We thank Ellen Ytebrouck for her valuable information regarding method application in rat. This work was supported by the Research Council of the KU Leuven (OT09/22) and the Fund for Scientific Research-Flanders (FWO Vlaanderen). Jeroen Aerts is supported by a Ph.D. fellowship of the Agency for Innovation through Science and Technology Flanders (IWT Vlaanderen) and Julie Nys by a Ph.D. grant from FWO Flanders.

Todos los experimentos fueron aprobados por el comité de ética de la investigación de la KU Leuven y eran estrictamente de acuerdo con las Comunidades Europeas la Directiva del Consejo de 22 de septiembre 2010 (2010/63 / UE) y con la legislación belga (KB de 29 de mayo de 2013). Se hicieron todos los esfuerzos posibles para minimizar el sufrimiento de los animales y reducir el número de animales. 1. Animal Tratamiento y Anestésicos <ol><li> Anestesiar al ratón con una inyección intraperitoneal de una mezcla de clorhidrato de ketamina (75 mg / ml) e hidrocloruro de medetomidina (1 mg / kg) en solución salina. </li><li> Compruebe los reflejos apretando los dedos de los pies con una pinza para asegurar el ratón está completamente sedado. </li><li> Aplicar etanol al 70% para la desinfección de los párpados y la región que rodea el ojo con una punta de algodón. Compruebe el reflejo palpebral para evaluar adicionalmente el grado de sedación. </li></ol> 2. Eliminar el Ojo <ol><li> Asegúrese de que el animal reside en un plano, seco ysuperficie lisa. </li><li> Esterilizar un fórceps con una punta dentada (tamaño de la punta preferido: 0,5 x 0,4 mm) curvada. </li><li> Presione suavemente sobre el canto (en la esquina del ojo) con las pinzas hasta que el globo ocular es desplazado de la toma y el nervio óptico es alcanzable. </li><li> Guía de las pinzas detrás del ojo. Mantenga pulsado el nervio óptico firmemente, preferiblemente con el comienzo de la curva y no la punta de la pinza. Esto ayudará a levantar el globo de la toma y para sujetar el nervio óptico completo. </li><li> Haga movimientos circulares con la mano que sostiene la pinza en la dirección con la menor resistencia mientras que el ratón permanece en la superficie plana. El ratón se moverá a lo largo de la superficie de acuerdo a la dirección del movimiento de la mano. </li><li> Lleve a cabo esta acción con el aumento de la velocidad gradualmente hasta que el nervio óptico está restringido en dos (por lo general entre 7 a 15 movimientos circulares, aproximadamente de media a una vuelta completa por segundo). Por lo tanto, el globo ocular es independienteeliminado. </li></ol> Cuidado 3. postoperatoria <ol><li> En caso de sangrado (poco frecuente), llenar la órbita con un coagulado viscoso y agente hemostático. </li><li> Invertir la anestesia mediante la inyección de 1 mg / kg de clorhidrato de atipamezol en solución salina por vía intraperitoneal. </li><li> Administrar 1 mg / kg de meloxicam por vía intraperitoneal cada 24 horas para aliviar el dolor. </li><li> Aplicar pomada ocular al ojo restante para prevenir la deshidratación de la córnea. </li><li> Deje que el animal se recupere en una placa calefactora o envuelva el animal en material aislante en una jaula separada para controlar la temperatura corporal. </li><li> Medir el peso del ratón cada día durante al menos 2 días. La pérdida de peso puede indicar sufrimiento y en este caso, continuar con el tratamiento Meloxicam hasta que el animal está totalmente recuperado. </li></ol>

<ol>
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The authors have nothing to disclose.

