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Keywords:

  • cromolyn;
  • Duchenne dystrophy;
  • mdx mice;
  • muscle degeneration;
  • sarcolemma leakiness

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

In dystrophin-deficient fibers of mdx mice and in Duchenne dystrophy, the lack of dystrophin leads to sarcolemma breakdown and muscle degeneration. We verified that cromolyn, a mast-cell stabilizer agent, stabilized dystrophic muscle fibers using Evans blue dye as a marker of sarcolemma leakiness. Mdx mice (n = 8; 14 days of age) received daily intraperitoneal injections of cromolyn (50 mg/kg body weight) for 15 days. Untreated mdx mice (n = 8) were injected with saline. Cryostat cross-sections of the sternomastoid, tibialis anterior, and diaphragm muscles were stained with hematoxylin and eosin. Cromolyn dramatically reduced Evans blue dye–positive fibers in all muscles (P < 0.05; Student's t-test) and led to a significant increase in the percentage of fibers with peripheral nuclei. This study supports the protective effects of cromolyn in dystrophic muscles and further indicates its action against muscle fiber leakiness in muscles that are differently affected by the lack of dystrophin. Muscle Nerve, 2007

In mdx mice and in Duchenne muscular dystrophy (DMD) a lack of the subsarcolemmal protein dystrophin initiates muscle pathology.4, 32 The lack of dystrophin is associated with changes in membrane stability and increased levels of calcium in muscle fibers, which leads to myonecrosis.2, 38 The identification of pharmacological therapies that can slow the process of muscle degeneration is of great importance,17, 21, 35 and cell and gene therapies to replace the defective dystrophin gene are under development.37

Disodium cromoglycate (cromolyn) is a mast-cell stabilizing agent due to its ability to delay Ca2+ mobilization and the degranulation process.5, 10 Functional studies have shown that cromolyn increases muscle strength in exercised adult mdx mice15, 16 and reduces the extent of myonecrosis in both young and exercised adult mdx mice due to its direct action on mast-cell degranulation.30

We hypothesized that cromolyn would act as a muscle-fiber stabilizing agent, retarding calcium mobilization in the muscle fiber and reducing the extent of muscle-fiber leakiness/necrosis. We used Evans blue dye as a sensitive label for the early detection of increased myofiber permeability or leakiness and sarcolemmal damage and disruption, typically seen in necrotic fibers.18, 22, 23 Considering that mechanical activity leads to muscle breakdown,9, 11 we studied axial (sternomastoid and diaphragm) and limb (tibialis anterior) muscles to verify whether differences in functional activities would result in different responses. Cromolyn dramatically reduced membrane leakiness in all muscles studied, supporting its protective effects in dystrophinopathies.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Animals.

Male and female mdx mice obtained from a breeding colony maintained by our institutional animal care facility were used in all experiments. The mice were housed according to institutional guidelines, with free access to food and water. Pregnant females were isolated and monitored daily. The date of birth was designated postnatal day 0. Cromolyn treatment was initiated on postnatal day 14 before the cycles of muscle degeneration–regeneration had started.8 Mice were weaned at 4 weeks of age. The animal experiments described here were performed in accordance with the guidelines of the Brazilian College for Animal Experimentation and our institutional guidelines.

Drug Administration.

Cromolyn-treated mdx mice (n = 8) were injected intraperitoneally with disodium cromoglycate (cromolyn; Galena, Campinas, Brazil) daily at a dose of 50 mg/kg body weight in saline (final volume of 0.01 ml for a 25-g mouse) for 15 days. Each mouse was weighed daily so that the drug dosage could be adjusted accurately. Control litter mdx mice (n = 8) were injected with an equivalent amount of saline.

Evans Blue Dye Analysis.

For visualization of muscle fiber leakiness/necrosis, treated and untreated mdx mice were injected with Evans blue dye (EBD; Sigma, St. Louis, Missouri).18, 22, 23 EBD was dissolved in phosphate-buffered saline (PBS; 0.15 M NaCl, 10 mM phosphate buffer, pH 7.4) and injected into the peritoneal cavity. The animals (three cromolyn-treated and three untreated) received an intraperitoneal injection of 1% EBD in PBS at a dose of 100 μl per 10 g body weight. The mice were visually inspected for dye uptake. Discoloration of all mice was observed within 50–60 min after intraperitoneal injection of EBD and successful injection of the dye was indicated by the blue color of the ears and paws.

