七氟烷预处理对兔脊髓缺血/再灌注损伤保护作用的研究
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摘要
【背景】尽管临床上已采用多种方法减轻脊髓缺血损伤,但是胸腹主动脉瘤手术患者不可避免发生脊髓缺血,有数据显示3%～18%患者发生永久性损伤(急性截瘫和/或延迟性截瘫)。除此之外,脊髓肿瘤压迫,脊柱畸形,椎管狭窄等患者围手术期也会出现脊髓缺血/再灌注损伤。截瘫造成患者失去工作能力并担负巨额的医疗费用,给患者本人及家庭造成深痛的打击和巨大的经济负担,同时加重整个社会的医疗保险的负担。
七氟烷具有刺激性低,诱导、苏醒迅速,麻醉维持平稳,肝、肾毒性低,心律失常发生率低等优点,被广泛应用于临床麻醉。已有研究报道,七氟烷预处理可诱导缺血耐受,对缺血、缺氧的心、脑起保护作用,但其对脊髓缺血/再灌注损伤是否具有保护作用尚不清楚。
本研究采用兔肾下腹主动脉阻闭20min缺血/再灌注模型,研究单次七氟烷预处理是否可诱导快速缺血耐受,减轻脊髓缺血/再灌注损伤,并探讨其可能的作用机制。
【方法】雄性新西兰大白兔,2.0千克～2.9千克。
实验一:随机分为2组,每组各6只动物。I/R组动物行20min脊髓缺血后再灌注48h。Sham组动物接受同样的麻醉和手术准备,但不行缺血/再灌注损伤。于再灌注后4h,8h,12h,24h及48h行后肢运动功能评分,再灌注后48h取L5节段脊髓行HE染色、p-ERK1/2染色、Fluoro-Jade B染色和TUNEL染色。计数脊髓前角正常神经元、Fluoro-Jade B阳性神经元和TUNEL阳性神经元。
实验二:随机分为4组,每组10只动物。Sev+W1h组和Sev+W15min组动物面罩密闭吸入3.7%(1.0MAC)七氟烷+96%氧气预处理30min,分别洗脱1h或15min;O2+W1h组和O2+W15min组动物面罩密闭吸入96%氧气30min,分别洗脱1h或15min;4组动物均行20min脊髓缺血后再灌注48h。于再灌注后4h,8h,12h,24h及48h行后肢运动功能评分,再灌注后48h取L5节段脊髓行HE染色和脊髓前角正常神经元计数。
实验三:随机分为2大组,Sev组和O2组。Sev组动物面罩密闭吸入3.7%(1.0MAC)七氟烷+96%氧气预处理30min;O2组动物面罩密闭吸入96%氧气30min,两组动物均洗脱1h后行脊髓缺血20min后再灌注。每组分别按最后观察点为再灌注后8h,1d,2d,3d,5d,7d分为六组,每组6只动物。于再灌注后8h,1d,2d,3d,5d,7d行后肢运动功能评分,取L5节段脊髓行HE染色、Fluoro-Jade B染色和TUNEL染色。计数脊髓前角正常神经元、Fluoro-Jade B阳性神经元和TUNEL阳性神经元。
实验四:随机分为4组,每组8只动物。Vehicle+Sev组和Vehicle+O2组动物静脉注射溶剂DMSO 50μL/kg,20min后分别吸入七氟烷和氧气预处理;U0126+Sev组和U0126+O2组动物静脉注射50μL/kg 0.4% U0126(溶于DMSO),20min后分别吸入七氟烷和氧气预处理;预处理方法和兔脊髓缺血/再灌注方法同实验三。于再灌注后4h,8h,12h,24h及48h行后肢运动功能评分,再灌注后48h取L5节段脊髓行HE染色、Fluoro-Jade B染色和TUNEL染色。计数脊髓前角正常神经元、Fluoro-Jade B阳性神经元和TUNEL阳性神经元。
【结果】
实验一:再灌注48h时,与Sham组相比,I/R组动物后肢运动功能评分降低,脊髓前角正常神经元数量显著减少,脊髓前角Fluoro-Jade B阳性神经元数量和TUNEL阳性神经元数量显著增多(P < 0.01);脊髓组织白质p-ERK1/2大量表达,并与星形胶质细胞标记物GFAP复染,但脊髓前角神经元没有表达p-ERK1/2。
实验二:再灌注48h时,与O2+W1h组相比,Sev+W1h组动物后肢运动功能评分增高,脊髓前角正常神经元数量显著增多(P<0.01),而Sev+W15min组和O2+W15min组比较差异无显著意义(P > 0.05)。
