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Context: Sepsis leads to multiple organ dysfunction and negatively impacts patient outcomes. Skeletal muscle disuse is a significant comorbidity in septic patients during their ICU stay due to prolonged immobilization., Hypothesis: Combination of sepsis and muscle disuse will promote a unique proteomic signature in skeletal muscle in comparison to disuse and sepsis separately., Methods and Models: Following cecal ligation and puncture (CLP) or Sham surgeries, mice were subjected to hindlimb suspension (HLS) or maintained normal ambulation (NA). Tibialis anterior muscles from 24 C57BL6/J male mice were harvested for proteomic analysis. Proteomic profiles were assessed using nano-liquid chromatography with tandem mass spectrometry, followed by data analysis including Partial Least Squares Discriminant Analysis (PLS-DA), to compare the differential protein expression across groups., Results: A total of 2876 differentially expressed proteins were identified, with marked differences between groups. In mice subjected to CLP and HLS combined, there was a distinctive proteomic signature characterized by a significant decrease in the expression of proteins involved in mitochondrial function and muscle metabolism, alongside a marked increase in proteins related to muscle degradation pathways. The PLS-DA demonstrated a clear separation among experimental groups, highlighting the unique profile of the CLP/HLS group. This suggests an important interaction between sepsis-induced inflammation and disuse atrophy mechanisms in sepsis-induced myopathy., Interpretations and Conclusions: Our findings reveal a complex proteomic landscape in skeletal muscle exposed to sepsis and disuse, consistent with an exacerbation of muscle protein degradation under these combined stressors. The identified proteins and their roles in cellular stress responses and muscle pathology provide potential targets for intervention to mitigate muscle dysfunction in septic conditions, highlighting the importance of addressing both sepsis and disuse concurrently in clinical and experimental settings., Competing Interests: The authors have disclosed that they do not have any potential conflicts of interest., (Copyright © 2024 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the Society of Critical Care Medicine.)

Vertebrate limbs start to develop as paired protrusions from the lateral plate mesoderm at specific locations of the body with forelimb buds developing anteriorly and hindlimb buds posteriorly. During the initiation process, limb progenitor cells maintain active proliferation to form protrusions and start to express Fgf10, which triggers molecular processes for outgrowth and patterning. Although both processes occur in both types of limbs, forelimbs (Tbx5), and hindlimbs (Isl1) utilize distinct transcriptional systems to trigger their development. Here, we report that Sall1 and Sall4, zinc finger transcription factor genes, regulate hindlimb initiation in mouse embryos. Compared to the 100% frequency loss of hindlimb buds in TCre; Isl1 conditional knockouts, Hoxb6Cre; Isl1 conditional knockout causes a hypomorphic phenotype with only approximately 5% of mutants lacking the hindlimb. Our previous study of SALL4 ChIP-seq showed SALL4 enrichment in an Isl1 enhancer, suggesting that SALL4 acts upstream of Isl1. Removing 1 allele of Sall4 from the hypomorphic Hoxb6Cre; Isl1 mutant background caused loss of hindlimbs, but removing both alleles caused an even higher frequency of loss of hindlimbs, suggesting a genetic interaction between Sall4 and Isl1. Furthermore, TCre-mediated conditional double knockouts of Sall1 and Sall4 displayed a loss of expression of hindlimb progenitor markers (Isl1, Pitx1, Tbx4) and failed to develop hindlimbs, demonstrating functional redundancy between Sall1 and Sall4. Our data provides genetic evidence that Sall1 and Sall4 act as master regulators of hindlimb initiation., Competing Interests: Conflicts of interest: The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Hindlimb ischaemia-reperfusion (IR) is among the most prominent pathophysiological conditions observed in peripheral artery disease (PAD). An exaggerated arterial blood pressure (BP) response during exercise is associated with an elevated risk of cardiovascular events in individuals with PAD. However, the precise mechanisms leading to this exaggerated BP response are poorly elucidated. The P2X 3 signalling pathway, which plays a key role in modifying the exercise pressor reflex (EPR), is the focus of the present study. We determined the regulatory role of P2X 3 on the EPR in a rat model of hindlimb IR. In vivo and in vitro approaches were used to determine the expression and functions of P2X 3 in muscle afferent nerves and EPR in IR rats. We found that in IR rats there was (1) upregulation of P2X 3 protein expression in the L4-6 dorsal root ganglia (DRG); (2) amplified P2X currents in isolated isolectin B4 (IB4)-positive muscle DRG neurons; and (3) amplification of the P2X-mediated BP response. We further verified that both A-317491 and siRNA knockdown of P2X 3 significantly decreased the activity of P2X currents in isolated muscle DRG neurons. Moreover, inhibition of muscle afferents' P2X 3 receptor using A-317491 was observed to alleviate the exaggerated BP response induced by static muscle contraction and P2X-induced BP response by α,β-methylene ATP injection. P2X 3 signalling pathway activity is amplified in muscle afferent DRG neurons in regulating the EPR following hindlimb IR., (© 2024 The Authors. Experimental Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

This study aims to investigate the effect of Buyang Huanwu Decoction on blood flow recovery and arteriogenesis after hindlimb ischemia in mice via the platelet-derived growth factor(PDGF) signaling pathway. Forty C57BL/6 mice were randomized into model(clean water, 10 mL·kg~(-1)·d~(-1)), beraprost sodium(positive control, 18 μg·kg~(-1)·d~(-1)), and low-, medium-, and high-dose(10, 20, and 40 g·kg~(-1)·d~(-1), respectively) Buyang Huanwu Decoction groups(n=8). The hindlimb ischemia model was established by femoral artery ligation. The mice were administrated with corresponding agents by gavage daily for 14 days after ligation. For laser Doppler perfusion imaging, the mice were anesthetized and measured under a Periscan PSI imager. The density of capillary and arterio-le in the ischemic gastrocnemius was measured using immunofluorescence staining of the frozen tissue sections. Western blot was employed to determine the expression of PDGF subunit B(PDGFB), phosphorylated mitogen extracellular kinase(p-MEK), MEK, phosphorylated extracellular signal-regulated kinase(p-ERK), and ERK. Real-time PCR was employed to determine the mRNA level of PDGFB. The Buyang Huanwu Decoction-containing serum was used to treat the vascular smooth muscle cells(VSMCs) in hypoxia at doses of 10% and 20%. The proliferation and migration of VSMCs was assessed in vitro. The results showed that compared with the model group, beraprost sodium and Buyang Huanwu Decoction enhanced the blood flow recovery, increased the capillary and arteriole density, and up-regulated the protein levels of PDGFB, p-MEK, p-ERK, and mRNA levels of PDGFB, with the medium-dose Buyang Huanwu Decoction demonstrating the most significant effect. The 10% Buyang Huanwu Decoction-containing serum enhanced the proliferation and migration of VSMCs. Our findings demonstrate that Buyang Huanwu Decoction up-regulates PDGFB transcription and activates PDGF signaling pathway to promote arteriogenesis and blood flow recovery in ischemic gastrocnemius.

Disuse muscle atrophy occurs consequent to prolonged limb immobility or bed rest, which represents an unmet medical need. As existing animal models of limb immobilization often cause skin erosion, edema, and other untoward effects, we here report an alternative method via thermoplastic immobilization of hindlimbs in mice. While significant decreases in the weight and fiber size were noted after 7 days of immobilization, no apparent skin erosion or edema was found. To shed light onto the molecular mechanism underlying this muscle wasting, we performed the next-generation sequencing analysis of gastrocnemius muscles from immobilized versus non-mobilized legs. Among a total of 55,487 genes analyzed, 787 genes were differentially expressed (> fourfold; 454 and 333 genes up- and down-regulated, respectively), which included genes associated with muscle tissue development, muscle system process, protein digestion and absorption, and inflammation-related signaling. From a clinical perspective, this model may help understand the molecular/cellular mechanism that drives muscle disuse and identify therapeutic strategies for this debilitating disease., (© 2023. The Author(s).)

Critical limb ischemia (CLI) occurs when blood flow is restricted through the arteries, resulting in ulcers, necrosis, and chronic wounds in the downstream extremities. The development of collateral arterioles (i.e. arteriogenesis), either by remodeling of pre-existing vascular networks or de novo growth of new vessels, can prevent or reverse ischemic damage, but it remains challenging to stimulate collateral arteriole development in a therapeutic context. Here, we show that a gelatin-based hydrogel, devoid of growth factors or encapsulated cells, promotes arteriogenesis and attenuates tissue damage in a murine CLI model. The gelatin hydrogel is functionalized with a peptide derived from the extracellular epitope of Type 1 cadherins. Mechanistically, these "GelCad" hydrogels promote arteriogenesis by recruiting smooth muscle cells to vessel structures in both ex vivo and in vivo assays. In a murine femoral artery ligation model of CLI, delivery of in situ crosslinking GelCad hydrogels was sufficient to restore limb perfusion and maintain tissue health for 14 days, whereas mice treated with gelatin hydrogels had extensive necrosis and autoamputated within 7 days. A small cohort of mice receiving the GelCad hydrogels were aged out to 5 months and exhibited no decline in tissue quality, indicating durability of the collateral arteriole networks. Overall, given the simplicity and off-the-shelf format of the GelCad hydrogel platform, we suggest it could have utility for CLI treatment and potentially other indications that would benefit from arteriole development., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ethan Lippmann reports financial support was provided by National Institutes of Health. Ethan Lippmann reports financial support was provided by The Chan Zuckerberg Initiative. Corinne Curry, Andrew Kjar reports financial support was provided by National Science Foundation. Ethan Lippmann and Brian O'Grady have a patent pending to Vanderbilt University., (Copyright © 2023. Published by Elsevier Ltd.)

