The Neurotoxic Effect And Mechanism Of Intrathecal Ropivacaine At Long Time In Rat Spinal Cord

Objective:1. To observe ultrastructural changes and mechanisms of spinal cords and nerve roots of intrathecal administration of0.5%or1%concentrations of ropivacaine after twenty-eight days in rats, and also to investigate whether ERK and Akt signaling pathways and mitochondrial opotosis were involved in spinal neurotoxicity of ropivacaine. To observe the ultrastructural changes of spinal cords and nerve roots of intrathecal administration of1%concentrations of ropivacaine and NT.-3after days in rats, and investigate the therapeutic potential of NT-3in local anesthetic toxicity.2. To examine the effects of NT-3on ropivacaine cell toxicity in vitro, providing theoretical and therapeutic exploration for application of ropivacaine in intrathecal administration.Methods:1. One hundred and forty-four/144male Sprague Dawley rats weighing280-320g were successfully implant with microspinal cather following the methods of Yaksh. Rats were randomly divided into four groups:group saline (Group NS, n=36、group0.5%ropivacaine (Group M, n=36)、group1%ropivacaine (Group R, n=36) and group ropivacaine+NT-3(Group T, n=36). Rats in group M were received0.5%ropivacaine or rats in group R and T were received1%ropivacaine, or rats in group NS were received only saline0.12ml/kg intrathecal injection with1.5h interval lasted for12h. And at the meantime Group T gives NT-30. lmg/kg (30μg) again. Each group was observed on1d、3d、5d、7d、14d、28d. The group NS was respectively signed with NS1、 NS3、NS5、NS7、NS14、NS28(n=6). The group M was respectively signed with M1、M3、M5、M7、M14、M28(n=6). The group R was respectively signed with R1、R3、R5、R7、R14、R28(n=6). The groupT was respectively signed with T1、T3、T5、T7、T14、T28(n=6). Moter function assessment was investigated after the last injection, while sensory threshold tested by effect of the paw withdrawal latency to heat (PWHL) and mechanical stimulate (PWML) were investigated both before injection and1d、3d、5d、7d、24d、28d after the last injection in all groups. After finishing all these neurofunctional tests, rats were sacrified. Their lumber spinal cords and posterior roots were examined by light and electron microscopy. The spinal cords were also evaluated for apoptosis by an in situ TUNEL assay. Expression cleaved ERK、Akt proteind by western blot. Spinal caspase-3、ERK1、Akt expression were determined by immunohistochemistry.2. Cell experiments were done in order to examine the effects of NT-3on ropivacaine cell toxicity in vitro. Rat pheochromocytoma cells (PC12) were divided into three group, group control (Group NS) without any special treatment, just routine incubation for48h, group ropivacaine (Group R) with incubation of15mmol/L ropivacaine for48h, group ropivacaine+NT-3(Group T) with incubation of15mmol/L ropivacaine and NT-3100ng/ml for48h. Cell counts and cell survival rate detected by MTT were observed after treatment in each group. Measurement of cell viability trypan was observed after treatment in each group. Changes of Akt、ERK protein expression were evaluated by western blot.Results:1The changes of PWML and PWHL in each groups: PWML increased in group R1、R3、R5and T1、T3compared with that of group NS and M in each time-course (P<0.05). Group R1showed the most significant increase.Group T5showed the significant decrease compared with R groups in each time-course (P<0.05). PWHL increased in group R1、R3、R5and T1、T3compared with that of group NS and M in each time-course (P<0.05). Group R1showed the most significant increase. Group T5showed the significant decrease compared with R groups in each time course (P<0.05).2.The results of Electron and optical microscope technique:Electron microscope result:different dgrees of changes appeared in group R1、R3、 R5、R7、R14、R28after admistration. Significant edema, mostly turned to pyknosis、karyorrhexis and vacuole, apoptotic body appeared in group R1. Clear mitochondrion, Nuclear shrinkage, focal edema in the nerve cells appeared in group R3. Parts of nerve cells in group R5showed nuclear shrinkage、slight expansion of the endoplasmic reticulum、partly swelling. Group R7showed focal edema, fuzzy structure of several mitochondrion、slight expansion of the endoplasmic reticulum and several cells shrinkage. Group R14showed nerve cells swelling, edema, Loss and vacuole of mitochondrial, slight expansion of the endoplasmic reticulum. Group R28showed nomal ranvier’s node. While correspondingly, the group T1showed lace shaped and wizened nucleus, emerging apoptotic bodies, normal chondriosomes. Group T3showed round neurons nucleus clear structure of apoptotic bodies but slightly edema. Group T5showed clear structure of apoptotic bodies, slightly expandation of endoplasmic reticulum, normal nucleus but slightly edema. Group T7showed slightly wizened neurons nucleus. Partial apoptotic bodies were vesicular but no edema. Group T14were almost