| In recent years, the ratio of obstetric anesthesiologists applying intrathecal anesthesia for cesarean section anesthesia, labor analgesia and postoperative analgesia increased year by year at home and abroad. But the clinical doctors discovered that some maternal had occur the neurological complications after spinal anesthesia. The data from abroad reported that the incidence of neurological complications was about 0.01%-0.05%in the general population after spinal anesthesia, while that was 0.58%-0.92%in the mother. According to available data analysis reports, that was caused by:①anesthesia-related factors;②Obstetric-related factors;③Oher factors. However, that is determined by a particular factor, or interaction of multiple factors the result is still unknown. Particularly Anesthesiology and Pain Management in the field all focus on whether the change in sensitivity and neurotoxicity of local anesthetics during pregnancy.The research on sensitivity and neurotoxicity of local anesthetics during pregnancy was much controversy. According to literature reported, nerve electrophysiology studies suggested that the sensitivity and neurotoxicity of local anesthetics increased during pregnancy, so that reduced the requirements for such drugs. But a few researchers had raised objections, who thought pregnancy-related changes in diffusion barriers and activation of endogenous analgesic systems without changes in the electrophysiologic properties of spinal root axons. The subsequent investigation found that the transient neurological syndrome incidence of maternal was different (0.015%-8.8%). The incidence of TNS associated with a variety of factors. If excluded other factors, the incidence of TNS in mother is not higher than those of non-pregnancy. Because there is no adequate randomized controlled study of patients or animals, and the large sample surveys on two different concepts, we need the further researches.Recently, local anesthetics had been reported to trigger apoptosis, although the underlying mechanisms remain unknown. Apoptosis is largely controlled by a family of aspartatespecific cysteine proteases, called caspases, which are initiators and executioners of the apoptotic process. Caspases are activated by two major signaling routes, the extrinsic death receptor and the intrinsic mitochondrial pathway, which both depend on the formation of large multiprotein complexes. Initiator caspase-8 is the key mediator of the extrinsic pathway. The intrinsic pathway, in contrast, is regulated at mitochondria, which release cytochrome c and other proapoptotic factors during different forms of cellular stress. In the cytosol, cytochrome c together with caspase-9 induces the formation of the apoptosome, thereby triggering the caspase cascade and subsequent apoptosis.It is important for maternal to control the occurrence of non-obstetric neurological complications. The research about prevention and control of local anesthetic neurotoxicity had not achieved the exact results. Astragalus polysaccharide is one of the main active ingredients of the traditional Chinese medicine Astragalus, which can not only eliminate the radical, resist lipid peroxidation and reduce the apoptosis of in nerve cells, but activate cellular immunity, increase the level of IL-1βto promote the spinal nerve injury. Extract from rabbit skin inflamed by vaccinia virus for injection is a nonprotein extract from inflamed rabbit skin inoculated with vaccinia virus. It has widely been used in clinical settings as an analgesic drug. It has effects of analgesic, immune regulation, improve autonomic nerve function, promote axonal growth, which can also decrease intracellular ROS and inhibit apoptotic cell death. APS and Extract from rabbit skin inflamed by vaccinia virus for injection can reduce the toxicity of bupivacaine for spinal nerve damage remains to be further examination.This study produced a model of intrathecal bupivacaine in pregnant rats. The objective is to investigate the sensitivity and neurotoxicity of local anesthetics during pregnancy, the apoptosis signal transduction pathway of intrathecal bupivacaine and the protective effect of pretreatment with APS and extract from rabbit skin inflamed by vaccinia virus for injection on the neurotoxicity of intrathecal bupivacaine in pregnant rats.Part 1 Effect of intrathecal bupivacaine on sensor motor function and on expression of caspase-9 and caspase-8 in dorsal root ganglion in pregnant rats.Objective To investigate the effect of intrathecal (IT) bupivacaine on sensor motor function and on expression of caspase-9 and caspase-8 in dorsal root ganglion (DRG) in pregnant rats or nonpregnant rats.Methods Eighteen female SD rats weighting 200-220g and eighteen late pregnant rats weighting 350-400g were used in this study. All the rats in which PE-10 catheter were successfully placed without complication were divided into 6 groups randomly (n=6 each). Normal pregnant control group (group PN),2% bupivacaine group (group P2B) and 4%bupivacaine group (group P4B); Normal nonpregnant-control group (group N),2%bupivacaine group (group 2B) and 4% bupivacaine group (group 4B). Normal group:received normal saline 30μl IT, others four groups:received 2%or 4%bupivacaine 30μl IT. Tail-flick test and motor function were performed at 10min,20min,30min, 1h,2h,4h, 1d,2d,3d and 4d after IT administration. TF