实验三:再灌注后1d起,Sev组动物后肢运动功能评分显著高于O2组(P < 0.05),再灌注后2d～7d,每组动物后肢运动功能评分随时间变化无显著差异(P > 0.05); 2组动物脊髓前角神经元计数均从再灌注后8h开始减少,48h时计数减到最少,随后没有显著变化(P > 0.05),各时间点Sev组动物脊髓前角正常神经元数量显著多于O2组(P < 0.05); Sev组动物的脊髓前角变性神经元计数从再灌注后8h开始从0增加,3d时计数增至最多,随后逐渐减少;O2组动物脊髓前角变性神经元计数8h时已增加,至3d时增至最多,随后逐渐减少,7d时2组动物脊髓前角Fluoro-Jade B阳性神经元计数均为0,其余各时间点Sev组动物脊髓前角Fluoro-Jade B阳性神经元数量显著少于O2组(P < 0.05); 2组动物脊髓前角凋亡神经元计数均从再灌注后8h以后开始从0增加,2d时计数增至最多,随后逐渐减少;7d时均减至0点,再灌注后1d～5d时Sev组动物脊髓前角TUNEL阳性神经元数量显著少于O2组(P < 0.05)。
实验四:再灌注48h时,与Vehicle+O2组和U0126+Sev组相比,Vehicle+Sev组动物后肢运动功能评分增高,脊髓前角正常神经元数量显著增多,脊髓前角Fluoro-Jade B阳性神经元数量和TUNEL阳性神经元数量显著减少(P≤0.01),而U0126+Sev组与U0126+O2组比较实验结果差异无统计学意义(P > 0.05)。
【结论】
1.脊髓缺血20min再灌注后5d内脊髓前角运动神经元发生变性和凋亡,证实神经元变性和凋亡是缺血/再灌注后脊髓前角运动神经元损伤、死亡的重要形式。
2.脊髓缺血20min再灌注48h时星形胶质细胞p-ERK1/2表达阳性,脊髓神经元无p-ERK1/2表达。证实ERK1/2的激活在脊髓缺血/再灌注中扮演重要角色。
3.缺血前1h给予30min 3.7%(1.0MAC)七氟烷预处理可以诱导快速缺血耐受,减轻兔脊髓缺血20min再灌注神经损伤。
4.缺血前1h给予30min 3.7%(1.0MAC)七氟烷预处理诱导快速缺血耐受的保护效果可以持续至再灌注后7d,此时神经元变性和凋亡均已结束,证实七氟烷预处理是通过减轻神经损伤而不是延迟神经损伤对脊髓缺血/再灌注起保护作用。
5. ERK1/2抑制剂U0126可以抑制七氟烷预处理对脊髓缺血/再灌注损伤的保护作用,同时也抑制了七氟烷预处理减轻脊髓组织缺血/再灌注后前角运动神经元变性和凋亡的作用。一定程度上说明七氟烷预处理通过激活ERK1/2磷酸化,抑制其下游神经元变性和凋亡对脊髓缺血/再灌注损伤起保护作用。
【Background】Despite many strategies have been developed to increase the ischemic tolerance of spinal cord and minimize the incidence of neurological complications, spinal cord ischemia occurred in the patients undergoing thoracic aorta or thoracoabdominal aorta aneurysm surgery. The quoted figure 3% to 18% was incidence of residual permanent injury (acute and/or delayed paraplegia) caused by the ischemic episode. Spinal cord ischemia/reperfusion (I/R) injury could also take place during spinal surgery of tumor, disc decompression and deformity. The social expense associated with the medical management and lost earnings of this disorder over the course of one’s life was significant.
