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Basic Science Aspects

INCREASED MORTALITY AND ALTERED IMMUNITY IN NEONATAL SEPSIS PRODUCED BY GENERALIZED PERITONITIS

Wynn, James L.*†; Scumpia, Philip O.*; Delano, Matthew J.*; O'Malley, Kerri A.*; Ungaro, Ricardo*; Abouhamze, Amer*; Moldawer, Lyle L.*

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doi: 10.1097/shk.0b013e3180556d09
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Abstract

INTRODUCTION

Sepsis is defined as the systemic inflammatory response to a microbial infection (1). Severe sepsis, characterized by hypotension, inadequate tissue perfusion, and organ dysfunction (2), is associated with increased mortality in all age populations (3) but is 10 times more likely to occur in infants compared with older children (4). Neonatal sepsis is a common occurrence in premature and term infants affecting up to 1 in 200 live births (5, 6), with subsequent mortality approaching 30% (5, 7, 8). Surprisingly, advances for the past 20 years in antimicrobials and pulmonary and circulatory support have not significantly improved survival in severe sepsis. The lack of any dramatic improvements in outcome to severe sepsis in neonates or adults is in large part because of the lack of understanding of the pathophysiology of sepsis and the resultant immune responses in vivo. Known deficits in the innate and adaptive immune response in neonates have been characterized primarily using in vitro studies because of, in large part, the absence of a well-accepted neonatal animal model of sepsis.

Bacterial peritonitis produced by cecal ligation and puncture (CLP) is widely used in young adult rodents to model generalized sepsis seen clinically after bowel perforation (9, 10). However, CLP is technically challenging in the neonatal mouse secondary to its small size, gut friability, and the increased risk for maternal cannibalization after the procedure. Cecal ligation and puncture has also been associated with a large degree of undesired investigator variability because of the required size and number of enterotomies and location of cecal ligation. An alternative model was originally proposed in adult pigs, the cecal slurry method (11), which consists of an intraperitoneal injection of cecal contents suspended in dextrose. Additional studies in adult rats have shown that this model recapitulates the time course and many of the physiological changes associated with human clinical sepsis, namely hypotension, initial and prolonged elevation in lactate, leukopenia with subsequent leukocytosis, and continuation of the septic process through formation of abdominal abscesses (12). Although all animal models have their limitations with respect to the human condition (13), they can be powerful research tools to better characterize the immunological disturbances in sepsis in vivo and to explore new therapeutic options. The current study was undertaken to validate cecal slurry as a model of polymicrobial sepsis in the murine neonate, with a subsequent characterization of its inflammatory and cellular immune responses. The murine neonatal response to cecal slurry was compared with the sepsis response seen in young adult mice after both cecal slurry administration and CLP. The results clearly demonstrate that cecal slurry can produce dose-dependent mortality in both neonatal and young adult mice and is associated with a systemic inflammatory response and alterations in splenic leukocyte populations. Neonatal mice were more sensitive to cecal slurry-induced mortality than young adults and showed significant differences in their immunological response. In particular, neonatal mice had a markedly attenuated systemic inflammatory response and did not exhibit the well-described loss of splenic CD3+CD4+ T cells seen in septic adult mice.

MATERIALS AND METHODS

Mice and monitoring

All studies were approved by the Institutional Animal Care and Use Committee at the University of Florida, College of Medicine before their initiation. The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals. Specific pathogen-free male and female C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, Me) between 6 and 10 weeks of age and allowed a minimum of 7 days to equilibrate to their environment before any experimental use. Mice were maintained on standard rodent food and water ad libitum. To generate neonatal mice, paired matings were established twice weekly, and females were isolated from males as soon as they were visually identified as pregnant and followed closely thereafter, so an accurate date of birth was recorded. Litters aged 5 to 7 days were used for experimental procedures and were defined as neonates (14, 15).

