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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) Changes in the density of intramuscular nerves in muscle tissue of mice 3 and 28 days after injection of either PBS, B. asper venom, Mtx or BaP1. Axons in nerves were visualized by immunostaining with a mouse anti-human neurofilament protein, as described in Materials and Methods. The number of intramuscular nerves per mm2 of tissue area showing at least one immunostained axon was quantified. Results are presented as mean±SD (n = 5). A significant drop (* p < 0.05) in nerves per area was observed at 3 days in samples injected with either venom, Mtx or BaP1, as compared to samples injected with PBS, whereas no differences in the number of nerves per area were detected between treatments at 28 days. (B) Axonal density in intramuscular nerves 28 days after injection of the various agents. The number of axons within each nerve was determined and expressed in terms of axons per nerve area. Results are presented as mean±SD (n = 11). * p < 0.05 when compared with axonal density in control muscles injected with PBS. **p < 0.05 when compared with axonal density in muscles injected with Mtx. (C to F) Light micrograph sections of mouse muscle tissue collected 28 days after injection of (C) PBS, (D) B. asper venom, (E) Mtx, and (F) BaP1. Sections were immunostained for neurofilament protein to detect axons in nerves (arrows). Notice the evident drop in the number of axons in samples from tissue injected with venom or BaP1. Bar represents 50 µm.
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pone-0019834-g005: (A) Changes in the density of intramuscular nerves in muscle tissue of mice 3 and 28 days after injection of either PBS, B. asper venom, Mtx or BaP1. Axons in nerves were visualized by immunostaining with a mouse anti-human neurofilament protein, as described in Materials and Methods. The number of intramuscular nerves per mm2 of tissue area showing at least one immunostained axon was quantified. Results are presented as mean±SD (n = 5). A significant drop (* p < 0.05) in nerves per area was observed at 3 days in samples injected with either venom, Mtx or BaP1, as compared to samples injected with PBS, whereas no differences in the number of nerves per area were detected between treatments at 28 days. (B) Axonal density in intramuscular nerves 28 days after injection of the various agents. The number of axons within each nerve was determined and expressed in terms of axons per nerve area. Results are presented as mean±SD (n = 11). * p < 0.05 when compared with axonal density in control muscles injected with PBS. **p < 0.05 when compared with axonal density in muscles injected with Mtx. (C to F) Light micrograph sections of mouse muscle tissue collected 28 days after injection of (C) PBS, (D) B. asper venom, (E) Mtx, and (F) BaP1. Sections were immunostained for neurofilament protein to detect axons in nerves (arrows). Notice the evident drop in the number of axons in samples from tissue injected with venom or BaP1. Bar represents 50 µm.

Mentions: Since an adequate innervation is a requisite for successful muscle regeneration, the integrity of intramuscular nerves was assessed by immunostaining with anti-neurofilament protein. A similar drop in nerve density was observed at 3 days in tissue injected with either venom, Mtx or BaP1, as compared with tissue injected with PBS (Fig 5A), thus reflecting acute damage to axons. A reduction in the density of nerves in control samples injected with PBS was observed at 28 days, as compared with 3 days, probably reflecting the normal growth of muscle tissue in mice. The number of nerves per area showing immunostained axons in tissue injected with venom, Mtx or BaP1 increased at 28 days, as compared with the same treatments at 3 days, and no difference was detected between control muscle and muscles injected with venom, Mtx or BaP1 at 28 days (Fig 5A). However, a conspicuous difference was noticed in the density of axons within nerves in the various treatments observed in immunostained sections. At 28 days, the density of axons within nerves was significantly lower in all treatments, as compared with muscle injected with PBS (Fig 5 B, C, D, E and F). However, the reduction in axons in venom-treated muscles was more pronounced than in Mtx-and BaP1-injected muscle (Fig 5B).


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) Changes in the density of intramuscular nerves in muscle tissue of mice 3 and 28 days after injection of either PBS, B. asper venom, Mtx or BaP1. Axons in nerves were visualized by immunostaining with a mouse anti-human neurofilament protein, as described in Materials and Methods. The number of intramuscular nerves per mm2 of tissue area showing at least one immunostained axon was quantified. Results are presented as mean±SD (n = 5). A significant drop (* p < 0.05) in nerves per area was observed at 3 days in samples injected with either venom, Mtx or BaP1, as compared to samples injected with PBS, whereas no differences in the number of nerves per area were detected between treatments at 28 days. (B) Axonal density in intramuscular nerves 28 days after injection of the various agents. The number of axons within each nerve was determined and expressed in terms of axons per nerve area. Results are presented as mean±SD (n = 11). * p < 0.05 when compared with axonal density in control muscles injected with PBS. **p < 0.05 when compared with axonal density in muscles injected with Mtx. (C to F) Light micrograph sections of mouse muscle tissue collected 28 days after injection of (C) PBS, (D) B. asper venom, (E) Mtx, and (F) BaP1. Sections were immunostained for neurofilament protein to detect axons in nerves (arrows). Notice the evident drop in the number of axons in samples from tissue injected with venom or BaP1. Bar represents 50 µm.
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Related In: Results  -  Collection

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pone-0019834-g005: (A) Changes in the density of intramuscular nerves in muscle tissue of mice 3 and 28 days after injection of either PBS, B. asper venom, Mtx or BaP1. Axons in nerves were visualized by immunostaining with a mouse anti-human neurofilament protein, as described in Materials and Methods. The number of intramuscular nerves per mm2 of tissue area showing at least one immunostained axon was quantified. Results are presented as mean±SD (n = 5). A significant drop (* p < 0.05) in nerves per area was observed at 3 days in samples injected with either venom, Mtx or BaP1, as compared to samples injected with PBS, whereas no differences in the number of nerves per area were detected between treatments at 28 days. (B) Axonal density in intramuscular nerves 28 days after injection of the various agents. The number of axons within each nerve was determined and expressed in terms of axons per nerve area. Results are presented as mean±SD (n = 11). * p < 0.05 when compared with axonal density in control muscles injected with PBS. **p < 0.05 when compared with axonal density in muscles injected with Mtx. (C to F) Light micrograph sections of mouse muscle tissue collected 28 days after injection of (C) PBS, (D) B. asper venom, (E) Mtx, and (F) BaP1. Sections were immunostained for neurofilament protein to detect axons in nerves (arrows). Notice the evident drop in the number of axons in samples from tissue injected with venom or BaP1. Bar represents 50 µm.
Mentions: Since an adequate innervation is a requisite for successful muscle regeneration, the integrity of intramuscular nerves was assessed by immunostaining with anti-neurofilament protein. A similar drop in nerve density was observed at 3 days in tissue injected with either venom, Mtx or BaP1, as compared with tissue injected with PBS (Fig 5A), thus reflecting acute damage to axons. A reduction in the density of nerves in control samples injected with PBS was observed at 28 days, as compared with 3 days, probably reflecting the normal growth of muscle tissue in mice. The number of nerves per area showing immunostained axons in tissue injected with venom, Mtx or BaP1 increased at 28 days, as compared with the same treatments at 3 days, and no difference was detected between control muscle and muscles injected with venom, Mtx or BaP1 at 28 days (Fig 5A). However, a conspicuous difference was noticed in the density of axons within nerves in the various treatments observed in immunostained sections. At 28 days, the density of axons within nerves was significantly lower in all treatments, as compared with muscle injected with PBS (Fig 5 B, C, D, E and F). However, the reduction in axons in venom-treated muscles was more pronounced than in Mtx-and BaP1-injected muscle (Fig 5B).

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