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 vendor and mouse strain which show a consistent response. For the experiments here, C57BL/6 mice obtained from Charles River exhibited consistent changes in behavior after exposure to mid-gestational MIA, while those purchased from Taconic show a greater magnitude response, with some differences across treatment groups compared to Charles River mice8.</li>
		<li>Strain: C57BL/6J mice are the most commonly utilized, but BTBR mice and other strains show differential responses to mid-gestational MIA9. Note these differential responses, as these enhance reproducibility of the method and can be a potential variable in contributing to differential outcomes in offspring.</li>
		<li>To ensure minimal variability, use only virgin females for MIA studies8 and clearly note details in methods.</li>
		<li>Age at shipping and acclimation period: mice shipped before 7 weeks show dysregulated endocrine systems15. Allow animals to acclimate for a minimum of 48 h16,17. Order mice to be shipped at 7 weeks (&#177; 2 days) and inject for BIR at 8 weeks (&#177; 2 days).</li>
		<li>Age at mating: animals&#39; immune systems are dynamic over their lifespan. Take care to minimize variability by keeping age at mating/injection as consistent as possible18,19,20. Mate female mice at 9 weeks (&#177; 2 days). Do not use males over 6 months of age for mating.</li>
	</ol>
	</li>
</ol>
2. Poly(I:C) lot testing and preparation
<ol>
	<li>Prepare high molecular weight poly(I:C) as described below.
	<ol>
		<li>Autoclave 1.5 mL microcentrifuge tubes for storage. Resuspended poly(I:C) can be stored at -20 &#176;C, but repeated freeze thaws can impact potency. Heat water bath to 70 &#176;C.</li>
		<li>Using sterile technique, add 10 mL of sterile physiological saline (NaCl 0.9%) to lyophilized poly(I:C) using a syringe. Heat in 70 &#176;C water bath for 15 min to allow full annealing. Remove and allow to cool to room temperature.</li>
		<li>In a sterile hood, add an additional 40 mL of physiological saline to the bottle and invert several times to mix. Remove the top of the poly(I:C) bottle or use a syringe to aliquot into 1.5 mL microcentrifuge tubes. Store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare mixed molecular weight poly(I:C) as described below.
	<ol>
		<li>Autoclave 1.5 mL microcentrifuge tubes for storage. Resuspended poly(I:C) can be stored at -20 &#176;C but repeated freeze thaws can impact potency. Set water bath to 50 &#176;C.</li>
		<li>Using sterile technique, add 10 mL of sterile 0.9% NaCl to lyophilized poly(I:C) and secure the lid. Heat in 50 &#176;C water bath for 25 min to allow full annealing. Remove and allow to cool to room temperature.</li>
		<li>Using sterile technique, aliquot into 1.5 mL microcentrifuge tubes and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Administer poly(I:C) through intraperitoneal (i.p.) injections as described below.
	<ol>
		<li>Weigh the mouse to determine accurate dosing. Utilizing a 0.5 cc insulin needle, draw up resuspended poly(I:C). Scruff the mouse and flip so the abdomen is exposed.</li>
		<li>Using the other hand, insert the needle to a depth of approximately 0.5 cm between the anterior two nipples at an angle of about 45&#176;.</li>
		<li>Draw up to determine that no blood or urine enters the syringe before injecting. If either occurs, reposition the needle and try again. Inject slowly. If the poly(I:C) bubbles out, the injection was likely subcutaneous. A successful injection placement will result in nothing being drawn up once the needle is inserted, and no leaking once it is removed.</li>
	</ol>
	</li>
	<li>Test MMW poly(I:C) lot potency as described below8.
	<ol>
		<li>Obtain desired form of poly(I:C). Some manufacturers will allow researchers to place a hold on a full or partial lot while potency is tested so that multiple bottles can be obtained later simultaneously. Typically, these can be stored lyophilized at -20 &#176;C for several years if freeze-thaw is avoided.</li>
		<li>Obtain or breed 30 pregnant dams for testing. At E12.5, perform i.p. injections of 20, 30, and 40 mg/kg in a minimum of 10 mice per dose.</li>
		<li>At 2.5 h post-injection, collect blood via tail bleed. Note that peripheral blood and trunk blood can differ in cytokine levels, so keep the collection method consistent within a study.</li>
		<li>Allow blood to clot overnight at room temperature. After 12-24 h, spin down blood samples at 3,768 x g at 4 &#176;C for 8 min. Collect serum and store at -80 &#176;C until analyzed.</li>
		<li>Isolate serum and measure IL-6 levels via ELISA or Luminex. Keep measurement tools consistent as there is significant variability in the total concentration measured with different modalities and manufacturers. Determine magnitude of IL-6 response needed to induce phenotypes utilizing a pilot cohort.</li>
	</ol>
	</li>
</ol>
3. Baseline immunoreactivity (BIR) testing
NOTE: Figure 1 shows the schematic of the steps. Use a different molecular weight poly(I:C) for BIR testing as compared to gestational to lower the likelihood of adaptive immune responses to the compound.