Study designs

Sepsis was induced in young adult female (age range, 7-10 weeks) and mixed sex (sexual dimorphism not present in prepubertal neonates) neonatal mice using two methods: CLP and cecal slurry (described below). Adult mice underwent either CLP or cecal slurry, whereas neonates underwent cecal slurry alone. Cecal ligation and puncture could not be performed on neonatal mice because of their small size, incomplete intestinal development, and postoperative cannibalization by their dams. All experimental sepsis groups were matched to sham-treated animals that had undergone identical procedures without cecal slurry administration or ligation and enterotomies in the cecum. Neonates were compared with age-appropriate control and sham animals. In response to each model of sepsis, one group of animals was followed for long-term survival (5 days), whereas a second group was killed at timed intervals for blood cytokine, bacteremia, and cell phenotype measurements. Groups of adult and neonatal mice were euthanized at 2, 12, and 24 h, and their blood and tissues underwent further processing as discussed below. All experiments were performed by the same investigator.

Pregnant dams were prepared for interruption of their natural postpartum routine by gentle handling for 3- to 5-min intervals once daily-2 to 3 days before delivery, placing a cotton ball with isopropyl alcohol in the cage for 1 to 2 min per day before delivery, and placing chow in the cage after delivery to decrease cannibalization.

Cecal slurry-induced generalized peritonitis

Polymicrobial sepsis was induced in both adult and neonatal mice using a modification of the cecal slurry method, as described by Sam et al. (12). Briefly, an adult C57Bl/6 female mouse aged 7 to 10 weeks was killed, and a midline laparotomy was performed to isolate the cecum. Using scissors, the cecum was opened at its most distal point, and the cecal contents were expressed and weighed. A suspension of the cecal contents and 5% dextrose were used for injection with a final concentration of 400-mg cecal contents per 5 mL. The cecal slurry was briefly vortexed before injection to create a homogenous suspension and was used within 2 h of preparation. For each experiment, new cecal slurry was prepared from an adult donor. The abdomen was prepared with 70% ethanol before injection. Adult and neonatal animals were gently restrained and received intraperitoneal administration of varying doses of the suspension based on the body weight. Using this method, a dose-response curve was created in adult and neonatal mice, and an approximate LD70 dose was subsequently used for inducing sepsis. Sham mice of the same age received an identical intraperitoneal volume of 5% dextrose and were killed at the same time intervals or followed for survival.

For all experimental and sham procedures, neonates were removed from their mothers as a group and placed on a warming blanket at 37°C. After the procedures were performed, neonates were returned as a group to their mothers. Adult mice were returned to their respective cages after the experiment. All mice were closely monitored for signs of sepsis. In adult mice, signs of sepsis consisted of lethargy, hunching posture, piloerection, and hypothermia. According to the Institutional Animal Care and Use Committee requirements, adult mice were euthanized if they became moribund (as defined by an inability to right themselves when placed on their backs or sides). In neonatal mice, signs of impending death that required euthanasia included scattering (positioned away from remainder of litter) or absence of milk in the stomach (failure to feed). Our past experience has been that either of these symptoms indicates that the pup is sufficiently moribund that it can neither feed nor obtain maternal warmth and will die within hours. All septic mice were monitored every 6 h for the first 2 days after the procedure, then every 8 h on the third day, and then daily thereafter. Mice that were euthanized for animal welfare concerns were considered to be nonsurvivors.

Cecal ligation and puncture

Adult animals undergoing CLP were briefly anesthetized with isoflurane. For induction of polymicrobial sepsis, mice were subjected to CLP, as previously described (16, 17). In brief, the abdomen was prepared with 70% ethanol, and a midline laparotomy was performed. The cecum was isolated, ligated 1 cm from the distal end, and punctured through and through with a 22-gauge needle; then, the abdomen was closed with staples in a single layer. This procedure resulted in an approximate LD70 lethality at 7 days. Sham-treated animals had the same procedure except for the ligation and puncture.

Blood cytokine analyses and bacteremia

Whole blood was obtained at sacrifice for the purpose of determining plasma cytokine concentrations and bacterial growth. Whole blood was obtained via intracardiac puncture using a heparinized syringe and a 25-gauge needle while the animal was under deep isoflurane anesthesia. The animals were killed immediately after the blood was obtained. Whole blood was centrifuged at 5,000 rpm for 5 min, and individual (not pooled) plasma samples were evaluated for cytokine concentrations. Plasma cytokine levels were measured using Luminex technology via a multiplex assay for 21 cytokines (Beadlyte Mouse 21-Plex Multi-Cytokine Beads; Upstate Cell Signaling Solutions, Charlottesville, Va). Cytokines evaluated using the 21-plex included interleukin (IL) 1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p40, IL-12p70, IL-13, IL-17, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN) γ, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1β, regulated on activation, normal T expressed and secreted (RANTES), tumor necrosis factor (TNF) α, and vascular endothelial growth factor (VEGF).

