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Improvement of endurance of DMD animal model using natural polyphenols.

Sitzia C, Farini A, Colleoni F, Fortunato F, Razini P, Erratico S, Tavelli A, Fabrizi F, Belicchi M, Meregalli M, Comi G, Torrente Y - Biomed Res Int (2015)

Bottom Line: Different mechanisms were hypothesized such as reduced hydroxyl radicals, nuclear factor-κB deactivation, and NO protection from inactivation.We observed a reduction of muscle fibrosis deposition and myofiber necrosis together with an amelioration of vascularization.Our data confirmed that ProAbe-based diet may represent a strategy to coadjuvate the treatment of DMD.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio Cellule Staminali, Dipartimento di Patofisiologia e dei Trapianti, Universitá degli Studi di Milano, Fondazione IRCCS Cá Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Via Francesco Sforza 35, 20122 Milano, Italy.

ABSTRACT
Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is characterized by muscular wasting caused by dystrophin deficiency that ultimately ends in force reduction and premature death. In addition to primary genetic defect, several mechanisms contribute to DMD pathogenesis. Recently, antioxidant supplementation was shown to be effective in the treatment of multiple diseases including muscular dystrophy. Different mechanisms were hypothesized such as reduced hydroxyl radicals, nuclear factor-κB deactivation, and NO protection from inactivation. Following these promising evidences, we investigated the effect of the administration of a mix of dietary natural polyphenols (ProAbe) on dystrophic mdx mice in terms of muscular architecture and functionality. We observed a reduction of muscle fibrosis deposition and myofiber necrosis together with an amelioration of vascularization. More importantly, the recovery of the morphological features of dystrophic muscle leads to an improvement of the endurance of treated dystrophic mice. Our data confirmed that ProAbe-based diet may represent a strategy to coadjuvate the treatment of DMD.

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Related in: MedlinePlus

H&E analysis of treated and untreated mice. H&E staining was performed on 10 μm-thick frozen sections from TA and QA muscles (a). In treated mice reduced signs of muscular wasting were observed in comparison to untreated mice. In particular reduced inflammatory infiltrates between myofibers, reduced fat deposition, and reduced necrotic fibers were assessed. In both treated and untreated mice small centronucleated regenerating fibers were observed. For each muscle analyzed with H&E staining we showed the myofiber area and their distribution frequency (b). The curve of TA/QA muscles of treated mice shifted to the right related to untreated mice, thus demonstrating an increase in myofiber area of treated mice (CSA QA: 25° percentile of treated mice 1221,26 and of untreated mice 903,152; CSA TA: 25° percentile of treated mice 1120,39 and of untreated mice 874,068; CSA QA: 75° percentile of treated mice 3107,04 and of untreated mice 1946,06; and CSA TA: 75° percentile of treated mice 2978,59 and of untreated mice 1883,39). Moreover, we indicated the coefficient of variance (graphs showing Min and Max values and mean + SEM) ((c) for QA and (d) for TA). Amelioration of dystrophic phenotype following ProAbe treatment was demonstrated by decrease of fibrosis by measuring area of connective tissue (AM) (e), of the percentage of necrotic fibers (f), of macrophage infiltration area (CD11b staining) (g), and of the number of centronucleated myofibers per section (h).
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fig1: H&E analysis of treated and untreated mice. H&E staining was performed on 10 μm-thick frozen sections from TA and QA muscles (a). In treated mice reduced signs of muscular wasting were observed in comparison to untreated mice. In particular reduced inflammatory infiltrates between myofibers, reduced fat deposition, and reduced necrotic fibers were assessed. In both treated and untreated mice small centronucleated regenerating fibers were observed. For each muscle analyzed with H&E staining we showed the myofiber area and their distribution frequency (b). The curve of TA/QA muscles of treated mice shifted to the right related to untreated mice, thus demonstrating an increase in myofiber area of treated mice (CSA QA: 25° percentile of treated mice 1221,26 and of untreated mice 903,152; CSA TA: 25° percentile of treated mice 1120,39 and of untreated mice 874,068; CSA QA: 75° percentile of treated mice 3107,04 and of untreated mice 1946,06; and CSA TA: 75° percentile of treated mice 2978,59 and of untreated mice 1883,39). Moreover, we indicated the coefficient of variance (graphs showing Min and Max values and mean + SEM) ((c) for QA and (d) for TA). Amelioration of dystrophic phenotype following ProAbe treatment was demonstrated by decrease of fibrosis by measuring area of connective tissue (AM) (e), of the percentage of necrotic fibers (f), of macrophage infiltration area (CD11b staining) (g), and of the number of centronucleated myofibers per section (h).

