Este trabajo fue apoyado por el NIH subvención 1R01EY022901, el Premio de Desarrollo de Carreras de Investigación para Prevención de la Ceguera (RPB), CMReeves &amp; MA Fundación Reeves, Fundación E. Matilda Ziegler para Ciegos, Caballeros Templarios Eye Foundation, y una subvención sin restricciones al Departamento de Oftalmología en la Universidad de Utah de RPB. Agradecemos Balamurali Ambati para la asistencia técnica en la multimodalidad Imaging System Spectralis y Tao Zhang para los debates y los comentarios sobre el manuscrito.

1. Preparación de Instrumentos <ol><li> El procedimiento se realiza en una sala de procedimientos en una instalación para animales. </li><li> Use mascarillas, gorros de pelo, batas quirúrgicas, los pies cubiertas estériles y guantes antes de comenzar el experimento. </li><li> Caliente el agua en un vaso de precipitados a ~ 40 ° C sobre una placa caliente. </li><li> Coloque una almohadilla azul estéril en la parte superior de una almohadilla eléctrica que se utilizará más adelante para mantener la temperatura corporal del ratón durante la exploración. Encienda la almohadilla térmica. </li><li> Prepare el sistema de imágenes: <ol><li> Quite la cubierta de polvo y encender el láser. </li><li> Saque la lente de 55 ° y montarlo en la máquina. </li><li> Abra el software de imagen de la computadora e ingrese la información del ratón para obtener imágenes bajo la hoja de un nuevo paciente (por ejemplo, el genotipo, la edad, etc.). En &quot;Tipo de dispositivo&quot;, seleccione el modo de infrarrojos (IR). </li></ol></li></ol> Nota: Se ha informado de que el uso Of una lente asférica de doble externa puede mejorar la calidad de imagen 17-20 aunque no tenemos ningún problema en la obtención de imágenes de alta calidad por IV-ICGA sin usar lentes externas (ver resultados representativos, Figuras 1-4). 2. Vena de la cola de inyección de ICGA <ol><li> Dilata los ojos de ratones con 1% solución oftálmica de tropicamida y esperar 5 min. </li><li> Pesar el ratón para determinar la cantidad de anestesia (ketamina / xilazina / Acepromazina 65-100/10-20/1-3 mg / kg) es necesario. </li><li> Recuperar una jeringa estéril de 1 ml junto con una aguja de calibre 32. Inyectar el ratón por vía intraperitoneal con los anestésicos (13 mg / ml de ketamina, 2,6 mg / ml de xilazina 0,3 mg / ml de Acepromazina en PBS estéril). Espere hasta que el ratón está totalmente anestesiado (~ 5 min). </li><li> Coloque la cola del ratón en 40 ° C el agua caliente para causar vasodilatación de la vena. </li><li> Recuperar una jeringa de 1 ml con una aguja de calibre 32. Extraer la cantidad deseada de ICG, típicamente 50 l de 1 mg / ml de ICG, que es estéril filtrada con un filtro de jeringa de 0,2 M en un tubo estéril, por un 25 g de ratón (2 mg / kg). Tenga cuidado de no introducir el aire en la jeringa. </li><li> Limpie la cola con un algodón con alcohol para esterilizar el área a ser inyectada. </li><li> Sostenga la cola con una mano para que la vena lateral de la cola es ascendente. Con el bisel de la aguja hacia arriba, inyectar la aguja ~ 2 mm en la vena en un ángulo mínimo. Tenga cuidado de no perforar la vena. Dibujar atrás de la jeringa ligeramente y buscar los rastros de flujo sanguíneo en el cubo de la aguja, lo que indica que la aguja se inserta con éxito en la vena. </li><li> Lentamente inyectar ICG en la vena. Debe haber un mínimo de resistencia al inyectar. Retire la aguja y aplique una gasa con alcohol directamente en el sitio de inyección para ~ 5-10 segundos para detener cualquier sangrado. El ratón está entonces listo para formación de imágenes. Con el fin de captar la primera fase (0-4 min después de la inyección), es essential a la imagen con el ratón rápidamente. </li></ol> Nota: los ojos del ratón pueden conseguir fácilmente en seco y pueden desarrollar cataratas bajo anestesia. Es importante mantener el ojo húmedo mediante la aplicación de PBS estéril durante el procedimiento. Limpie el exceso de PBS con un hisopo de algodón estéril antes de la grabación ICGA. Otros laboratorios han utilizado una lente de contacto para evitar la deshidratación de la córnea 17-20. 