Hindlimb Research Articles

Enhancing cellular regeneration through targeted pharmacological reprogramming of senescent endothelial cells

Abstract Funding Acknowledgements None. Background Senescent endothelial cells (EC) are key players in the pathophysiology of cardiovascular diseases and characterized by a reduced angiogenic and regenerative potential. Therefore, reversing EC senescence represents a promising therapeutic strategy to restore vascular integrity and increase health and lifespan. Purpose Here, we show a reversal of EC senescence following the application of a pharmacological, non-genetic, partial reprogramming strategy to induce a timely restricted induction of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc (OSKM). Methods Methods to characterize the effects of pharmacological reprogramming included the quantification of gene expression as well as functional analysis of EC in vitro. In addition, the regenerative capacity of EC was evaluated in a hind-limb ischemia model in vivo in young and old C57BL/6 mice. Results The application of a pharmacological reprogramming cocktail led to a strong but timely restricted activation of the Yamanaka-factors Oct3/4, Sox2, Klf4 and c-Myc in replicative senescent ECs. This activation occurred on both mRNA and protein levels (p<0.0001), concurrently significantly reducing markers of cellular aging as p16ink4a, p14arf, TNFα, IL-1b, IL-6 and CD44 (p<0.01 respectively p<0.0001), while enhancing their cellular function. Pharmacological reprogramming notably augmented the migratory (p<0.001) and proliferative capacities (p<0.01) of the replicate senescence EC and positively influenced their angiogenic functions such as sprouting (p<0.0001) and tube formation (p<0.01). Telomere length was stabilized (p<0.05) and production of reactive oxygen species decreased (p<0.05). Non-senescent EC were not influenced by the pharmacological treatment (p>0.05). Over prolonged periods, the treated ECs maintained consistently low expression of aging markers (p16ink4a and p14arf, p<0.01) while preserving their migratory capabilities (p<0.05). In vivo, a significantly improved blood flow was observed after hind limb ischemia in 22 months old C57BL/6 mice after 7 and 14 days (p<0.001), alongside a significant increase in capillary density in the gastrocnemius muscle quantified by CD31 staining (p<0.05). In 3 months old C57BL/6 mice blood flow was not influenced by the treatment (p>0.05). Conclusion In conclusion, we demonstrate that a short induction of OSKM via a pharmacological approach holds the potential to reverse cellular senescence in EC in vitro with improved regenerative and angiogenic capacities and thus to enhance endothelial regenerative capacity in vivo. This might present a promising strategy for ischemic diseases.

Design and Optimisation of Bionic Frog Hind Limbs

With the deepening of human exploration of the Earth, more and more survey tasks are being carried out in unstructured environments. Biomimetic robots that can replace humans in completing related tasks under harsh conditions have gradually become a hot topic in robot development. Therefore, developing a biomimetic robot with a wide range of activities, strong mobility, excellent obstacle crossing ability, and rapid avoidance response has important theoretical research significance and broad application prospects. This paper focuses on frogs as the research object,research is conducted from the perspective of institutional design, aiming to design a frog like jumping robot with strong obstacle crossing ability and good environmental adaptability. In terms of overall structural design, a M-shaped structure based on linear bearings was proposed to ensure the stability of the robot during takeoff and landing; In terms of leg structure design, C-shaped flexible springs are used to integrate the calf, ankle joint, and foot into one, which can play a buffering role when the robot lands; In terms of transmission structure design, Propose to control the compression cycle of the jumping leg through a cam to achieve continuous jumping of the robot. The relevant research results indicate that the structural principle of the designed frog like jumping robot is correct, and the jumping effect is relatively good.For the sake of ideals, this paper provides a certain theoretical basis and basis for further research on bionic jumping robots.

An In Vivo and In Silico Approach Reveals Possible Sodium Channel Nav1.2 Inhibitors from Ficus religiosa as a Novel Treatment for Epilepsy.

