| 1BackgroundsSpinal cord injury (SCI) is a common clinical disease. The incidence of SCI is increasing in recent years, due to rapid economic development and traffic accidents occurred frequently. It often causes loss of sensory-motor functions and bowel and bladder dysfunction, so the patients not only have a great deal of inconvenience in their lives, but also the patient’s families and the community bear heavy burdens. However, effective comprehensive treatments have not yet been discovered, the studies of SCI have been a focus in the medical profession.The studies of SCI are based on animal models and the contusion model is widely used. In1911, Reginald Allen developed a SCI model, and he reported that midline myelotomy reduced progressive tissue damage in contused spinal cord in1914. In1985, Wrathall and colleagues described morphological and behavioral changes in a rat weight-drop contusion model. In1989, Young developed a rat contusion model at New York University, initially called the "NYU Impactor". In recent years, many researchers continue to improve contusion model and this model applied to clinical relevant cervical spinal cord injury, in2005, Pearse developed a electromagnetic-driven spinal cord contusion device (ESCID) base on OSU, which produced0.80,0.95and1.10mm contusion at C5level of spinal cord, and he found that three levels of contusion generated light, moderate and severe degree of SCI, respectively.Vertebral fracture-dislocation has been reported as the most common cause of SCI in human adults. The vertebral dislocation models of SCI were established in recent years, which were more relevant with clinical. Fiford initially developed a thoracolumbar dislocation model of SCI, a clamp attached to the stationary beam of the device was attached to T12, and the other translating beam and clamp assembly was attached to L2. The stationary beam remained fixed, whilst the translating beam underwent a linear lateral displacement to the left at different displacements, and he reported the displacement was greater, the secondary injury at6hours was more severe. Choo developed a cervical anterior dislocation model of SCI, and found the character of SCI due to fracture-dislocation was different to those caused by contusion and distraction either in primary injury or secondary injury at3hours. In1983, Tator found anterior fracture-dislocation was more common than lateral fracture-dislocation. It has been reported that anterior fracture-dislocation produces a higher percentage of complete cord injuries in clinical, Clarker compared the lateral dislocation with the anterior dislocation in rat thoracolumbar spine, and found hemorrhage volume, the injured axons and degenerating neurons were more pronounced following the anterior dislocation. In addition, there was a different spatial distribution of axonal damage between the anterior and lateral dislocation.However, in the previous studies, the anterior dislocation was2.5mm at C4-C5level, shearing the spinal cord as high as90%. The rats were injured too sever to survive for studying the secondary SCI over a longer time. There is a need to investigate this innovative SCI model at different lower levels of dislocation associated with mild or moderate SCI.2Objectives2.1To establish a primary SCI model of rat cervical spinal dislocation fracture;2.2Compare severity of the primary SCI under different dislocation fracture displacement;2.3Evaluate effect of speed of dislocation on the primary SCI in this model.3Methods3.1The establishment of SCI modelThis study used16male, Sprague-Dawley (SD) rats with the body weight of295g to315g. The animals were divided into2groups for the study:a sham control group (n=8) and in vivo group (n=8). A rostral clamp connected to a stereotaxic apparatus held C3and C4and kept stationary during injury, while a caudal clamp held C5and C6and was connected to a material testing machine, the caudal clamp was driven dorsally up to1.90mm at the speed of2mm/s and returned to the original position, producing C4～5dislocation fracture. The displacement and force were recorded continuously during injury. After dislocation, the spinal cord and the fracture at the C4-C5were observed, and the volume of hemorrhage in spinal cord were calculated. The process of the control group was the same as the in vivo group, except dislocation.8male SD rat cervical specimens (the weight295g-315g) were collected in the in vitro group, The caudal clamp was driven dorsally up to1.60mm at the speed of2mm/s and returned to the original position, producing C4～5dislocation fracture. The displacement and force were recorded and the fracture at C4-C5was observed. Differences of the mechanical variables between the in vivo group and the in vitro group were determined using independent-samples t test. 3.2Effect of dislocation displacement on the primary SCI28male SD rats with the body weight of295g to315g were divided into4groups for the study:a sham control group (n=7),1.3mm group (n=6),1.6mm group (n=7) and1.9mm group (n=8). The caudal clamp was driven dorsally up to1.30mm,1.60mm and1.90mm at the speed of200mm/s and returned to the original position, producing C4～5dislocation fracture. The displacement and force were recorded continuously during injury. After dislocation, the spinal cord and the fracture at the C4-C5were observed, and volumes of hemorrhage in the spinal cord in three experimental groups were calculated. We compare the three groups about the mechanical variables and the volume of the hemorrhage using one-way ANOVA. Differences of the volume of hemorrhage between the white matter and gray matter in the same group were determined using paired-samples t test. The degree of the injured axons was observed with immunochemistry.3.3The effect of dislocation speed on the primary SCI24male SD rats with the body weight of295g to315g were divided into3groups for the study:a sham control group (n=8),1.3mm group (n=8),1.6mm group (n=8). The caudal clamp was driven dorsally up to1.3mm and1.6mm at the speed of2mm/s and returned to the original position, producing C4～5dislocation fracture. Combined with previous data, we compare the mechanical variables and the volume of the hemorrhage at the same level of displacement between the slow group (2mm/s) and the fast group (200mm/s) using independent-samples t test. Under the speed