Effects Of Polydatin On The Neurotoxicity Induced By Ropivacaine Injected Intrathecally In Rats

ObjectiveLocal anesthetics(LA) is anesthetic agent commonly used in clinic,however concerns have risen about the direct or indirect neurotoxicity of it that can induce transient and permanent neurotoxic lesions. Although the exact mechanism is not fully understood,the neurotoxicity correlate closely with concentrations,doses,time exposed in the nervous system. Some anesthetists have taken several measures to prevent and cure it,but the result is uncertain. So more and more anesthetists set to find some medicine to prevent the neurotoxity induced by LA in research..At present,anesthetists have found 4 kinds of medicine through a lot of tests.①Neurotrophic factors can support the recovery of neurons after exposure to LA in vitro. but whether they have same protective action in vivo, it remain elusive to a considerable degree.②AMPA receptor antagonist can reduce the neurological and histopathological injury caused by LA,however ,it does not completely eliminate the neuronal injury.③p38 MAPK inhibitor: Recent investigations demonstrate that LA induce apoptosis in neuron cultures in vitro. p38 MAPK inhibitor is effective in attenuating LA-induced neurotoxicity.④Chinese herbal medicine: someone have found Shenfu injection itself had no effect on spinal neurons, but it was able to reduce the bupivacaine-induced neurotoxicity in vitro.Polydatin(PD) can take pharmacologic action through hydrolization of resveratrol(Res).Some researchers have found that Res can attenuate spinal cord injury to promote nerve regeneration and functional restoration. Whether Res can be used to prevent the neurotoxicity induced by LA or not,it is unknown and need to be proved through further research. .So we want to do some tests to investigate effects of intravenous(i.v.) PD on the neurotoxicity induced by ropivacaine injected intrathecally (i.t.) in rats.Methods1,Surgical procedure for intrathecal catheterizationPolyethylene tube,PE-10,was sterilized to use. SD rats were anesthetized by chloral hydrate(100mg/kg).A 2 cm longitudinal skin incision was made above vertebrae L3 and L4. The rats were held firmly by the pelvicgirdle in one hand .Then processus articularis superior,inferior articular process and ligamentum flavum were exposed. The intervertebral space,dura of spinal cord and arachnoid were explored by a 7-gauge needle. When the sign of dura penetration (sudden movement of tail or the hindlimb) was observed, the catheter was pushed gently caudal ward to reach the lumbar enlargement. The catheter was tunneled under the skin. All incisions were then carefully sutured and the outer end of the catheter was sealed by melting. Heat melting was also used during the following experiments after each injection. After the surgery ,the rats were given benzylpenicillin sodium(i.m.) and fed in the cage alone for 2 days.We checked the performance of the rats every day .The rats which had behavior disorder were excluded.2,Group and intrathecal administrationFifty SD rats without behavior disorder were randomly assigned to five groups (n=10 in each):group NS received 40μl saline i.t and served as control;group R received 2% ropivacaine 40μl i.t;group R + PD1,R + PD2,R + PD3 received polydatin i.v. 5mg/kg,10mg/kg,15mg/kg,respectively,then received 2% ropivacaine 40μl i.t.The weight and time of intrathecal catheterization were expressed as mean±SD. To determine the differences in the weight and time of intrathecal catheterization among 5 groups, A one-way analysis of variance was used. All statistical procedures were performed using SPSS software version 13.0.A value of P≤0.05 was considered statistically significant.3,Behavior evaluation and paw-stimulation testSeven days later,animals were evaluated for sensory function and motor function by a technician blinded to the animal groups. The sensory function and motor function were analyzed among 5 groups using a nonparametric method(Kruskal-Wallis test) followed by the Mann-Whitney U-test with Bonferroni correction. All statistical procedures were performed using SPSS software version 13.0.A value of P≤0.05 was considered statistically significant.4,Tissue preparationAfter evaluations of 2 functional tests,the animals were deeply anesthetized by chloral hydrate (100mg/kg,i.p.) and then injected 6μl methylene blue i.t. to confirm the tip location of the catheter. The rats were perfused transcardially with 37℃saline and (4℃) 4% paraformaldehyde(0.1mol/L, pH7.35).The spinal cord,nerve roots and cauda equina were dissected and fixed in 4% paraformaldehyde and decalcifying fluid for 24h,respectively.All specimens were stained with hematoxylin and eosin(HE).Morphological changes of the tissues were assessed using light microscopy by a technician blinded to the animal groups.The lesions were analyzed among 5 groups using a nonparametric method(Kruskal-Wallis test) followed by the Mann-Whitney U test with Bonferroni correction. All statistical procedures were performed using SPSS software version 13.0.A value of P≤0.05 was considered statistically significant.Results1,There were no significant differences among 5 groups for the weight P=0.996(P>0.05).The weight of the rats was about (227±12)g.2,There were no significant differences among 5 groups for the time of intrathecal catheterization, P=0.427(P>0.05).The surgery time was about (30±4)min. No infection and nervous lesion were observed.3,The surgery of intrathecal catheterization were confirmed to be successful by 2 tests. First, 6μl methylene blue injected i.t. can proved the fact that PE-10 catheter was in the subarachnoid space.Second,after 2% ropivacaine was injected i.t.,all the rats were paralysis of both lower limbs.4,After intrathecal administration, MAP of the rats decreased,but were still above 60mmHg.All animals did not accepted fluid replacement.5,There were significance differences in sensory function score among each groups, P=0.000(P<0.05).Mann-Whitney U-test indicated that differences were significant between group NS and R,NS and R+PD1, R and R+PD2,R+PD1 and R+PD2, respectively (P<0.005). There were no significant differences in others. The results suggested that sensory function scores of group NS or R+PD2 were less than group R and R+PD1, respectively.6,There were no changes in the motor function for all animals.7,The histopathologic changes were seen only in the posterior white matter,nerve root and cauda equina. The lesions were characterized by edema and axonal degneneration including appearance of swelling,atrophy,and loss of axon with macrophage infiltration. There were no distinctive changes in the motor neurons of anterior horn.8,There were significance differences in histopathologic score among 5 groups, P=0.000(P < 0.05).Mann-Whitney U-test indicated that there were significance differences between group NS and R,NS and R+PD1,R and R+PD2, R and R+PD3,R+PD1 and R+PD2,respectively (P< 0.005). There were no significance differences in others. The histopathologic scores of group NS or R+PD2 were less than group R and R+PD1,and one of group R+PD3 were less than group R, respectively.Conclusion1,Surgical procedure for intrathecal catheterization is simple,furthermore,it can produce less lesion.PE-10 cather is difficult to escape.2,The lesion induced by large doses of ropivacaine i.t. in rats is positive. The histopathologic changes are seen only in the posterior white matter,nerve root and cauda equina. They are characterized by edema and axonal degneneration including appearance of swelling,atrophy,and loss of axon with macrophage infiltration. There are no distinctive changes in the motor neurons of anterior horn.3,Polydatin for the dose of 10mg/kg and 15mg/kg is protective for the the neurotoxicity induced by 2% ropivacaine 40μl i.t in rats,but it is not for the dose of 5mg/kg.