In vitro neurotoxicity by ropivacaine is reduced by silencing Cav3.3 T-type calcium subunits in neonatal rat sensory neurons

Abstract

Neurotoxicity of local anaesthetics has been alerted by more and more peoples. Cav3.1 and Cav3.2 T-type calcium channels were closely related with local anaesthetics toxicity. However, the role of Cav3.3, another subtype of the T-type calcium channel, on the neurotoxicity induced by local anaesthetics remains unclear. CaMKII_γ is a kind of multifunctional kinase and associated with a variety of physiological and pathological process. T-type calcium channel is closely related with CaMKII_γ. Up-regulation CaMKII_γ can increase T-type currents at the dorsal root ganglia (DRG). On the contrary, down-regulation results in the T-type currents decrease. Is the relation between Cav3.3 T-type channel calcium and CaMKII_γ involved with the ropivacaine hydrochloride neurotoxicity? In this study, we generated pAd-Cav3.3 and pAd-shRNA adenovirus vector to up-regulate and down-regulate Cav3.3 mRNA expression of the DRG. The cells treated or untreated with ropivacaine hydrochloride (3 mM) for 4 h were used to evaluate the neurotoxicity. Cell viability, cell death rate and apoptosis rate, Cav3.3 and CaMKII_γ expression were detected with MTT method, Hoechst–PI, flow cytometry, qRT-PCR and western blotting. Results showed that the cell viability of the DRG treated with ropivacaine hydrochloride markedly decreased, death rate and apoptosis rate, Cav3.3 and CaMKII_γ mRNA and protein expression significantly increased. Cav3.3 overexpression aggravated DRG injury induced by ropivacaine hydrochloride and inhibition of Cav3.3 expression improved the cell damages. Cav3.3 can regulate CaMKII_γ mRNA and protein expression. In conclusion, Cav3.3 regulated CaMKII_γ in DRG, which was involved with the cell injury induced by ropivacaine hydrochloride.

Keywords:

• Local anaesthetics
• neurotoxicity
• DRG
• Cav3.3
• ropivacaine hydrochloride

Introduction

With visualization technology development, regional block technique is becoming more and more precise. For example, ultrasound guided nerve block technique has been widely used in clinical anaesthesia and pain management [1–4]. Although local anaesthetics nano-formulated liposomal drug delivery systems technique was developed [5], the neurotoxicity of the local anaesthetics also has been concerned by people and it is often reported in clinic [6–9]. Ropivacaine hydrochloride is one of the amide local anaesthetics and widely used in clinical anaesthesia or pain management. However, there are some neurotoxicity cases of the ropivacaine hydrochloride reported in clinical anaesthesia [10–12]. Ropivacaine hydrochloride also resulted the cultured dorsal root ganglia (DRG) death and injury in vitro [13]. T-type calcium channel belongs to low voltage dependent calcium channel with rapid activation and slow inactivation characteristics [14–19]. It can be activated at resting potential, and be regarded as the pacemaker of the neuronal activity. T-type calcium channel is divided into Cav3.1, Cav3.2 and Cav3.3 subtype. It has been detected in small- and medium-size DRG [20]. In our previous study, we found that mibefradil, one kind of T-type calcium channel inhibitor, can improve the cells damages induced by the local anaesthetics [21]. We further tested that down-regulation Cav3.1 mRNA expression protected the SH-SY5Y cells damages induced by local anaesthetics [22]. Cav3.2 and Cav3.3 are the dominant subtype in DRG [23]. In this study, we generated pAd-Cav3.3 and pAd-shRNA adenovirus vector to up-regulation and down-regulation the Cav3.3 mRNA expression of the rat DRG. We investigated the Cav3.3 roles in the DRG damage induced by ropivacaine hydrochloride.