Background and Aims: Peripheral arterial disease (PAD) is a leading cause of morbimortality worldwide. Lipocalin-2 (LCN2) has been associated with higher risk of amputation or mortality in PAD and might be involved in muscle regeneration. Our aim is to unravel the role of LCN2 in skeletal muscle repair and PAD., Methods and Results: WT and Lcn2 -/- mice underwent hindlimb ischemia. Blood and crural muscles were analyzed at the inflammatory and regenerative phases. At day 2, Lcn2 -/- male mice, but not females, showed increased blood and soleus muscle neutrophils, and elevated circulating pro-inflammatory monocytes (p < 0.05), while locally, total infiltrating macrophages were reduced (p < 0.05). Moreover, Lcn2 -/- soleus displayed an elevation of Cxcl1 (p < 0.001), and Cxcr2 (p < 0.01 in males), and a decrease in Ccl5 (p < 0.05). At day 15, Lcn2 deficiency delayed muscle recovery, with higher density of regenerating myocytes (p < 0.04) and arterioles (αSMA + , p < 0.025). Reverse target prediction analysis identified miR-138-5p as a potential regulator of LCN2, showing an inverse correlation with Lcn2 mRNA in skeletal muscles (rho = -0.58, p < 0.01). In vitro, miR-138-5p mimic reduced Lcn2 expression and luciferase activity in murine macrophages (p < 0.05). Finally, in human serum miR-138-5p was inversely correlated with LCN2 (p ≤ 0.001 adjusted, n = 318), and associated with PAD (Odds ratio 0.634, p = 0.02, adjusted, PAD n = 264, control n = 54)., Conclusions: This study suggests a possible dual role of LCN2 in acute and chronic conditions, with a probable role in restraining inflammation early after skeletal muscle ischemia, while being associated with vascular damage in PAD, and identifies miR-138-5p as one potential post-transcriptional regulator of LCN2., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Postbiotics made from lactic acid bacteria may ameliorate sarcopenia via the metabolic reprogramming of gut dysbiosis. This study investigated the anti-sarcopenic effect of postbiotics (WDK) produced from polyphenol-rich melon peel extract (Cucumis melo L. var. makuwa, KEE) and whey with Lentilactobacillus kefiri DH5 (DH5) in C2C12 skeletal muscle cells and hindlimb-immobilized mice. WDK significantly ameliorated palmitate-induced atrophy of C2C12 cells, restoring myotube length and diameter. It also upregulated the expression of myogenic genes including Atrogin-1, Igf-1, and MyoD. Hindlimb-immobilized C57BL/6J mice were randomly divided and orally administered 10 mL/kg body weight of saline (CON), Whey, Whey + DH5 (WD), DH5 + KEE, Whey + DH5 + KEE postbiotic (WDK) for three weeks (n = 10/group). Interestingly, WDK significantly improved muscle function in hindlimb-immobilized mice by restoring both the grip strength and the mass of the soleus muscle, which was closely related to the upregulation of the myoD gene. WDK increased microbial diversity and modulated the distribution of intestinal bacteria, particularly those involved in protein synthesis and the production of butyrate. There was a significant correlation between myogenic biomarkers and butyrate producing gut microbiota. Restoration of muscle mass and function following postbiotic WDK is strongly related to the regulation of myogenic genes by in part remodulating gut microbiota. In conclusion, these findings suggest that polyphenol- and whey-based postbiotics WDK may have potential as an effective manner to combat the progression of sarcopenia., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Disuse-induced muscle atrophy is a common clinical problem observed mainly in older adults, intensive care units patients, or astronauts. Previous studies presented biological sex divergence in progression of disuse-induced atrophy along with differential changes in molecular mechanisms possibly underlying muscle atrophy. The aim of this study was to perform transcriptomic profiling of male and female mice during the onset and progression of unloading disuse-induced atrophy. Male and female mice underwent hindlimb unloading (HU) for 24, 48, 72, and 168 h ( n = 8/group). Muscles were weighed for each cohort and gastrocnemius was used for RNA-sequencing analysis. Females exhibited muscle loss as early as 24 h of HU, whereas males after 168 h of HU. In males, pathways related to proteasome degradation were upregulated throughout 168 h of HU, whereas in females these pathways were upregulated up to 72 h of HU. Lcn2 , a gene contributing to regulation of myogenesis, was upregulated by 6.46- to 19.86-fold across all time points in females only. A reverse expression of Fosb , a gene related to muscle degeneration, was observed between males (4.27-fold up) and females (4.57-fold down) at 24-h HU. Mitochondrial pathways related to tricarboxylic acid (TCA) cycle were highly downregulated at 168 h of HU in males, whereas in females this downregulation was less pronounced. Collagen-related pathways were consistently downregulated throughout 168 h of HU only in females, suggesting a potential biological sex-specific protective mechanism against disuse-induced fibrosis. In conclusion, females may have protection against HU-induced skeletal muscle mitochondrial degeneration and fibrosis through transcriptional mechanisms, although they may be more vulnerable to HU-induced muscle wasting compared with males. NEW & NOTEWORTHY Herein, we have assessed the transcriptomic response across biological sexes during the onset and progression of unloading disuse-induced atrophy in mice. We have demonstrated an inverse expression of Fosb between males and females, as well as differentially timed patterns of expressing atrophy-related pathways between sexes that are concomitant to the accelerated atrophy in females. We also identified in females signs of mechanisms to combat disuse-induced mitochondrial degeneration and fibrosis.

Background: Protection of cardiac function following myocardial infarction was largely enhanced by bradykinin-pretreated cardiac-specific c-kit + (BK-c-kit + ) cells, even without significant engraftment, indicating that paracrine actions of BK-c-kit + cells play a pivotal role in angiogenesis. Nevertheless, the active components of the paracrine actions of BK-c-kit + cells and the underlying mechanisms remain unknown. This study aimed to define the active components of exosomes from BK-c-kit + cells and elucidate their underlying protective mechanisms., Methods: Matrigel tube formation assay, cell cycle, and mobility in human umbilical vein endothelial cells (HUVECs) and hindlimb ischemia (HLI) in mice were applied to determine the angiogenic effect of condition medium (CM) and exosomes. Proteome profiler, microRNA sponge, Due-luciferase assay, microRNA-sequencing, qRT-PCR, and Western blot were used to determine the underlying mechanism of the angiogenic effect of exosomes from BK-c-kit + ., Results: As a result, BK-c-kit + CM and exosomes promoted tube formation in HUVECs and the repair of HLI in mice. Angiogenesis-related proteomic profiling and microRNA sequencing revealed highly enriched miR-3059-5p as a key angiogenic component of BK-c-kit + exosomes. Meanwhile, loss- and gain-of-function experiments revealed that the promotion of angiogenesis by miR-3059-5p was mainly through suppression of TNFSF15-inhibited effects on vascular tube formation, cell proliferation and cell migration. Moreover, enhanced angiogenesis of miR-3059-5p-inhibited TNFSF15 has been associated with Akt/Erk1/2/Smad2/3-modulated signaling pathway., Conclusion: Our results demonstrated a novel finding that BK-c-kit + cells enrich exosomal miR-3059-5p to suppress TNFSF15 and promote angiogenesis against hindlimb ischemia in mice., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mechanical unloading during microgravity causes skeletal muscle atrophy and impairs mitochondrial energetics. The elevated production of reactive oxygen species (ROS) by mitochondria and Nox2, coupled with impairment of stress protection (e.g., SIRT1, antioxidant enzymes), contribute to atrophy. We tested the hypothesis that the SIRT1 activator, SRT2104 would rescue unloading-induced mitochondrial dysfunction. Mitochondrial function in rat gastrocnemius and soleus muscles were evaluated under three conditions (10 days): ambulatory control (CON), hindlimb unloaded (HU), and hindlimb-unloaded-treated with SRT2104 (SIRT). Oxidative phosphorylation, electron transfer capacities, H 2 O 2 production, and oxidative and antioxidant enzymes were quantified using high-resolution respirometry and colorimetry. In the gastrocnemius, (1) integrative (per mg tissue) proton LEAK was lesser in SIRT than in HU or CON; (2) intrinsic (relative to citrate synthase) maximal noncoupled electron transfer capacity (E CI+II ) was lesser, while complex I-supported oxidative phosphorylation to E CI+II was greater in HU than CON; (3) the contribution of LEAK to E CI+II was greatest, but cytochrome c oxidase activity was lowest in HU. In both muscles, H 2 O 2 production and concentration was greatest in SIRT, as was gastrocnemius superoxide dismutase activity. In the soleus, H 2 O 2 concentration was greater in HU compared to CON. These results indicate that SRT2104 preserves mitochondrial function in unloaded skeletal muscle, suggesting its potential to support healthy muscle cells in microgravity by promoting necessary energy production in mitochondria.

Cerebral edema following cerebral infarction can be severe and directly affect mortality and mobility. Exercise therapy after cerebral infarction is an effective therapeutic approach; however, the molecular mechanism remains unclear. Myokines such as interleukin-1 receptor antagonist (IL-1RA) are released during skeletal muscle contraction with effects on other organs. We hypothesized that myokine release during exercise might improve brain edema and confirmed the hypothesis using transient middle cerebral artery occlusion (tMCAO) model rats. Rats subjected to tMCAO were divided according to the severity of illness and further assigned to exercise and non-exercise groups. Treadmill exercises were performed at a speed of 2-8 m/min for 10 min from 1-6 days post-reperfusion after tMCAO. Exercise significantly reduced edema and neurological deficits in severely ill rats, with a reduction in aquaporin-4 (AQP4) expression in the ischemic core and increased blood IL-1RA release from the stroke-unaffected hindlimb muscle after tMCAO. Administration of IL-1RA into the lateral ventricles significantly reduced edema and AQP4 expression in the ischemic core. In conclusion, treadmill exercise performed in the early phase of stroke onset alleviated the decrease in blood IL-1RA following ischemic stroke. IL-1RA administration decreased astrocytic AQP4 expression in the ischemic core, suppressing brain edema.

