Preclinical Models of Traumatic, Hemorrhagic Shock

Trauma is the leading cause of death between the ages 1 and 45. Between birth and age 36, trauma exceeds all other causes of death combined. Most “survivable” patients die when tissue trauma and shock act in concert to trigger inflammation that leads to organ failure or sepsis. Thus, clinical inflammation after injury is a major public health problem, and therapeutic advances in this field will depend upon animal models that predict the clinical effects of interventions accurately. Poor preclinical modeling will lead to therapeutic failure. Modeling traumatic, hemorrhagic shock (T/HS) in animal systems requires a precise understanding of clinical trauma and shock syndromes, as well as the mechanisms that transduce trauma and tissue ischemia into physiologic responses to injury, and of the animal models that can reconstitute these clinical responses accurately. The majority of clinical trauma results from blunt force injuries that result in extensive energy transfer to the patient. The resultant tissue injuries are accompanied to some degree by hemorrhage that can be rapid and exsanguinating, but more commonly, it is moderate in rate. Because tissue hemorrhage tends to be diffuse, however, it can be difficult to treat. Thus, shock from hemorrhage from blunt trauma tends to develop gradually, but tissue hypoperfusion and acidosis can persist for significant periods. Penetrating injury is more likely to cause direct injury to major blood vessels and rapid hemorrhagic shock. Conversely, penetrating injuries are seldom associated with major energy transfer and tissue destruction, but the operative procedures required to control bleeding do constitute a major tissue injury. Whereas tissue trauma is a mechanical event and hemorrhagic shock is a physiologic syndrome, the clinical entities that determine prognosis in patients who survive their initial insults are immune and inflammatory. This combination of mechanical tissue injury, hemorrhage, shock, ischemia, resuscitation, reperfusion, and later, sepsis, all interact to activate systemic immunity. Local tissue trauma and tissue necrosis are widely believed to unmask “pattern recognition” neo-antigens that lead to wound invasion by phagocytes that increase subsequent local mediator production. These events create an inflammatory wound environment that is a potent stimulus for systemic innate immune activation. The coagulation cascade is locally activated by tissue injury with hemorrhage and coagulation at injury sites. Activated platelets may represent an especially potent source of inflammatory lipids. Complement activation is mediated by soft-tissue trauma, burns, and local ischemia/reperfusion (I/R), and is an important determinant of later organ injury. Global I/R is also a key initiator of systemic inflammation. The mechanisms by which I/R leads to inflammation are complex. Gut ischemia may be important in initiating inflammation because the splanchnic circulation is preferentially hypoperfused during shock. Thus, the etiology, degree, duration, and location of tissue ischemia in shock models are all crucial. We are only now beginning to understand the cell-signaling mechanisms that transduce tissue injuries and shock syndromes into immune dysfunction syndromes. Effective preclinical models of human hemorrhagic shock and resuscitation must take into account many complex variables to create biologic systems that model the events and end points of interest in the most biologically relevant way possible. Successful models of T/HS require a detailed appreciation of all facets-mechanical, physiologic, cell biologic, and immunologic-of these complex relationships. Moreover, inflammatory responses to T/HS vary over time and models must take into account that the early and late responses tend to diverge. Response to t/HS can also be dichotomous, with decreased organ injury predicting increased susceptibility to sepsis. Thus, the appropriate time to study different end points of interest may vary substantially. CONSTRUCTING T/HS MODELS Trauma (energy transfer and tissue injury) Clinically relevant T/HS models include some component of tissue damage. Trauma contributes to the T/HS syndrome via wound creation. Wounds are extraordinarily complex environments with variable effects upon the organism over time. Initially, wounds contain multiple active agents such as complement fragments, necrotic cell debris, red blood cells, platelets, and their breakdown products. Later, they may become hyperosmolar, and become sites of leukocyte activation and mediator synthesis. Such factors can act as “danger” signals and activate innate immunity. The degree of wounding is a critical element in the creation of T/HS preparations that is often underappreciated as a source of experimental variability. Moreover, the degree of wounding in blunt trauma T/HS models may be much more difficult to control than the size of an abdominal incision. The experimental wounds typically used to model blunt trauma include fractures, soft-tissue trauma, injuries to the abdominal viscera, and direct lung injuries (i.e., pulmonary contusion). Although most penetrating injuries (outside of military wounds) do not result in significant tissue trauma per se, they commonly lead to operations (laparotomy or thoracotomy) to control hemorrhage or treat hollow viscous injuries. Thus, experimental operative trauma can be a valid recreation of clinical events.Table 1: Available protocols for modeling relevant injuries in test animalsHemorrhage and tissue I/R Hemorrhage acts globally by decreasing the circulating blood volume, thus diminishing cardiac output and tissue oxygen delivery (DO2). Decreased perfusion and oxygenation lead to rapid decreases in cellular oxygen consumption (VO2), ATP and “energy charge.” Such effects may be global or localized to tissues that normally have low PO2 environments such as the bone marrow or the hepatic parenchyma, or that are particularly susceptible to redistribution of perfusion such as the gut. Later, secondary ischemic injuries are typically incurred via I/R. The activation of I/R can cause direct oxidant injury or the activation of immunity via oxidant signaling. I/R need not be global to elicit systemic inflammation. In many models, the effects of I/R are mediated by the tissue (or tissues) that are most affected by shock and/or most capable of mounting an inflammatory response. The well-known preferential preservation of blood flow to the heart and brain in shock causes gut ischemia, activating enterocytes and leading to the production of inflammatory mesenteric