We thank Dr. Myka Estes for her persistence in addressing variability in the mouse MIA model and all of the contributors in Estes et al.8 for their work that led to the development of the methods protocol described here. The research reported here was supported by NIMH 2P50 MH106438-06 (A.K.M.) and NIMH T32MH112507 (K.P.).

All protocols are performed under the approval of the University of California-Davis Institutional Animal Care and Use Committee (IACUC).
1. Animal preparation
<ol>
	<li>When acquiring animals, keep the following parameters consistent to ensure maximal reproducibility.
	<ol>
		<li>Vendor and vendor location: as previously reported, wild type C57BL/6J mice exhibit different responses to the same dose of poly(I:C) depending on the vendor8. Choose a vend...

Maternal infection alters the course of brain development in humans and in both rodents and nonhuman primates4,5,7. Here, a procedure to induce MIA in mice at a mid-gestational time point using poly(I:C) is outlined. This method incorporates assessment of BIR before pregnancy, which increases reproducibility and offers the chance to mechanistically investigate mechanisms that lead to resilience and susceptibility of offspring to...

Epidemiological evidence links maternal infection to increased risk of psychiatric and NDDs, including schizophrenia (SZ) and autism spectrum disorder (ASD)1,2,3,4,5,6,7. The MIA mouse model was developed to test causality and the mechanistic role of MIA in the etiology of these disorders, as well as to identify molecular biomarkers and develop both diagnostic and therapeutic tools4,6. Despite the utility of this model and its increasing popularity, there is considerable variability in MIA induction protocols within the field, making it difficult to compare results across studies and replicate findings8,9. In addition, most iterations of the model do not investigate two important translational aspects of MIA: (i) many offspring are resilient to MIA, and (ii) susceptible offspring can exhibit distinct combinations of phenotypes8.
To generate a reproducible MIA model, investigators should report at least one quantitative measure of the magnitude of MIA induced in dams. To induce MIA during gestation, our lab performs intraperitoneal (i.p.) injections of the double stranded RNA viral mimic polyinositic: polycytidilic acid [poly(I:C)]. Poly(I:C) induces an immune cascade similar to influenza viruses as it is recognized by toll-like receptor 3 (TLR3)10. As a result, poly(I:C) activates the acute phase response that results in rapid elevation of proinflammatory cytokines8,11,12. Previous studies have demonstrated that the elevation of proinflammatory cytokines, including interleukin-6 (IL-6), is necessary to produce behavioral abnormalities and neuropathology in offspring as a result of MIA11,12,13. Thus, the level of IL-6 in maternal serum collected during its peak at 2.5 h following poly(I:C) injection is a compelling quantitative measure of MIA that can be used to compare results across laboratories within the field.
In order to generate an MIA model that addresses the translationally essential elements of resilience and susceptibility with a single induction protocol8,14, researchers can combine typical induction approaches with characterization of the dam&#39;s baseline immunoreactivity (BIR) before pregnancy8. Recently, it was discovered that virgin female C57BL/6 mice show a wide range of IL-6 responses to a low-dose exposure to poly(I:C) before pregnancy8. It is only a subset of these females that go on to produce susceptible offspring, and only at certain magnitudes of immune activation as dictated by the combination of BIR and poly(I:C) dose8. MIA induces phenotypes in an inverted U pattern; offspring show the greatest behavioral and molecular aberrations when dams are moderately immunoreactive, and the magnitude of maternal inflammation reaches, but does not exceed, a critical range8. Here, a detailed method of how to reliably create both resilient and susceptible offspring with divergent behavioral phenotypes as a result of mid-gestational injection of poly(I:C) is provided.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.9% NaCl physiological endotoxin free saline</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td>Control and vehicle for Poly(I:C)</td>
		</tr>
		<tr>
			<td>35mm petri dish</td>
			<td>Thomas Scientific</td>
			<td>1219Z45</td>
			<td>Used to hold oil during tail bleed</td>
		</tr>
		<tr>
			<td>7.5% TGX gels</td>
			<td>Bio-rad</td>
			<td>4561084</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Ancare Nestlets</td>
			<td>Fisher Scientific</td>
			<td>NC9365966</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>anti-&#946;-tubulin</td>
			<td>Millipore</td>
			<td>MAB3408</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Bio-Plex Pro Mouse Cytokine Standards Group I</td>
			<td>Bio-rad</td>
			<td>171I50001</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Plex Pro Reagent Kit with Flat Plate</td>
			<td>Bio-rad</td>
			<td>171304070M</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>ThermoFisher</td>
			<td>23209</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Covidien Monoject 1/2 mL Insulin Syringe with 28G x 1/2 in. Needle</td>
			<td>Spectrum</td>
			<td>552-58457-083</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>D9779-10G</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Environmental enrichment</td>
			<td>Bio-serv</td>
			<td>K3327 and K3322</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Ethovision</td>
			<td>Noldus</td>
			<td>Ethovision</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Fluorsecent-tagged seondary ntibodies</td>
			<td>Li-cor</td>
			<td>925-32213 and 925-68072</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Food-grade edible oil (like olive, canola or grapeseed)</td>
			<td>Various vendors</td>
			<td></td>
			<td>Use to lubricate tail during tail bleeds</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>14060040</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>High molecular weight polyinositic:polycytidilic acid</td>
			<td>Invivogen</td>
			<td>#tlrl-pic-5</td>
			<td>Used to establish females&#39; BIR</td>
		</tr>
		<tr>
			<td>Humane Mouse Restrainer</td>
			<td>AIMS</td>
			<td>1000</td>
			<td>Used to restrain mouse during tail bleeds</td>
		</tr>
		<tr>
			<td>Image Studio Software</td>
			<td>Licor</td>
			<td>5.2</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Laemmli buffer</td>
			<td>Bio-rad</td>
			<td>1610737EDU</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Luminex200</td>
			<td>ThermoFisher</td>
			<td>APX10031</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvette CB300 300&#956;l Serum capillary tube</td>
			<td>Sarstedt</td>
			<td>16.440.100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mixed molecular weight polyinositic:polycytidilic acid</td>
			<td>Sigma-Aldrich</td>
			<td>#P0913</td>
			<td>Gestational induction of MIA</td>
		</tr>
		<tr>
			<td>monoclonal anti-MEF2A</td>
			<td>AbCam</td>
			<td>ab76063</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>monoclonal anti-STAT3</td>
			<td>Cell signaling</td>
			<td>12640S</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Observer</td>
			<td>Noldus</td>
			<td>Observer</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Odyssey blocking buffer (TBS)</td>
			<td>Li-cor</td>
			<td>927-50003</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Odyssey CLx imaging system</td>
			<td>Li-cor</td>
			<td>9140</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Omnipure PBS</td>
			<td>Millipore</td>
			<td>65054L</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher</td>
			<td>23227</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>polyclonal anti_TH</td>
			<td>Pel-Freez</td>
			<td>P4101-150</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Bio-rad</td>
			<td>162-0177</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Qsonica Sonicator Q500</td>
			<td>Fisher Scientific</td>
			<td>15-338-282</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Quick blood stopper</td>
			<td>Petco</td>
			<td>17140</td>
			<td></td>
		</tr>
		<tr>
			<td>Seal-Rite 1.5 ml microcentrifuge tube, natural non-sterile</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>Soldering stand</td>
			<td>Amazon</td>
			<td>B08Y12QC73</td>
			<td>Used to hold capillary tube during tail bleeds</td>
		</tr>
		<tr>
			<td>Sunflower seeds</td>
			<td>Bio-serv</td>
			<td>S5137-1</td>
			<td>Use to increase breeding efficiency</td>
		</tr>
		<tr>
			<td>The Bio-Plex Pro Mouse IL-6 set,</td>
			<td>Bio-rad</td>
			<td>171G5007M</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher Scientific</td>
			<td>BP152-1</td>
			<td>Optional</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Bio-rad</td>
			<td>23209</td>
			<td>Optional</td>
		</tr>
	</tbody>
</table>

