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Skeletal muscles play important roles in innate immunity. However, in vitro, their sensitivity to lipopolysaccharide (LPS) is low. In other tissues, LPS sensing is facilitated by the presence of plasma, LPS binding protein (LBP) or soluble CD14 (sCD14). This study addressed whether these are critical for LPS sensitivity in skeletal muscle and whether LPS responsiveness is different between slow vs. fast muscle. Soleus (SOL) or extensor digitorum longus (EDL) muscle from adult male C57bl/6 mice were mounted in 1 ml oxygenated baths containing: 1) buffer only; 2) buffer+1% mouse plasma; 3) buffer+1 μg/ml LBP; or 4) buffer+1% plasma from sCD14(−/−) mice. In each condition, muscles were exposed to LPS from 0–1.0 μg/ml. Bath samples were collected at 0, 1 and 2 h, and analyzed using cytokine multiplex arrays. In both SOL and EDL the predominant responding cytokines/chemokines were KC(CXCL1), IL-6 and MCP-1(CCL2) and their average responses were amplified by ~10 fold in the presence of 1% plasma. Overall, SOL and EDL exhibited similar secretory responses in the presence of 1% plasma, with a lower limit of sensitivity to LPS of 0.01 μg/ml. LBP supplementation did not augment secretion; however, 1% plasma from CD14(−/−) mice suppressed cytokine/chemokine secretion from EDL muscle. In conclusion, intact slow and fast mouse muscles have similar cytokine/chemokine responses to LPS but depend on the presence of low levels of plasma constituents. Though sCD14 plays some role in EDL muscle, neither sCD14 nor LBP can fully account for the strong effects of plasma on LPS sensitivity.

Diminished bone perfusion develops in response to disuse and has been proposed as a mechanism underlying bone loss. Bone blood flow (BF) has not been investigated within the unique context of severe contusion spinal cord injury (SCI), a condition that produces neurogenic bone loss that is precipitated by disuse and other physiological consequences of central nervous system injury. Herein, 4-mo-old male Sprague–Dawley rats received T(9) laminectomy (SHAM) or laminectomy with severe contusion SCI (n = 20/group). Time course assessments of hindlimb bone microstructure and bone perfusion were performed in vivo at 1- and 2-wk postsurgery via microcomputed tomography (microCT) and intracardiac microsphere infusion, respectively, and bone turnover indices were determined via histomorphometry. Both groups exhibited cancellous bone loss beginning in the initial postsurgical week, with cancellous and cortical bone deficits progressing only in SCI thereafter. Trabecular bone deterioration coincided with uncoupled bone turnover after SCI, as indicated by signs of ongoing osteoclast-mediated bone resorption and a near-complete absence of osteoblasts and cancellous bone formation. Bone BF was not different between groups at 1 wk, when both groups displayed bone loss. In comparison, femur and tibia perfusion was 30%–40% lower in SCI versus SHAM at 2 wk, with the most pronounced regional BF deficits occurring at the distal femur. Significant associations existed between distal femur BF and cancellous and cortical bone loss indices. Our data provide the first direct evidence indicating that bone BF deficits develop in response to SCI and temporally coincide with suppressed bone formation and with cancellous and cortical bone deterioration. NEW & NOTEWORTHY We provide the first direct evidence indicating femur and tibia blood flow (BF) deficits exist in conscious (awake) rats after severe contusion spinal cord injury (SCI), with the distal femur displaying the largest BF deficits. Reduced bone perfusion temporally coincided with unopposed bone resorption, as indicated by ongoing osteoclast-mediated bone resorption and a near absence of surface-level bone formation indices, which resulted in severe cancellous and cortical microstructural deterioration after SCI.

Spinal cord injury (SCI) is associated with obesity and is a risk factor for type 2 diabetes mellitus (T2DM). Immobilization, muscle atrophy, obesity, and loss of sympathetic innervation to the liver are believed to contribute to risks of these abnormalities. Systematic study of the mechanisms underlying SCI-induced metabolic disorders has been limited by a lack of animal models of insulin resistance following SCI. Therefore, the effects of a high-fat diet (HFD), which causes weight gain and glucose intolerance in neurologically intact mice, was tested in mice that had undergone a spinal cord transection at thoracic vertebra 10 (T10) or a sham-transection. At 84 days after surgery, Sham-HFD and SCI-HFD mice showed impaired intraperitoneal glucose tolerance when compared with Sham control (Sham-Con) or SCI control (SCI-Con) mice fed a standard control chow. Glucose tolerance in SCI-Con mice was comparable to that of Sham-Con mice. The mass of paralyzed skeletal muscle, liver, and epididymal, inguinal, and omental fat deposits were lower in SCI versus Sham groups, with lower liver mass present in SCI-HFD versus SCI-Con animals. SCI also produced sublesional bone loss, with no differences between SCI-Con and SCI-HFD groups. The results suggest that administration of a HFD to mice after SCI may provide a model to better understand mechanisms leading to insulin resistance post-SCI, as well as an approach to study pathogenesis of glucose intolerance that is independent of obesity.

