低體溫急救之心臟保護作用

Hypothermia induces Cardioprotective Effects in Resuscitation

許瓊元(Chiung-Yuan Hsu)

指導教授 ： 陳文鍾

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01164

摘要

如何對心跳終止病患進行有效的急救使其恢復心跳及循環是目前急救醫學的領域中相當重要且熱門的課題。現今急救醫學所強調的生命之鍊、以及以恢復自發性血液循環（Return of Spontaneous Circulation, ROSC）為主要目標的急救精神與急救方法，雖然可以帶來較多初步急救成功的機會，但是長期來看病患的存活率與存活的品質仍未臻理想，尚有相當改善的空間。整體平均而言，所有到院前心跳停止病患能夠存活至出院的機會大約在2-5%左右，能夠維持意識清楚、腦部功能運作良好的只有1%；在醫院內心跳停止經過急救病患其存活率較高，平均存活至出院的比例可達16-18%，意識清楚者約為14%左右。心跳停止病患經過急救後恢復血液循環，但出院存活率不佳，原因在於急救後症候群（Post-Resuscitation Syndrome）導致心臟及中樞神經衰竭。大約有50-70％ 初步急救成功病患在短期內主要因為急救後心臟功能缺損導致血行動力學無法維持生命基本需求，進一步引發多發性器官損傷而死亡，其餘病患則大部分必須面對因為嚴重腦部的損傷造成病患意識無法恢復而被迫面對長期照顧之下後續產生的併發症。因此我們實在有必要對於急救成功之後的病患進行更進一步的醫療處置或藥物治療，以求延長病患生命及提升病患生命品質。
急救後病患面對的窘境不止源於缺血性傷害，很可能與循環停止及恢復之間器官及組織處於大量的缺血-再灌流（ischemia/ reperfusion）狀態有關。缺血再灌流傷害相關的機制複雜，但是作用於心肌細胞則導致心臟表現出急救後心臟功能缺損，並以全面性的收縮及舒張能力下降作為表現。目前由心肌梗塞及缺血性腦中風的研究發現：缺血再灌流傷害所引發之反應之中，包括自由基及許多細胞激素會進一步擴大細胞受損的範圍，除此之外細胞凋亡（Apoptosis）也是器官組織在缺血後重新灌流時細胞及組織受損的主要兇手之一，因為細胞凋亡在缺血後重新灌流當時會迅速且大量產生，並且隨細胞凋亡的產生而擴大細胞壞死的範圍，更進一步影響到器官功能表現。
然而生理上存有許多保護性的機制。例如IL-10可以抑制傷害性的細胞激素產生；亦有許多蛋白可以透過缺血再灌流傷害及細胞凋亡訊息傳導途徑中不同關鍵分子進行活化或抑制作用，以達到細胞保護的效果。其中包括由ERK （Extracellular Signal- Regulated Kinase），P38（p38 MAP Kinase），JNK （c-jun N-Terminal Kinase）所組成的MAPK（mitogen activation protein kinase）家族，可以將外來氧化壓力、細胞激素、生長激素等等不同刺激訊息帶入細胞核內對基因進行活化或抑制等調控作用。一般而言MAPK家族中ERK 1/2屬於RISK pathway（Reperfusion Injury Salvage Kinase pathway）的一份子，和缺血再灌流傷害引發的細胞凋亡關係密切，可以在缺血再灌流傷害中扮演保護性的角色；pP 38、JNK 1/2則反應環境影響調控細胞的蛋白生成，和細胞後續的反應息息相關。在臨床上也的確觀察和發現到包括預處理（Preconditioning）、延遲性預處理（Delayed Preconditioning）、後處理（Post-conditioning）以及許多藥物（例如血管生成素轉化酶抑制劑），皆可以減緩缺血再灌流後續傷害而誘發保護作用，對心臟功能缺損有很大的幫助。
低體溫治療（Therapeutic Hypothermia）已經經由臨床試驗證實可以對心因性疾病產生突發性心跳終止的病患、以及嚴重腦部外傷之病患具有腦細胞保護的效果；動物實驗上也證實低體溫治療對處於缺血再灌流傷害的腦細胞及心肌細胞有相當良好的保護作用，可以減緩缺血再灌流傷害及細胞凋亡的程度，對器官功能的恢復及預後有顯著的幫助。但是急救成功之後個體包括心臟及體內器官皆面臨大規模的缺血再灌流傷害，並且可能必須面對嚴重的急救後心臟功能缺損，因此低體溫治療對於急救後心臟功能缺損是否亦同樣有心臟保護的效果則尚未釐清。若低體溫對急救後的個體可以提供心臟保護效果，不失為改善急救後病患多發性器官衰竭的利器。

