Deletion of endothelial IGFBP5 protects against ischaemic hindlimb injury by promoting angiogenesis.

BACKGROUND: Angiogenesis is critical for forming new blood vessels from antedating vascular vessels. The endothelium is essential for angiogenesis, vascular remodelling and minimisation of functional deficits following ischaemia. The insulin-like growth factor (IGF) family is crucial for angiogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5), a binding protein of the IGF family, may have places in angiogenesis, but the mechanisms are not yet completely understood. We sought to probe whether IGFBP5 is involved in pathological angiogenesis and uncover the molecular mechanisms behind it. METHODS AND RESULTS: IGFBP5 expression was elevated in the vascular endothelium of gastrocnemius muscle from critical limb ischaemia patients and hindlimb ischaemic (HLI) mice and hypoxic human umbilical vein endothelial cells (HUVECs). In vivo, loss of endothelial IGFBP5 (IGFBP5EKO) facilitated the recovery of blood vessel function and limb necrosis in HLI mice. Moreover, skin damage healing and aortic ring sprouting were faster in IGFBP5EKO mice than in control mice. In vitro, the genetic inhibition of IGFBP5 in HUVECs significantly promoted tube formation, cell proliferation and migration by mediating the phosphorylation of IGF1R, Erk1/2 and Akt. Intriguingly, pharmacological treatment of HUVECs with recombinant human IGFBP5 ensued a contrasting effect on angiogenesis by inhibiting the IGF1 or IGF2 function. Genetic inhibition of IGFBP5 promoted cellular oxygen consumption and extracellular acidification rates via IGF1R-mediated glycolytic adenosine triphosphate (ATP) metabolism. Mechanistically, IGFBP5 exerted its role via E3 ubiquitin ligase Von Hippel-Lindau (VHL)-regulated HIF1α stability. Furthermore, the knockdown of the endothelial IGF1R partially abolished the reformative effect of IGFBP5EKO mice post-HLI. CONCLUSION: Our findings demonstrate that IGFBP5 ablation enhances angiogenesis by promoting ATP metabolism and stabilising HIF1α, implying IGFBP5 is a novel therapeutic target for treating abnormal angiogenesis-related conditions.

Anti-oxidative and anti-inflammatory effects of Ginkgo biloba extract (EGb761) on hindlimb skeletal muscle ischemia-reperfusion injury in rats.

In posterior spine surgery, retractors exert pressure on paraspinal muscles, elevating intramuscular pressure and compromising blood flow, potentially causing muscle injury during ischemia-reperfusion. Ginkgo biloba extract (EGb 761), known for its antioxidant and free radical scavenging properties and its role in treating cerebrovascular diseases, is investigated for its protective effects against muscle ischemia-reperfusion injury in vitro and in vivo. Animals were randomly divided into the control group, receiving normal saline, and experimental groups, receiving varying doses of EGb761 (25/50/100/200 mg/kg). A 2-h hind limb tourniquet-induced ischemia was followed by reperfusion. Blood samples collected pre-ischemia and 24 h post-reperfusion, along with muscle tissue samples after 24 h, demonstrated that EGb761 at 1000 µg/mL effectively inhibited IL-6 and TNF-α secretion in RAW 264.7 cells without cytotoxicity. EGb761 significantly reduced nitric oxide (NO) and malondialdehyde (MDA) levels, myeloperoxidase (MPO) activity, and increased glutathione (GSH) levels compared to the control after 24 h. Muscle tissue sections revealed more severe damage in the control group, indicating EGb761's potential in mitigating inflammatory responses and oxidative stress during ischemia-reperfusion injury, effectively protecting against muscle damage.

Effect of Goreisan, a Traditional Japanese Medicine, on Rat Hindlimb Lymphedema.

