Este estudio fue parcialmente apoyado por el NIH subvención RO1NS053407 a JH Zhang. Nos gustaría dar las gracias al Sr. Damon Klebe por sus valiosas contribuciones.

Todos los procedimientos se realizaron de acuerdo con la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio y aprobados por el Cuidado de Animales y el empleo Comisión de Loma Linda University. 1. Preparación prequirúrgica Técnicas asépticas se recomiendan para todos los procedimientos quirúrgicos. Desinfecte el aparato estereotáxico y preparar instrumentos quirúrgicos estériles antes de la cirugía. Use el equipo de protección personal (EPP) durante todo el manejo animal. Use una almohadilla eléctrica durante la cirugía para mantener la temperatura corporal del animal fisiológico. <ol><li> Pesar el ratón 8-12 semanas de edad mediante una escala haz animales triple. </li><li> Co-inyectar ketamina (100 mg / kg) y xilazina (10 mg / kg) por vía intraperitoneal luego permitir 7-10 minutos a que la anestesia surta efecto (monitor para una adecuada sedación). </li><li> Coloque el ratón sobre una manta térmica y afeitarse la cabeza. </li><li> Aplique ungüento oftálmico en ambos ojos. </li><li> Asegure ellas vías respiratorias, moviendo suavemente la lengua lateralmente, y asegurar cuidadosamente la cabeza del ratón sobre el aparato estereotáxico. Nota: La cabeza debe estar asegurado horizontalmente a la base del marco estereotáxico. </li><li> Desinfecte el área quirúrgica con Betadine y enjuague con un 70% de etanol. Repita alternando aplicaciones de Betadine y etanol al 70% para un total de tres veces. Aplicadores con punta de algodón se puede utilizar para este propósito. </li></ol> 2. Sangre modelo de inyección <ol><li> Haz un 1 cm de largo incisión de línea media del cuero cabelludo con una hoja de bisturí # 10. </li><li> Utilice aplicadores con punta de algodón para eliminar el tejido blando que cubre el cráneo, con el fin de exponer el punto de intersección perpendicular de la sutura coronal y sagital (bregma). </li><li> Montar la jeringa de Hamilton (250 l) a la bomba de inyección, y estereotáxicamente dirigir la aguja (26 Gauge) durante bregma. </li><li> A continuación, ajuste los brazos manipuladores estereotácticas para colocar la aguja de 0,2 mm por delante y 2mm lateralmente a la derecha. En estas coordenadas hacer un pequeño agujero craneal rebaba, utilizando un taladro de velocidad variable con una broca de 1 mm. </li><li> Suspender la cola del animal y desinfectar su superficie inferior con 70% de etanol. </li><li> La punción de la arteria central de la cola con una aguja estéril (por ejemplo, 26 Gauge) y recoger la sangre arterial en un tubo capilar unheparinized. </li><li> Transfiera la sangre rápidamente del tubo capilar en el cuerpo de vidrio de la jeringa Hamilton, a continuación, insertar el émbolo. </li><li> Vuelva a colocar el l ahora de 30 años o más de sangre arterial que contiene una jeringa Hamilton en la bomba de inyección e inserte la aguja (con el borde biselado hacia la sutura sagital) a través del orificio de trepanación sólo hasta que su bisel ya no es visible. </li><li> Desde este punto de avance de la aguja 3 mm ventralmente e inyectar 5 l de sangre autóloga a razón de 2 l / min. </li><li> Después de la terminación de la primera avance de inyección de la aguja 0,7 mm más en profundidad. </li><li> Esperar5 min y después se inyectan 25 l de sangre en el cuerpo estriado derecho. </li><li> Al término de la segunda inyección, deje la aguja en la posición de 10 minutos adicionales, antes de retirarlo a una velocidad de 1 mm / min. </li><li> Selle el orificio de trepanación con cera de hueso y suturar la piel. </li><li> Para la analgesia postoperatoria inyectar 0,05 mg / kg de buprenorfina por vía subcutánea en precalentadas líquidos (solución salina normal). </li></ol> 3. La colagenasa modelo de inyección <ol><li> A raíz de los preparativos prequirúrgicos, repita los pasos 1-4 como se describe para el modelo de inyección de sangre. </li><li> Llene la jeringa Hamilton (10 l) con 0.075U de bacterias (clostridios) colagenasa VII-S disolvió en 0,5 l de solución salina. Evitar la formación de burbujas de aire. </li><li> Vuelva a colocar la jeringa Hamilton en la bomba de inyección e inserte la aguja (26 Gauge), a través del orificio de trepanación sólo hasta que su bisel ya no es visible. </li><li> Avance la aguja de 3,7 mm ventral e inyectar el 0.075U de la colagenasa into el cuerpo estriado derecho a un ritmo de 2 l / min. </li><li> Una vez completada la inyección, deje la aguja en la posición de 10 minutos adicionales, antes de retirarlo a una velocidad de 1 mm / min. </li><li> Selle el orificio de trepanación con cera de hueso y suturar la piel. </li><li> Inyectar 0,05 mg / kg de buprenorfina por vía subcutánea en precalentadas post-operatorias fluidos. </li></ol> 4. Sham operación <ol><li> A raíz de los preparativos prequirúrgicos, repita los pasos 1-4 como se describe para el modelo de inyección de sangre. </li><li> Inserte la aguja (26 Gauge) 3,7 mm ventral a través del orificio de trepanación. La aguja debe permanecer en posición durante 10 min antes de ser retirado a una velocidad de 1 mm / min. </li><li> Selle el orificio de trepanación con cera de