EPA protects against muscle damage in the mdx mouse model of Duchenne muscular dystrophy by promoting a shift from the M1 to M2 macrophage phenotype

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Heart Failure-Induced Diaphragm Myopathy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Electric Ventilation: indications for and technical aspects of diaphragm pacing stimulation surgical implantation

Objective: Patients with high cervical spinal cord injury are usually dependent on mechanical ventilation support, which, albeit life saving, is associated with complications and decreased life expectancy because of respiratory infections. Diaphragm pacing stimulation (DPS), sometimes referred to as electric ventilation, induces inhalation by stimulating the inspiratory muscles. Our objective was to highlight the indications for and some aspects of the surgical technique employed in the laparoscopic insertion of the DPS electrodes, as well as to describe five cases of tetraplegic patients submitted to the technique. Methods: Patient selection involved transcutaneous phrenic nerve studies in order to determine whether the phrenic nerves were preserved. The surgical approach was traditional laparoscopy, with four ports. The initial step was electrical mapping in order to locate the "motor points" (the points at which stimulation would cause maximal contraction of the diaphragm). If the diaphragm mapping was successful, four electrodes were implanted into the abdominal surface of the diaphragm, two on each side, to stimulate the branches of the phrenic nerve. Results: Of the five patients, three could breathe using DPS alone for more than 24 h, one could do so for more than 6 h, and one could not do so at all. Conclusions: Although a longer follow-up period is needed in order to reach definitive conclusions, the initial results have been promising. At this writing, most of our patients have been able to remain ventilator-free for long periods of time.

Ultrastructural changes in diaphragm neuromuscular junctions in a severe mouse model for Spinal Muscular Atrophy and their prevention by bifunctional U7 snRNA correcting SMN2 splicing

In Spinal Muscular Atrophy (SMA), the SMN1 gene is deleted or inactivated. Because of a splicing problem, the second copy gene, SMN2, generates insufficient amounts of functional SMN protein, leading to the death of spinal cord motoneurons. For a "severe" mouse SMA model (Smn -/-, hSMN2 +/+; with affected pups dying at 5-7 days), which most closely mimicks the genetic set-up in human SMA patients, we characterise SMA-related ultrastructural changes in neuromuscular junctions (NMJs) of two striated muscles with discrete functions. In the diaphragm, but not the soleus muscle of 4-days old SMA mice, mitochondria on both sides of the NMJs degenerate, and perisynaptic Schwann cells as well as endoneurial fibroblasts show striking changes in morphology. Importantly, NMJs of SMA mice in which a modified U7 snRNA corrects SMN2 splicing and delays or prevents SMA symptoms are normal. This ultrastructural study reveals novel features of NMJ alterations - in particular the involvement of perisynaptic Schwann cells - that may be relevant for human SMA pathogenesis.

Relative impact of respiratory muscle activity on tidal flow and end expiratory volume in healthy neonates

INTRODUCTION: It has been suggested that infants dynamically regulate their tidal flow and end-expiratory volume level. The interaction between muscle activity, flow and lung volume in spontaneously sleeping neonates is poorly studied, since it requires the assessment of transcutaneous electromyography of respiratory muscles (rEMG) in matched comparison to lung function measurements. METHODS: After determining feasibility and repeatability of rEMG in 20 spontaneously sleeping healthy neonates, we measured the relative impact of intercostal and diaphragmatic EMG activity in direct comparison to the resulting tidal flow and FRC. RESULTS: We found good feasibility, repeatability and correlation of timing indices between rEMG activity and flow. The rEMG amplitude was significantly dependent on the resistive load of the face mask. Diaphragm and intercostal muscle activity commenced prior to the onset of flow and remained active during the expiratory cycle. The relative contribution of intercostal and diaphragmatic activity to flow was variable and changed dynamically. CONCLUSION: Using matched rEMG, air flow and lung volume measurements, we have found good feasibility and repeatability of intercostal and diaphragm rEMG measurements and provide the first quantitative measures of the temporal relationship between muscle activity and flow in spontaneously sleeping healthy neonates. Lung mechanical function is dynamically regulated and adapts on a breath to breath basis. So, non-invasive rEMG measurements alone or in combination with lung function might provide a more comprehensive picture of pulmonary mechanics in future studies. The data describing the timing of EMG and flow may be important for future studies of EMG triggered mechanical ventilation.

The craniosacral progression of muscle development influences the emergence of neuromuscular junction alterations in a severe murine model for spinal muscular atrophy.

