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Poor regenerative outcome after skeletal muscle necrosis induced by Bothrops asper venom: alterations in microvasculature and nerves.

Hernández R, Cabalceta C, Saravia-Otten P, Chaves A, Gutiérrez JM, Rucavado A - PLoS ONE (2011)

Bottom Line: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America.A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature.In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Químicas y Farmacia, Universidad de San Carlos de Guatemala, Guatemala. [corrected].

ABSTRACT

Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood.

Methodology/principal findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A(2) (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration.

Conclusions/significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.

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Light micrographs of sections of mouse skeletal muscle at 1, 7 and 28 days after the injection, in the gastrocnemius muscle, of phosphate-buffered saline solution (PBS), B. asper venom, Myotoxin (Mtx), and metalloproteinase BaP1.First, second and fourth horizontal rows of figures correspond to hematoxylin-eosin-stained sections, whereas the third row corresponds to sections stained with Sirius Red and counterstained with Fast Green FCF. Bar represents 100 µm.
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pone-0019834-g001: Light micrographs of sections of mouse skeletal muscle at 1, 7 and 28 days after the injection, in the gastrocnemius muscle, of phosphate-buffered saline solution (PBS), B. asper venom, Myotoxin (Mtx), and metalloproteinase BaP1.First, second and fourth horizontal rows of figures correspond to hematoxylin-eosin-stained sections, whereas the third row corresponds to sections stained with Sirius Red and counterstained with Fast Green FCF. Bar represents 100 µm.

Mentions: Histological observations showed that all treatments, except PBS, induced evident myonecrosis in the injected gastrocnemius one day after injection (Fig 1). In the case of muscle injected with venom or BaP1, hemorrhage was also observed, whereas no hemorrhage was induced by Mtx. An inflammatory infiltrate was present in muscles injected with venom or toxins by 1 and 3 days. Small regenerating fibers, characterized by centrally-located nuclei, were present at 3 days and thereafter, although differences were noticed among the various treatments. In the case of muscle injected with Mtx, by 7 days, regenerating fibers were distributed uniformly in the tissue (Fig 1). In contrast, muscle injected with venom or BaP1 showed areas of regenerating fibers intermixed with areas of remnants of necrotic cells, which had very low numbers of phagocytes, and areas of fibrosis (Fig 1). Therefore, the histological pattern at 7 days in muscle injected with venom was characterized by heterogeneity, whereas in the case of tissue affected by Mtx, a homogeneous pattern predominated (Fig 1). Fibrosis was corroborated 7 days after injection, by staining with Sirius Red, in the endomysium and perimysium in muscle injected with venom or BaP1 (Fig 1), whereas the extent of collagen deposition was less evident in Mtx-injected muscle, being nevertheless higher than in PBS-injected muscle. By 28 days, tissue injected with Mtx presented a pattern of successful regeneration, with abundant regenerating fibers of similar size having centrally-located nuclei, and with little fibrosis, albeit with an increment in the interstitial area as compared with PBS-injected muscle (Fig 1). In contrast, in the case of muscle injected with venom or BaP1, which induced hemorrhage, regenerating fibers of small size were observed, and were intermixed with areas of fibrosis and, in some cases, with areas in which the remnants of necrotic fibers had apparently become calcified (Fig 1).


Poor regenerative outcome after skeletal muscle necrosis induced by Bothrops asper venom: alterations in microvasculature and nerves.

Hernández R, Cabalceta C, Saravia-Otten P, Chaves A, Gutiérrez JM, Rucavado A - PLoS ONE (2011)