Bacterial colonization was determined by homogenizing lungs and spleens from neonatal animals that underwent cecal slurry and sham procedures (18). Briefly, a midline thorax and abdominal incision was performed, and the spleen and lungs were removed. The organs were briefly immersed in 70% ethanol to kill any bacteria on the organ surface; the organs were then disrupted using a 70-μm pore-size cell strainer (Falcon type), and the contents were suspended in 300 μL of sterile phosphate-buffered saline. Fifty microliters of the suspension were plated on each side of eosin-methylene blue/sheep's blood agar split plates and incubated at 37°C for 36 h. Whole blood samples (50 μL) were diluted 1:1 with sterile phosphate-buffered saline, plated, and incubated in the same manner. Colonies were counted, and results are reported in colony-forming units (CFU) per milliliter of blood or organ suspension. For bacterial growth results greater than 6,000 CFU/mL, the value 6000 CFU/mL was used for statistical analyses.

Splenocyte cell phenotypes

Spleens were harvested at sacrifice, and single-cell suspensions were created by passing the cells through a 70-μm pore-size cell strainer (Falcon type). Contaminating erythrocytes were lysed with an ammonium chloride lysis solution. After washing twice with buffer (1% bovine serum albumin and 1 mM EDTA [Fisher, Pittsburg, Pa]) and 0.1% sodium azide (NaN3; Sigma-Aldrich, St. Louis, Mo) in Hanks' buffered salt solution without phenol red, calcium, and magnesium (Cellgro; Mediatech, Herndon, Va), cells were resuspended in 4% Hanks' azide buffer (Hanks' buffered salt solution without calcium, magnesium, or phenol red, with 4% bovine serum albumin, 0.1% sodium azide, 0.2% anti-CD16/32, and 1 mM EDTA) and then stained. All antibodies were purchased from BD Pharmingen except the anti-plasmacytoid dendritic cell Ag-1 (PDCA-1) antibody, which was purchased from Miltenyi Biotec (Auburn, Calif). Dendritic cells were characterized using anti-CD11c conjugated to allophycocyanin, anti-PDCA-1 conjugated to phycoerythrin, and anti-B220 or anti-CD11b conjugated to allophycocyanin. Natural killer (NK) cells were characterized using anti-NK1.1 conjugated to fluorescein isothiocyanate, anti-DX5 conjugated to allophycocyanin, and anti-CD69 conjugated to phycoerythrin that were used as a measure of NK activation. Myeloid suppressor cells were characterized using anti-CD11b conjugated to fluorescein isothiocyanate, anti-CD31 conjugated to phycoerythrin, and anti-Gr1 conjugated to allophycocyanin. The T cells were characterized using anti-CD4 conjugated to allophycocyanin, anti-CD8 conjugated to phycoerythrin, and anti-CD3 conjugated to fluorescein isothiocyanate. Regulatory T cells were characterized using anti-CD4 conjugated to fluorescein isothiocyanate (BD Pharmingen), anti-CD25 conjugated to phycoerythrin (eBioscience, San Diego, Calif), and intracellular anti-FoxP3 conjugated to allophycocyanin (eBioscience). Intracellular FoxP3 staining was performed per protocol using the eBioscience intracellular FoxP3 staining kit. Cell samples were acquired and analyzed on one of two six-parameter FACSCalibur machines with CellQuest software (BD Biosciences) at the University of Florida Flow Cytometry Core Laboratory. In those cases where clearly identified cell populations could be obtained (CD8+ T cells), gates were established to capture identical cell populations. In cases where gating did not yield defined populations (NK cells), congruent gates were used. A minimum of 3 × 104 nondebris live cells (7-aminoactinomycin D) were used for analysis.

Statistics

Cytokine concentrations and leukocyte phenotypes were compared using Student t test. If the descriptive analyses failed normality or tests of equal variance, the Mann-Whitney U test was performed. Flow cytometry results are reported as the mean ± SD. For values failing tests of normality, median and range of (maximum/minimum) values are presented.

