Esther Bloem and Thomas Nygaard are acknowledged for support with measurements of FVIII in plasma. Bo Alsted is acknowledged for drawing and machining the template and cutting blocks.

All procedures described in this protocol have been approved by the Animal Welfare Body at Novo Nordisk A/S, and the Danish Animal Experiments Inspectorate, The Danish Ministry of Food, Agriculture, and Fisheries. The optimized 40 minutes method includes anesthesia and dosing time in the design (Figure 1). Hemophilic mice of both genders between 10-16 weeks of age are required for this procedure.
1. Preparations before the study
<ol>
	<li>Prepare the dosing solutions in the correct concentrations.</li>
	<li>Start the water bath and heat to 37 &#176;C. Fill the 15 mL centrifuge tubes for blood collection with saline (0.9% NaCl).</li>
	<li>Place the 15 mL saline tubes in the holes in the warmed base plate at least 15 min prior to the start of the experiment.</li>
	<li>Identify the mice and record their weight. Avoid handling mice more than necessary as this can cause stress and affect the study.</li>
	<li>Prepare the workstation in the fume hood before proceeding so that everything is within reach: napkins, tail holder, gauze, syringes, scalpels, stopwatches, and blood flow notation paper.</li>
	<li>Place the tail mark and cutting blocks on the heating plate - cold blocks will make the veins contract and thereby affect the bleeding.</li>
</ol>
2. Anesthesia
<ol>
	<li>Conduct the isoflurane anesthetic procedure inside a fume hood.</li>
	<li>Set the gas vaporizer to initially 5% isoflurane in 30% O2/70% N2O in the anesthesia chamber with 1 L/min flow. Allow sufficient time for the anesthesia chamber to fill (about 5 min depending on chamber volume and gas flow rate).&#160;Ensure rapid induction (less than one minute).</li>
	<li>Place the mice in the anesthesia chamber until they lose consciousness. 
	NOTE: This should occur within a minute or less if the chamber is sufficiently filled.</li>
	<li>Ensure proper anesthetization by the absence of painful response to pedal reflex (firm toe pinch).</li>
	<li>Place the mice on the heating plate, making sure that the nose is in the nose cone.</li>
	<li>Reduce the anesthesia to a maintenance level of 2% isoflurane in 30% O2/70% N2O and place a plastic cover above the mice to reduce the loss of heat. Apply a suitable eye ointment to prevent dryness while under anesthesia.</li>
	<li>Mark the tail at a diameter of 2.5 mm using the tail mark block. Do not force the tail into the slit in the block - it must fit snugly (Supplemental Figure 1)</li>
	<li>Place the tail in the saline tube for at least 5 min to ensure a warm tail vein that is optimal for intravenous (i.v.) dosing.</li>
</ol>
3. Dosing of test solution
<ol>
	<li>Place one mouse in the tail holder with the nose in an anesthesia mask.</li>
	<li>Dose the animal with the compound of interest (in this case, rFVIII) and immediately start the stopwatch (t = 0).</li>
	<li>Place the mouse back on the heating plate with the tail in the saline tube. Repeat the procedure with the other mice.</li>
</ol>
4. Performing tail vein transection
<ol>
	<li>Perform the tail vein transection exactly 5 min after dosing. Place the tail in the cutting block and turn 90&#176; to expose the vein (Supplemental Figure 2).</li>
	<li>Perform the cut on the opposite side/vein from where the test solution was&#160;dosed.</li>
	<li>Draw the #11 scalpel blade through the slit of the cutting block holding the tail to create bleeding. Reset the stopwatch and return the tail immediately to the saline.</li>
</ol>
5. Observation time and challenges
<ol>
	<li>Observe the bleeding and annotate the start and stop of the bleeding throughout 40 min; annotate it on the blood flow notation paper. 
	NOTE: This visual assessment of bleeding may vary slightly due to subjectivity.</li>
	<li>The primary bleeding must stop within 3 min after the cut is made. If this is not the case, disqualify the mouse, euthanize, and replace (failure to stop the primary bleed can indicate a too severe injury or lacking primary hemostasis, as in vWF KO mice).</li>
	<li>If there is no bleeding at 10 min, 20 min, and 30 min post-injury, challenge the tail cut as described in steps 4-5.</li>
	<li>Use a gauze swab soaked in warm saline from a separate tube kept in the water bath. Lift the tail out of the saline and softly wipe twice with the wet gauze in a distal direction over the tail cut.</li>
	<li>After each challenge, immediately re-submerge the tail into the saline tube again.</li>
	<li>At t=40 min, stop the blood collection by removing the tail from the saline tube.</li>
</ol>
6. Blood sampling
<ol>
	<li>After t = 40 min, obtain blood samples from the supraorbital vein.</li>
</ol>
7. Euthanasia
<ol>
	<li>Euthanize the mice by cervical dislocation while still under full anesthesia.</li>
</ol>
8. Treatment of samples
<ol>
	<li>Centrifuge the 15 mL blood collection tubes with saline at 4000 x g for 5 min at room temperature.</li>
	<li>Discard the supernatant from the 15 mL tubes, resuspend the pellet in 2-14 mL of erythrocytes (RBC) lysing solution, and then dilute it until it reaches a light coffee color.</li>
	<li>Note the total volume (volume of blood + volume of erythrocytes (RBC) lysing solution added using the graduation marks on the tube).</li>
	<li>Transfer 2 mL of the dilution to a hemoglobin tube and refrigerate it until the hemoglobin analysis.</li>
	<li>Determine the blood loss by measuring the hemoglobin concentration in the saline. Measure the absorbance at 550 nm on a microplate reader (Table of Materials).</li>
	<li>Convert the absorbance to nmol hemoglobin using a standard curve prepared from human hemoglobin (Table of Materials) and correct for the dilution with RBC lysing solution.</li>
</ol>
9. Statistical analyses
<ol>
	<li>Analyze the data using appropriate software. Here GraphPad Prism software was used. Over a range of studies, the following statistical methods were found to perform well. 
	NOTE: To analyze blood loss, bleeding time, exposure, platelet counts, and hematocrit; Brown-Forsythe and Welch ANOVA test was used, (as the data were continuous but without variance homogeneity of the residuals) applying Dunnett&#39;s test to adjust for multiple comparisons. A Pearson&#39;s test was used to test for correlations between bleeding time, blood loss, and doses. To determine ED50 values, a four-parameter inverse log (dose) response equation was fitted to bleeding- and blood loss data. To analyze the gender effect, a two-way ANOVA test was used, applying Bonferroni correction to adjust for multiple comparisons. The significance level was defined as P &#60; 0.05. Data are displayed as means &#177; SEM.</li>
</ol>