3. ICG angiografía <ol><li> Comience a tomar imágenes de 30-40 segundos después de la inyección de ICG, que permite la captura de la primera fase de llenado de la coroides hasta circulaciones retina y la coroides son a su máximo brillo (0-4 min). La vasculatura retinal se visualiza mejor en foco ~ 35-45 dioptrías y la vasculatura coroidea se visualiza al 10-15 dioptrías. Nota: Durante el primer examen de un modelo animal, se recomienda para capturar imágenes desde todos los ángulos (nasal, temporal, dorsal y ventral) para identificar todos los posibles abnormalities en la vasculatura. Durante la primera fase, las arterias y las venas coroideas medianas y grandes son bien visualizadas. En el modelo animal utilizado en este protocolo, las lesiones coroideas (por ejemplo polipoideas dilataciones) pueden comenzar a aparecer 1 min en la fase temprana. </li><li> Ajuste el enfoque de la imagen sobre la vasculatura. Control para el brillo y enfoque utilizando el módulo de control y mando de enfoque, respectivamente. Estos valores son ajustables digitalmente y son fácilmente mantienen constantes. Mantenga la distancia desde el ojo del ratón a la constante de lente de la cámara para asegurar la calidad de la imagen es reproducible utilizando la técnica que sigue. Nota: Dado que el dispositivo sólo puede imagen una porción de la parte posterior del ojo, tratamos de mantener el enfoque, el brillo y la distancia entre la lente de la cámara y el ojo del ratón constante a medida que la imagen de toda la parte posterior del ojo desde distintos ángulos. La clave para esto es para alinear la luminiscencia de forma circular emitida por el ICG a través del ojo con el campo de la vieW de la cámara. Esto se logra haciendo que de izquierda a derecha, de arriba a abajo, y en-y-hacia fuera los ajustes de la posición de la cámara hasta que la imagen tiene áreas oscuras. Cuando la luminiscencia y el campo de visión de la cámara están alineados, la distancia desde el ojo a la lente será reproducible para el siguiente grupo de imágenes, así como a una distancia óptima para la formación de imágenes de calidad. </li><li> Una vez que la vasculatura está enfocado, capturar los cuadros de imagen pulsando el botón redondo de color negro en el módulo de adquisición. El botón redondo de color negro también se puede utilizar para reducir o aumentar la señal de ICG para la mejor calidad de imagen. </li><li> Determinar el ángulo de visión óptimo y la profundidad de enfoque para imagen lesiones coroideas. Es importante mantener la posición del ojo, la profundidad de enfoque, y otros ajustes de dispositivos fijos para toda la ICGA tiempo-curso. Las imágenes se guardan pulsando el botón adquieren en el panel de la pantalla táctil del módulo de adquisición. </li><li> Adquirir imágenes en la fase media de 6-15 min popainyección er. Nota: Tanto la coroides y los vasos de la retina se distinguen menos. Vasculatura coroidea aparece fluorescencia difusa. Lesiones coroideas expositoras hiperfluorescencia emergen en contraste con el desvanecimiento normal circundante fluorescencia de fondo. </li><li> Adquirir imágenes en la fase tardía en 17-25 min después de la inyección. Nota: Hiperfluorescencia desvanece. Ambos vasos coroideos y la retina ya no son visibles. La cabeza del nervio óptico se vuelve negro. Lesiones coroideas hiperfluorescentes tienen máximo contraste con el fondo decoloración. </li><li> Después de terminar la adquisición de las imágenes, aplicar un gel transparente lubricante ocular para los ojos de ratones y dejar el ratón sobre una almohadilla térmica para la recuperación. </li><li> Ratones Vuelva a sus jaulas y zona de espera. Exportación de imágenes como archivos TIFF o JPEG para su posterior análisis. </li></ol> Nota: El tiempo de cada fase no es absoluta. Encontramos que el momento de eafase ch podría cambiar dependiendo de la cantidad de ICG inyectado. Más ICG tiende a prolongar cada fase. La mejor manera de definir una fase es de acuerdo con las características principales de cada fase que aparece arriba.