Secondary lymphedema occurs after cancer surgery involving lymph node dissection owing to the lymphatic system dysfunction. However, the pathophysiology of lymphedema and the molecular pathways involved remain unknown. This study aimed to develop a rat hindlimb lymphedema model and investigate the mechanisms that drive pathophysiology and the effects of the traditional Japanese medicine goreisan on lymphedema. The rat lymphedema model was induced by combination surgeries of popliteal lymph node dissection, skin cautery incision, and fascial ablation coagulation in the right hindlimb using male Wistar rats. The foot volume was significantly increased, and recovery was delayed by combination surgeries. Dermal thickness and dilated lymphatic vessels of the hindlimb were observed on postoperative day 2. The number of infiltrating leukocytes (CD45+ cells), including CD4+ T-cells, increased in the lymphedema group compared with that in the sham group. The relative mRNA expression and protein levels of interleukin-6 (IL-6), CC chemokine ligand 2 (CCL2), transforming growth factor ß1 (TGF-ß1), and Fms-related receptor tyrosine kinase 4 (FLT4) were significantly higher in the lymphedema group than in the sham group. Foot volume was decreased by goreisan, furosemide, and prednisolone treatments. Goreisan diminished the increase in CD4+ T-cells, and the same trend was observed for CCL2 and FLT4 expression. In conclusion, the rat hindlimb lymphedema model in this study exhibited increased foot volume, skin-infiltrating cells, and pathological changes accompanied by inflammatory and fibrotic responses, suggesting that the model presented significant clinical features of lymphedema. Goreisan may exert a therapeutic effect on lymphedema by inhibiting CD4+ T-cell infiltration.

Efficacy of Neonatal Porcine Bone Marrow-Derived Mesenchymal Stem Cell Xenotransplantation for the Therapy of Hind Limb Lymphedema in Mice.

Lymphedema is an intractable disease with few effective therapeutic options. Autologous mesenchymal stem cell (MSC) transplantation is a promising therapy for this disease. However, its use is limited by the cost and time for preparation. Recently, xenotransplantation of porcine MSCs has emerged as an alternative to autologous MSC transplantation. In this study, we aimed to clarify the usefulness of neonatal porcine bone marrow-derived MSC (NpBM-MSC) xenotransplantation for the treatment of lymphedema. One million NpBM-MSCs were xenotransplanted into the hind limbs of mice with severe lymphedema (MSC transplantation group). The therapeutic effects were assessed by measuring the femoral circumference, the volume of the hind limb, the number and diameter of lymphatic vessels in the hind limb, and lymphatic flow using a near-infrared fluorescence (NIRF) imaging system. We compared the effects using mice with lymphedema that did not undergo NpBM-MSC transplantation (negative control group). The condition of the transplanted NpBM-MSCs was also evaluated histologically. The femoral circumference and volume of the hind limb had been normalized by postoperative day (POD) 14 in the MSC transplantation group, but not in the negative control group (P = 0.041). NIRF imaging revealed that lymphatic flow had recovered in the MSC transplantation group by POD 14, as shown by an increase in luminance in the hind limb. Histological assessment also showed that the xenotransplantation of NpBM-MSC increased the proliferation of lymphatic vessels, but they had been rejected by POD 14. The xenotransplantation of NpBM-MSCs is an effective treatment for lymphedema, and this is mediated through the promotion of lymphangiogenesis.

VCA supercooling in a swine partial hindlimb model.

Vascularized composite allotransplantations are complex procedures with substantial functional impact on patients. Extended preservation of VCAs is of major importance in advancing this field. It would result in improved donor-recipient matching as well as the potential for ex vivo manipulation with gene and cell therapies. Moreover, it would make logistically feasible immune tolerance induction protocols through mixed chimerism. Supercooling techniques have shown promising results in multi-day liver preservation. It consists of reaching sub-zero temperatures while preventing ice formation within the graft by using various cryoprotective agents. By drastically decreasing the cell metabolism and need for oxygen and nutrients, supercooling allows extended preservation and recovery with lower ischemia-reperfusion injuries. This study is the first to demonstrate the supercooling of a large animal model of VCA. Porcine hindlimbs underwent 48 h of preservation at - 5 °C followed by recovery and normothermic machine perfusion assessment, with no issues in ice formation and favorable levels of injury markers. Our findings provide valuable preliminary results, suggesting a promising future for extended VCA preservation.

Transcriptome profiling of immune rejection mechanisms in a porcine vascularized composite allotransplantation model.