因此本研究提出假說為：適度之全身性低體溫對於急救後之心臟細胞可因為減緩缺血再灌流傷害所誘發之心肌細胞傷害而提供心臟保護功能。我們成功發展了一個穩定的合併溫度控制以及以窒息法誘發大鼠心跳終止後進行心肺復甦術的動物實驗急救模式，在急救成功之後評估低體溫治療對急救後動物短期存活率與急救後心臟功能缺損的影響，並進一步以不同的溫度及時間設計來評估低體溫治療對於缺血再灌流傷害相關細胞激素、MAPK家族相關訊息傳導及心肌細胞凋亡之影響。

研究材料及方法：
本研究分為動物實驗及實驗室操作兩大部分，首先利用窒息法誘發雄性大鼠心跳終止後進行心肺復甦術的動物實驗急救模式，並在急救成功之後分別進行低體溫及正常體溫治療。在動物實驗部分依照不同時間設計分別觀察不同溫度治療下總體存活率（4小時組）、操作心臟超音波及紀錄血行動力學變化分析心臟功能變化（2小時組）、並收集心臟檢體進行實驗室操作分析（1小時組、2小時組、對照組）。
實驗室操作部分則分為三大部分進行，以瞭解低體溫治療可能相關之機制：
1. 針對心肌缺血再灌流傷害相關細胞激素以ELISA法進行IL-10、TNF-α偵測
2. 以西方點墨法分析MAPK家族相關蛋白pERK, pP38, pJNK活性變化
3. 以caspase 3為代表，定量心肌細胞進行細胞凋亡程度。

初步研究結果：
1. 短期存活率：
經過低體溫治療之公鼠在急救後4小時內比正常體溫控制下之公鼠有顯著較佳的短期存活率（低體溫 13/14, （92.3%），正常體溫5/15 （33.3%），p<0.001）。

2. 急救後心臟功能缺損
分析不同體溫控制之下心臟功能之變化。正常體溫治療之下，61.5%公鼠在急救後30分鐘仍然持續發生心臟功能的衰退, 最後導致實驗動物提早死亡。反觀低體溫治療之下，在急救結束之後低體溫的治療有助於穩定左心室收縮比例及血液動力學表現，並且在急救30-60分鐘出現明顯的差別。
至於急救後心臟舒張功能的變化，可以發現低體溫治療之公鼠左心室舒張功能有較佳的表現趨向，但在統計上與正常體溫治療者無法達到顯著差異。

3. 缺血再灌流傷害相關細胞激素
偵測心肌組織中相關細胞激素IL-10、TNF-α。實驗結果初步發現正常溫度治療下歷經急救之後之公鼠，其心臟組織內的IL-10濃度在急救後短時時間內出現明顯降低，尤其是發生急救後心臟功能缺損之公鼠，其心臟組織中IL-10的下降程度比順利存活者明顯及快速。然而急救後經低體溫治療組其心肌組織中保護性的IL-10濃度變化無明顯影響。至於TNF-α在急救之後因為缺血再灌流傷害會出現大量活化，在低體溫治療之公鼠此活化現想隨時間穩定而持續表現，但是低體溫治療之公鼠心臟組織中的TNF-α則與急救前無明顯變化。因此由IL-10及TNF-α的研究可以發現低體溫治療對於缺血再灌流傷害能夠提供相當的保護效應。

4. MAPK家族成員
分析MAPK家族成員ERK1/2, pP38, JNK於不同體溫控制下的變化。 低體溫治療對於ERK活性的增加呈現正向的幫助，即使在急救之前低體溫治療即可顯著增加ERK的活性。若在急救後進行低體溫治療，公鼠pERK會隨時間持續放大活化程度，但急救後維持正常體溫治療之公鼠，pERK雖被活化但與低體溫治療比較起來，兩者的差別在急救後2小時會出現相當顯著的差異。
另一方面，急救之後正常體溫治療之公鼠pP38 隨著時間顯著增加活化的程度，急救後低體溫治療之公鼠則有抑制pP38活化的傾向，兩者於急救後1小時有相當顯著的分別; 在急救後2小時雖然因為統計關係無法達到顯著差別的程度，但是低體溫治療者仍有抑制pP38活化的傾向。
至於pJNK只能短暫的在急救成功之後相當短時間內偵測到活性的增加，但隨時間逐漸延長JNK的活性快速消失，低體溫治療對於pJNK在急救後的影響無法由本實驗明顯觀察出變化。

5. 細胞凋亡分析
分析不同體溫控制之下對細胞凋亡蛋白caspase 3活性的影響。實驗結果發現正常體溫控制之下caspase3活性在急救之後會顯著增加，而低體溫治療治療則有助於減緩急救前後細胞凋亡相關蛋白caspase 3活性上昇的速度，與正常體溫治療比較起來兩者在急救後1小時左右有顯著的差異。