CaMKII is a kind of multifunctional kinase and associated with a variety of physiological and pathological process, It is widely distributed in the muscles, nervous and immune tissues and divided into four subtypes, alpha, beta, gamma, delta, respectively [24–30]. In our previous study, we found all the above four subtype of CaMKII can be detected at rat DRG [31]. T-type calcium channel is closely related with CaMKII_γ. Up-regulation CaMKII_γ can increase T-type currents at the DRG [32]. On the contrary, down-regulation results in the T-type currents decrease. So we supposed that Cav3.3 induced local anaesthetics injury by the regulation to CaMKII_γ. In this study, we investigated whether Cav3.3 calcium channel regulated CaMKII_γ in the DRG neurotoxicity induced by ropivacaine hydrochloride.

Materials and methods

Isolation of rat DRG cells

All animal procedures were in accordance with national and international animal care and ethical guidelines and were approved by the Institutional Animal Care and Usage Committee at the first people`s hospital of Foshan city, Guangdong province, China. The neonatal Sparague–Dawley rats, purchased from experimental animal centre of Guangdong province, China, were used to isolate the DRG. After anesthetized with sevoflurane, the rats were being executed. Isolated the spinal cord and DRG. Put the DRG into pre-cold PBS buffer and centrifuged at 1000 bpm for 2 min at 4 °C, discarded the supernatant, washed with PBS buffer and repeated two times. Added 0.125% trypsin (4 ml) to digest the DRG at 37 °C for 20 min. Terminated the digestion with 4 ml DMEM complete culture medium and centrifuged at 1000 bpm for 2 min at 4 °C, discarded the supernatant, washed with PBS buffer and repeated two times. Suspended the DRG with 2 ml Neurobasal medium (4.5 g/L containing d-glucose, 2 mmol/L L-, 1%FBS 20 ml/L B-27, glutamine additive, 10 μg/ml NGF penicillin 100 U/ml, streptomycin, 100 μg/ml). Filtered the DRG with 400 mesh stainless steel mesh filter and seeded into 24 well plates with 2–3 × 10⁵/ml cells density and incubated at 37 °C and 5% CO₂ for 48 h. The culture media with 5 mM cytosine arabinoside were used to inhibit non neuronal cell proliferation and incubated in 37 °C and 5% CO₂ for 96 h. Then changed the culture media without cytosine arabinoside every three days.

Generation of pAd-shRNA and pAd-Cav3.3 adenovirus vector

To investigate the role of Cav3.3 on ropivacaine hydrochloride neurotoxicity, we generated pAd-shRNA and pAd-Cav3.3 adenovirus vector to down-regulate and up-regulate Cav3.3 mRNA expression. According to the previous method [33–35], in brief, we designed one shRNA primer according to the Cav3.3 gene of the rats (NM_020084.3) in Genebank: 5′-TTTGGCCAGGAAGTTCAACAGTATTCAAGACGTACTGTTGAACTTCCTGGCTTTTTTG-3′; 5′-AGCTCAAAAAAGCCAGGAAGTTCAACAGTACGTCTTGAATACTGTTGAACTTCCTGGC-3′ (synthesized by Jinsirui Ltd., Nanjing, China). The shRNA primers were annealed to form double strand and connected with pYr-1.1 plasmid (Wuhan Biobuffer Biotech Service Co. Ltd., Wuhan, China) with ligase. The pYr-1.1-shRNA was recombined with pAd/PL-DEST adenovirus vector (Invitrogen Technology Co., Ltd., CA) to generate pAd-shRNA and transfected into the HEK 293 cells for virus amplification and infection of DRG. To generate pAd-Cav3.3 adenovirus vector, we synthesized the full size cDNA of the rats Cav3.3 (NM_020084.3) by Jinsirui Ltd., Nanjing, China and connected with pYr-adshuttle-4 vector (Wuhan Biobuffer Biotech Service Co. Ltd., Wuhan, China) to generate pYr-Cav3.3 plasmid. Then pYr-Cav3.3 plasmid was recombined with pAd/PL-DEST adenovirus vector to generate pAd-Cav3.3 adenovirus vector. pAd-Cav3.3 adenovirus vector was transfected to DRG and Cav3.3 were detected to evaluate the transfection effects.

