Research Of Correlation Between Cervical Syringomyelia And Thoracic Scoliosis Though Exploring Asymmetrical Denervation Characters Of Apical Region Paravertebral Sacrospinous Muscle And Hydrodynamics Changes Of Spinal Canal Cerebrospinal Fluid Flow

Research of correlation between cervical syringomyelia and thoracic scoliosis though exploring asymmetrical denervation characters of apical region paravertebral sacrospinous muscle and hydrodynamics changes of spinal canal cerebrospinal fluid flow----Based on detecting of AChR expression level on postsynaptic membrane and measurement of CSF hydrodynamic parametersCandidate for PH.D Wang YingsongSupervior Li ShiheMember of Supervisor team Zhao XuelingDepartment of Orthopaedics, the 1st Clinical Medical College of Kunming Medical UniversityObjective1. To establish Kaolin induced non-communicating cervical syringomyelia associated with structured and progressed thoracic scoliosis animal model, then focused on early stage of scoliosis onset, compared the differences of spinal bilateral sides though counting of motor nerve terminal branches, detecting of receptor expression level on neuromuscular conjunction, and observation of muscle fiber morphological abnormal, in order to investigate whether imbalanced denervation in bilateral para-vertebral sacrospinous muscle on apex region played a key role on early scoliosis onset.2. Moreover, exploring hydrodynamics changes of the Cerebrospinal fluid (CSF) though tracer observation and fluid quantitative measurement of CSF flow, to analyze the interaction of CSF hydrodynamics changes and deformities occurrence or progression.3. Based on exploring asymmetrical denervation characters of apical region paravertebral sacrospinous muscle and hydrodynamics changes of spinal canal cerebrospinal fluid flow, offering basic clues contributed to stepwise and in-depth research on communication of deformities and pathogenetical relationship.MethodsPart 1 Established a modified model of Kaolin induced non-communicating cervical syringomyelia associated with thoracic scoliosis in rabbits.1. Animal groups. A total of 46 Japanese white rabbits were chosen for experimental object, and randomized divided into three groups, including sham operation group (8), physiological saline group (8) and experimental model group (30).2. Established animal model. Concerning sham operation group, the procedure of model establish involved:intravenous anesthesia by 2% pentobarbital sodium with a dosage of 10-15mg/kg was performed, and underwent dural sac exposure in a range of 0.3-0.4cm2 after C7 laminectomy though posterior approach. After that, fine needle had been punctured into medullae spinalis about 1.0-1.5mm depth though dura sac on the left side and lmm adjacency of posterior middle line. The position of needles needed be kept within 3 minutes, before withdrew it slowly. Concerning saline group, physiological saline 60μl was injected into medullae spinalis within 2 minutes while puncture progress finished, then the position of needle was kept within 1 minute, and withdrew it slowly. Concerning experimental model group, equivalence dosage 25% Kaolin was substituted for saline.3. Observation. Modified Tarlov score for evaluated rabbit's limb motion were launched at pre-operation,3 days post-operation,2weeks,4weeks and 8weeks post-operation, respectively.4. Detection of cervical syringomyelia. Magnetic resonance imaging (MRI) scanning on saggital plane of cervical spine and axial plane of C1-T6 were performed with a scan situation of TR/ TE as 230/ 26ms and 8000/ 264ms by 1.5T supraconduction MRI analyser at the time point of 4 weeks,6 weeks,8 weeks and 12 weeks, postoperatively, to detect occurrence or progression of syringomelia. In addition, though axial pathological sections obtained on the spinal cord level of needle puncture and Kaolin injection from MRI positive and paraformaldehyde perfusion rabbits, at 6 weeks,8 weeks and 12 weeks postoperatively, morphological changes of the central canal, ependyma and medullae spinalis were observed under HE staining and though X 100 amplification light microscope.5. Detection of thoracic scoliosis. Anterior-posterior radiographies of spine for Cobb's angle measurement were performed and morphological characters of scoliosis were recorded, at 4 weeks,6 weeks,8 weeks and 12 weeks postoperatively.Part 2 Comparative research of asymmetrical denervation characters of apical region paravertebral sacrospinous muscle in the early stage of non communicating cervical syringomyelia associated with thoracic scoliosis