Myocardial fibrosis is unaltered by long-term administration of L-arginine in dystrophin deficient mdx mice: a histomorphometric analysis.

Cardiac failure secondary to myocardial fibrosis (MF) significantly contributes to death in Duchenne muscular dystrophy (DMD), a fatal form of muscle disease. In aging, the mdx mice, an animal model of DMD, MF is similar to that observed in humans. Nitric oxide-based therapy has been proposed to retard MF in DMD and a candidate is L-arginine (L-arg). In this study we evaluated the effects of long-term therapy with L-arg in the MF of mdx mice. mdx mice (6 months old) were treated with L-arg in drinking water. Control mdx mice received water only. After 15 months of treatment, hearts were stained with Masson's trichrome for analysis of MF and with hematoxilyn and eosin for analysis of inflammation and cardiomyocyte damage. We observed that MF was not affected (29.5 +/- 2.5% of MF area for control vs 31.4 +/- 2% for L-arginine-treated animals; P > 0.05). The density of inflammatory cells was reduced (169 +/- 12 cells/mm 2 in control vs 102 +/- 9 cells/mm 2 in L-arg-treated; P < 0.05). The present study shows that long-term administration of L-arg is not effective in retarding MF in mdx dystrophinopathy.

Alterations in the permeability of dystrophic fibers during neuromuscular junction development.

In the mdx mice, lack of dystrophin leads to increases in calcium influx and myonecrosis, followed by muscle regeneration. Synapse elimination is faster in mdx than in controls, suggesting that increases in calcium influx during development could be involved. In the present study, we evaluated whether dystrophic fibers display changes in permeability to Evans Blue Dye (EBD) during development of the neuromuscular junction. EBD is a sensitive label for the early detection of increased myofiber permeability and sarcolemmal damage. After intraperitoneal injection of EBD, sternomastoid (STN) and tibialis anterior (T. anterior) muscles were analyzed with fluorescence microscopy. At 01, 07 and 14 days of age, STN and TA mdx myofibers were not stained with EBD. At 21 days of age, positive labeling of TA and STN mdx myofibers was seen, suggesting permeability modification and myonecrosis. Adult muscles showed a decrease (T. anterior) or no changes (STN) in the amount of EBD-positive fibers. These results suggest that there is no sarcolemmal damage detected by EBD during development of dystrophic neuromuscular junctions and other factors may contribute to the earlier synapse elimination seen in dystrophic muscle.

Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells.

Duchenne muscular dystrophy is one of the most devastating myopathies. Muscle fibers undergo necrosis and lose their ability to regenerate, and this may be related to increased interstitial fibrosis or the exhaustion of satellite cells. In this study, we used mdx mice, an animal model of Duchenne muscular dystrophy, to assess whether muscle fibers lose their ability to regenerate after repeated cycles of degeneration-regeneration and to establish the role of interstitial fibrosis or exhaustion of satellite cells in this process. Repeated degenerative-regenerative cycles were induced by the injection of bupivacaine (33 mg/kg), a myotoxic agent. Bupivacaine was injected weekly into the right tibialis anterior muscle of male, 8-week-old mdx (N = 20) and C57Bl/10 (control, N = 10) mice for 20 and 50 weeks. Three weeks after the last injection, the mice were killed and the proportion of regenerated fibers was counted and reported as a fibrosis index. Twenty weekly bupivacaine injections did not change the ability of mdx muscle to regenerate. However, after 50 weekly bupivacaine injections, there was a significant decrease in the regenerative response. There was no correlation between the inability to regenerate and the increase in interstitial fibrosis. These results show that after prolonged repeated cycles of degeneration-regeneration, mdx muscle loses its ability to regenerate because of the exhaustion of satellite cells, rather than because of an increase in interstitial fibrosis. This finding may be relevant to cell and gene therapy in the treatment of Duchenne muscular dystrophy.

Impaired regeneration of dystrophin-deficient muscle fibers is caused by exhaustion of myogenic cells

Duchenne muscular dystrophy is one of the most devastating myopathies. Muscle fibers undergo necrosis and lose their ability to regenerate, and this may be related to increased interstitial fibrosis or the exhaustion of satellite cells. In this study, we used mdx mice, an animal model of Duchenne muscular dystrophy, to assess whether muscle fibers lose their ability to regenerate after repeated cycles of degeneration-regeneration and to establish the role of interstitial fibrosis or exhaustion of satellite cells in this process. Repeated degenerative-regenerative cycles were induced by the injection of bupivacaine (33 mg/kg), a myotoxic agent. Bupivacaine was injected weekly into the right tibialis anterior muscle of male, 8-week-old mdx (N = 20) and C57Bl/10 (control, N = 10) mice for 20 and 50 weeks. Three weeks after the last injection, the mice were killed and the proportion of regenerated fibers was counted and reported as a fibrosis index. Twenty weekly bupivacaine injections did not change the ability of mdx muscle to regenerate. However, after 50 weekly bupivacaine injections, there was a significant decrease in the regenerative response. There was no correlation between the inability to regenerate and the increase in interstitial fibrosis. These results show that after prolonged repeated cycles of degeneration-regeneration, mdx muscle loses its ability to regenerate because of the exhaustion of satellite cells, rather than because of an increase in interstitial fibrosis. This finding may be relevant to cell and gene therapy in the treatment of Duchenne muscular dystrophy

Acetylcholine receptors and neuronal nitric oxide synthase distribution at the neuromuscular junction of regenerated muscle fibers.

