Changes in neurological behavior, radiology, and pathological outcomes

Establishment of a rat model of chronic cervical spinal cord compression: changes in neurological behavior, radiology, and pathological findings  
Summary:  
Cervical myelopathy is caused by compression of the chronic part of the spinal cord caused by degeneration of the spine. In any case, the exact mechanism of chronic cervical spinal cord compression is not fully understood. The aim of this study was to establish an effective new animal model of chronic spinal cord compression without cone and plate resection to replicate the clinical course. A polyethylene wire tied to a plastic plate that was tied to a C4 vertebral body of a one-month-old rat and wound three times. After surgery, as the spinal canal and vertebral body grow, the polyethylene wire gradually grows into the bone on the dorsal side of the spinal canal, resulting in progressive compression of the spinal cord. The results showed that after 9 months of cervical spinal canal stenosis, rats with cervical spinal stenosis showed dyskinesia and sensory disturbance; however, clinical symptoms did not appear until 6 months. After 12 months, 70% of the cervical spinal stenosis model rats showed high intraspinal signal. In the pathological section, the entire circumference of the spinal cord was compressed 12 months after the formation of cervical spinal canal stenosis. The number of motor neurons is reduced, and white matter shows Waller's degeneration. This model may be able to replicate the characteristic clinical features of clinical chronic cervical spinal cord compression, including the appearance of latent and invisible neural clouds, progressive motor and sensory function, and represent human chronic cervical spinal cord compression.  

Keywords: animal studies; behavioral assessment; nuclear magnetic resonance; neuronal death; spinal cord injury.  

