Murine Models in the Evaluation of Heparan Sulfate-Based Anticoagulants

ABSTRACT/SUMMARY.

Evaluating prospective anticoagulants therapies in animal thrombosis and bleeding models are standard pre-clinical approaches. Mice are frequently used for initial evaluations because a variety of models have been developed in this well characterized species, and mice are relatively inexpensive to maintain. Because mice seem to be resistant to forming “spontaneous” thrombosis, vessel injury is used to induce intravascular clot formation. For the purpose of testing heparin-based drugs, we adapted a well-established model in which thrombus formation in the carotid artery is induced by exposing the vessel to ferric chloride. For studying anticoagulant effects on venous thrombosis, we use a model in which the inferior vena cava is ligated and the size of the resulting clots are measured. The most common adverse effect of anticoagulation therapy is bleeding. We describe a simple tail bleeding time that has been used for many years to study the effects of anticoagulants on hemostasis. We also describe a more reproducible, but more technically challenging, saphenous vein bleeding model that is also used for this purpose.

GENERAL INTRODUCTION

Work with mouse models has provided a wealth of information that is central to our understanding of normal and pathologic blood coagulation (1-9). Because of their small size, short gestation period, high reproductive capacity, and relatively low cost of maintenance, mice are often used in initial in vivo analyses of antithrombotic compounds intended for therapeutic use in humans. With a few exceptions, mice and humans have similar complements of plasma coagulation factors and regulatory proteins (10-13). Furthermore, human coagulation proteins usually demonstrate reasonable activity (with a few exceptions) when added to mouse blood, and vice versa (10-13).

Heparin, heparan sulfate, and dermatan sulfate produce anticoagulant effects by enhancing the inhibition of the coagulation protease thrombin and proteases responsible for thrombin generation by serine protease inhibitors (serpins), particularly antithrombin (14). These glycosaminoglycans bind to the serpins and proteases, increasing rates of protease inhibition through allosteric and template-based mechanisms (serpin-dependent effects) [15]. Glycosaminoglycans may also have direct inhibitory effects on some coagulation proteases(serpin-independent effect) [16]. Anion binding sites on coagulation proteases and serpins required for productive interactions with glycosaminoglycans are similar in mice and humans, suggesting similar mechanisms for regulating thrombin generation.

Similarities in the hemostatic mechanisms of humans and mice make the latter species useful for studying anticoagulants even when the compound interacts only with the human version of a protein. Mouse genomes are easily manipulated to produce animals with constitutive or conditional deficiencies of a protein of interest (10-12). The animals can be reconstituted with the human counterpart of a missing plasma protein that can then be targeted with the study drug. Most human coagulation proteases will restore the wild type phenotype in hemostasis and thrombosis models in knockout mice (17-20).

There are some important differences between mice and humans that must be considered when interpreting results for of hemostasis and thrombosis models. The several thousand-fold size difference alone leads to different magnitudes of force on tissues, presenting different types of challenges for the respective hemostatic systems. Humans lacking coagulation factor IX have a condition (hemophilia B) characterized by recurrent hemorrhage into large joints (hemarthrosis) such as knees, ankles, and elbows (21). Factor IX deficient mice also have a bleeding disorder, but hemarthrosis is not a prominent feature, perhaps because of the smaller mechanical forces on their joints (22). Atherosclerotic changes in large arteries in humans develop over decades. Mice, with their shorter life-spans and high plasma levels of high density lipoprotein, are resistant to atherosclerotic changes, and require dietary or genetic manipulation to produce plaques (23-25). Even mice that do develop atherosclerosis, such as those lacking apolipoprotein E, due not often develop occlusive thrombi at sites of plaque rupture in the same manner as humans (26,27). In humans, venous thrombi form preferentially in the deep veins of the legs and pelvis, with blood stasis aggravated by upright posture serving as a major contributor (28). The long-term effects of hydrostatic forces on leg veins are less important in mice, which rarely develop spontaneous venous thrombi in their extremities.

