The African killifish: A short-lived vertebrate model to study the biology of sarcopenia and longevity.

Sarcopenia, the age-related decline in muscle function, places a considerable burden on health-care systems. While the stereotypic hallmarks of sarcopenia are well characterized, their contribution to muscle wasting remains elusive, which is partly due to the limited availability of animal models. Here, we have performed cellular and molecular characterization of skeletal muscle from the African killifish-an extremely short-lived vertebrate-revealing that while many characteristics deteriorate with increasing age, supporting the use of killifish as a model for sarcopenia research, some features surprisingly reverse to an "early-life" state in the extremely old stages. This suggests that in extremely old animals, there may be mechanisms that prevent further deterioration of skeletal muscle, contributing to an extension of life span. In line with this, we report a reduction in mortality rates in extremely old killifish. To identify mechanisms for this phenomenon, we used a systems metabolomics approach, which revealed that during aging there is a striking depletion of triglycerides, mimicking a state of calorie restriction. This results in the activation of mitohormesis, increasing Sirt1 levels, which improves lipid metabolism and maintains nutrient homeostasis in extremely old animals. Pharmacological induction of Sirt1 in aged animals was sufficient to induce a late life-like metabolic profile, supporting its role in life span extension in vertebrate populations that are naturally long-lived. Collectively, our results demonstrate that killifish are not only a novel model to study the biological processes that govern sarcopenia, but they also provide a unique vertebrate system to dissect the regulation of longevity.

Utility and Limitations of TALLYHO/JngJ as a Model for Type 2 Diabetes-Induced Bone Disease.

Type 2 diabetes mellitus (T2DM) increases risk of fractures due to bone microstructural and material deficits, though the mechanisms remain unclear. Preclinical models mimicking diabetic bone disease are required to further understand its pathogenesis. The TALLYHO/JngJ (TH) mouse is a polygenic model recapitulating adolescent-onset T2DM in humans. Due to incomplete penetrance of the phenotype ~25% of male TH mice never develop hyperglycemia, providing a strain-matched nondiabetic control. We performed a comprehensive characterization of the metabolic and skeletal phenotype of diabetic TH mice and compared them to either their nondiabetic TH controls or the recommended SWR/J controls to evaluate their suitability to study diabetic bone disease in humans. Compared to both controls, male TH mice with T2DM exhibited higher blood glucose levels, weight along with impaired glucose tolerance and insulin sensitivity. TH mice with/without T2DM displayed higher cortical bone parameters and lower trabecular bone parameters in the femurs and vertebrae compared to SWR/J. The mechanical properties remained unchanged for all three groups except for a low-energy failure in TH mice with T2DM only compared to SWR/J. Histomorphometry analyses only revealed higher number of osteoclasts and osteocytes for SWR/J compared to both groups of TH. Bone turnover markers procollagen type 1 N-terminal propeptide (P1NP) and tartrate-resistant acid phosphatase (TRAP) were low for both groups of TH mice compared to SWR/J. Silver nitrate staining of the femurs revealed low number of osteocyte lacunar and dendrites in TH mice with T2DM. Three-dimensional assessment showed reduced lacunar parameters in trabecular and cortical bone. Notably, osteocyte morphology changed in TH mice with T2DM compared to SWR/J. In summary, our study highlights the utility of the TH mouse to study T2DM, but not necessarily T2DM-induced bone disease, as there were no differences in bone strength and bone cell parameters between diabetic and non-diabetic TH mice. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

When Cortical Bone Matrix Properties Are Indiscernible between Elderly Men with and without Type 2 Diabetes, Fracture Resistance Follows Suit.

Type 2 diabetes mellitus (T2DM) is a metabolic disease affecting bone tissue and leading to increased fracture risk in men and women, independent of bone mineral density (BMD). Thus, bone material quality (i.e., properties that contribute to bone toughness but are not attributed to bone mass or quantity) is suggested to contribute to higher fracture risk in diabetic patients and has been shown to be altered. Fracture toughness properties are assumed to decline with aging and age-related disease, while toughness of human T2DM bone is mostly determined from compression testing of trabecular bone. In this case-control study, we determined fracture resistance in T2DM cortical bone tissue from male individuals in combination with a multiscale approach to assess bone material quality indices. All cortical bone samples stem from male nonosteoporotic individuals and show no significant differences in microstructure in both groups, control and T2DM. Bone material quality analyses reveal that both control and T2DM groups exhibit no significant differences in bone matrix composition assessed with Raman spectroscopy, in BMD distribution determined with quantitative back-scattered electron imaging, and in nanoscale local biomechanical properties assessed via nanoindentation. Finally, notched three-point bending tests revealed that the fracture resistance (measured from the total, elastic, and plastic J-integral) does not significantly differ in T2DM and control group, when both groups exhibit no significant differences in bone microstructure and material quality. This supports recent studies suggesting that not all T2DM patients are affected by a higher fracture risk but that individual risk profiles contribute to fracture susceptibility, which should spur further research on improving bone material quality assessment in vivo and identifying risk factors that increase bone fragility in T2DM. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Rapid Genotyping of Nothobranchius furzeri Embryos, Larvae, and Adult Fish via High-Resolution Melt Analysis (HRMA).

