A single chemotherapy administration induces muscle atrophy, mitochondrial alterations and apoptosis in breast cancer patients.

BACKGROUND: Breast cancer patients are commonly treated with sequential administrations of epirubicin-cyclophosphamide (EC) and paclitaxel (TAX). The chronic effect of this treatment induces skeletal muscle alterations, but the specific effect of each chemotherapy agent is unknown. This study aimed to investigate the effect of EC or TAX administration on skeletal muscle homeostasis in breast cancer patients. METHODS: Twenty early breast cancer patients undergoing EC followed by TAX chemotherapies were included. Two groups of 10 women were established and performed vastus lateralis skeletal muscle biopsies either before the first administration (pre) of EC (50 ± 14 years) or TAX (50 ± 16 years) and 4 days later (post). Mitochondrial respiratory capacity recording, reactive oxygen species production, western blotting and histological analyses were performed. RESULTS: Decrease in muscle fibres cross-sectional area was only observed post-EC (-25%; P < 0.001), associated with a reduction in mitochondrial respiratory capacity for the complex I (CI)-linked substrate state (-32%; P = 0.001), oxidative phosphorylation (OXPHOS) by CI (-35%; P = 0.002), CI&CII (-26%; P = 0.022) and CII (-24%; P = 0.027). If H2 O2 production was unchanged post-EC, an increase was observed post-TAX for OXPHOS by CII (+25%; P = 0.022). We found a decrease in makers of mitochondrial content, as shown post-EC by a decrease in the protein levels of citrate synthase (-53%; P < 0.001) and VDAC (-39%; P < 0.001). Despite no changes in markers of mitochondrial fission, a decrease in the expression of a marker of mitochondrial inner-membrane fusion was found post-EC (OPA1; -60%; P < 0.001). We explored markers of mitophagy and found reductions post-EC in the protein levels of PINK1 (-63%; P < 0.001) and Parkin (-56%; P = 0.005), without changes post-TAX. An increasing trend in Bax protein level was found post-EC (+96%; P = 0.068) and post-TAX (+77%; P = 0.073), while the Bcl-2 level was decreased only post-EC (-52%; P = 0.007). If an increasing trend in TUNEL-positive signal was observed post-EC (+68%; P = 0.082), upregulation was highlighted post-TAX (+86%; P < 0.001), suggesting activation of the apoptosis process. CONCLUSIONS: We demonstrated that a single administration of EC induced, in only 4 days, skeletal muscle atrophy and mitochondrial alterations in breast cancer patients. These alterations were characterized by reductions in mitochondrial function and content as well as impairment of mitochondrial dynamics and an increase in apoptosis. TAX administration did not worsen these alterations as this group had already received EC during the preceding weeks. However, it resulted in an increased apoptosis, likely in response to the increased H2 O2 production.

Peripheral Blood Mononuclear Cells Mitochondrial Respiration and Superoxide Anion after Heart Transplantation.

INTRODUCTION: The mitochondrial function of circulating peripheral blood mononuclear cells (PBMCs) is an interesting new approach to cardiac diseases. Thus, PBMC's mitochondrial respiration decreases in relation to heart failure severity. However, no data are available on heart-transplanted patients (Htx). POPULATION AND METHODS: We determined PBMCs mitochondrial respiration by high-resolution respirometry (Oroboros Instruments) and superoxide anion production using electron paramagnetic resonance (Bruker-Biospin) in 20 healthy subjects and 20 matched Htx and investigated clinical, biological, echocardiographic, coronarography and biopsy characteristics. RESULTS: PBMCs mitochondrial respiratory chain complex II respiration was decreased in Htx (4.69 ± 0.84 vs. 7.69 ± 1.00 pmol/s/million cell in controls and Htx patients, respectively; p = 0.007) and complex IV respiration was increased (24.58 ± 2.57 vs. 15.68 ± 1.67 pmol/s/million cell; p = 0.0035). Superoxide anion production was also increased in Htx (1.47 ± 0.10 vs. 1.15 ± 0.10 µmol/min; p = 0.041). The leucocyte-to-lymphocyte ratio was increased in Htx, whom complex II correlated with leucocyte number (r = 0.51, p = 0.02) and with the left ventricular posterior wall peak early diastolic myocardial velocity (r = -0.62, p = 0.005). Complex IV was increased in the two patients with acute rejection and correlated negatively with Htx's isovolumetric relation time (r = -0.45, p = 0.045). DISCUSSION: Although presenting with normal systolic function, Htx demonstrated abnormal PBMC's mitochondrial respiration. Unlike immunosuppressive therapies, subclinical diastolic dysfunction might be involved in these changes. Additionally, lymphopenia might reduce complex II, and acute rejection enhances complex IV respirations. CONCLUSION: PBMC's mitochondrial respiration appears modified in Htx, potentially linked to cellular shift, mild diastolic dysfunction and/or acute rejection.

