Injection of rodents with cecal slurry (CS) is a preclinical model of polymicrobial peritoneal infection that results in a rapid systemic inflammatory response and can induce circulatory shock, multiple organ dysfunction, and death. The technique consists of collecting cecal contents from donor animals, creating a suspension and then injecting the suspended slurry into the peritoneum of experimental animals (1, 2). Cryogenic preservation techniques allow the bacterial slurry to be frozen and used in multiple experiments over time, thus avoiding variations in the bacterial content (2). However, survival experiments are required for each new preparation of slurry, to determine the appropriate dose, and then for determining the impact of interventions. Using death as an endpoint raises important ethical concerns of animal discomfort, pain, and suffering and using commonly employed clinical signs of morbidity as a humane endpoint is often imprecise and subject to wide ranges of interpretation by practitioners (3) despite previous attempts to reduce bias (4). Therefore, we set out to find a practical, non-moribund end-point that would reduce suffering of our mice.
Mice become hypothermic in sepsis, proportional to the severity of infection (5, 6), and changes in core temperature have previously been employed as markers of recovery (7–10). In most cases the experimental methods have been impractical, as core temperature was monitored by the insertion of a rectal probe, which can induce additional stress, or by surgical placement of a telemetric thermometer. However, studies have successfully used near infrared (NIR) thermometry in models of fungal (10) and gram-negative bacteria (11) infections. This approach was very practical, noninvasive, and promising, but it could not directly translate to polymicrobial intraperitoneal sepsis because one study required experimentally induced immunosuppression and the latter used gastric infusion of a specific marine bacterium similar to cholera. Therefore, we tested the feasibility of this NIR approach in a more general model of peritoneal infection. We found that this refinement was a practical alternative in survival protocols that greatly reduced animal suffering and yet offered an accurate means of assessing survival rates in the murine CS model of septic shock.
MATERIALS AND METHODS
Animals and temperature measurement accuracy
We studied a cohort of 154 mice (100 males and 54 females, C57BL/6J background) aged 4 to 6 months old, weighing 20 g to 25 g. Animals were purchased from The Jackson Laboratory or bred, in house, and singly housed at the University of Florida Animal Care Facilities where they remained for the whole duration of the experiment. The temperature and relative humidity range of the room were 20°C to 25°C and 31% to 67%, respectively. Animals were under a 12:12-h light–dark cycle and had access to standard chow (2,918, Envigo) and automatic tap water ad libitum. All the procedures were previously approved by the University of Florida Institutional Animal Care and Use Committee and reported information about the procedures conformed to the Animal Research Reporting of in vivo Experiments—ARRIVE guidelines (12).
In our lab, we study the impact of skeletal muscles on innate immune responses to septic shock and we have developed two transgenic colonies of inducible, skeletal muscle specific knockout mice. The mice used in the current study were of wild type or two different genetic mutations on C57BL/6J background. One transgenic strain was bred to have skeletal muscle specific knockout of interleukin-6, whereas another strain was designed to have skeletal muscle-specific myeloid differentiation protein 88 knockout. The observations herein reported were made while performing survival experiments to define the appropriate dose of CS.
Approximately 1 month prior to the experiment, transgenic animals were either treated (30/154 mice) or untreated (30/154 mice) with a single intraperitoneal injection of 110 mg/g of body weight of a selective estrogen receptor modulator (Raloxifene Hydrochloride) suspended in polyethylene glycol (PEG400) to induce the CRE-LOX recombinase gene (13). Untreated animals received an intraperitoneal volume of PEG-400 (vehicle) only. Wild-type mice (94/154 mice) did not receive any treatment prior to CS injections. We did not observe a clear impact of these mutations or treatments on the relationship between temperature profile after CS and survival; therefore, we combined these groups into a single cohort for analyses.
