figs. 4, 5, 6) and the choroid measured thinner as age increased, showing statistically significant differences. This decrease was found to be progressive until 40 years, at which 65 SS-OCT and CT in a Healthy Population IOVS j June 2014 j Vol. 55 j No. 6 j 3541 point the most significant variation took place, from 313.9 lm in the 21- to 40-year-old group to 264.6 lm in the 41- to 60- year-old group (49-lm difference). Interestingly, from 40 years on (41–60 vs. >60 years) the differences in CT were not found to be statistically significant, either mean MCT or SFCT. Variations when only one eye per patient (as in Ray and O’Day53) or both were taken into account were minimal, showing calculated decreases of 9.4 vs. 10 lm per decade in MCT and 8.5 vs. 8.87 lm per patient in SFCT. One of the limitations of this study is the fact that the CT was manually determined. There is now an automated software commercially available, Topcon DRI OCT-1 software (version 9.01.003.02), for automated segmentation and thickness measurements in three-dimensional or radial scan mode; however, its accuracy in clinical practice has yet to be widely tested. According to our results, the macular CT profile in a healthy population is similar between different age groups, with the choroid in healthy eyes getting thinner with age, particularly when adults older than 40 years are compared to children and younger adults. 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Characterization of macular lesions in punctate inner choroidopathy with spectral domain optical coherence tomography. J Ophthalmic In- flamm Infect. 2012;2:113–120. 30. Fong AH, Li KK, Wong D. Choroidal evaluation using enhanced depth imaging spectral-domain optical coherence tomography in Vogt-Koyanagi-Harada disease. Retina. 2011; 31:502–509. 31. Nakai K, Gomi F, Ikuno Y, et al. Choroidal observations in Vogt- Koyanagi-Harada disease using high-penetration optical coher- ence tomography. Graefes Arch Clin Exp Ophthalmol. 2012; 250:1089–1095. 32. Say EA, Shah SU, Ferenczy S, Shields CL. Optical coherence tomography of retinal and choroidal tumors [published online ahead of print July 18, 2011]. J Ophthalmol. doi:10.1155/ 2011/385058. 33. Shields CL, Perez B, Materin MA, Mehta S, Shields JA. Optical coherence tomography of choroidal osteoma in 22 cases: evidence for photoreceptor atrophy over the decalcified portion of the tumor. Ophthalmology. 2007;114:e53–e58. 34. Ding X, Li J, Zeng J, et al. Choroidal thickness in healthy Chinese subjects. Invest Ophthalmol Vis Sci. 2011;52:9555– 9560. 35. Ho J, Branchini L, Regatieri C, Krishnan C, Fujimoto JG, Duker JS. Analysis of normal peripapillary choroidal thickness via spectral domain optical coherence tomography. Ophthalmol- ogy. 2011;118:2001–2007. 36. Ikuno Y, Kawaguchi K, Nouchi T, Yasuno Y. Choroidal thickness in healthy Japanese subjects. Invest Ophthalmol Vis Sci. 2010;51:2173–2176. 37. Manjunath V, Taha M, Fujimoto JG, Duker JS. Choroidal thickness in normal eyes measured using Cirrus HD optical coherence tomography. Am J Ophthalmol. 2010;150:325– 329. 38. Margolis R, Spaide RF. A pilot study of enhanced depth imaging optical coherence tomography of the choroid in normal eyes. Am J Ophthalmol. 2009;147:811–815. 39. Ouyang Y, Heussen FM, Mokwa N, et al. Spatial distribution of posterior pole choroidal thickness by spectral domain optical coherence tomography. Invest Ophthalmol Vis Sci. 2011;52: 7019–7026. 40. Brown JS, Flitcroft DI, Ying GS, et al. In vivo human choroidal thickness measurements: evidence for diurnal fluctuations. Invest Ophthalmol Vis Sci. 2009;50:5–12. 41. Tan CS, Ouyang Y, Ruiz H, Sadda SR. Diurnal variation of choroidal thickness in normal, healthy subjects measured by spectral domain optical coherence tomography. Invest Oph- thalmol Vis Sci. 2012;53:261–266. 42. Usui S, Ikuno Y, Akiba M, et al. Circadian changes in subfoveal choroidal thickness and the relationship with circulatory factors in healthy subjects. Invest Ophthalmol Vis Sci. 2012; 53:2300–2307. 43. Ikuno Y, Maruko I, Yasuno Y, et al. Reproducibility of retinal and choroidal thickness measurements in enhanced depth imaging and high-penetration optical coherence tomography. Invest Ophthalmol Vis Sci. 2011;52:5536–5540. 44. Hirata M, Tsujikawa A, Matsumoto A, et al. Macular choroidal thickness and volume in normal subjects measured by swept- source optical coherence tomography. Invest Ophthalmol Vis Sci. 2011;52:4971–4978. 45. Wei WB, Xu L, Jonas JB, et al. Subfoveal choroidal thickness: the Beijing Eye Study. Ophthalmology. 2013;120:175–180. 46. Yamashita T, Yamashita T, Shirasawa M, Arimura N, Terasaki H, Sakamoto T. Repeatability and reproducibility of subfoveal choroidal thickness in normal eyes of Japanese using different SD-OCT devices. Invest Ophthalmol Vis Sci. 2012;53:1102– 1107. 47. Shin JW, Shin YU, Cho HY, Lee BR. Measurement of choroidal thickness in normal eyes using 3D OCT-1000 spectral domain optical coherence tomography. Korean J Ophthalmol. 2012; 26:255–259. 48. Flores-Moreno I, Lugo F, Duker JS, Ruiz-Moreno JM. The relationship between axial length and choroidal thickness in eyes with high myopia. Am J Ophthalmol. 2013;155:314–319. 49. Xu J, Xu L, Du KF, et al. Subfoveal choroidal thickness in diabetes and diabetic retinopathy. Ophthalmology. 2013;120: 2023–2028. 50. Park KA, Oh SY. Choroidal thickness in healthy children. Retina. 2013;33:1971–1976. 51. Agawa T, Miura M, Ikuno Y, et al. Choroidal thickness measurement in healthy Japanese subjects by three-dimen- sional high-penetration optical coherence tomography. Graefes Arch Clin Exp Ophthalmol. 2011;249:1485–1492. 52. Ishii K, Iwata H, Oshika T. Quantitative evaluation of changes in eyeball shape in emmetropization and myopic changes based on elliptic fourier descriptors. Invest Ophthalmol Vis Sci. 2011;52:8585–8591. 53. Ray WA, O’Day DM. Statistical analysis of multi-eye data in ophthalmic research. Invest Ophthalmol Vis Sci. 1985;26: 1186–1188. 67 4.3 Morphologic Features of the Choroidoscleral Interface in a Healthy Population Using Swept-Source Optical Coherence Tomography 68 I Morphologic Features of the Choroidoscleral Interface in a Healthy Population Using Swept-Source Optical Coherence Tomography JORGE RUIZ-MEDRANO, IGNACIO FLORES-MORENO, JAVIER A. MONTERO, JAY S. DUKER, AND JOSÉ M. RUIZ-MORENO • OBJECTIVE: To analyze the morphologic features of the choroidoscleral interface in a healthy population using swept-source optical coherence tomography (SS OCT). • DESIGN: Retrospective data analysis of a subgroup of eyes from a previous single-center, prospective, cross- sectional, noninterventional study. • METHODS: A total of 276 healthy eyes from 154 sub- jects were evaluated using SS OCT. Inclusion criteria were best-corrected visual acuity between 20/20 and 20/25, spherical equivalent between ±3 diopters, and no systemic or ocular diseases. Two independent investiga- tors analyzed the morphologic features of the choroidoscl- eral interface in a masked fashion, classifying the contour and shape as concave (bowl-shaped) or inflective (S-shaped contour with ‡1 inflection point). • RESULTS: The presence of a temporal choroidoscleral interface inflection was identified in 12.8% of the eyes. The mean choroidal thickness was 372.1 ± 76.8 mm and the average distance from the inflection point to the fovea was 4427.3 ± 627.9 mm. Nine patients showed an inflective profile in both eyes. No changes in the retinal profile were found in any of these cases. The mean age of the patients with an inflective profile was 16 ± 19 years (range 4–82) vs 36 ± 25 years (range 3–95) in the group with a concave contour (P [ .001). The temporal choroidal thickness at 4000 and 5000 mm from the fovea was thicker in the group with a concave contour. • CONCLUSIONS: Temporal choroidoscleral interface in- flection or S-shaped profile of the choroidoscleral inter- face with focal thinning of the choroid can be considered a normal variation without clinical signifi- cance, especially in younger populations. (Am J Ophthalmol 2015;160(3):596–601. © 2015 by Elsevier Inc. All rights reserved.) Accepted for publication May 26, 2015. From the Clı́nico San Carlos University Hospital, Ophthalmology Unit, Madrid, Spain (J.R.M.); Department of Ophthalmology, Castilla La Mancha University, Albacete, Spain (I.F.M., J.M.R.M.); Ophthalmology Unit, Pı́o del Rı́o Hortega University Hospital, Valladolid, Spain (J.A.M.); New England Eye Center, Tufts Medical Center, Boston, Massachusetts (J.S.D.); and Insituto Europeo de la Retina (IER), Baviera, Spain (J.M.R.M.). Inquiries to Jorge Ruiz-Medrano, c/ Meléndez Valdés, 38, 28015 Madrid, Spain; e-mail: jorge.ruizmedrano@gmail.com N RECENT YEARS, STUDIES OF THE CHOROIDAL ANAT- omy in posterior segment disease using optical coher- ence tomography (OCT) have increased our knowledge of the role of choroidal changes in various con- ditions such as central serous chorioretinopathy,1–4 age- related macular degeneration,1,4–7 polypoidal choroidal vasculopathy,1,4–7 myopic maculopathy,8–10 posterior uveitis,1,3,4,11,12 and choroidal tumors.1,13,14 Spaide and associates15 introduced a technique for improving choroidal imaging using spectral-domain OCT (SD OCT) devices called enhanced depth image (EDI) OCT. EDI OCT pro- vides improved choroidal visualization in most eyes, permit- ting quantitative and reproducible determination of choroidal thickness. Longer-wavelength swept-source OCT (SS OCT)16–19 is an innovative technology available for OCT imaging that further improves choroidal imaging compared to EDI SD OCT. Copete and associates,20 Ruiz-Moreno and associates,21 and our group22 reported that reliable measurement of choroidal thickness is possible in 100% of eyes using an SS OCT de- vice. Age-related reduction of choroidal thickness in normal eyes,23–28 choroidal thickness differences in adult and pediatric eyes,21,22 and differences between right and left eye29 are reported findings using this technique. However, knowledge of choroidal morphology is not complete yet. Adhi and associates30 described significant alterations in choroidal morphologic features in eyes with advanced dia- betic retinopathy (DR). These alterations included an irregular or a temporal choroidoscleral interface inflecting in most DR eyes. In addition, a recent paper reported a focal inferotemporal scleral bulge with choroidal thin- ning31 in normal eyes modifying the normal contour and shape (bowl-shaped or concave when looking from the in- side out) of the choroidoscleral interface.30 The aim of this study is to analyze the morphologic features of the choroidoscleral interface in a healthy popu- lation using SS OCT. PATIENTS AND METHODS THE DATA AND IMAGES OF A SPECIFIC POPULATION SUB- group from a previously published prospective study were 596 © 2015 BY ELSEVIER INC. ALL RIGHTS RESERVED. 0002-9394/$36.00 http://dx.doi.org/10.1016/j.ajo.2015.05.027 69 VOL. 160, NO. 3 MORPHOLOGY OF THE CHOROIDOSCLERAL INTERFACE 597 analyzed.22 This study followed the tenets of the Declara- tion of Helsinki and the original study was approved by the institutional review board of Vissum Alicante. Data were collected from December 2011 to January 2013. The subjects provided informed consent to participate in the research to study the thickness and morphology of the choroid in a healthy population. Participation was offered to subjects who were attending routine ocular ex- aminations and voluntarily agreed to participate, provided they met the inclusion criteria, with no age limitations, and signed an informed consent. Inclusion criteria were best- corrected visual acuity (BCVA) between 20/20 and 20/ 25, spherical equivalent (SE) between 63 diopters (D), and no systemic or ocular diseases (other than cataract). Patients with prior history of any retinal condition in either eye were not included. All examinations were obtained in the afternoon (between 4 PM and 8 PM) to avoid diurnal variations.32–34 Two independent investigators performed an analysis of the morphologic features of the choroidoscleral interface in a masked fashion, classifying and labeling the contour and shape of the horizontal choroidoscleral interface as previ- ously published.30 Specifically, the choroidal morphology was defined as being concave (or bowl-shaped) or with tem- poral choroidoscleral interface inflecting (having an irreg- ular or concave-convex-concave shape with >_1 inflection point). If the contour was labeled as temporal choroidoscl- eral interface inflecting, choroidal thickness at the level of the inflection point was measured (as described below), as well as the distance to the fovea (Figure 1). The mean choroidal thickness measurements in healthy eyes reported previously22 were used as a reference to define focal thinning/thickening. According to Adhi and associates, eyes were considered to have focal thin- ning/thickening if choroidal thickness at the measured location was 50% less/more than that of the mean choroidal thickness of normal eyes at the corresponding location.30 The procedure followed to analyze and mea- sure choroidal thickness profile has been previously described.22 The macular horizontal choroidal thickness profile (choroidal thickness measured using a horizontal line centered at the fovea, containing 1024 axial scans and with a length of 12 mm)22 was created with an SS OCT system (Topcon Corporation, Tokyo, Japan), after the patients provided informed consent. A horizontal choroidal thickness profile of the macula was manually created by measuring the choroidal thickness from the posterior edge of retinal pigment epithelium (RPE) to the choroid/sclera junction under the fovea using the pro- totype software. Eight further determinations were performed every 1000 mm temporal (T1, T2, T3, T4, and T5) and nasal (N1, N2, and N3) to the fovea. The outer aspect of the lamina fusca, rather than the outer limit of the choroidal vessels, was the landmark used to determine the most distal aspect of the choroid and the in- ner edge of the sclera. FIGURE 1. Choroidoscleral interface of healthy patients analyzed using swept-source optical coherence tomography. (Top) Bowl-shaped/concave choroid: The choroid grows steadily thinner toward nasal and temporal sectors. White line represents the bowl-shaped contour to the choroidoscleral interface. (Mid- dle) Temporal choroidoscleral interface inflection: Evident choroidal thickening (inflection point) in relation to the 2 adja- cent measured points at both sides of the inflection point, without any other relevant choroidal finding, in a 7-year-old boy. (Bot- tom) White line represents the temporal choroidoscleral inter- face; arrow pointing at choroidal inflection (bottom). For this study, the reference mirror of SS OCT was placed at the deeper position of the retina so that the sensi- tivity was higher at the choroidal area in macular imaging. A 1-line scanning mode, which produces an OCT image containing 1024 axial scans with a scan length of 12 mm, was employed. Two observers determined choroidal thick- ness independently and in a masked fashion. An experienced technician determined refractive errors and BCVA using an autorefractometer (Nidek, Gamagori, Japan) that was later checked by a certified optometrist. Data obtained were statistically analyzed using a licensed version of SPSS 17.0 for Windows (SPSS, Chicago, Illi- nois, USA). The Kolmogorov-Smirnov test was applied for all data samples in order to check normality. Intergroup analysis was performed using the Student t test when sam- ples were normally distributed or Mann-Whitney test when parametric statistics were not possible. The level of signif- icance used was P < .05. The interobserver reproducibility was evaluated using intraclass correlation coefficient (ICC) for each variable measured (mean and 95% confidence 70 interval), coefficient of variation between graders, and Bland-Altman plots. RESULTS WE ANALYZED 276 EYES FROM 154 PATIENTS. NINETY-THREE patients (60.4%) were male and 61 (39.6%) were female. The presence of the concave or bowl-shaped contour of the horizontal choroidoscleral interface was found in 240 of the 276 eyes analyzed (87.2%). The presence of a tempo- ral choroidoscleral interface inflection was identified in 36 out of 276 eyes (12.8%), 19 times in the left eye and 17 in the right eye. All the irregularities or concave-convex- concave shapes with >_1 inflection point were found in the temporal aspect of the macular choroidal thickness pro- file. Only 1 inflection point was found in all eyes with a mean distance to the fovea of 4427.3 6 627.9 mm (range 2531–5492), and it was always located between 2000 and 5000 mm temporal the fovea, except in 6 eyes in which it was located more than 5000 mm away from it (Figure 2). Mean choroidal thickness at the site of the inflection point was 372.1 6 76.8 mm (range 168–538 mm). In 9 cases the temporal choroidoscleral interface inflection was bilateral (Figure 3). No changes in retinal profile were found in any of these cases. Mean age of the patients with temporal choroidoscleral interface inflection was 16 6 19 years (range 4–82 years) vs 36 6 25 years (range 3–95 years) in the group with bowl-shaped contour (P ¼ .001; Student t test for unpaired data). Temporal choroidal thickness was also different be- tween both groups. Mean choroidal thickness in the tem- poral choroidoscleral interface inflection group at the site of T4 was 317.6 6 80.2 mm (range 120–440 mm) vs 266.3 6 76.8 mm (range 105–504 mm) in the group with bowl-shaped contour (P ¼ .001; Mann-Whitney U test). Choroidal thickness in the temporal choroidoscleral inter- face inflection group was 286.0 6 71.7 mm (range 160– 466 mm) at the site of T5, vs 252.5 6 77.2 mm (range 66–483 mm) in the bowl-shaped group (P ¼ .01; Mann- Whitney U test). In contrast, the difference of mean choroidal thickness at T1, T2, and T3 was not statistically different (P ¼ .26, P ¼ .39, and P ¼ .11, respectively; Mann-Whitney U test). The prevalence of a temporal choroidoscleral interface inflection profile was 30 out of 120 (12.5%) in the group of patients aged 15 or younger, vs 6 out of 156 (3.8%) in the group of patients aged 16 or older (P < .01; x2 test). A high agreement in the measures taken by the 2 ob- servers was found. The ICC values obtained for the vari- ables evaluated were within the range 0.966–0.987. The Bland-Altman plots also confirmed high agreement be- tween measures, using 62 standard deviations as limits of reproducibility. FIGURE 2. Choroidal inflection point in the temporal side of the right eye in a healthy 8-year-old girl analyzed with swept- source optical coherence tomography. An image was identified (white arrow) that could fit the superior edge of the insertion of the inferior oblique muscle in the sclera, together with a thin, hyporeflective line parallel to the choroidoscleral limit, which could match the