Experimental design
Model of aging
The age of the most frequently treated septic patients has been reported to be 65 years [
15]. Based on the US Actuarial database, about 80% of the human male population reaches 65 years of age [
16]. Moreover, about 80% of male wild-type C57BL/6 mice reach 22 months of age [
17]. Male wild-type C57BL/6 mice were therefore aged to 22 months in our institutional animal facility under controlled conditions (i.e., standard light-dark cycle, temperature, humidity, food, and bedding). Experimental protocols, including induction of sepsis, were performed in accordance with the Canadian Council on Animal Care guidelines for the care and handling of animals. The institutional Animal Care Committee approved our protocols, except for survival studies of septic aged mice (Approval # 2011-062).
Mice presented with unrestricted regular food during aging typically become obese. Because obesity worsens the inflammatory response in septic mice [
18], our aging mice were food-restricted to focus the study on the effects of aging and exercise, thus avoiding the confounding effect of obesity with age. Mice were ordered from Charles River laboratories (Sherbrooke, QC, Canada) at 2–3 months of age, in batches of 10 at approximately 1–2 month intervals. Upon arrival, mice were weighed and housed in groups of four to six per cage. Mice in the initial batch were provided with unrestricted standard food plus water, and food consumption was measured. The amount of food provided to the mice was then reduced by 10% (of the measured amount) every 2–3 weeks until reaching 70% (i.e., 2.5 g/mouse/day) of the measured amount, which was maintained for the duration of the aging. Subsequent batches of mice followed the same protocol but started at 90% of the initial measured amount [
19,
20]. As found previously, under this regime, aging mice did not gain weight [
19]. Mice were weighed weekly to ensure they maintained body weight during aging. If a mouse in a particular cage began losing weight (i.e., its mate(s) fed more avidly), the mouse was then housed alone to allow weight recovery and subsequent weight maintenance during aging.
Model of running exercise
There are numerous animal models of running exercise, including voluntary running on a wheel accessible 24 h each day [
21] and treadmill running where an animal’s running is controlled [
22]. In general, the intensity and duration of exercise in these models have been used at levels high enough to ensure an effect of exercise.
The present study utilized a model of running that aimed to mimic the physical activity of elderly individuals (i.e., approaching retirement) who have begun moderate but regular aerobic exercise later in life (i.e., to get health benefits from exercise when they are older). Because aging increases the potential for muscle and joint injury during exercise [
23‐
25], these individuals may wish to include days of rest within their routine, to minimize this potential injury. Thus, between 20 and 22 months, a subgroup of mice ran voluntarily on wheels, alternating days of running with days of rest, to allow recovery from exercise. We hypothesized that the present model at submaximal duration and intensity (i.e., starting exercise later in life and including days of rest) would still yield beneficial effects against sepsis.
Specifically, at age 20 months a subset of the aging mice were put in cages with running wheels to which a miniature magnet was attached (Mouse Igloo Fast-Trac, BioServ, Flemington, NJ, USA). Each cage housed two mice and two wheels, or one mouse and one wheel, on which the mice could run voluntarily 24 h per day, and mice were monitored daily for running activities. Further, a sensor attached to the outside wall of the cage near the wheel measured the number of wheel revolutions during mouse running, permitting determination/estimation of the distance run by each mouse. The physical activity protocol consisted of (1) an initial period of 7 days when mice could run voluntarily every day, and (2) an 8-week period when 1–3 days of running were alternated with 1–2 days of recovery from running. To facilitate recovery, the wheel-igloo assembly was disengaged so that the wheel could not turn. To maintain body weight, running mice were provided with 90% (i.e., 3.2 g/mouse/day) of measured food between 20 and 22 months. Control non-running mice had no wheel in their cage; their physical activity continued to be the same as that for all mice prior to 20 months.
Model of sepsis
In the present study, we chose a model of sepsis involving fecal injection into the peritoneum (FIP) at the dose of 3.75 g/kg [
26,
27]. Using this model of sepsis permitted comparison of our previously accumulated data in young mice at 7 h post-FIP [
26,
27] with data from the present aged mice. The FIP dose in young mice resulted in 20% survival at 24 h post-FIP ([
26]). Based on the reported worsening of survival when comparing young and old CLP mice [
8], we predicted our aged mice would not survive past 12–14 h post-FIP at this dose. Thus, the focus of the present study in septic aged mice was on the early stage of the inflammatory response (i.e., 7 h post-FIP, as in our young mice).