Chemotherapy impairs skeletal muscle mitochondrial homeostasis in early breast cancer patients.

BACKGROUND: Chemotherapy is extensively used to treat breast cancer and is associated with skeletal muscle deconditioning, which is known to reduce patients' quality of life, treatment efficiency, and overall survival. To date, skeletal muscle mitochondrial alterations represent a major aspect explored in breast cancer patients; nevertheless, the cellular mechanisms remain relatively unknown. This study was dedicated to investigating overall skeletal muscle mitochondrial homeostasis in early breast cancer patients undergoing chemotherapy, including mitochondrial quantity, function, and dynamics. METHODS: Women undergoing (neo)adjuvant anthracycline-cyclophosphamide and taxane-based chemotherapy participated in this study (56 ± 12 years). Two muscle biopsies were collected from the vastus lateralis muscle before the first and after the last chemotherapy administration. Mitochondrial respiratory capacity, reactive oxygen species production, and western blotting analyses were performed. RESULTS: Among the 11 patients, we found a decrease in key markers of mitochondrial quantity, reaching -52.0% for citrate synthase protein levels (P = 0.02) and -38.2% for VDAC protein levels (P = 0.04). This mitochondrial content loss is likely explained by reduced mitochondrial biogenesis, as evidenced by a decrease in PGC-1α1 protein levels (-29.5%; P = 0.04). Mitochondrial dynamics were altered, as documented by a decrease in MFN2 protein expression (-33.4%; P = 0.01), a key marker of mitochondrial outer membrane fusion. Mitochondrial fission is a prerequisite for mitophagy activation, and no variation was found in either key markers of mitochondrial fission (Fis1 and DRP1) or mitophagy (Parkin, PINK1, and Mul1). Two contradictory hypotheses arise from these results: defective mitophagy, which probably increases the number of damaged and fragmented mitochondria, or a relative increase in mitophagy through elevated mitophagic potential (Parkin/VDAC ratio; +176.4%; P < 0.02). Despite no change in mitochondrial respiratory capacity and COX IV protein levels, we found an elevation in H2 O2 production (P < 0.05 for all substrate additions) without change in antioxidant enzymes. We investigated the apoptosis pathway and found an increase in the protein expression of the apoptosis initiation marker Bax (+72.0%; P = 0.04), without variation in the anti-apoptotic protein Bcl-2. CONCLUSIONS: This study demonstrated major mitochondrial alterations subsequent to chemotherapy in early breast cancer patients: (i) a striking reduction in mitochondrial biogenesis, (ii) altered mitochondrial dynamics and potential mitophagy defects, (iii) exacerbated H2 O2 production, and (iv) increased initiation of apoptosis. All of these alterations likely explain, at least in part, the high prevalence of skeletal muscle and cardiorespiratory deconditioning classically observed in breast cancer patients.

Deleterious Effects of Remote Ischaemic Per-conditioning During Lower Limb Ischaemia-Reperfusion in Mice.

OBJECTIVE: The aim of this study was to investigate whether remote ischaemic per-conditioning might protect skeletal muscle during lower limb ischaemia-reperfusion (IR). METHODS: Twenty-three male C57BL/6 mice were randomised into three groups: sham group (n = 7), IR group (unilateral tourniquet induced three hours of ischaemia followed by 24 hours of reperfusion, n = 8), and remote ischaemic per-conditioning group (RIPerC) (three cycles of 10 minute IR episodes on the non-ischaemic contralateral hindlimb, n = 8). Oxygraphy, spectrofluorometry, and electron paramagnetic resonance spectroscopy were performed in order to determine mitochondrial respiratory chain complexes activities, mitochondrial calcium retention capacity (CRC) and reactive oxygen species (ROS) production in skeletal muscle. RESULTS: IR impaired mitochondrial respiration (3.66 ± 0.98 vs. 7.31 ± 0. 54 µmol/min/g in ischaemic and sham muscles, p = .009 and p = .003 respectively) and tended to impair CRC (2.53 ± 0.32 vs. 3.64 ± 0.66 µmol/mg in ischaemic and sham muscles respectively, p = .066). IR did not modify ROS production (0.082 ± 0.004 vs. 0.070 ± 0.004 µmol/min/mg in ischaemic and sham muscles respectively, p = .74). RIPerC failed to restore mitochondrial respiration (3.82 ± 0.40 vs. 3.66 ± 0.98 µmol/min/g in ischaemic muscles from the RIPerC group and the IR group respectively, p = .45) and CRC (2.76 ± 0.3 vs. 2.53 ± 0.32 µmol/mg in ischaemic muscles from the RIPerC group and the IR group respectively, p = .25). RIPerC even impaired contralateral limb mitochondrial respiration (3.85 ± 0.34 vs. 7.31 ± 0. 54 µmol/min/g in contralateral muscles and sham muscles respectively, -47.3%, p = .009). CONCLUSION: RIPerC failed to protect ischaemic muscles and induced deleterious effects on the contralateral non-ischaemic muscles. These data do not support the concept of RIPerC.