Hindlimb ischemia (HLI), in which blood perfusion to the hindlimb is obstructed, is one of the major complications of diabetes. Skeletal muscle cells are crucial for revascularization as they can secrete various angiogenic factors; however, hyperglycemia impairs their viability and subsequently their angiogenic potential. Salidroside can promote skeletal muscle cell viability under hyperglycemia; however, the molecular mechanism is still poorly understood. Here we revealed that salidroside could suppress hyperglycemia-induced ferroptosis in skeletal muscle cells by promoting GPX4 expression, thereby restoring their viability and paracrine functions. These in turn promoted the proliferation and migration potentials of blood vessel-forming cells. Furthermore, we showed that salidroside/GPX4-mediated ferroptosis inhibition is crucial for promoting angiogenesis and blood perfusion recovery in diabetic HLI mice. Together, we reveal a novel molecular mechanism of salidroside in enhancing skeletal muscle cells-mediated revascularization and blood perfusion recovery in diabetic HLI mice, further highlighting it as a potential compound for treating diabetic HLI., Competing Interests: Conflict of interest statement The authors declared that they have no conflicts of interest., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Hindlimb ischemia (HLI) is an arterial occlusive disease that exposes the patients to the risk of limb gangrene and loss. Polarization of macrophages is related to HLI-induced inflammation. Period circadian regulator 1 (PER1) is a core component of the circadian clock. We first showed, based upon bioinformatics analysis of microarray data, that PER1 expression was reduced in monocytes from patients with critical limb ischemia. The proximal femoral artery in the left hindlimb of male mice was ligated and then the femoral artery and its collateral branches were removed to establish the HLI mouse model. After modeling, a single intramuscular injection of 1 × 10 9  pfu Ad-PER1 was performed at the adductor and gastrocnemius muscles. The gastrocnemius muscle tissues were collected at day 0, 3, 7, 14, 21 post-HLI. There was obvious pathological necrosis, accompanied with reduced expression of PER1 in the muscle tissues of HLI mice. Expression of CD68 and CD31 seemed to be corresponded to PER1 in gastrocnemius muscle, implying the potential of PER1 in regulating macrophage-related inflammation and angiogenesis. PER1 overexpression diminished myocyte damage, promoted blood flow restoration and improved behavioral scores of HLI mice. Immunostaining of CD31 and α-SMA revealed that PER1 upregulation reversed HLI-induced decreases in capillary and arteriole density. In vitro, RAW264.7 cells were cultured in hypoxia (1% O 2 ) for 24 h. The percentage of pro-inflammatory CD86 + macrophages (M1 type) was decreased and that of anti-inflammatory CD206+ macrophages (M2 type) was increased when PER1 was overexpressed. Moreover, the expression levels of TNF-α, IL-6 and M1-type marker iNOS were decreased, and levels of IL-10 and M2-type marker Arg-1 were increased by PER1 in gastrocnemius muscle of HLI mice and hypoxia-treated RAW264.7 cells. PER1 might reduce M1 macrophage polarization and promote M2 macrophage polarization, and thus exert anti-inflammatory and pro-angiogenic actions. Our findings suggest that PER1 overexpression promotes functional recovery of mice with HLI through regulating macrophage polarization., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yang Ding reports financial support was provided by Scientific Research Fund Project of Anhui Medical University. Yang Ding reports financial support was provided by Scientific Research Project of Anhui Province Colleges and Universities., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In this research, we introduced a novel strategy for fabricating cell sheets (CSs) prepared by simply adding a fibrinogen solution to growth medium without using any synthetic polymers or chemical agents. We confirmed that the fibrinogen-based CS could be modified for target tissue regardless of size, shape, and cell types. Also, fibrinogen-based CSs were versatile and could be used to form three-dimensional (3D) CSs such as multi-layered CSs and those mimicking native blood vessels. We also prepared fibrinogen-based spheroid sheets for the treatment of ischemic disease. The fibrinogen-based spheroid sheets had much higher in vitro tubule formation and released more angiogenic factors compared to other types of platform in this research. We transplanted fibrinogen-based spheroid sheets into a mouse hindlimb ischemia model and found that fibrinogen-based spheroid sheets showed significantly improved physiological function and blood perfusion rates compared to the other types of platform in this research., (© 2023 IOP Publishing Ltd.)

We investigated the role played by bradykinin 2 (B2) receptors in the exaggerated exercise pressor reflex in rats with a femoral artery ligated for 72 h to induce simulated peripheral artery disease (PAD). We hypothesized that in decerebrate, unanesthetized rats with a ligated femoral artery, hindlimb arterial injection of HOE-140 (100 ng, B2 receptor antagonist) would reduce the pressor response to 30 s of electrically induced 1 Hz hindlimb skeletal muscle contraction, and 30 s of 1 Hz hindlimb skeletal muscle stretch (a model of mechanoreflex activation isolated from contraction-induced metabolite production). We hypothesized no effect of HOE-140 in sham-operated "freely perfused" rats. In both freely perfused ( n = 4) and "ligated" ( n = 4) rats, we first confirmed efficacious B2 receptor blockade by demonstrating that HOE-140 injection significantly reduced ( P < 0.05) the peak increase in mean arterial pressure (peak ΔMAP) in response to hindlimb arterial injection of bradykinin. In subsequent experiments, we found that HOE-140 reduced the peak ΔMAP response to muscle contraction in ligated ( n = 14; control: 23 ± 2; HOE-140: 17 ± 2 mmHg; P = 0.03) but not freely perfused rats ( n = 7; control: 17 ± 3; HOE-140: 18 ± 4 mmHg; P = 0.65). Furthermore, HOE-140 had no effect on the peak ΔMAP response to stretch in ligated rats ( n = 14; control: 37 ± 4; HOE-140: 32 ± 5 mmHg; P = 0.13) but reduced the integrated area under the blood pressure signal over the final ∼20 s of the maneuver. The data suggest that B2 receptors contribute to the exaggerated exercise pressor reflex in rats with simulated PAD, and that contribution includes a modest role in the chronic sensitization of the mechanically activated channels/afferents that underlie mechanoreflex activation.

Cardiac arrest (CA) and return of spontaneous circulation (ROSC), a global ischemia and reperfusion event, lead to neuronal damage and/or death in the spinal cord as well as the brain. Hypothermic therapy is reported to protect neurons from damage and improve hindlimb paralysis after resuscitation in a rat model of CA induced by asphyxia. In this study, we investigated roles of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the lumbar spinal cord protected by therapeutic hypothermia in a rat model of asphyxial CA. Male Sprague-Dawley rats were subjected to seven minutes of asphyxial CA (induced by injection of 2 mg/kg vecuronium bromide) and hypothermia (four hours of cooling, 33 ± 0.5 °C). Survival rate, hindlimb motor function, histopathology, western blotting, and immunohistochemistry were examined at 12, 24, and 48 h after CA/ROSC. The rats of the CA/ROSC and hypothermia-treated groups had an increased survival rate and showed an attenuated hindlimb paralysis and a mild damage/death of motor neurons located in the anterior horn of the lumbar spinal cord compared with those of the CA/ROSC and normothermia-treated groups. In the CA/ROSC and hypothermia-treated groups, expressions of cytoplasmic and nuclear Nrf2 and HO-1 were significantly higher in the anterior horn compared with those of the CA/ROSC and normothermia-treated groups, showing that cytoplasmic and nuclear Nrf2 was expressed in both motor neurons and astrocytes. Moreover, in the CA/ROSC and hypothermia-treated group, interleukin-1β (IL-1β, a pro-inflammatory cytokine) expressed in the motor neurons was significantly reduced, and astrocyte damage was apparently attenuated compared with those found in the CA/ROSC and normothermia group. Taken together, our results indicate that hypothermic therapy after CA/ROSC attenuates CA-induced hindlimb paralysis by protecting motor neurons in the lumbar spinal cord via activating the Nrf2/HO-1 signaling pathway and attenuating pro-inflammation and astrocyte damage (reactive astrogliosis).

Electrical stimulation (ES) is effective for disuse-induced muscle atrophy. However, the acute effect of ES on muscle protein synthesis (MPS) and muscle protein breakdown (MPB) remains unclear. We investigated the effect of a single-session ES treatment on mTORC1 signaling, MPS, and MPB in the soleus muscle of 2-week hindlimb unloaded rats. Sprague Dawley rats (n = 12 male) were randomly divided into control (CON) and hindlimb unloaded (HU) groups. After 2 weeks, the right soleus muscle was percutaneously stimulated and underwent supramaximal isometric contractions. The left soleus muscle served as an internal control. We collected soleus muscle samples 6 h after ES. Two weeks of HU decreased p70S6K and S6rp activation, downstream factors for mTORC1 signaling, and SUnSET method-assessed MPS, but increased the LC3-II/I ratio, an indicator of autophagy. ES on disused muscle successfully activated mTORC1 signaling but did not affect MPS. Contrary, ES decreased ubiquitinated proteins expression and LC3B-II/I ratio. HU might affect mTORC1 activation and MPS differently in response to acute ES possibly due to excessive ROS production caused by ES. Our findings suggest that ES applied to disused skeletal muscles may suppress MPB, but its effect on MPS appears to be attenuated.

Hind limb ischemia (HLI) is the most severe form of peripheral arterial disease, associated with a substantial reduction of limb blood flow that impairs skeletal muscle homeostasis to promote functional disability. The molecular regulators of HLI-induced muscle perturbations remain poorly defined. This study investigated whether changes in the molecular catabolic-autophagy signaling network were linked to temporal remodeling of skeletal muscle in HLI. HLI was induced in mice via hindlimb ischemia (femoral artery ligation) and confirmed by Doppler echocardiography. Experiments were terminated at time points defined as early- (7 days; n = 5) or late- (28 days; n = 5) stage HLI. Ischemic and nonischemic (contralateral) limb muscles were compared. Ischemic versus nonischemic muscles demonstrated overt remodeling at early-HLI but normalized at late-HLI. Early-onset fiber atrophy was associated with excessive autophagy signaling in ischemic muscle; protein expression increased for Beclin-1, LC3, and p62 ( P < 0.05) but proteasome-dependent markers were reduced ( P < 0.05). Mitophagy signaling increased in early-stage HLI that aligned with an early and sustained loss of mitochondrial content ( P < 0.05). Upstream autophagy regulators, Sestrins, showed divergent responses during early-stage HLI (Sestrin2 increased while Sestrin1 decreased; P < 0.05) in parallel to increased AMP-activated protein kinase (AMPK) phosphorylation ( P < 0.05) and lower antioxidant enzyme expression. No changes were found in markers for mechanistic target of rapamycin complex 1 signaling. These data indicate that early activation of the sestrin-AMPK signaling axis may regulate autophagy to stimulate rapid and overt muscle atrophy in HLI, which is normalized within weeks and accompanied by recovery of muscle mass. A complex interplay between Sestrins to regulate autophagy signaling during early-to-late muscle remodeling in HLI is likely.

