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Levels of Cytokines and Matrix Metalloproteinases 2 and 9 in the Synovial Fluid of Osteoarthritic Horses Treated With Pamidronate
Accepted Manuscript Levels of Cytokines and Matrix Metalloproteinases 2 and 9 in the Synovial Fluid of Osteoarthritic Horses Treated with Pamidronate Emilio A. De Simone, Gustavo Perrone, Nicolás Caggiano, Yael Lastra, Florencia Rubatino, Julieta Díaz, Araceli Ferreto, Cristian Montes de Oca, Emilio Roldán, María Angelina Chiappe Barbará PII:
S0737-0806(15)00341-X
DOI:
10.1016/j.jevs.2015.03.194
Reference:
YJEVS 1874
To appear in:
Journal of Equine Veterinary Science
Received Date: 30 October 2014 Revised Date:
12 March 2015
Accepted Date: 25 March 2015
Please cite this article as: De Simone EA, Perrone G, Caggiano N, Lastra Y, Rubatino F, Díaz J, Ferreto A, de Oca CM, Roldán E, Chiappe Barbará MA, Levels of Cytokines and Matrix Metalloproteinases 2 and 9 in the Synovial Fluid of Osteoarthritic Horses Treated with Pamidronate, Journal of Equine Veterinary Science (2015), doi: 10.1016/j.jevs.2015.03.194. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Levels of Cytokines and Matrix Metalloproteinases 2 and 9 in the Synovial Fluid of Osteoarthritic Horses Treated with Pamidronate Authors: Emilio A. De Simonea, Gustavo Perroneb, Nicolás Caggianoa, Yael Lastraa, Florencia a a a a c Rubatino , Julieta Díaz , Araceli Ferreto , Cristian Montes de Oca , Emilio Roldán , María ad Angelina Chiappe Barbará .
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Affiliations: a Department of Animal Physiology, School of Veterinary Sciences, University of d
Buenos Aires, Argentina. Department of Equine Production, School of Veterinary Sciences, University of Buenos Aires, Argentina. c Gador S.A. d Corresponding author's E-mail address:
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[email protected]
Postal address: Department of Animal Physiology, School of Veterinary Sciences, University of
Argentina Tel.: (54-9 11) 4524-8456 Abstract
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Buenos Aires, Argentina. Chorroarín 280, Ciudad Autónoma de Buenos Aires, C1427CWO -
The aim of this study is to evaluate the effect of pamidronate on the clinical score and the secretory profile of inflammatory biomarkers (IL-6, TNFα, MMP-2 and MMP-9) in
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the synovial fluid in clinically healthy horses and in horses with joint disease. Healthy horses and horses with joint symptoms were examined and subjected to a standardised clinical evaluation of the locomotor system. The clinical condition was
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evaluated by a global score. MMP-2 and MMP-9 were measured by gel zymography. The concentration of cytokines (IL-6 and TNF-α) in synovial fluid was determined by
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ELISA. Pamidronate treatment significantly improved the clinical condition of horses with osteoarthritis (OA). Values of IL-6 (pg/ml) were similar (ns) in the healthy control group (102.2±26.94) and at day 3 of treated group (TD3) (113.9±18.33). TNF-α level, at day 3 of treatment, was significantly lower in treated groups than in untreated osteoarthritis (UOA). TD group registered a fast increase in MMP-9 activity but till day 21 and 60 it was not detectable. No significant differences were found in the MMP-2
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activity between groups. We concluded that treatment with pamidronate has a beneficial effect on the clinical score of horses with OA, and can reduce proinflammatory cytokines (IL-6 and TNF-alpha) and MMP-9 at different stages after
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treatment.
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Keywords: horses, pamidronate, cytokines; matrix metalloproteinases; osteoarthritis.
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Levels of Cytokines and Matrix Metalloproteinases 2 and 9 in the Synovial Fluid of Osteoarthritic Horses Treated with Pamidronate 1. Introduction Osteoarthritis (OA) is an inflammatory disease which evolves to painful and
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degenerative joint damage. OA usually occurs as a result of physical overtraining,
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dietary imbalances or growth disorders -including osteochondrosis- during
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development [1, 2]. OA is one of the major causes of economic loss in sport horses
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because it results in either temporary or permanent, premature retirement from
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sports competitions and racing [3]. The above mentioned risk factors lead to the
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occurrence of repeated and incompletely resolved articular micro trauma that
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maintain and increase the severity of the inflammatory process. Furthermore, an
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abnormal maturation of the cartilage might result in the formation of cartilage flaps
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and intra-articular bone fragments that can predispose to articular damage [2]. OA is
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characterized by joint damage that affects the articular cartilage, the adjacent
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subchondral bone and the synovial membrane. Pain is another important factor
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associated with the inflammatory process leading to progressive loss of joint function
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and turning into a performance-limiting factor.
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During the onset of OA, the changes in the properties of normal cartilage impact on
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subchondral bone, causing an increase of the resorptive activity of osteoclasts and
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bone turnover [4]. In addition, the production of anabolic factors by chondrocytes
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decreases, together with an increase in the release of proteases and other catabolic
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factors involved in joint and bone damage [5]. Vascular changes and up-regulation of
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inflammatory cytokines and nitric oxide production in the subchondral region and
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9, 10, 11]. In turn, the inflammatory cytokines induce the release of matrix
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metalloproteinases (MMPs) [4, 12] which are known to be involved in articular
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cartilage degradation [13].
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synovial fluid are also observed in OA [6, 7]. During the initial phase of OA, macrophages and neutrophils participate in the cascade of inflammatory response by secreting proinflammatory cytokines, TNF-α and IL-6 [8,
TNF-α is a key cytokine in the inflammatory process and is known to increase vascular
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permeability by inducing the endothelium to express adhesion molecules and cellular
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migration factors that promote leukocyte diapedesis [14]. Moreover, IL-6 is involved in
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the degradation of proteoglycans in the articular cartilage [15].
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Although MMPs are zinc-dependent endopeptidases involved in tissue repair they are
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also associated with the development of arthritis in humans [16] and in horses [17].