intrascleral portion of the temporal long posterior ciliary artery (black arrow). FIGURE 3. Bilateral temporal choroidoscleral interface inflec- tion in a healthy 11-year-old boy analyzed with swept-source op- tical coherence tomography. (Top) Right eye; (Bottom) left eye. DISCUSSION THE HORIZONTAL MACULAR CHOROIDAL THICKNESS PRO- file in healthy people has been described by our group22 and by other authors.21,23–28 The choroid normally is thickest in the subfoveal region, decreasing slowly and progressively toward the temporal aspect and more steeply toward the optic nerve,22,35,36 with a bowl-shaped contour.30 Adhi and associates described significant alterations in choroidal morphologic features in the majority of eyes with DR. They found an irregular temporal choroidoscleral interface inflection point in 8 of 9 eyes with nonprolifera- tive DR (89%), 9 of 10 eyes with proliferative DR (90%), and 13 of 14 eyes with diabetic macular edema (93%) compared with 0 of 24 in controls.30 The presence of an 71 irregular temporal choroidoscleral interface inflection con- tour with focal thinning of the choroid has been detected in some diseases (diabetic retinopathy and age-related macu- lar degeneration).30,37,38 Dolz-Marco and associates31 reported a bilateral case of focal inferotemporal scleral bulge with a concave choroidal thinning surrounded by normal choroidal tissue. The au- thors hypothesized that this finding could be related to the inferior oblique muscle leading to inward compression of the choroid, not being able to study the scleral wall, as it was not entirely visible.31 No anomalies in the fundus examination were found that could justify this thickening in our series of patients with temporal choroidoscleral interface inflection. SS OCT anal- FIGURE 4. Swept-source optical coherence tomography performed inferior to the fovea of a young patient. Temporal long posterior ciliary artery can be seen at the level of the choroi- doscleral inflection point. A thin, hyporeflective line parallel to the choroidoscleral limit that could match the intrascleral portion of the temporal long posterior ciliary artery can be identified (arrow). ysis did not detect any alteration in the choroid of these patients other than 1 inflection point (Figures 1–3). On the other hand, an image that could represent the superior edge of the insertion of the inferior oblique muscle in the sclera was identified (Figure 2), together with a thin, hypo- reflective line parallel to the sclera/choroid limit that could match the intrascleral portion of the temporal long posterior ciliary artery (Figures 2 and 4). The reason why retinal vessels seem to be hyperreflective in OCT images while choroidal vessels look hyporeflective is related to the velocity of blood cells through them. The blood flow velocity inside choroidal vessels is much higher than that of retinal vessels. OCT imaging is based on inter- ferometry, where an interference fringe is detected to construct the intensity of the signal. The fast speed of the blood cells makes the interference fringe vanish; thus no signal is observed inside the choroidal vessels. On the other hand, blood cell speed is relatively slow in the case of retinal vascular structures, which contributes to the inter- ference signal as a hyperreflective signal. Reported studies on the anatomy of the posterior aspect of the globe and the vascular and muscular structures and its relation to the sclera show that the anterior and superior limit of the posterior insertion of the inferior oblique mus- cle coincides with the horizontal line/meridian, within 5.7 mm of the fovea.39 The mean distance from the inflection point to the fovea was 4427 mm in our series and always located temporal to the fovea. The presence of the inflection point is probably related to the insertion of the inferior oblique muscle, which is believed to be 5.7 mm from the fovea. The traction applied to the sclera by the tendon might cause a separation of the scleral wall, leading to its deformation and a thickening of the choroid. This theory is supported by Figures 2 and 4, and is compatible with the tractional theory. Based on the anatomic descriptions of this region,39 the parallel, hyporeflective line at this level could corre- spond to the intrascleral portion of the temporal long pos- terior ciliary artery (Figures 2 and 4). The fact that this finding is more frequent in younger healthy people, especially those under age 15, could be explained by the reduced scleral stiffness in younger age,40 which could facilitate the deformation induced by the traction of the inferior oblique muscle tendon. The limitations of our study are the manual determina- tion of the choroidal thickness and the fact that it only shows the horizontal choroidal thickness profile. Auto- matic software is now commercially available: Topcon DRI OCT-1 software (Ver. 9.01.003.02) for automated seg- mentation and thickness measurements in 3-dimensional or radial scan mode. However, its accuracy in clinical prac- tice is yet to be widely tested. In conclusion, this temporal choroidoscleral interface inflection with focal thinning of the choroid can be consid- ered a normal variation without clinical significance, espe- cially among young people. Further studies of the whole macular profile, including the complete insertion of the inferior oblique muscle, and with a larger number of pa- tients will be necessary in order to establish the final char- acteristics of these variations and their incidence in a healthy population. ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST. Financial disclosures: Jay S. Duker is a consultant for and receives research support from Carl Zeiss Meditech, Inc (Dublin, California, USA) and Optovue Inc (Fremont, California, USA). José M. Ruiz-Moreno receives research support from Topcon Co (Tokyo, Japan). 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Invest Ophthalmol Vis Sci 2012; 53(2):975–985. 37. Regatieri CV, Branchini L, Carmody J, Fujimoto JG, Duker JS. Choroidal thickness in patients with diabetic reti- nopathy analyzed by spectral-domain optical coherence to- mography. Retina 2012;32(3):563–568. 38. Manjunath V, Goren J, Fujimoto JG, Duker JS. Analysis of choroidal thickness in age-related macular degeneration using spectral-domain optical coherence tomography. Am J Ophthalmol 2011;152(4):663–668. 39. Siam AL, El-Mamoun TA, Ali MH. A restudy of the surgical anatomy of the posterior aspect of the globe: an essential topography for exact macular buckling. Retina 2011;31(7): 1405–1411. 40. Pallikaris IG, Kymionis GD, Ginis HS, Kounis GA, Tsilimbaris MK. Ocular rigidity in living human eyes. Invest Ophthalmol Vis Sci 2005;46(2):409–414. 74 601.e1 AMERICAN JOURNAL OF OPHTHALMOLOGY SEPTEMBER 2015 Biosketch Jorge Ruiz-Medrano, MD, is an Ophthalmology resident at Clı́nico San Carlos University Hospital in Madrid, Spain. He received his MD from Universidad Autó noma de Madrid Medical College in June 2011. His research interests include advanced retinal and choroidal image analysis and he is currently working on a PhD on the study of the choroid using Swept-Source optical coherence tomography. 75 4.4 Asymmetry in Macular Choroidal Thickness Profile between both eyes in a healthy population measured by Swept-Source Optical Coherence Tomography 76 ASYMMETRY IN MACULAR CHOROIDAL THICKNESS PROFILE BETWEEN BOTH EYES IN A HEALTHY POPULATION MEASURED BY SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY JORGE RUIZ-MEDRANO, MD,* IGNACIO FLORES-MORENO, MD, PHD,† PABLO PEÑA-GARCÍA, MSC,† JAVIER A. MONTERO, MD, PHD,‡ JAY S. DUKER, MD,§ JOSÉ M. RUIZ-MORENO, MD, PHD†¶ Purpose: To determine the difference in macular choroidal thickness (CT) profile between eyes in healthy individuals using swept-source optical coherence tomography. Design: Cross-sectional noninterventional study. Participants: One hundred and forty eyes from 70 healthy patients with spherical equivalent between ±3 D and with difference #0.25 D between eyes were scanned using a swept-source optical coherence tomography (Topcon Corporation). Methods: Cross-sectional noninterventional study. One hundred and forty eyes from 70 healthy patients with spherical equivalent between ±3 D and with difference #0.25 D between eyes were scanned using a swept-source optical coherence tomography (Topcon Corporation). A horizontal CT profile of the macula was created in both eyes by manually measuring the subfoveal CT from the posterior edge of retinal pigment epithelium (RPE) to the choroid/sclera junction. Three determina − tions were performed at successive points 1,000 mm nasal to the fovea and 5 more temporal to the fovea. The differences in CT between both eyes were analyzed. Results: Mean age was 25.4 ± 19.9 years (from 4 to 75). The mean spherical equivalent was 0.18 ± 1.37 D (from 3 to +3). Mean macular nasal CT was thicker in the right eye (RE) than in the left eye (LE) (228.11 ± 69.23 mm vs. 212.27 ± 62.71 mm; P = 0.0002; Student’s t-test paired data). Mean subfoveal CT and mean temporal CT was not statistically signifi- cantly different between the eyes. No statistically significant differences were observed comparing spherical equivalent in the RE compared with the LE. Both men and women showed a thicker mean nasal choroid in the RE versus the left (men, 226.97 ± 61.56 mm vs. 209.87 ± 60.31 mm; women, 229.63 ± 79.39 mm vs. 215.47 ± 66.68 mm, P = 0.003 and P = 0.03, respectively; Student’s t-test paired data). At each nasal determination, CT in the RE was statistically significantly thicker than the LE (N1: 283.72 ± 81.10 mm vs. 269.76 ± 75.81 mm [P = 0.001]; in N2: 230.45 ± 73.47 mm vs. 211.33 ± 66.92 mm [P = 0.0002]; and in N3: 170.16 ± 61.00 mm vs. 155.72 ± 53.87 mm [P = 0.008], respectively). Conclusion: To the best of our knowledge, this is the first report suggesting thicker macular nasal choroid in the RE compared with the LE. In contrast, subfoveal CT and temporal CT were not found to be different between eyes. RETINA 35:2067–2073, 2015 The choroid and its involvement in a variety of ocular in glaucoma,23–26 and even in neurologic pathologies, pathologies has been the subject of intensive study during the past few years. The choroid plays a role in or is affected by pathologies, such as central serous chorior- etinopathy,1–4 age-related macular degeneration,1,5–10 posterior segment tumors,1,11–13 myopic maculop- athy,14–17 posterior uveitis1,6,18–22 and polypoidal choroidal vasculopathy.1,7,8 It may also have influence such as migraine27 and Alzheimer disease.28 Advances in optical coherence tomography (OCT) in recent years allows deep high-resolution imaging of the posterior eye layers “in vivo.”1 Optical coherence tomography’s brief acquisition time29,30 makes the examination easier for patients who are less cooperative, such as children, the elderly, and those with poor vision.17,31 2067 77 Recently, with the improvements in spectral domain optical coherence tomography provided by enhanced- depth imaging32 and swept-source optical coherence tomography (SS-OCT) technology,33–36 which is able to image deeper into the choroid, the choroidal thick- ness (CT) profiles of healthy populations have been studied.31,37–39 The aim of this study is to determine whether there are differences in horizontal macular CT profile between the two eyes in a large population of healthy individuals using SS-OCT. Patients and Methods This is a cross-sectional noninterventional study, performed at Castilla La Mancha University Spain. This study followed the tenets of the Declaration of Helsinki. The institutional review board approved this study. All examinations were obtained in the afternoon to avoid diurnal variations.40–42 Choroidal thickness was manually measured in 140 eyes from 70 patients with no known ocular or systemic disease. Their macular area was studied with an SS-OCT system (Topcon Corporation, Tokyo, Japan), after they provided informed consent. Inclusion criteria were best- corrected visual acuity of 20/20, spherical equivalent (SE) between ±3 diopters (D), with difference #0.25 D between eyes and no systemic diseases. Eyes with any history of mild retinal diseases were not included. The SS-OCT device used to image the full-thickness choroid and sclera43 uses a tunable laser as a light source operated at 100,000 Hz, A-scan repetition rate in the 1-mm wavelength region. The device can per- form image averaging of up to 96 B-scans at each location. For the macular imaging performed in this study, the reference mirror was placed at the deeper From the *Ophthalmology Unit, Clínico San Carlos University Hospital, Madrid, Spain; †Department of Ophthalmology, Castilla La Mancha University, Albacete, Spain; ‡Ophthalmology Unit, Pío del Río Hortega University Hospital, Valladolid, Spain; §New England Eye Center, Tufts Medical Center, Boston, Massachusetts; and ¶Instituto Europeo de la Retina, Baviera, Spain. Supported in part by a grant of the Spanish Ministry of Health, Instituto de Salud Carlos III, Red Temática de Investigación Co- operativa en Salud “Prevención, detección precoz y tratamiento de la patología ocular prevalente, degenerativa y crónica” (RD12/ 0034/0011), and by a Research to Prevent Blindness. Unrestricted Grant to the New England Eye Center/Department of Ophthalmol- ogy, Tufts University School of Medicine and the Massachusetts Lions Clubs. J. S. Duker is a consultant for and receives research support from Carl Zeiss Meditech, Inc and receives research support from Opto- vue. J. M. Ruiz-Moreno receives research support from Topcon, Co. The other authors have no conflicting interests to disclose. Reprint requests: Jorge Ruiz-Medrano, MD, Ophthalmology Unit, Clínico San Carlos University Hospital, Madrid, Spain; e-mail: jorge.ruizmedrano@gmail.com position of the retina so that the sensitivity was higher in the choroid. A one-line scanning mode, which pro- duces an OCT image containing 1,024 axial scans with a scan length of 12 mm was used. This sampling space in object space corresponds to 11.7 mm/pixel. Lateral resolution was set to be 20 mm with 24-mm axial eye length, whereas axial resolution was 8 mm in retina.44 Lateral and axial resolution were independent. Acquisition time for the scan protocol was 1 second. Both eyes were scanned consecutively. A horizontal CT profile of the macula was manually created measuring CT from the posterior edge of RPE to the choroid/sclera junction under the fovea, in a line perpendicular to the retinal surface. The outer aspect of the lamina fusca/inner border of the sclera rather than the outer limit of the choroidal vessels was determined as the outer limit of the choroid.38,45 Five determinations were performed every 1,000 mm temporal (T1, T2, T3, T4 and T5) and 3 more nasal (N1, N2 and N3) to the fovea (Figure 1). Fig. 1. Example of choroidal thickness measures at SFCT and N3, N2, and N1 in two eyes of one patient (top RE and bottom LE), from the posterior edge of RPE to the choroid/sclera junction. 78 Eyes with SE beyond ±3 D and with difference .0.25 D between eyes were excluded. An experi- enced technician determined refractive errors and best-corrected visual acuity using an auto- refractometer (Nidek, Gamagohri, Japan) that was later checked by a certified optometrist. The differences in mean CT and CT at each independent location between both eyes were ana- lyzed. Two observers determined CT independently and in a masked fashion. Statistical Analysis For the statistical treatment of the data, the program used was version 17.0 of SPSS for Windows (SPSS, Chicago, IL). The interobserver reproducibility was evaluated using intraclass correlation coefficient for each variable measured (mean and 95% confidence interval [CI]), coefficient of variation between graders Fig. 2. Mean horizontal choroidal thickness (mm) profile. RE versus 294.15 ± 54.69 mm (95% CI: 280.58– 307.72) in the LE (P − = 0.602; Student’s t-test paired data). No statistically significant differences were observed comparing SE between the REs (0.18 ± and Bland–Altman plots. The mean of the measures 1.33 D; 95% CI: 0.14 to 0.50) versus the LEs obtained by the two observers was the data used for the rest of calculations. Kolmogorov–Smirnov test was applied for all data samples to check normality. Com- parison between groups was performed using the Stu- dent’s t-test when samples were normally distributed or Mann–Whitney test when parametric statistics were not possible. The level of significance used was always the same (P , 0.05). The homogeneity of variances was checked using the Levene’s test. Bivar- iate correlations were evaluated using Spearman cor- relation coefficients. For the development of predictive models, linear regression was used. Mean macular nasal CT was determined by the arithmetic mean of the values obtained at N3, N2, and N1, whereas the mean macular temporal CT was calculated by the mean of T1, T2, T3, T4, and T5. Results Forty patien − ts (57.1%) were male (80 eyes) and 30 patients (42.9%) were female (60 eyes). Mean age was 25.4 ± 19.9 years (from 4 to 75). Mean SE was 0.18 ± (0.19 ± 1.37 D; 95% CI: –0.13 to 0.51) (P = 0.517; Wilcoxon’s test). With respect to sex, there were no statistically significant differences between eyes for SFCT or mean temporal CT (Table 1). The cohort of males in this population studied had a mean age of 19 years (range, 4 to 74 years). When analyzing mean nasal CT by sex, men manifested a nasal choroid thicker in the RE, 226.97 ± 61.56 mm (95 CI: 207.29–246.66) versus 209.87 ± 60.31 mm in the LE (95% CI: 190.58–229.16) (P = 0.003, Student’s t-test paired data). In the female cohort group, the mean age was 32.8 ± 18.3 years (from 8 to 75) statistically signifi- cantly older than men (P , 0.05). The mean nasal CT of 229.63 ± 79.39 mm in the RE (95% CI: 199.99–259.28) versus 215.47 ± 66.68 mm in the LE (95% CI: 190.57– 240.368) (P = 0.03; Student’s t-test paired data). Studying the CT at each individual location in the nasal sector, CT in the RE at N1 was 283.72 ± 81.10 mm (95% CI: 264.39–303.069) versus 269.76 ± 75.81 mm in the LE (95% CI: 251.68–287.83) (P = 0.001, Student’s t-test paired data); at N2, RE CT was 230.45 ± 73.47 mm (95% CI: 212.93–247.97) versus 211.33 ± 66.92 mm in the LE (95% CI: 195.37– 1.37 D (from 3 to +3). Mean macular nasal horizontal 227.29) (P = 0.0002, Student’s t-test paired data); CT was statistically thicker in the right eye (RE) 228.11 ± 69.23 mm, range 105.33 to 391.33 mm (95% CI: 211.61–244.62) on average, than in the left eye (LE) 212.27 ± 62.71 mm, range 84.17 to 356.83 mm (95% CI: 197.32–227.22) (P = 0.0002; Student’s t-test paired data) (Figure 2). Mean subfoveal CT (SFCT) was 315.86 ± 76.78 mm (95% CI: 297.42– 334.41) in the RE versus 308.41 ± 75.51 mm (95% CI: 290.99–325.83) in the LE (P = 0.138, Student’s t-test paired data) and mean macular temporal CT was 292.02 ± 63.68 mm (95% CI: 276.62–307.31) in the and at N3, RE CT was 170.16 ± 61.00 mm (95% CI: 155.62–184.71) versus 155.72 ± 53.87 mm in the LE (95% CI: 142.88–168.57) (P = 0.008, Student’s t-test paired data). The choroid was thicker in the RE at every nasal location for both sexes except for women at N1, where differences were not statistically significant (P = 0.259; Table 2). The correlation of nasal CT between RE and LE at each location (N3, N2, and N1) was r = 0.853, 0.842 and 0.822, respectively (P , 0.001; Spearman 79 Table 1. Choroidal Thickness (mm) at SF, Mean Temporal, T1, T2, T3, T4, and T5 by Eye RE LE P* SFCT (95% CI) 315.86 ± 76.78 (297.42–334.41) 308.41 ± 75.51 (290.99–325.83) 0.138 Mean TCT (95% CI) 292.02 ± 63.68 (276.62–307.31) 294.15 ± 54.69 (280.58–307.72) 0.602 T1 (95% CI) 311.75 ± 78.14 (292.96–330.51) 312.12 ± 68.42 (295.68–328.55) 0.883 T2 (95% CI) 306.57 ± 73.05 (286.02–321.12) 311.46 ± 63.42 (296.23–312.39) 0.188 T3 (95% CI) 293.42 ± 71.96 (276.14–310.71) 291.99 ± 61.01 (277.34–306.65) 0.961 T4 (95% CI) 282.46 ± 68.21 (266.08–298.85) 284.04 ± 67.24 (267.89–300.20) 0.626 T5 (95% CI) 269.71 ± 72.66 (251.39–287.04) 272.20 ± 75.76 (255.68–291.81) 0.585 *Student’s t-test paired data. TCT, temporal choroidal thickness. correlation test). The correlation between the mean nasal CT of RE and LE is shown by this equation: Mean  nasal  CT   RE   ðmmÞ ¼ 23:437 þ 0:964 · mean  nasal  CT   LE   ðmmÞ: r2 ¼ 0:763: A high agreement in the measures taken by the 2 observers was found as can be seen in Table 3. The intraclass correlation coefficient values obtained for the variables evaluated were within the range 0.976 to 0.988. The Bland–Altman plots (Figure 3) also con- firmed high agreement between measures. Ikuno et al47 studied CT and its profile with an SS-OCT device in 86 eyes of 43 healthy Japanese sub- jects as well as the correlation with axial length, refrac- tive error and age, but they also published no data concerning any differences between right and LEs. In contrast, Chen et al48 reported the factors influ- encing topographical and interocular variations in CT in a 50 healthy adult population using enhanced-depth imaging spectral domain optical coherence tomogra- phy. They measured SFCT and also performed CT measurements at 4 paramacular loci (3 mm superior, inferior, temporal, and nasal to the foveal center). They − analyzed the relationship between interocular differences in CT. The differences in CT between RE and LE were 1.0 mm at SFCT, 13.6 mm at nasal and 2.9 mm at temporal location, which was not Discussion Several studies have been published about CT in healthy populations. Ding et al analyze the CT in 210 healthy volunteers (420 eyes) with no ophthalmic disease history using enhanced-depth imaging spectral domain optical coherence tomography at multiple locations: SFCT and 1 mm and 3 mm temporal, nasal, superior, and inferior to the fovea. However, the authors did not analyze any differences between the eyes.46 statistically significant different. Of note, there was a trend toward a thicker nasal CT (14 mm) in REs. When comparing these data with the series reported in this article, at the same locations (SCFT, N3 CT and T3 CT), we observed that the difference comparing RE and LE was 7.45 mm in mean SFCT, 1.43 mm at T3, and 14.44 mm at N3. The differences between mean nasal CT (P = 0.0002), mean nasal CT in men (P = 0.003) and in women (P = 0.03), and mean CT at each nasal location (P = 0.001, 0.002 and 0.08, Table 2. Choroidal Thickness (mm) at N1, N2, and N3 by Eye and Sex RE LE P* N1 Men (95% CI) 284.08 ± 68.49 (262.17–305.98) 265.51 ± 68.89 (243.48–287.54) 0.006 Women (95% CI) 283.25 ± 96.63 (247.17–319.33) 275.42 ± 85.06 (243.65–307.18) 0.259 Total group (95% CI) 283.72 ± 81.09 (111.00–475.00) 269.76 ± 75.81 (251.68–287.83) 0.008 N2 Men (95% CI) 228.98 ± 64.97 (208.19–249.75) 208.73 ± 65.64 (187.73–229.72) 0.001 Women (95% CI) 232.42 ± 84.62 (200.82–264.02) 214.80 ± 69.58 (188.82–240.78) 0.026 Total group (95% CI) 230.45 ± 73.48 (212.93–247.97) 211.33 ± 66.93 (195.37–227.29) ,0.001 N3 Men (95% CI) 167.86 ± 58.71 (149.09–186.64) 155.38 ± 53.62 (138.23–172.52) 0.007 Women (95% CI) 173.23 ± 64.81 (149.03–197.44) 156.18 ± 55.12 (135.60–176.77) 0.016 Total group (95% CI) 170.16 ± 61.00 (155.62–184.71) 155.72 ± 53.87 (142.88–168.57) 0.001 *Student’s t-test paired data. 80 Table 3. Intraclass Coefficient and Coefficients of Variation of Each Observer at Each Measurement Point ICC 95% CI CV Observer 1 CV Observer 2 N3 0.976 0.962–0.985 0.349 0.355 N2 0.979 0.967–0.987 0.316 0.330 N1 0.988 0.982–0.993 0.282 0.292 SF 0.985 0.975–0.990 0.251 0.254 T1 0.982 0.972–0.989 0.236 0.236 T2 0.976 0.962–0.985 0.213 0.228 T3 0.977 0.964–0.986 0.226 0.233 T4 0.979 0.967–0.987 0.254 0.247 T5 0.976 0.962–0.985 0.284 0.272 CV, coefficient of variation; ICC, intraclass correlation coeffi- cient. respectively) were statistically significant. The choroid was thicker in the RE at every nasal location for both sexes except for women at N1 (P = 0.259; Table 2). The correlation of the CT obtained by Chen et al between eyes was strongest for SFCT (r = 0.90; P , 0.001, Spearman correlation test), for nasal locations was r = 0.83 (P , 0.001, Spearman correlation test), and weakest for temporal locations (r = 0.49; P , 0.001, Spearman correlation test).40 In our patients, the study of correlation for mean CT at SF, N3, and T3 was similar (r = 0.860, 0.853 and 0.754, respec- tively, P , 0.001; Spearman correlation test). The CT profile described in their article48 is very similar to the CT profile obtained in our work com- paring REs to LEs (Figure 2). It is difficult to explain the strong and consistent evidence that REs have a thicker nasal choroid than LEs in a healthy young population. One possibility is a differential in blood flow between the two eyes due to lack of anatomic symmetry at the aortic arch. Such asymmetry has been suggested to explain the differ- ences in incidence/prevalence of vascular patholo- gies between REs and left eys with respect to metastatic bacterial endophthalmitis49 and retinal artery occlusion.49–53 Choroidal circulation is generated from short pos- terior ciliary arteries that penetrate through the sclera around of optical nerve in a variable number between 10 and 20; so the nasal choroid studied in our cases (choroid between fovea and optical nerve) is directly supplied from these short posterior ciliary arteries. Furthermore, short posterior ciliary arteries are branches of the ophthalmic artery, which is a branch of the internal carotid artery, which is a branch of the common carotid artery.53 The difference generated by the fact that the origin of the right common carotid artery lays in the brachiocephalic trunk instead of emerging directly from the aorta (as the left common carotid artery) is Fig. 3. Bland–Altman plots for interobserver correlation in measurement location from N3 to SFCT. N1 (top left), N2 (top right), N3 (bottom left), and SF (bottom right). 81 presumably responsible of a more proximal and direct blood flow to the right carotid.49 Given that most of the choroidal structure is vascular tissue (the mean vessel density in outer choroid is 87%54), a supposed higher blood flow in the right versus left short posterior ciliary arteries may explain why the nasal choroid is thicker in RE, as stated in our study, and as described previously by Chen et al.48 In this study, as in previous articles,31 we used SE instead of axial length determinations since the pro- cedure is less invasive and previous indications from the literature show that refraction, which is more con- venient to obtain, provides equivalent modeling capa- bility as axial length.55 The limitations of this article are the relatively small number of eyes (140, 70 per group), study of the thickness limited to a line beneath the fovea, the use of SE instead of axial length, the fact that no correction was made for other variables, such as central corneal thickness, intraocular pressure, systemic medication, and blood pressure, and the impossibility to determine whether the vessels of the nasal choroid are wider in the RE than in the left. 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A pilot study of morphometric analysis of choroidal vasculature in vivo, using en face optical coherence tomopraphy. PLoS One 2012;7:e48631. 55. Nishida Y, Fujiwara T, Imamura Y, et al. Choroidal thickness and visual acuity in highly myopic eyes. Retina 2012;32:1229–1236. 83 4.5 Analysis of age-related Choroidal Layers thinning in Healthy Eyes Using Swept-Source Optical Coherence Tomography 84 85 86 87 88 89 90 91 92 93 4.6 Age-related changes in choroidal vascular density of healthy subjects based on image binarization of Swept-Source Optical Coherence Tomography 94 95 Title: Age-related changes in choroidal vascular density of healthy subjects based on image binarization of Swept-Source Optical Coherence Tomography. Short title: Normal subjects choroid vascular density. Jorge Ruiz-Medrano1, José M Ruiz-Moreno2, 3, Abhilash Goud4, Kiran Kumar Vupparaboina5, Soumya Jana5, Jay Chhablani4. 1 Jules Gonin Eye Hospital. Fondation Asile des Aveugles. Lausanne. SWITZERLAND. 2 Department of Ophthalmology, Castilla La Mancha University. Albacete. SPAIN. 3 Vissum Corporation. SPAIN. 4 Smt. Kanuri Santhamma Retina Vitreous Centre. L V Prasad Eye Institute Hyderabad. INDIA. 5 Department of Electrical Engineering, Indian Institute of Technology Hyderabad, Telangana, INDIA. 96 Acknowledgments Conception and design of the work/project: Jay Chhablani, José M Ruiz-Moreno. Acquisition of data: Jorge Ruiz-Medrano, José M Ruiz-Moreno. Conceptualization of the manuscript and review and synthesis of the literature: Jorge Ruiz-Medrano, José M Ruiz-Moreno. Analysis and interpretation of data: Abhilash Goud, Kiran Kumar Vupparaboina, Soumya Jana, Jay Chhablani, José M Ruiz-Moreno. Critical review and revision of the manuscript: Jorge Ruiz-Medrano, José M Ruiz- Moreno, Jay Chhablani, Abhilash Goud, Kiran Kumar Vupparaboina, Soumya Jana. Drafting of the manuscript: Jorge Ruiz-Medrano, José M Ruiz-Moreno. Final approval of the version to be published: Jorge Ruiz-Medrano, Abhilash Goud, Kiran Kumar Vupparaboina, Soumya Jana, Jay Chhablani, José M Ruiz-Moreno. a) FUNDING/SUPPORT This study has been supported in part by a grant of the Spanish Ministry of Health, Instituto de Salud Carlos III, Red Temática de Investigación Cooperativa en Salud "Prevención, detección precoz y tratamiento de la patología ocular prevalente, degenerativa y crónica" (RD12/0034/0011, SPAIN). b) FINANCIAL DISCLOSURES None of the other authors have any financial interest to disclose. Correspondence to: Jorge Ruiz-Medrano Meléndez Valdés, 38. 28015. Madrid. SPAIN Phone. 0034 647447796. E-mail: jorge.ruizmedrano@gmail.com Key words: Swept-Source optical coherence tomography, SS-OCT, choroidal vascular density, choroidal thickness, vascular area, stromal area. Word count: 2720 Summary statement: Choroidal vascular density in healthy patients based on image binarization of Swept- Source Optical Coherence Tomography. Jorge Ruiz-Medrano, José M Ruiz-Moreno, Abhilash Goud, Kiran Kumar Vupparaboina, Soumya Jana, Jay Chhablani. Based on the analysis of choroidal stroma and vessel area using a new automated segmentation and binarization software based on validated algorithms across a wide span of ages (3 to 85), the luminal area and the percentage of vascular/total area decrease with increasing age while stromal area remains stable. 97 Abstract Objective: To analyze the vascular density of the choroid in a healthy population using swept-source optical coherence tomography (SS-OCT). Methods: Cross-sectional, non-interventional study. Inclusion criteria: Best Corrected Visual Acuity (BCVA) between 20/20 to 20/25, spherical equivalent (SE) between ± 3 diopters (D), no systemic or ocular diseases and ages ranging from 3 to 85 years old. 136 eyes from 136 subjects were analyzed, 86 eyes (63.2%) were from male and 50 eyes (36.8%) from female subjects. The eyes were divided into different age groups to analyse the possible age-related changes. Twelve mm horizontal, fovea-centered B-scans were used. Choroidal stroma and vessel area analysis involved automated segmentation and binarization using validated algorithms. Results: Mean age was 33.1±24.5 years. Mean choroidal area was 0.5554±0.1377 mm2. Mean stromal area was 0.2524±0.0762 mm2, and mean vascular region area was 0.3029±0.0893 mm2. The percentage of choroidal vascularity (vascular area/total area) was 54.40±8.35 %. Choroid area, vascular region and percentage of choroidal vascular density were statistically higher in the <18-year-old group vs. >18- year-old group (p<0.001). Stromal region was not different (p=0.46). In the same way, choroid area, vascular region and percentage of choroidal vascular density between the five age groups was statistically different (p<0.001), showing larger figures in the 0-10 years old group; but not stromal region (p=0.71). There were no gender-related differences. Conclusions: The luminal area and the percentage of vascular/total area decrease with increasing age while stromal area remains stable. 98 Introduction Since Spaide et al introduced enhanced depth imaging (EDI) technique which improved choroidal imaging using spectral domain Optical Coherence Tomography (SD-OCT) devices, our knowledge of the role of choroidal changes in various conditions such as central serous chorioretinopathy1-4, age-related macular degeneration1, 4-10, polypoidal choroidal vasculopathy1, 4-6, 9, myopic maculopathy11-15, posterior uveitis1, 3, 4, 16-19 and choroidal tumors1, 20, 21. Swept-source longer-wavelength23-26 is a technology for OCT (SS-OCT) imaging that permits further improvement in choroidal study. Copete et al27 and Ruiz-Moreno et al28, 29 reported that reliable measurement of choroidal thickness (CT) is possible in 100% of eyes using an SS-OCT device. Age-related reduction of CT in normal eyes30-35, CT differences in adult and paediatric eyes28, 29 and differences between right and left eye36 have been reported using this technique. However, knowledge of choroidal morphology is not complete yet. Adhi et al37 described significant alterations in choroidal morphological features in eyes with advanced diabetic retinopathy. In addition, a recent paper reported a focal inferotemporal scleral bulge with choroidal thinning in normal eyes modifying the normal contour and shape (convex or bowl-shaped) of the choroidoscleral interface.38 Sonoda et al, using a binarization technique to analyse the luminal and stromal areas separately, show that both these areas decreased with increasing age and axial length. They affirm that the method should be of great help in analysing the choroid, and the normative data collected should provide basic information about the normal choroid.39 Agrawal et al introduced a novel OCT term: Choroidal vascularity index (CVI), to assess vascularity of the subfoveal choroid. They showed that on a single cross sectional EDI-OCT image, two-thirds (~66%) of the subfoveal choroid is vascular in healthy eyes.40 The aim of this paper is to analyse the vascular density of the choroid in a healthy population using SS-OCT and compare the differences in choroidal vascularity with increasing age. Subjects and Methods This was a cross-sectional, non-interventional study. It followed the tenets of the Declaration of Helsinki. All examinations were obtained in the afternoon to avoid diurnal variations (16:00-20:00).41-43 Inclusion criteria were: Best Corrected Visual Acuity (BCVA) between 20/20 to 20/25, spherical equivalent (SE) between ± 3 diopters (D), no systemic or ocular diseases (other than cataract). Eyes with any history of any retinal diseases in the fellow eye were not included. Eyes with spherical equivalent (SE) beyond ±3 D were excluded. Only the left eye was used for the study (image quality allowed for a proper binarization in 136 out of 141 eyes). The data and images of a specific population subgroup from a previously published prospective study were analyzed.38 The original study was approved by the institutional review board of Vissum Alicante. Data were collected from December 2011 to January 2013. The subjects provided informed consent to participate in the research to study the thickness and morphology of the choroid in a healthy population. Participation was offered to subjects who were attending routine ocular 99 examinations and voluntarily agreed to participate, provided they met the inclusion criteria, with no age limitations, and signed an informed consent.38 Image analysis: four parameters were calculated: choroid area, vascular and stromal area and percentage of choroidal vascularity (vascular area/total area). Choroidal stroma and vessel area analysis involved (i) automated binarization of an OCT B-scan and (ii) automated segmentation of the binarized choroid layer using previously validated algorithm.44 We used the central 9 mm from the 12 mm b scans obtained. The automated binarization involved (a) preprocessing, (b) exponential and non-linear enhancement, and (c) thresholding. OCT images were de- noised using the BM3D (block matching and 3D filtering) technique. Subsequently, adaptive histogram equalization considering 8x8 blocks was performed to increase the image contrast to better visualize various structures, especially choroidal vessels. Thresholding was based on Otsu’s thresholding algorithm.45 Next, exponential enhancement (exponentiation factor=6, empirically obtained) was performed on raw OCT images to increase the dynamic range of pixel intensities. This was followed by non-linear enhancement where each row of the resulting image was multiplied by the square of its row number, which ensures the uniform distribution of pixel intensities among stroma and vessel regions. Finally, thresholding, based on a histogram, was employed to obtain the binarized OCT B-scan. At this point, we defined choroid’s inner and outer boundaries to segment the binarized choroid layer. Choroidal segmentation was obtained using a recent method proposed by our group,46 which reported reproducibility of 98-99%. The choroid was localized in two steps, by first identifying the choroid inner boundary (CIB) and then the choroid outer boundary (COB) from the de-noised images. The CIB was found by locating the RPE inner boundary based on the comparative brightness of the RPE, and then using a gradient-based Canny edge operator (threshold 0.4, standard deviation 2) for delineation. Then, the COB was defined by structural similarity (SSIM) index, comparing a scleral window to the neighbourhood of every pixel in the potential COB area. Subsequently, the detected CIB and COB were used in segmenting the choroid layer from the previously obtained binarized images. Finally, the binarized choroid layer was used to quantify the stromal area, vessel area and percentage choroidal vascularity (Figures 1 and 2). The data were correlated with mean choroidal thickness (CT), subfoveal CT (SFCT), age, SE and gender. A horizontal CT profile of the macula was manually created measuring CT (from the posterior edge of RPE to the choroid/sclera junction) under the fovea (SFCT) using the prototype software. The outer aspect of the lamina fusca, rather than the outer limit of the choroidal vessels, was the landmark used to determine the most distal aspect of the choroid. Five further determinations were performed every 1000 μm temporal (T1, T2, T3, T4 and T5) and three more nasal (N1, N2 and N3) to the fovea (Figure 3). An experienced technician determined refractive errors and BCVA using an auto- refractometer (Nidek, Gamagohri, Japan) that was later checked by a certified optometrist. To study the possible age-related evolution of vascular density the study group was divided into 2 sub-groups according to age distribution: Subjects aged 18 or younger (59 eyes) and subjects older than 18 (77 eyes). Mean age was 33.1±24.5 years (from 100 3 to 85). The sample was further divided into 5 groups (0-10, 11-20, 21-40, 41-60 and more than 60 years old) to analyse the possible age-related changes like in previous papers.29 Mean SE was 0.11±1.38 D (from +3 to -3). 