RESULTS

Dose-response and kinetics of survival

To determine whether the cecal slurry method mimicked the increased susceptibility of neonates to sepsis mortality, we compared the dose-response and survival curves in neonatal and adult mice in whom cecal slurry was administered. Reproducible dose-dependent increases in mortality rate were seen in both adult and neonatal animals in whom increasing quantities of the cecal slurry were administered (Fig. 1A). The calculated LD50 for neonates was significantly less than that for the young adult mice (confidence intervals [CIs] did not overlap-see Figure 1A). Figure 1B shows the timing of death in neonatal and adult mice after an approximate LD70 dose of the cecal slurry administration. Adult animals received 1.5 mg/g body weight of cecal slurry, whereas neonates received 1.3 mg/g to produce comparable 70% lethality. As shown, mortality produced by cecal slurry developed for several days after injection in both groups in a pattern consistent with an ongoing septic process. During necropsy, neonatal and adult animals exhibited abdominal abscesses similar to those seen after a CLP (data not shown). Mortality was reproducible, and results shown in Figures 1, A and B are representative of multiple experiments. Animals that received dextrose via intraperitoneal injection as a control for the cecal slurry did not exhibit any mortality, nor did animals that received an identical volume of heat-killed slurry (80°C for 30 min) (data not shown).

Fig. 1
Fig. 1:
A, Dose-response for survival in neonates (▪) and young adults (♦) after administration of the cecal slurry (n = 5-10 per group at each dose; survival was followed for 5 days in each group). The calculated LD50 in the neonates and the adults was 1.085 mg/g (CI, 1.000-1.190 mg/g) and 1.342 mg/g (CI, 1.271-1.417 mg/g), respectively, which were statistically significant. B, Kaplan-Meier survival curve for neonates (▪) and adults (♦) after administration of an approximate LD70 quantity of cecal slurry (1.3 mg/g for neonates [n = 30], 1.5 mg/g for young adults [n = 15]). Mortality after the cecal slurry administration was consistent with a septic process, and the time course was similar to the survival curve produced by CLP (data not shown).

Bacteremia

To assess for the presence of bacteremia and organ colonization after administration of an approximate LD70 dose of the cecal slurry, neonatal animals were killed at specific time intervals to determine the degree of blood and organ colonization. As seen in Figure 2, bacteremia was observed at 2 h after injection of cecal slurry. Blood bacterial counts peaked at the 12-h sampling time, with concurrent evidence of organ colonization in both the spleen and the lung. Organ colonization followed the increase in organisms recovered from the blood and was sustained after bacterial levels in the blood had declined. Use of split plates (eosin-methylene blue and sheep's blood) allowed for the partial identification of aerobic gram-negative organisms. From each source (blood, lung, and spleen), isolated colonies exhibited a trend toward increased numbers of aerogenes-type gram-negative organisms, with increasing time after cecal slurry administration (data not shown).

Fig. 2
Fig. 2:
Bacteremia and organ colonization in neonates after administration of an LD70 dose of cecal slurry. Solid bars represent medians with rectangles representing quartiles (75%/25%). Blood colonization (n = 5) was detectable 2 h after cecal slurry administration and increased with time until the 24-h sampling. Lung (n = 5) and spleen (n = 5) colonization were delayed when compared with the measurements in blood and persisted at elevated concentrations for 24 h.

Blood cytokine analyses

Plasma cytokine concentrations were evaluated in neonatal mice 12 and 24 h after administration of the LD70 dose of cecal slurry (Table 1). Neonatal mice that received cecal slurry exhibited a robust inflammatory cytokine response as compared with sham-treated animals. Plasma levels of IL-1β, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-13, IL-17, MIP-1β, RANTES, TNF-α, KC, IFN-γ, and MCP-1 were all significantly elevated at 12 h, and most of these remained elevated at 24 h after cecal slurry administration (Table 1). The plasma concentrations of the remaining cytokines were not significantly elevated in cecal slurry as compared with sham-treated animals (data not shown).

