Jordana Miranda de Souza Silva

Muscle wasting in osteoarthritis model induced by anterior cruciate ligament transection

This study aimed to investigate the molecular pathways involved in muscle wasting in an animal model of osteoarthritis (OA) induced by anterior cruciate ligament transection (ACLT) in rats. Reduction of protein syntheses, increased proteolysis and impaired muscle regeneration are important pathways related to muscle wasting, and myogenin, MyoD, myostatin and MuRF-1 are some of their markers. Female Wistar rats were allocated into two groups: OA (submitted to the ACLT) and SHAM (submitted to surgery without ACLT). Nociception, spontaneous exploratory locomotion and body weight of animals were evaluated weekly. Twelve weeks after the disease induction, animals were euthanized, and the right knee joints were collected. Gastrocnemius muscle of the right hind paw were dissected and weighed. Gastrocnemius was used for evaluation of muscle atrophy and expression of IL-1β, TNF-α, Pax7, myogenin, MyoD, myostatin and MuRF-1. Histopathology of the knee confirmed the development of the disease in animals of OA group. Gastrocnemius of OA animals showed a reduction of about 10% in area and an increased IL-1β expression compared to animals of SHAM group. Expression of myostatin was increased in OA group, while myogenin expression was decreased. TNF-α, Pax7, MuRF-1 and MyoD expression was similar in both OA and SHAM groups. Nociception was significantly elevated in OA animals in the last two weeks of experimental period. Spontaneous exploratory locomotion, body weight and weight of gastrocnemius showed no difference between OA and SHAM groups. Gastrocnemius atrophy in OA induced by ACLT involves elevated expression of IL-1β within the muscle, as well as increased expression of myostatin and decreased expression of myogenin. Therefore, muscle wasting may be linked to impaired muscle regeneration.

Collagen-induced arthritis as an animal model of rheumatoid cachexia: CIA as an animal model of RA

Rheumatoid arthritis is characterized by chronic polyarticular synovitis and presents systemic changes that impact quality of life, such as impaired muscle function, seen in up to 66% of the patients. This can progress to severely debilitating state known as rheumatoid cachexia—without loss of fat mass and body weight—for which there is little consensus in terms of diagnosis or treatment. This study aims to evaluate whether the collagen‐induced arthritis (CIA) animal model also develops clinical and functional features characteristic of rheumatoid cachexia.

AB0110 The Proteasome Is Related To Muscle Wasting in Experimental Arthritis and Is Altered by Etanercept Treatment

Background Rheumatoid arthritis is an autoimmune inflammatory disease associated with systemic complications like fatigue and muscle wasting. Muscle wasting could be related to the activation of the ubiquitin-proteasome system. Objectives To evaluate muscle loss and involvement of the proteasome in collagen-induced arthritis (CIA), with or without treatment with methotrexate or a TNF inhibitor (etanercept). Methods Male DBA1/J mice were divided into 4 groups (n=8 each): CIA (saline); ETN (etanercept, 5.5 mg/kg) and MTX (methotrexate, 35 mg/kg), treated twice a week for 6 weeks, and a healthy control group (CO). Treatments started one week after booster injection. Clinical score, hind paw oedema, and body weight were analysed during the experimental period. Gastrocnemius muscles (GA) were weighted after death and used to quantify proteasome activity, protein levels and mRNA expression of its subunits by Western blot and rtPCR. Significance was considered when p≤0.05. Results Treatments slowed disease development, observed through smaller clinical score and hindpaw edema in ETN and MTX. ETN presented higher body weight (21±1.0g) compared to MTX (19±1.3) at weeks 5 and 7. GA weight was heavier in ETN (105±12g) than CIA and MTX (80±10 and 79±10g, respectively), a result also observed after normalization of muscle with body weight. Of note, the catalytic properties of 26S proteasome showed an increase of caspase-like activity in CIA and MTX groups (150 and 200% of activity, respectively). Furthermore, muscles of MTX treated animals showed higher protein levels for proteasomal subunits PSMB8 and PSMB9 and increased gene expression for Psmb5, Psmb8 and Psmb9. In contrast, expression of Psmb6 was decreased and of Psmb9 was enhanced in CIA. Conclusions Although both drugs improved the disease score, ETN presented a stronger anti-arthritic effect and was the only treatment able to partially prevent muscle wasting. In contrast to ETN, MTX treatment did not prevent muscle loss due to CIA accompanied by persistent up-regulation of proteasome expression and activity. Acknowledgement Financial support: CAPES, CNPq, FIPE-HCPA. Disclosure of Interest None declared

AB0136 Can Nitric Oxide (NO) Regulator Drugs be Used as Treatment for Muscle Loss in Collagen-Induced Arthritis (CIA)?