AIMS As 4-day-old mice of the severe spinal muscular atrophy (SMA) model (dying at 5-8 days) display pronounced neuromuscular changes in the diaphragm but not the soleus muscle, we wanted to gain more insight into the relationship between muscle development and the emergence of pathological changes and additionally to analyse intercostal muscles which are affected in human SMA. METHODS Structures of muscle fibres and neuromuscular junctions (NMJs) of the diaphragm, intercostal and calf muscles of prenatal (E21) and postnatal (P0 and P4) healthy and SMA mice were analysed by light and transmission electron microscopy. NMJ innervation was studied by whole mount immunofluorescence in diaphragms of P4 mice. RESULTS During this period, the investigated muscles still show a significant neck-to-tail developmental gradient. The diaphragm and calf muscles are most and least advanced, respectively, with respect to muscle fibre fusion and differentiation. The number and depth of subsynaptic folds increases, and perisynaptic Schwann cells (PSCs) acquire a basal lamina on their outer surface. Subsynaptic folds are connected to an extensive network of tubules and beaded caveolae, reminiscent of the T system in adult muscle. Interestingly, intercostal muscles from P4 SMA mice show weaker pathological involvement (that is, vacuolization of PSCs and perineurial cells) than those previously described by us for the diaphragm, whereas calf muscles show no pathological changes. CONCLUSION SMA-related alterations appear to occur only when the muscles have reached a certain developmental maturity. Moreover, glial cells, in particular PSCs, play an important role in SMA pathogenesis.

The FgfrL1 receptor is required for development of slow muscle fibers.

FgfrL1, which interacts with Fgf ligands and heparin, is a member of the fibroblast growth factor receptor (Fgfr) family. FgfrL1-deficient mice show two significant alterations when compared to wildtype mice: They die at birth due to a malformed diaphragm and they lack metanephric kidneys. Utilizing gene arrays, qPCR and in situ hybridization we show here that the diaphragm of FgfrL1 knockout animals lacks any slow muscle fibers at E18.5 as indicated by the absence of slow fiber markers Myh7, Myl2 and Myl3. Similar lesions are also found in other skeletal muscles that contain a high proportion of slow fibers at birth, such as the extraocular muscles. In contrast to the slow fibers, fast fibers do not appear to be affected as shown by expression of fast fiber markers Myh3, Myh8, Myl1 and MylPF. At early developmental stages (E10.5, E15.5), FgfrL1-deficient animals express slow fiber genes at normal levels. The loss of slow fibers cannot be attributed to the lack of kidneys, since Wnt4 knockout mice, which also lack metanephric kidneys, show normal expression of Myh7, Myl2 and Myl3. Thus, FgfrL1 is specifically required for embryonic development of slow muscle fibers.

Central and peripheral defects in motor units of the diaphragm of spinal muscular atrophy mice.

Spinal muscular atrophy (SMA) is characterized by motoneuron loss and muscle weakness. However, the structural and functional deficits that lead to the impairment of the neuromuscular system remain poorly defined. By electron microscopy, we previously found that neuromuscular junctions (NMJs) and muscle fibres of the diaphragm are among the earliest affected structures in the severe mouse SMA model. Because of certain anatomical features, i.e. its thinness and its innervation from the cervical segments of the spinal cord, the diaphragm is particularly suitable to characterize both central and peripheral events. Here we show by immunohistochemistry that, at postnatal day 3, the cervical motoneurons of SMA mice receive less stimulatory synaptic inputs. Moreover, their mitochondria become less elongated which might represent an early stage of degeneration. The NMJs of the diaphragm of SMA mice show a loss of synaptic vesicles and active zones. Moreover, the partly innervated endplates lack S100 positive perisynaptic Schwann cells (PSCs). We also demonstrate the feasibility of comparing the proteomic composition between diaphragm regions enriched and poor in NMJs. By this approach we have identified two proteins that are significantly upregulated only in the NMJ-specific regions of SMA mice. These are apoptosis inducing factor 1 (AIFM1), a mitochondrial flavoprotein that initiates apoptosis in a caspase-independent pathway, and four and a half Lim domain protein 1 (FHL1), a regulator of skeletal muscle mass that has been implicated in several myopathies.

Experimental muscle pain changes feedforward postural responses of the trunk muscles

Many studies have identified changes in trunk muscle recruitment in clinical low back pain (LBP). However, due to the heterogeneity of the LBP population these changes have been variable and it has been impossible to identify a cause-effect relationship. Several studies have identified a consistent change in the feed-forward postural response of transversus abdominis (TrA), the deepest abdominal muscle, in association with arm movements in chronic LBP. This study aimed to determine whether the feedforward recruitment of the trunk muscles in a postural task could be altered by acute experimentally induced LBP. Electromyographic (EMG) recordings of the abdominal and paraspinal muscles were made during arm movements in a control trial, following the injection of isotonic (non-painful) and hypertonic (painful) saline into the longissimus muscle at L4, and during a 1-h follow-up. Movements included rapid arm flexion in response to a light and repetitive arm flexion-extension. Temporal and spatial EMG parameters were measured. The onset and amplitude of EMG of most muscles was changed in a variable manner during the period of experimentally induced pain. However, across movement trials and subjects the activation of TrA was consistently reduced in amplitude or delayed. Analyses in the time and frequency domain were used to confirm these findings. The results suggest that acute experimentally induced pain may affect feedforward postural activity of the trunk muscles. Although the response was variable, pain produced differential changes in the motor control of the trunk muscles, with consistent impairment of TrA activity.