Epilepsy is a neurological disease that affects approximately 50 million people worldwide. Despite an existing abundance of antiepileptic drugs, lifelong disease treatment is often required but could be improved with alternative drugs that have fewer side effects. Given that epileptic seizures stem from abnormal neuronal discharges predominately modulated by the human sodium channel Nav1.2, the quest for novel and potent Nav1.2 blockers holds promise for epilepsy management. Herein, an in vivo approach was used to detect new antiepileptic compounds using the maximum electroshock test on mice. Pre-treatment of mice with extracts from the Ficus religiosa plant ameliorated the tonic hind limb extensor phase of induced convulsions. Subsequently, an in silico approach identified potential Nav1.2 blocking compounds from F. religiosa using a combination of computational techniques, including molecular docking, prime molecular mechanics/generalized Born surface area (MM/GBSA) analysis, and molecular dynamics (MD) simulation studies. The molecular docking and MM/GBSA analysis indicated that out of 82 compounds known to be present in F. religiosa, seven exhibited relatively strong binding affinities to Nav1.2 that ranged from -6.555 to -13.476 kcal/mol; similar or with higher affinity than phenytoin (-6.660 kcal/mol), a known Na+-channel blocking antiepileptic drug. Furthermore, MD simulations revealed that two compounds: 6-C-glucosyl-8-C-arabinosyl apigenin and pelargonidin-3-rhamnoside could form stable complexes with Nav1.2 at 300 K, indicating their potential as lead antiepileptic agents. In summary, the combination of in vivo and in silico approaches supports the potential of F. religiosa phytochemicals as natural antiepileptic therapeutic agents.

Region-specific activation in the accumbens nucleus by itch with modified scratch efficacy in mice – a model-free multivariate analysis

Itch is a protective/defensive function with divalent motivational drives. Itch itself elicits an unpleasant experience, which triggers the urge to scratch, relieving the itchiness. Still, it can also result in dissatisfaction when the scratch is too intense and painful or unsatisfactory due to insufficient scratch effect. Therefore, it is likely that the balance between the unpleasantness/pleasure and satisfaction/unsatisfaction associated with itch sensation and scratching behavior is determined by complex brain mechanisms. The physiological/pathological mechanisms underlying this balance remain largely elusive. To address this issue, we targeted the "reward center" of the brain, the nucleus accumbens (NAc), in which itch-responsive neurons have been found in rodents. We examined how neurons in the NAc are activated or suppressed during histamine-induced scratching behaviors in mice. The mice received an intradermal injection of histamine or saline at the neck, and the scratching number was analyzed by recording the movement of the bilateral hind limbs for about 45 min after injection. To experimentally manipulate the scratch efficacy in these histamine models, we compared histamine's behavioral and neuronal effects between mice with intact and clipped nails on the hind paws. As expected, the clipping of the hind limb nail increased the number of scratches after the histamine injection. In the brains of mice exhibiting scratching behaviors, we analyzed the expression of the c-fos gene (Fos) as a readout of an immediate activation of neurons during itch/scratch and dopamine receptors (Drd1 and Drd2) using multiplex single-molecule fluorescence in situ hybridization (RNAscope) in the NAc and surrounding structures. We performed a model-free analysis of gene expression in geometrically divided NAc subregions without assuming the conventional core–shell divisions. The results indicated that even within the NAc, multiple subregions responded differentially to various itch/scratch conditions. We also found different clusters with neurons showing similar or opposite changes in Fos expression and the correlation between scratch number and Fos expression in different itch/scratch conditions. These regional differences and clusters would provide a basis for the complex role of the NAc and surrounding structures in encoding the outcomes of scratching behavior and itchy sensations.

