PRECLINICAL MODELS OF TRAUMATIC, HEMORRHAGIC SHOCK
2005; Lippincott Williams & Wilkins; Volume: 24; Issue: Supplement 1 Linguagem: Inglês
10.1097/01.shk.0000191387.18818.43
ISSN1540-0514
Autores Tópico(s)Traumatic Brain Injury and Neurovascular Disturbances
ResumoTrauma 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 larger animals before clinical studies. The most common form of tissue trauma in mouse T/HS models is the laparotomy. In general, investigators have used a 2.5-cm midline incision closed in two layers with running 3-0 silk. However, many other forms of tissue trauma are also used. Wichmann et al. (1) have described a mouse femur fracture model. Although fractures are excellent general models of blunt trauma, ethical animal use concerns may be expected unless close attention is paid to chronic pain management. Mouse pulmonary contusion and blunt chest trauma models have also been described and offer the advantage of accurately modeling pulmonary dysfunction in the setting of chest trauma and shock (2, 3). The major drawbacks of such models are that they may require special instrumentation and experience to accurately deliver the needed external force to the chest wall, and that the resultant pulmonary lesion represents the combined effects of systemic T/HS and direct pulmonary injury. Chang (4) has described a technique for delivering a controlled head injury (cortical impact) in mice. This has not been reported as a component of a T/HS model. Last, Eckhoff (5) has described a hepatic injury model that uses a clamp to deliver an ischemic injury to the left lobe of the liver. This, of course, requires laparotomy, but may be an appropriate complement to systemic hemorrhage to model intra-abdominal injury and shock in man. A wide variety of hemorrhage models are available in mice. Probably the most commonly used model is a fixed pressure model where the animal is held at a mean arterial pressure (MAP) of 35 ± 5 mm Hg for 60 to 90 min and is then resuscitated (6). Fixed volume hemorrhage models used include the removal of 35% to 40% of the estimated blood volume (2, 7). Blood volume in the adult mouse has been assessed at 72 (range 63-80) mL/kg or about 2.2 mL for a 30 g mouse (8). Claridge et al. (9) has used a fixed volume model removing 0.025 mL of blood per gram of body weight. An excellent review comparing the effects of different hemorrhage preparations in the mouse is available (10). An important extension of the use of complex T/HS models is the modeling of sepsis after T/HS. Because sepsis is the most common late complication of T/HS and early systemic inflammatory response syndrome, this is a crucial issue for investigation. Cecal ligation and puncture has been used almost universally as the infective component of such complex models (6, 11, 12). There is no reason, however, that other infections could not be used to complement mouse models of T/HS. Because pneumonia is the most common infection after major injury, this might be especially appropriate. The rat- Experimentation using rat models has many advantages, including size, cost, ease of care, availability, and ethical acceptability. Some immunologic assay reagents are available, although fewer than are available for the mouse. Shock models are technically easier in rats than in mice. Moreover, some immune responses to T/HS in the rat may parallel the human. Nonetheless, rats are still genetically distant from humans. Also, small animal cardiovascular responses may not parallel those in larger animals and in general, hemodynamic studies are technically more challenging in small animals like the rat than in large-animal systems. As in the mouse, the midline laparotomy is by far the most common tissue trauma preparation in rats. Most authors report a 2.5- to 3-cm incision closed in layers. Many more models are available in the rat for inducing tissue trauma by long-bone fractures. Methods exist for producing reproducible fractures of the femur (13), tibia (14), and the heel (15). Guan et al. (16) produced fractures at multiple sites. This may be a useful means of creating an experimental grading system for the severity of soft tissue trauma in T/HS. Other models that can be applied to the study of T/HS include the addition of a lung blast injury (17), the creation of a closed head injury by use of a fluid wave (18), and the creation of a bleeding splenic injury by splenic crush (19). It should be noted that although clinically relevant and intuitively appealing, the use of massive splenic injuries to model hemorrhage will also decrease the extent to which the shock insult applied to the animals can be standardized. As with the mouse, the most popular rat models of T/HS incorporate a fixed-pressure hemorrhage wherein the rat is maintained at an MAP of 30 to 45 mm Hg for 30 to 90 