Precision Profiling of Disease Progression in Murine Models of Sepsis and Septic Shock

Abstract

Septic shock has an unacceptably high mortality rate and unmet need for new therapeutics. Murine models are crucial for research, yet methodologies often differ. This study characterised standard- and high-grade caecal ligation and puncture (CLP) murine models of septic shock by integrating ultraminiature arterial telemetry with comprehensive plasma biomarker analysis. Standard-grade and high-grade CLP was performed in 8-10 week old, male C57BL/6 mice (n = 98), with a subset implanted with arterial telemetry to monitor real-time circulatory function. Plasma markers of inflammation and organ damage were measured at multiple intervals up to 168 h post-CLP. Standard-grade and high-grade CLP showed distinct progressions; episodes of hypotension began 5-6 h after CLP in 30% of standard-grade and all high-grade CLP mice, with respective 168 h mortality of 40% and 71%. Recurrent episodes of hypotension 5-39 h after CLP were universally lethal. The coincidence of hypotension and elevated plasma lactate defined the onset of septic shock after high-grade CLP, which was always lethal. Inflammatory cytokines and markers of liver, renal, and cardiac damage were markedly elevated to 168 h after high-grade CLP, in contrast to standard-grade CLP, which returned to baseline by 48 h. Elevated plasma IL-6, TNFα, and corticosterone, along with reduced albumin, were significantly correlated with mortality. In conclusion, this research refines murine CLP models by providing a precise, dynamic map of the progression to septic shock. The high-grade CLP model consistently models early and late-stage physiological deterioration and serves as a robust model for evaluating the efficacy of novel therapies aimed at human septic shock.

Keywords: sepsis; septic shock; telemetry.

Figure 1

Survival, weight change, and morbidity of CLP mice. (A) 168 h Kaplan–Meier survival curve of male C57BL/6 mice subject to standard- (n = 10) or high-grade CLP (n = 28). (B) Weight change % following standard- and high-grade CLP. (C) Cumulative disease index score following standard- and high-grade CLP (high score indicates high morbidity). Data are presented as mean ± SEM; * p ≤ 0.05.

Figure 2

Real-time mean arterial pressure (MAP) in standard- and high-grade CLP. (A) MAP (mmHg) in standard-grade CLP survivors over (i) 168 h (each colour represents a mouse) and (ii) mean MAP in the first 36 h after CLP (n = 6). (B) MAP in standard-grade CLP non-survivors over (i) 168 h and (ii) mean MAP over the first 36 h (n = 4; vertical inset lines indicate death time). (C) MAP in high-grade CLP survivors (n = 12) over (i) 168 h and (ii) mean MAP in the first 36 h (n = 4). (D) MAP in high-grade CLP non-survivors over (i) 168 h and (ii) mean MAP in the first 36 h (n = 8). Horizontal dotted line depicts the 120 mmHg median MAP in the 48 h prior to CLP (n = 22).

Figure 3

Real-time heart rate in standard- and high-grade CLP mice. (A) Heart rate (bpm) in standard-grade CLP survivors over (i) 168 h (each colour represents a mouse) and (ii) mean heart rate in the first 36 h after CLP (n = 6). (B) Heart rate in standard-grade CLP non-survivors over (i) 168 h and (ii) mean heart rate in the first 36 h (n = 4; vertical inset line indicates a death). (C) Heart rate in high-grade CLP survivors fitted with telemetry (n = 12/28) over (i) 168 h and (ii) mean heart rate in the first 36 h (n = 4). (D) Heart rate in high-grade CLP non-survivors over (i) 168 h and (ii) mean heart rate in the first 36 h (n = 8). Horizontal dotted line depicts the median heart rate (590 bpm) in the 48 h prior to CLP (n = 22).

Figure 4

Real-time core temperature in standard- and high-grade CLP mice. (A) Core temperature (°C) in standard-grade CLP survivors over (i) 168 h (each colour represents a mouse) and (ii) mean temperature in the first 36 h after CLP (n = 6). (B) Core temperature in standard-grade CLP non-survivors over (i) 168 h and (ii) mean temperature in the first 36 h (n = 4; vertical inset line indicates a death). (C) Core temperature in high-grade CLP survivors fitted with telemetry (n = 12/28) over (i) 168 h and (ii) mean temperature in the first 36 h (n = 4). (D) Core temperature in high-grade CLP non-survivors over (i) 168 h and (ii) mean temperature in the first 36 h (n = 8). Horizontal dotted line depicts the median core temperature (37 °C) in the 48 h prior to CLP (n = 22).

Figure 5

Plasma cytokine responses in standard- and high-grade CLP mice. Plasma (A) interleukin (IL)-6 (ng/mL), (B) IL-10 (ng/mL), (C) macrophage inflammatory protein-2 (MIP-2; ng/mL), and (D) tumour necrosis factor alpha (TNFα; pg/mL) concentrations at 6, 12, 24, 48, 96, and 168 h after standard- and high-grade CLP (n = 6 per time point). Horizontal grey bar depicts the diurnal range of each cytokine measured in unoperated control mice (n = 24). Data are presented as mean ± SEM; * p ≤ 0.05.

Figure 6

Plasma lactate, albumin, and total corticosterone in standard- and high-grade CLP mice. Plasma (A) lactate (nmol), (B) albumin (mg/mL), and (C) total corticosterone (ng/mL) concentrations at 6, 12, 24, 48, 96, and 168 h after standard- and high-grade CLP (n = 6 per time point). Horizontal grey bar depicts the diurnal range of individual plasma markers measured in unoperated control mice (n = 24). Data are presented as mean ± SEM; * p ≤ 0.05.

Figure 7

Plasma markers of organ damage in standard- and high-grade CLP mice. Plasma (A) cystatin C (ng/mL), (B) troponin I (ng/mL), (C) aspartate transaminase (AST; pg/mL), and (D) alanine transaminase (ALT; pg/mL) concentrations at 6, 12, 24, 48, 96, and 168 h after standard- and high-grade CLP (n = 6 per time point). Horizontal grey bar depicts the diurnal range of individual damage marker measured in unoperated control mice (n = 24). Data are presented as mean ± SEM; * p ≤ 0.05.

Figure 8

Heat map summary of telemetry and plasma biomarker progression following CLP. Blue indicates low reading (telemetry) or plasma marker levels; red indicates low reading (telemetry) or plasma marker levels; black indicates normal physiological range. MAP, mean arterial pressure; HR, heart rate; ALT, alanine aminotransferase. Note: The blue shades for telemetry data from 40 h onwards denote progression to septic shock.

Figure 9

Caecal ligation and puncture (CLP) procedure. (A) Mouse caecum depicting ligation lengths and puncture/s related to standard (blue) and high-grade (green) CLP. (B) In-operative and 168 h post-operative image of high-grade CLP.