Background: Vascularized composite allotransplantation (VCA) offers the potential for a biological, functional reconstruction in individuals with limb loss or facial disfigurement. Yet, it faces substantial challenges due to heightened immune rejection rates compared to solid organ transplants. A deep understanding of the genetic and immunological drivers of VCA rejection is essential to improve VCA outcomes. Methods: Heterotopic porcine hindlimb VCA models were established and followed until reaching the endpoint. Skin and muscle samples were obtained from VCA transplant recipient pigs for histological assessments and RNA sequencing analysis. The rejection groups included recipients with moderate pathological rejection, treated locally with tacrolimus encapsulated in triglycerol-monostearate gel (TGMS-TAC), as well as recipients with severe end-stage rejection presenting evident necrosis. Healthy donor tissue served as controls. Bioinformatics analysis, immunofluorescence, and electron microscopy were utilized to examine gene expression patterns and the expression of immune response markers. Results: Our comprehensive analyses encompassed differentially expressed genes, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathways, spanning various composite tissues including skin and muscle, in comparison to the healthy control group. The analysis revealed a consistency and reproducibility in alignment with the pathological rejection grading. Genes and pathways associated with innate immunity, notably pattern recognition receptors (PRRs), damage-associated molecular patterns (DAMPs), and antigen processing and presentation pathways, exhibited upregulation in the VCA rejection groups compared to the healthy controls. Our investigation identified significant shifts in gene expression related to cytokines, chemokines, complement pathways, and diverse immune cell types, with CD8 T cells and macrophages notably enriched in the VCA rejection tissues. Mechanisms of cell death, such as apoptosis, necroptosis and ferroptosis were observed and coexisted in rejected tissues. Conclusion: Our study provides insights into the genetic profile of tissue rejection in the porcine VCA model. We comprehensively analyze the molecular landscape of immune rejection mechanisms, from innate immunity activation to critical stages such as antigen recognition, cytotoxic rejection, and cell death. This research advances our understanding of graft rejection mechanisms and offers potential for improving diagnostic and therapeutic strategies to enhance the long-term success of VCA.

Machine Perfusion Enables 24-h Preservation of Vascularized Composite Allografts in a Swine Model of Allotransplantation.

The current gold standard for preserving vascularized composite allografts (VCA) is 4°C static cold storage (SCS), albeit muscle vulnerability to ischemia can be described as early as after 2 h of SCS. Alternatively, machine perfusion (MP) is growing in the world of organ preservation. Herein, we investigated the outcomes of oxygenated acellular subnormothermic machine perfusion (SNMP) for 24-h VCA preservation before allotransplantation in a swine model. Six partial hindlimbs were procured on adult pigs and preserved ex vivo for 24 h with either SNMP (n = 3) or SCS (n = 3) before heterotopic allotransplantation. Recipient animals received immunosuppression and were followed up for 14 days. Clinical monitoring was carried out twice daily, and graft biopsies and blood samples were regularly collected. Two blinded pathologists assessed skin and muscle samples. Overall survival was higher in the SNMP group. Early euthanasia of 2 animals in the SCS group was linked to significant graft degeneration. Analyses of the grafts showed massive muscle degeneration in the SCS group and a normal aspect in the SNMP group 2 weeks after allotransplantation. Therefore, this 24-h SNMP protocol using a modified Steen solution generated better clinical and histological outcomes in allotransplantation when compared to time-matched SCS.

Detecting viscosity changes in the limb ischemia-reperfusion in mice with a near-infrared fluorescence probe.

BACKGROUND: Limb ischemia-reperfusion is a common phenomenon in clinical surgery, which disrupts the balanced physiological response process and ultimately leads to changes in intracellular viscosity. Intracellular viscosity is an important microenvironmental parameter that affects the normal function of organisms, and its level is closely related to many diseases. In addition, oxidative stress in the lower limbs can impair body function, and changes in pressure can lead to changes in the viscosity of limb tissues. Therefore, it is necessary to develop effective tools to detect changes in intracellular viscosity and visualize the progression of hind limb ischemia-reperfusion injury. RESULTS: In order to solve this problem, a near infrared viscometry sensitive fluorescence probe (PH-XQ) with long emission wavelength and stable luminescence performance was designed and synthesized by using oxanthracene derivatives and malononitrile. The fluorescence probe (PH-XQ) has excellent selectivity, high sensitivity, low toxicity, high biocompatibility and excellent detection performance. The fluorescence intensity of the PH-XQ probe at 667 nm is highly sensitive to the change of viscosity. With the increase of viscosity, the fluorescence intensity of probe PH-XQ was significantly enhanced, and the fluorescence enhancement ratio was about 14-fold. In addition, PH-XQ can detect not only changes in viscosity between normal cells and drug-induced inflammatory cells, but also changes in the viscosity of the hind limbs of normal mice and mice after ischemia reperfusion. SIGNIFICANCE: In particular, we are the first to successfully detect changes in handlimb viscosity after ischemia-reperfusion in mice using a probe. This study clearly elucidates changes in viscosity during ischemia-reperfusion of mouse limbs, providing favorable support for the relationship between viscosity and related diseases, and further providing a potential tool for the diagnosis of viscosity-related diseases.

