In the postconference phase, the work was primarily focused on the finalization of the MQTiPSS recommendations and considerations. This task was accomplished by teleconferences and electronic-based discussion among WGs using a modified Delphi method. Finally, a writing committee (formed at the conference) together with all participants developed an Executive Summary for MQTiPSS (4) and 3 full-length publications (5, 7). Each (of the 3) publication focuses on 2 related WGs; the current Part II paper provides detailed discussion on the guideline points for Types of Infections and Organ Injury/Dysfunction Endpoints.
Sepsis 3.0 defines human sepsis as life-threatening organ dysfunction caused by a dysregulated host response to infection (2). Septic shock is defined as a subset of sepsis in which particularly profound circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than with sepsis alone (2). Thus, the presence of an infection constitutes one of the defining elements for the development and progression of sepsis. Infection in sepsis is usually bacterial, most often located in the lungs, peritoneum, or urogenital system (9). When using animal models that replicate human sepsis, therefore, infectious challenges using bacteria are most appropriate and preferred over bacterial components such as molecules from the cell wall. The conference carefully reviewed the most highly cited papers using preclinical models of sepsis to ascertain the types of infection(s) that were used (Table 1). This analysis showed that most studies used the cecal ligation and puncture (CLP) peritonitis model (44%), followed by the lipopolysaccharide (LPS) model (40%), and single infection models such as pneumonia (16%). Our subsequent smaller review of 190 murine sepsis studies in the years 2013 to 2017 showed that CLP was employed in 64% of the studies while LPS was the second most common model used in 23% (both as single and 2-hit combination). The popularity of the CLP model was obvious in this review, but the extensive use of LPS (as a sepsis model) should be discouraged. Moreover, the relatively few studies using organ-specific sepsis models (lung, urogenital system, blood stream) resulted in a specific recommendation by the working group. The relatively simple model of septic peritonitis induced by CLP (10, 11) was viewed favorably, especially when performed in combination with resuscitation, antibiotics, and infectious burden control. The model begins with gut flora, which may or may not be in a dysbiotic condition (12). This model replicates some but not all features of human sepsis (e.g., no acute lung injury in some studies (13)).
Specific infection models may be more informative, because they allow pathogen dosing, selection of the route of infection, and strain of bacteria. Because not all animal species respond with the same intensity to given bacterial strains, some bacterial species and strains are only applicable in certain mammalian model systems (14).
The choice of the bacterial strain requires careful consideration. Although the bacterial isolates from septic patients differ between neonatal, pediatric, and adult sepsis patients, a number of the same species are frequently found in clinical isolates. These include the gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, P. aeruginosa, and Bacteroides fragilis and the gram-positive bacteria Streptococcus pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus. Using these species as infectious agents in sepsis animal models would be a logical choice. One should realize that many strains of a certain species are available to the researcher; however, in comparison to clinical isolates, these strains may differ greatly in their virulence (15), their capacity to form biofilms, and their antibiotic-resistance profile (16, 17). Thus, working with bacterial isolates from sepsis patients, provided that these are well characterized and pure populations, may be a choice to consider. The route of infection is important and will depend on the specific interest of the study, but should also follow simple concepts, e.g., infecting an animal intratracheally with a bacterial species exclusively found in the lungs of human with pneumonia.
The conference discussed several specific recommendations for preclinical models of sepsis. The following recommendations and considerations from the Types of Infections WG are numbered consecutively from the preceding companion paper Part I and start with recommendation 8.