#2026 Hypokalemia associated to neuroendocrine carcinoma of the cervix

Abstract Background and Aims Hypokalemia is one of the most common electrolyte disorders seen in clinical practice. Hypokalemia can be caused by decreased intake of potassium, redistribution of potassium (K+) in cells or by excessive gastrointestinal losses or renal losses. In this setting, hypokalemia may also be caused by the paraneoplastic production of ectopic adrenocorticotropic hormone (ACTH) by some tumors. Cancer of the uterine cervix is the fourth common cancer affecting women worldwide and the fourth most common gynecologic cancer in Spain. Neuroendocrine carcinoma of the cervix represents about 2% of all cervical neoplasms. Method We report the case of a 44-year-old Peruvian woman with hypokalemia who was diagnosed with Cushing syndrome secondary to paraneoplastic production of ACTH by neuroendocrine carcinoma of cervix. Results Patient was overweight and affected by uterine myoma and anemia without further investigations. She was admitted to our hospital due to lower back pain, leg swelling and constipation. Blood pressure was normal, chest radiography did not show abnormal findings and ECG showed the U wave. Laboratory tests at admission revealed hypokalemia (2.3 mmol/L), metabolic alkalosis, anemia (hemoglobin 9.5 g/dL), hyperkaliuria (K+ urine/Creatinine urine 70 mmol/g) with an elevated transtubular potassium gradient (TTKG=12) and normal renal function. Laboratory data are presented in Table 1. She developed hypertension in the first day of hospitalization. Renin, aldosterone and cortisol tests was performed. Hypercortisolism was observed and confirmed with 24-hour urinary cortisol excretion. Because of an elevated ACTH (263 pg/mL), pituitary magnetic resonance imaging was performed without lesions. Whole-body computed tomography revealed a cervical mass. PET scan showed pelvic, adrenal, hepatic and local lymphadenopathy activity (see Fig. 1). After the histopathological study, diagnostic of small cell neuroendocrine carcinoma of the cervix was made. Chemotherapy therapy was initiated with cisplatin/etoposide regimen. The patient decide to return to Peru because of the poor prognosis of her illness. At discharge, potassium level was 3 mmol/L despite the treatment with spironolactone, ramipril, losartan and oral potassium chloride (160 mmol). Conclusion Hypokalemia is a common clinical disorder. In some patients it may be caused by an increased mineralocorticoid activity due to ectopic ACTH-secreting tumors. Applying diagnostic algorithms to electrolyte disorders, beyond their simple treatment, can unmask secondary causes and serious diseases.

A new abelisaurid dinosaur from the end Cretaceous of Patagonia and evolutionary rates among the Ceratosauria.

Gondwanan dinosaur faunae during the 20 Myr preceding the Cretaceous-Palaeogene (K/Pg) extinction included several lineages that were absent or poorly represented in Laurasian landmasses. Among these, the South American fossil record contains diverse abelisaurids, arguably the most successful groups of carnivorous dinosaurs from Gondwana in the Cretaceous, reaching their highest diversity towards the end of this period. Here we describe Koleken inakayali gen. et sp. n., a new abelisaurid from the La Colonia Formation (Maastrichtian, Upper Cretaceous) of Patagonia. Koleken inakayali is known from several skull bones, an almost complete dorsal series, complete sacrum, several caudal vertebrae, pelvic girdle and almost complete hind limbs. The new abelisaurid shows a unique set of features in the skull and several anatomical differences from Carnotaurus sastrei (the only other abelisaurid known from the La Colonia Formation). Koleken inakayali is retrieved as a brachyrostran abelisaurid, clustered with other South American abelisaurids from the latest Cretaceous (Campanian-Maastrichtian), such as Aucasaurus, Niebla and Carnotaurus. Leveraging our phylogeny estimates, we explore rates of morphological evolution across ceratosaurian lineages, finding them to be particularly high for elaphrosaurine noasaurids and around the base of Abelisauridae, before the Early Cretaceous radiation of the latter clade. The Noasauridae and their sister clade show contrasting patterns of morphological evolution, with noasaurids undergoing an early phase of accelerated evolution of the axial and hind limb skeleton in the Jurassic, and the abelisaurids exhibiting sustained high rates of cranial evolution during the Early Cretaceous. These results provide much needed context for the evolutionary dynamics of ceratosaurian theropods, contributing to broader understanding of macroevolutionary patterns across dinosaurs.

Minimally Invasive versus Open Transforaminal Lumbar Interbody Fusion in Obese Patients: A Propensity Score-Matched Study.

