34 Animal Models of Resuscitation

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From: Contemporary Cardiology: Cardiopulmonary Resuscitation Edited by: J. P. Ornato and M. A. Peberdy © Humana Press Inc., Totowa, NJ

34 Animal Models of Resuscitation

Kevin R. Ward, ^MD

and R. Wayne Barbee, ^P h D

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INTRODUCTION

The ultimate goal of cardiopulmonary resuscitation (CPR) is the total reanimation of the cardiac arrest (CA) victim back to their pre-arrest status. Much of what we know and do regarding human CPR is based on animal modeling of the many components of CA and its treatment. Ideally, hypotheses regarding mechanisms of injury caused by arrest and treatments to improve outcome should first be tested in robust preclinical models of this disease followed by clinical testing. Although some aspects of the disease and treatment lend themselves to computational, cell culture, and isolated organ modeling, whole animal experimentation remains the standard for preclinical testing (1–4). To this end, the proper design and use of the preclinical model is crucial to ensure that clinical trials are warranted and optimally designed for ultimate validation of the hypotheses.

It is not the purpose of this chapter to review all known animal models of CPR but rather to provide an overview of the principles and challenges of animal modeling of CA and CPR. An understanding of these principles will assist the reader in the inter- pretation of past, current, and future preclinical literature and in the design of future clinically relevant preclinical models.

UNIQUENESS OF PROBLEMS AND QUESTIONS ASKED IN CA RESEARCH

Similar to other shock states such as hemorrhagic, cardiogenic, and septic shock, the

major pathophysiologic challenge that the body faces from CA hinges on the processes

associated with ischemia and reperfusion. However, unlike other shock states, the

ischemia of CA is absolute and affects each major organ system, especially the heart and

brain immediately. Thus, there is no active compensatory redistribution of blood flow to the heart and brain during the arrest period. The tissue blood flow produced by CPR is not sufficient to restore oxidative metabolism and membrane potentials to cells routinely and this period of intervention can be viewed as additional ischemia. This degree of total body ischemia is unique to the shock state of CA and the resulting challenges in regards to achieving return of spontaneous circulation (ROSC) and neu- rological recovery are similarly unique.

It is difficult to draw conclusions from studies of isolated organ ischemia and apply or mix them with those of CA. For example, global brain ischemia is studied widely using non-CA models. These results are applied frequently to CA because it is by far the entity most responsible for producing global brain ischemia (5). On the surface, results from models of isolated global brain ischemia might seem to be fully relevant to the global brain ischemia of CA. However, there may be significant confounding variables (e.g., ischemia to the brain itself), which might negate the major similarity between the two models. CA is associated with total body ischemia, which will drastically change the postresuscitation biochemical milieu that will reperfuse the brain. This includes a tre- mendous difference in the acid–base status of the blood, creation of other circulating mediators (e.g., cytokines produced by remote organ ischemia), immune and stress regu- lating events triggered by the arrest, and additional cerebral hypoperfusion that might be caused in the postresuscitation phase from a stunned and dysfunctional heart (6–8).

Similar arguments can be made in the case of isolated ischemia-reperfusion models of the heart, which are not routinely perfused or reperfused with acidotic blood returning from ischemic organs. In such models, the heart does not contain the important component of neutrophil and endothelial interactions, which may impact on the final damage caused by the arrest (1,9).

The lack of models that incorporate causative conditions of CA such as coronary artery stenosis and myocardial ischemia or important comorbid conditions such as hypertension, congestive heart failure (CHF), or diabetes also severely hamper to ability to apply results of animal models to clinical CA. As with other models, issues of anesthesia, preparatory surgery, and monitoring are debated to their effects on clini- cally relevant outcomes or pathophysiologic processes that are being studied. If, for example, brain protein synthesis is believed to be a vitally important cellular process adversely affected by ischemia and reperfusion, the effects of various anesthetics should be considered first (10,11). Ketamine, which is a popular anesthetic used in CA models examining neuronal protein synthesis, depresses protein synthesis severely compared to other anesthetics such as sodium pentobarbital (12). Even what might be deemed minor or inconsequential preparatory issues such as anticoagulation to keep moni- toring catheters patent are potentially controversial. The effects of heparin provide significant protection against endothelial cell dysfunction (not simply from its antico- agulant effects) in the ischemia-reperfusion injury of trauma (13,14). It is likely to effect microvascular function in the setting of CA as well and will need further study.