Table 1
Table 1:
Plasma cytokine concentrations (pg/mL) in the neonatal inflammatory response at 12 and 24 h after cecal slurry administration (n ≥ 5 for all groups) as compared with sham-treated animals

Splenocyte cell phenotypes in neonatal sepsis

Splenocyte changes associated with administration of the cecal slurry in neonatal mice are shown in Figures 3, 4, 5, 6, and 7. Sepsis induced by the administration of an LD70 dose of cecal slurry decreased NK cell percentages at 12 and 24 h in the neonatal mice (Fig. 3). The activation of NK cells at 12 and 24 h was dramatic, with 5-fold increases in CD69 expression (Fig. 4). Surprisingly, the percentage and absolute numbers of splenic CD3+CD4+ cells in the neonate after cecal slurry remained constant or increased in the neonatal animals (Fig. 5). In addition, there was an increase in the percentages and absolute numbers of CD3+CD8+ cell populations (Fig. 6) and an increase in the percentages and absolute numbers of CD4+CD25+FoxP3+ regulatory T cell population (Fig. 7) in the spleens of neonates and young adults 24 h after an LD70 cecal slurry administration. The CD4+CD25FoxP3+ cell populations experienced a similar expansion with a doubling in percentages and absolute numbers noted at 24 h after cecal slurry. The CD4+CD25+FoxP3+ regulatory T cell percentages are reported in Figure 7 as percent of total (living and dead) cells because the process of intracellular FoxP3 requires fixation, and concordant 7-AAD staining cannot be performed. There were also reductions in the splenic CD11chighCD11b+ myeloid and CD11c+PDCA-1+plasmacytoid dendritic cell populations (percentages and absolute numbers) after cecal slurry administration (data not shown).

Fig. 3
Fig. 3:
Natural killer cells from neonatal spleen after administration of cecal slurry compared with sham-treated animals. A, Scatter plots shown are representative of samples obtained from splenocytes 12 h after either a sham or an LD70 cecal slurry administration. The NK cell population (NK 1.1+DX5+) is identified within the box (R3). Numeric values within plots represent percentages of live splenocytes in the box (R3) that are NK 1.1+DX5+. B, Histogram compares NK splenocyte percentages from sham- (n = 5) and cecal slurry-administered (n = 5) mice at 12 and 24 h. *P < 0.05, P < 0.05 comparing 12 and 24 cecal slurry time points using Student t test. Error bars represent SD. CS indicates cecal slurry administration.
Fig. 4
Fig. 4:
Percentages of activated NK cells from neonatal spleen after administration of an LD70 dose of cecal slurry at 12 and 24 h. A, Scatter plot shown is from 12-h sample of splenocytes harvested from animals either sham or cecal slurry-administered. The CD69 expression was used as a marker of NK cell activation (R4). The scatter plot is gated on only NK1.1+DX5+ splenocytes, as shown in Figure 3. Numeric values within plots represent percentages of live NK1.1+DX5+CD69+ splenocytes. B, Histogram compares activated NK splenocyte percentages from sham- (n = 5) and cecal slurry-administered (n = 5) neonatal animals at 12 and 24 h. *P < 0.05, P < 0.05 comparing 12 and 24 cecal slurry time points. Error bars represent SD. CS indicates cecal slurry administration.
Fig. 5
Fig. 5:
CD3+CD4+ cell percentages from neonatal mice after administration of an LD70 dose of cecal slurry at 12 and 24 h. A, Scatter plot shown is from a representative 24-h sample of neonatal splenocytes harvested from either sham- and cecal slurry-administered mice. The CD3+CD4+ cell population is represented within the box (R4). Numeric values within plots represent percentages of live CD3+CD4+ cells in the box (R4). B, Histogram compares percentage of CD3+CD4+ splenocytes from sham- (n = 5) and cecal slurry-administered (n = 5) mice at 12 and 24 h. *P < 0.05, P < 0.05 comparing 12 and 24 cecal slurry time points. Error bars represent SD. CS indicates cecal slurry administration.
Fig. 6
Fig. 6:
Neonatal CD3+CD8+ cell percentages from splenocytes of neonatal mice after administration of cecal slurry, compared with sham administration at 12 and 24 h. A, Scatter plot shown is from a representative 24-h sample of splenocytes from sham- and cecal slurry-treated neonatal mice capturing the CD3+CD8+ cell population within the box (R3). Numeric values within plots represent the percentages of live CD3+CD8+ cells in the box (R3). B, Histogram compares CD3+CD8+ splenocyte percentages from sham- (n = 5) and cecal slurry-treated (n = 5) animals at 12 and 24 h. *P < 0.05, P < 0.05 comparing 12 and 24 cecal slurry time points. Error bars represent SD. CS indicates cecal slurry administration.
Fig. 7
Fig. 7:
Neonatal T regulatory cell percentages in splenocytes from mice after administration of cecal slurry, compared with sham at 12 and 24 h. A, Scatter plot shown is from a representative 24-h sample of splenocytes obtained from sham- and cecal slurry-treated neonatal animals showing CD4+FoxP3+ cell population within the box (R5). Although the scatterplot shows only the CD4+FoxP3+ populations, they were previously selected from the CD4+CD25+ populations. Numeric values within plots represent percentages of CD4+CD25+FoxP3+ cells in the box (R5). B, Histogram compares T regulatory cell percentages from sham- (n = 5) and cecal slurry-treated (n = 5) neonatal mice at 12 and 24 h. *P < 0.05, P < 0.05 comparing 12 and 24 cecal slurry time points. Error bars represent SD. CS indicates cecal slurry administration.

