恢復心跳血壓幫助意識清醒：以胺基酸溶液做神經保護及其分子機制之探討

Abstract

背景：病人在心跳停止的情況下，腦部的灌流停止，使得中樞神經系統開始缺氧，即使以目前的高等心肺復甦術施以急救，腦部的灌流能仍無法達成正常的供氧。一旦腦部缺氧的時間過久，就會引發缺氧性病變造成腦部損傷使得腦部功能的喪失。這樣的病人即使在急救後恢復了心跳血壓，可能會有認知功能的障礙，如果合併腦部的損傷太嚴重，甚至到無法恢復意識的程度，則一旦維生系統撤除，也會因為其他的併發症過世。心臟外科醫師在執行某些心臟大血管手術的時候，會需要將全身的血液流動暫停，會影響到全身器官甚至腦部的灌流，時間太久也會引起神經學的併發症。在心臟外科手術的時候，常常須使用心肌保護液要將心臟停止下來，讓心臟在缺氧過後能夠順利恢復功能。這樣的保護液，在器官移植的過程中也用來避免缺氧再灌流的傷害。由此，我們推測心肌保護液也能同樣的在腦部缺氧在灌流的時候提供保護。 方法：本篇研究先以健保資料庫的數據分析，觀察台灣使用葉克膜的原因、存活率以及產生的併發症，以證實我們的臨床觀察並不侷限於單一醫院。然後設計實驗以證實我們的假說。在實驗的第一部份採用老鼠胚胎的腦部神經元培養來進行體外實驗，在嚴重缺氧或是加入過氧化氫的環境下，將神經元培養基液的1/2到1/4體積置換成HTK溶液(Histidine-Tryptophan-Ketoglutarate solution)，以觀察因細胞受傷而釋出的乳酸去氫酶比例，細胞存活性分析，神經元細胞在低氧誘導因子、細胞凋亡蛋白脢3總量及其活化型的量、以及NADPH氧化酶2及NADPH氧化酶4的表現，在整個觀察時序第0到第72小時過程中的變化。在實驗的第二部份中，使用窒息所引發心跳停止的老鼠模型，並分別在左頸動脈輸注生理食鹽水或是HTK溶液，在窒息後4分30秒或是6分30秒開始施予標準化的心肺復甦術，觀察老鼠的存活率以及神經學表現。並在存活的第三天收集老鼠的血液並予以安樂死，之後取出老鼠的腦部並做切片，在相同的切面染色以評估神經細胞壞死的面積比例。在另一個相同的切面做過氧化氫濃度，NADPH氧化酶4基因表現以及活性的分析。 結果：在神經元嚴重缺氧的研究顯示，使用HTK溶液可以降低乳酸去氫酶的釋放比例，提昇神經元細胞在嚴重低氧環境中的存活能力，並且藉由將低氧誘導因子維持在較高的濃度，也讓細胞凋亡蛋白脢原3雖然表現增加但維持在沒有活化的狀態，以及讓NADPH氧化酶4的表現反而下降，並且在使用低氧誘導因子拮抗劑之後，恢復NOX4的表現。在第二部份因窒息誘發心跳停止的動物實驗中，使用HTK溶液的老鼠在缺氧後有較小的腦部壞死面積，以及顯著下降的NOX4訊息核糖核酸表現以及NOX4酶活性的表現，同時反應到腦部過氧化氫濃度的下降，以及對老鼠的存活率上升，以及神經受損分數的明顯改善。 結論：HTK溶液對於腦部的保護作用，在神經元的細胞研究以及老鼠的動物實驗中得到正面的結果，因此對於之後發展腦部保護液提供良好的基礎。另外，由於研究中加入了觀察各個時間點對各項因子的測量，所以擴展了低氧誘導因子在持續性高濃度的狀況下對於細胞內缺氧再灌流訊息傳遞所表現出不同的結果。這也讓我們對於生理上訊息傳遞的理解，從單一時間點訊息傳遞因子濃度之間的相對關係，到觀察整個時序上因為訊息傳遞因子濃度的變化可能會造成的不同後果。