Retrospective review of prospectively collected data. This study aimed to compare the clinical outcomes of obese patients undergoing minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) and open TLIF. The perioperative benefits of minimally invasive surgery in obese patients have been described. However, there is limited literature on the patient-reported outcomes (PROs), satisfaction and return to work following MIS-TLIF and open TLIF in this subgroup of patients. Obese patients (BMI ≥30.0kg/m2) who underwent a primary, one- to two-level open and MIS-TLIF were stratified and matched using propensity scoring. Operative time, length of stay and perioperative outcomes were recorded. Patient-reported outcomes (PROs) including Oswestry Disability Index, Short Form-36 Physical Component Score, Mental Component Score, Visual Analogue Scale for back pain and leg pain were compared at each postoperative time point. Achievement of minimal clinically important difference (MCID), patient satisfaction and return to work were also assessed. Revision procedures were recorded at mean 10±3.3 years follow-up. In total, 236 obese patients were included: 118 open TLIF and 118 MIS-TLIF. Length of stay was longer in the Open TLIF cohort and there was a trend towards a higher complication rate. However, there was no difference in operative time, transfusions or readmissions. Patients who underwent open TLIF reported worse ODI (P=0.043) and VAS leg pain at 2 years, although the latter did not reach statistical significance (P=0.095). Achievement of MCID for each PRO, patient satisfaction and return to work were also comparable. Obese patients who underwent MIS-TLIF had a shorter length of stay and improved functional disability at 2 years compared those who underwent open TLIF. However, a similar proportion achieved a clinical meaningful improvement. Patient satisfaction and return to work were also comparable at 2 years. Level III, retrospective cohort study.

Combination of proton- or X-irradiation with anti-PDL1 immunotherapy in two murine oral cancers

Combining radiation therapy with immunotherapy is a strategy to improve both treatments. The purpose of this study was to compare responses for two syngeneic head and neck cancer (HNC) tumor models in mice following X-ray or proton irradiation with or without immune checkpoint inhibition (ICI). MOC1 (immunogenic) and MOC2 (less immunogenic) tumors were inoculated in the right hind leg of each mouse (C57BL/6J, n = 398). Mice were injected with anti-PDL1 (10 mg/kg, twice weekly for 2 weeks), and tumors were treated with single-dose irradiation (5–30 Gy) with X-rays or protons. MOC2 tumors grew faster and were more radioresistant than MOC1 tumors, and all mice with MOC2 tumors developed metastases. Irradiation reduced the tumor volume in a dose-dependent manner. ICI alone reduced the tumor volume for MOC1 with 20% compared to controls, while no reduction was seen for MOC2. For MOC1, there was a clear treatment synergy when combining irradiation with ICI for radiation doses above 5 Gy and there was a tendency for X-rays being slightly more biologically effective compared to protons. For MOC2, there was a tendency of protons being more effective than X-rays, but both radiation types showed a small synergy when combined with ICI. Although the responses and magnitudes of the therapeutic effect varied, the optimal radiation dose for maximal synergy appeared to be in the order of 10–15 Gy, regardless of tumor model.

Phylogenetic patterns of leg morphometric traits in vespid wasps (Hymenoptera: Vespidae)

AbstractVespid wasps use legs primarily for locomotion and in contexts such as grooming, handling of food and nest materials. Studies on leg form and function are relatively scarce. We analysed measurements of legs of 127 species of all genera of Polistinae, and of 18 species of other subfamilies, with the intent of investigating the occurrence and phylogenetic locations of morphometric patterns. Body‐size variation across Polistinae was determinant as to the absolute dimensions of legs, but allometric effects are generally small when comparing proportional length differences between differently sized species. Quite differently, size‐independent shape aspects are notably distinct as expressed by regression residuals and are clearly phylogenetically structured. Most distinctive are the very long mid and hind legs (and simultaneously exceedingly short forelegs) of Mischocyttarus species, and the comparatively shorter legs of some Epiponini. We found that forelegs have evolved with relative independence of the mid and hind legs, which is probably related to functions associated with the transport and manipulation of food and materials. Most examined solitary species showed mid and hindlegs shorter than most social polistines and vespines. Forefemur shape in some solitary species (euparagiines, masarines and some eumenines) may be functionally related to fossorial habits.

