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Effects of Bothrops asper snake venom on lymphatic vessels: insights into a hidden aspect of envenomation.

Mora J, Mora R, Lomonte B, Gutiérrez JM - PLoS Negl Trop Dis (2008)

Bottom Line: B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow.In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat.Moreover, fucoidan significantly reduced venom-induced footpad edema.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitología, Universidad de Costa Rica, San José, Costa Rica.

ABSTRACT

Background: Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied.

Methodology/principal findings: B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A(2) (PLA(2)) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA(2) homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells.

Conclusions/significance: Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA(2)s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations.

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Related in: MedlinePlus

Effect of B. asper venom on the caliber of mouse mesentery collecting lymphatic vessels.The mesentery was exposed as described in Methods, and 50 µL of either PBS or various concentrations of B. asper venom, dissolved in 50 µL PBS, were applied onto the preparation. A video was recorded before venom application and then during 15 min after venom addition. Snap shots were taken at 30 second intervals. A section of the lymphatic was selected and the area of its lumen determined by an image analysis program for each snap shot. Changes in the area were expressed as percentage, considering 100% the area of the lymphatic vessel lumen before the application of venom or PBS. Results are presented as mean±S.E.M. of five different preparations. Open circles represent control samples treated with PBS alone and closed circles represent samples treated with venom (A: 20 µg, B: 50 µg, C: 100 µg of venom from adult specimens, D: 100 µg of venom from neonate specimens). *p<0.05 when compared with control preparations treated with PBS alone.
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pntd-0000318-g002: Effect of B. asper venom on the caliber of mouse mesentery collecting lymphatic vessels.The mesentery was exposed as described in Methods, and 50 µL of either PBS or various concentrations of B. asper venom, dissolved in 50 µL PBS, were applied onto the preparation. A video was recorded before venom application and then during 15 min after venom addition. Snap shots were taken at 30 second intervals. A section of the lymphatic was selected and the area of its lumen determined by an image analysis program for each snap shot. Changes in the area were expressed as percentage, considering 100% the area of the lymphatic vessel lumen before the application of venom or PBS. Results are presented as mean±S.E.M. of five different preparations. Open circles represent control samples treated with PBS alone and closed circles represent samples treated with venom (A: 20 µg, B: 50 µg, C: 100 µg of venom from adult specimens, D: 100 µg of venom from neonate specimens). *p<0.05 when compared with control preparations treated with PBS alone.

Mentions: In order to study the effects of crude venom on lymphatic vasculature, collecting lymphatics, located in the mesentery and presenting the characteristic valves, were identified using intravital microscopy (Fig 1). For analysis of the effect of venom, only collecting lymphatics having a regular lymph flow were used. Application of PBS alone did not induce any effect in the lumen of these vessels, as evidenced by the maintenance of a constant area in the lumen (Fig 2). When venom was applied onto the mesentery, a dose-dependent effect was observed, characterized by a significant contraction of the vessel wall, resulting in a reduction of the lumen; such effect occurred within 2–5 min of application of venom doses of 50 and 100 µg, but not with the dose of 20 µg (Fig 2A, B and C). The effect remained at the end of the 15 min observation period. When using doses of 50 and 100 µg, a complete halting of lymph flow occurred between 5 and 7 min after application of venom (Table 1). When using 50 µg of venom, there was a restart of lymph flow at later time intervals, whereas with the dose of 100 µg the flow was not recovered (not shown). In addition, a prominent hemorrhage developed within the first 5 min of application of the venom, evidenced by abundant areas of extravasation. Many erythrocytes were observed flowing through the lymphatics at early time periods, before the halting of lymph flow. Histological observations corroborated the presence of erythrocytes inside lymphatics (Fig 3). When a dose of 100 µg venom of neonate B. asper specimens was tested, there was no effect on the lumen of lymphatic vessels nor halting in lymph flow (Fig 2D), but a prominent hemorrhage developed in the mesentery.


Effects of Bothrops asper snake venom on lymphatic vessels: insights into a hidden aspect of envenomation.