CRISPR-Cas9 has eased the induction of sequence-specific mutations and has therefore become a powerful tool to generate mutant lines for studying the role of specific genes. The cellular repair of Cas9-induced double-stranded DNA breaks by the error-prone nonhomologous end-joining pathway can result in various indel mutations. Having identified and chosen a specific mutation in a target gene, the establishment of a respective mutant line requires a feasible and precise method to differentiate the genotypes of the offspring. Here, we provide a protocol that allows genotyping of large numbers of Nothobranchius furzeri embryos, larvae, and adults harboring a previously identified indel or point mutation in a short time via high-resolution melt analysis (HRMA).

Generation of a transparent killifish line through multiplex CRISPR/Cas9mediated gene inactivation.

Body pigmentation is a limitation for in vivo imaging and thus for the performance of longitudinal studies in biomedicine. A possibility to circumvent this obstacle is the employment of pigmentation mutants, which are used in fish species like zebrafish and medaka. To address the basis of aging, the short-lived African killifish Nothobranchius furzeri has recently been established as a model organism. Despite its short lifespan, N. furzeri shows typical signs of mammalian aging including telomere shortening, accumulation of senescent cells, and loss of regenerative capacity. Here, we report the generation of a transparent N. furzeri line by the simultaneous inactivation of three key loci responsible for pigmentation. We demonstrate that this stable line, named klara, can serve as a tool for different applications including behavioral experiments and the establishment of a senescence reporter by integration of a fluorophore into the cdkn1a (p21) locus and in vivo microscopy of the resulting line.

Human tibial cortical bone with high porosity in type 2 diabetes mellitus is accompanied by distinctive bone material properties.

Diabetes mellitus is a metabolic disease affecting bone tissue at different length-scales. Higher fracture risk in diabetic patients is difficult to detect with common clinical fracture risk assessment due to normal or high bone mineral density in diabetic patients. The observed higher fracture risk despite normal to high areal bone mineral density in diabetic patients points towards impaired bone material quality. Here, we analyze tibial bone from individuals with type 2 diabetes mellitus using a multiscale-approach, which includes clinical and laboratory-based bone quality measures. Tibial cortical bone tissue from individuals with type 2 diabetes mellitus (T2DM) and age-matched healthy controls (n = 15 each) was analyzed with in situ impact indentation, dual energy X-ray absorptiometry (DXA), high resolution peripheral microcomputed tomography (HR-pQCT), micro-computed tomography (microCT), cyclic indentation, quantitative backscattered electron microscopy (qBEI), vibrational spectroscopy (Raman), nanoindentation, and fluorescence spectroscopy. With this approach, a high cortical porosity subgroup of individuals with T2DM was discriminated from two study groups: individuals with T2DM and individuals without T2DM, while both groups were associated with similar cortical porosity quantified by means of microCT. The high porosity T2DM group, but not the T2DM group, showed compromised bone quality expressed by altered cyclic indentation properties (transversal direction) in combination with a higher carbonate-to-amide I ratio in endocortical bone. In addition, in the T2DM group with high cortical porosity group, greater cortical pore diameter was identified with HR-pQCT and lower tissue mineral density using microCT, both compared to T2DM group. Micromechanical analyses of cross-sectioned osteons (longitudinal direction) with cyclic indentation, qBEI, and nanoindentation showed no differences between the three groups. High tibial cortical porosity in T2DM can be linked to locally altered bone material composition. As the tibia is an accessible skeletal site for fracture risk assessment in the clinics (CT, indentation), our findings may contribute to further understanding the site-specific structural and compositional factors forming the basis of bone quality in diabetes mellitus. Refined diagnostic strategies are needed for a comprehensive fracture risk assessment in diabetic bone disease.

