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Sheep are frequently used animal models of musculoskeletal diseases and orthopedic procedures due to their docility, size and body weight, and similar joint biomechanics to humans. Estimation of body segment inertial properties (BSIPs) is a crucial step in development of biomechanical models, but few resources exist for BSIPs in sheep. The goal of this study was to develop predictive models to estimate the mass, center of mass, and inertia tensor of the hindlimbs of sheep from easily obtainable morphometric data. In addition, this study presents a more comprehensive and repeatable method for defining each hindlimb body segment when directly calculating BSIPs from tomographic imaging. Briefly, CT scans from 16 sheep of varying age, weight, sex, and phenotype were used to calculate BSIPs for the pelvis, thigh, crus, metatarsus, and pastern segments. Those measurements were then used to develop predictive models to estimate the BSIPs for those segments. The predictive models developed showed similar prediction errors to models developed in human populations.

Background Post-surgical lymphedema frequently occurs following lymph node dissection. The murine tail is the most commonly used model to study secondary lymphedema. The murine hindlimb model offers a more clinically translatable approach but results in the literature have been inconsistent. The purpose of this study is to optimize the murine hindlimb lymphedema to achieve consistent results and assess the utility of radiation. Methods C57BL/6 mice underwent either 20-Gy irradiation of one hindlimb 7 days prior to surgery ( n  = 11) or no radiation ( n  = 9). For all mice, a circumferential skin incision was created at the proximal hindlimb. Lymphatics were identified and disrupted. Popliteal lymph nodes were excised. Paw thickness was measured and near-infrared laser lymphangiography was used to assess lymphatic function. Results The average paw thickness of the operated hindlimb in irradiated mice on postoperative day (POD) 14 was 3.5 ± 0.3 cm compared to 2.1 ± 0.05 cm on the contralateral limb ( p  = 0.0001). Lymphangiography on POD-42 showed significantly worse lymphatic function in the operated hindlimb compared to the control hindlimb ( p  = 0.003). For the non-radiated mice, the paw thickness was 2.5 ± 0.2 cm on POD-42 compared to the contralateral limb (2.1 ± 0.1 cm) ( p  = 0.0002) but smaller than radiated hindlimb group (3.2 ± 0.1 cm) ( p  = 0.0002). The nonradiated mice had significantly greater paw thickness than the control limb until POD-56 whereas the radiated mice sustained significant paw thickness to POD-90. Conclusion Radiation of the murine hindlimb model results in sustained lymphedema compared to non-irradiated mice. The murine hindlimb lymphedema model is clinically more translatable than the murine tail model with consistent results.

Ischemic injury represents the most frequent cause of death and disability, and it remains unclear why, of all body organs, the brain is most sensitive to hypoxia. In many tissues, type 4 NADPH oxidase is induced upon ischemia or hypoxia, converting oxygen to reactive oxygen species. Here, we show in mouse models of ischemia in the heart, brain, and hindlimb that only in the brain does NADPH oxidase 4 (NOX4) lead to ischemic damage. We explain this distinct cellular distribution pattern through cell-specific knockouts. Endothelial NOX4 breaks down the BBB, while neuronal NOX4 leads to neuronal autotoxicity. Vascular smooth muscle NOX4, the common denominator of ischemia within all ischemic organs, played no apparent role. The direct neuroprotective potential of pharmacological NOX4 inhibition was confirmed in an ex vivo model, free of vascular and BBB components. Our results demonstrate that the heightened sensitivity of the brain to ischemic damage is due to an organ-specific role of NOX4 in blood–brain-barrier endothelial cells and neurons. This mechanism is conserved in at least two rodents and humans, making NOX4 a prime target for a first-in-class mechanism-based, cytoprotective therapy in the unmet high medical need indication of ischemic stroke.

Mouse hind limb ischemia is the most common used preclinical model for peripheral arterial disease and critical limb ischemia. This model is used to investigate the mechanisms of neovascularization and to develop new therapeutic agents. The literature shows many variations in the model, including the method of occlusion, the number of occlusions, and the position at which the occlusions are made to induce hind limb ischemia. Furthermore, predefined end points and the histopathological and radiological analysis vary. These differences hamper the correlation of results between different studies. In this review, variations in surgical methods of inducing hind limb ischemia in mice are described, and the consequences of these variations on perfusion restoration and vascular remodeling are discussed. This study aims at providing the reader with a comprehensive overview of the methods so far described, and proposing uniformity in research of hind limb ischemia in a mouse model.

Generating universal human umbilical mesenchymal stem cells (UMSCs) without immune rejection is desirable for clinical application. Here we developed an innovative strategy using CRISPR/Cas9 to generate B2M‐UMSCs in which human leucocyte antigen (HLA) light chain β2‐microglobulin (B2M) was deleted. The therapeutic potential of B2M‐UMSCs was examined in a mouse ischaemic hindlimb model. We show that B2M‐UMSCs facilitated perfusion recovery and enhanced running capability, without inducing immune rejection. The beneficial effect was mediated by exosomes. Mechanistically, microRNA (miR) sequencing identified miR‐24 as a major component of the exosomes originating from B2M‐UMSCs. We identified Bim as a potential target of miR‐24 through bioinformatics analysis, which was further confirmed by loss‐of‐function and gain‐of‐function approaches. Taken together, our data revealed that knockout of B2M is a convenient and efficient strategy to prevent UMSCs‐induced immune rejection, and it provides a universal clinical‐scale cell source for tissue repair and regeneration without the need for HLA matching in the future.