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MMP-2, MMP-3, MMP-9, and MMP-13 are especially involved in cartilage matrix
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degradation [18].
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Many different pharmaceutical products have been used to treat OA with the aim of
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reducing associated inflammation and pain; however, these drugs have adverse effects
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on the extracellular matrix and bone metabolism. For example, corticosteroids are
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widely used in the management of equine OA for their highly effective anti-
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inflammatory properties, but are associated with enhancement of cartilage
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proteolysis, subchondral bone resorption, microfractures and direct inhibition of
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osteoblast function. The aim of this paper is to evaluate the effects of pamidronate in
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horses with OA. While tiludronate was the first bisphosphonate licensed to treat
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navicular disease in horses [19], pamidronate, an aminobisphosphonate, is widely used
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in human medicine for the treatment of metabolic bone disease. Information on the
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anti-inflammatory effects of these drugs in equines is scarce [20].
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Pamidronate belongs to the family of aminobisphosphonates and is extensively used
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for the palliative treatment of cancer metastasis due to its osteoclast inhibitory effect
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[21, 22, 23, 24]. Aminobisphosphonates have a more potent anti-resorptive effect
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proliferation of osteoblasts [20, 26, 27]. Another mechanism by which pamidronate
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inhibits bone resorption is by stimulating osteoblast inhibitory activity on osteoclasts
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[28]. Pamidronate acts by inhibiting the mevalonate pathway [29]. Due to its analgesic
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compared to other bisphosphonates. Pamidronate has been used in the treatment of osteolysis associated to Paget's disease [25] and is known to have analgesic effects in patients suffering from cancer and other related disorders. In addition, bisphosphonates can inhibit apoptosis of osteocytes and osteoblasts and induces the
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effect and its anti-resorptive capacity, pamidronate could be an alternative treatment
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for OA [30].
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Bisphosphonates might have anti-inflammatory properties, particularly during the
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onset of arthritis [31]. The aim of this study is to evaluate the effect of pamidronate on
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the clinical score and the secretory profile of inflammatory biomarkers (IL-6, TNFα,
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MMP-2 and MMP-9) in the synovial fluid in clinically healthy horses and in horses with
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joint disease.
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2. Materials and Methods
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2.1 Animals, synovial samples and experimental design
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At the beginning of the study, healthy horses and horses with joint symptoms were
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examined and subjected to a standardised clinical evaluation of the locomotor system.
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The clinical condition was evaluated by a global score comprising 6 items: i) lameness
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degree (0-5) ii) tenderness (0-3), iii) presence of pain under forced flexion (0-3), iv)
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volume of synovial fluid extracted (0-3), v) colour of synovial fluid (0-5) and vi) synovial
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fluid viscosity (0-4). The score was based in the AAEP lameness score [32] with the
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addition of a wide evaluation methodology focused in the tibio-tarsal joint. This joint is
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an important site of OA prevalence in jumping horses [33, 34].
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The following groups were considered: i) control group (n = 8), clinically healthy young
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animals (2-4 years). Young animals were chosen to be certain that since birth they had
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no history of joint disease or disorders of bone metabolism. The clinical score of this
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group was ≤ 4. ii) animals with joint disease which were treated (TD) (n = 8, with
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clinical score > 5 at the beginning of treatment, aged between 4-8 years). This group
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was evaluated and analysed at different stages: baseline or pre-treatment group (TD0), day 3 (TD3); day 10 (TD10); day 21 (TD21) and day 60 (TD60). Animals included in TD group were animals with clinical signs and clinical history of chronic recurrent episodes of active OA with poor outcome to conventional treatment modalities.
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Radiographic analysis of both tibio-tarsal joint was performed in all animals included in
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the experiment, control and TD group. TD group had radiographics alterations of
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articular cartilage in the joints and subchondral bone resorption.
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On days 0 and 9, horses with joint disease were treated with 90 mg of intravenous
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disodium pamidronate (Aminomux® 90mg, Gador). There is no detailed information of
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pamidronate dosage and posology for horses in the bibliography, however divided
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doses was recommended. In this paper we used low doses of pamidronate (0.4-0.8
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mg/kg EV) administered in two doses 9 days apart [20, 35]. Synovial fluid samples were
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obtained on days 0, 3, 10, 21 and 60 by sterile aspiration from the tibio-tarsal joint.
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Samples were centrifuged at 2,000 x g for 10 min and the supernatant was kept at -
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70°C. The final clinical score in the pamidronate-treated group was considered on day
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60.
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2.2 Gel Zymography
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The gelatinolytic activities of MMP-2 and MMP-9 were measured by zymography as
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described by Gruber et al [36]. All samples were analyzed for protein concentration by
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the Bradford method [37]. Aliquots of 100 µg of each sample were briefly mixed with a
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buffer and loaded on a 10% SDS-PAGE with 0.2 % porcine skin gelatin (Sigma). After
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electrophoresis, gels were washed with 50mM Tris-HCl pH 7.5 plus 2.5 % Triton X-100
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for 45 min and then with a solution of 5mM CaCl2, 1µM ZnCl2, 50mM TrisHCl pH7.5
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plus 2.5 % Triton X-100 pH 7.5 for another 45 min. Finally gels were incubated with a
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solution containing 50mM TrisHCl, 10mM CaCl2 and 200mM NaCl pH 7.5 at 37 °C for
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24 h. Gels were stained with 0.2% (w/v) Coomassie Brilliant Blue R-250 for 2 h and
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then bleached with decolorizing solution (25% v/v isopropanol plus 10% v/v acetic
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acid). Inhibition controls were performed by incubating with 5 mmol/ l EDTA. The
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densitometric study of the bands generated by the gelatinolytic activity was done
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using the Image J software (National Institutes of Health, Maryland, USA). Zymographic activity was expressed as a % of the control group. Data for different gels was standardized with the help of control samples. As MMP-9 is not always present in
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synovial fluid, we worked with MMP-9 data in a qualitative scale (negative, lightly
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positive and strong positive).
