Tissue Triage and Freezing for Models of Skeletal Muscle Disease

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal results from functional, cellular, molecular, and pathological evaluations. Due to the subtlety of some pathological abnormalities seen in congenital muscle disorders and the potential for fixation to interfere with the recognition of these features, pathological evaluation of frozen muscle is preferable to fixed muscle when evaluating skeletal muscle for congenital muscle disease. Additionally, the potential to produce severe freezing artifacts in muscle requires specific precautions when freezing skeletal muscle for histological examination that are not commonly used when freezing other tissues. This manuscript describes a protocol for rapid freezing of skeletal muscle using isopentane (2-methylbutane) cooled with liquid nitrogen to preserve optimal skeletal muscle morphology. This procedure is also effective for freezing tissue intended for genetic or protein expression studies. Furthermore, we have integrated our freezing protocol into a broader procedure that also describes preferred methods for the short term triage of tissue for (1) single fiber functional studies and (2) myoblast cell culture, with a focus on the minimum effort necessary to collect tissue and transport it to specialized research or reference labs to complete these studies. Overall, this manuscript provides an outline of how fresh tissue can be effectively distributed for a variety of phenotypic studies and thereby provides standard operating procedures (SOPs) for pathological studies related to congenital muscle disease

Effect of Muscle Length on Cross-Bridge Kinetics in Intact Cardiac Trabeculae at Body Temperature

Dynamic force generation in cardiac muscle, which determines cardiac pumping activity, depends on both the number of sarcomeric cross-bridges and on their cycling kinetics. The Frank–Starling mechanism dictates that cardiac force development increases with increasing cardiac muscle length (corresponding to increased ventricular volume). It is, however, unclear to what extent this increase in cardiac muscle length affects the rate of cross-bridge cycling. Previous studies using permeabilized cardiac preparations, sub-physiological temperatures, or both have obtained conflicting results. Here, we developed a protocol that allowed us to reliably and reproducibly measure the rate of tension redevelopment (ktr; which depends on the rate of cross-bridge cycling) in intact trabeculae at body temperature. Using K+ contractures to induce a tonic level of force, we showed the ktr was slower in rabbit muscle (which contains predominantly β myosin) than in rat muscle (which contains predominantly α myosin). Analyses of ktr in rat muscle at optimal length (Lopt) and 90% of optimal length (L90) revealed that ktr was significantly slower at Lopt (27.7 ± 3.3 and 27.8 ± 3.0 s−1 in duplicate analyses) than at L90 (45.1 ± 7.6 and 47.5 ± 9.2 s−1). We therefore show that ktr can be measured in intact rat and rabbit cardiac trabeculae, and that the ktr decreases when muscles are stretched to their optimal length under near-physiological conditions, indicating that the Frank–Starling mechanism not only increases force but also affects cross-bridge cycling kinetics

Nf-κb Inhibition Rescues Cardiac Function By Remodeling Calcium Genes In A Duchenne Muscular Dystrophy Model

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function

2 days versus 5 days of postoperative antibiotics for complex appendicitis:a pragmatic, open-label, multicentre, non-inferiority randomised trial

Background: The appropriate duration of postoperative antibiotics for complex appendicitis is unclear. The increasing global threat of antimicrobial resistance warrants restrictive antibiotic use, which could also reduce side-effects, length of hospital stay, and costs. Methods: In this pragmatic, open-label, non-inferiority trial in 15 hospitals in the Netherlands, patients with complex appendicitis (aged ≥8 years) were randomly assigned (1:1) to receive 2 days or 5 days of intravenous antibiotics after appendicectomy. Randomisation was stratified by centre, and treating physicians and patients were not masked to treatment allocation. The primary endpoint was a composite endpoint of infectious complications and mortality within 90 days. The main outcome was the absolute risk difference (95% CI) in the primary endpoint, adjusted for age and severity of appendicitis, with a non-inferiority margin of 7·5%. Outcome assessment was based on electronic patient records and a telephone consultation 90 days after appendicectomy. Efficacy was analysed in the intention-to-treat and per-protocol populations. Safety outcomes were analysed in the intention-to-treat population. This trial was registered with the Netherlands Trial Register, NL5946. Findings: Between April 12, 2017, and June 3, 2021, 13 267 patients were screened and 1066 were randomly assigned, 533 to each group. 31 were excluded from intention-to-treat analysis of the 2-day group and 30 from the 5-day group owing to errors in recruitment or consent. Appendicectomy was done laparoscopically in 955 (95%) of 1005 patients. The telephone follow-up was completed in 664 (66%) of 1005 patients. The primary endpoint occurred in 51 (10%) of 502 patients analysed in the 2-day group and 41 (8%) of 503 patients analysed in the 5-day group (adjusted absolute risk difference 2·0%, 95% CI −1·6 to 5·6). Rates of complications and re-interventions were similar between trial groups. Fewer patients had adverse effects of antibiotics in the 2-day group (45 [9%] of 502 patients) than in the 5-day group (112 [22%] of 503 patients; odds ratio [OR] 0·344, 95% CI 0·237 to 0·498). Re-admission to hospital was more frequent in the 2-day group (58 [12%] of 502 patients) than in the 5-day group (29 [6%] of 503 patients; OR 2·135, 1·342 to 3·396). There were no treatment-related deaths. Interpretation: 2 days of postoperative intravenous antibiotics for complex appendicitis is non-inferior to 5 days in terms of infectious complications and mortality within 90 days, based on a non-inferiority margin of 7·5%. These findings apply to laparoscopic appendicectomy conducted in a well resourced health-care setting. Adopting this strategy will reduce adverse effects of antibiotics and length of hospital stay. Funding: The Netherlands Organization for Health Research and Development.</p

Prevalence of Age-Related Macular Degeneration in Europe: The Past and the Future

Purpose Age-related macular degeneration (AMD) is a frequent, complex disorder in elderly of European ancestry. Risk profiles and treatment options have changed considerably over the years, which may have affected disease prevalence and outcome. We determined the prevalence of early and late AMD in Europe from 1990 to 2013 using the European Eye Epidemiology (E3) consortium, and made projections for the future. Design Meta-analysis of prevalence data. Participants A total of 42 080 individuals 40 years of age and older participating in 14 population-based cohorts from 10 countries in Europe. Methods AMD was diagnosed based on fundus photographs using the Rotterdam Classification. Prevalence of early and late AMD was calculated using random-effects meta-analysis stratified for age, birth cohort, gender, geographic region, and time period of the study. Best-corrected visual acuity (BCVA) was compared between late AMD subtypes; geographic atrophy (GA) and choroidal neovascularization (CNV). Main Outcome Measures Prevalence of early and late AMD, BCVA, and number of AMD cases. Results Prevalence of early AMD increased from 3.5% (95% confidence interval [CI] 2.1%–5.0%) in those aged 55–59 years to 17.6% (95%