Even postsurgical antibiotics such as tetracycline may have downstream protective effects on tissue injury not related to their antimicrobial actions (15).

Researchers must remain cognizant of the unique features of CA and how the

downstream effects of other organ systems or model preparation will influence the

results. There is perhaps no other experimental model containing as many unknowns

as CA.

MODELING VARIABLES

The goal of all animal research is to measure an end-point (dependent variable). The two major end-points in CPR animal research are process and outcome variables (16).

Process variables in CPR research are innumerable and measure or describe events at the subcellular, cellular, organ, organ system, or whole-body level. Specific examples of these might include effects of CA on production of reactive oxygen species, the role of protein synthesis in contributing to neuronal necrosis or apoptosis, and the effects of CPR on coronary perfusion pressure (CPP), acid–base chemistry, and regional organ blood flow. Data obtained on process variables helps in solving the puzzle of CA and CPR in terms of pathophysiology and treatment mechanisms. In terms of their effects on human trials, process variables are used infrequently to support human trials examining out- comes. Process variables in animal experimentation are used to refine mechanistic hy- potheses, which can be tested in humans to substantiate mechanisms. For example, sampling tissue or hemodynamic parameters during arrest, CPR, or the postresuscitation period may confirm in humans a proposed mechanism of injury or potential benefits of a technique demonstrated in animals.

Outcome variables in resuscitation research usually describe an organism’s function after resuscitation with the most common variables being actual ROSC and neurologic outcome. Although debate exists, an argument can be made that human outcome trials are warranted when properly designed animal trials demonstrate an improvement in ROSC even if neurologic outcome is not improved because improvements in the rate of ROSC in humans is the first step toward improving neurologic function after CA resuscitation.

Species

Not all aspects of CA, CPR, and postresuscitation care need to be modeled simulta- neously when asking certain questions. However, it would be nice if the same model could be used by all so that questions asked and studied would come as close as possible to the clinically relevant. Ideally, animal models used to study process or outcome vari- ables would be amenable to modeling all aspects of the disease including the ischemia of the arrest itself (down time), the period of low flow produced by CPR, and the postresuscitation period after ROSC. This means that if the cause of the arrest could be incorporated in the model, the model would have the same metabolic profile allowing clinically relevant durations of arrest and resuscitation to be performed. Clearly defined end-points such as neurological outcome could be used if desired. Additionally, the models would incorporate important underlying conditions such as coronary heart dis- ease or comorbid conditions such as hypertension and/or CHF if adult CA is being modeled. Finally, the ability to manipulate the model genetically to assist in identifying and understanding various pathophysiologic mechanisms would greatly add to the sci- ence of CA research. Unfortunately, no such model exists and is unlikely to exist for some time to come. When it is finally developed, it is likely to be quite expensive. Despite this, it is incumbent that researchers balance issues of realism and cost to match the importance of the question asked with the most appropriate model.

Models of CA and CPR have included rodents, rabbits, cats, canines, swine, and primates. Each has its advantages and disadvantages ranging from price to complexity.

Of these, the models using rodents, canines, and swine predominate. A brief overview of

each of these species along with primates follows.

R^ODENTS

Although rodents are the most commonly studied animal species in biomedical research, their use in CA research is relatively new (17,18). Rats have been used most frequently for CA research. The use of mice has been reported only recently (19). The most obvious advantages of using rodents for CA research are their relatively low cost and maintenance, the controlled genetic variability, and most recently the potential to examine cellular mechanisms of pathophysiology and treatment through the use of genetic knock-out strains in mice and soon for rats.

