Effects Of Velocity And Contusive Displacement On The Primary Spinal Cord Injury Of Cervical Spine In Rats

1.BackgroundsSpinal cord injury (SCI) is a devastating disease and seriously hazards to human’s health, but effective comprehensive treatments have not been discovered so far. Spinal cord injury, nearly40percent patients occurred in the cervical spinal cord, most of which due to the traffic accidents, high fall and sports injuries,which leads to the vertebral fractures and spinal cord compression or contusion. After spinal cord injury, most of the patients will feel the disorder of sensory and motor function, urine abnormalities and so on, which causes huge burden to patients and their families. Therefore, the study of spinal cord injury is still one of the hot point in medical area.Currently, the study of spinal cord injury are based on animal models.Since Allen developed the controlled weight drop model in1911.The primary limitation of the weight drop technique is that the systems can not detect the biomechanical parameters,and the big variability of a second impact.In1989,Young improved it on the basis of weight drop model, and developed a new spinal cord contusion model at New York University (NYU Impactor). At Ohio State University, an unique device was set up at the same time which can better control some of the mechanical variables during the impact(OSU Model).In2005, Pearse developed a new contusion model called electromagnetic spinal cord injury device (ESCID) on the basis of OSU Model, producing0.80mm、0.95mm and1.10mmcontusion respectively in C5,and found that the three different contusive displacement can result in a mild, moderate, and severe degree of spinal cord injury respectively.Contusion models of traumatic spinal cord injury were majorly controlled the injury degree by contusive displacement and force.Recently years, the improvement of contusion model is mainly through a better control of displacement and the force during the spinal cord injury.Besides, researchs on the effect of contusive velocity were seldom. However, the contusive velocity played a very important role in the mechanism of SCI.In a variety of SCI animal models, such as contusion in thoracic vertebra and fracture dislocation in cervical vertebra, confirmed a higher impact velocity lead to a more serious primary spinal cord injury.For example,in2008,sparrey investgated the effect of contusive velocity at300mm/s and3mm/s at T10,foundthat fast contusion result in a more heavier hameorrhage and axonal injury.Approved that the contusive velocity as a parameter of spinal cord injury. In2013,Australian researcher Lau found severity of spinal cord injury of rats after vertebral dislocation depends upon displacement but not speed.So far,there is no contusion model of spinal cord injury of cervical under different impact velocity.We required to study the primary SCI result from a higher impact velocity, even secondary SCI.2. ObjectivesThis study utilization spinal cord contusion model by the servo-electromagnetic material testing machine,investigating the effect of primary spinal cord injury by different contusive velocity(5mm/s or500mm/s)under1.1mm or1.5mm respectively, analyzing the hemorrhage and axonal injury of the spinal cord after contusion,to verify the effect of different contusive velocity on cervical primary spinal cord injury in rats.3.Materials and Methods3.1To establish a contusion model of cervical spinal cordSelected10male Sprague-Dawley rats,weighing260±10g,2-3months of age. Randomly divided into experimental group(n=5) and sham control group(n=5).Use the small animal anesthesia machine.After anesthetized with isoflurane. Removal the back hair of the rat with an animal hair shaving machine.The rats were fixed on the stereotaxic instrument by ear cephalic rod fixed to the cervical spine,use a soft cushion to raise the cervical spine and make the head flexion90°to eliminate the cervical curvature.A vertical incision was made along the cervical vertebra and the superficial muscle and skin retracted.Cleaning up C4-C7lamina, exposed the grooves between the transverse and facet.A laminectomy performed at cervical vertebra C5exposed the dorsal surface of the spinal cord underneath (C5) without damaging the dura mater. After opening the C5lamina, stabled the C6and C7on the stereotaxic instrument by vertebra clips, and then connected to servo-electromagnetic material testing machine(Instron E1000). The contusion tip was a cone-shape flat head with a diameter of4mm,a small force sensor above the tip connected to servo-electromagnetic material testing machine to detect the load during the contusion.Move the tip head of the dura mater of C5,slowly fall down the tip until the touch force of0.03N.Defined it as the initial position of the spinal cord contusion.The study investigated contusion at500mm/sand produced1.5mm contusion at C5,after hitting the spinal cord,the contusion tip rising quickly leave from the spinal cord surface.The sham control group rats were the same with the experimental group without contusion.Recording injury biomechanical parameters(time,velocity and force) by servo-electromagnetic material testing machine during the contusion. A small load sensor (range0～10N)measuring the impact force during the contusion.The impact force including inertia force from fast impact and the actual force of spinal cord surface. This study used the force from empty impact as the force of inertia compensation at the same state,to eliminate the effect of inertial force in the process of actual impact.And the contusive displacement and velocity were received from displacement sensor of material testing machine.All rats were perfused intracardially in5minutes after contusion, then observe the inspection of the gross morphology. The harvested cord sections were postfixed in4%paraformaldehyde solution overnight to ensure complete fixation of the tissue..The tissue was cryoprotected using a series of sucrose solutions (10%,20%,30%).Serial, parasagittal frozen sections of the cord were cut,observe the inspection of the gross morphology after stained with hematoxylin and eosin,and hemorrhage volume of white matter、gray matter and