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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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Apoptosis in regenerating skeletal muscle after the injection of either B. asper venom or Mtx.Sections from muscle tissue samples collected 3, 5 and 7 days after injection of venom or Mtx were immunostained with anti-desmin antibodies and with TUNEL (see Methods for details). (A) The percentage of desmin-positive regenerating muscle fibers showing at least one TUNEL-positive nuclei, in relation to the total number of desmin-positive regenerating muscle fibers, was estimated. Results are presented as mean±SD (n = 9). (B) TUNEL-positive nuclei in desmin-positive cells 5 days after injection of either venom or Mtx. To highlight the pattern of spatial heterogeneity, each point corresponds to the density of TUNEL-positive nuclei per area in separate microscopic fields in different areas of the tissue. (C) and (D) Micrographs of muscle tissue sections from mice 5 days after injection of Mtx (C) or B. asper venom (D) immunostained for desmin (green fluorescence) and with TUNEL (reddish coloration in nuclei). No TUNEL-positive regenerating fibers, showing centrally-located nuclei, are observed in muscle injected with Mtx, whereas several regenerating fibers present TUNEL-positive nuclei in tissue injected with venom (arrows). Bar represents 50 µm.
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pone-0019834-g004: Apoptosis in regenerating skeletal muscle after the injection of either B. asper venom or Mtx.Sections from muscle tissue samples collected 3, 5 and 7 days after injection of venom or Mtx were immunostained with anti-desmin antibodies and with TUNEL (see Methods for details). (A) The percentage of desmin-positive regenerating muscle fibers showing at least one TUNEL-positive nuclei, in relation to the total number of desmin-positive regenerating muscle fibers, was estimated. Results are presented as mean±SD (n = 9). (B) TUNEL-positive nuclei in desmin-positive cells 5 days after injection of either venom or Mtx. To highlight the pattern of spatial heterogeneity, each point corresponds to the density of TUNEL-positive nuclei per area in separate microscopic fields in different areas of the tissue. (C) and (D) Micrographs of muscle tissue sections from mice 5 days after injection of Mtx (C) or B. asper venom (D) immunostained for desmin (green fluorescence) and with TUNEL (reddish coloration in nuclei). No TUNEL-positive regenerating fibers, showing centrally-located nuclei, are observed in muscle injected with Mtx, whereas several regenerating fibers present TUNEL-positive nuclei in tissue injected with venom (arrows). Bar represents 50 µm.

Mentions: As described above, both histological and immunohistochemical analyses evidenced profound differences during the first 7 days after injection between muscles treated with Mtx and those injected with venom. Thus, it is likely that critical events occur during these early stages in the reparative and regenerative scenario. In order to assess whether myogenic and regenerating fibers undergo cell damage, tissue sections were stained with TUNEL, together with Pax7 or desmin, which are markers of activated satellite cells/myoblasts and regenerating muscle cells, respectively [36]. No significant differences were observed in the number of Pax7-positive cells per area, at 3 days, between muscles injected with venom and with Mtx (not shown), thus revealing a similar extent of activation of satellite cells after these treatments. Moreover, there were no significant differences in the percentage of Pax7-positive cells showing positive TUNEL staining at this time interval (venom: 7.3±4.9 %; Mtx: 4.3±2.4 % (mean±SEM, n = 9); p > 0.05). When the percentage of desmin-positive cells having at least one TUNEL-positive nucleus was quantified, no significant differences were observed between treatments at 3, 5 and 7 days, despite a trend showing more TUNEL-positive nuclei in venom-injected muscle at 3 and 5 days (Fig 4A). Examination of tissue sections revealed a noticeable spatial heterogeneity in the pattern of TUNEL staining, especially in muscle injected with venom, since there were areas in which many myonuclei were TUNEL-positive, whereas this staining was lower or largely absent in other areas in venom-injected and, especially in Mtx-injected tissue (Fig 4B, C and D).


