甲基强的松龙对深低温停循环大鼠脑保护作用的研究
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摘要
【目的】
深低温停循环技术(DHCA)自上世纪50年代诞生以来，已广泛应用于复杂和严重的先天性心脏病及大血管手术中，但其可能并发脑损伤的问题尚未得到较好的解决，深低温停循环术后中枢神经系统的发病率高达4％～25％。因此，研究DHCA脑损伤机制，及如何延长DHCA安全时限就显得尤为重要。甲基强的松龙(MP)具有减少炎症病灶周围免疫细胞浸润、促进水肿的吸收等多种功效。近年来研究发现，MP具有抑制深低温停循环后脑水肿，炎症反应等脑保护作用，但其具体作用机制尚不清楚。本研究采用新的深低温停循环大鼠脑损伤模型，分别观察MP对深低温停循环大鼠脑组织NMDAR1蛋白、NMDAR1mRNA以及脑含水量、组织超微结构的影响。
【方法】
第一部分深低温停循环大鼠脑保护模型的建立和评价：雄性SD大鼠30只，随机分为3组，即颈动脉阻断深低温停循环组、颈内动脉引流深低温停循环组和假手术组。于深低温停循环后60min时监测脑电图变化，停循环60min后恢复循环并升温。所有大鼠于术后24h处死，并取脑组织测脑含水量。
第二部分甲基强的松龙对深低温停循环大鼠脑NMDAR1蛋白表达的影响：采用大鼠DHCA模型。雄性SD大鼠240只，随机分为A组(假手术组)、B组(DHCA模型组)、C组(甲强龙处理组)。A组16只；B组和C组各112只，每组再分为DHCA后2h、6h、12h、1d、2d、3d、7d共7个小组。观察甲强龙对DHCA60min，再灌注2h、6h、12h、1d、2d、3d及7d时脑含水量和NMDAR1蛋白表达的变化。
第三部分甲基强的松龙对深低温停循环大鼠脑NMDAR1 mRNA表达的影响：采用大鼠深低温停循环(DHCA)模型。雄性SD大鼠144只，随机分为A组(假手术组)、B组(DHCA模型组)、C组(甲强龙处理组)。观察DHCA60min，再灌注2h、6h、12h、1d、2d、3d时NMDAR1mRNA表达的变化以及脑组织超微结构变化。
【结果】
第一部分颈内动脉引流DHCA组大鼠α波相对功率值明显低于颈动脉阻断DHCA组(P＜0.01)，而且两组大鼠α波相对功率值均明显低于假手术组(P＜0.01)；颈内动脉引流DHCA组大鼠θ波相对功率值明显低于颈动脉阻断DHCA组和假手术组(P＜0.01)，而后两组大鼠θ波相对功率值无差异。脑含水率结果显示颈内动脉引流DHCA组大鼠脑含水量高于颈动脉阻断DHCA组(P＜0.01)。
第二部分B组和C组大鼠在深低温停循环再灌注6h起出现脑水肿，到24h水肿到达高峰，于3d后逐渐恢复至正常水平，其中C组在12h、1d、2d水肿明显低于B组(P＜0.01)。免疫组化结果显示，DHCA再灌注2h起，B组和C组大鼠NMDAR1蛋白开始升高，于1d到达高峰，后逐渐降低，于再灌注7d恢复至正常水平。C组大鼠再灌注后2h、6h、12h、1d、2d五个时间点NMDAR1表达明显低于B组(P＜0.05)。
第三部分假手术组NMDAR1mRNA呈基础水平表达。甲基强的松龙组NMDAR1mRNA表达量与模型组相比，在各时相点均显著降低(P＜0.01)。脑组织超微结构观察发现甲基强的松龙能抑制DHCA脑细胞凋亡。
【结论】
(1)颈内动脉引流深低温停循环模型较颈动脉阻断深低温停循环模型脑缺血更完全，是一种更为理想的深低温停循环大鼠脑保护模型。
(2)甲基强的松龙对DHCA大鼠脑组织具有保护作用，其对NMDAR1蛋白表达的抑制作用可能是其脑保护作用的重要机制。
(3)甲基强的松龙可能通过下调NMDAR1mRNA的过量表达，对深低温停循环脑损伤起到保护作用。
Objective:
The technique of deep hypothermic circulatory arrest has been widely applied in cardiac surgery after 1950s, but the temporarily or permanent neural complications were high to 4％～25％. Therefore, it is very important to investigate the mechanism of brain scathe and how to prolong the safe time of DHCA. Recent researches find that Methylprednisolone can restrain the hydrocephalus and the inflammation after DHCA, but the detailed mechanism is not clear. In the present study, we used a new brain protection model of deep hypothermic circulatory arrest in rats to explore the influence of methylprednisolone on the expression of NMDAR1 protein and NMDAR1mRNA.