hueso y suturar la piel. </li><li> Inyectar 0,05 mg / kg de buprenorfina por vía subcutánea en precalentadas post-operatorias fluidos. </li></ol> 5. Los resultados representativos Hemorragia intraestriatal Experimental evoca morfológica comoasí como los cambios de comportamiento en los roedores. Estos cambios pueden ser evaluados para asegurar una adecuada ejecución del procedimiento, o para investigar los efectos de los tratamientos potenciales. La generación de la hemorragia en un área del cerebro específica (ganglios de la base, por ejemplo) es más esencial para un enfoque reproducible, y puede ser verificada en las secciones del cerebro brutos o teñido histológico (Figura 1-2). La lesión de los resultados de los ganglios basales en los déficits sensoriomotoras, que pueden ser cuantificados a través de diversas evaluaciones del comportamiento. Resultados de la prueba de esquina a su vez mostraron que, después experimental del lado derecho ICH, los ratones resultó significativamente más a menudo ipsilateralmente y lejos de la alteración contralateral (izquierda), con operación simulada que los animales a las 24 y 72 h después de la cirugía (Figura 3 A). Además, la capacidad de colocar adecuadamente la alteración (a la izquierda) extremidad anterior en una superficie, después de la estimulación vibrisas, se evaluó mediante el test de extremidad anterior de colocación. A las 24 y 72 horas después de la cirugía, ratones subproyectada para el lado derecho ICH mostraron las colocaciones significativamente menos que los animales operación simulada. La medición de edema cerebral se emplea con frecuencia para cuantificar la extensión de la lesión cerebral después de ICH experimental. Las inyecciones intracerebrales de sangre autóloga (30 l) o colagenasa bacteriana (0,075 U) condujo a un aumento significativo del contenido de agua del cerebro en la corteza ipsilateral y los ganglios basales en 24 (Figura 4 A) y 72 h (Figura 4 B) después de la cirugía ( en comparación con el tratamiento simulado). El resultado de las pruebas de comportamiento (Figura 3) y el grado de edema cerebral (Figura 4) no mostraron diferencias entre la sangre y los modelos de inyección de colagenasa en volúmenes dados. <img alt="Figura 1" src="/files/ftp_upload/4289/4289fig1.jpg" /> Figura 1. Modelado de la HIC en ratones. (A) El esquema simplificado de una sección coronal del cerebro 0,2 mm anterior del bregma ilustra la proposed ubicación de sangre autóloga o inyección de colagenasa. El ventrículo lateral se marca LV. CPU significa caudado-putamen, una parte del cuerpo estriado, y GP identifica el globus pallidus. Ambos, el cuerpo estriado, así como el globus pallidus pertenecen a un grupo de sub-cortical núcleos, también conocida como los ganglios basales. (B) Representante microfotografía de una sección coronal del cerebro anterior de 0,2 mm bregma, obtenido a las 24 h después de la inyección intraestriatal de sangre entera autóloga. <img alt="Figura 2" src="/files/ftp_upload/4289/4289fig2.jpg" /> Figura 2. Manifestación histológica del hematoma. Representante hematoxilina y eosina (H &amp; E) criosección manchado coronal (10 micras) de un cerebro de ratón, que ilustra el tamaño del hematoma a las 24 h después de la inyección intraestriatal de colagenasa bacteriana (0,075 U). LH = hemicuerpo izquierdo, HR = hemisferio derecho. &lt;<html> img alt = &quot;Figura 3&quot; src = &quot;/ files/ftp_upload/4289/4289fig3.jpg&quot; /&gt; Figura 3. Evaluaciones neurofuncionales siguientes ICH experimental en ratones. Inyección intraestriatal de sangre autóloga (30 l) o colagenasa bacteriana (0,075 U) causada reproducibles déficit neurofuncionales. (A) ratones después de ICH experimental mostró vueltas significativamente más derecho que los animales operación simulada a 24 y 72 h después de la cirugía. (B) La capacidad de colocar las patas delanteras de la extremidad izquierda se vio afectada después de ICH a las 24 y 72 horas después de la cirugía. Los valores se expresan como media ± SEM y analizados con el test de Kruskal-Wallis de análisis de varianza unidireccional en Ranks, seguido por el método de Student-Newman-Keuls. Un valor de P &lt;0,05 fue considerado estadísticamente significativo, n = 6-12 por grupo, * p &lt;0,05 en comparación con el tratamiento simulado. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig3large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> . igura 4 &quot;src =&quot; / files/ftp_upload/4289/4289fig4.jpg &quot;/&gt; Figura 4. Evaluación del contenido de agua del cerebro después de ICH experimental en ratones. Inyección intracerebral de sangre autóloga (30 l) o colagenasa bacteriana (0,075 U) llevó a un aumento significativo del contenido de agua del cerebro en la corteza ipsilateral y los ganglios basales en 24 (A) y 72 hr (B) después de ICH-inducción. Los valores se expresan como media ± SEM y analizados con una forma de análisis de varianza, seguido de Tukey test post hoc. Un valor de P &lt;0,05 fue considerado estadísticamente significativo, n = 6-10 por grupo, * p &lt;0,05 en comparación con el tratamiento simulado. <a href="http://www.jove.com/files/ftp_upload/4289/4289fig4large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> .

