類鐸受體8在缺血性腦中風的角色：以改良之小鼠缺血性腦中風模式探討

Abstract

背景：目前認為Toll-like receptors(TLRs)可能在缺血性中風的局部發炎反應之源頭扮演一個決定性的角色。TLRs不僅與先天免疫(innate immunity)及後天免疫(adaptive immunity)有關，體內細胞的傷害也會引發其作用。在腦部，神經膠細胞與神經元細胞皆會表現數種TLRs，目前已知TLR2和TLR4會加重缺血性中風時的腦細胞傷害。至於TLR8，除了在對抗病毒上有其角色之外，也會促使神經細胞走向apoptosis。然而，TLR8在缺血性腦中風時是否增強表現、並且影響神經元細胞的apoptosis則是未知。 目的：在於確認TLR8在缺血性中風時是否有基因表現量的上升，以及TLR8之蛋白質含量是否也會有局部增加之現象。 方法：以「單側中大腦動脈及雙側總頸動脈暫時阻斷法」作為改良之腦中風小鼠模式，先以TTC染色證實此中風模式之中風位置。再以RT-PCR與real-time quantitative PCR的方式，分析單側中風小鼠之兩側大腦皮質組織的TLR8 mRNA含量，並探討缺血後不同的再灌流時間是否會有不同的基因表現量。此外，以免疫組織化學染色法(immunohistochemistry)確認中風時腦部是否有表現TLR8之蛋白質，以及會表現TLR8的細胞種類。並以Western blot定量TLR8蛋白質之表現量，比較中風側大腦之表現量是否比對側以及對照組小鼠來得高。 結果：我們完成了改良的小鼠缺血性腦中風模式，此模式所造成之缺血性中風是位於右側中大腦動脈灌流範圍之大腦皮質區。與只夾右側中大腦動脈之中風模式比較起來，其所造成之梗塞體積大，死亡率較高。在中風小鼠之神經學障礙方面，我們選用Longa 評分法來評估，發現在同樣的缺血時間之下，再灌流時間越長，則神經學症狀越輕微。在TLR8 mRNA 的表現量方面，缺血90分鐘後再灌流(reperfusion) 1小時及6小時者，皆在中風側之大腦皮質有表現量上升之趨勢，且缺血後再灌流6小時者之中風側與sham對照組之差別已達統計顯著意義(p = 0.021)。在免疫組織化學染色法的結果部分，發現再灌流6小時者，腦部缺血性中風區中有許多細胞具有細胞核皺縮之現象，暗示其正在進行細胞凋亡(apoptosis)，且在其細胞質呈現很強的陽性TLR8染色反應；相對的，對側腦部相對應之部位並無類似之TLR8染色反應。而再灌流18小時的腦部，缺血區的細胞數量已經大量減少，殘存的少數細胞大多有細胞核皺縮之現象，且也有陽性TLR8染色反應。此外，也證實了缺血性中風時會表現TLR8之細胞種類包括了 神經元細胞與神經膠細胞。在Western blot的結果中，於再灌流6小時之後，雙側皆會增加表現TLR8，但雙側沒有明顯差別。 結論：本研究以小鼠中風模式，發現缺血性中風時，局部腦組織之細胞會增加表現TLR8之mRNA及蛋白質；而且進一步確認中風時會表現TLR8蛋白質之細胞包括了神經元細胞及神經膠細胞。至於TLR8增加表現所代表之意義對神經細胞而言是保護性或是傷害性，則有待更進一步的研究證實。

Background: Toll-like receptors (TLRs) had been regard to play an important role in inducing stroke-related focal inflammatory reaction. TLRs are not only related to innate immunity and adaptive immunity, but also activated by damaged cells. In brain, glia cells and neurons can express several types of TLRs, and it has been proved that TLR2 and TLR4 can worsen the ischemic damage during ischemic stroke. As for TLR8, besides its role in defending virus, it can also induce apoptosis as well. However, it is unknown whether TLR8 is upregulated during ischemic stroke and affecting apoptosis of neuron. Aim: The purpose of this study is to evaluate whether TLR8 gene is upregulated during ischemic stroke, and whether the protein level of TLR8 is increased. Methods: In this study, the modified stroke mouse model was created by “transient unilateral middle cerebral artery and bilateral common carotid arteries occlusion method”, and the stroke area was confirmed by TTC stain. The neurological deficit of stroke mice was evaluated by Longa 5 grading method. We analyzed the level of TLR8 mRNA of bilateral cerebral cortex by RT-PCR and real-time quantitative PCR, and compared the genetic expression at different time points of reperfusion. Besides, we used immunohistochemistry to verify whether ischemic stroke increases local expression of TLR8 protein, and to clarify the cell types with increased TLR8 expression. Besides, Western blot was used to evaluate the expression level of TLR8 protein, and to compare whether the expression level at stroke lesion side would be higher than contralateral side and sham-control mice. Results: The modified mouse stroke model was successfully created, which had an infarction at the cerebral cortex of the right middle cerebral artery territory. As compared with the model made by occlusion of the right middle cerebral artery, this modified model had larger infarct volume and higher mortality rate. Longer reperfusion time was associated with fewer neurological deficits. In the analysis of mRNA expression level, there were trends of increased expression in the ipsilateral cerebral cortex of stroke after ischemia for 90 min followed by reperfusion for 1 hour and 6 hours, and the differences between the group with reperfusion for 6 hours and sham-operated group had statistical significance (p = 0.021). In the immunohistochemistry, there were many cells in the ischemic region after reperfusion for 6 hours, which was characterized with condensed nuclei indicating apoptosis. These cells also had strong positive TLR8 stain in their cytoplasm. On the contrary, there was no TLR8 stain in the contralateral hemisphere. In the mouse brain with reperfusion for 18 hours, the cellularity in ischemic region reduced markedly, and these residual cells were also characterized by condensed nuclei and positive TLR8 stain. Besides, we also revealed that neurons and glia cell could express TLR8 during ischemic stroke. In the result of Western blot, the brain of mice underwent ischemia and reperfusion for 6 hours had increased expression of TLR8 in bilateral hemispheres, but there was no obvious side-to-side difference. Conclusion: This study revealed that, in the mouse stroke model, the cells in ischemic region had upregulation of mRNA and protein level of TLR8 during ischemic stroke. Furthermore, the study demonstrated that the cell types expressing TLR8 in ischemic stroke were neurons and glia cells. Further study is needed to clarify the function of TLR8 upregulation is protective or damage for neurons.