Background: When patient’s heart stop beating, the blood flow to brain stopped and the insult of ischemia started. Even the advanced cardiopulmonary resuscitation or extracorporeal cardiopulmonary resuscitation could be commenced immediately, we still could not restore the full blood supply to all organs, including brain. As the ischemic time increased, the possibility of ischemic brain injury became higher and might resulted in permanent brain damage and loss of function. After spontaneous circulation regained, these patients might still pose some neurological deficits, and some of them would not regain consciousness, and might pass away if life-maintaining equipments withdrawn. When cardiovascular surgeon performing surgery, the whole body circulation might need to be hold for a while. If the duration became too long, the patient might complicated with neurological defects. We used cardioprotective solution to protect heart during ischemic period, and also use organ preservation solution during organ transplantation. Thus we expect that amino-acid based cardioprotective solution might as well protect brain during ischemic period. Method: We first conducted a retrospective study using national health insurance research database. Patients used ECMO were identified and the patient’s diagnosis, prior medical condition, mortality rate and the morbidities were evaluated. We verified our clinical experience to all hospitals of Taiwan. In the first part of laboratory study, we used rat embryonic cortical neurons culture, and challenged with severe hypoxia or hydrogen peroxide to mimic the oxidative stress during ischemia and reperfusion. The neuronal culture medium was replaced ½ to ¼ with HTK solution (Histidine-Tryptophan-Ketoglutarate) to test the effect of neuron protection. Lactate dehydrogenase, cell survival analysis were performed at each time point, and the level of hypoxia-inducible factor 1(HIF-1), total and cleaved caspase 3, the gene expression and enzymatic activity of NADPH oxidase 2(NOX2) and NADPH oxidase 4(NOX4) were measured during the observation time points at 0, 1, 2, 4, 8, 24, 48, and 72 hours. In the second part of laboratory study, we used rat asphyxial cardiac arrest model and clamp endotracheal tube for 4 minutes and 30 seconds, or 6 minutes and 30 seconds. Phosphate-buffered saline(PBS) or HTK solution was infused into left carotid artery at the beginning of asphyxia. Standardized chest compression, ventilation and epinephrine infusion were given at the end of asphyxia. The mortality rate and neurological deficit score of survived animals were observed after return to spontaneous circulation and on day 1, 2, and 3 after then. At 72 hours after experiment, the rats were sacrificed humanely. Blood were obtained for hydrogen peroxide analysis and brain were harvested for slices, which were sent for infarction area analysis and NOX4 mRNA quantification and NOX4 activity evaluation. Results: The cortical neuron studies revealed that the usage of HTK solution reduced LDH release during severe hypoxia or when challenged with hydrogen peroxide, also, viability increased. With the add of HTK solution, HIF-1 could maintain relatively high level, which made procaspase-3 expression increased but remained in inactive form. Also, the persisted high HIF-1 level resulted in reduced NOX4 expression, and when the antagonist of HIF-1, 400083, was used, NOX4 level returned to the level when challenged with severe hypoxia. In the second part of this study, we used rat asphyxial cardiac arrest model. The infusion of HTK solution made the infarction size of rate brain mild decreased, reduced NOX4 mRNA expression and decreased NOX4 enzyme activity. The hydrogen peroxide concentration from brain slice homogenate were also lowerd when HTK solution was used. The survival rate and neurological deficit score both improved if HTK were used. Conclusion: The protective effect of HTK solution to brain were proved by our study on the cortical neuron cells and animal studies. This proves our hypothesis were correct and build a foundation for future development of proper neurological protective solution. Our study observed the concentration changed for HIF-1 and the response of cells to it. The signal transduction of HIF-1 axis might not only depend on the concentration of HIF-1 at one single timepoint, but might depend on the change of HIF-1 concentration with a time-dependent manner. This would be a new point of view for cellular signal transduction.