<ol>
	<li>Order virgin female mice to be shipped at 7 weeks old. Upon arrival, group and house four to five mice in a cage and keep group housed until mated. Use ear notch or any another identification system.</li>
	<li>Inject females intraperitoneally with 5 mg/kg of poly(I:C) 1 week after arrival. At 2.5 h post-injection, when circulating IL-6 is highest6, collect whole blood from injected animals via tail snip.</li>
	<li>Allow blood to clot overnight at room temperature. After 12-24 h, spin down blood samples at 3,768 x g at 4 &#176;C for 8 min.</li>
	<li>Collect a minimum of 32 &#181;L of serum from each sample. Freeze at -80 &#176;C until ready to test for cytokines. To measure IL-6 levels most consistently, utilize a multiplex assay such as Luminex. Keep measurement tools consistent as there is significant variability in the total concentration measured with different modalities and manufacturers.
	<ol>
		<li>For Luminex assay protocol, refer to Bruce et al.21.</li>
	</ol>
	</li>
	<li>Using relative IL-6 levels, divide animals into low (bottom quartile), medium (middle two quartiles), and high (highest quartile) BIR groups.</li>
</ol>
4. Tail bleed method for blood collection
NOTE: To avoid use of potentially immunomodulatory sedatives, use the tail bleed method of blood collection.
<ol>
	<li>To set up, place a soldering stand and restraint cup on a surface on the side of the non-dominant hand. In a 35 mm Petri dish, add 1-2 mL of food-grade, edible oil. Remove the cap from the quick blood stopper and place near the setup.</li>
	<li>Place a few layers of paper towel on the soldering stand and the first capillary tube in a clip, positioning it near where the mouse&#39;s tail tip will be held and kept parallel to the table&#39;s surface. Have a razor blade handy.</li>
	<li>To collect the blood, perform the following steps.
	<ol>
		<li>At the required time, remove the mouse from the cage and place under the cup with its tail coming out of the notch at the base. Using a fresh razor blade, clip the very end (1-2 mm) of the tail off and collect the first drop of blood in the capillary tube clipped to the soldering stand.</li>
		<li>Dip fingers of dominant hand into the edible oil and use to squeeze from the base of the tail to the tip, guiding the tail tip to the capillary tube to collect resulting drops of blood. Continue until ~200 &#181;L of blood has been collected.</li>
		<li>Put a small end cap on the tapered end of capillary tube before the top cap. If the top cap is put on first, sample will be expelled from the tapered end of the tube. Put tube in protective outer shell.</li>
		<li>Allow to clot overnight at room temperature. Cool a microcentrifuge to 4 &#176;C and spin down blood as stated in step 3.3.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64095/64095fig01.jpg" /> 
Figure 1. The timeline for testing virgin females&#39; baseline immunoreactivity and mating. Order mice to arrive at 7 weeks old and allow to acclimate to facility for 1 week. Inject animals with 5 mg/kg of poly(I:C) and 2.5 h later draw blood. Allow blood to clot overnight, then centrifuge at 3,768 x g, 4 &#176;C for 8 min. Collect serum and assess relative IL-6 levels via ELISA or Multiplex. At 9 weeks old, set up mating pairs. Created using BioRender.com <a href="https://www.jove.com/files/ftp_upload/64095/64095fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Weight based method for mating and gestational E12.5 injection
NOTE: Figure 2 shows the schematic of the steps. Two methods can be used to set up mating pairs and determine the E12.5 time point. The first, timed-mating, is described elsewhere22. Weight-based calculations can also be used to assess for an E12.5 pregnancy23. The benefit of this approach is that it allows time-locking of the dam&#39;s age at mating, decreasing variability in the immune response. This procedure is used here.
<ol>
	<li>Place males in clean cages and allow them to acclimate for a minimum of 2 h. This decreases the likelihood of female aggression as males will already form a dominant scent in the cage.</li>
	<li>Set up single male, single female breeding pairs by adding the female to the male&#39;s cage. Before placing her in the cage, weigh her and record the weight. Add a small handful of sunflower seeds to each cage to increase mating efficiency.</li>
	<li>To determine weight gain range, perform the following steps.
	<ol>
		<li>Obtain a test group of females and set up mating pairs, recording weight at time of mating.</li>
		<li>When females begin to appear visibly pregnant, weigh them and divide in subsets of 8.5 g, 9.5 g, 10.5 g, and 11.5 g of weight gained. Fetuses at E12.5 have just begun to develop distinct digits in their paws. Use fetal morphology to determine average weight gain to reach E12.5.</li>
	</ol>
	</li>
	<li>At 12 days after mating, weigh females and determine weight gain. In a test facility, females consistently gain 9.5-10.5 g from time of mating to E12.5. Inject via i.p. the dose of solubilized poly(I:C) determined in step 2.3.2 when the female&#39;s weight gain falls within the predetermined range.</li>