of2mm/s, differences of the mechanical variables in the three groups were determined using one-way ANOVA, differences of the volume of hemorrhage between the1.6mm group and1.9mm group were determined using independent-samples t test, differences of the volume of hemorrhage between the white matter and gray matter in the same group were determined using paired-samples t test.4Results4.1The establishment of SCI modelUnder the speed of2mm/s, all C4～5intervertebral disks were ruptured at the C4inferior endplate. In the in vivo group and the in vitro group, on average, maximal force was12.7N and9.7N, and rupture displacement1.00mm and1.11mm, respectively. There was no significant difference in maximal forces and rupture displacements between the in vivo group and the in vitro group.Two symmetrical hemorrhage strips or dark points were observed on all spinal cords in the in vivo group. The hemorrhage was mainly located in the gray matter and scattered in the white matter more dorsally than ventrally. In slices of the spinal cord with HE staining. On average, hemorrhage volume in white matter, in gray matter and in total spinal cord was0.79mm3,1.63mm3and2.42mm3.There were no injury in spinal cord and intervertebral disks in the control group.4.2Effect of dislocation displacement on the primary SCIDuring the dislocation at the speed of200mm/s, all C4～5intervertebral disks were ruptured at C4inferior endplate, the maximal force was14.7N,13.5N and16.4N, the rupture displacement0.85mm,1.02mm and1.03mm, and the velocity199mm/s,209mm/s and202mm/s in the1.3mm,1.6mm and1.9mm group, respectively. There was no significant difference between the three groups about the maximal force and the rupture displacement. There was significant difference between the three groups about the velocity.There were two symmetrical light hemorrhage points on all spinal cords in the1.3mm group, two symmetrical dark hemorrhage points in the1.6mm group, and two symmetrical hemorrhage strips in the1.9mm group. The hemorrhage was almost located in the gray matter for the1.3mm group. The hemorrhage was mainly located in the gray matter and scattered in the white matter more dorsally than ventrally in the1.6mm and1.9mm groups. Moreover, the hemorrhage in the1.9mm group was more than in the1.6mm group. In the1.3mm,1.6mm and1.9mm group, average volume of hemorrhage was0.04mm3,0.10mm3and0.53mm3in the white matter,0.21mm3,0.62mm3and1.12mm3in the gray matter, and0.25mm3,0.72mm3and1.65mm3in the spinal cord. There was significant difference between the1.6mm group and the1.9mm group about the hemorrhage in gray matter and total spinal cord. There was significant difference between the1.3mm group and the1.6mm group about the hemorrhage in the gray matter and the spinal cord. There was significant difference between the1.3mm group and the1.9mm group about the hemorrhage in the gray matter, in the white matter and the spinal cord.Neurofilament200and β-Tubulin Isotype Ⅲ staining showed that the dorsal and ventral columns were predominantly spared while damage was localized to the gray matter in1.3mm group. In the1.6mm group, the damage was almost within the gray matter with few axons ruptured in the dorsal column. In the1.9mm group, the damage in the gray matter was extended to the dorsal and ventral column, and server dorsally than ventrally.There were no injury in spinal cord and intervertebral disks in control group.4.3The effect of dislocation speed on the primary SCIDuring the dislocation at the speed of2mm/s, all C4～5intervertebral disks were ruptured at C4inferior endplate, the maximal force was14.7N and11.3N, the rupture displacement0.99mm and0.99mm, the maximal displacement1.31mm and1.62mm in the1.3mm group and1.6mm group. There was no significant difference between slow group and fast group about the maximal force, the rupture displacement and the maximal displacement at the level of1.3mm and1.9mm displacement. There was no significant difference between slow group and fast group about the maximal force, the rupture displacement at the level of1.6mm displacement, except the maximal displacement. There was no significant difference between the three groups at the speed of2mm/s about the maximal force and the rupture displacement.Under1.3mm dislocation, there were two symmetrical light hemorrhage points in the fast group, while no hemorrhage in the slow group, there were two symmetrical dark hemorrhage points in the slow group and fast group under1.6mm dislocation, and two symmetrical hemorrhage strips in the slow group and fast group under1.9mm dislocation. Under1.3mm dislocation, the hemorrhage was almost in the gray matter in the fast group, while no hemorrhage in slow group. The hemorrhage was mainly located in the gray matter and scattered in the white matter more dorsally than ventrally in the slow group and fast group under1.6mm and1.9mm dislocation. Moreover, the hemorrhage in the slow group was more than in the fast group and the hemorrhage in the1.9mm group was more than in the1.6mm group at the speed of2mm/s. In the slow group and fast group, average volume of hemorrhage was0.12mm3and0.10mm3in the white matter,0.82mm3and0.62mm3in the gray matter,0.94mm3and0.72mm3in the spinal cord under1.6mm dislocation, average volume of hemorrhage was0.79mm3and0.53mm3in the white matter,1.63mm3and1.12mm3in the gray matter,2.42mm3and1.65mm3in the spinal cord under1.9mm dislocation. However, there was no significant difference between the slow group and fast group under the1.6mm and1.9mm dislocation. Under the speed of2mm/s, there was significant difference between the1.6mm group and the1.9mm group about the hemorrhage in white matter, in gray matter and in total spinal cord.There were no injury in spinal cord and intervertebral disks in the control group. 5ConclusionsA cervical spine fracture dislocation of SCI model was established successfully in this study. The dislocation resulted in hemorrhage in the spinal cord, and ruptured the spinal column, which was clinical relevant.The present results suggested that different dislocation displacement could generate the primary spinal cord injury from light to moderate injury, and even severe injury at1.9mm dislocation.We found that high speed dislocation could generate the primary spinal cord injury at lesser displacement. |
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