We investigated whether the changes in acetylcholine receptor (AChR) distribution and neuronal nitric oxide synthase (nNOS) expression reported for the skeletal muscle of mdx mice were a consequence of muscle fiber regeneration rather than of the absence of dystrophin. Degenerative-regenerative changes in muscle fibers of the sternomastoid muscle of normal mice were induced by injecting lidocaine hydrochloride. Twenty-one days later, AChRs were labeled with alpha-bungarotoxin and nNOS with anti-nNOS antibody, and observed under a confocal microscope. AChRs were distributed in continuous branches in normal fibers. Regenerated fibers showed disruption of AChRs distribution similar to that seen in muscle of mdx mice. This suggests that changes in AChRs distribution seen in mdx mice were probably a consequence of muscle fiber degeneration and regeneration, rather than a symptom of dystrophin deficiency. Conversely, there were no changes in nNOS distribution and expression in normal regenerated fibers, suggesting that the decrease in nNOS expression reported for mdx mice might be attributed to the absence of dystrophin.

Effects of methylene blue and indomethacin on sodium nitroprusside-induced relaxations of the guinea pig tracheal smooth muscle.

The aim of the present investigation was to evaluate the potential modulatory effect of the guinea pig tracheal smooth muscle intrinsic tone on the relaxant responses to sodium nitroprusside and the effects of methylene blue on this response. Paired tracheal chains were mounted for isotonic contractions under 500 mg of tension in Krebs-Henseleit solution. The intrinsic modulatory tone was inhibited by indomethacin, in a concentration that did not have any effect over carbachol induced contractions. Sodium nitroprusside-induced relaxations were the same in the absence or presence of the modulatory tone. Methylene blue inhibited 50% sodium nitroprusside-induced relaxations, in the presence or absence of the intrinsic system. This suggests that sodium nitroprusside-induced relaxations are mediated through guanylyl-cyclase activation and that these are not under the modulation of the intrinsic prostaglandinergic tone.

Imaging neuromuscular junctions by confocal fluorescence microscopy: individual endplates seen in whole muscles with vital intracellular staining of the nerve terminals.

The mammalian neuromuscular junction has been extensively studied by different methods to understand better the biological aspects of its normal development, ageing and pathological conditions, such as disorders of neuromuscular transmission. In the present report, a new technique is described that combines confocal microscopy with the use of a vital nerve terminal dye (4-Di-2-ASP) and rhodamine-alpha-bungarotoxin to stain postsynaptic acetylcholine receptors in the same endplate. Nerve terminals in the sternomastoid muscles of living adult mice were stained with 4-Di-2-ASP, which labels intracellular compartments of the nerve terminal containing mitochondria. Slides of these muscles were viewed by confocal microscopy and images were stored on magnetic optical discs. This procedure was compatible with subsequent acetylcholine receptor staining with rhodamine-alpha-bungarotoxin and observation under the confocal microscope. Classical features of the adult neuromuscular junction were displayed, such as the branched-pattern distribution of the nerve terminals and receptors and their complete colocalisation. In addition, nerve fibres from intramuscular nerve branches with their neighbouring cells, nuclei and muscle fibre striations could also be visualised. We conclude that the present technique can complement existing methods of investigation of nerve terminal anatomy and pathology, particularly where preservation of 3-dimensional relationships is required and intracellular disturbances involving mitochondrial organisation, such as ageing or other degenerative disorders, may be present.

On the incidence of the biceps brachii third head in Brazilian white and blacks.

The incidence of a third head of the biceps brachii muscle has been reported in several articles, and there is a general idea that it is a race-dependent variation. The aim of this investigation was to study the biceps brachii muscle with regard to the incidence of its third head in a mixed white and black Brazilian population. A total of 200 upper arms from adult white and black cadavers (100 whites and 100 blacks) fixed in a 10% formol solution were examined and compared. It was observed that for white subjects the incidence of the third head was 20% against 9% for the black subjects: a statistically significant difference. We suggest that other factors, in addition to racial ones, might play a part in determining the incidence of the biceps brachii third head in a population.