The authors are or were employees and/or shareholders of Novo Nordisk A/S at the time this research was carried out.

This optimized method of tail vein transection (TVT) has several advantages compared to the TVT survival method. The animals are fully anesthetized for the entire study duration, which makes mouse handling easier and increases animal wellbeing. Further, unlike the TVT survival model, overnight observation is not required, and this optimized model offers the possibility of measuring blood loss and observing the exact bleeding time over 40 min. Also, longer periods of bleeding in conscious animals can cause death by exsang...

Animal models are essential for understanding the pathogenesis of hemophilia and developing and testing treatment regimens and therapies. The Factor VIII knock-out mouse (F8-KO) is a widely used model for the study of hemophilia A1,2. These mice recapitulate key features of the disease and have been widely used for development of treatments, such as recombinant FVIII products3,4,5 and gene therapy strategies6,7.
There are various bleeding injury models for evaluating the pharmacological effects of different hemostatic compounds in vivo. One of these coagulation models is the tail vein transection survival model in mice8,9,10,11,12,13,14, measuring the ability of hemophilic mice to survive exsanguination after tail transection. This method was introduced more than four decades ago15 and is still used9,16,17. However, the model utilizes survival as an endpoint and requires observation of the animals over a period of up to 24 h, during which the animals are conscious and hence can experience pain and distress.
Bleeding models of shorter duration and under full anesthesia have been described previously, such as the tail clip model (also known as the tail tip)8,18,19,20,21,22,23,24,25,26,27,28. Nevertheless, for a complete normalization of blood loss after the bleeding challenge, these models require doses of procoagulant compounds (e.g., FVIII) far higher than those administered clinically29. A different injury model under anesthesia, the vena saphena bleeding method, is sensitive to lower doses of procoagulant compounds30 but requires a high level of experimenter intervention since the clots must be disrupted frequently (as opposed to 3 times in the presented model).
Standardization towards a common protocol to test new procoagulant compounds would greatly&#160;facilitate data comparison between laboratories31,32,33. In TVT models, there is not yet a common agreement on studied endpoints (blood loss7,26, bleeding time9,34, and survival rate35,36), and experimental length varies between studies13.
Our primary objective is to describe and characterize an optimized model with high reproducibility, the possibility to study on-demand as well as a prophylactic treatment, sensitivity to pharmacological intervention equivalent to the survival model, yet not using death or near-death as endpoints. In order to reduce pain and distress, the animals should not be conscious during bleeding and a more ethical endpoint needs to be implemented37.
Tail clip models are generally conducted in one of two variants, either amputating the tip of the tail, e.g., amputation of 1-5 mm18,19,20,21,23,24 or, in a more severe variant, transected at a tail diameter around 1-3 mm8,22,25. This causes a combined arteriovenous bleed, as the lateral and dorsal veins and ventral artery are usually severed, and in general, the larger the amputation, the lower the sensitivity to a procoagulant compound. Furthermore, since the tail tip is amputated, the arteriovenous injury is exposed without any opposing tissue; thus, at least in theory, it is dissimilar to the most common hemophilic bleeds.
