硫化氢联合治疗性低温对心脏骤停大鼠血脑屏障结构与功能的影响
发布时间：2018-07-16 12:02
【摘要】：目的:心脏骤停(cardiac arrest,CA)及心肺复苏(cardiopulmonary resuscitation,CPR)后居高不下的病残率和病死率主要源于全脑缺血再灌注损伤。血脑屏障(blood-brainbarrier,BBB)损伤与脑水肿是脑缺血再灌注损伤的重要病理改变,血脑屏障破坏又可加重缺血后的继发性脑损伤,因而减轻BBB损伤与脑水肿是减轻脑损伤的重要环节。治疗性低温(therapeutic hypothermia,TH)(32℃～34℃)是复苏后一种安全和有效的治疗方法,低温可通过多种机制发挥脑保护作用。但不少试验研究发现单一低温疗法在临床上很难达到预期的脑复苏效果,因此寻求一种联合手段,已成为目前提高脑复苏成功率的一个研究方向。有研究表明,硫化氢(hydrogen sulfide,H2S)可通过神经调节与神经保护作用,减轻中枢神经系统缺血再灌注损伤。因此本研究采用经食道电刺激法建立大鼠心脏骤停及心肺复苏模型,经静脉泵注H2S供体NaHS,并联合治疗性低温疗法观察其对心肺复苏后大鼠血脑屏障的影响,并初步探讨其可能的作用机制。方法:实验共分两部分进行;第一部分探讨硫化氢和(或)治疗性低温对心肺复苏后大鼠生存率及神经功能的影响。雄性Sprague-Dawley(SD)大鼠随机分为5组:(1)假手术组(Sham组,n=5):仅进行麻醉、经口腔气管插管和经左股动、静脉置管等操作;(2)心脏骤停模型组(CA+Normothermia+Saline组,n=15),采用经食道电刺激法建立大鼠心脏骤停模型,并进行心肺复苏,心脏骤停缺血时间为4min;(3)单独硫化氢处理组(CA+Normothermia+NaHS组,n=15),成功复苏后予硫化氢供体NaHS处理;(4)单独低温处理组(CA+Hypothermia+Saline组,n=15),成功复苏后予治疗性低温处理;(5)硫化氢联合低温处理组(CA+Hypothermia+NaHS组,n=15),成功复苏后予硫化氢与低温联合处理。硫化氢供体NaHS的给药方案:于自主循环恢复即刻经静脉注射剂量为的0.5mg/kg的NaHS(货号:13590,Sigma-Aldrich公司,美国)(浓度为0.3 mg/ml),随后以1.5 mg · kg-1 · h-1速率静脉输注,持续3 h;其它组静脉注射生理盐水1.67 ml/kg,随后以5 ml · kg-1 · h-1速率静脉输注3 h;低温的实施:于自主循环恢复即刻用乙醇涂擦大鼠体表,15 min内将直肠温度降至34℃,用冰袋和加热毯维持该体温6 h,随后进行升温处理,4 h内将体温升至38～39°C;其它组维持直肠温度在38～39℃。观察复苏成功后大鼠7天生存情况,使用条带移除实验(Tape removal test,TRT)评价大鼠感觉运动功能;第二部分探讨硫化氢和(或)治疗性低温对复苏后大鼠血脑屏障通透性、脑水肿、血脑屏障超微结构、紧密连接蛋白Occludin表达、基质金属蛋白酶-9(Matrix metalloproteinase-9,MMP-9)表达以及活性影响。分组及处理情况同实验一,每组25只大鼠。于复苏后24 h,采用干湿重法检测脑含水量,代表脑水肿程度;于复苏后24 h,检测脑组织对伊文思兰(Evans blue,EB)与对4 kDa荧光素异硫氰酸酯-葡聚糖(fluorescein isothiocyanate-dextran,FD4)的通透性来反应血脑屏障功能的变化;采用透射电镜技术检测血脑屏障超微结构改变;采用蛋白质免疫印迹法(Western blot)检测紧密连接蛋白Occludin表达;采用免疫组织化学法检测MMP-9表达与分布,采用明胶酶谱法检测MMP-9活性变化。结果:与Sham组比较,其他四组大鼠7天生存率明显降低,条带移除时间延长,脑含水量与血脑屏障通透性增加,血脑屏障超微结构破坏明显,Occludin表达减少,MMP-9表达增加(P0.05);与心脏骤停模型(CA+Normothermia+Saline组)比较,单独硫化氢处理组(CA+Normothermia+NaHS组)、单独低温处理组(CA+Hypothermia+Saline组)与硫化氢联合低温处理(CA+Hypothermia+NaHS组)大鼠7天生存率增加,条带移除时间缩短,脑含水量与血脑屏障通透性减轻,血脑屏障超微结构破坏减轻,Occludin表达增加,MMP-9表达降低,MMP-9活性降低(P0.05);其中硫化氢联合低温处理组(CA+Hypothermia+NaHS组)的各指标改善最明显,且差异均有统计学意义(P0.05)。结论:硫化氢联合治疗性低温能更有效地减轻心肺复苏大鼠血脑屏障通透性、保护血脑屏障结构与功能,发挥更好的脑复苏作用。其机制可能与共同抑制MMP-9表达与活性,减少紧密连接蛋白Occludin降解丢失相关。
[Abstract]:Objective: the morbidity and mortality of cardiac arrest (CA) and cardiopulmonary resuscitation (cardiopulmonary resuscitation, CPR) are mainly caused by cerebral ischemia reperfusion injury. The injury of blood brain barrier (blood-brainbarrier, BBB) and brain edema are important pathological changes of cerebral ischemia reperfusion injury, and the destruction of blood brain barrier can be added. Secondary brain injury after severe ischemia, thus reducing BBB injury and brain edema is an important link in reducing brain damage. Therapeutic hypothermia (therapeutic hypothermia, TH) (32 ~ 34 C) is a safe and effective treatment after resuscitation. Low temperature can play a role in brain protection through a variety of mechanisms. But a number of experimental studies have found a single hypothermia therapy. It is difficult to achieve the expected effect of cerebral resuscitation in clinical, so seeking a combined method has become a research direction to improve the success rate of cerebral resuscitation. Research shows that hydrogen sulfide (hydrogen sulfide, H2S) can alleviate the ischemia reperfusion injury in the central nervous system through neural regulation and neuroprotective effect. The rat model of cardiac arrest and cardiopulmonary resuscitation was established by esophagus electrical stimulation. The effect of H2S donor NaHS on the blood brain barrier of rats after cardiopulmonary resuscitation was observed by intravenous infusion of H2S donor, and the possible mechanism of its action was preliminarily discussed. Methods: the experiment was divided into two parts; the first part discussed hydrogen sulfide and (or) treatment. The effect of hypothermia on the survival rate and nerve function of rats after