Specifically, feces were collected from the cecum of a minimum of two donor mice, suspended in sterile saline at a concentration of 75 mg/mL, and stored overnight at 4 °C. The following day, mice were injected intraperitoneally with 3.75 g/kg of the feces slurry. Following feces injection, mice were fluid resuscitated by subcutaneous injection of 1 mL of sterile saline containing 4 μg/mL buprenorphine. For control sham mice, sterile saline was substituted for feces solution. In mice subjected to the running protocol, sepsis was induced 1–2 days after the last running bout.
Intravital microscopy of skeletal muscle, collection of fluids and tissues, and biochemical analyses
Sepsis-induced inflammation leads to activation of the coagulation pathway [
5]. We used the skeletal muscle as a bioassay to examine this aspect of sepsis in terms of capillary bed plugging, a well-known indicator of sepsis involving pro-coagulation responses [
28]. Capillary plugging, reported in animal and human organs during sepsis, leads to inadequate oxygenation of the tissue and organ failure [
28].
At 6 h post-FIP, mice were anesthetized with ketamine (80 mg/kg) and xylazine (4 mg/kg) and injected via the intrapenile vein with 0.1 mL of sterile saline. At 6.5 h post-FIP, surgery was begun to expose the right extensor digitorum longus (EDL) muscle, thereby allowing its surface to be imaged at 7 h post-FIP using intravital microscopy and epi-illumination as described previously [
26,
27]. Importantly, because of the epi-illumination aspect of this microscopy, the EDL muscle was not touched by surgical tools during this process [
29], resulting in little or no surgical injury to the muscle. Intravital images were used to determine capillary plugging. To this end, we measured the density of perfused capillaries and the density of stopped-flow capillaries seen within a 100-μm-deep surface layer of the muscle. Specifically, in each mouse, 4–5 fields of view of the muscle were used for determination of capillary plugging. In each field, a test line was drawn across muscle fibers, and capillaries with moving and stationary red blood cells (RBC) crossing the test line were counted. The total density was computed from the sum of capillaries with moving RBC and stationary RBC. Capillary plugging was determined as the ratio of stopped-flow to total capillaries (perfused plus stopped-flow) as previously reported [
26]. The values of total densities and of capillary plugging were averaged among the 4–5 fields. During the period from 6 h post-FIP to the end of the intravital experiment at ~7.5 h post-FIP, the mice were kept anesthetized with supplemental ketamine/xylazine doses approximately every 30 min.
Shortly after the intravital study was completed, the following procedures were carried out: (1) a blood sample from a punctured carotid artery was collected into a heparinized syringe containing the anticoagulant acid citrate dextrose (25–50 μL per sample) for subsequent systemic platelet count and lactate analyses; (2) the peritoneal cavity was lavaged with 2 mL sterile saline to assess bacterial count in the peritoneal fluid; (3) bronchoalveolar lavage (BAL) was done (detailed below); and (4) the lung, liver, and left hindlimb skeletal muscle (including the EDL) were harvested and flash-frozen in liquid N2. Frozen tissue samples were subjected to further biochemical analyses.
To determine the systemic blood platelet count, blood was diluted 200-fold in saline, and platelets were labeled with rhodamine 6G (0.4 μg/mL, Sigma-Aldrich) or calcein-AM (8 μM, Sigma-Aldrich) and counted in a hemocytometer chamber under a microscope. Plasma lactate was determined with the iSTAT system, cartridge CG4+ (Abbott, Mississauga, ON, Canada). To determine the bacterial count, peritoneal lavage samples were serially diluted in 10-fold fashion, plated on Columbia Blood Agar containing 5% sheep blood (MP0351, Oxoid, Nepean, ON, Canada) and grown overnight at 37 °C. The bacterial colony-forming units (CFU) were counted and expressed per μL of the peritoneal lavage fluid.
