Gabriel Borges Delfino

Effects of alternagin-C from Bothrops alternatus on gene expression and activity of metalloproteinases in regenerating skeletal muscle

This study evaluated the effects of alternagin-C (ALT-C) on mRNA levels of VEGF, MyoD and matrix metalloproteinase 2 (MMP-2) and on activity of MMPs in injured tibialis anterior (TA) muscle induced by cryolesioning in rats. Thirty-six Wistar rats (3 months old, 258.9+/-27 g) were divided into five groups: (1) control group; (2) injured TA and analyzed 3 days later; (3) injured TA treated with ALT-C and analyzed 3 days later; (4) injured TA and analyzed 7 days later and (5) injured TA treated with ALT-C and analyzed 7 days later. The injured muscle received 25 microl of ALT-C (50 nM). The injured and uninjured muscle areas were quantified by light microscopy. The MMP activity was evaluated through zymography, and mRNA of MyoD, VEGF and MMP-2 was assessed by quantitative polymerase chain reaction. ALT-C neither reduced the muscle injury area nor altered the pattern of MyoD and VEGF expression in injured muscles. However, ALT-C reduces both MMP-2 mRNA and gelatinolytic activity in injured muscles. The study indicates that ALT-C, at the tested concentrations, did not improve muscle regeneration process in rats. The effect on MMP-2 mRNA and gelatinolytic activity suggests that ALT-C changes the overall balance of ECM protein turnover during muscle regeneration.

Cryotherapy Reduces Inflammatory Response Without Altering Muscle Regeneration Process and Extracellular Matrix Remodeling of Rat Muscle

The application of cryotherapy is widely used in sports medicine today. Cooling could minimize secondary hypoxic injury through the reduction of cellular metabolism and injury area. Conflicting results have also suggested cryotherapy could delay and impair the regeneration process. There are no definitive findings about the effects of cryotherapy on the process of muscle regeneration. The aim of the present study was to evaluate the effects of a clinical-like cryotherapy on inflammation, regeneration and extracellular matrix (ECM) remodeling on the Tibialis anterior (TA) muscle of rats 3, 7 and 14 days post-injury. It was observed that the intermittent application of cryotherapy (three 30-minute sessions, every 2 h) in the first 48 h post-injury decreased inflammatory processes (mRNA levels of TNF-α, NF-κB, TGF-β and MMP-9 and macrophage percentage). Cryotherapy did not alter regeneration markers such as injury area, desmin and Myod expression. Despite regulating Collagen I and III and their growth factors, cryotherapy did not alter collagen deposition. In summary, clinical-like cryotherapy reduces the inflammatory process through the decrease of macrophage infiltration and the accumulation of the inflammatory key markers without influencing muscle injury area and ECM remodeling.

Quadriceps Muscle Atrophy After Anterior Cruciate Ligament Transection Involves Increased mRNA Levels of Atrogin-1, Muscle Ring Finger 1, and Myostatin

ObjectiveThe aim of this study was to assess the mRNA levels of atrogin-1, muscle ring finger 1, and myostatin in rat quadriceps after anterior cruciate ligament (ACL) transection. DesignWistar rats were randomized into three different groups: ACL (surgery and ACL transection), sham (surgery without ACL transection), and control. Vastus medialis, rectus femoris, and vastus lateralis muscles were harvested at 1, 2, 3, 7, and 15 days after ACL transection. The mRNA levels of atrogin-1, muscle ring finger 1, and myostatin, as well as the ubiquitinated protein content, muscle mass, and cross-sectional area of the muscle fibers, were evaluated. ResultsElevated levels of atrogin-1, muscle ring finger 1, and myostatin mRNA were detected in all tested muscles at most time points. The ubiquitinated protein content was increased at 3 days in the ACL and sham groups. The muscle mass of the ACL group was reduced at 3, 7, and 15 days (vastus lateralis and vastus medialis) and at 7 and 15 days (rectus femoris), whereas it was reduced in the sham group at 3 and 7 days (vastus lateralis and vastus medialis) and at 7 days (rectus femoris). The cross-sectional area of vastus medialis was reduced at 3, 7, and 15 days in the ACL group and at 3 and 7 days in the sham group. The cross-sectional area of the vastus lateralis was reduced at 7 and 15 days in the ACL group and at 7 days in the sham group. Whereas muscle mass and cross-sectional area recovery was noted in the sham group, no recovery was observed in the ACL group. ConclusionsQuadriceps atrophy after ACL transection involves increased levels of myostatin, atrogin-1, and muscle ring finger 1 mRNA and the accumulation of ubiquitinated protein.