Digestive n-6 Lipid Oxidation, a Key Trigger of Vascular Dysfunction and Atherosclerosis in the Western Diet: Protective Effects of Apple Polyphenols.

SCOPE: A main risk factor of atherosclerosis is a Western diet (WD) rich in n-6 polyunsaturated fatty acids (PUFAs) sensitive to oxidation. Their oxidation can be initiated by heme iron of red meat leading to the formation of 4-hydroxy-2-nonenal (4-HNE), a cytotoxic aldehyde. An increased 4-HNE production is implicated in endothelial dysfunction and atherosclerosis. By contrast, a diet rich in proanthocyanidins reduces oxidative stress and arterial diseases. This study evaluates the effects of a WD on vascular integrity in ApolipoproteinE (ApoE-/- ) mice and the protective capacity of apple extract and puree rich in antioxidant proanthocyanidins. METHODS AND RESULTS: ApoE-/- mice are fed during 12 weeks with a WD with or without n-6 PUFAs. Moreover, two WD + n-6 PUFAs groups are supplemented with apple puree or phenolic extract. An increase in digestive 4-HNE production associated with a rise in plasmatic 4-HNE and oxidized LDL concentrations is reported. Oxidizable n-6 PUFAs consumption is associated with a worsened endothelial dysfunction and atherosclerosis. Interestingly, supplementations with apple polyphenol extract or puree prevented these impairments while reducing oxidative stress. CONCLUSION: n-6 lipid oxidation during digestion may be a key factor of vascular impairments. Nevertheless, an antioxidant strategy can limit 4-HNE formation during digestion and thus durably protect vascular function.

Intraoperative Perfusion Assessment in Enhanced Reality Using Quantitative Optical Imaging: An Experimental Study in a Pancreatic Partial Ischemia Model.

To reduce the risk of pancreatic fistula after pancreatectomy, a satisfactory blood flow at the pancreatic stump is considered crucial. Our group has developed and validated a real-time computational imaging analysis of tissue perfusion, using fluorescence imaging, the fluorescence-based enhanced reality (FLER). Hyperspectral imaging (HSI) is another emerging technology, which provides tissue-specific spectral signatures, allowing for perfusion quantification. Both imaging modalities were employed to estimate perfusion in a porcine model of partial pancreatic ischemia. Perfusion quantification was assessed using the metrics of both imaging modalities (slope of the time to reach maximum fluorescence intensity and tissue oxygen saturation (StO2), for FLER and HSI, respectively). We found that the HSI-StO2 and the FLER slope were statistically correlated using the Spearman analysis (R = 0.697; p = 0.013). Local capillary lactate values were statistically correlated to the HSI-StO2 and to the FLER slope (R = -0.88; p < 0.001 and R = -0.608; p = 0.0074). HSI-based and FLER-based lactate prediction models had statistically similar predictive abilities (p = 0.112). Both modalities are promising to assess real-time pancreatic perfusion. Clinical translation in human pancreatic surgery is currently underway.

Computer-assisted quantification and visualization of bowel perfusion using fluorescence-based enhanced reality in left-sided colonic resections.

BACKGROUND: Fluorescence-based enhanced reality (FLER) is a computer-based quantification method of fluorescence angiographies to evaluate bowel perfusion. The aim of this prospective trial was to assess the clinical feasibility and to correlate FLER with metabolic markers of perfusion, during colorectal resections. METHODS: FLER analysis and visualization was performed in 22 patients (diverticulitis n = 17; colorectal cancer n = 5) intra- and extra-abdominally during distal and proximal resection, respectively. The fluorescence signal of indocyanine green (0.2 mg/kg) was captured using a near-infrared camera and computed to create a virtual color-coded cartography. This was overlaid onto the bowel (enhanced reality). It helped to identify regions of interest (ROIs) where samples were subsequently obtained. Resections were performed strictly guided according to clinical decision. On the surgical specimen, samplings were made at different ROIs to measure intestinal lactates (mmol/L) and mitochondria efficiency as acceptor control ratio (ACR). RESULTS: The native (unquantified) fluorescent signal diffused to obvious ischemic areas during the distal appreciation. Proximally, a lower diffusion of ICG was observed. Five anastomotic complications occurred. The expected values of local capillary lactates were correlated with the measured values both proximally (3.62 ± 2.48 expected vs. 3.17 ± 2.8 actual; rho 0.89; p = 0.0006) and distally (4.5 ± 3 expected vs. 4 ± 2.5 actual; rho 0.73; p = 0.0021). FLER values correlated with ACR at the proximal site (rho 0.76; p = 0.04) and at the ischemic zone (rho 0.71; p = 0.01). In complicated cases, lactates at the proximal resection site were higher (5.8 ± 4.5) as opposed to uncomplicated cases (2.45 ± 1.5; p = 0.008). ACR was reduced proximally in complicated (1.3 ± 0.18) vs. uncomplicated cases (1.68 ± 0.3; p = 0.023). CONCLUSIONS: FLER allows to image the quantified fluorescence signal in augmented reality and provides a reproducible estimation of bowel perfusion (NCT02626091).