Objective: To explore the mechanism of nerve growth factor (NGF) in the skeletal muscle fiber remodeling in ischemic limbs during therapeutic angiogenesis. Methods: Eighteen female mice with SPF grade, 6 weeks old and 25-30 g weighed were randomly allocated to sham-operated group ( n =6), blank control group ( n =6) and NGF gene transfection group ( n =6). The left hindlimb ischemia models were established by ligating the femoral artery in blank control group and NGF gene transfection group. Seven days after the operation, mice in the three groups were separately injected with normal saline, empty plasmids, and NGF plasmids. Gastrocnemius of left hindlimbs was harvested after the blood perfusion assessment of the ischemic limb on the 21st postoperative day. The gastrocnemius muscle specimens were stained with HE, CD31 and proliferating cell nuclear antigen (PCNA) immunohistochemistry staining, the mRNA expressions of myosin heavy chain-Ⅰ(MHC-Ⅰ), MHC-Ⅱa and MHC-Ⅱb were measured by real-time PCR, and the protein level of NGF and peroxisome proliferator-activated receptors-β/δ (PPAR β/δ) were detected by Western blot. The expression of cytochrome C oxidase (COX), isocitrate dehydrogenase (IDH) and adenosine triphosphate (ATP) were examined by enzyme-linked immunosorbent assay (ELISA). Results: On the 21st day after operation, the blood perfusion of the ischemic limb in NGF gene transfection group was (195.70±9.99)PU, which was lower than that in sham-operated group (312.15±17.32)PU ( P =0.001), while it was higher than that in blank control group (82.11±8.55)PU ( P =0.001). The degree of muscle atrophy in the NGF gene transfection group was lower than that in the blank control group. The capillary density of NGF gene transfection group (0.34±0.05) was higher than that of sham-operated group (0.11±0.03) and blank control group (0.27±0.04) ( P <0.05). The endothelial cell proliferation index in NGF gene transfection group (0.39±0.19) was significantly higher than that in sham-operated group (0.18±0.01) and blank control group (0.25±0.14) ( P <0.05). The expression of NGF, PPAR β/δ, COX, IDH, ATP, and MHC-Ⅰ mRNA in NGF gene transfection group were significantly higher than those in sham-operated group and blank control group ( P <0.05). Conclusions: NGF gene transfection can promote angiogenesis in the ischemic limbs of mice, increase the blood perfusion, and thus induce the remodeling of skeletal muscle fibers to type Ⅰ. This process may be related to NGF-induced PPAR β/δ expression and promote the cellular aerobic metabolism in skeletal muscle.

Endothelial progenitor cells (EPCs) are reduced in number and impaired in function in diabetic patients. Whether and how Nrf2 regulates the function of diabetic EPCs remains unclear. In this study, we found that the expression of Nrf2 and its downstream genes were decreased in EPCs from both diabetic patients and db/db mice. Survival ability and angiogenic function of EPCs from diabetic patients and db/db mice also were impaired. Gain- and loss-of-function studies, respectively, showed that knockdown of Nrf2 increased apoptosis and impaired tube formation in EPCs from healthy donors and wild-type mice, while Nrf2 overexpression decreased apoptosis and rescued tube formation in EPCs from diabetic patients and db/db mice. Additionally, proangiogenic function of Nrf2-manipulated mouse EPCs was validated in db/db mice with hind limb ischemia. Mechanistic studies demonstrated that diabetes induced mitochondrial fragmentation and dysfunction of EPCs by dysregulating the abundance of proteins controlling mitochondrial dynamics; upregulating Nrf2 expression attenuated diabetes-induced mitochondrial fragmentation and dysfunction and rectified the abundance of proteins controlling mitochondrial dynamics. Further RNA-sequencing analysis demonstrated that Nrf2 specifically upregulated the transcription of isocitrate dehydrogenase 2 (IDH2), a key enzyme regulating tricarboxylic acid cycle and mitochondrial function. Overexpression of IDH2 rectified Nrf2 knockdown- or diabetes-induced mitochondrial fragmentation and EPC dysfunction. In a therapeutic approach, supplementation of an Nrf2 activator sulforaphane enhanced angiogenesis and blood perfusion recovery in db/db mice with hind limb ischemia. Collectively, these findings indicate that Nrf2 is a potential therapeutic target for improving diabetic EPC function. Thus, elevating Nrf2 expression enhances EPC resistance to diabetes-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia likely via transcriptional upregulating IDH2 expression and improving mitochondrial function of diabetic EPCs., Competing Interests: Declaration of competing interest No potential conflicts of interest relevant to this article were reported., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Skeletal muscle unloading leads to muscle atrophy. Ribosome synthesis has been implicated as an important skeletal muscle mass regulator owing to its translational capacity. Muscle unloading induces a reduction in ribosome synthesis and content, with muscle atrophy. Percutaneous electrical muscle stimulation (pEMS)-induced muscle contraction is widely used in clinics to improve muscle mass. However, its efficacy in rescuing the reduction in ribosomal synthesis has not been addressed thus far. We examined the effects of daily pEMS treatment on ribosome synthesis and content during mouse hindlimb unloading. Male C57BL/6J mice were randomly assigned to sedentary (SED) and hindlimb unloading by pelvic suspension (HU) groups. Muscle contraction was triggered by pEMS treatment of the right gastrocnemius muscle of a subset of the HU group (HU + pEMS). Hindlimb unloading for 6 days significantly lowered 28S rRNA, rpL10, and rpS3 expression, which was rescued by daily pEMS treatment. The protein expression of phospho-p70S6K and UBF was significantly higher in the HU + pEMS than in the HU group. The mRNA expression of ribophagy receptor Nufip1 increased in both the HU and HU + pEMS groups. Protein light chain 3 (LC3)-II expression and the LC3-II/LC3-I ratio were increased by HU, but pEMS attenuated this increase. Our findings indicate that during HU, daily pEMS treatment prevents the reduction in the levels of some proteins associated with ribosome synthesis. In addition, the HU-induced activation of ribosome degradation may be attenuated. These data provide insights into ribosome content regulation and the mechanism of attenuation of muscle atrophy by pEMS treatment during muscle disuse. NEW & NOTEWORTHY Muscle inactivity reduces ribosome synthesis and content during atrophy. Whether percutaneous electrical muscle stimulation (pEMS)-induced muscle contraction rescues the ribosome synthesis and content during muscle unloading is unclear. Using a mouse hindlimb-unloading model with pelvic suspension, we provide evidence that daily pEMS-induced muscle contraction during hindlimb unloading rescues the reduction in the expression of some ribosome synthesis-related proteins and ribosome content in the gastrocnemius muscle.

Limb ischemia occurs due to obstruction of blood perfusion to lower limbs, a manifestation that is associated with peripheral artery disease (PAD). Angiogenesis is important for adequate oxygen delivery. The present study investigated a potential role for chrysin, a naturally occurring flavonoid, in promoting angiogenesis in hindlimb ischemia (HLI) rat model. Rats were allocated into four groups: (1) sham-operated control, (2) HLI: subjected to unilateral femoral artery ligation, (3) HLI + chrysin: received 100 mg/kg, i.p. chrysin immediately after HLI, and (4) HLI + chrysin + rapamycin: received 6 mg/kg/day rapamycin i.p. for 5 days then subjected to HLI and dosed with 100 mg/kg chrysin, i.p. Rats were killed 18 h later and gastrocnemius muscles were collected and divided into parts for (1) immunohistochemistry detection of CD31 and CD105, (2) qRT-PCR analysis of eNOS and VEGFR2, (3) colorimetric analysis of NO, (4) ELISA estimation of TGF-β, VEGF, ATG5 and Beclin-1, and (5) Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, and HIF-1α. Chrysin significantly enhanced microvessels growth in HLI muscles as indicated by increased CD31 and CD105 levels and decreased TGF-β. Chrysin's proangiogenic effect is potentially mediated by increased VEGF, VEGFR2 and activation of PI3K/AKT/mTOR pathway, which promoted eNOS and NO levels as it was reversed by the mTOR inhibitor, rapamycin. Chrysin also inhibited autophagy as it decreased ATG5 and Beclin-1. The current study shows that chrysin possesses a proangiogenic effect in HLI rats and might be useful in patients with PAD., (© 2022 Wiley Periodicals LLC.)