Mentions: Fibrosis is considered the most devastating consequence of the progression of disease in DMD patients: due to the lack of dystrophin, satellite cell proliferation cannot compensate constant myofiber breakdown so that inflammatory processes that follow muscular necrosis lead to fibrotic remodelling and finally fatty cell replacement. As in DMD children, the muscle pathology progressed in mdx mice as a function of age. This way, we fed 3-month-old mdx mice (n = 5) with ProAbe and we performed H&E analysis of muscle sections to verify whether this diet could delay the onset of the pathology. In tibialis anterior (TA) and quadriceps (QA) of treated mice, we observed the presence of degenerating and small centrally nucleated regenerating muscle fibers, such as in untreated mice; however reduced signs of degeneration (consisting in hypertrophic fibers, fiber splitting, and fat replacement) were seen in treated mice versus untreated ones (n = 5) (Figure 1(a)). To verify whether antioxidants supplementation could bear an effect on muscle mass we measured cross-section fiber area (CSA) of both treated and untreated mdx mice. We found that the distribution curves of treated mice shifted to the right in comparison to that one related to mdx control group, thus proving that there was a significant increase in fiber CSA in both muscles examined (TA and QA muscles) (F test to compare variance was significant for P < 0,0001) (Figures 1(b)–1(d)). In particular there was a reduction in the percentage of smaller fibers (CSA of QA in treated 2274 ± 32,59 and untreated mice 1535 ± 20,08; CSA of TA in treated 1681 ± 23,76 and untreated mice 1486 ± 19,44; t-test to compare mean was significant for P < 0,0001) (Figures 1(c)-1(d)). To better elucidate that ProAbe-dependent increase of muscle size was not due to fibrotic deposition, other morphological features of the muscles of treated mice were measured. We demonstrated a diminished percentage of fibrosis in both muscles treated with ProAbe (treated QA 6,255 ± 0,632 and untreated QA 14,67 ± 0,66 P < 0,0001; treated TA 11,29 ± 0,736 and untreated TA 20,62 ± 1,521 P < 0.0001) (Figure 1(e)) and we observed that the percentage of necrotic fibers was significantly smaller in both treated muscles (treated QA 0,8944 ± 0,06 and untreated QA 2,015 ± 0,376, P = 0,045; treated TA 3,867 ± 0,4884 and untreated TA 11,35 ± 1,926, P = 0,0093) (Figure 1(f)). As clearly described by different works [49–51] inflammatory cells in DMD can interact with resident muscular stem cells and cause the fibroadipogenic degeneration of muscular fibers. According to these evidences, CD11b staining was performed to identify macrophage infiltrates as an indicator of muscle inflammation and we assessed a reduction in inflammatory infiltrates both in TA and in QA muscles of treated mice (untreated TA: 21130 ± 4909 and treated TA: 6187 ± 1460, P = 0,0154; untreated QA 24610 ± 2217 and treated QA 10080 ± 1559, P = 0,0017) (Figure 1(g)). Moreover, we counted the number of centronucleated myofibers—that are the fibers in regeneration—and we demonstrated that their number was smaller in both treated muscles (untreated TA: 39,79 ± 4,571 and treated TA: 23,64 ± 2,833; P = 0,0133; untreated QA 40,42 ± 8,645 and treated QA 18,05 ± 3,981, P = 0,0369) (Figure 1(h)).


Improvement of endurance of DMD animal model using natural polyphenols.