Sarcopenia and peripheral arterial disease: a systematic review.

BACKGROUND: Patients with lower extremity peripheral arterial disease (PAD) and sarcopenia are a population at risk requiring specific and targeted care. The aim of this review is to gather all relevant studies associating sarcopenia and PAD and to identify the underlying pathophysiological mechanisms as well as potential therapeutic strategies to improve skeletal muscle function. METHODS: A systematic review was carried out following the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). RESULTS: Data extraction allowed the evaluation of 140 publications; 87 met the inclusion criteria; of which 79 were included in the final review, reporting sufficient data for epidemiological and diagnostic criteria, mechanical analysis, and therapeutic approaches. Epidemiological analysis and diagnostic criteria were based on 18 studies following 2362 PAD patients [31.39% (SD 7.61) women], aged 72.42 (SD 2.84); sarcopenia was present in 34.63% (SD 12.86) of the patients. Mechanical and pathway analysis were based on five animal studies and 29 clinical reports, showing significantly altered muscle strength and function in 1352 PAD patients [26.49% (SD 17.32) women], aged 67.67 (SD 5.14) years; impaired muscle histology in 192 PAD patients (9.2% (SD 11.22) women), aged 64.3 (SD 0.99) years; +58.63% (SD 25.48) of oxidative stress in 69 PAD patients [16.96% (SD 8.10) women], aged 63.17 (SD 1.43) years; mitochondriopathy in 153 PAD patients [29.39% (SD 28.27) women], aged 63.50 (SD 1.83) years; +15.58% (SD 7.41) of inflammation in 900 PAD patients [40.77% (SD 3.71) women], aged 74.88 (SD 2.76) years; and altered signalling pathways in 51 PAD patients [34.45% (SD 32.23) women], aged 72.25 (SD 5.25) years. Therapeutic approaches analysis was based on seven animal studies and 21 clinical reports. In total, 884 patients followed an exercise therapy, and 18 received an angiogenesis treatment; 30.84% (SD 17.74) were women. Mean ages of patients studied were 66.85 (SD 3.96). CONCLUSIONS: Sarcopenia and lower extremity PAD have musculoskeletal consequences that directly impair patients' quality of life and prognosis. Although PAD is primarily a vascular disease, all etiological factors of sarcopenia identified so far are present in PAD. Indeed, both sarcopenia and PAD are accompanied by oxidative stress, skeletal muscle mitochondrial impairments, inflammation, inhibition of specific pathways regulating muscle synthesis or protection (i.e. IGF-1, RISK, and SAFE), and activation of molecules associated with muscle degradation. To date, besides revascularization, the best therapeutic strategy includes exercise, but approaches targeting the underlying mechanisms still deserve further studies.

HYPerspectral Enhanced Reality (HYPER): a physiology-based surgical guidance tool.

BACKGROUND: HSI is an optical technology allowing for a real-time, contrast-free snapshot of physiological tissue properties, including oxygenation. Hyperspectral imaging (HSI) has the potential to quantify the gastrointestinal perfusion intraoperatively. This experimental study evaluates the accuracy of HSI, in order to quantify bowel perfusion, and to obtain a superposition of the hyperspectral information onto real-time images. METHODS: In 6 pigs, 4 ischemic bowel loops were created (A, B, C, D) and imaged at set time points (from 5 to 360 min). A commercially available HSI system provided pseudo-color maps of the perfusion status (StO2, Near-InfraRed perfusion) and the tissue water index. An ad hoc software was developed to superimpose HSI information onto the live video, creating the HYPerspectral-based Enhanced Reality (HYPER). Seven regions of interest (ROIs) were identified in each bowel loop according to StO2 ranges, i.e., vascular (VASC proximal and distal), marginal vascular (MV proximal and distal), marginal ischemic (MI proximal and distal), and ischemic (ISCH). Local capillary lactates (LCL), reactive oxygen species (ROS), and histopathology were measured at the ROIs. A machine-learning-based prediction algorithm of LCL, based on the HSI-StO2%, was trained in the 6 pigs and tested on 5 additional animals. RESULTS: HSI parameters (StO2 and NIR) were congruent with LCL levels, ROS production, and histopathology damage scores at the ROIs discriminated by HYPER. The global mean error of LCL prediction was 1.18 ± 1.35 mmol/L. For StO2 values > 30%, the mean error was 0.3 ± 0.33. CONCLUSIONS: HYPER imaging could precisely quantify the overtime perfusion changes in this bowel ischemia model.

Critical Limb Ischaemia Exacerbates Mitochondrial Dysfunction in ApoE-/- Mice Compared with ApoE+/+ Mice, but N-acetyl Cysteine still Confers Protection.

