Implementing Refinements in Preclinical Sepsis Modeling (2015)

INTRODUCTION This issue of Shock features yet another important voice regarding the design and execution of mouse-based studies in the area of sepsis and septic shock. In their work, Lilley et al. (1) have focused on an effective implementation of Refinement—the third component (after Replacement and Reduction) of the famous 3R tenet introduced by Drs. Russel and Burch in 1959. Both the timing of this elaborative piece and the country of its origin is well suited. The new European Union directive 2010/63/EU enforcing maximal compliance with the 3R principle in animal studies has come into force in January 2014, and the United Kingdom has been consistently on the forefront of voluntary implementation of those guidelines long before they have become legally binding. The context of the mouse species itself is equally fitting. Since the publication of the new (and entirely opposing) interpretation of the same comparative GLUE grant genomic data (and dozens of related commentaries) (2), we have been solidly anchored in the “post-PNAS reality.” This new reality provokes, among other things, a painful and frank reevaluation of our mouse modeling and study design habits. In the specific perspective of the in vivo sepsis research, the Refinement component has a unique gist and its implementation is perplexing. Given that human and animal sepsis syndromes are highly lethal and feature numerous severe pathophysiological sequelae, the typical humane end points self-evident in areas outside of the critical care disease experimentation are frequently inapplicable in the former field (3). Realistic adherence (or lack thereof) to the classically perceived animal welfare is further aggravated by additional confounders, for example, i) a superior resistance of rodents to infections/trauma, ii) the comorbidity-rich environment of clinical sepsis, and iii) frequently pronounced patients’ immunosuppression at sepsis diagnosis/onset. There is a growing consensus that relevant recapitulation of human sepsis syndromes in rodents requires complex modeling that incorporates the above (and other) characteristics (4). Given that any added comorbidity (as a primary/secondary chronic/acute hit etc.) creates an added burden to the animal, we face an excruciating moral dilemma: is it better to avoid comorbidities (or use their “mild” versions) to reduce this burden but likely generate less relevant data or include them expecting that a given comorbid setting might produce novel and translatable findings? In the opinion of Lilley et al. (1), there is currently insufficient evidence to promote the comorbidity-based experimentation. We have a contrasting view and offer two supportive examples: i) the majority of highly translatable experimental evidence pertaining to the key role of the immunosuppression component in sepsis pathophysiology was produced in various two-hit mouse model studies (e.g., combining cecal ligation and puncture with trauma, hemorrhage, and/or pneumonias). The existing literature provides a detailed coverage of this phenomenon (4, 5). The second just-published example shows that preexisting chronic lung disease caused by cigarette smoke exposure aggravated the posttraumatic inflammatory response and subsequent organ dysfunction after blunt chest trauma, even though cigarette smoke exposure alone without trauma was not overly detrimental (6). This closely reproduces clinical data demonstrating that the degree of acute lung injury in blunt trauma patients is directly related to their pretraumatic tobacco smoke exposure. A recent review on sepsis syndromes explicitly stresses the need for comorbidity experiments, stating that in vivo research should “…include the study of animals that are …older or have preexisting disease.” (7). Lilley et al. (1) also argue that incorporating the intensive care unit measures, in particular mechanical ventilation, should only be considered under “…appropriate anaesthetic depth…” and if “…ventilation does not cause additional welfare problems or confound the experimental outcome….” We would like to emphasize that the omission of the intensive care unit measures bears a comparable risk of confounding the experimental outcomes. The late Prof Daniel Traber (Galveston, Tex) aptly raised this issue already two decades ago: “…Would… a critical care physician accept data on a septic patient … not resuscitated? Would you accept data … on an intensive care patient who was not only not resuscitated … but who did not even have his blood pressure and heart rate monitored?….” (8). The previously mentioned 2013 review echoes the same sentiments stating that “…experiments with more advanced supportive care would allow … permitting the testing of drugs in a more realistic setting…” (7). From the 3R perspective, the preclinical use of an advanced mechanical ventilation more likely promotes rather than compromises animal welfare given that: i) mechanical ventilation typically requires deeper anesthesia/sedation, consequently reducing pain; ii) an adequate mechanical ventilation will relieve the respiratory distress resulting from sepsis-related acute lung injury; and iii) the lung-protective mechanical ventilation in the mouse was shown to attenuate lung dysfunction and organ damage (9). Undeniably, maximal adherence to animal welfare and assessment of the harm/benefit aspect discussed by Lilley et al. (1) should be focal points of each planned/performed preclinical study. Yet, in the preclinical research context, the current shortage of evidence (and the abundance of blunder) cannot be overcome other than by a painstaking trial-and-error approach, in which researchers will identify, retain, and use only the most relevant experimental models while discarding the suboptimal ones. In this approach, the emphasis on comorbidities in sepsis research occupies a central place. Proactive and conscious adherence to the 3Rs as a whole will not only alert the researchers to the welfare issues in animal studies but also steer them toward many other “winner fixes” that are built-in in this principle. They are all explicitly articulated in the ARRIVE guidelines (http://www.nc3rs.org.uk/arrive-guidelines; recently adapted by the Shock journal, “Instructions for Authors” Shock 41[1], January 2014), and the main examples include diligent power analysis, removal of bias, maximal “end point exploitation” of in vivo studies, full disclosure in data reporting followed by realistic interpretations. Thus, following the sentiments of the 2014 review by J. Everitt (10), the classical 3Rs principle that focused on the animals’ welfare should be simultaneously shadowed by an alternative (and equally important) 3Rs concept: Relevance, Robustness, and Reproducibility. Implementation of such tactics in animal studies will likely keep the “benefit” arm acceptably high above the “harm” arm. The grim truth is that the animals are always on the losing end. Yet, fewer of them will lose their lives, the quality of in vivo experiments will gradually improve, and benefits to patients will be undeniable.