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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Toutes les expériences in vivo (7221.3-1-006 / 15) ont été menées conformément à la législation allemande sur la protection des animaux et le NIH Guide pour les soins et l&#39; utilisation des animaux de laboratoire (Institut des ressources animales de laboratoire, Conseil national de recherches). 1. Tenue générale des animaux <ol><li> Effectuez les expériences avec des souris mâles hr SKH1-HR de 4 à 6 semaines. Utiliser des animaux d&#39;un poids compris en...

<ol>
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Il y a plusieurs étapes critiques pour l&#39;induction de thrombus réussie dans le lobe de l&#39; oreille de souris SKH1-Hr. Pour le dépannage, les étapes respectives du protocole sont indiqués entre parenthèses. conditions d&#39;examen sont idéales chez les jeunes animaux à l&#39;âge de 4 - 6 semaines et avec une faible kératinisation de l&#39;épiderme. Chez les animaux plus âgés, la qualité de la visualisation des vaisseaux est pire et moins comparables en raison d...

Le but de l&#39;article est de décrire la technique de microscopie intravitale appliquée à l&#39;auricule de la souris sans poils pour l&#39;observation directe et l&#39;analyse de la microcirculation, la formation de thrombus et de l&#39;évolution de thrombus. Avec un taux d&#39;incidence de 1 sur 1000, la thrombose veineuse est encore une cause fréquente de morbidité. Bien que le diagnostic, les stratégies de prévention et les thérapies ont été développées ces dernières années, un tiers de la thrombose veineuse se manifeste par une embolie pulmonaire 1. La thrombose artérielle joue un rôle essentiel dans les maladies cardio-vasculaires, qui sont la cause la plus fréquente de décès dans les pays industrialisés. thromboses artérielles basée sur la rupture des plaques d&#39;athérosclérose est impliqué dans les crises cardiaques, infarctus mésentérique, et apoplexie. Chaque chirurgie expose les structures sous-endothéliales aux composants sanguins, modifie la dynamique de la circulation sanguine, et immobilise le patient. En chirurgie endoprothétique du membre inférieur, organe transplantation et thrombose chirurgie du lambeau sont des causes fréquentes de complications. thrombose microvasculaire en particulier provoque souvent des dommages irréversibles, en raison de l&#39;absence de symptômes cliniques. De même, la thrombose microvasculaire joue une règle essentielle dans plusieurs maladies, y compris purpura thrombotique thrombocytopénique, la septicémie, la coagulation intravasculaire disséminée, syndrome des antiphospholipides, et l&#39;insuffisance veineuse chronique, entre autres. Plusieurs nouveaux médicaments pour le traitement et la prévention de la thrombose ont été développées ces dernières années, mais les médicaments antiplaquettaires et les anticoagulants encore avoir des effets secondaires, les antagonistes manquent, et présentent des effets de longue durée. Ces carences entraînent des problèmes dans les soins médicaux d&#39;urgence. Ainsi, il faut plus de recherches pour découvrir les processus complexes qui se produisent pendant la thrombose, qui peut difficilement être simulé in vitro. La souris sans poils SKH1-Hr a été découvert 1926 dans un zoo à Londres.En raison d&#39;un défaut de gène sur le chromosome 14, l&#39;animal perd sa fourrure après jour post-natal 10. Cela rend le bien vascularisé auricule accessible à la microscopie intravitale des vaisseaux. L&#39;épaisseur moyenne de l&#39;oreille est de 300 um. Il se compose de deux couches de derme, qui sont séparés par du cartilage. Sur la face dorsale convexe du cartilage, 3 faisceaux vasculaires entrent dans le lobe de l&#39;oreille. arcs vasculaires apical et shunts de base relient les trois faisceaux. Les veinules ont des diamètres compris entre 200 pm (de base) et 10 um (apical). Capillaires gros maillés entourent les follicules pileux vide 2. L&#39;anatomie de la souris sans poils SKH1-Hr fait l&#39;auricule un modèle puissant et rentable pour la recherche sur la thrombose.

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			<td height="19" style="height:19px;width:216px;">SKH-1/hr mice</td>
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			<td height="19" style="height:19px;width:216px;">intravital microscope</td>
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			<td>Axiotech Vario 100&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
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			<td style="width:231px;">BM081R</td>
			<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>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<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

complications thrombotiques des maladies vasculaires sont une des principales causes de morbidité et de mortalité dans les pays industrialisés. En raison des interactions complexes entre les composants sanguins cellulaires et non cellulaires pendant la formation de thrombus, des études fiables de la physiologie et physiopathologie de la thrombose ne peut être effectuée in vivo. Par conséquent, cet article présente un modèle de l&#39; oreille chez les souris sans poil et se concentre sur l&#39;analyse in vivo de la microcirculation, la formation de thrombus et de l&#39; évolution de thrombus. En utilisant la microscopie à fluorescence intravitale et l&#39;application par voie intraveineuse (iv) des colorants fluorescents respectifs, une analyse répétitive de la microcirculation dans l&#39;oreillette peut facilement être réalisé sans la nécessité d&#39;une préparation chirurgicale. En outre, ce modèle peut être adapté pour des études in vivo de différentes questions, y compris la cicatrisation des plaies, des lésions de reperfusion, ou angiogenèse. En résumé, l&#39;oreille des souris sans poils est un modèle idéal pour la viv eno Etude de la microcirculation cutanée dans des conditions physiologiques ou physiopathologiques et pour l&#39;évaluation de sa réaction à différents traitements systémiques ou topiques.

Effets du traitement cannabinoïdes sur thrombogenèse Lors de l&#39; injection de 0,05 ml de FITC-dextran, l&#39; induction phototoxique de thrombus conduit à une lésion endothéliale et la formation d&#39;un bouchon de plaquettes pariétal (figures 2 et 3). Dans la présente étude, l&#39; induction d&#39; un thrombus après l&#39;injection ip de cannabinoïdes (5 mg / kg de poids corporel) o...

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

Le modèle de l&#39; oreille de la souris sans poils SKH1-Hr permet la microscopie à fluorescence intravitale de la microcirculation et phototoxique induction de thrombus sans préparation chirurgicale préalable dans le lit microvasculaire examiné. Par conséquent, l&#39;oreille de la souris sans poil est un excellent modèle in vivo pour étudier les interactions complexes pendant la formation de thrombus microvasculaire, l&#39; évolution de thrombus et thrombolyse.

Microscopie intravitale et thrombus induction dans le lobe de l&#39;oreille d&#39;une souris sans poils

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

Aspects techniques de la fistule aorto-cave de la souris

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

Recherche

Médecine

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

Induction pharmacologique de mélanine épidermique et la protection contre les coups de soleil dans un modèle humanisé de souris

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

Un modèle de souris orthotopique glioblastome maintien contraintes physiques et parenchyme cérébral Convient pour intravitale deux microscopie biphotonique

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

Mesures microscopie intravitale Vidéo de la rétine débit sanguin chez les souris

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

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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

Combiné la microscopie intravitale et échographie de contraste accentué des membres postérieurs souris pour étudier la vasodilatation induite par l’insuline et la Perfusion du Muscle

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

Induire ischémie-reperfusion chez la souris Ear Skin pour Intravitale multiphotonique Imaging des réponses immunitaires

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

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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

Induction et Micro-CT Imaging of Cerebral Cavernous Malformations dans le modèle murin

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

Résonance magnétique cardiaque pour l'évaluation du thrombus cardiaque présumé : techniques conventionnelles et émergentes

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