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.

Effects of resistance training and estrogen replacement on adipose tissue inflammation in ovariectomized rats.

Estrogen deficiency is directly related to central obesity and low-grade inflammation. Hormonal replacement and exercise training are both able to decrease fat accumulation and inflammation in postmenopausal women. However, the efficiency of resistance training (RT) and estrogen replacement (ER) in minimizing adiposity and inflammation in the visceral adipose tissue (VAT) of ovariectomized (OVX) rats has not yet been elucidated. In this study, Sprague-Dawley rats were divided into the following 6 groups: sham-operated sedentary (Sham-Sed), OVX-Sed, Sham-RT, OVX-RT, OVX-Sed-ER, and OVX-RT-ER groups. ER was performed by implanting silastic capsules containing 17ß-estradiol. For RT, the animals were required to climb a 1.1-m vertical ladder with conical flasks containing weights attached to their tails for 12 weeks. Histological analyses were used to evaluate morphological changes. Gene expression levels were determined by quantitative real-time reverse transcriptase polymerase chain reaction, and protein concentrations were determined using Multiplex/Luminex assays. Ovariectomy increased the body mass (BM), adipocyte area, and inflammation in the VAT, the latter of which was indicated by reduced interleukin-10 (48%) and increased tumor necrosis factor (TNF)-α concentration (∼3%). RT efficiently decreased BM, adipocyte area, and inflammation in the OVX groups. The combination of RT and ER decreased BM (19%) and the TNF-α concentration (18%) and increased the gene and protein expression levels of adiponectin (173% and 18%). These results indicate that RT and the combination of RT and ER are efficient strategies for reducing the BM and improving the inflammatory status of OVX rats.

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

OBJECTIVE: The 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. DESIGN: Wistar 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. RESULTS: The 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. CONCLUSIONS: This 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.

Efeito de um programa de fisioterapia funcional em crianças com paralisia cerebral associado a orientações aos cuidadores: estudo preliminar / Effect of a functional physical therapy program on cerebral palsy children, associated to guidance for their caregivers: a preliminary study

O objetivo foi verificar o efeito de um programa de fisioterapia funcional para crianças com paralisia cerebral, associado a orientações aos pais e/ou cuidadores; e verificar a correlação entre as habilidades funcionais e a assistência do cuidador, utilizando o Inventário de Avaliação Pediátrica de Incapacidade (PEDI). Participaram quatro crianças entre 24 e 43 meses de idade, hemiplégicas, espásticas e nível I no sistema de classificação da função motora ampla (GMFCS). Foram realizadas quatro avaliações – uma antes do início do programa, as demais aos 30, 60 e 90 dias após a primeira –, empregando-se as partes I (Habilidades funcionais) e II (Assistência do cuidador) do PEDI. As crianças foram submetidas a sessões de uma hora de fisioterapia funcional três vezes por semana, durante três meses: duas vezes a sessão era de fisioterapia com base no conceito neuroevolutivo Bobath e uma vez, treino de atividades da vida diária. Também foram dadas orientações por escrito aos pais e/ou cuidadores quanto à assistência à criança, incentivando-os a praticá-la em casa. A análise dos resultados mostrou que, na última avaliação, as crianças obtiveram escores significativamente maiores que na primeira...

The purpose was to verify the effect of a functional physical therapy program on children with cerebral palsy, associated to guidance to parents and/or caregivers; and to search for correlations between the child’s functional abilities and caregivers’ assistance, by means of the Pediatric Evaluation Disability Inventory (PEDI). Four hemiplegic, spastic children between 24 and 43 months old, classified at the Gross Motor Function Classification System level I, were submitted to four evaluations, the first prior to program onset, and the others 30, 60, and 90 days after the first one. PEDI parts I (Functional abilities) and II (Caregivers’ assistance) were used. Caregivers were provided with written instructions on how to best deliver care at home. The physical therapy program consisted of three weekly 1-hour sessions for three months; two sessions were of physical therapy based on Bobath concept, and one, of daily activities training. The analysis of results showed children obtained a significantly higher score at the last assessment as compared to the first. A high, significant correlation was found between PEDI parts I and II (r=1.0; p=0.083). The functional physical therapy program associated to instructions to caregivers proved thus effective to improve the functional performance of level-I children with spastic hemiplegia...