Neuromuscular electrical stimulation alters gene expression and delays quadriceps muscle atrophy of rats after anterior cruciate ligament transection

Introduction: Neuromuscular electrical stimulation (NMES) is used to improve quadriceps mass after anterior cruciate ligament (ACL) injury. We studied the effect of NMES on mRNA levels of atrophy genes in the quadriceps muscle of rats after ACL transection. Methods: mRNA levels of atrogin‐1, MuRF‐1, and myostatin were assessed by quantitative PCR and the polyubiquitinated proteins by Western blot at 1, 2, 3, 7, and 15 days postinjury. Results: NMES minimized the accumulation of atrogenes and myostatin according to time period. NMES also prevented reduction in muscle mass in all muscles of the ACLES group at 3 days. Conclusions: Use of NMES decreased the accumulation of atrogenes and myostatin mRNA in the quadriceps muscles, inhibiting early atrophy at 3 days, although it did not prevent atrophy at 7 and 15 days after ACL transection. This study highlights the importance of therapeutic NMES interventions in the acute phase after ACL transection. Muscle Nerve 49: 120–128, 2014

Neuromuscular electrical stimulation induces beneficial adaptations in the extracellular matrix of quadriceps muscle after anterior cruciate ligament transection of rats.

ObjectiveThe aim of this study was to assess the effect of neuromuscular electrical stimulation (NMES) on the extracellular matrix remodeling of the quadriceps muscle after anterior cruciate ligament (ACL) transection in rats. The hypothesis of this study was that ACL transection would induce maladaptive modifications in the extracellular matrix through the increase in connective tissue (CT) accumulation and net degradation of type IV collagen of the quadriceps muscle. In addition, clinical-like NMES, applied to the quadriceps muscle immediately after the ACL transection, would reduce the accumulation of the CT content and net degradation of type IV collagen. DesignWistar male rats were randomized into five different groups: ACL (surgery and ACL transection), Sham (surgery without ACL transection), ACLES (surgery, ACL transection, and NMES), ShamES (surgery without ACL transection, but NMES), and Control (intact animals). The vastus medialis, rectus femoris, and vastus lateralis muscles of the quadriceps were harvested 1, 2, 3, 7, and 15 days after surgery. Matrix metalloproteinase–2 (MMP-2) (messenger RNA [mRNA] levels and activity), collagen IV (mRNA and protein levels), and CT density were assessed. ResultsThe ACL transection increased the CT content and MMP-2 mRNA levels and decreased collagen IV mRNA and protein levels. NMES minimized the CT density in all muscles and reduced the MMP-2 mRNA levels mainly in the vastus lateralis muscle at 7 days. Moreover, type IV collagen mRNA levels were increased in all muscles at 7 days, as was the protein level only at 15 days, in the NMES groups. ConclusionsThis study showed that ACL transection increases CT content and MMP-2 mRNA levels and induces rapid changes in basement membranes, causing net degradation of type IV collagen during the first 2 wks after ACL injury. Furthermore, clinical-like NMES minimized the accumulation of CT density, regulated the MMP-2 mRNA levels, and increased both type IV collagen mRNA and protein levels.