Effects of Different Target Distances on the Kinematics of Hip, Knee, and Ankle Joints in the Fencing Lunge

This study aimed to evaluate the effects of different target distances on various leg joints in the fencing lunge (lunge). Fifteen fencers performed the lunges from three different target distances (normal, short, and long). Joint angle data in the sagittal plane of the hip, knee, and ankle of the front and rear legs were measured using a 3D motion analysis system (Miqus M3). Joint angle variables were compared between each distance using a one-way repeated-measures analysis of variance and Friedman tests (p < 0.05). The results showed significant differences in various extensions, ranges of motion, and flexion angles in the measured joints for all distances. As the distances increased, there was greater flexion of the rear knee joint early during the lunge, followed by greater extension of the rear hip and knee joints, greater plantar flexion of the rear ankle joint, and higher peak velocity of the body center of mass. Furthermore, target distance extension was suggested to significantly affect front hip and knee joint flexion during the braking phase of the lunge. This study provides insight and information valuable to coaches and fencers operating in actual competition settings.

Dam–calf contact rearing in Switzerland: Aspects of management and milking

Cow-calf contact (CCC) rearing is becoming an increasingly popular alternative to the common practice of early separation of cow and calf in dairy management. Milkability can be impaired in nursing cows, which contributes to the loss of machine milk yield caused by calf intake, especially in pure dam-calf contact (DCC) systems. The aims of this study were (1) to describe the current status quo of DCC rearing regarding management and milking and (2) to evaluate the effects of DCC (suckling and milking vs. milking alone) and the effects of different types of DCC on milkability parameters, teat condition, and behavior during milking on Swiss DCC farms. By means of 17 telephone interviews with DCC farmers, we collected data on DCC management, housing, separation and weaning processes, milking procedures and techniques, and perceived milkability problems. Subsequently, we collected data on 10 of the interviewed DCC farms (183 cows): 4 DCC farms with a whole-day contact (WDC) system, 3 farms with DCC before milking (CBM), and 3 farms with DCC after milking (CAM). Five farms on which calves had no contact with dams were chosen as reference farms (178 cows). Using a milk flow meter, the occurrence of ejection disorders, bimodality of the milk flow curve, machine milk yield, duration of the decline phase, and duration of prestimulation were measured. The average mouthpiece chamber vacuum during the main milking phase and hind leg activity during milking were measured using a pressure sensor and an accelerometer, respectively. After cluster removal, the teat condition was evaluated, and a stripping milk sample was taken for fat content analysis. The interview results revealed that 8 of the 17 farms surveyed had a WDC system, and 2 farms operated a daytime DCC system. Contact before milking was applied by 3 farms, and 3 farms allowed CAM. On one farm, calves had access to dams 3 times a day. A great diversity in cow-calf management was found. In the on-farm data collection, 20 milkings of a total of 701 milkings examined met the criteria for a clear ejection disorder, with 17 of these observations occurring on WDC farms and none on reference farms. The stripping milk fat content was lower in nursing cows, indicating a lower degree of udder emptying. Machine milk yield was higher in nursing CAM cows than in nursing WDC and CBM cows. Farm types did not differ regarding teat condition, hind leg activity, or the occurrence of bimodal milk flow curves. In conclusion, the large variation in individual management approaches to DCC rearing even within DCC types, such as calf housing or cow breeds, implies caution when interpreting results. Contact after milking may be the system most beneficial for some productivity parameters, but adequate calf supply must be ensured. Higher amounts of milk remaining in the udder after cluster removal indicate that nursing can affect milkability, but future research should consider the effects of udder filling before milking to better interpret the fat content of stripping milk.

Inhibiting SHP2 reduces glycolysis, promotes microglial M1 polarization, and alleviates secondary inflammation following spinal cord injury in a mouse model.

JOURNAL/nrgr/04.03/01300535-202503000-00030/figure1/v/2024-06-17T092413Z/r/image-tiff Reducing the secondary inflammatory response, which is partly mediated by microglia, is a key focus in the treatment of spinal cord injury. Src homology 2-containing protein tyrosine phosphatase 2 (SHP2), encoded by PTPN11, is widely expressed in the human body and plays a role in inflammation through various mechanisms. Therefore, SHP2 is considered a potential target for the treatment of inflammation-related diseases. However, its role in secondary inflammation after spinal cord injury remains unclear. In this study, SHP2 was found to be abundantly expressed in microglia at the site of spinal cord injury. Inhibition of SHP2 expression using siRNA and SHP2 inhibitors attenuated the microglial inflammatory response in an in vitro lipopolysaccharide-induced model of inflammation. Notably, after treatment with SHP2 inhibitors, mice with spinal cord injury exhibited significantly improved hind limb locomotor function and reduced residual urine volume in the bladder. Subsequent in vitro experiments showed that, in microglia stimulated with lipopolysaccharide, inhibiting SHP2 expression promoted M2 polarization and inhibited M1 polarization. Finally, a co-culture experiment was conducted to assess the effect of microglia treated with SHP2 inhibitors on neuronal cells. The results demonstrated that inflammatory factors produced by microglia promoted neuronal apoptosis, while inhibiting SHP2 expression mitigated these effects. Collectively, our findings suggest that SHP2 enhances secondary inflammation and neuronal damage subsequent to spinal cord injury by modulating microglial phenotype. Therefore, inhibiting SHP2 alleviates the inflammatory response in mice with spinal cord injury and promotes functional recovery postinjury.

