Cav3.3 Down-Regulates CaMKII Beta to Ameliorate Neurotoxic Injury to the DRG Induced by Ropivacaine Hydrochloride

Background: The neurotoxicity of local anaesthetics is often reported. The present study aimed to investigate the relationship between Cav3.3 and CaMK (cid:0) beta in local anaesthetic neurotoxicity. Methods: An in vitro ropivacaine-induced model of neurotoxic injury to the dorsal root ganglion neurons was used. After specically inhibiting the expression of Cav3.3 mRNA in DRG neurons by RNAi, cell viability, CaMK (cid:0) beta expression and the intracellular calcium ion level were detected in DRG neurons. Results: The results showed that the expression of CaMK (cid:0) beta decreased after the expression of Cav3.3 was inhibited. At the same time, in a model of ropivacaine hydrochloride-induced neurotoxic injury, inhibiting the expression of Cav3.3 mRNA improved cell viability and reduced the intracellular calcium level. Conclusions: These results suggest that Cav3.3 may be involved in neurotoxic injury induced by local anaesthetics by regulating the expression of CaMK (cid:0) beta. Both Cav3.3 and CaMK (cid:0) beta are therapeutic targets for neuronal injury induced by local anaesthetics. local

CaMK gamma in neurons. In contrast, the expression of CaMK gamma decreases after inhibition of Cav3.3 expression (8).
CaMK beta is widely distributed in neurons, is related to cerebral ischaemia-reperfusion injury, and participates in the processes of learning and memory (12)(13)(14). A previous study indicated that Cav3. 3 and CaMK beta are involved in local anaesthetic neurotoxicity (7,11). Additionally, CaMK can regulate the Ttype calcium current density (10,15). Therefore, we speculate that there may be some connection between Cav3.3 and CaMK beta in the neurotoxicity of local anaesthetics. The effect of Cav3.3 expression on CaMK beta remains unclear.
In this study, we constructed a pAd-shRNA-Cav3.3-DRG plasmid to investigate the effects of Cav3.3 inhibition on CaMK II beta in local anaesthetic neurotoxicity. We evaluated cell viability, the cell apoptosis rate, Cav3.3 and CaMK II beta expression and calcium ion levels.

Isolation of DRG neurons
The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies (16). The protocols for animal use were approved by the ethics committee of the A liated Foshan Hospital of the Southern Medical University. After 1-3 days of acclimatization and sevo urane (2% vol) inhalation anesthesia, their spinal cords and ganglia of the rats were exposed rapidly at 4°C, and the ganglia were separated. After digestion with 0.125% trypsin for 20 min, the samples were centrifuged at 2000 rpm for 2 min, and the supernatant was discarded.
Neurobasal medium (containing 4.5 g/L D-glucose, 2 mmol L-glutamine, 1% FBS, 20 ml/L B-27 supplement, 10 µg/ml NGF, 100 U/ml penicillin, and 100 µg/ml streptomycin) was added to the separated DRG samples. The samples were ltrated with 400 mesh stainless steel mesh, seeded into a cell culture plate and incubated at 37°C and 5% CO 2 . Cytarabine (5 mM nal concentration) was added to inhibit the proliferation of nonneuronal cells. The cell density was seeded with 2× 10 5 / L. After 96 h, the medium was replaced with cytarabine-free culture solution, and the medium solution was changed once every 3 days.
To construct pAd-shRNA-Cav3.3-DRG neurons, the pAd-Cav3.3-shRNA adenovirus (DNA:lipid=1:2) was transfected into HEK 293 cells with the Lipo 2000 system to amplify the virus, which was then used to infect the DRG neurons. The expression of Cav3.3 in the pAd-shRNA-Cav3.3-DRG neurons was identi ed by real-time PCR and western blotting. Additionally, we constructed empty vector DRG cells as the control group.