Molecular Sexing of Nothobranchius furzeri Embryos and Larvae.

Differences between the sexes are of increasing interest in basic and applied research with regard to development, behavior, aging, and diseases. Although the African turquoise killifish Nothobranchius furzeri, a model for aging research well known for its remarkably short life span, develops strong sexual dimorphism in adulthood, there is no visible indicator of its sex in embryonic and juvenile stages. To address this issue, we developed a molecular sexing assay exploiting two large sequence polymorphisms in the minimal sex-determining region (SDR) of N. furzeri These polymorphisms are sequence deletions on the Y chromosome that involve the lack or truncation of one or multiple microsatellites. The simple polymerase chain reaction (PCR) readout of the assays described here allows the sexing of N. furzeri embryos and larvae in a medium- to high-throughput and cost-efficient manner.

Reorganization of the osteocyte lacuno-canalicular network characteristics in tumor sites of an immunocompetent murine model of osteotropic cancers.

Mechanosensitive osteocytes are central regulators of bone resorption and formation. However, during the formation of bone metastases, which arise as consequences of breast and prostate cancer and skew homeostatic bone remodeling to favor osteolytic, osteosclerotic or mixed lesions, only a paucity of data exists on tumor-associated osteocyte interaction. Herein, we used a suite of high-resolution imaging and histological techniques to evaluate the effect of osteotropic cancer on cortical bone microarchitecture. Confocal imaging highlighted a direct contact between tumor cells residing in the bone marrow and osteocytes. High-resolution microcomputed tomography revealed a 10-12% larger osteocyte lacuna volume in the presence of tumor cells at day 21 after intratibial injection of EO771-Luc breast and RM1-Luc prostate cancer cells. The 3D representative of the spatial distribution of cortical bone microporosity showed i) a regional accumulation of vascular canals and large lacunae with low connectivity in osteosclerotic regions of interest and ii) an absence of vascular canals and large lacunae in osteolytic regions. These findings pinpoint the relationship between the presence of tumor cells in the bone marrow microenvironment and osteocyte lacunar characteristics and cortical bone blood vessel structure.

Correction to: RF-ablation pattern shaping employing switching channels of dual bipolar needle electrodes: ex vivo results.

The original version of this article was published without funding note. The funding note is given below.

RF-ablation pattern shaping employing switching channels of dual bipolar needle electrodes: ex vivo results.

PURPOSE: Radiofrequency (RF) ablation with mono- or bipolar electrodes is a common procedure for hepatocellular carcinoma (HCC) with a low rate of recurrence for small size tumors. For larger lesions and/or non-round/ellipsoid shapes RF ablation has some limitations and generally does not achieve comparable success rates to microwave ablation or high-intensity focused ultrasound therapies. MATERIALS AND METHODS: To shape RF ablations for matching a tumor size and geometry, we have developed an electronic channel switch box for two bipolar needles that generates multiple selectable ablation patterns. The setup can be used with commercially available mono- or bipolar RF generators. The switch box provides ten selectable ablation procedures to generate different ablation patterns without a relocation of a needle. Five patterns were exemplary generated in ex vivo tissue of porcine liver and chicken breast and visually characterized. RESULTS: Different ablation patterns, e.g., in a L- or U-shape, were achieved. In chicken breast a maximum ablation with a diameter of [Formula: see text] was obtained and in porcine liver [Formula: see text] with electrodes of [Formula: see text] length. CONCLUSION: The resulting ablations with the electronic switch box and two bipolar needles show the potential ability to manage RF therapies of complex and large tumor geometries. Next steps would be to validate the actual tissue ablation volumes in further ex vivo and preclinical studies and against simulation results.

Filtering of ECG signals distorted by magnetic field gradients during MRI using non-linear filters and higher-order statistics.

The electrocardiogram (ECG) is the state-of-the-art signal for patient monitoring and gating in cardiovascular magnetic resonance (CMR) imaging applications. However, ECG signals are severely distorted during MRI scans due to the effects of static magnetic fields, radio frequency pulses and fast-switching gradient magnetic fields. Gradient-induced artifacts that cause high frequency peaks in the ECG signal especially hamper a correct and reliable QRS detection. To cope with this problem, a new median-based real-time gradient filter (M1) approach was developed. To improve the filter results, a preprocessing step based on higher-order statistics (M2) was added to this. For the evaluation of the filtering techniques, ECG signals were acquired in a 3T MRI scanner during different MR sequences. A qualitative comparison was made using the mean square error as well as the signal power before and after filtering and the results of the QRS detection. Here, reliable results were achieved (detection error rate [DER] M1: 0.23%, DER M2: 0.74%). It was shown that the two developed techniques allowed a reliable suppression of the gradient artifacts in real time.