Methods:
PartⅠ: To establish and appraise the brain protection model of deep hypothermic circulatory arrest in rats: 30 SD rats were divided randomly into three groups: Carotid occlusion DHCA group; inner carotid shunt DHCA group and Shame group. Recording the electroencephalograph (EEG) 24 hours after DHCA, then separating from DHCA and rewarming. All rats were killed 24 hours after DHCA, then measure the brain moisture content.
PartⅡ: Influence of methylprednisolone on the expression of NMDAR1 after DHCA: Using wistar rat model of deep hypo-thermic circulatory arrest, 240 SD rats were divided randomly into three groups: Sham group; Methylprednisolone(MP) group and Model group. After separation from DHCA, rats were killed at 2h, 4h, 12h, 1d, 2d and 3d and 1 week, the expressions of N-methyl-D-aspartate receptor 1 protein were detected by immunohistochemistry.
PartⅢ: Influence of methylprednisolone on the expression of NMDAR1 mRNA after DHCA: Using wistar rat model of deep hypothermic circulatory arrest, 144SD rats were divided randomly into three groups: Sham group(group A); Model group(group B) and Methylprednisolone (MP) group(group C). After separation from DHCA, rats were killed at 2h, 4h, 12h, 1d, 2d and 3d, the expressions of N-methyl-D-aspartate receptor 1 mRNA were detected by RT-PCR.
Results:
PartⅠ: Theαwave relative power of EEG in inner carotid shunt DHCA group was lower than that in Carotid occlusion DHCA group(P＜0.01), they were both lower than that in Sham group(P＜0.01); Theθwave relative power of EEG in inner carotid shunt DHCA group was lower than that in Carotid occlusion DHCA group and Sham group(P＜0.01), and there was no difference between Carotid occlusion DHCA group and Sham group. The brain moisture content was higher in inner carotid shunt DHCA group than Carotid occlusion DHCA group(P＜0.01).
PartⅡ: Hydrocephalus can be observed at 6h after separation from DHCA, reached peak at 24h, and gradually come back to normal level at 3d in MP group and Model group. In contrast to the Model group, hydrocephalus reduced significantly in MP group(P＜0.01). The expressions of N-methyl-D-aspartate receptor 1 protein was increased at 2h after separa-tion from DHCA, reached peak at 24h, and gradually come back to normal level at 7d in MP group and Model group. However, Preconditioning with Methylprednisolone re-sulted in a significant reduction in the expressions of NMDAR1 at 2h, 6h, 12h, 1d and 2d after separation from DHCA comparing with Model group(P＜0.05).
PartⅢ: The expressions of N-methyl-D-aspartate receptor 1 mRNA were on basal level in Sham group and the expressions of NMDAR1 mRNA were significantly higher in Model group than MP treatment group(P＜0.01).
Conclusion:
(1) Inner carotid shunt DHCA group is a more perfect brain protection model of DHCA.
(2) Preconditioning with Methylprednisolone inhibits the expression of N-methyl-D-aspartate receptor 1 protein after DHCA in rats. This maybe a important mechanism of cerebral protection of preconditioning with Methylprednisolone.
(3) Methylprednisolone maybe decrease the expression of NMDAR1 mRNA. This maybe a important mechanism of cerebral protection of preconditioning with Methylprednisolone.
