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

Gene expression and immune response kinetics using electroporation-mediated DNA delivery to muscle

Background Injection of DNA encoding exogenic proteins into muscle tissue combined with electroporation often results in a transient increase of the encoded protein concentration in the muscle and the blood. The reduction is normally due to an immune response against the exogenic protein but other factors may also be involved. How various electroporation parameters affect the concentration kinetics of syngenic and exogenic proteins is studied in relation to immune response and muscle damage after electroporation-mediated DNA transfer to muscle. Methods Electroporation was applied to mouse quadriceps muscles after injection of DNA encoding either secreted alkaline phosphatase (SEAP) or a mouse IgG molecule. Protein concentrations in blood or muscle and antibody responses were measured for a period up to 3 months. Tissue inflammation and muscle cell damage were studied on muscle cross-sections and assessed by measuring the concentrations of creatine phosphokinase (CPK) in blood. Results Mice with the highest SEAP concentration in blood at day 7 also had the highest rate of decrease afterwards, the strongest antibody responses against SEAP and the highest acute levels of CPK in blood. DNA-transfected muscle fibers were significantly reduced in number from days 7 to 14. Mononuclear cells surrounded the reporter gene expressing muscle fibers, thus indicating a cellular immune response. When using DNA encoding a syngenic protein the protein concentration in blood was relatively stabile over a 3-month period, but showed different kinetics for various electroporation parameters. Conclusions Our findings suggest that the optimal electroporation. parameters for DNA vaccination may be different from the optimal parameters for long-term expression of genes encoding syngenic proteins

‘Fast’ and ‘slow’ muscle fibres in hindlimb muscles of adult rats regenerate from intrinsically different satellite cells

Myosin heavy chain (MyHC) expression was examined in regenerating fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of adult rats. Myotoxic bupivacaine was injected into SOL and EDL and the muscles were either denervated or neuromuscularly blocked by tetrodotoxin (TTX) on the sciatic nerve. Three to 10 or 30 days later, denervated SOL or EDL, or innervated but neuromuscularly blocked EDL received a slow 20 Hz stimulus pattern through electrodes implanted on the muscles or along the fibular nerve to EDL below the TTX block. In addition, denervated SOL and EDL received a fast 100 Hz stimulus pattern. Denervated EDL and SOL stimulated with the same slow stimulus pattern expressed different amounts of type 1 MyHC protein (8%versus 35% at 10 days, 13%versus 87% at 30 days). Stimulated denervated and stimulated innervated (TTX blocked) EDL expressed the same amounts of type 1, 2A, 2X and 2B MyHC proteins. Cross-sections treated for in situ hybridization and immunocytochemistry showed expression of type 1 MyHC in all SOL fibres but only in some scattered single or smaller groups of fibres in EDL. The results suggest that muscle fibres regenerate from intrinsically different satellite cells in EDL and SOL and within EDL. However, induction by different extrinsic factors arising in extracellular matrix or from muscle position and usage in the limb has not been excluded. No evidence for nerve-derived trophic influences was obtained