Ferric Chloride-induced Murine Thrombosis Models

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 diabetes^1,2. 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 matrix^3,4. 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 cells^6-8, 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 rabbits^6-15.

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 microscopy^6,7,9. A range of times between 5 to 30 min has been reported in different studies in C57Bl6 mice^7-10,16, 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 variation^6-10,16-19. In this paper we describe and share the skills and report several representative experimental examples that can benefit from this model.

Protokół

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:

1. Fluorescent Dye for Labeling Platelets
   1. Prepare rhodamine 6G solution, 0.5 mg/ml, in saline and sterilize the solution with 0.22 µm filter.
2. FeCl₃ Solution
   1. Make a fresh stock solution of 30% FeCl₃ (Anhydrous, equals 1.85 M) in pure water, and filter with 0.45 µm filter. Prepare 5 ml fresh FeCl₃ solution at the desired concentration [e.g., 2.5% (0.154 M), 5% (0.308 M), 7.5% (0.463 M), 10% (0.617 M) and 12.5% (0.771 M)] by diluting the 30% FeCl₃ solution with water in a 6 cm tissue culture dish and keep the lid closed.
      NOTE: Using the culture dish makes it easier to pick up the filter paper saturated with FeCl₃ solution than to pick it up from a narrow container, such as a 1.5 ml microcentrifuge tube.  FeCl3 is extremely hazardous and that precautions, such as wearing gloves and lab coat etc., Personal Protective Equipment, should always be taken.
3. Filter Papers
   1. Cut the filter paper to 1 mm x 2 mm size. Soak enough pieces of the cut filter paper in the FeCl₃ solution in the same dish.
4. Papaverine Hydrochloride Solution
   1. Prepare papaverine hydrochloride solution (25 mg/ml) in water and sterilize the solution with 0.22 µm filter.
5. Agarose Gel
   1. Prepare 2% agarose gel in PBS or saline in 6-well tissue culture plate, ~ 1 cm thickness, and cut it to half-circle with ~ 3 cm diameter.