Numerical evaluation of internal femur osteosynthesis based on a biomechanical model of the loading in the proximal equine hindlimb.

Femoral fractures are often considered lethal for adult horses because femur osteosynthesis is still a surgical challenge. For equine femur osteosynthesis, primary stability is essential, but the detailed physiological forces occurring in the hindlimb are largely unknown. The objective of this study was to create a numerical testing environment to evaluate equine femur osteosynthesis based on physiological conditions. The study was designed as a finite element analysis (FEA) of the femur using a musculoskeletal model of the loading situation in stance. Relevant forces were determined in the musculoskeletal model via optimization. The treatment of four different fracture types with an intramedullary nail was investigated in FEA with loading conditions derived from the model. The analyzed diaphyseal fracture types were a transverse (TR) fracture, two oblique fractures in different orientations (OB-ML: medial-lateral and OB-AP: anterior-posterior) and a "gap" fracture (GAP) without contact between the fragments. For the native femur, the most relevant areas of increased stress were located distally to the femoral head and proximally to the caudal side of the condyles. For all fracture types, the highest stresses in the implant material were present in the fracture-adjacent screws. Maximum compressive (-348 MPa) and tensile stress (197 MPa) were found for the GAP fracture, but material strength was not exceeded. The mathematical model was able to predict a load distribution in the femur of the standing horse and was used to assess the performance of internal fixation devices via FEA. The analyzed intramedullary nail and screws showed sufficient stability for all fracture types.

3D-Printed proangiogenic patches of photo-crosslinked gelatin and polyurethane hydrogels laden with vascular cells for treating vascular ischemic diseases.

Engineering vascularized tissues remains a promising approach for treating ischemic cardiovascular diseases. The availability of 3D-bioprinted vascular grafts that induce therapeutic angiogenesis can help avoid necrosis and excision of ischemic tissues. Here, using a combination of living cells and biodegradable hydrogels, we fabricated 3D-printed biocompatible proangiogenic patches from endothelial cell-laden photo-crosslinked gelatin (EC-PCG) bioink and smooth muscle cell-encapsulated polyurethane (SMC-PU) bioink. Implantation of 3D-bioprinted proangiogenic patches in a mouse model showed that EC-PCG served as an angiogenic capillary bed, whereas patterned SMC-PU increased the density of microvessels. Moreover, the assembled patterns between EC-PCG and SMC-PU induced the geometrically guided generation of microvessels with blood perfusion. In a rodent model of hindlimb ischemia, the vascular patches rescued blood flow to distal tissues, prevented toe/foot necrosis, promoted muscle remodeling, and increased the capillary density, thereby improving the heat-escape behavior of ischemic animals. Thus, our 3D-printed vascular cell-laden bioinks constitute efficient and scalable biomaterials that facilitate the engineering of vascular patches capable of directing therapeutic angiogenesis for treating ischemic vascular diseases.

Forelimb movements contribute to hindlimb cutaneous reflexes during locomotion in cats.