Sevoflurane, as a novel inhalational anesthetic with minimal pungency, low solubility, less hepatic toxicity, and absence of arrhythmogenicity is a preferable choice in clinic. Recently, studies demonstrated that sevoflurane preconditioning could induce ischemic tolerance in brain and heart, and alleviate hypoxic and ischemic injury. However, few study attempting to describe the protective effect of this agent on spinal cord I/R appeared.
The present study was designed to investigate whether sevoflurane preconditioning could induce rapid ischemic tolerance to spinal cord in a rabbit model of transient spinal cord ischemia, and the potential mechanism of the protection.
【Methods】New Zealand White male rabbits (2.0 kg - 2.9 kg) were used in this experiment.
Protocol 1: Animals were randomly assigned to 2 groups (n = 6, each). Animals in the I/R group received spinal cord I/R induced by infrarenal aorta occlusion for 20 min; Sham group received the same anesthesia and surgical preparation but no preconditioning or spinal cord I/R. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for HE staining, Fluoro-Jade B staining, TUNEL staining and p-ERK1/2 immunofluorescent staining.
Protocol 2: Animals were randomly assigned to 4 groups (n = 10, each). Animals in the Sev+W1h group and Sev+W15min group received preconditioning with 3.7% sevoflurane (1.0 MAC) in 96% oxygen for 30 min 1 h or 15 min after preconditioning, animals were subjected to spinal cord I/R respectively. Animals in the O2+W1h group and O2+W15min group as control inhaled only 96% oxygen for 30 min instead. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for histopathological examination.
Protocol 3: Animals were randomly assigned to 2 groups. Animals in the Sev group received preconditioning with 3.7% sevoflurane (1.0 MAC) in 96% oxygen for 30 min, while animals in the O2 group as control inhaled only 96% oxygen for 30 min instead. One hour after preconditioning, the animals were subjected to spinal cord I/R. Animals in each group were randomly assigned to 6 small groups by their end point of investigation (8 h, 1 d, 2 d, 3 d, 5 d, 7 d after reperfusion, n = 6, each). All animals were assessed at 8 h, 1 d, 2 d, 3 d, 5 d, 7 d after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested for HE staining, Fluoro-Jade B staining and TUNEL staining.
Protocol 4: Animals were randomly assigned to 4 groups (n = 8). U0126, an ERK1/2 inhibitor, was administered intravenously 20 min before the beginning of preconditioning in the U0126 + O2 and U0126 + Sev groups. DMSO was administered intravenously at the same time in the Vehicle + O2 and Vehicle + Sev groups. Animals were subjected to spinal cord I/R 1 h after preconditioning. The methods of preconditioning and spinal cord I/R procedure were the same as described in protocol 3. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for HE staining, Fluoro-Jade B staining and TUNEL staining.
Normal motor neurons, Fluoro-Jade B-positive neurons and TUNEL-positive neurons in the anterior of the L5 spinal cord were counted.
【Results】
Protocol 1: Compared with Sham group, animals in the I/R group had lower neurological scores, less normal motor neurons, more Fluoro-Jade B-positive neurons and TUNEL-positive neurons in the anterior of spinal cord (P < 0.01) 48 h after reperfusion. There were many p-ERK1/2-positive astroglias (double labled with GFAP, the marker of astroglia) in the white matter of spinal cord in I/R group, while all neurons were p-ERK1/2-negative.
Protocol 2: 48 h after reperfusion, compared with O2+W1h group, animals in the Sev+W1h group had higher neurological scores and more normal motor neurons in the anterior of spinal cord (P < 0.01). There was no difference between the Sev+W15min group and O2+W15min group (P > 0.05).
