Sepsis remains a public health problem of epidemic proportions. Nearly a million cases of sepsis are diagnosed each year with a mortality similar to coronary artery disease (1). The Centers for Disease Control (CDC) issued an announcement in 2016 that sepsis should be considered a medical emergency (2). The Agency for Health Care Quality published a statistical report on the National Inpatient Hospital Costs in 2016 documenting that sepsis was the most expensive in-hospital condition (3). This report showed that in 2013 the costs of treating sepsis were more than $23 billion and accounted for 6.2% of the total costs of healthcare in US hospitals. While it is true that in-hospital mortality declined, the long-term morbidity and mortality can only be described as abysmal (1, 4, 5).
Given this background there was enthusiasm when Marik et al. (6) reported in 2017 that treatment with hydrocortisone, ascorbic acid and thiamine (HAT), dramatically reduced sepsis mortality in patients. This small study generated substantial interest and there are currently more than a dozen clinical trials recruiting patients (registered in clinicaltrials.gov as of May, 2019). While the paper has been cited 36 times in the literature, there are no papers published using HAT therapy in a preclinical sepsis model which could examine potential mechanisms at the time of this writing. It has been postulated that the improved survival with HAT therapy is due to reduced oxidative stress (7).
To address these issues, the efficacy of HAT therapy was tested using the well-described murine model of sepsis induced by cecal ligation and puncture (CLP). Previous reports established that noninvasive physiologic measurements may be used to monitor the response to therapy in this model (8, 9). An innovative approach of stratifying the mice based on cardiovascular parameters into predicted to live or die was used to randomize mice to receive HAT treatment or vehicle. The current study examined if HAT therapy would reduce mortality and determine potential mechanisms by measuring oxidative stress and cardiovascular parameters. Stratification of sepsis patients, in effect a precision medicine approach, was recently advocated (10), validating the clinical significance of the proposed studies.
Mice and chemicals
Female, adult outbred ICR mice, 22 to 25 g were purchased from Envigo (Indianapolis, Ind). Hydrocortisone, ascorbic acid, and thiamine were obtained from Sigma-Aldrich (St. Louis, Mo).
The standard CLP sepsis model (11) was used with minor modifications (8). The studies were conducted over 10 months by performing CLP on groups of 10 mice each week. Following recommendations from our Institutional Animal Care and Use Committee (IACUC), buprenorphine (0.05 mg/kg) was given 20 min prior to surgery. After isoflurane anesthesia, mice were subjected to CLP. To obtain 40% mortality over 7 days, mice were randomized and half the mice had the cecum punctured with a 16-gauge needle and the other half with an 18-gauge needle. Immediately following surgery mice were resuscitated with 1.0 mL of warmed normal saline. Analgesia was provided with buprenorphine (0.05 mg/kg) which was continued every 12 h for a total of 5 doses. Antibiotic therapy was provided with imipenem (25 mg/kg, Merck, West Point, Pa) in Lactated Ringers with 5% dextrose starting 2 h after CLP and continued every 12 h for a total of 6 doses. The second dose of buprenorphine was included with the imipenem. These studies were approved the Boston University IACUC and follow the ARRIVE guidelines.
Experimental design—survival studies
Prior to CLP, baseline information was collected from individual mice, to follow the recent recommendations concerning preclinical sepsis models (12). Mice were weighed, a small portion of the abdominal fur shaved to allow accurate recording of body temperature using an FDA-approved infrared body temperature thermometer (Model Temp1, TempIR). Physiologic measurements were obtained noninvasively with a cervical collar (MouseOx Plus, Starr Life Sci, Oakmont, Pa) that collects heart rate, respiratory rate, O2 saturation, and pulse distension. Pulse distension measures the increase in the size of the cervical blood vessel which becomes distended with each heart beat and may be considered a surrogate for cardiac output. To ensure data reproducibility, after obtaining a stable signal, data were collected five separate times for 5 s each, and the results averaged. For these survival studies, CLP was performed and physiologic parameters measured at 6, 24, and 48 h post CLP. Blood was collected from the facial vein for hematologic analysis, after the physiologic parameters were measured. Mice were monitored for survival twice per day for at least 7 days post CLP. Euthanasia was performed if the body temperature was less than 30°C or the animals failed to right after being placed in a recumbent position.
