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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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(A) Quantitative assessment of the extent of myonecrosis and regeneration in mouse gastrocnemius muscle injected with either B. asper venom, Mtx or BaP1. Histological sections stained with hematoxylin and eosin were analyzed one and 28 days after injections. The extent of necrosis was estimated in samples collected one day after injection as the percentage of the examined area corresponding to necrotic fibers, whereas the extent of regeneration was estimated in samples collected at 28 days as the percentage of the examined area corresponding to regenerating fibers, i.e. fibers having centrally-located nuclei. *p < 0.05 when comparing the percentage of necrosis and of regeneration for a single treatment. (B) Quantitative assessment of the diameter of regenerating muscle fibers, i.e. fibers presenting centrally-located nuclei, 28 days after intramuscular injection in the gastrocnemius of PBS, B. asper venom, Mtx or BaP1. Regenerating fibers in tissue injected with venom and BaP1 showed a reduced diameter when compared with control fibers in tissue injected with PBS (p < 0.05), whereas no significant difference was observed in the diameter of regenerating fibers in muscle injected with Mtx (p > 0.05). In both graphs, results are presented as mean±SD (n = 9).
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pone-0019834-g002: (A) Quantitative assessment of the extent of myonecrosis and regeneration in mouse gastrocnemius muscle injected with either B. asper venom, Mtx or BaP1. Histological sections stained with hematoxylin and eosin were analyzed one and 28 days after injections. The extent of necrosis was estimated in samples collected one day after injection as the percentage of the examined area corresponding to necrotic fibers, whereas the extent of regeneration was estimated in samples collected at 28 days as the percentage of the examined area corresponding to regenerating fibers, i.e. fibers having centrally-located nuclei. *p < 0.05 when comparing the percentage of necrosis and of regeneration for a single treatment. (B) Quantitative assessment of the diameter of regenerating muscle fibers, i.e. fibers presenting centrally-located nuclei, 28 days after intramuscular injection in the gastrocnemius of PBS, B. asper venom, Mtx or BaP1. Regenerating fibers in tissue injected with venom and BaP1 showed a reduced diameter when compared with control fibers in tissue injected with PBS (p < 0.05), whereas no significant difference was observed in the diameter of regenerating fibers in muscle injected with Mtx (p > 0.05). In both graphs, results are presented as mean±SD (n = 9).

Mentions: The extent of muscle necrosis and regeneration was quantitatively assessed by measuring the area corresponding to necrotic fibers in gastrocnemius muscle one day after injection of venom or isolated toxins, and the area of regenerating fibers, i.e. fibers with centrally located nuclei, 28 days after injection. As shown in Fig 2, all treatments induced acute muscle damage which ranged from 30% necrotic muscle in the case of SVMP BaP1 to 55% necrotic muscle in tissue injected with of Mtx. Muscle regeneration was significantly affected in the case of treatments that induced hemorrhage besides local myonecrosis, i.e. venom and BaP1 (Fig 2A). In contrast, a successful regeneration was observed in tissue injected with Mtx alone (Fig 2A). In addition, the diameter of regenerative fibers 28 days after injection greatly differed among the various treatments. The diameters of regenerating fibers in tissue injected with Mtx did not differ from those of control, PBS-injected muscle fibers (Fig 2B). In contrast, the diameter of regenerating fibers in tissue treated with either venom or BaP1, both of which induced hemorrhage, were smaller than those of control fibers (Fig 2B). On the basis of these results, the rest of the study was focused mostly on the comparison of two experimental groups: muscle injected with venom, as a model of poor regeneration, and muscle injected with Mtx, as a model of successful regeneration.


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)

(A) Quantitative assessment of the extent of myonecrosis and regeneration in mouse gastrocnemius muscle injected with either B. asper venom, Mtx or BaP1. Histological sections stained with hematoxylin and eosin were analyzed one and 28 days after injections. The extent of necrosis was estimated in samples collected one day after injection as the percentage of the examined area corresponding to necrotic fibers, whereas the extent of regeneration was estimated in samples collected at 28 days as the percentage of the examined area corresponding to regenerating fibers, i.e. fibers having centrally-located nuclei. *p < 0.05 when comparing the percentage of necrosis and of regeneration for a single treatment. (B) Quantitative assessment of the diameter of regenerating muscle fibers, i.e. fibers presenting centrally-located nuclei, 28 days after intramuscular injection in the gastrocnemius of PBS, B. asper venom, Mtx or BaP1. Regenerating fibers in tissue injected with venom and BaP1 showed a reduced diameter when compared with control fibers in tissue injected with PBS (p < 0.05), whereas no significant difference was observed in the diameter of regenerating fibers in muscle injected with Mtx (p > 0.05). In both graphs, results are presented as mean±SD (n = 9).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101212&req=5

pone-0019834-g002: (A) Quantitative assessment of the extent of myonecrosis and regeneration in mouse gastrocnemius muscle injected with either B. asper venom, Mtx or BaP1. Histological sections stained with hematoxylin and eosin were analyzed one and 28 days after injections. The extent of necrosis was estimated in samples collected one day after injection as the percentage of the examined area corresponding to necrotic fibers, whereas the extent of regeneration was estimated in samples collected at 28 days as the percentage of the examined area corresponding to regenerating fibers, i.e. fibers having centrally-located nuclei. *p < 0.05 when comparing the percentage of necrosis and of regeneration for a single treatment. (B) Quantitative assessment of the diameter of regenerating muscle fibers, i.e. fibers presenting centrally-located nuclei, 28 days after intramuscular injection in the gastrocnemius of PBS, B. asper venom, Mtx or BaP1. Regenerating fibers in tissue injected with venom and BaP1 showed a reduced diameter when compared with control fibers in tissue injected with PBS (p < 0.05), whereas no significant difference was observed in the diameter of regenerating fibers in muscle injected with Mtx (p > 0.05). In both graphs, results are presented as mean±SD (n = 9).
Mentions: The extent of muscle necrosis and regeneration was quantitatively assessed by measuring the area corresponding to necrotic fibers in gastrocnemius muscle one day after injection of venom or isolated toxins, and the area of regenerating fibers, i.e. fibers with centrally located nuclei, 28 days after injection. As shown in Fig 2, all treatments induced acute muscle damage which ranged from 30% necrotic muscle in the case of SVMP BaP1 to 55% necrotic muscle in tissue injected with of Mtx. Muscle regeneration was significantly affected in the case of treatments that induced hemorrhage besides local myonecrosis, i.e. venom and BaP1 (Fig 2A). In contrast, a successful regeneration was observed in tissue injected with Mtx alone (Fig 2A). In addition, the diameter of regenerative fibers 28 days after injection greatly differed among the various treatments. The diameters of regenerating fibers in tissue injected with Mtx did not differ from those of control, PBS-injected muscle fibers (Fig 2B). In contrast, the diameter of regenerating fibers in tissue treated with either venom or BaP1, both of which induced hemorrhage, were smaller than those of control fibers (Fig 2B). On the basis of these results, the rest of the study was focused mostly on the comparison of two experimental groups: muscle injected with venom, as a model of poor regeneration, and muscle injected with Mtx, as a model of successful regeneration.

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