Remobilization with whole-body vibration improves functionality, histomorphometric parameters, and AQP1 expression in the soleus muscle of Wistar rats.

Background: Whole-body vibration (WBV) is used to enhance physical performance in sports and rehabilitation. The present study analyzed the effects of remobilization with WBV on the soleus muscle of Wistar rats. Methods: Twenty-eight animals were separated into four experimental groups (n = 7): CON (control); IM (immobilized); FR (immobilization and free remobilization); and WBV (immobilization and remobilization with WBV). The immobilization of the pelvic limb was carried out according to the standard protocol using a plaster cast for 15 days. For remobilization with WBV, a Frequency of 60 Hz was applied for 10 min, five days a week, for two weeks. After the remobilization period, the animals were euthanized, and the right soleus muscle was dissected followed by processing for histomorphometric analysis and immunolocalization of Aquaporin 1 (AQP1). Results: We observed a reduced larger diameter in IM compared to CON, with restored values in WBV. For the estimation of connective tissue, a significant increase was observed in the immobilized groups, while a reduction was noted in the remobilized groups. AQP1 expression decreased significantly in IM and increased in WBV. Conclusion: Immobilization caused morphofunctional damage to the soleus muscle, and remobilization with WBV is efficient and offers advantages over free remobilization.

Costly immunometabolic remodelling in disused muscle buildup through physical exercise.

The mechanisms underlying the immunometabolic disturbances during skeletal muscle atrophy caused by a plethora of circumstances ranging from hospitalization to spaceflight missions remain unknown. Here, we outline the possible pathways that might be dysregulated in such conditions and assess the potential of physical exercise to mitigate and promote the recovery of muscle morphology, metabolism and function after intervals of disuse. Studies applying exercise to attenuate disuse-induced muscle atrophy have shown a pivotal role of circulating myokines in the activation of anabolic signalling pathways. These muscle-derived factors induce accretion of contractile proteins in the myofibers, and at the same time decrease protein breakdown and loss. Regular exercise plays a crucial role in re-establishing adequate immunometabolism and increasing the migration and presence in the muscle of macrophages with an anti-inflammatory phenotype (M2) and T regulatory cells (Tregs) after disease-induced muscle loss. Additionally, the switch in metabolic pathways (glycolysis to oxidative phosphorylation [OXPHOS]) is important for achieving rapid metabolic homeostasis during muscle regeneration. In this review, we discuss the molecular aspects of the immunometabolic response elicited by exercise during skeletal muscle regeneration. There is not, nevertheless, consensus on a single optimal intensity of exercise required to improve muscle strength, mass and functional capacity owing to the wide range of exercise protocols studied so far. Despite the absence of agreement on the specific strategy, physical exercise appears as a powerful complementary strategy to attenuate the harmful effects of muscle disuse in different scenarios.

Severe bone microarchitecture deterioration in a family with hereditary neuropathy: evidence of the key role of the mechanostat.

In this report, we present three cases of individuals from the same family with a diagnosis of CMT with severe tibia bone microarchitecture deterioration assessed by HR-pQCT. Charcot-Marie-Tooth disease (CMT) or hereditary neuropathy involves both motor and sensory nerves. Falls are often the first manifestation in these patients and represent an important risk factor for fracture. The reduction of mechanical input on bone inhibits bone formation by osteoblasts and accelerates bone resorption by osteoclasts, leading to disuse osteoporosis. We report three cases of individuals from the same family with a diagnosis of CMT with severe tibia bone microarchitecture deterioration assessed by high-resolution peripheral quantitative computed tomography (HR-pQCT). This affectation was exclusive to the tibia; the radius remained undamaged, showing the consequences of the lack of mobility and mechanical stimulation. Physical activity and rehabilitation, in addition to adequate calcium and vitamin D supplementation, may play an essential role in the management of this disease.

Experimental Model of HindLimb Suspension-Induced Skeletal Muscle Atrophy in Rodents.

Due to the difficulty of performing research protocols that reproduce human skeletal muscle disuse conditions, an experimental animal model of "hindlimb suspension" (or hindlimb unloading) was developed. This method was created in the 1970s and utilizes rats and mice to mimic space flight and bed rest in humans. It provides an alternative to investigate mechanisms associated with skeletal muscle mass loss and interventions designed to attenuate atrophy induced by hindlimb unloading. The mentioned protocol also allows investigating quality of bones and changes in several physiological parameters such as blood pressure, heart rate, plasma or tissue lipid composition, and glycemia.

