Voluntary running exercise protects against sepsis-induced early inflammatory and pro-coagulant responses in aged mice

Abstract

Background: Despite many animal studies and clinical trials, mortality in sepsis remains high. This may be due to the fact that most experimental studies of sepsis employ young animals, whereas the majority of septic patients are elderly (60 - 70 years). The objective of the present study was to examine the sepsis-induced inflammatory and pro-coagulant responses in aged mice. Since running exercise protects against a variety of diseases, we also examined the effect of voluntary running on septic responses in aged mice.

Methods: Male C57BL/6 mice were housed in our institute from 2-3 to 22 months (an age mimicking that of the elderly). Mice were prevented from becoming obese by food restriction (given 70-90% of ad libitum consumption amount). Between 20 and 22 months, a subgroup of mice ran voluntarily on wheels, alternating 1-3 days of running with 1-2 days of rest. At 22 months, mice were intraperitoneally injected with sterile saline (control) or 3.75 g/kg fecal slurry (septic). At 7 h post injection, we examined (1) neutrophil influx in the lung and liver by measuring myeloperoxidase and/or neutrophil elastase in the tissue homogenates by spectrophotometry, (2) interleukin 6 (IL6) and KC in the lung lavage by ELISA, (3) pulmonary surfactant function by measuring percentage of large aggregates, (4) capillary plugging (pro-coagulant response) in skeletal muscle by intravital microscopy, (5) endothelial nitric oxide synthase (eNOS) protein in skeletal muscle (eNOS-derived NO is putative inhibitor of capillary plugging) by immunoblotting, and (6) systemic blood platelet counts by hemocytometry.

Results: Sepsis caused high levels of pulmonary myeloperoxidase, elastase, IL6, KC, liver myeloperoxidase, and capillary plugging. Sepsis also caused low levels of surfactant function and platelet counts. Running exercise increased eNOS protein and attenuated the septic responses.

Conclusions: Voluntary running protects against exacerbated sepsis-induced inflammatory and pro-coagulant responses in aged mice. Protection against pro-coagulant responses may involve eNOS upregulation. The present discovery in aged mice calls for clinical investigation into potential beneficial effects of exercise on septic outcomes in the elderly.

Keywords: Aging; Capillary plugging; Pulmonary inflammation; Voluntary running; sepsis.

Fig. 1

Effect of sepsis and running exercise on myeloperoxidase (MPO) and neutrophil elastase (NE), measures of inflammatory neutrophil infiltration, in lung homogenates of aged mice (22 months). Sepsis induced by fecal injection into the peritoneum (7 h post-injection) increased lung MPO (a) (n = 6, 5, 6, and 6 for bars left to right, respectively) and NE (b) (n = 4 per bar) in non-exercised mice. Running exercise protected against these increases. *Effect of sepsis versus control, ^#effect of exercise versus non-exercise; P < 0.05

Fig. 2

Effect of sepsis and running exercise on concentrations of interleukin 6 (IL6) (a) and KC (b) in lung lavage of aged mice: n = 6, 5, 6, and 6 for bars, left to right, respectively (a and b). Sepsis (7 h post fecal injection into the peritoneum) increased lung IL6 and KC in both exercised and non-exercised mice. Running exercise reduced these increases. *Effect of sepsis versus control, ^#effect of running exercise versus non-exercise; P < 0.05

Fig. 3

Effect of sepsis and running exercise on surfactant pool size in lung lavage from aged mice. a The total was the sum of functional large aggregates and the inactive small aggregates of phospholipids (PL) measured in the bronchoalveolar lavage fluid. The large aggregate (b) and small aggregate sub-fractions were measured to assess the large aggregate percentage (c), a measure of the relative amount of functional lung surfactant. ^#Effect of running exercise versus non-exercise; P < 0.05: n = 6, 5, 6, and 6 for bars, left to right, respectively (a, b, c)

Fig. 4

Effect of sepsis and running exercise on myeloperoxidase (MPO) in liver homogenates of aged mice. Sepsis (7 h post fecal injection into the peritoneum) increased liver MPO in non-exercised mice. Running exercise protected against this increase. *Effect of sepsis versus control, ^#effect of running exercise versus non-exercise; P < 0.05, n = 6 per bar

Fig. 5

Effect of sepsis and running exercise on capillary bed plugging in the extensor digitorum longus muscle, endothelial nitric oxide synthase (eNOS) protein, and systemic arterial blood platelet count, in aged mice. a Capillary density in the extensor digitorum longus muscle was computed from the sum of capillaries with moving red blood cells (RBC) and stationary RBC. There was no effect of sepsis or exercise on total capillary density: n = 9, 6, 7, and 6 for bars, left to right, respectively. b Capillary plugging, computed as the percentage of capillaries with stationary RBC from the total of capillaries (perfused plus stationary RBC capillaries). Sepsis (7 h post fecal injection into the peritoneum (FIP)) increased plugging in both exercised and non-exercised mice. Running exercise reduced this increase. *Effect of sepsis versus control, ^#effect of running exercise versus non-exercise; P < 0.05: n = 9, 6, 7, and 6 for bars, left to right, respectively. c Sepsis (7 h post-FIP) did not significantly alter eNOS levels assessed by immunoblot. Running exercise, however, significantly increased eNOS in skeletal muscle of both controls and septic mice: ^#effect of running exercise versus non-exercise; P < 0.05; n = 4 per bar. d Sepsis (7 h post-FIP) reduced platelet count in non-exercised mice, but not in exercised mice: *effect of sepsis versus control; P < 0.05; n = 9, 6, 7, and 6 for bars, left to right, respectively