normal, round nucleus, clear mitochondrialcristae and normal endoplasmic reticulum. Group T28were normal. Optical microscope technique:nerve cells in Group R1、R3、R5、R7were rarely, nucleus showed significant karyopyknosis and darkstainednucleus. Some nerve cell appeared necrosis and partial glial cells appeared hyperplasia. There were more nerve cells, some of which had axone and some of which appeared atrophia and karyopyknosis and darkstainednucleus. The cells in Group T1、T3and T5were decreased in mount and reduced in size. They appeared karyopyknosis and darkstainednucleus. The number of nerve cell in Group T5was more than that in Group R5, and the cells showed spotty necrosis. Partial nerve cells in Group T7were normal, but most cells had long axone and some appeared atrophia, karyopyknosis and darkstainednucleus, glial cells hyperplasia. Some nerve cell in Group T4showed no axone and the little hyalomitome. Compared with R28, nerve cells are more than that in T28, and the cell shape were normal. Compared with Group NS and M in each corresponding time-points, the histopathological of group Rand group T were higher in the1d、3d、5d、7d and14d, the group R was the highest (p<0.05). Compared with Group R in each corresponding time-points, the histopathological assessment scores of Group T5、T7and T14were significantly decreased (p<0.05).3.The results of Postivie-staining cells counts by TUNEL staining: Positive-staining cells in group T1、T3、T5as well as group R1、R3、 R5、R7were more than that in group NS and M (P<0.05) in the same time-point; However, positive cells in group R1、R5、R7were more than that in corresponding T groups.(P<0.05)4.The detectived Akt、ERK1、caspase-3by Immunohistochemistry (IHC) Akt:compared group R1、R3、R5as well as group T1、T3with the group NS or M in the same time-point, the positive expression of Akt in Posterior horn of the spinal cord gray matter increased significantly (P<0.05).The positive expression of Akt protein decreased significantly in group T3、T5、T8compared with group R (P<0.05).ERK1:compared group R1、R3、R5、R7、R14as well as group T1、 T3、T5、T7with the group NS or M in the same time-point, the positive expression of ERK1in the spinal cord gray neurons matter increased significantly (P<0.05). The positive expression of ERK1protein decreased significantly in groupT1、T3、T5、T14compared with group R (P<0.05).Caspase-3:compared group R1、R3、R5、R7、R14as well as group T1、T3、T5、T7with the group NS in the same time-point, the positive expression of caspase-3in Posterior horn of the spinal cord gray matter increased significantly (P<0.05). The positive expression of caspase-3protein decreased significantly in group T1、T3、T5、T14compared with group R (P<0.05).5. The examined results of Phosphorylation of Akt and ERK in spinal cord by western blot:Compared to group NS or M in each time-course, group R1、R3、 R5、R7、R14. R28and group T1、T3、T5、T7、T14showed a decreased expression of Akt protein (P<0.05), and the group R1、R3were the most significantly. Compared to group R in each time-course, there was a significant increase of Akt protein expression in group T5、T7and T28(p<0.05).Compared to group NS or M in each time-course, group R1、R3、 R5、R7and group T1、T3、T5、T7showed a decreased expression of Akt protein (P<0.05), and the group R1、R3were the most significantly. Compared to group R in each time-course, there was a significant increase of ERK protein expression in group T1、T3(p<0.05).6. Cell manipulation in vitro:there are decreases of Cell counts and cell survival rate and cell viability in group R and group T compared with group NS (P<0.05). However, the protein expression decreased also in all groups (P<0.05). Compared to group R, the cell counts and cell survival rate and cell viability increased in group T (p<0.05), protein expression increased also in all groups (P<0.05).Conclusion:1. Repeatedly intrathecal administration of0.5%ropivacaine for12h does not cause ultramicrostructure and histology changes in spinal cords and nerve root of rats from lth day to28th day.2. Repeatedly intrathecal administration of1%ropivacaine for12h may cause the heavier ultramicrostructure and histology changes and also could cause more apoptosis cells in spinal cords and nerve root of rats from lth day to7th day. And along with the time extension, most pathological changes are back to normal on28th day.3. Repeatedly intrathecal administration of1%ropivacaine for12h may cause the lighter ultramicrostructure and histology changes and also could cause less apoptosis cells in spinal cords and nerve root of rats from lth day to7th day. And along with the time extension, all pathological changes are back to normal on14th day.4. Phosphorylation of Akt and ERK apoptosis pathway may be involved in spinal neurotoxicity of ropivacaine.5. NT-3may decline the spinal neurotoxicity of ropivacaine through the inhibition of Akt and ERK apoptosis pathway. There are figures201and tables3and references135.