Protocol 3: From 1 d after reperfusion, animals in Sev group had higher neurological scores than those in O2 group (P < 0.05). There was no significant change of neurological scores from 2 d to 7 d after reperfusion in every group (P > 0.05). The number of normal neurons in the anterior of spinal cord decreased from 8 h after reperfusion in 2 groups, and downed to the bottom at 48 h after reperfusion, then the number had no significant change (P > 0.05). Animals in Sev group had more normal motor neurons than O2 group at every investigative time point (P < 0.05). The number of Fluoro-Jade B-positive neurons in the anterior of spinal cord increased from 8 h after reperfusion in Sev group, while it increased before 8 h after reperfusion in the O2 group, and rose to the top at 3 d after reperfusion, then decreased to zero at 7 d after reperfusion gradually. Animals in Sev group had less Fluoro-Jade B-positive neurons than O2 group at every investigative point except 7 d after reperfusion (P < 0.05). The number of TUNEL-positive neurons in the anterior of spinal cord elevated from 8 h after reperfusion in 2 groups, and rose to the top at 2 d after reperfusion, then decreased to zero at 7 d after reperfusion gradually. Animals in Sev group had less TUNEL-positive neurons than O2 group from 1 d to 5 d after reperfusion (P < 0.05).
Protocol 4: Compared with Vehicle+Sev group, U0126+Sev group presented worse neurological outcomes, fewer viable neurons and more degenerative neurons and more apoptotic neurons (P≤0.01 for each comparison). There were no significant differences in the outcomes among Vehicle+O2, U0126+O2, and U0126+Sev groups (P > 0.05).
【Conclusion】
1. This study demonstrates that neuronal degeneration and apoptosis are important mechanisms of spinal cord I/R injury, as they presented within 5 d after spinal cord I/R.
2. This experiment indicates that ERK1/2 plays a crucial role in spinal cord I/R, because there were many p-ERK1/2-positive astroglias in the white matter of spinal cord in animals suffered spinal cord I/R, while all neurons were negative staining.
3. The investigation displays that 3.7% (1.0 MAC) sevoflurane preconditioning 1 h before ischemia induces rapid tolerance to spinal cord I/R in rabbits.
4. The research proves that the protection of sevoflurane preconditioning is caused by attenuating neurological injury instead of delaying the injury. The beneficial effect could last 7 d after reperfusion, and there were no neuronal degeneration or apoptosis at that time.
5. This test indicates that sevoflurane preconditioning induced rapid tolerance to spinal cord I/R in rabbits is possibly mediated through the activation of ERK1/2 and the pathway of ERK1/2 mediated for anti-apoptosis in the spinal cord.
