| Spinal anesthesia is often used in clinical anesthesia, to keep the patient awake, anesthesia stable, postoperative analgesia easy, and also save medical costs. Since 1991, emerging nerve complications were reported after the spinal anesthesia with the local anesthetic.Through a variety of recent studies (prospective, retrospective, major case investigations, etc.) concluded that spinal anesthesia risk of neurological dysfunction was 1/1000～1/1,000,000. Rigler reported four patients had occurred cauda equina syndrome (CES) after continuous spinal anesthesia in 1991. Thus the safety of intrathecal local anesthetics has been questioned. Two years later, Schneider, who found that intrathecal with 5% lidocaine, had postoperative transient neurology syndrome.(TNS) in 1993. What local anesthetic led to a transient radicular pain is also considered one of the symptoms of spinal cord neurotoxicity. The large number of clinical studies' results suggests that:the incidence of TNS is higher than the incidence of CES. Lidocaine compared with Bupivacaine, the high incidence probability of Lidocaine is twice of Bupivacaine. CES symptom include perineal area numbness hypoesthesia, abnormal bowel and bladder function as well.as barriers associated with lower extremity motor function. The TNS symptoms of the pain from the hip to the two lower limbs radiation, intensity of pain ranging from mild to severe associated with lower limb weakness.numb, paresthesias or urinary retention.Compared to the CES, TNS is not only the performance in different symptoms and prognoses are not the same:TRI usually self-healing in 5 days, while the CES may lead to permanent neurological dysfunction. Puncture directly or catheter leaded to the nerve injury, spinal cord ischemia, infection can be diagnosed by conventional clinical methods, such as CT, MRI and other for diagnostic and troubleshooting. These factors may lead to nerve damage,whice caused by TNS and the CES,was not found in the spinal cord and nerve root lesions, thus:Neurotoxicity of local anesthetics were the largest possible. However, there is a dispute on this point of view: Some scholars believe that CES is the result of nerve injury, while TNS is a muscle disease. Rasner found that TNS is an early and mild expression form of CES. In 1997, Hartrick found that TNS is radicular pain and is a just sense of the patient, and no hard objective evidence to support it. The "radicular pain" to describe such a phenomenon has some limitations, renamed TNS, it is a transient neurological symptoms (TNS). Relationship between the TRI and TNS has not been determined, but the methods used to study the clinical trials of their exact causes have some limitations. Therefore, it required to further clarify the neurotoxicity of local anesthetic in the spinal cord. LA leaded to the ultrastructure of nerve tissue, the metabolic and electrical injury of physiological aspects and how to reduce the damage caused by LA had become the research points to make out clinical related disciplines.Local anesthetics used in spinal block anesthesia access to very good effect, at the same time, there have been some serious neurological complications. Although many studies of various in vitro and animal experiments have found that there was the spinal nerve toxicity by local anesthetic. However, in the actual complication incidence of clinical is much lower than that of local anesthetic in studies. Spinal cord mechanism of neurotoxicity caused by local anesthesia (LA) is unclear. It needs further study.Therefore, this was studied in the overall level of animals to find the LA nerve tissue injury caused by inflammatory cytokines and the relationship between local anesthetic toxicity and inflammatory cytokines. The aim is to investigate the neurotoxic effect of LA and the relationship between inflammatory cytokines and the toxic effects between LA and spinal cord by puncture injury. To solve LA neurotoxicity and to provide a theoretical basis for the prevention and treatment spinal puncture injury in clinic.PART ONEEstablish and Evaluation of the animal spinal cord injury model by punctureObjectiveTo study local anesthetics spinal neurotoxicity mechanisms, the ideal animal model of spinal cord puncture injury model should be established. We will find animal model, whitch is reliable, sensitive, to study local anesthetics spinal neurotoxicity by observe pathophysiology, movement and electric changes after injury.MethodsTotal 144 SD rats were empolied, weighing 150-200g, regardless male or female, to