2015-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/646367摘要：背景：心肺停止併心肺復甦急救為一特殊且嚴重的全身性缺血/再灌流傷害之例子。在所 有的器官組織中，腦部為最不能耐受此傷害之器官，往往造成病患復甦後永久性的神經 缺損及障礙。心肺停止暨急救復甦後腦部血液循環與組織灌流往往受到影響而顯著下 降，不可避免地延長了缺血傷害，進而對神經預後與存活造不利的影響。遠處肢體缺血 後處置(remote limb ischemic post-conditioning, RLIP)繼前處置及後處置之後在近年被廣 為探討，不但可以減少缺血暨再灌流傷害，在臨床實用上亦合理可行。一氧化氮(NO) 在後處置的保護機轉中扮演重要角色，它不僅是個強力的血管擴張介質，在抗凋亡的訊 息傳導如Akt-eNOS-NO路徑中亦重要的訊息傳導因子。因此吾人計晝以遠處肢體缺血 後處置的介入治療，探討其對急救復甦後的神經保護作用，尤其著重於其在增進腦部血 行循環、組織灌流的角色，以及生理上與分子機制上的保護機轉。方法：吾人將以大鼠窒息導致心肺停止/心肺復甦急救之模型，探討RLIP對神經保護之 作用。動物分組如下：（1)控制組，（2)標準心肺停止/心肺復甦暨復甦後照護，（3) RLIP， (4) RLIP加上一氧化氮生成酶抑制劑L-NAME，(5) RLIP控制組（未心肺停止/急救復 甦），（6) L-NAME控制組。RLIP係在大鼠恢復自發性循環後在其左後腿以血管夾阻斷 股動脈血流五分鐘，再予以鬆開使其灌流五分鐘，如此重複三次。動脈血將於初始以及 復甦後1、2、4小時抽取以測定過氧化物(ROS)以及NO下游產物(nitrate/nitrite)。腦部 的血管及微血管將用MicroScan記錄並分析。腦組織灌流及腦氧分壓則以 OxyFlo/OxyLite導管連續監測並記錄。此外，吾人將以核磁共振掃描(MRI)技術(TOF MRI & 3D AR2-mMRA)呈現腦部的血管與微循環，同時以DCE MRI評估腦血屏障(BBB)之 破損情形，用MR spectroscopy量測神經的存活性與腦部代謝產物的變化。至於神經傷 害則以Fluoro-Jade染色以及TUNEL stain所代表的細胞凋亡測定。預後指標包括動物存 活率及神經學預後。對於RLIP生理保護機轉的探討則聚焦在系統循環中NO的濃度， 及其上游Akt-eNOS訊息傳導路徑-Akt、eNOS的磷酸化活化之程度在RLIP肢體動脈 的變化。至於腦部本身的Akt-eNOS路徑因事關神經的抗凋亡保護，吾人亦會測定腦組 織Akt、eNOS的磷酸化活化程度，並與TUNEL stain凋亡的結果作比較。而為了區分 RLIP對血管内皮細胞及神經細胞個別之作用，進一步將利用培養的大鼠内皮細胞及神 經細胞進行模擬缺血暨再灌流傷害，測定各自Akt及eNOS的活化情形，以了解其對内 皮細胞NO釋放，及神經細胞内Akt-eNOS相關抗凋亡機制的影響。最後，吾人將用RLIP 併用L-NAME觀測其對NO、腦部循環、組織灌流、Akt-eNOS訊息傳導、神經凋亡， 及存活與神經學預後之影響，輔助証明NO在缺血傷害後神經保護之角色。預期結果：在初步結果中吾人發現RLIP可大幅改善復甦後腦血循環及組織灌流不佳之 情形，而這可能可以改善大鼠復甦後的存活與神經學預後。至於MRI影像的呈現將使 吾人更能親眼目睹復甦後腦血管收縮與循環障礙的情形，以及RLIP改善此一窘境的生 理效果。基於NO在前處置與後處置的關鍵角色，吾人亦可預期RLIP可以增加血中NO 的濃度，並與腦血循環的改善有關。而藉由測定RLIP肢體動股脈的Akt及eNOS的磷 酸活化程度，則可追朔血中所增加NO的來源，說明其生理保護的機制。此外，RLIP 組的腦組織Akt及eNOS的磷酸活化程度亦可能增加，且與神經傷害與凋亡的減少有 關，說明RLIP亦透過Akt-eNOS-NO訊息傳導路徑達到抗凋亡保護的分子機轉。最後， L-NAME預期會逆轉上述的生理作用與保護效果，更輔助證明NO在此間之重要角色。 臨床應用：本系列研究不僅提供RLIP增進復甦後存活及神經學預後的重要角色，亦探 討了其對腦部循環及組織灌流的生理保護機制，以及抗凋亡的神經保護機轉。由於RLIP 在臨床上可用量測血壓之脈壓帶輕易執行，亦無重大的倫理爭議，此研究成果具有雄厚 潛力進一步轉譯至臨床研究與應用，以促進心肺停止急救病患的整體活及神經預後。 <br> Abstract: Backgrounds: Cardiac arrest and cardiopulmonary resuscitation (CPR) is a specific model of global ischemia/reperfusion (I/R) injury. Among all organ systems, brain is least tolerable to such insult and the resultant injury greatly impacts the survival and neurological prognoses. Cerebrovascular circulation is usually compromised after cardiac arrest and CPR, which inevitably prolongs ischemic insult and aggravates survival and neurological prognoses. Remote limb ischemic post-conditioning (RLIP) is clinically feasible and can potentially mitigate post-resuscitation neurological deficits. Nitric oxide (NO) has been shown to play a key role in post-conditioning protection. NO is not only a potent vasodilator regulating the vasotonus, but serves as an important mediator in anti-apoptotic pathway such as Akt-eNOS-NO signaling. We therefore aim to investigate the role of RLIP in post-resuscitation neuroprotection, with focus put on its impact on cerebrovascular circulation, brain tissue perfusion, and the underlying physical and molecular protective mechanisms. Methods: Using a well-established rat model of asphyxia cardiac arrest and CPR, we divide the rats into 6 groups: (1) Sham