rabbit model1. Animal groups. A total of 20 Japanese white rabbits involved in model establishment were chosen and divided into 3 groups according to different deformities verified by MRI and x-ray, including control group (8) with syrinx or scoliosis negative, syringomyelia alone group (3) and scoliosis group (9) with non-communicating cervical syringomyelia and thoracic scoliosis.2. Specimen drawing. Para-vertebral sacrospinous muscle of both side were harvested as size of 0.5×0.5×3.0cm3 on the level of T8 or apex of main curve, at the time of 2 weeks or 4 weeks after scoliosis detected.3. Observation of muscle fiber morphological abnormal. The myogenic and neurogenic pathological changes of muscle fibers observed under HE staining and though X 100-200 amplification light microscope, and ultramicrostructure characters though electronic microscopy.4. Counting of motor nerve terminal branches. Calculating the terminal innervation ratio (TIR) of axons under muscle end-plate silver staining though light microscope.5. Detecting of receptor expression level on neuromuscular conjunction. After extracted total RNA, its concentration and purity were measured. Then cDNA 1st chain was synthesis based on total RNA as a template. At last, the expression level of Acetylcholine receptor-γsubunit, which located on end plate postsynaptic membrane, was detected by SYBR GREEN relative quantification algorithms though ABI fluorescence PCR-7000, and 2-ΔΔCT values were calculated.6. All results were crossly compared between concave and convex sides.Part 3 Tracer observation of the cerebrospinal fluid flow intra spinal cord in the early stage of non-communicating cervical syringomyelia associated with thoracic scoliosis rabbit model1. Animal groups. A total of 20 Japanese white rabbits involved in model establishment were chosen and divided into 3 groups according to different deformities verified by MRI and x-ray, including control group (8), syringomyelia alone group (3) and scoliosis group (9).2. Percutaneous cisterna magna puncture and Horseradish peroxidase injection. At 2 weeks or 4 weeks after scoliosis detected, physiological tracer 3% HRP 500μl was injected into the Subarachnoid space (SAS) of animals spinal canal within 2 minutes.3. Detection. Nearly 10 minutes after injection finished, spinal cord rapid perfusion and fixation was proceeded by 4% paraformaldehyde, then cervical spinal cord slices were taken for 3,3'-diaminobenzidine staining. CSF flow variation from SAS though perivascular spaces (PVS) to spinal cord and central canal were observed.Part 4 Research of the interaction between the hydrodynamic of cerebrospinal fluid and the procedure of posterior vertebral column resection (PVCR) in cervical syringomyelia with thoracic scoliosis adolescents.1. Research groups. Adolescents suffered from cervical syringomyelia with thoracic scoliosis were chosen for experimental group (8). Meanwhile respectively syringomyelia alone group (5) and blank control group (8) were designed.2. Measurement. Basic parameters of CSF flow in SAS were measured on each axial planes of foramen magnum, C7, T7 (or apex), L1 level by synchronism electrocardio-gating phase contrast cine Magnetic Resonance Image (PC-cine MRI) scanning, with a scan situation of TR/ TE as 32-50 /6.9 ms and 75/16ms by 1.5T supraconduction MRI analyzer. Argus software for flow analysis calculated the detail parameters including:Duration of CSF down flow(DF), Duration of CSF up flow(UF), DF/Duration of one cycle(DF%), UF/Duration of one cycle(UF%), DF starting time point/Duration of one cycle(DS%), Duration of stationary phase/ Duration of one cycle(SP%), Maximum peak velocity of CSF down flow(VDmax), Maximum peak velocity of CSF up flow(VUmax), and curve of peak velocity vs time.3. Interference. All adolescents of experimental group were avoided prior suboccipital decompression and underwent one-stage posterior vertebral column resection (PVCR) procedure for vertebral column shortening correction.4. Measurement under interference. On the time of three weeks after operation, PC-cine MRI scanning on foramen magnum and C7 level were undertaken, for compared with pre-operative parameters.Statistical analysis. Statistical analysis was applied by SPSS 11.0 software. Group t test was chosen for inter group comparison of measurement data, and paired t test for intra group. Once P<0.05 indicated significant differences.Results1. Part 1 There were three rabbits died within 2 weeks post-operation. In addition, six rabbits gradually shown weakness of posterior limbs with