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YF es un inventor de dos patentes pendientes que son relevantes para el modelo de ratón AMD usado en este trabajo. SK, ZB y ADJ tienen nada que revelar.

En este estudio, hemos demostrado el uso de ICGA a imagen lesiones coroideas en ratones transgénicos HtrA1. Las características de los principios, mediados y finales de las fases de ICGA en nuestro modelo de ratón coincide con el curso temporal bien en los estudios humanos 1. Esto es importante para hacer mejores comparaciones entre patología y animales fenotipos humanos, que son de gran valor para la investigación sobre los mecanismos fisiopatológicos y las estrategias de tratamiento de condiciones rel...

Angiografía con verde indocianina (ICGA) es una prueba de diagnóstico para problemas de imagen relacionados con los vasos sanguíneos en el ojo. El espectro de absorción de ICG oscila desde 790 hasta 805 nm, mientras que el espectro de emisión oscila desde 770 hasta 880 nm con la emisión máxima a 835 nm 1. Esto es diferente de la otra tinte popular, fluoresceína de sodio, cuyo espectro cae en el rango visible. El espectro infrarrojo permite ICG para penetrar a través de epitelio pigmentario de la retina (RPE), de fluido serosanguinolento, y exudados lipídicos, todo lo cual puede bloquear fácilmente la visualización por angiografía con fluoresceína base de sodio-fluoresceína (FA). ICG es del 98% unido a proteínas en el sistema vascular que resulta en menos extravasación, lo que permite mejorar la imagen de los vasos de la coroides y lesiones coroideas 1,2. ICGA es casi la única opción para visualizar la vasculatura coroidea, que es posterior al RPE. La figura 1 muestra la comparación de ICGA y FA en la vasculatura de formación de imágenes en ojos de ratón. FA puede be utilizado para la imagen de la vasculatura de la retina así pero no la vasculatura coroidea. En contraste, ICGA se puede utilizar para la imagen tanto de la retina y la vasculatura coroidea. ICGA se realiza con sistemas de imágenes digitales de alta resolución o oftalmoscopios láser de barrido (SLO), junto con las cámaras de vídeo sensibles a los infrarrojos, que vamos a utilizar en este estudio. En la clínica, ICGA se ha recomendado en el diagnóstico de una serie de trastornos coriorretinianas involucra la vasculatura coroidea incluyendo vasculopatía coroidal polipoidea (PCV), Retina angiomatosas Proliferación (RAP), estrías angioides, viteliforme distrofia macular, coriorretinopatía serosa central, hemangioma de coroides, la retina hemorragia macroaneurismas arteriolares, tumores coroideos, y ciertas formas de uveítis posterior 1,3. La combinación de ICGA con FA y la tomografía de coherencia óptica (OCT) proporciona potentes herramientas para los médicos en el diagnóstico y manejo de exudativa macular relacionada con la edaddegeneración (AMD) 4-10. ICGA es especialmente útil para el diagnóstico de afecciones que implican la coroides. De hecho, ICGA es considerado el estándar de oro para el diagnóstico de PCV, una variante de la DMAE exudativa 11-13. PCV se caracteriza por una red de vasos ramificados con dilataciones polipoideas terminales en la vasculatura coroidea 11-13. PCV se asocia frecuentemente con destacamentos serosanguinolento recurrentes del EPR y la retina con fugas y el sangrado de los componentes polipoideas 11,14,15. Recientemente hemos informado de la generación del primer modelo animal PCV por transgénica que expresa HTRA1 humana, una serina proteasa multifuncional, en el ratón epitelio pigmentario retiniano (EPR) 16. Hemos demostrado que el aumento de HTRA1 indujo rasgos característicos del PCV, por ejemplo, lesiones polipoideas. Aquí hemos demostrado el uso del tiempo-por supuesto ICGA por inyección en la vena de la cola en la investigación de AMD utilizando nuestro modelo de ratón HTRA1. Nuestros datos sugieren que losIV-ICGA es superior a IP (o subcutánea (SC))-ICGA que se utilizan actualmente en el campo de 17,18 para la caracterización de las lesiones en la coroides. Declaración sobre Investigación Animal Los experimentos con animales se realizaron de acuerdo a los protocolos aprobados por el Cuidado de Animales institucional y el empleo Comisión (IACUC) y se llevaron a cabo de conformidad con la Declaración de ARVO para el uso de animales en Oftálmica y Vision Research.