Short-term creatine supplementation changes protein metabolism signaling in hindlimb suspension

The effect of a short-term creatine supplementation on hindlimb suspension (HS)-induced muscle atrophy was investigated. Creatine monohydrate (5 g/kg b.w. per day) or placebo, divided in 2 daily doses, was given by oral gavage for 5 days. Rats were maintained in HS with dietary supplementation concomitantly for 5 days. Body weight, soleus and EDL muscle masses, and cross-sectional areas (CSA) of the muscle fibers were measured. Signaling pathways associated with skeletal muscle mass regulation (FST, MSTN, FAK, IGF-1, MGF, Akt, mTOR, atrogin-1, and MuRF1 expressions, and Akt, S6, GSK3B, and 4EBP1 proteins) were evaluated in the muscles. Soleus muscle exhibited more atrophy than the EDL muscle due to HS. Creatine supplementation attenuated the decrease of wet weight and increased p-4EBP1 protein in the EDL muscle of HS rats. Also, creatine increased mTOR and atrogin-1 expressions in the same muscle and condition. In the absence of HS, creatine supplementation increased FAK and decreased MGF expressions in the EDL muscle. Creatine attenuated the increase in FST expression due to HS in the soleus muscle. MuRF1 expression increased in the soleus muscle due to creatine supplementation in HS animals whereas atrogin-1 expression increased still further in this group compared with untreated HS rats. In conclusion, short-term creatine supplementation changed protein metabolism signaling in soleus and EDL muscles. However, creatine supplementation only slightly attenuated the mass loss of both muscles and did not prevent the CSA reduction and muscle strength decrease induced by HS for 5 days.

Synaptophysin and caspase-3 expression on lumbar segments of spinal cord after sensorimotor restriction during early postnatal period and treadmill training.

The purpose of the current study was to investigate whether locomotor stimulation training could have beneficial effects on spinal cord plasticity consequent to sensorimotor restriction (SR). Male Wistar rats were exposed to SR from postnatal day 2 (P2) to P28. Control and experimental rats underwent locomotor stimulation training in a treadmill from P31 to P52. The intensity of the synaptophysin and caspase-3 immunoreaction was determined on ventral horn of spinal cord. The synaptophysin immunoreactivity was lower in the ventral horn of sensorimotor restricted rats compared to controls animals and was accompanied by an increased caspase-3 immunoreactivity. Those alterations were reversed at the end of the training period. Our results suggest that immobility affects the normal developmental process that spinal cord undergoes in early postnatal life influencing both pro-apoptotic and synapse markers. Also, we demonstrated that this phenomenon was reversed by 3 weeks of treadmill training.

Sarcomere structure: the importance of desmin protein in muscle atrophy / Estructura de sarcómera: la importancia de proteína desmina en atrofia muscular

Knowing the ultrastructure of skeletal muscle is critical to understand how it works under normal situation and the disorders caused by extreme or pathological conditions. Sarcomere is the basic structural unit of striated muscle tissue. An important element of sarcomere architecture are the intermediate filaments, including the desmin protein. Desmin protein contributes to maintenance of cell integrity, efficient transmission of force and mechanochemical signaling within the myocyte. Because of this, desmin protein has constantly been a focus of research that investigates its alterations associated to damage and muscle atrophy under different conditions. The purpose of the following literature review is to describe the basic concepts of muscle ultrastructure, emphasizing the desmin protein role under conditions of muscle disuse atrophy and aging.

Conocer la ultraestructura del músculo esquelético es crítico para entender cómo trabaja bajo situaciones normales y en desórdenes causados por condiciones extremas o patológicas. La sarcómera es la unidad de estructura básica del tejido muscular estriado. Elementos importantes en la arquitectura de la sarcómera son los filamentos intermedios, incluyendo la proteína desmina. La proteína desmina contribuye en mantener la integridad celular, la transmisión eficiente de fuerza y la señalización mecanoquímica dentro del miocito. Debido a lo anterior, la proteína desmina ha sido constante foco de investigación en trabajos que estudian sus alteraciones asociadas a daño y atrofia muscular bajo diferentes condiciones. El propósito de la siguiente revisión de la literatura es describir los conceptos básicos de la ultraestructura muscular, enfatizando en el rol de la proteína desmina bajo condiciones de atrofia muscular por desuso y envejecimiento.

Balanced Diet-Fed Fat-1 Transgenic Mice Exhibit Lower Hindlimb Suspension-Induced Soleus Muscle Atrophy.

The consequences of two-week hindlimb suspension (HS) on skeletal muscle atrophy were investigated in balanced diet-fed Fat-1 transgenic and C57BL/6 wild-type mice. Body composition and gastrocnemius fatty acid composition were measured. Skeletal muscle force, cross-sectional area (CSA), and signaling pathways associated with protein synthesis (protein kinase B, Akt; ribosomal protein S6, S6, eukaryotic translation initiation factor 4E-binding protein 1, 4EBP1; glycogen synthase kinase3-beta, GSK3-beta; and extracellular-signal-regulated kinases 1/2, ERK 1/2) and protein degradation (atrophy gene-1/muscle atrophy F-box, atrogin-1/MAFbx and muscle RING finger 1, MuRF1) were evaluated in the soleus muscle. HS decreased soleus muscle wet and dry weights (by 43% and 26%, respectively), muscle isotonic and tetanic force (by 29% and 18%, respectively), CSA of the soleus muscle (by 36%), and soleus muscle fibers (by 45%). Fat-1 transgenic mice had a decrease in the ω-6/ω-3 polyunsaturated fatty acids (PUFAs) ratio as compared with C57BL/6 wild-type mice (56%, p < 0.001). Fat-1 mice had lower soleus muscle dry mass loss (by 10%) and preserved absolute isotonic force (by 17%) and CSA of the soleus muscle (by 28%) after HS as compared with C57BL/6 wild-type mice. p-GSK3B/GSK3B ratio was increased (by 70%) and MuRF-1 content decreased (by 50%) in the soleus muscle of Fat-1 mice after HS. Balanced diet-fed Fat-1 mice are able to preserve in part the soleus muscle mass, absolute isotonic force and CSA of the soleus muscle in a disuse condition.