Background Rheumatoid arthritis is an autoimmune disease of unknown etiology associated with progressive disability, systemic complications like fatigue and muscle weakness, early death, and socioeconomic costs [1,2]. Nitric oxide (NO) have been related with inflammation and its regulation can lead to anti-inflammatory and anti-arthritic effects in CIA as well as induce muscle repair in muscle injury [3,4]. The role of NO in CIA muscle loss has not yet been studied. Objectives How NO synthase inhibitor (NG-nitroL-arginie methy ester: L-NAME) and the NO donor (3-morpholinosydnonimine: SIN-1) affects CIA muscle loss? Methods Female Wistar rats with CIA [5] were separated in four groups: CIA (saline, n=10); L-NAME (30mg kg-1 n=10); and SIN-1 (0.3 mg kg-1, n=13), treated twice a day for 10 days after the onset of the disease, and a wildtype group (WT, n=8). Clinical score (arbitrary units – au), hind paw edema (mm), spontaneous locomotion (cm), and body weight (g) were analyzed. Ankle was collected and used for histological confirmation of the disease. Tibialis anterior (TA), gastrocnemious and soleus muscles were weighted (g). TA was used for histological analysis and immune stained for TNF-alfa, TGF-beta and IL-1beta. Serum was collected for albumin (g dL-1), total iron (μg dL-1) and ionized calcium (mg dL-1) analysis. Proper statistics were performed and p<.05 was set for critical limit. Data are in Mean ± SEM. Results Ankle histology confirmed that all CIA groups developed arthritis. In vivo analysis of hindpaw edema, clinical score, body and muscle weight, and spontaneous locomotion showed no difference among CIA groups. On the other hand, both L-NAME (48±6,5) and SIN-1 (48±6,5) groups have shown statistically decreased clinical score than saline (77±9,2) when analyzed by the area under curve. Muscle cross sectional area were higher in L-NAME (1074±315 μm) and SIN-1 (1115±303 μm) than saline (786±243 μm), however it did not reach WT diameter (1755±278 μm). Blood vessels diameter were smaller in L-NAME (287±121 μm) group compared to SIN-1 (524±169 μm) and saline (445±165 μm). Albumin was lower in all CIA groups (saline 3,8±0,2; L-NAME 3,9±0,1; SIN-1 3,7±0,1; WT 4,2±0,1), and ionized calcium had no difference among all groups. Iron was decreased in L-NAME (229±42) and SIN-1 (226±62) than WT (353±53). All CIA groups had shown increased immune cells infiltration shown by TNF-alfa, TGF-beta and IL-1beta immune staining. Conclusions The data above mentioned suggests that nitric oxide regulator drugs show good prospects as intervention for muscle loss. As was observed, even a simply drug that have main influence in vessel pressure shows preventive clinical score development and ameliorates muscle cross sectional area. The mechanism behind such findings will soon be depicted by molecular analysis. References McInnes IB, Schett G. NEJM 2011;365(23):2205-19. Alver A, et al. Clinical Biochemistry 2011;44(17-18):1385-9. Gomaa A, et al. BJP 2009;158(7):1835-47. Filippin LI, et al. Nitric Oxide 2009;21(3-4):157-63. Rosloniec EF, et al. Curr Protoc Immunol 2010; Chapter 15: Unit 15.5.1-25. Acknowledgements Financial support: CAPES, CNPq, FAPERGS, FIPE-HCPA. Disclosure of Interest None declared

AB0135 Aerobic Exercise in Inclined Treadmill Reduce Fatigue in Collagen-Induced Arthritis