Experimental design—sample collection
Mice were sacrificed with an overdose of isoflurane and blood collected from the retro orbital venous plexus into EDTA tubes containing 50 μL of 169 mM K3EDTA. The peritoneum was opened and washed with 10 mL of Hanks Balanced Salt Solution (HBSS, Mediatech, Manassa, Va) with 10 U/mL Heparin (McKesson Packaging, Irving Tex) as described in our previous publication (9). Briefly, the collected fluid was filtered through a 70 μm nylon filter (BD Biosciences, San Jose, Calif) to remove debris. The filtered peritoneal fluid was centrifuged at 450 × g for 5 min to isolate cell pellets. The resuspended cell pellets were counted using a Beckman-Coulter particle counter model ZF (Coulter Electronics, Hialeah, Fla) and cell numbers adjusted to 2 × 107 cell/mL in ice-cold PBS containing protease inhibitor cocktail (Complete, Roche Diag, Manheim, Germany) and 0.05% Triton X-100 (Sigma). Three 10-s rounds of homogenization and sonication were followed by centrifugation at 15,000 × g for 15 min at 4°C. Total protein concentration in the supernatant was quantified by the Bradford assay using Coomassie Plus protein assay reagent (Thermo Sci, Rockford, Ill) as described in manufacturer's guide. The protein concentration of each supernatant was adjusted to 10 μg/mL for analysis for oxidative stress markers.
Oxidative stress markers
Oxidative stress was measured in the exudative cells using ELISA kits. Lipid peroxidation was measured by ELISA measuring 8-isoprostaglandin F2α (Cell Biolabs, STA-337-T, San Diego Calif) and protein oxidation by protein carbonyl derivatives by ELISA (Cell Biolabs, STA-310-T).
Hydrocortisone, ascorbic acid, and thiamine (HAT) doses were determined based on the original treatment protocol for patients (6). Specifically, hydrocortisone: 1.5 mg/kg, vitamin C: 45 mg/kg and thiamine: 3 mg/kg. The drugs were dissolved in 1 mL of Lactated ringer's solution and given every 12 h for 96 h subcutaneously for a total of 8 doses. The controls received the vehicle (Lactated Ringers). The compounds were included in the analgesia and antibiotic subcutaneous injections.
Physiologic data were collected at 6 and 24 h post CLP. The mice were divided into two groups based on the actual status of the animal (alive or dead) on day 4 post-CLP. The day of CLP was considered to be day 1. The physiology data were analyzed by calculating the area under the curve of the receiver operator characteristic (AUC-ROC) for heart rate (HR), pulse distension (PD), respiratory rate (RR), and oxygen saturation.
All statistical analysis was performed with Prism Version 8 (Graphpad, San Diego, Calif). Differences in survival were determined using Log-Rank survival analysis. Differences between groups were analyzed using unpaired t tests while changes in parameters between time points for individual animals were analyzed using a paired t test. A two-way ANOVA was used to determine the effect of time on different parameters. A one-way ANOVA with multiple comparisons was used to determine differences between the four different groups after HAT treatment. The statistical tests used are described in the figure legends.
CLP was performed on 59 mice and the mortality over 7 days was 46% (Fig. 1), similar to previous publications (13, 14), and demonstrating the ability to reproduce a standardized sepsis model (15).
Decline in physiological and hematological parameters after CLP
Physiologic parameters including heart rate (HR), pulse distension (PD), respiratory rate (RR), and O2 saturation were measured using a noninvasive cervical collar as previously described (8, 9). These parameters were used to characterize cardiac and pulmonary organ function\injury. Measurements were taken at baseline, prior to CLP, as well as 6 and 24 h post CLP and survival was monitored for at least 7 days. The data were then grouped based on the survival of the mouse on day 4 post CLP since prior work demonstrates that the early measurements predict survival during the first 1 to 4 days post CLP sepsis (14, 16). Figure 2 shows that there were no differences between the dead (D) and alive (A) groups in any of the parameters at baseline, i.e., prior to CLP. Within 6 h after CLP the mice who will die have a significantly lower heart rate than those who will survive (Fig. 2A). It should be noted that the mice are grouped based on their actual status at day 4 (dead or alive). The PD showed similar, early declines between the dead and alive groups (Fig. 2B). The RR was significantly different at 6 h but not 24 h (Fig. 2C). Similar to prior reports, there was no change in O2 saturation (Fig. 2D) (17).
To further highlight the decline in organ function, the data were plotted as the change from baseline for each individual mouse. Figure 3A shows the change from baseline to 6 h for the mice who die while Figure 3B has a similar plot for the mice who survive. It is apparent that the decline was greater in those who would subsequently die. The decrease in HR (Fig. 3C), PD (Fig. 3D), and RR (Fig. 3E) was significantly greater for each parameter in the mice who would die.
