Funding for this work was provided by: AG007218 and NIH F32 AG031606.
Images were generated in the Core Optical Imaging Facility, which is supported by UTHSCSA, NIH-NCI P30 CA54174 (CTRC at UTHSCSA) and NIH-NIA P01AG19316.

Nota: Todos los animales procedimientos fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Texas Health Science Center en San Antonio y fueron consistentes con los lineamientos llegar. 1. La anestesia para la Preparación cortical <ol><li> Coloca el ratón en una cámara de inducción con 3.2% isofluorano mezclado con oxígeno para inducir anestesia. Observe la disminución de la tasa de respiración como el ratón se induce. Apriete la pata del ratón para determinar si el ratón está listo para pasar al cono de la nariz. Nota: El nivel de anestesia es un paso crítico en cualquier preparación in vivo y se debe tener cuidado de no inducir a un nivel que hará que la isquemia global. </li><li> Una vez que el ratón se anestesia suficiente, traslado del animal a la plataforma de la cirugía / imagen y colocar la nariz del ratón en el cono de la nariz y aplicar 1-1,2% isofluorano para mantener un estado anestesiado. Asegúrese de que el ratón está mintiendo en una co temperaturaalmohadilla térmica ntrolled para mantener la temperatura corporal (37 ° C +/- 0,5 ° C) a través de los procedimientos restantes. Coloca un ungüento veterinario sobre los ojos para evitar la sequedad mientras que bajo anestesia. </li><li> Monitorear la fisiología del ratón utilizando un sistema de oximetría de pulso usando la cola o el pie de clip proporcionado con el sistema. Comprobar que se mantiene la frecuencia respiratoria entre 50 a 65 respiraciones / min. Compruebe que el ritmo cardíaco se mantiene entre 300 a 450 latidos por minuto y la saturación de oxígeno se mantiene entre los 97 a 98% para asegurar la supervivencia a largo plazo del animal. </li><li> Cuando el ratón se anestesia adecuada, afeitar el pelo sobre el cráneo usando una maquinilla eléctrica, eliminar el vello residual y limpio con betadine, seguido de un hisopo de etanol. Repita este procedimiento hasta tres veces para asegurar un ambiente quirúrgico estéril. </li></ol> 2. Procedimiento Quirúrgico <ol><li> Con el cuero cabelludo totalmente limpia y afeitada, hacer una incisión de 5 mm en el cuero cabelludo del ratón para revelar la fissu cranealres y para localizar bregma. </li><li> Use un aplicador de algodón estéril para eliminar cualquier resto de fascia recubre el cráneo. </li><li> Pegar un anillo hecho de encargo de acero inoxidable (Figura 1) con adhesivo de tejido al hueso que recubre la corteza parietal utilizando las coordenadas estereotáxicas de Bregma: -1 a -3 mm y lateral: 2-4 mm. Nota: El pegamento normalmente establece dentro de 2 min después de la colocación del anillo sobre el hueso. </li><li> Coloque el anillo a un titular estereotáxica (Figura 2) para estabilizar el ratón y para evitar el movimiento durante la exploración. </li><li> Bajo un microscopio de disección de grado quirúrgico, perforar lentamente una sección de 1-2 mm en el cráneo usando una herramienta dremel como la velocidad controlada (Meisinger 3.9 mm broca) asegurándose de mantener el nivel de zona, ya que se perfora. Lograr esto usando un patrón de zig-zag. Para evitar la acumulación de calor, ajuste la velocidad de perforación a la baja y tomar descansos frecuentes. </li><li> Cuando el cráneo craneal convierte shinny en apariencia, continuará el adelgazamientodel cráneo utilizando una hoja de bisturí utilizando el mismo patrón de zig-zag para mantener el nivel de la