Twenty-four hours later the mice were sacrificed with an overdose of chloral hydrate and the sternomastoid (STN), tibialis anterior (TA), and diaphragm (DIA) muscles were dissected out and snap-frozen in isopentane cooled in liquid nitrogen and stored at −80°C. These muscles were chosen because they are affected differently in DMD, with diaphragm and limb muscles being more severely impaired than other muscles.11

Cryostat cross-sections (7-μm thick) were incubated in ice-cold acetone at −20°C for 10 min, washed three times for 10 min with PBS, and mounted in DABCO (mounting medium for fluorescence microscopy; Sigma). EBD staining shows a bright red emission upon fluorescence microscopy. Fiber counts of EBD-positive muscle fibers were performed with a hand counter in all sections and photographed under a Nikon fluorescence microscope connected to a Hamamatsu video camera. The number of EBD-positive muscle fibers is expressed as the percentage of the total number of muscles fibers counted in each section. Comparisons between groups and values were made using Student's t-test (P < 0.05).

Quantitative and Morphometric Analysis.

Cryostat cross-sections of STN, TA, and DIA from cromolyn-treated (n = 5) and untreated (n = 5) were stained with hematoxylin–eosin (HE). Slides were placed in a Nikon Eclipse E 400 microscope connected to a personal computer and attached to a video camera (Nikon Express Series, Tokyo, Japan). Nonoverlapping images of the entire cross-section were taken and tiled together using the ImagePro-Express software (Media Cybernetic, Silver Spring, Maryland). For each cross-section the numbers of central nucleated fibers, fibers with peripheral nuclei, and fibers undergoing necrosis were counted using a hand counter and expressed as the percentage of the total number of fibers. Comparisons between groups and values were made using Student's t-test.

The following areas within each cross-section were measured with the ImagePro-Express software: (1) areas with inflammatory cell infiltrate and myotubes (inflammation/regeneration area; Fig. 1B); and (2) areas containing mainly myotubes and myofibers with a small diameter and poor inflammatory cell infiltration (regeneration area; Fig. 1C). Myotubes-myofibers appeared in cross-sections as plump basophilic cells with at least one centrally located nucleus. The remainder of the cross-sectional area (remaining area) contained fully regenerated fibers, necrotic fibers, and fibers with peripheral cell nuclei (asterisk in Fig. 1A,C). The inflammation/regeneration, regeneration, and remaining areas are expressed as proportions of the total cross-sectional area. Statistical analysis was performed with Student's t-test. All the counting and measurements (EBD, HE, and areas analysis) were done by a blinded observer.

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Figure 1. Histological appearance of mdx tibialis anterior (TA) and sternomastoid (STN) muscles in untreated (C,D; saline) and cromolyn-treated (A,B;E,F) mice. In A, a representative inflammation/regeneration area is indicated by the outline. B: Detail of the inflammation/regeneration area showing inflammatory cells and myotube (arrows). C: Regeneration area, indicated by the line. D: Detail of the regeneration area showing myotubes and myofibers (arrows) with a small diameter and poor inflammatory cell infiltration. Myotubes-myofibers are seen as plump basophilic cells with at least one centrally located nucleus. Asterisk in A,C: areas with central nucleated fibers, necrosis and peripheral cell nuclei. E,F: Central nucleated fibers (arrow) and fibers with peripheral cell nuclei. Scale bar (shown only in F) is 25 μm (B,D–F) or 100 μm (A,C).

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RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

No significant differences in weight gain were observed between cromolyn-treated and untreated (saline-injected) mdx mice during the study period. The mean body weight was 7.1 ± 0.9 g for cromolyn-treated mdx mice and 8.0 ± 0.9 g for untreated animals (P > 0.05; Student's t-test). Thus, cromolyn treatment did not interfere with the growth rate of young mdx mice.

The histological appearance of muscles from cromolyn-treated and saline-treated mdx mice is shown in Figure 1. Inflammatory cells, myoblasts, and myotubes predominated in the inflammation/regeneration area (Fig. 1A,B). Myotubes and myofibers were mainly seen in the regeneration area (Fig. 1C,D). Fully regenerated muscle fibers were char acterized by central nucleation and an apparent normal diameter (Fig. 1E,F). In addition, round or roughly polygonal muscle fibers were observed, whose nuclei presented a random peripheral location directly below the sarcolemma (Fig. 1F).

EBD-positive fibers could be seen in groups or isolated, and some EBD-stained myofibers corresponded to necrotic foci visualized by HE staining (Fig. 2). Cromolyn caused a significant decrease in Evans blue dye staining in STN, TA, and DIA mdx muscles (Table 1). Cromolyn increased the percentage of fibers with peripheral nuclei in all muscles studied (Table 1). A significant decrease in the percentage of central nucleated fibers was observed in cromolyn-treated TA and DIA muscles but not in STN compared to untreated muscle (Table 1).

