Animal Models of Depression - Chronic Despair Model (CDM)

Podsumowanie

The chronic despair mouse model (CDM) of depression consists of repetitive forced swim sessions and another delayed swim phase as a read-out. It represents a suitable model for induction of a chronic depressive-like state stable for at least 4 weeks, amendable to evaluate subchronic and acute treatment interventions.

Streszczenie

Major depressive disorder is one of the most prevalent forms of mental illnesses and causes tremendous individual suffering and socioeconomic burden. Despite its importance, current pharmacological treatment is limited, and novel treatment options are urgently needed. One key factor in the search for potential new drugs is evaluating their anti-depressive potency in appropriate animal models. The classical Porsolt forced swim test was used for this purpose for decades to induce and assess a depressive-like state. It consists of two short periods of forced swimming: the first to induce a depressed state and the second on the following day to evaluate the antidepressant effect of the agent given in between the two swim sessions. This model might be suitable as a screening tool for potential antidepressive agents but ignores the delayed onset of action of many antidepressants. The CDM was recently established and represented a modification of the classical test with notable differences. Mice are forced to swim for 5 consecutive days, following the idea that in humans, depression is induced by chronic rather than by acute stress. In a resting period of several days (1-3 weeks), animals develop sustained behavioral despair. The standard read-out method is the measurement of immobility time in an additional delayed swim session, but several alternative methods are proposed to get a broader view of the mood status of the animal. Multiple analysis tools can be used targeting behavioral, molecular, and electrophysiological changes. The depressed phenotype is stable for at least 4 weeks, providing a time window for rapid but also subchronic antidepressant treatment strategies. Furthermore, alterations in the development of a depressive-like state can be addressed using this approach. CDM, therefore, represents a useful tool to better understand depression and to develop novel treatment interventions.

Wprowadzenie

Affective disorders, such as major depressive disorder, are among the most frequent and challenging mental illnesses and are associated with high individual suffering¹, an increase of suicide risk², and cause a considerable socioeconomic burden³ for society. Despite its impact, treatment options are limited, and there is an urgent need for the development of novel antidepressive interventions, especially due to the innovation crisis in psychopharmacology over the last decades. In order to understand the pathophysiology of depression and test potential new agents, rational and valid animal models are urgently needed⁴. For almost half a century, the classical forced swim test (FST), originally described by Porsolt⁵, was used as induction and read-out for screening of potential novel antidepressants. It consists of a forced swim period for 5-15 min on day 1, subsequent one-time drug application, and evaluation of the portion mice spend immobile in water in another swim period on the following day. The immobility time was considered to represent a missing natural escape behavior and was thought to correlate with the degree of a depression-like state in the mice⁵.

The classical FST has been heavily criticized, not only in the scientific community^6,7,8 but also in public media⁸. Most controversies around the FST are due to the short induction and treatment periods of only 1 day in the classical paradigm. It was argued that FST represents rather an acute trauma model than a state comparable to human depression. Moreover, the Porsolt test might be suitable as a screening tool for potential antidepressive agents, but it ignores the delayed onset of action of many antidepressants.

The chronic despair model (CDM)^{9,10,11,12,13,14,15}, which is derived from the original FST, represents a more appropriate animal model for depression. In CDM, repeated swim stress over 5 consecutive days avoids acute traumatic effects. By failing to escape from a repeated and ongoing stressful situation, mice are thought to develop a state of helplessness, surrender, and ultimately despair. This paradigm is more comparable to current psychological theories for the development of depression in humans than a single acute trauma, which is commonly experienced at the onset of a posttraumatic stress disorder. The resulting depression-like state in CDM is stable for up to 4 weeks⁹ and therefore opens the possibility for longer treatment periods, which are better comparable to clinical conditions, where antidepressants usually need 2-4 weeks to show a benefit¹⁶.

The evaluation of the depressive-like state should then be multidimensional. The measurement of immobility time, such as in the classical FST, is useful, but should not be used as the only outcome parameter. Various methods, which are described below, should be able to map different dimensions of a depressive state in line with symptoms usually found in depressed humans. Suitable read-out assessments could include escape behavior (immobility time^9,10,17), tail suspension test (TST)⁹, anhedonia (classical sucrose preference test (SPT)¹⁸), motivation-oriented behavior (nose-poke sucrose preference test (NPSPT)¹⁰), expectation/exploration-behavior (response to ambiguous signal¹⁹; Y-maze test⁹), electrophysiology (measurements of long-term plasticity (long-term potentiation, LTP; long-term depression, LTD)²⁰), molecular assessments (activation patterns of immediate early genes (IEGs); further stress patterns²¹).

Theoretically, a repeated swim test can be used to induce a depressed state without any assessment of immobility time. However, it is strongly recommended to provide at least a proof-of-concept experimental series with immobility times. Additionally, CDM represents a suitable model to assess the development of a depressive-like state by measuring immobility time during the induction phase. Specific mouse strains or mice treated before swimming can be evaluated with respect to resilience or vulnerability to stress and the induction of behavioral despair.