superficie adelgazada lisa para facilitar la eliminación de capas delgadas de cráneo craneal. Con la punta de la hoja de bisturí hacer pequeños trazos lineales con una ligera presión para eliminar las capas delgadas de hueso a la vez. Continúe hasta la vasculatura es claramente visible a través del microscopio de disección. </li><li> Si el experimentador perfora a través o romper el cráneo durante el proceso de adelgazamiento, la eutanasia del animal debido al daño probable que la corteza subyacente. Nota: El cráneo del ratón es de aproximadamente 300 micras de espesor y está compuesto de dos capas delgadas de hueso compacto (uno externo y uno interno de la capa) y una capa de hueso esponjoso intercalada entre las dos capas de hueso compacto. La capa externa del hueso compacto y la mayoría del hueso esponjoso se retiran dentro de la zona de perforación de 5 mm resulta en una capa de 50 micras aproximada de hueso compacto restante (ver Figura 2B). Visualización de la vasculatura se asegurará de que el cráneo final de adelgazado intacta es de aproximadamente 50 micras de espesor. Por consiguiente, el cráneo todavía está presente cuando adelgazado a este espesor. </li></ol> 3. Microscopio Set-up <ol><li> Utilice un sistema de microscopio invertido (sistemas de dos fotones convencional, confocal o) que tiene un inversor objetivo. Nota: También es posible utilizar un microscopio vertical estándar. El factor limitante será el espacio entre el escenario y los objetivos. Las modificaciones de la etapa puede ser necesario llevar a cabo esta configuración. </li><li> Asegure la plataforma quirúrgica / formación de imágenes a una etapa por encargo que se encuentra a un lado de la base del microscopio. Nota: La plataforma se realiza mediante un conector de laboratorio para permitir el movimiento vertical de la plataforma quirúrgica / imagen bajo el objetivo. El jack de laboratorio está montado en una placa fijada a cuatro polos cilíndricos. (Ver Figura 2). </li><li> Coloque el inversor objetivo contiene un 20Xobjetiva sobre la ventana craneal. Utilice una fuente de luz externa para encontrar la ventana craneal mirando a través de los oculares del microscopio y coloque el objetivo en el área de imagen. Nota: El área de formación de imágenes se denota por la presencia de la vasculatura. </li><li> Para objetivos a base de agua, usar fluido cerebral espinal artificial (LCRa) (NaCl 130 mM; KCl 30 mM; 12 mM KH 2 PO 4; 200 mM NaHCO 3; 30 mM HEPES; y glucosa 100 mM) como el medio debido a la potencial fugas en la cavidad craneal durante la exploración (Figura 3). </li></ol> 4. Rosa de Bengala Tinte Preparación, Administración y de inducción de Stroke <ol><li> Preparar una solución / ml 20 mg fresca de Rosa de Bengala en el líquido cefalorraquídeo artificial (LCRa); filtrar y esterilizar antes de la administración. No vuelva a utilizar o almacenar la Rosa de Bengala, una vez que se ha mezclado. Prepare una solución fresca para cada experimento. </li><li> Dar una inyección en la vena de la cola 0,1 ml de Rosa de Bengala, mientras que senlatado de la ventana craneal con un láser de 561 nm para asegurar la inyección adecuada de la solución. Nota: Rosa de Bengala se visualizará dentro de 5 segundos después de la inyección en la vasculatura del cerebro. Todo el recipiente debe estar lleno de Rosa de Bengala. </li><li> Después de la inyección adecuada de tinte Rosa de Bengala elegir un recipiente apropiado para la trombosis basado en el diámetro del vaso (40-80 micras) para asegurar la reproducibilidad de un volumen de la lesión en