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Figure 2. Histological appearance of saline-treated tibialis anterior (TA) muscle from mdx mice. Myofibers positive for Evans blue dye (A, arrows) indicating sarcolemmal leakiness-damage. Some Evans blue dye–labeled cells correspond to necrotic fibers seen in HE-stained sections (B, arrows). Scale bar, 90 μm.

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Table 1. Effects of cromolyn treatment on the percentage of muscle fibers with peripheral cell nuclei, centrally located nuclei, and fibers stained with Evans blue dye (EBD).
  Peripheral nuclei (%)Central nuclei (%)EBD (%)
  • Values are expressed as the percentage (mean ± SD; n = 5 mice) of the total number of fibers of the sternomastoid (STN), tibialis anterior (TA), and diaphragm (DIA) muscles.

  • *

    Significantly different from untreated mdx mice within the same muscle group (P < 0.05, Student t-test).

  • Significantly different from untreated STN and TA muscles (P < 0.05, Student t-test).

  • Significantly different from untreated STN muscle (P < 0.05, Student t-test).

STNUntreated49.0 ± 3.130.3 ± 1.621.0 ± 6.0
 Treated64.2 ± 2.2*29.0 ± 6.24.5 ± 2.0*
TAUntreated55.0 ± 5.716.4 ± 2.928.6 ± 13.2
 Treated86.8 ± 6.6*10.9 ± 6.0*2.3 ± 1.4*
DIAUntreated92.7 ± 6.27.4 ± 3.07.3 ± 6.0
 Treated98.7 ± 0.4*2.4 ± 1.0*1.3 ± 0.4*

In untreated muscles the area containing cells with peripheral nuclei and central nuclei predominated (about 60% to 70% of the whole cross-sectional area), followed by the regeneration area (about 20% of the whole cross-sectional area) and the inflammation/regeneration area (about 10% of the whole cross-section; Fig. 3). Cromolyn resulted in a significant increase in the percentage of the inflammation/regeneration area in the TA and STN muscles (Fig. 3). In the DIA there was a decrease in the regeneration area in cromolyn-treated muscles compared to untreated control (Fig. 3).

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Figure 3. Effect of cromolyn on the inflammation/regeneration and regeneration areas of the sternomastoid (STN), tibialis anterior (TA), and diaphragm (DIA) muscles. The remaining area contains central nucleated fibers, fibers under necrosis, and fully regenerated fibers, with central nuclei. The areas are expressed as proportions (n = 5 mice) of the total muscle cross-sectional area. The graphs show both cromolyn-treated and untreated mdx mice. *Significantly different (P < 0.05, Student's t-test) from nontreated mdx mice in the same area.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Mast-cell stabilizing agent is a term used for drugs that retard calcium mobilization in mast cells, thus retarding their activation and making mast cells insensitive. Cromones, especially disodium cromoglycate (cromolyn), are well known as mast-cell stabilizing agents for their ability to retard calcium mobilization and the degranulation process.5 Since the action of cromolyn on its cellular targets is not specific,20, 36 we hypothesized that cromolyn would have a direct action on the dystrophic muscle fiber, as previously suggested,15 acting as a muscle-fiber stabilizing agent, retarding calcium mobilization and the necrosis process.

The lack of dystrophin or a normal dystrophin–glycoprotein complex renders the sarcolemma more susceptible to stresses imposed during contractions.29, 24 It has been suggested that transient membrane tears develop, allowing extracellular calcium to enter the fiber along its electrochemical gradient. Changes in membrane permeability, or leakiness, may progress to complete disorganization and disruption of sarcolemma, which characterize a necrotic fiber. We used EBD as an in vivo nontoxic marker of fibers during the initial changes in membrane permeability, being able to detect permeable myofibers in a muscle affected by muscular dystrophy that were not detected by standard histological techniques.18, 22, 23

We demonstrated a dramatic decrease in EBD uptake in the muscles studied. In addition, there was a concomitant increase in the percentage of fibers with peripheral nuclei, evidence that less myonecrosis occurred. The reduction in the percentage of central nucleated fibers further supports the lack of muscle degeneration. Taken together, these results show that cromolyn treatment has a protective effect on myofiber breakdown in limb (TA) and axial (STN and DIA) muscles of young mdx mice. These results further support the protective action of cromolyn on muscle dystrophy30 and corroborate the hypothesis that cromolyn acts as a muscle-fiber stabilizing agent, reducing the extent of muscle-fiber leakiness and necrosis. Previous studies had shown that cromolyn increases muscle strength in exercised mdx mice15 and significantly reduce myonecrosis in young and exercised mdx mice,30 showing that cromolyn is also able to protect muscle-fiber damage associated with contractile activity induced by exercise.