136 left eyes from 136 subjects were analysed, 86 eyes (63.2%) were from male and 50 eyes (36.8%) from female subjects (Table 1). For this study, the reference mirror of SS-OCT was placed at the deeper position of the retina so that the sensitivity was higher at the choroid area in macular imaging. A one-line scanning mode, which produces an OCT image that contains 1024 axial scans with a scan length of 12 mm, was employed. Two observers determined CT independently and in a masked fashion. Data obtained were statistically treated using a licensed version of SPSS 17.0 for Windows (SPSS, Chicago, IL). Kolmogorov-Smirnov test was applied for all data samples in order to check normality. Inter groups analysis was performed using the Student t test when samples were normally distributed or Mann-Whitney test when parametric statistics were not possible. The level of significance used was P<0.05. For study correlation Pearson correlation test was used. Results Mean SFCT was 305.77±78.29 mµ (range 144.50 to 539.50) and mean CT was 261.66±61.66 mµ (range 134.6 to 455.28). Mean choroidal area was 0.5554±0.1377 mm2 (range 1.0057 to 0.2738). Mean stromal area was 0.2524±0.0762 mm2 (range 0.1074 to 0.4601), and mean vascular region area was 0.3029±0.0893 mm2 (range 0.1104 to 0.5537). The percentage of choroidal vascularity (vascular area/total area) of the choroid area studied was 54.40±8.35 % (range 33.34 to 77.04, Table 1). Choroid area, vascular region and percentage of choroidal vascular density were statistically higher in the <18-year-old group vs. >18-year-old group (p<0.001 for all three comparisons; Student t test). Stromal region was not different (p=0.46 Student t test), (Table 2). In the same way, there were statistically significant differences when comparing choroid area, vascular region and percentage of choroidal vascular density between all five age groups (p<0.001 for all three comparisons; ANOVA test), being higher in the 0-10-year-old group; but not for stromal region (p=0.71; ANOVA test) (Table 3). There were no differences between men and women. A negative statistical significant correlation between choroid area, vascular region and percentage of choroidal vascular density were found for age (r=-0.396, p=0.000; r=-0.664, p=0.000 and r=-.653, p=0.000 respectively, Pearson correlation) but not for stromal area (r=0.063, p=0.46; Pearson correlation). No significant correlations were found between the four parameters studied, gender and SE. Mean CT and mean SFCT were significant correlated with choroid area (r=0.872, p=0.000 and r=0.802, p=0.000; Pearson correlation). The coefficients of variation (the ratio of the standard deviation [σ] to the mean [μ]) were: choroidal area 26%, stromal region 30%, vascular region 31%, percentage of vascularity 15%, CT 24% and SFCT 26%. Discussion 101 It is necessary to increase our knowledge on age-related variations of the choroidal structure in normal eyes so certain findings in everyday patients can be correctly interpreted. CT, its variations and the modifications of each of its layers have been already studied. 29,46 It would be interesting to understand the changes in stroma or vascular region separately to further explore the pathogenesis of its various diseases. Histologically, the choroid is composed of blood vessels and stroma (pigment cells, smooth muscles, neurons, vascular walls, and connective tissue).47 Using SD- OCT and even SS-OCT, which provides higher penetrance and resolution, it is not possible to differentiate between both these structures. Sonoda et al used a binarization technique to differentiate vascular/luminal areas from stromal areas of the choroid.48 Even though certainty that dark areas represent vascular structures and clearer areas represent stroma is not complete, this has been empirically accepted.49,50 On the other hand, it is known that the amount of eNOS is reduced and the vascular tone decreases with increasing age, which leads to a decrease of circulating blood volume.51,52 Thus, this should also be expected at the level of the choroid, with decreasing vascular areas. This may also be a consequence of a reduction of the number of choroidal vessels with age, as oxidative stress is known to damage endothelial cells. A histologic study showed that the volume of the cellular and interstitial components of the choroid decrease with increasing age.53 More recently, some groups performed a detailed biochemical analysis of the choroid and reported that the CT decreased with increasing age, accompanied by significant decreases in a serine protease inhibitor, fibrillar collagen, and the amount of celular components.54,55 These findings would speak against the findings of no age-related reduction of stromal areas found in this series. Nevertheless, the possibility of inducting changes during sample management should be taken into account, so more studies are necessary to confirm this discrepancy. Sonoda et al 39 studied the proportion of luminal and stromal areas of normal choroids in the OCT images obtained by EDI-OCT, in a prospective study. They reported that the luminal area, the stromal area and the ratio of luminal/stromal area also decreased significantly with increasing age, showing that the area of the vascular lumen decreased more than the stromal area with increasing age. They also found that the age was significantly and negatively correlated with the total choroid area, luminal area, stromal area and the luminal to stromal ratio. However, interpretation of these data is difficult in their study as they used subjects with different ages and SE (from -6 to +3.5D), and both these two are key factors influencing CT. The SE range of our series was of ±3D, with no significant correlation with any of the 4 parameters studied. There was no significant difference in SE between different age groups either. Furthermore, they did not evaluate children. Agrawal et al 40 stated that CVI may potentially be used to assess vascular status of the choroid, after analysing 345 healthy eyes (55% females) from 45 to 85 years old, with EDI-OCT and the binarization technique proposed by Snoda et al.39,48 CT was automatically measured and only right eyes were used. The area of 102 the choroid studied was 1.5 mm wide in the subfoveolar position and CVI was calculated as the proportion of luminal area to total choroid area, being similar to our percentage of vascularity. They concluded that CVI is less variable than SFCT and that it is less influenced by other factors, especially by age. They proposed CVI as a more stable index that could be useful to analyse variations in the choroid when compared to the more classic CT. In their series SFCT was the only factor associated with CVI, whereas stromal area did not have a significant association with SFCT. Clinically, measuring the proportion of vascularity of the eye would provide more information compared to CT measurements alone.40 Our study group was younger than in previous reports (33.1±24.5 vs. 55.9±18.8 vs. 61.5±8.7 years respectively) and with a broader age-span towards younger subjects (from 3 to 85 vs. 22 to 90 vs. 45 to 85 respectively), which could influence our results. Our SE was 0.11±1.38 D (from +3 to -3) vs. -1.23±2.3 D (-6 to 3.5) in Sonoda’s study. Mean SFCT was 305.77±78.29 µm vs. 241.34±97.11 in Agrawal’s study. This difference could be justified by our younger sample.29 Mean choroid area measured it is not comparable between the three studies given the different B-scan lengths used in each study: 9 mm in our study vs. 7.5 or 1.5 mm in the others; and the same happens with stromal and vascular areas. Agrawal’s CVI is a very similar concept to our percentage of vascularity, being of 65.61±2.3% vs. 54.40±8.35% in our sample. In Sonoda’s study stromal and, more significantly vascular area, are correlated with age. However, our main input in relation to previous studies is that stromal area and age are not correlated. OCT angiography and en-face SS-OCT have been used to perform quantitative and qualitative studies of choroidal vascularity.56-59 Nevertheless, our results about choroidal vascular density cannot be compared with the results obtained using OCT angiography or en-face SS-OCT given that only subfoveal axial scans were used to carry our study. Our study has several limitations though. Our study results are limited to normal healthy eyes, therefore cannot be applicable to pathologic eyes. We report results of single fovea-centered, horizontal 12 mm scans, so as the running directions of the choroidal blood vessels are known to be irregular further studies using differently oriented scans would be necessary to prove our results. 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Ferrara D, Waheed NK, Duker JS. Investigating the choriocapillaris and choroidal vasculature with new optical coherence tomography technologies. Prog Retin Eye Res 2016;52:130-55. Figure legends Figure 1: Swept source Optical coherence tomographic images of choroid converted to binarization images of two healthy eyes. The B-scan image (a) is converted to a binary image using software. Choroid limits are delineated (b). The luminal area and the stromal area can be seen. The binarized image and the margin of the traced area are merged, which shows that the traced areas coincide with the dark areas of the choroidal areas of the SS-OCT image (c). Figure 2: Swept source Optical coherence tomographic images of choroid converted to binarization images of two healthy eyes. The B-scan image (a) is converted to a binary image using software. Choroid limits are delineated (b). The luminal area and 107 the stromal area can be seen. The binarized image and the margin of the traced area are merged, which shows that the traced areas coincide with the dark areas of the choroidal areas of the SS-OCT image (c). Figure 3: SS-OCT image of an example of choroidal thickness measures in all different nine locations, from the posterior edge of RPE to the choroid/sclera junction; from N3 position to T5 position. Table legends Table 1: Sample demographics. Age: years. SE: spherical equivalent (diopetrs). BCVA: Best Corrected Visual Acuity (decimal). SFCT: SubFoveal Choroidal Thickness (mµ). MCT: Mean Choroidal Thickness (µm). Choroidal area, Stromal region, Vascular region: mm2. *% Table 2: Choroid area, vascular region, stromal region and percentage of choroidal vascular density in the <18-year-old group vs. >18-year-old group. Table 3: Choroid area, vascular region, stromal region and percentage of choroidal vascular density comparison between all five age groups. 108 Figure 1 Figure 2 109 Figure 3 Table 1 n Mean Standard deviation Minimum Maximum Age 136 33.11 24.53 3 85 SE 134 0.11 1.38 -3 3 BCVA 135 0.97 0.06 0.8 1 SFCT 136 305.77 78.29 144.50 539.50 MCT 136 261.66 61.66 134.67 455.28 Choroidal Area 136 0.5554 0.1377 0.2738 1.0057 Stromal Region 136 0.2524 0.0762 0.1074 0.4601 Vascular Region 136 0.3029 0.0893 0.1104 0.5537 Percentage of vascularity* 136 54.40 8.33 33.34 77.04 Age: years. SE: spherical equivalent (diopetrs). BCVA: Best Corrected Visual Acuity (decimal). SFCT: SubFoveal Choroidal Thickness (mµ). MCT: Mean Choroidal Thickness (µm). Choroidal area, Stromal region, Vascular region: mm2. *% 110 Table 2. ≤18 years >18 years P* N 59 77 Choroidal Area 0.6149± 0.1202 0.5098± 0.1334 <0.001 Stromal Region 0.2480± 0.0727 0.2558± 0.0791 0.55 Vascular Region 0.3668± 0673 0.2540± 0715 <0.001 Percentage of vascularity** 60.16± 6.51 49.98± 6.79 <0.001 Mean ± standard deviation mm2 * Student t test **% Table 3. 0-10 years 11-20 years 21-40 years 41-60 years >60 years P* N 38 22 26 20 30 Choroidal Area 0.6246± 0.1000 0.5919± 0.1500 0.5470± 0.1533 0.4764± 0.0920 0.5010± 0.1369 <0.001 Stromal Region 0.2473± 0.0568 0.2478± 0.948 0.2540± 0.0769 0.2405± 0.0645 0.2690± 0.0935 0.712 Vascular Region 0.3772± 0.0641 0.3441± 0.0711 0.2930± 0.0848 0.2358± 0.0484 0.2320± 0.0598 <0.001 Percentage of vascularity** 60.56± 5.80 59.25± 7.60 53.67± 5.30 49.76± 7.09 46.76± 6.32 <0.001 Mean ± standard deviation mm2 * ANOVA test **% 111 5. DISCUSIÓN 112 5. DISCUSIÓN La investigación de la coroides ha sido tradicionalmente difícil. La AVI permite la visualización de los vasos coroideos,27,59 y los avances más recientes en tecnología OCT han añadido información de la anatomía de coroides en forma de cortes trasversales.72 La mejora de las imágenes de capas más profundas proporcionada por el SD-OCT ha permitido el estudio de la coroides en todo su espesor, incrementando nuestro conocimiento sobre la fisiopatología y la etiología de multitud de enfermedades oculares.45,57,58,60–63,66–69,73 Prototipos de SS-OCT con mayor longitud de onda (1050-1060 nm) se han utilizado en los últimos años mejorando la calidad de las imágenes obtenidas. Programas más rápidos y con mayor definición pueden ayudar a superar el efecto barrera del EPR y las alteraciones inducidas por el movimiento.74,75 Las publicaciones sobre grosor coroideo establecen un adelgazamiento progresivo de la coroides con la edad.41,74,76–79 Margolis describió un adelgazamiento de 1,56 µm por año de vida.41 Por otra parte, Agawa75 y Li80 reportaron que la correlación entre el grosor coroideo y la edad no existía en ojos con longitudes axiales por debajo de los 25 mm. El efecto de la edad en el grosor coroideo de la población pediátrica no había sido estudiado con anterioridad. En nuestra serie de pacientes, el OCT Swept-Source permitió la visualización de la coroides hasta la esclerótica en todos los casos con imágenes de buena calidad, permitiendo la correcta medida del grosor coroideo. El grosor coroideo medio subfoveal fue muy parecido entre los dos grupos (312.9±65.3 µm en el grupo de estudio vs. 305.6±102.6 µm en el grupo control) (P=0.19). El grosor coroideo macular 113 medio fue ligeramente diferente (285.2±56.7 µm en el grupo de estudio vs. 275.2±92.7 µm en el grupo control) (P=0.08). El grosor coroideo subfoveal y nasal fue similar en los dos grupos; sin embargo, el grosor coroideo temporal fue mayor en el grupo de estudio en la población pediátrica alcanzando la significación estadística en los puntos de medida T2 y T3 (P=0.014 y P=0.002 respectivamente), quedándose cerca de la misma también en T1 (P=0,059). Estos datos sugieren que el grosor coroideo temporal parece reducirse con la edad, llegando a ser menor que el grosor subfoveal en el adulto. Este hallazgo se ve reforzado por la correlación negativa estadísticamente significativa que existe entre el grosor coroideo y la edad para todo el grupo en los puntos de medida T1, T2 y T3. Los resultados de grosor coroideo en nuestro grupo control son similares a los reportados previamente en la literatura científica. La edad media de nuestro grupo control fue de 53,2±15,6 años, similar a la publicada en otras series, haciendo las muestras comparables entre ellas.41,76,77 De todas formas, también existen publicaciones con poblaciones de estudio más jóvenes que nuestro grupo control;74,75,79,80 esta información debe de considerarse con precaución dada la fuerte correlación hallada entre la edad y el grosor coroideo en los adultos.41 El grosor coroideo medio subfoveal de nuestro grupo control (312,9±65,3 µm) es mayor que el hallado por otros autores: Margolis describe un grosor subfoveal medio de 287±76 µm,41 Manjunath77 272±81 µm y Ouyang 297±82 µm.79 El grosor coroideo subfoveal fue mayor en otras series publicadas: Ikuno 354±111µm;74 Agawa 348±73 µm;75 Branchini 337-347 µm;72 Li 342±118 µm.80 La 114 edad media de los pacientes de estos estudios es menor que la edad media de los pacientes de nuestro grupo control. Ikuno determinó el grosor coroideo macular (en lugar del subfoveolar) usando dos aparatos diferentes.45 Según sus datos, el grosor coroideo medio es 292,7 µm (utilizando un SS-OCT clásico) y de 283,7 µm (con EDI-OCT), en una población 37,6 años vs. 275,2 µm en una muestra de 53,2 años en nuestra serie. Branchini determinó el grosor coroideo en un grupo de pacientes de 35,2 años de edad utilizando tres plataformas diferentes. El grosor coroideo en posición N1 fue de 313,3 µm con un OCT RTVue, 319,8 µm con Spectralis y 320,4 µm con Cirrus; en nuestro grupo control fue de 290 µm en N1. Branchini encontró un grosor 335,9 µm con RTVue, 346,8 µm con Spectralis y 339,3 µm con Cirrus en posición T1; en esa posición el grosor fue de 299 µm en nuestro grupo control. Según Ikuno,74 el grosor coroideo medio fue de 227 µm a 3 mm nasal a la fóvea y 337 µm a 3 mm temporal a la fóvea en un grupo de pacientes de 39,4 años. En la serie de Margolis41 el grosor coroideo fue de 203 µm 2 mm nasal a la fóvea y 268 µm 2 mm temporal a la fóvea en una población que promediaba los 50,4 años de edad. En nuestra serie el grosor coroideo fue de 205 µm en N3 (2,25 mm nasal a la fóvea) y 281 µm en T3 (2,25 mm temporal a la fóvea).Sin embrago, la mayoría de estas determinaciones son muy similares dada la corrección por edad de 1,56 µm por año que establece Margolis. No se pudieron comparar estos datos con nuestro grupo de estudio dado que este tipo de medidas en pacientes pediátricos no habían sido publicadas hasta el momento. Sin embargo, en nuestra serie no hemos encontrado diferencias entre niños y adultos a excepción de la coroides temporal a la fóvea. La edad a la que la 115 coroides comienza a adelgazarse como algún autor ha sugerido aún está por determinar. Estaría en probable relación con las alteraciones y modificaciones vasculares propias de la edad. Hallamos una correlación estadísticamente significativa entre el grosor coroideo y la edad, el grosor coroideo y el equivalente