改进球囊导管法构建大鼠急性压迫型上颈脊髓损伤模型及模型大鼠伤后早期血液流变学指标观察

Experimental research on acute-compression-type upper cervical spinal cord injury rat models created by using improved Foley's tube method and early observation on post-traumatic hemorheological indexes in rat models

目的:探讨改进球囊导管法构建大鼠急性压迫型上颈脊髓损伤模型的有效性,并观察模型大鼠伤后早期血液流变学指标的变化。方法:将30只Wistar大鼠随机分为空白组、假手术组和模型组,每组10只。空白组不进行手术干预。假手术组和模型组大鼠经寰枕间隙置入球囊导管;操作时对谭明生教授等前期提出的方法进行改进,以2 mL注射器针头自制神经剥离子,分离寰枕覆膜及寰枢椎间黄韧带;以缝合针和铜丝代替导丝,引导球囊导管经寰枕间隙置入椎管内。术后24 h以手推压力泵向模型组大鼠球囊内注射生理盐水至球囊内压力达300 kPa,使球囊匀速膨胀,对颈脊髓产生压迫。分别于球囊压迫1 h、3 h、6 h、12 h、24 h后采用BBB评分法对各组大鼠的运动功能进行评价,最后一次运动功能评定结束后对各组大鼠进行颈椎MRI检查、血液流变学指标测定及受压节段脊髓组织病理学观察。结果:(1)模型验证结果。实验期间共废弃并补充4只大鼠。3组大鼠颈脊髓受到球囊压迫后不同时间BBB评分结果显示,时间因素和分组因素不存在交互效应(F=0.310,P=0.736);空白组和假手术组大鼠的BBB评分均高于模型组,即存在分组效应(F=0.310,P=0.000);球囊压迫后不同时点之间BBB评分的差异无统计学意义,即不存在时间效应(F=0.324,P=0.860);3组大鼠的BBB评分随时间推移均无明显变化。MRI示各组大鼠颈椎序列均正常;空白组大鼠颈部未见到球囊;假手术组和模型组球囊压迫系统稳定、无松脱,球囊呈高信号改变,与脊髓等信号对比明显;模型组球囊膨胀好,向椎管内单向均匀压迫。受压脊髓组织病理学观察结果显示,空白组和假手术组脊髓组织中未见或仅见极少数Fas阳性凋亡细胞;Fas阳性凋亡细胞在模型组脊髓组织中较多,广泛分布于脊髓灰、白质中。(2)模型大鼠伤后早期血液流变学指标测定结果。脊髓受压24 h后,除50/s外(F=0.602,P=0.663),3组大鼠血液在切变率为200/s、100/s、5/s、1/s时的全血黏度比较,组间差异均有统计学意义(F=2.713,P=0.042;F=6.358,P=0.000;F=18.893,P=0.000;F=7.320,P=0.000)。切变率为200/s时,模型组的全血黏度高于空白组和假手术组(P=0.028,P=0.043),空白组与假手术组的全血黏度比较,差异无统计学意义(P=0.851);切变率为100/s时,模型组的全血黏度高于空白组和假手术组(P=0.044,P=0.003),空白组与假手术组的全血黏度比较,差异无统计学意义(P=0.972);切变率为5/s时,模型组的全血黏度高于空白组和假手术组(P=0.001,P=0.000),空白组与假手术组的全血黏度比较,差异无统计学意义(P=0.865);切变率为1/s时,模型组的全血黏度高于空白组和假手术组(P=0.010,P=0.011),空白组与假手术组的全血黏度比较,差异无统计学意义(P=0.972)。3组大鼠的血浆黏度比较,差异无统计学意义(F=0.734,P=0.574)。3组大鼠红细胞聚集指数比较,差异有统计学意义(F=5.335,P=0.001);模型组的红细胞聚集指数高于空白组和假手术组(P=0.003,P=0.007);空白组与假手术组比较,差异无统计学意义(P=0.696)。3组大鼠红细胞变形指数比较,差异有统计学意义(F=18.288,P=0.000);模型组的红细胞变形指数高于空白组和假手术组(P=0.000,P=0.000);空白组与假手术组比较,差异无统计学意义(P=0.948)。结论:采用改进球囊导管法构建的大鼠上颈脊髓损伤模型,符合急性压迫型上颈脊髓损伤的特点;模型大鼠的血液在受伤后24 h时呈高凝状态。