深低温停循环技术(DHCA)自上世纪50年代诞生以来，已广泛应用于复杂和严重的先天性心脏病及大血管手术中，但其可能并发脑损伤的问题尚未得到较好的解决，深低温停循环术后中枢神经系统的发病率高达4％～25％。因此，研究DHCA脑损伤机制，及如何延长DHCA安全时限就显得尤为重要。甲基强的松龙(MP)具有减少炎症病灶周围免疫细胞浸润、促进水肿的吸收等多种功效。近年来研究发现，MP具有抑制深低温停循环后脑水肿，炎症反应等脑保护作用，但其具体作用机制尚不清楚。本研究采用新的深低温停循环大鼠脑损伤模型，分别观察MP对深低温停循环大鼠脑组织NMDAR1蛋白、NMDAR1mRNA以及脑含水量、组织超微结构的影响。
【方法】
第一部分深低温停循环大鼠脑保护模型的建立和评价：雄性SD大鼠30只，随机分为3组，即颈动脉阻断深低温停循环组、颈内动脉引流深低温停循环组和假手术组。于深低温停循环后60min时监测脑电图变化，停循环60min后恢复循环并升温。所有大鼠于术后24h处死，并取脑组织测脑含水量。
第二部分甲基强的松龙对深低温停循环大鼠脑NMDAR1蛋白表达的影响：采用大鼠DHCA模型。雄性SD大鼠240只，随机分为A组(假手术组)、B组(DHCA模型组)、C组(甲强龙处理组)。A组16只；B组和C组各112只，每组再分为DHCA后2h、6h、12h、1d、2d、3d、7d共7个小组。观察甲强龙对DHCA60min，再灌注2h、6h、12h、1d、2d、3d及7d时脑含水量和NMDAR1蛋白表达的变化。
第三部分甲基强的松龙对深低温停循环大鼠脑NMDAR1 mRNA表达的影响：采用大鼠深低温停循环(DHCA)模型。雄性SD大鼠144只，随机分为A组(假手术组)、B组(DHCA模型组)、C组(甲强龙处理组)。观察DHCA60min，再灌注2h、6h、12h、1d、2d、3d时NMDAR1mRNA表达的变化以及脑组织超微结构变化。
【结果】
第一部分颈内动脉引流DHCA组大鼠α波相对功率值明显低于颈动脉阻断DHCA组(P＜0.01)，而且两组大鼠α波相对功率值均明显低于假手术组(P＜0.01)；颈内动脉引流DHCA组大鼠θ波相对功率值明显低于颈动脉阻断DHCA组和假手术组(P＜0.01)，而后两组大鼠θ波相对功率值无差异。脑含水率结果显示颈内动脉引流DHCA组大鼠脑含水量高于颈动脉阻断DHCA组(P＜0.01)。
第二部分B组和C组大鼠在深低温停循环再灌注6h起出现脑水肿，到24h水肿到达高峰，于3d后逐渐恢复至正常水平，其中C组在12h、1d、2d水肿明显低于B组(P＜0.01)。免疫组化结果显示，DHCA再灌注2h起，B组和C组大鼠NMDAR1蛋白开始升高，于1d到达高峰，后逐渐降低，于再灌注7d恢复至正常水平。C组大鼠再灌注后2h、6h、12h、1d、2d五个时间点NMDAR1表达明显低于B组(P＜0.05)。
第三部分假手术组NMDAR1mRNA呈基础水平表达。甲基强的松龙组NMDAR1mRNA表达量与模型组相比，在各时相点均显著降低(P＜0.01)。脑组织超微结构观察发现甲基强的松龙能抑制DHCA脑细胞凋亡。
【结论】
(1)颈内动脉引流深低温停循环模型较颈动脉阻断深低温停循环模型脑缺血更完全，是一种更为理想的深低温停循环大鼠脑保护模型。
(2)甲基强的松龙对DHCA大鼠脑组织具有保护作用，其对NMDAR1蛋白表达的抑制作用可能是其脑保护作用的重要机制。
(3)甲基强的松龙可能通过下调NMDAR1mRNA的过量表达，对深低温停循环脑损伤起到保护作用。
Objective:
The technique of deep hypothermic circulatory arrest has been widely applied in cardiac surgery after 1950s, but the temporarily or permanent neural complications were high to 4％～25％. Therefore, it is very important to investigate the mechanism of brain scathe and how to prolong the safe time of DHCA. Recent researches find that Methylprednisolone can restrain the hydrocephalus and the inflammation after DHCA, but the detailed mechanism is not clear. In the present study, we used a new brain protection model of deep hypothermic circulatory arrest in rats to explore the influence of methylprednisolone on the expression of NMDAR1 protein and NMDAR1mRNA.
Methods:
PartⅠ: To establish and appraise the brain protection model of deep hypothermic circulatory arrest in rats: 30 SD rats were divided randomly into three groups: Carotid occlusion DHCA group; inner carotid shunt DHCA group and Shame group. Recording the electroencephalograph (EEG) 24 hours after DHCA, then separating from DHCA and rewarming. All rats were killed 24 hours after DHCA, then measure the brain moisture content.
PartⅡ: Influence of methylprednisolone on the expression of NMDAR1 after DHCA: Using wistar rat model of deep hypo-thermic circulatory arrest, 240 SD rats were divided randomly into three groups: Sham group; Methylprednisolone(MP) group and Model group. After separation from DHCA, rats were killed at 2h, 4h, 12h, 1d, 2d and 3d and 1 week, the expressions of N-methyl-D-aspartate receptor 1 protein were detected by immunohistochemistry.
