Finally, for extracellular matrix quantification, we used Sirius red staining. Specifically, we quantified the minimum Feret diameter, centrally nucleated fibers and the number of macrophages, starting from multiple images. Our pipelines consist of running image-processing modules in CellProfiler with the aim of quantifying different histopathological muscle hallmarks in mdx mice compared to their wild-type littermates.
#Cellprofiler fiber analysis software
Here, we describe a simple automatized computational approach to quantify muscle parameters with specific pipelines to be run by CellProfiler software in an open-source and well-defined fashion.
Indeed, non-automatic methods are time-consuming and prone to error. A well-defined and standardized approach for histological and morphometric analysis of muscle samples is necessary in order to measure and quantify specific regenerative parameters in myopathies. DMD is characterized by different features, such as continuous cycles of degeneration/regeneration, fiber heterogeneity, chronic inflammation and fibrosis. Therefore, here, we summarise and explain the rationale behind different histological techniques used in the literature to assess degeneration and regeneration in the field of dystrophinopathies, focusing especially on those related to DMD.Īdult skeletal muscle is capable of active and efficient differentiation in the event of injury in both physiological and pathological conditions, such as in Duchenne muscular dystrophy (DMD). Nevertheless, the complexity of the molecular events that take place in dystrophic muscles, together with the rise of a multitude of markers for each of the phases of the process, makes the histological assessment a challenging task. In this regard, the later advances in microscopy and image acquisition systems and the current expansion of antibodies for immunohistological evaluation together with the development of different spectrum fluorescent dyes have made histology a crucial tool. In addition to in vivo functional tests, ex vivo molecular evaluation aids assess to what extent the therapy has contributed to the regenerative process. Advances in genetic and exon-skipping therapies are the most promising intervention, the safety and efficiency of which are tested in animal models. Presently, there is no cure for DMD, but different treatments help manage some of the symptoms. Moreover, absence of the functional protein affects the expression and function of proteins within the DAPC, leading to molecular events responsible for myofibre damage, muscle weakening, disability and, eventually, premature death. This protein contributes to the stabilisation of striated cells during contraction, as it anchors the cytoskeleton with components of the extracellular matrix through the dystrophin-associated protein complex (DAPC). Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback.Duchenne muscular dystrophy (DMD) is a progressive disease caused by the loss of function of the protein dystrophin. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites.
0 kommentar(er)