Para realizar una enucleación exitosa de acuerdo con nuestro método, los pasos más importantes a considerar son: 1) el uso de unas pinzas con una punta curvada y dentada del tamaño adecuado; 2) la realización de la enucleación en una superficie suave y seco; y 3) acelerar gradualmente los movimientos circulares en la dirección con la menor fricción. Para un resultado eficaz, es esencial utilizar un fórceps adecuados caracterizados por una punta curvada y dentada (tamaño de la punta preferido: ratón: 0,5 x 0,4 mm; rata: 2,15 x 1,3 mm). La curvatura permite un fácil acceso al nervio óptico después del desplazamiento globo ocular y es necesario para la colocación correcta de la mano al realizar los movimientos circulares. Puntas suaves no son recomendables ya que carecen de la necesaria agarre al sostener el nervio óptico. El fracaso para mantener el nervio óptico correctamente durante los movimientos circulares resulta en la ruptura de la arteria oftálmica, pobres desprendimiento del ojo y por lo tanto mala reproducibilidad.Por lo tanto se recomienda primera práctica esta técnica en animales sacrificados para la optimización de las pinzas de manipulación con el fin de garantizar el bienestar animal máxima una vez que la aplicación del método in vivo. El éxito de la enucleación del ojo también ha sido recientemente realizado en la rata en nuestro laboratorio utilizando la misma técnica, excepto para girar manualmente el cuerpo del animal y mantener estacionario el fórceps. Una limitación de la técnica es que posiblemente podría dañar la retina. Por lo tanto este método es menos adecuado para recoger retinas para llevar a cabo la histología 5. Por otra parte, nuestro método se limita a publicar la apertura del ojo edades ya que el globo ocular debe ser desplazado de la toma sin quitar o cortar los párpados. Enucleación del ojo en diferentes especies, incluyendo roedores, se realiza de forma rutinaria utilizando métodos alternativos, que a menudo conllevan la eliminación de los párpados y el corte del nervio óptico 18,23-25. Estos methods tienden a ser más invasivos y tienen una curva de aprendizaje más alta que la técnica descrita aquí. Sin la necesidad de retirar o sutura de los párpados, el tiempo de recuperación después de la cirugía se reduce al mínimo, lo que resulta en un mayor bienestar de los animales y resultados más reproducibles.

Extracción de un ojo y con ello destruyendo irreversiblemente la superficie del receptor sensorial (retina), impone una considerable pérdida de información sensorial a lo largo de la vía visual. El modelo de la enucleación en el sistema visual juveniles y adultos ha demostrado ser valiosa para comprender el desarrollo, la plasticidad y la función de diferentes centros visuales 1-4. Las consecuencias moleculares, celulares y fisiológicos de esta privación sensorial pueden ayudar a comprender cómo se regula el desarrollo normal y Cómo se forman circuitos corticales hacer frente y cambian su estructura y función en respuesta a un amplio cambio de esta naturaleza en la experiencia. Existen diferentes métodos de privación visual y todos ellos tienen sus ventajas específicas en la investigación relacionada con la visión. Por ejemplo la cría oscuro específicamente elimina la actividad impulsada visualmente, sin embargo, no afecta a la actividad retinal espontáneo. Visua Del mismo modo, las suturas tapa o parches de los ojos Quitar modeladosl entrada sin perturbar la actividad espontánea, sino que permiten la penetración de luz dispersa a través de los ojos cerrados. Estos métodos son reversibles y se ha demostrado ser valiosa en la comprensión del papel de la visión y de bajo nivel modelado correlación de entradas binoculares en esculpir circuitos corticales durante el desarrollo 6-8. En la investigación glaucoma, el modelo de aplastamiento del nervio óptico en los animales adultos se ha utilizado ampliamente debido a que establece una pérdida progresiva de entradas de células ganglionares de la retina que constituyen el nervio óptico 9,10. Por otro lado, la enucleación, donde el ojo y por lo tanto la retina se elimina completamente y al instante, es la elección apropiada de la privación cuando el objetivo es eliminar de manera irreversible la visión tanto espontánea como patrón a la vez. También induce una robusta actividad intraocular desequilibrio que puede mejorar la relación señal a ruido en estudios de mapeo de la actividad 11,12. La comparación de los cambios funcionales y estructurales en respuesta a la enucleación con la famiose después de la privación por métodos menos drásticos tales como sutura tapa por ejemplo, también podría exponer nuevas ideas sobre el papel de la actividad espontánea de la retina en ambos tipos homeostáticos y de la plasticidad sináptica. La enucleación desencadena una pérdida de influencias tróficas en objetivos directos de la retina. Por ejemplo, los niveles de BDNF están regulados a la baja de manera significativa en el núcleo lateral geniculado (LGN) y colículo superior de ratas adultas enucleados 13. Especies reactivas de oxígeno, que funcionan como moléculas mensajeras para mediar remodelación estructural, también se detectaron en las estructuras subcorticales del sistema visual 14 de rata adulta. Además, microglial y astroglial de activación a través de diferentes estructuras subcorticales objetivo visual en el ratón se producen en un marco de tiempo post-enucleación específico de una semana 15. Juntos, los resultados de deaferenciación óptica en diferentes respuestas subcorticales en el glial, nivel estructural y molecular. A pesar de estas subcorticalefectos, que no implican necesariamente efectos a nivel cortical 16. Digno de mención, la plasticidad cortical cross-modal, incluyendo modificaciones en otras áreas sensoriales junto al fortalecimiento de las entradas no visuales a la corteza visual privados se producen después de tanto monocular (ME) 3,4,17,18 y binocular (BE) 1,17 enucleación. Además de contribuir a la neurociencia visual, la enucleación como un tipo de deaferentación se puede utilizar para estudiar el equilibrio entre neuroprotectores 19 y neurodegenerativas 20-22 propiedades del sistema nervioso central. Los diferentes procedimientos para llevar a cabo la enucleación ya se han descrito en la literatura. Algunas metodologías para in vivo ME en ratas y ratones son menos sencillo debido a seccionamiento innecesaria de los músculos orbitales y tejido 23-25. Otras publicaciones como Mahajan et al. (2011) 5 proporcionan un protocolo detallado utilizando<html> disección roma para una colección de alto rendimiento de los ojos para estudiar las correlaciones genotipo-fenotipo, probablemente post-mortem. Para su propósito, el método es conveniente y rápido. Sin embargo, este método es menos adecuado para la enucleación in vivo cuando uno opta para estudiar la vía visual aferente después de la enucleación (en los animales vivos) en lugar del propio ojo. En tal escenario, la supervivencia después de la enucleación es de gran importancia. Además, minimal daños vivo y preservación del nervio óptico y el tejido orbital es favorable. A continuación, presentamos un método enucleación alternativa, más similar a la descrita por Faguet et al (2008) 26, que ofrece ciertas propiedades ventajosas:. Se asocia con una recuperación postoperatoria rápida y se caracteriza por un umbral muy bajo de aprendizaje para los investigadores. En general, los diferentes métodos son complementarios dependiendo del enfoque de la investigación posterior: la morfología de los ojos o la investigación de las vías ópticas. ve_content &quot;&gt; En suma, la enucleación se puede aplicar de investigación de la visión hacia investigaciones de plasticidad homeostática y la cruz-modal cerebro, propiedades de respuesta gliales, y la estabilidad axón. En este artículo visualizado, se demuestra un método factible y fiable para la enucleación del ojo in vivo en el ratón.