Experimental protocol

There were four group in this study: the normal DRG (Normal group), the empty vector in DRG (Empty vector group), the pAd-shRNA adenovirus vector transfected into DRG (pAd-shRNA group) and the pAd-Cav3.3 adenovirus vector transfected into DRG (pAd-Cav3.3 group). The DRG in all above four group were treated or untreated with 3 mM ropivacaine hydrochloride for 4 h. Cell viability, cell death rate, cell apoptosis rate, Cav3.3 and CaMKII_γ were detected.

Cell viability detected with MTT method

The DRG were seeded into 96-well plates with 1 × 10⁵/ml density and incubated at 37 °C and 5% CO2 for two days. According to the experiment protocol, the DRG were treated or untreated with 3 mM ropivacaine hydrochloride for 4 h and changed new culture media. Added 5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-tetrazolium bromide (MTT; Beyotime Biotech Co. Ltd., Shanghai, China) 20 μl and incubated at the same conditions for 4 h. Discarded the culture media and added 150 μl DMSO into the cells. Detected the optical density at 570 nm and 630 nm absorbance wavelengths (OD₅₇₀ and OD₆₃₀) and calculated the difference between the two. The DRG in normal group untreated with ropivaciane hydrochloride was set as 100% and other groups were normalized to it.

Detection of cell deaths with Hoechst/PI

DRG isolated from the SD rats and cultured for 5 d were adapted to this detection of cell deaths with Hoechst/PI. In brief, the cultured DRG were seeded into one 12 well plate with 2 × 10⁵ density and were treated with 3 mM ropivacaine hydrochloride for 4 h. Discarded the media, added 1 ml PBS with 5 µg (1 µg/µl final concentration) Hoechst 33342 (Sigma-Aldrich, Inc. USA) and Propidium Iodide (PI; Sigma-Aldrich, Inc. MO) every well. After incubated for 15 min in 5% CO₂ incubator at 37 °C, the cells were detected under the fluorescence microscope (Leica, Heidelberg, German) and photographed. The red fluorescence positive cells were the dead cells and were calculated as a percentage of the total cells.

Cell apoptosis rate detected by flow cytometry

All the DRG were treated or untreated with 3 mM ropivacaine hydrochloride for 4 h. Cell apoptosis rates were detected by flow cytometry according to Annexin V apoptosis detection kit-APC (Affymetrix Inc., San Diego, CA, USA). In brief, the cells were digested and collected to centrifuge at 3000 bpm for 2 min. Discarded the supernatant, added 1 ml 1 × binding buffer to suspend the cells. Centrifuged at 3000 bpm for 2 min and discarded the supernatant, added 100 µl Annexin-5 antibody (1:20) and incubated at room temperature in dark place for 15 min. Then added 1 ml 1 × binding buffer to suspend the cells. Centrifuged at 3000 bpm for 2 min and discarded the supernatant, added 2% 7-A-A-D 10 μl and incubated at room temperature in dark place for 5 min. Added 190 μl 1 × binding buffer to suspend the cells, detected cell apoptosis rate with flow cytometry instrument.