During quadrupedal locomotion, interactions between spinal and supraspinal circuits and somatosensory feedback coordinate forelimb and hindlimb movements. How this is achieved is not clear. To determine whether forelimb movements modulate hindlimb cutaneous reflexes involved in responding to an external perturbation, we stimulated the superficial peroneal nerve in six intact cats during quadrupedal locomotion and during hindlimb-only locomotion (with forelimbs standing on stationary platform) and in two cats with a low spinal transection (T12-T13) during hindlimb-only locomotion. We compared cutaneous reflexes evoked in six ipsilateral and four contralateral hindlimb muscles. Results showed similar occurrence and phase-dependent modulation of short-latency inhibitory and excitatory responses during quadrupedal and hindlimb-only locomotion in intact cats. However, the depth of modulation was reduced in the ipsilateral semitendinosus during hindlimb-only locomotion. Additionally, longer-latency responses occurred less frequently in extensor muscles bilaterally during hindlimb-only locomotion, whereas short-latency inhibitory and longer-latency excitatory responses occurred more frequently in the ipsilateral and contralateral sartorius anterior, respectively. After spinal transection, short-latency inhibitory and excitatory responses were similar to both intact conditions, whereas mid- or longer-latency excitatory responses were reduced or abolished. Our results in intact cats and the comparison with spinal-transected cats suggest that the absence of forelimb movements suppresses inputs from supraspinal structures and/or cervical cord that normally contribute to longer-latency reflex responses in hindlimb extensor muscles.NEW & NOTEWORTHY During quadrupedal locomotion, the coordination of forelimb and hindlimb movements involves central circuits and somatosensory feedback. To demonstrate how forelimb movement affects hindlimb cutaneous reflexes during locomotion, we stimulated the superficial peroneal nerve in intact cats during quadrupedal and hindlimb-only locomotion as well as in spinal-transected cats during hindlimb-only locomotion. We show that forelimb movement influences the modulation of hindlimb cutaneous reflexes, particularly the occurrence of long-latency reflex responses.

Lymphaticovenous anastomosis in rabbits: A novel live experimental animal model for supermicrosurgical training.

BACKGROUND: Lymphaticovenous anastomosis is widely used in lymphedema management. Although its effectiveness in reducing edema in patients can be clinically observed, evaluating the long-term outcomes of this technique can be complex. This study established an animal model to assess the outcomes of lymphaticovenous anastomosis technique at 15 and 30-days post-surgery using indocyanine green lymphography, Patent Blue V dye injection, and histopathological examination. METHODS: An experimental model was established in the hindlimbs of 10 rabbits using the popliteal vein and afferent lymphatic vessels in the popliteal area. The subjects were divided into two groups: the first group (n = 5) underwent patency assessment at 0 and 15 days, and the second group (n = 5) at 0 and 30-days, resulting in 20 anastomoses. Patency was verified at 0, 15, and 30-days using indocyanine green lymphography and Patent Blue V injection. Histopathological examinations were performed on the collected anastomosis samples. RESULTS: The patency rate was 90% (19/20) initially, 60% (6/10) at 15 days post-surgery, and 80% (8/10) at 30-days. The average diameter of lymphatic vessels and veins was 1.0 mm and 0.8 mm, respectively. The median number of collateral veins was 3; the median surgical time was 65.8 min. Histopathology revealed minimal endothelial damage and inflammatory responses due to the surgical sutures, with vascular inflammation and thrombosis in a single case. Local vascular neoformations were observed. CONCLUSION: This study highlights the reliability and reproducibility of using rabbits as experimental models for training in lymphaticovenous anastomosis technique owing to the accessibility of the surgical site and dimensions of their popliteal vasculature.

Nitric oxide releasing nanofiber stimulates revascularization in response to ischemia via cGMP-dependent protein kinase.