The BAL procedure and processing was performed as previously reported [
30]. Briefly, after securing an endotracheal tube, lungs were lavaged with three boluses of 1 mL of saline. The total lavage volume was centrifuged at 150 g for 10 min to remove cells and debris, and the supernatant was used for measurement of interleukin 6 (IL6) and KC concentrations by ELISA (BD Bioscience, San Diego, CA, USA). A separate aliquot of the supernatant was used to isolate the two sub-fractions of pulmonary surfactant, the large aggregates (LA), which are the functional components of surfactant, and the small aggregates (SA), which are inactive. The amounts of phospholipids (PL) in LA and SA were determined by phosphorus assay after an organic solvent extraction [
31,
32].
Myeloperoxidase (MPO) assay
Tissue MPO accumulation is well-known to be associated with the degree of inflammation (i.e., neutrophil infiltration), as we have observed both in sepsis and lung injury [
33,
34]. To assess lung and liver inflammation following sepsis, we examined total MPO abundance within these tissues. To this end, frozen tissues were homogenized in 20 mM potassium phosphate buffer and centrifuged at 6000 g for 20 min. After discarding the supernatant, the pellet was resuspended in 50 mM acetic acid with 0.5% hexadecyltrimethylammonium hydroxide detergent. The samples were then sonicated for 10 s and centrifuged for 5 min. The supernatants were mixed with a 1 mM tetramethylbenzidine (TMB) and 0.2 M acetic acid solution, warmed to 37 °C, and the reaction initiated with the addition of 3 mM H
2O
2. The samples were left to react for 5–30 min and then quenched using 1000 U/mL beef catalase and 0.2 M acetic acid. Spectrophotometric analysis (655 nm) was conducted using the Model 680 microplate reader and software version 5.2.1 (Bio-Rad), and compared to an MPO standard (catalogue # M6908; Sigma).
Neutrophil elastase (NE) ELISA
NE is a serine protease found primarily within the azurophilic granules of the neutrophil [
35]. To confirm neutrophil influx into the lungs, we examined total NE abundance by DuoSet ELISA according to the manufacturer’s protocol (catalogue # DY4517-05; R&D Systems). Briefly, frozen lung tissue was homogenized in CellLytic M lysis buffer with proteinase inhibitor (Sigma). Wells of a 96-well plate were incubated with capture antibody overnight at room temperature and then washed three times with PBS and 0.05% Tween (PBST). Wells were blocked with PBS and 1% bovine serum albumin (BSA) for 1 h, washed with PBST, and incubated with lung sample or standard (12.5 − 800 pg/mL) overnight at 4 °C. Wells were again washed with PBST and then incubated with detection antibody. After 2 h, wells were washed with PBST and then incubated with streptavidin conjugated to horseradish peroxidase (HRP) for 20 min. Wells were once again washed with PBST, and TMB substrate was then added to each well and incubated for 20 min. The reaction was stopped using 1 M H
2SO
4 and plates read at 450 nm using the Model 680 microplate reader and software version 5.2.1 (Bio-Rad).
Immunoblotting of endothelial nitric oxide synthase (eNOS)
A total of 50 μg protein from the left skeletal muscle was loaded and separated by 10% SDS-PAGE, followed by electrotransfer to a nitrocellulose membrane. The blots were probed with specific primary antibodies against mouse eNOS (BD Transduction Laboratories) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Sigma). Each band was visualized by enhanced chemiluminescence detection and exposure to X-ray film. Optical density for individual bands was examined using the FluorChem 8000 (Alpha Innotech, San Leandro, CA, USA). The densitometry ratios of eNOS to GAPDH were then computed.
Heart function
In some aged mice, the heart rate (HR) and cardiac output (CO) were examined at 6 h after FIP or sham injection. Mice were lightly anesthetized with 1–2% isoflurane, and HR and CO measured using the Vevo 2100 ultrasound imaging system (Visual Sonics, Canada) as previously reported [
36‐
38].
Mouse attrition/usage
From 69 mice delivered to our institution at 2–3 months of age, 63 mice reached 22 months (i.e., 91% survival). However, 20 of these mice were excluded from the experimental analyses because of an erroneous feces dose injected (7 mice), death during intravital microscopy caused by ketamine/xylazine overdose (6 mice), tumors found in the liver (3 mice), and an unidentified illness/lethargy in the mice (4 mice).