Fibrosis of diaphragm muscle from 1-year-old <i>mdx</i> and wild type control mice after 22 weeks of treatment with saline or P-188 NF.

<p>Sections of diaphragms from the same groups of mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134832#pone.0134832.g001" target="_blank">Fig 1</a> were stained with Picosirius Red to visualize collagen deposition. All staining was done on diaphragm muscle from 12-month old mice with the exception of Panel B. Shown in the figure are cross sections of diaphragm muscle from: Panel A, wild type control saline-treated mouse; Panel B, a 7 month old <i>mdx</i> mouse; Panel C, an <i>mdx</i> saline-treated mouse; Panel D, an <i>mdx</i> mouse treated with 3 mg/Kg P-188 NF; Panel E, an <i>mdx</i> mouse treated with 1 mg/Kg prednisone.</p

Chronic Dosing with Membrane Sealant Poloxamer 188 NF Improves Respiratory Dysfunction in Dystrophic <i>Mdx and Mdx/Utrophin^-/-</i> Mice

<div><p>Poloxamer 188 NF (national formulary (NF) grade of P-188) improves cardiac muscle function in the <i>mdx</i> mouse and golden retriever muscular dystrophy models. However <i>in vivo</i> effects on skeletal muscle have not been reported. We postulated that P-188 NF might protect diaphragm muscle membranes from contraction-induced injury in <i>mdx</i> and mdx/utrophin^-/- (dko) muscular dystrophy models. In the first study 7-month old <i>mdx</i> mice were treated for 22 weeks with subcutaneous (s.c.) injections of saline or P-188 NF at 3 mg/Kg. In the second, dkos were treated with saline or P-188 NF (1 mg/Kg) for 8 weeks beginning at age 3 weeks. Prednisone was the positive control in both studies. Respiratory function was monitored using unrestrained whole body plethysmography. P-188 NF treatment affected several respiratory parameters including tidal volume/BW and minute volume/BW in mdx mice. In the more severe dko model, P-188 NF (1 mg/Kg) significantly slowed the decline in multiple respiratory parameters compared with saline-treated dko mice. Prednisone’s effects were similar to those seen with P-188 NF. Diaphragms from P-188 NF or prednisone treated <i>mdx</i> and dko mice showed signs of muscle fiber protection including less centralized nuclei, less variation in fiber size, greater fiber density, and exhibited a decreased amount of collagen deposition. P-188 NF at 3 mg/Kg s.c. also improved parameters of systolic and diastolic function in <i>mdx</i> mouse hearts. These results suggest that P-188 NF may be useful in treating respiratory and cardiac dysfunction, the leading causes of death in Duchenne muscular dystrophy patients.</p></div

Effects of P-188 NF and prednisone on tidal volume/BW in the <i>mdx</i> mouse over time.

<p><i>Mdx</i> mice were treated QD, <i>s</i>.<i>c</i>. with P-188 NF (3 mg/Kg) or prednisone (1 mg/Kg) from age 7 months to 12 months ± 2 weeks. The black line represents wild-type mice (C57BL/10 SnJ) treated with saline. The red line represents <i>mdx</i> mice treated with saline. The blue line represents mice treated with P-188 NF (Panel A) or 1 mg/Kg prednisone (Panel B). Data points are means +/- S.D. N = 11 for <i>mdx</i> 3 mg/Kg P-188 NF at 20 and 22 weeks, N = 12/group for prednisone treatment. Panel A, P < 0.0001 for the overall effect of P-188 NF 3 mg/Kg group vs. <i>mdx</i> saline and wild type saline. The <i>mdx</i> 3 mg/Kg group was also significantly greater than <i>mdx</i> saline at 22 weeks (P < 0.0001). Panel B, P = 0.001 to 0.0001 for <i>mdx</i> 1 mg/Kg prednisone group vs. <i>mdx</i> saline and P < 0.0001 vs. wild type saline. The <i>mdx</i> prednisone group was also significantly greater than <i>mdx</i> saline at 22 weeks (P < 0.0001).</p

Histological parameters from diaphragm muscle from saline, P-188 NF, or prednisone treated dko mice at 11 weeks of age.

