The number of podocyte and slit diaphragm is decreased in experimental diabetic nephropathy

Purpose: To determine the number of podocyte, slit diaphragms, slit diaphragm extensions and GBM thickness in diabetic nephropathy. Methods: Sixty Rattus Wistarof both sexes weighing 200-300g were divided in two experimental groups: normal group 10 animals, and alloxan diabetic rats - 50 animals. Alloxan was administered in a single IV dose of 42mg/kg body weight. Body weight, water and food intake, diuresis, and blood and urine glucose were determined in both groups before alloxan injection and two weeks, six and twelve months after alloxan injection. Proteinuria was measured at 12 months in both groups. After 12 months animals were sacrificed, and the right kidney processed for electron microscopy. Results: Clear clinical and laboratory signs of severe diabetes were seen, in all alloxan-diabetic rats at all follow-up times. Glomerular basement membrane (GBM) thickening, podocyte number, and slit diaphragm number and extension were determined. GBM of all diabetic rats was significantly thicker (median=0.29μm; semi-interquartile range=0.065μm) than in the normal rats (0.23μm; 0.035μm). Diabetic rat podocyte number (8; 1), slit diaphragm number (4; 1), and slit diaphragm extension (0.021μm; 0.00435μm) were significantly lower than in normal rats (11; 1) and (7; 1.5), and (0.031μm; 0.0058μm). Diabetic rat proteinuria (0.060mg/24h; 0.037mg/24h) was higher than in normal rats (0.00185mg/24h; 0.00055mg/24h). Conclusion: Experimental diabetes is associated with significant (p<0.05) changes in podocyte foot process, slit number, slit diaphragm extension, and GBM thickness.

Ultrastructural Study of the Denervated Diaphragm in Rats

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Heart Failure-Induced Diaphragm Myopathy

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Aerobic training attenuates nicotinic acetylcholine receptor changes in the diaphragm muscle during heart failure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Aerobic training attenuates nicotinic acethylcholine receptor changes in the diaphragm muscle during heart failure

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Muscle reorganisation through local injection of stem cells in the diaphragm of mdx mice

Background: The diaphragm is the major respiratory muscle affected by Duchenne muscular dystrophy (DMD) and is responsible for causing 80% of deaths. The use of mechanical forces that act on the body or intermittent pressure on the airways improves the quality of life of patients but does not prevent the progression of respiratory failure. Thus, diseases that require tissue repair, such as DMD, represent a group of pathologies that have great potential for cell therapy. The application of stem cells directly into the diaphragm instead of systemic application can reduce cell migration to other affected areas and increase the chances of muscle reorganisation. The mdx mouse is a suitable animal model for this research because its diaphragmatic phenotype is similar to human DMD. Therefore, the aim of this study was to assess the potential cell implantation in the diaphragm muscle after the xenotransplantation of stem cells. Methods: A total of 9 mice, including 3 control BALB/Cmice, 3 5-month-old mdx mice without stem cell injections and 3 mdx mice injected with stem cells, were used. The animals injected with stem cells underwent laparoscopy so that stem cells from GFP-labelled rabbit olfactory epithelium could be locally injected into the diaphragm muscle. After 8 days, all animals were euthanised, and the diaphragm muscle was dissected and subjected to histological and immunohistochemical analyses. Results: Both the fresh diaphragm tissue and immunohistochemical analyses showed immunopositive GFP labelling of some of the cells and immunonegativity of myoblast bundles. In the histological analysis, we observed a reduction in the inflammatory infiltrate as well as the presence of a few peripheral nuclei and myoblast bundles. Conclusion: We were able to implant stem cells into the diaphragm via local injection, which promoted moderate muscle reorganisation. The presence of myoblast bundles cannot be attributed to stem cell incorporation because there was no immunopositive labelling in this structure. It is believed that the formation of the bundles may have been stimulated by cellular signalling mechanisms that have not yet been elucidated.

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.

Computational and experimental characterization of a low-cost piezoelectric valveless diaphragm pump

Flow pumps act as important devices in areas such as Bioengineering, Medicine, and Pharmacy, among other areas of Engineering, mainly for delivering liquids or gases at small-scale and precision flow rate quantities. Principles for pumping fluids based on piezoelectric actuators have been widely studied, since they allow the construction of pump systems for displacement of small fluid volumes with low power consumption. This work studies valveless piezoelectric diaphragm pumps for flow generation, which uses a piezoelectric ceramic (PZT) as actuator to move a membrane (diaphragm) up and down as a piston. The direction of the flow is guaranteed by valveless configuration based on a nozzle-diffuser system that privileges the flow in just one pumping direction. Most research efforts on development of valveless flow pump deal either with computational simulations based on simplified models or with simplified physical approaches based on analytical models. The main objective of this work is the study of a methodology to develop a low-cost valveless piezoelectric diaphragm flow pump using computational simulations, parametric study, prototype manufacturing, and experimental characterization. The parametric study has shown that the eccentricity of PZT layer and metal layer plays a key role in the performance of the pump.

