The Analgetic Roles Of Ziconotide And/or Midazolane In Neuropathic Pain In Rats

Part One:The analgetic roles of domestic and imported ziconotide in rat's neuropathic painObjectiveTo investigate the efficacy of domestic and imported ziconotide in treating the neuropathic pain induced by L5 spinal nerve ligation in rats, and compare their bioequivalence at the same time.MethodsEighty-eight male SD rats (weighted from 180 to 220g) were chosen for raising adaptabiltity and adapting monitoring environment for 5 days. Then, they were tested on their 50% paw withdrawal threshold (50%PWT) and thermal withdrawal duration(TWD) as the basic pain threshold on the sixth and eighth day(preoperative 3d and 1d). The qualified rats were sorted out for the operation experiment according to the basic pain threshold of 50%PWT>4g, which had been operated with the lumber 5 spinal nerve ligation (L5 SNL) and spinal catheterization on the ninth day(sham-operated rats were only exposed and isolated in L5 spinal nerve, but not damaged in the nerve), At least 2 days were allowed for recovery from surgery before initiating further experimental procedures. Next, we detected the changes of the 50%PWT and TWD 4,1d after operation and assessed the location of the catheter tip by injecting 10 ul of 2% lidocaine intrathecally on the third postoperative day. Animals showing signs of 50% PWT<4g and the catheter tip in the correct spinal position were eligible for the treatment experiment,and were randomly divided into eleven groups, including sham controlled group(Group SC):0.9%NS 20ul; L5SNL group(Group L5SNL):0.9%NS 20ul; domestic ZIClgroup(Group LZ1,MZ1, HZ1):ZIC130,100,300ng; domestic ZIC2 group(Group LZ2,MZ2,HZ2):ZIC2 30,100,300ng; imported ZIC group(Group LTC,MTC,HTC):imported ziconotide 30? 100?300ng.The drugs were given by intrathecal bolus injection on the seventh postoperative day for all rats, and the 50%PWT and TWD were measured 1h before intrathecal administration andl,2,4,8h after intrathecal administration. We observed the changes of behavior and pain threshold in these rats, and calculated the maximum possible effect (MPE) to evaluate the analgetic efficacy of domestic and imported ziconotide in relieving the neuropathic pain.Using SPSS 13.0 software for statistical treatment, measurement data were expressed as mean± standard deviation. Analysis of rank-sum test and variance (ANOVA) were used to compare the differences in these time spots and groups for the 50%PWT;Analysis of paired t-test and variance (ANOVA) were used to compare the differences in these time spots and groups for TWD, P<0.05 was considered significant.Results(1) Compared with the Group SC and baseline, the pain threshold of all the other groups were signifieantly decreased (P<0.01); (2) Compared with the Group L5SNL, both 50% PWT and TWD were significantly increased by intrathecal injection 30,100,300ng of domestic and imported ziconotide respectively(P<0.05 or P<0.01), which reached maximal values within 1hr post-treatment, and persisted at near maximum levels for at least 4 hr after dosing. It showed a significant dose-effect relationship among the groups at different dose; (3) Compared with the same dose of the positive control group, the 50% PWT and MPE of ZIC 1 and ZIC2 at 30ng were increased more significant(P<0.01),but the TWD was lower(P<0.05).There were no significant different of the 50% PWT,MPE and TWD between all other dose groups(P>0.05).ConclusionCompared with isodose inported ziconotide, domestic ziconotide has an equivalent or better analgetic effect on the neuropathic pain induced by L5 SNL in rats.Part Two:The analgetic roles of midazolam in rat's neuropathic painObjectiveTo investigate the efficacy of midazolam in treating the neuropathic pain induced by L5 spinal nerve ligation in rats.MethodsForty male SD rats (weighting from 180 to 220 g) were chosen for raising adaptability and adapting monitoring environment for 5 days. Then, they were tested on their 50% paw withdrawal threshold (50%PWT) as the basic pain threshold on the sixth and eighth day(preoperative 3d and 1d). The qualified rats were sorted out for the operation experiment according to the basic pain threshold of 50%PWT>4g, which had been operated with the lumber 5 spinal nerve ligation (L5 SNL) and spinal catheterization on the ninth day(sham-operated rats were only exposed and isolated in L5 spinal nerve, but not damaged in the nerve), At least 2 days were allowed for recovery from surgery before initiating further experimental procedures. Next, we detected the changes of the 50%PWT 4,6d after operation and assessed the location of the catheter tip by injecting 10 ul of 2% lidocaine intrathecally on the third postoperative day. Animals showing signs of 50%PWT<4g and the catheter tip in the correct spinal position were eligible for the treatment experiment,and randomly divided into five groups, including sham controlled group(Group SC):0.9% NS 20ul; modle controlled group(Group MC):0.9%NS 20ul; low dose of midazolam group(Group ML):midazolam lug; moderate dose of midazolam group(Group MM): midazolam 5ug; high dose of midazolam group(Group MH):midazolam 25ug. The drugs were given by intrathecal bolus injection on the seventh postoperative day for all rats, and the 50%PWT was measured 1h before intrathecal administration and 1,2, 4 and 8h after intrathecal administration. We observed the changes of behavior and pain threshold of these rats, and calculated the maximum possible effect (MPE) to evaluate the analgetic efficacy of midazolam in relieving the neuropathic pain.Using SPSS 13.0 software for statistieal treatment, measurement data were expressed as mean ? standard deviation. Analysis of rank-sum test and variance (ANOVA) were used to compare the differences in these time spots and groups, P<0.05 was considered significant.Results(1) Before the operation, the 