To test for accuracy of the Ts measurements against a gold standard temperature monitoring system, a group of 12 wild-type mice (no CS injection) were instrumented with radio-telemetry transmitters (TA-E-Mitter; Starr Life Sciences, Oakmont, Pa) for real-time monitoring of core temperature. For this experiment, mice were monitored both at rest and 15 min after exercising on a forced running wheel (Lafayette Instruments, Lafayette, Ind), in the heat, until exhaustion using methodologies described previously (14).
We induced sepsis by intraperitoneal injection of CS collected previously from donor mice. The batch of CS used in this study was prepared and stored according to the method described by Starr et al. (2). In brief, cecal contents were filtered and mixed with 15% glycerol solution in phosphate-buffered saline. CS was then aliquoted in 2 mL cryovials and kept in freezing containers (Nalgene Mr. Frosty, Thermo Scientific, Waltham, Mass) at −80°C. Immediately prior to injection, cryovials were thawed to room temperature, mixed well, and then injected. Our initial goal was to determine the dose of CS in adult male and female mice that would induce a 40% to 60% survival over the course of 5 days. Trial CS dosages ranged from 0.9 mg/g to 1.8 mg/g, which corresponded to volumes of CS ranging from 225 (0.9 mg/g) to 450 μL (1.8 mg/g) for a 25 g mouse. All dosing took place at approximately 5 PM. Animals were restrained briefly by tail and scruff handling and CS injected IP into the lower left quadrant of the peritoneal cavity while the animal was held in the supine position with the head below the body. For consistency, all injections were performed by the same experienced lab member. Mice were monitored at 12, 18, 24, 30, 36 h and then every 12 h for 5 days. Nineteen animals that met a humane endpoint (moribund criteria, absence of righting reflexes after being laid in a lateral recumbent position) were euthanized by CO2 inhalation during recovery from CS injection.
Surface temperature recordings
Before CS injection and at each monitoring period, NIR temperature was measured at the level of the xiphoid process using a non-contact, infrared thermometer (Etekcity Lasergrip 774) (Fig. 1A). We performed parallel experiments in subgroups of non-septic mice to compare surface temperature measurements at different sites (e.g., forehead vs. xiphoid process). During the measurement at the xiphoid and at forehead, animals were restrained briefly by hand scruffing. The thermometer is equipped with a target laser, and the beam was aimed approximately 3 inches from the surface. This instrument works in a range of −50°C to 380°C. Manufacturer's instructions did not indicate that calibration, beyond initial factory settings, was necessary. Results in one group of animals were compared against a more sophisticated NIR monitoring device with calibration capability (Fluke 62 Max, set with an emissivity setting = 0.95). That system also provided visual laser feedback of the outer limits of the NIR detection area as demonstrated in Figure 1B.
Statistical approach and calculations
Normality of data was tested by analyzing skewness and kurtosis profile of data distribution. For parametric data, we used t test for independent samples for comparisons between forehead and xiphoid process surface mean temperatures and one-way ANOVA to compare mean surface temperature at different time points between survivors and non-survivors. Repeated measures ANOVA was used to compare means between IR thermometry and radio telemetry temperature monitoring system. Data were considered statistically significant when P < 0.05.
Sensitivity, specificity, and Youden's Index were determined for surface temperatures varying from 29°C to 31°C, 18 to 36 h postinjection of CS. The sensitivity of the proposed cutoff values was calculated based on the proportion of animals for whom the outcome was positive (i.e., a true positive response was identified when a set of test parameters accurately predicted eventual death within 5 days, as shown in Table 1). A true negative response was when a set of parameters predicted that animals would survive the experiment. A receiver operator curve (ROC) was then created based on the outcome of these analyses. The ROC was fit to an exponential relationship using nonlinear regression software (GraphPad Prism 5). We utilized Youden's Index (J = sensitivity + specificity – 1) as a traditional indicator to decide which value should be used to discriminate between animals according to outcomes (15). We defined the best predictor cutoff as the one displaying the highest Youden's Index (16). We created a Distress Index (Eq. 1) for estimating the accumulated distress and for evaluating the impact of each proposed threshold on the non-surviving animals.
