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

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.