Disc1異型合子基因型小鼠施打完LPS後的表現及反應

Responses to LPS treatment in heterozygous Disc1 mutant mice

張瑜芳(Yu-Fang Chang)

指導教授 ： 李立仁

國立臺灣大學/醫學院/解剖學暨細胞生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201701398

摘要

Disrupted-in-Schizophrenia 1 (DISC1)是一種與精神疾病相關的基因，它的缺損與許多精神疾病有關，例如:思覺失調症(schizophrenia)、重度憂鬱症(major depression)、躁鬱症(bipolar disorder)及自閉症(autisum)。我們使用Disc1基因缺損的小鼠，作為精神疾病的動物模式。這種C57BL/6背景的Disc1基因缺損小鼠，帶有從129S6/SvEv背景小鼠來的變異，在第六個exon中缺少了25bp，並在第七的exon中出現stop codon。先前的實驗中已經發現， Disc1基因缺損的異型合子 (heterozygote, Het) 小鼠腦中，內側前額葉皮質(medial prefrontal cortex, mPFC)細胞的樹突棘密度比起野生型 (wildtype, WT) 小鼠，有明顯的降低，而這些小鼠也有工作記憶(working memory)的功能異常。
本實驗中，我們使用8至12週齡之雄性WT以及Disc1 Het小鼠。首先觀察在mPFC中，神經膠細胞 (microglia) 的分布情況、密度及細胞形態在兩種基因型的老鼠上是否有差異。我們利用Iba1組織免疫染色 (Immunohistochemistry)來標定神經膠細胞，並使用neurolucida軟體來三維重建神經膠細胞的細胞體與分枝結構。與WT小鼠比較，我們發現到Het小鼠內側前額葉皮質中的神經膠細胞分枝較少，複雜度較低而總分枝長度也比較短。這種形態上的不同，意味著Het小鼠腦中的神經膠細胞可能處於較為活化的狀態。
接下來，我們對WT和Het小鼠給予腹腔注射0.5 mg/kg的細菌內毒素lipopolysaccharide (LPS) 或生理食鹽水。24小時之後，觀察Het小鼠在受到LPS的感染(發炎)刺激下，其行為、神經膠細胞及生化表現，是否與WT小鼠有所不同？行為實驗方面，在曠野實驗 (open field test)中，我們發現不論是WT還是Het小鼠，在施打LPS的組別中，其活動力都比施打生理食鹽水組有大幅的下降。仔細分析後也發現，施打LPS的WT小鼠相較於打生理食鹽水的WT小鼠，在探索中間區域(central area) 的時間顯著的下降。在架高十字迷宮 (elevated plus maze) 中，施打LPS的WT小鼠探索開放區域 (open arm area) 的時間，比打生理食鹽水的WT小鼠更短。這些結果顯示，施打LPS的WT小鼠正處於一種焦慮的狀態。然而特別的是，Het小鼠在曠野實驗探索中間區域的時間，以及在架高十字迷宮開放區域的時間，在施打LPS或生理食鹽水的組別中，並沒有看到差異性，這顯示LPS的施打，並不會使Het小鼠產生焦慮的行為表現。在神經膠細胞方面，不論是WT還是Het組，小鼠腦中的神經膠細胞密度都會因為施打LPS而上升；除了Het小鼠的杏仁基底外側核 (basolateral amygdala, BLA)之外。在Het小鼠杏仁基底外側核，這個跟情緒非常相關的腦區，其神經膠細胞密度，並不會因施打LPS而改變。
在形態方面，與施打生理食鹽水組別比較，施打LPS組動物內側前額葉皮質中神經膠細胞的分支變少，分枝長度及整體分枝的總長度都明顯的減少，而複雜度也明顯的下降。在WT小鼠中，神經膠細胞在施打完LPS後有非常明顯的變化，然而在Het小鼠中，變化並沒有像WT小鼠明顯。
最後我們觀察細胞激素 (cytokines) 在內側前額葉皮質的表現量。發現施打完LPS後，TNF-α, IL1-β, TLR4的表現量有明顯的上升，但在兩個基因型之間並沒有太大的差異性。綜合我們的結果可以發現Het小鼠神經膠細胞處於比較活化的狀態，因此在施打LPS後的行為表現比起正常WT小鼠會有不同的表現。