As the name implies, only the vein is injured in tail vein transection models such as described in this paper, thus resulting in an exclusively&#160;venous bleed. Since the vessel is not fully severed, the injury is expected to be smaller than in the amputation models, and the tissue around the cut, which a clot may adhere to, is retained. In addition, there is lower blood pressure in the vein as opposed to the artery. These factors contribute to an increased sensitivity relative to amputation models, such that normalization of bleeding can be achieved with clinically relevant doses of replacement therapy, e.g., with rFVIII in hemophilia A, which is useful for evaluating the magnitude and durability of effects of procoagulant treatment26,38,39.

<table><tbody><tr><td>#11 Scalpel blade</td><td>Swann-Morton</td><td>503</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Greiner Bio-One, Austria</td><td>188271</td><td></td></tr><tr><td>30 G needles connected to 300 &amp;micro;L precision (insulin) syringes for dosing</td><td>BD Micro-Fine + U-100 insulin syringe</td><td>320830</td><td></td></tr><tr><td>Advate</td><td>Takeda, Japan</td><td></td><td>Recombinant factor VIII replacement therapy (rFVIII)</td></tr><tr><td>Alcohol pads 70% ethanol</td><td>Hartmann, Soft-Zellin</td><td>999 979</td><td></td></tr><tr><td>Centrifuge</td><td>Omnifuge 2.0 RS, Heraus Sepatech</td><td></td><td></td></tr><tr><td>Cutting template (Stainless steel)</td><td>Self produced, you are welcomed to contact the authors for the exact drawings</td><td></td><td>Supplementary Figure 2: Size specifications: 20 mm x 40 mm x 10 mm (L x B x H). Groove: 3 mm depth and 3 mm width; radius 1.5 mm</td></tr><tr><td>Erythrocytes (RBC) lysing solution</td><td>Lysebio, ABX Diagnostics</td><td>906012</td><td></td></tr><tr><td>Gauze</td><td></td><td></td><td></td></tr><tr><td>Haematological analyser</td><td>Sysmex</td><td>CT-2000iv</td><td></td></tr><tr><td>Heating lamp on stand</td><td>Phillips</td><td>IR250</td><td></td></tr><tr><td>Heating pad with thermostat</td><td>CMA</td><td>model 150</td><td></td></tr><tr><td>Hemoglobin standards and controls - 8.81 mmol / l batch dependent</td><td>HemoCue, Denmark</td><td>HemoCue calibrator, 707037</td><td>Standards and controls are made from 2 different glasses of HemoCue calibrator. The value is determined against the International Reference Method for Hemoglobin (ICSH).</td></tr><tr><td>Isofluorane anaesthesia system complete with tubes, masks and induction box</td><td>Sigma Delta Dameca</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>26675-46-7</td><td></td></tr><tr><td>Magnifier with lights</td><td>Eschenbach</td><td></td><td></td></tr><tr><td>Measuring template (Aluminum)</td><td>Self produced, you are welcomed to contact the authors for the exact drawings</td><td></td><td>Supplementary Figure 1: Size specifications: 20 mm x 40 mm x 10 mm (L x B x H). Groove: 2.5 mm depth and 2.5 mm width; radius 1.25 mm</td></tr><tr><td>Micropipettes + tips</td><td>Finnpipette</td><td></td><td></td></tr><tr><td>Photometer</td><td>Molecular Devices Corporation, CA, USA</td><td>SpectraMax 340 photometer</td><td></td></tr><tr><td>Prism Software</td><td>GraphPad, San Diego, CA, USA</td><td>Version 9.0.1</td><td></td></tr><tr><td>Saline 0.9% NaCl</td><td>Fresenius Kabi, Sweden</td><td>883264</td><td></td></tr><tr><td>Special tail marker block for TVT tail cut</td><td></td><td></td><td></td></tr><tr><td>Tail holder</td><td></td><td></td><td></td></tr><tr><td>Vacuum liquid suction</td><td>Vacusafe comfort, IBS</td><td></td><td></td></tr><tr><td>Waterbath and thermostat</td><td>TYP 3/8 Julabo</td><td></td><td></td></tr></tbody></table>

