| Local anesthetics are commonly used for local anesthesia and pain therapy, but they have the potential neurotoxicity. With the wide application in nerve block anesthesia, reports increase gradually hat local anesthetics cause neurotoxicity, and clinical workers pay high attention to this phenomenon. Many research found that this neurotoxicity caused by local anesthetics cause was associated with cell apoptosis, but the exact mechanism is still not fully understood.Research found that the mitochondrial membrane function change, inner membrane potential disappeared and protease activation thing in the mitochondria released, and arouse all kinds of apoptosis related metabolic changes, then the apoptotic cells were induced to produce characteristic form change and DNA degradation. Burst of reactive oxygen species (ROS) is one of the main factors causing the nerve cells acute injury. ROS accumulation can affect oxidoreduction balance and damage the mitochondrial membrane in nerve cells, In addition, ROS can directly affect protein, lipid, and nucleic acid make them lose function. Moreover, ROS can also be a messenger, further cause nerve damage.Cell apoptosis pathways consist of two signal transduction pathways. One is the death receptor pathway. The death receptor of cell surface binds the death ligand by the ectodomain, introduces the death signal into cell, and activates caspase-8and caspase-3, leading to apoptosis. The other is mitochondria pathway. Stimulating factors, such as oxygen free radical and calcium overload, increase the permeability of mitochondria, decrease mitochondria membrane potential, release cytochrome C and apoptosis-inducing factor(AIF), and subsequently activate caspase-9and caspase-3, leading to apoptosis. Cell apoptosis is related to the opening of mitochondrial permeability transition pore(mPTP). The mPTP is composed of voltage dependent anion channel(VDAC), adenine nueleotide translocator(ANT) and cyclophilin D(Cyp-D). The influx of chloridion (Cl-) into mitochondria can occur through the open VDAC and Can cause mitochondfial membrane damage and collapse the membrane potential, leading to release of pro-apoptotic molecule such as cytochrome C and AIF. Accordingly, the increase in intracellular ROS induced by bupivacaine prompts the opening of VDAC, causing the influx of Cl-into mitochondria, increase of mitoehondrial osmotic pressure, swell of matrix, decrease of mitochondrial membrane potential and augmentation of permeability, and release of pro-apoptotic factors, which leads to cell apoptosis.P38mitogen-activated protein kinase (MAPK) is an important member of MAPK family. The p38MAPK signaling pathway is activated by different stimuli associated with stress, such as H2O2, TNF-a, lipopolysaccharide and hyperosmotic fluid. Previous studies demonstrated that the p38MAPK signaling pathway controls much gene expression activity of transcription factor, affects the production of many cytokines, and regulates the synthesis of NO and cytoskeleton protein. p38MAPK participates in immunological regulation, inflammatory reaction and cell apoptosis under the circumstance of stress. Studies found that peripheral nerve toxicity of lidocaine is related to the activation of p38MAPK. Lirk found that p38MAPK inhibitor4-(4-Fluorophenyl)-2-[4-(methylsulfinyl)phenyl]-5-(4-pyridyl)-1Hmidazole (SB203580) Can inhibit neurocyte axon degeneration.In previous studies, Our team found bupivacaine may interfere with oxidative phosphorylation and inhibit the complex I of the mitochondrial respiratory chain, decrease the production of ATP, lead to single phosphoric acid active adenosine protein kinase (AMP-activated protein kinase, AMPK) continuous activation, and cause the mitochondria create and release more ROS in the cells, then cause cell damage and apoptosis through the mechanisms of ROS-induced-ROS release, further study showed bupivacaine may induce nerve cell apoptosis by via p38MAPK and mitochondrial Cl-channel pathway.Levobupivacaine (LB) is a novel, long-acting amide local anesthetics. Due to similar physical and chemical properties and anesthetic potency to that of bupivacaine with less and lower toxicity in cardiovascular system and central nervous system, it shows a wide promising application potential in clinic and becomes a hot study project to investigate its mechanism in research field. Whether Levobupivacaine induced neurotoxicity is related to ROS production and mediation, or Levobupivacaine also induced neuronal apoptosis through activation of the p38MAPK pathway and mitochondrial Cl-channel, is still not clear,In this study, we compared the effects of SH-SY5Y cells producing ROS and apoptosis induced by different concentrations of levobupivacaine via cell biology and molecular biology methods at the cellular level. The chloride channel blocker (44’the-diisothiocyanatostilbene-2,2’-disulfonic