At 30 days of age the extent of damage in the diaphragm was lower than in the limb and neck muscles, in agreement with other reports.26, 31 This low level of active damage in the DIA was improved by cromolyn, where about 98% of the fibers remained undamaged. In the untreated neck and limb muscles the disease was already established. The neck muscle showed a trend for reduced active muscle damage at this age, with less EBD-positive fibers compared to untreated TA. The untreated TA showed a higher extent of active muscle leakiness/necrosis, with more fibers positive to EBD than untreated STN and DIA. The STN muscle from treated mice showed a decrease in permeability to EBD without a concomitant decrease in central nucleated fibers, i.e., myogenic damage and subsequent repair. This supports the fact that EBD labeling does not necessarily correlate with fiber damage and myogenesis, as already observed,1 at least in the STN muscle.

It has been demonstrated that cromolyn, by preventing mast-cell degranulation,5, 10, 13 led to a reduction in proinflammatory cytokines produced by inflammatory cells, such as mast cells, which have a direct effect on muscle-fiber degeneration.30 Tumor necrosis factor alpha (TNFα) is a potent proinflammatory cytokine and blocking its activity in young mdx mice prevented myofiber necrosis.17, 19 Thus, it is possible that the protection from myonecrosis observed here may also be related to the inhibition of proinflammatory cytokines production by mast cells. Alternatively, by inhibiting TNFα production cromolyn may interfere with insulin-like growth factor 1 signaling,12 which specifically reduces sarcolemmal damage and myonecrosis of dystrophic muscle.31

The inflammation/regeneration area described here is characterized by inflammatory cell infiltrates associated with myoblasts and myotubes, possibly representing an early stage of muscle regeneration. The regeneration area might represent a later step in the process of regeneration, containing more mature myofibers with no inflammatory cells. In untreated mdx mice the regeneration area is increased in relation to the inflammation–regeneration area. In cromolyn-treated mdx mice this situation is reversed, suggesting that cromolyn increased inflammation. Ibuprofen, a nonsteroidal antiinflammatory drug, also led to an increase in inflammatory cells in a model of muscle inflammation.7 Cromolyn could also hold back the process of muscle regeneration, possibly by inhibiting the inflammatory cells associated with muscle repair, such as macrophages, to release regenerative factors.33, 34 In dystrophic muscle, mast cells seem to be localized to areas of myonecrosis,25 whereas macrophages are the major leukocyte present between 24 and 48 h after injury,14 during the period of muscle regeneration. Therefore, besides its protection against myonecrosis, we cannot exclude a potential long-term effect of cromolyn on the inflammation–regeneration response, at least in some muscles.

In the DIA there were no differences in the inflammation/regeneration area between treated and untreated muscles, possibly due to the low level of active degeneration in this muscle. In addition, the regeneration area in the DIA was significantly decreased by cromolyn treatment, which further supports its action on muscle-fiber stabilization and prevention of muscle degeneration. Taken together, these results further demonstrate the variations in pathophysiological responses of different dystrophic muscles. The reasons for this are unclear, but it could be related to differences in the involvement of stress proteins and the activation of stretch-activated channels (SAC). These channels may be the primary way through which calcium enters the fibers, contributing to the increased membrane permeability following stretched contractions of mdx fibers.38 It had been demonstrated that SAC mediate the increases in heat shock proteins (HSP) in in vitro preparations of rat hearts.6 Interestingly, heat shock proteins are expressed in neuromuscular disorders3, 27 and are inhibited by cromolyn.28

In conclusion, the present investigation demonstrates that cromolyn decreases sarcolemmal leakiness and myonecrosis of dystrophic fibers, as measured by EBD uptake. The differences in the extent of muscle damage in the muscles studied here allowed the observation that cromolyn treatment is effective prior to significant fiber damage (diaphragm muscle) and also when the disease is already established (tibialis anterior and sternomastoid). In addition to its protective effects against myonecrosis, acting as an inhibitor of the inflammatory response,30 cromolyn may have a direct action on membrane stabilization or calcium mobilization in the muscle fiber.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grants 95/6110-2, 01/00570-4 and 04/15526-9). H.S.N. and M.J.M. are recipients of fellowships from Conselho Nacional de Pesquisas (CNPq; 301286/03-5; 302880/04-6; 474708/06-3). R.V.M was the recipient of a Capes fellowship. We thank Prof. Miranda Grounds, T. Shavlakadze, and H. Radley (School of Anatomy and Human Biology, University of Western Australia, Perth, Western Australia) for critical review of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES