Poor Regenerative Outcome after Skeletal Muscle Necrosis Induced by Bothrops asper Venom: Alterations in Microvasculature and Nerves

artículo (arbitrado) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2011Background: 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 A2 (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.This study was supported by NeTropica (grant 2-N-2008), by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A7-604). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Figure 5

<p>(A) Changes in the density of intramuscular nerves in muscle tissue of mice 3 and 28 days after injection of either PBS, <i>B. asper</i> venom, Mtx or BaP1. Axons in nerves were visualized by immunostaining with a mouse anti-human neurofilament protein, as described in Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019834#s2" target="_blank">Methods</a>. 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) <i>B. asper</i> 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.</p

Figure 2

<p>(A) Quantitative assessment of the extent of myonecrosis and regeneration in mouse gastrocnemius muscle injected with either <i>B. asper</i> 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, <i>B. asper</i> 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).</p

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), <i>B. asper</i> venom, Myotoxin (Mtx), and metalloproteinase BaP1.

<p>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.</p