Diabetic vasculopathy is a major health problem worldwide. Peripheral arterial disease (PAD), and in its severe form, critical limb ischemia is a major form of diabetic vasculopathy with limited treatment options. Existing literature suggested an important role of PPARδ in vascular homeostasis. It remains elusive for using PPARδ as a potential therapeutic target due to mostly the side effects of PPARδ agonists. To explore the roles of PPARδ in endothelial homeostasis, endothelial cell (EC) selective Ppard knockout and controlled mice were subjected to hindlimb ischemia (HLI) injury. The muscle ECs were sorted for single-cell RNA sequencing (scRNA-seq) analysis. HLI was also performed in high fat diet (HFD)-induced obese mice to examine the function of PPARδ in obese mice with delayed vascular repair. Adeno-associated virus type 1 (AAV1) carrying ICAM2 promoter to target endothelium for overexpressing PPARδ was injected into the injured muscles of normal chow- and HFD-fed obese mice before HLI surgery was performed. scRNA-seq analysis of ECs in ischemic muscles revealed a pivotal role of PPARδ in endothelial homeostasis. PPARδ expression was diminished both after HLI injury, and also in obese mice, which showed further delayed vascular repair. Endothelium-targeted delivery of PPARδ by AAV1 improved perfusion recovery, increased capillary density, restored endothelial integrity, suppressed vascular inflammation, and promoted muscle regeneration in ischemic hindlimbs of both lean and obese mice. Our study indicated the effectiveness of endothelium-targeted PPARδ overexpression for restoring functional vasculature after ischemic injury, which might be a promising option of gene therapy to treat PAD and CLI., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Hypoxia and inflammation play a major role in revascularization following ischemia. Sildenafil inhibits phosphodiesterase-5, increases intracellular cGMP and induces revascularization through a pathway which remains incompletely understood. Thus, we investigated the effect of sildenafil on post-ischemic revascularization. The left femoral artery was ligated in control and sildenafil-treated (25 mg/kg per day) rats. Vascular density was evaluated and expressed as the left/right leg (L/R) ratio. In control rats, L/R ratio was 33 ± 2% and 54 ± 9%, at 7- and 21-days post-ligation, respectively, and was significantly increased in sildenafil-treated rats to 47 ± 4% and 128 ± 11%, respectively. A neutralizing anti-VEGF antibody significantly decreased vascular density (by 0.48-fold) in control without effect in sildenafil-treated animals. Blood flow and arteriolar density followed the same pattern. In the ischemic leg, HIF-1α and VEGF expression levels increased in control, but not in sildenafil-treated rats, suggesting that sildenafil did not induce angiogenesis. PI3-kinase, Akt and eNOS increased after 7 days, with down-regulation after 21 days. Sildenafil induced outward remodeling or arteriogenesis in mesenteric resistance arteries in association with eNOS protein activation. We conclude that sildenafil treatment increased tissue blood flow and arteriogenesis independently of VEGF, but in association with PI3-kinase, Akt and eNOS activation.

Serotonin (5-HT) is a multifaceted neurotransmitter that has been described to play a role as a peripheral inflammatory mediator when released in ischemic or injured muscle. Dorsal root ganglia (DRG) neurons are key sensors of noxious stimuli that are released under inflammatory conditions or mechanical stress. Little information is available on the specific 5-HT receptor subtypes expressed in primary afferents that help regulate reflex pressor responses. In the present study, the whole-cell patch-clamp technique was employed to examine the modulation of voltage-gated calcium channel (Ca V ) 2.2 currents by 5-HT and to identify the 5-HT receptor subtype(s) mediating this response in acutely dissociated rat DRG neurons innervating triceps surae muscle. Our results indicate that exposure of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled DRG neurons to 5-HT can exert three modulatory effects on Ca V currents: high inhibition, low inhibition, and enhancement. Both 5-HT-mediated inhibition responses were blocked after pretreatment with pertussis toxin (PTX), indicating that 5-HT receptors are coupled to Ca V 2.2 via G α i/o protein subunits. Application of selective serotonin receptor type 1 (5-HT1) agonists revealed that modulation of Ca V 2.2 currents occurs primarily after 5-HT1A receptor subtype stimulation and minimally from 5-HT1D activation. Finally, the intrathecal administration of the selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), significantly ( P < 0.05) decreased the pressor response induced by intra-arterial administration of lactic acid. This suggests that 5-HT1A receptors are expressed presynaptically on primary afferent neurons innervating triceps surae muscle. Our findings indicate that preferential stimulation of 5-HT1 receptors, expressed on thin fiber muscle afferents, serves to regulate the reflex pressor response to metabolic stimuli. SIGNIFICANCE STATEMENT: The monoamine serotonin (5-HT), released under ischemic conditions, can contribute to the development of inflammation that negatively affects the exercise pressor reflex. The 5-HT receptor subtype and signaling pathway that underlies calcium channel modulation in dorsal root ganglia afferents, innervating hindlimb muscles, are unknown. We show that 5-HT can either block (primarily via serotonin receptor type 1 (5-HT1)A subtypes) or enhance voltage-gated calcium channel (Ca V 2.2) currents. Our findings suggest 5-HT exhibits receptor subtype selectivity, providing a complexity of cellular responses., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Peripheral vascular disease (PVD) is a common clinical manifestation of atherosclerosis. Vascular endothelial growth factor (VEGF) gene therapy is a promising approach for PVD treatment. However, due to single-gene therapy limitations and high H 2 O 2 pathological microenvironment, VEGF gene therapy are not as expectations and its clinical application are limited. Synergistic effects of Nerve factors and vascular factors in angiogenesis have attracted attention in recent years. In this study, VEGF and nerve growth factor (NGF) genes co-delivery nanoparticles (VEGF/NGF-NPs) were prepared by using H 2 O 2 responsive 6s-PLGA-Po-PEG as a carrier. 6s-PLGA-Po-PEG could react with H 2 O 2 specifically due to the internal peroxalate bond. Angiogenic effects of VEGF/NGF-NPs has been evaluated in cells and hindlimb ischemia mice model. Results showed that VEGF/NGF-NPs promoted VEGF and NGF co-expression simultaneously, eliminated excessive H 2 O 2 , strengthened reactions between SH-SY5Ys and HUVECs, and finally enhanced migration, tube formation, proliferation and H 2 O 2 damage resistance of HUVECs. VEGF/NGF-NPs also recovered blood perfusion, promoted the expression of VEGF, NGF, eNOS and NO, and enhanced vascular coverage of pericytes. Treatment effects of VEGF/NGF-NPs may related to VEGF/eNOS/NO pathway. Altogether, VEGF/NGF-NPs eliminated excessive H 2 O 2 while achieving gene co-delivery, and promoted stable angiogenesis. It's a promising way for PVD treatment by using VEGF/NGF-NPs., (© 2022. The Author(s).)

Harmonia axyridis presents remarkable appendage regeneration capacity and can therefore be considered as an emerging regeneration research model. Amino acid sequences of the Janus kinase Hopscotch (Hahop) and the transcription factor STAT (HaStat), the main components of the JAK/STAT signaling pathway, conserved with their homologs in other models. The expression levels of these two genes were continuously up-regulated during the appendage regeneration process. To identify the functions of JAK/STAT signaling, we performed RNAi experiments of Hahop and HaStat in H. axyridis, and found regeneration defects following in Hahop RNAi and HaStat RNAi treatments at different regeneration stages. Additionally, we confirmed that regeneration defects caused by the low-level of JAK/STAT activity were due to the inhibition of cell proliferation. The results of the current study suggest that JAK/STAT signaling regulates the entire regeneration process by coordinating cell proliferation of regenerating appendages., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The cetacean hindlimb skeleton massively decreased to only vestigial limb elements as cetaceans evolved from land to aquatic lifestyles; however, the molecular mechanism underlying this major morphological transition remains unclear. In this study, four deletions and specific substitutions were detected in cetacean hindlimb enhancer A (HLEA), an enhancer that can regulate Tbx4 expression in hindlimb tissues to control hindlimb development. Transcriptional activation of HLEA was significantly weaker in bottlenose dolphin than mice, and this was found to be closely associated with cetacean-specific deletions. Furthermore, deletions in cetacean HLEA might disrupt HOX and PITX1 binding sites, which are required for enhancer activation. The ancestral state of these deletions was investigated, and all four specific deletions were found to have occurred after the species diverged from their common ancestor, suggesting that the deletion occurred recently, during a secondary aquatic adaptation. Taking these findings together, we suggest that cetacean-specific sequence changes reduced the Tbx4 gene expression pattern, and consequently drove the gradual loss of hindlimb in cetaceans., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

A concurrent reduction in muscle mass and strength is frequently observed in numerous conditions, including neuromuscular disease, ageing, and muscle inactivity due to limb immobilization or prolonged bed rest. Thus, identifying the molecular mechanisms that control skeletal muscle mass and strength is fundamental for developing interventions aimed at counteracting muscle loss (muscle atrophy). It was recently reported that muscle atrophy induced by denervation of motor nerves was associated with increased expression of Ca 2+ /calmodulin-dependent protein serine/threonine kinase II β (CaMKIIβ) in muscle. In addition, treatment with KN-93 phosphate, which inhibits CaMKII-family kinases, partly suppressed denervation-induced muscle atrophy. Therefore, to test a possible role for CaMKIIβ in muscle mass regulation, we generated and injected recombinant adeno-associated virus (AAV) vectors encoding wild-type (AAV-WT), inactive (AAV-K43 M), or constitutively active (AAV-T287D) CaMKIIβ into the left hindlimb tibialis anterior muscle of mice at three months of age. Although AAV-WT infection induced expression of exogenous CaMKIIβ in the hindlimb muscle, no significant changes in muscle mass and strength were observed. By contrast, AAV-K43 M or AAV-T287D infection induced exogenous expression of the corresponding mutants and significantly increased or decreased the muscle mass and strength of the infected hind limb, respectively. Together, these findings demonstrate the potential of CaMKIIβ as a novel therapeutic target for enhancing muscle mass and strength., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objective: To study the effects of vibration on the expression of mitochondrial fusion and fission genes and ultrastructure of skeletal muscle in rabbits. Methods: Thirty-two 3.5-month-old New Zealand rabbits were randomly divided into low-intensity group, medium-intensity group, high-intensity group and control group, with 8 rabbits in each group. The rabbits in the experimental group were subjected to hind limb vibration load test for 45 days. The vibration intensity of the high intensity group was 12.26 m/s(2), the medium intensity group was 6.13 m/s(2), and the low intensity group was 3.02 m/s(2) according to the effective value of weighted acceleration[a(hw (4))] for 4 hours of equal energy frequency. The control group was exposed to noise only in the same experimental environment as the medium-intensity group. The noise levels of each group were measured during the vibration load experiment. After the test, the mRNA expression of mitochondrial fusion gene ( Mfn1/Mfn2 ) and fission gene ( Fis1 , Drp1 ) by RT-PCR in the skeletal muscles were measured and the ultrastructure of the skeletal muscles were observed in high intensity group. Results: The mRNA expression of mitochondrial in the skeletal muscle tissues of control group, low intensity group, medium intensity group and high intensity group were Mfn1 : 3.25±1.36, 3.85±1.90, 4.53±2.31 and 11.63±7.68; Mfn2 : 0.68±0.25, 1.02±0.40, 0.94±0.33 and 1.40±0.45; Fis1 : 1.05±0.62, 1.15±0.59, 1.53±1.06 and 2.46±1.51 and Drp1 : 3.72±1.76, 2.91±1.63, 3.27±2.01 and 4.21±2.46, respectively. Compared with the control group, the expressions of Mfn1 mRNA, Mfn2 mRNA and Fis1 mRNA in the high-intensity group increased significantly ( P <0.05) , and the expressions of Mfn2 mRNA in the medium-intensity group and the low-intensity group increased significantly ( P <0.05) . Compared with the control group, the ultrastructure of skeletal muscle of high intensity group showed mitochondrial focal accumulation, cristae membrane damage, vacuole-like changes; Z-line irregularity of muscle fibers, and deficiency of sarcomere. Conclusion: Vibration must be lead to the abnormal mitochondrial morphology and structure and the disorder of energy metabolism due to the expression imbalance of mitochondrial fusion and fission genes in skeletal muscles of rabbits, which may be an important target of vibration-induced skeletal muscle injury.