min, followed by resuscitation (13, 20-23). Some have based the depth of hemorrhagic shock induced on phlebotomy to 50% of pre-existing MAP for 60 min (24). Kretchmar (15) has proposed a fixed-volume model that involves fracturing a heel bone and then aiming for removal of one-third of the rat's blood volume for 15 to 120 min. Measurements of the rat's blood volume have averaged 64 mL/kg (range 58-70) (8). The rat has also been a popular model for comparisons of various resuscitation fluids in shock. Most, if not all, resuscitation fluids will have some immune effect. The regimen with the least effect in the rat is probably reinfusion of shed blood only at body temperature without further bolus resuscitation. In addition to the "component" approach to creating T/HS models, a number of well-defined "off the shelf" T/HS models have been described. Perhaps the most widely used is the combination of laparotomy, fixed-pressure hemorrhage to MAP 35 ± 5 mm Hg for 90 min, and then resuscitation with shed blood only (21, 22, 25, 26). Femur fracture has also been combined with fixed-pressure hemorrhage to a MAP of 45 ± 5 mm Hg for 60 min (23). Pasquale (14) has described using a bilateral femur fracture followed by a laparotomy with aspiration of 2 mL of blood from the vena cava (14). The combination of rat T/HS plus cecal ligation and puncture was described by Jarrar et al. (27) where MAP was decreased to 40 mmHg until 40% of the maximal shed blood volume was returned in the form of Ringer's lactate with cecal ligation and puncture then performed subsequently. This preparation is essentially a variant on the Wiggers preparation (28). The rabbit- Like the rat, experimentation using rabbit models would have the advantages of larger size, cost, ease of care, and availability. The acceptability of rabbit experimentation is generally high, although perhaps not as universal as the mouse and rat. Some differences do exist in the requirements for caging that can increase expenses (29). Some immunologic assay reagents are now available, and some reagents developed for rodents may cross-react. Immune assays and bioassays for mediator species that are species independent are also available (30). Shock preparations in the rabbit are technically easier to perform than those done in the mouse, and more sophisticated hemodynamic studies can be performed in rabbits with greater ease and lower cost than is possible in smaller species. Femur fracture in the rabbit is a standard model for orthopedic purposes. Although the impact of shock on fracture healing has been studied, the impact of fracture trauma on the rabbit's response to hemorrhagic shock has not. There are also several rabbit models that use chest wall trauma (31-33). Standardized abdominal trauma models have also been described in the rabbit (34). Fixed-pressure models of shock reported include hemorrhage to MAP 30 to 40 mm Hg for 5 min (35) and 60 min (36), to 30 to 35 mm Hg for 2 h (37), or to 50% of baseline blood pressure (30). In systems using hemodynamic monitoring, HS has been created by phlebotomy to 40% of baseline cardiac output for 90 min (38) or to an MAP of 45 mmHg and mean cardiac output 30% of baseline for 1 h (39). The rabbit's blood volume has been estimated at 56 ml/kg (range 44-70) (8). A fixed-volume model of hemorrhagic shock was produced by Boura et al. (40) by withdrawal of blood at 1.5 mL/min for 60 min and maintaining that pressure for 15 min. The authors estimated that this volume corresponded to 50% of the blood volume. Fixed-volume hemorrhagic shock has also been induced by withdrawal of 25 mL/kg of blood for 60 min (41). It would seem that the rabbit could be used more commonly for complex studies modeling T/HS. Certainly, the advantages of rabbits over small rodents in terms of studying hemodynamic responses are considerable. Lu (42) studied the effects of combined shock and abdominal compartmental hypertension on rabbit PMN, and Koch (43) has studied the interactions of shock with hypoxia and "trauma." In the latter study, complement activation was used to replace mechanical trauma, and the protocol was used to assess organ specific bacterial clearance after subsequent Escherichia coli challenge. Nonetheless, rabbits may tolerate shock less well and recover less reliably than other small animals. Other small animals- The only other small animal used with any regularity for the study of shock has been the guinea pig. Fixed-volume hemorrhagic shock has been induced by controlled hemorrhage of 50% to 60% of the estimated blood volume (44, 45). The blood volume of the guinea pig has been measured at about 55 ml/kg (46). Fixed-pressure hemorrhage has also been induced by phlebotomy to an MAP of 30 mm Hg for 2 h. Thermistor catheters can be placed for assessments of cardiac output by thermodilution (47). The guinea pig has also proved useful for studies of respiratory mechanics (48), wound healing (49), and hemorrhagic shock. Well-described models of standardized tissue trauma exist in the guinea pig, including hind limb fracture (50), burn injury (51), smoke inhalation (52), and head trauma (53). Chest wall trauma models have not been described in the guinea pig. Large-animal models Canine models- Canine preparations are some of the most venerable and best-studied systems in shock physiology. Conditioned and inbred dogs are easily available. The shock preparations widely used are technically easy to perform. In addition, a number of highly relevant hemorrhage models have been developed in the dog that might be extraordinarily useful in other models. Unfortunately, canine hemodynamic responses to shock may not parallel human physiology. Consistent hemodynamic responses to phlebotomy require splenectomy, which may alter immune responses to injury and shock. In addition, dogs are genetically distant from humans. Ethical concerns may be very serious for chronic preparations. Little if any information is available concerning whether canine inflammatory responses parallel those in humans. Moreover, essentially no immunologic reagents are available. A variety of canine tissue trauma models were developed in the 1970s and 1980s that could be of use in shock research. Gelin and associates (54) created a hind limb crush model without shock that appeared to model secondary lung injury particularly well. Redl (55) created a similar model that uses bilateral femur fractures in conjunction with fixed-pressure hemorrhagic shock to an MAP of 50 mm Hg. This model appeared to be very useful for assessments of extravascular lung water and albumin flux after injury. Poole et al. (56) created a combined model of T/HS and brain injury to study the effects of fluid resuscitation on cerebral perfusion. The Wiggers hemorrhagic shock preparation and a wide variety of modifications have been used as standard shock research tools for more than half a century (28). These models are all variations on a constant-pressure shock model with the blood pressure maintained at 40 to 50 mm Hg and using the "reuptake" of shed blood over time to determine the depth of shock. The rate and amount of reuptake is highly variable in Wiggers preparations, but can be standardized some by previous splenectomy. Measurements of the blood volume in "conditioned beagles" have averaged 85 mL/kg (range 79-90) (8). In general, dogs bled approximately 40% of their blood volume over 30 min will reach an MAP of 50 mm Hg, which can then be maintained for at least 60 min (57). Constant-pressure models can be made more accurate by the addition of an aortic reservoir (58). A major advantage of canine model is the variety of relevant methods of recreating traumatic hemorrhage that have been developed. Some that may be of special interest include the retroperitoneal iliac artery injury models described by Rocha y Silva et al. (59, 60). These are superb clinical HS models, although they create little in the way of tissue damage. Such models accurately recreate the hemodynamics as well as the coagulopathy and inflammation associated with retroperitoneal bleeding and hematoma formation. They could easily be combined with other tissue trauma or used in other species to model fluid resuscitation and the inflammatory response to trauma. Another important issue in hemorrhagic shock research is the effect of rebleeding. Although we often think of "two-hit" inflammation as an episode of shock followed by an episode of sepsis, successive bleeding episodes may act similarly. The tandem hemorrhage model developed in Gann's laboratory is well suited to study this issue. Splenectomized animals are bled 10% of estimated blood volume with reinfusion of shed blood at 30 min. The hemorrhage is then repeated 5 h later (61). Obviously, the volume of hemorrhage can be tailored to the effects sought, and again, this protocol could be easily adapted to use in other species. Porcine models- Pigs are reasonably inexpensive for a large animal and are widely available. There are many well-established shock preparations, and porcine cardiovascular and hemodynamic responses often parallel human responses rather closely. Also, responses to and metabolism of drugs are often similar to those in humans. An increasing number of immune reagents are now available for porcine studies, and some studies can be performed with reagents developed for human molecular species. Another potentially important advantage of porcine shock studies is that wound healing is so similar in pig and human skin. There are also disadvantages of porcine shock studies. Ethical issues still arise, especially where the use of chronic injury preparations is considered. Pigs are also