新生兒窒息腦病變是新生兒死亡以及造成日後神經發展障礙之主要原因之一。新生兒窒息缺氧腦病變之幼兒約有45％死亡或產生永久性之神經後遺症，神經系統方面的後遺症，常見如癲癇、腦性麻痺及智力發展遲緩。此缺氧腦病變目前仍無有效之臨床用藥。研究透過前置處理（preconditioning）所產生之腦部保護作用可被用來探討對新生兒缺氧腦病變可能之神經保護藥物。
　　環單磷酸腺苷反應分子結合蛋白（CREB）是一種轉錄因子，其主要扮演的關鍵角色乃是接受細胞外環境刺激引發胞內至細胞核內訊息之傳遞，例如在神經系統中之學習與記憶之形成及突觸可塑性，皆與此一蛋白有密切關係。在組織遭受缺氧攻擊時，會有許多細胞外刺激作用會經由各種不同之因子傳遞訊息管道共同來活化磷酸化環單磷酸腺苷反應分子結合蛋白( pCREB )；過去文獻顯示，在缺氧刺激下，CREB可迅速地被活化，而持續活化之CREB將可使神經細胞更具抗缺氧傷害。本實驗室發現若預先經24小時單側頸動脈結紮前置處理之幼鼠，在水迷宮行為測試及腦傷程度上較未經前置處理之幼鼠有大幅度改善之現象；並且我們發現隨著前置處理時間的延長，保護程度愈發明顯；這樣的保護效果藉由分子生物技術之分析探討得知，持續表現磷酸化之CREB扮演非常重要的角色；反之，我們利用反義脫氧寡核苷酸技術（antisense-oligodeoxynucleotides; As-ODNs) 降低CREB之表現，此時因前置處理引發之保護亦隨之減少。所以，證據顯示持續活化CREB在前置處理所引發的腦部保護是必須的。我們利用rolipram減少cyclic AMP被破壞或利用forskolin加強cAMP 之表現活化CREB；此二者皆能達到明顯之腦部保護，是故，我們的證據顯示活化cAMP-CREB之pathway足以引發腦部的保護機制；並且藥物性之前置處理實驗亦提供臨床預防或治療上一個重要的契機！
　　我們發現在缺氧傷害前24小時而非前1小時，給予單側頸動脈結紮可在神經傷害及神經行為測試上達非常顯著之腦部保護作用，而經頸動脈結紮24小時後再行缺氧之幼鼠腦部在缺氧後較結紮1小時即行缺氧之幼鼠有更多之腦血流灌注，因此缺氧後反而有更多之腦血流灌注之前置處理過之幼鼠腦部或許具有血管新生之情形。除了pCREB在缺氧中扮演重要的因子之外；血管再生在腦部發展過程中亦扮演重要角色。
　　血管生成因子(Vascular endothelial growth factor; VEGF)與血管及神經之再生過程中均具有重要影響作用，VEGF除了刺激血管新生形成之血管保護作用外，對神經細胞亦具有生長、保護及再生刺激之作用。過去文獻指出，在成鼠之實驗，若靜脈注射VEGF可改善腦血管阻塞-中風後之神經後遺症；反之若在中風老鼠腦部打入抗VEGF之抗體則會加重腦病變之傷害。在成鼠腦部各種傷害之模式中不少研究均證明VEGF之神經保護作用，但VEGF在發展中之腦部傷害是否也具有保護作用則未明。
　　在神經系統中，VEGF主要藉由刺激結合其二種不同之受體VEGF receptors (VEGFR)進而活化細胞內tyrosine kinase domains來使細胞產生反應。過去研究指出VEGF是經由活化其VEGFR-2來產生神經再生作用，而VEGF是透過VEGFR-1來刺激astrocytes之生長。在缺氧前置處理(hypoxic preconditioning)所產生之神經保護作用中亦發現VEGF是透過活化VEGFR-2來產生作用。在幼鼠模擬早產兒之視網膜病變之病變上卻發現若要預防氧氣引發之視網膜血管退化病變上，乃藉由刺激VEGFR-1；而非VEGFR-2，來產生血管保護作用。因此，在發展中的新生兒腦部之傷害保護作用是否可透過VEGF-VEGFR-1或VEGF-VEGFR-2之活化路徑來達成，也是未知之重要問題。
　　我們在新生鼠腦部組織染色中發現單側頸動脈結紮24小時能夠產生血管新生；並且這樣的前置處理下會引發VEGF之活化，而我們利用反義脫氧寡核苷酸技術降低VEGF之表現發現，保護亦隨之減少。我們並發現VEGF能藉由活化VEGFR-2之訊息傳遞來啟動CREB持續磷酸化達到保護腦部的效果，而非是VEGFR-1。我們亦發現給予幼鼠VEGF作為前置處理的條件，亦足以誘發VEGF活化下游保護機制以減少腦傷；相似的實驗證據亦顯示，給予幼鼠VEGFR-2之活化物(VEGF-E)作為前置處理的條件，亦具引發CREB持續磷酸化表現增加而減少新生鼠因缺血-缺氧性之腦傷，但是，VEGFR-1之活化物(PlGF)卻沒有明顯的腦部保護效果。
　　從各實驗證據充分顯示，持續性增加pCREB表現在單側頸動脈結紮引發之腦部保護，扮演非常重要的角色，而這樣的保護一方面可以由VEGF在神經細胞上結合其受體VEGFR-2並活化CREB的表現，另一方面，相似之機制亦發生在血管之內皮細胞上；這樣雙重的保護機制啟動下，可以解釋為什麼經過24小時前置處理所誘發之腦部保護效果會如此明顯；我們希望藉由實驗所證實之機制探討，未來在臨床上，可以提供一個可行的治療方向，特別是腦部因缺血缺氧引發的嚴重傷害。　

　　Perinatal hypoxic-ischemic brain injury is a major cause of permanent neurological dysfunction in children. An approach to study the treatment of neonatal hypoxic-ischemic encephalopathy that allows for neuroprotection is to investigate the states of tolerance to hypoxic-ischemia. Twenty-four-hour carotid-artery-ligation preconditioning established by delaying the onset of hypoxia for 24 h after permanent unilateral carotid ligation in neonatal rats markedly diminished the cerebral injury. Although much has been learned about the ischemic preconditioning mechanisms in adult rats, the signaling mechanisms of this 24-h-carotid-artery-ligation preconditioning in neonatal rats remain unknown.