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

BackgroundLimb 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. ResultsIn 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. SignificanceIn 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.

Pathogenicity and transcriptomic profiling reveals immunology molecular hallmarks after CA10 virus infection.

Hand, foot and mouth disease (HFMD) is a common infectious disease caused by viral infection by a variety of enteroviruses, with coxsackievirus A 10 (CA10) having become more prevalent in recent years. In this study, models of CA10 infection were established in 7-day-old Institute of Cancer Research (ICR) mice by intraperitoneal injection to analyze the pathogenicity of the virus. RNA sequencing analysis was used to screen the differentially expressed genes (DEGs) after CA10 infection. Coxsackievirus A 16 (CA16) and enterovirus 71 (EV71) infections were also compared with CA10. After CA10 virus infection, the mice showed paralysis of the hind limbs at 3 days post infection and weight loss at 5 days post infection. We observed viral replication in various tissues and severe inflammatory cell infiltration in skeletal muscle. The RNA-sequencing analysis showed that the DEGs in blood, muscle, thymus and spleen showed heterogeneity after CA10 infection and the most up-regulated DEGs in muscle were enriched in immune-related pathways. Compared with CA16 and EV71 infection, CA10 may have an inhibitory effect on T helper (Th) cell differentiation and cell growth. Additionally, the common DEGs in the three viruses were most enriched in the immune system response, including the Toll-like receptor pathway and the nucleotide-binding and oligomerization domain (NOD)-like pathway. Our findings revealed a group of genes that coordinate in response to CA10 infection, which increases our understanding of the pathological mechanism of HFMD.

Effects of dry and wet ageing on culled goat meat quality

This study aims to evaluate the effects of ageing time (0, 1, 2, 3, 4 and 5 weeks) and of two different ageing techniques (dry and wet ageing) on the rheological, colorimetric, oxidative, volatile, and sensory attributes of meat (hind limbs) sourced from culled Rossa Mediterranea breed goats. Twenty-four goats were involved in the trial. The hind limbs were divided in the two experimental groups: the right ones were subjected to dry ageing and the left ones to wet ageing. Dry-aged (DA) meat showed higher water-holding capacity (WHC) and lower cooking loss (CL) after ageing compared to wet aged (WA) meat (p < 0.05). Among the colorimetric parameters, only lightness showed to be affected by ageing time, decreasing at week 4 (p < 0.05), with lower values in DA then WA (p < 0.05). Warner Bratzler shear force (WBSF) decreased at week 1 of DA and at week 2 of WA (p < 0.05), with no further changes probably due to the water loss during ageing. Hexanal was the most produced volatile compound, with higher amount in DA meat from 1 to 3 weeks of ageing (p < 0.01) The first two weeks of dry ageing led to an enhancement in sensory and instrumental tenderness, along with an improvement in flavour, characterised by a heightened release of aldehydes and ketones. However, it is noteworthy that beyond this initial phase, dry ageing has led to a substantial decline in the overall quality.