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	<tbody>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">Spectralis Multi-Modality Imaging System</td>
			<td style="width:152px;">Heidelberg Engineering, Germany</td>
			<td style="width:132px;">SPECTRALIS HRA+OCT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tropicamide ophthalmic solution (1%)</td>
			<td style="width:152px;">Bausch &#38; Lomb</td>
			<td style="width:132px;">NDC 24208-585-64</td>
			<td>for dilation of pupils</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenTeal Gel</td>
			<td style="width:152px;">Genteal</td>
			<td style="width:132px;">NDC 58768-791-15</td>
			<td>&#160;clear lubricant eye gel&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-996-06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:152px;">Lloyd Laboratories</td>
			<td style="width:132px;">NADA 139-236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:152px;">Vedco Inc</td>
			<td style="width:132px;">NDC 50989-160-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">32-G Needle</td>
			<td>Steriject</td>
			<td style="width:132px;">PRE-32013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-ml syringe</td>
			<td style="width:152px;">BD</td>
			<td style="width:132px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Indocyanine Green</td>
			<td style="width:152px;">Pfaltz &#38; Bauer</td>
			<td style="width:132px;">I01250</td>
			<td></td>
		</tr>
	</tbody>
</table>

detecting abnormalities in choroidal vasculature in a mouse model of age-related macular degeneration by time-course indocyanine green angiography

Indocianina verde Angiografía (o ICGA) es una técnica realizada por los oftalmólogos para diagnosticar anormalidades de la coroides y la vasculatura de la retina de varias enfermedades oculares como la degeneración macular asociada a la edad (DMAE). ICGA es especialmente útil para la imagen de la vasculatura coroidea posterior del ojo debido a su capacidad de penetrar a través de la capa pigmentada con su espectro infrarrojo. Curso temporal ICGA puede dividirse en etapas temprana, media y fases finales. Las tres fases proporcionan información valiosa sobre la patología de los problemas de los ojos. Aunque el tiempo-curso ICGA por vía intravenosa (IV) de inyección se utiliza ampliamente en la clínica para el diagnóstico y manejo de problemas coroideo, ICGA por inyección intraperitoneal (IP) se utiliza comúnmente en la investigación con animales. Aquí hemos demostrado la técnica para obtener imágenes de alta resolución ICGA tiempo-por supuesto en ratones mediante la inyección de cola vena y confocal de barrido láser oftalmoscopia. Se utilizó esta técnica para la imagen de la coroides lesiones en un modelo de ratón de la degeneración macular relacionada con la edad. Aunque es mucho más fácil introducir ICG a la vasculatura del ratón a través de IP, nuestros datos indican que es difícil obtener imágenes reproducibles curso temporal ICGA por IP-ICGA. Por el contrario, a través de inyección ICGA vena de la cola proporciona imágenes tiempo-por supuesto ICGA alta calidad comparables a los estudios en humanos. Además, demostramos que ICGA realizó en ratones albinos da imágenes más claras de los vasos coroideos que la realizada con ratones pigmentados. Nosotros sugerimos que el tiempo-por supuesto-IV ICGA debería convertirse en una práctica habitual en la investigación AMD basado en modelos animales.

Se realizó curso de tiempo ICGA en HtrA1 ratones transgénicos y sus compañeros de camada WT de control, ambos de los cuales están en el fondo CD1. El fondo CD1 albino fue seleccionado para facilitar la angiografía con verde indocianina (ICGA) de imágenes (ver Discusión). Algunos aneurisma como dilataciones comenzó a aparecer en la primera fase en el ratón HTRA1 (Figura 2, una flecha roja indica la dilatación en el extremo de un recipiente y un círculo rojo indica una lesión polipoidea tipo c...

Watch this Scientific Journal Video about Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography at JoVE.com

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocianina verde Angiografía (o ICGA) realizado por inyección en la vena de la cola proporciona imágenes de curso temporal ICGA alta calidad para caracterizar las anormalidades en la coroides ratón.