lymph. Thus, gut hypoperfusion in systemic shock states can cause a systemic inflammatory response syndrome. This can be mimicked by shock limited exclusively to the gut such as superior mesenteric artery occlusion models. I/R acts synergistically with tissue trauma to induce the T/HS insult. But like tissue trauma, the effects of HS are highly variable and are dependent on multiple factors. The degree and distribution of tissue ischemia varies markedly with the rapidity with which shock is induced, the use of fixed-pressure versus fixed-volume loss shock models, open artery (low systemic vascular resistance) versus closed vessel (hypovolemic) hypotension models, and the duration of the shock episode. Resuscitation The amount, type, and timing of resuscitation are also key parameters that must be controlled to achieve reproducible, clinically relevant T/HS shock models. Resuscitation choices will alter the rate of vascular refilling, the degree of hemodilution, viscosity changes, and flow distribution, all of which will affect the distribution and degree of tissue ischemia and I/R, and thus the inflammatory response to T/HS. In addition, the type of fluid used for resuscitation (for instance, lactated Ringer's solutions versus albumin) will affect the inflammatory response to T/HS. Other conditions Numerous other general conditions must play a role in the construction of T/HS models. Animals need to be conditioned to a reproducible preshock state and excessive stress must be avoided. Body temperature must be controlled within close limits because inflammatory responses are highly temperature dependent. The specific anesthetic used may also be of importance: opioids and ketamine have been noted to have anti-inflammatory properties. Systemic anticoagulation with heparin has also been used in shock preparations to suppress thrombosis and the loss of vascular catheters. Heparins are now known to act as potent anti-inflammatory drugs. Platelets are important sources of inflammatory phospholipids and their functional suppression is likely to result in profound changes in the formation of these important signaling molecules at sites of mechanical trauma. Conclusions T/HS models are highly relevant to clinical shock in the setting of injury, but the experimenter needs to choose and calibrate the model that is most appropriate for the specific clinical scenario being modeled. Blunt trauma is associated with far more tissue injury than penetrating trauma is, but operative trauma will be more common in penetrating injuries. Blunt trauma bleeding is commonly moderate in rate, and is diffuse and persistent. Although shock develops gradually with blunt trauma, and is uncommonly rapidly exsanguinating, there is a prolonged period of hypoperfusion and acidosis. In penetrating injury, major blood vessels are more frequently injured, leading to more rapid exsanguination but less tissue trauma. Experimental systems that model the degree of mechanical injury accurately, replicate related shock physiology and immunobiology at time points of interest, and thus model T/HS in most exact and biologically relevant way possible are most likely to lead to successful interventions.Table 2: Parameters for Inducing Hemorrhagic Shock in Animal ModelsIt should be recognized in this regard that existing models concentrate on acute responses to trauma and shock. Long-term outcome studies in animal models are uncommon. They create enormous ethical problems and moreover may require the use of long-term intensive care to mimic human responses to treatment. Thus, they are usually extraordinarily expensive. This may limit their use to late-stage studies, done immediately before human studies. In any case, there is insufficient published experience concerning chronic T/HS shock models to comment upon here. Last, specific studies will often be based upon similarities of animal and human immunology or physiology and will ignore the underlying differences, which may be very significant. Thus, investigators need to interpret the experimental data derived from even the most cogent animal models with an eye to the similarities of the specific systems studied and to the possible differences between the overall biology of the animal model and man. MODELS OF T/HS Small animal models The mouse- The use of mice to model T/HS has many inherent advantages. These include the size, cost, ease of care, and availability of mice. Two important advantages of mice are that they can be genetically modified relatively easily and that a wide variety of reagents is available for the performance of immunologic assays. Thus, mouse models are especially useful for studies of molecular mechanisms and for the performance of proof of concept studies. Conversely, the small size of the mouse leads to difficulty in operative techniques and in creating reproducible hemodynamic preparations, although recently developed micropressure-volume catheters now allow quite detailed hemodynamic studies in mice. In addition, the mouse is genetically distant from humans. Thus, preclinical studies often require follow-up in before clinical studies. The most common of tissue trauma in mouse T/HS models is the In investigators have used a closed in with many other of tissue trauma are also have a mouse Although are general models of blunt trauma, ethical animal use may be close is to chronic pulmonary and blunt trauma models have also been and the of modeling pulmonary dysfunction in the setting of trauma and shock The major of such models are that they may require and experience to the force to the and that the resultant pulmonary the effects of systemic T/HS and direct pulmonary injury. has a for a controlled injury in mice. This has not been as a component of a T/HS Last, has a hepatic injury model that a to an ischemic injury to the of the of requires but may be an appropriate complement to systemic hemorrhage to model injury and shock in man. wide variety of hemorrhage models are available in mice. the most commonly used model is a model the animal is at a of for to and is hemorrhage models used include the of to of the blood in the mouse has been at or for a mouse has used a model of blood per of the effects of different hemorrhage preparations in the mouse is available important of the use of complex T/HS models is the modeling of after T/HS. Because is the most common late of T/HS and early systemic inflammatory response syndrome, this is a for and has been used as the component of such complex models is however, that other not be used to complement mouse models of T/HS. 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