Background Rheumatoid arthritis (RA) patients suffer from joint pain and decreased physical capacity, like muscle wasting and fatigue. Fatigue is a clinical manifestation reported by 40-80% of patients with RA and is regarded as an important feature of the disease. Aerobic exercise may be beneficial for treating this feature in RA patients, however the mechanisms involved are still unclear. Objectives To evaluate the effect of aerobic exercise training on the endurance exercise performance in collagen-induced arthritis (CIA) mice. Methods Male DBA1/J mice with CIA [1] were randomly divided into 3 groups: wildtype with exercise (WT-EXE, n=4), CIA exercised (CIA-EXE, n=5) and CIA non-exercised (CIA, n=4). Endurance exercise performance test (fatigue) was analyzed in all groups prior to booster injection and each 15 days after protocol started. Eighteen days after the disease induction (booster), WT and CIA-EXE were submitted to training on an inclined treadmill (θ=5°), 45 minutes a day, 5 days per week for 6 weeks at 60% of their own endurance exercise performance. Variables analyzed were disease score, hindpaw nociception, body weight (g), fatigue (by endurance exercise performance in min) and relative muscle weight (muscle weight in mg divided by total animal weight in g). Data was analyzed with ANOVA Two-Way followed by Bonferroni and independent sample t-test and p<0.05 was considered significant. All data are represented as Mean ± SEM. Results Body weight was significantly higher in WT-EXE compared with CIA after 4 and 6 weeks of exercise. At week 6 of exercise, CIA-EXE had higher body weight than CIA. Fatigue test at 4 and 6 weeks of experiment was significantly different among all experimental groups; WT-EXE and CIA had, respectively, the highest and the lowest fatigue velocity. Gastrocnemius muscle weight was significantly heavier in control group than in CIA-EXE and CIA. Nociception and clinical score of arthritis did not differ between CIA-EXE and CIA. Conclusions Inclined aerobic exercise appears as an interesting intervention in RA to treat decreased physical capacity. Collagen-induced arthritis animals demonstrated decreased endurance, and consequently increased fatigue, characteristics of a good animal model to study fatigue. The exercise protocol used in this study was able to improve this feature, demonstrating that interventions used to treat physical disabilities in RA are also valid in this model. Further studies are necessary to clarify the mechanisms behind fatigue, especially when combining exercise training and common treatments of RA. References Rosloniec EF, et al. Curr Protoc Immunol 2010; Chapter 15: Unit 15.5.1-25. Acknowledgements Financial support: CAPES, CNPq, FAPERGS, FIPE-HCPA. Disclosure of Interest None declared

AB0094 Histopathological Muscle Findings in Experimental Osteoarthritis

Background Osteoarthritis (OA) is a chronic joint disease characterized by progressive loss of articular cartilage and abnormal bone formation. Furthermore, there are changes in periarticular muscles, such as loss of muscle mass, strength and function. These features may contribute to functional impairment among patients. Objectives To study the effects of OA induced by anterior cruciate ligament transection (ACLT) on muscle histopathology in rat. Methods Female Wistar rat underwent ACLT surgery (n=9) or sham surgery (n=8) (1) on the right knee only. Body weight and spontaneous exploratory locomotion were evaluated weekly. Twenty weeks after the surgery, animals were euthanized. Joints from the right knee were collected for histological analyses. Gastrocnemius muscles from the right paw were collected for weighing and histological and molecular analyses. Data were analyzed with ANOVA Two-Way followed by Bonferroni and independent samples t-test and p<0.05 was considered significant. Results Body weight and spontaneous exploratory locomotion were not statistically different between groups, during the experimental period. Histological evaluation of the right knee joint proved the development of OA in animals which underwent ACLT surgery (p<0,01), accordingly to OARSI histologic scoring system (2). The weight of gastrocnemius muscle was not statistically different between groups at the end of the experimental period (p<0,14). Histological evaluation of gastrocnemius muscle fiber cross-sectional area (CSA) (3) showed a reduction in muscle fiber CSA in ACLT animals (p=0,0006). Conclusions ACLT induced chronic degenerative articular changes and muscle fiber CSA reduction in rats. It is yet to be studied what factors are related to atrophy, evaluating the expression of muscle factors related to muscle growth, differentiation, and mass regulation. References Elsaid KA, Machan JT, Waller K, Fleming BC, Jay GD. The Impact of Anterior Cruciate Ligament Injury on Lubricin Metabolism and the Effect of Inhibiting Tumor Necrosis Factor alpha on Chondroprotection in an Animal Model. Arthritis and Rheumatism. 2009;60(10):2997-3006. Gerwin N, Bendele AM, Glasson S, Carlson CS. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the rat. Osteoarthritis and Cartilage. 2010;18:S24-S34. Filippin LI, Teixeira VN, Viacava PR, Lora PS, Xavier LL, Xavier RM. Temporal development of muscle atrophy in murine model of arthritis is related to disease severity. Journal of Cachexia Sarcopenia and Muscle. 2013;4(3):231-8. Acknowledgements Financial support: CAPES, FIPE-HCPA. Disclosure of Interest None declared