In septic patients who die there is a progressive increase in organ injury. The HR, PD, and RR were also measured at 24 h post CLP and that data analyzed based on the actual mortality at day 4. The data from Figure 2 were regraphed to better illustrate these changes. Figure 4A shows that the HR continued to decline in those mice who would die. The PD stabilized although it remained low and was significantly different 24 h post CLP in the mice who would die (Fig. 4B). In contrast, the RR recovered in the mice that would die and was not different than those that would live (4C). These data show that mice that would die have progressively decreasing HR. By two-way ANOVA there was a significant interaction between time and survival status for all three measured parameters.
Previous reports have shown that mice that die in the first few days of sepsis have higher lymphocyte counts (14) and lower body temperature (18). To demonstrate the reproducibility of the model (15), the absolute lymphocyte count and body temperature were measured. The body temperature was lower at 6 and 24 h in the mice that would die (Fig. 4D). The lymphocyte count was also lower 24 h post-CLP in the mice that would live (1.01 ± 0.18 Die-P vs 0.45 ± 0.06, Live-P, mean ± SEM) confirming the prior observations. Plasma levels of IL-6 were also increased in the Die-P mice (data not shown).
Physiological parameters that predict early mortality
The data were then analyzed to determine if any of the physiology parameters would predict mortality. It should be noted that HR and RR predict clinical deterioration in the Modified Early Warning Score (MEWS (19)), the National Early Warning Score (NEWS (20)), and the original description of the Systemic Inflammatory Response Syndrome (SIRS (21)). Oxygen saturation was not evaluated since there were never any differences between the alive or dead groups in the current study (Fig. 2D) or previous reports (17). The data were first analyzed by calculating the area under the curve for each parameter at both 6 and 24 h post CLP. The highest AUC-ROC was determined to be the heart rate measured 6 h after CLP (Fig. 5A). The ROC curves from the 6 h post-CLP data were then analyzed to determine the optimal discrimination values to differentiate mice alive at day 4 from those dead at day 4, and the mortality curves of the different groups plotted. Figure 5B compares the mortality for mice with a HR >620 beats per minute (bpm) 6 h after CLP to those with an HR below 620 bpm. Those mice with a lower HR have significantly greater mortality. The discrimination value for PD was 300 μm and for RR it was 150 breaths per minute. The mortality curves for these parameters also showed that mice with a lower 6 h PD had higher mortality (Fig. 5C). Mice with a lower RR also had greater mortality (Fig. 5D). The 6 h HR was used to predict mortality in the subsequent studies, since this parameter had the best AUC-ROC (Fig. 5A). Using the 6 h time point also allows detection of sepsis earlier than the 24 h time point, which increases the time when therapy may be initiated.
Increased oxidative stress in mice predicted to die
Oxidative stress has been suggested as a potential mechanism for the increased organ injury and mortality in sepsis (22). The site of active infection, the peritoneum, should have increased oxidative stress as the recruited inflammatory cells attempt to eradicate the bacteria. We hypothesized that mice predicted to die, based on a lower heart rate, would have greater oxidative stress compared to those predicted to live. HR was measured 6 h after CLP and mice were sacrificed 24 h post-CLP. The peritoneal exudative cells were harvested and oxidative stress measured in these cells. Two separate indices of oxidative stress were measured, 8-isoprostaglandin F2α to quantify lipid peroxidation and protein carbonyl derivatives to measure protein oxidative stress. Since the mice were sacrificed the data were divided into those predicted to die (Die-P) and those predicted to live (Live-P). There was a significant increase in both indices of oxidative stress in the Die-P mice (Fig. 6).
The data demonstrate that CLP induces significant physiologic deterioration in those mice that will die within the first 4 days of sepsis which correlated with increased measures of oxidative stress in the peritoneal exudative cells. These data provide the premise for the subsequent studies. HAT treatment improved survival in a small study with sepsis patients (6) and it has been proposed that the improved survival is due to reduced oxidative stress (7). We tested this hypothesis with an innovative experimental design.
HAT reduces oxidative stress
Mice were subjected to CLP and the heart rate measured 6 h later. Mice with a HR less than 620 beats per minute (bpm) were stratified into the Die-P group while those with an HR >620 bpm were categorized as Live-P. After stratification mice in each group were randomized to receive HAT therapy or vehicle in a 1:1 ratio. Effectively this created four groups: Die-P treated with HAT, Die-P receiving only vehicle, Live-P treated with HAT, and Live-P receiving only vehicle. HAT therapy was initiated within an hour of measuring heart rate, i.e., approximately 7 h after CLP. Groups of mice were sacrificed 24 h after CLP to measure 8 isoPG F2α or protein carbonyl derivatives as measures of oxidative stress. Figure 7 demonstrates that HAT therapy significantly decreased cellular levels of 8 isoPG and protein carbonyl derivatives in the Die-P mice. HAT treatment did not alter these levels in the Live-P mice. It is important to note that therapy was not only delayed until after the onset of sepsis, HAT therapy was initiated after the mice had been stratified into a predicted high mortality group.