esférico (EE) y el grosor coroideo subfoveal y el EE en el grupo de estudio. El perfil topográfico de grosor coroideo del grupo control en nuestra serie es más grueso en posición subfoveal, seguido por el sector temporal y nasal en este orden. Este perfil es similar al descrito previamente en otras series. Pero el perfil del grupo estudio fue diferente (mayor en el sector temporal con 320, 322 y 324 µm, seguido por el subfoveal con 312 µm y por el nasal con 281, 239 y 195 µm). Las diferentes plataformas de OCT utilizadas para las investigaciones que aparecen en la literatura proporcionan diferentes niveles de calidad de imagen, permitiendo una visualización más o menos adecuada de la línea que separa la coroides de la esclerótica. En nuestra muestra todos los pacientes fueron examinados con un SS-OCT, mostrando de forma clara el límite posterior de la coroides. Los porcentajes sobre la proporción de coroides que fueron capaces de ser medidas correctamente en los diferentes estudios publicados ha sido de 74%77 a 90%76 de los ojos examinados por un Cirrus HD-OCT y de 95.8% de los ojos examinados por un Heildeberg EDI-OCT. Dos artículos que comparan diferentes OCT aportan una capacidad de medida de 96.,4% y 90.,7% respectivamente.72,81 La elevada correlación intraclase entre los tres observadores independientes (de 0,91 a 0,98) remarca la precisión de medida que proporciona el SS-OCT. El adelgazamiento progresivo de la coroides con la edad es un factor clave 116 para establecer el papel que juega la coroides en la patología retiniana. Aunque Ikuno74 llevó a cabo un estudio similar en el que describió el grosor de la coroides en sujetos japoneses sanos, no incluyó pacientes de un rango de edad amplio. Además, la pérdida de grosor coroideo subfoveal que puede deducirse de nuestro estudio es de 0,852 µm al año, lejos de las 4,32 µm por año predichas por otros estudios anteriores, alcanzando pues diferentes conclusiones. La primera de nuestras publicaciones representó la serie más grande publicada hasta la fecha sobre las mediciones del grosor coroideo con tecnología SS- OCT. Se eligió esta plataforma para llevar a cabo el estudio ya que sus características permiten un estudio de la coroides con más precisión que la anteriormente publicada en otros estudios que utilizaron otros modelos de OCT desde un punto de vista teórico. Esta precisión ha sido demostrada en artículos publicados por Copete47 o Ruiz-Moreno,46 utilizando este mismo prototipo. El grosor de la coroides pudo ser determinado en el 100% de los pacientes de sus respectivos estudios. En nuestro caso, usando el SS-OCT la coroides pudo medirse con éxito en el 100% de los ojos estudiados. Wei82 midió con éxito el 93,2% de 3.468 ojos (el estudio más grande publicado hasta la fecha) utilizando un OCT de Heidelberg Spectralis con la modalidad EDI. La elección de Manjunah77 fue el Cirrus HD-OCT sin tecnología EDI, y fue capaz de medir la coroides en sólo el 74% de los ojos. Yamashita81 y Shin83 utilizaron un Topcon SD-OCT y fueron capaces de medir correctamente un 63,3 % y un 90,7% de los ojos respectivamente. Copete47 comparó la capacidad del SD-OCT y el SS-OCT obteniendo buenas imágenes en el 70,4 y el 100% respectivamente. 117 Margolis41 estudió un grupo de 54 ojos con un EE medio de -1,3 dioptrías (D) hallando un grosor coroideo subfoveal medio 287 µm, similar a los resultados de Flores-Moreno64 y a los de Manjunath77 que publicó grosores de 272 µm en 34 ojos de una muestra de 51,1 años de edad. Xu84 comparó diabéticos frente a no diabéticos, hallando un grosor coroideo subfoveal de 266 µm en el grupo control (1.795 sujetos). Como se ha comentado con anterioridad, la coroides también se ha estudiado en la población pediátrica con tecnología SS-OCT.46 Park85 en un estudio más reciente con pacientes pediátricos encontró un grosor coroideo subfoveal medio de 348,4 µm en los 48 ojos que evaluó. En este estudio el grosor coroideo subfoveal medio fue de 301,89 µm. Este dato es entorno a 10 µm mayor que el encontrado por otros autores que utilizaron tecnología SD-OCT. Es lógico pensar que la introducción de pacientes en edad pediátrica en nuestra muestra pueda haber llevado a este ligero incremento. De manera interesante, estos resultados coinciden con los encontrados por nuestro grupo, que usando el mismo OCT hallaron prácticamente el mismo grosor en su grupo de pacientes adultos. El SS-OCT puede dar lugar a unas medidas ligeramente superiores a las proporcionadas por los SD-OCT. Esto puede ser explicado por una mayor calidad de las imágenes y por el hecho de que nuestras medidas fueron tomadas desde límite más posterior del EPR hasta el límite más externo de la lámina fusca, en lugar de hasta el límite externo de los vasos coroideos. Ruiz-Moreno con un SS-OCT y Flores-Moreno con un SD-OCT proporcionaron datos sobre grosores coroideos maculares medios. Ruiz-Moreno obtuvo un grosor de 118 275 µm en los adultos que estudió frente a las 285 µm que obtuvo en los pacientes pediátricos. Flores-Moreno obtuvo grosores de 257 µm en los pacientes de su grupo control, frente a 115 µm en los altos miopes estudiados. Nosotros hallamos un grosor coroideo macular medio de 258,69 µm, similar a los resultados proporcionados por estos autores. Sin embargo, nuestro estudio fue el primero en analizar grosores a lo largo de un corte de 8 mm de la mácula, centrado en la fóvea de los pacientes, por los 6 mm estudiados con anterioridad por otros autores. Esto fue gracias a que el SS-OCT proporciona imágenes de gran calidad también en el sector más temporal, que es precisamente donde se produjo este incremento de 2 mm de la zona estudiada. Esto podría ser clínicamente relevante dado que es en este sector temporal donde se producen las variaciones más significativas en términos de grosor. No se encontraron diferencias estadísticamente significativas entre hombres y mujeres. Los sujetos de ambos sexos mostraron perfiles prácticamente idénticos con diferencias mínimas en el sector más temporal, en el que las mujeres mostraron tener una coroides ligeramente más delgada. El grosor subfoveal medio fue 303,9 µm en hombres vs. 296,4 µm en mujeres. Esta pequeña diferencia no significativa, se explica con facilidad dada la diferencia de edad entre ambos grupos (30,39 años en el caso de los hombres y 37,48 en las mujeres). El grosor fue cercano a 7 µm mayor de media en el caso de los hombres, pero en esta muestra eran 7 años más jóvenes. Si hacemos caso a las fórmulas de correlación que se obtienen de nuestra base de datos, el grosor coroideo en un grupo pareado por edad y sexo hubiera sido prácticamente idéntico. 119 Hay estudio previos que utilizan OCT de alta penetración para el estudio del grosor coroideo en adultos, pero no llevaron a cabo comparaciones entre diferentes grupos de edad. En el presente estudio se compararon las diferencias de grosor entre diferentes grupos de edad, a intervalos de 20 años. Sin embargo, los dos grupos más jóvenes se formaron a intervalos de 10 años (0-10 y 10-20 años) dado que el crecimiento ocular ocurre teóricamente en dos fases: la primera hasta los 6 años de edad, alcanzando la emetropización; y la segunda de los 7 a los 19 años, momento en el que tiene lugar la expansión global de los tejidos oculares.86 El perfil de grosor coroideo fue diferente en cada grupo y la coroides se adelgazó progresivamente en cada grupo de edad, mostrando diferencias estadísticamente significativas entre los diferentes grupos. Esta reducción fue claramente progresiva hasta los 40 años, momento en el que tuvo lugar la reducción más llamativa, pasando de las 313,9 µm en el grupo de 21 a 40 años de edad a las 264,6 µm en el grupo de 41 a 60 años (49 µm de diferencia). De manera interesante, a partir de los 40 años (41-60 vs. >60) las diferencias de grosor coroideo no fueron estadísticamente significativas en lo que respecta al grosor macular medio ni al grosor subfoveal medio. Si tenemos en cuenta las diferencias halladas al considerar un solo ojo por paciente (como recomienda en su artículo Ray87) o ambos, éstas fueron mínimas. Se obtuvieron predicciones de reducción del grosor coroideo de 9,4 vs. 10 µm por década de vida en grosor macular medio; y de 8,5 vs. 8,87 µm por década en grosor coroideo subfoveal. Sabemos pues que la coroides es más gruesa en localización subfoveal y que este grosor se reduce de manera progresiva hacia el sector temporal, reduciéndose 120 de una manera más acusada hacia el nervio óptico,88,89 con un contorno o un perfil de forma cóncava.90 Adhi describió alteraciones significativas en las características morfológicas de la coroides de la mayoría de los ojos de pacientes con retinopatía diabética que estudió. Hallaron que la interfaz esclero-coroidea presentaba una forma irregular, con un punto de inflexión en 8 de los 9 ojos estudiados con retinopatía diabética no proliferativa (89%), en 9 de los 10 ojos estudiados con retinopatía diabética proliferativa (90%) y en 13 de 14 ojos (93%) con edema macular diabético. Por el contrario, no hallaron esta alteración en ninguno de los 24 controles que se emplearon para su estudio.90 La presencia de un perfil esclero-coroideo irregular con un punto de inflexión y un adelgazamiento focal de la coroides se ha descrito en algunas enfermedades como la retinopatía diabética y la DMAE.73,90,91 Dolz-Marco reportó un caso de protrusión focal inferotemporal de la esclerótica con un adelgazamiento cóncavo de la coroides, rodeado de coroides normal.92 Los autores plantearon la hipótesis de que este hallazgo podría estar en relación con la inserción del músculo oblicuo inferior, que podría dar lugar a una compresión de la coroides, sin ser capaces de pronunciarse sobre la situación de la esclerótica, al no ser completamente visible. En nuestra serie de pacientes no se halló ninguna anomalía en el fondo de ojo que pudiera justificar este engrosamiento focal en los pacientes que presentaban la inflexión esclero-coroidea. El análisis con SS-OCT no detectó ninguna alteración de la coroides en estos pacientes, aparte de la inflexión. Por otro lado se identificó una imagen que podría representar el borde superior de la inserción del músculo oblicuo 121 inferior en la esclerótica, junto con un fina línea hiper-reflectiva, paralela a la esclerótica que podría corresponder la porción intraescleral de una arteria ciliar posterior larga. La razón por la cual los vasos de la retina aparecen como hiper-reflectivos en las imágenes de OCT mientras que los vasos coroideos aparecen hipo-reflectivos está en relación con la velocidad de las células sanguíneas que los recorren. La velocidad del flujo sanguíneo en los vasos de la coroides es mucho mayor que en los vasos de la retina. La elevada velocidad de los eritrocitos en la coroides hace que la señal de interferencia desaparezca, resultando en una señal nula en el interior de sus vasos. Por el contrario, la velocidad de los mismos es mucho menor en la circulación retiniana, lo que lleva a que la señal de interferencia produzca una imagen hiper- reflectiva. Los estudios publicados sobre la anatomía de la parte más posterior del globo ocular, los músculos y sus vasos indican que el límite superior de la inserción del músculo oblicuo inferior, coincide con el meridiano horizontal del ojo a unos 5,7 mm de la fóvea. 93 La distancia media del punto de inflexión a la fóvea fue de 4.427 µm en nuestra muestra y situado siempre en el sector temporal de la mácula. La presencia de este punto de inflexión está probablemente en relación con la inserción del músculo oblicuo inferior, que como se ha comentado con anterioridad se halla a unos 5,7 mm de la fóvea. La tracción ejercida sobre la esclerótica por el tendón de este músculo puede ser la responsable de una separación de la pared de la esclerótica y el consiguiente engrosamiento coroideo. Basándose en las 122 descripciones anatómicas de la región, la línea paralela, hipo-reflectiva a este nivel puede corresponder con el segmento intraescleral de una arteria ciliar posterior larga temporal. El hecho de que este hallazgo sea más frecuente entre los pacientes más jóvenes, especialmente entre los menores de 15 años, se puede explicar por la menor rigidez de la esclerótica entre la población más joven,94 lo que podría facilitar la deformación inducida por la tracción muscular inducida por el tendón del músculo oblicuo inferior. Son varios los estudios publicados sobre el grosor coroideo en poblaciones sanas. Ding estudió el grosor coroideo de 210 voluntarios sanos (420 ojos) sin enfermedades oftalmológicas utilizando un EDI-OCT y en múltiples localizaciones de la mácula: En posición subfoveal y a 1 y 3 mm en dirección superior, inferior nasal y temporal a la fóvea. Sin embargo, los autores no estudiaron la existencia de diferencias entre los ojos.78 Ikuno estudió el grosor y el perfil de la coroides de 86 ojos de 43 pacientes sanos con un SS-OCT, así como la correlación entre el mismo con la longitud axial, el error refractivo y la edad, pero no analizó las diferencias existentes entre ojos derechos e izquierdos.74 Por el contrario, Chen89 describió la variación inter-ocular en lo que respecta al grosor coroideo en una población de 50 adultos sanos usando tecnología EDI-OCT. Midió el grosor subfoveal y realizó medidas en 4 localizaciones paramaculares: 3 mm superior, inferior, nasal y temporal. Las diferencias encontradas fueron de 1 µm en localización subfoveal, 13,6 µm nasal a la fóvea y -2,9 µm temporal a la misma (diferencias no estadísticamente significativas). Lo que parece claro es que muestran 123 una tendencia hacia un mayor grosor del sector nasal de la mácula en ojos derechos (14 µm). Al comparar estos resultados con los obtenidos en el presente estudio en las mismas localizaciones (subfoveal, N3 y T3), se observa una diferencia entre ojos derechos e izquierdos de 7,45 µm en grosor subfoveal medio, 1,43 µm en T3 y 14,44 µm en N3. Las diferencias entre grosor coroideo nasal medio (p=0,0002), grosor coroideo nasal medio en hombres (p=0,003) y en mujeres (p=0,03), y el grosor coroideo medio en cada punto de medida nasal a la fóvea (p=0,001; 0,002 and 0,08 respectivamente) fueron estadísticamente significativas. La coroides resultó ser más gruesa en los ojos derechos en cada punto de medida nasal para ambos sexos, a excepción de las mujeres en N1 (p=0,259). La correlación entre el grosor coroideo inter-ocular obtenido por Chen fue más fuerte para el grosor coroideo subfoveal (r=0,90; p<0,001, test de correlación de Spearman), mientras que para los puntos nasales fue de r=0,83 (p<0,001). Para los puntos de medida temporales fue más débil (r=0,49; p<0,001). En la presente serie el estudio de correlación para el grosor coroideo medio en posición subfoveal, N3 y T3 fue similar (r=0,860; 0,853 y 0,754 respectivamente, p<0,001; test de correlación de Spearman). El perfil de grosor coroideo descrito en su artículo es muy similar al perfil descrito en el presente trabajo comparando ojos derechos e izquierdos. Es difícil explicar la evidencia fuerte y consistente que existe sobre un mayor grosor coroideo en ojos derechos en el sector nasal de la mácula en la población sana. Una posible explicación podría ser un distinto flujo sanguíneo entre ambos ojos dada la falta de simetría del arco aórtico. Dicha asimetría ha sido usada como la 124 hipótesis que podría explicar las diferencias de incidencia y prevalencia de determinadas patologías vasculares entre ojos derechos e izquierdos, como es el caso de la endoftalmitis bacteriana metastásica95 y las oclusiones arteriales retinianas.96-99 La circulación coroidea tiene su origen en las arterias ciliares posteriores cortas, que penetran la esclerótica alrededor del nervio óptico en un número que varía entre 10 y 20; la mitad nasal de la coroides macular estudiada en este trabajo está pues directamente suministrada por estas arterias ciliares posteriores cortas. Éstas son ramas derivadas de la arteria oftálmica, que es una rama de la carótida interna, que a su vez es rama de la arteria carótida común.99 La diferencia generada por el hecho de que el origen de la arteria carótida común derecha yace en el tronco braquiocefálico, mientras que la carótida común izquierda sale directamente de la aorta, es posiblemente la responsable de un flujo más directo en la mitad derecha de este árbol vascular.95 Dado que la mayor parte de la estructura coroidea está formada por tejido vascular (la densidad vascular media de la coroides externa ronda el 87%),100 un teórico flujo sanguíneo más elevado en el ojo derecho con relación al ojo izquierdo a través de las arterias ciliares posteriores podría explicar por qué la coroides nasal es más gruesa en los ojos derechos como se afirma en este estudio y como fue sugerido previamente por Chen.89 En este estudio, así como en trabajos precedentes se utilizó el EE en lugar de la longitud axial dado que el procedimiento es menos invasivo y más rápido, ya que anteriores indicaciones en la literatura demuestran que la refracción, más fácil de 125 obtener, posee la misma capacidad para generar los modelos necesario que la longitud axial.101 La coroides, como se ha mencionado anteriormente, se adelgaza con la edad.102 Nuestro grupo de investigación analizó la coroides de individuos sanos a través de un amplio rango de edades (de 3 a 95 años), encontrando que el perfil de grosor de la coroides era similar entre los diferentes grupos de edad, adelgazándose progresivamente, especialmente a partir de los 40.88 Esmaeelpour utilizó un SD-OCT (longitud de onda de 840 nm) con un protocolo de exploración en mapa que contenía 512 A-scans y 512 B-scans a través de un campo de 36º x 36º para analizar de manera automática las capas de la coroides de 45 ojos de pacientes sanos con una edad media de 44 años (de 23 a 84 años).103 El grosor coroideo medio del área analizada fue de 228 µm y el grosor de la capa de Haller y de la capa de Sattler + coriocapilar fueron de 141 µm y de 87 µm respectivamente. Adhi90 estudió 24 ojos de 24 pacientes de una media de edad de 64 años. Las medidas de grosor coroideo fueron tomadas de manera manual mediante el software con el que está equipado el SD-OCT que utilizó y se alcanzó a ver correctamente el límite entre la esclerótica y la coroides en el 79% de los casos. El protocolo de imagen empleado consistió en escáneres de 1 línea centrados en la fóvea. El grosor coroideo subfoveal fue de 276 µm, el grosor de la capa de Haller subfoveal fue de 224 µm y el grosor de la capa de Sattler + coriocapilar subfoveal fue de 52 µm. 126 Empleando este mismo método Branchini estudió la coroides de 42 ojos de 42 pacientes sanos de una edad media de 52 años, hallando el límite entre esclerótica y coroides en el 92% de ellos.104 Midieron de manera manual el grosor de la coroides y el de sus capas en localización subfoveal, utilizando a su vez dos puntos más de medida a 750 µm a cada lado de la fóvea. Encontraron