Rodents are valuable for testing of interventions expected to produce small but impor- tant changes in outcome thus necessitating the study of a greater number of animals. They are also valuable for hypothesis testing of process variables at the cellular level. There are, however, very important limitations in the use of rodents for CA research. The anatomy and physiology of rodents are, markedly different from humans. For example, rodents defibrillate themselves spontaneously, and thus are difficult to use if ventricular fibrillation (VF) is the mode of arrest. Another major potential difference that has not been adequately studied is the issue of metabolism and individual organ tolerance to ischemia. Fundamental questions of basal oxygen delivery and consumption of indi- vidual organs and the level of oxygen delivery that results in ischemia and accumulation of oxygen debt have not been well defined (20). This lack of information prevents basic comparisons of the effect of arrest times on process and outcome variables between rodents, larger animals, and humans. Is a 4-minute arrest in a rat or mouse equivalent to a 10-minute arrest in humans? Can anything but basic process variables at a cellular level be compared to humans? These are basic questions that should be asked and studied if rodents are to become an important link in the chain of CA research.

Neurological outcome studies in rodents is appealing because they can be trained easily (compared to large animals) to perform tasks requiring memory prior to the arrest and then be tested postarrest. However, provision of postresuscitation care to rodents is difficult in terms of providing clinically relevant ventilation and cardiovascular support.

This is an important but often overlooked issue when examining neurological outcome several days after the arrest. Similar to other species, postresuscitation process and out- come variables may be misinterpreted if animals are left to their own resuscitation after CA. For example, something as simple as postresuscitation hypoxemia that occurs from tracheal or bronchial secretions might result in death or a worsened neurologic outcome and negate any positive findings proposed as the result of an experimental intervention.

Rodents can be obtained that are bred to have important comorbid conditions associ- ated with CA such as hypertension and age (21,22). Although tempting, the use of genetic knock-out strains of rodents to study cellular processes associated with CA and CPR should be viewed with caution. Nature will have a tendency to compensate for removal of a gene. Good examples of this are (a) the upregulation of various isoforms of nitric oxide synthase when the gene for the endothelial form of the synthase to maintain micro- vascular vasodilation is removed, or (b) the increase in capillary density that occurs in response to removal of the gene responsible for making myoglobin to maintain oxygen delivery (23).

In terms of animal husbandry, there are differences between inbred and outbred

species of rodents. Viruses and bacterial pathogens such as rat respiratory virus or

clinical signs are not present (24). Thus, attempts should be made to assure that animals

are pathogen-free. Even what might be minor pre-experimental events such as cold or transport stress should be taken into account when planning experiments examining both process and outcome variables as these events may significantly alter the animals’

immune system and response to the stress of surgery.

CANINES

Until the mid-1980s, canines were the favored large animal model used in CA research.

Although canine cardiovascular function in general is similar to humans, the canine heart contains extensive collateral circulation. Additionally, the thoracic dimensions of most canines (which are keel chested) are substantially different from humans, making com- parison of chest compression techniques to humans difficult. Between breeds there is significant difference in the size and shape of the chest, heart, and brain that may poten- tially affect outcome. Additionally, some feel that the cardiac output and regional blood flow in canines are higher before and during resuscitation compared to humans (25).

Despite these shortcomings, canines remain a valuable model and there exists exten- sive neurologic and histopathologic outcome data for the species in the setting of CA and CPR. Specific neurological outcome scores have been developed for canines, which appear to correlate well with histopathology (26). Canines can also be “engineered”

(nongenetically) to develop via diet and other means to have clinically relevant causal or comorbid conditions such as atherosclerosis and CHF.

Canines can be obtained from colonies that are purpose-bred, which indicates that animals were bred specifically for research in a regulated facility. Such animals are conditioned and should thus be free of clinical disease and vaccinated from such entities as heartworms. Use of canines from local sources such as shelters or pounds is discour- aged because uniform health and size among animals cannot be guaranteed.

SWINE

Swine use in CA research has greatly increased and appears to be currently favored over canines for a variety reasons. They are generally less expensive than canines and can be obtained in more uniform sizes and ages for experiments modeling infants, children, and adults. From an anatomical standpoint, the swine thorax has more similarities to humans and the swine heart has less collateral circulation than canines. Humans and swine are also very similar in terms of metabolism and cardiovascular function. The electrophysiology of the heart and the central hemodynamic and organ-specific blood flow of the swine in response to CPR and pharmacology appear to be more similar to humans and may allow for better preclinical studies on techniques and interventions, which take place during the period of CPR than with rodents and canines (27). Similar to canines, the size of swine allows for easier measurement of central hemodyanics such as cardiac output and organ-specific hemodynamics such as regional blood flow than rodents. As with canines, this is especially important in the postresuscitation phase in which a level of critical care similar to humans can be provided to examine longer term variables such as neurological outcome and histopathology or shorter term variables such as myocardial function (28,29).