cardiopulmonary resuscitation. Male Sprague-Dawley (SD) rats were randomly divided into 5 groups: (1) sham operation group (group Sham, n=5): only anesthesia, oral tracheal intubation, left femoral movement, venous catheterization, and (2) cardiac arrest model group (CA+Normothermia+Saline group, n=15), using transesophageal spiny Rat cardiac arrest model was established and cardiopulmonary resuscitation was established and cardiopulmonary resuscitation was carried out. The ischemic time of cardiac arrest was 4min; (3) separate hydrogen sulfide treatment group (group CA+Normothermia+NaHS, n=15), after successful resuscitation, the hydrogen sulfide donor NaHS treatment; (4) single cryogenic treatment group (CA+ Hypothermia+Saline group, n=15), after successful resuscitation, treatment of low temperature treatment; (5) vulcanization. Hydrogen combined with cryogenic treatment group (group CA+Hypothermia+NaHS, n=15), combined with hydrogen sulfide and low temperature after successful resuscitation. Hydrogen sulfide donor NaHS administration scheme: 0.5mg/kg NaHS (13590, Sigma-Aldrich public division, US) (concentration of 0.3 mg/ml), and then 1.5 mg. Kg-1 H-1 Rate intravenous infusion lasted 3 h; the other groups were intravenously injected with normal saline 1.67 ml/kg, followed by intravenous infusion of 3 h at 5 ml. Kg-1. H-1 rate; low temperature was carried out: the body surface of the rat was immediately retreated with ethanol, and the rectal temperature was reduced to 34 C in 15 min, and the temperature 6 h was maintained with ice bag and heat blanket, then heating treatment, 4 h The body temperature was increased to 38~39 C; the other groups maintained the rectal temperature at 38~39 degrees C. Observe the 7 day survival of the rats after the successful resuscitation, using the strip removal experiment (Tape removal test, TRT) to evaluate the sensory motor function of the rats; and the second part discussed the blood brain barrier permeability, brain edema and blood brain of the rats after resuscitation after the recovery of hydrogen sulfide and / or therapeutic low temperature. The ultrastructure of the barrier, the expression of close connexin Occludin, the expression of matrix metalloproteinase -9 (Matrix metalloproteinase-9, MMP-9) and the effect of activity. The grouping and treatment were the same as experiment one, 25 rats in each group. The brain water content was detected by dry wet weight method after 24 h, and the degree of brain edema was represented by the dry wet weight method; the brain tissue was detected after 24 h after resuscitation. Evans Lan (Evans blue, EB) reacts to the change of blood brain barrier function with the permeability of 4 kDa fluorescein isothiocyanate (fluorescein isothiocyanate-dextran, FD4); the ultrastructural changes of the blood brain barrier are detected by transmission electron microscopy, and the protein immunoblotting (Western blot) is used to detect the close connexin Occludin. The expression and distribution of MMP-9 were detected by immunohistochemistry. The changes of MMP-9 activity were detected by gelatin zymogram. Results: compared with group Sham, the 7 natural survival rates of the other four groups were obviously decreased, the time of strip removal was prolonged, the permeability of brain water content and blood brain barrier increased, the ultrastructure of blood brain barrier was obviously destroyed and the expression of Occludin decreased. MMP-9 expression increased (P0.05); compared with cardiac arrest model (group CA+Normothermia+Saline), single hydrogen sulfide treatment group (group CA+Normothermia+NaHS), single cryogenic treatment group (group CA+Hypothermia+Saline) and hydrogen sulfide combined with low temperature treatment (group CA+Hypothermia+NaHS) increased the 7 natural survival rate of rats, the time of strip removal shortened, the water content of the brain was increased. The permeability of blood brain barrier decreased, the ultrastructural damage of blood brain barrier lightened, the expression of Occludin increased, the expression of MMP-9 decreased and the activity of MMP-9 decreased (P0.05). The improvement of each index of hydrogen sulfide combined with cryogenic treatment group (CA+Hypothermia+NaHS group) was most obvious, and the difference had the significance of overall planning (P0.05). Conclusion: hydrogen sulfide combined with therapeutic low temperature can be more effective. It can effectively reduce the permeability of blood brain barrier in the cardiopulmonary resuscitation rats, protect the structure and function of the blood brain barrier, and play a better role in the brain recovery. The mechanism may be related to the common inhibition of MMP-9 expression and activity, and the reduction of the degradation and loss of the close connexin Occludin.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.78
，
本文编号：2126330
[Abstract]:Objective: the morbidity and mortality of cardiac arrest (CA) and cardiopulmonary resuscitation (cardiopulmonary resuscitation, CPR) are mainly caused by cerebral ischemia reperfusion injury. The injury of blood brain barrier (blood-brainbarrier, BBB) and brain edema are important pathological changes of cerebral ischemia reperfusion injury, and the destruction of blood brain barrier can be added. Secondary brain injury after severe ischemia, thus reducing BBB injury and brain edema is an important link in reducing brain damage. Therapeutic hypothermia (therapeutic hypothermia, TH) (32 ~ 34 C) is a safe and effective treatment after resuscitation. Low temperature can play a role in brain protection through a variety of mechanisms. But a number of experimental studies have found a single hypothermia therapy. It is difficult to achieve the expected effect of cerebral resuscitation in clinical, so seeking a combined method has become a research direction to improve the success rate of cerebral resuscitation. Research shows that hydrogen sulfide (hydrogen sulfide, H2S) can alleviate the ischemia reperfusion injury in the central nervous system through neural regulation and neuroprotective effect. The rat model of cardiac arrest and cardiopulmonary resuscitation was established by esophagus electrical stimulation. The effect of H2S donor NaHS on the blood brain barrier of rats after cardiopulmonary resuscitation was observed by intravenous infusion of H2S donor, and the possible mechanism of its action was preliminarily discussed. Methods: the experiment was divided into two parts; the first part discussed hydrogen sulfide and (or) treatment. The effect of hypothermia on the survival rate and nerve function of rats after cardiopulmonary resuscitation. Male Sprague-Dawley (SD) rats were randomly divided into 5 groups: (1) sham operation group (group Sham, n=5): only anesthesia, oral tracheal intubation, left femoral movement, venous catheterization, and (2) cardiac arrest model group (CA+Normothermia+Saline group, n=15), using transesophageal spiny Rat cardiac arrest model was established and cardiopulmonary resuscitation was established and cardiopulmonary resuscitation was carried out. The ischemic time of cardiac arrest was 4min; (3) separate hydrogen sulfide treatment group (group CA+Normothermia+NaHS, n=15), after successful resuscitation, the hydrogen sulfide donor NaHS treatment; (4) single cryogenic treatment group (CA+ Hypothermia+Saline group, n=15), after successful resuscitation, treatment of low temperature treatment; (5) vulcanization. Hydrogen combined with cryogenic treatment group (group CA+Hypothermia+NaHS, n=15), combined with hydrogen sulfide and low temperature after successful resuscitation. Hydrogen sulfide donor NaHS administration scheme: 0.5mg/kg NaHS (13590, Sigma-Aldrich