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Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes
Abstracts 2016 / Neuromuscular Disorders 26 (2016) S88–S212 that TRPV2 inhibition can be effective for cardiomyopathy of muscular dystrophy. Further studies are needed to certify the effects and safety of it. http://dx.doi.org/10.1016/j.nmd.2016.06.429
P.401 Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes H. Kim 1, J. Shin 1, D. Kim 1, C. Kim 1, C. Kim 2 1 Pusan National University Yangsan Hospital, Yangsan, Republic of Korea; 2 Pusan National University, Yangsan, Republic of Korea Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1 to 10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of 4E-BP and P70S6K which initiate translation as downstream of mTOR pathway were upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mTOR/P70S6K and 4E-BP signaling pathway in human myotubes. SF can be a useful natural dietary supplement increasing skeletal muscle mass, especially with the aged with sarcopenia and the patients with disuse atrophy. http://dx.doi.org/10.1016/j.nmd.2016.06.430
S205
C. Timson, D. Chambers, L. Feng, F. Muntoni, C. Sewry, R. Phadke Dubowitz Neuromuscular Centre, UCL and Great Ormond Street Hospital for Children, London, UK Reprocessed frozen tissue (FT) or formalin-fixed paraffin-embedded (FFPE) tissue for transmission electron microscopy (TEM) has been used in general pathology but there is little information on its use in diagnostic muscle pathology. A recent study of re-processed stored frozen muscle biopsies from healthy volunteers showed morphology almost comparable to that of freshly fixed muscle (FFM). This is a retrospective study of 8 muscle biopsies referred to the DNC pathology service where TEM was required to confirm/exclude diagnosis and only FFPE/FT blocks were available. A small strip parallel to the fibres was cut from each of the FT blocks in the cryo-cabinet at −25°C and immediately placed into cold 4% glutaraldehyde in cacodylate buffer and some also into a 2% glutaraldehyde/2% paraformaldehyde mixture, allowed to thaw at room temperature, then cut into smaller pieces approximately 1.5 × 0.5 mm2. After 1.5 hours the samples were thoroughly rinsed, dehydrated through an ethanol series to propylene oxide and embedded in Araldite resin. Stained slides were reviewed to select an area of interest for FFPE blocks; these were cut and de-waxed overnight in chloroform, then re-hydrated, fixed and processed as above. Resin semi-/ultra-thin sections were cut and stained as per standard procedures and viewed with a Phillips CM10 or CM12 electron microscope. Reprocessed FT/FFPE tissue morphology was somewhat poor compared to FFM, but there were better-preserved foci giving good diagnostic yield. Mitochondrial morphology was poorer in reprocessed cases. Diagnostic pathology identified included reducing bodies, granulofilamentous aggregates, nemaline rods, polyglucosan aggregates and abnormal mitochondria (5/9), nonspecific pathology (2/9) and exclusion of macrophagic myofascitis (1/9). In conclusion, in instances where FFM processed tissue for TEM is not available, it is possible to produce adequate results using re-processed FT/FFPE material for diagnostic muscle biopsies. http://dx.doi.org/10.1016/j.nmd.2016.06.432
P.402 The development of new equations to estimate ventilator setting in patients with neuromuscular disease D. Park 1, G. Lee 2, H. Kim 2, J. Ryu 2 1 Daegu Fatima Hospital, Daegu, Republic of Korea; 2 Bundang Hospital, Seoul National University, Seongnam-si, Republic of Korea The purpose of this study to investigate the appropriate setting values for home ventilators in patients with neuromuscular disease. We retrospectively reviewed the home ventilator settings of 71 patients with neuromuscular disease, who had received either a non-invasive positive pressure ventilation (NIPPV) or tracheostomy positive pressure ventilation (TPPV) when both the arterial blood gas and transcutaneous blood gas analyses showed a stable CO2 level. We identify appropriate home ventilator settings to improve hypercapnia and its symptoms in patients with neuromuscular disease. To measure main outcome, we also investigated patient’s body weight and height, and the following setting values of ventilators: inspired tidal volume (VTi), minute ventilation (MV), peak inspiratory pressure (PIP), and positive end expiratory pressure (PEEP), and inspiratory time (Tins). In the results, VTi and MV had significantly positive correlations with height of patients in TPPV. By using new equations, we categorized patients according to height for easy application of VTi and MV in clinical setting. In conclusion, these new equations, which allow for the categorization of VTi and MV according to height, may offer a new valid option as the initial ventilator settings for patients with neuromuscular disease, especially to those undergoing TPPV. http://dx.doi.org/10.1016/j.nmd.2016.06.431