Angiotensin-(1-7) attenuates disuse skeletal muscle atrophy in mice via its receptor, Mas.

Immobilization is a form of disuse characterized by a loss of strength and muscle mass. Among the main features are decreased IGF-1/Akt signalling and increased ubiquitin-proteasome pathway signalling, which induce greater myosin heavy chain degradation. Activation of the classical renin-angiotensin system (RAS) causes deleterious effects in skeletal muscle, including muscle wasting. In contrast, angiotensin-(1-7) [Ang-(1-7)], a peptide of the non-classical RAS, produces beneficial effects in skeletal muscle. However, the role of Ang-(1-7) in skeletal muscle disuse atrophy and independent of classical RAS activation has not been evaluated. Therefore, we assessed the functions of Ang-(1-7) and the Mas receptor in disuse muscle atrophyin vivousing unilateral cast immobilization of the hind limb in male, 12-week-old wild-type (WT) and Mas-knockout (Mas KO) mice for 1 and 14 days. Additionally, we evaluated the participation of IGF-1/IGFR-1/Akt signalling and ubiquitin-proteasome pathway expression on the effects of Ang-(1-7) immobilization-induced muscle atrophy. Our results found that Ang-(1-7) prevented decreased muscle strength and reduced myofiber diameter, myosin heavy chain levels, and the induction of atrogin-1 and MuRF-1 expressions, all of which normally occur during immobilization. Analyses indicated that Ang-(1-7) increases IGF-1/IGFR-1/Akt pathway signalling through IGFR-1 and Akt phosphorylation, and the concomitant activation of two downstream targets of Akt, p70S6K and FoxO3. These anti-atrophic effects of Ang-(1-7) were not observed in Mas KO mice, indicating crucial participation of the Mas receptor. This report is the first to propose anti-atrophic effects of Ang-(1-7) via the Mas receptor and the participation of the IGF-1/IGFR-1/Akt/p70S6K/FoxO3 mechanism in disuse skeletal muscle atrophy.

Loss of Ovarian Function Results in Increased Loss of Skeletal Muscle in Arthritic Rats / Perda da função ovariana resulta em perda de músculo esquelético aumentada em ratos artríticos

Objective We studied the effects of loss of ovarian function (ovariectomy) onmuscle mass of gastrocnemius and themRNA levels of IGF-1, atrogin-1, MuRF-1, andmyostatin in an experimental model of rheumatoid arthritis in rats. Methods We randomly allocated 24 female Wistar rats (9 weeks, 195.3±17.4 grams) into four groups: control (CT-Sham; n = 6); rheumatoid arthritis (RA; n = 6); ovariectomy without rheumatoid arthritis (OV; n = 6); ovariectomy with rheumatoid arthritis (RAOV; n = 6). We performed the ovariectomy (OV and RAOV) or Sham (CTSham or RA) procedures at the same time, fifteen days before the rheumatoid arthritis induction. The RA and RAOV groups were immunized and then were injected with Met- BSA in the tibiotarsal joint. After 15 days of intra-articular injections the animals were euthanized. We evaluated the external manifestations of rheumatoid arthritis (perimeter joint) as well as animal weight, and food intake throughout the study. We also analyzed the cross-sectional areas (CSA) of gastrocnemius muscle fibers in 200 fibers (H&E method). In the gastrocnemius muscle, we analyzed mRNA expression by quantitative real time PCR followed by the Livak method (ΔΔCT). Results The rheumatoid arthritis induced reduction in CSA of gastrocnemius muscle fibers. The RAOV group showed a lower CSA of gastrocnemius muscle fibers compared to RA and CT-Sham groups. Skeletal muscle IGF-1 mRNA increased in arthritics and ovariectomized rats. The increased IGF-1 mRNA was higher in OV groups than in the RA and RAOV groups. Antrogin-1 mRNA also increased in the gastrocnemius muscle of arthritic and ovariectomized rats. However, the increased atrogin-1 mRNA was higher in RAOV groups than in the RA and OV groups. Gastrocnemius muscle MuRF-1 mRNA increased in the OVand RAOVgroups, but not in the RA and Shamgroups. However, the RAOV group showed higher MuRF-1 mRNA than the OV group. The myostatin gene expression was similar in all groups. Conclusion Loss of ovarian function results in increased loss of skeletal musclerelated ubiquitin ligases atrogin-1 and MuRF-1 in arthritic rats.