particular. Diferenciar entre las arterias y las venas mirando a la dirección del flujo sanguíneo: arterias se moverán de diámetro más grande para los vasos de menor diámetro, las venas se mueven de menor a vasos de mayor diámetro. Esto se logra fácilmente mediante la visualización una vez que se inyecta Rosa de Bengala. </li><li> Cambie el ajuste del microscopio de la siguiente manera: <ol><li> Aumentar el tiempo de permanencia. Nota: Esto variará dependiendo del sistema de microscopio que se utilizan. </li><li> Aumentar la potencia del láser a 100%. </li><li> Recoge imágenes de la secuencia de tiempo en 1 cuadro / s utilizando lavelocidad de exploración máxima. </li></ol></li><li> Analiza el ratón hasta que se forme un coágulo estable dentro de la embarcación. Nota: Normalmente, esto se logra a 5 minutos de la exploración continua (Ver Figura 4). </li><li> Después de la inducción de la formación de coágulos usando Rosa de Bengala, quite el ratón de la zona de formación de imágenes de nuevo a la microscopio de disección. Retire con cuidado el anillo de acero inoxidable del cráneo craneal. Examine la ventana craneal para cualquier sangrado. Si se produce sangrado, dar por terminado el experimento. </li><li> Utilice 6,0 monofilamento de sutura para cerrar la incisión sobre el cráneo. Coloca un ungüento antibiótico lo largo de la línea de sutura para prevenir la infección. Inyectar Buprenex (0,05 mg / kg) por vía subcutánea cada 12 horas durante tres días para el manejo del dolor. </li><li> Devuelva el ratón a una cámara de recuperación después de la retirada de la anestesia hasta que esté completamente despierto y se mueve libremente. </li><li> Devuelva el ratón a una jaula limpia para una mayor investigación en un momento posterior. </li></ol> 5. Imaging longitudinal en los días siguientes <ol><li> Emplear los siguientes métodos para realizar las imágenes longitudinal en los días posteriores post-photothrombosis. <ol><li> Anestesiar el ratón como se describe en la Sección 1 de los métodos. </li><li> Tras la anestesia adecuada re-abrir el cuero cabelludo, eliminando las suturas residuales para reabrir la piel que cubre el campo de la imagen previamente perforado. </li><li> Use un aplicador de algodón estéril para eliminar cualquier resto de fascia recubre el cráneo. </li><li> Pegue un anillo hecho a medida de acero inoxidable (Figura 1) con adhesivo tisular en el hueso que cubre el campo de la imagen anterior. </li><li> Coloque el anillo a un titular estereotáxica (Figura 2) para estabilizar el ratón y para evitar el movimiento durante la exploración. </li><li> Busque la vasculatura que subyace el cráneo previamente adelgazada. Utilice inyección en la vena de la cola de FITC-dextrano para verificar la presencia del coágulo previamente inducido. </li><li> Utilice 6,0 monofilamento de sutura para cerrar la encisión sobre el cráneo. Coloca un ungüento antibiótico lo largo de la línea de sutura para prevenir la infección. Inyectar Buprenex (0,05 mg / kg) por vía subcutánea cada 12 horas durante tres días para el manejo del dolor. </li><li> Devuelva el ratón a una cámara de recuperación después de la retirada de la anestesia hasta que esté completamente despierto y se mueve libremente. </li></ol></li></ol> 6. Verificación de Inducción Stroke (post mortem) <ol><li> A la conclusión de un estudio, verificar el volumen sistólico utilizando cloruro (TTC) tinción 2,3,5-trifeniltetrazolio como se muestra en la Figura 5. El método completo se puede encontrar en 12. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

