The overall goal of this procedure is to establish a mouse model of sub chronic and mild social defeat stress or S-C-S-D-S to study the mechanisms of stress-induced symptoms. This is accomplished by first habituating individual male black six mice and ICR mice to the environment. The second step is to screen and select aggressive ICR mice.
Next, the S-C-S-D-S mice are exposed to black six mice individually, repeatedly over several days. Ultimately measured social behavior scores, body weight, food intake, and water intake all reveal the stress-induced changes in the S-C-S-D-S mouse model. In order to find biomarkers and develop objective tics for the various types of depression in humans, it is important to establish several different animal models of deterioration.
For this demonstration, seven week old male black six mice and older male ICR mice are utilized. Have two groups of a dozen black six mice and one group of a dozen ICR mice Independently move the groups to the housing facility. One black six group is for screening the ICR mice and the other is for developing the S-C-S-D-S model.
House the mice individually for a week under standard conditions with around 40%relative humidity between the cages. Put opaque white barricades so the mice cannot see each other on a daily basis. Measure each animal's food and water intake as well as each animal's body mass.
After the habituation week, perform resident intruder tests with the ICR mice as the residents during the mid-afternoon hours. Conduct the test for three minutes under 300 looks in the housing room during the test record the latency to attack and the duration of rapid motions with biting which defines an attack After each trial, check the black six mice for lacerations. Use this as an index of hyperaggressive behavior in the ICR mice for the first aggression index.
Use the ratio of trials with attack latency of less than 30 seconds. Select the most aggressive ICR mice that are not hyper aggressive. Use these mice in the S-C-S-D-S model.
Repeat the test three times per day for five consecutive days. Each of the 12 ICR mice should meet each of the 12 black six mice at least once. Introduce the black six test mice to the housing room.
Barricaded from view of neighboring mice. Let them habituate like this for seven days. Two black six mice are needed for each aggressive ICR mouse in the study.
Three days before beginning the experiment, move the aggressive ICR mice into one compartment of each SD cage. An SD cage contains a movable acrylic divider. The SD cages should all be barricaded so that neighbors cannot be seen.
Next house, the non-stressed control black six mice so that each pair occupies opposite compartments of SD cages Every day of the study. Monitor the weight, change water and food consumption of all the mice on the first day of the trial. After taking the regular measurements, place a subject black six mouse into the compartment occupied by an ICR mouse.
Do this mid-afternoon and under 300 locks in the housing room. Once the first attack is made, start scoring the attacks for five minutes. Tally the number of attacks directed to the back or flank of the intruder mouse.
After five minutes, remove the black six mouse and check it for wounds. Then place the black six mouse into the opposite compartment from the same ICR mouse until the next testing day. Thus the black six mouse will still be able to see, hear, and smell the ICR mouse to experience cumulative stress around the clock.
On the second day after taking weight measurements, introduce each black six mouse to a different ICR mouse territory. After the first attack, let the mice interact for four and a half minutes. Take the same measurements as day one.
After the interaction check for wounds on the black six mouse. Return it to the same cage but opposite of the aggressive mouse. With each consecutive day of testing.
Reduce the interaction time between the mice by 30 seconds on the last session. The interaction time is just 30 seconds after the first attack. If on any day after five minutes an ICR mouse doesn't make an attack, remove it from the study and replace it with a spare.
Introduce the black six mouse to that spare mouse on the same test today on the morning of day 11, perform a test with all the black six mice on an open field half an hour before it begins. Place the cages of the control black six and subject black six on a shelf under less than one locks of lighting the room lighting should be around 20 locks near the arena. Keep a single unfamiliar aggressive ICR mouse.
Begin the trial by placing a black six mouse in an open field box with an empty interaction box for two and a half minutes. The behavior should be monitored by an automated system. After two and a half minutes, return the black six mouse to its home cage and place the ICR mouse in the interaction box in the open field.
This mouse is the target and is the center point of the target zone in the open field. Then reintroduce the black six to the open field with the target for two and a half minutes. After the trial is completed, return both mice to their home cages and clean the open field arena.
Proceed with giving each black six mouse this experience afterwards. Determine the time spent by the black six mice in each interaction zone and in the corner zones from these values. Calculate the social interaction score using the described protocol.
Attacks were scored for a 10 day period. There was considerable variability early on, but this gateway to consistency. The mice had less time to interact each day.
Hence the downward trend. The average number of attack bites received gradually decreased over time because the duration of the physical contact decreased from five minutes to 30 seconds. Stressed mice seemed to gain more weight over the 10 days than control mice.
There wasn't much difference in food consumption. However, stressed mice consumed twice the volume of water and this was quite significant in the social interaction test conducted after the 10 day stress regime. There was a significant difference between control and stressed mice as might be expected.
The control mice were much more explorative when the target mouse was present. The use of sub chronic social disease stress model of mice is advantageous because this model mice can help us further understand the mechanism underlying stress induced weight gain and poly dip and hyperphagia right symptoms.