We tested the hypothesis that there are sex differences in hindlimb unloading-induced activation of the forkhead box subfamily O3a (FoxO3a) signaling pathway in rat soleus muscle. Age-matched male and female Wistar rats were subjected to hindlimb unloading, and the soleus muscle was removed before or 1 or 7 days after unloading. Female rats showed greater percent changes in relative soleus muscle weight than males. FoxO3a phosphorylation was lower in females than in males and was associated with higher levels of protein ubiquitination 7 days after unloading. Heat shock protein 72 (Hsp72) levels were lower in female rats and increased in males during unloading. Female rats showed slightly higher myostatin levels, which showed a non-significant decline in male rats following unloading. Thus, males and females show different responses to the FoxO3a/ubiquitin–proteasome pathway following hindlimb unloading in rat soleus muscle, which may be associated with differences in Hsp72 expression and myostatin signaling.

BPC 157, a pentadecapeptide with extensive healing effects, has recently been suggested to contribute to angiogenesis. However, the underlying mechanism is not yet clear. The present study aimed to explore the potential therapeutic effect and pro-angiogenic mechanism of BPC 157. As demonstrated by the chick chorioallantoic membrane (CAM) assay and endothelial tube formation assay, BPC 157 could increase the vessel density both in vivo and in vitro, respectively. BPC 157 could also accelerate the recovery of blood flow in the ischemic muscle of the rat hind limb as detected by laser Doppler scanning, indicating the promotion of angiogenesis. Histological analysis of the hind limb muscle confirmed the increased number of vessels and the enhanced vascular expression of vascular endothelial growth factor receptor 2 (VEGFR2) in rat with BPC 157 treatment. In vitro study using human vascular endothelial cells further confirmed the increased mRNA and protein expressions of VEGFR2 but not VEGF-A by BPC 157. In addition, BPC 157 could promote VEGFR2 internalization in vascular endothelial cells which was blocked in the presence of dynasore, an inhibitor of endocytosis. BPC 157 time dependently activated the VEGFR2-Akt-eNOS signaling pathway which could also be suppressed by dynasore. The increase of endothelial tube formation induced by BPC 157 was also inhibited by dynasore. This study demonstrates the pro-angiogenic effects of BPC 157 that is associated with the increased expression, internalization of VEGFR2, and the activation of VEGFR2-Akt-eNOS signaling pathway. BPC 157 promotes angiogenesis in CAM assay and tube formation assay. BPC 157 accelerates the blood flow recovery and vessel number in rats with hind limb ischemia. BPC 157 up-regulates VEGFR2 expression in rats with hind limb ischemia and endothelial cell culture. BPC 157 promotes VEGFR2 internalization in association with VEGFR2-Akt-eNOS activation. Key message BPC 157 promotes angiogenesis in CAM assay and tube formation assay. BPC 157 accelerates the blood flow recovery and vessel number in rats with hind limb ischemia. BPC 157 up-regulates VEGFR2 expression in rats with hind limb ischemia and endothelial cell culture. BPC 157 promotes VEGFR2 internalization in association with VEGFR2-Akt-eNOS activation.

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.

Purpose To explore the microcirculation formation mechanism of contrast-enhanced (CE) ultrasonography imaging performance in rabbits with limb muscle crush injury. Methods Seventy-two New Zealand white rabbits were randomly divided into two groups. A limb muscle crush injury model was created by airing a balloon cuff device with a force of 40 kpa. CE ultrasonography parameters were detected in the first group. In vivo microcirculation parameters were detected in the second group. Fine blood vessel diameter and blood flow velocity were calculated before extrusion and 0.5, 2, 6, 24 h, and 3 days after decompression. Results Compared with the uninjured muscle, reperfusion of the injured muscles showed early and high enhancement in CE ultrasonography images. The time-intensity curve showed a trend of rapid elevation and gradual drop. Compared with the control group, fine artery and vein diameters in the experimental group were wider and the blood flow velocity was slower, especially in the fine veins. Conclusion In vivo microcirculation detection can reflect changes in muscle microvascular diameter and blood flow velocity, which have a correlation with quantitative ultrasound imaging parameters.

Controlled activation or release of biomolecules is very crucial in various biological applications. Controlling the activity of biomolecules have been attempted by various means and controlling the activity by light has gained popularity in the past decade. The major hurdle in this process is that photoactivable compounds mostly respond to UV radiation and not to visible or near-infrared (NIR) light. The use of UV irradiation is limited by its toxicity and very low tissue penetration power. In this study, we report the exploitation of the potential of NIR-to-UV upconversion nanoparticles (UCNs), which act as nanotransducers to absorb NIR light having high tissue penetration power and negligible phototoxicity and emit UV light locally, for photoactivation of caged compounds and, in particular, used for photo-controlled gene expression. Both activation and knockdown of GFP was performed in both solution and cells, and patterned activation of GFP was achieved successfully by using upconverted UV light produced by NIR-to-UV UCNs. In-depth photoactivation through tissue phantoms and in vivo activation of caged nucleic acids were also accomplished. The success of this methodology has defined a unique level in the field of photo-controlled activation and delivery of molecules.