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.

All procedures and manipulations of animals have been approved by Institutional Animal Care and Use Committees (IACUC) of The Cleveland Clinic in accordance with the United States Public Health Service Policy on the Humane Care and Use of Animals, and the NIH Guide for the Care and Use of Laboratory Animals.
1. Preparations:
<ol>
	<li>Fluorescent Dye for Labeling Platelets
	<ol>
		<li>Prepare rhodamine 6G solution, 0.5 mg/ml, in saline and sterilize the solution with 0.22 &#181...

<ol>
	<li>Sachs, U. J., Nieswandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+thrombus+formation+in+murine+models.">In vivo thrombus formation in murine models.</a> Circ Res. 100, 979-991 (2007).</li><li>Libby, P., Lichtman, A. H., Hansson, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+effector+mechanisms+implicated+in+atherosclerosis:+from+mice+to+humans.">Immune effector mechanisms implicated in atherosclerosis: from mice to humans.</a> Immunity. 38, 1092-1104 (2013).</li><li>Ruggeri, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+adhesion+under+flow.">Platelet adhesion under flow.</a> Microcirculation. 16, 58-83 (2009).</li><li>Watson, S. 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The FeCl3-induced model is one of the most widely used thrombosis models, which can not only provide valuable information about genetic modifications on platelet function and thrombosis7,8,16,19,31-33, but can also be a valuable tool for evaluation of therapeutic compounds and strategies for treatment and prevention of atherothrombotic diseases11,17,34-37. Here we have shown our modifications and refinements of this model and showed additional evidence of the utility of this technique, wh...

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 that occur in the arterial circulation stem from inappropriate platelet activation, aggregation and subsequent coagulatory mechanisms, and are implicated in heart attacks, strokes and extremity loss. The vessel wall is a complex system that includes multiple cell types and is influenced by a multitude of extrinsic factors including shear stress, circulating blood cells, hormones and cytokines, as well as expression of antioxidant proteins in the vessel wall. In vitro experiments cannot replicate this complex environment and therefore in vivo studies using animal models are critical to allow better understanding of mechanisms involved in thrombotic disorders.
Mice have been shown to have similar mechanisms to humans in terms of thrombosis, atherosclerosis, inflammation and diabetes1,2. Furthermore, transgenic and knockout mice can be created to test the function of specific gene products in a complex physiologic or pathologic environment. Such studies mimic human pathology and may provide important mechanistic information related to discovery of new pathways and therapies, as well as provide important details in characterizing drug effects on thrombosis.
Pathological arterial thrombi occur due to endothelial layer injury or dysfunction and exposure of the blood stream to the subendothelial matrix3,4. Various thrombosis models have been developed to induce this endothelial damage such as mechanical injury, photoreactive compound Rose Bengal-based oxidative injury and laser injury5. In this spectrum, Ferric chloride (FeCl3)-induced vascular injury is a widely used model of thrombosis. This reagent when applied to the outer aspect of vessels induces oxidative damage to vascular cells6-8, with loss of endothelial cell protection from circulating platelets and components of the coagulation cascade. The FeCl3 model is simple and sensitive to both anticoagulant and anti-platelets drugs, and has been performed on carotid and femoral arteries, jugular veins, and mesenteric and cremasteric arterioles and venules in mice, rats, guinea pigs and rabbits6-15.
One measurable parameter in this model is the elapsed time from injury to complete vessel occlusion, measured as blood flow cessation with a Doppler flow meter or under direct observation with intravital microscopy6,7,9. A range of times between 5 to 30 min has been reported in different studies in C57Bl6 mice7-10,16, suggesting that FeCl3 concentrations, types of anesthesia, surgical techniques, mouse age, genomic background, method of measuring blood flow, and other environmental variables have significant effects in this model. This wide variability makes it difficult to compare studies from different research groups and may make detection of subtle differences difficult.
With a vision to minimize such variabilities and establish a uniformly reproducible in vivo model system, we have refined the FeCl3-induced carotid artery model that makes the data highly reproducible with minimal variation6-10,16-19. In this paper we describe and share the skills and report several representative experimental examples that can benefit from this model.