qRT-PCR detection CaMKIIγ, Cav3.3 mRNA expression

After treated or untreated with 3 mM ropivacaine hydrochloride, the DRG were collected to detect CaMKIIγ, Cav3.3 mRNA. In brief, added 200 μl Trizol to lysis the cells for 5 min on ice. Five folds volume chloroform was added into the lysis cells and mixed. Centrifuged at 14,000 rpm and 4 °C for 15 min. Collected the upper layer RNA and added the same volume isopropanol to precipitate RNA for 10 min. Centrifuged at 14,000 rpm and 4 °C for 10 min. Washed with 70% ethyl alcohol and centrifuged at 1000 rpm and room temperature for 10 min. Discarded the supernatant and dried in the air for 15 min. Dissolved the total RNA with 30 μl nuclear enzyme free water. To generate the cDNA products from the total RNA, total RNA 5 μg, 2 mM dNTP 5 μl, 10pM Radom primer 1 μl and nuclear enzyme free water to 37 μl and incubated at 65 °C for 5 min. Added 5 × first stand buffer 10 μl, 0.1 M DTT 2 μl, mLv reverse transcriptase 1 μl (Bioscience Inc., USA) to the mixture and incubated at 42 °C for 1 h, 70 °C for 15 min. Mixed cDNA product 5 μl, 10pM β-actin, CaMK II γ, and Cav3.3 mRNA primer 0.5 μl (synthesized by Jinsirui Ltd., Nanjing, China, see Table 1) and 6 μl sybergreen (Bioscience Inc., USA) to PCR reaction. The reaction parameter was as following: set 95 °C 10 min one repeat, 95 °C 15 s, 60 °C 30 s, 95 °C 15 s 40 repeats. Collected the fluorescence signal and calculated the Ct value. 2^−ΔCt (Ct cycle threshold) was set as the amount of gene expression. ΔCt = [Ct (target gene)−Ct (beta-actin)], the normal group DRG mRNA expression was set as 1, other groups were normalized to the normal group.

Table 1. β-Actin, CaMKIIγ, Cav3.3 mRNA primer sequence.

Gene	Primer sequence	Product size (bp)
β-actin	F: 5′-CACGATGGAGGGGCCGGACTCATC-3′ R: 5′-TAAAGACCTCTATGCCAACACAGT-3′	240
Cav3.3	F: 5′-GACCAGCAGCCAGTGACGAA-3′ R: 5′-CACGACCACGCCCACAAACA-3′	108
CaMKIIγ	F: 5′-TTGCTGCTGGCGAGTAAATG-3′ R: 5′-TAGGGATCTTTCCTCAAGACCTCA-3′	149

Western blotting detection CaMKIIγ, Cav3.3 protein expression

After treated or untreated with ropivacaine hydrochloride, collected the cells to lysis with 200 μl pre-cold lysis solution and incubated for 30 min on ice. The cells were with ultrasonic grind and centrifuged at 12,000 rmp and 4 °C for 20 min. Took 20 μg protein in every group to the SDS electrophoresis. According to the position of the protein Marker, the target strip was cut and transferred to the PVDF membrane. Then closed the PVDF membrane at room temperature on shaking bed for 1 h. Washed and incubated in antibody (1:200) of CaMKII γ, Cav3.3 and β-actin (Sigma-Aldrich, Inc. MO) at 4 °C overnight. Next day, washed the film and incubated in the second antibody (1:1000) for 1 h. Washed and added the chemiluminescence reagent and incubated for 1 min, quickly wrapped and placed in a cassette and X-film attached to the exposure. Quantity one image analysis software was used to analyse the absorbance value of the target band, taking the absorbance value of β-actin band as the reference, and the ratio was the target protein expression level.

Statistical analysis

Data were expressed as mean ± SD. Factorial design was adopt for the statistical analysis. Two-way analysis of variance (two-way ANOVA) was used in comparison among groups, LSD method was used in multiple comparisons.

Results

Cell viability

The cells viability was detected with MTT method. After treated with ropivacaine hydrochloride, the viability of the cells in every group significantly decreased. It indicated that ropivacaine hydrochloride caused the DRG injury. The cell viability of the normal DRG treated with 3 mM ropivacaine hydrochloride for 4 h was 49%.The viability of the cells in the pAd-Cav3.3 group sharply reduced to 33%, however, the viability of the cells in the pAd-shRNA group was 65%, see Figure 1. Those results showed that Cav3.3 up-regulation caused the DRG cell damage and Cav3.3 down-regulation improved cell damage induced by the ropivacaine hydrochloride.