The natural resistance of mice to forming occlusive thrombi is most easily overcome by acutely injuring a vessel in specific ways. It is important to remember, however, that thrombi in such models form in vessels that were healthy immediately prior to injury. This is distinctly different from the situation in humans, where thrombosis typically involves diseased arteries and veins. A variety of techniques are employed to induce thrombosis in mice. Here, ferric chloride-induced injury of the carotid artery and ligature-induced venous stasis in the inferior vena cava are described. Both techniques are widely used, and are sensitive to heparin. The major adverse side effect of anticoagulants, including heparin-based anticoagulants is bleeding. The last section two of this chapter compare a simple tail bleeding time assay to a more sophisticated saphenous vein bleeding model for studying the anti-hemostatic effects of these drugs.

FERRIC CHLORIDE-INDUCED ARTERIAL THROMBOSIS MODEL

1. INTRODUCTION

A variety of approaches are used to induce acute thrombus formation in large (e.g. carotid), medium (e.g. mesenteric) and small (e.g. cremaster) diameter arteries in mice. Injury to vascular endothelium to promote thrombus formation can be induced by chemical exposure (e.g. ferric chloride) (1-3,29-31), photochemical techniques (e.g. Rose Bengal-laser injury) (1-3), direct laser injury (1-4), or mechanical methods (2). Thrombus formation is monitored by following changes in blood flow with a Doppler flow-probe, by visualizing thrombi with intravital microscopy, or by observing histologic changes in fixed tissue specimens. It is not established which injury type most closely recapitulate processes that occur during arterial thrombosis in humans. Many groups find ferric chloride-induced injury to be a reproducible method for generating clots that resemble human platelet-rich arterial thrombi (29-34). Typically one or more small pieces of paper saturated with FeCl₃ solution are applied to a vessels adventitial surface. FeCl₃ defuses through the vessel wall to the luminal surface. Initially, it was thought that thrombosis was the result of denudation of endothelium, exposing subendothelial matrix to flowing blood. While this occurs with high FeCl₃ concentrations, work from Barr et al. suggests the endothelium remains largely intact after most FeCl₃ applications (31). They observed that erythrocytes initially bind to endothelium, with ferric ions primarily interacting with erythrocyte and erythrocyte-derived structures and not endothelial cells themselves. Platelets subsequently bind to adherent erythrocytes in a manner dependent on the platelet receptor glycoprotein 1bα, and then platelet aggregates to eventually occlude the vessel lumen.

We use ferric chloride-induced carotid artery occlusion to study the antithrombotic effects of heparin (33) and heparan-based anticoagulants (35-36). Thrombus formation in mice induced by FeCl₃ requires contributions from tissue factor-initiated coagulation (extrinsic pathway) (37) and from factors XII and XI (intrinsic pathway) (19,20,33,38). Our approach involves establishing the lowest FeCl₃ concentration that reproducibly induces vessel occlusion in untreated wild type mice, and then testing an anticoagulant to determine if it prevents vessel occlusion at that FeCl₃ concentration (18,33,34). If an antithrombotic effect is observed, the drug is then tested at progressively higher FeCl₃ concentrations until the drug is no longer able to prevent vessel occlusion.

2. MATERIALS

1. Several inbred and mixed lines of mice are used to study hemostasis and thrombosis. C57Bl/6 mice are used extensively in this regard, and our work with the FeCl₃ arterial thrombosis model is standardized with this readily available line (18,33,34). Assay reproducibility is enhanced by attention to several animal-related factors (see Note 1).

2. Pentobarbital. Mice are placed under general anesthesia with pentobarbital. This drug is a controlled substance and will require DEA licensure to obtain and use. Other general anesthetics may be used, but the sensitivity of the assay to vessel injury may be different than with pentobarbital (see Note 2).

3. Ferric Chloride. A 20% stock solution is prepared by bringing 200 mg of FeCl₃ up to 1 ml with de-ionized water. Subsequent dilutions of the stock are prepared with deionized water. We prepare fresh stock solution every one or two weeks.

4. Filter paper for application of FeCl₃. Whatman 3MM Chromatography paper (catalog # 3030-917) is cut into small rectangles measuring ~1 x 1.5 millimeters.