Experimental protocol
There were six groups in this study. In the normal group, DRG cells were cultured with normal medium; in the vector group, empty vector DRG cells were treated with normal medium; in the pAd-shRNA-Cav3.3-DRG group, pAd-shRNA-Cav3.3-DRG cells were treated with normal medium; in the normal +R group, DRG cells were cultured with 3 mM ropivacaine hydrochloride for 4 h; in the vector+R group, empty vector DRG cells were treated with 3 mM ropivacaine hydrochloride for 4 h; in the pAd-shRNA-Cav3.3-DRG +R group, the pAd-shRNA-Cav3.3-DRG cells were treated with 3 mM ropivacaine hydrochloride for 4 h. Cell viability, cellular ultrastructure changes, the expression of Cav3.3 and CaMK beta, and the intracellular calcium ion level were detected. The concentration (3 mM) and exposure time (4 h) to ropivacaine hydrochloride were based on a previous study (7,11). The titration pH value of the cell culture medium in each group was 4.0-6.0, which was consistent with that of ropivacaine hydrochloride.

Cell viability as detected by an MTT assay
Cell viability was detected using an MTT assay. In brief, after treatment with or without ropivacaine hydrochloride, the cells from each group were collected, MTT solution (5 mg/ml) was added and the samples were incubated for 4 h. The supernatant was discarded, and DMSO was added to the samples.
After the purple crystals dissolved, the absorbance at 570 and 630 nm (OD570 and OD630) was measured. The ratio of OD570 to OD630 of the cells in the normal group was regarded as 100%. The difference in the OD570/OD630 ratio between the cells in the normal group and other groups represented the cell viability.

Cellular ultrastructure changes observed by electron microscopy
Cells from each group were collected and xed at 4°C. Then, the cells were dehydrated with 50%, 70% and 90% acetone overnight at room temperature with an embedding agent. The cell mass was moved into the centre of the bottom of the capsule for embedding. Ultrathin slices of 50-70 nm were cut with an ultrathin microtome. The slices were stored in a drying vessel for dyeing and cleaned with sodium acetate and lead citrate. After dyeing, the slices were dried, and the mitochondrial structure of the neurons was observed under a transmission electron microscope (Hitchi, HT7700, Japan). mRNA detected by real-time PCR After treatment with or without ropivacaine hydrochloride, the cells were collected to detect Cav3.3 and CaMK beta mRNA expression. In brief, total RNA was isolated by the TRIzol method. After lysis, isolation, and washing, the samples were air-dried, and 30 μl of nuclease-free water was used to dissolve the RNA.
The reaction for generating cDNA contained 5 µg of total RNA, 5 μl of 2 mM dNTPs, 1 μl of random primer 1, and DEPC H 2 O was added to reach a nal volume of 37 μl. The mixture was incubated at 65°C for 5 min. Then, 10 μl of 5× rst strand buffer, 2 μl of 0.1 M DTT, and 1 μl of MLV reverse transcriptase was added to the reaction. The total volume was 50 μl, and the mixture was incubated at 42°C for 1 h and then at 70°C for 15 min. The product was cDNA.
The PCR mixture included 5 µl of cDNA, 6 μl of SYBRGreen, and 1 μl of 10 pmol/l CaMK beta and Cav3.3 mRNA forward-and-reverse primers ( The cells were inoculated on coverslips in 12-well plates. After treatment with or without ropivacaine hydrochloride, cells from each group were xed with 4% polyformaldehyde for 15 min and washed 3 times with PBS. Normal goat serum was added to the slides at room temperature for 30 min. Cav3.3 and CaMK beta antibodies (1:100) were added, and the slides were incubated at 4°C overnight. A uorescentlabelled (Cy3) sheep anti-rabbit IgG (1:100) antibody was added to the slides, and the slides were incubated at 20-37°C for 1 h and then incubated with DAPI for 5 min. The coverslips were sealed with an anti uorescence quenching agent, and uorescence images were collected with a uorescence microscope. Image analysis was carried out with ImageJ 5.0 software. The ratio of the integrated density to the visual eld area was calculated as the mean integrated density. The samples from each group were measured three times.