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			<td height="20" style="height:20px;width:351px;">Surgical Scissors - Tungsten Carbide</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td style="width:119px;">14502-14</td>
			<td style="width:325px;">cut and hold skin</td>
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			<td height="20" style="height:20px;width:351px;">Micro-Adson Forceps - Serrated/Straight/12cm</td>
			<td style="width:159px;">Fine Science Tools&#160;</td>
			<td>11018-12</td>
			<td>cut and hold skin</td>
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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>
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			<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>
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			<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>
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			<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>
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			<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>
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			<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.

Carotid Artery Thrombosis Model 
In mice with C57BL6 background, we recommend using 7.5% FeCl3 to treat the vessel for 1 min as a starting point. Under treatment of 7.5% FeCl3, borders of the injured area and normal vessel wall are easily identified under microscope (See online video 1), suggesting that the endothelial layer was significantly damaged. The thrombi formed immediately upon FeCl3 treatment, and are observ...

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

Ferric Chloride-induced Murine Thrombosis Models

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

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

The overall goal of this procedure is to produce ferric chloride-induced thrombosis in a mouse carotid artery model to obtain highly reproducible results as analyzed by intravital microscopy. These mice can help answer key questions about various disease or genetic disorders associated with platelet activation and the aggregation of closed vascular system. The main advantage of this technique are that it is simple and sensitive to both anticoagulant and antiplatelet drugs, and that it can be combined with other techniques.This method can be used to gain valuable insight into the pathophysiology of thrombosis, discover new mechanisms and therapies, and evaluate the effects of Novel drugs, including targeted nanomedicine on thrombosis. Visualization of this method is critical for learning the strategies for obtaining accurate blood flow cessation time. After confirming a lack of response to toe pinch, use small electric animal clippers to remove the fur from the neck and upper chest of an eight to 12-week-old mouse.Apply neutral petroleum eye ointment to animal's eyes, and tape the mouse in the supine position on the lid of a 15-centimeter tissue culture plate. Wrap a 4-0 suture around the incisors, and tape the ends of the suture to the lid to keep the neck straight. Then, move the animal under a surgical light source, and use an alcohol pad to sterilize the surgical site.Next, use Micro-Adson forceps to grasp the skin, and use surgical scissors to make a two to three-millimeter incision. Holding the incision with forceps, insert the scissors with the blades closed, and push the scissors subcutaneously toward the manubrium. Then, open the blades to free the skin from the subcutaneous layer.Now, make a midline incision from the manubrium to the hyoid bone, and use a fine hemostat and Graefe forceps to bluntly dissect the thin fascia between the submaxillary gland. When the trachea is visible, use the forceps to hold the soft tissue to the right of the manubrium, and insert the hemostat under the fascia toward the two o'clock position. Open the jaws of the hemostat to free the jugular vein from the surrounding tissue.Then, continue the incision through the fascia, soft tissue, and skin toward the two o'clock position until the right jugular vein is exposed. Now, attach a 30-gauge needle attached to a one-milliliter syringe containing 100 microliters of rhodamine 6G solution, and use a needle-holder to bend two to three millimeters of the tip 90 degrees. Then, holding the syringe with one hand and the soft tissue with the distal site of the jugular vein with the Graefe forceps in the other, insert the needle into the vessel.Stabilize the needle with the forceps, and inject the rhodamine. Once all of the platelet labeling solution has been administered, clamp the injection site with the forceps to prevent bleeding by opening the hemostat and inserting the instrument under the forceps to clamp the vessel wall of the injection site. Then, use a 6-0 suture to ligate the injection site.Bluntly dissect the soft tissue and fascia around the left submaxillary gland, and pull the gland toward the seven o'clock position to expose the left sternocleidomastoid muscle. Then, bluntly dissect the fascia between the left sternocleidomastoid muscle and the omohyoid and sternohyoid muscles to the left side of the trachea. When the fascia has been displaced, pass a needle with a 15-centimeter, 6-0 silk suture under the middle of the sternocleidomastoid muscle, and pull the two ends of the suture laterally toward the 10 o'clock position.Bluntly separate the thing sternohyoid and omohyoid muscles to expose the carotid artery. Then, use the