七氟烷具有刺激性低,诱导、苏醒迅速,麻醉维持平稳,肝、肾毒性低,心律失常发生率低等优点,被广泛应用于临床麻醉。已有研究报道,七氟烷预处理可诱导缺血耐受,对缺血、缺氧的心、脑起保护作用,但其对脊髓缺血/再灌注损伤是否具有保护作用尚不清楚。
本研究采用兔肾下腹主动脉阻闭20min缺血/再灌注模型,研究单次七氟烷预处理是否可诱导快速缺血耐受,减轻脊髓缺血/再灌注损伤,并探讨其可能的作用机制。
【方法】雄性新西兰大白兔,2.0千克～2.9千克。
实验一:随机分为2组,每组各6只动物。I/R组动物行20min脊髓缺血后再灌注48h。Sham组动物接受同样的麻醉和手术准备,但不行缺血/再灌注损伤。于再灌注后4h,8h,12h,24h及48h行后肢运动功能评分,再灌注后48h取L5节段脊髓行HE染色、p-ERK1/2染色、Fluoro-Jade B染色和TUNEL染色。计数脊髓前角正常神经元、Fluoro-Jade B阳性神经元和TUNEL阳性神经元。
实验二:随机分为4组,每组10只动物。Sev+W1h组和Sev+W15min组动物面罩密闭吸入3.7%(1.0MAC)七氟烷+96%氧气预处理30min,分别洗脱1h或15min;O2+W1h组和O2+W15min组动物面罩密闭吸入96%氧气30min,分别洗脱1h或15min;4组动物均行20min脊髓缺血后再灌注48h。于再灌注后4h,8h,12h,24h及48h行后肢运动功能评分,再灌注后48h取L5节段脊髓行HE染色和脊髓前角正常神经元计数。
实验三:随机分为2大组,Sev组和O2组。Sev组动物面罩密闭吸入3.7%(1.0MAC)七氟烷+96%氧气预处理30min;O2组动物面罩密闭吸入96%氧气30min,两组动物均洗脱1h后行脊髓缺血20min后再灌注。每组分别按最后观察点为再灌注后8h,1d,2d,3d,5d,7d分为六组,每组6只动物。于再灌注后8h,1d,2d,3d,5d,7d行后肢运动功能评分,取L5节段脊髓行HE染色、Fluoro-Jade B染色和TUNEL染色。计数脊髓前角正常神经元、Fluoro-Jade B阳性神经元和TUNEL阳性神经元。
实验四:随机分为4组,每组8只动物。Vehicle+Sev组和Vehicle+O2组动物静脉注射溶剂DMSO 50μL/kg,20min后分别吸入七氟烷和氧气预处理;U0126+Sev组和U0126+O2组动物静脉注射50μL/kg 0.4% U0126(溶于DMSO),20min后分别吸入七氟烷和氧气预处理;预处理方法和兔脊髓缺血/再灌注方法同实验三。于再灌注后4h,8h,12h,24h及48h行后肢运动功能评分,再灌注后48h取L5节段脊髓行HE染色、Fluoro-Jade B染色和TUNEL染色。计数脊髓前角正常神经元、Fluoro-Jade B阳性神经元和TUNEL阳性神经元。
【结果】
实验一:再灌注48h时,与Sham组相比,I/R组动物后肢运动功能评分降低,脊髓前角正常神经元数量显著减少,脊髓前角Fluoro-Jade B阳性神经元数量和TUNEL阳性神经元数量显著增多(P < 0.01);脊髓组织白质p-ERK1/2大量表达,并与星形胶质细胞标记物GFAP复染,但脊髓前角神经元没有表达p-ERK1/2。
实验二:再灌注48h时,与O2+W1h组相比,Sev+W1h组动物后肢运动功能评分增高,脊髓前角正常神经元数量显著增多(P<0.01),而Sev+W15min组和O2+W15min组比较差异无显著意义(P > 0.05)。
实验三:再灌注后1d起,Sev组动物后肢运动功能评分显著高于O2组(P < 0.05),再灌注后2d～7d,每组动物后肢运动功能评分随时间变化无显著差异(P > 0.05); 2组动物脊髓前角神经元计数均从再灌注后8h开始减少,48h时计数减到最少,随后没有显著变化(P > 0.05),各时间点Sev组动物脊髓前角正常神经元数量显著多于O2组(P < 0.05); Sev组动物的脊髓前角变性神经元计数从再灌注后8h开始从0增加,3d时计数增至最多,随后逐渐减少;O2组动物脊髓前角变性神经元计数8h时已增加,至3d时增至最多,随后逐渐减少,7d时2组动物脊髓前角Fluoro-Jade B阳性神经元计数均为0,其余各时间点Sev组动物脊髓前角Fluoro-Jade B阳性神经元数量显著少于O2组(P < 0.05); 2组动物脊髓前角凋亡神经元计数均从再灌注后8h以后开始从0增加,2d时计数增至最多,随后逐渐减少;7d时均减至0点,再灌注后1d～5d时Sev组动物脊髓前角TUNEL阳性神经元数量显著少于O2组(P < 0.05)。
实验四:再灌注48h时,与Vehicle+O2组和U0126+Sev组相比,Vehicle+Sev组动物后肢运动功能评分增高,脊髓前角正常神经元数量显著增多,脊髓前角Fluoro-Jade B阳性神经元数量和TUNEL阳性神经元数量显著减少(P≤0.01),而U0126+Sev组与U0126+O2组比较实验结果差异无统计学意义(P > 0.05)。
【结论】
1.脊髓缺血20min再灌注后5d内脊髓前角运动神经元发生变性和凋亡,证实神经元变性和凋亡是缺血/再灌注后脊髓前角运动神经元损伤、死亡的重要形式。
2.脊髓缺血20min再灌注48h时星形胶质细胞p-ERK1/2表达阳性,脊髓神经元无p-ERK1/2表达。证实ERK1/2的激活在脊髓缺血/再灌注中扮演重要角色。
3.缺血前1h给予30min 3.7%(1.0MAC)七氟烷预处理可以诱导快速缺血耐受,减轻兔脊髓缺血20min再灌注神经损伤。
4.缺血前1h给予30min 3.7%(1.0MAC)七氟烷预处理诱导快速缺血耐受的保护效果可以持续至再灌注后7d,此时神经元变性和凋亡均已结束,证实七氟烷预处理是通过减轻神经损伤而不是延迟神经损伤对脊髓缺血/再灌注起保护作用。
5. ERK1/2抑制剂U0126可以抑制七氟烷预处理对脊髓缺血/再灌注损伤的保护作用,同时也抑制了七氟烷预处理减轻脊髓组织缺血/再灌注后前角运动神经元变性和凋亡的作用。一定程度上说明七氟烷预处理通过激活ERK1/2磷酸化,抑制其下游神经元变性和凋亡对脊髓缺血/再灌注损伤起保护作用。
