Name: uncontrolled hemorrhagic shock modeled via liver laceration in mice with real time hemodynamic monitoring
Uploaded: 2017-05-21T13:00:00
Description: Watch this Scientific Journal Video about Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring at JoVE.com

The work of this manuscript was supported by funding to Dr. Neal by the Vascular Medicine Institute Pilot Project Program in Hemostasis and Vascular Biology (P3HVB) and the AAST Research Fellowship. This work is supported by U.S. National Institutes of Health grants 1 R35 GM119526-01 and UM1HL120877-01.

Mice were housed in accordance with University of Pittsburgh (Pittsburgh, PA, USA) and National Institutes of Health (NIH; Bethesda, MD, USA) animal care guidelines in specific pathogen-free conditions with 12 h light-dark cycles and free access to standard feed and water. All animal experiments were approved and conducted in accordance with the guidelines set forth by the Animal Research and Care Committee at the University of Pittsburgh.
1. Surgical Field and Instrument Setup
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The murine liver laceration model described here provides a reliable, consistent model of uncontrolled hemorrhage. This model is straightforward to perform but there are important steps that require meticulous consideration. The most technically challenging part of the model is cannulation of the femoral vessels for hemodynamic monitoring and fluid/drug administration. Care must be taken during the dissection of the nerve and the arteriotomy/venotomy. It is important to not touch the nerve during the dissection of the ve...

Trauma is the leading cause of death and disability among young people worldwide.1 Uncontrolled hemorrhage remains a leading cause of mortality among severely injured trauma patients.2 Management of the hemorrhaging trauma patient is two-fold: control of surgical bleeding, and resuscitation and replacement of lost blood.
Animal models of hemorrhagic shock have been the cornerstone in trauma research and can be used in the evaluation of the pathophysiology and treatment of traumatic/hemorrhagic shock.3,4 Shock in animal models can be achieved broadly by two methods: controlled-hemorrhage and uncontrolled-hemorrhage.5,6 Controlled-hemorrhage is performed by removal of a fixed volume of blood or by blood removal to achieve a certain blood pressure (fixed-pressure). While these models are useful in the evaluation in the mechanisms and immune alterations in hemorrhagic shock, they are not applicable to the testing of hemostatic agents and do not mimic the clinical scenario of hemorrhage following trauma. To this degree, we sought to develop a model of uncontrolled hemorrhage that would allow us to test hemostatic changes and pro-coagulant agents in a murine model. The liver is an attractive option for uncontrolled hemorrhage in part because of the dual blood supply to the liver and it is one of the most commonly injured intrabdominal organs in both blunt and penetrating trauma. Given the high clinical relevance, the liver has been utilized as a model of uncontrolled hemorrhage, most commonly in rat and porcine models but recently in primates as well.7,8,9,10,11,12 Murine models have also incorporated liver injury, such as a crush model or blunt trauma; however, these models do not result in hemorrhagic shock secondary to the liver injury.13,14
The rat and porcine models of uncontrolled liver hemorrhage, while valuable in looking at resuscitation practices and hemodynamic monitoring, are less advantageous than a murine model for various reasons such as cost, number of animals utilized, and importantly the relative lack transgenic lines available for analysis of specific cellular and molecular signaling. The present murine model shares important similarities to existing liver hemorrhage models including standardized liver laceration, blood loss quantification, hemodynamic monitoring, and the ability to perform survival analysis. Many existing models only incorporate some of these aspects whereas our model was developed to measure many of the physiologic variables simultaneously and in multiple mice. As well, development of a murine model opens the door to investigations beyond resuscitation and into greater pathophysiology mechanisms in uncontrolled hemorrhage with the potential of a cost efficient, high-throughput model using advanced molecular techniques.

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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.

The liver laceration model results in reproducible and consistent blood loss in mice. Figure 1A demonstrates the consistent weight of lacerated liver that can be obtained with a standard deviation of only 0.02 g. This consistency in lacerated liver weight allows the ability to reproduce the model between mice and in different experimental set-ups such as different resuscitative protocols. As well, the reproducible weight of the lacerated liver, with a standard error of on...

Watch this Scientific Journal Video about Uncontrolled Hemorrhagic Shock Modeled via Liver Laceration in Mice with Real Time Hemodynamic Monitoring at JoVE.com

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

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 ...