Detection of intracellular calcium ion level by laser confocal microscopy
The cells were incubated in 12-well migration plates. After treatment, the cells from each group were washed with PBS and incubated in a 5% CO 2 incubator at 37°C for 30 min with Rhod-2 AM ( nal concentration, 5 μM). The cells were washed twice with PBS and observed under a laser scanning confocal microscopy (Leica SP8 STED). The uorescence intensity (excitation wavelength of 549 nm and emission wavelength of 578 nm) was recorded. ImageJ 5.0 software was used for image analysis. The total uorescence intensity of all cells in the eld of vision was analysed. The total uorescence intensity/the total cell area was regarded as the average optical density.

Statistical analysis
The data are expressed as the Mean±SD. A factorial design was adopted for the statistical analysis. Normality and variance homogeneity were determined with SPSS17.0. For the normality and variance homogeneity data, one-way analysis of variance (one-way ANOVA) was used for comparisons among groups, and the LSD method was used for multiple comparisons.

Cell viability
There was no difference in the cell viability among the cells from the normal, vector and pAd-shRNA-Cav3.3-DRG groups. However, compared with that of the cells from the normal group, the cell viability of the cells from the normal +R group, the vector +R group and the pAd-shRNA-Cav3.3-DRG+R group were decreased.As well as, compared with that of the cells from the normal +R group, the cell viability of the cells from the pAd-shRNA-Cav3.3-DRG+R group was increased. These data suggest that the inhibition of Cav3.3 expression improved cell viability, as shown in Figure 1.

Cellular ultrastructure changes
A large number of nerve laments were observed in the normal group cells by electron microscopy, \as well as,the shape of the mitochondria was regular, and the structure of the mitochondria was complete. There were no signi cant differences between the cells from the vector group and the pAd-shRNA-Cav3.3-DRG group.
However, after treated with ropivacaine hydrochloride, the mitochondria of the cells from the normal +R group, the vector +R group and the pAd-shRNA-Cav3.3-DRG+R group, , were destroyed and vacuolated, the mitochondrial ridge was incomplete or absentand bilateral membranes were observed in the organelles. Additionally, cytophagosomes were present. However, it is gratifying that those changes of the cells from pAd-shRNA-Cav3.3-DRG+R group, inhibiting the expression of Cav3.3 mRNA, is signi cant smaller than those from the normal +R group and the vector +R group (Figure 2).

CaMK beta
Compared with the cells from the normal group, the CaMK beta protein expression in the cells from the vector group was not different, but the CaMK beta protein expression in the cells from the pAd-shRNA-Cav3.3-DRG group was downregulated. After treatment with ropivacaine hydrochloride, CaMK beta protein expression was upregulated. There was no difference between the cells from the normal+R group and the vector+R group. Additionally, CaMK beta protein expression in the pAd-shRNA-Cav3.3-DRG+R group was downregulated ( Figure 5).
Calcium ion level, as detected by laser confocal microscopy Compared with the cells from the normal group, there was no difference in intracellular calcium levels in the cells from the vector group and the pAd-shRNA-Cav3.3-DRG group. After ropivacaine hydrochloride treatment, the intracellular calcium level of the cells increased. Compared with the normal+R group, the intracellular calcium ion level in the vector+R group was not different, while that in the pAd-shRNA-Cav3.3-DRG+R group was decreased ( Figure 6).