tail vein transection bleeding model in fully anesthetized hemophilia a mice

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) survival model has been preferred in many settings due to sensitivity to clinically relevant doses of FVIII, whereas other established models, such as the tail clip model, require higher levels of procoagulant compounds. To avoid using survival as an endpoint, we developed a TVT model establishing blood loss and bleeding time as endpoints and full anesthesia during the entire experiment. Briefly, anesthetized mice are positioned with the tail submerged in temperate saline (37&#176;C) and dosed with the test compound in the right lateral tail vein. After 5 min, the left lateral tail vein is transected using a template guide, the tail is returned to the saline, and all bleeding episodes are monitored and recorded for 40 min while collecting the blood. If no bleeding occurs at 10 min, 20 min, or 30 min post-injury, the clot is challenged gently by wiping the cut twice with a wet gauze swab. After 40 min, blood loss is quantified by the amount of hemoglobin bled into the saline. This fast and relatively simple procedure results in consistent and reproducible bleeds. Compared to the TVT survival model, it uses a more humane procedure without compromising sensitivity to pharmacological intervention. Furthermore, it is possible to use both genders, reducing the total number of animals that need to be bred, in adherence with the principles of 3R&#39;s. A potential limitation in bleeding models is the stochastic nature of hemostasis, which can reduce the reproducibility of the model. To counter this, manual clot disruption ensures that the clot is challenged during monitoring, preventing primary (platelet) hemostasis from stopping bleeding. This addition to the catalog of bleeding injury models provides an option to characterize procoagulant effects in a standardized and humane manner.

To assess the applicability of the optimized model, a study was performed in F8-KO (C57BL genetic background) mice administered with a commercially available recombinant factor VIII replacement therapy (rFVIII); four different doses were tested: 1 IU/kg, 5 IU/kg, 10 IU/kg, and 20 IU/kg. Furthermore, we tested the corresponding vehicle (negative) control in F8-KO mice and wild-type (WT) group using C57BL mice as a positive control group to assess the response range in the model.
Following the o...

Watch this Scientific Journal Video about Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice at JoVE.com

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

The refined tail vein transection (TVT) bleeding model in anesthetized mice is a sensitive in vivo method for the assessment of hemophilic bleeding. This optimized TVT bleeding model uses blood loss and bleeding time as endpoints, refining other models and avoiding death as an endpoint.

Tail bleeding models are important tools in hemophilia research, specifically for the assessment of procoagulant effects. The tail vein transection (TVT) ...

tail-vein-transection-bleeding-model-fully-anesthetized-hemophilia

Research

JoVE Journal

Biology

6.0K Views.  Novo Nordisk A/S. Staartbloedingsmodellen kunnen belangrijke hulpmiddelen zijn om farmacologische effecten van hemostatische verbindingen in vivo te beoordelen om de reproduceerbaarheid tussen experimenten te garanderen. In vergelijking met andere gevoelige bloedingsmodellen gebruikt ons model bloedverlies en bloedingstijd als eindpunten in plaats van overleving. Ook wordt het model uitgevoerd onder anesthesie, waardoor pijn en angst voor de dieren worden verminderd.Deze techniek is vooral nuttig voor d...