Light micrographs of sections of mouse skeletal muscle at 1, 7 and 28 days after the injection, in the gastrocnemius muscle, of phosphate-buffered saline solution (PBS), B. asper venom, Myotoxin (Mtx), and metalloproteinase BaP1.First, second and fourth horizontal rows of figures correspond to hematoxylin-eosin-stained sections, whereas the third row corresponds to sections stained with Sirius Red and counterstained with Fast Green FCF. Bar represents 100 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3101212&req=5

pone-0019834-g001: Light micrographs of sections of mouse skeletal muscle at 1, 7 and 28 days after the injection, in the gastrocnemius muscle, of phosphate-buffered saline solution (PBS), B. asper venom, Myotoxin (Mtx), and metalloproteinase BaP1.First, second and fourth horizontal rows of figures correspond to hematoxylin-eosin-stained sections, whereas the third row corresponds to sections stained with Sirius Red and counterstained with Fast Green FCF. Bar represents 100 µm.
Mentions: Histological observations showed that all treatments, except PBS, induced evident myonecrosis in the injected gastrocnemius one day after injection (Fig 1). In the case of muscle injected with venom or BaP1, hemorrhage was also observed, whereas no hemorrhage was induced by Mtx. An inflammatory infiltrate was present in muscles injected with venom or toxins by 1 and 3 days. Small regenerating fibers, characterized by centrally-located nuclei, were present at 3 days and thereafter, although differences were noticed among the various treatments. In the case of muscle injected with Mtx, by 7 days, regenerating fibers were distributed uniformly in the tissue (Fig 1). In contrast, muscle injected with venom or BaP1 showed areas of regenerating fibers intermixed with areas of remnants of necrotic cells, which had very low numbers of phagocytes, and areas of fibrosis (Fig 1). Therefore, the histological pattern at 7 days in muscle injected with venom was characterized by heterogeneity, whereas in the case of tissue affected by Mtx, a homogeneous pattern predominated (Fig 1). Fibrosis was corroborated 7 days after injection, by staining with Sirius Red, in the endomysium and perimysium in muscle injected with venom or BaP1 (Fig 1), whereas the extent of collagen deposition was less evident in Mtx-injected muscle, being nevertheless higher than in PBS-injected muscle. By 28 days, tissue injected with Mtx presented a pattern of successful regeneration, with abundant regenerating fibers of similar size having centrally-located nuclei, and with little fibrosis, albeit with an increment in the interstitial area as compared with PBS-injected muscle (Fig 1). In contrast, in the case of muscle injected with venom or BaP1, which induced hemorrhage, regenerating fibers of small size were observed, and were intermixed with areas of fibrosis and, in some cases, with areas in which the remnants of necrotic fibers had apparently become calcified (Fig 1).

Bottom Line: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America.A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature.In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.

View Article: PubMed Central - PubMed

Affiliation: Facultad de Ciencias Químicas y Farmacia, Universidad de San Carlos de Guatemala, Guatemala. [corrected].

ABSTRACT

Background: Viperid snakebite envenoming is characterized by prominent local tissue damage, including muscle necrosis. A frequent outcome of such local pathology is deficient skeletal muscle regeneration, which causes muscle dysfunction, muscle loss and fibrosis, thus provoking permanent sequelae that greatly affect the quality of life of patients. The causes of such poor regenerative outcome of skeletal muscle after viperid snakebites are not fully understood.

Methodology/principal findings: A murine model of muscle necrosis and regeneration was adapted to study the effects of the venom and isolated toxins of Bothrops asper, the medically most important snake in Central America. Gastrocnemius muscle was injected with either B. asper venom, a myotoxic phospholipase A(2) (Mtx), a hemorrhagic metalloproteinase (SVMP), or saline solution. At various time intervals, during one month, tissue samples were collected and analyzed by histology, and by immunocytochemical and immunohistochemical techniques aimed at detecting muscle fibers, collagen, endothelial cells, myoblasts, myotubes, macrophages, TUNEL-positive nuclei, and axons. A successful regenerative response was observed in muscle injected with Mtx, which induces myonecrosis but does not affect the microvasculature. In contrast, poor regeneration, with fibrosis and atrophic fibers, occurred when muscle was injected with venom or SVMP, both of which provoke necrosis, microvascular damage leading to hemorrhage, and poor axonal regeneration.

Conclusions/significance: The deficient skeletal muscle regeneration after injection of B. asper venom is likely to depend on the widespread damage to the microvasculature, which affects the removal of necrotic debris by phagocytes, and the provision of nutrients and oxygen required for regeneration. In addition, deficient axonal regeneration is likely to contribute to the poor regenerative outcome in this model.

Show MeSH
Related in: MedlinePlus