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)

Apoptosis in regenerating skeletal muscle after the injection of either B. asper venom or Mtx.Sections from muscle tissue samples collected 3, 5 and 7 days after injection of venom or Mtx were immunostained with anti-desmin antibodies and with TUNEL (see Methods for details). (A) The percentage of desmin-positive regenerating muscle fibers showing at least one TUNEL-positive nuclei, in relation to the total number of desmin-positive regenerating muscle fibers, was estimated. Results are presented as mean±SD (n = 9). (B) TUNEL-positive nuclei in desmin-positive cells 5 days after injection of either venom or Mtx. To highlight the pattern of spatial heterogeneity, each point corresponds to the density of TUNEL-positive nuclei per area in separate microscopic fields in different areas of the tissue. (C) and (D) Micrographs of muscle tissue sections from mice 5 days after injection of Mtx (C) or B. asper venom (D) immunostained for desmin (green fluorescence) and with TUNEL (reddish coloration in nuclei). No TUNEL-positive regenerating fibers, showing centrally-located nuclei, are observed in muscle injected with Mtx, whereas several regenerating fibers present TUNEL-positive nuclei in tissue injected with venom (arrows). Bar represents 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0019834-g004: Apoptosis in regenerating skeletal muscle after the injection of either B. asper venom or Mtx.Sections from muscle tissue samples collected 3, 5 and 7 days after injection of venom or Mtx were immunostained with anti-desmin antibodies and with TUNEL (see Methods for details). (A) The percentage of desmin-positive regenerating muscle fibers showing at least one TUNEL-positive nuclei, in relation to the total number of desmin-positive regenerating muscle fibers, was estimated. Results are presented as mean±SD (n = 9). (B) TUNEL-positive nuclei in desmin-positive cells 5 days after injection of either venom or Mtx. To highlight the pattern of spatial heterogeneity, each point corresponds to the density of TUNEL-positive nuclei per area in separate microscopic fields in different areas of the tissue. (C) and (D) Micrographs of muscle tissue sections from mice 5 days after injection of Mtx (C) or B. asper venom (D) immunostained for desmin (green fluorescence) and with TUNEL (reddish coloration in nuclei). No TUNEL-positive regenerating fibers, showing centrally-located nuclei, are observed in muscle injected with Mtx, whereas several regenerating fibers present TUNEL-positive nuclei in tissue injected with venom (arrows). Bar represents 50 µm.
Mentions: As described above, both histological and immunohistochemical analyses evidenced profound differences during the first 7 days after injection between muscles treated with Mtx and those injected with venom. Thus, it is likely that critical events occur during these early stages in the reparative and regenerative scenario. In order to assess whether myogenic and regenerating fibers undergo cell damage, tissue sections were stained with TUNEL, together with Pax7 or desmin, which are markers of activated satellite cells/myoblasts and regenerating muscle cells, respectively [36]. No significant differences were observed in the number of Pax7-positive cells per area, at 3 days, between muscles injected with venom and with Mtx (not shown), thus revealing a similar extent of activation of satellite cells after these treatments. Moreover, there were no significant differences in the percentage of Pax7-positive cells showing positive TUNEL staining at this time interval (venom: 7.3±4.9 %; Mtx: 4.3±2.4 % (mean±SEM, n = 9); p > 0.05). When the percentage of desmin-positive cells having at least one TUNEL-positive nucleus was quantified, no significant differences were observed between treatments at 3, 5 and 7 days, despite a trend showing more TUNEL-positive nuclei in venom-injected muscle at 3 and 5 days (Fig 4A). Examination of tissue sections revealed a noticeable spatial heterogeneity in the pattern of TUNEL staining, especially in muscle injected with venom, since there were areas in which many myonuclei were TUNEL-positive, whereas this staining was lower or largely absent in other areas in venom-injected and, especially in Mtx-injected tissue (Fig 4B, C and D).

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