【Background】Despite many strategies have been developed to increase the ischemic tolerance of spinal cord and minimize the incidence of neurological complications, spinal cord ischemia occurred in the patients undergoing thoracic aorta or thoracoabdominal aorta aneurysm surgery. The quoted figure 3% to 18% was incidence of residual permanent injury (acute and/or delayed paraplegia) caused by the ischemic episode. Spinal cord ischemia/reperfusion (I/R) injury could also take place during spinal surgery of tumor, disc decompression and deformity. The social expense associated with the medical management and lost earnings of this disorder over the course of one’s life was significant.
Sevoflurane, as a novel inhalational anesthetic with minimal pungency, low solubility, less hepatic toxicity, and absence of arrhythmogenicity is a preferable choice in clinic. Recently, studies demonstrated that sevoflurane preconditioning could induce ischemic tolerance in brain and heart, and alleviate hypoxic and ischemic injury. However, few study attempting to describe the protective effect of this agent on spinal cord I/R appeared.
The present study was designed to investigate whether sevoflurane preconditioning could induce rapid ischemic tolerance to spinal cord in a rabbit model of transient spinal cord ischemia, and the potential mechanism of the protection.
【Methods】New Zealand White male rabbits (2.0 kg - 2.9 kg) were used in this experiment.
Protocol 1: Animals were randomly assigned to 2 groups (n = 6, each). Animals in the I/R group received spinal cord I/R induced by infrarenal aorta occlusion for 20 min; Sham group received the same anesthesia and surgical preparation but no preconditioning or spinal cord I/R. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for HE staining, Fluoro-Jade B staining, TUNEL staining and p-ERK1/2 immunofluorescent staining.
Protocol 2: Animals were randomly assigned to 4 groups (n = 10, each). Animals in the Sev+W1h group and Sev+W15min group received preconditioning with 3.7% sevoflurane (1.0 MAC) in 96% oxygen for 30 min 1 h or 15 min after preconditioning, animals were subjected to spinal cord I/R respectively. Animals in the O2+W1h group and O2+W15min group as control inhaled only 96% oxygen for 30 min instead. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for histopathological examination.
Protocol 3: Animals were randomly assigned to 2 groups. Animals in the Sev group received preconditioning with 3.7% sevoflurane (1.0 MAC) in 96% oxygen for 30 min, while animals in the O2 group as control inhaled only 96% oxygen for 30 min instead. One hour after preconditioning, the animals were subjected to spinal cord I/R. Animals in each group were randomly assigned to 6 small groups by their end point of investigation (8 h, 1 d, 2 d, 3 d, 5 d, 7 d after reperfusion, n = 6, each). All animals were assessed at 8 h, 1 d, 2 d, 3 d, 5 d, 7 d after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested for HE staining, Fluoro-Jade B staining and TUNEL staining.