2. FeCl₃ Induced Carotid Arterial Injury Thrombosis Model

1. Surgical Procedures for the Thrombosis Model
   1. Use 8 - 12 weeks old C57Bl6 mice (or other strains as necessary). Anesthetize mice with the mixture of ketamine (100 mg/kg)/xylazine (10 mg/kg) via intraperitoneal injection. Confirm the depth of the anesthesia by toe pinching.
      NOTE: This amount of ketamine/xylazine is sufficient to keep the animal in stable anesthesia and no pain (response to toe pinching) for at least 1 hr.
   2. Remove fur on the neck and upper chest with a small animal electric clipper. Depilatory cream is not necessary. Apply neutral petroleum eye ointment on the eyes to prevent dryness during anesthesia.
   3. Secure the mouse in the supine position on the lid of 15 cm tissue culture plate. Use one long tape (~ 10 cm) to secure hind limbs and lower body, and two pieces of small tape (2 cm x 0.5 cm) to secure forelegs. Use a 4-0 suture to wrap around the incisors, tape the two ends of the suture to the lid to keep mouse neck straight (Figure 2).
   4. Place plate with mouse head toward the operator under a surgical light source.
   5. Sterilize the surgical site with alcohol pad. Use the Micro-Adson Forceps to hold the skin and use a surgical scissors to make a small incision (2 - 3 mm).
   6. Hold the skin of the incision with the forceps and insert surgical scissors with jaws closed into the incision. Push the scissors subcutaneously toward the manubrium or chin, and then open the scissors to free the skin from the subcutaneous layer.
   7. Cut the skin with the surgical scissors to make a midline incision from the manubrium to the level of hyoid bone (Figure 3A and 3B). Bluntly dissect the thin fascia (dotted line, in Figure 3B) between the submaxillary glands with a fine hemostat and a Graefe forceps (Figure 3B and 3C; blue arrows).
      NOTE: The manubrium (black arrow in Figure 3C) and the trachea (T in Figure 3C) will be seen after this step.
   8. Hold the soft tissue and the skin seen at the right of the manubrium with the Graefe forceps, insert the hemostat under the fascia toward the 2 o'clock position (yellow dotted line in Figure 3C), open the jaws of the hemostat to free the jugular vein from surrounding tissue.
   9. Cut the fascia, soft tissue and skin toward the 2 o'clock position (Figure 3C) with the surgical scissors to expose the right jugular vein (Figure 3D, arrow).
   10. Draw about 200 - 300 µl rhodamine 6G solution using a 1 ml syringe with 22 G needle, and then change it to 30 G needle.
       NOTE: This protects the 30 G needle from damage of its fine tip.  Directly drawing the rhodamine 6G solution with the 30 G needle will not damage the tip; however touching the container wall accidentally will cause damage, which may make injection difficult.
   11. Flush the 30 G needle by slowly pushing out the rhodamine 6G solution, make sure no air bubbles remain in the syringe or the needle. Keep 100 µl rhodamine 6G solution for injection.
   12. Bend 2 - 3 mm tip of the needle to a 90° angle with the needle holder, which will prevent inserting the needle too deep to the jugular vein and makes the injection more easily controlled (Figure 3D).
   13. Inject the rhodamine 6G solution into the right jugular vein to label platelets. To stabilize the syringe and keep the needle in position, hold the syringe with one hand and the needle with the Graefe forceps with another hand during the injection.After injection, clamp the injection site with the Graefe forceps to avoid bleeding.
   14. Open the jaws of the hemostat and insert it under the Graefe forceps to clamp the vessel wall of the injection site, and then ligate the injection site with a 6-0 suture.
       NOTE: As an alternative of step 2.1.15, applying pressure to the injection site with a finger for 2 min will stop the bleeding; however, this method has a risk of causing further bleeding if the clot at the injection site is removed accidently.
   15. Bluntly dissect the soft tissue and fascia around the left submaxillary gland with the hemostat and the Graefe forceps and pull the gland toward the 7 o'clock position (Figure 3E) to expose the left sternocleidomastoid muscle (blue arrow in Figure 3E).
   16. Bluntly dissect the fascia (Figure 3E, dotted line) between the left sternocleidomastoid muscle and the omohyoid muscle or the sternohyoid muscle (Figure 3E, green arrow) located to the left site of the trachea with the hemostat.
   17. Pass a needle with 6-0 silk suture (about 15 cm long) under the middle of the sternocleidomastoid muscle (blue arrow in Figure 3E), put the two ends of the suture together and pull the suture laterally toward the 10 o'clock position.
       NOTE: This procedure should be performed carefully to avoid injury of the left jugular vein, which is located outside of the sternocleidomastoid muscle.
   18. Bluntly separate the thin sternohyoid muscle and/or omohyoid muscle to expose the carotid artery (CA) (Figure 3F arrow) after pulling away the sternocleidomastoid muscle. Cut the sternohyoid muscle and/or omohyoid muscle as necessary. Use the hemostat to separate the soft tissue from CA without touching the CA.
       NOTE: CA is accompanied by the vagus nerve, the white structure seen in Figure 3G (green arrow), and in most cases it is under the thin sternohyoid muscle and/or omohyoid muscle (between the yellow dotted lines in Figure 3F).