Sitzia C, Farini A, Colleoni F, Fortunato F, Razini P, Erratico S, Tavelli A, Fabrizi F, Belicchi M, Meregalli M, Comi G, Torrente Y - Biomed Res Int (2015)

H&E analysis of treated and untreated mice. H&E staining was performed on 10 μm-thick frozen sections from TA and QA muscles (a). In treated mice reduced signs of muscular wasting were observed in comparison to untreated mice. In particular reduced inflammatory infiltrates between myofibers, reduced fat deposition, and reduced necrotic fibers were assessed. In both treated and untreated mice small centronucleated regenerating fibers were observed. For each muscle analyzed with H&E staining we showed the myofiber area and their distribution frequency (b). The curve of TA/QA muscles of treated mice shifted to the right related to untreated mice, thus demonstrating an increase in myofiber area of treated mice (CSA QA: 25° percentile of treated mice 1221,26 and of untreated mice 903,152; CSA TA: 25° percentile of treated mice 1120,39 and of untreated mice 874,068; CSA QA: 75° percentile of treated mice 3107,04 and of untreated mice 1946,06; and CSA TA: 75° percentile of treated mice 2978,59 and of untreated mice 1883,39). Moreover, we indicated the coefficient of variance (graphs showing Min and Max values and mean + SEM) ((c) for QA and (d) for TA). Amelioration of dystrophic phenotype following ProAbe treatment was demonstrated by decrease of fibrosis by measuring area of connective tissue (AM) (e), of the percentage of necrotic fibers (f), of macrophage infiltration area (CD11b staining) (g), and of the number of centronucleated myofibers per section (h).
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4377377&req=5