Estimation of the number and size of human flexor digiti minimi muscle motor units using histological methods.

Motor unit (MU) number and size estimates were obtained from the human flexor digiti minimi muscle using histological methods. Ten adult fresh cadavers (33-74 years old) were used. The number of MUs was 130 +/- 15 and the MU size was 108 +/- 10. These values are similar to those reported for other hypothenar muscles, using the same criteria. The results here described are in best agreement with those reported by the incremental and automated incremental methods.

Axonal regeneration through muscle autografts submitted to local anaesthetic pretreatment.

Freeze-thawed muscle grafts (FTMG) have been used as an alternative to nerve grafts for the reconstruction of peripheral nerve defects, although their use has some disadvantages. For instance, FTMG may fragment easily after freeze-thawing, a fact impairing their use for surgery. In this study we describe a method to obtain muscle autografts based on the myotoxic properties of local anaesthetics. Fifteen adult rats had their left tibialis anterior muscles injected transcutaneously with 0.3 ml of 2% lidocaine hydrochloride. Twenty-four hours later the injected muscle was removed and a lidocaine muscle graft (LMG) was obtained by trimming the muscle to a rectangular block of approximately 1.0 cm in length. The left sciatic nerve was exposed in the mid-thigh region and a segment removed so that a final 1.0 cm-long gap was produced. The LMG was coaxially autografted to the epineurium between the proximal and distal nerve stumps. In another 15 rats, the sciatic nerve gap was repaired with FTMG obtained from the tibialis anterior muscle. Surgical procedures were similar in both groups. Axonal regeneration and muscle reinnervation were studied quantitatively and ultrastructurally 60 days after the insertion of LMG and FTMG. The results showed that axonal regeneration with the LMG was qualitatively similar to that observed with the FTMG, with no significant differences between groups. We conclude that LMG was a successful muscle graft and a suitable alternative to other denaturing methods, without the disadvantages of FTMG.

Effects of methylene blue and indomethacin on sodium nitroprusside-induced relaxations of the guinea pig tracheal smooth muscle

The aim of the present investigation was to evaluate the potential modulatory effect of the guinea pig tracheal smooth muscle intrinsic tone on the relaxant responses to sodium nitroprusside and the effects of methylene blue on this response. Paired tracheal chains were mounted for isotonic contractions under 500 mg of tension in Krebs-Henseleit solution. The intrinsic modulatory tone was inhibited by indomethacin, in a concentration that did not have any effect over carbachol induced contractions. Sodium nitroprusside-induced relaxations were the same in the absence or presence of the modulatory tone. Methylene blue inhibited 50 per cent sodium nitroprusside-induced relaxations, in the presence or absence of the intrinsic system. This suggests that sodium nitroprusside-induced relaxations are mediated through guanylyl-cylase activation and that these are not under the modulation of the intrinsic prostaglandinergic tone. (AU)

Effects of methylene blue and indomethacin on sodium nitroprusside-induced relaxations of the guinea pig tracheal smooth muscle

The aim of the present investigation was to evaluate the potential modulatory effect of the guinea pig tracheal smooth muscle intrinsic tone on the relaxant responses to sodium nitroprusside and the effects of methylene blue on this response. Paired tracheal chains were mounted for isotonic contractions under 500 mg of tension in Krebs-Henseleit solution. The intrinsic modulatory tone was inhibited by indomethacin, in a concentration that did not have any effect over carbachol induced contractions. Sodium nitroprusside-induced relaxations were the same in the absence or presence of the modulatory tone. Methylene blue inhibited 50 per cent sodium nitroprusside-induced relaxations, in the presence or absence of the intrinsic system. This suggests that sodium nitroprusside-induced relaxations are mediated through guanylyl-cylase activation and that these are not under the modulation of the intrinsic prostaglandinergic tone.

Effects of methylene blue on the development of intrinsic contractile responses of the guinea pig tracheal smooth muscle.

The aim of the present study was to investigate the effects of methylene blue, a guanylyl cyclase inhibitor, on the development of intrinsic contractile responses of the guinea pig tracheal smooth muscle. Paired tracheal chains were mounted for isotonic contractions under 500 mg of tension in Krebs-Henseleit solution. The intrinsic contractile tone modulated carbachol-induced contractions and was inhibited by indomethacin, suggesting the involvement of cyclooxygenase products on this tone. Methylene blue (5 x 10(-5)M) irreversibly inhibited the intrinsic contractile responses. Considering that methylene blue prevents any endogenous production of cGMP, it would be expected to enhance the contractions. However, since methylene blue has effects over nitric oxide synthase and nitric oxide itself, we suggest that guanylyl cyclase activation is not important for the development of the intrinsic tone.

