Name: ferric chloride-induced murine thrombosis models
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Description: Watch this Scientific Journal Video about Ferric Chloride-induced Murine Thrombosis Models at JoVE.com

This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta), R01 HL129179 (PI: Sen Gupta, Co-I: Li) and R01 HL098217 (PI: Nieman). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Alle procedurer og manipulationer af dyr er blevet godkendt af Institutional Animal Care og Brug udvalg (IACUC) af The Cleveland Clinic i overensstemmelse med USA Public Health Service Policy på human måde og anvendelse af dyr, og NIH Guide til pleje og anvendelse af forsøgsdyr. 1. Forberedelser: <ol><li> Fluorescerende farvestof til mærkning blodplader <ol><li> Forbered rhodamin 6G opløsning, 0,5 mg / ml, i saltvand og sterilisere opløsningen med 0,22 um filter. </li><...

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Den FeCl3-induceret model er en af de mest udbredte trombose modeller, som ikke alene kan give værdifulde oplysninger om genetiske modifikationer på trombocytfunktionen og trombose 7,8,16,19,31-33, men kan også være et værdifuldt redskab til vurdering af terapeutiske forbindelser og strategier for behandling og forebyggelse af aterotrombotiske sygdomme 11,17,34-37. Her har vi vist vores ændringer og forbedringer af denne model og viste yderligere bevis for anvendeligheden af ​​...

Arteriel trombose (blodprop) er en almindelig komplikation ved mange systemiske sygdomme associeret med kronisk inflammation, herunder aterosklerose, diabetes, fedme, cancer og kroniske autoimmune reumatologiske sygdomme. Tromber, der forekommer i det arterielle cirkulation stammer fra uhensigtsmæssig blodpladeaktivering, aggregering og efterfølgende coagulatory mekanismer, og er impliceret i hjerteanfald, slagtilfælde og ekstremitet tab. Karvæggen er et komplekst system, der omfatter multiple celletyper og påvirkes af en lang række ydre faktorer, herunder forskydningsspænding, cirkulerende blodceller, hormoner og cytokiner, såvel som ekspression af antioxidant proteiner i karvæggen. In vitro forsøg kan ikke replikere dette komplekse miljø, og derfor in vivo-undersøgelser med dyremodeller er kritiske for at tillade bedre forståelse af mekanismer, der er involveret i trombotiske lidelser. Mus har vist sig at have simma- mekanismer til mennesker i form af trombose, åreforkalkning, inflammation og diabetes 1,2. Endvidere kan transgene og knockout-mus være skabt til at teste funktionen af ​​specifikke genprodukter i et komplekst fysiologisk eller patologisk miljø. Sådanne undersøgelser efterligne menneskelige patologi og kan give vigtige mekanistisk information relateret til opdagelsen af ​​nye veje og behandlingsformer samt give vigtige detaljer karakterisere lægemiddelvirkninger på trombose. Patologiske arterielle tromber forekomme på grund af endothellag skade eller dysfunktion og eksponering af blodstrømmen til subendoteliale matrix 3,4. Forskellige trombose modeller er blevet udviklet til at fremkalde denne endotel skader såsom mekanisk skade, fotoreaktiv sammensatte Rose Bengal-baserede oxidativ skade og laser skade fem. I dette spektrum, Ferrichlorid (FeCl3) -induceret vaskulær skade er en meget anvendt model for trombose. Dette reagens nårpåført på den ydre aspekt af fartøjer inducerer oxidative skader på vaskulære celler 6-8, med tab af endotel cellebeskyttelse fra cirkulerende blodplader og komponenter i koagulationskaskaden. Den FeCl3-modellen er enkel og følsom over for både antikoagulerende og anti-blodplader narkotika, og er blevet udført på carotis og femorale arterier, halsvener, og mesenteriale og cremasteric arterioler og venuler i mus, rotter, marsvin og kaniner 6-15. En målbar parameter i denne model er den forløbne tid fra skade til at fuldføre fartøj okklusion, målt som blodgennemstrømningen ophør med en Doppler flow meter eller under direkte observation med intravital mikroskopi 6,7,9. En række gange mellem 5 til 30 min er blevet rapporteret i forskellige undersøgelser i C57BL6-mus 7-10,16, hvilket antyder, at FeCl3 koncentrationer, typer af anæstesi, kirurgiske teknikker, mus alder, genomisk baggrund, fremgangsmåde til måling Blood flow, og andre miljømæssige variabler få væsentlig indvirkning på denne model. Denne brede variation gør det vanskeligt at sammenligne undersøgelser fra forskellige forskergrupper og kan gøre påvisning af subtile forskelle vanskelig. Med en vision om at minimere sådanne variabilitet og etablere en ensartet reproducerbar in vivo model-system, har vi forfinet FeCl3-induceret halspulsåren model, der gør data meget reproducerbare med minimal variation 6-10,16-19. I dette papir beskriver vi og dele de færdigheder og rapportere flere repræsentative eksperimentelle eksempler, der kan drage fordel af denne model.