In preliminary studies, we evaluated different locations for Ts measurements and found that Ts measured at the base of the sternum, just over the xiphoid process and heart, was the most reliable location. This location also resulted in a value closest to actual core temperature, an approach utilized previously (10). In one group of animals, we made direct comparisons between Ts measured at the xiphoid versus the forehead (Fig. 2A). The xiphoid measurements provided the most reliable and reproducible measurement.
In another group of animals, we tested xiphoid Ts using two different IR thermometers (Etekcity [IR1] vs. Fluke [IR2]) and against measurements made using implanted radio-telemetry devices in the same animals (Fig. 2B). The core temperatures measured with the two IR thermometers were not significantly different at rest. At rest, average Ts at the xiphoid was ≈2°C lower than core temperature, measured by telemetry. These animals were then tested immediately following a prolonged moderate exercise test in hyperthermia. The measurements with the two contrasting NIR systems were not significantly different from each other, but during exercise-imposed hyperthermia the gradient between xiphoid Ts and core temperature widened to nearly 3.5°C (P < 0.05).
Eighty animals died and 74 survived during the sepsis experiments, for an overall mortality rate of 52%. Regardless of the experimental group, there was a definite pattern to the frequency of deaths occurring over the first 78 h of recovery, with the large majority of animals dying between 30 and 48 h, as shown in Figure 3. Surface temperature profiles of survivors and non-survivors are reported in Figure 4 and follow a clear bimodal distribution between outcomes. The general temperature profiles for wild-type and transgenic males and females, when treated separately, were not different from the overall combined group. Average baseline Ts before CS injection was 34.0 ± 3.0°C and within the first 12 h, post CS injection, fell to 30.9 ± 4.0 and 26.6 ± 1.1°C in survivors and non-survivors, respectively (P < 0.05). For survivors, Ts returned toward baseline within 18 h and remained elevated throughout the remaining experiment. For non-survivors, Ts remained lower throughout the experiment, sometimes rebounding briefly but never fully recovering. Sixty-one non-surviving animals were discovered dead in their cage, while 19 were euthanized after reaching a humane endpoint (lack of righting reflex).
Table 2 reports the individual sensitivity, specificity, and Youden's index for each set of results for proposed humane endpoint criteria at specific measurement times (18—36 h) and threshold temperatures from 29°C to 31°C. The overall outcome of the ROC analysis is summarized in Figure 5A and the calculated range of Youden's Index values is shown in Figure 5B. The highest Youden's index (J = 0.74) was identified at 24 h, with a threshold Ts of ≤30.5°C. This condition predicted mortality with 84% sensitivity and 91% specificity. Other threshold conditions with strong predictive specificity are also highlighted in Table 3 to distinguish desirable endpoint criteria that may be of equal value to Youden's index for survival studies. Comparisons of these candidate endpoint criteria can be found in Table 3, which reports the reductions in stress, based on calculated Distress Index, that would occur in similar cohorts when select temperature and time criteria are chosen for euthanasia. If the condition with highest Youden's index (Ts ≤ 30.5°C at 24 h) was used as the threshold, there would be a 41% reduction in mortality. However, if the threshold Ts ≤ 29°C at 24 h was used, a more accurate estimate of mortality is obtained as specificity is 96%, but with a smaller reduction in Distress Index (31.3%) of the non-surviving mice. Condition Ts ≤ 29°C at 36 h has some advantages in that the specificity is 100% and the prediction of mortality rate is perfect, but the approach would have even less impact on reducing animal suffering (26.5%).
Our results demonstrate that the drop in xiphoid Ts during the first 24 h of recovery from sepsis is a strong predictor of mortality in this murine CS model. We employed a ROC characteristic approach to detect the Ts and time after induction of sepsis that most accurately predicts mortality in this model. This is the first study to refine a predictive marker for mortality in this particular preclinical model of septic shock using IR thermometry.