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No hay conflictos de interés declarado.

Los modelos animales de hemorragia intracerebral (ICH), contribuyen en gran medida a una comprensión avanzada de la fisiopatología de la enfermedad, y se utilizan para desarrollar y evaluar nuevas estrategias terapéuticas en un ambiente preclínica. Intraparenquimatosa inyecciones de sangre autóloga o colagenasa bacteriana son métodos bien establecidos para generar ICH en los roedores. Ambos métodos se desarrollaron inicialmente en la rata, sin embargo, debido a la disponibilidad creciente de cepas transgénicas y...

<table border="1"><tbody><tr><td> Material </td><td> Empresa </td><td> Número de catálogo </td><td> Comentario </td></tr><tr><td> Marco de Head estereotáctica </td><td> Stoelting Co. </td><td> 51600 </td><td></td></tr><tr><td> Nanomite bomba de jeringa </td><td> Harvard Apparatus </td><td> PY2 70-2217 </td><td></td></tr><tr><td> Jeringa Hamilton </td><td> Hamilton Company </td><td> 1725RN (250 l) 1701 RN (10 l) </td><td> 26 Indicador de aguja de 250 l y 10 jeringas mu l. </td></tr><tr><td> Microtaladro </td><td> Herramientas Artes Ciencias </td><td> 18000-17 </td><td></td></tr><tr><td> Microtaladro rebaba </td><td> Herramientas Artes Ciencias </td><td> 19007-09 </td><td> 0,9 mm de diámetro </td></tr><tr><td> Colagenasa de tipo VII-S </td><td> Sigma-Aldrich </td><td> C2399 </td><td></td></tr><tr><td> Tubos capilares de microhematocrito </td><td> Fisher Scientific </td><td> 22-362-574 </td><td> unheparinized </td></tr><tr><td> Bone Wax </td><td> Ethicon </td><td> W31 </td><td></td></tr><tr><td> Suture </td><td> Ethicon </td><td> 1676G </td><td></td></tr><tr><td> La ketamina </td><td> JHP Farmacia </td><td> 42023-115-10 </td><td> Ketalar </td></tr><tr><td> Xilazina </td><td> LLOYD Laboratorios </td><td> 139-236 </td><td> AnaSed </td></tr></tbody></table>

modeling intracerebral hemorrhage in mice: injection of autologous blood or bacterial collagenase