Skeletal muscles secrete a wide variety of immunologically active cytokines, but the functional significance of this response to in vivo innate immunity is not understood. We addressed this by knocking out the toll receptor adapter protein, Myd88, only in skeletal muscle fibers (skmMyd88KO), and followed male and female mice at 6 and 12 h after peritoneal injection of cecal slurry (CS), a model of polymicrobial sepsis. Because of a previously identified increase in mortality to CS injection, males received ~ 30% lower dose. At 12 h, skmMyd88KO caused significant reductions in a wide variety of pro- and anti-inflammatory plasma cytokines, e.g. TNFα, IL-1β and IL-10, compared to strain-matched controls in both males and females. Similar reductions were observed at 6 h in females. SkmMyd88KO led to ~ 40–50% elevations in peritoneal neutrophils at 6 and 12 h post CS in females. At 12 h post CS, skmMyd88KO increased peritoneal monocytes/macrophages and decreased %eosinophils and %basophils in females. SkmMyd88KO also led to significantly higher rates of mortality in female mice but not in males. In conclusion, the results suggest that skeletal muscle Myd88-dependent signal transduction can play functionally important role in normal whole body, innate immune inflammatory responses to peritoneal sepsis.

Interleukin-6 (IL-6) is a major cytokine released by skeletal muscle. Although IL-6 plays complex but well-known roles in host defense, the specific contribution of skeletal muscle IL-6 to innate immunity remains unknown. We tested its functional relevance by exposing inducible skeletal muscle IL-6 knockdown (skmIL-6KD) mice to a cecal slurry model of polymicrobial peritonitis and compared responses to strain-matched controls and skeletal muscle Cre-matched controls at 3, 6, and 12 h postinfection. In both sexes, skmIL-6KD mice at 6 h of infection exhibited marked changes to leukocyte trafficking in the peritoneum, characterized by ∼1.75-fold elevation in %neutrophils, a ∼3-fold reduction in %lymphocytes and a ∼2 to 3-fold reduction in %basophils. A similar pattern was seen at 12 h. No changes were observed in plasma leukocyte counts. Circulating cytokines in female skmIL-6KD mice at 6 h consistently showed modest reductions in IL-6, but marked reductions in a broad range of both pro- and anti-inflammatory cytokines, e.g., TNFα and IL-10. In both sexes at 12 h, a generalized suppression of plasma cytokines was also seen after the effects of Cre-induction with raloxifene were addressed. There were no significant effects of skmIL-6KD on mortality in either sex. Collectively, our results are consistent with skmIL-6 playing an important and previously unrecognized role in immune cell trafficking and cytokine regulation during septic shock.

Intestinal injury is one of the most prominent features of organ damage in exertional heat stroke (EHS). However, whether damage to the intestine in this setting is exacerbated by ibuprofen (IBU), the most commonly used nonsteroidal anti-inflammatory drug in exercising populations, is not well understood. PURPOSE We hypothesized that IBU would exacerbate intestinal injury, reduce exercise performance, and increase susceptibility to heat stroke. METHODS To test this hypothesis, we administered IBU via diet to male and female C57/BL6J mice, over 48 h before EHS. Susceptibility to EHS was determined by assessing exercise response using a forced running wheel, housed inside an environmental chamber at 37.5°C. Core temperature (Tc) was monitored by telemetry. Mice were allocated into four groups: exercise only (EXC); EHS + IBU; EXC + IBU; and EHS only. Exercise performance and Tc profiles were evaluated and stomachs, intestines and plasma were collected at 3 h post-EHS. RESULTS The EHS + IBU males ran approximately 87% longer when Tc was above 41°C (P < 0.03) and attained significantly higher peak Tc (P < 0.01) than EHS-only mice. Histological analyses showed decreased villi surface area throughout the small intestine for both sexes in the EXC + IBU group versus EXC only. Interestingly, though EHS in both sexes caused intestinal injury, in neither sex were there any additional effects of IBU. CONCLUSIONS Our results suggest that in a preclinical mouse model of EHS, oral IBU at pharmacologically effective doses does not pose additional risks of heat stroke, does not reduce exercise performance, and does not contribute further to intestinal injury, though this could have been masked by significant gut injury induced by EHS alone.