Predicting altered bone biomechanics in juvenile mice: insights from microgravity simulation, loading interventions, and Raman Spectroscopy

BackgroundMicrogravity, a condition experienced in a spatial environment, poses unique challenges to the skeletal system, particularly in juvenile organisms. This study aimed to investigate alterations in bone biomechanics of juvenile mice due to unloading – that simulates microgravity in the laboratory—and the effects of a bone-loading intervention. We compared bone compositional and mechanical properties between 21-six-week-old C57Bl/6 from a control group (wild type) and a group that underwent a tail-suspension unloading protocol to mimic microgravity (MG). The second group (MG) experienced additional in vivo loading protocol (MG + LDG) on the right hind leg, where dynamic compressive loading was applied to the right knee using a custom-built loading device.ResultsOur results show that after two weeks, we successfully induced bone alterations by (i) decreasing the energy dissipated before fracture and (ii) decreasing the yield and maximum stress. In addition, we showed that Mineral to matrix component [ν1PO4/Amide I], Carbonate to Amide [CO3/Amide I], and Crystallinity [1/FWHM(ν1PO4)] are strongly linked in physiological bone but not in microgravity even after loading intervention. While Crystallinity is very sensitive to bone deformation (strain) alterations coming from simulated microgravity, we show that Carbonate to Amide [CO3/Amide I] – a common marker of turnover rate/remodeling activity—is a specific predictor of bone deformation for bone after simulated microgravity. Our results also invalidate the current parameters of the loading intervention to prevent bone alterations entirely in juvenile mice.ConclusionsOur study successfully induced bone alterations in juvenile mice by using an unloading protocol to simulate microgravity, and we provided a new Raman Spectroscopy (RS) dataset of juvenile mice that contributes to the prediction of cortical bone mechanical properties, where the degree of interrelationship for RS data for physiological bone is improved compared to the most recent evidence.

Proton FLASH: Impact of Dose Rate and Split Dose on Acute Skin Toxicity in a Murine Model

PurposePreclinical studies have shown a preferential normal tissue sparing effect of FLASH radiotherapy with ultra-high dose rates. The aim of the present study was to use a murine model of acute skin toxicity to investigate the biological effect of varying dose rates, time structure, and of introducing pauses in the dose delivery. Materials and MethodsThe right hind limbs of non-anaesthetized mice were irradiated in the entrance plateau of a PBS proton beam with 39.3 Gy. Experiment 1 with varying field dose rates (0.7- 80 Gy/s) without repainting, Experiment 2 with varying field dose rates (0.37- 80 Gy/s) with repainting, and Experiment 3 where the dose was split into 2, 3, 4 or 6 identical deliveries with 2 minutes pauses. In total 320 mice were included with 6-25 mice per group. The endpoints were skin toxicity of different levels up to 25 days after irradiation. ResultsThe Dose rate50, dose rate to induce response in 50% of the animals, depended on the level of skin toxicity, with the higher toxicity levels displaying a FLASH effect at 0.7-2 Gy/s. Repainting resulted in higher toxicity for the same field dose rate. Splitting the dose into two deliveries reduced the FLASH effect, and for three or more deliveries, the FLASH effect was almost abolished for lower grades of toxicity. ConclusionsThe dose rate that induced a FLASH effect varied for different skin toxicity levels, which are characterized by a differing degree of sensitivity to radiation dosage. Conclusions on a threshold for the dose rate needed to obtain a FLASH effect can therefore be influenced by the dose sensitivity of the used endpoint. Splitting the total dose into more deliveries compromised the FLASH effect. This can have an impact for fractionation as well as for regions where two or more FLASH fields overlap within the same treatment session.

Mechanism of acupotomy on chondrocyte ferroptosis in rabbits with knee osteoarthritis based on HSPA5/GPX4 signaling pathway