La capacidad de traducir experimental ictus fisiopatología de animal a la aplicación humana ha estado plagado de fracaso. Sin embargo, el uso de modelos animales, tales como el modelo photothrombosis, permite una mejor comprensión de la fisiopatología de carrera y la exploración de nuevos enfoques terapéuticos para proporcionar neuroprotección después de un accidente cerebrovascular. Accidentes cerebrovasculares y microinfartos producidos por el modelo photothrombotic corticales pequeños son clínicamente relev...

La técnica descrita permite visualizar in vivo respuestas celulares inmediatamente después de la inducción de la Rosa de Bengala photothrombosis en un ratón intacto. Rosa de Bengala (4,5,6,7-tetracloro-2 &#39;, 4&#39;, 5 &#39;, 7&#39;-tetrayodofluoresceína) es un colorante fotosensible utilizado para inducir el accidente cerebrovascular isquémico en modelos animales (ratón y rata). Después de una inyección en bolo de RB a través de la vena de la cola y la iluminación posterior a través de un cráneo adelgazado con una luz láser de 564 nm, un trombo se induce causando un derrame cerebral fisiológica 1. El método fue descrito originalmente por Rosenblum y El-Sabban en 1977, y más tarde fue adaptada por Watson a mediados de 1980 1,2. En breve, Rosa de Bengala se irradia con luz verde de excitación (láser de 561 nm en nuestro caso), que genera la producción de especies reactivas de oxígeno, que activa posteriormente factor tisular, un iniciador de la cascada de coagulación. La inducción de la cascada de coagulación produce un isquémico lesion que es patológicamente relevantes para accidente cerebrovascular clínico 3. Stroke tiene una fisiopatología compleja debido a la interacción de muchos tipos diferentes de células, incluyendo neuronas, glia, endotelio y el sistema inmunológico. La elección de la mejor técnica para el estudio de un proceso celular particular requiere múltiples consideraciones. Las técnicas experimentales caer en términos generales en una de tres categorías: in vitro, in vivo e in silico con cada uno con ventajas y desventajas Estudios in vitro tienen la desventaja principal de la eliminación de células de su entorno natural y por lo tanto no pueden reproducir los efectos observados en un intacta,. animal vivo. Las técnicas in vivo para la replicación proporcionar experimental mejorada de estados de enfermedad con una mayor importancia de la traducción. En silico generalmente se refiere a modelado por ordenador de una enfermedad o proceso celular, y mientras que cada vez más utilizado para estudiar las posibles interacciones farmacológicas para el examenPLE, cualquier información obtenida todavía debe ser probado en células o tejidos vivos. El modelo ideal de accidente cerebrovascular en el laboratorio debe demostrar características patológicas similares a los observados en la población humana. Si bien hay características fisiológicas comunes de accidente cerebrovascular en la población humana, también hay muchas diferencias dependiendo del tipo de lesión experimentada. Carrera en la población humana se produce como pequeñas o grandes oclusiones de los vasos, lesiones hemorrágicas, y la arteria a arteria o cardio-embolias que dan lugar a los volúmenes de infarto variadas, así como las diferencias en los mecanismos relacionados con cada patología. La ventaja de la utilización de modelos de accidente cerebrovascular animal es la generación de infartos reproducibles que imitan las características de ictus humano. Los modelos más comunes de accidente cerebrovascular animales incluyen oclusión de la arteria usando: oclusión de la arteria cerebral media (métodos de incandescencia o embólicos endovasculares) que modelos MCAO distal y el modelo photothrombosis. Las ventajas de unad desventajas de cada modelo se han revisado en otro lugar (ver 4 y 5). Los modelos globales isquémicos (MCAO), si bien es relativamente fácil de realizar son menos relevantes para acariciar humano que son modelos de accidente cerebrovascular focales. Además, estos métodos son muy variables en la inducción de lesiones infarto cerebral reproducibles. El modelo photothrombosis es altamente reproducible siempre que el experimentador controla sus experimentos bien, proporcionando una clara ventaja sobre los modelos de MCAO. Sin embargo, debido a insulto microvasculatura el modelo ha sido descrito para mostrar una penumbra isquémica mínimo, el área donde las células se cree que son rescatable 6,7. Además, edema vasogénico y la formación de edema citotóxico también pueden ser inducidas después de la irradiación de la superficie de imágenes. A pesar de estas limitaciones de la técnica ha proporcionado una nueva visión de muchos procesos fisiológicos después del accidente cerebrovascular 8, 9, 10, 11.