public division, US) (concentration of 0.3 mg/ml), and then 1.5 mg. Kg-1 H-1 Rate intravenous infusion lasted 3 h; the other groups were intravenously injected with normal saline 1.67 ml/kg, followed by intravenous infusion of 3 h at 5 ml. Kg-1. H-1 rate; low temperature was carried out: the body surface of the rat was immediately retreated with ethanol, and the rectal temperature was reduced to 34 C in 15 min, and the temperature 6 h was maintained with ice bag and heat blanket, then heating treatment, 4 h The body temperature was increased to 38~39 C; the other groups maintained the rectal temperature at 38~39 degrees C. Observe the 7 day survival of the rats after the successful resuscitation, using the strip removal experiment (Tape removal test, TRT) to evaluate the sensory motor function of the rats; and the second part discussed the blood brain barrier permeability, brain edema and blood brain of the rats after resuscitation after the recovery of hydrogen sulfide and / or therapeutic low temperature. The ultrastructure of the barrier, the expression of close connexin Occludin, the expression of matrix metalloproteinase -9 (Matrix metalloproteinase-9, MMP-9) and the effect of activity. The grouping and treatment were the same as experiment one, 25 rats in each group. The brain water content was detected by dry wet weight method after 24 h, and the degree of brain edema was represented by the dry wet weight method; the brain tissue was detected after 24 h after resuscitation. Evans Lan (Evans blue, EB) reacts to the change of blood brain barrier function with the permeability of 4 kDa fluorescein isothiocyanate (fluorescein isothiocyanate-dextran, FD4); the ultrastructural changes of the blood brain barrier are detected by transmission electron microscopy, and the protein immunoblotting (Western blot) is used to detect the close connexin Occludin. The expression and distribution of MMP-9 were detected by immunohistochemistry. The changes of MMP-9 activity were detected by gelatin zymogram. Results: compared with group Sham, the 7 natural survival rates of the other four groups were obviously decreased, the time of strip removal was prolonged, the permeability of brain water content and blood brain barrier increased, the ultrastructure of blood brain barrier was obviously destroyed and the expression of Occludin decreased. MMP-9 expression increased (P0.05); compared with cardiac arrest model (group CA+Normothermia+Saline), single hydrogen sulfide treatment group (group CA+Normothermia+NaHS), single cryogenic treatment group (group CA+Hypothermia+Saline) and hydrogen sulfide combined with low temperature treatment (group CA+Hypothermia+NaHS) increased the 7 natural survival rate of rats, the time of strip removal shortened, the water content of the brain was increased. The permeability of blood brain barrier decreased, the ultrastructural damage of blood brain barrier lightened, the expression of Occludin increased, the expression of MMP-9 decreased and the activity of MMP-9 decreased (P0.05). The improvement of each index of hydrogen sulfide combined with cryogenic treatment group (CA+Hypothermia+NaHS group) was most obvious, and the difference had the significance of overall planning (P0.05). Conclusion: hydrogen sulfide combined with therapeutic low temperature can be more effective. It can effectively reduce the permeability of blood brain barrier in the cardiopulmonary resuscitation rats, protect the structure and function of the blood brain barrier, and play a better role in the brain recovery. The mechanism may be related to the common inhibition of MMP-9 expression and activity, and the reduction of the degradation and loss of the close connexin Occludin.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R541.78
，
本文编号：2126330
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