limitation of motion, and average Tarlov score was 2.50 at 8 weeks post-operation. Cervical syringomyelia induced by inflammation adhesion and central canal obstruction could be noticed in most experienced rabbits who were observed continuously for 12 weeks, and there were 0(0%),7(38.9%),9(50.0%) and 12(66.7%) animals detected syrinx formation at 4 weeks,6 weeks,8 weeks and 12weeks, respectively. The diameters of syringomyelia were increased gradually. Moreover, there were 4(22.2%),9(50.0%) and 9(50.0%) animals detected thoracic scoliosis at 8 weeks,10 weeks and 12weeks, respectively, with average Cobb angle of 17.75°±4.37°(12°-22°), 20.55°±9.05°(11°-35°) and 35.09°±13.22°(18°-68°). All thoracic curves were associated with cervical syringomyelia, detected at the time including synchronism (2), delay 2 weeks (5) and 4 weeks (2) accordance with syrinx be noticed, and progressed gradually. Meanwhile, all curves were shown extremely similar shape as clinical features. No syringomyelia or scoliosis appeared in other groups.2. Part 2. During 4 weeks after scoliosis onset, scoliosis rabbits were shown denervation phenotype in para-vertebral muscle, characterized by atrophy fibers as horn shape, ultramicrostructure disorder, increasing terminal branches of end plate axons, and expression up-regulation of AChR-γsubunit on postsynaptic membrane. However, neither TIR value (convex 1.14±0.18, concave 1.17±0.36) nor 2-ΔΔCT value(convex 1.628±0.002, concave 1.729±0.003) shown statistics difference between concave and convex sides in scoliosis animals.3. Part 3. All animals were observed HRP reaction products gathering around PVS in cervical spinal cord in CSF flow tracer research. But HRP reaction products gathering in different regions of medullary substance, in control group they located in dorsal white matter of the spinal cord, in syringomyelia alone group they diffused into grey matter, and in scoliosis group they even distributed widely in central gray matter, ventral and dorsal white matter, with apparent tropism towards the central canal.4. Part 4. Patients with cervical syringomyelia alone shown low dynamic characteristics of CSF flow though PC-cine MRI scanning, and blockage of the flow from intracranial to spinal canal. Once associated with scoliosis, low dynamic down flow on foramen magnum would be more notable. Meanwhile, down flow starting delay on cervical segment and high dynamic flow could be noticed. CSF dynamic situation turned to be approximate normal on craniocervical junction after PVCR procedures, and syringomyelia shrink could be detected during follow-up.Conclusion1. Established the rabbit model of non-communicating cervical syringomyelia associated with thoracic structured and progressed scoliosis, induced by Kaolin injected into medullary substance of C7 corresponding spinal cord. The animal model was closed to clinical features, established by simple proceeding, and validated by maturity iconographic techniques. It provided an idea platform for further research involving syringomyelia associated scoliosis.2. Although denervation phenotype of para-vertebral muscle was detected, scoliosis associated with syringomyelia may not originate in disequilibrium denervation on both sides of spine. It was necessary to explore another possible pathogenic mechanism.3. In the early stage of rabbit thoracic scoliosis onset, in-flow of CSF though PVS pathway of cervical spinal cord into syringomyelia was increased obviously. The dynamic change of CSF though PVS pathway influenced formation and enlargement of syringomyelia in rabbits. In addition, scoliosis intensified CSF dynamic dysfunction in rabbit cervical spinal cord.4. The first time investigated the CSF dynamic parameters intra SAS in patients with syringomyelia and scoliosis. CSF dynamic dysfunction on craniocervical junction was confirmed in patients with cervical syringomyelia alone. When associated with scoliosis, CSF dynamic obstruction on craniocervical junction turned to worse due to the spinal cord distraction then influenced formation and enlargement of syringomyelia.5. The first time performed one-stage PVCR procedure for vertebral column shortening correction avoided prior suboccipital decompression in patients with syringomyelia and scoliosis. Though PVCR procedure, effective correction of spinal deformities can be achieved. Meanwhile, the proceeding of vertebral column shortening released the tension of spinal cord, then improved CSF dynamic abnormal situation on craniocervical junction, ultimately be benefited to shrink of syringomyelia.