que el grosor coroideo subfoveal de su muestra fue de 256 µm. El grosor de la capa de Haller y el de la capa de Sattler + la coriocapilar fue de 204 y 53 µm respectivamente. En el presente estudio se analizaron 169 ojos de 169 pacientes sanos utilizando un SS-OCT, siendo la mayor muestra empleada para el estudio de las capas de la coroides hasta donde alcanza nuestro conocimiento. La edad media fue algo menos que en los otros estudios similares, siendo de 33,5 años. Por otra parte la visualización del límite entre la esclerótica y la coroides fue posible en el 100% de los casos. Las cifras obtenidas para grosor subfoveal, capa de Haller y capa de Sattler + coriocapilar (305,8, 208,6 y 87,3 µm respectivamente) son mayores que las publicadas por otros autores. Un hallazgo similar fue presentado en otra publicación previa de nuestro grupo en la que se comparó el grosor coroideo obtenido con un SS-OCT en relación al publicado por otras series que emplearon SD-OCT, obteniendo una diferencia de entorno a las 10 µm a favor de la tecnología Swept-Source.88 Este hecho junto a que nuestra muestra es considerablemente más joven que en las otras publicaciones podría explicar la diferencia en los resultados obtenidos. Con el objetivo de evaluar el grosor de la coroides y de sus capas a través de la mácula se añadieron 8 puntos de medida adicionales (3 en sentido nasal y 5 más en sentido temporal). Los valores medios de grosor coroideo, capa de Haller y capa 127 de Sattler + coriocapilar fueron de 260,1; 180,2 y 78,2 respectivamente. El perfil de grosor coroideo mostró una morfología similar entre los diferentes grupos de edad, reduciéndose progresivamente con la edad, al igual que el grosor de la capa de Haller (p<0,001). La capa de Sattler + coriocapilar mostró una tendencia hacia la reducción con la edad sin alcanzar la significación estadística (p=0,124). Nuestros modelos de regresión lineal predijeron una reducción de 0,93 µm de grosor coroideo por año de edad; 0,69 de las cuales se debieron al adelgazamiento de la capa de Haller, que fue responsable del 75% del adelgazamiento ligado a la edad de la coroides. No se hallaron diferencias relacionadas con el sexo de los pacientes ni en grosor coroideo ni en ninguna de sus capas en ninguno de los grupos. Nuestro estudio muestra que la mayoría de la pérdida de grosor debida a la edad es a expensas de los grandes vasos. Estos hallazgos en ojos sanos van en desacuerdo con los hallazgos histopatológicos en ojos con una evidente esclerosis senil, que muestran mayoritariamente una atrofia de los pequeños y medianos vasos coroideos.102, 105 Spaide102 publicó que la pérdida de vasos sanguíneos coroideos asociada a la edad estaba en directa relación con la pérdida de los vasos visibles, concluyendo que es una enfermedad de pequeño vaso que afecta a la coroides. Sin embargo, sus datos no son comparables con nuestros resultados ya que Spaide sólo incluyó en su estudio a pacientes con coroides por debajo de 125 µm. Un artículo de Sarks describe únicamente dos casos cuya retinoscopia mostraba áreas bien demarcadas de esclerosis coroides que estaban en probable relación con una DMAE atrófica, mientras que en nuestro estudio sólo se analizaron pacientes sanos.105 Este 128 adelgazamiento ligado a la edad podría ser un factor clave para establecer el posible papel de la coroides en multitud de patologías retinianas. Entre las limitaciones de este estudio se puede mencionar el hecho de que las medidas del grosor coroideo fueron realizadas en todos los casos de una forma manual, por dos observadores experimentados en el análisis de imágenes de la coroides. Actualmente existe un software comercialmente disponible para el Topcon DRI OCT-1 (Ver. 9.01.003.02) que permite la segmentación automática y la medida del grosor coroideo en los modos 3D y escáner radial, sin embargo su precisión en la práctica clínica aún está por demostrar. Este trabajo incluye el primer estudio publicado sobre población infantil y el cálculo de su grosor coroideo utilizando tecnología SS-OCT. Son pocos los estudios que han sido llevados a cabo hasta la fecha en adultos de diferentes rangos de edad empleando esta plataforma. Otra limitación de este estudio es el hecho de que sólo se estudiaron los perfiles coroideos en el plano horizontal, el uso del EE en lugar de la longitud axial, el no haber realizado correcciones según otras variables como puedan ser la paquimetría, la presión intraocular, medicación sistémica o presión arterial y la imposibilidad de determinar si los vasos de la coroides nasal son más gruesos o bien más numerosos en ojos derechos que en izquierdos. Esto se debe a que la tecnología actual no permite un análisis adecuado y preciso del grosor de los vasos de la coroides, y no hay forma de saber si se está estudiando el diámetro máximo de un vaso dada la irregularidad anatómica de los mismos. 129 Como otra limitación, la capa coriocapilar no pudo ser diferenciada de la capa de Sattler debido a las limitaciones en la tecnología de imagen actual descritas con anterioridad, a pesar del elevado índice de correlación intraclase. En conclusión y según nuestros resultados podemos dar respuesta a las hipótesis iniciales planteadas afirmando que el grosor coroideo de la mácula es similar entre la población pediátrica y adulta. El perfil de grosor coroideo es distinto en la población infantil, con un grosor máximo en posición temporal. Este perfil es muy similar entre los distintos grupos de edad en la población adulta, con la coroides adelgazándose progresivamente con la edad, especialmente cuando se compara a los pacientes mayores de 40 años con adultos más jóvenes o con la población infantil. La mayor variación de grosor coroideo con la edad tiene lugar en el sector temporal. Una inflexión en la interfaz esclero-coroidea con un adelgazamiento focal de la coroides puede considerarse como una variación de la normalidad sin significación clínica, especialmente entre la población joven. Más estudios que incluyan el área macular al completo incluyendo la inserción del músculo oblicuo inferior en su totalidad y con mayor número de pacientes serán necesarios para establecer las características finales de estas alteraciones, así como su incidencia en la población normal. La zona nasal del perfil de grosor coroideo de la mácula de la población normal es más gruesa en ojos derechos que en ojos izquierdos. Por el contrario, el grosor subfoveal y temporal no fueron estadísticamente diferentes al comparar ambos ojos entre sí. 130 La coroides y la capa de Haller sufren un adelgazamiento progresivo con la edad y sus perfiles muestran características similares entre los diferentes grupos etarios. Este adelgazamiento asociado a la edad va a ser principalmente a expensas de la capa de Haller. Serán necesarios más estudios para probar los resultados de esta investigación. 131 5. DISCUSSION Choroidal research has always been difficult. Indocyanine green angiography (ICGA) permits visualization of choroidal vessels,27,59 and recent advances in OCT technology have added cross-sectional information about the choroid.72 Improved deep imaging provided by SD-OCT has permitted cross-sectional research of the choroid, increasing our knowledge of the pathophysiology and etiology of several ocular conditions. 45,57,58,60–63,66–69,73 Longer wavelength SS-OCT prototypes (1050-1060 nm) have been used in patients improving image quality. Faster and higher quality software may overcome retinal pigment epithelium (RPE) barrier effect and movement artifacts.74,75 Papers on choroid thickness (CT) report a progressive choroidal thinning with age.41,74,76–79 Margolis described 1.56 µm thinning for each year of life.41 On the other side, Agawa75 and Li80 reported that correlation between CT and age did not exist in eyes with axial length < 25 mm. The effect of age on CT in children has not been studied previously. In our series of eyes, SS-OCT allowed visualization of CT in all the cases with high quality images permitting CT determination. Mean subfoveal CT was very similar in both groups (312.9±65.3 µm in SG vs. 305.6±102.6 µm in CG) (P=0.19). Mean macular CT was slightly different (285.2±56.7 µm in SG vs. 275.2±92.7 µm in CG) (P=0.08). Subfoveal and nasal CT was similar in both groups; however temporal CT was higher in SG with statistical significance in T2 (P=0.014) and in T3 (P=0.002) and near significance in T1 (P=0.059). These data suggest that temporal CT might decrease with age and become thinner than subfoveal CT in the adult. This finding is 132 reinforced by the significant inverse correlation between CT and age in the whole group at T1, T2 and T3. The results of CT in our CG are similar to those previously reported in the literature. Mean age in our CG was 53.2±15.6 years, similar to that of other series.41,76,77 We have found reports with patient populations younger than our CG;74,75,79,80 this information should be carefully considered due to the strong correlation found between age and CT among adults.41 Mean subfoveal CT in our CG (312.9±65.3 µm) is higher than those reported in other series. Margolis reported an average subfoveal CT of 287±76 µm,41 Manjunath77 272±81 µm and Ouyang 297±82 µm.79 Subfoveal CT was higher in other series: Ikuno 354±111µm;74 Agawa 348±73 µm;75 Branchini 337 to 347 µm;72 Li 342±118 µm.80 Patients in these last five series were younger than those in our CG. Ikuno determined macular CT (instead of subfoveal CT) using two different equipments.45 According to Ikuno, average CT is 292.7 µm (using SS-OCT) and 283.7 µm (with EDI-OCT), in a population averaged 37.6 years of age vs. 275.2 µm and 53.2 years in our series. Branchini et al. determined CT in a group of patients aged 35.2 years using three different systems. CT at N1 position was 313.3 µm with RTVue, 319.8 µm with Spectralis and 320.4 µm with Cirrus; in our CG CT was 290 µm at N1. Branchini found CT to be 335.9 µm with RTVue, 346.8 µm with Spectralis and 339.3 µm with Cirrus at T1 position; in our CG CT was 299 µm at T1. According to Ikuno,74 average CT was 227 µm 3 mm nasal to the fovea and 337 µm at 3 mm temporal to the fovea in a group of patients averaged 39.4 years of age. In Margolis’ series41 CT was 203 µm at 2 mm nasal to the fovea and 268 µm at 2 mm temporal to the fovea 133 in a population averaged 50.4 years. In our series, CT was 205 µm at N3 (2.25 mm nasal to the fovea) and 281 µm at T3 (2.25 mm temporal to the fovea). However, most of these determinations are very similar considering Margolis’ age-correction for CT (1.56 µm reduction per year). We are unable to compare these data with those from our SG, since such data have not been previously reported. However, in our series we have not found such differences between adults and youngsters except for the choroid temporal to the fovea. The age at which subfoveal CT starts to decrease as has been suggested by some authors is still to be determined. This decline is probably related to aging vascular changes. We have found a significant correlation between macular CT and age, macular CT and spherical equivalent (SE) and between SE and subfoveal CT among the SG. The topographic profile of CT in the CG in our series is highest in the subfoveal area, followed by the temporal and the nasal areas. This profile is similar to those previously published in other series. However, CT profile was different among the SG (highest in the temporal choroid with 320, 322 and 324 µm, followed by subfoveal choroid with 312 µm and the nasal choroid with 281, 239 and 195 µm). OCT devices reported in the literature provide different quality of imaging, permitting a more or less adequate visualization of the line delimiting the choroid and the sclera. In our series all the patients examined by SS-OCT, showed a clearly defined, measurable posterior portion of the choroid. Measurable choroidal thickness has been reported in 74%77 to 90%76 of the eyes examined by Cirrus HD- OCT and in 95.8% of the eyes examined by Heildeberg EDI-OCT. Two papers comparing OCT equipment report 96.4% and 90.7% measurability.72,81 Choroidal 134 visualization was better in those studies using longer wavelength equipment. The high intraclass correlation coefficient (0.91 to 0.98) for the three independent observers highlights SS-OCT accuracy in CT determination. Age dependant thinning is a key factor to establish the role of the choroid in retinal pathology. Although Ikuno et al74 performed a similar study in which they described the choroidal thickness in healthy Japanese subjects, their study did not include a wide variation of ages. Furthermore, the loss of SFCT predicted by our studies is of 0.852 µm per year, far from the 4.32 µm per year predicted by previous papers, reaching different conclusions. This was the largest series to date of CT measured with SS OCT and it confirms again the accuracy with which SS OCT can measure the choroid. SS-OCT was chosen to perform this study because its characteristics allow the analysis of the choroid with more precision than previous OCT technology from a theoretical point of view. This accuracy has been shown in other papers by Copete47 or Ruiz- Moreno,46 using the same device. Full choroidal thickness was successfully measured in 100% of the patients of their studies. Using SS-OCT we successfully measured the choroidal thickness of 100% of the eyes in this study as well. Wei et al82 successfully measured 93.2% of 3,468 (largest study reported to date) using Heidelberg Spectralis OCT on the EDI setting. The choice of Manjunah et al77 was Cirrus HD OCT without EDI which was able to successfully measure the choroid in only 74% of the eyes. Yamashita81 and Shin83 used Topcon SD OCT and were able to image 63.3 % and 90.7% respectively. Copete et al47 compared SD OCT and SS OCT obtaining good measurements in 70.4 and 100% respectively. 135 Margolis et al41 studied a group of 54 eyes with a mean SE of -1.3 D finding a mean SFCT of 287 µm, similar to Flores-Moreno’s64 results and to Manjunath’s77 who published a mean SFCT of 272 µm in 34 eyes of 51.1-year-old patients. Xu et al84 compared diabetic with non-diabetic patients, finding a mean SFCT of 266 µm in the control group (1,795 subjects). As noted before, the choroid has also been studied in pediatric population with SS OCT.46 Park et al,85 in a recent study with pediatric subjects, find a mean SFCT of 348.4 µm in the 48 eyes studied. In the present study the mean SFCT was found to be 301.89 µm. This figure is about 10 µm thicker than that found by other authors using SD OCT. It is likely that the introduction of pediatric eyes into the study group resulted in a slight increase in the mean SFCT. Interestingly, these results match those of Ruiz-Moreno et al, who using the same device found almost the same CT in their patient group consisting of adults only. SS-OCT may result in a CT measurement slightly greater than that seen with SD-OCT. This may be explained by the higher quality of the images and by the fact that the measures were taken from the posterior edge of the RPE to the outer aspect of the lamina fusca, rather than the outer limit of the choroidal vessels. Ruiz-Moreno with SS OCT and Flores-Moreno with SD-OCT provide data about mean macular choroidal thickness (MCT). Ruiz-Moreno found it to be of 275 µm in adults compared to 285 µm in pediatric subjects. Flores-Moreno obtained results of 257 µm in healthy patients and of 115 µm in highly myopic patients. We found that mean MCT was 258.69 µm, similar to these authors’ results. Nevertheless the present study is the first employing a subfoveal 8 mm line of the macula 136 compared to the 6 mm studied by other authors. This was possible due to SS-OCT technology, which provides a more robust imaging of the temporal choroid, as this 2 mm increase was performed on this sector. This may prove to be a clinically important measure as it is in the temporal sector where greater variations of CT with age take place. We found no statistically significant difference when comparing men to women. Subjects of both sexes showed almost identical choroidal profiles with a minimal difference in the temporal sector, in which the females usually had slightly thinner choroids. Mean SFCT was 303.9 µm in men vs. 296.4 µm in women. These slight, non-statistically significant differences can be explained by the age differences of the two groups, 30.39 in men and 37.48 in women. CT was about 7 µm thicker in men, on average, but the men in this study were, on average 7 years younger. According to the correlation formulas established by this database, CT in a sex and age-matched group would like have been almost identical. A few studies have been previously performed in adults using high penetration OCT but without comparing different age groups. We analysed CT comparing several ranges of age, with a 20-year gap. The first two age groups spanned 0 to 10 and from 10 to 20 years old. These times were selected because eye growth undergoes two phases, first until age 6 years, reaching emmetropization, and second from 7 to 19 years of age, when global expansion of the eye takes place.86 CT profile changed in every group and the choroid measured thinner as age increases, showing statistically significant differences. This decrease was found to be progressive until 40 years old, at which point the most significant variation takes 137 place, from 313.9 µm in the 21-40 years old group to 264.6 µm in the 41-60 years old one (49 µm difference). Interestingly from 40 years on (41-60 vs. >60 years) the differences in CT were not found to be statistically significant in either mean MCT or SFCT. Variations when taking into account one eye per patient only (as stated by Ray et al87) or both were minimal, obtaining calculated decreases of 9.4 vs. 10 µm per decade in MCT and 8.5 vs. 8.87 µm per patient in SFCT. The choroid normally is thickest in the subfoveal region decreasing slowly and progressively towards the temporal aspect and more steeply towards the optic nerve,88,89 with a bowl-shaped contour.90 Adhi et al described significant alterations in choroidal morphological features in the majority of eyes with DR. They found an irregular temporal choroidoscleral interface inflection point in 8 of 9 eyes with non-proliferative DR (89%), 9 of 10 eyes with proliferative DR (90%), and 13 of 14 eyes with diabetic macular oedema (93%) compared with 0 of 24 in controls.90 The presence of an irregular temporal choroidoscleral interface inflection contour with focal thinning of the choroid has been detected in some diseases (diabetic retinopathy and age- related macular degeneration).73,90,91 Dolz-Marco et al92 reported a bilateral case of focal inferotemporal scleral bulge with a concave choroidal thinning surrounded by normal choroidal tissue. The authors hypothesized that this finding could be related to the inferior oblique muscle leading to inward