	<li>Observe the response to MIA in dams using the following parameters.
	<ol>
		<li>Sickness behavior: Collect subjective scores on a scale of 1-3 for how active dams become in response to being handled, where 1 is little or no movement in response to being handled and 3 is a normal response to capture and restraint. Animals with greater immune responses will show less resistance to handling8.</li>
		<li>Febrile response: Using an IR thermometer, collect pre-injection and 2.5 h post-injection temperatures. Animals with greater magnitude immune responses often display hypothermia in response to greater immune activity8.</li>
		<li>Weight change: Weigh animals at 24 h post-injection. Animals with greater magnitude immune responses generally lose more weight8.</li>
		<li>Measure gestational IL-6 levels as follow8.
		<ol>
			<li>At 2.5 h post-injection, collect blood with the preferred method. Allow blood to clot overnight at room temperature. After 12-24 h, spin down blood samples at 3,768 x g at 4 &#176;C for 8 min.</li>
			<li>Collect serum and store at -80 &#176;C until analyzed. Isolate serum and measure IL-6 levels via ELISA or Luminex. Keep measurement tools consistent as there is significant variability in the total concentration measured with different modalities and manufacturers.</li>
			<li>Singly house the dam after injection with appropriate enrichment like nestlets and enrichment devices. Keep all enrichment consistent as alterations in enrichment can have significant impacts on rodent behavior24,25,26,27,28,29.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Gestation time for C57 mice ranges from 18.5-20.5 days. Perform litter checks to determine if animals were born within this range to ensure injection was performed at the correct time point. When checking for litters, disturb the cage as little as possible. Stress immediately after the litter is born can increase the risk of cannibalization.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64095/64095fig02.jpg" /> 
Figure 2. MIA induction. MIA induction requires assessment of pregnancy, i.p. injection of poly(I:C), and litter checks to ensure correct timing of maternal inflammation. After assessing gestational day either via timed mating or the weight-gain method, deliver an i.p. injection of poly(I:C) at E12.5. Collect a blood sample at 2.5 h after injection to confirm immune activation and determine level of IL-6 activation. Litters will be born at approximately E18.5-E20.5. Created using BioRender.com <a href="https://www.jove.com/files/ftp_upload/64095/64095fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Investigation of alterations in behavior in adult MIA and control offspring (optional)
<ol>
	<li>Starting at P60 and before conducting behavioral tests, acclimate animals to human contact with gentle handling for 1 min a day for 3 consecutive days. Make sure that cage change days do not occur on the same day that behavioral tests are performed.</li>
	<li>Always allow mice to acclimate to the testing room for 30-60 min before starting behavioral tests. Use dimly lit (15-20 lux) rooms to minimize anxiety.</li>
	<li>For repetitive grooming, place mice alone in clean, bedding-free cages with lids. Using a camera, record the mice in these cages for 20 min. The first 10 min function as an acclimation period, the latter 10 min are the test period.</li>
	<li>Using saved videos and a stopwatch, score cumulative grooming time for each mouse during the 10 min test period. Other behaviors that can be scored from these videos include rearing (standing on hind legs), freezing, and jumping8.</li>
	<li>Use other common tests for the MIA model such as prepulse inhibition (PPI) 14,30,31,32, open field12,33,34, 3-chamber social approach13,35,36, novel object recognition37, y-maze30, elevated plus maze33, and context/cued fear conditioning38.</li>
	<li>Postnatal immunoblotting8 (optional)
	<ol>
		<li>At P0, decapitate rapidly and dissect fetal brain tissue in HBSS, freeze in liquid nitrogen, and store at &#8722;80 &#176;C.</li>
		<li>Disrupt samples using a probe sonicator with an amplitude of 20% for 5 s in 2x Laemmli buffer, then denature at 85 &#176;C for 5 min. Centrifuge lysate at 16,000 x g for 10 min at room temperature. Collect supernatant and store at -80 &#176;C.</li>
		<li>Measure total protein content using a commercial BCA protein assay kit, following manufacturer&#39;s instruction, and use bovine serum albumin as the calibration standard.</li>
		<li>Add dithiothreitol as a reducing agent to the samples as a final concentration of 100 mM. Heat to 85 &#176;C for 2 min before loading onto a gel.</li>
		<li>Run 5 &#181;g/lane of protein under reducing conditions on 7.5% TGS gels and transfer electrophoretically onto PVDF membranes. Block membranes with blocking buffer and incubate with chosen antibodies.</li>
		<li>Wash three times with TBS + 0.05% Tween 20 and incubate membranes for 45 min with fluorescent-tagged secondary antibodies.</li>
		<li>Wash an additional four times in TBS/Tween 20 and image results. Standardize results using &#946;-tubulin, detected using anti-&#946;-tubulin.</li>
	</ol>
	</li>
</ol>