Early prediction of mortality during survival protocols allows for early euthanasia and reduces the level of suffering throughout the remainder of the protocol. It is thus a more humane approach to obtaining accurate survival characteristics of an animal population. As observed in Figure 3, mortality rates peak at approximately 48 h after CS administration. Based on this figure and Table 3, we calculated estimates of the “cost” in terms of stress exposure and suffering (Distress Index) in the non-surviving animals. When no criteria except lack of righting reflex were used, this study accumulated a Distress Index of 3,120 (mice × hours) in just the non-surviving mice. Note that ideally one would estimate this value for all surviving mice as well, but the surviving mice recover from their infection within 24 to 36 h, making it more difficult to determine the hours that they suffered during the infection.
Refinement of mortality indicators to reduce pain and suffering is not the single advantage of these results. For instance, studies involving the use of CS as a preclinical model of sepsis require survival experiments to determine the dose to be injected in subsequent trials (2). This is extremely costly in both investigator time and animal housing costs. Here, we injected doses ranging from 0.9 mg/g to 1.8 mg/g of body mass in a cohort of 154 mice of both sexes and monitored the animals periodically over the course of 5 days. By employing our proposed threshold at 24 h (Ts ≤ 30.5°C or Ts ≤ 29°C at 24 h, Table 2) researchers can readily estimate mortality rate within 24 h, rather than after many days. For this application, it may be desirable to utilize Ts ≤ 29°C at 24 h, because it has a more ideal specificity (lower number of false positives) and predicted mortality rate, despite a smaller reduction in Distress Index.
Depending on the purpose of the experiment, however, different criteria might be more ideally suited to the needs of the trial. For instance, if the accuracy of the mortality rate is absolutely critical, choosing Ts ≤ 29°C at 36 h and allowing the remaining animals to continue until death might be appropriate in some cases. It still reduces the suffering by approximately 27% and allows investigators to evaluate the shape of the remaining mortality curve. As shown in Figure 6, if the decision on eventual survival is selected at the 24 h time point, additional information is lost in the tail of the actual survival curve. This tail can contain information on treatment effects (i.e., prolonging or shortening life) that may be independent of raw survival. In this case, choosing Ts ≤ 29°C at 36 h might be ideal because the investigator could follow this tail for the remainder of the animals and thus possibly have greater fidelity for treatment effects. Nonetheless, our approach may not be useful if the purpose of the experiment is to prolong survival with a given intervention for longer than 36 h after CS injection.
Our proposed approach to reducing animal suffering and predicting mortality by using NIR surface temperature is specific to the murine model of CS, but it shares some interesting features with previous reports (10, 11, 17). For instance, Trammell and Toth (17) surgically implanted an intra-abdominal radiofrequency transmitter to measure body temperature and to establish its potential to predict mortality in mice exposed to multiple infectious organisms. They studied different mouse strains such as A/J, DBA/2J, C57BL/6J, BALB/cByJ, and monitored body weight over the course of infection. Although no threshold for a humane endpoint was described, hypothermia was the most valuable characteristic for distinguishing mice that would survive or succumb to the infection. Importantly, they suggested that the endpoint threshold should be determined for each specific experimental model. Another study (10) used a NIR surface temperature approach in a fungal infection model in the CD1 strain of mice. They established a cutoff of 33.3°C at any time point that was 97% specific and 68% sensitive as a predictor of death. A recently published investigation suggested a threshold of 23.5°C at 18 h as a predictor of mortality in a murine model of gram-negative bacteria infection (11), which greatly reduced animal suffering in that model. We also know that the temperature profiles of mice that undergo cecal ligation and puncture often show less dramatic and less rapid reductions in temperature, with clear male and female differences (18). However, core temperatures of less than 30°C are commonly reported in severe cecal ligation models (6). Since cecal ligation and puncture is such a common and now well-described procedure, it may be of benefit to establish a similar NIR criterion in that model.
