控制挫伤深度建立大鼠颈脊髓半侧挫伤模型

Establishment of hemi-contusive cervical spinal cord injury model in rats and its characters in function and histology

目的 :建立大鼠颈脊髓半侧挫伤模型,评价不同程度脊髓损伤大鼠的行为学和组织学改变。方法 :选取32只成年雄性Sprague-Dawley大鼠,体重为280~320g。按挫伤深度分为1.2mm组(n=12)和2mm组(n=12),并设立假手术组(n=8)对照。麻醉状态下后路手术暴露大鼠C5左侧脊髓,特制椎夹固定颈椎并连于大鼠立体定位架后置于脊髓打击器平台上,然后应用直径为1.5mm的圆柱形打击头在500mm/s的速度下分别以1.2mm和2mm打击深度背侧垂直挫伤大鼠C5脊髓左半侧,监测最大打击力和挫伤能量。每只大鼠损伤后进行前肢运动功能评分,圆柱攀爬试验和梳理试验。伤后8周取材,在脊髓横断面组织学切片上测量损伤中心处脊髓损伤面积,以及灰、白质残存面积。结果:中度损伤组最大打击力和挫伤能量分别为0.99±0.14N和(1.08±0.01)×10-5J,重度损伤组为1.53±0.22N和(8.56±0.02)×10-5J,两组间打击力和损伤能量具有统计学差异(P<0.05)。术后对照组大鼠均未见明显神经功能障碍,前肢运动功能评分为16.29±0.49。损伤组大鼠均出现单侧神经功能障碍,损伤同侧前肢运动功能评分中度损伤组为1.40±0.52,重度损伤组为0.83±0.25,两组无统计学差异(P>0.05)。伤后8周前肢运动功能评分中度损伤组为12.40±1.15,重度损伤组为8.41±0.83,对照组为17.00±0.00,梳理试验评分中度损伤组为3.80±0.60,重度损伤组为2.51±0.54,对照组为5.00±0.00,损伤同侧前肢使用比例中度损伤组为(32.31±5.70)%,重度损伤组为(4.92±1.21)%,对照组为(49.42±1.20)%,各组间均有统计学差异(P<0.05)。镜下观察损伤组大鼠脊髓组织都出现了明显的半侧空洞和结构破坏,而对侧脊髓结构基本完整。在损伤中心处,中度损伤组脊髓背侧束、背外侧束和脊髓后角有破坏,重度损伤组脊髓仅残留部分的腹外侧束,对照组脊髓组织结构完整。灰质和白质残存面积在中度损伤组分别为1.45±0.11mm^2和4.13±0.29mm^2,重度损伤组分别为1.10±0.05mm^2和3.84±0.19mm^2,组间均存在统计学差异(P<0.05)。结论:本研究成功建立了不同程度的大鼠颈脊髓半侧挫伤模型,不同挫伤深度表现出不同程度的单侧前肢运动功能障碍和脊髓组织破坏,为颈脊髓半侧损伤模型提供研究基础。

Objectives： To establish a unilateral cervical spinal cord contusion injury model in rats, and to evaluate the behavioral and histological changes with different degrees of cervical spinal cord injury. Methods： Thirty-two male adult Sprague-Dawley rats were divided into 1.2mm group（n=12） defined as moderate injury group, 2mm group（n=12） defined as severe injury group and the control group（n=8）, which was based on contusive displacement. The left spinal cord of C5 was exposed under anesthesia. The cervical vertebra was held by custom-made vertebral clamps and connected to the stereo positioning frame. Contusion injuries were applied to the left dorsal cord of C5 by using a 1.5mm-diameter impounder at 500mm/s. The maximum impact force and the contusion energy were recorded. Forelimb motor function was evaluated by using the forelimb locomotor scores, the rearing and grooming tests. All outcome measures were estimated weekly prior to and up to 8 weeks following injury. The spared areas of gray and white matter at the epicenter were analyzed on cross-sections of spinal cord. Results： The peak force and contusion energy were 0.99±0.14N and （1.08±0.01）×10-5J in the moderate group, and 1.53±0.22N and （8.56±0.02）×10-5J in the severe group, respectively. Significant difference was observed in the peak force and contusion energy between groups. No obvious neurological dysfunction was observed in the control group after surgery, and the functional score of forelimbs was 16.29±0.49. There was unilateral neurological dysfunction in the rats of the injury group, and the forelimb function score was 1.40±0.52 in the moderate group and 0.83±0.25 in the severe group. The two experimental groups had no statistical difference（P〉0.05）. At 8 weeks after injury, forelimb motor function score was 12.40±1.15 in the moderate injury group, 8.41±0.83 in the severe group, 17.00±0.00 in the control group. Grooming test score was 3.80±0.60 in the moderate injury group, 2.51±0.54 in the severe group, 5.00±0.00 in the control group. Proportion of ipsilateral forelimb use was （32.31±5.70）% in the moderate injury group, （4.92±1.21）% in severe group, （49.42±1.20）% in the control group. There were statistical differences among the groups（P〈0.05）. Unilateral structural failure and cavity were presented in spinal cord at 8 weeks after injury. The moderate contusion caused primarily dorsal column, spinal dorsal horn and part of dorsolateral fasciculus damage while the severe contusion produced additional damage to lateral and ventral tissue, while the structure of spinal cord was intact in the control group. The spared areas of gray and white matter in the moderate group at lesion epicenter were 1.45±0.11mm2 and 4.13±0.29mm2, and in the severe group were 1.10±0.05mm2 and 3.84±0.19mm2 respectively. There was significant difference in the spared areas of gray and white matter between groups（P〈0.05）. Conclusions： This study successfully establishes a different degree of unilateral cervical spinal cord contusion model in rats, which shows persistent unilateral neurological dysfunction and structural destruction of the injured spinal cord and the motor impairment, and it can be used for basic research of spinal cord injury.