Paulo Vinicius Gil Alabarse

Pulmonary antioxidant defences and protein damage during the ageing process of both sexes.

The free radical theory holds that the senescence is caused by oxidative damage that results from an imbalance between reactive oxygen and nitrogen species (RONS) and antioxidant defences. Hence, it plays an important role in the field of gerontology. We evaluated, in male and female rats, the activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and total superoxide dismutase (tSOD), as well as oxidative protein damage in pulmonary tissue at 3, 6, 12, and 20 months of age. The results show an increase in the activities of all antioxidant enzymes at 12 months of age in female rats, suggesting an association with the reproductive life cycle. Protein damage in female pulmonary tissues did not change significantly throughout the ageing process. In male rats, the activity of GPx in 20 months of age showed an inter‐gender increase, while the tSOD and GPx showed higher activities in 20 months of age in the intra‐gender analysis. The male lung showed higher protein damage at 6 months of age. These findings suggest that antioxidant enzymatic activity is connected to the reproductive life cycle. Copyright

Oxidative stress in testis of animals during aging with and without reproductive activity

The free radical theory of aging postulates that an imbalance between reactive oxygen species (ROS) and reactive nitrogen species (RNS) and antioxidant defenses is important in senescence. To address this issue and gain insight into the aging process, we have evaluated the antioxidant defenses and have assessed oxidative damage in testis tissues in aging male rats. In order to relate aging and reproduction, animals with and without reproductive activity were studied. In reproductive animals the results showed a progressive increase in antioxidant enzyme activity until 12 months of age followed by an abrupt fall at 24 months. In non-reproductive animals, antioxidant activity was stable through 12 months of age, but again, fell abruptly at 24 months of age. In addition, increased aconitase activity and increased testosterone levels were found among reproductively active animals. The data demonstrate the existence of metabolic differences in testis of reproductively experienced animals and reproductively naïve animals.

Differential Resistance to Oxidants and Production of Hydrolytic Enzymes in Candida albicans

Resistance to the toxic effects of reactive oxygen species produced by phagocytes and production of hydrolytic enzymes are important aspects of Candida albicans virulence. In this report, we compared twelve C. albicans isolates for their in vitro capacity to resist oxidants—hydrogen peroxide, menadione and paraquat; and to produce hydrolytic enzymes—phospholipase and protease. Different C. albicans isolates showed different degrees of resistance to oxidants as well as differences in production of hydrolytic enzymes. Resistance to oxidative stress did not correlate with production of hydrolytic enzymes. This reinforces the view that C. albicans differentially regulates the expression of virulence factors in response to local environmental conditions.

Blood markers of oxidative stress predict weaning failure from mechanical ventilation

Patients undergoing mechanical ventilation (MV) often experience respiratory muscle dysfunction, which complicates the weaning process. There is no simple means to predict or diagnose respiratory muscle dysfunction because diagnosis depends on measurements in muscle diaphragmatic fibre. As oxidative stress is a key mechanism contributing to MV‐induced respiratory muscle dysfunction, the aim of this study was to determine if differences in blood measures of oxidative stress in patients who had success and failure in a spontaneous breathing trial (SBT) could be used to predict the outcome of MV. This was a prospective analysis of MV‐dependent patients (≥72 hrs; n = 34) undergoing a standard weaning protocol. Clinical, laboratory and oxidative stress analyses were performed. Measurements were made on blood samples taken at three time‐points: immediately before the trial, 30 min. into the trial in weaning success (WS) patients, or immediately before return to MV in weaning failure (WF) patients, and 6 hrs after the trial. We found that blood measures of oxidative stress distinguished patients who would experience WF from patients who would experience WS. Before SBT, WF patients presented higher oxidative damage in lipids and higher antioxidant levels and decreased nitric oxide concentrations. The observed differences in measures between WF and WS patients persisted throughout and after the weaning trial. In conclusion, WF may be predicted based on higher malondialdehyde, higher vitamin C and lower nitric oxide concentration in plasma.

Oxidative stress in the kidney of reproductive male rats during aging.

Reproduction alters the male physiology. We performed a comprehensive examination of oxidative stress in the kidneys of male rats with (experienced) or without (naïve) reproductive activity during aging. Oxidative stress was assessed by measuring the activity of catalase, glutathione peroxidase, glutathione S-transferase, and superoxide dismutase, and by measuring protein carbonylation, lipid peroxidation, nitrite and nitrate levels, vitamin C levels, and glutathione (total, reduced, and oxidized forms) levels, and metabolism was accessed by aconitase activity in kidney tissue, as well as testosterone and estradiol levels in serum. Reproductively active animals exhibited increased testosterone levels and altered metabolism. Aging affects tissues and organs and contributes to their functional decline. Elderly naïve rats showed high nitrite and nitrate levels. The experienced rats had less damage in elderly ages, probably because they had higher antioxidant amount and antioxidant enzyme activities at earlier ages, which would have avoided oxidative damage seen in naïve group, and because of the metabolism decline. Glutathione increase in naïve elder rats probably was induced for direct protection against oxidative damage and indirect protection by higher glutathione peroxidase and glutathione S-transferase activities. Linear regression shows that lipid peroxidation levels explained vitamin C levels (B standardized value of 0.42), indicating that vitamin C was properly produced or recruited into kidneys to combat lipid peroxidation. Catalase activity reflected the protein carbonylation and lipid peroxidation levels (B standardized values of 0.28 and 0.48). These results add comprehensive data regarding changes in oxidative stress during aging, and suggest an explanation for the costs of reproduction.

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