The widespread use of LPS can be attributed, among other things, to the fact that its administration mimics some of the acute clinical features of the sepsis syndrome, including a systemic inflammatory response syndrome (SIRS) (e.g., fever, leukocytosis, cytokine release). Our evaluation of the most cited recent literature on animal sepsis models showed that LPS was used in 40% of the reviewed studies (Table 1). The current body of evidence, however, clearly demonstrates that administration of LPS should not be accepted as a relevant model of sepsis given the many differences between the sepsis phenotype and that induced by LPS. For example, the robust but transient character of the inflammatory response after LPS strongly differs from milder and protracted release of cytokines in the CLP model (20). Studies demonstrated that anti-tumor necrosis factor (TNF) treatment was beneficial in an LPS but not a CLP mouse model (21). Furthermore, comparison of TNF-α release profiles in human volunteers injected with a low LPS dose (22, 23) and severely septic patients (24, 25) shows that peak TNF-α concentration in the latter group never exceeded the one in the former. An emergence of a new patient phenotype of “persistent critical illness” (26, 27), further negates the translational usefulness of (acute) LPS-based protocols. At best, researchers may claim that some characteristics of the early septic reaction can be replicated by a challenge with LPS.
However, there continue to be studies using LPS as a model of sepsis. A researcher with limited time, money, and perhaps understanding of clinical sepsis may be tempted to use LPS-based models, arguing that at least some patients with a gram-negative infection causing sepsis have been shown to have an elevated level of LPS in the blood (28), and that such an LPS-exposure is an important part of the illness. Species differences in sensitivity to LPS exist (29), with humans being very sensitive. A way forward is to continue accepting data from research on LPS as relevant for the study of the first 24 h of the septic response. However, funding agencies and the readership should always be reminded of the unavoidable limitations in extrapolating any conclusions drawn in a study on LPS to clinical sepsis.
However, preclinical sepsis modeling with nonidentical pathogens should not be completely dismissed. In some cases, nonidentical pathogens can produce similar disease phenotypes in animals when compared to patients. For example, a renal disease caused by a Citrobacter infection in animals is identical to the one caused by E. coli-produced Shiga toxin in humans. Similarly, Plasmodium berghei ANKA in C57BL/6 mice (partly) recapitulates cerebral malaria caused by Plasmodium falciparum in humans.
Sepsis in patients may originate from infections in different body compartments. Over the past 20 years, the sites of infection in humans have remained essentially unchanged, with lungs being the most frequent site followed by the abdominal cavity, urinary tract, and soft tissue (41, 42). In sharp contrast to these clinical data, nearly 60% of preclinical sepsis models rely on infections originating from the abdominal cavity (Table 1). As such, the polymicrobial models of CLP and colon ascendens stent peritonitis or mono microbial settings including the direct i.p. or i.v. injections of bacteria are frequently employed (42, 43). Although these models provide important information, peritonitis models do not replicate the most frequent site of infection in patients (the lung), a problem has been addressed in prior publications (10, 44).
Considering these epidemiological clinical data, we propose that multiple preclinical models would be appropriate for the study of sepsis. The specific model should attempt to closely mirror clinical reality to identify relevant pathways and pathophysiological changes at specific body sites. A better understanding of the dysregulated pathways will aid in the design of more appropriate therapeutic interventions (1). Sepsis models where the infection occurs in the lung have the greatest clinical relevance and such infections should be initiated with causative organs such as S. pneumoniae, K. pneumoniae, or P. aeruginosa (44) to potentially identify distinct features. In a similar fashion, models of urinary tract infection (45) or soft tissue infections, e.g., Group A Streptococcus (46), will provide valuable information. Using different models with different sites of infection will allow the study of the natural course of infection(s) that may culminate in sepsis.
The following recommendations from the Organ Injury and Dysfunction Endpoints WG are numbered consecutively from the preceding chapter and start with recommendation 10.
Organ dysfunction may be defined as a maladaptive response to a potentially harmful danger signal or pathogen. Importantly, organ dysfunction caused by sepsis fundamentally differs from adaptive responses that attempt to minimize harm. It is essential to develop objective, readily measured criteria for dysfunction in individual organ systems and utilize these criteria as outcome variables.