5. Flow probe. We use a Transonic (Ithaca, New York) TS4020 transit time perivascular flow meter fitted with a 0.5VB504 Doppler flow probe (Catalog Mao-5VB). The flow meter is connected to an ML866 PowerLab 4/30 data acquisition system (AD Instruments, Dunedin, New Zealand) interfaced with a computer.

6. Phosphate buffered saline (PBS). PBS is used to keep tissues moist during the procedure and as a vehicle for diluting heparan-based anticoagulants for intravenous administration. We use 1X Cellgro Dulbecco’s phosphate buffered saline without calcium or magnesium (Mediatech, Manassas, VA), but any source of sterile PBS should work.

3. METHODS

1. Mice are anesthetized by an intraperitoneal injection of pentobarbital (50 mg/kg). The injection is given into the right side of the abdomen to avoid injuring the cecum or spleen.

2. Animals under anesthesia are placed on their backs on a 37 °C warming pad, and the extremities are immobilized with tape. The neck is opened along the midline and the carotid artery and jugular vein are exposed on one side of the neck. The artery is separated from surrounding tissues by blunt dissection (Figure 1A) (see Note 3).

3. The flow probe is attached to the artery, and the area is bathed with PBS to insure proper signal transduction from vessel to probe (Figure 1B). A baseline flow rate is established (typically 0.5 to 0.8 ml/min in an adult mouse). The flow probe is then removed.

4. Anticoagulant compounds are diluted in 100 μl of PBS. The drug (or vehicle) is infused into the jugular vein using a 300 μl tuberculin syringe fitted with a 30-gauge needle. The needle tip pointing toward the heart (the direction of blood flow) (Figure 1C) (Note 4).

5. Five minutes after drug infusion, the area around the carotid artery is dried with cotton Q-tips. Two Whatman chromatography paper pads are each saturated with 50 μl of FeCl₃ solution (Figure 1D). Initial studies are typically performed with 3.5% FeCl₃. The pad cannot hold all 50 μl of solution. Non-absorbed solution is discarded. Pads are applied on opposite sides of the carotid artery from each other. After three minutes, the pads are removed, the area is washed with PBS, the Doppler flow probe is replaced (Figure 1B) and flow is monitored for up to 30 minutes. Changes in flow over time, and time to vessel occlusion are measured. Mice are sacrificed prior to recovering from anesthesia (Note 5).

Figure 1. Ferric Chloride Carotid Artery Thrombosis Model.

(A) Anatomy of ventral surface of the mouse neck showing the relative positions of the carotid arteries and jugular vein to the trachea, which runs along the midline. The positions of the animal’s head and tail relative to the drawing are indicated. The black arrows indicate the direction of blood flow in the major vessels. (B) A Doppler flow probe is placed on the carotid artery to monitor blood flow. (C) Anticoagulant compounds to be tested are administered through an intravenous injection into the jugular vein in the direction of blood flow (toward the heart). (D) Pieces of filter paper (two total) saturated with ferric chloride solution are applied beneath and on top of the carotid artery

INFERIOR VENA CAVA STASIS-INDUCED VENOUS THROMBOSIS MODEL

1. INTRODUCTION

Venous thrombi that cause symptoms in humans form primarily in the deep veins of the legs and pelvis (28). Embolization of clot to the pulmonary circulation is a major cause of mortality in such patients. While platelets contribute to venous thrombus formation, the clots are predominantly comprised of fibrin and erythrocytes, and differ significantly in their histology from the platelet-rich thrombi that form in the arterial circulation. Hydrostatic forces cause blood to pool in deep leg veins in humans as a consequence of our upright posture. These features of human venous thrombosis are difficult to reproduce in mice. Chemical injury to a vein with a substance such as FeCl₃ results in platelet-rich thrombi that are more similar to arterial clots than venous clots in humans (5,39). Venous stasis models involving partial or complete ligation of the inferior vena cava in the abdomen have been developed to study the effects of anticoagulants on thrombus formation (5-7,40). Here we present a method that involves incomplete ligation of the inferior vena cava (40).