compression of the choroid, not being able to study the scleral wall, as it was not entirely visible. No anomalies in the fundus examination were found that could justify this 138 thickening in our series of patients with temporal choroidoscleral interface inflection. SS-OCT analysis did not detect any alteration in the choroid of these patients other than one inflection point. On the other hand, an image that could represent the superior edge of the insertion of the inferior oblique muscle in the sclera was identified, together with a thin, hypo-reflective line parallel to the sclera/choroid limit that could match the intra-scleral portion of the temporal long posterior ciliary artery. The reason why retinal vessels seem to be hyper-reflective in OCT images, while choroidal vessels look hypo-reflective is related to the velocity of blood cells through them. The blood flow velocity inside choroidal vessels is much higher than that of retinal vessels. OCT imaging is based on interferometry, where an interference fringe is detected to construct the intensity of the signal. Fast speed of the blood cells makes the interference fringe vanish, thus no signal is observed inside the choroidal vessels. On the other hand blood cell speed is relatively slow in the case of retinal vascular structures, which contributes to the interference signal as a hyper-reflective signal. Reported studies on the anatomy of the posterior aspect of the globe and the vascular and muscular structures and its relation to the sclera show that the anterior and superior limit of the posterior insertion of the inferior oblique muscle coincides with the horizontal line/meridian, within 5.7 mm of the fovea.93 The mean distance from the inflection point to the fovea was 4427 µm in our series and always located temporal to the fovea. The presence of the inflection point 139 is probably related to the insertion of the inferior oblique muscle, which is believed to be 5.7 mm from the fovea. The traction applied to the sclera by the tendon might cause a separation of the scleral wall leading to its deformation and a thickening of the choroid. Based on the anatomical descriptions of this region, the parallel, hypo- reflective line at this level could correspond to the intra-scleral portion of the temporal long posterior ciliary artery. The fact that this finding is more frequent in younger healthy people, especially those under age 15, could be explained by the reduced scleral stiffness in younger age,94 that could facilitate the deformation induced by the traction of the inferior oblique muscle tendon. Several studies have been published about CT in healthy populations. Ding et al. analysed the CT in 210 healthy volunteers (420 eyes) with no ophthalmic disease history using EDI SD-OCT at multiple locations: SFCT and 1 mm and 3 mm temporal, nasal, superior and inferior to the fovea. However, the authors did not analyse any differences between the eyes.78 Ikuno et al. studied CT and its profile with a SS-OCT device in 86 eyes of 43 healthy Japanese subjects as well as the correlation with axial length, refractive error and age but they also published no data concerning any differences between right and left eyes.74 In contrast, Chen et al.89 reported the factors influencing topographical and inter-ocular variations in CT in a 50 healthy adult population using EDI SD-OCT. They measured SFCT and also performed CT measurements at four paramacular loci (3 mm superior, inferior, temporal and nasal to the foveal centre). They analysed the relationship between inter-ocular differences in CT. The differences in CT between 140 right eye and left eye were 1.0 µm at SFCT, 13,6 µm at nasal and -2.9 µm at temporal location, which was not statistically significant different. Of note, there was a trend towards a thicker nasal CT (14 µm) in right eyes. When comparing these data with the series reported in this study, at the same locations (SCFT, N3 CT and T3 CT), we observed that the difference comparing right eye and left eye was 7.45 µm in mean SFCT, 1.43 µm at T3 and 14.44 µm at N3. The differences between mean nasal CT (p=0.0002), mean nasal CT in men (p=0.003) and in women (p=0.03), mean CT at each nasal location (p=0.001, 0.002 and 0.08 respectively) were statistically significant. The choroid was thicker in the right eye at every nasal location for both sexes except for women at N1 (p=0.259). The correlation of the CT obtained by Chen et al. between eyes was strongest for SFCT (r=0.90; p<0.001, Spearman correlation test), for nasal locations was r=0.83 (p<0.001, Spearman correlation test) and weakest for temporal locations (r=0.49; p<0.001, Spearman correlation test). In our patients, the study of correlation for mean CT at SF, N3 and T3 was similar (r=0.860, 0.853 and 0.754 respectively, p<0.001; Spearman correlation test). The CT profile described in their paper is very similar to the CT profile obtained in our work comparing right eyes to left eyes. It is difficult to explain the strong and consistent evidence that right eyes have a thicker nasal choroid than left eyes in a healthy, young population. One possibility is a differential in blood flow between the two eyes due to lack of anatomic symmetry at the aortic arch. Such asymmetry has been suggested to explain the differences in incidence/prevalence of vascular pathologies between 141 right eyes and left eyes with respect to metastatic bacterial endophthalmitis95 and retinal artery occlusion.96–99 Choroidal circulation is generated from short posterior ciliary arteries that penetrate through the sclera around of optical nerve in a variable number between 10 and 20; so the nasal choroid studied in our cases (choroid between fovea and optical nerve), is directly supplied from these short posterior ciliary arteries. Furthermore, short posterior ciliary arteries are branches of the ophthalmic artery, which is a branch of the internal carotid artery, which is a branch of the common carotid artery.99 The difference generated by the fact that the origin of the right common carotid artery lays in the brachiocephalic trunk instead of emerging directly from the aorta (as the left common carotid artery) is presumably responsible of a more proximal and direct blood flow to the right carotid.95 Given that most of the choroidal structure is vascular tissue (the mean vessel density in outer choroid is 87%)100, a supposed higher blood flow in the right vs. left short posterior ciliary arteries may explain why the nasal choroid is thicker in RE, as stated in our study, and as described previously by Chen et al.89 In the present study, as in previous papers88 we used spherical equivalent instead of axial length determinations since the procedure is less invasive and previous indications from the literature show that refraction, which is more convenient to obtain, provides equivalent modeling capability as axial length.101 The choroid has been proven to become thinner with age.102 Our research group evaluated the choroid of healthy individuals across a wide age span (3 to 95 142 years old) finding that macular CT profile in healthy population was similar among the different age groups, and that healthy choroid showed a trend to become thinner with age, particularly comparing adults older than 40 years to children and younger adults. These differences were not attributable to gender and greater age- related CT variations appeared in the temporal sectors.88 Analysing our results, it can be stated that the choroid has a wide extent of normal thickness, with under 5% of healthy population presenting CT thicker than 320 µm and no differences in CT between genders. Esmaeelpour et al. used a SD-OCT (840 nm) with a raster protocol image consisting of 512 A-scans and 512 B-scans across a 36x36º field to automatically analyse the choroidal layers of 45 healthy eyes with a mean age of 44 years (ranging from 23 to 84 years old), although using a different method.103 Mean CT through the areas analysed was 228 µm and the thickness of the Haller’s layer and Sattler’s layer + choriocapillaris were 141 µm and 87 µm respectively. Adhi et al90 studied 24 eyes of 24 patients with a mean age of 64 years. CT measurements were manually performed using the caliper provided by the SD-OCT device and the choroid-sclera limit was well delimited in 79% of the eyes. The imaging protocol used consisted of 1-line rasters centred at the fovea. Subfoveal CT was 276 µm, subfoveal Haller’s layer thickness was 224 µm and subfoveal Sattler’s layer + choriocapillaris was 52 µm. Using the same method, Branchini analysed the choroidal layers of 42 eyes of 42 patients with a mean age of 52 years, locating the choroid-sclera limit in 92% of them.104 They manually measured CT and its layers at the subfoveal location using 143 two more measurement points 750 µm away from the fovea in both the nasal and the temporal sectors. They found the subfoveal CT of their sample to be of 256 µm. Haller’s layer and Sattler’s layer + choriocapillaris thickness were 204 and 53 µm respectively. In the present study, 169 eyes of 169 patients were analysed using a SS-OCT device, being the largest sample analysing choroidal layers to the best of our knowledge. The mean age was lower that other studies’ at 33.5 years old, while visualization of the choroidoscleral interface was possible in all of the eyes. Our figures for subfoveal CT, Haller’s layer and Sattler’s layer + choriocapillaris (305.8, 208.6 and 87.3 µm respectively) are larger than those presented by other authors. A similar finding was observed when in a previous paper our group compared CT measurements with other series using SD-OCT devices, with a difference of roughly 10 µm.88 This difference and the fact that the mean age of our sample is considerably younger that that of other series could explain the different results. In order to evaluate the choroid and its layers across the macula we added 8 more measure points to the subfoveal location (3 nasally and 5 more temporally, although N3 was not taken into account as described before). The mean values for CT, Haller’s layer and Sattler’s layers + choriocapillaris thickness were 260.81, 180.2 and 78.2 µm respectively. CT profile showed a similar morphology across different age groups with both CT and Haller’s layer thickness decreasing significantly with age (p<0.001). Choriocapillaris plus Sattler’s layer thickness showed an age-dependent reduction trend, but was not statistically significant (p=0.124). Our linear regression models led to a predicted loss of 0.93 µm per year in CT, 0.69 of those were due to 144 the thinning of Haller’s layer, which explained 75% of the age-related thinning of the choroid. No differences due to gender were found in CT, or any of its layers in any age group. Our study shows that most of the age-related thinning of the choroid of normal eyes is due to the reduction of large vessels. These findings in healthy eyes disagree with histopathological findings in eyes with marked senile sclerosis, that show atrophy of small and medium vessels mostly.102,105 Spaide102 stated that age-related loss of choroidal thickness was directly related to the loss of visible vessels, implying that it is a manifestation of small-vessel disease affecting the choroid. These data are not comparable to ours, as Spaide only included eyes with CT under 125 µm. Sarks’ paper describes only two cases with choroidal sclerosis whose retinoscopy showed well-demarcated areas of choroidal sclerosis that are probably in relation with atrophic age-related macular degeneration, while our study analyses healthy patients.105 This age-related choroidal thinning could be a key factor to establish the role of the choroid in retinal pathology. Among the limitations of this study we may mention that CT had to be manually determined. There is now an automated software commercially available, Topcon DRI OCT-1 software (Ver. 9.01.003.02) for automated segmentation and thickness measurements in 3D or radial scan mode, however its accuracy in clinical practice is yet to be widely tested. To our best knowledge this was the first report of CT determination in youngsters using SS-OCT. Few studies had been previously performed in adults using CT SS-OCT with different mean ages, and others using different methodology (no 145 vertical scans and no differences in CT were analysed). Our results may also differ from those previously performed with SD-OCT. Another limitation is the fact that only the horizontal choroidal thickness profiles were studied, the use of SE instead of axial length, the fact that no correction was made for other variables such as central corneal thickness, intraocular pressure, systemic medication and blood pressure, and the impossibility to determine whether the vessels of the nasal choroid are wider in the right eye than in the left. This is due to the fact the resolution provided by the current technology does not allow a proper and accurate analysis of choroidal vessel thickness and there is no way to make sure you are analysing the largest diameter of a vessel because of the irregularity of choroidal vascularization. As a further limitation, the choriocapillaris could not be differentiated from Sattler’s layer due to the limitations imaging technology and that despite the high ICC. In conclusion, according to our results, an answer can be given to the initial hypothesis affirming that macular CT is similar in the healthy young and adult population. CT profile is different and temporal choroid is thicker. The macular CT profile in healthy population is similar between different age groups, with the choroid in healthy eyes getting thinner with age, particularly comparing adults older than 40 years to children and younger adults. There are no differences due to gender. The greater CT variation due to age takes place in temporal sectors. A temporal choroidoscleral interface inflection with focal thinning of the choroid can be considered a normal variation without clinical significance, especially 146 among young people. Further studies of the whole macular profile including the complete insertion of the inferior oblique muscle and with a larger number of patients will be necessary in order to establish the final characteristics of these variations and their incidence in healthy population. The nasal area of the macular CT profile is thicker in RE when compared to LE. In contrast, subfoveal CT and temporal CT were not found to be different between eyes. CT and Haller’s layer grow progressively thinner as patients grow older and their profiles are similar between different groups of age. Age-related choroidal thinning seems to be mostly at the expense of Haller’s layer. There are no differences due to gender. Further studies would be necessary in order to confirm our results. 147 6. CONCLUSIONES POR OBJETIVOS 148 6. CONCLUSIONES POR OBJETIVOS 6.1. CONCLUSIÓN 1 El grosor coroideo del área macular en la población pediátrica no es estadísticamente mayor que en adultos sanos. Sin embargo el perfil de la coroides no es igual, siendo las diferencias más apreciables en el sector temporal, donde hallamos el máximo grosor coroideo en niños, al contrario que en adultos, donde la coroides más gruesa se halla bajo la fóvea. 6.2. CONCLUSIÓN 2 Según nuestros resultados el perfil de grosor coroideo macular es similar entre los diferentes grupos de edad. La coroides se adelgaza progresivamente con la edad, especialmente a partir de los 40 años. Las mayores variaciones tienen lugar en el sector temporal de la mácula. No existen diferencias en relación al sexo de los pacientes. 6.3. CONCLUSIÓN 3 Hallamos una inflexión en la interfaz esclero-coroidea temporal, que puede ser considerada como una variación normal sin significación clínica, especialmente frecuente entre la población joven. 6.4. CONCLUSIÓN 4 Según nuestros resultados existe una diferencia estadísticamente significativa entre el grosor coroideo macular de el ojo derecho y el ojo izquierdo. Por otra parte, el 149 grosor coroideo subfoveal y temporal no son diferentes cuando comparamos los dos ojos entre sí. 6.5. CONCLUSIÓN 5 El grosor de la coroides y el de la capa de Haller se adelgazan de manera progresiva con la edad, pero sus perfiles de grosor mantienen una forma similar cuando comparamos diferentes grupos de edad. La mayoría del adelgazamiento de la coroides parece ser a expensas del adelgazamiento de la capa de Haller. No se hallaron diferencias en relación al sexo de los pacientes. 6.6. CONCLUSIÓN 6 El área de luz vascular y el porcentaje de vasos/área total disminuye con la edad, mientras que el área estromal se mantiene estable. 150 6. CONCLUSIONS CONCLUSION 1 Macular choroidal thickness in the pediatric population is not significantly thicker than that of healthy adults. Differences are more remarkable in the temporal side of the fovea. CONCLUSION 2 To our knowledge, this is the first population study of CT of healthy eyes across a broad range of age groups using SS-OCT. As has been determined using spectral- domain OCT, CT decreases with advancing age, especially after age 40. There were no differences due to gender. The greatest CT variation takes place in temporal sectors. CONCLUSION 3 Temporal choroidoscleral interface inflection or S-shaped profile of the choroidoscleral interface with focal thinning of the choroid can be considered a normal variation without clinical significance, especially in younger populations. CONCLUSION 4 To the best of our knowledge, this is the first report suggesting thicker macular nasal choroid in the RE compared with the LE. In contrast, subfoveal CT and temporal CT were not found to be different between eyes. 151 CONCLUSION 5 According to our results, CT and Haller’s layer grow progressively thinner as patients grow older and their profiles are similar between different groups of age. Age- related choroidal thinning seems to be mostly at the expense of Haller’s layer. There were no differences due to gender. CONCLUSION 6 The luminal area and the percentage of vascular/total area decrease with increasing age while stromal area remains stable. 