<ol><li>Adams, W., Kendell, R. E., Hare, E. H., Munk-Jørgensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidemiological+evidence+that+maternal+influenza+contributes+to+the+aetiology+of+schizophrenia.+An+analysis+of+Scottish%2C+English%2C+and+Danish+data%5BTitle%5D)+AND+%22The+British+Journal+of+Psychiatry%3A+The+Journal+of+Mental+Science%22%5BJournal%5D)">Epidemiological evidence that maternal influenza contributes to the aetiology of schizophrenia. An analysis of Scottish, English, and Danish data.</a> The British Journal of Psychiatry: The Journal of Mental Science. 163 (4), 522-534 (1993).</li>
<li>Brown, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Serologic+evidence+of+prenatal+influenza+in+the+etiology+of+schizophrenia%5BTitle%5D)+AND+%22Archives+of+General+Psychiatry%22%5BJournal%5D)">Serologic evidence of prenatal influenza in the etiology of schizophrenia.</a> Archives of General Psychiatry. 61 (8), 774-780 (2004).</li>
<li>Brown, A. S., Derkits, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prenatal+infection+and+schizophrenia%3A+a+review+of+epidemiologic+and+translational+studies%5BTitle%5D)+AND+%22The+American+Journal+of+Psychiatry%22%5BJournal%5D)">Prenatal infection and schizophrenia: a review of epidemiologic and translational studies.</a> The American Journal of Psychiatry. 167 (3), 261-280 (2010).</li>
<li>Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+involvement+in+schizophrenia+and+autism%3A+etiology%2C+pathology+and+animal+models%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">Immune involvement in schizophrenia and autism: etiology, pathology and animal models.</a> Behavioural Brain Research. 204 (2), 313-321 (2009).</li>
<li>Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+infection+and+immune+involvement+in+autism%5BTitle%5D)+AND+%22Trends+in+Molecular+Medicine%22%5BJournal%5D)">Maternal infection and immune involvement in autism.</a> Trends in Molecular Medicine. 17 (7), 389-394 (2011).</li>
<li>Estes, M. L., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immune+mediators+in+the+brain+and+peripheral+tissues+in+autism+spectrum+disorder%5BTitle%5D)+AND+%22Nature+Reviews.+Neuroscience%22%5BJournal%5D)">Immune mediators in the brain and peripheral tissues in autism spectrum disorder.</a> Nature Reviews. Neuroscience. 16 (8), 469-486 (2015).</li>
<li>Estes, M. L., McAllister, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation%3A+Implications+for+neuropsychiatric+disorders%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Maternal immune activation: Implications for neuropsychiatric disorders.</a> Science. 353 (6301), 772-777 (2016).</li>
<li>Estes, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Baseline+immunoreactivity+before+pregnancy+and+poly%28I%3AC%29+dose+combine+to+dictate+susceptibility+and+resilience+of+offspring+to+maternal+immune+activation%5BTitle%5D)+AND+%22Brain%2C+Behavior+and+Immunity%22%5BJournal%5D)">Baseline immunoreactivity before pregnancy and poly(I:C) dose combine to dictate susceptibility and resilience of offspring to maternal immune activation.</a> Brain, Behavior and Immunity. 88, 619-630 (2020).</li>
<li>Kentner, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation%3A+reporting+guidelines+to+improve+the+rigor%2C+reproducibility%2C+and+transparency+of+the+model%5BTitle%5D)+AND+%22Neuropsychopharmacology%22%5BJournal%5D)">Maternal immune activation: reporting guidelines to improve the rigor, reproducibility, and transparency of the model.</a> Neuropsychopharmacology. 44 (2), 245-258 (2019).</li>
<li>Zhou, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TLR3+activation+efficiency+by+high+or+low+molecular+mass+poly+I%3AC%5BTitle%5D)+AND+%22Innate+Immunity%22%5BJournal%5D)">TLR3 activation efficiency by high or low molecular mass poly I:C.</a> Innate Immunity. 19 (2), 184-192 (2013).</li>
<li>Hsiao, E. Y., Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Activation+of+the+maternal+immune+system+induces+endocrine+changes+in+the+placenta+via+IL-6%5BTitle%5D)+AND+%22Brain%2C+Behavior+and+Immunity%22%5BJournal%5D)">Activation of the maternal immune system induces endocrine changes in the placenta via IL-6.</a> Brain, Behavior and Immunity. 25 (4), 604-615 (2011).</li>
<li>Smith, S. E., Li, J., Garbett, K., Mirnics, K., Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation+alters+fetal+brain+development+through+interleukin-6%5BTitle%5D)+AND+%22The+Journal+of+Neuroscience%22%5BJournal%5D)">Maternal immune activation alters fetal brain development through interleukin-6.</a> The Journal of Neuroscience. 27 (40), 10695-10702 (2007).</li>
<li>Choi, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+maternal+interleukin-17a+pathway+in+mice+promotes+autism-like+phenotypes+in+offspring%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The maternal interleukin-17a pathway in mice promotes autism-like phenotypes in offspring.</a> Science. 351 (6276), 933-939 (2016).</li>
<li>Meyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurodevelopmental+resilience+and+susceptibility+to+maternal+immune+activation%5BTitle%5D)+AND+%22Trends+in+Neurosciences%22%5BJournal%5D)">Neurodevelopmental resilience and susceptibility to maternal immune activation.</a> Trends in Neurosciences. 42 (11), 793-806 (2019).</li>
<li>Laroche, J., Gasbarro, L., Herman, J. P., Blaustein, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduced+behavioral+response+to+gonadal+hormones+in+mice+shipped+during+the+peripubertal%2Fadolescent+period%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">Reduced behavioral response to gonadal hormones in mice shipped during the peripubertal/adolescent period.</a> Endocrinology. 150 (5), 2351-2358 (2009).</li>