Spinal cord injury (SCI) produces paralysis and a unique form of neurogenic disuse osteoporosis that dramatically increases fracture risk at the distal femur and proximal tibia. This bone loss is driven by heightened bone resorption and near-absent bone formation during the acute post-SCI recovery phase and by a more traditional high-turnover osteopenia that emerges more chronically, which is likely influenced by the continual neural impairment and musculoskeletal unloading. These observations have stimulated interest in specialized exercise or activity-based physical therapy (ABPT) modalities (e.g., neuromuscular or functional electrical stimulation cycling, rowing, or resistance training, as well as other standing, walking, or partial weight-bearing interventions) that reload the paralyzed limbs and promote muscle recovery and use-dependent neuroplasticity. However, only sparse and relatively inconsistent evidence supports the ability of these physical rehabilitation regimens to influence bone metabolism or to increase bone mineral density (BMD) at the most fracture-prone sites in persons with severe SCI. This review discusses the pathophysiology and cellular/molecular mechanisms that influence bone loss after SCI, describes studies evaluating bone turnover and BMD responses to ABPTs during acute versus chronic SCI, identifies factors that may impact the bone responses to ABPT, and provides recommendations to optimize ABPTs for bone recovery.

With increasing participation of females in endurance athletics and active military service, it is important to determine if there are inherent sex-dependent susceptibilities to exertional heat injury or heat stroke. In this study we compared responses of male and female adult mice to exertional heat stroke (EHS). All mice were instrumented for telemetry core temperature measurements and were exercise-trained for 3 wk before EHS. During EHS, environmental temperature was 37.5°C (35% RH) while the mice ran on a forced running wheel, using incremental increases in speed. The symptom-limited endpoint was loss of consciousness, occurring at ~42.2°C core temperature. Females ran greater distances (623 vs. 346 m, P < 0.0001), reached faster running speeds (7.2 vs. 5.1 m/min, P < 0.0001), exercised for longer times (177 vs. 124 min, P < 0.0001), and were exposed to greater internal heat loads (240 vs.160°C·min; P < 0.0001). Minimum Tc during hypothermic recovery was ~32.0°C in both sexes. Females lost 9.2% body weight vs. 7.5% in males ( P < 0.001). Females demonstrated higher circulating corticosterone (286 vs 183 ng/ml, P = 0.001, at 3 h), but most plasma cytokines were not different. A component of performance in females could be attributed to greater body surface area/mass and greater external power performance. However, there were significant and independent effects of sex alone and a crossed effect of “sex × power” on performance. These results demonstrate that female mice have greater resistance to EHS during exercise in hyperthermia and that these effects cannot be attributed solely to body size.NEW & NOTEWORTHY Female mice are surprisingly more resistant to exertional heat stroke than male mice. They run faster and longer and can withstand greater internal heat loads. These changes cannot be fully accounted for by increased body surface/mass ratio in females or on differences in aerobic performance. Although the stress-immune response in males and females was similar, females exhibited markedly higher plasma corticosteroid levels, which were sustained over 14 days of recovery.