The mechanisms by which hypothermia affects mortality in septic shock in humans and rodents are still unclear, but may involve an impaired host response to infection, which can halt the ability of the immune system to prevent multi-organ damage and/or dysfunction (19). An important aspect to consider is that, in some instances, hypothermia may be considered an adaptive and protective response to sepsis (20). Rodents are endotherms and can regulate their core temperature via metabolism as suggested by a study demonstrating that hypometabolism can prevent hypoxia in rodents undergoing endotoxic shock (21). This could be paradoxically interpreted as hypothermia being an indication of good prognosis in response to infection. Nevertheless, our findings as well as reports of other research groups using different models of infection (10, 11, 17, 22) indicated that hypothermia was a sensitive predictor of morbidity and mortality in response to distinct murine models of sepsis/infection and was an effective, practical, and noninvasive way to reduce animal suffering.
Body temperature of mice can be influenced by a number of factors such as ambient temperature, the time of day, the presence and type of bedding (23), and the number of cage mates (24). Additionally, as previously shown (25, 26), different strains of mice can differ widely with regards to temperature responses to the same experimental model. In our current study, mice were housed individually with corncob bedding. They were also kept at the animal facility for the whole duration of the experiment, which ensured an exposure to a controlled environmental temperature and humidity. Importantly, mice thermoregulatory response to sepsis is influenced by age (27). Therefore, whether our results hold true to groups of animals within a different age span remains to be determined. These considerations must be contemplated when using NIR surface temperature as a predictor of infection severity and mortality in this murine model of CS.
In conclusion, our results support the use of xiphoid Ts as a surrogate marker of infection severity and a predictor of mortality. For most applications, we recommend a cutoff criterion of Ts ≤ 30.5°C at 24 h, which in this study proved an accurate predictor of mortality rate within 4% of actual mortality rate. Most importantly, the use of this or similar criteria can drastically reduce the amount of animal suffering (by 41%) required for estimates of severity of infection, effects of dosages, differences in susceptibility of populations, and the effects of treatment interventions.
1. Wynn JL, Scumpia PO, Delano MJ, O’Malley KA, Ungaro R, Abouhamze A, Moldawer LL. Increased mortality and altered immunity in neonatal sepsis
produced by generalized peritonitis. Shock
28 6: 675–683, 2007.
2. Starr ME, Steele AM, Saito M, Hacker BJ, Evers BM, Saito H. A new cecal slurry preparation protocol with improved long-term reproducibility for animal models of sepsis
. PLoS One
9 12:e115705, 2014.
3. Krarup A, Chattopadhyay P, Bhattacharjee AK, Burge JR, Ruble GR. Evaluation of surrogate markers of impending death in the galactosamine-sensitized murine model of bacterial endotoxemia. Lab Anim Sci
49 5: 545–550, 1999.
4. Shrum B, Anantha RV, Xu SX, Donnelly M, Haeryfar SM, McCormick JK, Mele T. A robust scoring system to evaluate sepsis
severity in an animal model. BMC Res Notes
5. Monroe LL, Armstrong MG, Zhang X, Hall JV, Ozment TR, Li C, Williams DL, Hoover DB. Zymosan-induced peritonitis: effects on cardiac function, temperature regulation, translocation of bacteria, and role of dectin-1. Shock
46 6: 723–730, 2016.
6. Ebong S, Call D, Nemzek J, Bolgos G, Newcomb D, Remick D. Immunopathologic alterations in murine models of sepsis
of increasing severity. Infect Immun
67 12: 6603–6610, 1999.
7. Soothill JS, Morton DB, Ahmad A. The HID50 (hypothermia
-inducing dose 50): an alternative to the LD50 for measurement of bacterial virulence. Int J Exp Pathol
73 1: 95–98, 1992.
8. Wong JP, Saravolac EG, Clement JG, Nagata LP. Development of a murine hypothermia
model for study of respiratory tract influenza virus infection
. Lab Anim Sci
47 2: 143–147, 1997.