HAT improves HR in Die-P mice
Heart rate is one of the parameters used to predict mortality in patients (19–21) and HR deteriorated between 6 and 24 h in the mice predicted to die (Fig. 4A). Mice were stratified based on HR at 6 h, treated and then HR measured again at 24 h. This experiment specifically sought to determine if HAT therapy would prevent the decline in HR in those mice predicted to die. Figure 8 shows an increased HR in the mice treated with HAT therapy compared to the vehicle group. The data also show that the HR in the vehicle treated Die-P mice was significantly lower than the HR in the Live-P mice, reproducing the data in Figure 4. HAT therapy had no effect on the HR in the Live-P group.
The primary goal of the experiments was to determine if HAT therapy would improve survival. Mice were subjected to CLP, stratified on the basis of HR and randomized to HAT therapy or vehicle. Therapy was initiated approximately 7 h after the onset of sepsis. We first examined if HAT therapy would improve survival without stratification. A total of 79 mice were studied and HAT did not significantly change survival (Supplemental Figure 1, Supplemental Digital Content, https://links.lww.com/SHK/A898). However, in those mice predicted to die, HAT significantly reduced mortality (Figure 9A). In contrast, in those mice predicted to live there was no change in survival (Fig. 9B).
The current studies were based on the premise that hydrocortisone, ascorbic acid, and thiamine (HAT) therapy would improve survival in murine sepsis, which would replicate the small clinical trial (6). If the clinical results could be reproduced, then the animal model could be used to examine potential mechanisms. The mortality in the experimental model (40%) was similar to that observed in the clinical trial, adding to the significance of the current report. An innovative experimental design was used where physiologic measurements stratified the mice into those predicted to die and those predicted to live. The current studies identified that a decline in heart rate after sepsis predicts subsequent mortality. In the present manuscript we showed that the actual heart rate predicted mortality and HR was used to stratify mice. The actual HR is more clinically relevant rather than the change from baseline since many patients will not have baseline data. For example, an emergency room patient presenting with sepsis may not have a baseline HR.
Clinical studies have shown that early myocardial dysfunction predicts sepsis mortality (23, 24). Cardiac depression after CLP-induced sepsis has been well documented with a reduction in the ejection fraction within 24 h (25) and decreases in HR (26). In one study, CLP induced significant impairment of cardiac function (27) within 6 h. In another study, CLP-induced sepsis resulted in a decreased heart rate in mice, from 595 ± 96 to 461 ± 86 within 24 h as measured with intravascular catheters (28), results similar to the current manuscript. However, it should be noted that another report did not show any reduction in HR in CLP mice, although there was a decrease in mean arterial pressure (29).
In a rat fecal peritonitis model with a mortality similar to our study, changes in the heart rate within the first 4 h predicted death (30). The mortality was 78% in rats with an increase of 50 bpm compared to 7% in those with less than 50 bpm increase. The change in heart rate in the non-survivors was significantly different within 4 h after the induction of sepsis with an AUC for the ROC at 6 h was 0.732.
Animal studies allow careful examination of potential mechanisms that drive disease, since tissues may be harvested for in-depth investigation. Oxidative stress (OS) has been suggested as a mechanism for causing organ injury and mortality in sepsis (22) and septic patients with decreased plasma antioxidants have greater mortality (31, 32). It has been postulated that the mechanism of how HAT therapy works is through reducing OS (33). The current studies show that HAT therapy significantly reduced oxidative stress in the peritoneal exudative cells, providing support for this hypothesis. Future studies should be performed to more fully define the oxidative stress parameters and determine if oxidative stress is the mechanism responsible for the decline in heart rate.
An important innovation in the current study is the stratification of mice based on sepsis physiology, using parameters that predict mortality in patients (19–21). Without stratification, HAT therapy did not improve survival. A sample size analysis using the unstratified data showed that an additional 23 mice would have been required to show an improvement in survival. The current study reduced the number of animals necessary to achieve the major goal of the study, consistent with the ARRIVE guidelines.
There are limitations to the current study. We measured oxidative stress in the peritoneal cells but did not measure these parameters in the injured organ, the heart. Further studies will need to explore if there is increased oxidative stress in the heart and myocardial damage. Only female mice were used in this initial study and the findings will need to be extended to male mice. Finally, recent recommendations advocate for collecting substantial amounts of data from each experiment (12, 34, 35). Additional data have been collected and will be analyzed in future papers.
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