Effects of methylene blue on the development of intrinsic contractile responses of the guinea pig tracheal smooth muscle

The aim of the present study was to investigate the effects of methylene bue, a guanylyl cyclase inhibitor, on the development of intrinsic contractile responses of the guinea pig tracheal smooth muscle. Paired tracheal chains were mounted for isotonic contractions under 500 mg of tension in Krebs-Henseleit solution. The intrinsic contractile tone modulated carbachol-induced contractions and was inhibited by indomethacin, suggesting the involvement of cyclooxygenase products on this tone. Methylene blue (5x10(-5)M) irreversibly inhibited the intrinsic contractile responses. considering that methylene blue prevents any endogenous production of cGMP, it would be expected to enhance the contractions. However, since methylene blue has effects over nitric oxide synthase and nitric oxide itself, we suggest that guanylyl cyclase activations is not important for the development of the intrinsic tone. (AU)

Effects of methylene blue on the development of intrinsic contractile responses of the guinea pig tracheal smooth muscle

The aim of the present study was to investigate the effects of methylene bue, a guanylyl cyclase inhibitor, on the development of intrinsic contractile responses of the guinea pig tracheal smooth muscle. Paired tracheal chains were mounted for isotonic contractions under 500 mg of tension in Krebs-Henseleit solution. The intrinsic contractile tone modulated carbachol-induced contractions and was inhibited by indomethacin, suggesting the involvement of cyclooxygenase products on this tone. Methylene blue (5x10(-5)M) irreversibly inhibited the intrinsic contractile responses. considering that methylene blue prevents any endogenous production of cGMP, it would be expected to enhance the contractions. However, since methylene blue has effects over nitric oxide synthase and nitric oxide itself, we suggest that guanylyl cyclase activations is not important for the development of the intrinsic tone.

Non-metric cranial variants in Brazilian skulls.

The incidence of 27 non-metric cranial variants is studied in a sample of 124 skulls from Brazil. The unilateral and bilateral frequencies of the variants are determined. The Brazilian population sample was compared with 28 population samples from different parts of the world.

Motor units and functional anatomy of the human musculus opponens digiti minimi.

The present anatomical investigation showed that, in 4 musculus opponens digiti minimi beings studied, we found an average value of 158 motor units and 100 muscle fibres per motor unit. These data are in accordance with the execution of the complex action of the m. opponens as an articular stabilizer.

Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin.

A myotoxin, bothropstoxin (BthTX), showing no detectable phospholipase A2 activity, was purified to homogeneity from the venom of the Brazilian snake Bothrops jararacussu by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on SP-Sephadex C-25. Four phospholipases (Sm-SP1 to Sm-SPIV) were also isolated, the latter showing, similarly to BthTX (Sm-SPv) myonecrotic activity. Approximate mol. wts, as determined by SDS-PAGE, and pI of Sm-SPI to Sm-SPIV are: 22,400-4.2; 15,500-4.8; 13,800-6.9; and 13,200-7.7, respectively. BthTX is a single chain protein, approximate mol. wt 13,000, with 16 half-cystine residues, pI = 8.2 and LD50 = 7.5 mg/kg (i.p.) and 4.8 mg/kg (i.v.) for 20 g mice. The ten first N-terminal amino acid residues show a significant homology to other toxins with phospholipase structure. BthTX is specifically myotoxic, contrary to crude B. jararacussu venom which, although also myotoxic, affects intramuscular arteries and veins leading to thrombosis. BthTX and Sm-SPIV also differ from toxins isolated from the venom of other Brazilian snakes which are strongly hemorrhagic.

Muscular lesions induced by a hemorrhagic factor from Bothrops neuwiedi snake venom.

The local tissue effects of crude Bothrops neuwiedi snake venom and of its hemorrhagic factor (NHF) were studied on mouse tibialis anterior muscle in vivo. After 6 h, 8 days and 6 weeks the muscles were examined in paraffin sections stained with hematoxylin and eosin. Both NHF and crude venom produced hemorrhage and myonecrosis, later followed by muscle fiber regeneration. Intramuscular arteries also suffered necrosis. The minimal dose of NHF necessary to produce detectable hemorrhage and myonecrosis was 50 ng, while the minimal venom dose needed to produce the same effect was 20 times higher. The results indicate that NHF is one of the major factors responsible for the local effects of B. neuwiedi venom.

Pathological changes in muscle caused by haemorrhagic and proteolytic factors from Bothrops jararaca snake venom.

Haemorrhagic factor HF2 and bothropasin, two metalloproteins isolated from the venom of Bothrops jararaca, caused haemorrhage followed by myonecrosis and arterial necrosis after i.m. injection in mice. The effects of HF2 were qualitatively similar to those of bothropasin and crude B. jararaca venom, but its potency was about 20 times higher. The haemorrhagic and necrotizing actions of these components are unrelated to their proteolytic activity on casein.