　　We demonstrated that carotid artery ligation 24 h before hypoxia on postnatal day 7 (P7) rat pups provided complete neuroprotection, while artery ligation 6 h produced intermediate benefit, at behavioral and pathological levels compared to ligation 1 h before hypoxia. We first showed that the 24-h-carotid-artery-ligation preconditioning was associated with a robust and sustained activation of cAMP response element-binding protein (CREB), a transcription factor that acts as a key mediator of stimulus-induced nuclear responses underlying learning and memory, survival, and synaptic plasticity of the nervous system. Intracerebroventricular infusions of antisense CREB oligodeoxynucleotides significantly reduced the 24-h-carotid-artery ligation-induced neuroprotection by decreasing CREB expression. Pharmacological activation of the cAMP-CREB signaling with rolipram 24 h prehypoxia protected rat pups at behavioral and pathological levels by sustained increased CREB phosphorylation. These findings suggest that CREB activation provides important mechanism for potential pharmacological treatment against neonatal hypoxic-ischemic brain injury.
　　The upstream signaling mechanisms leading to CREB activation, however, in this 24 h carotid-artery ligation preconditioning of neonatal rat brain remained unknown. We next found that vascular endothelial growth factor (VEGF)-A and VEGF receptor-2 (VEGFR-2) instead of VEGFR-1 were expressed in vessels and neurons of the P7 rat brain. Increased angiogenesis and upregulated expression of VEGF-A and VEGFR-2, but not VEGFR-1, was also found in vessels and neurons in the ipsilateral cerebral cortex 24 h after carotid artery ligation. A blockade of VEGF-A or VEGFR-2, instead of VEGFR-1, by antisense oligodeoxynucleotides decreased VEGFR-2 and pCREB expression and abolished the neuroprotective effect of carotid artery ligation preconditioning. In contrast, VEGF-A treatment or selective activation of VEGFR-2 before hypoxic-ischemia selectively upregulated VEGFR-2 and pCREB expression and provided neuroprotection against neonatal hypoxic-ischemic brain injury. Furthermore, selective activation of VEGFR-2 but not VEGFR-1 after hypoxic-ischemia also significantly protected P7 rat pups against hypoxic-ischemic brain injury. Further in vitro oxygen-glucose deprivation (OGD) study confirmed that VEGFR-2 and CREB activation was required for VEGF-A-induced neuroprotection against oxygen glucose deprivation neuronal death in differentiated H19-7 cells.
　　Taken together, these in vivo and in vitro evidences suggest that VEGF-A/VEGFR-2 signaling leading to CREB activation is an important event in neuroprotection against hypoxic-ischemic injury in the neonatal brain. Pharmacological activation of VEGFR-2 might be an important strategy for the treatment of neonatal hypoxic-ischemic brain injury.

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