Similar to canines, swine also allow for the potential advantage of imputing other important variables into a model of CA including coronary atherosclerosis and CHF(30–

33). It is also likely that swine will be the first large animal to have its genome sequenced

and to undergo knock-out experimentation although the same compensatory caveats will

exists with swine as exists with rodents.

Swine can be obtained from herds accredited by a national agency and can be indicated as such by the term specific-pathogen-free. However, even in such circumstances, such pre-existing disease states as pneumonia can be present and are difficult to diagnoses unless animals are radiographed prior to use. Use of swine that are contracted out from local farms is discouraged because it will be more difficult to ensure uniform health among animals prior to experimentation.

N^ONHUMAN P^RIMATES

Despite their similarity to humans and ability to be bred to have human cardiac disease, primates have not been used frequently for CA research (34–36). The ability to observe and test for functional neurological changes produced by the arrest and treatment and their extrapolation to humans is enticing. However, the expense of purchase and upkeep of these animals are major deterrents. Additionally, among animal models, primates will have the greatest degree of genetic variability that may or may not be valuable when looking for an initial treatment effect. Many CA clinical trials have been based on data obtained from species other than nonhuman primates (37–40). It is unclear what benefit or changes would have occurred if nonhuman primates had been used prior to human trials in these instances.

Lessons and Examples From the Literature

Following are a number of studies that have been performed in CA research, which serve to stress the importance of clinical relevance and the sometimes counterintuitive results of clinical studies that were not supported by animal studies.

Perhaps one of the greatest stumbling blocks in CA research is the clinical relevance of the model chosen. If outcome variables of an animal model such as ROSC and neu- rologic outcome are chosen to mimic out-of-hospital CA as a prelude to clinical studies, as many clinical variables should be incorporated as possible that mimic the clinical scenario so that any improvement in the outcome variable is more likely to be observed in a clinical study.

This is not specific to CA research but permeates research in other shock states. For example, models of hemorrhagic shock examining the effects of a treatment on out- come can be criticized because none contains all of the common clinical elements of severe soft tissue injury, pain, hypoxemia, resuscitation with stored packed cells, pro- longed mechanical ventilation, and potentially intra-abdominal hypertension in addi- tion to volume hemorrhage leading to severe oxygen debt. In this situation, events concomitant to hemorrhage that occur in the individual of multisystem trauma as a result of both injury and treatment have the potential to effect outcome profoundly. For instance, stored packed red cells (commonly used in the clinical setting) have been demonstrated to adversely effect microcirculatory tissue oxygen delivery adversely compared to citrated or heparinized fresh whole blood (never clinically used [41]).

Pain from sever soft tissue injury has been demonstrated to significantly increase

oxygen consumption and decrease splanchnic blood flow, both of which have the

potential to potentiate cumulative oxygen debt (42,43). This can be compounded by

increases in intra-abdominal pressure, which further decreases splanchnic perfusion

and cardiac output (44). Prolonged (>24 hours) mechanical ventilation is injurious to

the lungs, increasing the chance of acute lung injury after hemorrhage (45). It is easy

to envision how a model designed to test the ability of a new blood substitute vs

crystalloid fluid and fresh hemorrhaged whole blood on 24 to 48 hours survival could significantly underestimate the severity of injury and illness if the model was one of simple pressure hemorrhage. Significant benefits of the blood substitute found in such a simple experiment may not be present when applied to the more complex everyday clinical scenario. Failure to model all of the above entities is also likely to be respon- sible for why there are no animal models of trauma-induced multisystem organ failure.