Nitric oxide (NO) promotes angiogenesis via various mechanisms; however, the effective transmission of NO in ischemic diseases is unclear. Herein, we tested whether NO-releasing nanofibers modulate therapeutic angiogenesis in an animal hindlimb ischemia model. Male wild-type C57BL/6 mice with surgically-induced hindlimb ischemia were treated with NO-releasing 3-methylaminopropyltrimethoxysilane (MAP3)-derived or control (i.e., non-NO-releasing) nanofibers, by applying them to the wound for 20 min, three times every two days. The amount of NO from the nanofiber into tissues was assessed by NO fluorometric assay. The activity of cGMP-dependent protein kinase (PKG) was determined by western blot analysis. Perfusion ratios were measured 2, 4, and 14 days after inducing ischemia using laser doppler imaging. On day 4, Immunohistochemistry (IHC) with F4/80 and gelatin zymography were performed. IHC with CD31 was performed on day 14. To determine the angiogenic potential of NO-releasing nanofibers, aorta-ring explants were treated with MAP3 or control fiber for 20 min, and the sprout lengths were examined after 6 days. As per either LDPI (Laser doppler perfusion image) ratio or CD31 capillary density measurement, angiogenesis in the ischemic hindlimb was improved in the MAP3 nanofiber group; further, the total nitrate/nitrite concentration in the adduct muscle increased. The number of macrophage infiltrations and matrix metalloproteinase-9 (MMP-9) activity decreased. Vasodilator-stimulated phosphoprotein (VASP), one of the major substrates for PKG, increased phosphorylation in the MAP3 group. MAP3 nanofiber or NO donor SNAP (s-nitroso-n-acetyl penicillamine)-treated aortic explants showed enhanced sprouting in an ex vivo aortic ring assay, which was partially abrogated by KT5823, a potent inhibitor of PKG. These findings suggest that the novel NO-releasing nanofiber, MAP3 activates PKG and promotes therapeutic angiogenesis in response to hindlimb ischemia.

Depletion of donor dendritic cells ameliorates immunogenicity of both skin and hind limb transplants.

Acute cellular rejection remains a significant obstacle affecting successful outcomes of organ transplantation including vascularized composite tissue allografts (VCA). Donor antigen presenting cells (APCs), particularly dendritic cells (DCs), orchestrate early alloimmune responses by activating recipient effector T cells. Employing a targeted approach, we investigated the impact of donor-derived conventional DCs (cDCs) and APCs on the immunogenicity of skin and skin-containing VCA grafts, using mouse models of skin and hind limb transplantation. By post-transplantation day 6, skin grafts demonstrated severe rejections, characterized by predominance of recipient CD4 T cells. In contrast, hind limb grafts showed moderate rejection, primarily infiltrated by CD8 T cells. Notably, the skin component exhibited heightened immunogenicity when compared to the entire VCA, evidenced by increased frequencies of pan (CD11b-CD11c+), mature (CD11b-CD11c+MHCII+) and active (CD11b-CD11c+CD40+) DCs and cDC2 subset (CD11b+CD11c+ MHCII+) in the lymphoid tissues and the blood of skin transplant recipients. While donor depletion of cDC and APC reduced frequencies, maturation and activation of DCs in all analyzed tissues of skin transplant recipients, reduction in DC activities was only observed in the spleen of hind limb recipients. Donor cDC and APC depletion did not impact all lymphocyte compartments but significantly affected CD8 T cells and activated CD4 T in lymph nodes of skin recipients. Moreover, both donor APC and cDC depletion attenuated the Th17 immune response, evident by significantly reduced Th17 (CD4+IL-17+) cells in the spleen of skin recipients and reduced levels of IL-17E and lymphotoxin-α in the serum samples of both skin and hind limb recipients. In conclusion, our findings underscore the highly immunogenic nature of skin component in VCA. The depletion of donor APCs and cDCs mitigates the immunogenicity of skin grafts while exerting minimal impact on VCA.

Gold Nanorod-Loaded Nano-Contrast Agent with Composite Shell-Core Structure for Ultrasonic/Photothermal Imaging-Guided Therapy in Ischemic Muscle Disorders.