Reduced inspiratory muscle endurance following successful weaning from prolonged mechanical ventilation

Study objectives: Respiratory muscle weakness and decreased endurance have been demonstrated following mechanical ventilation. However, its relationship to the duration of mechanical ventilation is not known. The aim of this study was to assess respiratory muscle endurance and its relationship to the duration of mechanical ventilation. Design: Prospective study. Setting: Tertiary teaching hospital ICU. Patients: Twenty subjects were recruited for the study who had received mechanical ventilation for a 48 h and had been discharged from the ICU. Measurements: FEV1 FVC, and maximal inspiratory pressure (Pimax) at functional residual capacity were recorded. The Pimax attained following resisted inspiration at 30% of the initial Pimax for 2 min was recorded, and the fatigue resistance index (FRI) [Pimax final/Pimax initial] was calculated. The duration of ICU length of stay (ICULOS), duration of mechanical ventilation (MVD), duration of weaning (WD), and Charlson comorbidities score (CCS) were also recorded. Relationships between fatigue and other parameters were analyzed using the Spearman correlations (p). Results: Subjects were admitted to the ICU for a mean duration of 7.7 days (SD, 3.7 days) and required mechanical ventilation for a mean duration of 4.6 days (SD, 2.5 days). The mean FRI was 0.88 (SD, 0.13), indicating a 12% fall in Pimax, and was negatively correlated with MVD (r = -0.65; p = 0.007). No correlations were found between the FRI and FEV1, FVC, ICULOS, WD, or CCS. Conclusions: Patients who had received mechanical ventilation for > 48 h have reduced inspiratory muscle endurance that worsens with the duration of mechanical ventilation and is present following successful weaning. These data suggest that patients needing prolonged mechanical ventilation are at risk of respiratory muscle fatigue and may benefit from respiratory muscle training.

Glycinergic and GABAergic synaptic activity differentially regulate motoneuron survival and skeletal muscle innervation

GABAergic and glycinergic synaptic transmission is proposed to promote the maturation and refinement of the developing CNS. Here we provide morphological and functional evidence that glycinergic and GABAergic synapses control motoneuron development in a region-specific manner during programmed cell death. In gephyrin-deficient mice that lack all postsynaptic glycine receptor and some GABA(A) receptor clusters, there was increased spontaneous respiratory motor activity, reduced respiratory motoneuron survival, and decreased innervation of the diaphragm. In contrast, limb-innervating motoneurons showed decreased spontaneous activity, increased survival, and increased innervation of their target muscles. Both GABA and glycine increased limb-innervating motoneuron activity and decreased respiratory motoneuron activity in wild-type mice, but only glycine responses were abolished in gephyrin-deficient mice. Our results provide genetic evidence that the development of glycinergic and GABAergic synaptic inputs onto motoneurons plays an important role in the survival, axonal branching, and spontaneous activity of motoneurons in developing mammalian embryos.

The toxic effects of anticholinesterases on muscle

It has been shown that acute administration of ecothiopate iodine in vivo caused an approximate 80% depression of acetylcholinesterase activity in the diaphragms of mice. Inhibition of acetylcholinesterase was accompanied by an influx of calcium at the junctional region of the diaphragm, which continued during subsequent progressive development of a severe myopathy located in the same region. Myopathy was accompanied by loss of creatine kinase from the muscle and was represented, at the light microscope level, by hypercontraction, Procion Yellow staining and loss of cross striations within the muscle fibres. It appeared to reach a point of maximum severity approximately 3-6 hours after ecothiopate administration and then, by means of some repair/regeneration process, regained an apparently normal morphology within 72 hours of the intoxication. At the ultrastructural level, ecothiopate-induced myopathy was recognised by loss of Z-lines, swelling and vacuolation of mitochondria and sarcoplasmic reticulum, dissarray of myofilaments, crystal formation, and sometimes, by the complete obliteration of sarcomeric structure. The development of myopathy in vitro was shown to be nerve-mediated and to require a functional acetylcholine receptor for its development It was successfully treated therapeutically in vivo by pyridine-2-aldoxime methiodide and prophylactically by pyridostigmine bromide. However, the use of a range of membrane-on channel blockers, and of leupeptin, an inhibitor of calcium-activated-neutral-protease, have been unsuccessful in the prevention of ecothiopate-induced myopathy.