hemostat to separate the soft tissue from the carotid artery without touching the artery. Using a fine-tipped forceps, lift the lateral fascia around the carotid artery while avoiding the vagus nerve and the artery.Then, use another fine-tipped forceps to puncture the fascia between the carotid artery and the vagus nerve. Now, pass a blunt hook through the hole to gently lift the carotid artery, and slide the fine-tipped forceps under the artery. Moving the hook and forceps in opposite directions along the artery, strip about five millimeters of advantigial soft tissue from the vessel from the surrounding tissue.Then, rinse a piece of U-shaped plastic with saline, and use the hook to gently lift the artery to allow for the placement of the plastic under the artery. The plastic will block the background fluorescence, preventing diffusing of the ferric chloride solution to the surrounding tissue, and they ensure that only the carotid artery is visible under the microscope. Immediately hydrate the carotid artery with two to three drops of saline.Then, transfer the mouse onto a fluorescent microsope stage under the 10X water lens, and fill the space between the lens and the artery with saline. Now, start the digital video recording software application, and obtain images of the normal vessel. If the vessel wall is integrated and uninjured, transfer the mouse to the Gibraltar platform, and use the corner of a paper towel to carefully blot the saline around the carotid artery in the surgical field without touching the artery.Then, use fine-tipped forceps to transfer a piece of ferric chloride-saturated filter paper directly on top of the carotid artery close to the distal end of the exposed artery. Make sure to place the filter paper close the distal end of the exposed carotid artery, which we will leave a short segment at the proximal side for observing the blood flow during the later phase of the injury. After one minute, remove the filter paper, and rinse the artery with at least two milliliters of saline.Return the mouse to the microscope stage, and obtain images of the vessel. The formation of the thrombus can be identified by the accumulation of the fluorescent platelets. Continue to image the vessel for 10 seconds every minute for the first 10 minutes, followed by 10 seconds every other minute until the end of the experiment.Tissue-type plasminogen activator profusion five minutes after ferric chloride injury demonstrates the initiation of thrombolysis about four minutes after the treatment. With the formed thrombi observed to undergo repeated size variations during the 30 minute observation period, but without a complete ablation of the platelet-mediated thrombus. Treatment with a polyclonal PAR4 antibody that delays PAR4 cleavage significantly prolongs the time to occlusive thrombus formation in the ferric chloride-induced carotid artery thrombosis model.Non-platelet red blood cell labeling experiments indicate that, while ferric chloride-induced thrombi are made up exclusively of vessel wall-adherent platelets, red blood cells do become trapped within the thrombus, contributing to the shape and occlusive property of the clot over time. Interestingly, while the initial thrombus is visualized within about 15 seconds of the injury to the mesenteric artery and vein, the formation of the thrombus within the mesenteric vessels is dramatically delayed to approximately 17 minutes in the mesenteric vein. And to nearly 24 minutes in the mesenteric artery, due at least in part to diffusion of the ferric chloride through the adipose tissue surrounding the mesenteric vessels.Once mastered, this refund ferric chloride injury induced thrombosis model can be finished in about a 30 to 60 minutes depending on the mastery used, if it is performed properly. While attempting this procedure, it is important to remember that the measure it will permit us is the elapsed attempt from the injury to the complete ways of occlusion, and a precise observation of the blood flow is critical for obtaining accurate results. This model may be combined with a transfusion of ex-vivo treated wild-type platelets to mimic platelet-specific knockouts for defining the function of regulatory pathways and platelets.This technique paved the way for researchers in the field of vascular biology to explore not only the role of platelets in thrombosis, but also under various other endothelium inflammatory conditions. After watching this video, you should have a good understanding of how to set up a ferric chloride-induced carotid artery thrombosis model in mice to achieve highly reproducible result using intravital microscope. Don't forget that ferric chloride can be extremely hazardous, and that precautions such as wearing the proper personal protective equipment should always be taken while performing this model.

ferric-chloride-induced-murine-thrombosis-models

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JoVE Journal

Medicine

20.9K 조회수.  Lerner Research Institute, Cleveland Clinic. The overall goal of this procedure is to produce ferric chloride-induced thrombosis in a mouse carotid artery model to obtain highly reproducible results as analyzed by intravital microscopy. These mice can help answer key questions about various disease or genetic disorders associated with platelet activation and the aggregation of closed vascular system. The main advantage of this technique are that it is simple and sensitive to both anticoagulant and antiplatelet drugs, and that it can be ...