Young adult versus neonate response

Differences in the plasma cytokine response to an LD70 dose of cecal slurry between neonatal and adult mice were compared at 12 and 24 h postinjection (Table 2). There were dramatic differences in the inflammatory response at 12 h in young adult animals, with 13- to 60-fold increases in plasma concentrations of IL-1α, IL-1β, IL-12p40, IL-13, TNF-α, GM-CSF, MIP-1β, RANTES, and IFN-γ versus those in the neonates. Conversely, the neonatal concentration of IL-6 was more than double that found in the young adults after cecal slurry; unlike in the adults, IL-10 concentrations were significantly elevated in the neonates at 24 h after injection of the cecal slurry.

Table 2
Table 2:
Plasma cytokine concentrations (pg/mL) at 12 and 24 h between neonatal (n = 24) and adult (n = 5) inflammatory responses after cecal slurry

The changes in splenic leukocyte populations were also compared among neonatal and young adult mice administered with a dose of cecal slurry aimed at producing comparable mortality (LD70). In addition to the marked differences in the plasma cytokine response between neonatal and young adult mice (Table 2), splenic CD3+CD4+ cells in the neonate exhibited a significant increase at 24 h that was not recapitulated in the young adult animals in whom the cecal slurry was given (Fig. 8). In the young adult animals, sepsis induced by the cecal slurry resulted in a significant loss of CD3+CD4+ cells at 12 h. There were no other differences in the splenic cell populations between neonatal and young adult mice in response to the cecal slurry sepsis (data not shown).

Fig. 8
Fig. 8:
Differences in splenocyte CD3+CD4+ cell percentages from splenocytes of neonatal versus young adult mice at 12 and 24 h after administration of cecal slurry as compared with sham (* P < 0.05, P < 0.05 comparing 12 and 24 cecal slurry time points). Error bars represent SD. CS indicates cecal slurry administered; Sh, sham treated.

Sepsis induced by cecal slurry and CLP in young adult mice

Because CLP are the most generally accepted model of sepsis in the adult mouse, the immunological responses produced by the administration of cecal slurry were compared with those seen after CLP in the young adult mice. In both cases, the response to an approximate LD70 was evaluated at 12 and 24 h before the onset of significant mortality (Fig. 1). After cecal slurry, adult mice exhibited a much more intense inflammatory reaction as compared with CLP (Table 3). Elevations of IL-1β, IL-5, IL-12p40, IL-12p70, IL-17, GM-CSF, TNF-α, MIP-1β, MCP-1, RANTES, and IFN-γ were 2- to 50-fold greater in mice in whom the cecal slurry was administered than the levels exhibited after CLP at 12 h. Levels of IL-6, IL-10, IL-13, and KC were similarly elevated at 12 h in both groups (Table 3). At 24 h, there were similar elevations of IL-1β, IL-12p40, MCP-1, MIP-1β, and KC between cecal slurry and CLP groups. Levels of IL-2, IL-3, IL-4, and VEGF were statistically different but were at the limits of detectable values for both groups (data not shown). Remaining cytokines tested were not statistically different when compared.