<li>Aguila, H. N., Pakes, S. P., Lai, W. C., Lu, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+transportation+stress+on+splenic+natural+killer+cell+activity+in+C57BL%2F6J+mice%5BTitle%5D)+AND+%22Laboratory+Animal+Science%22%5BJournal%5D)">The effect of transportation stress on splenic natural killer cell activity in C57BL/6J mice.</a> Laboratory Animal Science. 38 (2), 148-151 (1988).</li>
<li>Landi, M. S., Kreider, J. W., Lang, C. M., Bullock, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+shipping+on+the+immune+function+in+mice%5BTitle%5D)+AND+%22American+Journal+of+Veterinary+Research%22%5BJournal%5D)">Effects of shipping on the immune function in mice.</a> American Journal of Veterinary Research. 43 (9), 1654-1657 (1982).</li>
<li>Menees, K. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sex-+and+age-dependent+alterations+of+splenic+immune+cell+profile+and+NK+cell+phenotypes+and+function+in+C57BL%2F6J+mice%5BTitle%5D)+AND+%22Immunity+%26+Ageing%22%5BJournal%5D)">Sex- and age-dependent alterations of splenic immune cell profile and NK cell phenotypes and function in C57BL/6J mice.</a> Immunity & Ageing. 18 (1), 3(2021).</li>
<li>Shaw, A. C., Goldstein, D. R., Montgomery, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-dependent+dysregulation+of+innate+immunity%5BTitle%5D)+AND+%22Nature+Reviews+Immunology%22%5BJournal%5D)">Age-dependent dysregulation of innate immunity.</a> Nature Reviews Immunology. 13 (12), 875-887 (2013).</li>
<li>Starr, M. E., Saito, M., Evers, B. M., Saito, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-associated+increase+in+Cytokine+production+during+systemic+inflammation-II%3A+the+role+of+IL-1beta+in+age-dependent+IL-6+upregulation+in+adipose+tissue%5BTitle%5D)+AND+%22The+Journals+of+Gerontology.+Series+A%2C+Biological+Sciences+and+Medical+Sciences%22%5BJournal%5D)">Age-associated increase in Cytokine production during systemic inflammation-II: the role of IL-1beta in age-dependent IL-6 upregulation in adipose tissue.</a> The Journals of Gerontology. Series A, Biological Sciences and Medical Sciences. 70 (12), 1508-1515 (2015).</li>
<li>Bruce, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acute+peripheral+immune+activation+alters+cytokine+expression+and+glial+activation+in+the+early+postnatal+rat+brain%5BTitle%5D)+AND+%22Journal+of+Neuroinflammation%22%5BJournal%5D)">Acute peripheral immune activation alters cytokine expression and glial activation in the early postnatal rat brain.</a> Journal of Neuroinflammation. 16 (1), 200(2019).</li>
<li>Mader, S. L., Libal, N. L., Pritchett-Corning, K., Yang, R., Murphy, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Refining+timed+pregnancies+in+two+strains+of+genetically+engineered+mice%5BTitle%5D)+AND+%22Lab+Animal%22%5BJournal%5D)">Refining timed pregnancies in two strains of genetically engineered mice.</a> Lab Animal. 38 (9), 305-310 (2009).</li>
<li>Heyne, G. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+simple+and+reliable+method+for+early+pregnancy+detection+in+inbred+mice%5BTitle%5D)+AND+%22Journal+of+the+American+Association+for+Laboratory+Animal+Science%22%5BJournal%5D)">A simple and reliable method for early pregnancy detection in inbred mice.</a> Journal of the American Association for Laboratory Animal Science. 54 (4), 368-371 (2015).</li>
<li>Hutchinson, E., Avery, A., VandeWoude, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+enrichment+for+laboratory+rodents%5BTitle%5D)+AND+%22ILAR+Journal%22%5BJournal%5D)">Environmental enrichment for laboratory rodents.</a> ILAR Journal. 46 (2), 148-161 (2005).</li>
<li>Bayne, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+enrichment+and+mouse+models%3A+Current+perspectives%5BTitle%5D)+AND+%22Animal+Models+and+Experimental+Medicine%22%5BJournal%5D)">Environmental enrichment and mouse models: Current perspectives.</a> Animal Models and Experimental Medicine. 1 (2), 82-90 (2018).</li>
<li>Toth, L. A., Kregel, K., Leon, L., Musch, T. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+enrichment+of+laboratory+rodents%3A+the+answer+depends+on+the+question%5BTitle%5D)+AND+%22Comparative+Medicine%22%5BJournal%5D)">Environmental enrichment of laboratory rodents: the answer depends on the question.</a> Comparative Medicine. 61 (4), 314-321 (2011).</li>
<li>Sparling, J. E., Barbeau, K., Boileau, K., Konkle, A. T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+enrichment+and+its+influence+on+rodent+offspring+and+maternal+behaviours%2C+a+scoping+style+review+of+indices+of+depression+and+anxiety%5BTitle%5D)+AND+%22Pharmacology+Biochemistry+and+Behavior%22%5BJournal%5D)">Environmental enrichment and its influence on rodent offspring and maternal behaviours, a scoping style review of indices of depression and anxiety.</a> Pharmacology Biochemistry and Behavior. 197, 172997(2020).</li>
<li>Xiao, R., Ali, S., Caligiuri, M. A., Cao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhancing+effects+of+environmental+enrichment+on+the+functions+of+natural+killer+cells+in+mice%5BTitle%5D)+AND+%22Frontiers+in+Immunology%22%5BJournal%5D)">Enhancing effects of environmental enrichment on the functions of natural killer cells in mice.</a> Frontiers in Immunology. 12, 695859(2021).</li>
<li>Girbovan, C., Plamondon, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+enrichment+in+female+rodents%3A+considerations+in+the+effects+on+behavior+and+biochemical+markers%5BTitle%5D)+AND+%22Behavioural+Brain+Research%22%5BJournal%5D)">Environmental enrichment in female rodents: considerations in the effects on behavior and biochemical markers.</a> Behavioural Brain Research. 253, 178-190 (2013).</li>