generating a reproducible model of mid-gestational maternal immune activation using poly(i:c) to study susceptibility and resilience in offspring

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in offspring. Animal models of MIA are used to test causality, investigate mechanisms, and develop diagnostics and treatments for these disorders. Despite their widespread use, many MIA models suffer from a lack of reproducibility and almost all ignore two important aspects of this risk factor: (i) many offspring are resilient to MIA, and (ii) susceptible offspring can exhibit distinct combinations of phenotypes. To increase reproducibility and model both susceptibility and resilience to MIA, the baseline immunoreactivity (BIR) of female mice before pregnancy is used to predict which pregnancies will result in either resilient offspring or offspring with defined behavioral and molecular abnormalities after exposure to MIA. Here, a detailed method of inducing MIA via intraperitoneal (i.p.) injection of the double stranded RNA (dsRNA) viral mimic poly(I:C) at 12.5 days of gestation is provided. This method induces an acute inflammatory response in the dam, which results in perturbations in brain development in mice that map onto similarly impacted domains in human psychiatric and neurodevelopmental disorders (NDDs).

Not all animals exposed to 30 mg/kg of poly(I:C) at E12.5 produce offspring with consistent behavioral abnormalities8,31. Though both 30 mg/kg and 40 mg/kg of poly(I:C) reliably produce sickness behaviors in dams, including decreased activity levels, hypothermic responses, and weight loss, and also cause significant elevations in IL-6, only a subset of litters exposed to MIA will go on to develop behavioral abnormalities in domains similar to those observed in hu...

Watch this Scientific Journal Video about Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring at JoVE.com

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using PolyI:C to Study Susceptibility and Resilience in Offspring

Maternal infection is a risk factor for neurodevelopmental disorders. Mouse models of maternal immune activation (MIA) may elucidate infection's impact on brain development and function. Here, general guidelines and a procedure are provided to produce reliably resilient and susceptible offspring exposed to MIA.

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

Maternal immune activation (MIA) during pregnancy is consistently linked to increased risk of neurodevelopmental and neuropsychiatric disorders in ...

generating-reproducible-model-mid-gestational-maternal-immune

Generating a Reproducible Model of Mid-Gestational Maternal Immune Activation using Poly(I:C) to Study Susceptibility and Resilience in Offspring

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Abstract

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