Video: Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

Autologous Blood Injection to Model Spontaneous Intracerebral Hemorrhage in Mice

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model. While ICH
accounts for 10-15% of all strokes, there remains no specific
effective therapy. The autologous blood injection model in mice
involves the stereotaxic injection of arterial blood into the basal
ganglia mimicking a spontaneous hypertensive hemorrhage in man. The
response to hemorrhage can then be studied in vivo and the
neurobehavioral deficits quantified, allowing for description of the
ensuing pathology and the testing of potential therapeutic agents. The
procedure described in this protocol uses the double injection
technique to minimize risk of blood reflux up the needle track, no
anticoagulants in the pumping system, and eliminates all dead space
and expandable tubing in the system.

The autologous blood injection model of intracerebral hemorrhage in mice described in this protocol uses the double injection technique to minimize risk of blood reflux up the needle track, no anticoagulants in the pumping system, and eliminates all dead space and expandable tubing in the system.

Investigation of the pathophysiology of injury after intracerebral
hemorrhage (ICH) requires a reproducible animal model.  While ICH
accounts for 10-15% ...

Neuroscience

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase 

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related hospitalizations and for which no effective treatments are available to date1,2. Because of the heterogeneity of ICH in humans, various preclinical models are needed to thoroughly explore prospective therapeutic strategies3. Experimental ICH is commonly induced in rodents by intraparenchymal injection of either autologous blood or bacterial collagenase4. The appropriate model is selected based on the pathophysiology of hemorrhage induction and injury progression. The blood injection model mimics a rapidly progressing hemorrhage. Alternatively, bacterial collagenase enzymatically disrupts the basal lamina of brain capillaries, causing an active bleed that generally evolves over several hours5. Resultant perihematomal edema and neurofunctional deficits can be quantified from both models. In this study, we described and evaluated a modified double injection model of autologous whole blood6 as well as an ICH injection model of bacterial collagenase7, both of which target the basal ganglia (corpus striatum) of male CD-1 mice. We assessed neurofunctional deficits and brain edema at 24 and 72 hr after ICH induction. Intrastriatal injection of autologous blood (30 &mu;l) or bacterial collagenase (0.075U) caused reproducible neurofunctional deficits in mice and significantly increased brain edema at 24 and 72 hr after surgery (p&lt;0.05). In conclusion, both models yield consistent hemorrhagic infarcts and represent basic methods for preclinical ICH research.

Modeling Intracerebral Hemorrhage in Mice: Injection of Autologous Blood or Bacterial Collagenase

Clinically relevant animal models of intracerebral hemorrhage (ICH) are needed to extend our knowledge of hemorrhagic stroke and to examine novel therapeutic strategies. In this study, we describe and evaluate two ICH models that implement unilateral injections of either autologous whole blood or bacterial collagenase into the basal ganglia (corpus striatum) of mice.

Spontaneous intracerebral hemorrhage (ICH) defines a potentially life-threatening neurological malady that accounts for 10-15% of all stroke-related ...

Medicine

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Optimized Venous Thrombosis Assay in a Mouse Cancer Model Using Vascular Clips

Venous Thrombosis Assay in a Mouse Model of Cancer

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

Author Spotlight: Advancing Cancer Associated Thrombosis Research in Rodent Models 

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis ...

Cancer Research

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.

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, ...

Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via a liver laceration that results in consistent blood loss, hemodynamic alterations, and survival.
Mice undergo a standardized resection of the left-middle lobe of the liver. They are allowed to bleed without mechanical intervention. Hemostatic agents can be administered as pre-treatment or rescue therapy depending on the interest of the investigator. During the time of hemorrhage, real-time hemodynamic monitoring via a left femoral arterial line is performed. Mice are then sacrificed, blood loss is quantified, blood is collected for further analysis, and organs are harvested for analysis of injury. Experimental design is described to allow for simultaneous testing of multiple animals.
Liver hemorrhage as a model of uncontrolled hemorrhage exists in the literature, primarily in rat and porcine models. Some of these models utilize hemodynamic monitoring or quantify blood loss but lack consistency. The present model incorporates quantification of blood loss, real-time hemodynamic monitoring in a murine model that offers the advantage of using transgenic lines and a high-throughput mechanism to further investigate the pathophysiologic mechanisms in uncontrolled hemorrhage.

Uncontrolled hemorrhage, an important cause of mortality among trauma patients, can be modeled using a standard liver laceration in a murine model. This model results in consistent blood loss, survival, and allows for testing hemostatic agents. This article provides the step-by-step process to perform this valuable model.