Table 3
Table 3:
Plasma cytokine concentrations (pg/mL) at 12 and 24 h between cecal slurry and CLP performed on adult mice (n = 5 per group)

In contrast, there were no significant differences in the splenocyte phenotypes in young adult mice after CLP or cecal slurry administration. The loss of CD3+CD4+ splenocytes and the increase in CD4+CD25+FoxP3+ regulatory T cells seen in young adult mice in whom the cecal slurry was administered were recapitulated in the animals subjected to a CLP (data not shown).

DISCUSSION

The present results with the cecal slurry administration suggest that neonatal mice are more susceptible to generalized peritonitis than young adult mice. These findings are consistent with clinical observations that human neonates are more susceptible to sepsis and have an increased mortality (4). The use of cecal slurry to model generalized peritonitis in neonates also mimics the clinical conditions of necrotizing enterocolitis or spontaneous bowel perforation. Both of these clinical entities, and most of the mortality associated with severe sepsis, occur primarily in premature infants (19, 20).

Previous in vivo models of neonatal sepsis have used various routes and pathogens for infection. Among those extensively studied has been group B Streptococcus, a gram-positive organism, which was responsible for considerable mortality in the neonatal period before the era of intrapartum antibiotic prophylaxis. Although coagulase-negative Staphylococcus is currently the most commonly isolated blood pathogen, mortality is substantially higher when the infant is infected with a gram-negative species or fungi (20). Therefore, the use of the cecal slurry as a means of generating peritonitis and sepsis with resultant gram-negative bacteremia should accurately mimic the clinical condition of sepsis currently seen in neonates.

Based on our results, the neonate is capable of generating a systemic inflammatory response to the cecal slurry administration. However, this inflammatory response is markedly attenuated when compared with the response seen in young adult animals given a similar semilethal challenge. For many of the inflammatory cytokines evaluated, the concentrations produced in the neonate were 10 to 50 times less than those seen in the young adult (Table 2). Specifically, the reduced TNF-α and elevated IL-6 levels observed in neonatal mice after sepsis have been demonstrated in human neonatal cord blood monocytes and may contribute to immature neonatal immunity (21). The increased IL-6 response in the neonate as compared with that in the adult has been suggested to be associated with a more innate inflammatory system, favoring anti-inflammatory cytokines (22), but we did not find a significant difference between adult and neonatal IL-10 levels. Other investigators have reported that blood monocytes and macrophages from neonatal patients and experimental animals also have a reduced inflammatory response to a microbial challenge, like bacterial lipopolysaccharide (23). The importance of this finding is unclear because an exaggerated and not an attenuated inflammatory response is generally assumed to contribute to the increased mortality in these sepsis models (24), and the neonatal mice had increased mortality associated with an attenuated inflammatory response.

The attenuated inflammatory response demonstrated by the septic neonate may reflect maternal antepartum immune system stimuli. In a recent report, levels of serum cytokines were measured in pregnant mice during gestation and after birth. Interestingly, many correlations occurred between the murine prepregnancy serum cytokine profiles and those we found in septic neonatal mice (25), specifically IFN-γ, IL-1α, IL-1β, and TNF-α. The maternal cytokine concentrations are likely decreased because of the risk of fetal loss because elevated levels of IFN-γ, IL-1α, IL-1β, and TNF-α are associated with abortion (26, 27).

There were, however, cellular responses to the cecal slurry administration that also differed between the neonate and the young adult mice that might explain the differential sensitivity to the cecal slurry administration. For example, CD4+ T cell percentages and absolute numbers actually increased in the septic neonatal mice at 24 h after sepsis (Fig. 5). This latter observation is in direct contradiction to the response seen in septic young adult animals. Decreased splenic CD4+ T cells have been reported with sepsis in adult humans and mice (28-30) and at necropsy in humans including children (31) and neonates after multiorgan failure because of sepsis (32). The differences in CD4+ cell populations in neonates reported here and those reported in the human studies may be explained in part by the sampling interval. We studied neonatal mice in the early septic period before their death, whereas the human studies were conducted at necropsy much later in the septic process. For example, Felmet et al. (31) reported lymphocyte depletion in pediatric cases after sepsis and multisystem organ failure, but the splenic and lymph node samples were obtained postmortem, and the time from onset of disease to death was at least 6 days. Toti et al. (32) reported postmortem spleen depletion of B and T lymphocytes as evidenced by immunohistochemical staining in human neonates after chorioamnionitis or sepsis, but samples were again processed between 4 and 12 h after death. More importantly, the increases in splenic CD4+and CD8+ populations that we saw early in the septic response (12 and 24 h after initiation of sepsis) may reflect a release of T cells from the neonatal thymus in response to the inflammatory challenge. Although the young adult mouse exhibits the expected decrease in CD4+ cells after sepsis when examined at the same time points as those in the neonate, examination of the neonatal murine spleen at later time intervals would help to clarify this apparent contradiction with the human data.