結論：
綜合本實驗結果，我們可以瞭解急救後進行低體溫治療可以增加急救短期的存活率，以及減緩急救後心臟功能缺損的發生，進一步在功能上維持急救後心臟功能的穩定。低體溫治療其保護機制可能與下列有關：
1. 低體溫治療有效減少心肌組織中相關細胞激素TNF-α的濃度，並且減緩保護性發炎前驅物IL-10在急救之後短時間內快速下降的速度。
2. 低體溫治療持續增進急救後pERK的活化程度，並進一步抑制pP38在急救後的活化，促使心肌組織在受到缺血再灌流傷害之時能夠減少細胞凋亡並朝向進一步存活發展。

關鍵字

低體溫 ； 心跳停止 ； 急救 ； 心臟保護 ； 後處理

並列摘要

Introduction
To restart spontaneous heart beat and restore spontaneous circulation are the very goals in the modern Resuscitation Medicine for decays. We have focused on how to improve the rate of Return Spontaneous Circulation (ROSC rate) so much, and the goal of successful resuscitation has been achieved after much effort on the resuscitation education and technique training. However, clinically the doctors, families and patients still face the tragedy that post-resuscitation multi-organs failure still exist and strongly threaten patient’s survival even initial successfully resuscitated. The discharge rate of these out-of-hospital cardiac arrest patients is still very unsatisfactory as decays ago, 2-5% on average, and the patients with good cortical function are still very rare. The in-hospital-cardiac-arrest （IHCA）patients have higher discharge rate but it is still limited to 16-18% only no matter in Taiwan or the west. Post-Resuscitation Syndrome is the most important reason which results in multiple organs failure because reperfusion is absolute necessary for life saving in the cardiac arrest patients, but global and large scaled ischemic reperfusion injury also starts when resuscitation begins. Numerous published researches had found even the patient is successfully resuscitated but the systemic ischemic reperfusion injury may induce severe organ dysfunction, including post-resuscitation myocardium dysfunction and unstable hemodynamic, and 50-70% patients died in the pos-resuscitation acute stages due to cardiac dysfunction fails to maintain homodynamic. Most of the survived patients need long tern nursing care due to sever brain injury and results in many complications. Therefore, to develop new managements or even medicines, which can attenuate post-resuscitation syndrome, is as important as to achieve ROSC when resuscitation starts, so that we could really help these patients to prolong their meaningful life but not waste medical resources or produce more vegetative only.

The pos-resuscitated patients not only experience the ischemic injury but also, the most important, the large scaled Ischemia/Reperfusion injury between cardiac arrest and circulation restore. Cell apoptosis is the central hypotheses but in fact there are many mechanisms like oxidant injury, pro-inflammatory cytokines, complex and cross-talked signal transduction pathways involved in the tissue response to Ischemic/Reperfusion injury. Many evidences from animal studies of myocardium infarction or brain stroke disclose the Ischemic – Reperfusion injury would augment the area and severity of initial ischemic injury and extend organ dysfunction. In the heart, they all result in global, both systolic and diastolic, cardiac dysfunctions and it is termed “post-resuscitation myocardium stunning” if it developed after cardiac arrest and resuscitation.

Clinically, organ protection is feasible. More and more evidences find the myocardium have protective windows when ischemic reperfusion injury attacks, the pre-conditioning、delayed - preconditioning and post-condition. There are many physiological protective responses against ischemic reperfusion injury under some strategies like brief and transient ischemia、exercise、Angiotensin Conversion Enzyme Inhibitors、angiotensin II type 1 receptor antagonist are all the examples and well known in these protective effects. The mechanisms of these protective effects are complex and including many proteins in different signal pathways but finally all leads to cell survival or decrease cell apoptotic response. For example, Interleukin-10 is the anti-proinflammatory cytokine which suppress the inflammatory cytokines. Other proteins attenuate Ischemic/Reperfusion injury related cell apoptosis process by modulate the key proteins like Bcl-2, Bax or caspases in the apoptosis pathways. Mitogen Activation Protein Kinase Family ( MAPK Family), including ERK、p38、JNK, could bring signals like oxidant pressure, cytokines stimulation, or signals from growth hormone and environment change from the receptors to the nucleus and then modulate cell function by activate or suppress gene transcription. ERK 1/2 participate in the signal transcription of Reperfusion Injury Salvage Kinase Pathway (RISK pathway) and have strong protective effects against cell apoptosis which is induced by ischemic reperfusion injury. JNK and p38 are the major stress signal transduction pathways and cell response to the outside depends on their effective work.