latencies were converted to the percent of maximal possible effect (MPE). Four days later, the animals were sacrificed and the DRG were removed for determination of caspase-9 and caspase-8 protein expression and mRNA using western blotting and RT-PCR. Using SPSS 13.0 software 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. Caspase-8, 9 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<0.05 was considered significant.Results (1) The impact on the MPE:MPE were difference among the groups in rats, which had the significant difference with time after IT, and which had the interaction between group and time. MPE in group P2B and P4B were significantly higher compared with those in group 2B and 4B after IT 2h,4 h, and 1d. After the IT 10min,2B,4B, P2B, and P4B group MPE reached the peak. MPE in group 2B and 4B respectively return the baseline values after IT 4h and 1d, in while group P2B and P4B return the baseline values after IT 2d. (2) The impact on the MF:MF scores were significantly different after IT 10min-4h in rats, Group P2B and P4B motor block time was longer than group 2B and 4B. Group 2B and 4B return to normal after IT 2h, then group P2B and P4B respectively return normal after IT 4h and Id. (3) The impact on the caspase-8 and caspase-9 protein and mRNA:Rats in group 2B,4B, P2B and P4B showed significantly increase the expression of caspase-9 protein and mRNA in DRG compared with those in group N and PN. No significant difference on the expression of caspase-8 protein and mRNA in DRG in all groups.Conclusion Intrathecal injection of the same concentration and dosage of bupivacaine, pregnant rats sensory, motor nerve block lasted significantly longer than nonpregnant rats, indicating that the sensitivity of bupivacaine increased in pregnancy rats. Bupivacaine induces apoptosis via the mitochondrial pathway activating caspase-9 gene in pregnancy rat DRG cells.Part 2 Effect of repeated intrathecal bupivacaine on sensor motor function and apoptosis in dorsal root ganglion in pregnant rats.Objective To investigate the effect of repeated intrathecal bupivacaine on sensor motor function and apoptosis in dorsal root ganglion (DRG) in pregnant rats.Methods Twelve female SD rats weighting 200-220g and twelve late pregnant rats weighting 350-400g were used in this study. All the rats in which PE-10 catheter were successfully placed without complication were divided into 4 groups randomly (n=6 each). Normal pregnant control group (group PN),2% bupivacaine group (group P2B); Normal nonpregnant-control group (group N),2% bupivacaine group (group 2B). Group P2B and 2B:respectively received 2% bupivacaine 30μl,20μl and 10μl, a total of three times, each time interval of 1h. Normal group:received normal saline. Tail-flick test and motor function were performed at 1d,2d,3d,4d and 5d after the last IT administration. TF latencies were converted to the percent of maximal possible effect (MPE). Five days later, the DRG were cut and used for HE staining and TUNEL for apoptosis and immunohistochemical analysis for caspase-9 protein. Using SPSS 13.0 software 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. Comparisons among groups MPE were made by Repeated Measures. The number of apoptotic cells and caspase-9 protein were made by analysis of variance (ANOVA). P<0.05 was considered significant.Results (1) The impact on the MPE:MPE were different among the groups in rats, which had the significant difference with time after IT, after the IT 2d 2B group MPE reached the peak, then dropped, however P2B group MPE sustained increased. There were no interaction between group and time. (2) The impact on the MF:MF scores were 0 in all the rats. (3) HE stain:Compared with the group 2B. group P2B increased neuronal degeneration, ischemia, cellular necrosis and interstitial edema. (4) The number of apoptotic positive cells:the number of apoptotic positive cells in group 2B and P2B was significantly higher compared with those in group N and PN. (5)The number of caspase-9 positive cells:the number of caspase-9 positive cells in group 2B and P2B was significantly higher compared with those in group N and PN.Conclusion The neurotoxicity of bupivacaine increased in pregnancy rats. Repeated intrathecal injection of the same concentration and dosage of bupivacaine, pregnant rats appeared sense of dysfunction and nerve cell damage, but motor function were not affected. Bupivacaine induces apoptosis via the mitochondrial pathway activating caspase-9 gene in pregnancy rat DRG cells. DRG cell apoptosis and caspase-9 gene activation are closely linked.Part 3 Effect of pretreatment with astragalus polysaccharides on neurotoxicity of intrathecal bupivacaine in pregnant rats.Objective To investigate the effect of pretreatment with APS on neurotoxicity of intrathecal bupivacaine in pregnant rats.Methods Thirty late pregnant rats weighting 350-400g were used in this study. All the rats in which PE-10 catheter were successfully placed without complication were divided into 5 groups randomly (n=6 each). Normal pregnant control group (group N),2%bupivacaine group (group 2B) and 2%bupivacaine APS pretreatment group (group 2H), repeated 2%bupivacaine group (group R2B) and repeated 2%bupivacaine APS pretreatment group (group R2H). Group N: received normal saline 30μl IT and intraperitoneal injection with normal saline 2ml. Group 2B:received 