fig1: H&E analysis of treated and untreated mice. H&E staining was performed on 10 μm-thick frozen sections from TA and QA muscles (a). In treated mice reduced signs of muscular wasting were observed in comparison to untreated mice. In particular reduced inflammatory infiltrates between myofibers, reduced fat deposition, and reduced necrotic fibers were assessed. In both treated and untreated mice small centronucleated regenerating fibers were observed. For each muscle analyzed with H&E staining we showed the myofiber area and their distribution frequency (b). The curve of TA/QA muscles of treated mice shifted to the right related to untreated mice, thus demonstrating an increase in myofiber area of treated mice (CSA QA: 25° percentile of treated mice 1221,26 and of untreated mice 903,152; CSA TA: 25° percentile of treated mice 1120,39 and of untreated mice 874,068; CSA QA: 75° percentile of treated mice 3107,04 and of untreated mice 1946,06; and CSA TA: 75° percentile of treated mice 2978,59 and of untreated mice 1883,39). Moreover, we indicated the coefficient of variance (graphs showing Min and Max values and mean + SEM) ((c) for QA and (d) for TA). Amelioration of dystrophic phenotype following ProAbe treatment was demonstrated by decrease of fibrosis by measuring area of connective tissue (AM) (e), of the percentage of necrotic fibers (f), of macrophage infiltration area (CD11b staining) (g), and of the number of centronucleated myofibers per section (h).
Mentions: Fibrosis is considered the most devastating consequence of the progression of disease in DMD patients: due to the lack of dystrophin, satellite cell proliferation cannot compensate constant myofiber breakdown so that inflammatory processes that follow muscular necrosis lead to fibrotic remodelling and finally fatty cell replacement. As in DMD children, the muscle pathology progressed in mdx mice as a function of age. This way, we fed 3-month-old mdx mice (n = 5) with ProAbe and we performed H&E analysis of muscle sections to verify whether this diet could delay the onset of the pathology. In tibialis anterior (TA) and quadriceps (QA) of treated mice, we observed the presence of degenerating and small centrally nucleated regenerating muscle fibers, such as in untreated mice; however reduced signs of degeneration (consisting in hypertrophic fibers, fiber splitting, and fat replacement) were seen in treated mice versus untreated ones (n = 5) (Figure 1(a)). To verify whether antioxidants supplementation could bear an effect on muscle mass we measured cross-section fiber area (CSA) of both treated and untreated mdx mice. We found that the distribution curves of treated mice shifted to the right in comparison to that one related to mdx control group, thus proving that there was a significant increase in fiber CSA in both muscles examined (TA and QA muscles) (F test to compare variance was significant for P < 0,0001) (Figures 1(b)–1(d)). In particular there was a reduction in the percentage of smaller fibers (CSA of QA in treated 2274 ± 32,59 and untreated mice 1535 ± 20,08; CSA of TA in treated 1681 ± 23,76 and untreated mice 1486 ± 19,44; t-test to compare mean was significant for P < 0,0001) (Figures 1(c)-1(d)). To better elucidate that ProAbe-dependent increase of muscle size was not due to fibrotic deposition, other morphological features of the muscles of treated mice were measured. We demonstrated a diminished percentage of fibrosis in both muscles treated with ProAbe (treated QA 6,255 ± 0,632 and untreated QA 14,67 ± 0,66 P < 0,0001; treated TA 11,29 ± 0,736 and untreated TA 20,62 ± 1,521 P < 0.0001) (Figure 1(e)) and we observed that the percentage of necrotic fibers was significantly smaller in both treated muscles (treated QA 0,8944 ± 0,06 and untreated QA 2,015 ± 0,376, P = 0,045; treated TA 3,867 ± 0,4884 and untreated TA 11,35 ± 1,926, P = 0,0093) (Figure 1(f)). As clearly described by different works [49–51] inflammatory cells in DMD can interact with resident muscular stem cells and cause the fibroadipogenic degeneration of muscular fibers. According to these evidences, CD11b staining was performed to identify macrophage infiltrates as an indicator of muscle inflammation and we assessed a reduction in inflammatory infiltrates both in TA and in QA muscles of treated mice (untreated TA: 21130 ± 4909 and treated TA: 6187 ± 1460, P = 0,0154; untreated QA 24610 ± 2217 and treated QA 10080 ± 1559, P = 0,0017) (Figure 1(g)). Moreover, we counted the number of centronucleated myofibers—that are the fibers in regeneration—and we demonstrated that their number was smaller in both treated muscles (untreated TA: 39,79 ± 4,571 and treated TA: 23,64 ± 2,833; P = 0,0133; untreated QA 40,42 ± 8,645 and treated QA 18,05 ± 3,981, P = 0,0369) (Figure 1(h)).

Bottom Line: Different mechanisms were hypothesized such as reduced hydroxyl radicals, nuclear factor-κB deactivation, and NO protection from inactivation.We observed a reduction of muscle fibrosis deposition and myofiber necrosis together with an amelioration of vascularization.Our data confirmed that ProAbe-based diet may represent a strategy to coadjuvate the treatment of DMD.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio Cellule Staminali, Dipartimento di Patofisiologia e dei Trapianti, Universitá degli Studi di Milano, Fondazione IRCCS Cá Granda Ospedale Maggiore Policlinico, Centro Dino Ferrari, Via Francesco Sforza 35, 20122 Milano, Italy.

ABSTRACT
Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is characterized by muscular wasting caused by dystrophin deficiency that ultimately ends in force reduction and premature death. In addition to primary genetic defect, several mechanisms contribute to DMD pathogenesis. Recently, antioxidant supplementation was shown to be effective in the treatment of multiple diseases including muscular dystrophy. Different mechanisms were hypothesized such as reduced hydroxyl radicals, nuclear factor-κB deactivation, and NO protection from inactivation. Following these promising evidences, we investigated the effect of the administration of a mix of dietary natural polyphenols (ProAbe) on dystrophic mdx mice in terms of muscular architecture and functionality. We observed a reduction of muscle fibrosis deposition and myofiber necrosis together with an amelioration of vascularization. More importantly, the recovery of the morphological features of dystrophic muscle leads to an improvement of the endurance of treated dystrophic mice. Our data confirmed that ProAbe-based diet may represent a strategy to coadjuvate the treatment of DMD.

Show MeSH
Related in: MedlinePlus