Uncontrolled hemorrhage is an important cause of preventable deaths among trauma patients. We have developed a murine model of uncontrolled hemorrhage via ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Stab-Wound Mouse Model for Studying Hemorrhage and Inflammation in Traumatic Brain Injury

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including concussions, brain contusions, hematomas, and skull fractures. To replicate these different causes, various TBI mouse models have been developed using distinct protocols. Physical brain injury leads to both primary and secondary brain injuries, which exacerbate neuronal loss. Primary injury occurs immediately after the damage, often due to hemorrhage, and subsequently triggers secondary injuries, including inflammation around the lesion. Developing a TBI model suitable for assessing hemorrhage extension and inflammatory severity is therefore crucial. This protocol introduces a method for mimicking penetrating brain injury, referred to as the stab-wound TBI mouse model, to study mechanisms of hemorrhage, inflammation, and neuronal loss associated with TBI pathology. This model is created by puncturing the skull and brain with needles and is simple to execute without the need for specialized experimental equipment. Additionally, the minor injury inflicted on the mouse cerebral cortex using a needle does not affect the animal's behavior post-surgery. This feature allows researchers to study the localized effects of brain injury without concerns about broader behavioral consequences. Sample data from stab-wounded mouse cerebral cortices demonstrate the model's effectiveness in assessing blood leakage into the parenchyma, glial activation, and inflammatory cytokine production. Furthermore, this protocol facilitates the evaluation of blood coagulants and anti-inflammatory compounds, aiding in the development of therapeutic agents for TBI.

Simplified traumatic brain injury (TBI) models have facilitated the development of therapeutic approaches. This protocol outlines the creation of a stab-wound mouse cortex using needles, enabling the analysis of hemorrhage and inflammation. The stab-wound TBI mouse model offers the advantage of being performed without requiring specialized equipment.

Traumatic brain injury (TBI) results from physical damage, often caused by accidents or sports-related incidents. The causes of TBI are diverse, including ...

Swine Axillary Artery Injury Model for Testing Pre-Hospital Hemostatic Products

A New Hybrid Quantitative Evaluation Model for Axillary Junctional Hemorrhage in Swine

In this study, we developed and validated a hybrid quantitative model for simulating upper extremity junctional hemorrhage in swine, aiming to advance the development of pre-hospital hemostatic products. Utilizing 12 healthy 8-month-old male Yorkshire swine, we demonstrated the feasibility of a swine axillary artery injury model for evaluating hemostatic efficacy. Animals were divided into three groups to undergo volume-controlled hemorrhage (VCH), mimicking Class I-III hemorrhagic shock by withdrawing blood at different rates. Subsequent external compression was applied using a novel device consisting of a mechanical arm and an inflatable hemostatic balloon, achieving controlled pressure to enhance clot formation. Hemodynamic parameters, including heart rate and blood pressure, were continuously monitored, highlighting the impact of controlled hemorrhage and external compression on physiological responses. The findings suggest that the combination of VCH with targeted external compression effectively simulates clinical scenarios of axillary artery injury, providing a valuable model for testing hemostatic interventions in a controlled, standardized manner. This study underscores the potential of the model in facilitating the development and evaluation of new hemostatic agents and devices for managing junctional hemorrhages.

This study presents a hybrid quantitative model for axillary junctional hemorrhage in swine, enhancing pre-hospital hemostatic intervention evaluation.

In this study, we developed and validated a hybrid quantitative model for simulating upper extremity junctional hemorrhage in swine, aiming to advance the ...

Blood Withdrawal II

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
The collection of blood from mice and rats for analysis can be done through a variety of methods. Each method of collection has variations in the type of restraint required, the invasiveness of the procedure, and the necessity of a general anesthetic.1Historically, the use of the retro-orbital sinus cavity has been used, but not without debate. The controversy related to the potential tissue damage,or even blindness,caused by retro-orbital bleeds has led to the development of facial and submandibular vein bleeding methods in mice.Blood collection from the saphenous vein in both mice and rats is another technique that has been developed. These procedures do not require anesthesia and therefore are suitable when the use of anesthetics may confound blood results or other data.

Collecting Blood from Facial, Submandibular, Saphenous &amp; Femoral Veins in Rodents

Source: Kay Stewart, RVT, RLATG, CMAR; Valerie A. Schroeder, RVT, RLATG. University of Notre Dame, IN
The collection of blood from mice and rats for ...

Tail Vein Transection Bleeding Model in Fully Anesthetized Hemophilia A Mice

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