João Luiz Quagliotti Durigan

Rat hindlimb joint immobilization with acrylic resin orthoses

The objective of the present study was to propose an orthosis of light material that would be functional for the animal and that would maintain only the ankle joint immobilized. Male Wistar rats (3 to 4 months old, 250-300 g) were divided into 2 groups (N = 6): control and immobilized for 7 days. Rats were anesthetized with sodium pentobarbital (40 mg/kg weight) and the left hindlimb was immobilized with the orthoses composed of acrylic resin model, abdominal belt and lateral supports. The following analyses were performed: glycogen content of the soleus, extensor digitorum longus, white gastrocnemius, red gastrocnemius, and tibialis anterior muscles by the phenol sulfuric method, and the weight, fiber area and intramuscular connective tissue of the soleus by the planimetric system. Data were analyzed statistically by the Kolmogorov-Smirnov, Student t and Wilcoxon tests. Immobilization decreased glycogen in all muscles (P < 0.05; soleus: 31.6%, white gastrocnemius: 56.6%, red gastrocnemius: 39%, extensor digitorum longus: 41.7%, tibialis anterior: 45.2%) in addition to reducing soleus weight by 34% (P < 0.05). Furthermore, immobilization promoted reduction of the fiber area (43%, P < 0.05) and increased the connective tissue (200%, P < 0.05). The orthosis model was efficient comparing with another alternative immobilization model, like plaster casts, in promoting skeletal muscle alterations, indicating that it could be used as a new model in other studies related to muscle disuse.

Electrical stimulation increases matrix metalloproteinase‐2 gene expression but does not change its activity in denervated rat muscle

Muscle‐fiber atrophy occurs concomitantly with intramuscular connective tissue proliferation following denervation. These events contribute to the impairment of mechanical and functional properties of denervated muscles and compromise their recovery. Electrical stimulation (ES) is used in human rehabilitation to treat denervated muscles. However, the effects of this therapy on the intramuscular extracellular matrix (ECM) remain uncertain. Metalloproteinases (MMPs) are responsible by remodeling ECM in many neuromuscular disorders. This study evaluates the effect of ES on the activity of two important MMPs, MMP‐2 and MMP‐9, both involved in ECM remodeling of rat denervated muscles. Thirty‐four Wistar rats (3 months old, 356 ± 38.7 g) were divided into five groups: denervated (D); D+ES; sham denervation; normal (N); and N+ES. Twenty maximal muscle contractions were stimulated every 48 h using surface electrodes, as generally used in the rehabilitation of human denervated muscle. Both zymographic analysis and real‐time polymerase chain reaction (PCR) of MMPs were used to evaluate muscle after denervation for 28 days. Both the D and D+ES groups showed increased MMP‐2 activity compared with the N group (P < 0.05). Furthermore, only the D+ES had increased MMP‐2 gene expression compared with the N group (P < 0.05). MMP‐9 activity was not detected in any of the groups. The results of this study indicate that denervation increases MMP‐2 activity, and ES regulates MMP‐2 gene expression in rat denervated skeletal muscle. These findings clarify the effects of ES on the ECM of denervated muscle and may be helpful in designing new therapeutic strategies for rehabilitation in patients with denervation of muscle. Muscle Nerve, 2008

Joint inflammation alters gene and protein expression and leads to atrophy in the tibialis anterior muscle in rats.

Objective: The aim of this study was to evaluate the effect of tibiotarsal joint inflammation in rat tibialis anterior muscle through muscle fiber cross-sectional area (CSA) and gene expression (atrogin-1, muscle ring finger-1 [MuRF1], myogenic differentiation-1 [MyoD], p38 mitogen-activated protein kinase [p38MAPK], nuclear factor kappa B-dependent [NF&kgr;B], tumor necrosis factor-alpha [TNF-&agr;]). Design: Wistar rats were randomly divided into three periods (2, 7, and 15 days) and assigned into four groups within each experimental period: control, sham, inflammation, and immobilization. Real-time polymerase chain reaction, Western blot, immunofluorescence, and muscle fiber CSA analyses were performed. Results: At 2 days, the inflammation group increased atrogin-1, MuRF1, and myostatin and reduced MyoD expression. At 7 days, the inflammation group increased atrogin-1, MuRF1, NF&kgr;B, p38MAPK, MyoD, myostatin, and TNF-&agr; expression and TNF-&agr; protein and reduced muscle fiber CSA. At 15 days, gene and protein expression in the inflammation group returned to basal levels, and CSA values were similar to those in control and sham groups. The immobilization groups have a similar pattern in all experimental periods, with increased atrogin-1, MuRF1, NF&kgr;B, and TNF-&agr; gene expression and reduced muscle fiber CSA. The sham group had increased myostatin and atrogin-1 expression at 2 days and increased MyoD and myostatin expression at 7 days. Conclusions: Joint inflammation stimulated the expression of muscle factors related to atrophy, growth, differentiation, and mass regulation followed by muscle atrophy.

