The Influence Of Oxygen-ozone Epidural Injection On Blood Biochemistry And Microstructure Of Nerve In The Rats Of Lumbar Nucleus Pulposus Prolapse

Objective: Ozone is known as one of the most powerful oxidants.Having a large excess of energy, ozone is an allotropic form of oxygen containing three oxygen automs, which are being defined and applied to biological systems as well as to clinical practice. Medical ozone (oxygen-ozone, mixture of oxygen and ozone) has been used to cure many diseases including wound , abscess , gangrene , hepatitis , herpes , osteo- myelitis,vulvovaginitis, thrombophlebitis, Raynaud's disease , AIDS , etc . For the last decade , oxygen-ozone has been used in some western-European countries for treatment of lumbar discal herniation , and good results were reported, but the animal experimental study is still less reported. During the process of percutaneous lumbar discectomy with oxygen-ozone injection, the toxicity of ozone has potential risk for the patient and becomes a reality and urgent issue . Therefore , the optimal injected dose of ozone is not defined .The aim of the study is to produce an animal model of lumbar nucleus pulposus prolapse and investigate the safty and toxicity of oxygen-ozone epidural injection with different concentration through observing blood biochemistry and microstructure of nerve root in the rats of lumbar nucleus pulposus prolapse.Moreover,to put forward constructive suggestions to clinical use according to the research results and lay the foundation for the further research .Methods : Seventy health adult male wistar rats weighing 300g were used for this study.The rat were randomly allocated into two groups :control group(group A,n=10) and experimental group(group B,n=60). The rats of lumbar nucleus pulposus prolapse were tested in group B . The rats of lumbar nucleus pulposus prolapse were further randomly allocated into four groups again:control group of group B (group BS), pure oxygen group of group B(group BO₂), oxygen-ozone 40 group of group B (group BO₂-O₃40)and oxygen-ozone 80 group of group B (group BO₂-O₃80). Pure oxygen and oxygen-ozone of different concentration including 40mg/L,80mg/L, were injected into the rat epidural cavum of three groups: group BO₂, group BO₂-O₃40 and group BO₂-O₃80 except group BS with only puncturation.The animals were anesthetized with intraperitoneal 1% ketamine 50mg/kg.Clip the caudal vertebrae .Each nucleus pulposus of coccygeal vertebra was about 10mg . Five nucleus pulposus were obtained from each animal in group B and added to 50μl of normal saline (NS) and made into autologous nucleus pulposus suspension.In group A NS 20μl and 2% lidocaine 30μl were injected into epidural space at L4～5 interspace . In group B autologous nucleus pulposus suspension 20μl and 2% lidocaine were injected into epidural space . 1 day before operation , 3 days and 6 days after operation, The cauda equine nerve conduction velocity was observed . 7 days after operation , the animals were anesthetized with intraperitoneal 1% ketamine 50mg/kg . First,NS 20μl and 2% lidocaine 30μl were injected into epidural space at L4～5 interspace . Pure oxygen and oxygen-ozone of different concentration including 40mg/L,80mg/L, were injected into the rat epidural cavum of three groups:group BO₂ , group BO₂-O₃40 and group BO₂-O₃80.Before and 1, 4 , 24 hours after puncturation or injection , 1ml blood serum was collected to detect the content of SOD , MDA and its ratio in rat . 24 hour after injection , the animals were anesthetized again and killed by intra-aortic injection of 4℃NS and 4% glutaraldehyde . The microstructure of L5 nerve root was observed by light microscope and electron microscope .Results:1 To produce an animal model of lumbar nucleus pulposus prolapse.The rats in group B appeared irritability and activity frequently after operation .The conduction velocity changes of cauda equina nerve roots was pronounced ,especially 6 days after the operation .Compared with group A , the sensory nerve conduction velocity and motor nerve conduction velocity of rats in group B stepped down ,3 days and 6 days after the operation (p<0.05).The conduction velocity stepped down more dramatically,6 days after the operation .2 The influence of oxygen-ozone epidural injection with different concentration on blood serum superoxide dismutase (SOD), malondialdehyde(MDA)and its ratio in rats of lumbar nucleus pulposus prolapse.(1)Compared with group A: SOD,MDA and its ratio of group BS were significantly lower (P<0.05); (2)Compared with group BS, SOD of group BO₂-O₃40 were significantly higher 4 and 24 hours after injection (p<0.05), group BO₂-O₃80 were significantly higher 4 hours after injection (p<0.05). MDA of group BO₂-O₃40 were significantly lower 4 and 24 hours after injection (p<0.05), MDA of group BO₂-O₃80 were significantly higher 4 hours after injection (p<0.05). The ratio on SOD/MDA of group BO₂-O₃40 were significantly higher 4 and 24 hours after injection (p<0.05) .3 The microstructure of L5 nerve root was observed .HE results illustrated that the structures of axon and myelin sheats were normal in the group A .There were edema of nerve root, inflammatory cell infiltration, the splitting and distortion of myelin sheaths in the group BS . We also found segmental demyelination in some nerve fibers of group BS .Compared with group BS, the kind,amount and,morphology of cells and nerve fiber have not been changed in three groups (group BO₂﹑group BO₂-O₃40 and group BO₂-O₃80) observed under HE stain . Electron microscopic examination showed that the structures of axon and myelin sheats were normal in the group A. In the ultrastructural observation of nerve root , we found that the nerve fiber was swelling,layering, solution, disintegration, fracture, denaturation and edema between myelin sheath in the group BS. Compared with BS, there were also not obvious changes in three groups (group BO₂﹑group BO₂-O₃40 and group BO₂-O₃80) observed by electron microscopic .Conclusion:1 The conduction velocity of of cauda equina nerve roots stepped down obviously after the autologous nucleus pulposus were transplanted . Indicates that the animal model of lumber pulposus prolapse was produced successfully .2 Compared with group A, SOD,MDA of group BS were significantly lower(p<0.05).Indicates that antioxidization capacity of the rats with lumbar nucleus pulposus prolapse decrease . Oxygen-ozone epidural injection with 40mg/L and 80mg/L can increase the level of blood serum SOD of the rats with lumbar nucleus pulposus prolapse . Indicates that Oxygen- ozone epidural injection can increase the antioxidization capacity to alleviate the damage by oxygen free radical . Reaction of lipid peroxide in the blood serum was transient increased following the dose increasing. Indicates that epidural injection with oxygen-ozone has potential toxicity in rats of lumbar nucleus pulposus prolapse.3 Rats'autologous nucleus pulposus displayed significant microstructural of nerve root changes. Indicates further that the animal model of lumber pulposus prolapse was produced successfully. 24 hours after oxygen-ozone epidural injection with 40mg/L and 80mg/L, there was not more serious damage observed by light microscopic and electron microscopic. Though the microstructural of nerve root observation, Oxygen-ozone epidural injection in short term is safe ,but the long term result need to research.