探討DWI-T2不一致區缺血神經細胞之分子標記

Exploring Molecular Signature of Ischemic Neurons in the DWI-T2 Mismatch Areas After Stroke

葉馨喬(Shin-Joe Yeh)

指導教授 ： 謝松蒼

國立臺灣大學/醫學院/解剖學暨細胞生物學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200618

摘要

缺血性腦中風之磁振造影若呈現擴散加權成像(diffusion-weighted imaging, DWI)與液體衰減反轉恢復序列(fluid-attenuated inversion recovery, FLAIR)或T2加權成像(T2-weighted images)之間有不一致區，則表示缺血可能發生於4.5小時內；然而，此不一致區所蘊含之病理意義尚屬未知。此研究使用蛋白質體分析Sprague-Dawley大鼠於中大腦動脈阻塞4.5小時內，(1)在腦部T2(+)區、不一致區及對側區之蛋白質表現量，以及(2)在T2(+)區最高表現之蛋白質作為候選蛋白質。候選蛋白質之表現則進一步於下列組織中驗證 (1)大鼠中大腦動脈阻塞模式、(2)大鼠大腦皮質神經細胞培養經歷缺氧缺糖(oxygen glucose deprivation)刺激、及(3)人類中風腦組織。此研究發現細胞凋亡(apoptosis)發生於T2(+)區及不一致區，而程序性壞死(necroptosis)則主要發生於T2(+)區。我們發現候選蛋白質capping protein regulator and myosin 1 linker 3 (CARMIL3)在T2(+)區及不一致區有增加表現，於腦部只表現於神經細胞，主要表現於細胞質，且在不一致區有最明顯之表現。在T2(+)區及不一致區之CARMIL3(+)神經細胞體及神經突觸皆比對側大腦者大，且伴隨(1)增加sulfonylurea receptor 1 (SUR1)表現，意指細胞水腫；(2) p62堆積，表示細胞自噬(autophagy)功能受損；(3) 8-hydroxy-2′-deoxyguanosine (8-OHdG)增加，代表細胞氧化壓力上升。對於此CARMIL3於缺血腦區表現上升之現象，此研究進一步於缺氧缺糖刺激培養的鼠腦神經細胞及人類缺血性腦中風組織證實。總而言之，此研究發現缺血性腦中風4.5小時內之特色為CARMIL3之表現於不一致區及T2(+)區之神經細胞有明顯增加，尤其在不一致區更為明顯，且CARMIL3於神經細胞之表現上升伴隨細胞水腫、自噬功能受損、及氧化壓力上升，表示CARMIL3為腦部缺血的神經細胞之重要指標。

關鍵字

缺血性腦中風 ； DWI-T2不一致區 ； DWI-FLAIR不一致區 ； CARMIL3 ； 水腫 ； 氧化壓力

並列摘要

Ischemic stroke with a mismatch between diffusion-weighted imaging (DWI) and fluid-attenuated inversion recovery (FLAIR) or T2-weighted images indicates onset within 4.5 h, but the pathological substrates in the DWI-T2 mismatch and T2(+) areas remain elusive. In this study, proteomics was used to explore (1) the protein expression profiles in the T2(+), mismatch, and contralateral areas, and (2) the protein with the highest expression in the T2(+) area in the brains of male Sprague-Dawley rats within 4.5 h after middle cerebral artery occlusion (MCAO). The expression of the candidate protein was further validated in (1) rat brain subjected to MCAO, (2) rat primary cortical neuronal culture with oxygen-glucose deprivation (OGD), and (3) infarcted human brain tissues. This study showed that apoptosis was observed in the T2(+) and mismatch regions and necroptosis in the T2(+) region of rat brains after MCAO. We identified capping protein regulator and myosin 1 linker 3 (CARMIL3) as the candidate molecule in the T2(+) and mismatch areas, exclusively in neurons, predominantly in the cytoplasm, and most abundant in the mismatch area. The CARMIL3(+) neurons and neurites in the mismatch and T2(+) areas were larger than those in the control area, and associated with (1) increased expression of sulfonylurea receptor 1 (SUR1), indicating edema, (2) accumulation of p62, indicating impaired autophagy, and (3) increase in 8-hydroxy-2′-deoxyguanosine (8-OHdG), indicating oxidative stress. The increased expression of CARMIL3 was validated in a cell model of cortical neurons after OGD and in infarcted human brain tissues. In conclusion, this study shows that the mismatch and T2(+) areas within 4.5 h after ischemia are characterized by upregulated expression of CARMIL3 in neurons, particularly the mismatch area, which is associated with neuronal edema, impaired autophagy, and oxidative stress, indicating that CARMIL3 serves as a molecular signature of brain ischemia.

並列關鍵字

ischemic stroke ； DWI-T2 mismatch ； DWI-FLAIR mismatch ； CARMIL3 ； edema ； oxidative stress

參考文獻

Buscemi, L., Price, M., Bezzi, P., and Hirt, L. (2019). Spatio-temporal overview of neuroinflammation in an experimental mouse stroke model. Sci. Rep. 9:507. doi: 10.1038/s41598-018-36598-4

Chen, A., Liao, W. P., Lu, Q., Wong, W. S., and Wong, P. T. (2007). Upregulation of dihydropyrimidinase-related protein 2, spectrin alpha II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats–a proteomics approach. Neurochem. Int. 50, 1078–1086. doi: 10.1016/j.neuint.2006.11.008

Collombet, J. M., Masqueliez, C., Four, E., Burckhart, M. F., Bernabé, D., Baubichon, D., et al. (2006). Early reduction of NeuN antigenicity induced by soman poisoning in mice can be used to predict delayed neuronal degeneration in the hippocampus. Neurosci. Lett. 398, 337–342. doi: 10.1016/j.neulet.2006.01.029

Gusel’nikova, V. V., and Korzhevskiy, D. E. (2015). NeuN as a neuronal nuclear antigen and neuron differentiation marker. Acta Nat. 7, 42–47.

Hahn, Y. K., Masvekar, R. R., Xu, R., Hauser, K. F., and Knapp, P. E. (2015). Chronic HIV-1 Tat and HIV reduce Rbfox3/NeuN: evidence for sex-related effects. Curr. HIV Res. 13, 10–20. doi: 10.2174/1570162x13666150311163733