PartⅢ: Influence of methylprednisolone on the expression of NMDAR1 mRNA after DHCA: Using wistar rat model of deep hypothermic circulatory arrest, 144SD rats were divided randomly into three groups: Sham group(group A); Model group(group B) and Methylprednisolone (MP) group(group C). After separation from DHCA, rats were killed at 2h, 4h, 12h, 1d, 2d and 3d, the expressions of N-methyl-D-aspartate receptor 1 mRNA were detected by RT-PCR.
Results:
PartⅠ: Theαwave relative power of EEG in inner carotid shunt DHCA group was lower than that in Carotid occlusion DHCA group(P＜0.01), they were both lower than that in Sham group(P＜0.01); Theθwave relative power of EEG in inner carotid shunt DHCA group was lower than that in Carotid occlusion DHCA group and Sham group(P＜0.01), and there was no difference between Carotid occlusion DHCA group and Sham group. The brain moisture content was higher in inner carotid shunt DHCA group than Carotid occlusion DHCA group(P＜0.01).
PartⅡ: Hydrocephalus can be observed at 6h after separation from DHCA, reached peak at 24h, and gradually come back to normal level at 3d in MP group and Model group. In contrast to the Model group, hydrocephalus reduced significantly in MP group(P＜0.01). The expressions of N-methyl-D-aspartate receptor 1 protein was increased at 2h after separa-tion from DHCA, reached peak at 24h, and gradually come back to normal level at 7d in MP group and Model group. However, Preconditioning with Methylprednisolone re-sulted in a significant reduction in the expressions of NMDAR1 at 2h, 6h, 12h, 1d and 2d after separation from DHCA comparing with Model group(P＜0.05).
PartⅢ: The expressions of N-methyl-D-aspartate receptor 1 mRNA were on basal level in Sham group and the expressions of NMDAR1 mRNA were significantly higher in Model group than MP treatment group(P＜0.01).
Conclusion:
(1) Inner carotid shunt DHCA group is a more perfect brain protection model of DHCA.
(2) Preconditioning with Methylprednisolone inhibits the expression of N-methyl-D-aspartate receptor 1 protein after DHCA in rats. This maybe a important mechanism of cerebral protection of preconditioning with Methylprednisolone.
(3) Methylprednisolone maybe decrease the expression of NMDAR1 mRNA. This maybe a important mechanism of cerebral protection of preconditioning with Methylprednisolone.
引文
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41. Heimburger M, Larfars G, Bratt J. Prednisolone inhibits cytokine induced adhesive and cytotoxic interactions between endothelial cells and neutrophils in vitro [J]. Clin Exp Immunol. 2000,119(3): 441-448.
1. Ferry PC. Neurologic Sequelae of open-heart surgery in Children. American Journal of Diseases of Children, 1990;144:369-373.
2. William LY, Michael TL, Dhanesh KG, et al. Anesthetic Management of deep hypothermic circulatory arrest for cerebral aneurysm clipping. Anesthesiology, 2002, 96:497-503.
3. Dennis C, Speng DS Jr, Nelson GE. Development of a pumpoxy-gerator to replace the heart and lungs; an apparatus applicable to human patients and application to one case. Ann Thorac Surg, 1951,134:709-721
4. Jonas RA, et al. Brain injury and pediatric cardiac surgery, 1995, 201-204.
5. Kristian T, Siesjo BK. Calcium-related damage in ischemia[J]. Life Science, 1996,59:357-367.
6. Karibe H,Chen SF, Zarow GJ , et al. Mild intra ischemic hypothermia suppresses consumption of endogenous antioxidants after temporary focal ischemia in rats [J]. Brain Res, 1994,649: 12-18.
7. Siesjo BK. Cell damage in the brain: a speculative synthase[J]. J Cereb Blood Flow M etab, 1981, 1: 155-185.
8. Bracken MB, Shepard MJ , Collins W F, et al. A randomized Controlled trial of methylprednisolone or naloxone in the treatment of acute spinal cord injury [J]. New Engl J M ed, 1990, 322:1405-1411.
9. Braughler JM, Lainer MJ. The effects of large doses of methylprednisolone on neurologic recovery and survival in the Mongolian gerbil following three hours of unilateral carotid occlusion.CN S Trauma, 1986,3:153
10.De Courten-M yers GM , Kleiholz M ,Wagner KR, et al. Efficacious experiment stroke treatment with high dose methylprednisolone. Stroke, 1994,25:487
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12.Langley SM, Chai PJ, Jaggers JJ, et al. Preoperative high dose methylprednisolone attenuates the cerebral response to deep hypothermic circulatory arrest. Eur J Cardiothorac Surg. 2000 Mar;17(3):279-86.