To observe the effect of acupotomy on heat shock protein A family member 5 (HSPA5)/glutathione peroxidase 4 (GPX4) signaling pathway in the chondrocytes of the rabbits with knee osteoarthritis (KOA) and explore the mechanism of acupotomy on chondrocyte ferroptosis in KOA. Twenty-seven New Zealand rabbits were randomly divided into a normal group, a model group and an acupotomy group, with 9 rabbits in each group. The left hind limb was fixed by the modified Videman method for 6 weeks to establish KOA model. After modeling, acupotomy was given in the acupotomy group, once a week and for consecutive 3 weeks. Using Lequesne MG score, the local symptoms, physical signs and functions of knee joint were evaluated. With HE staining and saffrane-solid green staining adopted, the morphology of chondrocytes and cartilage tissue was observed. Under transmission electron microscope, the mitochondrial structure of chondrocytes was observed. The iron content of cartilage tissue was detected by iron ion kit. The mitochondrial membrane potential (Δψm) and the reactive oxygen species (ROS) level in cartilage tissue were determined by flow cytometry, and the mitochondrial damage rate was calculated. The mRNA expression of HSPA5, GPX4, type Ⅱ collagen α1 chain (COL2A1), matrix metalloproteinases (MMP) 3 and MMP13 was detected by the real-time quantitative PCR; and the protein expression of HSPA5, GPX4, type Ⅱ collagen (COL-Ⅱ), MMP3 and MMP13 was detected by Western blot. The mean flourscence intensity of HSPA5 and GPX4 in cartilage tissue was determined by immunofluorescence. Before intervention, compared with the normal group, the Lequesne MG scores were increased in the model group and the acupotomy group (P<0.01). After intervention, the Lequesne MG score in the acupotomy group was decreased when compared with that in the model group. In comparison with that in the normal group, the number of chondrocytes was reduced and the cells were disarranged; the layers of cartilage structure were unclear, the tide lines disordered and blurred; the mitochondria were wrinkled and the mitochondrial crista decreased or even disappeared in the model group. Compared with the model group, the number of chondrocytes was increased, the layers of cartilage structure were clear, the tide lines recovered, the number of mitochondria elevated, with normal structure and more crista in the acupotomy group. The iron content of cartilage tissue was increased (P<0.01), the Δψm of chondrocytes was declined, the mitochondrial damage rate was increased (P<0.01), the average fluorescence intensity of ROS was increased (P<0.01); the mRNA and corresponding protein expression of HSPA5, GPX4 and COL2A1 was decreased (P<0.01), the mRNA and protein expression of MMP3 and MMP13 was increased (P<0.01) and the average fluorescence intensity of HSPA5, GPX4 was decreased (P<0.01) in the model group when compared with those in the normal group. Compared with the model group, the iron content in cartilage tissue was reduced (P<0.01), the Δψm of chondrocytes was increased, the mitochondrial damage rate was decreased (P<0.01), and the average fluorescence intensity of ROS was decreased (P<0.01); the mRNA and corresponding protein expression of HSPA5, GPX4 and COL2A1 was higher (P<0.01), and the mRNA and protein expression of MMP3 and MMP13 was lower, and the average fluorescence intensity of HSPA5, GPX4 was increased (P<0.01) in the acupotomy group. Acupotomy can alleviate cartilage injury of KOA rabbits, and its mechanism may be related to the regulation of HSPA5/GPX4 signaling pathway to maintain iron homeostasis in articular cartilage, thus inhibiting chondrocyte ferroptosis and relieving extracellular matrix degradation.

Transcriptomic analysis of the effect of remote ischaemic conditioning in an animal model of necrotising enterocolitis

Necrotising enterocolitis (NEC) has a complex pathophysiology but the common end-point is ischaemia reperfusion injury (IRI) and intestinal necrosis. We have previously reported that RIC significantly reduces the intestinal injury in a rat model of NEC. Here we describe the changes in intestinal mRNA occurring in the intestine of animals exposed to IRI, both with and without RIC. Related rat-pups were randomly assigned to four groups: SHAM, IRI only, RIC only and RIC + IRI. IRI animals, underwent 40 min of intestinal ischaemia, and 90 min of reperfusion. Animals that underwent RIC had three cycles of 5 min of alternating ischaemia/reperfusion by means of a ligature applied to the hind limb. Samples from the terminal ileum were immediately stored in RNA-preserving media for later next generation sequencing and transciptome analysis using R v 3.6.1. Differential expression testing showed that 868 genes differentially expressed in animals exposed to RIC alone compared to SHAM and 135 in the IRI and RIC group compared to IRI alone. Comparison between these two sets showed that 25 genes were differentially expressed in both groups. Pro-inflammatory molecules: NF-ĸβ2, Cxcl1, SOD2 and Map3k8 all show reduced expression in response to RIC. Targeted gene analysis revealed increased expression in PI3K which is part of the so-called RISK-pathway which is a key part of the protective mechanisms of RIC in the heart. Overall, this transcriptomic analysis shows that RIC provides a protective effect to the intestine via anti-inflammatory pathways. This could be particularly relevant to treating and preventing NEC.

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.