The Role Of Lysosomal And Mitochondrial Pathway In LAs-induced Death Of Rabbit Intervertebral Disc Cells

Part IComparison of toxicity effects of various local anesthetics on rabbit intervertebral disc cellsObjectiveThe purpose of this study was to evaluate the effect of various local anesthetics (LAs) on rabbit intervertebral disc (IVD) cells in vitro and further to compare the cytotoxicity of ropivacaine, bupivacaine, lidocaine and saline solution control.MethodsBoth annulus fibrosus (AF) and nucleus pulposus (NP) cells at the second generation maintained in monolayer were exposed to various concentrations of LAs (e.g. bupivacaine) or different durations of exposure and evaluated for cell viability by use of cell counting kit-8 (CCK-8). In addition, to compare the cytotoxicity of ropivacaine, bupivacaine, lidocaine and saline solution control in commercial concentration, the viability was analyzed by flow cytometry after 60-minute exposure, and the morphologic changes were observed by the phase-contrast microscopy. Apoptosis and necrosis of IVD cells were confirmed by using fluorescence microscopy with double staining of Hoechst 33342 and propidium iodide (PI).ResultsRabbit IVD cells death demonstrated a time and dose dependence in response to bupivacaine and lidocaine. However, ropivacaine only exerted a significant time-dependent effect on IVD cells. There was no significant difference in IVD viability after treatment with different doses of ropivacaine. In addition, the results showed that lidocaine was the most toxic of the three LAs, and that ropivacaine presented less cytotoxicity than lidocaine and bupivacaine. Fluorescence microscopy also confirmed that the short-term toxic effect of local anesthetics on both AF and NP cells was mainly caused by necrosis rather than apoptosis.ConclusionsResults show that bupivacaine and lidocaine decrease cell viability in rabbit IVD cells in a dose-and time-dependent manner. These results show all LAs should be avoided if at all possible. Ropivacaine may be a choice if necessary, but it is also toxic. The increase in cell death is more related with cell necrosis rather than cell apoptosis. If these results can be corroborated in tissue explant models or animal studies, caution regarding to diagnose, treat, and control spine-related pain with LAs is prompted.Part ?LAs-induced death of rabbit intervertebral disc cells involves ROS-mediated lysosomal membrane permeabilizationObjectiveThe purposes of this study were to assess the short-term effects of LAs (e.g. bupivacaine) on IVD cells in vitro and further to explore the precise mechanism of LAs-induced IVD cell death.MethodsBoth rabbit AF and NP cells at the second generation maintained in monolayer were exposed to various concentrations of bupivacaine for 60 minutes. Cell viability was evaluated by using CCK-8. Cell death was measured by flow cytometry and the LIVE/DEAD assay. Transmission electronic microscopy (TEM) was used to further characterize the type of cell death after exposure to bupivacaine. The integrity of the lysosomal compartment was evaluated by LysoTracker Red staining. Lysosomal membrane permeabilization (LMP) was confirmed by acridine orange (AO) staining. Moreover, the levels of reactive oxygen species (ROS) were determined with fluorescent probe DCFH-DA.ResultsBupivacaine treatment induced a dose-and time-dependent cytotoxic effect in rabbit IVD cells. Flow cytometry and fluorescence microscopy showed that the short-term toxic effect of bupivacaine on both AF and NP cells was mainly caused by necrosis. The morphological signs of necrosis such as disintegration in organelle and disruption in plasma membrane were observed under TEM. The key finding of this study was that bupivacaine is able to induce lysosomal membrane rupture, as evidenced by LysoTracker Red staining and AO staining. Finally, we found that bupivacaine resulted in an increase in intracellular ROS, and inhibition of ROS by N-acetyl-L-cysteine (NAC) effectively blocked the bupivacaine-induced LMP and cell death.ConclusionsThe results of this in vitro study indicate that a short-term exposure of bupivacaine can cause a rapid cell death, which is primarily due to necrosis, in cultured rabbit IVD cells. In addition, our results show that bupivacaine-induced cell death involves LMP, and ROS is a critical mediator in bupivacaine-induced LMP and cell death. Further investigation regarding in vivo cytotoxicity appears warranted.Part ?LAs-induced death of rabbit annulus fibrosus cells in vitro: involvement of the mitochondrial apoptotic pathwayObjectiveThe purposes of this study were to assess the long-term effects of LAs, such as ropivacaine and bupivacaine, on IVD cells in vitro. In addition, we would like to investigate whether LAs could induce apoptosis of rabbit IVD cells and further to explore the possible underlying mechanism.MethodsRabbit AF cells at second passage were treated with saline solution and various concentrations of LAs for 60min. After 60-min exposure, cells were immediately re-incubated in fresh culture medium to allow time for recovery for 24 and 120 h. Cell proliferation was evaluated by CCK-8 and EdU incorporation assays. Apoptosis of AF cells were examined by Annexin V/PI assays, Hoechst 33342 staining, and Caspase-3,-9 activity assays. The expression of apoptosis-related markers was detected by real-time PCR and Western Blot. The JC-1 staining was used to evaluate the change of mitochondrial membrane potential (MMP). Moreover, the levels of ROS were determined with fluorescent probe DCFH-DA.ResultsThe results of CCK-8 and EdU incorporation assays showed that ropivacaine and bupivacaine could inhibit the cell proliferation of rabbit AF cells in a dose-dependent manner, and bupivacaine is more toxic to AF cells than ropivacaine when diluted to the same concentrations. The results of flow cytometry indicated that the increase in cell death of AF cells at 24 h after exposure was more related with cell necrosis, but the decrease in viability on AF cells at 120 h following 1-h exposure to LAs was predominately due to apoptosis. Apoptosis was confirmed by cell morphology, condensed nuclei and activation of Caspase-3 and-9. In addition, the molecular data showed that LAs could significantly up-regulate the expression of Bax, accompanied by a significant down-regulation of Bcl-2 expression. LAs enhanced the release of cytochrome c from mitochondria and generation of activated fragments of the Caspase-3 and-9. Furthermore, we also observed that LAs resulted in alteration of MMP and accumulation of intracellular ROS in AF cells. Blockade of ROS production by NAC inhibited LAs-induced alteration of MMP and apoptosis.ConclusionsThese findings suggest that the long-term toxic effects of LAs in clinically relevant concentrations on AF cells was predominately due to apoptosis, and the mitochondrial pathway was, at least in part, involved in the LAs-mediated apoptosis. Further investigations focusing on the potential cytotoxicity of LAs on IVD cells are needed.