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2.3 Determination of IL-6 and TNF-α levels
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The concentration of cytokines (IL-6 and TNF-α) in synovial fluid was determined by
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commercial ELISA kits (OptEIA BD Biosciences, San Diego, CA, USA) as per
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manufacturer's instructions. Cytokines were analysed in synovial fluid since it has
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previously been demonstrated that this biological fluid is more suitable than serum [4].
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Cytokine levels were derived from a standard curve. Undiluted samples were analysed.
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Reactions were elicited by adding TMB substrate solution (Biosciences, San Diego, CA,
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USA) and interrupted by adding 1M sulphuric acid. Plates were read at 450 nm in a
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Rayto 2100C microplate reader (China).
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2.4 Statistical analysis:
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Data were expressed as mean ± SD. Comparisons were performed using a
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nonparametric ANOVA (Kruskal-Wallis’ test) followed by Dunn's post-test. Significant
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differences were indicated as *p <0.05, **p<0.01, ***p<0.001.
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3. Results
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Pamidronate treatment significantly improved the clinical condition of horses with OA,
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as measured by the clinical score. The comparative analysis showed that treatment
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with pamidronate 60 days after BP application significantly lowered the clinical score in
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animals with joint disease versus TD0 animals (p<0.05), and that there was no
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significant difference between the control group (C) and the treated group at this time
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(Figure 1) (Table 1).
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Cytokine levels were measured following synovial fluid extraction. Synovial values of
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IL-6 (pg/ml) were similar in the healthy control group (102±26.94), the TD3
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(113.9±18.33) and TD60 group (78.87±26.17) (ns); however TD0, TD10 and TD21
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groups had higher IL-6 values (p<0.05 vs control) (Figure 2). TNF-α dropped considerably by day 3 in the treated animals (TD3) (6.44pg/ml±4.71) and was statistically significant compared to TD0 group (p<0.05), but compared with the control
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group (15.2 pg/ml ±12.7) the difference was not statistically significant due to the wide
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scattering of data.
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Synovial MMP-2 levels showed no significant differences between groups (Figure 4).
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MMP-9 were scored as i) absent ii) slightly positive and iii) strong positive. The results
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in control group showed that only 3 animals were slightly positive for MMP-9 (Figure 5
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and Table 2). Only 1 animal of TD0 group had strong MMP-9 activity. Pamidronate
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seems to stimulate an acute increase in synovial MMP-9 activity because 6 of 8 animals
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of TD3 group were positive (3 slightly and 3 strong). However TD10 group only had a
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strong positive MMP-9 activity and TD21 and TD60 had only negative MMP-9 activity.
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4. Discussion
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In this study, we observed that animals treated with pamidronate showed a significant
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improvement in the clinical score compared with the TD0 group. The clinical changes
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were characterized by a decrease in the signs of inflammation, less or no pain under
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forced flexion and increased ability to work.
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It has previously been observed that pamidronate can reduce pain intensity and
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inflammation in humans with ankylosing spondylitis, [31]. Although OA is generally
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considered a degenerative gradual disorder it can also be considered an inflammatory
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process.
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In this study, no significant differences were found in MMP-2 activity between clinically
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healthy animals, TD0 and TD animals. Pamidronate would seem to stimulate a sharp
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increase in the activity of MMP-9 but at day 21 and 60 animals treated with
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pamidronate had an absence of MMP-9 activity, which indicated that it could have a
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beneficial effect in the medium term. These findings are in line with previous results in
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which the treatment with bisphosphonates inhibited MMP activity [38, 39]. This
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phenomenon will result in a lower degradation rate of the extracellular matrix and the
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articular cartilage surface. Furthermore, the low activity of MMPs in synovial fluid
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reduced leukocyte migration, inflammation and associated pain. The latter observation
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is related to the therapeutic prescription of pamidronate in humans, where this drug is
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known to inhibit subchondral bone resorption, thus leading to the amelioration of pain in cancer bone metastases [40]. Given that high osteoclast activity plays an important role in the degradation of the adjacent subchondral bone and in synovial inflammation causing pain adjacent to subchondral bone [41], pamidronate-induced osteoclast
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apoptosis will probably result in a decrease of bone turnover and the antinflammatory
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related effect.
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Our results show that TNFα levels in OA were significantly decreased at day 3 of
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treatment and elevated on days 10, 21 and 60. Moreover, on day 3 in the treated
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group IL-6 and TNF-α levels were similar to those in healthy animals.
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Studies carried out in cultures of peripheral leukocytes obtained from patients with
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osteoporosis treated with pamidronate showed an increase in IL-6 and TNFα
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production during the first 24 h after the first dose [42]. Cancer patients treated with
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IV pamidronate showed an increase in IL -6 levels that lasted 7 days [43]. These results
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differ from other published studies performed in rheumatoid arthritis patients, whose
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levels of IL -6 and TNFα were significantly increased for 30 days after the treatment
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with pamidronate [44] consistent with our own results at day 21. Other authors
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suggest an anti-inflammatory effect of pamidronate evidenced by decreased IL-6 levels
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[45]. We have also observed decreased levels of IL-6 after pamidronate treatment but
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only on day 3 and 60. In this study, the increased TNFα levels observed on days 10 and
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21 after treatment might be related to an exacerbation of the inflammatory process
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which could be beneficial for the resolution of chronic arthritis [46]. TNFα is a critical
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factor for NFkappa B expression which, in turn, is responsible for MMP-9 expression
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[47, 48]. In this study, it could be assumed that MMP-9 activities in synovial fluid did
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not increase after day 21 as a consequence of the elevated TNFα levels on days 10 and
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21; however, further studies are required to confirm this hypothesis.
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Many studies have focused on the role of certain MMPs (MMP-1, MMP -3 and MMP -
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13) with collagenase activity whereas in this study we have focused on the gelatinases
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(MMP-2 and MMP-9) which play a major role in the degradation of articular cartilage.