acid disodium DIDS) and p38MAPK antagonist SB203580were used to explore whether the apoptosis triggered by levobupivacaine is related to the p38MAPK signaling pathway and mitochondrial chloride ion channel activation. The further observation focused on the effects of different concentrations of levobupivacaine to the ROS increment and apoptosis of rat spinal nerve cells at the whole animal level. Meanwhile, the SB203580and DIDS were also used to investigate whether the nerve cells apoptosis in L4-5spinal cord segment of rats triggered by levobupivacaine is related to the p38MAPK signaling pathway and mitochondrial chloride ion channel activation. By studying mitochondria and p38MAPK pathways on spinal nerve cells apoptosis induced by Levobupivacaine, our study might provide a theoretical basis to prevent from neurotoxicity induced by levobupivacaine or develop an effective pharmaceutical treatment to relieve it.Chapter1Levobupivacaine induce ROS generation and apoptosis in SH-SY5Y cellObjective To explore the relationship of ROS level and apoptosis induced by different concentrations of levobupivacaine in SH-SY5Y cellsMethod We culture SH SY5Y cell as the research object, treated by0,0.5,1,1.5,2,2.5mM levobupivacaine culture medium for1,2,3,4,5,6h, respectively, observe the cell morphology, detect the cell vitality with CCK-8method, Hochest33258detect the changes of apoptosis morphology, detect intracellular average ROS fluorescence intensity and cell apoptosis rate using flow cytometry instrument.Statistical the measurement data were record mean±standard deviation, SPSS13.0statistics software was used to analysis the date. Two factors factorial analysis of variance was used to analysiscellular ROS level. The cell vitality, cell apoptosis rate and the comparison between groups was analysis by single factor analysis of variance (one-way ANOVA) in single effect analysis, multiple comparisons was analysis using LSD method for the homogeneity of variance and Welch or Dunnett’s T3for the unequal variances were adopted when separate effect analysis as necessary, P<0.05for the difference was statistically significant.Results Flow cytometry results showed that the intracellular ROS level gradually increased and peaked at4h,decreased at5and6h after the cells treated by different concentration of levobupivacaine. Compared to untreated group, intracellular ROS leveln of the groups after levobupivacaine treatment1,2,3,4,5, and6h were higher,the difference was statistically significant(P<0.01).; detect the cell vitality after treated by different concentration of levobupivacaine after4h using CCK-8method, the cell viability present a dose dependent decrease cell survival, the cell survival rate was the lowest when the LB is2.5mM as (23.15±1.60)%, the results show statistical difference among the groups processed by different LB (F=2043.476, P=0.000); the apoptosis rate detected by flow cytometry instrument was statistically difference among each group(F=614.342, P=0.000), and the apoptosis rate was highest when LB was2mM; SH SY5Y cells was observed highly agglutinated and showed strong blue fluorescence after treated with different concentrations of LB using Hoechst33258dyeing, the number of cells present strong blue fluorescence nucleus was most especially in LB=2mM.Conclusion Levobupivacaine can cause SH-SY5Y cells ROS dependence apoptosis in SH-SY5Y cells, and the cells apoptosis rate was the highest when LB=2mM, levobupivacaine can also cause dose dependent cell survival rate decreased in SH-SY5Y cells.Chapter2Reserch on Levobupivacaine induces type SH-SY5Y apoptosis via Cl-of moyochondria and p38MAPK signal conduction pathway.Objective To discuss the effect on Cl-pathway of moyochondria and p38MAPK caused by increased ROS of SH-SY5Y which result from LB, revealing the functions of Cl-of moyochondria and p38MAPK signal conduction pathway in the process that LB induces type SH-SY5Y apoptosis.Methods SH-SY5Y cells randomly divided into four groups:DIDS group, SB203580group, DIDS+SB203580group and non-pretreated group. Cells of DIDS group, SB203580group and DIDS+SB203580group were pretreated respectively with50μmol/L DIDS,10μmol/L SB203580, and50μmol/L DIDS plus10μmol/L SB20358030min prior to the treatment with the culture medium containing2mmol/L levobupivacaine and cells of non-pretreated group treated with the culture medium containing2mmol/L levobupivacaine. After4hours levobupivacaine treatment, intracellular ROS level was measured by FCM, the mitochondrial membrane potential was measured by5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl benzirnidazole-carbocyanideiodine(JC-1), the p38and p-p38MAPK protein was examined by western blots, and mitochondrial Cl-concentration was measured by laser scanning con_focal microScope.4h after levobupivaeaine treatment, the cell viability was examined