硫化氫聯(lián)合治療性低溫對(duì)心臟驟停大鼠血腦屏障結(jié)構(gòu)與功能的影響
發(fā)布時(shí)間：2018-07-16 12:02
【摘要】：目的:心臟驟停(cardiac arrest,CA)及心肺復(fù)蘇(cardiopulmonary resuscitation,CPR)后居高不下的病殘率和病死率主要源于全腦缺血再灌注損傷。血腦屏障(blood-brainbarrier,BBB)損傷與腦水腫是腦缺血再灌注損傷的重要病理改變,血腦屏障破壞又可加重缺血后的繼發(fā)性腦損傷,因而減輕BBB損傷與腦水腫是減輕腦損傷的重要環(huán)節(jié)。治療性低溫(therapeutic hypothermia,TH)(32℃～34℃)是復(fù)蘇后一種安全和有效的治療方法,低溫可通過多種機(jī)制發(fā)揮腦保護(hù)作用。但不少試驗(yàn)研究發(fā)現(xiàn)單一低溫療法在臨床上很難達(dá)到預(yù)期的腦復(fù)蘇效果,因此尋求一種聯(lián)合手段,已成為目前提高腦復(fù)蘇成功率的一個(gè)研究方向。有研究表明,硫化氫(hydrogen sulfide,H2S)可通過神經(jīng)調(diào)節(jié)與神經(jīng)保護(hù)作用,減輕中樞神經(jīng)系統(tǒng)缺血再灌注損傷。因此本研究采用經(jīng)食道電刺激法建立大鼠心臟驟停及心肺復(fù)蘇模型,經(jīng)靜脈泵注H2S供體NaHS,并聯(lián)合治療性低溫療法觀察其對(duì)心肺復(fù)蘇后大鼠血腦屏障的影響,并初步探討其可能的作用機(jī)制。方法:實(shí)驗(yàn)共分兩部分進(jìn)行;第一部分探討硫化氫和(或)治療性低溫對(duì)心肺復(fù)蘇后大鼠生存率及神經(jīng)功能的影響。雄性Sprague-Dawley(SD)大鼠隨機(jī)分為5組:(1)假手術(shù)組(Sham組,n=5):僅進(jìn)行麻醉、經(jīng)口腔氣管插管和經(jīng)左股動(dòng)、靜脈置管等操作;(2)心臟驟停模型組(CA+Normothermia+Saline組,n=15),采用經(jīng)食道電刺激法建立大鼠心臟驟停模型,并進(jìn)行心肺復(fù)蘇,心臟驟停缺血時(shí)間為4min;(3)單獨(dú)硫化氫處理組(CA+Normothermia+NaHS組,n=15),成功復(fù)蘇后予硫化氫供體NaHS處理;(4)單獨(dú)低溫處理組(CA+Hypothermia+Saline組,n=15),成功復(fù)蘇后予治療性低溫處理;(5)硫化氫聯(lián)合低溫處理組(CA+Hypothermia+NaHS組,n=15),成功復(fù)蘇后予硫化氫與低溫聯(lián)合處理。硫化氫供體NaHS的給藥方案:于自主循環(huán)恢復(fù)即刻經(jīng)靜脈注射劑量為的0.5mg/kg的NaHS(貨號(hào):13590,Sigma-Aldrich公司,美國)(濃度為0.3 mg/ml),隨后以1.5 mg · kg-1 · h-1速率靜脈輸注,持續(xù)3 h;其它組靜脈注射生理鹽水1.67 ml/kg,隨后以5 ml · kg-1 · h-1速率靜脈輸注3 h;低溫的實(shí)施:于自主循環(huán)恢復(fù)即刻用乙醇涂擦大鼠體表,15 min內(nèi)將直腸溫度降至34℃,用冰袋和加熱毯維持該體溫6 h,隨后進(jìn)行升溫處理,4 h內(nèi)將體溫升至38～39°C;其它組維持直腸溫度在38～39℃。觀察復(fù)蘇成功后大鼠7天生存情況,使用條帶移除實(shí)驗(yàn)(Tape removal test,TRT)評(píng)價(jià)大鼠感覺運(yùn)動(dòng)功能;第二部分探討硫化氫和(或)治療性低溫對(duì)復(fù)蘇后大鼠血腦屏障通透性、腦水腫、血腦屏障超微結(jié)構(gòu)、緊密連接蛋白Occludin表達(dá)、基質(zhì)金屬蛋白酶-9(Matrix metalloproteinase-9,MMP-9)表達(dá)以及活性影響。分組及處理情況同實(shí)驗(yàn)一,每組25只大鼠。于復(fù)蘇后24 h,采用干濕重法檢測(cè)腦含水量,代表腦水腫程度;于復(fù)蘇后24 h,檢測(cè)腦組織對(duì)伊文思蘭(Evans blue,EB)與對(duì)4 kDa熒光素異硫氰酸酯-葡聚糖(fluorescein isothiocyanate-dextran,FD4)的通透性來反應(yīng)血腦屏障功能的變化;采用透射電鏡技術(shù)檢測(cè)血腦屏障超微結(jié)構(gòu)改變;采用蛋白質(zhì)免疫印跡法(Western blot)檢測(cè)緊密連接蛋白Occludin表達(dá);采用免疫組織化學(xué)法檢測(cè)MMP-9表達(dá)與分布,采用明膠酶譜法檢測(cè)MMP-9活性變化。結(jié)果:與Sham組比較,其他四組大鼠7天生存率明顯降低,條帶移除時(shí)間延長,腦含水量與血腦屏障通透性增加,血腦屏障超微結(jié)構(gòu)破壞明顯,Occludin表達(dá)減少,MMP-9表達(dá)增加(P0.05);與心臟驟停模型(CA+Normothermia+Saline組)比較,單獨(dú)硫化氫處理組(CA+Normothermia+NaHS組)、單獨(dú)低溫處理組(CA+Hypothermia+Saline組)與硫化氫聯(lián)合低溫處理(CA+Hypothermia+NaHS組)大鼠7天生存率增加,條帶移除時(shí)間縮短,腦含水量與血腦屏障通透性減輕,血腦屏障超微結(jié)構(gòu)破壞減輕,Occludin表達(dá)增加,MMP-9表達(dá)降低,MMP-9活性降低(P0.05);其中硫化氫聯(lián)合低溫處理組(CA+Hypothermia+NaHS組)的各指標(biāo)改善最明顯,且差異均有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:硫化氫聯(lián)合治療性低溫能更有效地減輕心肺復(fù)蘇大鼠血腦屏障通透性、保護(hù)血腦屏障結(jié)構(gòu)與功能,發(fā)揮更好的腦復(fù)蘇作用。其機(jī)制可能與共同抑制MMP-9表達(dá)與活性,減少緊密連接蛋白Occludin降解丟失相關(guān)。
[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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R541.78
，
本文編號(hào)：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.
【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R541.78
，
本文編號(hào)：2126330
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