BackgroundSkeletal muscle aging is associated with a decreased regenerative potential due to the loss of function of endogenous stem cells or myogenic progenitor cells (MPCs). Aged skeletal muscle is characterized by the deposition of extracellular matrix (ECM), which in turn influences the biomechanical properties of myofibers by increasing their stiffness. Since the stiffness of the MPC microenvironment directly impacts MPC function, we hypothesized that the increase in muscle stiffness that occurs with aging impairs the behavior of MPCs, ultimately leading to a decrease in regenerative potential.ResultsWe showed that freshly isolated individual myofibers from aged mouse muscles contain fewer MPCs overall than myofibers from adult muscles, with fewer quiescent MPCs and more proliferative and differentiating MPCs. We observed alterations in cultured MPC behavior in aged animals, where the proliferation and differentiation of MPCs were lower and higher, respectively. These alterations were not linked to the intrinsic properties of aged myofibers, as shown by the similar values for the cumulative population-doubling values and fusion indexes. However, atomic force microscopy (AFM) indentation experiments revealed a nearly 4-fold increase in the stiffness of the MPC microenvironment. We further showed that the increase in stiffness is associated with alterations to muscle ECM, including the accumulation of collagen, which was correlated with higher hydroxyproline and advanced glycation end-product content. Lastly, we recapitulated the impaired MPC behavior observed in aging using a hydrogel substrate that mimics the stiffness of myofibers.ConclusionsThese findings provide novel evidence that the low regenerative potential of aged skeletal muscle is independent of intrinsic MPC properties but is related to the increase in the stiffness of the MPC microenvironment.
BackgroundThe stiffness of the myogenic stem cell microenvironment markedly influences the ability to regenerate tissue. We studied the effect of damaged myofibers on myogenic progenitor cell (MPC) proliferation and determined whether the structural integrity of the microenvironment contributes to phenotypic changes.MethodsIndividual myofibers were isolated and cultured for 6 days. During this period, the cytoskeleton of myofibers and transcription factors regulating MPC differentiation were characterized by immunostaining. Atomic Force Microscopy (AFM) was performed to measure stiffness of cultured myofibers. Healthy and damaged myofibers, and their associated MPCs, were studied in skeletal muscle from dystrophic and tenotomy mouse models. MPCs were cultured on stiffness-tunable substrates, and their phenotypes were assessed by immunostaining of myogenic transcription factors.ResultsWe showed that individual myofibers tend to shrink or collapse when cultured ex vivo starting from day 1 and that this is associated with a marked increase in the number of proliferative MPCs (Pax7+MyoD+). The myofibers collapsed due to a loss of viability as shown by Evans blue dye uptake and the disorganization of their cytoskeletons. Interestingly, collapsed myofibers in mdx skeletal muscles were similar to damaged myofibers in that they lose their viability, have a disorganized cytoskeleton (actin and α-actinin), and display local MPC (MyoD+) proliferation at their periphery. In a tenotomy model that causes loss of muscle tension, the cytoskeletal disorganization of myofibers also correlated with the activation/proliferation of MPCs. A deeper analysis of collapsed myofibers revealed that they produce trophic factors that influence MPC proliferation. In addition, collapsed myofibers expressed several genes related to the basal lamina. Immunostaining revealed the presence of fibronectin in the basal lamina and the cytoplasm of damaged myofibers. Lastly, using atomic force microscopy (AFM), we showed that collapsed myofibers exhibit greater stiffness than intact myofibers. Growing MPCs on a 2-kPa polyacrylamide-based substrate, exempt of additional microenvironmental cues, recapitulated proliferation and reduced spontaneous differentiation compared to growth on a 0.5-kPa substrate.ConclusionsOur results support the notion that collapsed or damaged myofibers increase the structural stiffness of the satellite cell microenvironment, which in addition to other cues such as trophic factors and changes in extracellular matrix composition, promotes the proliferation and maintenance of MPCs, required for myofiber repair.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0030-1) contains supplementary material, which is available to authorized users.
Background and Objective:Ambulatory total laparoscopic hysterectomy (TLH) could lead to significant cost savings, but some fear the effects of what could be premature postsurgical discharge. We sought to estimate the feasibility and safety of TLH as an outpatient procedure for benign gynecologic conditions.Methods:We report a prospective, consecutive case series of 128 outpatient TLHs performed for benign gynecologic conditions in a tertiary care center.Results:Of the 295 women scheduled for a TLH, 151 (51%) were attempted as an outpatient procedure. A total of 128 women (85%) were actually discharged home the day of their surgery. The most common reasons for admission the same day were urinary retention (19%) and nausea (15%). Indications for hysterectomy were mainly leiomyomas (62%), menorrhagia (24%), and pelvic pain (9%). Endometriosis and adhesions were found in 23% and 25% of the cases, respectively. Mean estimated blood loss was 56 mL and mean uterus weight was 215 g, with the heaviest uterus weighing 841 g. Unplanned consultation and readmission were infrequent, occurring in 3.1% and 0.8% of cases, respectively, in the first 72 hours. At 3 months, unplanned consultation, complication, and readmission had occurred in a similar proportion of inpatient and outpatient TLHs (17.2%, 12.5%, and 4.7% versus 18.1%, 12.7%, and 5.4%, respectively). In a logistic regression model, uterus weight, presence of adhesions or endometriosis, and duration of the operation were not associated with adverse outcomes.Conclusion:Same-day discharge is a feasible and safe option for carefully selected patients who undergo an uncomplicated TLH, even in the presence of leiomyomas, severe adhesions, or endometriosis.
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