13.Shum-Tim D, Tchervenkov CI, Jamal AM, et al. Systemic steroid pretreatment improves cerebral protection after circulatory arrest. Ann Thorac Surg. 2001 Nov; 72(5): 1465-71; discussion 1471-2.
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18.Schubert S, Stoltenburg-Didinger G, Wehsack A,et al. Large-dose pretreatment with methylprednisolone fails to attenuate neuronal injury after deep hypothermic circulatory arrest in a neonatal piglet model. Anesth Analg,2005 Nov; 101(5): 1311-8.
2. Ferry PC. Neurologic Sequelae of open-heart surgery in Children. American Journal of Diseases of Children, 1990;144:369-373.
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41. Heimburger M, Larfars G, Bratt J. Prednisolone inhibits cytokine induced adhesive and cytotoxic interactions between endothelial cells and neutrophils in vitro [J]. Clin Exp Immunol. 2000,119(3): 441-448.
1. Ferry PC. Neurologic Sequelae of open-heart surgery in Children. American Journal of Diseases of Children, 1990;144:369-373.
2. William LY, Michael TL, Dhanesh KG, et al. Anesthetic Management of deep hypothermic circulatory arrest for cerebral aneurysm clipping. Anesthesiology, 2002, 96:497-503.
3. Dennis C, Speng DS Jr, Nelson GE. Development of a pumpoxy-gerator to replace the heart and lungs; an apparatus applicable to human patients and application to one case. Ann Thorac Surg, 1951,134:709-721
4. Jonas RA, et al. Brain injury and pediatric cardiac surgery, 1995, 201-204.
5. Kristian T, Siesjo BK. Calcium-related damage in ischemia[J]. Life Science, 1996,59:357-367.
6. Karibe H,Chen SF, Zarow GJ , et al. Mild intra ischemic hypothermia suppresses consumption of endogenous antioxidants after temporary focal ischemia in rats [J]. Brain Res, 1994,649: 12-18.
7. Siesjo BK. Cell damage in the brain: a speculative synthase[J]. J Cereb Blood Flow M etab, 1981, 1: 155-185.
8. Bracken MB, Shepard MJ , Collins W F, et al. A randomized Controlled trial of methylprednisolone or naloxone in the treatment of acute spinal cord injury [J]. New Engl J M ed, 1990, 322:1405-1411.
9. Braughler JM, Lainer MJ. The effects of large doses of methylprednisolone on neurologic recovery and survival in the Mongolian gerbil following three hours of unilateral carotid occlusion.CN S Trauma, 1986,3:153
10.De Courten-M yers GM , Kleiholz M ,Wagner KR, et al. Efficacious experiment stroke treatment with high dose methylprednisolone. Stroke, 1994,25:487
11.Danegemez M,Kurt E, Cosar A,et al. Methylprednisolone and vitamin E therapy in perinatal hypoxic ischemic brain damage in rats. Neuroscience,1999, 92(2) : 693- 697.
12.Langley SM, Chai PJ, Jaggers JJ, et al. Preoperative high dose methylprednisolone attenuates the cerebral response to deep hypothermic circulatory arrest. Eur J Cardiothorac Surg. 2000 Mar;17(3):279-86.
13.Shum-Tim D, Tchervenkov CI, Jamal AM, et al. Systemic steroid pretreatment improves cerebral protection after circulatory arrest. Ann Thorac Surg. 2001 Nov; 72(5): 1465-71; discussion 1471-2.
14.Shum-Tim D, Tchervenkov CI, Laliberte E,et al. Timing of steroid treatment is important for cerebral protection during cardiopulmonary bypass and circulatory arrest: minimal protection of pump prime methylprednisolone. Eur J Cardiothorac Surg. 2003 Jul;24(1): 125-32.
15.Kim JS,Chopp M,Gautam SC. High dose methylprednisolone therapy reduces expression of JE/MCP-1 mRNA and macrophage accumulation in the ischemic rat brain. J Neurol Sci ,1995,128(1):28-35.
16.Lees GJ. The possible contribution of microglia and macrophages to delayed neurol death after ischemia. J Neurol Sci, 1993, 114: 109.
17.11dan F, Polat S, Oner A, et al. The effect of treatment of high dose methyprednisolone on Na~+-K~+/Mg~(2+) ATPase activity and lipid peroxidation and ultra- structural findings following cerebral contusion in rat. Surg Neurol, 1995, 44: 573-580.
18.Schubert S, Stoltenburg-Didinger G, Wehsack A,et al. Large-dose pretreatment with methylprednisolone fails to attenuate neuronal injury after deep hypothermic circulatory arrest in a neonatal piglet model. Anesth Analg,2005 Nov; 101(5): 1311-8.