Tese
Condrotoxicidade e efeitos inflamatórios in vitro e in vivo da ropivacaína em equinos
Autor
Silva, Gabriele Biavaschi da
Institución
Resumen
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.