Objetivo Foram estudados os efeitos da perda da função ovariana (ovariectomia) sobre músculo esquelético e os níveis de RNAm de IGF-1, atrogina-1, MuRF-1, e de miostatina em modelo experimental de artrite reumatóide em ratos. Métodos 24 ratos Wistar (9 semanas, 195,3±17,4 gramas) foram distribuídos aleatoriamente em quatro grupos: controle (CT-Sham, n = 6); artrite reumatóide (RA, n = 6); ovariectomia sem artrite reumatóide (OV; n = 6); ovariectomia com artrite reumatóide (RAOV; n = 6). Os procedimentos da ovariectomia (OV e RAOV) ou simulação da ovariectomia (CT-Shamou RA) foramrealizados aomesmo tempo, quinze dias antes da indução da artrite reumatóide. Os grupos RA e RAOV foramimunizados e, em seguida, foram injetados com Met-BSA na articulação tibiotársica. Após 15 dias das injeções intra-articulares, os animais foram eutanasiados. Foram avaliadas as manifestações externas da artrite reumatóide (perimetria articular), bem como o peso dos animais e a ingestão de alimentos ao longo do estudo. Além disso, as áreas de secção transversa (CSA) do músculo gastrocnêmio foram analisadas em 200 fibras (método H & E). No músculo gastrocnêmio, a expressão de RNAm foi analisada por PCR quantitativo em tempo real, seguido pelo método Livak (ΔΔCT). Resultados A artrite reumatoide reduziu a CSA das fibras do músculo gastrocnêmio. O grupo RAOV mostrou uma CSA menor nas fibras do músculo gastrocnêmio em comparação com os grupos RA e CT-Sham. O RNAm do IGF-1 do músculo esquelético aumentou nos ratos artríticos e ovariectomizados. O RNAm do IGF-1 foi maior nos grupos OV do que nos grupos RA e RAOV. A expressão de antrogina-1 também aumentou no músculo gastrocnêmio dos ratos artríticos e ovariectomizados. No entanto, o aumento do RNAm da atrogina-1 foi maior no grupo RAOV do que nos grupos RA e OV. O RNAm da MuRF-1 aumentou nos grupos OV e RAOV, mas não nos grupos RA e CT-Sham. Porém, o grupo RAOV apresentou maior expressão gênica de MuRF-1 do que o grupo OV. A expressão do gene da miostatina foi semelhante em todos os grupos. Conclusão A perda de função ovariana resulta em perda de músculo esquelético associado às ubiquitina-ligases atrogina-1 e MuRF-1 em ratos artríticos.

Efeitos da terapia combinada com ácido zoledrônico e propranolol na resistência mecânica em um modelo de rato com osteoporose por desuso / Effect of combined treatment with zoledronic acid and propranolol on mechanical strength in an rat model of disuse osteoporosis

Resumo Objetivos: Investigar os efeitos aditivos do agente antirreabsorção ácido zoledrônico (ZOL), isolado e em combinação ao propranolol (PRO), em um modelo de rato com osteoporose por desuso. Métodos: Usou-se um modelo de pata traseira direita de rato privada de descarga de peso para estudar as consequências da falta de descarga de peso sobre o esqueleto durante várias condições, como missões espaciais e repouso prolongado no leito em idosos. Ratos Wistar machos de três meses de idade foram submetidos à imobilização da pata traseira direita (IPTD) por 10 semanas para induzir à osteopenia; em seguida, foram divididos aleatoriamente em quatro grupos: 1 – IPTD para controle positivo; 2 – IPTD mais ZOL (50 μg/kg, dose única intravenosa); 3 – IPTD mais PRO (0,1 mg/kg, via subcutânea, cinco dias na semana); 4 – IPTD mais PRO (0,1 mg/kg, via subcutânea, cinco dias na semana) mais ZOL (50 μg/kg, dose única intravenosa) por outras 10 semanas. Um grupo de ratos não imobilizados foi usado como controle negativo. No fim do tratamento, os fêmures foram removidos e testaram-se a porosidade do osso e suas propriedades mecânicas, além do peso seco e das cinzas do osso. Resultados: No que diz respeito à melhoria da resistência mecânica da diáfise femoral média, a terapia combinada com ZOL mais PRO foi mais eficaz do que a monoterapia com ZOL ou PRO. Além disso, a terapia combinada com ZOL mais PRO foi mais eficaz na melhoria do peso seco do osso e preservou melhor a porosidade do osso cortical do que a monoterapia com ZOL ou PRO em ratos submetidos à imobilização da pata traseira direita. Conclusões: Esses dados sugerem que a terapia combinada com ZOL mais PRO deve ser recomendada para o tratamento da osteoporose por desuso.