Examination of FGFRL1 as a candidate gene for diaphragmatic defects at chromosome 4p16.3 shows that Fgfrl1 null mice have reduced expression of Tpm3, sarcomere genes and Lrtm1 in the diaphragm

Fgfrl1 (also known as Fgfr5; OMIM 605830) homozygous null mice have thin, amuscular diaphragms and die at birth because of diaphragm hypoplasia. FGFRL1 is located at 4p16.3, and this chromosome region can be deleted in patients with congenital diaphragmatic hernia (CDH). We examined FGFRL1 as a candidate gene for the diaphragmatic defects associated with 4p16.3 deletions and re-sequenced this gene in 54 patients with CDH. We confirmed six known coding single nucleotide polymorphisms (SNPs): c.209G > A (p.Pro20Pro), c.977G > A (p.Pro276Pro), c.1040T > C (p.Asp297Asp), c.1234C > A (p.Pro362Gln), c.1420G > T (p.Arg424Leu), and c.1540C > T (p.Pro464Leu), but we did not identify any gene mutations. We genotyped additional CDH patients for four of these six SNPs, including the three non-synonymous SNPs, to make a total of 200 chromosomes, and found that the allele frequency for the four SNPs, did not differ significantly between patients and normal controls (p > or = 0.05). We then used Affymetrix Genechip Mouse Gene 1.0 ST arrays and found eight genes with significantly reduced expression levels in the diaphragms of Fgfrl1 homozygous null mice when compared with wildtype mice-Tpm3, Fgfrl1 (p = 0.004), Myl2, Lrtm1, Myh4, Myl3, Myh7 and Hephl1. Lrtm1 is closely related to Slit3, a protein associated with herniation of the central tendon of the diaphragm in mice. The Slit proteins are known to regulate axon branching and cell migration, and inhibition of Slit3 reduces cell motility and decreases the expression of Rac and Cdc42, two genes that are essential for myoblast fusion. Further studies to determine if Lrtm1 has a similar function to Slit3 and if reduced Fgfrl1 expression can cause diaphragm hypoplasia through a mechanism involving decreased myoblast motility and/or myoblast fusion, seem indicated.

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.

Reduction of airspace after lung resection through controlled paralysis of the diaphragm

OBJECTIVES: Residual airspace following thoracic resections is a common clinical problem. Persistent air leak, prolonged drainage time, and reduced hemostasis extend hospital stay and morbidity. We report a trial of pharmacologic-induced diaphragmatic paralysis through continuous paraphrenic injection of lidocaine to reduced residual airspace. The objectives were confirmation of diaphragmatic paralysis and possible procedure related complications. METHODS: Six eligible patients undergoing resectional surgery (lobectomy or bilobectomy) were included. Inclusion criteria consisted of: postoperative predicted FEV1 greater than 1300 ml, right-sided resection, absence of parenchymal lung disease, no class III antiarrhythmic therapy, absence of hypersensitivity reactions to lidocaine, no signs of infection, and informed consent. Upon completion of resection an epidural catheter was attached in the periphrenic tissue on the proximal pericardial surface, externalized through a separate parasternal incision, and connected to a perfusing system injecting lidocaine 1% at a rate of 3 ml/h (30 mg/h). Postoperative ICU surveillance for 24h and daily measurement of vital signs, drainage output, and bedside spirometry were performed. Within 48 h fluoroscopic confirmation of diaphragmatic paralysis was obtained. The catheter removal coincided with the chest tube removal when no procedural related complications occurred. RESULTS: None of the patients reported respiratory impairment. Diaphragmatic paralysis was documented in all patients. Upon removal of catheter or discontinuation of lidocaine prompt return of diaphragmatic motility was noticed. Two patients showed postoperative hemodynamic irrelevant atrial fibrillation. CONCLUSION: Postoperative paraphrenic catheter administration of lidocaine to ensure reversible diaphragmatic paralysis is safe and reproducible. Further studies have to assess a benefit in terms of reduction in morbidity, drainage time, and hospital stay, and determine the patients who will profit.

Mice with a targeted disruption of the Fgfrl1 gene die at birth due to alterations in the diaphragm

FGFRL1 is a recently discovered member of the fibroblast growth factor receptor family that is lacking the intracellular tyrosine kinase domain. To elucidate the function of the novel receptor, we created mice with a targeted disruption of the Fgfrl1 gene. These mice develop normally until term, but die within a few minutes after birth due to respiratory failure. The respiratory problems are explained by a significant reduction in the size of the diaphragm muscle, which is not sufficient to inflate the lungs after birth. The remaining portion of the diaphragm muscle appears to be well developed and innervated. It consists of differentiated myofibers with nuclei at the periphery. Fast and slow muscle fibers occur in normal proportions. The myogenic regulatory factors MyoD, Myf5, myogenin and Mrf4 and the myocyte enhancer factors Mef2A, Mef2B, Mef2C and Mef2D are expressed at normal levels. Experiments with a cell culture model involving C2C12 myoblasts show that Fgfrl1 is expressed during the late stages of myotube formation. Other skeletal muscles do not appear to be affected in the Fgfrl1 deficient mice. Thus, Fgfrl1 plays a critical role in the development of the diaphragm.

The feasibility of non-viral gene transfer to the diaphragm in vivo

Gene transfer using electroporation is an essential method for the study of developmental biology, especially to understand the internal control of degeneration and apoptosis of the muscle cells that occurs earlier and quicker than the usual degeneration process occurring by aging. Such experimental studies may have a role in developing new strategies for treating patients suffering from inherited primary myopathies such as Duchenne muscular dystrophy (DMD). The present study was designed to evaluate the feasibility of electroporation mediated transfer of reporter genes to the diaphragm in vivo. This is the first report of gene transfer of naked plasmid DNA into the diaphragm muscle in vivo using electroporation. Our results showed that in vivo gene transfer of naked plasmid DNA into the diaphragm muscle using electroporation is feasible.

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.