母体免疫激活诱导小鼠的病毒性模仿聚妊娠中期阶段（I：C）
Summary
Maternal immune activation (MIA) is a model for an environmental risk factor of autism and schizophrenia. The goal of this article is to provide a step-by-step procedure of how to induce MIA in the pregnant mice in order to enhance the reproducibility of this model.
Abstract
Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.
Introduction
母体免疫激活（MIA）的概念从母体感染患有自闭症和精神分裂症1的关联流行病学研究起源。由于没有在胎盘或母体病毒感染2,3后胎儿大脑检测复制病毒病原体，感染的对后代的影响是假设由母体免疫系统的激活，而不是病原体本身引起的。
为了阐明MIA和精神障碍，注入化学合成，病毒模拟双链RNA聚肌苷之间的原因和结果关系：胞苷酸：成怀孕啮齿类动物（聚（I C））已被广泛地用作动物模型MIA 4,5。聚（I：C）通过toll样受体3（TLR3），和聚的全身给药确认（I：C）诱导病毒样急性炎症反应。一种机制，通过它的聚（I：C）公关oduces行为异常和neuropathologies的后代是由母体胎盘胎儿轴6使亲和抗炎细胞因子的不平衡。一些研究小组已经通过在MIA模型理解精神障碍7的病因，并且由于研究组之间的不同的利益，免疫激活的不同时间点已被用来实现对脑发育和行为7不同扰动。
在保罗·帕特森实验室技术加州理工学院采用注射聚的策略（I：C）为孕鼠胚胎在12.5天（E12.5），它已经成功地证明了MIA能够诱导行为，神经的，并且在与自闭症和精神分裂症相关8-11后代的免疫学变化。我们之前的工作表明，MIA后代显示行为异常（ 如社会impairmenT，通信逆差，重复的行为，焦虑样行为，以及潜在抑制赤字8,10,12），免疫失调和细胞因子失衡8,13,14，胎儿大脑的基因表达15的改变，在小叶第七浦肯野细胞的损失小脑11，海马突触9性质改变，基因与环境互作13，肠道通透性的改变，以及肠道菌群组成16。此外，治疗和预防策略也从该模型系统13,16,17的发展。通过在E12.5诱导MIA，其他人已经表明MIA产生脑突触分子17的胎儿胶质细胞活化和前脑基底胆碱能18发育改造，株特异性相互作用19，大脑突触脑超微结构异常，脑线粒体呼吸链功能亢进abnormalties，下调，抑郁样行为，认知和海马长时程增强（LTP）障碍，与成人海马神经20的赤字。
在这里，我们提供了如何通过聚诱导MIA在E12.5的详细方法（I：C），以及如何运用这一模型来研究自闭症和精神分裂症的病因学范式。要注意的是MIA为多种病症4的危险因素是很重要的，其结果是对时间和感应的方法，以及在怀孕水坝的饲养极为敏感。这样，实验室之间甚至轻微不一致常常导致低的再现性和/或后代不同的表型。我们的方法是专门为那些有兴趣在学习MIA为自闭症和精神分裂症的环境风险因素设计的，详细的描述中提供将有助于研究人员提高他们的数据的可重复性。
Protocol
Representative Results
Discussion
MIA感应在不同的时间窗扰乱在啮齿类动物不同脑发育的事件，并因此导致在后代不同行为异常和neuropathologies。这里，我们描述了一种协议以诱导MIA小鼠与聚（I：C）在E12.5注射。 MIA感应的此方法导致行为，神经病学，免疫学，并与自闭症和精神分裂症的后代8,10,11,16有关的胃肠道异常。要注意，这一点很重要，因为MIA是一个环境风险因素，它们的后代的表型，预计将有比来自神经发育障碍…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们要纪念已故保罗·帕特森博士对MIA模型，自闭症和精神分裂症研究的进步作出贡献。我们承认萨尔基斯K. Mazmanian就这一协议的大力支持;鲁本·M.巴戎寺，伊薇特·加西亚 – 弗洛雷斯，卡伦C.伦乔尼，和Leslie A.诺伊曼行政援助;阿里Khoshnan和Jan C.柯对拍摄的援助;伊莱恩Y.萧和Natalia Malkova它们对MIA感应意见;杰弗里·科克伦S.，华金·古铁雷斯，关F·李，海梅·罗德里格斯，洛雷娜C.桑多瓦尔，和Natalie A.贝尔杜斯科及专家畜牧业。这项工作是由美国国立卫生研究院康特中心奖（NIH 5P50MH086383-04，保罗·H·帕特森）的支持;自闭症（＃7670，保罗·H·帕特森）;西蒙斯基金会（＃322839，以萨尔基斯K. Mazmanian）;美国国立卫生研究院培训资助（NIH / NRSA T32GM07616到K-HC）;加州理工大学暑期大学生研究奖学金（SURF）（以ZY）;安进学者计划在加州理工学院（ZY到）;和博士后奖学金FROM国家科学委员会，台湾（NSC 101-2917-I-564-039，到W-LW）。