Abstract Objectives: A model that uses right hind-limb unloading of rats is used to study the consequences of skeletal unloading during various conditions like space flights and prolonged bed rest in elderly. This study was aimed to investigate the additive effects of antiresorptive agent zoledronic acid (ZOL), alone and in combination with propranolol (PRO) in a rat model of disuse osteoporosis. Methods: In the present study, 3-month-old male Wistar rats had their right hind-limb immobilized (RHLI) for 10 weeks to induce osteopenia, then were randomized into four groups: (1) RHLI positive control, (2) RHLI plus ZOL (50 μg/kg, i.v. single dose), (3) RHLI plus PRO (0.1 mg/kg, s.c. 5 days per week), (4) RHLI plus PRO (0.1 mg/kg, s.c. 5 days per week) plus ZOL (50 μg/kg, i.v. single dose) for another 10 weeks. One group of non-immobilized rats was used as negative control. At the end of treatment, the femurs were removed and tested for bone porosity, bone mechanical properties, and bone dry and ash weight. Results: With respect to improvement in the mechanical strength of the femoral mid-shaft, the combination treatment with ZOL plus PRO was more effective than ZOL or PRO monotherapy. Moreover, combination therapy using ZOL plus PRO was more effective in improving dry bone weight and preserved the cortical bone porosity better than monotherapy using ZOL or PRO in RHLI rats. Conclusions: These data suggest that this combined treatment with ZOL plus PRO should be recommended for the treatment of disuse osteoporosis.

Efeito combinado do ácido zoledrônico e do alfacalcidol no tratamento da osteoporose por desuso em ratos / Additive effect of zoledronic acid and alfacalcidol in the treatment of disuse osteoporosis in rats

Objetivos: O desuso pelo repouso no leito, pela imobilização de membros ou por missões espaciais provoca a perda óssea rápida. Fez-se este estudo para investigar os efeitos terapêuticos do ácido zoledrônico (ZOL), isoladamente e em combinação ao alfacalcidol (ALF), em um modelo de rato com osteoporose por desuso. Métodos: Ratos Wistar machos de três meses foram submetidos à imobilização da pata traseira direita (IPTD) por 10 semanas para induzir a osteopenia; em seguida, foram divididos em quatro grupos: 1 – IPTD para controle positivo; 2 – IPTD mais ZOL (50 µg/kg, dose única intravenosa); 3 – IPTD mais ALF (0,5 µg/kg, via oral diariamente); 4 – IPTD mais ALF (0,5 µg/kg, via oral diariamente) mais ZOL (50 µg/kg, dose única intravenosa) por outras 10 semanas. Um grupo de ratos não imobilizados foi usado como controle negativo. No fim do tratamento, os fêmures foram removidos e testaram-se a porosidade do osso e suas propriedades mecânicas, além do peso seco e das cinzas do osso. Resultados: A terapia combinada com ZOL mais ALF foi mais eficaz em reduzir a porosidade do osso do que a monoterapia com um dos fármacos administrado isoladamente em ratos submetidos à IPTD. No que diz respeito à melhoria da resistência mecânica da diáfise femoral média, o tratamento combinado com ZOL mais ALF foi mais eficaz do que a monoterapia com um dos fármacos administrado isoladamente. Além disso, a terapia combinada com ZOL mais ALF foi mais eficaz na melhoria do peso seco e das cinzas do osso do que a monoterapia com ZOL ou ALF em ratos submetidos à IPTD. Conclusões: Esses dados sugerem que a terapia combinada com ZOL mais ALF representa uma opção terapêutica potencialmente útil para o tratamento da osteoporose por desuso. .

Objectives: Disuse by bed rest, limb immobilization or space flight causes rapid bone loss. We conducted the present study to investigate the therapeutic effects of zoledronic acid (ZOL), alone and in combination with alfacalcidol (ALF) in a rat model of disuse osteoporosis. Methods: In the present study, 3-month-old male Wistar rats had their right hind-limb immobilized (RHLI) for 10 weeks to induce osteopenia, then were divided into four groups: 1 – RHLI positive control; 2 – RHLI plus ZOL (50 µg/kg, i.v. single dose); 3 – RHLI plus ALF (0.5 µg/kg, oral gauge daily); 4 – RHLI plus ALF (0.5 µg/kg, oral gauge daily) plus ZOL (50 µg/kg, i.v. single dose) for another 10 weeks. One group of non-immobilized rats was used as negative control. At the end of the treatment, the femurs were removed and tested for bone porosity, bone mechanical properties, and bone dry and ash weight. Results: Combination therapy with ZOL plus ALF was more effective in decreasing bone porosity than each drug administered as monotherapy in RHLI rats. With respect to improvement in the mechanical strength of the femoral mid-shaft, the combination treatment of ZOL plus ALF was more effective than each drug administered as a monotherapy. Moreover, combination therapy using ZOL plus ALF was more effective in improving dry bone and ash weight, than single-drug therapy using ZOL or ALF in RHLI rats. Conclusions: These data suggest that combination therapy with ZOL plus ALF represents a potentially useful therapeutic option for the treatment of disuse osteoporosis. .

[Additive effect of zoledronic acid and alfacalcidol in the treatment of disuse osteoporosis in rats]. / Efeito combinado do ácido zoledrônico e do alfacalcidol no tratamento da osteoporose por desuso em ratos.