Figure 1. The cell viability of the cell [

%, n=8]. Cell viability in every group was normalized to the normal group. ^ap < .05 vs normal group, ^bp < .05 vs normal + R group.

Figure 2. The cell death rate detected with Hoechst–PI method [

%, n=8]. (A) The representative figure of the death rate in every group with Hoechst-PI. B: The results of the death rate in every group, ^ap < .05 vs normal group, ^bp < .05 vs normal + R group.

Cell death rate

After cell death and membranolysis, propidium iodide can enter into intracellular and the cell nuclear was marked in red. The cell death rate (death cell to the whole cell) in untreated with ropivacaine hydrochloride group was 4%. The death rate in Normal + R group, pAd-Cav3.3 group and pAd-shRNA group were 18%, 25% and 9.5%, respectively. Those data showed that Cav3.3 expression in DRG was involved with the cell death rate induced by ropivacaine hydrochloride (Figure 2).

Cell apoptosis rate

Ropivacaine hydrochloride resulted in the DRG cell apoptosis. The apoptosis rate of the cells in Normal + R group was 25%. The cell in pAd-Cav3.3 + R group, which Cav3.3 expression up-regulated, caused the cell apoptosis rate rise to 38%. However, the cell in pAd-shRNA + R group, which Cav3.3 expression down-regulated, had a lower apoptosis rate than those in Normal + R group, Figure 3. Those data showed that Cav3.3 expression up-regulation aggravated the DRG cell apoptosis and inhibition of Cav3.3 expression improved the DRG damage induced by the ropivacaine hydrochloride.

Figure 3. Cell apoptosis rate [

%, n=8]. (A) The represent figure of the cell apoptosis rate detected with flow cytometry. (B) The results of the apoptosis rate in every group, ^ap < .05 vs normal group, ^bp < .05 vs normal + R group.

Expression of Cav3.3 and CaMKIIγ mRNA

To up-regulate or down-regulate Cav3.3 expression, we generated pAd-Cav3.3 and pAd-shRNA adenovirus vector to infect DRG. qRT-PCR results indicated that Cav3.3 mRNA expression of the DRG in pAd-Cav3.3 group or pAd-shRNA group up-regulated or down-regulated. Compared with the cells in Normal group, Cav3.3 mRNA expression of the cell in pAd-Cav3.3 group increased 4.8 fold, and reduced to 0.21fold in pAd-shRNA group. Cav3.3 mRNA expression in those treated with ropivacaine hydrochloride significantly increased, It was 7.1 fold in pAd-Cav3.3 + R group and 0.5 fold in pAd-shRNA + R to those in Normal group, Figure 4.

Figure 4. The effects of ropivacaine hydrochloride on Cav3.3 mRNA expression [

%, n=6]. ^ap < .05 vs normal group, ^bp < .05 vs normal + R group.

To investigate the effects of Cav3.3 on CaMKIIγmRNA, We detected the CaMKIIγmRNA expression. Compared with Normal group, CaMKIIγmRNA expression of the DRG in pAd-Cav3.3 group increased and in pAd-Cav3.3 group decreased. As well as, the DRG treated with 3 mM ropivacaine hydrochloride for 4 h resulted in CaMKIIγmRNA expression up-regulation. It was 5.9 fold in pAd-Cav3.3 + R group and 0.6 fold in pAd-shRNA + R to those in Normal group, Figure 5.

Figure 5. The effects of ropivacaine hydrochloride on CaMKIIγmRNA expression [

% n=6]. ^ap < .05 vs normal group, ^bp < .05 vs normal + R group.

Expression of Cav3.3 and CaMKIIγ protein

We detected the Cav3.3 and CaMKIIγ protein with western blotting and testified that Cav3.3 protein expression up-regulated in pAd-Cav3.3 group and down-regulated in pAd-shRNA group. And the similar results were acquired in CaMKIIγ protein expression. Compared with the cells in untreated with ropivacaine hydrochloride group, Cav3.3 and CaMKIIγ protein expression of the cells in treated with ropivacaine hydrochloride group increased, Figure 6. Those data indicated that Cav3.3 protein expression was involved with the cell damage induced with ropivacaine hydrochloride and Cav3.3 regulated CaMKIIγ expression in the ropivacaine hydrochloride neurotoxicity.