introduction:  
It is generally believed that the etiology of chronic compression myelopathy is related to the pathological features of the cervical spine, such as vertebral joint sclerosis, ligament ossification, spinal stenosis, and disc herniation, which may be caused by mechanical compression and vascular disease. Chronic oppressive myelopathy occult and progressively impairs motor and sensory kinetic energy. Many patients have no clinical symptoms for several years. Although there are spinal cord compressions and defects during this period, once the clinical symptoms of the disease appear, the dysfunction of the dysfunction is progressively aggravated and rarely reversed. A study of a randomly selected symptomatic cohort of cervical magnetic resonance imaging showed that 26% of subjects older than 64 years had spinal cord injury and 16% of subjects aged 45-64 years had spinal cord injury. Clinically, even those severely diagnosed by neuroimaging often show unpredictable findings of non-essential neurological function, and considerable neurological recovery after surgical decompression may be expected. Therefore, an acute spinal cord injury of chronic spinal cord compression is not characterized by spinal plasticity. So far, the clinical effect of chronic cervical spinal cord surgery has continued to increase. In any case, there are still some unsatisfactory results, even if adequate decompression surgery. This means that the cervical spinal cord is severely damaged so that it is less resilience during the course of the disease. The exact pathological mechanism of the injury process is still not fully understood, but hypoperfusion and vascular embolization have proven to be possible factors. Spinal pathology of cadaveric specimens revealed a decrease in motor neurons and vascular degeneration in the gray matter, as well as demyelination and axonal edema. A suitable model for a secondary pathological condition should demonstrate the occult phase of occult neurological dysfunction after compression introduction, followed by a progressive dysfunction phase. Further, it is important to have a model without spinal cord injury. The aim of this study was to develop a new model of chronic spinal cord compression injury to meet these clinical conditions, using MRI and pathology to assess motor sensory function.  
material:  
animal  
This experiment was conducted under the supervision of the local animal ethics committee and the University Animal Surveillance Regulations, as well as the Japanese Government Animal Protection and Management Regulations and the Japanese Government Animal Feed Notice. Twenty-seven male SD rats were used for the experiment, 3 weeks old, with an average body weight of 51 g. Animals are kept under laboratory conditions of 22 degrees humidity and 55% humidity, while free access to water and food.  
Production of cervical spinal stenosis model  
Before surgery, animals are kept in cages for a week to adapt to the environment. Anesthesia (35 mg/kg) was injected intraperitoneally with sodium pentobarbital. Each rat was placed in a prone position on a hot plate and the procedure was performed under strict aseptic conditions. The body temperature was maintained at 37 ° C, and the blood pressure was monitored with a non-invasive blood pressure monitor to maintain a stable physiological state during the operation. Centering on the C4 spinous process, make a longitudinal skin incision and blunt the paravertebral muscles on both sides of the cone. The bilateral cone plates were exposed, and the roots of C4 and C5 were separated under a microscope and excised. Then, the C4 spinous process is excised and laminectomy is performed. The left side of C4 was pulled from the side to one side, and the polyethylene was passed through the ventral side of the C4 vertebral body, and fixed on a plastic piece of 1*2*0.5 mm size against the ventral side of the C4 vertebral body, and then fixed three times. In this model, as the spinal canal and vertebral body grow, the polyethylene wire gradually grows into the lamina of the spinal canal. Thus a progressive spinal cord compression is formed. The control group was only exposed to the C4C5 lamina and was not tied. Ten rats in the control group and 10 rats in the control group were continued for 12 months after the laboratory conditions described above.  
Neurological function evaluation  
BBB score. Neuromotor function was assessed by BBB scores at 1, 3, 6, 9, and 12 months after surgery. The BBB score is mainly used for evaluation of motor function after experimental spinal cord injury. In short, the BBB is a 21-point sequence, from 0 (no hind limb movement) to 21 points (continuous coordinated hind limb movement). 0-7 is divided into the early stages of recovery, 8-13 points describe the intermediate stage of recovery, 14-21 represents the late stage of recovery, with staged toe cleaning, primary paws, gait stability, nail position . Two examiners observed and evaluated the 4-min movement of each rat. Neurological examination uses double-blind techniques.  
Nuclear magnetic resonance research  
MRI study: NMR study was performed with a 0.4T permanent magnet at 3, 6, 9, and 12 months postoperatively. The inspection series includes T1 (350/25), T2O (5000/119), field of view 150mm, 5mm thickness level, 256*256 matrix, and 4 excitation points. The scan time is 5-6 minutes. All animals were scanned in the prone position and the neck segment was placed in the center of the coil at the isocenter of the static magnetic field.  
The cross-sectional area and compression rate of the spinal cord were obtained from the region of the maximum compression level using a MR image digital converter with a T1-weighted sagittal plane. The compression rate is the diameter of the cross section of the anterior and posterior diameter spinal cord. The compression rate and cross-sectional area of ​​the spinal cord of the experimental group and the control group were compared.  
Whether or not the intramedullary high signal region is examined with a T2-weighted image. Quantitative measurements are measured by ROI techniques to measure the high signal of the cervical spinal cord. All sequences of SDNRS were used to evaluate the cervical spinal cord to evaluate the spinal cord and surrounding tissue within its spinal canal. SDNRS measures the signal intensity (mean ROI) of the spinal cord and the coding direction of the extra-neck phase, including ghosting artifacts. The difference between the two obtained variables is in the same image.  
Histopathological examination:  
After MR imaging studies, all rats were sacrificed for histopathological examination. All animals were fixed with 4% formalin infusion. The cervical spine was removed and fixed in the same solution for 1 week. Specimens were decalcified with 0.5 m ethylenediamine acid and embedded in stone standard procedure wax. Under the optical microscope, the specimens were cut from C2 to C7, and the cross section of the head to the end was cut out, a total of 600 pieces, each 5 μm thick. For each of the 10th Nissl stains, a micrograph of each stained fragment contained the entire ventral horn.  