Protocol 4: Animals were randomly assigned to 4 groups (n = 8). U0126, an ERK1/2 inhibitor, was administered intravenously 20 min before the beginning of preconditioning in the U0126 + O2 and U0126 + Sev groups. DMSO was administered intravenously at the same time in the Vehicle + O2 and Vehicle + Sev groups. Animals were subjected to spinal cord I/R 1 h after preconditioning. The methods of preconditioning and spinal cord I/R procedure were the same as described in protocol 3. All animals were assessed at 4 h, 8 h, 12 h, 24 h and 48 h after reperfusion with modified Tarlov criteria, and the spinal cord segments (L5) were harvested 48 h after reperfusion for HE staining, Fluoro-Jade B staining and TUNEL staining.
Normal motor neurons, Fluoro-Jade B-positive neurons and TUNEL-positive neurons in the anterior of the L5 spinal cord were counted.
【Results】
Protocol 1: Compared with Sham group, animals in the I/R group had lower neurological scores, less normal motor neurons, more Fluoro-Jade B-positive neurons and TUNEL-positive neurons in the anterior of spinal cord (P < 0.01) 48 h after reperfusion. There were many p-ERK1/2-positive astroglias (double labled with GFAP, the marker of astroglia) in the white matter of spinal cord in I/R group, while all neurons were p-ERK1/2-negative.
Protocol 2: 48 h after reperfusion, compared with O2+W1h group, animals in the Sev+W1h group had higher neurological scores and more normal motor neurons in the anterior of spinal cord (P < 0.01). There was no difference between the Sev+W15min group and O2+W15min group (P > 0.05).
Protocol 3: From 1 d after reperfusion, animals in Sev group had higher neurological scores than those in O2 group (P < 0.05). There was no significant change of neurological scores from 2 d to 7 d after reperfusion in every group (P > 0.05). The number of normal neurons in the anterior of spinal cord decreased from 8 h after reperfusion in 2 groups, and downed to the bottom at 48 h after reperfusion, then the number had no significant change (P > 0.05). Animals in Sev group had more normal motor neurons than O2 group at every investigative time point (P < 0.05). The number of Fluoro-Jade B-positive neurons in the anterior of spinal cord increased from 8 h after reperfusion in Sev group, while it increased before 8 h after reperfusion in the O2 group, and rose to the top at 3 d after reperfusion, then decreased to zero at 7 d after reperfusion gradually. Animals in Sev group had less Fluoro-Jade B-positive neurons than O2 group at every investigative point except 7 d after reperfusion (P < 0.05). The number of TUNEL-positive neurons in the anterior of spinal cord elevated from 8 h after reperfusion in 2 groups, and rose to the top at 2 d after reperfusion, then decreased to zero at 7 d after reperfusion gradually. Animals in Sev group had less TUNEL-positive neurons than O2 group from 1 d to 5 d after reperfusion (P < 0.05).
Protocol 4: Compared with Vehicle+Sev group, U0126+Sev group presented worse neurological outcomes, fewer viable neurons and more degenerative neurons and more apoptotic neurons (P≤0.01 for each comparison). There were no significant differences in the outcomes among Vehicle+O2, U0126+O2, and U0126+Sev groups (P > 0.05).
【Conclusion】
1. This study demonstrates that neuronal degeneration and apoptosis are important mechanisms of spinal cord I/R injury, as they presented within 5 d after spinal cord I/R.
2. This experiment indicates that ERK1/2 plays a crucial role in spinal cord I/R, because there were many p-ERK1/2-positive astroglias in the white matter of spinal cord in animals suffered spinal cord I/R, while all neurons were negative staining.
3. The investigation displays that 3.7% (1.0 MAC) sevoflurane preconditioning 1 h before ischemia induces rapid tolerance to spinal cord I/R in rabbits.
4. The research proves that the protection of sevoflurane preconditioning is caused by attenuating neurological injury instead of delaying the injury. The beneficial effect could last 7 d after reperfusion, and there were no neuronal degeneration or apoptosis at that time.
5. This test indicates that sevoflurane preconditioning induced rapid tolerance to spinal cord I/R in rabbits is possibly mediated through the activation of ERK1/2 and the pathway of ERK1/2 mediated for anti-apoptosis in the spinal cord.
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