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Original Article Lidocaine-Induced Neurotoxicity Via Upregulating TMEM16F (Ano6)

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Original Article Lidocaine-Induced Neurotoxicity Via Upregulating TMEM16F (Ano6) Int J Clin Exp Med 2020;13(12):9409-9418 www.ijcem.com /ISSN:1940-5901/IJCEM0115619 Original Article Lidocaine-induced neurotoxicity via upregulating TMEM16F (ano6) Hailiang Du1, Xinyuan Liu2, Wei Gao1, Shuyuan Liu1, Yaomin Zhu1 1Department of Anesthesiology, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China; 2Department of Nursing, The Second Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, People’s Republic of China Received June 1, 2020; Accepted September 29, 2020; Epub December 15, 2020; Published December 30, 2020 Abstract: Purpose: The aim of this study was to investigate the possible mechanism of lidocaine neurotoxicity. Meth- ods: Forty rats were randomly divided into four groups: the control, sham, saline, and lidocaine groups. Neurobe- havioral tests were performed on rats in each group before and 1-4 days after the intrathecal administration. On the fourth day, the spinal cords of all rats from L2 to L6 were taken out and immediately stored in liquid nitrogen. In the control and lidocaine groups, parts of the spinal cord tissues of three rats in each group were selected for gene microarray analysis, revealing that compared with the control group, TMEM16F (ano6) expression was upregulated in the lidocaine group. To verify the expression of ano6 at the mRNA and protein levels, five rats in each group un- derwent real-time quantitative polymerase chain reaction (RQ-PCR) and five underwent western blot analysis. The SK-N-SH cells were cultured and randomly assigned into control, lidocaine, and ano6 siRNA + lidocaine groups. The expression level of ano6 in each group was detected, and flow cytometry was used to detect apoptosis. Results: The expression of ano6 in spinal cord neurons was up-regulated for lidocaine neurotoxicity and induced neurobehavioral changes in rats. Knockdown of ano6 could alleviate the apoptosis of neurons induced by lidocaine. Conclusions: Neuronal apoptosis induced by lidocaine neurotoxicity might be due to the upregulation of ano6 expression. Inhibi- tion of ano6 expression alleviated the apoptosis induced by lidocaine neurotoxicity. Keywords: Neurotoxicity, apoptosis, lidocaine, TMEM16F (ano6) Introduction rotoxicity in the position regulation of apoptotic process were detected and verified. Cell experi- Represented by lidocaine, local anesthetics ments were conducted to investigate the poten- (LAs) are widely used to induce regional and tial mechanism of lidocaine neurotoxicity. intraspinal anesthesia [1, 2]. The mechanism of LAs is mainly to block the voltage-gated Material and methods sodium channel of neurons reversibly, thereby blocking the transmission of nerve impulses Ethical assessment [3]. The complications caused by LA neuroto- In the present study, all the plans were exam- xicity, such as transient neurologic symptoms ined and approved by the Animal Research and and cauda equina syndrome, are rare, but usu- Ethics Committee of the First Affiliated Hospital ally with severe outcomes. The mechanism of of Xi’an Jiaotong University, Shaanxi Province. LA neurotoxicity has always been the focus of All technical processes were implemented fol- research, but the “overall view” is still unclear lowing the Principles of the Helsinki Declaration. [4]. Animals Previous reports have shown that lidocaine is highly neurotoxic among commonly used LAs The experimental animals were produced by [5-7]. Now, gene chip technology is widely used the experimental animal center of the Medical to screen a variety of differentially expressed College of Xi’an Jiaotong University. They includ- genes. Through this technology, the differen- ed 40 rats of 260-280 g, which were all male tially expressed genes caused by lidocaine neu- Sprague Dawley rats. The animals were fed at Lidocaine neurotoxicity and TMEM16F (ano6) room temperature (25 ± 1°C), the light-dark Gaopigeon Industry and Trade Co. Ltd., China) cycle was 12 h and the relative humidity was at a single dose. 40%-60%. The animals were given standard rat feed and water at will and kept individually. Neurobehavioral tests They were randomly split into four groups: con- trol group; sham group, received only intrathe- A researcher who did not aware of the experi- cal catheter insertion; saline group, saline was mental conditions fulfilled all neurobehavioral injected intrathecally; and lidocaine group, 10% tests in rats. The sensory and motor functions lidocaine was injected intrathecally (n = 10 per of rats in each group were measured using the group). thermal withdrawal latency (TWL) and the score of hindlimb motor dysfunction. A ther- Intrathecal catheter insertion mal stimulator (PL-200, Chengdu Taimeng Software Co. Ltd., China) was used to measure Reference to previous studies, intrathecal cath- TWL. Under a 3 mm thick glass plate was a eterization was executed [2]. In brief, the plexiglass box. After the rats were placed on injection of 