| ObjectiveTo establish a rat model of graded Allen’s spinal cord injury (SCI), and evaluate the and provide theoretical evidence for a new model of contusive SCI. To investigate the relationship of endothelial progenitor cells (EPCs) and early neovascularization following spinal cord injury (SCI) in rats.MethodsUsing the modified Allen’s methods, the spinal cord contusion were made in rats. A total of70SPF (Specific pathogen Free) grade Sprague-Dawley rats were divided into surgical controls (n=10) that received laminectomy but did not receive a contusive injury, and experimental groups (n=60) ruined by modified Allen’s method and distinguished by the amount of weight in gram and height in millimeter dropped onto the impounder:group A(10g×12.5mm n=15), group B(10g×25mm n=15), group C (10g×50mm n=15) and group D (20g×50mm n=15). All rats were evaluated with the BBB locomotor rating scale pre-operation, and at2d,7d,14d,28d, and42d post operation. A total of60Sprague-Dawley rats were divided into experimental groups ruined by modified Allen’s method and surgical controls that received laminectomy but did not receive a contusive injury. Peripheral venous blood samples were taken on pre-operation and at3h,6h,24h,48,72h and168h post operation with capillary glass tubes. And the counts of EPCs marked with CD133-PE and CD34-FITC were determined by flow cytometry. The rats were sacrificed on day1,4,7and14. The morphologic changes in neovascularization were observed, and the expression of CD31that was a marker of vascular endothelial cell was detected by immunohistochemistry. Another30adult male SD rats were randomly divided into the spinal cord injury+atorvastatin treatment group, spinal cord injury+saline treated group and sham injuried group. Each rat in the SCI+Atorvastatin group administered atorvastatin just after SCI at a dose of1mg/kg/day for14consecutive days. Saline group given normal saline, given14consecutive days.2hours after injury,6hours,1day,3days,7days,14days,21days from the rat orbital venous plexus blood0.5ml, and then using flow cytometry in peripheral blood endothelial progenitor the number of cells. Another51rats were divided into spinal cord injury+Alto atorvastatin treatment group, spinal cord injury+saline group, sham operation group,1,4,7,14,21days BBB motor scores, slope experiments, and use with4%formaldehyde perfusion morphological observation and immunohistochemical staining of spinal cord vascular newborn rat spinal cord tissue.Results1. The BBB locomotor rating scale revealed a statistically significant difference between surgical controls and experimental groups after injury(P<0.01). There was no significant difference in BBB scales between group A and B(P>0.05). There were significant differences in group A or B, and group C and D at various determined time points(P<0.05). In group A、B and C, the neural function of the animals recovered to different extent. In group D, the recovery was not obvious.2. The levels of circulating EPCs within the first3h of injury were lower than normal subjects (t=7.959, P<0.01), but increased after6h (t=13.972, P<0.001) Reaching plateau around24h post-injury (F=67.349, P<0.01) at a level that was significantly higher than controls(t=3.166,P<0.05).3. The number of CD31+cells in the injury sites increased after SCI (F=58.115,P<0.01). The change in control group was not obvious (F=0.894,P>0.05). The change of CD31+vessels had obvious correlation with the change of CD31+cells in the region adjacent to the epicenter of the injury site.(r=0.906, tr=3.709, P<0.05)4. The BBB motor scores was performed that compared with the saline treated group,14and21days after injury, atorvastatin treated rats behavioral function has been effectively improved (P<0.05).5. The vivo studies showed that SCI induced a downregulation of the cireulating EPCs level at3hours after SCI and then an sharp upregulation was observed to peak at48hours post-SCI(P<0.05), which then decreased gradually to the normal level48hours later after SCI. While the level of the circulating EPCs was induced by the atorvastatin but not saline, which increased gradually and reaching a plateau around 7th day after atorvastatin treatment(P<0.05). And remained at a high level continued until more than21days after injury.6. Compared with the sham group, the number of microvessels of saline group after spinal cord injury and atovastatin group trauma area around were higher than sham operation group (P<0.05) in14,21days; each time point of14,21days after the injury, atorvastatin treatment group, the trauma area surrounding the microvessel number than the saline group were increased to varying degrees, there are statistically significant (P<0.05).Conclusions1. The impact device made by ourselves could produce reliable and controllable contusive spinal cord injury models in rats. The spinal cord contusion models could provide theory basis for SCI studies.2. After spinal cord injury in rats, the number of endothelial progenitor cells in short time decline (3hours after spinal cord injury), followed by significantly increased(6hours after spinal cord injury), and traumatic lesions gradually increased the number of microvascular.3. Atorvastatin significantly enhanced bone marrow progenitor cell mobilization after spinal cord injury of rat, promotion of angiogenesis in spinal cord injury around, and promote the recovery of neurological function. |
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