by MTT assay, and cell apoptosis was examined by FCM. Meanwhile, the same indicators were also be tested in the cells of each group untreated with levobupivacaine at the same time to be as contrast.Measurement data were presented as mean+standard deviation. SPSS13.0statistical software was used for analysis. Factorial design ANOVA was used to analyze the data from ROS measurement, Cl-fluorescence intensity mitochondrial membrane potential assay, cells survival rate assay, cell apoptosis rate assay, p38and p-p38MAPK protein expression. Comparisons among groups were performed by LSD (homogeneous variance)or Dunnett’S T3(heterogeneity of variance). A probability value of P<0.05was considered to be statistically significant.Results After levobupivacaine treatment, the ROS levels of DIDS group and DIDS+SB203580group were lower than that of non-pretreated group(<0.01). The results of fluorescence intensity measured by FCM showed that the ratios of mitochondrial membrane JC-1polymer/monomer in DIDS group and DIDS+SB203580group were0.82+0.01and0.79±0.01respectively, which were higher than that of non-pretreated group(0.52±0.01). The difference had statistical significance(P<0.01). Mitochondrial Cl-concentration measured by laser scanning confocal microscope showed that the Cl-concentration of DIDS group and DIDS+SB203580group were lower than that of non-pmtreated group(P<0.01). Western blot showed that the p-p38MAPK protein expression of DIDS group and DIDS+SB203580group were lower than that of non-pretreated group(P<0.01). CCK-8assay revealed that the survival rates of DIDS and SB203580groups were higher than that of non-pretreated group(P<0.01), but lower than that of DIDS+SB203580group (P<0.01). Cells apoptosis rates of group DIDS, DIDS+SB203580, SB203580and non-pretreated were23.42±1.58%,12.73±0.96%,24.53±0.93%and38.17±0.22%, which were measured by FCM after levobupivacaine treatment. The apoptosis rate of SB203580group was lower than that of non-pretreated group(P<0.01), but there were no distinct difference in ROS levels and the ratios of JC-1polymer/monomer fluorescence intensity between the SB203580group and non-pretreated group (P<0.05). Hoechst33258staining assay revealed that the apoptosis rates of group DIDS, DIDS+SB203580, SB203580and non-pretreated were22.88±1.13%,12.45±0.74%,24.68±0.85%and37.87±0.99%.Conclusion These findings indicate that the mitochondrial VDAC channel and activated p38MAPK pathway effected by levobupicavmne probably causes apoptosis. Increased intracellular ROS induced by Levobupivacaine opens mitochondrial VDAC channel and results in an influx of Cl-into mitochondria, which seems to be responsible for the mitochondrial depolarization, permeability enlargment of the mitochondria and the p38MAPK activation.Chapter3Effects of different concentrations of levobupivacaine on ROS increasement and cell apoptosis of spinal nerve in ratsObjective To compare the effects of the ROS increasement and cell apoptosis of spinal nerve in rats with different concentrations of levobupivacaine and to investigate the extent of spinal nerve injury.Methods After inserted intrathecal catheter, adult healthy male SD rats (weight220～250g) were divided into five groups (n=6):normal control group (C), the solvent group (D),0.5%levobupivacaine group (LB0.5),2.0%levobupivacaine the group (LB2.0) and5.0%levobupivacaine group (LB5.0) using a random number table. Group C, D, LB0.5, LB2.0and LB5.0were injected with saline, dimethyl sulfoxide,0.5%,2.0%,5.0%levobupivacaine20μl via intrathecal catheter respectively. Rats in each group were observed and recorded tail flick response latency (TFL) in the percentage of the maximum effect (MPE) and hind limb motor function score (MF) before intrathecal administration (basal state), after administration of lOmin,20min,30min,1h,2h,4h,8h,1d,2d,3d. After3d, rats were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation. The ROS level of nerve tissue was detected, and caspase-3and apoptosis of nerve cells were identified via immunohistochemical assay and TUNEL assay respectively.All of the data were analyzed using SPSS13.0statistical software and were expressed as mean±standard deviation (x±s). The data among the TFL groups before and after catheter were compared using one-way ANOVA, and paired t-test was used between before and after catheter. The MPE values were analyzed via repeated measures analysis of variance. Caspase-3-positive cell counts and apoptotic cell counts were compared via single-factor analysis of variance, and the Bonferroni method (homogeneity of variance) or Dunnett’s T3method (unequal variances) was used in multiple comparisons. Motor function scores were expressed with median (10,90bit point median percentile), and Kruskal-Wallis test was used among groups. P <0.05was considered statistically significant.Result1. Comparison of MPE among groups