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Clodronate is a first generation bisphosphonate, which acts as an inhibitor of MMP-1
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purified from human fibroblasts [49]. In vitro studies have shown that alendronate,
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could also inhibit leukocyte migration by reducing MMP-2 activity [50, 51]. Moreover, the administration of alendronate can lead to the inhibition of MMP-13 in patients with rheumatoid arthritis [52]. Osteoclasts, macrophage linage cells, are a major source of MMP-9 in subchondral
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bone in OA joints. The increased number and activity of osteoclasts in OA induce the
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appearance of subchondral bone resorption areas and are the reason for increased
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plasma activity of MMP-9 [53, 54]. Pamidronate inhibits the MMP-9 activity through
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the induction of osteoclast apoptosis. The activity of MMP-9 has been associated with
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rheumatoid arthritis and some other types of OA in humans [55]. In vitro studies have
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shown an inhibitory effect of alendronate on MMP-9 activity due to its chelating effect
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[56]. In this paper we registered a fast increase in MMP-9 activity but it was not
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detectable until day 21 and 60. The latter phenomenon could be important since the
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inhibition of MMP-9 is associated with the prevention of articular lesions.
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Potent MMP inhibitors, such as doxycycline delays the progression of OA lesions [57].
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Furthermore, it has been observed that glucosamine combined with chondroitin
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sulphate inhibits MMP-9 activity and the joint damage associated with adjuvant-
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induced arthritis in rats [58].
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According to Clegg [59], MMP-9 activity is elevated in joint pathological processes. In
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this paper, the absence in the activity of MMP-9 after 21 and 60 days of treatment
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suggests that bisphosphonates may be a safe alternative to corticoids and other anti-
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inflammatory therapies for the treatment of OA.
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5. Conclusion
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We concluded that pamidronate treatment has positive effect in the clinical score of
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horses with osteoarthritis, and reduces proinflammatory cytokines (IL-6 and TNF-
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alpha) and MMP-9 after treatment. Recent advances in the understanding of
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molecular events in OA allow us to use novel drugs in this disease. However, the real
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benefit of these treatments in horses should be further studied.
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Figure Legends
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Figure 1. Clinical Score (Mean ± SD) for control, pretreated (TD0), and treated at
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different stages (TD3, TD10, TD21 and TD60). TD0, TD3, TD10 and TD21 differ
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significantly vs control (**p<0.01) and vs TD60 (* p<0.05).
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Figure 2. IL-6 levels in synovial fluid (Mean ± SD) from control, pretreated (TD0), and
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treated at different stages groups (TD3, TD10, TD21 and TD60). Levels of TD0, TD10 and
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TD21 were significantly different versus control, TD3 and TD60 group (*p<0.05).
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Figure 3. TNFα levels in synovial fluid (Mean ± SD) for control, pretreated (TD0), and
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treated at different stages (TD3, TD10, TD21 and TD60). TD10 and TD21 groups
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presented significant differences from both the control and TD3 groups (**p<0.01).
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TD0 and TD60 presented significant differences versus the control, and TD3 groups
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(*p<0.05)
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Figure 4. MMP-2 activities in joint synovial fluid for control, pretreated (TD0), and
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treated at different stages (TD3, TD10, TD21 and TD60). No significant differences
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were found in the MMP-2 activity between groups.
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Figure 5. Gelatin zimography for detection of MMP-9 activity in a standard sample (line
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A), negative sample (B and E), slightly positive (D and F) and strong positive (C).
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Acknowledgment
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The authors thank UBACYT 20020120100092BA for financial support.
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The authors declared a conflict of interest (such as defined by JEVS policy). Emilio
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Roldán is employed in Gador SA, the company that provides Pamidronate
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(Aminomux®). All other authors have stated that they have no conflict of interest.
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Authors contributions: M.A.C.B and E.D.S. designed research, performed research,
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analyzed data and wrote paper. M.A.C.B performed the statistical analysis. E.R.
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participated in designed research. G.P. and C.M.D.O performed field research and
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acquisition of clinical data. N.C., Y.L., F.R., J.D. and A.F. performed laboratory activity.
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[53]. Kaneko M, Tomita T, Nakase T, Ohsawa Y, Seki H, Takeuchi E, et al.
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[55]. Kim KS, Choi HM, Lee YA, Choi IA, Lee SH, Hong SJ, Yang HI, Yoo MC.
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[56]. Farina AR, Cappabianca L, Di Ianni N, Ruggeri P, Ragone M, Merolle S, et al.
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inflammation, interleukin-1beta, matrix metalloprotease-9, and cartilage
450
damage in arthritis. Exp Biol Med 2005; 230: 255-62.
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[59]. Clegg PD, Burke RM, Coughlan AR, Riggs CM, Carter Sd.. Characterization of
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equine matrix metalloproteinase 2 and 9; and identification of the cellular
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sources of these enzymes in joints. Equine Vet J 1997. 29: 335-42.
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control
TD0
TD3
TD10
TD21
6.3±0.51
8.2±0.51
7.5±1.21
6.2±0.131
TD60 3.8±0.32
2.1 ± 0.2
IL-6 (pg/ml)
102.2±26.94
251.8±61.983
113.9±18.33
275±20.733
239.3±71.253
78.87±26.17
TNF-α(pg/ml)
15.2±12.7
30.02±24.254
6.44±4.71
48.2±5.25
51.33±22.525
24.43±16.964
MMP-2 (% of control)
100±66
148±45
107±62
181±116
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Clinical Score
124±6.6
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Table 1. Measured parameters (Mean ± SD) for control, pretreated (TD0), and treated at different stages (TD3, TD10, TD21 and TD60) . 1p<0.01 vs control and 2 p<0.05 vs TD0. 3p<0.05 vs control, TD3 and TD60.4 p<0.05 vs control and TD3. 5p<0.01 vs control and TD3 group. No significant differences were found in the MMP-2 activity between groups.
120±42
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MMP-9
control
TD0
TD3
TD10
TD21
TD60
5/8
7/8
2/8
7/8
8/8
8/8
Slightly positive
3/8
-
3/8
-
-
-
Strong positive
-
1/8
3/8
1/8
-
-
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Negative
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B
D
E
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Table 2. Number of animals with negative, slightly positive and strong positive gelatinolytic MMP-9 activity in synovial fluid in control, TD0, TD3, TD10, TD21 and TD60 group.
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Figure 5. Zimography showing gelatinolytic activity in a standard sample (line A), negative sample (B and E), slightly positive (D and F) and strong positive (C).
