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

저자는 어떤 승인이 없습니다.

모든 생체 실험 (7221.3-1-006 / 15) 동물의 보호 및 실험 동물의 관리 및 사용에 대한 NIH 가이드 (실험 동물 자원 연구소, 국립 연구위원회)에 독일 법률에 따라 실시 하였다. 동물의 1. 일반 상태 유지 <ol><li> 4 ~ 6 주 세 남성 SKH1 - 시간의 시간의 쥐 실험을 수행합니다. (20)와 25g 사이 무게 동물을 사용합니다. </li><li> 물과 음식을 자유로이에 지속적으로 액세?...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+coronary+atherosclerosis+in+swine+produced+by+combination+of+balloon-catheter+injury+and+cholesterol+feeding.">Advanced coronary atherosclerosis in swine produced by combination of balloon-catheter injury and cholesterol feeding.</a> Exp Mol Pathol. 23 (3), 491-499 (1975).</li><li>Callahan, A. B., Lutz, B. R., Fulton, G. P., Degelman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+and+thrombus+thresholds+to+unipolar+stimulation+of+small+blood+vessels.">Smooth muscle and thrombus thresholds to unipolar stimulation of small blood vessels.</a> Angiology. 11, 35-39 (1960).</li><li>Rosen, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+noninvasive+vascular+injury+models+in+mice+generate+platelet-+and+coagulation-dependent+thrombi.">Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi.</a> Am J Pathol. 158 (5), 1613-1622 (2001).</li><li>Agero, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+mutalysin+II+on+vascular+recanalization+after+thrombosis+induction+in+the+ear+of+the+hairless+mice+model.">Effect of mutalysin II on vascular recanalization after thrombosis induction in the ear of the hairless mice model.</a> Toxicon. 50 (5), 698-706 (2007).</li><li>Menger, M. D., R&#246;sken, M., R&#252;cker, M., Seiffge, D., Vollmar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithrombotic+and+thrombolytic+effectiveness+of+rhirudin+in+microvessels.">Antithrombotic and thrombolytic effectiveness of rhirudin in microvessels.</a> Langenbecks Arch Chir. 115 (1), 19-20 (1998).</li><li>Bilheiro, R. 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SKH1 - 시간의 시간 마우스의 귓불에서 성공적인 혈전 유도에 대한 몇 가지 중요한 단계가 있습니다. 문제 해결을 위해 프로토콜의 각 단계는 괄호 안에 표시됩니다. 육주와 표피의 낮은 각화로 - 시험 조건은 4 세의 나이에 젊은 동물에 이상적입니다. 이전 동물에서 혈관의 시각화 품질로 인해 피부 표면과 타겟 용기 (단계 1.1) 사이의 거리보다 더 적은 대등하다. <p ...

본 문서의 목적은 직접적인 관찰과 미세 분석, 혈전 형성 및 혈전 진화의 무모 마우스의 귓바퀴에 적용 생체 내에 현미경의 기술을 설명하는 것입니다. 1000 년 1의 발생율로, 정맥 혈전증 여전히 이환율의 일반적인 원인이다. 진단, 예방 전략 및 치료는 최근에 개발되었지만, 정맥 혈전증의 3 분의 1은 폐 색전증 1로 나타난다. 동맥 혈전증은 선진국에서 사망의 가장 흔한 원인이되는 심장 혈관 질환에 중요한 역할을한다. 동맥 경화성 플라크의 파열에 따라 동맥 혈전증은 심장 마비, 장간막 경색 및 뇌졸중에 참여하고있다. 모든 수술은, 혈액 성분에 피하 구조를 노출 혈류 역학을 변경하고 환자를 고정시킨다. 낮은 사지, 기관 t의 endoprosthetic 수술에서ransplantation 및 플랩 수술 혈전 합병증의 빈번한 원인입니다. 특히 미세 혈관 혈전증 자주 인해 임상 증상의 부족으로 돌이킬 수없는 손상을 야기한다. 마찬가지로, 미세 혈관 혈전증은 다른 사람의 사이에서 혈전 성 혈소판 감소 성 자반증, 패혈증, 파종 성 혈관 내 응고, 항 인지질 증후군, 만성 정맥 부전, 등 여러 질병에 중요한 규칙을한다. 치료 및 혈전증의 예방을위한 여러 가지 새로운 약물은 최근에 개발되었지만, 항 혈소판 약물과 항 응혈은 여전히 ​​부작용, 부족 길항제가 있고, 장기간 효과가 있습니다. 이 결함은 응급 의료의 문제로 이어집니다. 따라서, 더 많은 연구가 거의 시험 관내에서 모사 될 수 없다 혈전증 동안 발생하는 복잡한 프로세스를 발견 할 필요가있다. 털이없는 SKH1 - 시간의 시간 마우스 런던 동물원에서 1926 발견되었다.때문에 14 번 염색체에 유전자 결함, 동물이이 선박의 생체 내에 현미경에 액세스 잘 혈관 귓바퀴를 만드는 출생 후 하루 10 후 털을 잃는다. 귀의 평균 두께는 300 μm의입니다. 이것은 연골에 의해 분리되어 진피의 두 층으로 구성. 연골의 볼록 등쪽에서 3 개 혈관 번들 귓불을 입력한다. 혀끝 혈관 호 및 기초 션트는 세 개의 번들을 연결합니다. 세정맥 200 μm의 (기부) 10 ㎛의 (정점) 사이의 직경을 갖는다. 근접 메시 모세 혈관 빈 머리 2 여포 둘러싸고 있습니다. 털이없는 SKH1 - 시간의 시간 마우스의 해부학은 귓바퀴 혈전증 연구를위한 강력하고 비용 효율적인 모델을 만든다.

