脊髓冲击伤动物模型的建立及凋亡相关基因表达的初步研究
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摘要
目的:①研制一种小型冲击波发生装置,模拟爆炸冲击波的致伤作用。②建立实验条件下的兔不同程度脊髓冲击伤动物模型。③探讨兔脊髓冲击伤后脊髓组织中Bcl-2、Bax蛋白早期表达的变化规律,为进一步阐明脊髓冲击伤的发生机制和指导临床治疗提供实验依据。
方法:①根据气体动力学原理,采用破膜技术及传感器检测技术,研制出一种由储气设备、发射系统、冲击波测试分析系统所组成的小型冲击波发生装置。检测气源压力分别为400kPa、500kPa、600kPa、700kPa、800kPa时该装置所产生的冲击波的超压峰值、持续时间及传播速度,不同的气源压力分别重复5次。②将24只新西兰大白兔随机分为4组,每组6只。A组:对照组;B组:气源压力为0.4MPa损伤组;C组:气源压力为0.6MPa损伤组;D组:气源压力为0.8MPa损伤组。采用小型冲击波发生装置,将兔T9和T10全椎板切除显露硬膜,按分组预设气源压力实施单次冲击波致伤,对照组仅行椎板全切除,不设施冲击波致伤。48h后观察各组兔后肢运动、感觉功能变化以及脊髓病理学变化,综合评价脊髓损伤程度。③设定小型冲击波发生装置的气源压力为0.6MPa,单次冲击波致兔脊髓中度损伤,致伤后在不同时间点(4～72h),光镜观察脊髓组织形态学变化,免疫组织化学方法观察Bcl-2和Bax蛋白在脊髓组织的表达情况。
结果:①小型冲击波发生装置能模拟产生冲击波,其检测到的波形类似于Friedlander波形,即典型空气冲击波波形。随着气源压力的增大,冲击波超压峰值及速度同时增大,正压持续时间基本不变。各组数据重复性好。②各组随着气源压力的增大,兔后肢运动、感觉功能损害明显加重。兔脊髓冲击伤后48小时,各组动物运动及感觉功能评分差异显著。伤后48h发现受损节段脊髓水肿、变性坏死,脊髓灰质的病理损害严重。③兔脊髓中度冲击伤后4～72h,光镜下观察脊髓组织细胞肿胀、坏死改变增多,脊髓灰质的病理损害严重。冲击伤后脊髓灰、白质均有Bcl-2和Bax蛋白表达,但在时间分布上呈不均一性。Bcl-2在伤后12h呈阳性表达,表达高峰持续在伤后24h～48h,而Bax在伤后4h呈阳性表达,表达高峰也持续在伤后24h～48h,但表达程度始终强于Bcl-2。
结论:①小型冲击波发生装置能模拟产生冲击波,且性能稳定、安全,可重复性强。②小型冲击波发生装置建立的动物脊髓冲击伤模型安全、稳定、有效,能较好的模拟轻、中、重不同程度脊髓冲击伤,适用于实验条件下的脊髓冲击伤研究。③兔脊髓冲击伤后损伤早期局部促进凋亡的因子表达占主导优势,而保护性因子的表达不足,最终使脊髓神经细胞向凋亡的方向发展。
Objective:①To develop a small size device which generate shockwave and simulate blast injuries caused by the shock of explosion.②To establish a laboratory efficient wound model in rabbits for the study on spinal cord shockwave injury at different extents.③To observe potential changes in the Bcl-2 and Bax proteins expression in rabbit spinal cord after the blast injury, so as to provide the experimental basis for further clarifying the mechanism of spinal cord blast injury and for clinical therapy.