total.The axonal injury was detected with P-amyloid precursor protein immunohistochemistry.3.2fects of velocity and contusive displacement on the primary SCIThis study selected36male Sprague-Dawley rats,weighing260±10g,2-3months of age.According to the contusive displacement,divided into1.1mm or1.5mm group randomly, then divided into fast group(500mm/s) and slow group (5mm/s) according to the contusive velocity.Set the sham control group at the same time.The definite grouping were shown in the figure below. Surgical procedure and method of injury were the same with the former.Using two factor analysis of variance to compare the effect of velocity and contusive displacement to biomechanical parameters during the contusion and effect of velocity and contusive displacement to amount of hemorrhage in each part of spinal cord after contusion.4. Result4.1The establishment of cervical spinal cord injury modelAt500mm/s contusion,the maximal force in experimental group was5.7±1.2N,the actual contusive displacement was1.49±0.03mm and the contusive velocity was511±4mm/s.Proving that the control of velocity and contusion displacement were fairly accurate.Two darker symmetrical hemorrhage strips were observed on all spinal cords in the experimental group.None of rats in the sham control group showed any hemorrhage.After HE staining,we found that all spinal cords in the experimental group show hemorrhage.The hemorrhage was mainly located in the gray matter and few in the white matter,more dorsally than ventrally.On average, hemorrhage volume in the gray matter、white matter and total spinal cord was0.71、0.23and0.94mm3.None of rats in the sham control group showed any hemorrhage.Immunostaining of β-APP showed axonal disruption in both white matter and gray matter,we can find the represention of β-APP positive cell.And seldom axonal disruption in the sham control group, none of the rats show the represention of β-APP positive cell.4.2Effects of velocity and contusive displacement on the primary SCIUnder1.5mm contusion, the maximal force in fast group(500mm/s) and slow group(5mm/s) was5.3±1.2N and2.8±0.6N respectively. The contusive displacement in the two groups was1.50±0.05mm and1.51±0.04mm respectively. And the contusive velocity was516±11mm/s and4.8±0.5mm/s.Under1.1mm contusion, the maximal force in fast group(500mm/s) and slow group(5mm/s) was5.3±3.1N and2.3±0.1N respectively. The contusive displacement in the two groups was1.11±0.05mm and1.12±0.03mm respectively. And the contusive velocity was469±57mm/s and4.9±0.4mm/s.The maximal force was significant difference between different velocity(F=21.567, P=0.000),and the maximal force in fast group was significant bigger than slow group.There was no significant difference between different contusive displacement (F=0.199, P=0.660).And no interaction effect between the contusive velocity and displacement (P=0.63).The results above proved that the faster of the velocity,the bigger maximal force of the spinal cord was.The contusive velocity is a fact which result in the difference between the maximal force.Hemorrhage was observed on the dorsa of spinal cords after contusion. The forelimbs of rats have a transient twitch at the sudden of hit, which proved the existence of spinal cord injury.After intranscardially perfused, We can see a ribbon hemorrhage on the center of C5spinal cord surface after contusion. And the color of hemorrhage was darker in fast group compared to slow group at the same contusive displacement;And darker in the1.5mm group compared to1.1mm group at the same contusive velocity.After HE stained, we found that the hemorrhage area in1.5mm fast group were mostly located in the grey matter, while few scattered in the white matter.The hemorrhage area in1.5mm slow group were nearly all located in the grey matter, while seldom in the white matter.The1.1mm group were similar to the1.5mm group,but the hemorrhage was significant lower than1.5mm group. Under1.5mm contusion,the hemorrhage volume in the white matter in fast group and slow group was0.23mm3and0.12mm3,0.71mm3and0.42mm3in the grey matter. The total hemorrhage was0.94mm3and0.55mm3.Under1.1mm contusion, the hemorrhage volume in the white matter was0.18mm3and0.06mm3in fast and slow group, and in the grey matter was0.49mm3and0.25mm3, respectively. The total hemorrhage was0.67mm3and0.31mm3.Statistical analysis showed that the total hemorrhage, the white matter hemorrhage and the grey matter hemorrhage between different contusive velocity was significantly different,the volume of hemorrhage in fast group was significantly higher than slow group. The total hemorrhage and the grey matter hemorrhage between different contusive displacement was significantly different,the volume of hemorrhage in1.5mm group was higher than1.1mm group. There was no significant difference of the white matter hemorrhage between the two groups.The results above demonstrated both velocity and contusive displacement were the factors that lead to the difference among each part of the spinal cord hemorrhage.The faster the velocity was,the more hemorrhage in each part of spinal cord was;The bigger contusive displacement was,the more hemorrhage in each part of spinal cord was too.After β-APP immunohistochemical staining, we found that the axonal of gray matter and white matter of the spinal cord were disruption, and have β-APP positive cell represent.Injured axons typically formed retraction balls.Both in the1.5mm group and1.1mm group, the number of β-APP-positive cells in fast group were significantly more than slow group.5. ConclusionThis study confirmed that contusion at a500mm/s velocity at C5,producing1.5mm contusion can lead to hemorrhage in both gray matter and white matter and axonal disruption, result in the primary spinal cord injury.A cervical spine contusion of SCI model was established successfully in this study.The contusive velocity was a factor of primary spinal cord injury. Contusion at higher speed resulted in severer primary spinal cord injury, more hemorrhage in each part of spinal cord and axonal disruption.Contusion under bigger displacement, it can also lead to severer primary spinal cord injury, more hemorrhage and axonal disruption.