50% PWT was no significant difference of between left and right paws of all groups (P>0.05), but which was significantly decreased compared to baseline and group SC after L5SNL operation, and the left paws were more significant than the right. There were no significant different of the mechanical pain threshold between preoperative and postoperative in the group SC(P>0.05); (2) Compared with the Group L5SNL, intrathecal bolus injections of midazolam at 1,5,25ug produced a dose-dependent suppression of mechanical allodynia as measured by increased the 50%PWT and MPE(P<0.05or P<0.01). Allodynia thresholds were elevated at the first measured time point (1hr) and reached maximal values within 4hr post-treatment. The anti-allodynic effects of midazolam were long-lived and persisted at near maximum levels for at least 8hr after dosing.ConclusionIntrathecal bolus injections of midazolam could relieve the neuropathic pain significantly induced by L5 SNL in rats.Part Three:The analgetic roles of intrathecally administerded midazolam and ziconotide in rat's neuropathic painObjectiveTo investigate the efficacy of intrathecally administerded midazolam and ziconotide in treating the neuropathic pain induced by L5 spinal nerve ligation in rats.MethodsSixty-four male SD rats (weighted from 180 to 220 g) were chosen for raising adaptability and adapting monitoring environment for 5 days. Then, they were tested on their 50% paw withdrawal threshold (50%PWT) as the basic pain threshold on the sixth and eighth day(preoperative 3d and 1d). The qualified rats were sorted out for the operation experiment according to the basic pain threshold of 50%PWT>4g, which had been operated with the lumber 5 spinal nerve ligation (L5 SNL) and spinal catheterization on the ninth day(sham-operated rats were only exposed and isolated in L5 spinal nerve, but not damaged in the nerve), At least 2 days were allowed for recovery from surgery before initiating further experimental procedures. Next, we detected the changes of the 50%PWT 4,6d after operation and assessed the location of the catheter tip by injecting 10 ul of 2%lidocaine intrathecally on the third postoperative day. Animals showing signs of 50%PWT<4g and the catheter tip in the correct spinal position were eligible for the treatment experimental,and randomly divided into eight groups, including L5SNL group(Group L5SNL):0.9%NS 20ul; ziconotide group(Group Z):ziconotide 30ng; low, moderate and high dose of midazolam group(Group M1, M2, M3):midazolam 1,5,25ug; ziconotide+ midazolam group(Group Z+M1, Z+M2, Z+M3):ziconotide 30ng+midazolam lug, ziconotide 30ng+ midazolam 5ug, ziconotide 30ng+midazolam 25ug. The drugs were given by intrathecal bolus injection on the seventh postoperative day for all rats, and the 50%PWT was measured lh before intrathecal administration andl,2,4and 8h after intrathecal administration. We observed the changes of behavior and pain threshold in these rats, and calculated the maximum possible effect (MPE) to evaluate the analgetic efficacy of midazolam and ziconotide in relieving the neuropathic pain.Using SPSS 13.0 software for statistical treatment, measurement data were expressed as mean ? standard deviation. Analysis of rank-sum test and variance (ANOVA) were used to compare the differences in these time spots and groups, P<0.05 was considered significant.Results(1) Before the operation, the 50% PWT was no significant difference of between left and right paws of all groups (P>0.05), but which was significantly decreased compared to baseline after L5SNL operation(P<0.01), operation, and the left paws(1.20±0.47g) were more significant than the right (6.51±1.58g),P<0.01; (2) Compared with L5SNL group, the 50%PWT and MPE of M1, M2, M3, Z, Z+M1, Z+M2, Z+M3 group were icreased significantly (P<0.05or P<0.01). Intrathecal bolus injections of midazolam at 1,5.25ug produced a dose-dependent suppression of mechanical allodynia (P<0.05or P<0.01). Allodynia thresholds were elevated at the first measured time point (1hr) and reached maximal values within 4hr post-treatment. The anti-allodynic effects of midazolam were long-lived and persisted at near maximum levels for at least 8hr after dosing; The anti-allodynic effects of ziconotide were reached maximal values within 1hr post-treatment, and persisted at near maximum levels for 4hr after dosing(P>0.05); ones of ziconotide+midazolam were reached maximal values within lhr post-treatment, and persisted at near maximum levels for at least 8hr after dosing(P>0.05).(3) Compared with midazolam(M1, M2, M3) or ziconotide(Z) treated alone groups respectively, the 50%PWT and MPE of midazolzm-ziconotide combination groups(Z+M1, Z+M2, Z+M3) were elevated more significantly(P<0.01);That's more, compared with the former, combination groups can achieve the peak time of Analgesic effect early; compared with the latter, the maintenance of peak time is longer in combination groups. (4) Compared with Z+M1 group, the 50% PWT and MPE of Z+M2 group were icreased more significantly (P<0.01); There were no significant different of the 50%PWT and MPE between Z+M2 and Z+M3, but the side effects of the fomer lesser than that of latter.ConclusionIntrathecal bolus injections of midazolam and ziconotide could produce significant analgesic effects in neuropathic pain induced by L5 SNL in rats, midazolzm-ziconotide combination are superior to midazolam or ziconotide alone for analgesic effects in neuropathic pain rats. (2)After combination, the analgesic effect of drug is produced more earlier and maintained longer. (3) To decrease the side effects of ziconotide as many as possible, we can combinate it to midazolam with a appropriate dose, the best combination dose for neuropathic pain rats is combinating the minimum effect dose of ziconotide(30ng) to midazolam 5ug(0.02ug/g), the analgesic effects are significant but the side effects are little.