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*Department of Anesthesiology and Intensive Care, Hôpital de l’Hôtel-Dieu, Lyon; and
Laboratory of Glycobiology, Unité INSERM 346, Centre Hospitalier Lyon Sud, Pierre-Bénite, France
Address correspondence and reprint requests to Pr Dominique Chassard, Service d’Anesthésie-Réanimation, Hôpital de l’Hôtel-Dieu, 69002, Lyon, France. Address e-mail to dominique. chassard{at}chu-lyon.fr
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Abstract |
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IMPLICATIONS: This study suggests that lidocaine can induce apoptosis (detected by dual staining with Annexin V and propidium iodide) on T-cell line cultures in a time-dependent manner. This was not observed with ropivacaine.
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Introduction |
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Methods |
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Annexin V, fluorescein isothiocyanate (FITC), and propidium iodide (PI) were purchased from Zymed Laboratories (ApoDETECTTM Annexin V-FITC kit; San Francisco, CA). Lidocaine chlorydrate and ropivacaine chlorydrate were both purchased from Astra Zeneca Laboratories (Rueil Malmaison, France).
During the early stages of apoptosis, phosphatidylserine (PS) is translocated to the outer layer of the plasma membrane. This event can be detected by using a sensitive method to detect PS exposure (5). This phenomenon also occurs during cell necrosis; however, the cell becomes leaky and loses its integrity. Therefore, it is required to assess membrane integrity together with PS translocation. Based on its affinity for PS, Annexin V can be used as a sensitive probe for cell surface exposure of PS. To use Annexin V as a probe for apoptotic cells, the protein is labeled with FITC. In this form, the protein can be used directly for quantification of apoptotic cells. The measurement of Annexin V binding when performed simultaneously with a dye exclusion test, such as PI, can be used to effectively discriminate between apoptotic and necrotic cells (5).
After more than 95% cell viability was confirmed by trypan blue staining, suspensions of 4 x 105 cells were incubated in a 2-mL medium at 37°C with either lidocaine 6 mg/mL or ropivacaine 1 mg/mL for 1–6 h. Other samples were incubated in the 2-mL medium alone as a control. After incubation, cells were centrifuged for 1 min at 3000 rpm, then washed in ice-cold phosphate-buffered solution, with a pH value of 7.4, and again centrifuged at 3000 rpm. Cells were resuspended in 190 µL of binding buffer (10 mM of HEPES/NaOH, pH value of 7.4, 140 mM of NaCl, and 2.5 mM of CaCl2), and 10 µL of Annexin V-FITC was added. Cells were washed in binding buffer and centrifuged for 1 min at 3000 rpm and then resuspended in 190 µL of binding buffer, and 10 µL of 20-µg/mL PI stock solution was added.
Apoptotic cells can be recognized and distinguished from necrotic cells using flow cytometry (FACScan; Becton Dickinson Systems, San Jose, CA) after double staining by Annexin V-FITC and PI (5). Apoptotic cells exclude all those dyes that are in use for cell viability assays, such as PI, whereas necrotic cells do not. In cells with a damaged cell membrane, PI induces a red fluorescence of the DNA, whereas it is excluded by cells with a preserved cytoplasm in the membrane. Hence, during the initial phase of apoptosis, the cells are still able to exclude PI and therefore do not show any red fluorescence signal, which is similar to that of living cells. PS is exposed on the external surface of apoptotic cells but not viable cells and thus are positive for Annexin V-FITC binding, which is detected by a green fluorescence. This method allows one to distinguish apoptotic cells (FITC+/PI-, green fluorescence), necrotic cells (FITC+/PI+, red and green fluorescence), and viable cells (FITC-/PI-, no fluorescence).
All data were expressed as a mean ± SD percentage of apoptotic cells in each sample (Fig. 1). Statistical analyses for apoptotic cells (FITC+/PI-) were performed using the Statview software (SAS Institute, Cary, NC) with an analysis of variance for repeated measures. Significant differences (P < 0.05) were identified using Bonferroni adjusted comparison test.
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Results |
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Discussion |
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Cell toxicity induced by local anesthetics has been widely studied in animal and in vitro models, although its mechanism remains unclear (2). It seems that local anesthetics lead to a perturbation of energetic cell metabolism (1). Little is known about the induction of apoptosis by local anesthetics on cell cultures. Only one study has suggested the possible induction of apoptosis by cocaine on embryonic brain cells (6). In a recent study, published in an abstract form, the authors suggested that small doses of lidocaine (2.5 mg/mL) cause neuronal injury and apoptosis on neuronal cell lines derived from rat dorsal root ganglion (4). There is no information in the literature about the induction of apoptosis in cell cultures by ropivacaine.
The method of detection of apoptosis by dual staining with Annexin V-FITC was chosen, although there are more conventional methodologies such as the TUNEL (terminal deoxynucleotidyl transferase technique) method, and this quite elegant method has been widely used in recent studies devoted to the induction of apoptosis (7–9).
Our objective was to determine whether lidocaine and ropivacaine could induce apoptosis on cell cultures. The induction of apoptosis might be one explanation of cell toxicity caused by local anesthetics, especially with lidocaine. As a preliminary study, only one concentration for each local anesthetic was chosen, and the model chosen was a Jurkat T-cell line because this line can be cultivated with routine techniques, as compared with neurons. These results are encouraging, and further dose-response studies tested on a neuronal model and providing indications of the molecular mechanisms underlying these actions are required to determine the clinical relevance of these findings.
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