Mora J, Mora R, Lomonte B, Gutiérrez JM - PLoS Negl Trop Dis (2008)

Effect of B. asper venom on the caliber of mouse mesentery collecting lymphatic vessels.The mesentery was exposed as described in Methods, and 50 µL of either PBS or various concentrations of B. asper venom, dissolved in 50 µL PBS, were applied onto the preparation. A video was recorded before venom application and then during 15 min after venom addition. Snap shots were taken at 30 second intervals. A section of the lymphatic was selected and the area of its lumen determined by an image analysis program for each snap shot. Changes in the area were expressed as percentage, considering 100% the area of the lymphatic vessel lumen before the application of venom or PBS. Results are presented as mean±S.E.M. of five different preparations. Open circles represent control samples treated with PBS alone and closed circles represent samples treated with venom (A: 20 µg, B: 50 µg, C: 100 µg of venom from adult specimens, D: 100 µg of venom from neonate specimens). *p<0.05 when compared with control preparations treated with PBS alone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563035&req=5

pntd-0000318-g002: Effect of B. asper venom on the caliber of mouse mesentery collecting lymphatic vessels.The mesentery was exposed as described in Methods, and 50 µL of either PBS or various concentrations of B. asper venom, dissolved in 50 µL PBS, were applied onto the preparation. A video was recorded before venom application and then during 15 min after venom addition. Snap shots were taken at 30 second intervals. A section of the lymphatic was selected and the area of its lumen determined by an image analysis program for each snap shot. Changes in the area were expressed as percentage, considering 100% the area of the lymphatic vessel lumen before the application of venom or PBS. Results are presented as mean±S.E.M. of five different preparations. Open circles represent control samples treated with PBS alone and closed circles represent samples treated with venom (A: 20 µg, B: 50 µg, C: 100 µg of venom from adult specimens, D: 100 µg of venom from neonate specimens). *p<0.05 when compared with control preparations treated with PBS alone.
Mentions: In order to study the effects of crude venom on lymphatic vasculature, collecting lymphatics, located in the mesentery and presenting the characteristic valves, were identified using intravital microscopy (Fig 1). For analysis of the effect of venom, only collecting lymphatics having a regular lymph flow were used. Application of PBS alone did not induce any effect in the lumen of these vessels, as evidenced by the maintenance of a constant area in the lumen (Fig 2). When venom was applied onto the mesentery, a dose-dependent effect was observed, characterized by a significant contraction of the vessel wall, resulting in a reduction of the lumen; such effect occurred within 2–5 min of application of venom doses of 50 and 100 µg, but not with the dose of 20 µg (Fig 2A, B and C). The effect remained at the end of the 15 min observation period. When using doses of 50 and 100 µg, a complete halting of lymph flow occurred between 5 and 7 min after application of venom (Table 1). When using 50 µg of venom, there was a restart of lymph flow at later time intervals, whereas with the dose of 100 µg the flow was not recovered (not shown). In addition, a prominent hemorrhage developed within the first 5 min of application of the venom, evidenced by abundant areas of extravasation. Many erythrocytes were observed flowing through the lymphatics at early time periods, before the halting of lymph flow. Histological observations corroborated the presence of erythrocytes inside lymphatics (Fig 3). When a dose of 100 µg venom of neonate B. asper specimens was tested, there was no effect on the lumen of lymphatic vessels nor halting in lymph flow (Fig 2D), but a prominent hemorrhage developed in the mesentery.

Bottom Line: B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow.In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat.Moreover, fucoidan significantly reduced venom-induced footpad edema.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Parasitología, Universidad de Costa Rica, San José, Costa Rica.

ABSTRACT

Background: Envenomations by the snake Bothrops asper represent a serious medical problem in Central America and parts of South America. These envenomations concur with drastic local tissue pathology, including a prominent edema. Since lymph flow plays a role in the maintenance of tissue fluid balance, the effect of B. asper venom on collecting lymphatic vessels was studied.

Methodology/principal findings: B. asper venom was applied to mouse mesentery, and the effects were studied using an intravital microscopy methodology coupled with an image analysis program. B. asper venom induced a dose-dependent contraction of collecting lymphatic vessels, resulting in a reduction of their lumen and in a halting of lymph flow. The effect was reproduced by a myotoxic phospholipase A(2) (PLA(2)) homologue isolated from this venom, but not by a hemorrhagic metalloproteinase or a coagulant thrombin-like serine proteinase. In agreement with this, treatment of the venom with fucoidan, a myotoxin inhibitor, abrogated the effect, whereas no inhibition was observed after incubation with the peptidomimetic metalloproteinase inhibitor Batimastat. Moreover, fucoidan significantly reduced venom-induced footpad edema. The myotoxic PLA(2) homologue, known to induce skeletal muscle necrosis, was able to induce cytotoxicity in smooth muscle cells in culture and to promote an increment in the permeability to propidium iodide in these cells.

Conclusions/significance: Our observations indicate that B. asper venom affects collecting lymphatic vessels through the action of myotoxic PLA(2)s on the smooth muscle of these vessels, inducing cell contraction and irreversible cell damage. This activity may play an important role in the pathogenesis of the pronounced local edema characteristic of viperid snakebite envenomation, as well as in the systemic biodistribution of the venom, thus representing a potential therapeutical target in these envenomations.

Show MeSH
Related in: MedlinePlus