2. MATERIALS

1. C57Bl/6 mice. As in the FeCl₃ thrombosis model (see Note 1).

2. Pentobarbital. As in the FeCl₃ thrombosis model (see Note 2).

3. Phosphate buffered saline (PBS). As in the FeCl₃ thrombosis model.

4. Surgical supplies. 4-0 Vicryl suture is used to ligate the inferior vena cava. 4-0 Steelex metal monofilament suture (Braun Catalog #F1614037).

3. METHODS

1. Mice are anesthetized by intraperitoneal injection of pentobarbital (50 mg/kg) as described in the section on the FeCl₃ thrombosis model.

2. The mouse is placed on its back on a 37 °C warming pad, and the extremities are immobilized with tape. Heparan-based drug or vehicle in 100 μl of PBS is infused into a lateral tail vein using a 1 ml tuberculin syringe fitted with a 27-gauge needle. If the drug appears to compromise subsequent surgery, it can be administered shortly after the surgical procedure described below is complete (see Note 6).

3. A midline vertical incision is made through the skin and abdominal wall with a scalpel. The inferior vena cava is exposed between the iliac bifurcation and the renal veins by pushing the abdominal contents (bowel) to the left side of the animal (Figure 2A). The bowel is kept moist by covering it with cotton gauze soaked in PBS. The vena cava is gently separated from the aorta by blunt dissection (see note 7).

4. A 4-0 coated Vicryl suture is placed underneath the vena cava immediately below the renal veins, and a 4-0 Steelex metal monofilament suture is placed longitudinally over the IVC (Figure 2B – Step 1) (see note 8). The Vicryl suture is tied over the IVC and the metal suture to stop blood flow through the vessel (Figure 2B – Step 2). Then the metal suture is gently removed by sliding it out from ligature (Figure 2B – Step 3). Removal of the steel suture restores a small amount of blood flow through the vena cava (see note 9).

5. A sterile forceps with serrated tip is used to compress (crimp) the vena cava immediately below the suture (toward the tail) for 20 seconds (Figure 2B – Step 4). The procedure is repeated at a location 5 mm below the first compression site (Figure 2B – Step 5). The “crush” injury causes endothelial damage that serves as a nidus for thrombus formation (see note 9).

6. Bowel is returned to the abdominal cavity, and the abdominal wall is closed with sutures. The overlying skin is closed with surgical clips. The animal is observed until it recovers from anesthesia.

7. 24 hours post-surgery, the mouse is sacrificed with an intracardiac infusion of pentobarbital (100 mg/kg) and the abdomen is reopened. The vena cava is cut above the ligature and at the distal end above the iliac bifurcation (Figure 2C). Vessel contents are pushed out of the distal end of the vena cava by running forceps along the length of the vessel starting at the ligature. Expressed clot is placed in 10% formalin for 24 hours. After fixing, the clot is dried on a piece of filter paper and weighed (see note 10).

Figure 2. Inferior Vena Cava Venous Thrombosis Model.

(A) Anatomy of the retroperitoneum of the mouse as viewed from a ventral abdominal incision. The positions of the animal’s head and tail relative to the drawing are indicated. The black arrows indicate the direction of blood flow in the major vessels. (B) Thrombosis Model. Step 1 - A ligature is loosely placed around the inferior vena cava caudal to the left renal vein. A steel suture or other linear object such as a needle is also placed within the ligature. Step 2 – The ligature is tightened around the vessel and steel suture. Step 3 – The steel suture is gently removed from the ligature. This results in the ligature restricting flow through the vena cava without completely blocking it. Step 4 – Forceps are used to crimp the vena cava immediately below the suture, and (Step 5) 5 millimeters caudal to the suture to injure the vessel endothelium. (C) twenty-four hours after surgery, the abdomen is reopened, and the vena cava is excised for processing by cutting the vessel immediately above (cranial) to the ligature, and just above the iliac bifurcation.