Effect of tibiotarsal joint inflammation on gene expression and cross-sectional area in rat soleus muscle

BACKGROUND Joint inflammation is a common clinical problem in patients treated by physical therapists. The hypothesis of this study is that joint inflammation induces molecular and structural changes in the soleus muscle, which is composed mainly of slow-twitch muscle fibers. OBJECTIVE To study the effect of tibiotarsal joint inflammation on muscle fiber cross-sectional area (CSA), gene expression levels (atrogin-1, MuRF1, MyoD, myostatin, p38MAPK, NFκB, TNF-alpha), and TNF-alpha protein in the soleus muscle. METHOD Wistar rats were randomly divided into 3 periods (2, 7 and 15 days) and assigned to 4 groups (control, sham, inflammation, and immobilization). RESULTS In the inflammation group at 2 days, MuRF1 and p38MAPK expression had increased, and NFκB mRNA levels had decreased. At 7 days, myostatin expression had decreased. At 7 and 15 days, this group had muscle fiber CSA reduction. At 2 days, the immobilization group showed increased atrogin-1, MuRF1, NFκB, MyoD, and p38MAPK expressions and reduced muscle fiber CSA. At 7 and 15 days, myostatin mRNA levels had increased, and the CSA had decreased. The sham group showed increased p38MAPK and myostatin expressions at 2 and 7 days, respectively. No changes occurred in TNF-alpha gene or protein expression. CONCLUSION Acute joint inflammation induces gene expression related to the proteolytic pathway without reduction in muscle fiber CSA. Chronic joint inflammation induced muscle atrophy without up-regulation of important genes belonging to the proteolytic pathway. Thus, muscle adaptation may differ according to the stage of joint inflammation, which suggests that the therapeutic modalities used by physical therapists at each stage should also be different.

Efeito do sulfato de vanadil sobre o comprometimento metabólico muscular induzido pela imobilização de membro posterior de ratos

The purpose of this study was to evaluate the metabolic performance of immobilized skeletal muscle in rats treated with vanadyl sulphate. Male Wistar rats were divided in groups (n = 6): control (C), immobilized (I), treated with vanadyl sulphate (VS, 0,25 mM) and immobilized treated with vanadyl sulphate (I + VS) during seven days. The concentration of vanadyl sulphate diluted in water was 0,25 mM. After experimental stage, the glycogen content (GC) was evaluated in soleus (S), white gastrocnemius (WG), red gastrocnemius (RG), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles, besides S and EDL weight. The statistical analysis was realized by the ANOVA followed by Tukey test (p < 0,05). In VS group, the results showed a significant increase in GC (S 110%, WG 71%, RG 85%, TA 125%, EDL 108%) and in the weight (S 9%, EDL 11%). The immobilization reduced significantly the GC (S 31.6%, WG 56.6%, RG 39.1%, EDL 41.7%, TA 45.2%) and weight (S 34.2% and ELD 27%), and in I + VS group, there was a increase of the GC in all muscles (S 211%, WG 115%, RG 148%, EDL 161.9%, TA 147%), besides hindering the weight loss in S (75%) and EDL (46%). The vanadyl sulphate treatment promoted an increase in the glycogen content of control and immobilized groups, besides hindering the weight loss, showing that the insulino-mimetic effect is represented by glycogenic action associate to a possible anti-catabolic action.

Metformina minimiza as alterações morfométricas no músculo sóleo de ratos submetidos à imobilização articular

The aim of this study was to evaluate the effect of metformin treatment on the muscle mass, fibers area and connective tissue area density in soleus (S) muscle under articular immobilization. METHODS AND RESULTS: Male Wistar rats (250-300g) were divided in 4 groups (n=5): control, treated with metformin, immobilized and immobilized treated with metformin. Immobilization was performed by acrylic resin orthoses on the left hindlimb keeping the ankle in neutral position during 7 days. The animals were euthanatized and the S muscle was dissected and weighed. Samples of its ventral portion were treated for inclusion in paraffin and stained in Hematoxylin-Eosin (H:E). The results were obtained through analyses of the muscular fiber area (images analyzed - Image Pro-plus 4,0), as well as of intramuscular connective tissue by means of planimetry. The statistical analysis was performed by normality test followed by ANOVA and Tukey (p<0.05). Hindlimb immobilization during 7 days promoted significant reduction (p<0.05) of 35% in the muscular mass; 44% (p<0.05) in the fiber area and increase of 216% (p<0.05) in the intramuscular connective tissue. The metformin in immobilized group promoted significant alterations (p<0.05) in the muscular mass. In addiction, it was observed significant increase (p<0.05) of 29.6% in the fibers area and significant reduction (p<0.05) of 67% in the intramuscular connective tissue. CONCLUSION: The metformin treatment minimized the reduction of muscle fibers area, as well as the increase of connective tissue in hindlimb immobilized muscle. These results suggest that metformin should be used to increase fast muscle recovery after hindlimb immobilization.