control, (2) Standard CPR and post-CPR care, (3) CPR + RLIP, (4) CPR + RLIP + L-NAME, (5) RLIP control without CPR, (6) L-NAME control without CPR. RLIP will be done by 3 cycles of 5 min of left hind limb ischemia followed by 5 min of reperfusion using cyclic hemoclipping and release of femoral artery. Arterial blood will be sampled at baseline 1, 2 and 4 h post-CPR for measurement of ROS and colorimetric determination of nitrate/nitrite. The cerebral vasculature will be video-scanned by Microscan. Cerebral tissue perfusion and oxygenation will be continuously recorded by OxyFlo/OxyLite probe. In addition, we will employ MR imaging for demonstration cerebral macro- and micro-vasculatures. Dynamic contrast-enhanced (DCE) MRI will be used to evaluate blood-brain-barrier (BBB) disruption. MR spectroscopy will be adopted for measurement of neuronal viability and metabolite changes of the brain. Four hours after ROSC, the left femoral artery and brain will be harvested for measuring the phosphorylated endothelial NO synthase (p-eNOS at Ser1177) and protein kinase B (p-Akt at Ser473). In a subgroup of rats with minimal invasive procedures, the survival and neurological outcomes will be monitored up to 3 days. For further clarification of the effects of RLIP on endothelial cells and neuron cells, we will also employ cell models of simulated ischemia-reperfusion and apply 3 cycles of 5/5 min ischemic PC for verify the respective role of Akt-eNOS signaling in NO release (in endothelium) and anti-apoptotic protection (in neurons).Expected Results: The preliminary data show that RLIP significantly improves the compromised cerebrovascular circulation and tissue perfusion in the post-resuscitation phase, which could potentially improve survival and neurological outcomes. MRI would further demonstrate the vasoconstriction and BBB disruption post-CPR, and the physiological benefits of RLIP in ameliorating these derangements. Plasma NO as indicated by nitrate/nitrite would be significantly increased in RLIP. The p-eNOS and p-Akt may be increased in left femoral artery where RLIP is applied, suggesting that NO is originated from RLIP limb vessels. Further, p-eNOS and p-Akt could also be increased in the brain, indicating RLIP potentially confers anti-apoptotic neuroprotection via Akt-eNOS-NO signaling. If NOS inhibitor L-NAME (10 mg/kg) is used, not only NO increase would be reversed, all the physiological and survival/neurological benefits would be abrogated, suggesting the crucial roles NO plays in RLIP-mediated neuroprotection.Clinical Implications: This series of study offers not only the survival and neurological benefits of RLIP in post-resuscitation care, but brings insight into the protective mechanisms centered on NO. With the ease of use and potential application in clinical settings, RLIP could prove to be a novel way to improve post-resuscitation neurological injuries.心肺停止心肺復甦急救後處置前處置腦血管循環血管内皮一氧化 氮一氧化氮合成酶cardiac arrestcardiopulmonary resuscitationpost-conditioningpre-conditioningcerebrovascular circulationendotheliumnitric oxidenitric oxide synthase