ABSTRACT
Objective To evaluate the effect of ganglioside ( GM-1) preconditioning on endoplas-mic reticulum stress ( ERS)-dependent apoptosis during spinal cord injury induced by bupivacaine in rats. Methods One hundred and eight clean-grade healthy male Sprague-Dawley rats, aged 8 weeks, weighing 250-300 g, were divided into 3 groups ( n=36 each) using a random number table method: control group (group C), bupivacaine group (group B) and GM-1 pretreatment group (group G). In group B, 5％bupivacaine 0. 12 μl/g was intrathecally injected at 1. 5 h intervals, 3 times intotal. In group G, GM-120μl ( 30 mg/kg) was intrathecally injected, and 24 h later bupivacaine was intrathecally injected according to the method previously described in group B. Immediately after intrathecal injection and at 1, 3, 5, 7 and 14 days after intrathecal injection, the maximum percentage of anti-nociceptive effects (％MPE) was detected, the hindlimb motor function score ( BBB score) was evaluated, and spinal cord tissues were har-vested for determination of cell apoptosis ( using TUNEL) and expression of caspase-12 and CCAAT/enhan-cer-binding protein homologous protein ( CHOP ) protein and mRNA ( by Western blot or quantitative real-time polymerase chain reaction) . The apoptosis rate was calculated. Results Compared with group C, the％MPE and apoptosis rate were significantly increased, the BBB score was decreased, and the expression of CHOP and caspase-12 protein and mRNA was up-regulated in group B ( P＜0. 05) . Compared with group B, the ％MPE and apoptosis rate were significantly decreased, the BBB score was increased, and the ex-pression of CHOP and caspase-12 protein and mRNA was down-regulated in group G ( P＜0. 05) . Conclu-sion GM-1 preconditioning reduces bupivacaine-induced spinal cord injury possibly through inhibiting ERS-dependent apoptosis in rats.
ABSTRACT
Objective To discuss the impact of the neurotoxity of bupivacaine and bupivacaine-induced cellular neurotoxicity caused by pretreatment of ganglion (GM-1 )monoglyceride on the ex-pression of caspase-3.Methods The mouse neuroblastoma cells-N2a cells was used as a research object to carry out the following experiments:(1)To observe the damage effects of different concen-trations of bupivacaine on N2a cells and find out the most suitable damage concentration to establish cell damage model.The N2a cells were interacted with bupivacaine with different concentrations [0μmol/L (group C),600 μmol/L (group B1),900 μmol/L (group B2),1 200 μmol/L (group B3), 1 500 μmol/L (group B4),2 000 μmol/L (group B5)]for 6,12,24,36 h and then evaluated by CCK-8 cell survival.Each experiment was repeated three times.The protective function of GM-1 to bupiva-caine-induced N2a cells damage.The N2a cells were treated with different concentrations (0.1μmol/L (group BG1),1.0 μmol/L (group BG2),10 μmol/L (group BG3))of GM-1 pretreatment 24 h,CCK-8 was evaluated in cell viability,Western Blot method was used to detect damaged cells caspase-3 expression levels.Each experiment was repeated three times.Results (1)Bupivacaine could significantly damage N2a cells,the greater the bupivacaine concentration,the longer the action time,the stronger neurotoxicity.(2)GM-1 bupivacaine nerve injury had a significant protective effect in a dose-related manner.The maximum of protective dose of this experiment was 10 μmol/L.Conclusion Bupivacaine can significantly damage N2a cells,correlating with both dose and time double positively,while GM-1 pretreatment significantly reduced the expression of caspase-3 induced by bupivacaine.
