Generation of the Early-Gestational Maternal Immune Activation Mouse Model to Assess Prenatal Inflammation on Neurodevelopment

Our lab studies how prenatal inflammation alters the biology of microglia, the brain's immune cells contributing to adverse neurodevelopmental outcomes. Existing MI models focus on mid-gestation. However, we present an early gestational protocol that moreover incorporates quality control metrics to improve survival and consistency.

The model can be applied to any wild type or genetic mouse model to explore the impact of environmental inflammation on brain development. To begin, set up two eight to 10-week-old C57 BL6 female mice with C57 BL6 breeder males in the late afternoon or evening. On the next morning, assess the female mice for vaginal plugs and continue checking once daily until plugging occurs.

The plugs are temporary and may fall out by the afternoon, if not checked in the morning. After confirming a vaginal plug, weigh the female mouse and record the weight to monitor pregnancy status by tracking weight gain across gestation. Separate plugged females into individual cages and maintain them under standard housing conditions with ad libidum access to food and water.

Designate plugged females as embryonic day 0.5 and record the date of the vaginal plug to ensure accurate tracking of gestational age. To reconstitute lyophilized poly IC to a high concentration stock solution, dissolve it in sterile saline or sterile water according to the manufacturer's instructions. Vortex the high concentration stock solution.

Then, dilute the solution in saline to prepare a low concentration stock. To avoid repeated freeze thaw cycles, aliquot approximately 125 microliters of the low concentration stock into 1.5 milliliter tubes, and store the tubes at 20 degrees Celsius or 80 degrees Celsius. Immediately before injection on embryonic day 9.5.

Dilute the low concentration poly IC stock with sterile saline to obtain a working concentration. Heat the working stock at 65 degrees Celsius for 10 minutes to ensure proper reanealing of the double stranded ribonucleic acid structure. Then vortex or pipette the solution to ensure a homogeneous mixture before injection.

To confirm pregnancy, weigh the dam on the morning of embryonic day 9.5. Calculate the poly-IC injection volume for maternal immune activation, or MIA and the 0.9%saline injection volume for controls. Draw the appropriate volume of the prepared poly-IC working solution or saline into a sterile 27 gauge or smaller syringe.

Now, properly restrain the dam to minimize pain and distress. Inject the poly-IC acid or saline intraperitoneally in the lower left or right quadrant just above the hip level or at the second set of nipples. Then dispose of syringes in a BioSafety Sharps container.

Also, record the injection side to maintain consistency across the cohort. Immediately return the dam to the home cage and monitor for any signs of distress. Then supplement the cage with hydrogel for at least 24 hours after injection to prevent dehydration associated with sickness behavior.

After that, record the poly-IC dose injected volume, injection date and time, gestational age, and poly IC lot number. Three hours post-injection, observe each mouse individually for one minute and assess behavior like reduced activity, hunched posture, facial grimacing, or watery stool using a three-point scale. Classify mice exhibiting one or more of these behaviors as showing acute sickness behaviors.

Then, using a minimally invasive method such as tail bleed, carefully collect maternal blood from the injected dam. Collect approximately 50 microliters of blood. After confirming hemostasis, return the dam to the home cage.

Allow the collected blood to clot for 30 minutes at room temperature. Centrifuge the sample at 2000 G for five minutes at room temperature to separate the serum from the clot. After centrifuging the sample once again, carefully collect the clear upper serum layer without disturbing the clot.

Store the serum at 20 degrees Celsius, until ready for downstream cytokine analysis. After the injection, weigh the dams daily for three days to monitor the response to poly IC acid. On the day of expected parturation, record litter outcomes including litter size, pup viability, or no litter to indicate an unsuccessful pregnancy.

Promptly remove any deceased pups and dispose of them according to institutional biosafety guidelines. Use ELISA or multiplex assays to quantify inflammatory cytokines such as IL-6 and TNF-alpha in maternal serum after MIA or control injections following the manufacturer's instructions. After quantification, use the results to establish a representative threshold for the MIA response.

Finally, perform a statistical analysis using our software to assess the relationship between maternal serum IL-6 and TNF alpha levels. Following poly IC administration at embryonic day 9.5, MIA dams displayed significantly increased IL-6 levels relative to control dams. TNF alpha levels showed a strong positive correlation with maternal IL-6 levels.

Between embryonic day 0.5 and embryonic day 9.5, dams that successfully delivered a litter gained significantly more weight than dams that failed to produce a litter. One day after injection, MIA dams lost weight, while saline treated control dams gained weight. But by embryonic day 12, both groups showed similar weight gain.

Pre-pregnancy weight did not affect the litter efficacy in either saline treated control or MIA dams. However, C57 BL6 mice sourced from Jackson Labs had more successful litters than C57 BL6 mice sourced from Charles River during the MIA procedure. In contrast, the source of the poly IC did not affect litter efficacy.

Moreover, animals sourced from Jackson Labs had a more robust maternal inflammatory response as measured by serum IL-6 compared to animals sourced from Charles River. In contrast, the maternal inflammatory response was the same between different sources of poly IC.The protocol allows for reproducible generation of the early MIA model and correlation of the severity of maternal inflammation with offspring experimental outcomes. It is critical to maintain detailed records, track lot numbers of reagents, and evaluate maternal cytokine responses in all dams to ensure reproducible results.