Homoimmunity Of Neucleus Pulposus To Rabbits And The Clinical Forensic Significance: An Experimental Study On Lumbar Disc Herniation

AbstractLumber disc heniation (LDH) is one of the most common diseases in the clinic. It is also called annulus fibrosus disci intervertebralis disruption or prolapse of nucleus pulposus (NP) of lumbar spine, etc. In the pathogenesis of LDH, physiological cataplasia is the main reason, trauma or other causes are less minor. So that, there are lots of cases which are commonly related with the discri-ptionbetween truma and disease about LDH, and the concluding of the evoked ti me.During the work of forensic clinical identification, as a result of the specific psychoeffect of the person concerned, it is much more important to accurately c -onclude the happening time of LDH. At present, CT MRI and other imaging, electrophysiological techniques can only be used for the diagnosis of LDH. But neither they can tell the reversal point of the continuous process of physiologica lcataplasia and LDH, nor they could conclude the protrution time exactly.Most of the patients whose final diagnosis are LDH, have low back or radical pain and other symptoms which are caused by the nucleus pulpsus (NP) h-erniation. About how LDH evokes the pain, there have been a lot of disputes i-n the history. Nowadays, the autoimmune and the inflammatory reaction theories are the most popular ones.In 1965, Bobechko,etal. proposed that the neucleus pulposus (NP) of disc has the foundation of autoantigen. The largest diversity between this theory and the previous ones is that its key point is the disruption of the annulus fibrosus which results in the exposure of the neucleus pulposus and the autoimmune reaction. In fact, this is also the most importent key point of the continuous process of the physiological cataplasia and LDH. In our experiment, we use neucleus pulposus from the variant rabbits of the same race to immune the experimental animals. We made an animal model for the immunology mechanism of LDH. At last we verified this theory and checked whether or not the animals produce d specific antibodies to the neucleus pulposus.Materials1. Experimental animal: Male adult Newsland rabbits (n=50) weighing about 2.0-2.5 kg, purchased from the department of experimantal animals in China Medical Universtiy. Raised adaptively before the experiment.2. Experimental reagents: Freund's complete adjuvant; Freund's incomplete adjunvant(provided by the immunity department of China medical university); HRP-IgG, HRP-IgM( purchased from the Zhongshan reagent limited company); BSA(purchased from the Boster Biotechnology Co.LTD).3. Experimantal instruments: homogenating machine; deep freeze refrigerator; highspeed centrifuge; magnetic shaker; PH detection instrument.Methods1. Animal grouping: Experimental rabbits were housed for 7days to adapt the conditions of the lab. After that, they were divided into 4 groups randomly: 10 rabbits for antigen abstraction; 10 rabbits for NS control group; 10 rabbits for adjuvant control group; 20 rabbits for the experimental group.2. Antigen abstraction: Take the neucleus pulposus from every segment of the spine, pay attention not to infiltrate any of the blood, muscle, annular fibrosus or other foreign substances. Then, homogenate them in the icy water at a ratio of 4000r/min, centrifuge at 6000r/min, 20min, take the supernatant. Determ- ine A₂₆₀, A₂₈₀ with the spectrophotometer. Finally, calculate the protein density using Lowry-Kalckar formula, and keep the supernatant under -70℃in the deep fridge separatly (1.0-2.0ml) .3. Immunologic process: After living adaptively for 7 days, immune the rabbits with the mixture of antigen and Freund's complete adjuvant. There will be a booster with the incomplete adjuvant every 2 weeks, 3 times in total.Every 2 weeks, collect the venous blood of the rabbits'ears. After standing 1h, put the samples into the fridge at 4℃for 24h. Separate the blood serum at a ratio of 10000r/min, 10min. Then keep the them in the fridge at -20℃. Among these, group 0 is the control serum to every rabbit, and the others is the serum of the animals after immuning.4. Detect the content of IgG, IgM with indirect ELISA: Invest the ELISA plates with the antigen which is made by ourselves, use the indirect ELISA to check the antibodies for the neucleus pulposus.5. Statistics methods: Analyze the data with SPSS11.5 software.ResultsAfter collecting the venous blood at the end of the immune cycle, separate the serum of the control and the experimental groups, check the expression co-ntentof IgG, IgM in the two groups. Mean IgG content of the immunity group, own control group, adjuvant control group, NS control group is 0.7566±0.1607 (OD450) , 0.4936±0.0491 (OD₄₅₀) , 0.4690±0.0512 (OD₄₅₀) , 0.4339±0.0636 (O D₄₅₀).Mean IgM content of these groups is 0.0745±0.0139 (OD₄₅₀) , 0.0441±0. 0046 (OD₄₅₀) , 0.0438±0.0054 (OD₄₅₀) , 0.0426±0.0033 (OD₄₅₀). There is significant differences between the own control and the immunity groups, P <0.05; and there is no significant differences between the adjuvant and NS control grou ps. But, of course, the immunity group and the last 2 control gruoups are quit different (P<0.05). Conclusion1. Set up a new kind of animal experimental method to certificate the dysi-mmunity of LDH, and certified that after the exposure of neucleus pulposus (NP) to the peripheral circulation, the humoral immuity changes significantly.2. The neucleus pulposus (NP) of lumbar disc is a kind of autoimmune antigen.3. After the homoimmunity of neuleus pulposu (NP), the IgG, IgM content increased respectively.4. Through the subsequent research, the regular pattern of the IgG, IgM content changes is expected to be used as a new kind of index for the dating of LDH.