OBJECTIVES: Disuse by bed rest, limb immobilization or space flight causes rapid bone loss. We conducted the present study to investigate the therapeutic effects of zoledronic acid (ZOL), alone and in combination with alfacalcidol (ALF) in a rat model of disuse osteoporosis. METHODS: In the present study, 3-month-old male Wistar rats had their right hind-limb immobilized (RHLI) for 10 weeks to induce osteopenia, then were divided into four groups: 1- RHLI positive control; 2- RHLI plus ZOL (50 µg/kg, i.v. single dose); 3- RHLI plus ALF (0.5 µg/kg, oral gauge daily); 4- RHLI plus ALF (0.5 µg/kg, oral gauge daily) plus ZOL (50 µg/kg, i.v. single dose) for another 10 weeks. One group of non-immobilized rats was used as negative control. At the end of the treatment, the femurs were removed and tested for bone porosity, bone mechanical properties, and bone dry and ash weight. RESULTS: Combination therapy with ZOL plus ALF was more effective in decreasing bone porosity than each drug administered as monotherapy in RHLI rats. With respect to improvement in the mechanical strength of the femoral mid-shaft, the combination treatment of ZOL plus ALF was more effective than each drug administered as a monotherapy. Moreover, combination therapy using ZOL plus ALF was more effective in improving dry bone and ash weight, than single-drug therapy using ZOL or ALF in RHLI rats. CONCLUSIONS: These data suggest that combination therapy with ZOL plus ALF represents a potentially useful therapeutic option for the treatment of disuse osteoporosis.

Effect of combined treatment with zoledronic acid and propranolol on mechanical strength in an rat model of disuse osteoporosis.

OBJECTIVES: A model that uses right hind-limb unloading of rats is used to study the consequences of skeletal unloading during various conditions like space flights and prolonged bed rest in elderly. This study was aimed to investigate the additive effects of antiresorptive agent zoledronic acid (ZOL), alone and in combination with propranolol (PRO) in a rat model of disuse osteoporosis. METHODS: In the present study, 3-month-old male Wistar rats had their right hind-limb immobilized (RHLI) for 10 weeks to induce osteopenia, then were randomized into four groups: 1- RHLI positive control, 2- RHLI plus ZOL (50 µg/kg, i.v. single dose), 3- RHLI plus PRO (0.1mg/kg, s.c. 5 days per week), 4- RHLI plus PRO (0.1mg/kg, s.c. 5 days per week) plus ZOL (50 µg/kg, i.v. single dose) for another 10 weeks. One group of non-immobilized rats was used as negative control. At the end of treatment, the femurs were removed and tested for bone porosity, bone mechanical properties, and bone dry and ash weight. RESULTS: With respect to improvement in the mechanical strength of the femoral mid-shaft, the combination treatment with ZOL plus PRO was more effective than ZOL or PRO monotherapy. Moreover, combination therapy using ZOL plus PRO was more effective in improving dry bone weight and preserved the cortical bone porosity better than monotherapy using ZOL or PRO in right hind-limb immobilized rats. CONCLUSIONS: These data suggest that this combined treatment with ZOL plus PRO should be recommended for the treatment of disuse osteoporosis.

An experimental model of disuse in the alveolar rat bone: A histomorphometrical study

Lack of mechanical stimulation is known to cause disuse osteopenia in bones. However, experimental models for disuse osteopenia on bones other than jawbones are not applicable to jawbones. The little available information in this field has been applied to the study of overeruption of teeth lacking antagonists. However, the absence of an antagonist in the opposite jaw means that there is no stimulation by occlusion. Our hypothesis is that the lack of stimulation due to the absence of teeth causes disuse osteopenia in the interradicular bone of the antagonist teeth. Our aim was to develop a model of disuse osteopenia due to the absence of occlusal forces. We used male Wistar rats with 215-230 g body weight, divided into 2 groups: one absolute control group (C) and one experimental group in which the three right lower molars were extracted (E). The left side of the jaw in the experimental group was used as a paired control (PC). The animals were euthanized 7 days after extraction. The jaws were placed in occlusion, fastened and fixed in 10% formalin. The heads were cut in half and radiographs made of both jaws. The upper jaws were processed histologically. After decalcification, bucco-palatine oriented sections were cut at the level of the mesial root and distal roots of the first upper molars. On the radiographs, the distance from the tip of the cusp on the first upper molar to the antagonist edentulous ridge (DA) was measured. On the microphotographs, the following parameters were measured: passive eruption degree (PED), height of periodontal ligament at the level of the furcation (HPL) and interradicular bone volume (BVI). The data were compared statistically using ANOVA and Bonferroni's post-hoc test, considering p<0.05 as statistically significant. DA in experimental animals was 0.34}0.048 mm. PED in experimental animals was significantly greater than in the control groups, both for the buccal plate and for the palatal plate. HPL showed no significant difference between groups. BVI was significantly lower in the experimental group than in the control group. The results showed that the model used produces a condition of disuse osteopenia, shown by the statistically significant reduction in interradicular bone volume. The use of this model at different experimental times will enable the evaluation of cell responses in periodontal tissues, particularly bone tissue, e.g. to compare them to known responses such as the application of orthodontic forces.

