Our protocol is significant because it represents a new paradigm for evaluating the effects of chronic stress, specifically psychosocial stress, on pregnancy outcomes. In this protocol, we expose pregnant mice to various psychosocial insults of different intensities in an unpredictable fashion. Previous animal model paradigms employed physical stressors or a limited number of stressors administered repetitively, which does not reflect the experience of women during pregnancy.
Our chronic psychosocial stress model overcomes these limitations. This method has wide ramifications for various research areas, including maternal mental health and adverse infant development-related research, and more broadly, for understanding the developmental origins of adult disease through adverse intrauterine events. Before starting each stressor in the CGS paradigm, bring the CGS group mice from the housing room into the room designated for CGS.
Expose the mice to different stressors following an 11-day stressor regimen running from gestational day 6.5 to 17.5. To expose the mice to foreign objects, randomly distribute six marbles or six Legos of different shapes into a clean static cage with mouse bedding without the mouse nestlets. Place all mice from one home cage into the static cage with foreign objects for two hours, then return the mice to their home cage.
To expose the mice to predator odor, place one centimeter thick dirty bedding from female rats into a clean static cage with no mouse bedding or nestlets. Place all mice from one home cage into the static cage with dirty rat bedding for two hours, then return the mice to their home cage. To expose the mice to a cage tilt, place all mice from one home cage into a clean static cage with mouse bedding without the mouse nestlets.
Keep the cage tilted at a 30 degree angle against the wall for two hours, then return the mice to their home cage. To expose the mice to frequent bedding changes, place all mice from one home cage into a clean static cage with mouse bedding without the mouse nestlets. Every 10 minutes, gently move the mice to a different clean cage and replace the mouse bedding.
After two hours, return the mice to their home cage. To expose the mice to bedding removal, place all mice from one home cage into an empty, clean static cage with no mouse bedding or nestlets for two hours, then return the mice to their home cage. To expose the mice to movement on a shaker, place all mice from one home cage into a clean static cage with mouse bedding without the mouse nestlets.
Place the static cage atop a reciprocal lab shaker set to 140 strokes per minute for two hours, then return the mice to their home cage. To expose the mice to lights overnight, place all mice from one home cage into a clean static cage with mouse bedding without the mouse nestlets. Keep the lights on overnight to interfere with the dark cycle.
On the following day, return the mice to their home cage. To expose a mouse to new cage mates, transfer the mouse into a clean static cage with mouse bedding containing two unfamiliar female mice. Leave the mouse in the static cage with the unfamiliar cage mates overnight.
On the following day, returned the mouse to its home cage with familiar cage mates. To expose the mice to wet bedding, saturate the mouse bedding in a static cage with clean water kept at 24 degrees Celsius. Place all mice from one home cage into the static cage with wet bedding overnight.
On the following day, return the mice to their home cage. After the stressor regimen is complete on gestational day 17.5, single house all the experimental mice to prepare for parturition and downstream functional assessments. Exposing pregnant female mice to CGS causes changes in chronic stress-relevant parameters, such as reduction in body weight gain during pregnancy and increased adrenal gland weights in the early postpartum period.
Importantly, CGS dams exhibit postpartum abnormalities and maternal neuroendocrine function as evidenced by increased serum corticosterone levels following a novel acute insult. Furthermore, CGS dams display alterations in maternal care as reflected by an increase in the degree of fragmentation of maternal signals received by the pups. The average duration of licking/grooming bouts is reduced while the mean number of bouts is increased, indicating numerous short episodes of nurturing behavior.
Sucrose preference is also depressed in CGS dams when compared to control dams, suggesting the presence of anhedonia. Lastly, the CGS dams also display increased anxiety-related behaviors as measured by a reduction in the time spent in the open quadrants of the EZM when compared to control dams. In the offspring, exposure to CGS in utero results in decreased weight gain during the postnatal period from postnatal day 7 to 21, although there are no differences at birth.
This reduction in body weight gain is present in offspring of both sexes. When attempting this procedure, establishing a schedule for obtaining timed pregnancies and consistently implementing the entire series, variable unpredictable psychosocial stressors is critical. This technique can help answer interesting questions such as can maternal psychosocial stress cause sex-specific effects on placental function or offspring brain development and behavior?
And can these effects be reversed or modified by pharmacological intervention or postnatal maternal care?