in rats after intrathecal injection shows MPE were different among groups (F=14220.696, P=0.000), and there was significant MPE change (F=5358.641, P=0.000) after injection with MPE positively correlating to time. Also, an interaction between the groups and the time was observed (F=1410.001, P=0.000). The results showed the differences among groups differed in time points. Further separate effects analysis found that there was no significant difference of MPE (P>0.05) between Group C and Group D at all time points. When compared within each group at different time points, MPE in Group LB0.5, Group LB2.0and Group LB5.0achieved their peak and downward in10min after the intrathecal injection. The MPE in Group LB0.5and Group LB2.0returned to baseline levels at4h and8h after injection, respectively, and Group LB5.0returned to baseline levels after2d.2. The comparison of MF scores showed no motor dysfunction in Group C and Group D after intrathecal injection. The motor function was significantly different after lOmin-2h intrathecal injection in each group according to the mean rank analysis. The MF scores were significantly increased among Group LB0.5, LB2.0and LB5.0than that of Group C and Group D. The MF score of Group LB0.5resumed in20min, LB2.0resumed in one hour MF, and LB5.0resumed in4h.3. Group C, D and LB0.5shows intact neuronal cell morphology and structure of spinal cord tissue in H&E. Group LB2.0shows varying degrees of damage with nuclei lyses in the neuronal structures of spinal cord tissue. Group LB5.0shows obvious spinal cord neuronal apoptosis with scattered apoptotic cells and apoptotic bodies.4. The comparison of ROS levels in each spinal cord tissues of rats shows there were no significant changes in Group C and Group D. The ROS levels in Groups LB0.5, LB2.0and LB5.0increased with the increase of drug concentration. The difference was statistically significant (P<0.01), with a ROS level of192.93±10.05in Group LB5.0.5. The Immunohistochemical positive cells counts of caspase-3shows no statistically significant among Group C, D and LB0.5in the spinal cord nerve tissue of rats. However, the caspase-3positive cells were significantly (P<0.01) increased in Group LB2.0and Group LB5.0compared with Group C.6. The TUNEL positive cells percentage detection shows no statistically significant among Group C, D and LB0.5in the spinal cord nerve tissue of rats. However, the TUNEL positive cells percentage was significantly (P<0.01) increased in Group LB2.0and Group LB5.0compared with Group C.Conclusion Levobupivacaine can cause a concentration-dependent ROS increase and induce apoptosis in the rat spinal cord nerve tissue, resulting in nerve damage and sensory dysfunction at a certain extent. The higher of concentration of Levobupivacaine, the more delayed sensory function recovery was observed.Chapter4Effects of SB203580and DIDS on spinal nerve cells apoptosis induced by levobupivacaine in ratObjective To investigate the effect of SB203580and DIDS on apoptosis in rat spinal nerve injured by levobupivacaine and to explore the related spinal neurotoxicity mechanism.Methods After inserted intrathecal catheter, adult healthy male SD rats (weight220～250g) were divided into four groups (n=6):Group LB, Group DIDS+LB, Group SB203580+LB, Group (DIDS+SB203580)+LB. Group LB were slowly intrathecal injected with20μl of0.9%saline via micro-syringe, and then slowly (10’) injected with20μl of5.0%levobupivacaine via micro-syringe after2hours. Group DIDS+LB were given14mg/kg of DIDS through the femoral vein via the program-controlled micro-pump [4ml/(Kg-h)] within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Group SB203580+LB were intrathecal injected with20μl of0.1%SB203580(dissolved in2%DMSO) within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Group (DIDS+SB203580)+LB were intrathecal injected with20μl of0.1%SB203580, then given14mg/kg of DIDS through the femoral vein via the program-controlled micro-pump within2hours, and then slowly injected with20μl of5.0%levobupivacaine. Six rats in each group were separated using a random number table and the sensory-motor function was observed. The tail flick response latency (TFL) were observed and recorded using the maximum percentage effect (MPE) and hind limb motor function score (MF) after30min,1h,2h,4h,8h,12h,16h, Id,2d,3d of intrathecal administration. After3d, rats were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation, immunohistochemical detection of caspase-3, caspase-9positive cells and TUNEL in situ detection of apoptosis. The other six rats in each group were perfused with paraformaldehyde, and L4-5spinal cord segments were cut for pathological observation, ELISAL detection of he ROS level of nerve tissue, and Western blot analysis of p38MAPK and p-p38MAPK protein expression.Using SPSS13.0software for statistical treatment, measurement data