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•
We evaluate the effect of pamidronate on the clinical score and the secretory profile of inflammatory biomarkers (IL-6, TNFα, MMP-2 and MMP-9) in the synovial fluid in clinically healthy horses and in horses with joint disease. Pamidronate treatment significantly improved the clinical condition of horses
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•
with osteoarthritis (OA). •
Values of IL-6 and TNFα. In synovial fluid were similar with the control group at
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day 3 of treatment and MMP-9 activity was absent at day 10 and 21 of
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treatment.
S0737-0806(15)00341-X
DOI:
10.1016/j.jevs.2015.03.194
Reference:
YJEVS 1874
To appear in:
Journal of Equine Veterinary Science
Received Date: 30 October 2014 Revised Date:
12 March 2015
Accepted Date: 25 March 2015
Please cite this article as: De Simone EA, Perrone G, Caggiano N, Lastra Y, Rubatino F, Díaz J, Ferreto A, de Oca CM, Roldán E, Chiappe Barbará MA, Levels of Cytokines and Matrix Metalloproteinases 2 and 9 in the Synovial Fluid of Osteoarthritic Horses Treated with Pamidronate, Journal of Equine Veterinary Science (2015), doi: 10.1016/j.jevs.2015.03.194. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Levels of Cytokines and Matrix Metalloproteinases 2 and 9 in the Synovial Fluid of Osteoarthritic Horses Treated with Pamidronate Authors: Emilio A. De Simonea, Gustavo Perroneb, Nicolás Caggianoa, Yael Lastraa, Florencia a a a a c Rubatino , Julieta Díaz , Araceli Ferreto , Cristian Montes de Oca , Emilio Roldán , María ad Angelina Chiappe Barbará .
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Affiliations: a Department of Animal Physiology, School of Veterinary Sciences, University of d
Buenos Aires, Argentina. Department of Equine Production, School of Veterinary Sciences, University of Buenos Aires, Argentina. c Gador S.A. d Corresponding author's E-mail address:
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[email protected]
Postal address: Department of Animal Physiology, School of Veterinary Sciences, University of
Argentina Tel.: (54-9 11) 4524-8456 Abstract
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Buenos Aires, Argentina. Chorroarín 280, Ciudad Autónoma de Buenos Aires, C1427CWO -
The aim of this study is to evaluate the effect of pamidronate on the clinical score and the secretory profile of inflammatory biomarkers (IL-6, TNFα, MMP-2 and MMP-9) in
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the synovial fluid in clinically healthy horses and in horses with joint disease. Healthy horses and horses with joint symptoms were examined and subjected to a standardised clinical evaluation of the locomotor system. The clinical condition was
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evaluated by a global score. MMP-2 and MMP-9 were measured by gel zymography. The concentration of cytokines (IL-6 and TNF-α) in synovial fluid was determined by
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ELISA. Pamidronate treatment significantly improved the clinical condition of horses with osteoarthritis (OA). Values of IL-6 (pg/ml) were similar (ns) in the healthy control group (102.2±26.94) and at day 3 of treated group (TD3) (113.9±18.33). TNF-α level, at day 3 of treatment, was significantly lower in treated groups than in untreated osteoarthritis (UOA). TD group registered a fast increase in MMP-9 activity but till day 21 and 60 it was not detectable. No significant differences were found in the MMP-2
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activity between groups. We concluded that treatment with pamidronate has a beneficial effect on the clinical score of horses with OA, and can reduce proinflammatory cytokines (IL-6 and TNF-alpha) and MMP-9 at different stages after
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treatment.
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Keywords: horses, pamidronate, cytokines; matrix metalloproteinases; osteoarthritis.
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1 2 3
Levels of Cytokines and Matrix Metalloproteinases 2 and 9 in the Synovial Fluid of Osteoarthritic Horses Treated with Pamidronate 1. Introduction Osteoarthritis (OA) is an inflammatory disease which evolves to painful and
5
degenerative joint damage. OA usually occurs as a result of physical overtraining,
6
dietary imbalances or growth disorders -including osteochondrosis- during
7
development [1, 2]. OA is one of the major causes of economic loss in sport horses
8
because it results in either temporary or permanent, premature retirement from
9
sports competitions and racing [3]. The above mentioned risk factors lead to the
10
occurrence of repeated and incompletely resolved articular micro trauma that
11
maintain and increase the severity of the inflammatory process. Furthermore, an
12
abnormal maturation of the cartilage might result in the formation of cartilage flaps
13
and intra-articular bone fragments that can predispose to articular damage [2]. OA is
14
characterized by joint damage that affects the articular cartilage, the adjacent
15
subchondral bone and the synovial membrane. Pain is another important factor
16
associated with the inflammatory process leading to progressive loss of joint function
17
and turning into a performance-limiting factor.
18
During the onset of OA, the changes in the properties of normal cartilage impact on
19
subchondral bone, causing an increase of the resorptive activity of osteoclasts and
20
bone turnover [4]. In addition, the production of anabolic factors by chondrocytes
21
decreases, together with an increase in the release of proteases and other catabolic
22
factors involved in joint and bone damage [5]. Vascular changes and up-regulation of
23
inflammatory cytokines and nitric oxide production in the subchondral region and
26
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9, 10, 11]. In turn, the inflammatory cytokines induce the release of matrix
28
metalloproteinases (MMPs) [4, 12] which are known to be involved in articular
29
cartilage degradation [13].
24 25
synovial fluid are also observed in OA [6, 7]. During the initial phase of OA, macrophages and neutrophils participate in the cascade of inflammatory response by secreting proinflammatory cytokines, TNF-α and IL-6 [8,
TNF-α is a key cytokine in the inflammatory process and is known to increase vascular
31
permeability by inducing the endothelium to express adhesion molecules and cellular
32
migration factors that promote leukocyte diapedesis [14]. Moreover, IL-6 is involved in
33
the degradation of proteoglycans in the articular cartilage [15].
34
Although MMPs are zinc-dependent endopeptidases involved in tissue repair they are
35
also associated with the development of arthritis in humans [16] and in horses [17].