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rose bengal photothrombosis by confocal optical imaging in vivo: a model of single vessel stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

El objetivo de este método era para inducir un accidente cerebrovascular isquémico en modelos animales (ratón y rata) después de una inyección en bolo de RB a través de la vena de la cola y la iluminación posterior de un cráneo adelgazado con una luz láser de 561 nm. Las imágenes en la Figura 4 demuestran la progresión de la formación de coágulos dentro de un solo recipiente después de la irradiación de la zona en 0, 1, 1,5 y 2 min. Antes de la formación de coágulos de todo el recipient...

Watch this Scientific Journal Video about Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke at JoVE.com

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rosa de Bengala Photothrombosis por imagen óptica confocal<em&gt; En Vivo</em&gt;: Un modelo de solo recipiente Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

rose-bengal-photothrombosis-confocal-optical-imaging-vivo-model

Research

JoVE Journal

Medicine

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

14.9K vistas.  The University of Texas Health Science Center San Antonio. Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection. 

Video: Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

No invasiva de medición óptica del metabolismo cerebral en lactantes y Hemodinámica

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Investigación

Medicina

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Imágenes in vivo de la angiogénesis tumoral usando fluorescencia confocal videomicroscopia

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

Un modelo murino de hemorragia subaracnoidea

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

En vivo de imágenes óptico de tumores del cerebro y la artritis Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Combinado<em&gt; En vivo</em&gt; Óptica y μCT imágenes para vigilar la infección, inflamación y Hueso anatomía en una infección de implantes ortopédicos en ratones

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Inducida por la quimioterapia Vascular Toxicidad - En tiempo real<em&gt; En vivo</em&gt; Imagen de Deterioro del buque

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Photothrombosis-induced Focal Ischemia as a Model of Spinal Cord Injury in Mice

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) below the site of injury. The secondary ischemia that develops following the initial mechanical insult is a serious complication of the SCI and severely impairs the function and viability of surviving neuronal and non-neuronal cells in the SC. In addition, ischemia is also responsible for the growth of lesion during chronic phase of injury and interferes with the cellular repair and healing processes. Thus there is a need to develop a spinal cord ischemia model for studying the mechanisms of ischemia-induced pathology. Focal ischemia induced by photothrombosis (PT) is a minimally invasive and very well established procedure used to investigate the pathology of ischemia-induced cell death in the brain. Here, we describe the use of PT to induce an ischemic lesion in the spinal cord of mice. Following retro-orbital sinus injection of Rose Bengal, the posterior spinal vein and other capillaries on the dorsal surface of SC were irradiated with a green light resulting in the formation of a thrombus and thus ischemia in the affected region. Results from histology and immunochemistry studies show that PT-induced ischemia caused spinal cord infarction, loss of neurons and reactive gliosis. Using this technique a highly reproducible and relatively easy model of SCI in mice can be achieved that would serve the purpose of scientific investigations into the mechanisms of ischemia induced cell death as well as the efficacy of neuroprotective drugs. This model will also allow exploration of the pathological changes that occur following SCI in live mice like axonal degeneration and regeneration, neuronal and astrocytic Ca2+ signaling using two-photon microscopy.

Photothrombosis is a minimally invasive and highly reproducible procedure to induce focal ischemia in the spinal cord and serves as a model of spinal cord injury in mice.

Photothrombosis inducida por isquemia focal como un modelo de lesión de médula espinal en ratones

Spinal cord injury (SCI) is a devastating clinical condition causing permanent changes in sensorimotor and autonomic functions of the spinal cord (SC) ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualización Cuantitativa del Infiltrado de Leucocitos en un Modelo Murino de Miocarditis Fulminante por Microscopía de Hoja Ligera

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

El Método de Muestreo de 4 Barcos para Estudios Integrativos de la Fisiología Placentaria Humana<em&gt; In Vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...