P.403 Muscle biopsies reprocessed for electron microscopy from paraffin blocks and frozen tissue produce material of sufficient quality for diagnostic use
P.404 Muscle fibre size revisited: Updated age-stratified normative data in histologically normal/minimal change (HN/MC) paediatric quadriceps biopsies using a high-throughput automated digital script L. Feng 1, M. Ellis 2, E. Curtis-Wetton 2, D. Chambers 1, A. Matthews 3, A. Manzur 3, F. Muntoni 1, C. Sewry 1, R. Phadke 1 1 Dubowitz Neuromuscular Centre, UCL and Great Ormond Street Hospital for Children, London, UK; 2 UCL Institute of Neurology, London, UK; 3 Great Ormond Street Hospital for Children, London, UK Fibre size evaluation related to physiological development and disease is important in interpretation of diagnostic muscle biopsies. Existing literature on normative fibre size range for paediatric quadriceps muscle (pQM) (0–16 years) is sparse, dated, and based on small cohorts/sample sizes. We present an update on age-stratified normative pQM fibre size data employing a high throughput automated digital script. 42 archival HN/MC needle muscle biopsies were recruited from the DNC muscle biopsy service. 13/42 cases had no evidence of primary muscle disease after extensive testing. Whole spectrin-labelled frozen sections were digitally scanned and analysed using Definiens developer 2.4.2 software to extract lesser diameter of myofibres through a multi-step process of tissue identification, automatic thresholding, gradient and morphology based methods and noise exclusion. This dataset was compared with digital measurements obtained after careful manual selection of regions of interest (ROI). The age range was 0.03 to 16.5 years. Total fibre counts ranged from 449 to 160,751; mean = 16,858). Age stratified mean fibre size diameters were obtained (0–1: 10.7 μm; 1.5–2: 17.5 μm; 2.5–3.5: 24 μm; 4–6.5: 27 μm; 6.5–9: 36 μm; 10.5–13.5: 44 μm; 14–16.5: 61 μm). The samples within the cohort were normally distributed. There was strong positive correlation between age and mean fibre size (r2 = 0.8). Bland–Altman analysis showed a good measure of agreement between the fully automated and semi-automated methods (R2 = 0.63; p < <0.001). Co-variance analysis showed no significant difference
P.401 Schisandrae fructus enhances myogenic differentiation and inhibits atrophy through protein synthesis in human myotubes H. Kim 1, J. Shin 1, D. Kim 1, C. Kim 1, C. Kim 2 1 Pusan National University Yangsan Hospital, Yangsan, Republic of Korea; 2 Pusan National University, Yangsan, Republic of Korea Schisandrae fructus (SF) has recently been reported to increase skeletal muscle mass and inhibit atrophy in mice. We investigated the effect of SF extract on human myotube differentiation and its acting pathway. Various concentrations (0.1 to 10 μg/mL) of SF extract were applied on human skeletal muscle cells in vitro. Myotube area and fusion index were measured to quantify myotube differentiation. The maximum effect was observed at 0.5 μg/mL of SF extract, enhancing differentiation up to 1.4-fold in fusion index and 1.6-fold in myotube area at 8 days after induction of differentiation compared to control. Phosphorylation of 4E-BP and P70S6K which initiate translation as downstream of mTOR pathway were upregulated in early phases of differentiation after SF treatment. SF also attenuated dexamethasone-induced atrophy. In conclusion, we show SF augments myogenic differentiation and attenuates atrophy by increasing protein synthesis through mTOR/P70S6K and 4E-BP signaling pathway in human myotubes. SF can be a useful natural dietary supplement increasing skeletal muscle mass, especially with the aged with sarcopenia and the patients with disuse atrophy. http://dx.doi.org/10.1016/j.nmd.2016.06.430
S205
C. Timson, D. Chambers, L. Feng, F. Muntoni, C. Sewry, R. Phadke Dubowitz Neuromuscular Centre, UCL and Great Ormond Street Hospital for Children, London, UK Reprocessed frozen tissue (FT) or formalin-fixed paraffin-embedded (FFPE) tissue for transmission electron microscopy (TEM) has been used in general pathology but there is little information on its use in diagnostic muscle pathology. A recent study of re-processed stored frozen muscle biopsies from healthy volunteers showed morphology almost comparable to that of freshly fixed muscle (FFM). This is a retrospective study of 8 muscle biopsies referred to the DNC pathology service where TEM was required to confirm/exclude diagnosis and only FFPE/FT blocks were available. A small strip parallel to the fibres was cut from each of the FT blocks in the cryo-cabinet at −25°C and immediately placed into cold 4% glutaraldehyde in cacodylate buffer and some also into a 2% glutaraldehyde/2% paraformaldehyde mixture, allowed to thaw at room temperature, then cut into smaller pieces approximately 1.5 × 0.5 mm2. After 1.5 hours the samples were thoroughly rinsed, dehydrated through an ethanol series to propylene