La detección de anormalidades en la vasculatura coroidea en un modelo de ratón de la relacionada con la edad degeneración macular por Time-curso de indocianina verde Angiografía

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

detecting-abnormalities-choroidal-vasculature-mouse-model-age-related

Research

JoVE Journal

Medicine

13.5K vistas.  University of Utah Health Sciences Center.  Indocianina verde Angiografía (o ICGA) realizado por inyección en la vena de la cola proporciona imágenes de curso temporal ICGA alta calidad para caracterizar las anormalidades en la coroides ratón. 

Video: Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Evaluación no invasiva de la cardiaca Anomalías en la miocarditis autoinmune experimental por Resonancia Magnética de imágenes de microscopía en el ratón

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Investigación

Medicina

A Mouse Model for Laser-induced Choroidal Neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

Un modelo de ratón para inducida por láser neovascularización coroidea

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

El Modelo Monoiodoacetate del dolor de la osteoartritis en el ratón

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Detección de Anastasis en Vivo por CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

The goal of this study is to assess whether the suprachoroidal graft of adipose-derived stem cells included in the stromal vascular fraction and platelets derived from platelet-rich plasma by the Limoli Retinal Restoration Technique can improve visual acuity and retinal sensitivity responses in eyes affected by dry age-related macular degeneration.

Terapia regenerativa por autoinjerto Suprachoroidal célula en Degeneración Macular seca relacionada con la edad: informe preliminar en Vivo

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medición de la contractilidad isométrica de la arteria mesentérica de ratón con cable miografía

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Neonatal BALB/c Mouse Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight infants, with high mortality and unclear pathogenesis. The cause of NEC may be related to inflammatory immune regulatory system abnormalities. An NEC animal model is an indispensable tool for NEC disease immune research. NEC animal models usually use C57BL/6J neonatal mice; BALB/c neonatal mice are rarely used. Related studies have shown that when mice are infected, Th2 cell differentiation is predominant in BALB/c mice compared to C57BL/6J mice. Studies have suggested that the occurrence and development of NEC are associated with an increase in T helper type 2 (Th2) cells and are generally accompanied by infection. Therefore, this study used neonatal BALB/c mice to induce an NEC model with similar clinical characteristics and intestinal pathological changes as those observed in children with NEC. Further study is warranted to determine whether this animal model could be used to study Th2 cell responses in NEC.

Un modelo de ratón BALB/c neonatal de enterocolitis necrosante

Necrotizing enterocolitis (NEC) is the most severe gastrointestinal (GI) disease that often occurs in premature infants, especially very low birth weight ...

Robotic Left Hepatectomy using Indocyanine Green Fluorescence Imaging for an Intrahepatic Complex Biliary Cyst

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. Surgery is the cornerstone treatment for patients with BC. While total BC excision and Roux-Y hepaticojejunostomy is the treatment method of the choice in patients with extrahepatic BC (i.e., Todani I-IV), patients with intrahepatic BC (i.e., Todani V) benefit the most from a surgical liver resection. In recent years, minimally invasive liver surgery (MILS) including robotic MILS has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. Robotic major MILS is still considered technically demanding and a detailed description of the technical approach during robotic major MILS has only been limitedly discussed in the literature. The current article describes the main steps for a robotic left hepatectomy in a patient with a large BC Todani Type V. The patient is in French position with 5 trocars placed (4 robotic, 1 laparoscopic assistant). After mobilizing the left hemiliver, the left and right hepatic artery are dissected carefully followed by a cholecystectomy. Intraoperative ultrasound is performed to confirm localization and margins of the BC. The Left hepatic artery and left portal vein are isolated, clipped, and divided. Indocyanine green (ICG) fluorescence imaging is used regularly during the entire procedure to visualize and confirm biliary tract anatomy and the BC. Parenchymal transection is performed with robotic cautery hook for the superficial part and robotic cautery spatula, bipolar cautery, and vessel sealer for the deeper parenchyma. The postoperative course was uncomplicated. A robotic left hepatectomy is technically demanding, yet a feasible and safe procedure. ICG-fluorescence imaging aids in delineating the BC and bile duct anatomy. Further, comparative studies are needed to confirm clinical benefits of robotic MILS for benign and malignant indications.

Robotic liver surgery has gained more acceptance as a feasible, safe, and effective procedure for the treatment of both benign and malignant indications. However, robotic left hepatectomy is still technically demanding. We describe our surgical technique of a robotic left hepatectomy using indocyanine green fluorescence imaging for a large biliary cyst.