TAIL BLEEDING MODEL

1. INTRODUCTION

Most antithrombotic drugs, including heparin, produce therapeutic effects by inhibiting proteases that are required for the normal hemostatic responses to injury. The major trade-off for the beneficial drug effect is, therefore, an increased risk of bleeding (41). Newer oral agents, while exhibiting better safety profiles than older drugs such as heparin and warfarin, still target the key proteases thrombin and factor Xa and, therefore, increase bleeding risk. Compounds are now under development that target plasma proteases such as factor XIa and factor XIIa that do not play major roles in hemostasis but that contribute to thrombus growth (42-44). While such drugs may not show better efficacy than currently available agents, it is anticipated that they will have better safety profiles, permitting therapy to be applied to a wider range of patients. Mice lacking factor XI or factor XII exhibit resistance to injury-induced thrombosis in a number of thrombosis models but do not have obvious hemostatic abnormalities (12,19,33). Drugs specifically targeting factor XIa and factor XIIa should, therefore, prevent thrombus formation but not increase bleeding. Drugs based on heparan-like structures that specifically inhibit factor XIa are being developed (35,36). As heparans have a tendency to bind to multiple targets, it is important to test such compounds for off-target effects that compromise hemostasis.

The tail bleeding time (TBT) has been used extensively to study hemostasis in mice (45-49). The assay is easy to perform, and is sensitive to heparin (33). Removal of the tip of the tail with a scalpel transects several blood vessels including two large lateral veins and a ventral artery. The tail tip is usually immersed in normal saline and time to cessation of bleeding and/or total blood loss determined. While wild type mice usually bleed for one to three minutes, mice with certain bleeding disorders or mice receiving anticoagulation therapy may have prolonged bleeding that is punctuated by periods of 1 to 2 minutes in which no bleeding occurs. For example mice lacking factor VIII or factor IX (hemophilia A or B, respectively) have similar initial tail bleeding times as wild type mice. However, shortly (15 to 60 seconds) after initial cessation of bleeding, hemorrhage recurs and is usually severe (Figure 3A) (47). We observe mice for up to 30 minutes after tail transection to account for this “rebleeding”, with the recorded bleeding time being the time it takes for all bleeding (initial and rebleeding) to stop. Any bleeding model provides information about hemostasis in response to a specific-type of injury in a specific vascular bed, and does not necessarily reflect what will happen with injury to other tissues. Nevertheless, the simplicity of the tail bleeding time has made it a mainstay of evaluating hemostasis in mice.

Figure 3. Tail Bleeding Model and Saphenous Vein Bleeding Model.

(A) Tail bleeding time model. Shown are average tail bleeding times in wild type (WT) and factor IX deficient (f9−/−) C57Bl/6 mice. (B-D) Saphenous vein bleeding model. (B) Average bleeding times and (C) number of clots per minute in in wild type (WT) and factor IX deficient (f9−/−) C57Bl/6 mice tested in the saphenous vein bleeding model. (D) Technical aspects of the saphenous vein bleeding model. The image on the left shows the ventral surface of the mouse hind limb and the relative positions of the saphenous artery and vein. A 23 gauge needle is used to create an entry hole in the saphenous vein. The blue lines outline the borders of the vein in the middle and right hand images, and the red line indicates the position of the saphenous artery. The tip of a Student Vannas spring scissor is introduced into the entry hole and is used to make an incision in the vein wall running away from the direction of blood flow of about one millimeter in length.

2. MATERIALS

1. C57Bl/6 mice. As in the FeCl₃ thrombosis model (see Note 1)

2. Pentobarbital. As in the FeCl₃ thrombosis model (see Note 2).

3. Phosphate buffered saline (PBS). As in the FeCl₃ thrombosis model.

3. METHODS

1. Mice are anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg) as described in the section on the FeCl₃ thrombosis model.

2. Once under anesthesia, the animal is placed on a 37 °C heating pad. Heparan based drug, or vehicle, in 100 μl of PBS is infused into a lateral tail vein using a 1 ml tuberculin syringe fitted with a 27-gauge needle (see Note 6).

3. The tail is transected with a scalpel 2 millimeters above the tip (see Note 11).

4. The bleeding tail is immediately immersed in a 1.7 ml microfuge tube filled with 1.2 ml of PBS kept at 37 °C with a heating block.

5. The animal is observed for up to 30 minutes. The time to cessation of bleeding (including rebleeding) is noted (see Note 12). Mice are sacrificed before recovering from anesthesia.

