The Role Of Oxidative Stress In Neurotoxicity Caused By Ropivacaine In Spinal Cord Of Rats And Its Mechanism

Objective:To observe the situation of oxidative stress, apoptosis and expression of ERK1 in Spinal cord after repeatedly intrathecal administration of 1%ropivacaine and the change after application of antioxidant TEMPOL,and investigate the influence and mechanism of oxidative stress to neurotoxicity of ropivacaine.Methods:A polyurethane 10 microcatheter was inserted into the lumbar subarachnoid space of male SD rat.144 Successfully implanted rats were randomly divided into 4 groups, group S (Sham-operate), group N (control), group R (ropivacaine), group T(TEMPOL). The rats in group S received no agent,the rats in group N received saline 0.12μL/g for 8 times at 1.5 h interval through the catheter, the rats in the R group received 1%ropinacaine in the same way as group N,and the rats in the T group received 1%ropinacaine in the same way as group R first, then received TEMPOL 5μL(380 nmol)at 4h,8h,12h,24h,48h,72h. Each group was observed at Id,3d,5d,7d,14d,28d. The group S was respectively signed with S1, S3, S5, S7, S14, S28.The group N was respectively signed with N1, N3, N5, N7, N14, N28. The group R was respectively signed with R1, R3, R5, R7, R14, R28. And the group T was respectively signed with T1, T3, T5, T7, T14, T28.(n=6) The poster paw withdrawal latency to heat (PWHL) and mechanical stimulation (von Fray filament) (PWML) were measured at the day before the intrathecal administration, 1.5h,1 day,3d,5d,7d,14d,28d after intrathecal administration of ropivacaine. Then the rats were sacrificed and the lumber segments of the spinal cord were immediately removed for HE staining to observe the organization morphology change, TUNEL staining to test the cell apoptosis, immunohistochemical method to test the expression of ERK1, and amount of MDA and activity of SOD to test the level of ROS.Results:1.1.5h after repeatedly intrathecal administration of 1% ropivacaine, the pain threshold of rat's legs significantly rose compared to group N, and reached a high level in the 1st days then gradually declined, it hadn't returned to the level before administrition until the 28th day (P<0.05). Compared to group R, the pain threshold significantly decreased in 1-3d.Then it was lower than group R in rest groups until the 28th day.2. HE staining:There was no significant pathology change in group S and group N.Degeneration and apoptosis of spinal neurons occurred in group R in 1-3d. Then the damage developed and apoptotic cells increase gradually, even necrosis happened in part of cells. Lesions began to relieve and the apoptotic cells decline at the 14th day.Compared to group R, there were more normal neurons, less apoptosis cells and a bit of necrosis early in group T.Most of the cells were normal and only a few apoptotic cells could be seen at the 14th day.Spinal neurons was nearly normal at the 28th day.3. Compared to group N, positive cells of TUNEL staining were significantly increased in group R1, R3, R5 and T1, T3, T5.And then the number reduced gradually but still more than group N (P<0.05). Compared to group R, Positive cells were significantly decreased in group T3.Then the number was less than group R in the rest groups (P<0.05).4. Compared to group N,expression of ERK1 were significantly increased in group R1, R3, and T1, T3.And then it decreased gradually but still significantly more than group N (P<0.05) until the 28th day. Compared to group R, expression was significantly decreased in group T3.Then the expression was less than group R in the rest T groups (P<0.05).Until the 14th day, There was no obvious difference between group T and group N (P>0.05).Compared to group N,the amount of MDA were significantly increased in group R1, R3, R5 and T1, T3,T5,T7.And then it decreased gradually but still significantly more than group N (P<0.05) until the 28th day. Compared to group R, content of MDA was significantly decreased in group T3.Then it was less than group R in the rest T groups (P<0.05).Compared to group N, the activity of SOD were significantly increased in group R1, R3, R5 and T1, T3,T5,T7.And then it decreased gradually but still significantly more than group N (P<0.05). Compared to group R, the activity of SOD was significantly increased in group T3.Then it was less than group R in the rest T groups (P<0.05)Conclusion:1.After repeatedly intrathecal administration of 1% ropivacaine for 12h, ROS in spinal cord of rat increased significantly, pain threshold raised and apoptosis accured in neurons.Antioxidant TEMPOL could reduced the injury. It implies that oxidative stress may be involved in the formation and development of neurotoxicity induced by ropivacaine.2. After repeatedly intrathecal administration of 1% ropivacaine for 12h, increased ROS may participate in the neurotoxicity induced by ropivacaine through activation of ERK pathways to promote apoptosis of neurons.