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Neurociencias

使用聚（I：C）生成妊娠中期母体免疫激活的可重复模型，以研究后代的易感性和恢复力

Published: August 17, 2022
doi:
10.3791/64095
,
1Center for Neuroscience,UC Davis

Summary

母体感染是神经发育障碍的危险因素。母体免疫激活（MIA）小鼠模型可以阐明感染对大脑发育和功能的影响。在这里，提供了一般指南和程序，以产生暴露于MIA的可靠弹性和易感后代。

Abstract

怀孕期间的母体免疫激活（MIA）始终与后代神经发育和神经精神疾病的风险增加有关。MIA的动物模型用于测试因果关系，研究机制，并开发这些疾病的诊断和治疗方法。尽管它们被广泛使用，但许多MIA模型缺乏可重复性，并且几乎都忽略了该风险因素的两个重要方面：（i）许多后代对MIA具有弹性，以及（ii）易感后代可以表现出不同的表型组合。为了提高可重复性并模拟对MIA的易感性和恢复力，雌性小鼠在怀孕前的基线免疫反应性（BIR）用于预测哪些怀孕将导致有弹性的后代或暴露于MIA后具有明确行为和分子异常的后代。本文提供了在妊娠12.5天时通过腹膜内（ip）注射双链RNA（dsRNA）病毒模拟物poly（I：C） 诱导 MIA的详细方法。这种方法在大坝中诱导急性炎症反应，导致小鼠大脑发育的扰动，这些扰动映射到人类精神和神经发育障碍（NDD）中类似受影响的结构域。

Introduction

流行病学证据将母体感染与精神和NDD的风险增加联系起来，包括精神分裂症（SZ）和自闭症谱系障碍（ASD）1，2，3，4，5，6，7。开发MIA小鼠模型是为了测试MIA在这些疾病的病因中的因果关系和机制作用，以及鉴定分子生物标志物并开发诊断和治疗工具4，6。尽管该模型具有实用性并且越来越受欢迎，但该领域内MIA诱导方案存在相当大的差异，因此难以比较研究之间的结果并重复研究结果8，9。此外，该模型的大多数迭代都没有研究MIA的两个重要翻译方面：（i）许多后代对MIA具有弹性，以及（ii）易感后代可以表现出表型8的不同组合。

为了生成可重复的MIA模型，研究人员应报告至少一个关于大坝中诱导的MIA幅度的定量测量。为了在妊娠期间诱导MIA，我们的实验室进行腹膜内（ip）注射双链RNA病毒模拟聚肌醇：聚胞苷酸[poly（I：C）]。 Poly（I：C）诱导类似于流感病毒的免疫级联反应，因为它被toll样受体3（TLR3）10识别。结果，poly（I：C）激活急性期反应，导致促炎细胞因子8，11，12快速升高。先前的研究表明，由于MIA11，12，13，导致后代的行为异常和神经病理学，包括白细胞介素-6（IL-6）的升高是必要的。因此，在注射聚（I：C）后2.5小时达到峰值期间收集的母体血清中IL-6水平是MIA的一种令人信服的定量测量方法，可用于比较该领域内实验室的结果。

为了生成一个MIA模型，通过单一诱导方案解决弹性和易感性的翻译基本要素8，14，研究人员可以将典型的诱导方法与怀孕前大坝基线免疫反应性（BIR）的特征相结合8。最近，发现处女雌性C57BL / 6小鼠在怀孕前对低剂量暴露于poly（I：C）表现出广泛的IL-6反应8。只有这些雌性中的一个子集继续产生易感的后代，并且仅在BIR和poly（I：C）剂量8的组合所决定的一定程度的免疫激活下。MIA 以倒 U 型诱导表型;当DAM具有中等免疫反应性时，后代表现出最大的行为和分子畸变，并且母体炎症的程度达到但不超过临界范围8。本文提供了一种详细的方法，说明如何可靠地创建具有不同行为表型的弹性和易感后代，这些后代是由于妊娠中期注射poly（I：C）的结果。

Protocol

所有方案均在加州大学戴维斯分校机构动物护理和使用委员会（IACUC）的批准下进行。 1. 动物制备 采集动物时，请保持以下参数一致，以确保最大的可重复性。供应商和供应商位置：如前所述，野生型C57BL / 6J小鼠对相同剂量的poly（I：C）表现出不同的反应，具体取决于供应商8。选择显示一致响应的供应商和小鼠品系。对于这里的?…