6. Volume of PBS plus blood is recorded to establish the amount of blood lost (see Note 13).

SAPHENOUS VEIN BLEEDING MODEL

1. INTRODUCTION

The saphenous vein bleeding (SVB) model was originally developed to study hemostasis in hemophiliac mice with an assay that has better sensitivity and lower variability than the TBT [9,50-52]. The technique was subsequently adapted for the study of anticoagulation therapy [53]. We find that the SVB technique has advantages over the TBT, particularly in the area of intra-operator reproducibility. This may reflect the more standardized wound involved. However, investigators contemplating using the SVB method should be aware that it is more technically demanding than the TBT, and requires practice.

The saphenous vein is exposed on the anterior surface of the thigh, and the vessel is opened with a small incision made with scissors. The time it takes for a clot to form is then recorded. The clot is then gently removed to re-start bleeding. This is repeated each time a clot forms. The period of time it takes each clot to form, and the total number of clots that form over a thirty minute period are record. Data comparing results for C57Bl/6 wild type and factor IX deficient mice (mice with hemophilia B) are shown in (Figure 3B and 3C). Note the inverse relationship between the average individual bleeding times, and the total number of clots that form over thirty minutes.

2. MATERIALS

1. C57Bl/6 mice. As in the FeCl₃ thrombosis model (see Note 1)

2. Pentobarbital. As in the FeCl₃ thrombosis model (see Note 2).

3. Phosphate buffered saline (PBS). As in the FeCl₃ thrombosis model.

3. METHODS

1. Mice are anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg) as described in the section on the FeCl₃ thrombosis model.

2. Once under anesthesia, the animal is placed on a 37 °C heating pad. Heparan based drug, or vehicle, in 100 μl of PBS is infused into a lateral tail vein using a 1 ml tuberculin syringe fitted with a 27-gauge needle (see Note 6).

3. The ventral surface of one hind limb is shaved, and a longitudinal incision is made in the skin along the length of the limb to expose the saphenous vein.

4. The saphenous vein is punctured once with a 23-gauge needle to create an entry hole.

5. After initial bleeding stops (typically 1-2 minutes), a blade of a Student Vannas spring scissor is inserted through the entry hole into the distal part of the vessel. A longitudinal snip of ~ 1 millimeter is made in the vessel (see Note 14).

6. Gauze is used to wick away blood issuing from the injury site, without touching the edges of the wound. The time to cessation of bleeding is recorded.

7. The clot is gently disrupted to restart bleeding. The goal is to remove the clot with minimal manipulation of the vessel. We achieve this by gently running gauze along the clot surface in the direction of blood flow.

8. Steps 6 and 7 are repeated each time a clot forms, over a 30 minute observation period. The time it takes for each clot to form, and the total number of clots formed in 30 minutes are recorded.

NOTES

1. A number of animal-related factors produce variability in the FeCl₃ model. Different mouse strains vary in their propensity to form thrombi in response to FeCl₃. Each laboratory should determine the sensitivity of the mouse line (inbred or mixed) they use to different concentrations of FeCl₃ before testing anticoagulants. Older mice are larger than younger animals and have thicker vessel walls. This can alter the response to injury. We prefer to use mice that are in a relatively narrow age range (12-20 weeks) to limit this effect. Many investigators confine analysis to male mice, to avoid effects of variation in coagulation protein levels and other factors over the estrus cycle in females. We have not noticed a significant gender difference with the FeCl₃ carotid artery injury model, but it is possible that certain anticoagulants may be sensitive to changes due to the estrus cycle.

2. The response of the animal to FeCl₃ injury (i.e. the lowest concentration required to reproducibly induce thrombus formation) varies depending on the anesthetic. For example, 3.5% FeCl₃ is required to reproducibly induce occlusive thrombus formation in C57Bl/6 mice anesthetized with pentobarbital (33). The same mice anesthetized by isoflurane inhalation reproducibly occlude with 2.5% FeCl₃. For each anesthetic, a range of FeCl₃ concentrations should be tested to identify the lowest concentration that reproducibly induces vessel occlusion.