The overall goal of this murine liver laceration model is to induce uncontrolled hemorrhage for the study of resuscitation practices and the pathophysiologic mechanisms of uncontrolled hemorrhage. This method can help answer key questions in the hemorrhagic shock field about resuscitation practices, new hemostatic drugs, and the molecular mechanisms that follow uncontrolled hemorrhage. The main advantage of this technique are that it is reproducible, high throughput, and takes advantage of available transgenic mouse strains.Demonstrating the procedure will be Shannon Haldeman, a technician from the Neal Laboratory. Before beginning the procedure, place a sterile drape on top of a blue surgical pad over a 37 degree Celsius water-circulating heat pad. Secure the anesthetized mouse onto a surgical board with tape, and use a razor to shave the abdomen and bilateral groins.Using sterile gauze, disinfect the exposed skin with Betadine and insert a rectal temperature probe. Place the mouse under a dissecting microscope and make a four to five millimeter longitudinal incision over the groin muscle. Using Dumont forceps, grab the adipose tissue connected to the adductor muscle and pull the muscle laterally to cleanly expose the femoral bundle.Use the forceps to carefully dissect away from the artery and vein until the fat pad adjacent to the nerve is observed. Pull the pad laterally with the forceps to move the nerve away from the artery and use a second pair of Dumont forceps to bluntly dissect the connective tissue between the nerve and the artery. Next, loop one loose 6-0 silk suture around both vessels proximal to the profunda femoris takeoff, placing a second suture distal to the first.After immediately tying the second suture, place a third loose suture between the first two sutures and use microvascular scissors to make an arteriotomy on the ventral surface of the vessel. Insert a three-way catheter into the artery and tie the proximal and middle suture to secure the catheter. Connect the three-way catheter to a sterile transducer and collect the baseline blood pressure data.To lacerate the liver, first make a ventral midline laparotomy incision starting at the xiphoid process and extending caudally to allow complete exposure of the liver. Then insert one pre weighed absorption triangle against each of the right and left sides of the abdominal wall, taking care that the triangles are not against the liver to avoid liver hemostasis. Next, carefully grab the left middle lobe of the liver and use surgical Iris scissors to lacerate 75%of the tissue.Place the lacerated segment into a pre weighed tube containing 0.5 milliliters of PBS, and immediately use a staple applicator to close the skin and muscle together. Then, after the appropriate time of interest for hemorrhage, remove the staples and the absorption triangles. Use the third triangle to collect excess blood in the peritoneal cavity.Then weigh all three of the absorption filters to calculate the total blood loss. The liver laceration model results in reproducible and consistent blood loss in mice with a standard deviation of only 0.02 grams between animals, allowing the reproducible results to be obtained between mice and between different experimental setups. Pretreatment with heparin as a positive control for blood loss, an antifibrinolytic as a negative control, or a validated prohemostatic nanoparticle previously tested in murine tail vein bleeding assays demonstrates the ability of this model to be used to assess the hemostatic or anticoagulant effects on a hemorrhage setting.Uncontrolled hemorrhage is often accompanied by hemodynamic derangements that are important to monitor. For example, the mean arterial blood pressure of individual mice following liver laceration demonstrates precipitous and reproducible drops in pressure after laceration, resulting in a hemorrhagic shock state and allowing the hemodynamic effects of different resuscitative or interventional measures to be assessed. Although there are significant hemodynamic effects following liver laceration, the model can be used to evaluate survival effects out to 72 hours with a 56%survival rate at three days post laceration.Once mastered, this technique can be completed in 30 minutes if it is properly performed. This procedure readily combines with other models including soft tissue injury or polytrauma, and is easily adapted for testing topical hemostatic agents. While attempting this procedure, it's important to remember to avoid nerve damage during the vascular dissections and to avoid packing hemostasis when placing the filter triangles, and to quickly close the abdomen following the liver laceration.This technique provides a highly reproducible, high throughput uncontrolled hemorrhage model for the investigation of resuscitation practices similar to large animal models of uncontrolled hemorrhage, while allowing greater mechanistic investigations of the underlying pathophysiology following hemorrhagic shock. After watching this video, you should have a good understanding on how to cannulate the femoral vessels for hemodynamic monitoring and drug administration, and to reproducibly perform a liver laceration for the induction of hemorrhagic shock.

Research

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