Representative Results

并非所有在E12.5暴露于30mg / kg聚（I：C）的动物都会产生具有一致行为异常的后代8，31。尽管 30 mg/kg 和 40 mg/kg 的聚（I：C） 都能可靠地在大坝中产生疾病行为，包括活动水平降低、体温过低和体重减轻，并且还会导致 IL-6 显著升高，但只有一部分暴露于 MIA 的窝产仔会继续在类似于人类精神病学和 NDD 中观察到的领域出现行为异常（图 3A-…

Discussion

母体感染会改变人类以及啮齿动物和非人灵长类动物的大脑发育过程4，5，7。在这里，概述了使用poly（I：C）在妊娠中期时间点诱导小鼠MIA的程序。该方法结合了怀孕前对BIR的评估，这增加了可重复性，并提供了机械地研究导致后代对MIA8的弹性和易感性的机制的机会。MIA后，来自中等BIR组的母体（IL-6水平在?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们感谢Myka Estes博士坚持不懈地解决小鼠MIA模型中的变异性，并感谢Estes等人8的所有贡献者所做的工作 ，这些工作导致了此处描述的方法方案的开发。这里报告的研究得到了NIMH 2P50 MH106438-06（AKM）和NIMH T32MH112507（K.P.）的支持。

Materials

0.9% NaCl physiological endotoxin free saline Sigma-Aldrich 7647-14-5 Control and vehicle for Poly(I:C)
35mm petri dish Thomas Scientific 1219Z45 Used to hold oil during tail bleed
7.5% TGX gels Bio-rad 4561084 Optional
Ancare Nestlets Fisher Scientific NC9365966 Optional
anti-β-tubulin Millipore MAB3408 Optional
Bio-Plex Pro Mouse Cytokine Standards Group I Bio-rad 171I50001
Bio-Plex Pro Reagent Kit with Flat Plate Bio-rad 171304070M
Bovine Serum Albumin ThermoFisher 23209 Optional
Centrifuge Eppendorf 5810R Optional
Covidien Monoject 1/2 mL Insulin Syringe with 28G x 1/2 in. Needle Spectrum 552-58457-083
Dithiothreitol Sigma-Aldrich D9779-10G Optional
Environmental enrichment Bio-serv K3327 and K3322 Optional
Ethovision Noldus Ethovision Optional
Fluorsecent-tagged seondary ntibodies Li-cor 925-32213 and 925-68072 Optional
Food-grade edible oil (like olive, canola or grapeseed) Various vendors Use to lubricate tail during tail bleeds
HBSS ThermoFisher 14060040 Optional
High molecular weight polyinositic:polycytidilic acid Invivogen #tlrl-pic-5 Used to establish females' BIR
Humane Mouse Restrainer AIMS 1000 Used to restrain mouse during tail bleeds
Image Studio Software Licor 5.2 Optional
Laemmli buffer Bio-rad 1610737EDU Optional
Luminex200 ThermoFisher APX10031
Microvette CB300 300μl Serum capillary tube Sarstedt 16.440.100
Mixed molecular weight polyinositic:polycytidilic acid Sigma-Aldrich #P0913 Gestational induction of MIA
monoclonal anti-MEF2A AbCam ab76063 Optional
monoclonal anti-STAT3 Cell signaling 12640S Optional
Observer Noldus Observer Optional
Odyssey blocking buffer (TBS) Li-cor 927-50003 Optional
Odyssey CLx imaging system Li-cor 9140 Optional
Omnipure PBS Millipore 65054L Optional
Pierce BCA Protein Assay Kit ThermoFisher 23227 Optional
polyclonal anti_TH Pel-Freez P4101-150 Optional
PVDF membrane Bio-rad 162-0177 Optional
Qsonica Sonicator Q500 Fisher Scientific 15-338-282 Optional
Quick blood stopper Petco 17140
Seal-Rite 1.5 ml microcentrifuge tube, natural non-sterile USA Scientific 1615-5500
Soldering stand Amazon B08Y12QC73 Used to hold capillary tube during tail bleeds
Sunflower seeds Bio-serv S5137-1 Use to increase breeding efficiency
The Bio-Plex Pro Mouse IL-6 set, Bio-rad 171G5007M
Tris base Fisher Scientific BP152-1 Optional
Tween 20 Bio-rad 23209 Optional
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Maternal Immune ActivationPoly(I:C)Neurodevelopmental DisordersAnimal ModelReproducibilityResilienceSusceptibility

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Prendergast, K., McAllister, A. K. Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring. J. Vis. Exp. (186), e64095, doi:10.3791/64095 (2022).
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