Name: intravital microscopy and thrombus induction in the earlobe of a hairless mouse
Uploaded: 2017-04-02T14:00:00
Description: Watch this Scientific Journal Video about Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse at JoVE.com

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Todos los experimentos in vivo (7221.3-1-006 / 15) se llevaron a cabo de acuerdo con la legislación alemana sobre protección de los animales y la Guía del NIH para el Cuidado y Uso de Animales de Laboratorio (Instituto de Recursos Animales de Laboratorio, Consejo Superior de Investigaciones Científicas). 1. Mantener general de los animales <ol><li> Realice los experimentos con ratones hr SKH1-HR macho de 4 a 6 semanas. Utilizar animales con un peso entre 20 y 25 g. ...

<ol>
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Hay varios pasos críticos para el éxito de la inducción de trombos en el lóbulo de la oreja de los ratones hr SKH1-HR. Para la solución de problemas, las respectivas etapas del protocolo se indican entre paréntesis. condiciones de examen son ideales en los animales jóvenes a la edad de 4 - 6 semanas y con baja cornificación de la epidermis. En animales de mayor edad, la calidad de la visualización de los vasos es peor y menos comparable debido a la distancia mayor entre l...

El propósito del presente artículo es describir la técnica de la microscopía intravital aplicada a la aurícula del ratón sin pelo para la observación directa y el análisis de la microcirculación, la formación de trombos, y la evolución de trombos. Con una tasa de incidencia de 1 en 1000, trombosis venosa sigue siendo una causa común de morbilidad. Aunque el diagnóstico, estrategias de prevención y terapias se han desarrollado en los últimos años, un tercio de la trombosis venosa se manifiesta como una embolia pulmonar 1. La trombosis arterial juega un papel crítico en las enfermedades cardiovasculares, que son la causa más común de muerte en los países industrializados. La trombosis arterial basado en la ruptura de las placas ateroscleróticas está involucrado en ataques cardiacos, infartos mesentéricas, y apoplejía. Cada cirugía expone estructuras subendoteliales a componentes de la sangre, cambia la dinámica de flujo de sangre, e inmoviliza el paciente. En la cirugía de endoprótesis de la extremidad inferior, órgano transplantation y trombosis cirugía de colgajo son causas frecuentes de complicaciones. trombosis microvascular en particular, con frecuencia provoca daños irreversibles, debido a la falta de síntomas clínicos. Del mismo modo, la trombosis microvascular juega una regla fundamental en varias enfermedades, incluyendo la púrpura trombótica trombocitopénica, sepsis, coagulación intravascular diseminada, síndrome antifosfolípido, y la insuficiencia venosa crónica, entre otros. Varios de los nuevos medicamentos para la terapia y prevención de la trombosis se han desarrollado en los últimos años, pero los fármacos antiplaquetarios y anticoagulantes todavía tienen efectos secundarios, antagonistas de la falta, y cuentan con efectos de larga duración. Estas deficiencias conducen a problemas en la atención médica de emergencia. Por lo tanto, se necesita más investigación para descubrir los procesos complejos que se producen durante la trombosis, que difícilmente puede ser simulada in vitro. El ratón sin pelo HR SKH1-Hr fue descubierta 1926 en un zoológico de Londres.Debido a un defecto genético en el cromosoma 14, el animal pierde su piel después del día postnatal 10. Esto hace que la aurícula bien vascularizado accesible para microscopía intravital de los vasos. El espesor medio de la oreja es de 300 m. Se compone de dos capas de la dermis, que están separados por el cartílago. En el lado dorsal convexa del cartílago, 3 haces vasculares entran en el lóbulo de la oreja. arcos vasculares apical y shunts basales conectan los tres haces. Las vénulas tienen diámetros comprendidos entre 200 micras (basal) y 10 m (apical). Capilares de malla fina rodean el cabello folículos vacíos 2. La anatomía del ratón sin pelo HR SKH1-Hr hace que la aurícula un modelo potente y rentable para la investigación de la trombosis.