36
MMP-2, MMP-3, MMP-9, and MMP-13 are especially involved in cartilage matrix
37
degradation [18].
38
Many different pharmaceutical products have been used to treat OA with the aim of
39
reducing associated inflammation and pain; however, these drugs have adverse effects
40
on the extracellular matrix and bone metabolism. For example, corticosteroids are
41
widely used in the management of equine OA for their highly effective anti-
42
inflammatory properties, but are associated with enhancement of cartilage
43
proteolysis, subchondral bone resorption, microfractures and direct inhibition of
44
osteoblast function. The aim of this paper is to evaluate the effects of pamidronate in
45
horses with OA. While tiludronate was the first bisphosphonate licensed to treat
46
navicular disease in horses [19], pamidronate, an aminobisphosphonate, is widely used
47
in human medicine for the treatment of metabolic bone disease. Information on the
48
anti-inflammatory effects of these drugs in equines is scarce [20].
49
Pamidronate belongs to the family of aminobisphosphonates and is extensively used
50
for the palliative treatment of cancer metastasis due to its osteoclast inhibitory effect
51
[21, 22, 23, 24]. Aminobisphosphonates have a more potent anti-resorptive effect
52
55 56
proliferation of osteoblasts [20, 26, 27]. Another mechanism by which pamidronate
57
inhibits bone resorption is by stimulating osteoblast inhibitory activity on osteoclasts
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[28]. Pamidronate acts by inhibiting the mevalonate pathway [29]. Due to its analgesic
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compared to other bisphosphonates. Pamidronate has been used in the treatment of osteolysis associated to Paget's disease [25] and is known to have analgesic effects in patients suffering from cancer and other related disorders. In addition, bisphosphonates can inhibit apoptosis of osteocytes and osteoblasts and induces the
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effect and its anti-resorptive capacity, pamidronate could be an alternative treatment
60
for OA [30].
61
Bisphosphonates might have anti-inflammatory properties, particularly during the
62
onset of arthritis [31]. The aim of this study is to evaluate the effect of pamidronate on
63
the clinical score and the secretory profile of inflammatory biomarkers (IL-6, TNFα,
64
MMP-2 and MMP-9) in the synovial fluid in clinically healthy horses and in horses with
65
joint disease.
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2. Materials and Methods
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2.1 Animals, synovial samples and experimental design
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At the beginning of the study, healthy horses and horses with joint symptoms were
69
examined and subjected to a standardised clinical evaluation of the locomotor system.
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The clinical condition was evaluated by a global score comprising 6 items: i) lameness
71
degree (0-5) ii) tenderness (0-3), iii) presence of pain under forced flexion (0-3), iv)
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volume of synovial fluid extracted (0-3), v) colour of synovial fluid (0-5) and vi) synovial
73
fluid viscosity (0-4). The score was based in the AAEP lameness score [32] with the
74
addition of a wide evaluation methodology focused in the tibio-tarsal joint. This joint is
75
an important site of OA prevalence in jumping horses [33, 34].
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The following groups were considered: i) control group (n = 8), clinically healthy young
77
animals (2-4 years). Young animals were chosen to be certain that since birth they had
78
no history of joint disease or disorders of bone metabolism. The clinical score of this
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group was ≤ 4. ii) animals with joint disease which were treated (TD) (n = 8, with
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clinical score > 5 at the beginning of treatment, aged between 4-8 years). This group
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was evaluated and analysed at different stages: baseline or pre-treatment group (TD0), day 3 (TD3); day 10 (TD10); day 21 (TD21) and day 60 (TD60). Animals included in TD group were animals with clinical signs and clinical history of chronic recurrent episodes of active OA with poor outcome to conventional treatment modalities.
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Radiographic analysis of both tibio-tarsal joint was performed in all animals included in
86
the experiment, control and TD group. TD group had radiographics alterations of
87
articular cartilage in the joints and subchondral bone resorption.
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On days 0 and 9, horses with joint disease were treated with 90 mg of intravenous
89
disodium pamidronate (Aminomux® 90mg, Gador). There is no detailed information of
90
pamidronate dosage and posology for horses in the bibliography, however divided
91
doses was recommended. In this paper we used low doses of pamidronate (0.4-0.8
92
mg/kg EV) administered in two doses 9 days apart [20, 35]. Synovial fluid samples were
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obtained on days 0, 3, 10, 21 and 60 by sterile aspiration from the tibio-tarsal joint.
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Samples were centrifuged at 2,000 x g for 10 min and the supernatant was kept at -
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70°C. The final clinical score in the pamidronate-treated group was considered on day
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60.
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2.2 Gel Zymography
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The gelatinolytic activities of MMP-2 and MMP-9 were measured by zymography as
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described by Gruber et al [36]. All samples were analyzed for protein concentration by
102
the Bradford method [37]. Aliquots of 100 µg of each sample were briefly mixed with a
103
buffer and loaded on a 10% SDS-PAGE with 0.2 % porcine skin gelatin (Sigma). After
104
electrophoresis, gels were washed with 50mM Tris-HCl pH 7.5 plus 2.5 % Triton X-100
105
for 45 min and then with a solution of 5mM CaCl2, 1µM ZnCl2, 50mM TrisHCl pH7.5
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plus 2.5 % Triton X-100 pH 7.5 for another 45 min. Finally gels were incubated with a
107
solution containing 50mM TrisHCl, 10mM CaCl2 and 200mM NaCl pH 7.5 at 37 °C for
108
24 h. Gels were stained with 0.2% (w/v) Coomassie Brilliant Blue R-250 for 2 h and
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then bleached with decolorizing solution (25% v/v isopropanol plus 10% v/v acetic
110
acid). Inhibition controls were performed by incubating with 5 mmol/ l EDTA. The
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densitometric study of the bands generated by the gelatinolytic activity was done
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using the Image J software (National Institutes of Health, Maryland, USA). Zymographic activity was expressed as a % of the control group. Data for different gels was standardized with the help of control samples. As MMP-9 is not always present in
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synovial fluid, we worked with MMP-9 data in a qualitative scale (negative, lightly
116
positive and strong positive).
