Amikacin Cytotoxicity on Equine Joint Cells and Mesenchymal Stromal Cells
Presented by: Lynn Pezzanite
Authors: L. Pezzanite, L. Chow, S. Soontararak, N. Phillips, S. Dow, L. Goodrich
Affiliations: Colorado State University, Veterinary Teaching Hospital
Introduction: Equine veterinarians frequently inject aminoglycoside antibiotics intra-articularly, either in treatment of septic arthritis or prophylactically when injecting therapeutics in joints. Incidence of septic arthritis following joint injection in horses is low (<0.1%), and concurrent administration of antibiotics with joint injections has not been shown to reduce infection risk. However, prophylactic antibiotic use remains prevalent among practitioners, with 78% of practitioners reporting adding antimicrobials to joint injections in at least some situations. Although aminoglycosides are known to be toxic to various mammalian cells, their effect on native joint cells has not been investigated and appropriate intra-articular doses have not been defined.
Hypothesis / Objectives: The objective was to evaluate the effect of amikacin concentration, time of exposure and pH on four equine cell types and determine whether presence of synovial fluid influences this effect. We hypothesized that amikacin is cytotoxic to equine cells at concentrations typically used for intra-articular injections by inducing apoptosis.
Materials and Methods: Four cell types (chondrocytes, synoviocytes, adipose and bone marrow derived mesenchymal stromal cells) were harvested from 3 donor horses and plated on 96 well plates at 30,000 cells/well for 48h prior to the addition of amikacin. The effect of amikacin was assessed for different exposure times, concentrations, with pH buffered or unbuffered, and with synovial fluid. Viability and proliferation were assessed using live imaging, mitrochondrial staining, and colorimetric assay. Cells were assessed for occurrence of apoptosis using Annexin V and propidium iodide staining with flow cytometric quantification as well as for caspase expression to determine mechanism of cell death.
Results: Amikacin cytotoxicity occurred by apoptosis in a rapid, dose-dependent, pH-independent manner, at doses clinically relevant to those used in joints. Cells demonstrated reduced metabolism rapidly (1hr) using green fluorescent mitochondrial staining under confocal microscopy (Fig1). Amikacin reduced media pH, but buffering did not improve metabolism by MTT assay. (Fig2). Cell proliferation was suppressed in dose-dependent manner for ≥24h by live cell imaging (Fig3). Amikacin effect was not mitigated by synovial fluid. Amikacin dose at which 50% of cells were viable (LD50) was approximately 1mg/ml. Cells underwent apoptosis evidenced by caspase expression and annexin/PI staining using flow cytometric quantification (Fig4).
Conclusions: This study highlights the dose-dependent toxicity of amikacin to equine joint cells and provides an important guideline to clinicians on use of this commonly used drug. Current doses of intra-articular amikacin commonly used not only have the potential to cause iatrogenic harm to native joint cells and MSC if co-injected, but also raise questions of judicious use of antimicrobials. While the authors are cautious to extrapolate in vitro findings to the in vivo joint environment, it is recommended that doses of amikacin administered intra-articularly be reduced or eliminated pending further in vivo dose titration and toxicity studies.
Acknowledgements, Funding, and Conflicts of Interest: The authors would like to thank and acknowledge the USDA National Institute of Food and Agriculture Animal Health and Disease Grant NI18AHDRG010, the Verdad Foundation, the Colorado State University Young Investigator Award in Companion Studies, and the Colorado State University Veterinary Summer Scholars Program for financial support of this work. The authors would like to acknowledge Dr. Ann Hess and Dr. Gregg Griffenhagen for help with statistical analyses.