Condrotoxicidade e efeitos inflamatórios in vitro e in vivo da ropivacaína em equinos

Abstract

Local anesthetics are routinely used in lameness diagnosis and joint pain control in horses, but because of their potential toxicity, they should be used with caution in order to avoid the development of joint disease. Previous studies in humans demonstrate ropivacaine as a less toxic alternative, motivating interest in the use of this anesthetic by the intra-articular (IA) route in horses. The objective of this study was to evaluate the IA effects of ropivacaine in vitro and in vivo. For viability evaluation, monolayer chondrocytes were divided into six groups and exposed to 30 minutes of treatment. The groups were: chondrogenic medium (DMEM), ropivacaine 7.5mg/ml, ropivacaine 10mg/ml, saline 0.9%, mepivacaine 20mg/ml and mepivacaine 30mg/ml. Chondrocyte viability was assessed by trypan blue, MTT and flow cytometry methods. To evaluate gene expression in vitro, pellets cultures of chondrocytes were used and submitted to 30 minutes of the following treatments: mepivacaine 20mg/ml, ropivacaine 10mg/ml and saline 0.9%. The cells were evaluated by Real-Time Quantitative Polymerase Chain Reaction (qPCR) to investigate the expression of Ilb1, Il6, Mmp9, Mmp13, Acan and Col2a1. The in vivo study was performed with the administration of 10 mg/ml ropivacaine and saline in the tibiotarsal joints of eight ponies. Synovial fluid samples were collected before and 24 hours after treatment. Then, samples were evaluated for total nucleated cells count, total protein and mucin precipitate evaluation. After 24 hours, the ponies were anesthetized and samples of synovia and cartilage were collected through arthroscopy to evaluate by qPCR. In the synovia and cartilage samples Ilb1, Il6, Mmp9 and Mmp13 were evaluated; genes Acan and Col2a1 were also evaluated in the cartilage samples. The evaluation of cell viability produced different results in the different assays: in the trypan blue method there was no difference; the MTT method demonstrated less viability of cells treated with ropivacaine than DMEM. Differently, flow cytometry did not show differences between ropivacaine 7.5mg ml and DMEM. In chondrocytes cultured in pellets, the expression of the evaluated genes was similar in the mepivacaine, ropivacaine and saline treatments. In pellets treated with mepivacaine and ropivacaine there was downregulation of the expression of Il6 and Mmp13 before and after treatment. After exposure to ropivacaine, there was downregulation of Mmp9 levels. In turn, the short exposure of the pellets to mepivacaine caused an increase in Col2a1 expression. In vivo results demonstrated that ropivacaine and saline cause inflammation in the synovial fluid when applied IA. However, no differences were observed between anabolic and cartilage catabolic markers between treatments. Only Mmp9 levels in synovia were increased following ropivacaine treatment. The in vitro results of this study suggest that short exposure to ropivacaine may result in a lower cell death rate than exposure to mepivacaine; in addition, mepivacaine and ropivacaine did not cause unbalance of chondrocyte homeostasis and appear to have some anti-inflammatory effect. Finally, the in vivo study revealed similarity between the effects of the IA treatment of ropivacaine and saline, suggesting that this anesthetic is a safe option for IA use.