Materials
| Polyinosinic–polycytidylic acid potassium salt | SIGMA | P9582 | |
| 0.9% sodium chloride INJ. USP | HOSPIRA | NDC 0409-4888-10 | |
| MONOJECT insuline syrinage 3/10 mL 29G x 1/2" | COVIDIEN | 8881600145 | |
| 50 ml conical screw cap tubes | USA SCIENTIFIC | 1500-1211 | |
| Nanodrop 1000 spectrophotometer | THERMO SCIENTIFIC | 1000 | Optional |
| Stereomicroscope | Wild Heerbrugg | M5A | Optional |
| Dumont #5 Forceps Inox Tip Size .10 X .06mm | Roboz | RS-5045 | Optional |
| RNAlater RNA stabilization reagent | Qiagen | 76104 | Optional |
| TRIzol reagent | Life Technologies | 15596-026 | Optional |
| RQ1 Rnase-free DNase | Promega | M610A | Optional |
| iScript cDNA synthesis kit | Bio-Rad | 170-8891 | Optional |
| FastStart universal SYBR green master mix with ROX | Roche | 4913922001 | Optional |
| Real-time PCR | ABI | 7300 | Optional |
| Primer: Il6 forward | Life Technologies | TAGTCCTTCCTACCCCAATTTCC | Optional |
| Primer: Il6 Reverse | Life Technologies | TTGGTCCTTAGCCACTCCTTC | Optional |
| Primer: beta-actin forward | Life Technologies | AGAGGGAAATCGTGCGTGAC | Optional |
| Primer: beta-actin Reverse | Life Technologies | CAATAGTGATGACCTGGCCGT | Optional |
| MicroAmp optical 96-well reaction plate | Life Technologies | 4306737 | Optionl |
| MicroAmp optical adhesive film | Life Technologies | 4311971 | Optionl |
| EthoVision | Noldus | EthoVision | Optionl |
| SR-LAB apparatus (PPI) | San Diego Instruments | SR-LAB | Optionl |
| Marbles | PENN-PLAX | Blue gem stones marbles | Optionl |
| Dulbecco's Phosphate-Buffered Saline (DPBS) | Life Technologies | 21600-069 | Optionl |
| Paraformaldehyde | MACRON | 2621-59 | Optionl |
| Vibratome | Leica | VT1000 S | Optionl |
| Sodium azide | Sigma | S2002 | Optionl |
| Triton x-100 | Sigma | X100 | Optionl |
| Hydrogen peroxide solution | Sigma | 18312 | Optionl |
| Goat serum | Vector Laboratories | S-1000 | Optionl |
| Rabbit anti-calbindin antibody | Abcam | ab11426 | Optionl |
| Biotinlyated goat anti-rabbit IgGantibody | Vector Laboratories | BA-1000 | Optionl |
| VECTASTAIN ABC Kit | Vector Laboratories | PK-4000 | Optionl |
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