Stretching and electrical stimulation regulate the metalloproteinase-2 in rat denervated skeletal muscle

Abstract Objective: To evaluate the effects of electrical stimulation (ES), stretching and their combined effects in matrix metalloproteinases (MMPs) gene expression and activity during early denervation of rat tibialis anterior (TA) muscle by axonotmesis. Methods: Thirty adult male Wistar rats were divided into five groups: normal (N), denervated TA (D), denervated TA submitted to daily ES (D + ES), denervated TA submitted to daily stretching (D + St) and denervated TA submitted daily to both ES and stretching concurrently (D + ES + St). Both zimographic analysis and real time polymerase chain reaction of MMPs were used to muscular evaluation. In addition, muscle fiber cross-section area (CSA) was also evaluated. Results: Stretching increased MMP-2 activity in denervated muscle when performed alone as well as in association with ES (p<0·01). Both stretching and ES, individually and in association, increased MMP-2 gene expression in denervated muscle compared to N and D (p<0·05). All denervated groups decreased muscle fiber CSA compared to N (p<0·05). Discussion: While stretching is the main stimulus leading to the activation of MMP-2, both ES and stretching are able to increase MMP-2 gene expression in rat denervated muscle suggesting ECM remodeling.

Effects of electrical stimulation and stretching on the adaptation of denervated skeletal muscle: implications for physical therapy

BACKGROUND This review will describe the main cellular mechanisms involved in the reduction and increase of myoproteins synthesis commonly associated with muscle atrophy and hypertrophy, respectively. OBJECTIVE We analyzed the effects of electrical stimulation (ES) and stretching exercise on the molecular pathways involved in muscle atrophy and hypertrophy. We also described the main effects and limits of these resources in the skeletal muscle, particularly on the denervated muscle. DISCUSSION Recently, our studies showed that the ES applied in a similar manner as performed in clinical practice is able to attenuate the increase of genes expression involved in muscle atrophy. However, ES was not effective to prevent the loss of muscle mass caused by denervation. Regarding to stretching exercises, their mechanisms of action on the denervated muscle are not fully understood and studies on this area are scarce. Studies from our laboratory have found that stretching exercise increased the extracellular matrix remodeling and decreased genes expression related to atrophy in denervated muscle. Nevertheless, it was not enough to prevent muscle atrophy after denervation. CONCLUSIONS In spite of the use of stretching exercise and ES in clinical practice in order to minimize the atrophy of denervated muscle, there is still lack of scientific evidence to justify the effectiveness of these resources to prevent muscle atrophy in denervated muscle.

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.

Rat skin wound healing induced by alternagin-C, a disintegrin-like, Cys-rich protein from Bothrops alternatus venom

Alternagin‐C (ALT‐C) is a disintegrin‐like, Cys‐rich protein isolated from Bothrops alternatus snake venom, which has been shown to induce in vivo angiogenesis. Therefore, this protein could be interesting as a new approach for tissue regeneration studies. Here the effects of ALT‐C on fibroblasts and inflammatory cells, collagen type III and type I and TGF‐α expression in a rat wounded skin model were studied. Thirty‐five male Wistar rats (weight 270 ± 20 g) were divided into seven groups with five animals in each of the following groups: a control group which wounded animals received treatment with natrozol® gel only; ALT‐C10, ALT‐C60 and ALT‐C100 groups of wounded animals that were treated with the same amount of gel containing 10, 60 and 100 ng of ALT‐C, respectively. Animals were treated once a day with 20 µl of gel associated or not with ALT‐C for 1, 3, 5 or 7 days. ALT‐C treatment increased the fibroblast density, collagen deposition and accelerated the inflammatory process, mostly in the ALT‐C60 group. These results indicate that ALT‐C improves wound repair process in rat skin. Thus, ALT‐C could be a candidate to the development of a novel therapeutic strategy for wounded skin repair.

Efeitos da estimulação elétrica neuromuscular sobre o membro posterior imobilizado de ratos durante 15 dias: análises metabólicas e morfométricas

OBJECTIVE: To evaluate the effect of electrical stimulation on the metabolic and morphometric profile of rat hind limb muscles subjected to immobilization for 15 days. METHOD: Wistar rats were divided into three groups (n=5): control; immobilized for 15 days; and immobilized for 15 days with electrical stimulation. The glycogen reserves of the soleus, extensor digitorum longus (EDL), white gastrocnemius (WG), red gastrocnemius (RG) and tibialis anterior (TA) muscles were evaluated, along with the weight, fibrous area and conjunctive tissue of the soleus. The statistical analysis was performed using the Anova and Kruskal-Wallis tests (p<0.05). RESULTS: Immobilization promoted significant alterations (p<0.05), such as: reductions in the glycogen reserves (soleus: 44.73%, WG: 47.82%, RG: 46.34%, EDL: 41.66%, TA: 48.38%) and in the weight (7.2%) and fibrous area (35%) of the soleus, and also increased connective tissue density (160%). Electrical stimulation promoted a significant increase (p<0.05) in the glycogen reserves of all the immobilized muscles: (soleus: 90.47%, WG: 62.5%, RG: 95.45%, EDL: 76.19%, TA: 56.25%) and in the weight (20.94%) and fibrous area (19.65%) of the soleus, and also promoted a significant reduction (15.38%, p<0.05) in connective tissue density. CONCLUSION: Electrical stimulation minimized the reduction in glycogen reserves and prevented the reduction in fibrous area and proliferation of connective tissue in the muscles subjected to immobilization.