關鍵字

精神疾病 ； 動物模式 ； 神經膠細胞 ； 三維細胞重建 ； 焦慮行為 ； 神經發炎

並列摘要

Disrupted-in-Schizophrenia 1 (DISC1) is a susceptibility gene related to various mental disorders including schizophrenia, major depression, bipolar disorder and autism. In our Disc1 mutant mice, the normal Disc1 gene was replaced by 129S6/SvEv 25bp deletion variant. Mice carried one copy of mutant Disc1 gene (Het) resulted in reduced spine density in the mPFC and impaired working memory. In the present study, we first examined the status of microglia in the mPFC of wildtype (WT) and Disc1 Het mice. The morphology of microglia was revealed by Iba1 immunohistochemistry and three-dimensionally reconstructed. Compared with those in WT mice, Iba1-positive microglia in Het Disc1 mice had less branches and shorter branch length, signifying an activated condition. Secondly, we challenged the mice with lipopolysaccharide (LPS; 0.5 mg/kg). One day after LPS or saline injection, both WT and Disc1 Het mice exhibited reduced locomotor activity. The LPS-treated WT mice spent significantly less amount of time in the central region of the open field and less exploration time in the open arms of the elevated plus maze, indicating a LPS-induced anxiety-like behavior. However, in Het mice the time spent in the central region and in the open arms were not altered by LPS treatment. In WT mice the densities of microglia increased in the various forebrain areas after LPS encounter. However, in Disc1 Het mice, the density of microglia failed to increase in the basolateral amygdala (BLA).
The microglial morphology was greatly altered in LPS-treated mice of both genotypes. Compared with saline group, less branch, shorter branch length and lower complexity were found in LPS-treated mice. In WT mice, the changes of microglial morphology was dramatic. However, in Het mice the changes of microglial morphology were minimal.
Finally, we measured the levels of various cytokines in the mPFC. After LPS treatment TNF-α, IL-1β and TLR4 expression were significant increased. However, we did not find any difference between WT and Het mice. Our result indicated an altered microglial status which might contribute to the differential responses following LPS treatment.

並列關鍵字

psychiatric disorders ； animal model ； microglia ； 3D-reconstruction ； anxiety behavior

參考文獻

d'Avila JC, Lam TI, Bingham D, Shi J, Won SJ, Kauppinen TM, Massa S, Liu J, SwansonRA.Microglial activation induced by brain trauma is suppressed by post-injury treatmentwith a PARP inhibitor.J Neuroinflammation. 2012 9:31.

Ballinger MD, Saito A, Abazyan B, Taniguchi Y, Huang CH, Ito K, Zhu X, Segal H, Jaaro-Peled H, Sawa A, Mackie K, Pletnikov MV, Kamiya A. Adolescent cannabis exposure interacts with mutant DISC1 to produce impaired adult emotional memory.Neurobiol Dis. 2015 82: 176-184.

Biesmans S, Meert TF, Bouwknecht JA, Acton PD, Davoodi N, De Haes P, Kuijlaars J, Langlois X, Matthews LJ, VerDonck L, Hellings N, Nuydens R. Systemic immune activation leads to neuroinflammation and sickness behavior in mice.Mediators Inflamm. 2013: 271359.

Carola V, D'Olimpio F, Brunamonti E, Mangia F, Renzi P. Evaluation of the elevated plus-maze and open-field tests for the assessment of anxiety-related behaviour in inbred mice. Behav Brain Res. 2002 134:49-57.

Chung DW, Yoo KY, Hwang IK, Kim DW, Chung JY, Lee CH, Choi JH, Choi SY, Youn HY, Lee IS, Won MH. Systemic administration of lipopolysaccharide induces cyclooxygenase-2 immunoreactivity in endothelium and increases microglia in the mouse hippocampus. Cell MolNeurobiol. 2010 3:531-541