2%bupivacaine 30μl IT and intraperitoneal injection with normal saline. Group 2H:received 2%bupivacaine 30μl IT and intraperitoneal injection with APS. Group R2B:respectively received 2%bupivacaine 30μl,20μl and lOμl, a total of three times, each time interval of 1h and intraperitoneal injection with normal saline. Group R2H:respectively received 2%bupivacaine 30μl,20μl and 10μl, a total of three times, each time interval of 1h and intraperitoneal injection with APS. APS administration methods:25mg/kg, normal saline diluted to 2ml, in a continuous intraperitoneal injection from intrathecal administration 3 days ago to 4 days after, once a day. Tail-flick test and motor function were performed at 1 d,2d,3d,4d and 5d after the last IT administration. TF latencies were converted to the percent of maximal possible effect (MPE). Five days later, the DRG were cut and used for HE staining and TUNEL for apoptosis and immunohistochemical analysis for caspase-9 protein. Using SPSS 13.0 software 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. Comparisons among groups MPE were made by Repeated Measures. The number of apoptotic cells and caspase-9 protein were made by analysis of variance (ANOVA). P<0.05 was considered significant.Results (1) The impact on the MPE:MPE were different among the groups in rats, which had the significant difference with time after IT, and which had the interaction between group and time. At the same time point, the MPE of the groups were significantly different, group R2B had a higher MPE. After the IT 1d group 2B, 2H and R2H MPE reached the peak, then dropped, however R2B group MPE sustained increased. (2) The impact on the MF:MF scores were 0 in all the rats. (3) HE stain:Compared with the group 2B and R2H, group R2B increased neuronal degeneration, ischemia, cellular necrosis and interstitial edema. (4) The number of apoptotic positive cells:the number of apoptotic positive cells in group R2B was significantly higher compared with those in group 2B,2H and R2H. (5)The number of caspase-9 positive cells:the number of caspase-9 positive cells in group R2B was significantly higher compared with those in group 2B,2H and R2H.Conclusion Pretreatment with APS can reduce the neurotoxicity of intrathecal bupivacaine in rats, which is related with down-regulate caspase-9 expression.Part 4 Effect of pretreatment with extract from rabbit skin inflamed by vaccinia virus for injection on neurotoxicity of intrathecal bupivacaine in pregnant rats.Objective To investigate the effect of pretreatment with extract from rabbit skin inflamed by vaccinia virus for injection on neurotoxicity of intrathecal bupivacaine in pregnant rats.Methods Thirty late pregnant rats weighting 350--400g were used in this study. All the rats in which PE-10 catheter were successfully placed without complication were divided into 3 groups randomly (n=6 each). Normal pregnant control group (group N), repeated 2%bupivacaine group (group R2B) and repeated 2%bupivacaine AGC pretreatment group (group R2A). Group N:received normal saline 30μl,20μl and 10μl, three times, each time interval of 1h and intraperitoneal injection with normal saline. Group R2B:respectively received 2% bupivacaine 30μl,20μl and 10μl, a total of three times, each time interval of 1h and intraperitoneal injection with normal saline. Group R2A:respectively received 2% bupivacaine 30μl,20μl and 10μl, a total of three times, each time interval of 1h and intraperitoneal injection with AGC. AGC administration methods:AGC dilution (10u/ml), 100u/kg, in a continuous intraperitoneal injection from intrathecal administration 3 days ago to 4 days after, once a day. Tail-flick test and motor function were performed at 1d,2d,3d,4d and 5d after the last IT administration. TF latencies were converted to the percent of maximal possible effect (MPE). Five days later, the DRG were cut and used for HE staining and TUNEL for apoptosis and immunohistochemical analysis for caspase-9 protein. Using SPSS 13.0 software 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. Comparisons among groups MPE were made by Repeated Measures. The number of apoptotic cells and caspase-9 protein were made by analysis of variance (ANOVA). P<0.05 was considered significant.Results (1) The impact on the MPE:MPE were different among the groups in rats, which had not the significant difference with time after IT, and which had the interaction between group and time. At the same time point, the MPE of the groups were significantly different, group R2B had a higher MPE. After the IT Id group R2A MPE reached the peak, then dropped, however R2B group MPE sustained increased. (2) The impact on the MF:MF scores were 0 in all the rats. (3) HE stain:Compared with the group N and R2A, group R2B increased neuronal degeneration, ischemia, cellular necrosis and interstitial edema. (4) The number of apoptotic positive cells:the number of apoptotic positive cells in group R2B was significantly higher compared with those in group N and R2A. (5)The number of caspase-9 positive cells:the number of caspase-9 positive cells in group R2B was significantly higher compared with those in group N and R2A.Conclusion Pretreatment with AGA can reduce the neurotoxicity of intrathecal bupivacaine in rats, which is related with down-regulate caspase-9 expression.
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