randomly divided into control group (sham operation group,36 rats) and experimental group (n=108). They were divided by spinal cord injury (SCI) in the experimental as follow:A group (29G, n=36), B group (25G, n=36), C group (21G, n=36). The SD rats were weighed and recorded to sevoflurane anesthesia,3% sevoflurane anesthesia induction,2% sevoflurane anesthesia maintenance. At prone position, rat skin was slitted along the back midline after routine disinfection. The spinal muscles of both sides were separated, fully exposed the spinal process and find the L5 transverse process and foramen. After determining the location, use a fine rongeur to bite the right L4 process to expose L4-5 segment of dura mater of spinal cord. The rats L4-5 spinal were puctured with angle 60 along with spinal and rat head by different kinds of pin such as 21,25 and 29 gauge. After 2 seconds the pins were draw out. Incision was sutured. Operation time was recorded.4 million units of sodium penicillin were intramuscularly injected after surgery. Lamp lighted for keep warm during operation, single-cage, check the wounds of local infection daily. In the sham group, rats had a same operation without punture the spinal cord. The checking time:the BBB score were checked before surgery, and then at SCI 8h,24h,72h, 1w, 2w, the BBB score, motor evoked potential(MEP), HE staining. rats hindlimb motor function was detected, each group had 6 animals at the corresponding phase. Motor function (BBB score) of mice in each group 8h,24h,72h, 1w,2w after operetion were respectively scored. In this study, all data were recorded as (x±s) form. With PSS 13.0 statistical analysis package, ANOVA was used to compare group data with completely randomized design analysis of variance, and LSD was used to multiple compare in groups. P<0.05 was considered statistically significant.ResultsIn this surgical manipulation of the spinal cord exposed, bleeding quantity was about 0.2ml-0.3ml, normal saline 0.4ml-0.6ml was given to supplement lost blood volume. Spinal cord injury by puncture was different due to needle the size of in the different groups, so these were the degree of local congestion. Throughout the experiment,1 rat died of hypoxia in group C, cause of respiration depression by deep anesthesia. The mortality rate was up to 2.8%. No death in group A and B.8 hours after surgery, all rats were fully awake. BBB scores between control group and experimental was significantly different P<0.05. With time on, the spinal cord function has restored. The most significant recovery was at 72h after injury. BBB scores between the experimental groups were significantly different P<0.05. In group A,the rats recovered faster than the other two experimental groups after injury. There was no significant difference with the control group at 8h, P>0.05. Group B was the second, the score was stable on 72h, the BBB score was 19.50±0.55, and fully restored at 1w. Group C had the heaviest damage, motor recovery worst, 1w BBB score was 16.67±1.37, no improvement even to 2w. The lower body shrinking appeared in group C after two weeks, varying degrees of urethral erosion, ulcer happened because of the long-term unilateral contact and friction. The peak latency of MEP of each group restored to varying degrees after experimental. MEP latency test between the experiment groups was significantly different P<0.05. MEP of group C was longer than the group B, which appeared immediately after the injury in group B. The MEP of group C was the highest at 8h after injury. Group B and C MEP latency were prolonged in 24h after the injury, and then gradually recovered. After spinal puncture congestion, spinal tissue edema and swell were appeared mainly in the damage zone around the spinal puncture, and were obvious at 8h. Group C appeared hemorrhage and edema at 8h, and gradually subsided at 24h, recovery at 3d postoperative. A large number of lymphocytes and glial cell infiltrated at 3d, capillary proliferation, the phenomenon of neuronal satellite could be seen. The proliferation of inflammatory cells and the capillaries on 1w gradually returned to normal. Some of neuron shrinkage necrosis could be seen in group A. Group C had the least damage.ConclusionAccording to the movement, nerve physiology and pathological changes, this study use 29G to pucture the rat spinal cord just imitation the human being clinical pratice. The pucture rat model is good in repeat and low injury and locatin. It may be useful in study local analgesic neurotoxicity.PART TWOInfluence of ropivacaine