Methods:①The device composed of the gas storage facilities, launch systems and shock wave analysis system was developed based on gas-dynamical principles, rupture of membrane and sensor detection technology. Positive pressure peak, duration and propagation velocity were detected by oscilloscope at the different pressure of gas source. Each of the gas pressure 400kPa、500kPa、600kPa、700kPa、800kPa was repeated five times.②24 New Zealand white rabbits were randomly divided into four groups of six. A group: control group; B group: gas source pressure of 0.4MPa; C group: gas source pressure of 0.6MPa; D group: gas source pressure of 0.8MPa.The rabbit’s spinal cord of T9 and T10 was injured by different pressure of gas source with the device. The degrees of spinal cord shockwave injury were evaluated by histological analysis and the extent of function changes of feel and behavior were investigated after injury 48 hours.③Moderate spinal cord blast injury in rabbits were induced by the device at the gas source pressure of 0.6MPa.Light microscopy was used to observe the morphologic changes in rabbit spinal cord and immunochemical method was used to study the expression of Bcl-2 and Bax proteins in rabbit spinal cord from 4 h to 72 h after the spinal cord blast injury.
Results:①The waveform detected by the shock wave analysis system of the device is similar to the Friedlander waveform, a typical air-blast waveform. With the gas source pressure, positive pressure peak and propagation velocity increase at the same time, the duration of positive pressure remain basically unchanged. Data of each group keep good reproducibility.②The motor function of rabbit hind limbs was significantly worsened duo to the stronger shockwave pressure. It was found that the damaged spinal cord area edema, degeneration and necrosis, the pathological damage to the gray matter of spinal cord was serious 48h after injury. The score of rabbit hind limb sensory and motor function was significantly different and with good reproducibility.③During the post burst phase (4～72 h),the increased necrotic and swollen cell in spinal cord was observed by light microscopy and the pathological damage to the gray matter of spinal cord was serious. After the blast injury, expressions of protein Bcl-2 and protein Bax were observed all over the spinal cord, but it was not uniform in time distribution. The expressions of protein Bcl-2 were observed 12h after the spinal cord blast injury and reached peak value during 24h～48h. The expressions of protein Bax were observed 4h after the spinal cord blast injury and the degree of the expressions was stronger than protein Bcl-2.
Conclusion:①The device we developed can simulate the shockwave generation. It was safe and functionally stable.②The device was able to better simulate light, moderate and severe spinal cord blast injury in varying degrees. The present animal model of spinal cord blast injury is safe and effective and it is highly advantageous for the study of shockwave injury in laboratory.③The spinal cord blast injury may cause apoptosis of some neurons because the expressions of protective factor are less than the expression of the apoptosis factor.
方法:①根据气体动力学原理,采用破膜技术及传感器检测技术,研制出一种由储气设备、发射系统、冲击波测试分析系统所组成的小型冲击波发生装置。检测气源压力分别为400kPa、500kPa、600kPa、700kPa、800kPa时该装置所产生的冲击波的超压峰值、持续时间及传播速度,不同的气源压力分别重复5次。②将24只新西兰大白兔随机分为4组,每组6只。A组:对照组;B组:气源压力为0.4MPa损伤组;C组:气源压力为0.6MPa损伤组;D组:气源压力为0.8MPa损伤组。采用小型冲击波发生装置,将兔T9和T10全椎板切除显露硬膜,按分组预设气源压力实施单次冲击波致伤,对照组仅行椎板全切除,不设施冲击波致伤。48h后观察各组兔后肢运动、感觉功能变化以及脊髓病理学变化,综合评价脊髓损伤程度。③设定小型冲击波发生装置的气源压力为0.6MPa,单次冲击波致兔脊髓中度损伤,致伤后在不同时间点(4～72h),光镜观察脊髓组织形态学变化,免疫组织化学方法观察Bcl-2和Bax蛋白在脊髓组织的表达情况。
结果:①小型冲击波发生装置能模拟产生冲击波,其检测到的波形类似于Friedlander波形,即典型空气冲击波波形。随着气源压力的增大,冲击波超压峰值及速度同时增大,正压持续时间基本不变。各组数据重复性好。②各组随着气源压力的增大,兔后肢运动、感觉功能损害明显加重。兔脊髓冲击伤后48小时,各组动物运动及感觉功能评分差异显著。伤后48h发现受损节段脊髓水肿、变性坏死,脊髓灰质的病理损害严重。③兔脊髓中度冲击伤后4～72h,光镜下观察脊髓组织细胞肿胀、坏死改变增多,脊髓灰质的病理损害严重。冲击伤后脊髓灰、白质均有Bcl-2和Bax蛋白表达,但在时间分布上呈不均一性。Bcl-2在伤后12h呈阳性表达,表达高峰持续在伤后24h～48h,而Bax在伤后4h呈阳性表达,表达高峰也持续在伤后24h～48h,但表达程度始终强于Bcl-2。