Both clinical and experimental investigations in sepsis have focused on 2 outcome criteria; survival and/or biomarkers (51–54). In experimental animals, biomarkers are often the measure of immune function, such as cytokine concentrations (55), e.g., IL-6 (56, 57) or a white blood cell (WBC) count. Human biomarkers usually consist of composites of commonly measured variables such as vital signs or standard laboratory tests such as WBC counts. For example, the SIRS criteria measure body temperature, heart rate, respiratory rate, and the WBC count (19, 58, 59). The Sepsis-3 clinical criteria redirected the focus toward organ dysfunction by making it 1 of the 2 essential characteristics that define sepsis (2). Unfortunately, measures used to estimate the presence of organ dysfunction are poor, failing to provide specificity. For example, the Sepsis-3 task force used the Sequential Organ Failure Assessment (SOFA) score (60), a composite of clinical measures that has prognostic value (61), as a proxy for global “organ dysfunction.” However, the task force noted that points used by SOFA only linked to a given organ system and did not specifically identify dysfunction in that organ. Consequently, the task force also recommended derivation of an updated tool (2). Human studies are further hampered by the lack of “gold standard” criteria that unequivocally identify sepsis; instead surrogates that have either outcome (predicting mortality) or construct (identifying patients that develop characteristics that “look like” sepsis, e.g., refractory lung injury) validity are used to identify septic patients post hoc (62, 63). Animal studies eliminate the need to use an outcome to identify sepsis because both infection and sepsis are known to be present. Therefore, preclinical models may be used to identify potentially translatable criteria for organ dysfunction. In addition to assessing the effects of interventions on survival and the development of the long-term abnormalities that plague sepsis survivors, animal studies provide a unique opportunity to evaluate organ dysfunction, which is now required for the clinical diagnosis of sepsis (2).
Although the progression of acute illness to Multiple Organ Dysfunction Syndrome (MODS) and the pattern of organ dysfunction has been addressed in clinical studies since the first description of the syndrome by Tilney et al. (63) in 1973, many animal studies only measure survival. However, quantifying conventional markers (platelets, creatinine, bilirubin, etc.) of organ dysfunction similar to the SOFA score is feasible (64–67), even in rodents. If conventional biochemistry markers are used to describe organ dysfunction, they are typically only used as a single endpoint assessment, unlike the clinical SOFA score measured sequentially (the S in SOFA). Animal studies lack a validated and standardized panel of markers similar to SOFA.
The validity of conventional markers of organ impairment (platelets, creatinine, bilirubin, etc.) to quantify the injury to cells and tissues in this context is augmented by the ability to study organ-specific dysfunction. This measurement of injury may be done using histology, immunohistochemistry, and other modalities, allowing in-depth analysis of organ integrity as tissue samples would be available. These techniques allow assessment of the severity of dysfunction, to gather insights into the mode of injury, such as necrosis as opposed to various forms of programmed cell death (68–70). In addition, these techniques provide information regarding the spatial distribution of injury. The option of “reverse translation,” i.e., comparing patterns and severity of tissue damage in animal models, allows conventional clinical markers to be more useful when interpreting the degree of protection provided by a therapeutic intervention (65, 66, 69). Interestingly, in murine CLP, it was shown that although parameters indicative of organ dysfunction were greater in dying compared with surviving mice, they never exceeded the changes in surviving CLP mice in which cisplatin or carbon tetrachloride were used to induce nonlethal hepatotoxicity and nephrotoxicity (71). In other words, standard organ injury models which are non-lethal induce substantially more organ injury than that observed in mice dying from CLP-induced sepsis. Given the central role of the cardiovascular system in failing organs, a longitudinal assessment of clinical and hemodynamic parameters using wireless biotelemetry monitoring is particularly promising (72, 73). In the murine CLP model, telemetry has been used to define and validate criteria for acute deterioration to mimic human sepsis studies (73). In addition, in small animal models techniques applying in-vivo microscopy allow assessment of microcirculatory disturbances beyond macrohemodynamic assessment even in internal organs such as liver and kidney (67). Extrapolating from patients, reduced organ function in individual organs would meet the definition of an inappropriate host response, albeit experimental studies suggest that several tissues and organs are only mildly affected. ICU-specific interventions, including mechanical and cardiovascular support, might be required to allow the development of severe MODS (74).