152 7. FUTURO 153 7. FUTURO 7.1 ESTUDIO DE LA CORIOCAPILAR MEDIANTE ANGIOGRAFÍA OCT Ya ha sido comentado que la capa coriocapilar es la que finalmente nutre y ayuda en el metabolismo de las estructuras de la retina más externa. Hay multitud de estudios histopatológicos que hablan de cómo una pobre circulación coroidea puede ser un factor de riesgo para el desarrollo de enfermedades oculares tales como la DMAE y la retinopatía diabética. La mejoría en las técnicas de imagen de la vascularización coroidea podría ayudar a la detección precoz de una la reducción de perfusión coriocapilar, lo que podría ser un buen marcador temprano para diversas enfermedades de la retina y la coroides. La OCTa es una técnica no invasiva que permite la obtención de imágenes de la vascularización retiniana y de la superficie coroidea mediante un promedio de multitud de B-scans tomados en una misma posición. Se genera una imagen a partir de los componentes dinámicos de estos cortes (los glóbulos rojos del interior de los vasos sanguíneos), mientras que el tejido estático es sustraído para darnos una imagen final en la que sólo se aprecia el contenido de los vasos. Aunque la coriocapilar se ha intentado estudiar utilizando OCTa basada en tecnología SD-OCT, la pobre penetración de esta longitud de onda a través del EPR ha limitado su utilidad clínica. Sin embargo, los prototipos más recientes de SS-OCT con longitudes de onda de entorno a 1050 nm y mayor penetración tisular permiten visualizar de una manera más clara esta capa de la coroides, y permiten ver como su patrón vascular varía en función de la zona estudiada, mostrando una mayor 154 densidad vascular en el área foveal y mostrando un patrón más difuso hacia la periferia.106,107 7.2 ÓPTICA ADAPTATIVA OCT La óptica adaptativa OCT (OA-OCT) es una nueva tecnología que permite una captura mejorada de las estructuras microscópicas de la retina y de la vascularización coroidea que se encuentran habitualmente limitadas por las aberraciones que reducen la actual resolución trasversal de los sistemas de OCT. Gracias a la combinación de la gran resolución trasversal de la óptica adaptativa y la gran resolución vertical de la OCT, se consigue la imagen más precisa de todas las técnicas de imagen in vivo para estructuras celulares.108 Aunque ya se ha empleado para el estudio de las vascularización retiniana, la coroidea implica mayores dificultades al estar bajo el EPR, que dispersa la señal, impidiendo la obtención de imágenes de buena calidad.108 Estos problemas se han solucionado recientemente utilizando la OA-OCT Doppler, mejorando el contraste de la coriocapilar, las arteriolas y las vénulas de la capa de Sattler.109 Así, esta nueva técnica de imagen se podría utilizar para el estudio y la detección de alteraciones en las redes de la coriocapilar sin la necesidad de utilizar productos de contraste. Sin embargo, esta técnica por el momento requiere un estudio en mayor profundidad para poder relacionar sus hallazgos con el conocimiento histopatológico actual y dado que muestra importantes limitaciones como el pequeño campo de trabajo estudiado, el elevado tiempo requerido para la toma de la imagen y la facilidad a la hora de generar artefactos que esto conlleva. 155 7.3 OCT DE CAMPO AMPLIO Las imágenes de campo amplio para OCT que se habían obtenido hasta los últimos años de conseguían a base de usar protocolos de imagen destinados a áreas extra maculares y a hacer montajes de las imágenes a posteriori.110 Utilizando tecnología SD-OCT, los sistemas Spectralis (Heidelberg Engineering, Heidelberg, GERMANY) han incrementado su longitud máxima de B- scan desde los 9 mm gracias al uso de lentes de 30º hasta los 15 mm gracias a la adaptación de las lentes de 50º, anteriormente limitadas a la captura de imagen de autofluorescencia y de angiografías. La última generación de OCT con tecnología Swept-Source nos ofrece la posibilidad de capturar imágenes B-scan que pueden alcanzar los 12 mm de longitud, llegando según algunas publicaciones hasta los 100º de ángulo de visualización de la retina.57 7.4 ANGIOGRAFÍA CON VERDE DE INDODIANINA DE CAMPO ULTRA-AMPLIO La tecnología de angiografía de campo amplio ya había mostrado su utilidad para la captura de imágenes de AF, sobretodo para la evaluación del grado de isquemia o no-perfusión periférica en pacientes con retinopatía diabética y otras enfermedades vasculares de la retina. Una publicación reciente en la que los autores utilizan un sistema Optos modificado (Optos PLC, Dunfermline, SCOTLAND) para la obtención de imágenes de AVI de campo ultra-amplio, llegando hasta los 200º de campo.111 Sería de esperar que técnicas como ésta puedan resultar útiles a la hora de incrementar nuestro 156 conocimiento sobre la coroides periférica y mejorar la detección, seguimiento y tratamiento de patologías como la VCP, la CSC y las MNV.111 157 8. RESUMEN 158 8. RESUMEN Objetivos: Empleando la tomografía de coherencia óptica Swept-Source (SS-OCT): - Evaluar el grosor coroideo (GC) en la población infantil. - Determinar el perfil del GC en población sana. - Analizar las características morfológicas de la interfase coroides-esclerótica. - Determinar la diferencia interocular en el perfil del GC macular. - Estudiar el GC y sus variaciones con la edad, así como la morfología y cambios de las tres capas anatómicas de la coroides. - Analizar la densidad de la coroides de la población sana. Material y Métodos: Los criterios de inclusión fueron: mejor agudeza visual corregida entre 20/20 y 20/25, equivalente esférico (EE) entre ±3 dioptrías (D) y ausencia de enfermedades sistémicas u oculares. Las medidas y determinaciones fueron realizadas por dos o tres investigadores independientes y enmascarados según el caso, con un prototipo SS-OCT (Topcon Co., Japón). Para el estudio de la población infantil se realizó un estudio comparativo transversal no intervencionista del área macular de 83 ojos de 43 pacientes pediátricos (<18 años). El GC macular se determinó independientemente por 3 observadores manualmente a intervalos de 750 μm midiendo la distancia desde el borde posterior del epitelio pigmentario de la retina (EPR) hasta la unión coroides- esclerótica, a lo largo de una línea de 4500 μm centrada en la fóvea. El GC pediátrico se comparó con el de 75 ojos de 50 voluntarios adultos sanos (≥18 años). 159 Para el estudio del perfil coroideo y la morfología de la interfase esclerótica- coroides se hizo un estudio transversal, no intervencionista de 276 ojos. Dos investigadores de manera independiente y enmascarada analizaron las características morfológicas de la interfase coroides-esclerótica, clasificando su contorno y forma como cóncavo (bowl-shaped) o inflexión (S-shaped) y midieron de forma manual el GC en posición subfoveal desde el límite posterior del EPR hasta la interfase esclerótica-coroides. Se realizaron tres mediciones cada 1000 µm nasal a la fóvea, y cinco más en sentido temporal a la misma. Los sujetos estudiados fueron estratificados en 5 grupos de edad. Para buscar la posible existencia de diferencias en el GC entre ambos ojos se realizó un estudio transversal, no intervencionista en 140 ojos de 70 pacientes sanos con diferencias interoculares ≤0,25D. El perfil de la coroides macular se creo midiendo manualmente el GC subfoveal (GCSF) y realizando 3 mediciones más cada 1000 µm en sentido nasal y otras 5 en sentido temporal a la fóvea. Para el análisis de las capas de la coroides se realizó un análisis retrospectivo de un subgrupo de 169 ojos de 169 sujetos. Dos observadores determinaron el GC macular horizontal y de la capa de Haller en posición subfoveal; se repitió este procedimiento cada 1000 µm en dirección nasal y temporal a la fóvea obteniendo 9 mediciones por ojo. Para en análisis de la evolución de la densidad vascular de la coroides se llevó a cabo un estudio transversal, no intervencionista sobre 136 ojos de 136 pacientes. El análisis del estroma y de los vasos coroideos se realizó mediante una segmentación y una binarización automática empleando algoritmos validados. 160 Resultados: La edad media fue 10±3 años en la población pediátrica vs. 53±16 en la adulta (P<0,001). El equivalente esférico medio no fue diferente (P=0,06) entre los grupos. El GCSF medio fue de 312,9±65,3 μm en la población pediátrica vs. 305,6±102,6 μm en la adulta (P=0,19). El GC macular medio fue de 285,2±56,7 μm en la población pediátrica vs. 275,2±92,7 μm en la adulta (P=0,08). La distribución del GC fue distinto entre las poblaciones. La coroides temporal resultó ser más gruesa en la población pediátrica (320, 322 y 324 μm; P=0,002, P=0,001 y P=0,06 respectivamente), seguido por la subfoveal (312 μm) y la nasal (281, 239 y 195 μm). En la población sana el GCSF medio fue de 301,89±80,53 µm (95%: 292,34 a 311,43). El GC macular medio fue de 258,69±64,59 µm (95%: 251,04 a 266,35). No se hallaron diferencias de grosor entre hombres y mujeres. El GCSF medio de los diferentes grupos estudiados fue de 325,6±51,1 µm (0 a 10 años), 316,7±90,1 µm (11 a 20 años), 313,9±80,3 µm (21 a 40 años), 264,6±79,3 µm (41 a 60 años) y 276,3±88,8 µm en mayores de 60 respectivamente (P<0,001; ANOVA). El GC macular medio fue de 286,0±43,5 µm, 277,7±68,2 µm, 264,0±61,9 µm, 223,4±62,2 µm y 229,7±66,1 µm respectivamente (P<0,001; ANOVA). El GC fue estadísticamente diferente entre los diferentes grupos etarios. La presencia de inflexión coroideo-escleral fue identificada en 12,8% de los ojos. El GC en este punto fue de 372,1±76,8 µm y la distancia media a la fóvea fue de 4427,3±627,9 µm. Nueve pacientes mostraron esta inflexión en ambos ojos. No se hallaron cambios en el perfil retiniano en ninguno de los casos. La edad media de los pacientes con inflexión fue de 16±19 años vs. 36±25 años en el grupo de contorno 161 cóncavo (P=0,001). El GC temporal a 4000 y 5000 µm de la fóvea fue mayor en el grupo de contorno cóncavo. En el estudio de las diferencias entre ojos la edad media fue de 25,4±19,9 años. El EE medio fue de 0,18±1,37. El GC macular medio fue mayor en ojos derecho que en izquierdos, (228,11±69,23 µm vs. 212,27±62,71 µm; P=0,0002). El GCSF medio y el GC macular temporal medio no fueron diferentes entre ambos ojos. No hubo diferencias en EE entre ojos derechos e izquierdos. El GC nasal medio también fue mayor en ojos derechos independientemente del género (hombres P=0,003 y mujeres p=0,03). El grosor subfoveal de la capa de Haller fue 215,47±67,70 µm (207,30-227,86) y el de la capa de Sattler + coriocapilar fue 87,31±40,40 µm (83,38-95,65). No se hallaron diferencias significativas en ninguna de las capas al comparar hombres y mujeres. El perfil de GC fue similar entre los diferentes grupos de edad; el GC y de la capa de Haller disminuyó progresivamente con la edad (P=0,002). La capa de Sattler + coriocapilar mostró esta tendencia hacia el adelgazamiento, sin alcanzar la significación estadística (P=0,124). El porcentaje de vascularización de la coroides fue de 54,40±8,35 %. El área coroidea, vascular y el porcentaje de vascularización coroidea fueron mayores en el grupo de <18 años que en el de >18 años (p<0,001). El área estromal no fue diferente (p=0,46). De la misma manera el área coroidea, vascular y el porcentaje de vascularización coroidea fueron diferentes en los 5 grupos de edad (p<0.001), mientras que no se hallaron diferencias en el área estromal (p=0.71). 162 Conclusiones: El GC macular pediátrico no es significativamente más grueso que el de adultos sanos. Las diferencias son más destacables en la zona temporal. El GC disminuye con la edad, especialmente después de los 40 años. No hay diferencias relacionadas con el género. Las mayores variaciones de GC tienen lugar en los sectores temporales de la coroides. La inflexión coroideo-esclerótica temporal o perfil S-shaped con adelgazamiento focal de la coroides puede ser considerado una variante normal sin significación clínica, especialmente en la población joven. Existe un mayor GC macular nasal en ojos derechos en comparación con los izquierdos, mientras que el GCSF y el grosor temporal no fue diferente entre los dos ojos. El perfil de la coroides, Haller y Sattler + coriocapilar son similares entre diferentes grupos de edad; las dos primeras se adelgazan de manera progresiva con la edad. El área luminal y el porcentaje de área vascular/área total disminuyen con la edad, mientras que el área estromal permanece estable. 163 8. SUMMARY Objectives: While using Swept-Source optical coherence tomography (SS-OCT): - Study choroidal thickness of healthy pediatric population. - Study the choroidal thickness profile of healthy population. - Study the morphologic characteristics of the choroid-sclera interface. - Study interocular differences regarding the macular choroidal thickness profile. - Study choroidal thickness, its variations with age and the changes that appear within its three layers. - To analyse the vascular density of the choroid in a healthy population. Material y Methods: Inclusion criteria were: Best corrected visual acuity between 20/20 and 20/25, spherical equivalent (SE) between ±3 diopters (D) and absence of systemic or ocular diseases. Measures were performed by 2 or 3 independent, masked observers using a SS-OCT prototype (Topcon Co. Japan). To study pediatric population, a cross-sectional, non-interventional study was carried out on 83 eyes from 43 underage patients (<18 years old). Macular choroidal thickness was independently determined by 3 different observers who performed manual measures at 750 µm intervals, measuring the distance between the posterior edge of the RPE and the choroid-sclera junction through a 4500 µm fovea- centred line-scan. Pediatric choroidal thickness was compared to that of 75 eyes from 50 healthy, adult volunteers. 164 A cross-sectional, non-interventional study of 276 eyes was performed to analyse the choroidal thickness profile and the morphology of the choroid-sclera interface. Two independent, masked observers studied these characteristics and classified the shape into concave (bowl-shaped) or inflection (S-shaped), manually measuring choroidal thickness from the posterior edge of the RPE and the choroid- sclera junction under the fovea. They performed 8 more measures every 1000 µm towards temporal and nasal sectors. Analised subjects were stratified into 5 age groups. A cross-sectional, non-interventional study was performed using 140 eyes from 70 patients with interocular differences of SE < 0.25 D to analyse interocular differences of choroidal thickness. A retrospective analysis of 169 eyes from 169 patients was performed to study choroidal layers. Two independent, masked observers determined choroidal thickness, Haller’s layer thickness and Sattler’s layer + choriocapillary thickness under the fovea. They performed 8 more measures every 1000 µm towards temporal and nasal sectors. A cross-sectional, non-interventional study was performed on 136 eyes from 136 to analyse the possible age-related changes regarding the percentage of vascularization of the choroid. Choroidal stroma and vessel area analysis involved automated segmentation and binarization using validated algorithms. Results: Mean age was 10±3 years old in pediatric population vs. 53±16 in the adult group (P<0.001). SE was not different between groups (P=0.06). Mean subfoveal 165 choroidal thickness (SFCT) was 312.9±65.3 μm in pediatric population vs. 305.6±102.6 μm in adults (P=0.19). Mean macular choroidal thickness (MCT) was 285.2±56.7 μm in pediatric population vs. 275.2±92.7 μm in adults (P=0.08). Choroidal thickness profiles were different between both groups. Temporal choroid was shown to be thicker in pediatric subjects (320, 322 and 324 μm; P=0.002, P=0.001 y P=0.06 respectively), followed by subfoveal thickness (312 μm) and nasal (281, 239 y 195 μm). Healthy population showed a mean SFCT of 301.89±80.53 µm (95%: 292.34 to 311.43). Mean MCT was 258.69±64.59 µm (95%: 251.04 to 266.35). There were no statistically significant differences between men and women regarding choroidal thickness. Mean SFCT in the different groups studied was 325.6±51.1 µm (0 to 10), 316.7±90.1 µm (11 to 20), 313.9±80.3 µm (21 to 40), 264.6±79.3 µm (41 to 60) and 276.3±88.8 µm (>60) respectively (P<0.001; ANOVA). Mean MCT was 286.0±43.5 µm, 277.7±68.2 µm, 264.0±61.9 µm, 223.4±62.2 µm and 229.7±66.1 µm respectively (P<0.001; ANOVA). MCT was statistically different between different age groups. The presence of a temporal choroidoscleral interface inflection was identified in 12.8% of the eyes. The mean choroidal thickness was 372.1±76.8 µm and the average distance from the inflection point to the fovea was 4427.3±627.9 µm. Nine patients showed an inflective profile in both eyes. No changes in the retinal profile were found in any of these cases. The mean age of the patients with an inflective profile was 16±19 years (range 4 to 82) vs. 36±25 years (range 3 to 95) in the group with a concave contour (p=0.001). The temporal choroidal thickness at 4,000 and 5,000 µm from the fovea was thicker in the group with a concave contour. 166 In the interocular difference study mean age was 25.4±19.9 years (4 to 75). The mean SE was 0.18±1.37 D (-3 to +3). Mean macular nasal CT was thicker in the right eye (RE) than in the left eye (LE), (228.11±69.23 µm vs. 212.27±62.71 µm; p= 0.0002; Student t test paired data). Mean SFCT and mean temporal CT were not different between RE and LE. No statistically significant differences were observed comparing SE in the RE vs. LE. Mean nasal CT by gender, was also thicker in the RE vs. LE. At each nasal determination CT in the RE was thicker than LE (p= 0.001, p= 0.0002 and p= 0.008 respectively). Mean subfoveal thickness for Haller’s layer was 215.47 ± 67.70 mm (95% confidence interval: 207.30–227.86) and mean subfoveal thickness for choriocapillaris plus Sattler’s layer was 87.31 ± 40.40 mm (95% confidence interval: 83.38–95.65). No significant differences were found due to gender. Choroidal thickness profile was similar between groups with choroidal thickness and Haller’s layer thickness decreasing with age (P = 0.002). The percentage of choroidal vascularity was 54.40±8.35 %. Choroid area, vascular region and percentage of choroidal vascular density were statistically higher in the <18-year-old group vs. >18-year-old group (p<0.001). Stromal region was not different (p=0.46). In the same way, choroid area, vascular region and percentage of choroidal vascular density between the five age groups was statistically different (p<0.001), while stromal region was not (p=0.71). Conclusions: Mean MCT in pediatric population is not statistically thicker than that of healthy adults. Differences are more remarkable in temporal sectors. Choroidal 167 thickness is reduced with age, especially after age 40. There are no gender-related differences. Greatest variations in choroidal thickness take place in temporal sectors. Temporal choroidoscleral interface inflection or S-shaped profile of the choroidoscleral interface with focal thinning of the choroid can be considered a normal variation without clinical significance, especially in younger populations. Nasal macular choroid is thicker in the RE compared with the LE. In contrast, subfoveal CT and temporal CT were not found to be different between eyes. CT and Haller’s layer grow progressively thinner as patients grow older and their profiles are similar between different groups of age. Age-related choroidal thinning seems to be mostly at the expense of Haller’s layer. The luminal area and the percentage of vascular/total area decrease with increasing age while stromal area remains stable. 168 9. BIBLIOGRAFÍA 169 9. 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