Today, hypothermic resuscitation has been proved by prospective multi-centers randomized clinical trials to have strong protective effects in the post-resuscitated patients. It can improve long term survival and brain recovery in the cardiogenic originated cardiac arrest or even the severe to moderate traumatic brain injury patients. Many animal studies also revealed localized or systemic hypothermia have good protective effects against local ischemic reperfusion injury related cell apoptosis in the brain and myocardium which improve organ dysfunction significantly. However, severe hypothermia also has controversial negative inotropic effects. It has not been studied and well understood whether systemic hypothermia also has protective effect in the pot-resuscitation myocardium dysfunction. If the hypothermia has post-resuscitation cardioprotective effects, it would be the very powerful strategy to reduce multi-organs failure in post-resuscitation care.

Hypothesis and Study Designs
Therefore, we purposed that adequate systemic hypothermia may provide cardioprotective effect and attenuate post-resuscitation myocardium dysfunction by reduce ischemic reperfusion injury. We designed and set up the first temperature controlled asphyxia induced cardiac arrest and resuscitation model in the rat. Serial animal studies are designed to further observe if the hypothermia improved the short term survival and decline post-resuscitation myocardium dysfunction. The others are designed to evaluate the temperature effects in different Ischemia/Reperfusion injury related signals transduction especially focused on cytokines, MAPK family and cell apoptosis.

The Preliminary Results
1. Short-term Survival Rate:
Post-resuscitation hypothermia therapy would significantly improve 4 hours short term survival then Normothermia. (Hypothermia survival rate 92.9%; Normothermia 32.3%, p< 0.001)

2. Post-Resuscitation Myocardium Dysfunction:
In the study of LV fraction shortening, post-resuscitation hypothermia can significantly stabilize post -resuscitation systolic dysfunction and maintain hemodynamic if compared with Normothermia especially in post-resuscitation 30-60 minutes. The Normothermia individuals would results in early death if heart sunning ongoing deteriorate after post-resuscitation 30 minutes. However, in the study of positive dp/dt(max), the hypothermia therapy only results in trends of better systolic function but statistically not achieved significant level.

Besides, systemic hypothermia therapy has trends toward improving diastolic dysfunction in the negative dp/dt(max), but the statistics results neither do not achieve significant level in our studies and need more investigations.

3. Ischemia/Reperfusion injury related cytokines:
Global ischemic and resuscitation would lower myocardium protective IL-10 levels immediately in the post-resuscitation period. The individuals who developed post-resuscitation myocardium dysfunction under Normothermia would significantly develop more rapid attenuation in myocardium IL-10 level then Hypothermia or those without myocardium dysfunction. The hypothermia therapy would provide significant protective effect then normothermia therapy in the post-resuscitation 1 hours.

In the study of myocardium TNF-α, post-resuscitation hypothermia can effectively repress the elevation of myocardial TNF-α level after ischemia/reperfusion injury from global cardiac arrest and resuscitation then post-resuscitation Normothermia therapy. The results of TNF-α would achieve significant level in the post-resuscitation 2 hours

4. MAPK Family:
Hypothermia would increase pERK activations no matter ischemic reperfusion injury developed or not. The Hypothermia would persistently and significantly augment pERK activation after resuscitation if compared with Normothermia which maintains activated but stable level in the myocardium. The difference waxes and wanes especially at post-resuscitation 2 hours.

On the other way, pP38 activation is significantly found in the Normothermia after resuscitation. Post-resuscitation Hypothermia has trends toward suppress pP38 activation after ROSC and achieve significant difference level in post-resuscitation 1hr.

However, pJNK activation is only observed transiently and immediately after resuscitation. It is not detectable after post-resuscitation periods and the moderate hypothermia would not influence its effect.

5. Cell apoptosis
We detect myocardium Caspase 3 activity and found caspase 3 activities would increase much after ROSC under Normothermia. Hypothermia would decrease caspase 3 activities even before ischemic reperfusion injury, and post-resuscitation hypothermia would further maintain slower increase rate of caspase 3 then Normothermia after resuscitation and achieved significant difference in the first 1 hour.

Conclusion:
To summarize the results of this study, we could conclude that the Post-resuscitation Hypothermia would increase short term survival rate for stabilized post-resuscitation myocardium dysfunction. The mechanism of its cardioprotective effect may be related to:

1. Hypothermia could effectively decrease pro- inflammatory cytokine TNF-α, and attenuate the rapid deterioration of protective cytokine, IL-10, in the post-resuscitation periods.

2. Hypothermia could persistently induce pERK activation and suppress stress signal pP38 transduction in the post-resuscitation periods. The total effects of the MAPK lead the myocardium to survive under ischemia/Reperfusion injury.

並列關鍵字

Hypothermia ； cardiac arrest ； resuscitation ； cardioprotection ； post-conditioning

參考文獻

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