This specific recommendation states that not all functions of an organ need to be disrupted or abnormal to state that organ dysfunction is present. This recommendation is specifically included because preclinical models offer opportunities to study multiple parameters and provide tissue for in-depth studies. The standard for organ dysfunction would be too high if it was required that all functions were abnormal.
In disease and pathological conditions in patients, where individual or multiple organs are involved, e.g. sepsis, acute lung injury, or severe traumatic injury, diagnostic scoring systems have been developed and validated which describe the organ(s) morbid status. Scoring systems such as SOFA, qSOFA, Simplified Acute Physiology Score, Acute Physiology And Chronic Health Evaluation 2, (1, 2, 9) have been developed as an index of the organ(s) function. Such scoring systems largely serve to provide a uniform reference for the individual clinician's assessment of organ function. With this common basis, decisions can be made about the patient's status which drive clinical decisions. Databases of organ dysfunction have been created to allow study of the disease. In a similar manner, scoring systems could be developed for experimental models of sepsis to allow sharing of results. These scoring systems, similar to those used for patients, would consist of biochemical indices, physiological indices, blood chemistry, coagulation system status, discrete pathological changes (both gross and microscopic), and behavioral alterations. Comorbid conditions, such as age, gender, nutritional status, etc., could also be included (2, 60, 75–80). Such scoring systems have been especially useful for stratifying the severely injured (78, 79). Scoring systems for preclinical studies have not been applied in a consistent manner. Consequently, they provide limited data for detailing the clinical condition of sepsis they were attempting to model (8, 81). At the 9th Wiggers-Bernard Conference on “Pre-clinical Modeling in Sepsis” we reviewed 260 manuscripts looking at experimental sepsis which included a total of 374 experimental studies that assessed the organ(s) for dysfunction (see Table 3). There were 116 published studies that examined only a single organ injury/dysfunction endpoint, whereas 88 measured multiple organ dysfunction endpoints and 170 simply did not report any endpoints. Further, of those 374 experimental studies only 10 used some form of clinical-like scoring system, whereas 364 did not. Finally, of those 374 experimental studies 118 used histological assessment of organ damage and 256 did not. Although most of the recent studies did not use an organ injury score, guidelines have already been published on which parameters should be measured, such as the American Thoracic Society's guidelines on experimental acute lung injury (48). These guidelines were an attempt to encourage investigators to adopt standard approaches to measure dysfunction in experimental models of lung injury. Measuring a common set of organ injury parameters will allow better comparison between the animal models. If such guidelines exist or can be established through scientific consensus for not only an organ (76, 80) but also at a system level, such as a rodent sepsis severity score (82), investigators attempting to document their animal model of sepsis should incorporate such scoring systems.
Conducting research and developing therapies and treatments for sepsis relies heavily on in-vivo studies. Models of sepsis, however, can cause significant discomfort, pain, and distress to the animals. Therefore, investigators should attempt to collect as much information as their budgets allow and thus make the best possible use of the animals, while attempting to reduce the numbers of animals needed.
A number of simple behavioral and physiologic measurements can be obtained noninvasively and without the need for sophisticated equipment. Cage activity, body weight, and rectal temperature, for example, can be easily obtained and prior studies show that these correlate with hypothalamic levels of proinflammatory cytokines (83). Vital signs such as arterial blood pressure, heart rate, respiratory rate, and blood oxygen saturation can also be easily measured noninvasively (84). These vital signs can be used to indicate septic shock, increased sympathetic tone, metabolic acidosis, and pulmonary shunt. Furthermore, investigators can use a sepsis severity score for later correlation with other variables (82).