Purpose: This study aims to broaden the application of nano-contrast agents (NCAs) within the realm of the musculoskeletal system. It aims to introduce novel methods, strategies, and insights for the clinical management of ischemic muscle disorders, encompassing diagnosis, monitoring, evaluation, and therapeutic intervention. Methods: We developed a composite encapsulation technique employing O-carboxymethyl chitosan (OCMC) and liposome to encapsulate NCA-containing gold nanorods (GNRs) and perfluoropentane (PFP). This nanoscale contrast agent was thoroughly characterized for its basic physicochemical properties and performance. Its capabilities for in vivo and in vitro ultrasound imaging and photothermal imaging were authenticated, alongside a comprehensive biocompatibility assessment to ascertain its effects on microcirculatory perfusion in skeletal muscle using a murine model of hindlimb ischemia, and its potential to augment blood flow and facilitate recovery. Results: The engineered GNR@OCMC-liposome/PFP nanostructure exhibited an average size of 203.18±1.49 nm, characterized by size uniformity, regular morphology, and a good biocompatibility profile. In vitro assessments revealed NCA's potent photothermal response and its transformation into microbubbles (MBs) under near-infrared (NIR) irradiation, thereby enhancing ultrasonographic visibility. Animal studies demonstrated the nanostructure's efficacy in photothermal imaging at ischemic loci in mouse hindlimbs, where NIR irradiation induced rapid temperature increases and significantly increased blood circulation. Conclusion: The dual-modal ultrasound/photothermal NCA, encapsulating GNR and PFP within a composite shell-core architecture, was synthesized successfully. It demonstrated exceptional stability, biocompatibility, and phase transition efficiency. Importantly, it facilitates the encapsulation of PFP, enabling both enhanced ultrasound imaging and photothermal imaging following NIR light exposure. This advancement provides a critical step towards the integrated diagnosis and treatment of ischemic muscle diseases, signifying a pivotal development in nanomedicine for musculoskeletal therapeutics.

Locomotion and morphological adaptations in the glass lizard Ophiodes cf. fragilis (Raddi, 1820) (Squamata: Anguidae).

There are few studies related to the biological and ecological aspects of the glass snake, a limbless lizard and with a wide geographic distribution. The aim of this study was to analyze the locomotion mode of specimens of Ophiodes cf. fragilis in different substrates and to investigate the morphological adaptations associated with this type of behavior. We observed that the analyzed specimens presented slide-push locomotion modes and lateral undulation in different substrates, using their hind limbs to aid locomotion in three of the four substrates analyzed. The bones of the hind limbs (proximal - femur - and distal - tibia and fibula) were present and highly reduced and the femur is connected to a thin pelvic girdle. Our data support that hind limbs observed in species of this genus are reduced rather than vestigial. The costocutaneous musculature was macroscopically absent. This is the first study of locomotor behavior and morphology associated with locomotion in Ophiodes, providing important information for studies on morphological evolution in the genus.

Dapagliflozin Improves Angiogenesis after Hindlimb Ischemia through the PI3K-Akt-eNOS Pathway.

Recently, the vascular protective effect of anti-diabetic agents has been receiving much attention. Sodium glucose cotransporter 2 (SGLT2) inhibitors had demonstrated reductions in cardiovascular (CV) events. However, the therapeutic effect of dapagliflozin on angiogenesis in peripheral arterial disease was unclear. This study aimed to explore the effect and mechanism of dapagliflozin on angiogenesis after hindlimb ischemia. We first evaluated the effect of dapagliflozin on post-ischemic angiogenesis in the hindlimbs of rats. Laser doppler imaging was used to detect the hindlimb blood perfusion. In addition, we used immunohistochemistry to detect the density of new capillaries after ischemia. The relevant signaling pathways of dapagliflozin affecting post-ischemic angiogenesis were screened through phosphoproteomic detection, and then the mechanism of dapagliflozin affecting post-ischemic angiogenesis was verified at the level of human umbilical vein endothelial cells (HUVECs). After subjection to excision of the left femoral artery, all rats were randomly distributed into two groups: the dapagliflozin group (left femoral artery resection, receiving intragastric feeding with dapagliflozin (1 mg/kg/d), for 21 consecutive days) and the model group, that is, the positive control group (left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days). In addition, the control group, that is the negative control group (without left femoral artery resection, receiving intragastric feeding with citric acid-sodium citrate buffer solution (1 mg/kg/d), for 21 consecutive days) was added. At day 21 post-surgery, the dapagliflozin-treatment group had the greatest blood perfusion, accompanied by elevated capillary density. The results showed that dapagliflozin could promote angiogenesis after hindlimb ischemia. Then, the ischemic hindlimb adductor-muscle tissue samples from three rats of model group and dapagliflozin group were taken for phosphoproteomic testing. The results showed that the PI3K-Akt-eNOS signaling pathway was closely related to the effect of dapagliflozin on post-ischemic angiogenesis. Our study intended to verify this mechanism from the perspective of endothelial cells. In vitro, dapagliflozin enhanced the tube formation, migration, and proliferation of HUVECs under ischemic and hypoxic conditions. Additionally, the dapagliflozin administration upregulated the expression of angiogenic factors phosphorylated Akt (p-Akt) and phosphorylated endothelial nitric oxide synthase (p-eNOS), as well as vascular endothelial growth factor A (VEGFA), both in vivo and in vitro. These benefits could be blocked by either phosphoinositide 3-kinase (PI3K) or eNOS inhibitor. dapagliflozin could promote angiogenesis after ischemia. This effect might be achieved by promoting the activation of the PI3K-Akt-eNOS signaling pathway. This study provided a new perspective, new ideas, and a theoretical basis for the treatment of peripheral arterial disease.

