An Unpredictable Chronic Mild Stress Protocol for Instigating Depressive Symptoms, Behavioral Changes and Negative Health Outcomes in Rodents

The overall goal of the following experiment is to expose a rodent strain to a series of chronic, unpredictable stressors that are randomly determined, such that the animal cannot effectively compensate under growing severity of depressive symptoms develops. This is achieved by daily exposure to one of a list of possible stimuli that are designed to be stressful but not damaging or injurious to the animal. The animals are exposed to the stressors five to six days a week for eight weeks at regular intervals, the animals are monitored for the development of depressive symptoms by assessing code status and the animal responses to impose stimuli, such as a sucrose spray test.

The results show that imposition of chronic randomized unresolvable stressors to the animal's environment prevents the ability of the animal to adapt and causes the development of depressive symptoms over the course of several weeks. This method can help to answer key questions in the chronic disease field, such as how chronic depressive symptoms can lead to negative health outcomes in other tissue or organ systems. We first had the idea for utilizing this method when we learned about the profound comorbid relationships between advanced cardiovascular disease and disease risk in our clinical populations, and the concurrent presentation of profound depressive illnesses as well.

Demonstrating this procedure will be Mr.Steven Brooks and Ms.Kayla Brannon doctoral students from our laboratories. In order to produce mild stress house mice in separate cages and expose the mice to one or two stressors per day for a minimum of three to four hours for each stressor, the following list of stressors should be performed on a randomized schedule for the damp bedding stressor. Wet the bedding by pouring 10 to 20 ounces of clean water into a standard cage.

Keep the mouse in the damp cage for three to four hours. Remove bedding from the cage for three to four hours after which transition the mouse into another stressor requiring an empty cage, or place it into a clean cage with fresh bedding. Tilt bedding free cages to approximately 45 degrees for three to four hours using a sturdy object that will remain in place as the mouse moves around in the cage For this stressor, alter the normal 12 hour light, 12 hour dark cycles into successions of 30 minute periods, lasting a total of eight hours.

Transfer the mouse from its home cage to the cage of a neighboring mouse that has been removed for the three hour stressor period. Remove the bedding from the cage and add water to a depth of about 0.25 inches for mice, and leave the mouse for approximately four hours. Briefly, dry the mouse with a soft towel at the conclusion of the water exposure.

Expose mice to predator, smells or sounds by adding randomly distributed tufts or 10 to 20 milliliters of urine from a natural predator species into the cage, and leave the mouse for three to four hours. Alternatively, play growling or predatory noises from natural predator species ENC proximity to the cages for three hours at the end of each daily stress period. Place the mice into clean cages and return them to the housing facility daily.

Inspect each animal for the development of wounds or sores that may require veterinary treatment once a week, have two investigators trained in evaluating code grooming. Inspect each animal and assign a weekly code score on a scale from zero to one. Add a each individual score by assigning individual scores of zero meaning clean or one meaning dirty to each of eight body regions.

The head, the neck, the dorsal coat, the ventral coat, the tail, the four limbs, the hind limbs, and the genital region. Place the animal in a clean cage lined with a cotton pad or towel to prevent bedding from adhering to its coat. Then spray a 10%sucrose solution onto the head and dorsal coat of each animal and record the total grooming behavior for five minutes.

Measure the latency and frequency of the facial grooming activity during this time. The latency is the amount of time between the first spray and initiation of facial grooming, and the frequency should be measured as the number of times the face is groomed. Following eight weeks of unpredictable chronic mild stress, there were significant alterations to both behavioral and physiological outcomes in chronically stressed rodents relative to their non-stressed control strains.

Mice developed depressive like symptoms following unpredictable chronic mild stress, and showed a progressive decline in coat grooming scores. This marked degradation in the physical state of the coat suggests decreased grooming activity and may parallel a lack of motivation. Recent evidence has suggested that there is a substantial quantitative divergence in the responses to unpredictable chronic mild stress between male and female mice.

Specifically, data suggests that female mice exposed to the identical stress protocol as age-matched males develop more severe behavioral responses to the protocol than do males. Following this procedure, other methods that are appropriate to organ specific function can be readily performed in order to answer additional questions like how the presence of chronic depressive symptoms can impact normal function in the organ system of interest. After watching this video, you should have a good understanding of how to utilize the different steps and procedures in the UCMS protocol to progressively create the conditions of growing depressive symptoms in your rodent model.