Neurotoxicity induced by bupivacaine via T-type calcium channels in SH-SY5Y cells
Authors: Chenxiang Yang
Xianjie Wen
Hongzhen Liu
Quinguo Zhang
Shiyuan Xu
Hua Liang
Hanbing Wang
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: neurotoxicity; local anesthetics; T-type calcium channels; bupivacaine; SH-SY5Y cells
Affiliations: Department of Anesthesiology, Zhujiang Hospital of Southern Medical University, Guangzhou, Guangdong Province, China. 

Abstract:  There is concern regarding neurotoxicity induced by the use of local anesthetics. A previous study showed that an overload of intracellular calcium is involved in the neurotoxic effect of some anesthetics. T-type calcium channels, which lower the threshold of action potentials, can regulate the influx of calcium ions. We hypothesized that T-type calcium channels are involved in bupivacaine-induced neurotoxicity. In this study, we first investigated the effects of different concentrations of bupivacaine on SH-SY5Y cell viability, and established a cell injury model with 1 mM bupivacaine. The cell viability of SH-SY5Y cells was measured following treatment with 1 mM bupivacaine and/or different dosages (10, 50, or 100 ¬µM) of NNC 55-0396 dihydrochloride, an antagonist of T-type calcium channels for 24 h. In addition, we monitored the release of lactate dehydrogenase, cytosolic Ca(2+) ([Ca2+]i), cell apoptosis and caspase-3 expression. SH-SY5Y cells pretreated with different dosages (10, 50, or 100 ¬µM) of NNC 55-0396 dihydrochloride improved cell viability, reduced lactate dehydrogenase release, inhibited apoptosis, and reduced caspase-3 expression following bupivacaine exposure. However, the protective effect of NNC 55-0396 dihydrochloride plateaued. Overall, our results suggest that T-type calcium channels may be involved in bupivacaine neurotoxicity. However, identification of the specific subtype of T calcium channels involved requires further investigation. 

Retracted: No

PubPeer comments:
 2019 expression of concern. https: journals.plos.org plosone article?id=10.1371 journal.pone.0222623 Following publication of this article [1] concerns were raised regarding images in Figs 5 and 7. Specifically, In Fig 5A there are regions of similarity between the dot plots in the top left panel u201cSu201d and the top middle panel u201cS+NNC100u201d. In Fig 5A there are regions of similarity between the dot plots in the bottom middle panel u201cS+B+NNC50u201d and the bottom right panel u201cS+B+NNC100u201d. In the western blot in Fig 7, there are horizontal discontinuities in the background shading above and below the band in lane 5 of the cleaved caspase-3 panel. In the western blot in Fig 7, there are similarities between bands in lanes 1u20133 compared to lanes 4u20136 of the u03b2-actin panel. The corresponding author has clarified that the original flow cytometry experiments were carried out by a technician in another laboratory within the same institution. The corresponding author stands by the flow cytometry dot plots in Fig 5 as generated from independent samples. The original raw data files for the flow cytometry experiments in Figs 4 and 5 are no longer available. Higher resolution image files of the original dot plots are provided here as Supporting Information (S1 File). The authors have repeated the experiment and provide a revised Fig 5 using the new data. The underlying raw data files for the repeat experiment are provided as Supporting Information (S2 File). The corresponding author stands by the western blot images in Fig 7 as accurate; however, the original uncropped blot images are not available. The underlying data for the other parts of the article remain available upon request. The PLOS ONE Editors issue an Expression of Concern to inform readers about the concerns regarding the accuracy of the western blot and flow cytometry image data in the original article, which could not be fully resolved due to the unavailability of the original raw data files. Reference 1.Wen X, Xu S, Liu H, Zhang Q, Liang H, Yang C, et al. (2013) Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells. PLoS ONE 8(5): e62942. https: doi.org 10.1371 journal.pone.0062942 pmid:23658789 , 
 Here is the original Figure 5A, with the (redrawn) concerns reported to the journal in May 2014 (5 years before the Expression of Concern was issued). Boxes of the same color draw attention to areas that look more similar than expected.  , 
 2# Dear Prof. Elisabeth M Bik, Thanks very much for your comment, For the problem you mentioned, we have carried out the third-party identification. Through the analysis of high-definition figure (original figure) by beyond compare 4 software (version No.: 4.2.10), the generation time of S and S + nnc100, S + B + nnc50 and S + B + nnc100 in Fig5a is different. In order to verify the experimental results, we repeat the experiments mentioned above and get similar results with the original.Thank you very much for your comments!Because the attachment format cannot upload the original TIF format, the original TIF format is converted to PNG format.Sorry, the file uff0840muff09 of the identification report is too large to upload. [](http: )   peer.com storage image-1584486543073.png)    , 
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 Dear Dr. Wen, I am not familiar with the software Beyond Compare, but these high definition plots you have uploaded clearly have regions that are more similar than what could be expected by chance. If we compare two of the originally highlighted figures - panels 2 and 3 in the bottom row we can highlight a number of regions that are virtually identical.  I could highlight more clusters, but I hope I make my point. These appear almost like re-gatings re-samplings of the same population acquisition. Could this be possible? , 
 Dear Coniochaeta Malacotricha, Because the original flow cytometry experiments were carried out by a technician. I m not sure the possible of re-gatings re-samplings of the same population acquisition, but the identification results show that the generation time of these original pictures is different, indicating that these pictures have not been edited. In order to exclude the influence of what you said on the experimental results, we repeated this part of the experiment, which is similar to the original experiment results.Your opinion is very valuableuff0cThank you very muchuff01 , 
 #7 I believe the Beyond Compare software is being used to compare the two image files and it concludes they are not exact matches (which is obvious, and not the point) and they have different date stamps. If I am correct, I believe both of these facts would still be true if the photos were edited in Photoshop before saving them for use in the paper. This would mean that this particular analysis is not useful in addressing the suspicion that the original cytometry results may have been fabricated or manipulated. , 
 #9 Dear Actinopolyspora Biskrensis , the date of the stamp on the original identifyed picture were from the the flow cytometry. You can see from the identified picture that it is impossible to edit two pictures in a short time. Considering the experimental method, I am not sure Prof. Coniochaeta Malacotricha metioned the possible of re-gatings re-samplings of the same population acquisition, because we didn t have a flow cytometer at that time, we prepared the sample and asked other laboratory technicians to test it. It is possible that Flow cytometer on the machine for about 1min to acquisition. As well as,we repeated this part of the experiment to verify the original experiment results.Thanks!  , 
 2020 retraction. https: journals.plos.org plosone article?id=10.1371 journal.pone.0238400 Following publication of this article [1] concerns were raised regarding images in Figs 5 and 7, as detailed in an Expression of Concern published 12 September, 2019 [2]. The authors have carried out further repeat experiments. However, in view of the unavailability of the original underlying data, the PLOS ONE Editors consider that the issues raised cannot be fully resolved by the provision of repeat experimental data. The corresponding authors requested the article s retraction. In light of the unresolved issues with Figs 5 and 7, and in line with the corresponding authorsu2019 request, the PLOS ONE Editors retract this article [1]. XW, SX, QZ, and HLiang agreed with the retraction. XW, SX, and QZ, and HLiang stand by the article s findings. HLiu, CY, and HW either could not be reached or did not reply directly. References 1.Wen X, Xu S, Liu H, Zhang Q, Liang H, Yang C, et al. (2013) Neurotoxicity Induced by Bupivacaine via T-Type Calcium Channels in SH-SY5Y Cells. PLoS ONE 8(5): e62942. https: doi.org 10.1371 journal.pone.0062942 ,