<li>Mueller, F. S., Polesel, M., Richetto, J., Meyer, U., Weber-Stadlbauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+maternal+immune+activation%3A+Mind+your+caging+system%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Mouse models of maternal immune activation: Mind your caging system.</a> Brain, Behavior, and Immunity. 73, 643-660 (2018).</li>
<li>Mueller, F. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((neuroanatomical%2C+and+molecular+correlates+of+resilience+and+susceptibility+to+maternal+immune+activation%5BTitle%5D)+AND+%22Molecular+Psychiatry%22%5BJournal%5D)">neuroanatomical, and molecular correlates of resilience and susceptibility to maternal immune activation.</a> Molecular Psychiatry. 26 (2), 396-410 (2021).</li>
<li>Nyffeler, M., Meyer, U., Yee, B. K., Feldon, J., Knuesel, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation+during+pregnancy+increases+limbic+GABAA+receptor+immunoreactivity+in+the+adult+offspring%3A+implications+for+schizophrenia%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Maternal immune activation during pregnancy increases limbic GABAA receptor immunoreactivity in the adult offspring: implications for schizophrenia.</a> Neuroscience. 143 (1), 51-62 (2006).</li>
<li>Babri, S., Doosti, M. H., Salari, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Strain-dependent+effects+of+prenatal+maternal+immune+activation+on+anxiety-+and+depression-like+behaviors+in+offspring%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Strain-dependent effects of prenatal maternal immune activation on anxiety- and depression-like behaviors in offspring.</a> Brain, Behavior, and Immunity. 37, 164-176 (2014).</li>
<li>Vigli, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation+in+mice+only+partially+recapitulates+the+autism+spectrum+disorders+symptomatology%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Maternal immune activation in mice only partially recapitulates the autism spectrum disorders symptomatology.</a> Neuroscience. 445, 109-119 (2020).</li>
<li>Malkova, N. V., Yu, C. Z., Hsiao, E. Y., Moore, M. J., Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation+yields+offspring+displaying+mouse+versions+of+the+three+core+symptoms+of+autism%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Maternal immune activation yields offspring displaying mouse versions of the three core symptoms of autism.</a> Brain, Behavior, and Immunity. 26 (4), 607-616 (2012).</li>
<li>Shin Yim, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reversing+behavioural+abnormalities+in+mice+exposed+to+maternal+inflammation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Reversing behavioural abnormalities in mice exposed to maternal inflammation.</a> Nature. 549 (7673), 482-487 (2017).</li>
<li>Ito, H. T., Smith, S. E., Hsiao, E., Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation+alters+nonspatial+information+processing+in+the+hippocampus+of+the+adult+offspring%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Maternal immune activation alters nonspatial information processing in the hippocampus of the adult offspring.</a> Brain, Behavior, and Immunity. 24 (6), 930-941 (2010).</li>
<li>Zuckerman, L., Weiner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation+leads+to+behavioral+and+pharmacological+changes+in+the+adult+offspring%5BTitle%5D)+AND+%22Journal+of+Psychiatric+Research%22%5BJournal%5D)">Maternal immune activation leads to behavioral and pharmacological changes in the adult offspring.</a> Journal of Psychiatric Research. 39 (3), 311-323 (2005).</li>
<li>Mueller, F. S., Polesel, M., Richetto, J., Meyer, U., Weber-Stadlbauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+maternal+immune+activation%3A+Mind+your+caging+system%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Mouse models of maternal immune activation: Mind your caging system.</a> Brain, Behavior, and Immunity. 73, 643-660 (2018).</li>
<li>Careaga, M., Murai, T., Bauman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation+and+autism+spectrum+disorder%3A+from+rodents+to+nonhuman+and+human+primates%5BTitle%5D)+AND+%22Biological+Psychiatry%22%5BJournal%5D)">Maternal immune activation and autism spectrum disorder: from rodents to nonhuman and human primates.</a> Biological Psychiatry. 81 (5), 391-401 (2017).</li>
<li>Lazic, S. E., Essioux, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improving+basic+and+translational+science+by+accounting+for+litter-to-litter+variation+in+animal+models%5BTitle%5D)+AND+%22BMC+Neuroscience%22%5BJournal%5D)">Improving basic and translational science by accounting for litter-to-litter variation in animal models.</a> BMC Neuroscience. 14, 37(2013).</li>
<li>Spencer, S. J., Meyer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Perinatal+programming+by+inflammation%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Perinatal programming by inflammation.</a> Brain, Behavior, and Immunity. 63, 1-7 (2017).</li>
<li>Mouihate, A., Kalakh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+Interleukin-6+hampers+hippocampal+neurogenesis+in+adult+rat+offspring+in+a+sex-dependent+manner%5BTitle%5D)+AND+%22Developmental+Neuroscience%22%5BJournal%5D)">Maternal Interleukin-6 hampers hippocampal neurogenesis in adult rat offspring in a sex-dependent manner.</a> Developmental Neuroscience. 43 (2), 106-115 (2021).</li>
<li>Zhang, Z., van Praag, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maternal+immune+activation+differentially+impacts+mature+and+adult-born+hippocampal+neurons+in+male+mice%5BTitle%5D)+AND+%22Brain%2C+Behavior%2C+and+Immunity%22%5BJournal%5D)">Maternal immune activation differentially impacts mature and adult-born hippocampal neurons in male mice.</a> Brain, Behavior, and Immunity. 45, 60-70 (2015).</li>
</ol>