10% chloral hydrate at the dose of the glass plate quietly, one third of the an- 300-350 g/kg intraperitoneally was used to terolateral part of the hind sole of the rats was anesthetize rats. After the intervertebral spa- irradiated with the thermal stimulator. TWL was ce of L4-L5 and ligamentum flavum were the time between the beginning of irradiation exposed, a heat-connected polyethylene cath- and the occurrence of leg-raising avoidance in eter was inserted 2 cm within the subara- rats. The longest time of irradiation was 30 s chnoid space. Hind leg twitching or tail swing to prevent tissue damage. TWL was measured indicated that the catheter was inserted, and five times at 5-min intervals, and the test the cerebrospinal fluid outflowing indicated its results of each rat were taken as the average successful insertion. Then, 10 µL of saline was value of five tests. The standard for evaluating used to flush the catheter. Before suturing the hindlimb motor dysfunction was as follows: incision, the end of catheter was sealed off hindlimbs could move independently, gait was and subcutaneously fastened. Once the rats steady and could bear weight, score 0; hin- were awake after anesthesia, their movement dlimbs could not bear weight, gait was not and clamped foot were monitored to detect steady, and when pressed or clamped, the any nerve injury caused by the intubation pro- hindlimbs had evasive reactions, score 1; cess. The rats with nerve injury were excluded. hindlimbs could not move independently and One day after catheterization, the rats in each when pressed or clamped, no evasive reaction group, except group C, were injected with 20 occurred in the hindlimbs, score 2. Neurobe- µL of 1% lidocaine intrathecally. The lidocaine havioral tests were performed in each group screening test was positive for the paralysis of before injection (T0) and 1 day (T1), 2 days (T2), both lower limbs within 30 s. The exclusion cri- 3 days (T3), and 4 days (T4) after the intrathe- teria were as follows: paralysis of limbs, dyski- cal injection. nesia, negative lidocaine test, or paralysis of unilateral limbs after a puncture. Three rats Tissue preparation were excluded and three others supple- mented. The fourth day after subarachnoid injection, neurobehavioral tests were performed on rats Intrathecal drug injection in each group. Then, the injection of 10% chlo- ral hydrate at the dose of 300-350 g/kg intra- Behavioral tests were conducted 1 day after peritoneally was used to anesthetize rats. The catheterization. Then, each rat in the saline spinal cords (lumbar enlargement) of all rats group was injected 20 μL of saline into the sub- from L2 to L6 were taken out and immediately arachnoid space. Each rat in the lidocaine stored in liquid nitrogen. group was injected with 20 μL of 10% lidocaine (L5647-15G, Sigma, MO, USA), diluted with Microarray analysis and hierarchical clustering saline, into the intrathecal space [2]. Sub- sequently, 10 µL of saline was used to flush the Parts of spinal cord tissues of three rats were catheter. The saline and lidocaine solutions randomly selected from both the control and were injected manually at a rate of about 10 lidocaine groups for microarray analysis. The µL/15 s through a microinjector (Shanghai TRIzol reagent (Life Technologies, CA, USA) 9410 Int J Clin Exp Med 2020;13(12):9409-9418 Lidocaine neurotoxicity and TMEM16F (ano6) and the RNeasy mini kit (Qiagen, CA, USA) were toma cell line SK-N-SH for this study. All cell successively used to extract and purify the lines were cultured in the Hyclone minimum total RNA of spinal cord tissues. According to essential medium, added penicillin and stre- the standard Affymetrix protocol, an Ambion ptomycin, each at a concentration of 50 μg/ WT Expression Kit was utilized to prepare the mL, containing 10% fetal bovine serum. All biotinylated cDNA from 150 ng total RNA. cells were cultured at 37°C in a moist environ- Fragmented cDNA was labeled and then hy- ment containing 5% carbon dioxide. bridized on the gene chip Affymetrix Clariom D rat array (Affymetrix) at 45°C for 16 h. After Ano6 inhibition in SK-N-SH that, the Affymetrix Fluidics Station 450 was chosen to wash and stain the gene chips. All Ano6 siRNA (sc-96071, Santa Cruz Biotech- chips were scanned with the Affymetrix Gene- nology, Inc., TX, USA) or negative control Chip Operating Software (GCOS) installed in a siRNA-A (sc-37007, Santa Cruz Biotechnology, GeneChip Scanner 3000 7G. Robust multichip Inc.) was transfected into SK-N-SH cells using analysis (RMA) algorithm was used for data Lipofectamine 2000 (11668019, Invitrogen, analysis, and methods of normalization were MA, USA) in the light of the manufacturer’s global scaling and Affymetrix default analysis protocol. Transfection occurred when the cell growth covered 80% of the bottom of the settings. The values were presented as log2 RMA signal intensity. Differentially expressed Petri dish (60 mm in diameter). After 48 h of genes between the two groups were discern- siRNA transfection, the knockdown efficiency ed basing on the results of the Student t test.
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