Experimental Research On Degeneration, Nutritional Pathway, Morphology, And Cell Apoprosis Of The Intervertebral Disk

ObjectiveDegeneration of intervertebral disk is a term to describe the lose of normal structure of intervertebral disk accompanied with the progressive fibrosis. The manifestations are gelatinous disappearance of pulpiform nucleus, rough lamellar bone of anulus fibrosus and progressive fibrosis. Finally, fissure and senile chro-matosis ensue. Studies indicate that about 23% of patients with lumbocrural pain is related to degeneration of intervertebral disk. However, little is known a-bout the convinced cause of degeneration of intervertebral disk and the physio-pathologic mechanism. For these reasons, general basic researches are needed.Methods1. Animals and Grouping90 female and male Japanese big ear rabbits were selected 8 weeks after born. The weight varied from 20g to 22g. All the rabbits were fed individually and divided into 3 groups (internal fixation group, 30 rabbits; degeneration group, 30 rabbits; control group, 30 rabbits).2. InstrumentsSurgical instruments for laboratory; Light microscope, transmission electron microscope (TEM) , Scanning electron microscope (SEM) , X - ray , CT and MRI.3. Creation of Animal Model:3. 1 Creation of Internal Fixation Model: 0. 3 ml/kg anesthetic (846 fluid) was administered by muscular injection. The rabbit took prone position after anesthesia and the position disappeared with preserved skin. 12cm posteriomedianincision around T₁₂ was performed on skin and subcutaneous tissue . Then exposed the spinous process and erector spinal muscle and isolated the T₁₀ — L₃ spi-nous process and the relevant two - sided articular processes under the periosteum. With the help of L - like needle and guide pin, thread the steel wire through the articular processes of T₁₁ , T₁₂, L₁ and L₂ and connected them with L -like needle. The internal fixation was finished sutured after hemostasis. Under the same condition to feed the animals in the internal fixation group and in the control group for more than 6 months individually.3.2 Creation of Degeneration Model; 0. 3 ml/kg anesthetic (846 fluid) was administered by muscular injection. Then fix the rabbit on the table by prone position after anesthesia and sterilize the preserved skin. 12cm posterior incision was created around L₅. Cut off the two - sided erector spinal muscles horizontally and the zygapophysial joints completely. Suture after hemostasis. Feed the internal degeneration group and the control group under the same condition for more than 6 months individually.4. Experiment Methods4. 1 Observation of Animal Models: 6 months after the preparation of animal models, X - ray was taken for the animals in the internal fixation group and for the degeneration group, X - ray, CT scan and MRI scan were performed.4.2 Research on the Nutritional Pathway of Intervertebral Disk: Distribution figures and the changes of the peripheral structure in blood vessels of cartilaginous end plate were observed after perfusion of model groups and control group with prepared Chinese ink. The details were: Fix the rabbit on the table by prone position after anesthesia. Take thoracotomy along midline to ascending aorta. Then take ligature at proximal vessels of heart and intubation at distal vessels of heart. With assurance of no leakage, blood in the right auricle of heart was released. Then infuse with 12. 5u/ml heparin salt water. Be sure of no air bubble. Do not infuse the filtered prepared Chinese ink until the irrigating solution with achromatic color flowed from the right auricle of heart. Take ligature when prepared Chinese ink flowed out of the right auricle of heart. Then infuse 700 ml ink with high pressure and take ligature at the distal vessels of heart.Take samples 3 days later and immerse intervertebral disk T₁₀, T₁₁, T₁₂, L₁, L₂, L₃ and part vertebral body into 10% formaldehyde solution for 2 weeks. After 2 -week decalcification, enbed with paraffin get 100um serial section of sagittal plane from border to center. Observe the ink distribution in both model groups and control group at the same position and the ink distribution at fixation and non - fixation area.4.3 Histological Observation: (1)light microscope: Immobilize samples with 10% for maldehyde solution for 2 weeks, Decalcify with VonEbnerMethod. Get 8um thin slice after enbeding with paraffin. Observe the stained samples with HE,Mallomy, POD. (2) transmission electron microscope: Immobilize tissue with 2. 5% glutaraldehyde. Decalcify with 1% EDTA. Immobilize with 1% osmate. Dehydrate with ethyl alcohol gradually. Transit with ep-oxypropane. Enbed with epoxy resin; epikote 812 and get ultrathin section. Observe structure and form of nucleus pulposus cellsanulus fibrosus cells by binary stain with Citric Acid. (3) scanning electron microscope: Immobilize tissue mass with 2. 5% glutaraldehyde. Divide horizontally or in sagittal plane after dryness. Observe the surface structure of the golden tissue mass.4.4 Detection of the Cell Apoprosis: Cell apoprosis is characterized by the activation of endogenous endonuclease, division of the cell chromatosome or DNA, formation of single - or double - stranded nick and 3 ' - OH terminal. Terminal deoxynuleotidyl Transferase could tag 3 ' - OH terminal with dUTP (DIG - dUTP) tagged by cedoxin. DIG - dUTP, combined with DNA break point, responded to Anti - DIG - Biotin and combined with streptomycin avidin- biotin - complex (sABC)—peroxidase. And display with substrate DAB. Pigmented apoptotic cells could be observed under microscope because the apop-totic nucleus were yellow.Results1. Animal Models:X - ray taken for the two animal models 6 months later showed intervertebral space stenosis in the related area. Iatrogenic hyperplasia of spinal body wassimilar to the degeneration of intervertebral disk in human beings. Intervertebral space stenosis was responsible for the failure of image formation of CT scanning for intervertebral change model. The results of MRI scanning supported the degeneration of intervertebral disk accompanied by certain projections. 2. Histological Observation:2. 1 Results of light microscope observation: Obvious decrease and decay of pulpiform nucleus cells were founding in the animal model groups. Collagenous fibers increased. The space betweenanulus fibrosus and pulpiform nucleus was not clear. Cells of pulpiform nucleus decreased and collagen increased. Disorder , break, even ossification ofanulus fibrosus could be found partly.2.2 Results of transmission electron microscope observation: Pulpiform nucleus and internal cells ofanulus fibrosus occurred earlier thanthe external cells ofanulus fibrosus. Protein ficoll granule and special structure were found in pulpiform nucleus and matrix ofanulus fibrosus. However, the forms of the special structure in pulpiform nucleus and internal layer ofanulus fibrosus were different. In the degeneration matrix of intervertebral disk, obvious decreases of protein ficoll granule and special structure were found.2.3 Results of scanning electron microscope observation: Considering the characters of method and scanning electron microscope, obvious differences were not found in the animal model groups and the control group.3. Detection of cell decay:The intervertebral disk samples packed by paraffin were tested with reagent kit. The results indicated higher decay rate for the intervertebral disk cells with degeneration or after internal fixation. In the animal model groups, higher decay rate occurred in the external layer ofanulus fibrosus. For the animal model groups and control group, the decay rate occurred in the former group and had statistical significance.Conclusion1. Similarities existed in the two animal models and the degenerations of intervertebral disk in human beings. They are comparable. So the basic research...