Hepatectomía izquierda robótica con imágenes de fluorescencia verde de indocianina para un quiste biliar complejo intrahepático

Biliary cysts (BC) are rare congenital dilatations of intra- and extrahepatic parts of the biliary tract and bear a significant risk of carcinogenesis. ...

Fluorescent Laparoscopic Central Hepatectomy for Liver Cancer Using Indocyanine Green Negative Staining

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ultrasound, important vascular structures, and surgeon experience. With the development of anatomical hepatectomy, visual surgery technology has gradually been applied to this type of surgery, particularly indocyanine green (ICG)-guided anatomical hepatectomy. As ICG can be specifically ingested by hepatocytes and used for fluorescence tracing, negative staining techniques have been applied according to different tumor positions. Under ICG fluorescent guidance, the surface boundary and deep resection plane can be more accurately displayed during liver resection. Thus, the tumor-bearing liver segment can be anatomically removed, which helps to avoid damage to important vessels and reduce ischemia or congestion of the remaining liver tissue. Finally, the incidence of postoperative biliary fistula and liver dysfunction is reduced; therefore, a better prognosis is obtained after the resection of liver cancer. Centrally located liver cancer is usually defined as a tumor located at segments 4, 5, or 8 that requires resection of the middle section of the liver. These are among the most difficult hepatectomies to perform because of the large surgical wounds and multiple vessel transections. Based on the specific tumor location, we formulated the required resection ranges by designing personalized fluorescent staining strategies. By completing anatomical resection based on the portal territory, this work aims to achieve the best therapeutic effect.

The present protocol describes fluorescent negative staining in laparoscopic central hepatectomy. This technique can make hepatectomy more accurate and precise.

Hepatectomía central laparoscópica fluorescente para el cáncer de hígado con tinción negativa con verde de indocianina

Laparoscopic hepatectomy is an important treatment method for liver cancer. In the past, the resection boundary was usually determined by intraoperative ...

Explorando la autofluorescencia cuantitativa en la degeneración macular relacionada con la edad

A Workflow to Quantitatively Determine Age-Related Macular Degeneration Lesion-Specific Variations in Fundus Autofluorescence

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment epithelium (RPE), now quantifiable with the advent of confocal scanning laser ophthalmoscopy-based quantitative autofluorescence (QAF). QAF has been shown to be generally decreased at the posterior pole in age-related macular degeneration (AMD). The relationship between QAF and various AMD lesions (drusen, subretinal drusenoid deposits) is still unclear.
This paper describes a workflow to determine lesion-specific QAF in AMD. A multimodal in vivo imaging approach is used, including but not limited to spectral domain optical coherence tomography (SD-OCT) macular volume scanning and QAF. Using customized FIJI plug-ins, the corresponding QAF image is aligned with the near-infrared image from the SD-OCT scan (characteristic landmarks; i.e., vessel bifurcations). The foveola and the edge of the optic nerve head are marked in the OCT images (and transferred to the registered QAF image) for accurate positioning of the analysis grids.
AMD-specific lesions can then be marked on individual OCT BScans or the QAF image itself. Normative QAF maps are created to account for the varying mean and standard deviation of QAF values throughout the fundus (QAF images from a representative AMD group were averaged to build normative standard retinal QAF AMD maps). The plug-ins record the X and Y coordinates, z-score (a numerical measurement that describes the QAF value in relation to the mean of AF maps in terms of standard deviation from the mean), mean intensity value, standard deviation, and number of pixels marked. The tools also determine z-scores from the border zone of marked lesions.&#160;This workflow and the analysis tools will improve the understanding of the pathophysiology and clinical AF image interpretation in AMD.

Autor destacado: Comprensión de la fisiopatología de la degeneración macular relacionada con la edad con el flujo de trabajo QAF 

This research describes a workflow to determine and compare autofluorescence levels from individual regions of interest (e.g., drusen and subretinal drusenoid deposits in age-related macular degeneration [AMD]) while accounting for varying autofluorescence levels throughout the fundus.

Un flujo de trabajo para determinar cuantitativamente las variaciones específicas de la lesión de la degeneración macular relacionada con la edad en la autofluorescencia del fondo de ojo

Fundus autofluorescence (FAF) imaging allows the noninvasive mapping of intrinsic fluorophores of the ocular fundus, particularly the retinal pigment ...