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		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Metzenbaum Fino Scissors - Tungsten Carbide/Curved/Blunt-Blunt/14.5cm</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td style="width:119px;">14519-14&#160;&#160;</td>
			<td>to dissect and separate soft tissue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Ultra Fine Hemostat - Smooth/Curved/12.5cm</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>13021-12</td>
			<td>to dissect and separate soft tissue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Graefe Forceps - Serrated/Straight/10cm</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td style="width:119px;">11050-10</td>
			<td>to dissect and separate soft tissue</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Dumont #5 Fine Forceps - Biology Tips/Straight/Inox/11cm</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>11254-20&#160;</td>
			<td>Isolate vessel from surounding tissue</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Dumont #5XL Forceps - Standard Tips/Straight/Inox/15cm</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>11253-10</td>
			<td>Isolate vessel from surounding tissue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Blunt Hook- 12cm/0.3mm Tip Diameter</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>10062-12</td>
			<td>Isolate vessel from surounding tissue</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Castroviejo Micro Needle Holders</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>12061-02</td>
			<td>Needle holders</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Suture Thread 4-0</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>18020-40</td>
			<td>For fix the incisors to the plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Suture Thread 6-0</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>18020-60</td>
			<td>For all surgery and ligation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Kalt Suture Needles</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>12050-03</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rhodamine 6G&#160;</td>
			<td style="width:159px;">Sigma</td>
			<td>83697-1G</td>
			<td>To lebel platelets</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">FeCl3 (Anhydrous)</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:119px;">12321</td>
			<td>To induce vessel injury</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papaverine hydrochloride</td>
			<td style="width:159px;">Sigma</td>
			<td>P3510</td>
			<td>To inhibit gut peristalsis.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Medline Surgical Instrument Sterilization Steam Autoclave Tapes</td>
			<td style="width:159px;">Medline</td>
			<td>111625</td>
			<td>To fix the mouse to the plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Fisherbrand&#8482;&#160;Syringe Filters - Sterile 0.22&#181;m</td>
			<td style="width:159px;">Fisher</td>
			<td>09-720-004</td>
			<td>For sterlization of solutions injected to mice</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:351px;">Fisherbrand&#8482;&#160;Syringe Filters - Sterile 0.45&#181;m</td>
			<td style="width:159px;">Fisher</td>
			<td>09-719D</td>
			<td>To filter the FeCl3 solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Sterile Alcohol Prep Pad</td>
			<td style="width:159px;">Fisher</td>
			<td>06-669-62</td>
			<td>To sterilize the surgical site</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Agarose&#160;</td>
			<td style="width:159px;">BioExpress</td>
			<td>E-3120-500</td>
			<td>To make gel stage</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Leica DMLFS fluorescent microscope</td>
			<td style="width:159px;">Leica</td>
			<td style="width:119px;"></td>
			<td>Intravital microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">GIBRALTAR Platform and X-Y Stage System</td>
			<td style="width:159px;">npi electronic GmbH</td>
			<td style="width:119px;"></td>
			<td style="width:325px;">http://www.npielectronic.de/products/micropositioners/burleigh/gibraltar.html</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:351px;">Streampix version 3.17.2 software</td>
			<td style="width:159px;">NorPix</td>
			<td style="width:119px;"></td>
			<td>https://www.norpix.com/</td>
		</tr>
	</tbody>
</table>

ferric chloride-induced murine thrombosis models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

Arteria carotis Trombose Model I mus med C57BL6 baggrund, anbefaler vi at bruge 7,5% FeCl3 at behandle skibet i 1 min som udgangspunkt. Under behandlingen på 7,5% FeCl3 er grænser skadede område og normal karvæggen let identificeres under mikroskop (se online video 1), hvilket tyder på, at endotel lag betydeligt beskadiget. Den tromber dannet umiddelbart efter FeCl3 behandling, og observeres i alle WT C57BL6 m...

Watch this Scientific Journal Video about Ferric Chloride-induced Murine Thrombosis Models at JoVE.com

Ferric Chloride-induced Murine Thrombosis Models

Vi rapporterer en raffineret procedure af ferrichlorid (FeCl3) -inducerede trombose modeller på carotis og mesenterisk arterie samt vene, kendetegnet effektivt med intravital mikroskopi til at overvåge tid til okklusiv tromber formation.

Ferrichlorid-induceret murin Trombose Modeller

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Research

JoVE Journal

Medicine

Electrolytic Inferior Vena Cava Model (EIM) of Venous Thrombosis

Electrolytic Inferior Vena Cava Model EIM of Venous Thrombosis

Animal models serve a vital role in deep venous thrombosis (DVT) research in order to study thrombus formation, thrombus resolution and to test potential ...

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Segmentation and Measurement of Fat Volumes in Murine Obesity Models Using X-ray Computed Tomography

Segmentering og måling af tykke bind i murine Fedme-modeller vha. X-ray Computed Tomography

Obesity is associated with increased morbidity and mortality as well as reduced metrics in quality of life.1 Both environmental and genetic factors are ...

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

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

Intrastriatal Injektion af autologt blod eller Clostridial Collagenase som murine modeller af intracerebral blødning

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

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Ortotopisk Injektion af brystkræft celler i brystfedtpuden af ​​mus til at studere tumorvækst.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

Tilpasse det menneskelige Videofluoroscopic Swallow Study metoder til påvisning og karakterisere Dysfagi i murine sygdomsmodeller

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

Højfrekvente Ultrasound Imaging af Mouse Cervikal lymfeknuder

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal ...

A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis

En multicenter MRI-protokollen for Vurdering og kvantificering af dyb venetrombose

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Ferric Chloride-induceret Trombose Mouse Model på Arteria carotis og mesenterium Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...