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			<td height="19" style="height:19px;width:216px;">SKH-1/hr mice</td>
			<td style="width:161px;">Charles River</td>
			<td style="width:231px;">477</td>
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			<td height="17" style="height:17px;width:216px;">standard laboratory food</td>
			<td style="width:161px;">ssniff Spezialdiaeten</td>
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			<td>Leica&#160;</td>
			<td style="width:231px;">M651/M655&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
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			<td height="19" style="height:19px;width:216px;">intravital microscope</td>
			<td style="width:161px;">Zeiss</td>
			<td>Axiotech Vario 100&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
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			<td height="19" style="height:19px;width:216px;">objective (20x/0.95)&#160;</td>
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			<td>20x/0,50 W; Plan-NEOFLUAR&#160;</td>
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			<td>63x/0,95 W; ACHROPLAN&#160;</td>
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			<td height="19" style="height:19px;width:216px;">formaldehyde 4%</td>
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			<td>can be purchased from other vendors&#160;</td>
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			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">microforceps</td>
			<td style="width:161px;">Aesculap</td>
			<td style="width:231px;">BD331R</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">microscissors</td>
			<td style="width:161px;">Aesculap</td>
			<td style="width:231px;">OC496R</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">scalpel 21</td>
			<td style="width:161px;">Dahlhausen</td>
			<td style="width:231px;">11.000.00.511</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Prolene 7-0</td>
			<td style="width:161px;">Ethicon</td>
			<td style="width:231px;">XNEH7470</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Prolene 6-0</td>
			<td style="width:161px;">Ethicon</td>
			<td style="width:231px;">XN8706.P33</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">electrocautery</td>
			<td style="width:161px;">Servoprax</td>
			<td style="width:231px;">H40140</td>
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		<tr height="19">
			<td height="19" style="height:19px;width:216px;">acrylglass pad</td>
			<td style="width:161px;"></td>
			<td style="width:231px;"></td>
			<td>integrated heating, 0,5 cm high plane&#160;</td>
		</tr>
	</tbody>
</table>

intravital microscopy and thrombus induction in the earlobe of a hairless mouse

complicaciones trombóticas de las enfermedades vasculares son una de las principales causas de morbilidad y mortalidad en los países industrializados. Debido a las complejas interacciones entre los componentes sanguíneos celulares y no celulares durante la formación de trombos, estudios fiables de la fisiología y la patofisiología de la trombosis sólo pueden llevarse a cabo in vivo. Por lo tanto, este artículo presenta un modelo de oreja en ratones sin pelo y se centra en el análisis in vivo de la microcirculación, la formación de trombos, y la evolución de trombos. Mediante el uso de microscopía de fluorescencia intravital y la intravenosa (iv) la aplicación de los respectivos colorantes fluorescentes, un análisis repetitivo de la microcirculación en la aurícula fácilmente se puede realizar, sin la necesidad de la preparación quirúrgica. Además, este modelo puede ser adaptado para estudios in vivo de los diferentes temas, incluyendo la cicatrización de heridas, lesión por reperfusión, o angiogénesis. En resumen, la oreja de los ratones sin pelo es un modelo ideal para el en vivo estudio de la microcirculación de la piel en condiciones fisiológicas o fisiopatológicas y para la evaluación de su reacción a diferentes tratamientos sistémicos o tópicos.

Efectos del tratamiento sobre Cannabinoides trombogénesis Tras la inyección de 0,05 ml de FITC-dextrano, la inducción de trombos fototóxico conduce a una lesión endotelial y la formación de un tapón de plaquetas parietal (Figuras 2 y 3). En el presente estudio, la inducción de trombos después de la inyección ip de los cannabinoides (5 mg / kg de peso corporal) o vehículo dio lugar a un...

Watch this Scientific Journal Video about Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse at JoVE.com

Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

El modelo de oreja del ratón sin pelo SKH1-Hr hr permite microscopía de fluorescencia intravital de la microcirculación y la inducción de trombos fototóxico sin preparación quirúrgica previa en el lecho microvascular examinado. Por lo tanto, la oreja del ratón sin pelo es un excelente modelo in vivo para estudiar las interacciones complejas durante la formación de trombos microvasculares, la evolución de trombos, y la trombólisis.

Microscopia intravital y trombos de inducción en el lóbulo de la oreja de un ratón sin pelo

