| ObjectiveTo explore feasibility and its mechanisms of tanshinone totalor partial substitute for methylprednisolone on the animal modelof screw compressive upper cervical spinal cord injury to reduceside effects and ensure the efficacy of the drugs.MethodsPart one:36healthy male rabbits were randomly divided into twogroups,24in the model group and12in the control group. Therabbits in model group were subjected to contusion of upper cervicalspinal cord injury with continuing screw compression mothed inelectrophysiological monitoring, and the rabbits in control groupwere stitched immediately after exposing axis lamina without spinalcord injury. Postoperative somatosensory evoked was measured toverify whether the spinal cord injury had been formed or not, andthe CT examination was made to verify whether the screws had enteredinto the spinal canal. Behavioral evaluation was observed by Tarlovscore(1day,3days,7days and14days after operation), and thelevel of the neuronal apoptotic cells was detected by the terminal deoxynucleotidyl transferase mediated dUTP nick end labelingmethods.Part two:60healthy male rabbits were randomly divided into thesham operation group (group A), model group (group B), Tanshinonegroup (group C), methylprednisolone group (group D), Tanshinonecombined with methylprednisolone group (group E), and each groupwas12rabbits. The rabbits in sham operation group were stitchedimmediately after exposing axis lamina without spinal cord injury,and other ones in the rest groups were subjected to contusion ofupper cervical spinal cord injury (CSCI) with screw compressionmethod in electrophysiological monitoring. The rabbits in group A,Bwere received equal volume of normal saline, ones in group C wereadministered with Tanshinone injection (3mg/kg/d) after CSCI, onesin group D were injected methylprednisolone (30mg/kg) via ear veinimmediately after surgery and ones in group E were injectedTanshinone combined with methylprednisolone. Tarlov score was usedto assess the motor function of limbs at3,7and14days after CSCI,while superoxide dismutase (SOD) and malondialdehyde (MDA) changeswere observed at the same time, as well as it was deleted the levelof the neuronal apoptotic cells by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling methods. Histologicalchanges of spinal cord were observed by light microscopy with400times and electron microscopy with8000times at14days afteroperation.Part three: Experimental animal groups were the same asexperiment two. All experimental groups were assessed theexpression of Caspase－3, Bcl-2and Bax mRNA by reverse transcription-polymerase chain reaction and the Caspase－3proteinby Western blotting.SPSS17.0statistical software was used to analyze databases.Measurement data was recorded by terms of mean±standarddeviation (X±s). The two groups were compared with independentsamples t test, data of multiple groups was compared with univariateanalysis of variance, the SNK-q test was used to pairwise comparisonof data within the group. It indicated a significant difference forP<0.05.ResultsPart one: The usage of sustain screw compression method was aimedto establish the animal model of upper cervical spinal cord injury,which was verified by somatosensory evoked detection. The CT scanwas used to exam whether the screws was entered the spinal canal.Compared the model group with the control group at the time of1day,3days,7days and14days after operation, the Tarlov scoreand Tunel positive cells were significantly difference(P<0.05),and the expression of the two indicators was correlated in time.Part two:1. At the time of3days after operation, Tarlov scoreand the SOD activity in group B and group D were lower than thoseof group C and group E respectively, and the content of MDA, Tunelpositive cells in group B and group D were higher than that in groupC and group E respectively, while all of them didn’t have significantdifference (P>0.05). Tarlov score, SOD activity, MDA content andTunel positive cells in each group at the time of7and14days afterCSCI were statistically significant (P <0.05).2. The performance of spinal cord tissue by light microscopy (×400): Group A showed the normal structure of rabbit spinal cord, the myelin and axons were normal; Group B showed the myelinationwas thin and partly dissolved and disappeared in white matter, andit had a sponge-like change obviously, while a part of the vacuoleswere interconnected; Compared with Group B, the extent ofstructural damage to the myelin sheath in Group C was lighter; themyelin structure in group D was intact basically, and the nervefiber edema number in group D was less than that of group C; thenumber and the extent of myelinated nerve fiber edema in Group Ereduced futher.3. The performance of spinal cord tissue by transmissionelectron microscope (×8000): the morphology of myelin sheath ingroup A was normal; Group B showed lamellar structure disordered,onion-like change, most of the structure within the axon wasdissolved and severely edema; Group C showed layered anddegenerated myelin obviously, irregular shapes, partialdissolution of the axonal structure performance, and moderate edema;Group D showed partly layering of myelin, the basic structure intactaxons, and localized edema; Group E showed mild myelin layers andslightly irregular shapes.Part three: RT-PCR test results showed the amount of relativeexpression of Caspase-3from high to low was B,C,D,E and Asuccessively. while the results of the ratio of Bcl-2mRNA and BaxmRNA was on the contrary,and had significant differences (P<0.05).Conclusions1. This study that the animal model of upper cervical spinal cordinjury is established through sustain screw compression is assistedby electrophysiological monitoring and verified by CT scan. Thereis a corresponding animal limb movement dysfunction and abnormal apoptosis after modeling. This model could basically reflect thepathophysiology of spinal cord injury, and have better operabilityand repeatability. The research may be used on the basis ofexperimental spinal cord injury.2. Tanshinone injection improves the SOD activity, decreases thecontent of MDA, inhibits the peroxidation of the rabbits after uppercervical cord secondary injury. By the scanning of light andelectron microscopy, tanshinone reduces the degree of peroxide onmyelin, axon structure damage and edema of spinal cord injury. Ithas a synergistic protective effect on nerve cells when it combineswith methylprednisolone.3. Tanshinone injection reduces the number of apoptotic cells.The RT-PCR and Western blot test results show that tanshinoneinhibits the overexpression of Caspase-3, and raises the ratio ofBcl-2mRNA/Bax mRNA, thus it reduces the apoptosis effect of oxygenfree radical injury mediated. Tanshinone injection can be long-termused after the first time in the methylprednisolone pulse therapyto reduce side effects. It has a synergistic protective effect onearly secondary injury of spinal cord, but it is not a substitutefor therapeutic effect when methylprednisolone is used at the firsttime. |
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