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

The overall goal of this procedure is to induce maternal immune activation, or MIA, in mice by intraperitoneal injection of the viral mimic poly at the mid-gestational embryonic stage. This method can help us answer key questions in a neuro-immunology field, such as, how immune dysfunction can lead to perturbations in brain development and behavior. The main advantage of this technique is that it's specifically designed for researchers interested in studying MIA as an environmental risk factor for autism and schizophrenia.

Generally, individuals new to this method will struggle, because small differences in the MIA induction method can significantly alter the downstream response in the offspring. To set up a timed mating pair, begin by housing five female mice per cage next to each other in standard mouse cages to synchronize their estra cycles, and use an ear punch to label each animal. Zero to two hours before the dark cycle begins, house the female mice two to a cage, and weigh and record the body weight of each individual mouse, then add one male mouse per cage of two female mice.

Zero to four hours after the dark cycle ends, gently lift each female mouse by the base of the tail, allowing the animal to grip the cage grid with the forepaws, to determine the existence of a whitish mass in the vaginal opening. If a plug is not apparent, gently insert a 200 microliter pipette tip into the vaginal opening. Resistance from coagulation confirms the formation of a plug.

Group the plugged mice into a new cage, as the presence of a vaginal plug confirms only that copulation has occurred. On embryonic days 10.5 to 11.5, lift each plugged female mouse by the base of the tail, and look for a bulge in the abdomen, then weigh the mice to verify the pregnancy, and house the pregnant animals in individual clean cages. Weigh the poly(I:C)powder in a 50 milliliter conical.

On embryonic day 12.5, add the appropriate volume of 0.9%sodium chloride to the corresponding volume of poly(I:C)in a conical tube, and close the cap. Gently roll the saline droplet over the powder until the solution is clear. then centrifuge the tube, and transfer the poly(I:C)solution to a microcentrifuge tube.

Use a spectrophotometer to measure the 260 to 280 ratio of the poly(I:C)solution. The ratio should be between 1.54 and 1.82, as described by the manufacturer. Next, weigh the first mouse and load a 0.3 milliliter insulin syringe with the corresponding calculated volume of poly(I:C)solution.

Then, picking the mouse up by the tail, place it on the cage top, and secure it by the scruff. Now insert the needle, bevel side up, into the center of the two upper nipples at approximately 20 degrees relative to the mouse, and administer the poly(I:C)solution. Then place the mouse back into the cage.

After all of the mice have been injected, house the cages in a quiet, low-traffic room to avoid other confounding effects, and weigh the mice the next morning. At least three days before the behavioral testing, change the cage bedding of the eight to 12 week old adult maternal immune activation or MIA offspring. 30 minutes before the test, acclimate the mice to the testing room.

For a pre-pulse inhibition test, restrain the mice in plexiglass cylinders on top of a piezoelectric sensor. Acclimate the mouse to the pre-pulse inhibition chamber for five minutes, with six trials of 120 decibels of white noise. Then expose the mice to randomized mixtures of sound, as outlined in the table, and compare the results to the baseline startle response, to determine the pre-pulse inhibition of the MIA animals.

For an open field test, place a mouse in the corner of an open square arena, with a defined 17 by 17 centimeter center zone, and record the trajectory of the mouse for 10 minutes, through a ceiling-mounted camera. Then quantify the center zone entries, and the time spent in the center zone. For a marble burying test, acclimate a mouse to a test cage, with compressed five centimeter deep, clean aspen pine bedding for 10 minutes, then place the mouse back in the home cage, and gently arrange 20 navy blue 1.5 centimeter diameter glass marbles in a four by five grid in the test cage.

Now return the mouse to the test cage for 10 minutes, then place the animal back in the home cage, and count the number of marbles that were buried during the test period. Poly(I:C)injection at embryonic day 12.5 can evoke an acute inflammatory response in the maternal placental fetal axis, and precipitate a chronic effect on brain development and behavioral phenotypes. Indeed, poly(I:C)induces a higher Il6 gene expression in the maternal spleen, placenta, and fetal brain, three hours post-injection.

Generally, when compared to saline-treated adult offspring, MIA adult offspring exhibit fewer center zone entries, and a shorter center zone duration in the open field test. The poly(I:C)injected animals also display more frequent burying behaviors in the marble burying test, and a lower pre-pulse inhibition. The loss of Purkinje cells in the cerebellum lobule seven is another hallmark of MIA offspring, with fewer calbindin positive Purkinje cells observed in the cerebellum lobule seven of these animals, compared to saline injected controls.

While attempting this procedure, it's important to remember not to disturb the pregnant mice after MIA induction. Afterward, additional factors such as postnatal stress or infection can be induced to study additional environment environment interactions.