Late Therapeutic Intervention with Antibiotics and Fluid Resuscitation Allows for a Prolonged Disease Course with High Survival in a Severe Murine Model of Sepsis

Abstract

Current animal models of sepsis often incorporate antibiotics to be consistent with clinical standards for treatment of patients in the intensive care unit. However, such experimental intervention is commonly initiated very early after infectious insult, which likely blunts the progression of systemic inflammation and downstream pathology. The objective of this study was to establish an animal model of sepsis with delayed therapeutic intervention, allowing a longer disease course and downstream pathology, but still resulting in a high survival rate. Severe lethal abdominal infection was initiated in young adult (17-18-week-old) C57BL/6 mice by cecal slurry (CS) injection. When initiated early (1- or 6-h post-CS injection), antibiotic treatment (imipenem, 1.5 mg/mouse i.p., twice/day for 5 days) rescued the majority of mice; however, few of these mice showed evidence of bacteremia, cytokinemia, or organ injury. When antibiotic treatment was delayed until late time-points (12- or 24-h post-CS injection) the majority of animals did not survive beyond 48 h. When fluid resuscitation (physiological saline, s.c.) was performed in combination with antibiotic treatment (twice daily) beginning at these late time-points, the majority of mice survived (75%) and showed bacteremia, cytokinemia, organ dysfunction, and prolonged body weight loss (<90% for 4 weeks). We recommend that this new repeated combination treatment with antibiotics and fluids resuscitation be initiated at a late time point after bacteremia becomes evident because this model more closely mimics the downstream pathological characteristics of severe clinical sepsis yet maintains a high survival rate. This model would be advantageous for studies on severe sepsis and postintensive care illness.

Figure 1. Protocol flowchart for preparing cecal slurry (CS) stock

Cecal contents are collected and prepared for long-term cryopreservation according to the diagram.

Figure 2. Survival rate and disease severity correlate with time of intervention after CS injection

All mice were given a minimum lethal dose of cecal slurry (CS; 500μL). (A) Blood bacteria load was assessed 1, 6, and 12 hours after CS injection in animals (n=8) in animals which did not receive therapeutic intervention. (B-C) In another experiment, animals received antibiotic treatment (imipenem, IPM; 1.5mg i.p.) beginning 1, 6, 12, and 24 hours after CS injection. Antibiotics were continued twice daily for five days (n=5-6 per group). (B) Survival (star notates p<0.05 compared to no-antibiotics group) and (C) body temperature were monitored. Data represent mean ± standard deviation.

Figure 3. Late intervention with antibiotics and fluid resuscitation rescues the majority of mice from otherwise lethal sepsis

All animals were given a lethal dose of cecal slurry (CS). Mice received antibiotic treatment alone (imipenem, IPM; 1.5mg i.p.), fluid resuscitation alone (1mL physiological saline, s.c.), or a combination treatment of antibiotics and fluid resuscitation (n=5-9 per group) beginning 12 or 24 hours after CS injection. Therapeutic treatment was continued twice daily for five days. (A, D) Survival and (B, E) body temperature were monitored for multiple days. Data is represented as mean ± standard deviation. (C, F) Circulating bacteria load was assessed in the combination treatment groups by culturing blood obtained by micropuncture of the tail vein immediately before the first therapeutic treatment (12, 24h), after 3 treatments (48, 72h) and after 7 treatments (96, 108h). Symbols * and *** represent p<0.05 and p<0.001 respectively compared to no intervention group; † and †† represent p<0.05 and p<0.01 respectively compared to bacteria load before intervention (i.e. 12 and 24h).

Figure 4. Late, but not early, therapeutic intervention results in cytokinemia and prolonged reduction in body weight

Severe infection was induced by cecal slurry (CS) injection and mice were divided into groups (n=4-8). Mice received antibiotic treatment (imipenem, 1.5 mg/mouse, i.p.) beginning 1 or 6 hours after CS injection, or combination treatment with antibiotics and fluid resuscitation (physiological saline, s.c.) beginning 12 hours later. A group of animals (n=4) received vehicle injection (10% glycerol-PBS) for comparison. (A-C) Plasma samples obtained 24 hours after CS injection were subjected to IL-6, IL-1β, and TNFα quantification. (D) Body weight was monitored for 14 days after CS injection (n=5-9). (E) In a separate experiment, the body weight of animals resuscitated with antibiotics and fluid resuscitation beginning 12 hours after CS injection was monitored up to 30 days post-CS injection (n=7). * indicates p<0.05 by one-way ANOVA and †† indicates p<0.01 at all time-points compared to baseline (paired student's t-test).

Figure 5. Early, but not late, therapeutic intervention prevents sepsis-induced lung injury

Mice received either cecal slurry (CS; n=14) or vehicle (10% glycerol; n=4) injection (i.p.). CS-injected mice were divided among the following groups: nonresuscitated, 1h antibiotics, 6h antibiotics, or 12h combination treatment with antibiotics and fluid resuscitation (n=3-4 per group). All animals were euthanized 24 hours after CS or vehicle injection. (A-E) Lung tissue sections stained with H&E. Representative images (400X, scale bar represents 50 μm) are shown for each group. (F) Hisopathological scoring (n=3-4 per group). *** represents p<0.001.

Figure 6. Delayed therapeutic intervention results in heightened plasma ALT levels

Animals were divided among treatment groups as described in Figure 5. Plasma samples obtained 24 hours after CS injection were subjected to ALT quantification by colorimetric assay (n=3-4 per group). * indicates p<0.05.

Figure 7. Early vs. Late therapeutic intervention: A comparison of sepsis outcomes after severe infection in murine models

Late therapeutic intervention starting 12 hours after severe infection allows development of severe sepsis with organ injury and long-term weight reduction.