Muscle and nerve responses after different intervals of electrical stimulation sessions on denervated rat muscle.

OBJECTIVE: Electrical stimulation is a procedure used to treat denervated muscles. The number of electrical stimulation sessions varies across muscle rehabilitation protocols, from daily to certain days throughout the week. The purpose of this study was to evaluate how muscle and nerve respond to different intervals of electrical stimulation applied to denervated muscle. DESIGN: Denervation of rat gastrocnemius muscle was imposed via nerve crush, and electrical stimulation was applied to the muscle either daily (Monday through Friday) or on alternate days (Monday, Wednesday, and Friday). Four experimental groups were studied: denervated, denervated plus daily electrical stimulation, denervated plus alternate-day electrical stimulation, and control. RESULTS: On the 12th day after nerve crush, levels of MyoD, myostatin, and atrogin-1 gene expression, as well as muscle fiber and nerve morphometry, were evaluated. Expression levels of all three genes were higher in the denervated group when compared with control. Also, expression levels of MyoD and myostatin were higher in denervated plus alternate-day electrical stimulation and denervated plus daily electrical stimulation groups when compared with denervated. The denervated plus daily electrical stimulation group had lower atrogin-1 expression, lower density of intramuscular connective tissue, and better morphometric nerve characteristics when compared with the denervated and denervated plus alternate-day electrical stimulation. CONCLUSIONS: These results indicate that the responses of both muscle and nerve to electrical stimulation after muscle denervation depend on the intervals of electrical stimulation application.

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.

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.

Electrical stimulation based on chronaxie reduces atrogin-1 and myoD gene expressions in denervated rat muscle.

Denervation induces muscle fiber atrophy and changes in the gene expression rates of skeletal muscle. Electrical stimulation (ES) is a procedure generally used to treat denervated muscles in humans. This study evaluated the effect of ES based on chronaxie and rheobase on the expression of the myoD and atrogin-1 genes in denervated tibialis anterior (TA) muscle of Wistar rats. Five groups were examined: (1) denervated (D); (2) D+ES; (3) sham denervation; (4) normal (N); and (5) N+ES. Twenty muscle contractions were stimulated every 48 h using surface electrodes. After 28 days, ES significantly decreased the expression of myoD and atrogin-1 in D+ES compared to the D group. However, ES did not prevent muscle-fiber atrophy after denervation. Thus, ES based on chronaxie values and applied to denervated muscles using surface electrodes, as normally used in human rehabilitation, was able to reduce the myoD and atrogin-1 gene expressions, which are related to muscular growth and atrophy, respectively. The results of this study provide new information for the treatment of denervated skeletal muscle using surface ES.

Short bouts of stretching increase myo-D, myostatin and atrogin-1 in rat soleus muscle.

Stretching is widely used in rehabilitation and sports activities to improve joint range-of-motion and flexibility in humans, but the effect of stretching on the gene expression of skeletal muscle is poorly understood. We evaluated the effect of short bouts of passive stretching of rat soleus muscle on myo-D, myostatin, and atrogin-1 gene expressions. Six groups of animals were submitted to a single session of stretching (10 stretches of 1 minute with 30 seconds of rest between them, performed manually) and were evaluated immediately (I), and 8, 24, 48, 72, and 168 hours after the session. To evaluate the effect of repetitive sessions of stretching on the soleus muscle over 1 week, three groups of animals received a single session per day of stretching and the muscle was evaluated immediately after 2, 3, and 7 sessions. The mRNA levels of myo-D, myostatin, and atrogin-1 were determined by real-time polymerase chain reaction. A single session of stretching increased the mRNA levels of myo-D (after 24 h), myostatin (I, and 168 h later), and atrogin-1 (after 48 h). Repeated daily session of stretching over 1 week increased myostatin (after 7 sessions) and atrogin-1 expression (after 2, 3, and 7 sessions). Thus, short bouts of passive stretching are able to increase the gene expression of factors associated with muscle growth (myo-D), negative regulation of muscle mass (myostatin), and atrophy (atrogin-1), indicating muscle remodeling through different pathways.