Distortion indicator algorithm for simple artifact assessment of passive MRI markers.

The main purpose of this work is to present a simple quantitative approach for assessing artifacts in passive instruments when used in an MRI environment. It is mainly based on a quantitative indicator related with the amount of distortion produced by the instrument on a 3D surface obtained from the 2D MR image. After a preprocessing stage for attenuation of the 3D surface low frequency components, an indicator related with the volume of the background distortion is computed for each MRI slice. Then a monotonically decreasing curve is calculated for assessing artifact level using all the slices of a sequence. Results show that an indicator can be computed automatically from all the slices of a given MRI sequence and through this indicator it is possible to perform comparative studies between artifacts produced by different catheters and needles.

ECG-based gating in ultra high field cardiovascular magnetic resonance using an independent component analysis approach.

BACKGROUND: In Cardiovascular Magnetic Resonance (CMR), the synchronization of image acquisition with heart motion is performed in clinical practice by processing the electrocardiogram (ECG). The ECG-based synchronization is well established for MR scanners with magnetic fields up to 3 T. However, this technique is prone to errors in ultra high field environments, e.g. in 7 T MR scanners as used in research applications. The high magnetic fields cause severe magnetohydrodynamic (MHD) effects which disturb the ECG signal. Image synchronization is thus less reliable and yields artefacts in CMR images. METHODS: A strategy based on Independent Component Analysis (ICA) was pursued in this work to enhance the ECG contribution and attenuate the MHD effect. ICA was applied to 12-lead ECG signals recorded inside a 7 T MR scanner. An automatic source identification procedure was proposed to identify an independent component (IC) dominated by the ECG signal. The identified IC was then used for detecting the R-peaks. The presented ICA-based method was compared to other R-peak detection methods using 1) the raw ECG signal, 2) the raw vectorcardiogram (VCG), 3) the state-of-the-art gating technique based on the VCG, 4) an updated version of the VCG-based approach and 5) the ICA of the VCG. RESULTS: ECG signals from eight volunteers were recorded inside the MR scanner. Recordings with an overall length of 87 min accounting for 5457 QRS complexes were available for the analysis. The records were divided into a training and a test dataset. In terms of R-peak detection within the test dataset, the proposed ICA-based algorithm achieved a detection performance with an average sensitivity (Se) of 99.2%, a positive predictive value (+P) of 99.1%, with an average trigger delay and jitter of 5.8 ms and 5.0 ms, respectively. Long term stability of the demixing matrix was shown based on two measurements of the same subject, each being separated by one year, whereas an averaged detection performance of Se = 99.4% and +P = 99.7% was achieved.Compared to the state-of-the-art VCG-based gating technique at 7 T, the proposed method increased the sensitivity and positive predictive value within the test dataset by 27.1% and 42.7%, respectively. CONCLUSIONS: The presented ICA-based method allows the estimation and identification of an IC dominated by the ECG signal. R-peak detection based on this IC outperforms the state-of-the-art VCG-based technique in a 7 T MR scanner environment.

Simulation and experimental validation of resonant electric markers used for medical device tracking in magnetic resonance imaging.

Magnetic resonance imaging (MRI), which was traditionally used for patient diagnosis, has gained in importance in minimally invasive interventions in the recent past. Hence, there is an increasing demand for medical devices compatible with the MR environment. One of the challenges is to visualize the medical devices, e.g. catheters, within the MR image. Several methods exist to cope with this task.

MR-compatible RF ablation system for online treatment monitoring using MR thermometry.

RF ablation (RFA) is used for thermal ablation of tumors in which the RF electrode is placed in the tissue under image-guidance. Because of the good tumor visibility and the lack of ionizing radiation, MR-guided RFA is the method of choice. Additionally, with the help of MR thermometry the RF ablation can be monitored during the intervention. Unfortunately, the imaging of an MR scanner is highly sensitive to interferences caused by external electrical signals. In this paper the high-power RF ablation signal of a commercially available medical therapy device is made MR-compatible. A design of a low-pass filter with high-power compatibility is presented. The filter performance is demonstrated by means of simulations and measurements.