OBJECTIVES: The current study was performed in order to determine the influence of hypercholesterolaemia on critical limb ischaemia (CLI) and whether targeting oxidative stress by antioxidant therapies such as N-acetyl cysteine (NAC), considered to be a direct scavenger of reactive oxygen species, could confer muscle protection. METHODS: Apolipoprotein E (ApoE)-/- mice (n = 9, 29 weeks old) and their genetic controls ApoE+/+ mice (n = 9, 29 weeks old) were submitted to sequential right femoral and iliac ligations; the left limb served as control. ApoE+/+ mice were divided into two groups: Group 1 (n = 4) and Group 2 (n = 5); as well as ApoE-/- mice: Group 3 (n = 3), and Group 4 (n = 6). NAC treatment was administered to Groups 2 and 4 in drinking water. Mice were sacrificed on Day 40 and gastrocnemius muscles were harvested to study mitochondrial respiration by oxygraphy, calcium retention capacity by spectrofluorometry, and production of reactive oxygen species by electron paramagnetic resonance. RESULTS: CLI associated with ApoE deficiency resulted in more severe mitochondrial dysfunction: maximum oxidative capacity and calcium retention capacity were decreased (-42.9% vs. -25.1%, p = .010; and -73.1% vs. -40.3%, p = .003 respectively) and production of reactive oxygen species was enhanced (+63.6% vs. +41.4%, p = .03) in ApoE-/- mice compared with ApoE+/+ mice respectively. Antioxidant treatment restored oxidative capacity, calcium retention capacity and decreased production of reactive oxygen species in both mice strands. CONCLUSIONS: In this small murine study, hypercholesterolaemia exacerbated mitochondrial dysfunction, as clinically expected; but antioxidant therapy appeared protective, which is counter to clinical experience. Further work is clearly needed.

Effects of a High Fat Meal Associated with Water, Juice, or Champagne Consumption on Endothelial Function and Markers of Oxidative Stress and Inflammation in Young, Healthy Subjects.

Endothelial dysfunction (ED), often linked to hypertriglyceridemia, is an early step of atherosclerosis. We investigated, in a randomized cross-over study, whether high-fat meal (HFM)-induced ED might be reduced by fruit juice or champagne containing polyphenols. Flow-mediated dilatation (FMD) and biological parameters (lipid profile, glycemia, inflammation, and oxidative stress markers) were determined before and two and three hours after the HFM in 17 healthy young subjects (24.6 ± 0.9 years) drinking water, juice, or champagne. Considering the entire group, despite significant hypertriglyceridemia (from 0.77 ± 0.07 to 1.41 ± 0.18 mmol/L, p < 0.001) and a decrease in Low Density Lipoprotein (LDL), the FMD was not impaired. However, the FMD decreased in 10 subjects (from 10.73 ± 0.95 to 8.1 3± 0.86 and 8.07 ± 1.16%; p < 0.05 and p < 0.01; 2 and 3 hours, respectively, after the HFM), without concomitant change in concentration reactive protein or reactive oxygen species, but with an increase in glycemia. In the same subjects, the FMD did not decrease when drinking juice or champagne. In conclusion, HFM can impair the endothelial function in healthy young subjects. Fruit juice, rich in anthocyanins and procyanidins, or champagne, rich in simple phenolic acids, might reduce such alterations, but further studies are needed to determine the underlying mechanisms, likely involving polyphenols.

N-Acetyl Cysteine Restores Limb Function, Improves Mitochondrial Respiration, and Reduces Oxidative Stress in a Murine Model of Critical Limb Ischaemia.

OBJECTIVE/BACKGROUND: The aim of this study was to investigate whether antioxidant therapy might decrease oxidative stress related deleterious effects in the setting of critical limb ischaemia (CLI). METHODS: Twenty Swiss mice were submitted to sequential right femoral and iliac ligatures; the left limb served as control. The mice were assigned to two groups: in the first group (no-treatment group, n = 10) no treatment was administered; in the second group (N-acetyl cysteine [NAC] group, n = 10) NAC was administered by dissolution in drinking water for 4 weeks, starting on day 7, when CLI was effective. Clinical and functional scores were assessed by two blinded investigators. Mice were killed on day 40 and mitochondrial respiratory chain complex activities, calcium retention capacity, oxidative stress, and histological analysis were analysed. RESULTS: Ischaemic muscles in the no-treatment group showed significantly impaired mitochondrial respiration and calcium retention capacity, with increased production of reactive oxygen species; but no statistical difference was noticed when comparing ischaemic muscles in the NAC group (n = 10) to contralateral muscles (n = 10) and to control muscles in the no-treatment group (n = 10). Ischaemic muscles in the no-treatment group exhibited myopathic features such as wider range in fibre size, rounded shape, centrally located nuclei, and smaller cross sectional areas, but none of these features were observed in contralateral muscles or in NAC-group muscles (ischaemic or controls). CONCLUSION: Targeting inhibition of oxidative stress may be a potential therapeutic strategy for muscle protection in CLI and might be considered as potential adjunctive therapy to revascularisation procedures.