Objective：To explore the effectiveness of improved Foley＇s tube method in building acute-compression-type upper cervical spinal cord injury rat models, and to observe the early changes of hemorheological indexes after injury in rat models. Methods：Thirty Wistar rats were randomly divided into blank group, sham-operated group and model group, 10 cases in each group. The rats in blank group were not given any surgical intervention,while the rats in the other two groups were intervened by inserting a Foley＇s tube into atlanto-occipital space. The method proposed by professor TAN Mingsheng. was improved in the operation. A 2 mL gauge syringe needle was made into a neural detacher to separate atlanto-occipital tectorial membrane and atlantoaxial intervertebral ligamentum flavum, and a Foley＇s tube was inserted into spinal canal through the atlanto-occipital space under the guidance of surgical sewing needle and copper wire instead of guide wire. At 24 hours after the surgery,the normal saline were injected into the Foley＇s tube in model group using manumotive force-pumps till the pressure reached 300 kPa. The balloon of Foley＇s tube was expanded in uniform speed to oppress the cervical spinal cord. The motor function of rats in each group were evaluated by using BBB scoring method after 1-,3-,6-, 12- and 24-hour oppressign with balloon respec- tively. Cervical MRI examination, determination of hemorheological indexes and histopathological observation on oppressed spinal cord were performed on rats in each group after the last motor function evaluation. Results：Four rats were abandoned during the experiment and the experiment were supplemented with another 4 rats. The BBB scores of the 3 groups at different time-points after the cervical spinal cord was compressed by balloon demonstrated that（ 1 ）there was no interaction between time factor and group factor（ F = 0. 310 ,P = 0. 736） ;（2）the BBB scores were higher in blank group and sham-operated group compared to model group, in other words, there was group effect （ F = 0. 310,P = 0. 000） ; （3）there was no statistical difference in BBB scores between different timepoints, in other words, there was no time effect（F =0. 324,P =0. 860） ; （4） the BBB scores changed unconspicuously with time in the 3 groups. The results of MRI examination demonstrated that （ 1 ） the cervical spine sequences of rats were normal in each group ; （2） no balloon was found in the neck of rats of blank group;（3） the stable balloon compression system was found in rats of sham-operated group and model group and the balloon presented with high signal changes;（4）the balloon expanded well and unidirectional uniform compression of spinal canal was found in rats of model group. The results of histopathological observation on compressed spinal cord demonstrated that few Fas positive apoptotic cells were found in the spinal cord tissues of rats of blank group and sham-operated group, while many Fas positive apoptotic cells were found in the spinal cord tis- sues of rats of model group and they widely distributed in the spinal gray matter and white matter. After 24-hour spinal cord compression, there was statistical difference in the whole blood viscosity（WBV） between the 3 groups when the shear rate was 200/s, 100/s ,5/s and 1/s （ F = 2. 713 ,P =O. O42 ;F =6. 358,P =O. OOO;F =18. 893 ,P =O. OOO;F = 7. 320 ,P =O. OOO ） and there was no statistical difference when the shear rate was 50/s（ F = 0. 602 ,P = 0. 663 ）. When the shear rate was 200/s, the WBV was higher in model group compared to blank group and sham-operated group （ P = 0. 028, P = 0. 043 ） , and there was no statistical difference in the WBV between blank group and sham- operated group（P = 0. 851 ）. When the shear rate was 100/s, the WBV was higher in model group compared to blank group and sham-oper- ated group（ P = O. 044 ,P = 0. 003 ） , and there was no statistical difference in the WBV between blank group and sham-operated group（ P = 0. 972）. When the shear rate was 5/s, the WBV was higher in model group compared to blank group and sham-operated group （P = 0. 001, P = 0.000）, and there was no statistical difference in the WBV between blank group and sham-operated group（ P = 0.865 ）. When the shear rate was 1/s,the WBV was higher in model group compared to blank group and sham-operated group（P =0. 010,P =0. 011 ） ,and there was no statistical difference in the WBV between blank group and sham-operated group（P = 0. 972）. There was no statistical difference in plasma viscosity between the 3 groups （ F = 0. 734, P = O. 574 ）. There was statistical difference in erythrocyte aggregation index between the 3 groups（ F = 5. 335 ,P = 0. 001 ）. The erythrocyte aggregation index was higher in model group compared to blank group and sham-operated group （P = 0. 003, P = 0.007 ）, and there was no statistical difference in erythrocyte aggregation index between blank group and sham-opera- ted group （ P = 0. 696 ）. There was statistical difference in erythrocyte deformation index between the 3 groups （ F = 18. 288, P = 0.000）. The erythrocyte deformation index was higher in model group compared to blank group and sham-operated group （ P = 0. 000, P = 0. 000 ）, and there was no statistical difference in erythrocyte deformation index between blank group and sham-operated group （ P = 0. 948 ）. Conclu- sion：The upper cervical spinal cord injury rat models created by using improved Foley＇s tube method fit the characteristics of acute-com- pression-type upper cervical spinal cord injury. The blood of rat models presents hypercoagulabale state at 24 hours after injury.