Motor neurons are identified with large nucleoli and well-developed densely stained Nissl bodies. The nucleolus, the normal center, contains a normal boundary with the surrounding nuclei. in order to achieve,. In order to obtain accurate neuron counting, without missing or repeating, we chose to slice 5 μm thickness, leaving a distance of 5 μm, based on the following stereoscopic considerations: a typical large core is about 5 μm in diameter, and this diameter is consistent; the thickness of the slice is chosen. And a spherical nucleolus with a thickness of 5 μm, which means that each slice will contain the complete outline of the nucleolus, and their center is within 2.5 μm outside the slice. Therefore, the nucleoli of motor neurons were counted. These nucleoli appeared in the field of view with a thickness of 5 μm. These nucleoli were confined within 5 μm on each side of the center of each slice, so leaving a gap of 5 μm between the slices means stereo counting. It is possible to avoid omission or duplication of the number of motor neurons.  
Statistical Analysis:  
Data statistics: Data are expressed in terms of mean + standard. BBB scores, maximum treadmill speed, sensory evaluation, statistical data from MR image studies, and the number of motor neurons were measured using Wilcoxon's rank sum test using spss11. p<0.05.  
RESULTS: After surgery, the animals were healthy and free of any infection. The survival rates of the control group and the experimental group at 12 months after surgery were 76.9% and 71.4%, respectively. Their weight gain is rapid, from 51+-1.1 grams at the time of surgery, to three months later to 481.7 – 14.7 g (control rats), or to 465.0 – 19.8 g (CCS model rat). Subsequently, both groups gained weight, and after 12 months, the body weight of the control and experimental groups increased to 706.7 – 43.2 g and 679.1 – 43.9 g, respectively. There was no significant difference in growth between the two groups.  
Neurological results:  
Neurological function was measured at 3, 6, 9, and 12 months after surgery. At 3 months and 6 months, no rats developed symptoms of myelopathy, and the BBB score and maximum treadmill speed were similar to the control group. Two of the 10 experimental groups showed a decrease in the BBB score and the maximum treadmill speed. At 12 months after surgery, all the animals in the experimental group developed symptoms of myelopathy. The 12-month postoperative BBB score group (17.3 –+ 1.6) was lower than the control group (20.8 +– 0.4), and the experimental group had the maximum treadmill speed (21.7 – +4.5 m/min), which was lower than the control group. (33.3 – +3.5m/min). In the von frey fiber test, the rats in the experimental group had a similar procedure to the control rats until 6 months after surgery. 3 showed slight numbness in the forelimbs and hind limbs. Subsequently, 12 months later, in all experimental models, the frequency of response of the forelimbs decreased (26.0 – 12.2.6%) and the frequency of response of the hind limbs decreased (21.0 – + 23.8%).  
Nuclear magnetic resonance:  
Magnetic resonance imaging was performed 3/6/12 months after the cervical spine model. On the MR images, from 6 months onwards, the spinal cord of CCS model animals gradually flattened and deteriorated to 12 months. The cross-sectional area of ​​the spinal cord obtained from the sagittal T1 image of the spinal cord is reduced, and the number of motor neurons is reduced. (Table 1 and Picture 5). White matter in all CCS models showed myelin destruction, and spongy axonal degeneration. Pathological changes occur mainly below the compression zone.  
Histopathological findings: establishment of a rat model of chronic cervical spinal cord compression: changes in neurological behavior, radiology, and pathological findings  
discuss:  
Cervical myelopathy is the most serious consequence of oppressive myelopathy, such as tough joints, ossification of the ligaments, spinal stenosis, and disc herniation, but his pathological details are not understood. One reason is that the satisfactory chronic cervical spinal cord compression model of experimental animals has not been established. So far, a large number of methods have been used to generate spinal cord compression injuries; however, most useful models are only suitable for acute or subacute compression injuries, using cone and plate resection to form spinal cord injuries, thus dura mater, vascular injury and Scar tissue formation. There is no suitable animal model for analyzing the effects of chronic cervical spinal cord compression. Other models, including transplantation of tumor cells, progressive tightening of screws, transplantation of dilated slices, and ligament ossification of rat spinal ligaments. These models have some drawbacks, such as the tumor model growing too fast, the epidural tissue damage in the direct slice or screw transplant, the knockout mouse compression point lacks selectivity, and may occur in vertebral bodies other than C1-C2. In our model, the polyethylene line utilizes the growth of the spinal canal itself, deep in the enlarged spinal canal, which will progressively compress the spinal cord. The cervical spinal cord was compressed without performing surgery on the spinal canal, so the epidural tissue was not damaged, and the scar tissue removed by the cone and plate did not occur. The bone maturation of rats and humans is about 3-4 months old, and 13-16 years old, respectively. Rat and human spinal cord growth was completed approximately 3 months and 18 years after birth. Therefore, 3-week-sized rats and 12 months corresponded to human adolescents and old age. Some studies have found that the morphology of human compression spinal cord is related to clinical symptoms in CT or MRI images. According to reports, if the cross-sectional area of ​​the spinal cord is less than 55-75% when confirmed by CT imaging and MRI, the spinal cord cannot be tolerated. In addition, the typical feature of human cervical spinal cord compression is the occult and delayed appearance of clinical symptoms after spinal cord compression. In this study, cervical spinal cord compression did not result in acute persistent neurological dysfunction of the spinal cord. All animals returned to normal neurological status after surgery. On the MRI image, the spinal cord of the cervical spinal canal stenosis gradually flattens, starting from 6 months after surgery.  

Conclusion: This study established a new and effective model of chronic cervical spinal cord compression that does not directly damage the rat spinal cord. In this experiment, rats with bundled C4 vertebral bodies with polyethylene wire developed dyskinesia and sensory disturbance after 9 months; however, clinical symptoms did not appear until 6 months. This occult and delayed symptom is one of the most typical features of chronic spinal cord compression. Therefore, this model may have replicated the process of chronic cervical spinal cord compression in humans.  

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