Mitochondria: An Organelle of Bacterial Origin Controlling Inflammation.

Inflammation is a cellular and molecular response to infection and/or tissues injury. While a suited inflammatory response in intensity and time allows for killing pathogens, clearing necrotic tissue, and healing injury; an excessive inflammatory response drives various diseases in which inflammation and tissues damages/stress self-sustain each other. Microbes have been poorly implied in non-resolving inflammation, emphasizing the importance of endogenous regulation of inflammation. Mitochondria have been historically identified as the main source of cellular energy, by coupling the oxidation of fatty acids and pyruvate with the production of high amount of adenosine triphosphate by the electron transport chain. Mitochondria are also the main source of reactive oxygen species. Interestingly, research in the last decade has highlighted that since its integration in eukaryote cells, this organelle of bacterial origin has not only been tolerated by immunity, but has also been placed as a central regulator of cell defense. In intact cells, mitochondria regulate cell responses to critical innate immune receptors engagement. Downstream intracellular signaling pathways interact with mitochondrial proteins and are tuned by mitochondrial functioning. Moreover, upon cell stress or damages, mitochondrial components are released into the cytoplasm or the extra cellular milieu, where they act as danger signals when recognized by innate immune receptors. Finally, by regulating the energetic state of immunological synapse between dendritic cells and lymphocytes, mitochondria regulate the inflammation fate toward immunotolerance or immunogenicity. As dysregulations of these processes have been recently involved in various diseases, the identification of the underlying mechanisms might open new avenues to modulate inflammation.

Impact of valve-less vs. standard insufflation on pneumoperitoneum volume, inflammation, and peritoneal physiology in a laparoscopic sigmoid resection experimental model.

BACKGROUND: Standard insufflators compensate for intra-abdominal pressure variations with pressure spikes. Our aim was to evaluate the impact of a stable, low-pressure pneumoperitoneum induced by a valve-less insufflator, on working space, hemodynamics, inflammation, and peritoneal physiology, in a model of laparoscopic sigmoid resection. MATERIALS AND METHODS: Twelve pigs (47 ± 3.3 kg) were equipped for invasive hemodynamic monitoring and randomly assigned to Standard (n = 6) vs. valve-less (n = 6) insufflation. Animals were positioned in a 30° Trendelenburg on a CT scan bed. A low-pressure pneumoperitoneum (8 mmHg) was started and duration was set for 180 min. Abdominal CT scans were performed, under neuromuscular blockade, before, immediately after, and 1 and 3 h after insufflation. Pneumoperitoneum volumes were calculated on 3D reconstructed CT scans. After creation of a mesenteric window, capillary blood was obtained by puncturing the sigmoid serosa and local lactatemia (mmol/L) was measured using a handheld analyzer. Surgical resection was performed according to the level of lactates, in order to standardize bowel stump perfusion. IL-1 and IL-6 (ng/mL) were measured repeatedly. The peritoneum was sampled close to the surgical site and distantly for the oxygraphic assessment of mitochondrial respiration. A pathologist applied a semi-quantitative score to evaluate the anastomosis. RESULTS: Mean arterial pressure, pulse, body temperature, oximetry, systemic lactatemia, and local lactates were similar. IL-6 was lower in the valve-less group, reaching a statistically significant difference after 3 h of insufflation (64.85 ± 32.5 vs. 133.95 ± 59.73; p = 0.038) and 48 h (77.53 ± 68.4 vs. 190.74 ± 140.79; p = 0.029). Peritoneal mitochondrial respiration was significantly increased after the survival period, with no difference among the groups. The anastomoses in the valve-less group demonstrated a lower acute (p = 0.04) inflammatory infiltration. The mean anterior posterior thickness was slightly, yet significantly higher in the valve-less group, on all post-insufflation CT scans. CONCLUSIONS: Valve-less insufflation achieved a slightly higher working space and a lower systemic and localized inflammatory response in this experimental setting.

Moderate Exercise Allows for shorter Recovery Time in Critical Limb Ischemia.