   19. Use a fine tip forceps to pick up the lateral fascia around the CA while avoiding the vagus nerve and CA. Use another fine tip forceps to punch a hole on the fascia between the CA and vagus nerve.
   20. Pass the hook through the hole and gently lift the CA, and then place the fine tip forceps under the CA. Move the hook and forceps in opposite directions along the CA to strip adventitial soft tissue. Free at least ~ 5 mm length of CA (Figure 3H, blue arrow) from surrounding tissue.
   21. To block background fluorescence, press the end of a black plastic coffee stirrer to flat, crosscut the coffee stirrer into a 3 mm piece, and then cut longitudinally along the fold edges to get two pieces of "U" shape plastic (Figure 3H, green arrow).
   22. Rinse one piece of this "U" shape plastic with saline and hold it with forceps and place it under the CA while gently lifting the CA with the hook (Figure 3H, green arrow). Immediately apply 2-3 drops of saline to avoid drying the CA.
2. Real time Recording Video
   1. Transfer the mouse and plate lid together to the microscope stage and place in an appropriate position under the 10X water lens. Fill the space between the 10X lens and the CA with saline.
   2. Start the digital video recording software application, and launch a new file and name it accordingly. Record normal vessel images and write down the frame number when the video recording is stopped.
      NOTE: Reference the video recording software in the computer for recording. We use digital video recording software and the following descriptions are based on this application. 
      NOTE: Since mechanical trauma can injure the endothelium and lead to thrombus formation²⁰, it is necessary to confirm that there is no surgical injury to the vessel wall prior to the FeCl₃ injury. It is also necessary to confirm that there is no remaining tissue around the CA, which may form a barrier and attenuate the effect of FeCl₃-induced injury. Write down the frame number of the records will make it easy to analyze the videos according elapsed times later.
   3. Move the mouse with plate lid out of the 10X lens. Fold a corner of a paper towel to make a thin and fine tip and use it to carefully blot the saline around the CA (avoid touching CA) as well as in other places in the surgical field. This is important to avoid dilution of the FeCl₃ solution used.
   4. Use a fine tip forceps and pick up a piece of filter paper saturated with FeCl₃ solution and place it directly on the CA and keep it on site for 1 min.
      NOTE: To aid visualization of the bloodstream and harvest accurate data, place the filter paper close to the distal end of the exposed CA (Figure 3I) and leave a short segment at the proximal site (green arrow in Figure 3I) for observing blood flow at late phase (see below).
   5. Remove the filter paper (this time point is defined as the start of “after injury”) and rinse the CA with saline (at least 2 ml).  
   6. Put the mouse with plate lid back to the 10X lens; observe the vessel and start to record video images. Write down the video frame number at the end of the first minute of recording.
      NOTE: Thrombus formation is identified by accumulation of the fluorescent platelets, which is observed in real-time video images on computer screen or under microscope. Record video images immediately after putting the mouse back under the microscope. Keep recording to the end of the first minute after removing the filter paper. Observe the entire vessel from the distal to the proximal sites to obtain information about the injury, and then focus on the area of interest (usually is the center of the injured area) for further imaging.
   7. Record video images for 10 sec every minute for the first 10 min (e.g., 2:55 to 3:05) and then 10 sec every other minute until the end of the experiment. Write down the frame numbers when each recording is stopped.
      NOTE: The end points of the model are: 1) when blood flow has ceased for >  30 sec; or 2) if occlusion is not seen in 30 min after injury. In this case, use 30 min for statistical analysis. Set Exposure Time of the video record as 10 msec at the beginning, so it will be easy to observe the blood flow over the thrombi. When the thrombi become large and the fluorescence saturates the camera’s sensor, it becomes difficult to identify the blood flow over thrombi. To solve this issue, move the imaging center to the proximal uninjured site (Figure 3I, green arrow) where blood flow can still be clearly observed. We also increase the Expose Time to 15 or 20 msec when focusing on the proximal un-injured site, so the blood flow can be observed more clearly. When blood flow will cease, numerous larger cells (leukocytes) start to roll on the vessel wall at the proximal site of the thrombus. Flow usually stops within 2 - 3 min after the appearance of the large cells. Write down the Expose Time on the recording sheet if it is changed. It usually takes about 30 min from the anesthesia to the end of the experiment, if the normal wild type C57Bl6 mice were used.
      CAUTION NOTE: Don't use the white aggregated platelet clot as the sign of the blood flow cessation, which will not give an accurate data and it is affected by the exposure time. The white clot covered the vessel lumen does not mean blood flow ceased (see the representative video 1).
   8. At the end of experiment, euthanize the mice using overdose ketamine/xylazine (200/20 mg/Kg), followed by cervical dislocation after confirming no breath and heartbeat.