The authors have no conflicts of interest to disclose.

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><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-&amp;beta;-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&#039; 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&amp;mu;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

Research

JoVE Journal

Neuroscience

1.6K Views.  UC Davis. Maternale infectie is een risicofactor voor neurologische ontwikkelingsstoornissen. Muismodellen van maternale immuunactivatie (MIA) kunnen de impact van infectie op de ontwikkeling en functie van de hersenen ophelderen. Hier worden algemene richtlijnen en een procedure gegeven om betrouwbaar veerkrachtige en gevoelige nakomelingen te produceren die zijn blootgesteld aan MIA.

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

Using Chronic Social Stress to Model Postpartum Depression in Lactating Rodents

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals and humans. However, many animal models of depression were developed in males and are not applicable or effective in studies of postpartum females. Recent studies have reported significant effects of chronic social stress during lactation, an ethologically relevant and effective stressor, on maternal behavior, growth, and behavioral neuroendocrinology. This manuscript will describe this chronic social stress paradigm using repeated exposure of a lactating dam to a novel male intruder, and the assessment of the behavioral, physiological, and neuroendocrine effects of this model. Chronic social stress (CSS) is a valuable model for studying the effects of stress on the behavior and physiology of the dam as well as her offspring and future generations. The exposure of pups to CSS can also be used as an early life stress that has long term effects on behavior, physiology, and neuroendocrinology.

This article describes the use of chronic resident intruder social stress as an ethologically relevant paradigm to model postpartum depression and anxiety in lactating rodents.

Exposure to chronic stress is a reliable predictor of depressive disorders, and social stress is a common ethologically relevant stressor in both animals ...