117 118 119
2.3 Determination of IL-6 and TNF-α levels
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The concentration of cytokines (IL-6 and TNF-α) in synovial fluid was determined by
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commercial ELISA kits (OptEIA BD Biosciences, San Diego, CA, USA) as per
122
manufacturer's instructions. Cytokines were analysed in synovial fluid since it has
123
previously been demonstrated that this biological fluid is more suitable than serum [4].
124
Cytokine levels were derived from a standard curve. Undiluted samples were analysed.
125
Reactions were elicited by adding TMB substrate solution (Biosciences, San Diego, CA,
126
USA) and interrupted by adding 1M sulphuric acid. Plates were read at 450 nm in a
127
Rayto 2100C microplate reader (China).
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2.4 Statistical analysis:
130
Data were expressed as mean ± SD. Comparisons were performed using a
131
nonparametric ANOVA (Kruskal-Wallis’ test) followed by Dunn's post-test. Significant
132
differences were indicated as *p <0.05, **p<0.01, ***p<0.001.
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3. Results
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Pamidronate treatment significantly improved the clinical condition of horses with OA,
136
as measured by the clinical score. The comparative analysis showed that treatment
137
with pamidronate 60 days after BP application significantly lowered the clinical score in
138
animals with joint disease versus TD0 animals (p<0.05), and that there was no
139
significant difference between the control group (C) and the treated group at this time
140
(Figure 1) (Table 1).
141
Cytokine levels were measured following synovial fluid extraction. Synovial values of
142
IL-6 (pg/ml) were similar in the healthy control group (102±26.94), the TD3
143
(113.9±18.33) and TD60 group (78.87±26.17) (ns); however TD0, TD10 and TD21
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groups had higher IL-6 values (p<0.05 vs control) (Figure 2). TNF-α dropped considerably by day 3 in the treated animals (TD3) (6.44pg/ml±4.71) and was statistically significant compared to TD0 group (p<0.05), but compared with the control
147
group (15.2 pg/ml ±12.7) the difference was not statistically significant due to the wide
148
scattering of data.
149
Synovial MMP-2 levels showed no significant differences between groups (Figure 4).
150
MMP-9 were scored as i) absent ii) slightly positive and iii) strong positive. The results
151
in control group showed that only 3 animals were slightly positive for MMP-9 (Figure 5
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and Table 2). Only 1 animal of TD0 group had strong MMP-9 activity. Pamidronate
153
seems to stimulate an acute increase in synovial MMP-9 activity because 6 of 8 animals
154
of TD3 group were positive (3 slightly and 3 strong). However TD10 group only had a
155
strong positive MMP-9 activity and TD21 and TD60 had only negative MMP-9 activity.
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4. Discussion
158
In this study, we observed that animals treated with pamidronate showed a significant
159
improvement in the clinical score compared with the TD0 group. The clinical changes
160
were characterized by a decrease in the signs of inflammation, less or no pain under
161
forced flexion and increased ability to work.
162
It has previously been observed that pamidronate can reduce pain intensity and
163
inflammation in humans with ankylosing spondylitis, [31]. Although OA is generally
164
considered a degenerative gradual disorder it can also be considered an inflammatory
165
process.
166
In this study, no significant differences were found in MMP-2 activity between clinically
167
healthy animals, TD0 and TD animals. Pamidronate would seem to stimulate a sharp
168
increase in the activity of MMP-9 but at day 21 and 60 animals treated with
169
pamidronate had an absence of MMP-9 activity, which indicated that it could have a
170
beneficial effect in the medium term. These findings are in line with previous results in
171
which the treatment with bisphosphonates inhibited MMP activity [38, 39]. This
172
phenomenon will result in a lower degradation rate of the extracellular matrix and the
173
articular cartilage surface. Furthermore, the low activity of MMPs in synovial fluid
174
reduced leukocyte migration, inflammation and associated pain. The latter observation
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is related to the therapeutic prescription of pamidronate in humans, where this drug is
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known to inhibit subchondral bone resorption, thus leading to the amelioration of pain in cancer bone metastases [40]. Given that high osteoclast activity plays an important role in the degradation of the adjacent subchondral bone and in synovial inflammation causing pain adjacent to subchondral bone [41], pamidronate-induced osteoclast
180
apoptosis will probably result in a decrease of bone turnover and the antinflammatory
181
related effect.
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Our results show that TNFα levels in OA were significantly decreased at day 3 of
183
treatment and elevated on days 10, 21 and 60. Moreover, on day 3 in the treated
184
group IL-6 and TNF-α levels were similar to those in healthy animals.
185
Studies carried out in cultures of peripheral leukocytes obtained from patients with
186
osteoporosis treated with pamidronate showed an increase in IL-6 and TNFα
187
production during the first 24 h after the first dose [42]. Cancer patients treated with
188
IV pamidronate showed an increase in IL -6 levels that lasted 7 days [43]. These results
189
differ from other published studies performed in rheumatoid arthritis patients, whose
190
levels of IL -6 and TNFα were significantly increased for 30 days after the treatment
191
with pamidronate [44] consistent with our own results at day 21. Other authors
192
suggest an anti-inflammatory effect of pamidronate evidenced by decreased IL-6 levels
193
[45]. We have also observed decreased levels of IL-6 after pamidronate treatment but
194
only on day 3 and 60. In this study, the increased TNFα levels observed on days 10 and
195
21 after treatment might be related to an exacerbation of the inflammatory process
196
which could be beneficial for the resolution of chronic arthritis [46]. TNFα is a critical
197
factor for NFkappa B expression which, in turn, is responsible for MMP-9 expression
198
[47, 48]. In this study, it could be assumed that MMP-9 activities in synovial fluid did
199
not increase after day 21 as a consequence of the elevated TNFα levels on days 10 and
200
21; however, further studies are required to confirm this hypothesis.
201
Many studies have focused on the role of certain MMPs (MMP-1, MMP -3 and MMP -
202
13) with collagenase activity whereas in this study we have focused on the gelatinases
203
(MMP-2 and MMP-9) which play a major role in the degradation of articular cartilage.