genetically distant from humans and, thus far, little information exists on extent to which porcine inflammatory responses to T/HS parallel human. Tissue trauma can be modeled in the pig by a wide variety of means. Laparotomy can be used with shock (62), and liver injury can be added to the laparotomy (63). Standard chest trauma can be created using a bolt gun (64). Standardized musculoskeletal trauma can be delivered by mechanical means (65), and isolated femur fractures can be created with captive-bolt guns (66) or gunshot wounds (67). Standard traumatic brain injuries are created in the pig using cortical fluid percussion (68). Blood volume of standard size swine has been measured at about 76 mL/kg (69). In mini-pigs, it has generally been measured at 65 mL/kg (range 61-68) (8). Volume-controlled hemorrhagic shock is generally produced by phlebotomy of 25-30 mL/kg (70, 71). Animals are kept in shock for 1 to 3 h, and are then resuscitated. Hess (72) has described a volume-controlled 25% "graded hemorrhage" model: 7 mL/kg is withdrawn in the first 15 min, with 5, 3.5, and 2 mL/kg withdrawn in three succeeding 15-min intervals (total 17.5 mL/kg). Fixed-pressure hemorrhage in the pig has generally been produced by rapid bleeding to lower the MAP to 25 to 45 mm Hg. This is usually maintained for 30 to 90 min, depending upon the depth of shock and lethality desired (73-77). Several combined models of porcine T/HS have been described. Perhaps the most widely useful is the combination of laparotomy with hemorrhage to an MAP of 30 mm Hg for 1 h followed by an MAP of 45 mm Hg for 3 h. This model was used to assess the effects of interventions on pulmonary neutrophil sequestration (62). Of interest, the plasma taken from those pigs was biologically active in human neutrophil systems. Soft tissue trauma has also been combined with hemorrhagic shock in pigs by use of a captive bolt gun to create bilateral femur fractures followed by pressure-controlled hemorrhage to an MAP of 25 mmHg for 40 min (66). Brundage (63) has created a model of Grade V liver injury with hemorrhagic shock and resuscitation and used it to assess cytokine responses to trauma. Voelckel also studied high-grade experimental liver injuries with estimated 40% blood volume losses and MAP as low as 20 mmHg. Liver injuries are clinically important and can model tissue trauma. However, it may be difficult to control the extent of hemorrhage accurately in models that include large bleeding liver injuries. Porcine chest trauma (bolt gun) has also been combined with 10 to 12 mL/kg hemorrhage (64). This model may be of great use in evaluating strategies to modify shock and acute lung injury. Malhotra (68) created a standard traumatic brain injury using cortical fluid percussion and followed this with a 45% blood volume hemorrhage. This system will be especially useful in studies of cerebral resuscitation in the setting of hemorrhagic shock. The lethal groin transaction model created by Alam et al. (78) may be useful for specialized studies such as studies of field hemostatic agents and resuscitation from rapid exsanguination. Last, Eissner (79) has created a "two-hit" model of hemorrhagic shock followed by sepsis where an initial 50% reduction of the MAP or the cardiac index for 45 min was sufficient to prime pigs for worse organ failure after subsequent bacteremia. Although this model lacks a tissue trauma component, one could easily be added. Also, Pseudomonas bacteremia is a somewhat unrealistic model for infection in the trauma patient, but this model might still be useful in generating preclinical data on responses to septic insults after injury. Ovine models- The sheep is highly available and relatively well-studied with respect to hemorrhagic shock. Experiments using ruminants have clear cost advantages over primate studies. Moreover, the use of sheep is often more ethically acceptable. Well-established shock preparations exist, and hemodynamic responses to shock seem to parallel human responses. Lung injury in response to shock and sepsis may also model human responses well. Methods for assessing the humoral and cellular contents of lymph at pulmonary, intestinal, and peripheral sites are particularly well described (80-82). There are several potential disadvantages with sheep. As with most large animals, potential ethical and animal use issues may exist, but chronic injury preparations are likely to be more acceptable in ruminants than nonhuman primates. Sheep are also genetically distant from humans and there is little information available yet on the extent to which sheep inflammatory responses parallel those of humans. Tissue trauma in the sheep is usually modeled using fractures, but these have typically been produced by osteotomies (83-85). This approach lacks any significant element of soft tissue