Counting unstained, confluent cells by modified bright-field microscopy

We present a very simple procedure yielding high-contrast images of adherent, confluent cells such as human neuroblastoma (SH-EP) cells by ordinary bright-field microscopy. Cells are illuminated through a color filter and a pinhole aperture placed between the condenser and the cell culture surface. Refraction by each cell body generates a sharp, bright spot when the image is defocused. The technique allows robust, automatic cell counting from a single bright-field image in a wide range of focal positions using free, readily available image-analysis tools. Contrast may be enhanced by swelling cell bodies with a brief incubation in PBS. The procedure was benchmarked against manual and automated counting of fluorescently labeled cell nuclei. Counts from day-old and freshly seeded plates were compared in a range of densities, from sparse to densely overgrown. On average, bright-field images produced the same counts as fluorescence images, with less than 5% error. This method will allow routine cell counting using a plain bright-field microscope without cell-line modification or cell staining

Counting unstained, confluent cells by modified bright-field microscopy

We present a very simple procedure yielding high-contrast images of adherent, confluent cells such as human neuroblastoma (SH-EP) cells by ordinary bright-field microscopy. Cells are illuminated through a color filter and a pinhole aperture placed between the condenser and the cell culture surface. Refraction by each cell body generates a sharp, bright spot when the image is defocused. The technique allows robust, automatic cell counting from a single bright-field image in a wide range of focal positions using free, readily available image-analysis tools. Contrast may be enhanced by swelling cell bodies with a brief incubation in PBS. The procedure was benchmarked against manual and automated counting of fluorescently labeled cell nuclei. Counts from day-old and freshly seeded plates were compared in a range of densities, from sparse to densely overgrown. On average, bright-field images produced the same counts as fluorescence images, with less than 5% error. This method will allow routine cell counting using a plain bright-field microscope without cell-line modification or cell staining

Performance declines are accelerated in the oldest-old track and field athletes 80 to 94 years of age.

Physical performance declines with age, even in exercising, healthy individuals without major illnesses or orthopaedic issues. The rate of performance decline is often reported to accelerate after the age of 70 years, but almost no data are available on performance in the fittest oldest-old. To assess their rate of decline in performance, the biggest dataset of track and field athletes ≥80 years (1567 results) ever published was generated for different disciplines from German athletics Federations including 1997-2019. Performance at age 80 of athletes still participating at age 85 was compared to those who discontinued. Only one out of every 22 athletes competing at age 80 still competed at age 90. The performance decline was more than three times as steep in athletes ≥80 (on average 1.62%/year, men: 100m R=0.31, p<0.001; 200m R=0.17, p=0.037; long jump R=-0,37, p<0.001; shot put R=-0.32, p<0.001; discus R=-0.34, p<0.001; javelin R=-0.43, p<0.001; women: shot put R=-0.24, p=0.017; discus R=-0.33, p=0.010) compared to athletes 30-69 years (0.46%/year), and accelerated at an average of 67 years. This accelerated decline was most pronounced in the sprint disciplines and lowest in the throws. Performance at age 80 was similar in athletes still participating at age 85 to those who discontinued, and the variability in results was decreased after age 90. In conclusion, physical performance declines more than three times as fast after around the age of 67 years compared to before. This was particularly the case for sprinting, but was not a result of dropout of poorer performing athletes