Severe sepsis remains a clinical enigma. Although there have been dramatic improvements in our understanding of the pathogenesis of severe sepsis, there have been very few new therapeutics that have proven successful for its treatment in any age group (33). Activated protein C has been approved for the treatment of severe sepsis in adults but not in children or neonates (34, 35). Furthermore, its mode of action is still not clearly known (36). Even with activated protein C, outcome to severe sepsis in adults remains dismal, with mortality rate still hovering around 30%. Neonatal sepsis is even a greater challenge.

One of the difficulties with studying the immunological consequences of sepsis is the inadequacy of appropriate animal models. This is especially true for the in vivo study of neonatal sepsis. Many therapeutic approaches that have shown efficacy in rodent and primate models have failed in clinical trials, suggesting that the preclinical models do not effectively recapitulate the human syndrome (13, 37). Criticism has been primarily focused on models of sepsis produced by the intravenous administration of live bacteria or microbial products. For the past several years, there has been a trend toward the use of rodent models of sepsis based on bacterial peritonitis, produced by either CLP or creation of an enterocecal fistula with a stent (38). These models seem to reproduce several of the classic temporal responses to sepsis, characterized by an initial hyperdynamic response followed by decompensation, organ failure, and death (24). Many of the early T cell and dendritic cell responses seen in patients dying from sepsis can also be reproduced with these models (29, 39, 40).

However, these models are technically challenging in the neonate. We initially attempted to create bacterial peritonitis in the neonatal mouse by performing a CLP, but because of the small size and the immaturity of the gut, it was not technically possible. The neonatal mouse offers a number of other challenges beyond their small size and immaturity. Survival is dependent upon the pup being able to nurse and obtain warmth from its mother. Neonates are uniformly rejected by their mothers if their physical appearance is modified by surgical staples or sutures, and excessive human handling of a sick animal can lead to abandonment or cannibalism.

Kazarian et al. (11) had reported on a model of generalized peritonitis produced by the intraperitoneal injection of cecal contents in the pig. Sam et al. (12) refined this model in rats and demonstrated that, hemodynamically, the animals showed a reproducible hypotension and inadequate tissue perfusion. We have now modified the model further to adapt it to the neonatal mouse. As shown in Figure 1, by varying the dose of administered cecal slurry, we could reproducibly produce mortality varying anywhere from 0% to 100%. Importantly, the mortality from the cecal slurry followed a temporal pattern very similar to that seen in young adult mice after cecal slurry administration or with a CLP. Furthermore, in the young adult animals, the cecal slurry administration produced nearly identical changes in the splenocyte phenotype that were seen after CLP. The cecal slurry method tended to produce a greater early inflammatory response than was seen with the CLP (Table 3), although this is understandable, given the volume of cecal contents released into the peritoneal cavity with the two methods. Nevertheless, the cecal slurry method produced organ colonization, blood bacteremia, and similar phenotypic changes in the splenocyte populations.

CONCLUSIONS

The present results clearly demonstrate that intraperitoneal administration of cecal slurry can reproducibly generate sepsis in the neonatal mouse with dose-dependent lethality. Mortality is associated with abscess formation, bacterial colonization, systemic inflammatory response, and reproducible alterations in leukocyte phenotypes in the spleen. Neonatal mice are more sensitive to mortality than young adult animals, and this increased mortality is associated with a number of different inflammatory and immune responses. The cecal slurry model of neonatal sepsis may prove helpful in better understanding why neonates are more susceptible to an adverse outcome to sepsis and the subsequent development of novel therapeutics for the treatment of neonatal sepsis.

ACKNOWLEDGMENTS

The authors thank Professors Terrence Flotte and David Burchfield for their review of the manuscript and their continued support of the research program.

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Keywords:

Mouse; cecal ligation and puncture; SIRS; cytokines; T cells; dendritic cells; cecal slurry; neonatal

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