Se sabe que la falta de estimulo mecanico produce un cuadro de osteopenia por desuso en huesos de la economia. Los modelos experimentales de osteopenia por desuso utilizados en otros huesos no son aplicables a los huesos maxilares. La escasa informacion que existe en este campo se aplico al estudio de la sobreerupcion de dientes sin antagonista. Sin embargo, la ausencia de antagonista en el maxilar opuesto hace que falte el estimulo de la oclusion. Por tal razon, nuestra hipotesis es que la falta de estimulo por ausencia de piezas dentarias provoca un cuadro de osteopenia por desuso en el hueso interradicular de los dientes antagonistas. Nuestro objetivo fue poner a punto un modelo de osteopenia por desuso debido a la ausencia de fuerzas oclusales. Se emplearon ratas Wistar machos de entre 215-230 g de peso corporal divididas en 2 grupos, un grupo control absoluto (C) y un grupo experimental al que se le extrajeron los tres molares inferiores derechos (E). El lado izquierdo del maxilar del grupo experimental, fue utilizado como control apareado (CA). A los 7 dias de efectuadas las extracciones se realizo la eutanasia de los animales. Los maxilares se colocaron en oclusion, se precintaron y asi ubicados se fijaron en formol 10%. Se dividieron las hemicabezas y tomaron radiografias de ambos maxilares siendo procesados histologicamente los maxilares superiores. Luego de la descalcificacion se obtuvieron cortes orientados en sentido buco-palatino a nivel de la raiz mesial y de las raices distales de los 1o molares superiores. Sobre las radiografias se midio la distancia desde el vertice cuspideo del 1o molar superior al reborde antagonista desdentado (DA), y sobre microfotografias se midieron los siguientes parametros: grado de erupcion pasiva (GE), altura del ligamento periodontal a nivel de la furcacion (ALP) y volumen oseo interradicular (VOI). Los datos se compararon estadisticamente mediante el test ANOVA y prueba post hoc de Bonferroni, considerando una p<0,05 como estadisticamente significativa. La DA en los animales experimentales fue de 0,34}0,048 mm. La GE en los animales experimentales fue significativamente mayor que en los grupos control, tanto a nivel de la tabla vestibular como de la tabla palatina. La ALP no mostro diferencias significativas entre los grupos. El VOi fue significativamente menor en el grupo experimental con respecto a los controles. Los resultados mostraron que el modelo empleado logra una condicion de osteopenia por desuso manifestada por la disminucion del volumen oseo interradicular. La utilizacion de este modelo de desuso a diferentes tiempos experimentales permitira evaluar las respuestas celulares de los tejidos periodontales, especial - mente del tejido oseo, permitiendo por ejemplo, compararlas con respuestas conocidas como la aplicacion de fuerzas ortodoncicas.

Functional and morphological effects of resistance exercise on disuse-induced skeletal muscle atrophy

Abstract The reduction of skeletal muscle loss in pathological states, such as muscle disuse, has considerable effects in terms of rehabilitation and quality of life. Since there is no currently effective and safe treatment available for skeletal muscle atrophy, the search for new alternatives is necessary. Resistance exercise (RE) seems to be an important tool in the treatment of disuse-induced skeletal muscle atrophy by promoting positive functional (strength and power) and structural (hypertrophy and phenotypic changes) adaptive responses. Human and animal studies using different types of resistance exercise (flywheel, vascular occlusion, dynamic, isometric, and eccentric) have obtained results of great importance. However, since RE is a complex phenomenon, lack of strict control of its variables (volume, frequency, intensity, muscle action, rest intervals) limits the interpretation of the impact of the manipulation on skeletal muscle remodeling and function under disuse. The aim of this review is to critically describe the functional and morphological role of resistance exercise in disuse-induced skeletal muscle atrophy with emphasis on the principles of training.

Metabolic and morphometric alterations inherent to neuromuscular electric stimulation in the antagonist muscle submitted to ankle joint immobilization

The aim of this work was to investigate the effect of the neuromuscular electrical stimulation (ES) on the metabolic and morfometric profile of the tibialis anterior muscle, antagonist to the soleus muscle which was stimulate, under the joint immobilization condition of the ankle for 7 and 15 days. The immobilization promoted the reduction in the muscle mass (I7: 17.36 percent, I15: 20.83 percent), in the glycogen content (I7: 48 percent, I15: 48 percent), in the muscle fibers areas (I7: 27 percent, I15: 40 percent) and increase in intramuscular connective tissue density (I7: 122 percent, I15: 206 percent). The EE didn't promote significant alterations in the mass of the immobilized groups, however, promoted increase in the glycogen (IEE7: 31.25 percent; IEE15: 56.25 percent), reduction in the muscle fibers areas (IEE7: 14 percent, IEE15: 24.69 percent) and reduction in the connective tissue density (IEE7: 25.63 percent, IEE15: 49.09 percent) when compared with the respective immobilized groups.