Insulin treatment reverses the increase in atrogin-1 expression in atrophied skeletal muscles of diabetic rats with acute joint inflammation

Background The aim of this study was to evaluate the changes in biomarkers of skeletal muscle proteolysis (atrogin-1, muscle RING finger-1 protein [MuRF-1]) and inflammation (nuclear factor kappa-B) in skeletal muscles of rats under two catabolic conditions, diabetes mellitus (DM) and acute joint inflammation, and the effects of insulin therapy. Materials and methods Male Wistar rats were divided into groups without diabetes – normal (N), saline (NS), or ι-carrageenan (NCa) injection into the tibiotarsal joint – and groups with diabetes – diabetes (D), plus insulin (DI), saline (DS), or ι-carrageenan (DCa) injection into the tibiotarsal joint, or ι-carrageenan injection and treatment with insulin (DCaI). Three days after ι-carrageenan injection (17 days after diabetes induction), tibialis anterior (TA) and soleus (SO) skeletal muscles were used for analysis. Results DM alone caused a significant decrease in the mass of TA and SO muscles, even with low levels of atrogenes (atrogin-1, MuRF-1), which could be interpreted as an adaptive mechanism to spare muscle proteins under this catabolic condition. The loss of muscle mass was exacerbated when ι-carrageenan was administered in the joints of diabetic rats, in association with increased expression of atrogin-1, MuRF-1, and nuclear factor kappa-B. Treatment with insulin prevented the increase in atrogin-1 (TA, SO) and the loss of muscle mass (SO) in diabetic-carrageenan rats; in comparison with TA, SO muscle was more responsive to the anabolic actions of insulin. Conclusion Acute joint inflammation overcame the adaptive mechanism in diabetic rats to prevent excessive loss of muscle mass, worsening the catabolic state. The treatment of diabetic-carrageenan rats with insulin prevented the loss of skeletal muscle mass mainly via atrogin-1 inhibition. Under the condition of DM and inflammation, muscles with the prevalence of slow-twitch, type 1 fibers were more responsive to insulin treatment, recovering the ability to grow.

Effect of the vanadyl sulphate on the muscular metabolic compromising induced by immobilization of posterior limb of rats

The purpose of this study was to evaluate the metabolic performance of immobilized skeletal muscle in rats treated with vanadyl sulphate. Male Wistar rats were divided in groups (n = 6): control (C), immobilized (I), treated with vanadyl sulphate (VS, 0,25 mM) and immobilized treated with vanadyl sulphate (I + VS) during seven days. The concentration of vanadyl sulphate diluted in water was 0,25 mM. After experimental stage, the glycogen content (GC) was evaluated in soleus (S), white gastrocnemius (WG), red gastrocnemius (RG), tibialis anterior (TA) and extensor digitorum longus (EDL) muscles, besides S and EDL weight. The statistical analysis was realized by the ANOVA followed by Tukey test (p < 0,05). In VS group, the results showed a significant increase in GC (S 110%, WG 71%, RG 85%, TA 125%, EDL 108%) and in the weight (S 9%, EDL 11%). The immobilization reduced significantly the GC (S 31.6%, WG 56.6%, RG 39.1%, EDL 41.7%, TA 45.2%) and weight (S 34.2% and ELD 27%), and in I + VS group, there was a increase of the GC in all muscles (S 211%, WG 115%, RG 148%, EDL 161.9%, TA 147%), besides hindering the weight loss in S (75%) and EDL (46%). The vanadyl sulphate treatment promoted an increase in the glycogen content of control and immobilized groups, besides hindering the weight loss, showing that the insulino-mimetic effect is represented by glycogenic action associate to a possible anti-catabolic action.