<table><tbody><tr><td>Ketamine hydrochloride (Anesketin)</td><td>Dechra Veterinary Products (Eurovet)</td><td>BE-V136516</td><td></td></tr><tr><td>Medetomidine hydrochloride (Domitor)</td><td>Orion Corporation (Janssen Animal Health)</td><td>BE-V151742</td><td></td></tr><tr><td>Atipamezol hydrochloride (Antisedan)</td><td>Orion Corporation (Elanco Animal Health)</td><td>BE-V153352</td><td></td></tr><tr><td>Antibiotics (cefazolin, Kefzol)</td><td>Eurocept Pharmaceuticals</td><td>BE 106267</td><td></td></tr><tr><td>Eye ointment (Fucithalmic)</td><td>Leo Pharma nv-sa</td><td>BE 144654</td><td></td></tr><tr><td>Moria MC31 Forceps - Serrated Curved</td><td>Fine Science Tools</td><td>11370-31</td><td>For application in the mouse. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td></tr><tr><td>Narrow Pattern Forceps - curved</td><td>Fine Science Tools</td><td>11003-13</td><td>For application in the rat. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td></tr><tr><td>Hemostatic cotton wool</td><td>Qualiphar</td><td>N/A</td><td>Other hemostatic agents are equally suitable (&lt;em&gt;e.g.&lt;/em&gt;, Viscostat, #649, Ultradent Products)</td></tr></tbody></table>

a highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening

La enucleación o la extirpación quirúrgica de un ojo generalmente se pueden considerar como un modelo para deaferentación nervio. Proporciona una herramienta valiosa para estudiar los diferentes aspectos de la visual, cross-modal y la plasticidad del desarrollo a lo largo del sistema visual mamífero 1-4. Este sentido, demuestran una técnica elegante y sencillo para la extracción de uno o ambos ojos en el ratón, que se validó en ratones de 20 días de edad hasta adultos. Brevemente, un desinfectados pinzas curvas se utiliza para pinzar el nervio óptico detrás del ojo. Posteriormente, se realizan movimientos circulares para constreñir el nervio óptico y quitar el globo ocular. Las ventajas de esta técnica son de alta reproducibilidad, un mínimo o ningún sangrado, rápida recuperación post-operatorio y un umbral muy bajo de aprendizaje para el experimentador. Por lo tanto, una gran cantidad de animales puede ser manipulada y procesada con la cantidad mínima de esfuerzo. La naturaleza de la técnica puede inducir daños leves ala retina durante el procedimiento. Este efecto secundario hace que este método menos adecuado en comparación con Mahajan et al. (2011) 5, si el objetivo es recoger y analizar el tejido de la retina. Además, nuestro método se limita a post-oculares apertura de edades (de ratón: P10 - 13 en adelante) ya que el globo ocular necesita ser desplazados de la toma de corriente sin la eliminación de los párpados. La técnica de enucleación in vivo descrito en este manuscrito se ha aplicado recientemente con éxito con modificaciones menores en las ratas y parece útil para estudiar la vía visual aferente de los roedores en general.

La Figura 1 ilustra la eliminación con éxito del ojo utilizando el protocolo descrito y se caracteriza por la ausencia de sangrado o cualquier daño físico evidente para el tejido orbital o cuenca del ojo (Figuras 1A, 1B). Además, el ojo tiene una córnea eliminado lisa, la coroides y el disco óptico, indicativo de un globo completamente intacto (Figura 1C). Dado que nuestro protocolo incluye la sujeción del nervio óptico detrás del ojo y de giro mecánico, el nervio óptico del ojo eliminado es constreñido en la base de la retina (Figura 1D). Realización de los descritos procedimiento da como resultado un nervio óptico de corte limpio y sin ningún daño a la zona cerebral circundante (Figura 1E). Enucleación monocular, en combinación con el mapeo de la actividad (Figura 2), permite delinear fuertemente las regiones funcionales de entrada u ojo específicos en el visua contralaterall corteza del ratón 12,27 o columnas de dominancia ocular, incluso en los mamíferos de orden superior como los monos 28. En experimentos con ratones, la eliminación de una (ME) o ambos ojos (BE) en combinación con la estimulación visual específica y la detección de ARNm de zif268 o c-Fos los niveles de expresión de proteínas se aplicó para descubrir la activación neuronal regional en la corteza visual 12,27 . En contraste con los controles estimulados visualmente (Figura 2A), BE ratones mostraron actividad basal en la corteza visual debido a la completa falta de información visual (Figura 2B). Como tal, se descubrieron las fronteras entre visual con corteza no visual (es decir. Corteza somatosensorial en más secciones anterior y la corteza auditiva en más secciones posteriores). Los resultados de ME ratones con una semana de tiempo de supervivencia visualizaron las regiones de entrada específicos de los ojos en la corteza visual contralateral. Las dos regiones monocularmente impulsadas fueron hipoactivo y ubicados mediy al lateral de la zona binocular central (Figura 2C). <img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51936/51936fig1highres.jpg" width="500" /> Figura 1. Evaluación cualitativa del estado post-enucleación de la cuenca del ojo, el ojo eliminado y el nervio óptico. Después de la eliminación del ojo con unas pinzas curvas (A) no hay sangrado o daño se observa en la cuenca del ojo (B). El ojo es eliminado completamente intacto como se refleja por una apariencia normal de la córnea y la coroides (C, D). El nervio óptico se estrecha en el disco óptico donde sale del ojo (D). El examen de la parte ventral del cerebro revela un nervio corte limpio óptica (asterisco) y ningún daño aparente a otras estructuras (E). Las barras de escala en C, D: 1 mm. La barra de escala en E: 5 mm. A: anterior; L: izquierdo; P: posterior; R: derecha. Figura 2. funcionales subdivisiones específicas ojo-de entrada en la corteza visual del ratón según lo revelado por la enucleación. Las líneas negras y grises que conectan los ojos y la corteza representan el entrecruzamiento de las fibras aferentes de la retina y de las regiones de entrada específicos de los ojos. La actividad neuronal se visualiza en secciones coronales de control (A), BE (B) y ME (C) ratones por radiactiva de hibridación in situ (ISH) para zif268 (escala de grises) en torno al nivel Bregma -3.40 mm. En los animales control (a), la corteza visual en ambos hemisferios expresa una alta actividad después de la estimulación visual. Cuando uno o ambos ojos (s) se extirpa, una clara disminución de la señal de la actividad es visible en las regiones corticales privadas correspondientes. Monocular enucleado (C) ratones muestran una zona de alta actividad en la zona binocular de la corteza visual rodeado por una disminución de la señal en las zonas monoculares contralaterales al ojo eliminado. Barra de escala: 2 mm. Reproducido con permiso del Van Brussel et al 12.