to inflammatory cytokines IL-1β, IL-6, IL-10 and TNF-a after ropivacaine intra-spinal cord injectionObjectiveTo explore the ropivacaine neurotoxicity mechanisms by study spinal cytokines changes with ropivacaine intra-spinal cord injection to judge to use ropivacaine or not, and also get further understanding ropivacaine neurotoxiciy results.Methods144 SD rats were randomly divided into control group (n=36) and experimental group (n=108). The control group just had spinal puncure, no spinal injection. NS group (group A) spinal cord were exposed and then gave spinal injection of isotonic saline (n=36); 0.5% Ropivacaine group (group B) spinal cords were exposed, and then gave spinal injection of 0.5% ropivacaine (n=36); 2% Ropivacaine group (group C) spinal cords were exposed, and then gave spinal injection of 2% ropivacaine (n=36). Puncture model of spinal cord was same as part one. Immunohistochemical method was use to detecte expression of 4 cytokine proteins by peroxidase labeled streptavidin biotin complex pro (SABC). Real-time quantitative PCR was used to detecte expression of 4 cytokine mRNA, detection steps:①sterile collection of rat spinal cord tissue;②spinal cord total RNA extraction.③reverse reaction:the M-MLV reverse transcriptase under the action of the mRNA molecules transcribed into cDNA.④Quantitative PCR reaction.⑤establish the standard curve:Analysis data according to the FTC-2000 software.⑥To calculate the relative quantitative value of the tested genes according to the formula. Immunoblotting (Western bloting) detection of relative expression of 4 of cytokine protein of spinal cord tissue, detection steps:spinal cord total protein extraction, protein concentration determination, SDS-PAGE gel electrophoresis, transfer, immunoassay, protein band intensity with Kodaka Digital density scanner analysis. Data were recorded as mean±tandard deviation. Statistical software SPSS 13.0 was used. Among groups, statistics were used with one-way ANOVA. LSD test was used to do multiple statistics in multiple mean comparisons. P<0.05 was statistically significant.Results(1) The relative expression of 4 cytokine protein results with immunohistochemical method in spinal. The location and number of 1L-1β, IL-6, IL-10 and TNF-αprotein positive cells was analyzed. In the contral group and group A, IL-1β, IL-6, IL-10 and TNF-a protein expressed mainly in the inflammatory infiltration cytoplasm. In group B and C,8h,24h,72h, 1w and 2w after spinal cord injury, IL-1β, IL-6, IL-10 and TNF-αprotein expressed positively, and with time on, the positive cells gradually decreased. Compared with the control group, express of IL-1β, IL-6, IL-10 and TNF-a in spinal cord tissue of the experimental group B and C, were significantly increased. There was significant difference, P<0.05. The IL-1β, IL-6, IL-10 and TNF-αprotein expression and the cell number reduced in experimental group at 2w after spinal cord injury, which was close to the control group. Compared with the control group, IL-1β, IL-6, IL-10 and TNF-αprotein expression of spinal cord tissue in group A, was not significant. There was no significant difference, P>0.05.(2) Immunoblotting (Western blotting) detection of relative protein expression of 4 cytokines results. The IL-1β, IL-6, IL-10 and TNF-a protein levels in rat spinal cord tissue at 8h after the spinal cord injury in the rat model of the experimental group (group B represented) were respectively 16.38±1.83,14.65±2.28,19.09±1.68 and 21.67±2.43, which were higher than the control group, respectively 4.53±0.47, 3.89±0.43,4.77±0.39 and 5.31±0.38, (P<0.01)ConclusionThe IL-1β, IL-6, IL-10 and TNF-a cytokines in spinal cord with ropivacaine injection of intra-spinal were highly expressed with higher ropivacaine. The time of effective of ropivacaine with single intra-spinal injection on spinal cord to release cytokines is short. IL-1β, IL-6 and TNF-αmay have damaging effects on the spinal cord, and IL-10 may had a protective effect on the spinal cord, they may participated in the pathogenesis of spinal cord ropivacaine injection. Therefore, we believe that IL-1β, IL-6, IL-10 and TNF-αmay be an important immunological markers of spinal cord ropivacaine injection. The routine testing of IL-1β, IL-6, IL-10 and TNF-αin spinal cord ropivacaine injection in clinical contribute to the early diagnosis of spinal cord injury, and also help to determine the severity of spinal cord injury and provide a theoretical basis for the rational cure ropivacaine neurotoxicity.PART THREEInfluence of ropivacaine to