Late-onset neuromuscular disorders in the differential diagnosis of sarcopenia

BACKGROUND Sarcopenia is the age-related loss of muscle mass and strength. Undiagnosed late-onset neuromuscular disorders need to be considered in the differential diagnosis of sarcopenia. AIM Based on emblematic case reports and current neuromuscular diagnostic guidelines for three common late-onset neuromuscular disorders, a differential diagnostic approach for geriatric patients presenting with a sarcopenic phenotype is given. METHODS Patients over 65~years of age with sarcopenia, amyotrophic lateral sclerosis, inclusion body myositis and myotonic dystrophy type 2 were recruited. All patients were assessed for sarcopenia based on the revised European consensus definition. Patients with neuromuscular diseases were diagnosed according to the revised El Escorial criteria and the European neuromuscular centre criteria. Phenotypes and diagnostic criteria for all patients were summarized including their specific histopathological findings. RESULTS All patients with neuromuscular diseases were positively screened for sarcopenia and classified as severe sarcopenic by means of assessment. The clinical phenotype, the evolution pattern of weakness and muscle atrophy combined with laboratory finding including electromyography could unquestionably distinguish the diseases. DISCUSSION Neuromuscular disorders can manifest beyond the age of 65~years and misdiagnosed as sarcopenia. The most common diseases are inclusion body myositis, amyotrophic lateral sclerosis and myotonic dystrophy type 2. A diagnostic work-up for neuromuscular diseases ensures their correct diagnosis by clinical-, electrophysiological, histopathological, and genetic work-up. CONCLUSIONS In geriatric patients with a focal or asymmetrical muscular weakness and atrophy, sarcopenia assessment should be extended with patient's history of disease course. Furthermore, concomitant diseases, analysis of serum creatine kinase, electrophysiological examination, and in selected patients muscle biopsy and gene analysis is needed to rule out a late-onset neuromuscular disorder

Influence of IGF-I serum concentration on muscular regeneration capacity in patients with sarcopenia

BACKGROUND Previous research has described a neuroprotective effect of IGF-I, supporting neuronal survival, axon growth and proliferation of muscle cells. Therefore, the association between IGF-I concentration, muscle histology and electrophysiological markers in a cohort of patients with sarcopenia dares investigation. METHODS Measurement of serum concentrations of IGF-I and binding partners, electromyographic measurements with the MUNIX (Motor Unit Number Index) method and muscle biopsies were performed in 31 patients with acute hip fracture older age 60 years. Molecular markers for denervation (neural cell adhesion molecule NCAM) and proliferation markers (Ki67) were assessed by immunofluorescence staining of muscle biopsy tissue. Skeletal muscle mass by bioelectrical impedance analysis and hand-grip strength were measured to assess sarcopenia status according to EWGSOP2 criteria. RESULTS Thirty-one patients (20 women) with a mean age of 80.6 ± 7.4 years were included. Concentrations of IGF-I and its binding partners were significantly associated with sarcopenia (ß = - 0.360; p = 0.047) and MUNIX (ß = 0.512; p = 0.005). Further, expression of NCAM (ß = 0.380; p = 0.039) and Ki67 (ß = 0.424; p = 0.022) showed significant associations to IGF-I concentrations. CONCLUSIONS The findings suggest a pathogenetic role of IGF-I in sarcopenia based on muscle denervation

The Effect of Biochemical Remission on Bone Metabolism in Cushing's Syndrome: A 2‐Year Follow‐Up Study

Endogenous Cushing's syndrome (CS) is a rare cause of secondary osteoporosis. The long‐term consequences for bone metabolism after successful surgical treatment remain largely unknown. We assessed bone mineral density and fracture rates in 89 patients with confirmed Cushing's syndrome at the time of diagnosis and 2 years after successful tumor resection. We determined five bone turnover markers at the time of diagnosis, 1 and 2 years postoperatively. The bone turnover markers osteocalcin, intact procollagen‐IN‐propeptide (PINP), alkaline bone phosphatase, CTX‐I, and TrAcP 5b were measured in plasma or serum by chemiluminescent immunoassays. For comparison, 71 sex‐, age‐, and body mass index (BMI)‐matched patients in whom Cushing's syndrome had been excluded were studied. None of the patients received specific osteoanabolic treatment. At time of diagnosis, 69% of the patients had low bone mass (mean T‐score = −1.4 ± 1.1). Two years after successful surgery, the T‐score had improved in 78% of patients (mean T‐score 2 years postoperatively −1.0 ± 0.9). The bone formation markers osteocalcin and intact PINP were significantly decreased at time of diagnosis (p ≤ 0.001 and p = 0.03, respectively), and the bone resorption marker CTX‐I and TrAcP 5b increased. Postoperatively, the bone formation markers showed a three‐ to fourfold increase 1 year postoperatively, with a moderate decline thereafter. The bone resorption markers showed a similar but less pronounced course. This study shows that the phase immediately after surgical remission from endogenous CS is characterized by a high rate of bone turnover resulting in a striking net increase in bone mineral density in the majority of patients