3. An obvious concern with using model requiring surgery to test anticoagulants is peri-operative bleeding. We find that if care is taken not to injure structures underlying the skin, the FeCl₃ arterial injury model and the venous stasis model can be performed with minimal blood loss even on mice with severe hemophilia (factor VIII or IX deficiency) or in mice who have received heparin.

4. In our experience, FeCl₃ arterial injury models are less sensitive to anti-platelet agents than to inhibitors of thrombin generation. However, when using low FeCl₃ concentrations, anti-platelet agents can influence results (produce an antithrombotic effect). Given this, we avoid using non-steroidal anti-inflammatory analgesics that inhibit cyclo-oxygenase 1 to treat pain because the anti-platelet effect will alter results in the thrombosis model.

5. Changing the size or number of the FeCl₃-soaked filter papers, or the duration of exposure of the vessel to FeCl₃, changes the extent of vessel injury, and affects result. If these factors are standardized, the assay should have a high degree of reproducibility.

6. Administering drugs by tail vein injection is a skill that requires practice. The diameter of the target vessel is small and the skin and underlying tissue of the tail are tough and can be difficult to penetrate with a small-gauge needle. Warming the tail for a few minutes with a warm cloth to dilate the vessels can make injection easier.

7. While the anatomy of the carotid artery varies relatively little between mice, there can be considerable variation in the anatomy of the inferior vena cava, even among animals of the same strain. The number and size of collateral branches, and the positions of branch points of important vessels such as the renal veins vary. Some investigators ligate large collaterals so that flow to the vessel comes mostly from the lower extremities, but we have not found that this affects results appreciably. Rarely, an animal develops paralysis of the hind limbs a few hours post-procedure. This might be caused by trauma to the aorta or small arteries that branch off of it at numerous points to supply the spinal cord. It is can be difficult to distinguish the vena cava from the aorta, leading to inadvertent ligation of both vessels. Practice is required to obtain proficiency in isolating and manipulate the inferior vena cava without injuring the aorta.

8. The 4-0 Steelex metal monofilament suture used in step 4 under Methods can be replaced with another object of comparable diameter, such as a 26-gauge needle. The extent of the partial obstruction of blood flow can be adjusted by using needles of different gauge.

9. This model requires a significant amount of practice to perform reproducibly compared to the FeCl₃-induced arterial injury model, as the inferior vena cava is considerably more delicate than the carotid artery, the vessel is closely associated with the descending aorta in the abdominal cavity, and the required manipulations of the vessel are relatively complex. When crimping the vessel with forceps, excessive force can result in laceration.

10. A relatively large range of thrombus sizes should be expected, even in a control group of mice of the same strain. On occasion, a control mouse may even fail to develop a thrombus. For this reason, it is necessary to test a larger number of animals than would be used in a more reproducible assay such as the FeCl₃-induced arterial injury model.

11. While a common strategy is to transect the animal’s tail ~2 mm from the tip, some investigators use injuries at different points on the tail. It is important to note that tail anatomy varies between animals, resulting in different amounts of tissue being injury if a fixed distance from the tail tip is used to determine the point of injury. Some investigators chose to injure tails at a point where the cross-sectional areas of the tail are the same. A template (typically a piece of plastic) with an aperture of desired cross-sectional area can be used for this purpose. The animal’s tail is drawn through the aperture and transected using the surface of the template to guide the scalpel.

12. Bleeding time should always be time to cessation of all bleeding, including rebleeding. Some investigators will determine when bleeding has stopped for at least 60 seconds as an indication that all bleeding has stopped. We prefer to observe the animal for a full 30 minutes to insure that bleeding does not recur.

13. We feel that the bleeding time alone is not sufficient to accurately reflect the extent of bleeding. A mouse may have a prolonged bleeding time, but lose relatively little blood during that time, compared to another animal that bleeds more briskly for a short period of time. We recommend measuring total blood loss as well as time to cessation of bleeding.

14. In the saphenous vein bleeding model we usually make an ~1 millimeter snip with the scissors. However, the length can be altered, and this can be used to change the severity of bleeding and affect the sensitivity of the assay to specific drugs. The ability to adjust the performance of the assay by altering the initial scissor-induced injury is we feel, a strength of the procedure that allows it to be optimized for specific drugs.