Whether and how moderate exercise might allow for accelerated limb recovery in chronic critical limb ischemia (CLI) remains to be determined. Chronic CLI was surgically induced in mice, and the effect of moderate exercise (training five times per week over a 3-week period) was investigated. Tissue damages and functional scores were assessed on the 4th, 6th, 10th, 20th, and 30th day after surgery. Mice were sacrificed 48 h after the last exercise session in order to assess muscle structure, mitochondrial respiration, calcium retention capacity, oxidative stress and transcript levels of genes encoding proteins controlling mitochondrial functions (PGC1α, PGC1ß, NRF1) and anti-oxidant defenses markers (SOD1, SOD2, catalase). CLI resulted in tissue damages and impaired functional scores. Mitochondrial respiration and calcium retention capacity were decreased in the ischemic limb of the non-exercised group (Vmax = 7.11 ± 1.14 vs. 9.86 ± 0.86 mmol 02/min/g dw, p < 0.001; CRC = 7.01 ± 0.97 vs. 11.96 ± 0.92 microM/mg dw, p < 0.001, respectively). Moderate exercise reduced tissue damages, improved functional scores, and restored mitochondrial respiration and calcium retention capacity in the ischemic limb (Vmax = 9.75 ± 1.00 vs. 9.82 ± 0.68 mmol 02/min/g dw; CRC = 11.36 ± 1.33 vs. 12.01 ± 1.24 microM/mg dw, respectively). Exercise also enhanced the transcript levels of PGC1α, PGC1ß, NRF1, as well as SOD1, SOD2, and catalase. Moderate exercise restores mitochondrial respiration and calcium retention capacity, and it has beneficial functional effects in chronic CLI, likely by stimulating reactive oxygen species-induced biogenesis and anti-oxidant defenses. These data support further development of exercise therapy even in advanced peripheral arterial disease.

IFN-ß-induced reactive oxygen species and mitochondrial damage contribute to muscle impairment and inflammation maintenance in dermatomyositis.

Dermatomyositis (DM) is an autoimmune disease associated with enhanced type I interferon (IFN) signalling in skeletal muscle, but the mechanisms underlying muscle dysfunction and inflammation perpetuation remain unknown. Transcriptomic analysis of early untreated DM muscles revealed that the main cluster of down-regulated genes was mitochondria-related. Histochemical, electron microscopy, and in situ oxygraphy analysis showed mitochondrial abnormalities, including increased reactive oxygen species (ROS) production and decreased respiration, which was correlated with low exercise capacities and a type I IFN signature. Moreover, IFN-ß induced ROS production in human myotubes was found to contribute to mitochondrial malfunctions. Importantly, the ROS scavenger N-acetyl cysteine (NAC) prevented mitochondrial dysfunctions, type I IFN-stimulated transcript levels, inflammatory cell infiltrate, and muscle weakness in an experimental autoimmune myositis mouse model. Thus, these data highlight a central role of mitochondria and ROS in DM. Mitochondrial dysfunctions, mediated by IFN-ß induced-ROS, contribute to poor exercise capacity. In addition, mitochondrial dysfunctions increase ROS production that drive type I IFN-inducible gene expression and muscle inflammation, and may thus self-sustain the disease. Given that current DM treatments only induce partial recovery and expose to serious adverse events (including muscular toxicity), protecting mitochondria from dysfunctions may open new therapeutic avenues for DM.

Effects of cyclic nucleotide phosphodiesterases (PDEs) on mitochondrial skeletal muscle functions.

Mitochondria play a critical role in skeletal muscle metabolism and function, notably at the level of tissue respiration, which conduct muscle strength as well as muscle survival. Pathological conditions induce mitochondria dysfunctions notably characterized by free oxygen radical production disturbing intracellular signaling. In that way, the second messengers, cyclic AMP and cyclic GMP, control intracellular signaling at the physiological and transcription levels by governing phosphorylation cascades. Both nucleotides are specifically and selectively hydrolyzed in their respective 5'-nucleotide by cyclic nucleotide phosphodiesterases (PDEs), which constitute a multi-genic family differently tissue distributed and subcellularly compartmentalized. These PDEs are presently recognized as therapeutic targets for cardiovascular, pulmonary, and neurologic diseases. However, very few data concerning cyclic nucleotides and PDEs in skeletal muscle, specifically in mitochondria, are reported in the literature. The knowledge of PDE implication in mitochondrial signaling would be helpful for resolving critical mitochondrial dysfunctions in skeletal muscle.

Precision real-time evaluation of bowel perfusion: accuracy of confocal endomicroscopy assessment of stoma in a controlled hemorrhagic shock model.