Figure 6. The effects of ropivacaine hydrochloride on Cav3.3 and CaMKIIγ protein expression detected with western blotting [

% n=6]. (A) The present band detected with western blotting. Lane 1,7: normal group; Lane 2,8: empty vector group; Lane 3: pAd-Cav3.3 group; Lane 4,10: normal + R group; Lane 5,11: empty vector + R group; Lane 6: pAd-Cav3.3 + R group; Lane 9: pAd-shRNA group; Lane 12: pAd-shRNA + R group. (B) The statistical result of Cav3.3 protein. (C) The statistical result of CaMKIIγ protein. ^ap < .05 vs normal group, ^bp < .05 vs normal + R group.

Discussion

Ropivacaine hydrochloride is usually used clinic anaesthetics and pain management. The DRG treated with different concentration and exposure time of ropivaciane hydrochloride resulted in cell damage [31]. The cell viability of the DRG treated with 3 mM ropivacaine hydrochloride for 4 h reduced to 50%. The data showed that 3 mM ropivacaine hydrochloride caused significant DRG damage and did not result in a large number of the DRG cell death which effected the experiment results. So, in this study, we treated the cells with 3 mM ropivacaine hydrochloride for 4 h.

In this study, we found that Cav3.3 is closely related with the ropivacaine hydrochloride neurotoxicity. Cav3.3 mRNA and protein expression up-regulation aggravated DRG cells damage induced by ropivacaine hydrochloride and cell viability decreased, cell death rate and apoptosis rate increased. As well as, Cav3.3 regulated CaMKIIγ expression. CaMKIIγ is also involved with the neurotoxicity of the local anaesthetics [31,36].

T-type calcium channel – CaMKIIγ mutual regulation maybe an important mechanism of local anaesthetics neurotoxicity. Cav3.3 can regulated CaMKIIγ expression. Up-regulation Cav3.3 mRNA and protein expression increased CaMKIIγ mRNA and protein expression. As well as, down-regulation Cav3.3 expression also decreased CaMKIIγ expression. On the other hand, we found CaMKIIγ was also involved with ropivacaine hydrochloride neurotoxicity [31]. Inhibition of CaMKIIγ expression can improve DRG cell damage induced by ropivacaine hydrochloride. In fact, CaMKIIγ also can regulate Cav3.3 expression. Up-regulation or down-regulation CaMKIIγ can increase or decrease Cav3.3 expression [36]. CaMKIIγ depended on the intracellular calcium ion and T type calcium channel was one important factor regulating intracellular calcium ion [24,25,29]. At resting potential, extracellular Ca²⁺ was entered into the cells by T-type calcium channel, on the one hand, high voltage dependent calcium channel was active, on the other hand, activated intracellular calcium induced calcium release, intracellular Ca²⁺ concentration rose rapidly [15,30]. Intracellular Ca²⁺ concentration increased, calmodulin was in combination with calcium ion and formed Ca²⁺–CaM compound, activated CaMKIIγ, formed the Ca²⁺–CaM–CaMKIIγ signalling pathways, activated CaMKIIγ increased T-type calcium channel expression.

In brief, ropivaciane hydrochloride neurotoxicity maybe involved with Cav3.3 and CaMKIIγ, inhibition Cav3.3 and CaMKIIγ can improve the DRG damage induced by ropivaciane hydrochloride. However, the above results were from cell culture in vitro, they maybe some difference in vivo. In the future, experiment in vivo should be done to test the above results.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This study was supported by the National Natural Science Foundation of China (Nos. 81471278, 81100831) and the Medical Scientific Research Projects of Foshan City, Guangdong Province (No. 2014AB00275).