结论:①小型冲击波发生装置能模拟产生冲击波,且性能稳定、安全,可重复性强。②小型冲击波发生装置建立的动物脊髓冲击伤模型安全、稳定、有效,能较好的模拟轻、中、重不同程度脊髓冲击伤,适用于实验条件下的脊髓冲击伤研究。③兔脊髓冲击伤后损伤早期局部促进凋亡的因子表达占主导优势,而保护性因子的表达不足,最终使脊髓神经细胞向凋亡的方向发展。
Objective:①To develop a small size device which generate shockwave and simulate blast injuries caused by the shock of explosion.②To establish a laboratory efficient wound model in rabbits for the study on spinal cord shockwave injury at different extents.③To observe potential changes in the Bcl-2 and Bax proteins expression in rabbit spinal cord after the blast injury, so as to provide the experimental basis for further clarifying the mechanism of spinal cord blast injury and for clinical therapy.
Methods:①The device composed of the gas storage facilities, launch systems and shock wave analysis system was developed based on gas-dynamical principles, rupture of membrane and sensor detection technology. Positive pressure peak, duration and propagation velocity were detected by oscilloscope at the different pressure of gas source. Each of the gas pressure 400kPa、500kPa、600kPa、700kPa、800kPa was repeated five times.②24 New Zealand white rabbits were randomly divided into four groups of six. A group: control group; B group: gas source pressure of 0.4MPa; C group: gas source pressure of 0.6MPa; D group: gas source pressure of 0.8MPa.The rabbit’s spinal cord of T9 and T10 was injured by different pressure of gas source with the device. The degrees of spinal cord shockwave injury were evaluated by histological analysis and the extent of function changes of feel and behavior were investigated after injury 48 hours.③Moderate spinal cord blast injury in rabbits were induced by the device at the gas source pressure of 0.6MPa.Light microscopy was used to observe the morphologic changes in rabbit spinal cord and immunochemical method was used to study the expression of Bcl-2 and Bax proteins in rabbit spinal cord from 4 h to 72 h after the spinal cord blast injury.
Results:①The waveform detected by the shock wave analysis system of the device is similar to the Friedlander waveform, a typical air-blast waveform. With the gas source pressure, positive pressure peak and propagation velocity increase at the same time, the duration of positive pressure remain basically unchanged. Data of each group keep good reproducibility.②The motor function of rabbit hind limbs was significantly worsened duo to the stronger shockwave pressure. It was found that the damaged spinal cord area edema, degeneration and necrosis, the pathological damage to the gray matter of spinal cord was serious 48h after injury. The score of rabbit hind limb sensory and motor function was significantly different and with good reproducibility.③During the post burst phase (4～72 h),the increased necrotic and swollen cell in spinal cord was observed by light microscopy and the pathological damage to the gray matter of spinal cord was serious. After the blast injury, expressions of protein Bcl-2 and protein Bax were observed all over the spinal cord, but it was not uniform in time distribution. The expressions of protein Bcl-2 were observed 12h after the spinal cord blast injury and reached peak value during 24h～48h. The expressions of protein Bax were observed 4h after the spinal cord blast injury and the degree of the expressions was stronger than protein Bcl-2.
Conclusion:①The device we developed can simulate the shockwave generation. It was safe and functionally stable.②The device was able to better simulate light, moderate and severe spinal cord blast injury in varying degrees. The present animal model of spinal cord blast injury is safe and effective and it is highly advantageous for the study of shockwave injury in laboratory.③The spinal cord blast injury may cause apoptosis of some neurons because the expressions of protective factor are less than the expression of the apoptosis factor.
引文
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