9. Kort WJ, Hekking-Weijma JM, TenKate MT, Sorm V, VanStrik R. A microchip implant system as a method to determine body temperature of terminally ill rats and mice. Lab Anim
32 3: 260–269, 1998.
10. Warn PA, Brampton MW, Sharp A, Morrissey G, Steel N, Denning DW, Priest T. Infrared body temperature measurement of mice as an early predictor of death in experimental fungal infections. Lab Anim
37 2: 126–131, 2003.
11. Gavin HE, Satchell KJF. Surface hypothermia
predicts murine mortality in the intragastric Vibrio vulnificus infection
model. BMC Microbiol
17 1:136, 2017.
12. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol
8 6:e1000412, 2010.
13. McCarthy JJ, Srikuea R, Kirby TJ, Peterson CA, Esser KA. Inducible Cre transgenic mouse strain for skeletal muscle-specific gene targeting. Skelet Muscle
2 1:8, 2012.
14. King MA, Leon LR, Mustico DL, Haines JM, Clanton TL. Biomarkers of multiorgan injury in a preclinical model of exertional heat stroke. J Appl Physiol (1985)
118 10: 1207–1220, 2015.
15. Youden WJ. Index for rating diagnostic tests. Cancer
3 1: 32–35, 1950.
16. Schisterman EF, Perkins NJ, Liu A, Bondell H. Optimal cut-point and its corresponding Youden Index to discriminate individuals using pooled blood samples. Epidemiology
16 1: 73–81, 2005.
17. Trammell RA, Toth LA. Markers for predicting death as an outcome for mice used in infectious disease research. Comp Med
61 6: 492–498, 2011.
18. Leon LR, White AA, Kluger MJ. Role of IL-6 and TNF in thermoregulation and survival
in mice. Am J Physiol
275 (1 pt 2):R269–R277, 1998.
19. Remick DG, Xioa H. Hypothermia
. Front Biosci
11: 1006–1013, 2006.
20. Lilley E, Armstrong R, Clark N, Gray P, Hawkins P, Mason K, Lopez-Salesansky N, Stark AK, Jackson SK, Thiemermann C, et al. Refinement of animal models of sepsis
and septic shock. Shock
43 4: 304–316, 2015.
21. Corrigan JJ, Fonseca MT, Flatow EA, Lewis K, Steiner AA. Hypometabolism and hypothermia
in the rat model of endotoxic shock: independence of circulatory hypoxia. J Physiol
592 17: 3901–3916, 2014.
22. Miao P, Kong Y, Ma Y, Zeng H, Yu Z. Hypothermia
predicts the prognosis in colon ascendens stent peritonitis mice. J Surg Res
181 1: 129–135, 2013.
23. Gordon CJ. Effect of cage bedding on temperature regulation and metabolism of group-housed female mice. Comp Med
54 1: 63–68, 2004.
24. Gordon CJ, Becker P, Ali JS. Behavioral thermoregulatory responses of single- and group-housed mice. Physiol Behav
65 2: 255–262, 1998.
25. Stiles BG, Campbell YG, Castle RM, Grove SA. Correlation of temperature and toxicity in murine studies of staphylococcal enterotoxins and toxic shock syndrome toxin 1. Infect Immun
67 3: 1521–1525, 1999.
26. Toth LA, Hughes LF. Sleep and temperature responses of inbred mice with Candida albicans-induced pyelonephritis. Comp Med
56 4: 252–261, 2006.
27. Saito H, Sherwood ER, Varma TK, Evers BM. Effects of aging on mortality, hypothermia
, and cytokine induction in mice with endotoxemia or sepsis
. Mech Ageing Dev
124 (10–12):1047–1058, 2003.
Keywords:© 2018 by the Shock Society
Hypothermia; infection; infrared thermometry; ROC curve; sepsis; survival