zinc finger protein A20 expression in spinal cord after ropivacaine intra-spinal injectionObjectiveThe aim of this experiment was to study expression changes of zinc lipoprotein of A20 after ropivacaine spinal cord injection, and also to provide an experimental basis for further study the relationship between ropivacaine and of secondary neuronal damage.Methods159 SD rats were randomly divided into control group (n=36) and experimental group (n=123). The control group just had spinal puncure, no spinal injection.0.9% NS group (group A) spinal cord were exposed and then gave spinal injection of isotonic saline (n=41); 0.5% Ropivacaine group (group B) spinal cords were exposed, and then gave spinal injection of 0.5% ropivacaine (n=41); 2% Ropivacaine group (group C) spinal cords were exposed, and then gave spinal injection of 2% ropivacaine (n=41). Puncture model of spinal cord was same as part one. Intramuscular injection of surgery 4 million units of sodium penicillin were given after operation. Lamp lighted for keep warm during operation, single-cage, check the wounds of local infection daily. Help rats to urine 2 to 3 per days after operation, until the rats had restoration of spontaneous bladder.6 rats of in each group at 8h,24h,72h, 1w and 2w after operation were given histological observation, BBB scores, same as the first part. Use TFL machine to test the TFL. Histopathological changes:After sevofrane anesthesia with 2%-3%, left ventricular was cannulated, right atrial appendage was opened,50ml normal saline lavage and then 50ml 40% formaldehyde were perfused, and then the spinal cord was harvested about 10mm along the center of puncture site. The sample were fixed with neutral formalin for 48-72h, took the puncture site, paraffin embedded, then sliced, HE staining. Protein immunohistochemistry:the samples were used with biopsy specimens S-P immunohistochemistry kit, and stained according to the instructions. The anti-A20 monoclonal antibody (rat origin IG1, U.S. ACTIVEMOTIF Company) concentration was 1:300, then diaminobenzidine (DAB) color. When Brown cytoplasmic granules appear it was positive. Select each group at each time point to do biopsy staining, six animals of each time point were selected, each animal were removed three levels, counting the number of positive cells at every level under the light microscope by two staff members not related to the experiment value, and 5 animals at each time point were statistically analyzed. Data were recorded as mean±standard deviation. Statistical software SPSS 13.0 was used. Among groups, statistics were used with one-way ANOVA. LSD test was used to do multiple statistics in multiple mean comparisons. P<0.05 was statistically significant.ResultsNo rats died in all group, there was no significant changes in activities such as eating in each group. In experiment A, B and C, eating amount and spirits decreased at first day. Every group rats were awake at 8h. There were statistically significant in Group B (0.5%ropivacaine) and C (2%ropivacaine) comparison with control group at 8h,24hand 72h (P<0.05). There were no statistically different in group A and control group. With time go on, the spinal cord function has been restored to normal. HE staining:No significant neurocyte nuclear condensation, fragmentation was found in group A and control group. There were congestion and edema after spinal puncture mainly in the damage zone around the lumbar puncture. Spinal cord edema and injury were obvious during 8h. Hematoma gradually subsided at 24h after operation. A large number of lymphocytes, glial cell infiltration, and the phenomenon of neurocyte satellite can be seen at 72h. The number of lymphocytes gradually returned to normal at 1w. A few neuron shrinkage necrosis were seen in group C but not in group B. Immunohistochemical staining results:no obviously positive expression of zinc finger protein A20 was found in spinal cord neuron cytoplasm of the control group at each time point。A20 positive expressions were seen in experiments A, B, C groups at 8h. The zinc finger protein A20 expression levels:group C>group B>group A, decreased at 24h. No significant expression was found at 1w.ConclusionThe zinc finger protein A20 is an important endogenous protective anti-inflammatory substance. A20 expression may induce by ropivacaine, which was important to conservation of inflammatory respons by ropivacaine intra-spinal cord injection, but the time is too short to stop the inflammation reaction.
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