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

The overall goal of this experiment is to study the influence of potential vasoactive drugs on microvascular thrombus formation in vivo. This method can help answer key questions in thrombus formation such as identifying new antithrombotic substances that could help to prevent microvascular thrombus formation, which is one of the main complications in sepsis and during tissue transplantation. The main advantage of this technique is that the ear model of the hairless mouse enables intravital fluorescence microscopy and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed.Begin by weighing and recording the weight of the mouse. Then load the appropriate volume of the test substance into an insulin syringe. Administer the drug 30 minutes prior to thrombus induction.After anesthetizing the mouse 15 minutes prior to thrombus induction, place the mouse back into the cage until the onset of anesthesia. To verify sufficient anesthesia, pinch the tail with forceps and note the absence of a reaction. Next, 0.05 ml of defrosted 50 Dextran into an insulin syringe.After filling the syringe, ensure that no air bubbles remain, because even small intravenously administered air bubbles can be lethal for the animal. Place the anesthetized mouse on an acroglass pad with an integrated heating plate in the face-down position. Adjust the heating plate to 37 degress Celsius.Cover the mouse eye corneas with ophthalmic ointment. Then stitch two sutures of polypropylene 7.0 into the cranial and caudal edge of the right ear. Place the stitches as close to the edge and as proximal to the base as possible.Shift the mouse to a dorsal position. Fix all legs to the platform using adhesive bandage. Hook a suture under the front teeth and position the head in dorsiflexion by affixing the suture to the platform with adhesive bandage.Place the animal on the platform under the stereo microscope. Use 10X magnification. For microscopy of the right ear, prepare the left jugular vein by first creating a 5 mm incision in the skin of the neck in a craniocaudal direction.Then use microforceps and microscissors to dissect the subcutaneous tissue. Then, free the vein from its adventia without touching the vessel. Now, use the previously prepared insulin syringe for the injection of the fluoroscent dye.Carefully grab the vessel wall with the microforceps without perforating the vein. Penetrate the distended vessel wall with the syringe needle and inject 50 Dextran intravenously. Withdraw the needle and stop the bleeding using cotton swabs.Avoid blood and dye contamination of the ear. Transfer the animal on the heating plate to an acro glass construction with a slat for the hearing plate and 0.5 cm high plane for positioning the ear. Fix the animal in a prone position on the heating plate using adhesive bandage.Place the convex cartilage at the base of the ear beside the plane for the ear so that the apical part of the ear can be positioned flat on the plane. Add one drop of room temperature aqua to the acro glass plate. Using cotton swabs, absorb the drop of aqua, and let capillary forces attach the ear plane to the acro glass.The auricle must be positioned as flat as possible, even though the cartilage gives the auricle a convex shape. So only the more flexible distal part of the auricle should be positioned on the acrylic glass plate. To avoid tissue damage and extravasation of fluids and dye, touch the ear as little as possible with the forceps.Next, add drop of aqua to the convex dorsal side of the ear. Carefully put one cover slip on the ear without compressing the basal vessels entering the ear. Use cotton swabs to remove as much aqua as possible from under the cover slip To minimize the distance between the cover slip and the ear target vessels.Begin by adjusting the intravital fluorescence microscope for 50 Dextran visualization. Tranfer the prepared animal to the deck of the intravital fluorescence microscope. Using 5X, 10X and 20X magnification, and 20%light intensity, search for a venous vessel of 50 to 60 microns in diameter and with an anterograde blood flow.Add one drop of room-temperature water to the cover slip for water immersion of the 63X magnification objective. Immediately after the application of the water drop, begin recording the vessel for 20 seconds for the baseline assessment of the diameter and blood flow. Start thrombus induction five minutes after the injection of 50 Dextran.For this purpose, raise the light intensity to 100%During phototoxic thrombus induction, close the aperture of the microscope for two seconds within a 30-second period to check for occlusion of blood flow. If blood flow persists, open the aperture again. The vessel is classified as occluded if the flow stands still for 30 seconds or more, or if the blood flow is retrograde.Select and occlude five vessels per ear within a one hour period after injection of 50 Dextran. Here, animals were treated with vehicle or with a cannabinoids, anandamide, WIN 155, 212-2 or cannabidiol before injection of 50 Dextan prior to thrombus induction. Anandamide, but not CBD, and WIN 155, 212-2 significantly accelerated thrombus growth after one-time treatment as compared to vehicle control.In another setting, anandamide was combined with indomethacin to assess the impact of cyclooxygenase-dependent products on thrombus formation via anandamide. Co-administration with indomethacin neutralized the pro-thrombotic effect of anandamide. Therefore, acute exposure to anandamide elicits a cyclooxygenase-dependent pro-thrombotic effect in vivo.Once mastered, thrombus induction in tight vessels can be performed in 90 minutes, including preparation. While attempting this procedure, it's important to handle the auricle very carefully every time it is touched to avoid extravasation of the fluids and dye. Though this method can provide insight into thrombus formation, it can also be applied to other systems, such as wound healing, flap failure, and angiogenesis.After watching this video, you should have a good understanding of intravenous fluorescence dye application, position of the auricle, and illumination of specific vessels in order to perform intravital phototoxic thrombus induction.

Research

JoVE Journal

Medicine

Technical Aspects of the Mouse Aortocaval Fistula

Aspectos técnicos de la aortocava Fístula Ratón

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Investigación

Medicina

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

La inducción farmacológica de la melanina epidérmica y Protección contra las quemaduras solares en un modelo de ratón humanizado

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

El mantenimiento de un modelo de ratón ortotópico Glioblastoma Restricciones cerebral parenquimatosa físicas y Conveniente para intravital microscopía de dos fotones

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

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 ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Inducción de la Aterosclerosis Acelerada en Ratones: El Modelo de "Lesiones por Alambre"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

Combina la microscopia Intravital y ecografía de contraste de la trasera de ratón para estudiar la perfusión muscular y vasodilatación inducida por la insulina

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

La inducción de la lesión por isquemia-reperfusión en la piel de la oreja del ratón para intravital n fotones de imágenes de la respuesta inmune

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

Microscopia intravital de monocito autoguiado hacia el blanco y la angiogénesis tumorales en un modelo murino de la enfermedad Arterial periférica

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Inducción y Micro-CT en la proyección de imagen de malformaciones cavernosas cerebrales en modelo murino

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

Resonancia Magnética Cardíaca para la Evaluación del Trombo Cardíaco Sospechoso: Técnicas Convencionales y Emergentes

We present the conventional cardiac magnetic resonance (CMR) protocol for evaluating a suspected thrombus and highlight emerging techniques.&#160;The ...