Key points Exposure to exertional heat stroke (EHS) is associated with increased risk of long-term cardiovascular disorders in humans. We demonstrate that in female mice, severe EHS results in metabolic changes in the myocardium, emerging only after 9-14 days. This was not observed in males that were symptom-limited at much lower exercise levels and heat loads compared to females. At 14 days of recovery in females, there were marked elevations in myocardial free fatty acids, ceramides and diacylglycerols, consistent with development of underlying cardiac abnormalities. Glycolysis shifted towards the pentose phosphate and glycerol-3-phosphate dehydrogenase pathways. There was evidence for oxidative stress, tissue injury and microscopic interstitial inflammation. The tricarboxylic acid cycle and nucleic acid metabolism pathways were also negatively affected. We conclude that exposure to EHS in female mice has the capacity to cause delayed metabolic disorders in the heart that could influence long-term health. Abstract Exposure to exertional heat stroke (EHS) is associated with a higher risk of long-term cardiovascular disease in humans. Whether this is a cause-and-effect relationship remains unknown. We studied the potential of EHS to contribute to the development of a 'silent' form of cardiovascular disease using a preclinical mouse model of EHS. Plasma and ventricular myocardial samples were collected over 14 days of recovery. Male and female C57bl/6J mice underwent forced wheel running for 1.5-3 h in a 37.5°C/40% relative humidity until symptom limitation, characterized by CNS dysfunction. They reached peak core temperatures of 42.2 ± 0.3°C. Females ran ∼40% longer, reaching ∼51% greater heat load. Myocardial and plasma samples (n = 8 per group) were obtained between 30 min and 14 days of recovery, analysed using metabolomics/lipidomics platforms and compared to exercise controls. The immediate recovery period revealed an acute energy substrate crisis from which both sexes recovered within 24 h. However, at 9-14 days, the myocardium of female mice developed marked elevations in free fatty acids, ceramides and diacylglycerols. Glycolytic and tricarboxylic acid cycle metabolites revealed bottlenecks in substrate flow, with build-up of intermediate metabolites consistent with oxidative stress and damage. Males exhibited only late stage reductions in acylcarnitines and elevations in acetylcarnitine. Histopathology at 14 days showed interstitial inflammation in the female hearts only. The results demonstrate that the myocardium of female mice is vulnerable to a slowly emerging metabolic disorder following EHS that may harbinger long-term cardiovascular complications. Lack of similar findings in males may reflect their lower heat exposure.

Hyperthermia and dehydration can occur during exercise in hot environments. Nevertheless, whether elevations in extracellular osmolality contributes to the increased skeletal muscle tension, sarcolemmal injury, and oxidative stress reported in warm climates remains unknown. We simulated osmotic and heat stress, in vitro, in mouse limb muscles with different fiber compositions. Extensor digitorum longus (EDL) and soleus (SOL) were dissected from 36 male C57BL6J and mounted at optimal length in tissue baths containing oxygenated buffer. Muscles were stimulated with non-fatiguing twitches for 30 min. Four experimental conditions were tested: isotonic-normothermia (285 mOsm•kg-1 and 35°C), hypertonic-normothermia (300 mOsm•kg-1 and 35°C), isotonic-hyperthermia (285 mOsm•kg-1 and 41°C), and hypertonic-hyperthermia (300 mOsm•kg-1 and 41°C). Passive tension was recorded continuously. The integrity of the sarcolemma was determined using a cell-impermeable fluorescent dye and immunoblots were used for detection of protein carbonyls. In EDL muscles, isotonic and hypertonic-hyperthermia increased resting tension (P < 0.001). Whereas isotonic-hyperthermia increased sarcolemmal injury in EDL (P < 0.001), this effect was absent in hypertonic-hyperthermia. Similarly, isotonic-hyperthermia elevated protein carbonyls (P = 0.018), a response not observed with hypertonic-hyperthermia. In SOL muscles, isotonic-hyperthermia also increases resting tension (P < 0.001); however, these effects were eliminated in hypertonic-hyperthermia. Unlike EDL, there were no effects of hyperthermia and/or hyperosmolality on sarcolemmal injury or protein carbonyls. Osmolality selectively modifies skeletal muscle response to hyperthermia in this model. Fast-glycolytic muscle appears particularly vulnerable to isotonic-hyperthermia, resulting in elevated muscle tension, sarcolemmal injury and protein oxidation; whereas slow-oxidative muscle exhibits increased tension but no injury or protein oxidation under the conditions and duration tested.

