建立斑馬魚缺氧誘導神經損傷模型模擬新生兒缺氧缺血性腦病變

Abstract

新生兒缺氧缺血性腦病變 (hypoxic ischemic encephalopathy, HIE) 是因周產期新生兒窒息腦部缺氧導致的腦部傷害，具有高發生率、高死亡率和嚴重的後遺症。目前低溫治療(therapeutic hypothermia) 為臨床治療新生兒缺氧缺血性腦病變的標準策略，然而治療後死亡和殘疾的比率仍然相當高，且並不適用於所有的新生兒。因此迫切需要發展新的治療策略。目前新生兒缺氧缺血性腦病變的動物模型主要應用大型動物或囓齒動物，然而這些動物模型高成本、需要複雜手術操作且再現性較低，並不符合新治療策略初期開發階段所需。基於此，我們利用全身缺氧再復氧誘導斑馬魚神經損傷，建立新生兒缺氧缺血性腦病變的斑馬魚疾病模型。為了找尋最適條件，我們測試了受精後五和六天的斑馬魚幼魚在不同的缺氧時間下的存活率，發現受精後六天的斑馬魚缺氧十五分鐘，兩天後的存活率約六成，是測試治療策略的合適條件。為了評估斑馬魚幼魚缺氧後的神經功能，我們設計了一個行為觀察箱可隔絕外界光線和聲音對斑馬魚的干擾，透過箱內的數位顯微鏡可以直接紀錄斑馬魚受刺激後的反應;我們更進一步建立斑馬魚神經行為評分系統，包括反射反應、運動行為和協調性。我們觀察到復氧後一小時，根據斑馬魚對刺激的反應可分成嚴重傷害和中度傷害兩個組別。中度傷害的斑馬魚在復氧後二十四小時神經功能指標完全恢復正常，而嚴重傷害的組別神經功能未能恢復。此結果顯示早期 (復氧後一小時) 的神經功能指標可預測後期的結果。為了觀察腦部神經細胞死亡，我們也應用腦部運動神經表現綠色螢光蛋白轉殖基因魚之綠色螢光衰減來定量腦部神經細胞傷害程度。結果顯示，腦部神經細胞傷害程度與神經功能指標有高度相關性。應用血管內皮細胞表現紅色螢光蛋白的轉殖基因魚，我們發現斑馬魚在復氧後腦部血管有顯著收縮，且其收縮的程度也與神經功能指標有高度相關性。此發現顯示利用藥物減少腦部血管收縮或許可成為治療新生兒缺氧缺血性腦病變的策略。我們也將低溫治療法應用於此缺氧誘導斑馬魚神經損傷模型，發現復氧後短時間的低溫處理確實可以降低缺氧對腦神經的傷害。此結果展現未來應用此斑馬魚模型測試新治療策略的潛力。

Hypoxic ischemic encephalopathy (HIE), which is the brain injury caused by cerebral hypoxia due to perinatal asphyxia, has high morbidity, high mortality and severe sequelae. At present therapeutic hypothermia is a standard strategy for clinical treatment of HIE; however, the rates of death and disability remain high after treatment and therapeutic hypothermia is not amenable to all the newborns. Therefore, there is an urgent need to develop new treatment strategies. Current animal models of HIE mainly use large animals or rodents, but these animal models are high cost, require complex surgical procedure and are low reproducibility. As a result, they do not satisfy the requirements for early-stage development of new therapeutic strategies. According to this, we employed global hypoxia-reoxygenation to induce neuronal damage in zebrafish, and established a zebrafish disease model for HIE. In order to find the optimal condition, we tested the survival rate of zebrafish at 5 days post fertilization (dpf) and 6 dpf in different duration of hypoxia and found that the survival rate of 15 min hypoxia treatment in 6 dpf larvae was about 60% after 2 days, which is an appropriate condition for testing treatment strategies. To evaluate the neurological function of the zebrafish larvae after hypoxia, we designed a behavioral observation chamber, which can isolate the interference of the external light and the sound to the zebrafish and directly record the response of the zebrafish after stimulation through the digital microscope in the observation chamber; We further built the neurological scoring system for zebrafish, including reflex response, locomotor behavior and coordination. We observed 1 h post reoxygenation, according to the response to stimulation zebrafish can be divided into two groups: (1) serious injury and (2) moderate injury. Moderately injured zebrafish were all recovery of neurological function at 24 h post reoxygenation. In contrast, seriously injured zebrafish still failed to recover neurological function. The results showed that the earlier (1 h post reoxygenation) neurological function index can predict late outcome. To observe the neural cell death in brain, we employed decreased green fluorescence in the transgenic fish which express enhanced green fluorescent protein in the cranial motor neurons to quantify the extent of neural cell injury in brain. The results manifested that the extent of neural cell injury in brain was highly correlated with the neurological function index. Using the transgenic fish which express red fluorescent protein in the vascular endothelial cells, we observed that after reoxygenation cerebral vessels have significant constriction and the extent of vasoconstriction was also highly correlated with the neurological function index. This observation revealed that using drugs to reduce the extent of vasoconstriction in brain may become a strategy for the treatment of HIE. We also applied the therapeutic hypothermia to the zebrafish model of hypoxia-induced neuronal damage and found that treatment with short-term hypothermia after reoxygenation indeed reduced the neuronal damage in brain caused by hypoxia. This result demonstrates the potential of using the zebrafish model to test new therapeutic strategies in the future.