A large set of FoxOs-dependent genes play a primary role in controlling muscle mass during hindlimb unloading. Mitochondrial dysfunction can modulate such a process. We hypothesized that endurance exercise before disuse can protect against disuse-induced muscle atrophy by enhancing peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) expression and preventing mitochondrial dysfunction and energy-sensing AMP-activated protein kinase (AMPK) activation. We studied cross sectional area (CSA) of muscle fibers of gastrocnemius muscle by histochemistry following 1, 3, 7, and 14 days of hindlimb unloading (HU). We used Western blotting and qRT-PCR to study mitochondrial dynamics and FoxOs-dependent atrogenes' expression at 1 and 3 days after HU. Preconditioned animals were submitted to moderate treadmill exercise for 7 days before disuse. Exercise preconditioning protected the gastrocnemius from disuse atrophy until 7 days of HU. It blunted alterations in mitochondrial dynamics up to 3 days after HU and the expression of most atrogenes at 1 day after disuse. In preconditioned mice , the activation of atrogenes resumed 3 days after HU when mitochondrial dynamics, assessed by profusion and pro-fission markers (mitofusin 1, MFN1, mitofusin 2, MFN2, optic atrophy 1, OPA1, dynamin related protein 1, DRP1 and fission 1, FIS1), PGC1α levels, and AMPK activation were at a basal level. Therefore, the normalization of mitochondrial dynamics and function was not sufficient to prevent atrogenes activation just a few days after HU. The time course of sirtuin 1 (SIRT1) expression and content paralleled the time course of atrogenes' expression. In conclusion, seven days of endurance exercise counteracted alterations of mitochondrial dynamics and the activation of atrogenes early into disuse. Despite the normalization of mitochondrial dynamics, the effect on atrogenes' suppression died away within 3 days of HU. Interestingly, muscle protection lasted until 7 days of HU. A longer or more intense exercise preconditioning may prolong atrogenes suppression and muscle protection.

Developmental genes are frequently controlled by multiple enhancers sharing similar specificities. As a result, deletions of such regulatory elements have often failed to reveal their full function. Here, we use the Pitx1 testbed locus to characterize in detail the regulatory and cellular identity alterations following the deletion of one of its enhancers (Pen). By combining single cell transcriptomics and an in-embryo cell tracing approach, we observe an increased fraction of Pitx1 non/low-expressing cells and a decreased fraction of Pitx1 high-expressing cells. We find that the over-representation of Pitx1 non/low-expressing cells originates from a failure of the Pitx1 locus to coordinate enhancer activities and 3D chromatin changes. This locus mis-activation induces a localized heterochrony and a concurrent loss of irregular connective tissue, eventually leading to a clubfoot phenotype. This data suggests that, in some cases, redundant enhancers may be used to locally enforce a robust activation of their host regulatory landscapes., (© 2021. The Author(s).)

Strain-related differences in arteriogenesis in inbred mouse strains have already been studied excessively. However, these analyses missed evaluating the mouse strain-related differences in ischemia-induced angiogenic capacities. With the present study, we wanted to shed light on the different angiogenic potentials and the associated leukocyte infiltration of C57BL/6J and SV-129 mice to facilitate the comparison of angiogenesis-related analyses between these strains. For the induction of angiogenesis, we ligated the femoral artery in 8-12-week-old male C57BL/6J and SV-129 mice and performed (immuno-) histological analyses on the ischemic gastrocnemius muscles collected 24 h or 7 days after ligation. As evidenced by hematoxylin and eosin staining, C57BL/6J mice showed reduced tissue damage but displayed an increased capillary-to-muscle fiber ratio and an elevated number of proliferating capillaries (CD31 + /BrdU + cells) compared to SV-129 mice, thus showing improved angiogenesis. Regarding the associated leukocyte infiltration, we found increased numbers of neutrophils (MPO + cells), NETs (MPO + /CitH3 + /DAPI + ), and macrophages (CD68 + cells) in SV-129 mice, whereas macrophage polarization (MRC1 - vs. MRC1 + ) and total leukocyte infiltration (CD45 + cells) did not differ between the mouse strains. In summary, we show increased ischemia-induced angiogenic capacities in C57BL/6J mice compared to SV-129 mice, with the latter showing aggravated tissue damage, inflammation, and impaired angiogenesis.

Previous studies have shown that infiltration of capsaicin into the surgical site can prevent incision-induced spontaneous pain like behaviors and heat hyperalgesia. In the present study, we aimed to monitor primary sensory neuron Ca 2+ activity in the intact dorsal root ganglia (DRG) using Pirt-GCaMP3 male and female mice pretreated with capsaicin or vehicle before the plantar incision. Intraplantar injection of capsaicin (0.05%) significantly attenuated spontaneous pain, mechanical, and heat hypersensitivity after plantar incision. The Ca 2+ response in in vivo DRG and in in situ spinal cord was significantly enhanced in the ipsilateral side compared with contralateral side or naive control. Primary sensory nerve fiber length was significantly decreased in the incision skin area in capsaicin-pretreated animals detected by immunohistochemistry and placental alkaline phosphatase (PLAP) staining. Thus, capsaicin pretreatment attenuates incisional pain by suppressing Ca 2+ response because of degeneration of primary sensory nerve fibers in the skin. SIGNIFICANCE STATEMENT Postoperative surgery pain is a major health and economic problem worldwide with ∼235 million major surgical procedures annually. Approximately 50% of these patients report uncontrolled or poorly controlled postoperative pain. However, mechanistic studies of postoperative surgery pain in primary sensory neurons have been limited to in vitro models or small numbers of neurons. Using an innovative, distinctive, and interdisciplinary in vivo populational dorsal root ganglia (DRG) imaging (>1800 neurons/DRG) approach, we revealed increased DRG neuronal Ca 2+ activity from postoperative pain mouse model. This indicates widespread DRG primary sensory neuron plasticity. Increased neuronal Ca 2+ activity occurs among various sizes of neurons but mostly in small-diameter and medium-diameter nociceptors. Capsaicin pretreatment as a therapeutic option significantly attenuates Ca 2+ activity and postoperative pain., (Copyright © 2021 the authors.)

Background: The aim of this study was to investigate the mechanism of hydrogen gas on hind limb IR injury., Methods: Male C57BL/6 mice were randomly divided into three groups: sham group (Sham), ischemia-reperfusion group (IR), IR plus H 2 inhalation group (IR + H 2 ). IR was induced by interrupting hind limb blood flow for 3h, followed by 4h of reperfusion, and H 2 was administered by inhalation throughout the reperfusion process. Our data show that H 2 inhalation could significantly decrease the infarct-affected tissue volume (P < 0.05), attenuate the degree of morphological injury (P < 0.05), and suppress the level of oxidative stress damage (P < 0.05), compared with the IR group. In exploring the underlying mechanisms, we found that hydrogen could markedly mitigate the degree of IR-induced ER stress and apoptosis (P < 0.05). Additionally, hydrogen could markedly inhibit the IR injury by modulating the phosphorylated c-Jun N-terminal kinase (JNK) signaling pathway (P < 0.05)., Conclusions: Taken together, these results revealed the protective effect of hydrogen gas on hind limb ischemia reperfusion injury on mice by attenuating oxidative stress, impairing ER stress and apoptosis, and its ability to modulate JNK signaling pathway., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fetal skeletal muscle growth requires myoblast proliferation, differentiation, and fusion into myofibers in addition to protein accretion for fiber hypertrophy. Oxygen is an important regulator of this process. Therefore, we hypothesized that fetal anemic hypoxemia would inhibit skeletal muscle growth. Studies were performed in late-gestation fetal sheep that were bled to anemic and therefore hypoxemic conditions beginning at ∼125 days of gestation (term = 148 days) for 9 ± 0 days ( n = 19) and compared with control fetuses ( n = 16). A metabolic study was performed on gestational day ∼134 to measure fetal protein kinetic rates. Myoblast proliferation and myofiber area were determined in biceps femoris (BF), tibialis anterior (TA), and flexor digitorum superficialis (FDS) muscles. mRNA expression of muscle regulatory factors was determined in BF. Fetal arterial hematocrit and oxygen content were 28% and 52% lower, respectively, in anemic fetuses. Fetal weight and whole body protein synthesis, breakdown, and accretion rates were not different between groups. Hindlimb length, however, was 7% shorter in anemic fetuses. TA and FDS muscles weighed less, and FDS myofiber area was smaller in anemic fetuses compared with controls. The percentage of Pax7 + myoblasts that expressed Ki67 was lower in BF and tended to be lower in FDS from anemic fetuses indicating reduced myoblast proliferation. There was less MYOD and MYF6 mRNA expression in anemic versus control BF consistent with reduced myoblast differentiation. These results indicate that fetal anemic hypoxemia reduced muscle growth. We speculate that fetal muscle growth may be improved by strategies that increase oxygen availability.