oxide and embedded in Araldite resin. Stained slides were reviewed to select an area of interest for FFPE blocks; these were cut and de-waxed overnight in chloroform, then re-hydrated, fixed and processed as above. Resin semi-/ultra-thin sections were cut and stained as per standard procedures and viewed with a Phillips CM10 or CM12 electron microscope. Reprocessed FT/FFPE tissue morphology was somewhat poor compared to FFM, but there were better-preserved foci giving good diagnostic yield. Mitochondrial morphology was poorer in reprocessed cases. Diagnostic pathology identified included reducing bodies, granulofilamentous aggregates, nemaline rods, polyglucosan aggregates and abnormal mitochondria (5/9), nonspecific pathology (2/9) and exclusion of macrophagic myofascitis (1/9). In conclusion, in instances where FFM processed tissue for TEM is not available, it is possible to produce adequate results using re-processed FT/FFPE material for diagnostic muscle biopsies. http://dx.doi.org/10.1016/j.nmd.2016.06.432
P.402 The development of new equations to estimate ventilator setting in patients with neuromuscular disease D. Park 1, G. Lee 2, H. Kim 2, J. Ryu 2 1 Daegu Fatima Hospital, Daegu, Republic of Korea; 2 Bundang Hospital, Seoul National University, Seongnam-si, Republic of Korea The purpose of this study to investigate the appropriate setting values for home ventilators in patients with neuromuscular disease. We retrospectively reviewed the home ventilator settings of 71 patients with neuromuscular disease, who had received either a non-invasive positive pressure ventilation (NIPPV) or tracheostomy positive pressure ventilation (TPPV) when both the arterial blood gas and transcutaneous blood gas analyses showed a stable CO2 level. We identify appropriate home ventilator settings to improve hypercapnia and its symptoms in patients with neuromuscular disease. To measure main outcome, we also investigated patient’s body weight and height, and the following setting values of ventilators: inspired tidal volume (VTi), minute ventilation (MV), peak inspiratory pressure (PIP), and positive end expiratory pressure (PEEP), and inspiratory time (Tins). In the results, VTi and MV had significantly positive correlations with height of patients in TPPV. By using new equations, we categorized patients according to height for easy application of VTi and MV in clinical setting. In conclusion, these new equations, which allow for the categorization of VTi and MV according to height, may offer a new valid option as the initial ventilator settings for patients with neuromuscular disease, especially to those undergoing TPPV. http://dx.doi.org/10.1016/j.nmd.2016.06.431
P.403 Muscle biopsies reprocessed for electron microscopy from paraffin blocks and frozen tissue produce material of sufficient quality for diagnostic use
P.404 Muscle fibre size revisited: Updated age-stratified normative data in histologically normal/minimal change (HN/MC) paediatric quadriceps biopsies using a high-throughput automated digital script L. Feng 1, M. Ellis 2, E. Curtis-Wetton 2, D. Chambers 1, A. Matthews 3, A. Manzur 3, F. Muntoni 1, C. Sewry 1, R. Phadke 1 1 Dubowitz Neuromuscular Centre, UCL and Great Ormond Street Hospital for Children, London, UK; 2 UCL Institute of Neurology, London, UK; 3 Great Ormond Street Hospital for Children, London, UK Fibre size evaluation related to physiological development and disease is important in interpretation of diagnostic muscle biopsies. Existing literature on normative fibre size range for paediatric quadriceps muscle (pQM) (0–16 years) is sparse, dated, and based on small cohorts/sample sizes. We present an update on age-stratified normative pQM fibre size data employing a high throughput automated digital script. 42 archival HN/MC needle muscle biopsies were recruited from the DNC muscle biopsy service. 13/42 cases had no evidence of primary muscle disease after extensive testing. Whole spectrin-labelled frozen sections were digitally scanned and analysed using Definiens developer 2.4.2 software to extract lesser diameter of myofibres through a multi-step process of tissue identification, automatic thresholding, gradient and morphology based methods and noise exclusion. This dataset was compared with digital measurements obtained after careful manual selection of regions of interest (ROI). The age range was 0.03 to 16.5 years. Total fibre counts ranged from 449 to 160,751; mean = 16,858). Age stratified mean fibre size diameters were obtained (0–1: 10.7 μm; 1.5–2: 17.5 μm; 2.5–3.5: 24 μm; 4–6.5: 27 μm; 6.5–9: 36 μm; 10.5–13.5: 44 μm; 14–16.5: 61 μm). The samples within the cohort were normally distributed. There was strong positive correlation between age and mean fibre size (r2 = 0.8). Bland–Altman analysis showed a good measure of agreement between the fully automated and semi-automated methods (R2 = 0.63; p < <0.001). Co-variance analysis showed no significant difference