Similar disparities exist when modeling out-of-hospital CA. Epinephrine dosing is a prime example. The original recommendation by the American Heart Association (AHA) that epinephrine be administered to adults at a dose of 1 mg was based on the studies of Redding who used 1 mg of epinephrine on 10-kg weight dogs (46). Without critical evaluation, this dose was accepted before realizing that the human dose may not have been properly extrapolated from animal data. This in turn led to a large number of animal studies that examined the effects of much higher doses of epinephrine. The results of these animal studies demonstrated higher doses of epinephrine significantly improved variables such as CPP, which are associated with increases in ROSC (47,48). However, when tested in humans, high-dose epinephrine failed to improve outcome (37). Close examination of the clinical studies revealed several important issues that may not have been taken into account in animal studies (49). Chief among these was the timing of administration. Whereas most animal studies examined the use of the drug approx 10 minutes after onset of CA, analysis of human trails found that the time to first drug administration averaged 18 to 20 minutes, a time almost double that of animals. Addition- ally, postresuscitation care of humans after CA is not standardized and indeed had not been studied at that point in animals to understand what affects high doses of catechola- mines would have on postresuscitation myocardial function and microcirculatory blood flow (50). Clinicians were ill prepared to treat the postresuscitation systemic effects of massive catecholamine dosing that were found to produce significant tissue oxygen defects (51,52).

Another major issue not taken into account was whether higher doses of epinephrine would be able to increase myocardial blood flow in the face of significant coronary artery stenosis. This is an important question because the majority of adult arrest victims will have significant coronary artery disease (CAD) and a very important segment of these victims will have had a myocardial infarction (MI) as a cause of their arrest (53). In a remarkable series of studies performed by Kern and colleagues, coronary artery lesions of 50% or less were shown to drastically reduce distal myocardial blood flow during CPR for a given perfusion pressure (54–56). Although the effect of high-dose epinephrine was not examined in these studies, a distinct possibility exists that the benefits demonstrated in previous animal models of CPR without coronary lesions would have been less impres- sive and may not have reached significance if they had been studied in models incorpo- rating coronary artery stenosis. Additionally, MI-induced VF is rarely modeled (57).

Instead, VF is induced electrically. The ability to obtain ROSC in an infarcted heart with significant CAD and a prolonged post-arrest predrug administration time of 20 minutes will be much more difficult than that of a nondiseased, noninjured pre-arrest heart with a post-arrest predrug administration time of 10 minutes. Therapies that significantly improve ROSC and neurological outcome in the former model should have much less chance of failure in a clinical trial.

Use of asphyxial models that correspond to the clinical experience presents similar

challenges and pitfalls in attempting not to underestimate the severity of the insult.

Rodent models of asphyxia have used asphyxia times of 8 to 10 minutes to produce actual CA times of only 4 to 8 minutes (18,58,59). With the exception of drowning, asphyxia (at least in adults) is likely to be much more prolonged and produced by more insidious processes as exacerbations of acute airway diseases such as asthma or emphysema in which the individual fatigues and finally becomes hypoxemic enough to arrest. In these settings, the accumulated oxygen debts of the individuals will be significantly higher than the short durations of total asphyxia and arrest produced in current preclinical models.

Additionally, arrest times prior to interventions such as drug therapy will be prolonged much more clinically (49).

Using vasoactive agents again as an example, animal studies can be designed in such as way as not to see an affect at all or to see something artificial. Both human and animal studies have demonstrated the need to create a minimum CPP for ROSC to occur. In order to demonstrate that the new agent is helpful in achieving critical CPP, baseline chest compressions should be performed to create the suboptimal range of CPP that occurs clinically (60–62). If, however, chest compressions are performed in the animal model and in of themselves produce a CPP above the critical threshold for ROSC and produce ROSC without the need for exogenous vasoactive agents, it becomes less likely that the vasoactive agent(s) being tested will be demonstrated to superior over each other (63).

In fact, it might be possible in this model to find detrimental effects of one agent over another that would not be found ordinarily because administration of the agent such as high-dose epinephrine in this model would be similar to producing a model of simple catecholamine overdose.