204
Clodronate is a first generation bisphosphonate, which acts as an inhibitor of MMP-1
205
purified from human fibroblasts [49]. In vitro studies have shown that alendronate,
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could also inhibit leukocyte migration by reducing MMP-2 activity [50, 51]. Moreover, the administration of alendronate can lead to the inhibition of MMP-13 in patients with rheumatoid arthritis [52]. Osteoclasts, macrophage linage cells, are a major source of MMP-9 in subchondral
210
bone in OA joints. The increased number and activity of osteoclasts in OA induce the
211
appearance of subchondral bone resorption areas and are the reason for increased
212
plasma activity of MMP-9 [53, 54]. Pamidronate inhibits the MMP-9 activity through
213
the induction of osteoclast apoptosis. The activity of MMP-9 has been associated with
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rheumatoid arthritis and some other types of OA in humans [55]. In vitro studies have
215
shown an inhibitory effect of alendronate on MMP-9 activity due to its chelating effect
216
[56]. In this paper we registered a fast increase in MMP-9 activity but it was not
217
detectable until day 21 and 60. The latter phenomenon could be important since the
218
inhibition of MMP-9 is associated with the prevention of articular lesions.
219
Potent MMP inhibitors, such as doxycycline delays the progression of OA lesions [57].
220
Furthermore, it has been observed that glucosamine combined with chondroitin
221
sulphate inhibits MMP-9 activity and the joint damage associated with adjuvant-
222
induced arthritis in rats [58].
223
According to Clegg [59], MMP-9 activity is elevated in joint pathological processes. In
224
this paper, the absence in the activity of MMP-9 after 21 and 60 days of treatment
225
suggests that bisphosphonates may be a safe alternative to corticoids and other anti-
226
inflammatory therapies for the treatment of OA.
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5. Conclusion
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We concluded that pamidronate treatment has positive effect in the clinical score of
231
horses with osteoarthritis, and reduces proinflammatory cytokines (IL-6 and TNF-
232
alpha) and MMP-9 after treatment. Recent advances in the understanding of
233
molecular events in OA allow us to use novel drugs in this disease. However, the real
234
benefit of these treatments in horses should be further studied.
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Figure Legends
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Figure 1. Clinical Score (Mean ± SD) for control, pretreated (TD0), and treated at
240
different stages (TD3, TD10, TD21 and TD60). TD0, TD3, TD10 and TD21 differ
241
significantly vs control (**p<0.01) and vs TD60 (* p<0.05).
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Figure 2. IL-6 levels in synovial fluid (Mean ± SD) from control, pretreated (TD0), and
245
treated at different stages groups (TD3, TD10, TD21 and TD60). Levels of TD0, TD10 and
246
TD21 were significantly different versus control, TD3 and TD60 group (*p<0.05).
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247 248
Figure 3. TNFα levels in synovial fluid (Mean ± SD) for control, pretreated (TD0), and
250
treated at different stages (TD3, TD10, TD21 and TD60). TD10 and TD21 groups
251
presented significant differences from both the control and TD3 groups (**p<0.01).
252
TD0 and TD60 presented significant differences versus the control, and TD3 groups
253
(*p<0.05)
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Figure 4. MMP-2 activities in joint synovial fluid for control, pretreated (TD0), and
256
treated at different stages (TD3, TD10, TD21 and TD60). No significant differences
257
were found in the MMP-2 activity between groups.
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Figure 5. Gelatin zimography for detection of MMP-9 activity in a standard sample (line
260
A), negative sample (B and E), slightly positive (D and F) and strong positive (C).
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Acknowledgment
264 265
The authors thank UBACYT 20020120100092BA for financial support.
266
The authors declared a conflict of interest (such as defined by JEVS policy). Emilio
268
Roldán is employed in Gador SA, the company that provides Pamidronate
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(Aminomux®). All other authors have stated that they have no conflict of interest.
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Authors contributions: M.A.C.B and E.D.S. designed research, performed research,
272
analyzed data and wrote paper. M.A.C.B performed the statistical analysis. E.R.
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participated in designed research. G.P. and C.M.D.O performed field research and
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acquisition of clinical data. N.C., Y.L., F.R., J.D. and A.F. performed laboratory activity.
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control
TD0
TD3
TD10
TD21
6.3±0.51
8.2±0.51
7.5±1.21
6.2±0.131
TD60 3.8±0.32
2.1 ± 0.2
IL-6 (pg/ml)
102.2±26.94
251.8±61.983
113.9±18.33
275±20.733
239.3±71.253
78.87±26.17
TNF-α(pg/ml)
15.2±12.7
30.02±24.254
6.44±4.71
48.2±5.25
51.33±22.525
24.43±16.964
MMP-2 (% of control)
100±66
148±45
107±62
181±116
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Table 1. Measured parameters (Mean ± SD) for control, pretreated (TD0), and treated at different stages (TD3, TD10, TD21 and TD60) . 1p<0.01 vs control and 2 p<0.05 vs TD0. 3p<0.05 vs control, TD3 and TD60.4 p<0.05 vs control and TD3. 5p<0.01 vs control and TD3 group. No significant differences were found in the MMP-2 activity between groups.
120±42
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MMP-9
control
TD0
TD3
TD10
TD21
TD60
5/8
7/8
2/8
7/8
8/8
8/8
Slightly positive
3/8
-
3/8
-
-
-
Strong positive
-
1/8
3/8
1/8
-
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Table 2. Number of animals with negative, slightly positive and strong positive gelatinolytic MMP-9 activity in synovial fluid in control, TD0, TD3, TD10, TD21 and TD60 group.
F
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Figure 5. Zimography showing gelatinolytic activity in a standard sample (line A), negative sample (B and E), slightly positive (D and F) and strong positive (C).
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•
We evaluate the effect of pamidronate on the clinical score and the secretory profile of inflammatory biomarkers (IL-6, TNFα, MMP-2 and MMP-9) in the synovial fluid in clinically healthy horses and in horses with joint disease. Pamidronate treatment significantly improved the clinical condition of horses
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with osteoarthritis (OA). •
Values of IL-6 and TNFα. In synovial fluid were similar with the control group at
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day 3 of treatment and MMP-9 activity was absent at day 10 and 21 of
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treatment.