3. FeCl₃ Induced Mesenteric Artery/Vein Thrombosis Model

1. Anesthetize 8 − 12 week old C57Bl6 mice as mentioned in section 2.1.1. Apply vet ointment on the eyes to prevent dryness during anesthesia.
2. Inject Papaverine solution (total 0.4 mg/mouse) intraperitoneally to inhibit gut peristalsis.
3. Remove fur on the neck and abdomen with an animal electric clipper. Depilatory cream is not necessary.
4. Secure the mouse in the supine position on the lid of 15 cm tissue culture plate. Use four pieces of small tape (2 cm x 0.5 cm) to secure all legs. Use a 4 -0 suture to wrap around the incisors, tape the two ends of the suture to the lid to keep the neck straight (Figure 2).
5. Follow procedures 2.1.4 - 2.1.15 to expose the jugular vein for injection of rhodamine 6G to label platelets.
6. Perform a midline incision through the skin from xiphoid to lower abdomen using the surgical scissors (Figure 4A). Pick up the middle peritoneum (also called linea alba, Figure 4A, arrow) with Graefe forceps, which is clear and has no blood vessels, lift about 2 - 3 mm high, confirm no bowel under the cutting line, and cut the peritoneum open longitudinally with the fine scissors (Figure 4B).
7. Re-secure the mice to right lateral position. Place the half-circle agarose gel close to the abdomen, and gently exteriorize the intestines and spread it on the top of the gel with the Graefe forceps (Figure 4C). Use the second branches for the thrombosis experiment (Figure 4C, arrow).
   NOTE: Use the hemostat or Micro-Adson forceps to help to exteriorize the intestines. Avoid hurting the bowel and the vessel.
8. Place 5 - 6 drops saline to keep the moisture of the bowel and vessel. Transfer the mouse with the plate lid together to the microscope stage and place in appropriate position under the 10X dry lens.
   NOTE: Use a dry lens because the jejunoileal membrane cannot hold saline solution. Make sure to drop saline solution on the exposed tissues to keep them moist.
9. Blot the saline around the mesenteric artery and vein before treatment.
10. Use a fine tip forceps to pick up a piece of filter paper saturated with 12.5% FeCl₃ solution and place it directly on the mesenteric artery and vein for 1 min (Figure 4D).  Remove the filter paper (this time point is defined as the start of “after injury”) and rinse the injured mesenteric artery and vein with saline (at least 2 ml).  
11. Put the mouse with plate lid back under the 10X dry lens; observe the vessels and start to record video images as mentioned in 2.2).
    NOTE: The end points of this experiment are: 1) when blood flow has ceased for > 30 sec; or 2) if occlusion is not seen in 30 min after injury, and in this case use 30 min for statistical analysis.
12. At the end of experiment, euthanize the mice using overdose ketamine/xylazine (200/20 mg/Kg), followed by cervical dislocation after no breath and heart beating are confirmed.

Wyniki

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...

Dyskusje

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 thrombosis^{7,8,16,19,31-33}, but can also be a valuable tool for evaluation of therapeutic compounds and strategies for treatment and prevention of atherothrombotic diseases^11,17,34-37. Here we have shown our modifications and refinements of this model and showed additional evidence of the utility of this technique, wh...

Materiały

Name	Company	Catalog Number	Comments
Surgical Scissors - Tungsten Carbide	Fine Science Tools	14502-14	cut and hold skin
Micro-Adson Forceps - Serrated/Straight/12 cm	Fine Science Tools	11018-12	cut and hold skin
Metzenbaum Fino Scissors - Tungsten Carbide/Curved/Blunt-Blunt/14.5 cm	Fine Science Tools	14519-14	to dissect and separate soft tissue
Ultra Fine Hemostat - Smooth/Curved/12.5 cm	Fine Science Tools	13021-12	to dissect and separate soft tissue
Graefe Forceps - Serrated/Straight/10 cm	Fine Science Tools	11050-10	to dissect and separate soft tissue
Dumont #5 Fine Forceps - Biology Tips/Straight/Inox/11 cm	Fine Science Tools	11254-20	Isolate vessel from surounding tissue
Dumont #5XL Forceps - Standard Tips/Straight/Inox/15 cm	Fine Science Tools	11253-10	Isolate vessel from surounding tissue
Blunt Hook- 12 cm/0.3 mm Tip Diameter	Fine Science Tools	10062-12	Isolate vessel from surounding tissue
Castroviejo Micro Needle Holders	Fine Science Tools	12061-02	Needle holders
Suture Thread 4-0	Fine Science Tools	18020-40	For fix the incisors to the plate
Suture Thread 6-0	Fine Science Tools	18020-60	For all surgery and ligation
Kalt Suture Needles	Fine Science Tools	12050-03
rhodamine 6G	Sigma	83697-1G	To lebel platelets
FeCl3 (Anhydrous)	Sigma	12321	To induce vessel injury
Papaverine hydrochloride	Sigma	P3510	To inhibit gut peristalsis.
Medline Surgical Instrument Sterilization Steam Autoclave Tapes	Medline	111625	To fix the mouse to the plate
Fisherbrand™ Syringe Filters - Sterile 0.22 µm	Fisher	09-720-004	For sterlization of solutions injected to mice
Fisherbrand™ Syringe Filters - Sterile 0.45 µm	Fisher	09-719D	To filter the FeCl3 solution
Sterile Alcohol Prep Pad	Fisher	06-669-62	To sterilize the surgical site
Agarose	BioExpress	E-3120-500	To make gel stage
Leica DMLFS fluorescent microscope	Leica		Intravital microscope
GIBRALTAR Platform and X-Y Stage System with heating plate attached.	npi electronic GmbH		http://www.npielectronic.de/products/micropositioners/burleigh/gibraltar.html
Streampix version 3.17.2 software	NorPix		https://www.norpix.com/