were expressed as mean±standard deviation. Comparisons among groups TFL were made by analysis of variance (ANOVA) and paired-sample T test. MPE were made by analysis of Repeated Measures. p-p38MAPK and p38MAPK expression, caspase-3〠caspase-9positive cell counts and apoptotic cell counts were made by analysis of variance (ANOVA). MF scores are presented as median (10th—90th percentiles) and were compared using the Kruskal-Wallis test followed by the Mann-Whitney u-test. P<0.05was considered significant.Result1. Comparison of MPE among groups in rats after intrathecal injection shows MPE were different among groups (F=143.324, P=0.000), and there was significant MPE change (F=130.948, P=0.000) after injection with MPE positively correlating to time. Also, an interaction between the groups and the time was observed (F=179.714, P=0.000). The results showed the differences among groups differed in time points. When compared within each group at different time points, MPE in Group LB, Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB achieved their peak in30min and downward after4-8h after the intrathecal injection. The MPE in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB recovered at12h-ld after injection, which is faster than that of Group LB. The recovery of Group (DIDS+SB203580)+LB is faster than that of Group DIDS+LB and Group SB203580+LB. all the groups returned to baseline levels after2d.2. The comparison of MF scores of rats showed there was no significant difference of the hind limb motor function recovery at each time point among each groups after intrathecal injection. The MF scores in different groups were recovered in4hours.3. Histopathological observation found that the neurons morphology and structural were damaged, cytoplasmic Nissl body decreased and cracked with nucleus lyses in Group LB. The neuronal structures in Group DIDS+LB and Group SB203580+LB show varying degrees of damage. The neuronal structures in Group (DIDS+SB203580)+LB were Less damaged; the cytoplasmic Nissl body morphology was still intact and the ratio of nucleus to cytoplasm was normal when compared with the first three groups.4. The comparison of ROS levels in each spinal cord tissues of rats shows no statistically significant differences (P>0.05) between Group DIDS+LB and Group (DIDS+SB203580)+LB. There were also no statistically significant differences (P>0.05) between Group SB203580+LB and Group LB. The ROS levels in Groups DIDS+LB and Group (DIDS+SB203580)+LB were less than that in Group SB203580+LB and Group LB, and the difference was statistically significant (P<0.05).5. The Caspase-3positive cell counts in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The Caspase-3positive cell counts in Group DIDS+LB, Group SB203580+LB were higher than that in Group (DIDS+SB203580)+LB, the difference was statistically significant (P<0.05). 6. The Caspase-9positive cell counts showed no significant difference (P>0.05) between Group SB203580+LB and Group LB. It was also no significant difference (P>0.05) between Group DIDS+LB and Group (DIDS+SB203580)+LB. The Caspase-9positive cell counts in Group DIDS+LB and Group (DIDS+SB203580)+LB were lower than that in Group SB203580+LB and Group LB (P<0.05).7. The p-p38MAPK expression level is significant different (F=234.117, P=0.000) among groups. The p-p38MAPK expression levels in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The p-p38MAPK expression levels in Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that of in Group DIDS+LB (P <0.05). The p38MAPK expression level showed no differences (F=0.936, P=0.442) among groups.8. The apoptosis rate in Group DIDS+LB, Group SB203580+LB and Group (DIDS+SB203580)+LB were lower than that in Group LB (P<0.05). The apoptosis rate in Group DIDS+LB, Group SB203580+LB were higher than that in Group (DIDS+SB203580)+LB, the difference was statistically significant (P<0.05).Conclusion Levobupivacaine may interfere with oxidative phosphorylation and inhibit mitochondrial respiratory chain complex I, which decreased the production of ATP and triggered explosive increasement of ROS in the rat spinal cord nerve cells. On the one hand, the direct activation of p38MAPK pathway can induce apoptosis; On the other hand the mitochondrial Cl-channel opening and mitochondrial membrane dysfunction may lead to the production and release of more ROS, which activate p38MAPK protein and result in apoptosis. Mitochondrial Damage may also be caused by the peeling of inner and outer membrane, which may release cytochrome C and apoptosis-inducing factors. Thus it may activate caspase-9and caspase-3to form an activated complex, and ultimately induce apoptosis. Therefore, the mechanism of neurotoxicity caused by levobupivacaine in the rat spinal cord may relate to mitochondrial Cl-channels and p38MAPK signal transduction pathway. |
PDF Full Text Request