O objetivo desse trabalho foi investigar o efeito da estimulação elétrica neuromuscular (EE) sobre o perfil metabólico e morfométrico do músculo tibial anterior, antagonista ao músculo sóleo, o qual foi estimulado, sob a condição de imobilização articular de tornozelo durante 7 e 15 dias. A imobilização promoveu redução na massa muscular (I7: 17,36 por cento, I15: 20,83 por cento), no conteúdo de glicogênio (I7: 48 por cento, I15: 48 por cento), na área das fibras musculares (I7: 27 por cento, I15: 40 por cento) e aumento na densidade do tecido conjuntivo intramuscular (I7: 122 por cento, I15: 206 por cento). A EE não promoveu alterações significativas na massa muscular dos grupos imobilizados, porém, promoveu aumento no glicogênio (IEE7: 31,25 por cento; IEE15: 56,25 por cento), redução na área das fibras musculares (IEE7: 14 por cento, IEE15: 24,69 por cento) e redução na densidade do tecido conjuntivo de (IEE7: 25,63 por cento, IEE15: 49,09 por cento) quando comparado aos respectivos grupos imobilizados.

Metformina minimiza as alterações morfométricas no músculo sóleo de ratos submetidos à imobilização articular / Metformin minimizes the morphometric alterations in the soleus muscle of rats submitted to articular immobilization

A proposta deste trabalho foi avaliar o músculo sóleo (S) de ratos submetidos à imobilização articular por sete dias, associado ou não ao tratamento com metformina (MET, 1,4mg.ml-1) por meio de análises morfométricas. Ratos adultos Wistar (n = 5) foram divididos nos grupos: controle (C), imobilizado em posição neutra do tornozelo (I), tratado com metformina (M), imobilizado tratado com metformina (I + MET). Foram avaliadas a área das fibras, a densidade de área do tecido conjuntivo intramuscular e a massa muscular do S. A análise estatística foi realizada pelo teste de normalidade, ANOVA e de Tukey (p < 0,05). A imobilização reduziu o peso muscular (mg) do S (34 por cento). No grupo M não houve alteração significativa do peso muscular quando comparado com o grupo C. Já no grupo I + MET foi observado aumento do peso muscular em 29,6 por cento quando comparado com o grupo I. O tratamento com metformina não alterou a área da fibra muscular quando comparado com grupo C. Já no grupo I, houve redução de 44 por cento na área da fibra. Com relação ao grupo I + MET, houve aumento de 22 por cento quando comparado com o grupo I. Por outro lado, ao compararmos o grupo C com o grupo I + MET, houve redução de 31 por cento. Ao avaliar a densidade de área do tecido conjuntivo, observou-se que o grupo I apresentou elevação de 216 por cento quando comparado com o grupo C. No grupo I + MET, houve redução de 67 por cento comparado com o grupo I. O tratamento com metformina em músculos submetidos à imobilização minimizou a redução da área das fibras do S, bem como o aumento do tecido conjuntivo. Esses resultados sugerem que a metformina pode favorecer recuperação mais rápida na fase pós-imobilização.

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 Pró-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 percent in the muscular mass; 44 percent (p<0.05) in the fiber area and increase of 216 percent (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 percent in the fibers area and significant reduction (p<0.05) of 67 percent 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.

Metformin protects the skeletal muscle glycogen stores against alterations inherent to functional limitation

The aim of this study was to evaluate the glycogen content (GC) of the rat hind limb muscles submitted to joint immobilization, either associated with metformin treatment (M, 1,4mg.ml-1) or not. In the metformin group, there was a significant increase in the GC (soleus - S 65 percent , white gastrocnemius - WG 30.5 percent, red gastrocnemius- RG31.7 percent, extensor digitorum longus - EDL 44 percent, tibialis anterior- TA 77.4 percent). The immobilization significantly reduced the GC (S 31.6 percent, WG 56.6 percent, RG 39.1 percent, ELD 41.7 percent, TA 45.2 percent) and weight (S 34.2 percent and ELD 27 percent), whereas in the group immobilized with the metformin, there was an increase in the GC of all the muscles (S 177 percent, WG 290 percent, RG 172 percent,ELD 47 percent, TA 217 percent), in addition to minimizing the weight loss of S (29.6 percent) and ELD (27.8 percent).

O objetivo deste estudo foi avaliar o conteúdo de glicogênio (GLI) da musculatura da pata posterior de ratos submetidos à imobilização articular, associado ou não ao tratamento com metformina (MET, 1,4 mg.ml -1) no período de sete dias. No grupo metformina, houve elevação significativa nas RG (65 por cento no sóleo - S, 30.5 por cento no gastrocnêmio branco - GB, 31.7 por cento no gastrocnêmio vermelho - GV , 44 por cento no extensor longo dos dedos - EDL e de 77.4 por cento no tibial anterior - TA ). A imobilização reduziu significativamente as RG (S 31,6 por cento, GB 56,6 por cento, GV 39,1 por cento, ELD 41,7 por cento, TA 45,2 por cento) e peso (S 34,2 por cento e ELD 27 por cento), já no grupo imobilizado com metformina houve o aumento das RG de todos os músculos (S 177 por cento, GB 290 por cento, GV 172 por cento,EDL 47 por cento, TA 217 por cento), além de minimizar a perda de peso do S (29,6 por cento) e ELD (27,8 por cento).