Maximal oxygen uptake and fatty acid oxidation in athletic older men and women and healthy control

Introduction: Cardiopulmonary and musculoskeletal systems deteriorate through middle and into older age. This has a negative impact on physical capability and energy metabolism. The purpose of the present study was to determine the effects of ageing and exercise on peak rates of oxygen uptake (VO2peak) and fatty acid oxidation (PFO).Methods: All participants provided written, informed consent. Masters Athletes (MA: n=40, aged 37-90) specialised in endurance (n=10) or sprint running (n=30) were recruited during the 2012 European MA Championships in Zittau, Germany. Untrained (n=42, aged 18-67; 23 men and 16 women) were recruited from the general Manchester population (UK). The untrained participants also completed 12 weeks very high intensity sprint cycle training (4* 20s at 170% VO2max, 3/wk). VO2max and PFO were assessed using indirect calorimetry and incremental cycle ergometry. Statistical significance was gained by independent samples t-tests using IBM SPSS v.20.Results: The endurance and sprint trained MA were a similar age and had similar VO2max (Endurance MA: 47.22 ml/kg/min (±4.15) vs Sprint MA: 43.52 ml/kg/min (±2.21) p=0.416). Both MA groups were significantly higher than untrained people (38.86 ml/kg/min). MA sprinters and endurance runners had a VO2max similar to 19 years younger untrained, healthy people. Regression analysis showed that VO2max decreased by around 11% per decade after the age of 40 yrs in the MA group and 5% per decade after the age of 40 yrs in the untrained group. PFO was similar in endurance and sprint trained MA (Endurance: 8.09 mg/kg/min (±0.95) vs Sprint: 6.91 mg/kg/min (±0.53) p=0.284). In the untrained group, PFO was significantly lower than MA (p=0.006). Regression showed that PFO of MAs was similar to that of an untrained, healthy person 19 years younger. The sprint-training programme caused VO2max to increase by 10% (Pre: 38.86 ml/kg/min (±1.31) vs Post: 42.84 ml/kg/min (±1.24) p&lt;0.001) and PFO to increase by 18% (Pre: 5.57 mg/kg/min (±0.33) vs Post: 6.58 mg/kg/min (±0.41) p=0.050).Conclusion: These results show that MAs have a cardiopulmonary and metabolic fitness at levels equivalent to someone almost 20 yrs younger. Previously untrained middle-aged people can achieve substantial gains in fitness by completing relatively short duration, but high intensity sprint training and reach levels similar to those observed in the master athletes

Grip strength values and cut-off points based on over 200,000 adults of the German National Cohort - a comparison to the EWGSOP2 cut-off points

BACKGROUND: The European Working Group on Sarcopenia in Older People (EWGSOP) updated in 2018 the cut-off points for low grip strength to assess sarcopenia based on pooled data from 12 British studies. OBJECTIVE: Comparison of the EWGSOP2 cut-off points for low grip strength to those derived from a large German sample. METHODS: We assessed the grip strength distribution across age and derived low grip strength cut-off points for men and women (peak mean -2.5 × SD) based on 200,389 German National Cohort (NAKO) participants aged 19–75 years. In 1,012 Cooperative Health Research in the Region of Augsburg (KORA)-Age participants aged 65–93 years, we calculated the age-standardised prevalence of low grip strength and time-dependent sensitivity and specificity for all-cause mortality. RESULTS: Grip strength increased in the third and fourth decade of life and declined afterwards. Calculated cut-off points for low grip strength were 29 kg for men and 18 kg for women. In KORA-Age, the age-standardised prevalence of low grip strength was 1.5× higher for NAKO-derived (17.7%) compared to EWGSOP2 (11.7%) cut-off points. NAKO-derived cut-off points yielded a higher sensitivity and lower specificity for all-cause mortality. CONCLUSIONS: Cut-off points for low grip strength from German population-based data were 2 kg higher than the EWGSOP2 cut-off points. Higher cut-off points increase the sensitivity, thereby suggesting an intervention for more patients at risk, while other individuals might receive additional diagnostics/treatment without the urgent need. Research on the effectiveness of intervention in patients with low grip strength defined by different cut-off points is needed