miR-709 exerts an angiogenic effect through a FGF2 upregulation induced by a GSK3B downregulation.

The aim of this study was to identify angiogenic microRNAs (miRNAs) that could be used in the treatment of hindlimb ischemic tissues. miRNAs contained in extracellular vesicles (EVs) deriving from the plasma were analyzed in C57BL/6 mice, which have ischemia tolerance, and in BALB/c mice without ischemia tolerance as part of a hindlimb ischemia model; as a result 43 angiogenic miRNA candidates were identified. An aortic ring assay was employed by using femoral arteries isolated from BALC/c mice and EVs containing miRNA; as a result, the angiogenic miRNA candidates were limited to 14. The blood flow recovery was assessed after injecting EVs containing miRNA into BALB/c mice with hindlimb ischemia, and miR-709 was identified as a promising angiogenic miRNA. miR-709-encapsulating EVs were found to increase the expression levels of the fibroblast growth factor 2 (FGF2) mRNA in the thigh tissues of hindlimb ischemia model BALB/c mice. miR-709 was also found to bind to the 3'UTR of glycogen synthase kinase 3 beta (GSK3B) in three places. GSK3B-knockdown human artery-derived endothelial cells were found to express high levels of FGF2, and were characterized by increased cell proliferation. These findings indicate that miR-709 induces an upregulation of FGF2 through the downregulation of GSK3B.

A surgical technique for individual control of the muscles of the rabbit lower hindlimb.

Little is known regarding the precise muscle, bone and joint actions resulting from individual and simultaneous muscle activation(s) of the lower limb. An in situ experimental approach is described herein to control the muscles of the rabbit lower hindlimb, including the medial and lateral gastrocnemius, soleus, plantaris and tibialis anterior. The muscles were stimulated using nerve-cuff electrodes placed around the innervating nerves of each muscle. Animals were fixed in a stereotactic frame with the ankle angle set at 90 deg. To demonstrate the efficacy of the experimental technique, isometric plantarflexion torque was measured at the 90 deg ankle joint angle at a stimulation frequency of 100, 60 and 30 Hz. Individual muscle torque and the torque produced during simultaneous activation of all plantarflexor muscles are presented for four animals. These results demonstrate that the experimental approach was reliable, with insignificant variation in torque between repeated contractions. The experimental approach described herein provides the potential for measuring a diverse array of muscle properties, which is important to improve our understanding of musculoskeletal biomechanics.

Different impact of short-term and long-term hindlimb disuse on bone homeostasis.

Disuse osteoporosis is one of the major problems of bone health which commonly occurs in astronauts during long-term spaceflight and bedridden patients. However, the mechanisms underlying such mechanical unloading induced bone loss have not been fully understood. In this study, we employed hindlimb-unloading mice models with different length of tail suspension to investigate if the bone loss was regulated by distinct factors under different duration of disuse. Our micro-CT results showed more significant decrease of bone mass in 6W (6-week) tail-suspension mice compared to the 1W (1-week) tail-suspension ones, as indicated by greater reduction of BV/TV, Tb.N, B.Ar/T.Ar and Ct.Th. RNA-sequencing results showed significant effects of hindlimb disuse on cell locomotion and immune system process which could cause bone loss.Real-time quantitative PCR results indicated a greater number of bone formation related genes that were downregulated in short-term tail-suspension mice compared to the long-term ones. It is, thus, suggested while sustained hindlimb unloading continuously contributes to bone loss, molecular regulation of bone homeostasis tends to reach a balance during this process.