Behavior

Determining the Role of Maternally-Expressed Genes in Early Development with Maternal Crispants

Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary for development until zygotic genome activation. Typically, genetic targeting of these maternal factors requires an additional generation to identify maternal-effect phenotypes, hindering the ability to determine the role of maternally-expressed genes during development. The discovery of the biallelic editing capabilities of CRISPR-Cas9 has allowed screening of embryonic phenotypes in somatic tissues of injected embryos or "crispants," augmenting the understanding of the role zygotically-expressed genes play in developmental programs. This article describes a protocol that is an extension of the crispant method. In this method, the biallelic editing of germ cells allows for the isolation of a maternal-effect phenotype in a single generation, or "maternal crispants." Multiplexing guide RNAs to a single target promotes the efficient production of maternal crispants, while sequence analysis of maternal crispant haploids provides a simple method to corroborate genetic lesions that produce a maternal-effect phenotype. The use of maternal crispants supports the rapid identification of essential maternally-expressed genes, thus facilitating the understanding of early development.

Early development is dependent on maternally-inherited products, and the role of many of these products is currently unknown. Herein, we described a protocol that uses CRISPR-Cas9 to identify maternal-effect phenotypes in a single generation.

Early development depends on a pool of maternal factors incorporated into the mature oocyte during oogenesis that perform all cellular functions necessary ...

Genetics

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

Immunology and Infection

Using a Murine Model of Psychosocial Stress in Pregnancy as a Translationally Relevant Paradigm for Psychiatric Disorders in Mothers and Infants

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and offspring, including the development of neuropsychiatric disorders. Risk factors linked to the emergence of affective dysregulation in the maternal-infant dyad have been extensively studied. Exposure to psychosocial stress during pregnancy has consistently emerged as one of the strongest predictors. Several rodent models have been created to explore this association; however, these models rely on the use of physical stressors or a limited number of psychosocial stressors presented in a repetitive fashion, which do not accurately capture the type, intensity, and frequency of stressors experienced by women. To overcome these limitations, a chronic psychosocial stress (CGS) paradigm was generated that employs various psychosocial insults of different intensity presented in an unpredictable fashion. The manuscript describes this novel CGS paradigm where pregnant female mice, from gestational day 6.5 to 17.5, are exposed to various stressors during the day and overnight. Day stressors, two per day separated by a 2 h break, range from exposure to foreign objects or predator odor to frequent changes in bedding, removal of bedding, and cage tilting. Overnight stressors include continuous light exposure, changing cage mates, or wetting bedding. We have previously shown that exposure to CGS results in the development of maternal neuroendocrine and behavioral abnormalities, including increased stress reactivity, the emergence of fragmented maternal care patterns, anhedonia, and anxiety-related behaviors, core features of women suffering from perinatal mood and anxiety disorders. This CGS model, therefore, becomes a unique tool that can be used to elucidate molecular defects underlying maternal affective dysregulation, as well as trans-placental mechanisms that impact fetal neurodevelopment and result in negative long-term behavioral consequences in the offspring.

The chronic psychosocial stress (CGS) paradigm employs clinically relevant stressors during pregnancy in mice to model psychiatric disorders of mothers and infants. Here, we provide a step-by-step procedure of applying the CGS paradigm and downstream assessments to validate this model.

The peripartum period is considered a sensitive period where adverse maternal exposures can result in long-term negative consequences for both mother and ...

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic Poly(I:C)

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.

Induction of Maternal Immune Activation in Mice at Mid-gestation Stage with Viral Mimic PolyI:C

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.

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Medicine

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, termed chorioamnionitis. Chorioamnionitis exposes the developing fetus to a high risk of organ injuries, perinatal morbidity, and mortality, as well as life-long neurobehavioral impairments and other non-neurological developmental issues. The two most frequent subtypes of GBS isolates from maternal and fetal tissues are serotypes Ia (13%-23%) and III (25%-53%). Our lab has developed and characterized a rat model of GBS-induced chorioamnionitis to study subsequent impacts on the central nervous system of the developing fetus and to understand underlying mechanistic aspects. This article presents the design as well as uses of the preclinical rat model, which closely reproduces the hallmark of GBS-induced chorioamnionitis in humans. This article aims to help scientists reproduce the experimental design as well as to provide support through examples of troubleshooting. The present model may also contribute to potential discoveries through uncovering causes, mechanisms, and novel therapeutic avenues, which remain unsettled in many developmental impairments arising from chorioamnionitis. Furthermore, the use of this model may be extended to the studies of perinatal non-neurological common and severe morbidities affecting, for instance, the retina, bowel, lung, and kidney. The main interest of this research is in the field of GBS-induced fetal neurodevelopmental impairments such as cerebral palsy (CP), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). The rationale supporting this model is presented in this article, followed by procedures and results.

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus GBS

The goal of this protocol is to describe a preclinical animal model of Group B Streptococcus (GBS)-induced chorioamnionitis. The study is designed to investigate mechanistic processes, potential causal links with developmental impairments, and finally to develop translational anti-inflammatory placento- and neuro-protective treatments.

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, ...

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm&#160;model.

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

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

Samenvatting

Meer video's verkennen