Hemorragia intracerebral espontánea (ICH) define una enfermedad potencialmente mortal neurológico que representa el 10-15% de todas las hospitalizaciones relacionadas con la carrera y para los cuales no hay tratamientos efectivos disponibles hasta la fecha 1,2. Debido a la heterogeneidad de la HIC en los seres humanos, diferentes modelos preclínicos son necesarios para explorar a fondo posibles estrategias terapéuticas 3. ICH Experimental es comúnmente inducida en los roedores mediante inyección intraparenquimatosa de cualquiera de sangre autóloga o colagenasa bacteriana 4. El modelo apropiado se selecciona basándose en la fisiopatología de la inducción y progresión de la lesión hemorragia. El modelo de inyección de sangre imita una hemorragia rápidamente progresivo. Alternativamente, colagenasa bacteriana enzimáticamente interrumpe la lámina basal de los capilares del cerebro, causando una hemorragia activos que generalmente se desarrolla durante varias horas 5. Edema resultante perihematoma y déficits neurofuncionales se puede cuantificar vaivénm ambos modelos. En este estudio, hemos descrito y evaluado un modelo modificado de doble inyección de sangre autóloga conjunto 6, así como un modelo de inyección de ICH de colagenasa bacteriana 7, ambos de los cuales objetivo de los ganglios basales (corpus striatum) de machos CD-1 ratones. Se evaluaron los déficits neurofuncionales y edema cerebral a las 24 y 72 horas después de la inducción de ICH. Inyección intraestriatal de sangre autóloga (30 l) o colagenasa bacteriana (0.075U) causada reproducibles déficit neurofuncionales en ratones y edema cerebral aumentado significativamente a las 24 y 72 h después de la cirugía (p &lt;0,05). En conclusión, tanto los modelos de rendimiento consistentes infartos hemorrágicos y representan métodos básicos para la investigación preclínica ICH.

Watch this Scientific Journal Video about Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase at JoVE.com

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Modelos animales clínicamente relevante de hemorragia intracerebral (HIC) son necesarios para profundizar en el conocimiento de accidente cerebrovascular hemorrágico y examinar nuevas estrategias terapéuticas. En este estudio, se describe y evaluar dos modelos que implementan ICH inyecciones unilaterales de sangre autóloga o bien total o colagenasa bacteriana en los ganglios basales (corpus striatum) de ratones.

Modelado Hemorragia intracerebral en ratones: La inyección de sangre autóloga o colagenasa bacteriana

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

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24.0K vistas.  Loma Linda University School of Medicine. Modelos animales clínicamente relevante de hemorragia intracerebral (HIC) son necesarios para profundizar en el conocimiento de accidente cerebrovascular hemorrágico y examinar nuevas estrategias terapéuticas. En este estudio, se describe y evaluar dos modelos que implementan ICH inyecciones unilaterales de sangre autóloga o bien total o colagenasa bacteriana en los ganglios basales (corpus striatum) de ratones.

Video: Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modelado de accidente cerebrovascular en ratones - Oclusión arteria cerebral media con el modelo de filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Investigación

Medicina

Sequencing of Bacterial Microflora in Peripheral Blood: our Experience with HIV-infected Patients

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral and cellular mucosal immune system1,2. 
 Microbiota is shielded from the immune system via a strong mucosal barrier. Infections and antibiotics are known to alter both the normal 
	gastrointestinal tract barrier and the composition of resident bacteria, which may result in possible immune abnormalities3.
HIV causes a breach in the gastrointestinal barrier with progressive failure of mucosal immunity and leakage into the systemic circulation of bacterial bioproducts, such as lipopolysaccharide and 
	bacterial DNA fragments, which contribute to systemic immune activation4-7. Microbial translocation is implicated in HIV/AIDS immunopathogenesis and response to therapy 4,8.
We aimed to characterise the composition of bacteria translocating in peripheral blood of HIV-infected patients. To pursue our aim we set up a PCR reaction for the panbacteric 16S ribosomial gene 
	followed by a sequencing analysis.
Briefly, whole blood from both HIV-infected and healthy subjects is used. Given that healthy individuals present normal intestinal homeostasis no translocation of microflora is expected in 
	these patients. Following whole blood collection by venipuncture and plasma separation, DNA is extracted from plasma and used to perform a broad range PCR reaction for the panbacteric 
	16S ribosomial gene9. Following PCR product purification, cloning and sequencing analyses are performed.