BACKGROUND AND AIMS: Confocal laser endomicroscopy (CLE) can provide real-time evaluation of bowel perfusion. We aimed to evaluate CLE perfusion imaging in a hemorrhagic shock model. MATERIALS AND METHODS: Five pigs were equipped to ensure hemodynamic monitoring. Three ileostomies per animal (total n = 15) were randomly created (T0). Blood was withdrawn targeting a mean arterial pressure of 40 mmHg (shock phase, T1), for 90 min. Infusion of Ringer's lactate was started and continued for 90 min (T2). At the different time points: (a) stomas' mucosa was scanned with CLE; (b) capillary lactates were measured on blood obtained by puncturing stomas' mucosa; and (c) full-thickness stomas' biopsies were sampled for histology, mitochondrial respiratory rate (V 0 = basal and V ADP = respiratory rate in excess of adenosine diphosphate), and levels of superoxide anion evaluation. Functional capillary density (FCD) was measured using ad hoc software. RESULTS: Confocal scanning provided consistent and specific imaging of bowel hypoperfusion at T1: vascular hyperpermeability (blurred and enlarged capillaries) and edema (enhanced visualization of the brush border due to increased intercellular spaces and fluorescein leakage). At the end of T2, there was an improved capillary flow. FCD-A index expressed statistically significant correlation with (1) stoma capillary lactates (p = 0.023); (2) systemic capillary lactates (p = 0.031); (3) inflammation pathology score (p = 0.048); (4) central venous pressure (p = 0.0043); and (5) pulmonary artery pressure (p = 0.01). Stoma capillary lactates (mmol/L) were significantly increased at T1 (8.81 ± 4.23; p < 0.0001) and at T2 (4.77 ± 3.13; p < 0.01) when compared to T0 inclusion values (1.86 ± 0.56). V 0 and V ADP (pmol O2/min/mg tissue) were both significantly decreased at T1 versus T0 (p < 0.007 and p < 0.01, respectively) and recovered by the end of reanimation (T2 vs. T0, p = n.s.). Mean O 2·- production (µmol/min/mg/dry tissue) increased at T1 and significantly decreased at T2. CONCLUSIONS: Confocal laser endomicroscopy (CLE) imaging could identify morphological signs congruent with biochemical markers of bowel perfusion and could be useful for assessment of stomas.

Skeletal muscle ischemia-reperfusion injury and cyclosporine A in the aging rat.

Old patients exhibit muscle impairments and increased perioperative risk during vascular surgery procedures. Although aging generally impairs protective mechanisms, data are lacking concerning skeletal muscle in elderly. We tested whether cyclosporine A (CsA), which protects skeletal muscle from ischemia-reperfusion (IR) in young rats, might reduce skeletal muscle mitochondrial dysfunction and oxidative stress in aging rats submitted to hindlimb IR. Wistar rats aged 71-73 weeks were randomized to IR (3 h unilateral tourniquet application and 2 h reperfusion) or IR + CsA (10 mg/kg cyclosporine IV before reperfusion). Maximal oxidative capacity (VM ax ), acceptor control ratio (ACR), and relative contribution of the mitochondrial respiratory chain complexes II, III, IV (VS ucc ), and IV (VTMPD /Asc ), together with calcium retention capacity (CRC) a marker of apoptosis, and tissue reactive oxygen species (ROS) production were determined in gastrocnemius muscles from both hindlimbs. Compared to the nonischemic hindlimb, IR significantly reduced mitochondrial coupling, VMax (from 7.34 ± 1.50 to 2.87 ± 1.22 µMO2 /min/g; P < 0.05; -70%), and VS ucc (from 6.14 ± 1.07 to 3.82 ± 0.83 µMO2 /min/g; P < 0.05; -42%) but not VTMPD /Asc . IR also decreased the CRC from 15.58 ± 3.85 to 6.19 ± 0.86 µMCa(2+) /min/g; P < 0.05; -42%). These alterations were not corrected by CsA (-77%, -49%, and -32% after IR for VM ax, VS ucc , and CRC, respectively). Further, CsA significantly increased ROS production in both hindlimbs (P < 0.05; +73%). In old rats, hindlimb IR impairs skeletal muscle mitochondrial function and increases oxidative stress. Cyclosporine A did not show protective effects.

Metabolism-Guided Bowel Resection: Potential Role and Accuracy of Instant Capillary Lactates to Identify the Optimal Resection Site.

BACKGROUND: Strip-based handheld devices can measure lactatemia on capillary blood obtained by needle puncturing. We aimed to assess the kinetic of bowel capillary lactates, metabolomics profiling, and mitochondria respiratory rate in a prolonged model of bowel hypoperfusion. MATERIALS AND METHODS: In 6 pigs, a 3- to 4-cm ischemic segment was created in 6 small bowel loops (total = 36 loops) by clamping the vascular supply, for a duration of 1 to 6 hours. Hourly, 5 blood samples were obtained by puncturing the serosa, and lactates were measured using a handheld analyzer. Samples were made at the following regions of interest (ROIs): center of the ischemic area (1), proximal and distal clinical margins of resection (2a-2b), and vascularized zones (3a-3b). Every hour, surgical biopsies of ROIs were sampled. Activity of bowel mitochondria complexes was measured after 1, 3, and 5 hours of ischemia. Quantification of metabolites was performed on all samples (total N = 180). RESULTS: Capillary lactates were significantly higher at ROI 1 versus ROI 3ab at all time points. After 1 hour lactates at the margins were significantly higher than those at vascularized areas (P = .0095), showing a mismatch between visual assessment and actual perfusion status. From 2 to 6 hours, there was no difference in lactates between ROIs 2a-2b and 3a-3b. Maximal tissue respiration decreased significantly after 1 hour (ROI 1 vs ROI 3ab). Seven metabolites (lactate, glucose, aspartate, choline, creatine, taurine, and tyrosine) expressed significantly different evolutions between ROIs. CONCLUSIONS: Capillary lactates could help precisely estimate local bowel perfusion status.