Maximizing sample collection is also recommended. For example, if an animal is sacrificed, plasma or serum should be collected, frozen, and stored for subsequent studies. Animals euthanized because of humane endpoints or at the end of the study should be subjected to necropsy, including external examination, exsanguination, exploratory dissection, gross anatomy, weighing, and preservation of key organs, including the heart, lungs, spleen, gut, liver, and kidneys in fixative solution for later studies. Necropsy studies should be planned before each study, as some organs require unique collection and fixation techniques (85, 86). Ideally, the use of the animal data and samples can also be improved by collaboration among investigators in various fields. An investigator studying sepsis-associated kidney injury, for example, could share sepsis severity scores, blood oxygen saturation, and lung tissues with investigators interested in lung injury, as well as body weight, serum, and gut tissue with investigators interested in sepsis-associated gut dysfunction.
It is recognized that not all investigators will have the time, equipment, or expertise to fully define dysfunction in each organ. For example, it is possible to measure pulse oximetry and respiratory rate noninvasively in a mouse, although these measurements do not provide the same level of detail as arterial blood gases, whole body plethysmography, and histologic examination of the lungs. However, if an investigator's studies focus on the kidney, it would be unreasonable to expect a detailed pulmonary study. It may be reasonable for the investigator to noninvasively measure blood oxygenation and respiratory rate.
Metabolic disorders occurring in sepsis often lead to hyperglycemia, which is frequently associated with worsened outcome in septic patients (87) and experimental models of sepsis (88). The definition of acute hyperglycemia varies by study ranging from 8 mmol/L to 15 mmol/L (144–270 mg/dL) (89, 90). It has been shown that the severity of sepsis is impacted by the occurrence of hyperglycemia, which appears as a risk marker of morbidity and mortality (91). The latter is the reason for insulin therapy in septic patients. However, intensive insulin therapy has repeatedly been associated with increased incidence of hypoglycemia, defined as a blood glucose <70 mg/dL (92). Two studies in large observational cohorts have identified sepsis as an important risk factor for the development of severe hypoglycemia predominantly because of insulin therapy (93, 94). Even a single episode of severe hypoglycemia can substantially influence the severity of sepsis (95) and significantly increase risk of mortality. Apart from hyperglycemia and hypoglycemia, increased glycemic variability has been associated with substantially increased mortality rate in septic patients (95–97). The effect of increased glycemic variability was unexpectedly high; it was associated with nearly 10-fold increased mortality in critical care patients (95).
The available data suggest that the levels of blood glucose critically influence the sepsis outcome and should be considered in preclinical models with particular attention to the glucose variability. If the effects of hyperglycemia and hypoglycemia are not in the scope of a study, then it can be recommended keeping glucose levels in a normal range (79.2–110 mg/dL), and the daily variations should neither drop below 70 mg/dL nor exceed 140 mg/dL.
However, the glycemic response in septic mice is different from that in patients. CLP mice typically develop a prolonged and profound hypoglycemia, whereas the hyperglycemia often reported in patients does not occur in rodents (98). Hypoglycemia in rodents was associated with increased sensitivity to insulin resulting in hypoglycemia at normal insulin levels (99). However, even in rodents that are hypoglycemic insulin reduces inflammation and multiple organ dysfunction (100). For these reasons, investigators should consider preventing marked changes in blood glucose levels in preclinical models of sepsis. We recognize that other parameters are typically monitored in septic patients, e.g., blood pressure, urine output, and these issues should be addressed in future discussions.
This Part II manuscript details the recommendations and considerations of the 2 WGs from the Wiggers-Bernard conference on preclinical models of sepsis. The intent was to determine the current state of preclinical models by reviewing the most highly cited articles on preclinical models. Analysis of the data showed great heterogeneity with regards to types of models and how the animals were monitored to evaluate organ injury. The participants were aware of the 2016 Sepsis-3 definition stating that sepsis was organ dysfunction because of an infection (2). The WG made specific recommendations about the types of infections as well as monitoring organ dysfunction. We hope that these recommendations and considerations will serve to bring a level of standardization to preclinical models of sepsis and ultimately improve the translation of preclinical findings. We acknowledge that new challenges based on new information from the clinical and bench studies will continue to arise. A close collaborative work between basic scientists and clinicians is critical for a thoughtful (re)interpretation of any existing and newly posited principles.
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