<table border="0" cellpadding="0" cellspacing="0" width="846">
	<tbody>
		<tr height="19">
			<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>
			<td style="width:239px;">can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:216px;">standard laboratory food</td>
			<td style="width:161px;">ssniff Spezialdiaeten</td>
			<td>V1594-0&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">operation stereomicroscope</td>
			<td>Leica&#160;</td>
			<td style="width:231px;">M651/M655&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<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>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">objective (20x/0.95)&#160;</td>
			<td style="width:161px;">Zeiss</td>
			<td>20x/0,50 W; Plan-NEOFLUAR&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">objective (63x/0.95)</td>
			<td style="width:161px;">Zeiss</td>
			<td>63x/0,95 W; ACHROPLAN&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">black and white CCD-camera&#160;</td>
			<td>Pieper&#160;</td>
			<td>FK 6990 IQ-S&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">DVD-recorder</td>
			<td>Panasonic</td>
			<td>DMR-EX99V&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">sodium chloride</td>
			<td style="width:161px;">Braun</td>
			<td style="width:231px;">5/12612055/1011</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Ketamine 10%</td>
			<td style="width:161px;">Bela pharm</td>
			<td style="width:231px;">F3901-6</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Xylazine 2%</td>
			<td style="width:161px;">Bayer</td>
			<td style="width:231px;">6293841.00.00</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">FITC-dextran 5%</td>
			<td>Sigma&#160;</td>
			<td style="width:231px;">46945-100MG-F</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">dexapanthenol 5% eye ointment</td>
			<td style="width:161px;">Bayer</td>
			<td style="width:231px;">6029009.00.00</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">formaldehyde 4%</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:231px;">HT501128-4L</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">DMSO</td>
			<td style="width:161px;">Sigma</td>
			<td style="width:231px;">472301</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">coverslips 5 x 5 x 1 mm</td>
			<td style="width:161px;">Menzel</td>
			<td style="width:231px;">L4339</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">plasters</td>
			<td style="width:161px;">Leukosilk</td>
			<td style="width:231px;">4683400</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">centrifuge</td>
			<td style="width:161px;">Beckman Coulter</td>
			<td style="width:231px;">CLGS 15</td>
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intravital microscopy and thrombus induction in the earlobe of a hairless mouse

혈관 질환의 혈전 성 합병증은 선진국의 이환율과 사망률의 한 주요 원인이다. 때문에 혈전 형성시 셀룰러 및 비 셀룰러 혈액 성분 간의 복잡한 상호 작용의 생리 학적 및 병태 생리 혈전증의 신뢰성 시험은 생체 내에서 수행 될 수있다. 따라서이 문서는 털이없는 쥐의 귀 모델을 제시하고, 미세, 혈전 형성 및 혈전 진화의 생체 분석에 초점을 맞추고 있습니다. 생체 내에 형광 현미경 및 형광 염료 각각의 정맥 내 (IV) 애플리케이션을 이용함으로써, 귓바퀴의 미세 순환을 반복 분석을 용이하게 수술 준비 할 필요없이 수행 될 수있다. 또한,이 모델은 상처 치유, 재관류 손상, 또는 혈관 신생 등 다양한 문제의 생체 내 연구에 대해 적용 할 수 있습니다. 요약하면, 털이없는 쥐의 귀는에서 비비안을위한 이상적인 모델입니다생리 학적 또는 병태 생리 학적 조건에서 다른 전신 또는 국소 치료법과의 반응의 평가를 위해 피부의 미세 O 연구.

혈전의 치료 효과 카나비노이드 FITC 덱스 트란 0.05 mL를 주입하면, 광독성 혈전 유도는 (도 2 및도 3). 내피 병변 및 정수리 혈소판 플러그의 형성을 유도 본 연구에있어서, 칸 나비 노이드의 IP 주입 (5 ㎎ / ㎏ 체중) 또는 비히클 후의 혈전 유도는 모든 세정맥에서 혈전 혈관 폐색 (도 4) 결과. ?...

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

털이없는 SKH1 - 시간의 시간 마우스의 귀 모델은 검토 미세 혈관 침대에서 사전 수술 준비없이 미세과 광독성 혈전 유도의 생체 내에 형광 현미경을 할 수 있습니다. 따라서 털이 마우스의 귀는 미세 혈관 혈전 형성, 혈전 진화 및 혈전 용해시 복잡한 상호 작용을 연구하는 생체 모델의 우수합니다.

털이 마우스의 귓불에서 생체 내에 현미경 및 혈전 유도

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

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연구

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

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

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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

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

유도 및 마우스 모델에서 대뇌 동굴 같은 기형의 마이크로-CT 영상

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

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