<p>Wild type or dko mice were treated with saline, P-188 NF (1 mg/Kg) or prednisone (1 mg/Kg) QD, s.c. for 8 weeks beginning at 3 weeks of age. At termination, diaphragm muscle was harvested, washed and embedded in paraffin. Sections from across each diaphragm were stained with wheat germ agglutinin and DAPI (Panels A & C) or lectin antibody (Panel B) and photographed. Images were analyzed using Nikon Elements software, or by direct counting. (A) Percentage of fibers with centralized nuclei; (B) Variance coefficient of the mean minimum Ferets diameter of diaphragm fibers; and (C) Fiber density. * < 0.01 vs. dko saline, ** P < 0.001 vs. dko saline, ^# P < 0.001 vs. all other groups.</p

Histological parameters from diaphragm muscle from saline, P-188 NF, or prednisone treated <i>mdx</i> mice at 1 year of age.

<p><i>Mdx</i> and wild type control mice were age to seven months and then randomized into groups (N = 12) based on tidal volume measurements. Mice were treated QD by s.c injection with saline, P-188 NF at 3 mg/Kg or prednisone (1 mg/Kg). At termination, diaphragm muscle was harvested, washed and embedded in paraffin. Sections from across each diaphragm were stained with wheat germ agglutinin and DAPI (Panels A & C) or lectin antibody (Panel B) and photographed. Images were analyzed using Nikon Elements software, or by direct counting. (A) Percentage of fibers with centralized nuclei; (B) Variance coefficient of the mean minimum Ferets diameter of diaphragm fibers; and (C) Fiber density. A minimum of 10,000 fibers was assessed for each parameter. * < 0.001 vs. <i>mdx</i> saline, ^# P < 0.001 vs. all other groups.</p

Comparison of baseline respiratory function and body weight between groups of wild type and <i>mdx</i> mice at age 7 months^g.

<p>^a P < 0.05 vs. wild type saline.</p><p>^b P = 0.001–0.01 vs. wild type saline.</p><p>^c P < 0.001 vs. wild type saline.</p><p>^d P < 0.05 vs. <i>mdx</i> saline.</p><p>^e P = 0.001–0.01 vs. <i>mdx</i> saline.</p><p>^f < 0.001 vs. <i>mdx</i> saline.</p><p>^g all values are mean ± S.D. Analysis by 2-way ANOVA using a multiple comparison (simple effects within rows) post-hoc test.</p><p>Comparison of baseline respiratory function and body weight between groups of wild type and <i>mdx</i> mice at age 7 months^g.</p

Overall effect of P-188 NF and prednisone at 1 mg/Kg versus saline on respiratory parameters in the dko mouse model.

<p>Two-way ANOVA (treatment vs. time) vs. dko saline (main treatment effect) post-hoc test.</p><p>* P< 0.05.</p><p>** P = 0.01 to 0.001.</p><p>*** P < 0.001.</p><p>**** P < 0.0001.</p><p>Overall effect of P-188 NF and prednisone at 1 mg/Kg versus saline on respiratory parameters in the dko mouse model.</p

Effects of NF treatment on PEF and BW in dko mice over time.

<p>Dko mice were treated with 1 mg/Kg of P-188 NF QD, s.c, from ages 3–11 weeks. The black line represents wild-type mice (C57BL/10 SnJ) treated with saline; the red line represents <i>dko</i> mice treated with saline; the blue line represents dko treated with 1 mg/Kg P-188 NF. PEF and BW are shown in panels A & B, respectively. P < 0.0001 for wild type saline vs. all other groups. For panel A, P = 0.01 to 0.001 for dko 1 mg/Kg P-188 NF group vs. dko saline. For panel B, there is no significant difference between dko groups treated with saline or P-188 NF.</p

Effects of NF treatment on TV/BW and MV/BW in dko mice over time.

<p>Dko mice were treated with 1 mg/Kg of P-188 NF QD, s.c, from ages 3–11 weeks. The black line represents wild-type mice (C57BL/10 SnJ) treated with saline; the red line represents <i>dko</i> mice treated with saline; the blue line represents dko treated with 1 mg/Kg P-188 NF. TV/BW and MV/BW are shown in panels A, and B, respectively. Data points are means +/- S.D. N = 8/group wild type and 5/group dko saline and dko P-188 NF. For panels A and B, P < 0.0001 for dko 1 mg/Kg P-188 NF group vs. dko saline and wild type saline.</p

Effect of P-188 NF on collagen composition in diaphragm muscle.

<p>Measurements of picosirius red staining were determined from in <i>mdx</i> (panel A) and dko mice (panel B) at the end of the respective treatment periods. All surviving mice were use in the analysis. Staining was quantified using Nikon Elements software to determine the percentage of area stained in each section. A minimum of 6 sections/diaphragm was assessed. The wild type saline group was significantly different from all other groups, ^#P < 0.0001. Panel A, ^** P <0.01 vs. <i>mdx</i> saline; Panel B ** P < 0.01 vs. dko saline.</p