ABSTRACT
Objective To investigate the therapeutic effects of intravenous monosialo ganglio-sides(GM-1)on neurotoxicity of intrathecally administered bupivacaine in rats and its possible mecha-nism.Methods One hundred and eight adult male Sprague-Dawley rats,weighing 280-300 g,were randomly divided into 3 groups (n=36 each):sham operation group (group sham),group saline and group GM-1.Neurotoxicity model was performed by injecting 0.12μl/g body weight of bupivacaine at concentrations of 5% via an implanted intrathecal catheter at 90-minute intervals for 4.5 h in groups saline and GM-1.After observing 24 h,group GM-1 was administered GM-1 30 mg/kg by intrave-nous injection for 7 days,once a day;while groups saline and sham received equal volume of normal saline.The recovery of the locomotor function was evaluated with Basso,Beattie and Bresnahan (BBB)and tail-flick latency(TFL)before injection bupivacaine and days 1,3,5,7,14,28 after in-jection,TFL was converted to the percent maximum possible effect (%MPE).Six rats were sacri-ficed in each group at each time point,and spinal cord was taken to examine histological injury scores by light and electron microscopy at the L3 level,and neuron caspase-3 expression was evluated using immunohistochemistry and RT-PCR.Results Compared with group saline,%MPE,histological inju-ry score and caspase-3 mRNA expression were decreased on days 7,14 and 28;Caspase-3 protein ex-pression was decreased on days 5,7,14 and 28;while BBB score was higher on days 14 and 28 in group GM-1 (P < 0.05 ).Compared with group sham,% MPE,histological injury score,caspase-3 mRNA and protein expression in groups GM-1 and saline were significantly higher,while BBB score was lower on 1,3,5,7,14 and 28 d after injection (P <0.05).Conclusion GM-1 can promote neuro-functional recovery after bupivacaine neurotoxicity in rats through the possible mechanism of down-regulating neuron caspase-3 expression.
ABSTRACT
Objective To evaluate the efficacy of intrathecally administered monosialoganglioside (GM-1)for treatment of bupivacaine spinal anesthesia-induced neurotoxicity to rat spinal cord.Methods Adult male Sprague-Dawley rats,weighing 280-300 g,in which the intrathecal catheter was successfully inserted into the L3,4 intervertebral space and advanced toward the tail,were randomly divided into 4 groups (n =36 each):sham operation group (group S),GM-1 group,bupivacaine group (group B) and bupivacaine + GM-1 group (group BG).In B and BG groups,the rats received 5％ bupivacaine 20 μl via the intrathecal catheter 3 times at 1.5-hour intervals.GM-1 20 μg was injected intrathecally 24 h later once a day for 7 days in BG and GM-1 groups.Before bupivacaine injection and on days 1,3,5,7,14 and 28 after bupivacaine injection (T0-T6),tail flick latency (TFL) was measured,MPE (percentage of maximal possible effect) was calculated,and the locomotor recovery was evaluated using the Basso,Beattie,Bresnahan (BBB) Locomotor Rating Scale.Then six rats were randomly chosen and sacrificed in each group.Spinal cord was removed for histopathologic examination (with light and electronic microscope) and for determination of caspase-3 protein and mRNA expression (by immuno-histochemistry and RTPCR).The pathological changes of the spinal cord were scored.Results Compared with S and GM-1 groups,MPE,pathological scores,and caspase-3 protein and mRNA expression were significantly increased and BBB score was decreased at T1-6 in group B (P ＜ 0.05),and MPE was increased at T1-5 (P ＜ 0.05) and returned to the baseline value at T6 (P ＞ 0.05) and pathological scores,and caspase-3 protein and mRNA expression were significantly increased and BBB score was decreased at T1-6 in group BG (P ＜ 0.05).There were no significant differences in each parameter at each time point between S and GM-1 groups (P ＞ 0.05).Compared with group B,MPE and caspase-3 protein and mRNA expression were significantly decreased at T2-6,pathological scores were decreased at T3-6,and BBB score was increased at T4-6 in group BG (P ＜ 0.05).The pathological changes of spinal cord tissues were obvious at T1-6 in group B and at T1-3 in group BG,but the changes gradually recovered at T4-6 in group BG.Conclusion Intrathecally administered GM-1 has therapeutic effect against bupivacaine spinal anesthesia-induced neurotoxicity to rat spinal cord,and inhibition of neuronal apoptosis in the spinal cord may be involved in the underlying mechanism.