Effect of BaP1 and CsH1 on size and density of gaps between adjacent smooth muscle and pericytes on mouse cremaster muscle vasculature.

<p>Isolated cremaster muscles were incubated with either 30 μg of BaP1 or 15 μg of CsH1 (model without blood flow). In another experiment, anesthetized mice were injected by intrascrotal route with either 60 μg of BaP1 or 30 μg of CsH1 (model with blood flow). Controls were incubated or injected with PBS. After 15 min of exposition to toxin in each model, whole cremaster muscles were fixed and immunostained for observation by confocal microscopy and analysis of the gaps between adjacent smooth muscle and pericytes. Results are expressed as the mean ± SEM of the (A, B) gap size and (C, D) gap density (number of gaps per vessel area) of at least five images of arterioles and PCV per cremaster (n = 4). (E) Representative three-dimensional images of each vessel type immunostained for actin α smooth muscle are shown for BaP1 30 μg and control in the model without blood flow. Notice the increase in the gap size (arrows) in arterioles and PCV of treated tissues as compared to control. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. C: control; PCV: post-capillary venules.</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

Dose and time dependency of BaP1 effects on type IV collagen from vascular BM on isolated mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with different amounts of BaP1 (10, 30 and 100 μg) for either 5 or 15 min (model without blood flow). Control tissues were incubated with PBS. Whole tissues were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for type IV collagen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type: (A) arterioles, (B) capillaries, and (C) PCV per cremaster (n = 4). Below each graph, representative three-dimensional images of each vessel type immunostained for type IV collagen are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions) for BaP1 (30 μg) and control at 15 min. The images show a decrease in fluorescence intensity for type IV collagen in BM of capillaries of treated tissues as compared to control, whereas no significant reduction was observed in arterioles and PCV. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. C: control; Col IV: type IV collagen; PCV: post-capillary venules.</p

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

Effect of BaP1 on laminin and nidogen from vascular BM on isolated mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with 30 μg of BaP1 for 15 min (model without blood flow). Control tissues were incubated with PBS. Whole tissues were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for (A) laminin and (B) nidogen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type per cremaster (n = 4). (C) Representative three-dimensional images of each vessel type immunostained for laminin are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions). The images show a decrease in fluorescence intensity for laminin in BM of capillaries and PCV of treated tissues as compared to control, whereas no reduction in the fluorescence intensity was observed for nidogen. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. Lam: laminin; Nid: nidogen; PCV: post-capillary venules.</p

Apoptosis in regenerating skeletal muscle after the injection of either <i>B. asper</i> venom or Mtx.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019834#s2" target="_blank">Methods</a> 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 <i>B. asper</i> 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.</p

Effect of BaP1 and CsH1 on vascular permeability after intradermical application.

<p>Mice received an intravenous injection of 200 μl of Evans Blue dye (6 mg/ml). After 5 min, mice were injected by intradermal route, in the ventral abdominal region, with either 2 μg of BaP1 or 1 μg of CsH1. PBS was injected as control in another group of animals. After 15 min mice were sacrificed by cervical dislocation, their skin was removed and the area of extravasation was measured. (A) Results are expressed as the mean ± SEM (n = 5). **p < 0.001 as compared to CsH1. (B) Figure shows representative images from five animals analyzed.</p

Effect of BaP1 and CsH1 on type IV collagen from vascular BM on mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with either (A) 30 μg of BaP1 or (C) 15 μg of CsH1 (model without blood flow). In another experiment, anesthetized mice were injected by intrascrotal route with either (B) 60 μg of BaP1 or (D) 30 μg of CsH1 (model with blood flow). Controls were incubated or injected with PBS. After 15 min of exposition to toxin in each model, whole cremaster muscles were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for type IV collagen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type per animal (n = 4). (E) Representative three-dimensional images of each vessel type immunostained for type IV collagen are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions) for CsH1 30 μg and control applied in anesthetized mice (i.e. model with blood flow). The images show a decrease in fluorescence intensity for type IV collagen in BM of arterioles, capillaries, and PCV of treated tissues as compared to control. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. Col IV: type IV collagen; PCV: post-capillary venules.</p

Proteomic analysis of extracellular matrix proteins in exudates collected from mice injected with BaP1 or Leuc-a.

<p>*Values underlined correspond to proteins whose quantitative value differed by more than 3 fold when comparing the two SVMPs.</p