Sepsis continues to be a major challenge for modern medicine. Several preclinical models were developed to study sepsis and each has strengths and weaknesses. The cecal slurry (CS) method is a practical alternative because it does not require surgery, and the infection can be dosed. However, one disadvantage is that the dosage must be determined for each CS preparation using survival studies. Our aim was to refine a survival protocol for the CS model by determining a premonitory humane endpoint that would reduce animal suffering. Mice become hypothermic in sepsis; therefore, we tested whether reductions in surface temperature (T(s)), measured by non-invasive infrared thermometry, could predict eventual death. We injected 154 C57BL/6J mice with CS (0.9–1.8 mg/g) and periodically monitored T(s) at the xiphoid process over 5 days. We used, as predictors, combinations of temperature thresholds (29–31°C) and times, post injection (18–36 h). A receiver-operator curve, sensitivity, and specificity were determined. A Distress Index value was calculated for the threshold conditions. The optimum detection threshold (highest Youden’s index) was found at T(s) ≤ 30.5°C at 24 h (90% specific, 84% sensitive). This threshold condition reduced animal suffering by 41% while providing an accurate survival rate estimate. Using this threshold, only 13/154 mice would have died from sepsis; 67 would have been euthanized at 24 h, and only 7/154 would have been euthanized unnecessarily. In conclusion, using a humane endpoint of T(s) ≤ 30.5°C at 24 h accurately predicts mortality and can effectively reduce animal suffering during CS survival protocols.

Catecholamines stimulate interleukin‐6 (IL‐6) secretion in skeletal muscles. However, whether other cytokines are secreted is currently unknown. Skeletal muscle ex vivo preparations commonly used to study cytokine secretion have dealt with limitations including auto‐oxidation of catecholamines. The use of metal chelators could be an alternative to avoid auto‐oxidation and allow catecholamines to be used at physiological doses. We exposed isolated soleus muscles to 1 or 100 ng/mL epinephrine (EPI) and collected bath samples at 1 and 2 h for multiplex cytokine analysis. Keratinocyte chemoattractant (CXCL1), IL‐6, and IL‐1 α were significantly elevated by 100 ng/mL exposure, but not by 1 ng/mL (median [CXCL1] (2 h) = 83 pg/mL; [IL‐6] = 19 pg/mL; IL‐1 α = 7.5 pg/mL). CXCL1 and IL‐6 were highly correlated in each sample ( P = 0.0001). A second experiment combined the metal chelator, deferoxamine mesylate (DFO), to prevent EPI autoxidation, with 2 ng/mL EPI and 10.5 ng/mL norepinephrine (NOREPI) to mimic peak exercise. Unexpectedly, DFO alone stimulated both IL‐6 and CXCL1 secretion, but together with EPI and NOREPI had no additional effects. Stimulation of cytokine secretory responses from skeletal muscle cells in response to DFO thus precludes its use as a chelating agent in ex vivo models. In conclusion, 100 ng/mL EPI stimulates a robust secretory CXCL1 response, which together with IL‐6 and IL‐1 α , may constitute an adrenal‐muscle endocrine response system.

In inflammatory cells, hyperthermia inhibits lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) gene expression and protein secretion. Since hyperthermia alone stimulates IL-6 in skeletal muscle, we hypothesized that it would amplify responses to other receptor-mediated stimuli. IL-6 regulation was tested in C2C12 myotubes and in soleus during treatment with epinephrine (EPI) or LPS. In EPI-treated myotubes (100 ng/ml), 1 h exposure at 40.5°C-42°C transiently increased IL-6 mRNA compared to EPI treatment alone at 37°C. In LPS-treated myotubes (1 μg/ml), exposure to 41°C-42°C also increased IL-6 mRNA. In isolated mouse soleus, similar amplifications of IL-6 gene expression were observed in 41°C, during both low (1 ng/ml) and high dose (100 ng/ml) EPI, but only in high dose LPS (1 μg/ml). In myotubes, heat increased IL-6 secretion during EPI exposure but had no effect or inhibited secretion with LPS. In soleus there were no effects of heat on IL-6 secretion during either EPI or LPS treatment. Mechanisms for the effects of heat on IL-6 mRNA were explored using a luciferase-reporter in C2C12 myotubes. Overexpression of heat shock factor-1 (HSF-1) had no impact on IL-6 promoter activity during EPI stimulation, but elevated IL-6 promoter activity during LPS stimulation. In contrast, when the activator protein-1 (AP-1) element was mutated, responses to both LPS and EPI were suppressed in heat. Using siRNA against activating transcription factor-3 (ATF-3), a heat-stress-induced inhibitor of IL-6, no ATF-3-dependent effects were observed. The results demonstrate that, unlike inflammatory cells, hyperthermia in muscle fibers amplifies IL-6 gene expression to EPI and LPS. The effect appears to reflect differential engagement of HSF-1 and AP-1 sensitive elements on the IL-6 gene, with no evidence for involvement of ATF-3. The functional significance of increased IL-6 mRNA expression during heat may serve to overcome the well-known suppression of protein synthetic pathways occurring during heat shock.