新生兒缺氧窒息腦病變是造成嬰幼兒神經病變最常見的原因,除了嚴重威脅新生兒的生命，在存活者中也常導致永久性的神經缺陷諸如腦性麻痺、智能障礙和癲癇。另外，肥胖問題在現今社會中有日趨嚴重的傾向。先前的研究已顯示，肥胖的成人相較於非肥胖的成人更易發生腦部中風且預後較差。因此我們假設新生兒期肥胖對於新生兒缺氧窒息腦病變也有不良影響。在此篇研究中，我們利用已妥善建立於新生幼鼠的缺氧窒息模式來驗証由過度餵養所導致的過胖幼鼠比起正常餵養的幼鼠有更嚴重的缺氧窒息腦傷。
　　在幼鼠出生後的第一天，我們利用每胎育養隻數減半的方法定義過度餵養組(OF group)為每胎六仔，而正常餵養組(NF group)為每胎十二仔。於出生後第七天觀察幼鼠肥胖的情形。相較於正常餵養組，過度餵養組幼鼠有顯著的體重及脂肪增加(P<0.001)，且血中葡萄糖濃度也明顯升高(P<0.01)。
　　在缺氧窒息處理中，過度餵養組幼鼠的死亡率相對增高。此外，以行為測試及病理切片進行長期的腦傷評估。結果顯示，相較於正常餵養組，缺氧後倖存的過度餵養組成鼠其學習能力更差且有較嚴重的腦部缺損。
　　利用Nissl及TUNEL染色缺氧後24小時的腦部切片，我們觀察到過度餵養組的腦傷程度較高且TUNEL陽性的細胞多於正常餵養組。以western blotting進行細胞凋亡的分析，發現在過度餵養組經缺氧後24小時，大腦皮質中caspase-3分子的活化程度明顯高於正常餵養組。此外，藉由免疫螢光染色，我們也觀察到在過度餵養組缺氧後之大腦皮質與海馬迴中，有更多細胞出現核濃縮的現象，並呈現細胞核轉位(nulear-translocation)的凋亡相關因子AIF。這些研究結果顯示，粒線體依賴性的細胞凋亡參與在因新生期肥胖而惡化的缺氧窒息腦傷中。
　　接下來我們檢驗，是否在過度餵養導致的新生期肥胖伴隨有內質網壓力(ER stress)及JNK分子的過度激活。利用western blotting分析內質網陪護蛋白(chaperon protein)分子Grp78的表現及JNK磷酸化程度。結果顯示，不論缺氧前後，相較於正常餵養組，過度餵養組大腦皮質中的JNK活化程度均較高，而Grp78則在缺氧前後無明顯變化，於兩組亦無差異。進一步分析JNK下游促細胞凋亡的分子BimEL的活化情形發現，缺氧後過度餵養組中BimEL於Ser65位點的磷酸化較正常餵養組增加。然而另一上游分子—壓力激活蛋白磷酸酶p38則在缺氧前後無顯著活化。
　　為了証明新生期肥胖所誘發的JNK過度激活會惡化缺氧窒息性腦傷。我們在過度餵養組中，利用顱內注射的方法給予JNK的抑制劑SP600125阻斷JNK活化後的訊息傳遞。實驗結果顯示，相較於安慰劑，給予SP600125改善了缺氧處理中幼鼠的死亡率且降低缺氧後JNK及BimEL的活化，並在病理學上提供予過度餵養組幼鼠有效的腦部保護。根據以上的實驗，我們認為JNK的過度活化乃為新生期肥胖致使缺氧窒息性腦傷加重的一個重要因素。

　　Perinatal hypoxic–ischemic (HI) brain damage remains a major cause of acute mortality and chronic neurologic morbidity in children. Obesity is a growing problem in modern society. Evidence has shown that obese adult persons suffer a higher risk of stroke and have worse prognosis than non-obese adults. Here, using a well-established HI brain injury model in neonatal rats, we first tested the hypothesis that the over-fed (OF) obese rat pups had a higher degree of HI brain injury compared with the normal-fed (NF) rat pups.
　　The OF group, defined by reducing the litter size to 6 pups from postnatal day 1 (P1), had a significantly higher body-weight (P<0.001), excess fat deposition in the subcutaneous and perirenal space (P<0.001) and bore hyperglycemia (P<0.01) at P7 compared with the NF group defined by keeping the litter size of 12 pups. The OF-HI group had a higher mortality rate than the NF-HI group during HI. In addition, the OF-HI survivors showed a significantly poorer learning behavior performance assessed by water maze task and higher degree of brain damage measured by pathology at adulthood.
　　Twenty-four hours after HI, more brain damage (Nissl stain) and more TUNEL postive cells were observed in the OF-HI brain than in the NF-HI brain. Caspase-3 activation also significantly increased in cerebral cortex in the OF-HI group compared with the NF-HI group. Immunofluorescence imaging showed that the OF-HI group had increased number of injured cells that exhibited pyknotic morphology and cells that showed nuclear translocalization of apoptotic inducing factor in the cortex and hippocampus than the NF-HI group. These results suggest that mitochondria-dependent apoptosis is involved in the obesity-aggravated HI neuronal death in the neonatal rat brain.
　　Thus, we examined whether the over-nutrient status in the neonatal period would cause endoplasmic reticulum (ER) stress and hyperactivation of c-Jun N-terminal kinase (JNK). Our results showed that there was no difference in the ER chaperon protein Grp78 expression between the two groups. In contrast, the OF pups had higher levels of activated JNKs in the cortex than the NF group before and after HI. Furthermore, the OF-HI group had increased phosphorylation at Ser65 site of cell death molecular BIMEL, and the activation of stress-activated protein kinase p38 was not detected after HI in the two groups. Intracerebroventricular administration of JNK inhibitor SP600125 was operated to prove the causal relationship of JNK hyper-activation in neonatal obesity-aggravated HI brain injury. In the OF-HI group, JNK inhibition reduced the mortality during HI and BIMEL phosphorylation after HI, and also provided significant neuroprotection at pathological level. In conclusion, our study shows that obesity in the neonatal period could aggravate HI neuronal death and JNK activation plays a significant role in obesity-aggravated HI neuronal death in neonatal rats.

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