Discussion
T-type calcium channels are involved in the neurotoxic injury induced by local anaesthetics (7,17,18). Extracellular calcium ions enter into cells through low-voltage-dependent calcium channels (T-type calcium channels) and induce the opening of high-voltage-dependent calcium channels and liganddependent calcium channels (19)(20)(21). Additionally, extracellular calcium ion in ux increases intracellular calcium ion levels and causes cell injury. Cav3.2 and Cav3.3 calcium channels are mainly distributed in dorsal root ganglion cells (22). Both are involved in the spinal neurotoxicity of local anaesthetics. Inhibiting the expression of Cav3.2 and Cav3.3 calcium channels can alleviate neurotoxic injury induced by local anaesthetics (7). The mutual regulation of CaMK gamma and Cav3.3 participates in the neurotoxic damage induced by local anaesthetics (8). The calcium ion level increase induced by their positive regulation is one of the mechanisms by which neurotoxic injury is induced by local anaesthetics (23).
CaMK beta, a subtype of CaMK , is widely distributed in the central nervous system and is related to cerebral ischaemia-reperfusion injury, learning and the formation of memories (24)(25)(26)(27). Ropivacaine hydrochloride can upregulate the expression of CaMK beta in the spinal cord of rats in a manner that is related to the concentration of and exposure time to ropivacaine hydrochloride (11). As well as, our previous study show that inhibition of CaMK beta expression can down-regulate Cav3.3 expression and reduce the neurotoxicity induce by ropivacaine hydrochloride(28).This study showed that inhibiting the expression of Cav3.3 mRNA in the spinal cord results in downregulation of the expression of CaMK beta, which can alleviate neurotoxicity injury induced by ropivacaine hydrochloride. The mechanism may be related to the decrease of extracellular calcium in ux and the decrease of CaMK beta activity induced by the Cav3.3 mRNA downregulation. The cells exhibited an increase in cell viability and an intracellular calcium ion level decrease. These results suggested that Cav3.3 and CaMK beta are involved in the neurotoxic injury induced by ropivacaine hydrochloride. The CaV3.3 -Ca2 +/CaMK beta pathway may be one of the important mechanisms regulating the neurotoxicity induced by ropivacaine hydrochloride.
After the expression of Cav3.3 mRNA was reduced, calcium ions entered the cells through fewer Cav3.3 channels. Thus, the intracellular calcium ion level was reduced. Therefore, calcium-dependent calcium release mediated by Ca2+/CaMK beta was inhibited, which reduced the intracellular calcium overload and alleviated the neuronal damage caused by local anaesthetics.
On the other hand, we found that ropivacaine hydrochloride can cause mitochondrial damage in DRG cells. Mitochondrial membrane injury inhibits oxidative phosphorylation and reduces ATP production, which causes a shortage of energy supply for Ca 2 + -ATPases and promotes Ca 2+ overload (29). An insu cient energy supply for Na + -ATPases in the cell membrane also signi cantly increases the intracellular sodium ion concentration, which activates the Na + -Ca 2 + exchange proteins and increases the Ca 2+ in ux (30).In fact, our previous study show that that Cav3.3 is also closely related to CaMK gama in the neurotoxicity of ropivacaine hydrochloride, and inhibition of Cav3.3 can down regulate the expression of CaMK gama to reduce the neurotoxicity(31).
This study found a close relationship between Cav3.3 and CaMK beta in local anaesthetic neurotoxicity, but there were also some limitations. This study only explored the effect of the downregulation of Cav3.3 on CaMK beta in neurotoxic injury induced by local anaesthetics. The effects of the upregulation of CaMK beta needs to be further studied.

Conclusion
In this study, we observed the effects of the inhibition of Cav3.3 mRNA expression on CaMK beta in a model of local anaesthetic neurotoxicity. The data showed that the inhibition of Cav3.3 resulted in a decrease in CaMK beta expression and alleviated the injury induced by ropivacaine hydrochloride. On the contrary, inhibition of CaMK beta expression can down regulate Cav3.3 expression and reduce nerve injury induced by ropivacaine hydrochloride. The interaction between Cav3.3 and CaMK beta is involved in the neurotoxicity of ropivacaine hydrochloride.

Declarations
Ethics approval and consent to participate The study was approved by the ethics committee of the Second People`s Hospital of Foshan City. Availability of data and material