Our experiment will show how to perform a sequencing analysis of bacterial species translocating in peripheral blood of HIV positive patients.

La secuenciación de la microflora bacteriana en la sangre periférica: Nuestra experiencia con pacientes infectados por VIH

The healthy gastrointestinal tract is physiologically colonized by a large variety of commensal microbes that influence the development 
	of the humoral ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

La entrega de los agentes terapéuticos a través de intracerebroventricular (ICV) y la inyección intravenosa (IV) en ratones

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

Assessing Changes in Volatile General Anesthetic Sensitivity of Mice after Local or Systemic Pharmacological Intervention

One desirable endpoint of general anesthesia is the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is less straightforward than it is in human patients. A widely used behavioral surrogate for hypnosis in rodents is the loss of righting reflex (LORR), or the point at which the animal no longer responds to their innate instinct to avoid the vulnerability of dorsal recumbency. We have developed a system to assess LORR in 24 mice simultaneously while carefully controlling for potential confounds, including temperature fluctuations and varying gas flows. These chambers permit reliable assessment of anesthetic sensitivity as measured by latency to return of the righting reflex (RORR) following a fixed anesthetic exposure. Alternatively, using stepwise increases (or decreases) in anesthetic concentration, the chambers also enable determination of a population's sensitivity to induction (or emergence) as measured by EC50 and Hill slope. Finally, the controlled environmental chambers described here can be adapted for a variety of alternative uses, including inhaled delivery of other drugs, toxicology studies, and simultaneous real-time monitoring of vital signs.

Loss of the righting reflex has long served as a standard behavioral surrogate for unconsciousness, also called hypnosis, in laboratory animals. Alterations in volatile anesthetic sensitivity caused by pharmacological interventions can be detected with a carefully controlled high-throughput assessment system, which may be adapted for delivery of any inhaled therapeutic.

La evaluación de cambios en la volátil Anestésico general La sensibilidad de los ratones después de la intervención farmacológica local o sistémica

One desirable endpoint of general anesthesia is  the state of unconsciousness, also known as hypnosis. Defining the hypnotic state in animals is  less ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Intrastriatal Injection of Autologous Blood or Clostridial Collagenase as Murine Models of Intracerebral Hemorrhage

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective treatment and failure of large clinical trials aimed at hemostasis and clot removal demonstrate the need for further mechanism-driven investigation of ICH. This research may be performed through the framework provided by preclinical models. Two murine models in popular use include intrastriatal (basal ganglia) injection of either autologous whole blood or clostridial collagenase. Since, each model represents distinctly different pathophysiological features related to ICH, use of a particular model may be selected based on what aspect of the disease is to be studied. For example, autologous blood injection most accurately represents the brain&#39;s response to the presence of intraparenchymal blood, and may most closely replicate lobar hemorrhage. Clostridial collagenase injection most accurately represents the small vessel rupture and hematoma evolution characteristic of deep hemorrhages. Thus, each model results in different hematoma formation, neuroinflammatory response, cerebral edema development, and neurobehavioral outcomes. Robustness of a purported therapeutic intervention can be best assessed using both models. In this protocol, induction of ICH using both models, immediate post-operative demonstration of injury, and early post-operative care techniques are demonstrated. Both models result in reproducible injuries, hematoma volumes, and neurobehavioral deficits. Because of the heterogeneity of human ICH, multiple preclinical models are needed to thoroughly explore pathophysiologic mechanisms and test potential therapeutic strategies.

Preclinical models of intracerebral hemorrhage are utilized to mimic certain aspects of clinical disease. Thus, mechanisms of injury and potential therapeutic strategies may be explored. In this protocol, two models of intracerebral hemorrhage are described, intrastriatal (basal ganglia) injections of autologous blood or collagenase.

Inyección intraestriatal de sangre autóloga o clostridial colagenasa como modelos murinos de la hemorragia intracerebral

Intracerebral hemorrhage (ICH) is a common form of cerebrovascular disease and is associated with significant morbidity and mortality. Lack of effective ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Modelado espontánea metastásico Carcinoma de células renales (CCR metastásico) en ratones después de la nefrectomía

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Guiada por ecografía transtorácica intramiocárdica inyección en ratones

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Carrera Modelado en ratones: Coagulación permanente de la arteria cerebral media Distal

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.