Peripheral arterial disease (PAD) is an increasingly common narrowing of the peripheral arteries that can lead to lower limb ischemia, muscle weakness and gangrene. Surgical vein or arterial grafts could improve PAD, but may not be suitable in elderly patients, prompting research into less invasive approaches. Mesenchymal stem cells (MSCs) have been proposed as potential therapy, but their effectiveness and underlying mechanisms in limb ischemia are unclear. We tested the hypothesis that treatment with naive MSCs (nMSCs) or MSCs expressing CD146 ( CD146+ MSCs) could improve vascularity and muscle function in rat model of hind-limb ischemia. Sixteen month old Sprague-Dawley rats were randomly assigned to 4 groups: sham-operated control, ischemia, ischemia + nMSCs and ischemia+ CD146+ MSCs. After 4 weeks of respective treatment, rat groups were assessed for ischemic clinical score, Tarlov score, muscle capillary density, TUNEL apoptosis assay, contractile force, and vascular endothelial growth factor (VEGF) mRNA expression. CD146+ MSCs showed greater CD146 mRNA expression than nMSCs. Treatment with nMSCs or CD146+ MSCs improved clinical and Tarlov scores, muscle capillary density, contractile force and VEGF mRNA expression in ischemic limbs as compared to non-treated ischemia group. The improvements in muscle vascularity and function were particularly greater in ischemia+ CD146+ MSCs than ischemia + nMSCs group. TUNEL positive apoptotic cells were least abundant in ischemia+ CD146+ MSCs compared with ischemia + nMSCs and non-treated ischemia groups. Thus, MSCs particularly those expressing CD146 improve vascularity, muscle function and VEGF expression and reduce apoptosis in rat ischemic limb, and could represent a promising approach to improve angiogenesis and muscle function in PAD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

We previously identified the inhibitory serpin protease nexin-1 (PN-1) as an important player of the angiogenic balance with anti-angiogenic activity in physiological conditions. In the present study, we aimed to determine the role of PN-1 on pathological angiogenesis and particularly in response to ischemia, in the mouse model induced by femoral artery ligation. In wild-type (WT) muscle, we observed an upregulation of PN-1 mRNA and protein after ischemia. Angiography analysis showed that femoral artery perfusion was more rapidly restored in PN-1 -/- mice than in WT mice. Moreover, immunohistochemistry showed that capillary density increased following ischemia to a greater extent in PN-1 -/- than in WT muscles. Moreover, leukocyte recruitment and IL-6 and MCP-1 levels were also increased in PN-1 -/- mice compared to WT after ischemia. This increase was accompanied by a higher overexpression of the growth factor midkine, known to promote leukocyte trafficking and to modulate expression of proinflammatory cytokines. Our results thus suggest that the higher expression of midkine observed in PN-1- deficient mice can increase leukocyte recruitment in response to higher levels of MCP-1, finally driving neoangiogenesis. Thus, PN-1 can limit neovascularisation in pathological conditions, including post-ischemic reperfusion of the lower limbs.

Background: Recent studies have demonstrated that inhibition of CD26 potentiates stromal cell-derived factor-1α (SDF-1α), promotes tissue regeneration, and suppresses the rejection of organ transplants. This study investigated whether the combination of a CD26 inhibitor (CD26i) with granulocyte colony-stimulating factor (G-CSF) and short-term immunosuppressants modulates vascularized composite tissue allotransplant survival in a rodent orthotopic hindlimb allotransplant model., Methods: The hindlimb allotransplantation from Brown-Norway to Lewis rats was divided into 4 groups. Group 1 (controls) did not receive any treatment. Group 2 was treated with short-term antilymphocyte serum (ALS) and cyclosporine-A (CsA). Group 3 was administrated CD26i and G-CSF. Group 4 received a combination of CD26i/G-CSF/ALS/CsA. Each subgroup comprised 10 rats. Peripheral blood and sampling of transplanted tissues were collected for immunological and histological analysis., Results: The results revealed that allotransplant survival was found to be significantly prolonged in group 4 with CD26i/G-CSF/ALS/CsA treatment compared with those in the other groups. The interleukin-10 and transforming growth factor-βl levels, the percentage of CD4+/CD25+/FoxP3+ T cells, as well as the levels of SDF-1α expressions were significantly increased in group 4 compared with those in the other groups. Group 4 revealed a statistical increase in the percentage of donor cells (RT1n) expression in the recipient peripheral blood, and the mixed lymphocyte reaction showed hyporesponsiveness of the T cells to donor alloantigens., Conclusion: The combination of CD26i/G-CSF and short-term immunosuppressants prolongs allotransplant survival by inducing immunoregulatory effects and enhancing the percentage of SDF-1α expression. This immunomodulatory approach has great potential as a strategy to increase vascularized composite allotransplantation survival., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Developmental engineering strategies aim to recapitulate aspects of development in vitro as a means of forming functional engineered tissues, including cartilage and bone, for tissue repair and regeneration. Biophysical stimuli arising from fetal movements are critical for guiding skeletogenesis, but there have been few investigations of the biomechanical parameters which optimally promote cartilage and bone development events in in vitro explants. The effect of applied flexion-extension movement frequencies (0.33 and 0.67 Hz) and durations (2 h periods, 1, 2 or 3 × per day) on knee (stifle) joint cartilage shape, chondrogenesis and diaphyseal mineralisation of fetal chick hindlimbs, cultured in a mechanostimulation bioreactor, were assessed both quantitatively and qualitatively. It was hypothesised that increasing frequency and duration of movements would synergistically promote cartilage and bone formation in a dose-dependent manner. Increasing loading duration promoted cartilage growth, shape development and mineralisation of the femoral condyles and tibiotarsus. While increasing frequency had a significant positive effect on mineralisation, hyaline cartilage growth and joint shape were unaffected by frequency change within the ranges assessed, and there were limited statistical interactions between the effects of movement frequency and duration on cartilage or bone formation. Increased glycosaminoglycan deposition and cell proliferation may have contributed to the accelerated cartilage growth and shape change under increasing loading duration. The results demonstrated that frequencies and durations of applied biomechanical stimulation differentially promoted cartilage and bone formation, with implications for developmentally inspired tissue engineering strategies aiming to modulate tissue construct properties.

2-Arachidonyl-lysophosphatidylethanolamine, shortly 2-ARA-LPE, is a polyunsaturated lysophosphatidylethanolamine. 2-ARA-LPE has a very long chain arachidonic acid, formed by an ester bond at the sn -2 position. It has been reported that 2-ARA-LPE has anti-inflammatory effects in a zymosan-induced peritonitis model. However, it's action mechanisms are poorly investigated. Recently, resolution of inflammation is considered to be an active process driven by M2 polarized macrophages. Therefore, we have investigated whether 2-ARA-LPE acts on macrophages for anti-inflammation, whether 2-ARA-LPE modulates macrophage phenotypes to reduce inflammation, and whether 2-ARA-LPE is anti-inflammatory in a carrageenan-induced paw edema model. In mouse peritoneal macrophages, 2-ARA-LPE was found to inhibit lipopolysaccharide (LPS)-induced M1 macrophage polarization, but not induce M2 polarization. 2-ARA-LPE inhibited the inductions of inducible nitric oxide synthase and cyclooxygenase-2 in mouse peritoneal macrophages at the mRNA and protein levels. Furthermore, products of the two genes, nitric oxide and prostaglandin E 2 , were also inhibited by 2-ARA-LPE. However, 1-oleoyl-LPE did not show any activity on the macrophage polarization and inflammatory responses. The anti-inflammatory activity of 2-ARA-LPE was also verified in vivo in a carrageenan-induced paw edema model. 2-ARA-LPE inhibits LPS-induced M1 polarization, which contributes to anti-inflammation and suppresses the carrageenan-induced paw edema in vivo.

The study was designed to investigate the potential anti-arthritic effects of methyl palmitate in an adjuvant arthritis model in rats that shares many histopathological similarities with human RA. The underlying mechanism and its effect on CD68 macrophages were investigated, as a further argument to its possible efficacy in RA treatment. A normal control group was injected only with saline, arthritic group, and three treatment groups with CFA induced arthritis received methyl palmitate (MP) at three different doses (75, 150, 300 mg/kg/week for 3 weeks, intraperitoneal). The degree of ipsilateral paw swelling, ankle diameter, spleen index, thymus index and the expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β were measured. In addition, the underlying molecular mechanism was investigated using CD68 expression. Methyl palpitate significantly and dose dependently decreased the arthritic symptoms as measured by ipsilateral paw volume and ankle diameter. It showed no effect on body weight but significantly decreased splenic, thymus index, serum TNF-α and IL-1β. CD68 macrophages expression and the overall synovial inflammatory cellularity were halted. Methyl palmitate exhibits significant anti-inflammatory and exerts a potential anti-arthritic effect in a rat model of adjuvant induced arthritis. Furthermore, it inhibits expression of synovial CD68 macrophage that validate its therapeutic potential adjuvant arthritis., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Aim: The aim of this study was to evaluate the effects of irisin in a murine model of hind limb ischemia reperfusion (I/R)., Methods: The mice were divided into four groups (n = 6 in each group): control, irisin, ischemia reperfusion (I/R), and irisin-ischemia reperfusion (I-I/R). Irisin (0.5 µg.g -1 , intraperitoneally [i.p.]) was administered 30 min before the I/R procedure. After 2 h of ischemia and 2.5 h of reperfusion, blood and tissue samples were taken for biochemical and histopathological analysis. The results were analyzed by Kruskal-Wallis and Mann-Whitney U -tests., Results: There was a statistically significant difference in the total antioxidant status (TAS) and total oxidant status (TOS) levels in all the groups. The TAS level in the I/R group was significantly lower than that in the control, irisin, and I-I/R groups, whereas the TOS level was significantly higher in the I/R group as compared with that in the other groups. Caspase-3 activity and caspase-8 activity, indicators of inflammation, were significantly higher in the I/R and I-I/R groups as compared with those in the control and irisin groups., Conclusion: Irisin may have protective effects in skeletal muscle ischemia reperfusion injury., Competing Interests: The authors declare that they have no competing interests., (© 2021 Küçük et al.)