A much overlooked part of animal models of CA is the issue and importance of modeling the postresuscitation phase of care. For as much emphasis as has been placed on demonstrating the ability of experimental agents or techniques to improve neurologi- cal outcome, it is amazing how much this aspect of modeling has been neglected because neurologic outcome cannot be predicted immediately upon ROSC but instead takes several days to determine. The postresuscitation care of animals has ranged from liter- ally extubating comatose animals within several hours of ROSC and placing them into cages to providing clinically relevant intensive care from the onset of ROSC and lasting 96 hours (26,63–65). Failure to provide aggressive postresuscitation care to animals may lead to the inability to observe the ability of an intervention delivered during CPR to improve ROSC and long-term outcome because poor postresuscitation care may increases mortality or morbidity as a result of cardiovascular or respiratory events that would have ordinarily been avoided with proper monitoring in the clinical setting.

Conversely, aggressive postresuscitation care from a cardiovascular standpoint imme- diately after ROSC my also lead to the inability to observe differences in outcome because in the clinical environment, it may take several hours to instrument patients properly and optimize cardiovascular performance and cerebral blood flow.

Finally, there are animal models demonstrating benefit of an intervention on outcome

that have also significantly underestimated the benefit of the intervention in humans. A

prime example of this has been in the large animal studies of hypothermia to improve

neurological outcome after CA. A significant body of work examining the effects of

postresuscitation hypothermia in canines demonstrated that in order to be effective in

improving neurological outcome, hypothermia had to be instituted in less than 15 minutes

after ROSC (65). This would present many challenges in applying hypothermia as a

clinical postresuscitation treatment. Despite these findings, several large clinical trials

demonstrated a significant improvement in neurological outcome when hypothermia was instituted in the postresuscitation phase, even when the therapy could not be imme- diately instituted and the time to reach target temperature was several hours longer than animal studies suggested would be effective (39,66,67). It was also demonstrated that delays in instituting hypothermia in rat models of CA did not negate its effectiveness (68).

These findings appear to violate the hierarchical approach to clinical trials in which one would originally screen a therapy in rodents followed by movement to a higher species such as canines or swine, and then finally move to a clinical trial. Why canines demon- strated little improvement in neurological outcome after delayed cooling remains a mystery but again points out that there is no perfect preclinical model.

Utstein Guidelines

Historically, it has been difficult to compare the results of clinical studies with each other, the results of laboratory studies with each other and the results of clinical and laboratory studies to each other. One of the major reasons for this difficulty is the lack of standardization and the use of nonuniform terminology. In 1990, an international conference on the topic of out of hospital resuscitation was held at the Utstein Abbey in Norway to discuss the lack of standardized nomenclature and language in clinical research reports. That same year, an additional meeting was held to develop a consen- sus. This resulted in development of a uniform style of reporting and definitions of clinical resuscitation research and was termed the Utstein Style (69). That same year, the European Academy of Anaesthesiology developed a similar approach for animal mod- els that led to a series of conferences and workshops culminating in 1996 with an Utstein-Style guidelines for uniform reporting of laboratory CPR research (70,71). In a survey performed by Idris and colleagues, it had been noted that lack of simple uni- form definitions and item reporting made comparison between studies difficult. These included failure to define and report such basic but important issues as minute ventila- tion, CPP and ROSC (72). The aim of Utstein-type guidelines for laboratory study were not to encourage or discourage one species or model over another but simply to include enough uniform reporting information concerning aspects of the laboratory study to allow its comparison with other published studies. The guidelines are centered around nine templates and include the following:

• Study design

• Subjects

• Preparation of animals

• Methods for monitoring

• Experimental protocol

• Outcome variables

• Analytical approach

• Results

• Discussion and conclusions (71)

These templates cover almost every conceivable aspect of a laboratory CPR experi-

ment using animals and ranges form living conditions of the animals prior to the experi-

ment to mode of ventilation to comparing the statistical and biologic significance of

results. However, despite being published in 1996, the extent of adherence to these

guidelines remains unclear.

SUMMARY

In the quest to develop successful treatments for CA, animal research will remain an invaluable asset. However, researchers must critically evaluate the appropriateness of the model used based on the clinical relevance of the question asked as it relates to process vs outcome variables to be assessed. When placed in the greater context of the actual clinical experience, more robust animal models are likely to be created. Clinical trials based on such data will hopefully be at less risk of failure.

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