trauma and is therefore of lesser clinical relevance than a true blunt-force injury. More realistic but still controlled bony "operative trauma" can be produced by reaming an intact femur (85). High-energy ballistic fractures can also be produced (86). A graded pulmonary contusion model can be produced by using compressed air-driven shock tubes and a reflector plate, as was shown by Dodd (87). Standardized traumatic brain injuries can be induced by impact using a stunner or by freezing (88, 89). Adult ovine blood volume is approximately 75 mL/kg (90). Fixed-pressure models of hemorrhagic shock have been described using hemorrhage to an MAP of 50 mm Hg for 1.5 to 2 h (84, 85). In adult sheep weighing 30-47 kg, this required the phlebotomy of 1400 to 1700 mL of blood (91). A fixed-volume hemorrhage model has also been reported by Anderson et al. (89), who used rapid hemorrhage to achieve an MAP of 40 mm Hg, and then withdrew additional blood to maintain that pressure to a total volume of hemorrhage of approximately 35% of estimated total blood volume (89). Aguilar (83) has reported a combined model of hind limb fracture, hemorrhage, resuscitation, and then fracture fixation. This highly clinical model was combined with a previous chronic femoral lymph fistula to assess T cell function, but it could be adapted to the study of pneumonia or acute lung injury/acute respiratory distress syndrome (ARDS) by incorporating Staub's lung-lymph fistula (80). Pape (92) has created chronic lung lymph fistulas in sheep, followed by pulmonary contusions and hemorrhagic shock (92). These studies used rodding of the femur on day 3 to create a "second hit." This clinical model of late acute lung injury/ARDS could also be readily adapted to study the role of sepsis after trauma in the generation of ARDS. Primate models- Primate systems hold the most promise for accurate preclinical modeling the hemodynamic and immune responses of man. Clearly, primates have a high degree of genetic similarity with man and thus immunologic reagents targeting human systems may be usable in primate studies (93, 94). Larger primates may have similar physiologic responses to stresses, and their drug responses often parallel the human. Shock preparations are also fairly well established. The major drawbacks of primate systems are the ethical and animal use issues involved in creating acute and chronic injury models. Moreover, animal care can be very difficult. These factors make primate shock research very expensive and labor intensive. Many of the great apes closest to man have become endangered and smaller primates may offer limited or no advantages over studies using other systems. Thus, primate shock research has almost universally centered on the baboon, and has been quite limited in extent. Fracture and soft tissue injury models (95) have been described in the baboon, but these have given way to the use of complement activation by cobra venom factor (94) or zymosan-activated serum (96) to simulate tissue trauma. Although ostensibly more ethically acceptable, such models are limited in their ability to duplicate human disease. This is because tissue trauma has many more effects than simply to activate complement; moreover, cobra venom factor and zymosan-activated serum are likely to produce inflammatory effects not caused by tissue trauma. Reported hemorrhagic shock preparations in the baboon have used pressure controlled phlebotomy down to an MAP of 35 to 40 mmHg. This requires phlebotomy of about 50% of the blood volume and the presence of shock is assured by the presence of an oxygen debt or a base deficit (93, 96, 97). As noted above, complement activation was used in all the recent work described by Schlag et al. (94) to simulate tissue trauma because of ethical issues. Also, in chronic preparations, animals had to be anesthetized using EEG-controlled closed-loop feedback to insure appropriate levels of anesthesia and thus humane treatment. This system increases the expense and complexity of experiments, but animals can recover from shock and anesthesia, and have been allowed to survive for up to 3 days (94). There is no obvious reason that such humane experimental systems could not be applied to the production of true tissue T/HS models. CONCLUSIONS Properly constructed, complex animal models of T/HS are the most likely way to derive accurate preclinical predictions of therapeutic effect. A wide variety of choices of small and large animals exists. These can be tailored readily to meet the needs of early, proof-of-principle studies and of later preclinical studies. Clinical relevance to human disease will always be enhanced by choosing and adapting the animal model to mimic the disease process of interest in the greatest possible detail.
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