Toll-like receptors (TLR) are crucial for recognizing bacterial, viral or fungal pathogens and to orchestrate the appropriate immune response. The widely expressed TLR2 and TLR4 differentially recognize various pathogens to initiate partly overlapping immune cascades. To better understand the physiological consequences of both immune responses, we performed comparative lipidomic analyses of local paw inflammation in mice induced by the TLR2 and TLR4 agonists, zymosan and lipopolysaccharide (LPS), respectively, which are commonly used in models for inflammation and inflammatory pain. Doses for both agonists were chosen to cause mechanical hypersensitivity with identical strength and duration. Lipidomic analysis showed 5 h after LPS or zymosan injection in both models an increase of ether-phosphatidylcholines (PC O) and their corresponding lyso species with additional lipids being increased only in response to LPS. However, zymosan induced stronger immune cell recruitment and edema formation as compared to LPS. Importantly, only in LPS-induced inflammation the lipid profile in the contralateral paw was altered. Fittingly, the plasma level of various cytokines and chemokines, including IL-1β and IL-6, were significantly increased only in LPS-treated mice. Accordingly LPS induced distinct changes in the lipid profiles of ipsilateral and contralateral paws. Here, oxydized fatty acids, phosphatidylcholines and phosphatidylethanolamines were uniquely upregulated on the contralateral side. Thus, both models cause increased levels of PC O and lyso-PC O lipids at the site of inflammation pointing at a common role in inflammation. Also, LPS initiates systemic changes, which can be detected by changes in the lipid profiles., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Recently, the function of blood cells in remote ischemic conditioning (RIC) mediated neuroprotection was undoubtedly confirmed. In the present paper, we have focused on the role of blood elements in glutamate homeostasis. The blood of remote conditioned (tolerant) animals was incubated ex vivo with 100 μM glutamate, and the quantitative and qualitative changes of excitatory amino acid transporters (EAAT 1, 2, and 3) were determined. We confirmed RIC mediated accelerated sequestration of extracellular glutamate via EAATs and altered distribution of that amino acid between plasma and cell elements compared to non-tolerant counterparts. The activity of EAATs was elevated in erythrocytes and monocytes, while the density of transporters was not affected. Quantitative changes of EAAT1 density were detected solely in platelets where the forced scavenging was independent of EAATs inhibition. Surprisingly, the trafficking of immunovisualised EAAT2 and 3 raised at tolerant erythrocytes and monocytes. We have found that protein synthesis underlined this process. On the other hand, depletion of protein synthesis did not significantly affect the scavenging capacity of those cell populations. Our work has demonstrated that the elevated blood scavenging of glutamate overdose could be one of the potential mechanisms underlying RIC mediated tissue protection., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

New Findings: What is the central question of this study? Could skeletal muscle be involved in microgravity-induced iron misdistribution by modulating expression of hepcidin, the master regulator of iron metabolism? What is the main finding and its importance? We demonstrate, in rats, that hepcidin upregulation is not a transient adaptation associated with early exposure to microgravity and that intermittent reloading does not limit microgravity-induced iron misdistribution despite having a beneficial effect on soleus muscle wasting., Abstract: In humans, exposure to microgravity during spaceflight causes muscle atrophy, changes in iron storage and a reduction in iron availability. We previously observed that during 7 days of simulated microgravity in rats, hepcidin plays a key role in iron misdistribution, and we suggested that a crosstalk between skeletal muscle and liver could regulate hepcidin synthesis in this context. In the present study in rats, we investigated the medium-term effects of simulated microgravity on iron metabolism. We also tested whether intermittent reloading (IR) to target skeletal muscle atrophy limits iron misdistribution efficiently. For this purpose, Wistar rats underwent 14 days of hindlimb unloading (HU) combined or not combined with daily IR. At the end of this period, the serum iron concentration and transferrin saturation were significantly reduced, whereas hepatic hepcidin mRNA was upregulated. However, the main signalling pathways involved in hepcidin synthesis in the liver (BMP-small mothers against decapentaplegic (SMAD), interleukin-6-STAT3 and ERK1/2) were unaffected. Unlike what was observed after 7 days of HU, the iron concentration in the spleen, liver and skeletal muscle was comparable between control animals and those that underwent HU or HU plus IR for 14 days. Despite its beneficial effect on soleus muscle atrophy and slow-to-fast myosin heavy chain distribution, IR did not significantly prevent a reduction in iron availability and hepcidin upregulation. Altogether, these results highlight that iron availability is durably reduced during longer exposure to simulated microgravity and that the related hepcidin upregulation is not a transient adaptation to these conditions. The results also suggest that skeletal muscle does not necessarily play a key role in the iron misdistribution that occurs during simulated microgravity., (© 2020 The Authors. Experimental Physiology © 2020 The Physiological Society.)

Hydrogen sulfide (H 2 S) is the third gasotransmitter and is generated endogenously in hypoxic or inflammatory tissues and various cancers. We have recently demonstrated that endogenous H 2 S can be imaged with [ 99m Tc]Tc-gluconate. In the present study, we detected H 2 S generated in hypoxic tissue, both in vitro and in vivo, using [ 99m Tc]Tc-gluconate. In vitro uptake of [ 99m Tc]Tc-gluconate was measured under hypoxic and normoxic conditions, using the colon carcinoma cell line CT26, and was higher in hypoxic cells than that in normoxic cells. An acute hindlimb ischemia-reperfusion model was established in BALB/c mice by exposing the animals to 3 h of ischemia and 3 h of reperfusion prior to in vivo imaging. [ 99m Tc]Tc-gluconate (12.5 MBq) was intravenously injected through the tail vein, and uptake in the lower limb was analyzed by single-photon emission computed tomography/computed tomography (SPECT/CT). SPECT/CT images showed five times higher uptake in the ischemic limb than that in the normal limb. The standard uptake value (SUVmean) of the ischemic limb was 0.39 ± 0.03, while that of the normal limb was 0.07 ± 0.01. [ 99m Tc]Tc-gluconate is a novel imaging agent that can be used both in vitro and in vivo for the detection of endogenous H 2 S generated in hypoxic tissue.

The mechanism of severe pain occurring because of physical disuse, such as complex regional pain syndrome Type I, has not been elucidated so far. Therefore, to investigate this mechanism, we have developed a model called a chronic post-cast pain (CPCP) model. Oxidative stress-related factors generated in a fixed limb may be triggers for nociceptive signals due to physical disuse. On the basis of the results of our previous studies, we speculated that oxidative stress-related factors in immobilized hind limbs may also be triggers of nociceptive signals due to physical disuse. In this study, we aimed to clarify whether an oxidative stress-related factor is involved in the induction of nociceptive signals. The time course of oxidative damage in the soleus (slow-twitch fiber) and gastrocnemius (fast-twitch fiber) muscles was evaluated by immunostaining of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative damage in DNA). We also investigated the effects of tempol, a scavenger of superoxide, on oxidative damage in DNA, spontaneous pain-related behaviors (licking and/or biting and flinching), and the activation of spinal dorsal horn neurons (c-Fos). Systemic administration of tempol before cast removal attenuated oxidative damage to DNA in immobilized skeletal muscles, suppressed spontaneous pain-related behavior, and suppressed the activation of spinal dorsal horn neurons. We suggest that superoxide generated in immobilized skeletal muscles after cast removal is one of the peripheral factors that trigger nociceptive signals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction: A sufficient oxygen supply to ischemic limb tissue is the most important requirement for wound healing and limb salvage. We investigated whether partial venous occlusion in the common iliac vein (CIV) causes a further increase of venous oxygenation in a porcine model of acute hindlimb ischemia., Materials and Methods: In 7 pigs, the model of acute hindlimb ischemia was created with intra-vascular embolization of the common iliac artery (CIA). The arterial and venous oxygen saturation was evaluated at different moments. Oxygen saturation was evaluated at baseline (T0), just after the arterial embolization (T1), at 10 minutes (T2), at 20 minutes (T3), and at 40 minutes (T4). Next, an intentional partial venous occlusion was achieved by inflating the vascular balloon at the level of the right CIV. Then, blood sampling was repeated at 5 minutes (T5), at 15 minutes (T6), and at 25 minutes (T7)., Results: The arterial oxygen saturation in the right SFA was similar during all phases. In contrast, after arterial embolization, an immediate reduction of venous oxygen saturation was observed (from 85.57 ± 1.72 at T0 to 71.86 ± 7.58 at T4). After the partial venous occlusion, interestingly, the venous oxygen saturations (T5-T7) were significantly increased, again. The venous oxygen saturations evaluated in the hindlimb ischemia with partial venous occlusion and in the control limb (without partial venous occlusion) were significantly over time. Venous oxygen saturations in the experimental limbs were higher than those in the control limbs (79.28 ± 4.82 vs 59.00 ± 2.82, p-value <0.001, 79.71 ± 4.78 vs 60.00 ± 4.24 at T7, p-value <0.001)., Conclusions: Partial venous occlusion results in an increase of venous oxygen saturation in the ischemic limb, while significant changes in venous oxygen saturation are not observed in the control limb. An explanation for this may be that the oxygen consumption in the limb tissue is increased because it gets congested with the partial venous occlusion in the right CIV., Competing Interests: The authors have declared that no competing interests exist.