Watch this Scientific Journal Video about A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening at JoVE.com

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

La eliminación de los ojos, también llamado enucleación, ofrece una estrategia útil para estudiar los aspectos de la visual, cross-modal, y la plasticidad del desarrollo a lo largo del sistema visual de mamíferos ya que induce irreversible parcial (monocular) o completa (binocular) pérdida de la visión. Aquí se describe un enfoque altamente reproducible y fácil de realizar en la enucleación vivo.

Un método altamente reproducible y fácil de Perform<em&gt; En Vivo</em&gt; Ocular enucleación en el ratón después de la abertura del ojo

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

a-highly-reproducible-straightforward-method-to-perform-vivo-ocular

Research

JoVE Journal

Biology

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

23.2K vistas.  KU Leuven - University of Leuven.  La eliminación de los ojos, también llamado enucleación, ofrece una estrategia útil para estudiar los aspectos de la visual, cross-modal, y la plasticidad del desarrollo a lo largo del sistema visual de mamíferos ya que induce irreversible parcial (monocular) o completa (binocular) pérdida de la visión. Aquí se describe un enfoque altamente reproducible y fácil de realizar en la enucleación vivo. 

Video: A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

Ratón enucleación del ojo por remoto de alto rendimiento fenotipificación

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Investigación

Medicina

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Vídeo-oculografía en ratones

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Neurociencias

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

En vivo-como organotípicos Cultura murino wholemount retina

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

Biología

Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the cortex to the contralateral eye to a stimulus is much stronger than the response of the ipsilateral eye in the binocular segment of the mouse primary visual cortex (V1). During the mammalian critical period, suturing the contralateral eye will result in a rapid loss of responsiveness of V1 cells to contralateral eye stimulation. With the continuing development of transgenic technologies, more and more studies are using transgenic mice as experimental models to examine the effects of specific genes on ocular dominance (OD) plasticity. In this study, we introduce detailed protocols for monocular visual deprivation and calculate the change in OD plasticity in mouse V1. After monocular deprivation (MD) for 4 days during the critical period, the orientation tuning curves of each neuron are measured, and the tuning curves of layer four neurons in V1 are compared between stimulation of the ipsilateral and contralateral eyes. The contralateral bias index (CBI) can be calculated using each cell&#39;s ocular OD score to indicate the degree of OD plasticity. This experimental technique is important for studying the neural mechanisms of OD plasticity during the critical period and for surveying the roles of specific genes in neural development. The major limitation is that the acute study cannot investigate the change in neural plasticity of the same mouse at a different time.

Here, we present detailed protocols for monocular visual deprivation and ocular dominance plasticity analysis, which are important methods for studying the neural mechanisms of visual plasticity during the critical period and the effects of specific genes on visual development.

Medición de la plasticidad de la privación visual monocular y la dominación ocular en la corteza visual primaria del ratón

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the ...

Biología del Desarrollo

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

En Vivo La proyección de imagen multimodal y análisis del modelo de neovascularización coroidea inducida por láser Mouse

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

En Vivo Proyección de imagen de ratones de reportero Cx3cr1gfp/gfp con tomografía de coherencia óptica de dominio espectral y exploración oftalmoscopia de láser

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

Neovascularization del diafragma como un modelo de ratón de iridis del rubeosis inducida por la punción

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Microdissection of the Rodent Eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

Microdisección del ojo de roedor

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...

Un método de punción de biopsia modificado para quemaduras precisas de álcali en ratones

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Autor destacado: Avance de la investigación en el tratamiento y la regeneración de la opacidad corneal 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

Inducción de lesiones corneales y alcalinas mediante una técnica de punzón y trefina en un modelo de ratón

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

Un método altamente reproducible y fácil de Perform

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