Protokół

All experiments were performed in agreement with European guidelines (EU 2010/63) and in accordance with the German animal protection law (TierSchG), FELASA (www.felasa.eu/guidelines.php), the national animal welfare body GV-SOLAS (www.gv-solas.de/index.html) guide for the care and use of laboratory animals, and were approved by the animal welfare committee of the University of Freiburg and by the Comite d’Ethique en Matiere d’Experimentation Animale de Strasbourg (CREMEAS, CEEA35), as well as local authorities. Both sexes of C57Bl6N wild-type mice aged 10-14 weeks (70-98 post-natal days, PND) were used for wild-type (WT) indicated experiments. As a stress-resilient line, the transgenic mouse line with enhanced expression of adenosine A₁ receptors under the forebrain neuronal CaMKII promoter was used^9,15. After the experiments, mice were sacrificed by cervical dislocation.

1. Preparation

1. Obtain an animal research license, including thorough experimental planning.
2. Arrival: On arrival, raise the animals in the animal facility to perform the CDM. If the animals are bought from an external supplier, allow them at least 2 weeks to adapt to the new environment.
3. Housing: To house the animals, ensure that the cages are not occupied with the maximum number of animals to avoid additional stress. Guarantee that housing conditions are in line with international recommendations of mouse housing (for further information, see²²) and constantly maintain them at all times.
   NOTE: The most important standard housing conditions include individually ventilated cages with 25-120 air changes per hour, 12 h light-dark cycle, temperature as stable as possible (at least constant between 20-24 °C), humidity as stable as possible (at least between 45%-65%), gnawing material and shelter present, no individual housing.
4. Time point: Perform all experiments at the same time of the day.
   NOTE: No direct assessment has been made to verify the influence of the daytime on CDM, but most behavioral tests evaluating depressive-like states show variations depending on the time of the day^23,24,25, and it is highly probable that daytime also influences CDM.
5. Nesting material: Reduce the nesting material to a minimum. Ensure there are no running wheels, etc., present in the cage.
   NOTE: Enriched environment prevents the induction of a depressed state.
6. Group composition: Allow the animals to remain in the same group throughout the whole experiment. Group the female mice together even from different litters; group the male mice together with littermate male animals. Due to upcoming aggressiveness, especially of males, biting and barbering may become a problem, therefore give special emphasis to the group composition. Avoid single housing as deprivation is a major additional stressor.
7. Animals: Use different mouse strains, even though specific differences have been observed^9,10. A frequently used mouse strain is C57Bl6N. Label mice in order to perform paired statistical analysis (see step 3.2.4).
8. Animal sex: Equally use both male and female mice.
9. Animal age: Ensure that the animals are at least 10 weeks (70 PND) old. Do not use younger animals due to the exhaustion caused by swimming.
10. Equipment: Use a transparent glass cylinder/beaker with a capacity of at least 2 L, a diameter of 24-26 cm, and a minimum height of 30 cm. Further requirements include a thermometer to check the water temperature, paper towels, red light heating lamp/heating mat or comparable source of heating, timer, stopwatch, quiet surroundings. Videotape the swim sessions for offline analysis and documentation. Ensure that the date and the time are continuously visible on the tape/file, together with an identification code number for the individual animal. Store the files for later analysis and further reference. Film from the side of the glass cylinder, not from above, to facilitate analysis.

2. Induction phase

1. Before starting
   1. Visually observe the animals for abnormalities, including signs of biting or barbering. Exclude the whole cage from the experimental series if an animal shows any minimal injuries. Ensure that a veterinarian is available at any time as injuries will worsen during the experiment and will prevent continuation as mice become more aggressive under the influence of stress.
   2. Obtain the bodyweight for each animal before starting the experiment. Ensure that the weight loss often observed does not exceed 20% of the initial body weight. Exclude animals with a weight loss of over 20% and immediately euthanize them due to the assumed high suffering.
   3. Fill up a beaker or cylinder with water at room temperature (22-23 °C) to a height of at least 20 cm from the bottom, leaving a minimum of 10 cm between the water surface and the upper border of the vessel.
2. Performance
   1. Gently transfer the animals into the water. During the swim phase, keep the animal under continuous observation to prevent drowning. Observe from a position where the animal cannot see the experimenter (for instance, video observation from a room next door).
   2. Set a chronometer at the beginning of the experiment. Take the animals out of the water after 10 min by simply grabbing their tails. Gently dry them with a paper towel and put them either under a heating light or on a heating mat.
   3. Evaluate only one animal at a time. Ensure that animals cannot see each other (for example, separate the housing cage from the experimental set-up by a room-divider).
   4. Perform the forced swim session for 10 min each day for 5 consecutive days.
3. Finishing
   1. Transfer the animals back to their home cages after five swim sessions and allow them to rest for at least 2 days. Start specific treatment interventions subsequently.

3. Evaluation of an anti-depressive treatment

1. Time course
   1. Assess the acute and subchronic treatments with the CDM. Depending on the scientific question, adapt the resting period between the induction phase and the read-out.
   2. To evaluate the acute and rapid-acting potency of ketamine, choose a short resting period (a few days) after the induction phase of CDM. Apply the treatment (i.e., intraperitoneal injection), and then perform the evaluation (additional swim session or different evaluation method) shortly afterward.
   3. To evaluate the effects of a subchronic treatment, increase the treatment period up to 4 weeks (there is no data available for longer treatment periods). For example, give the oral treatment with imipramine to the animals during 4 weeks after the induction phase and evaluate thereafter.
   4. Start to evaluate the depressed state right after the end of the treatment period, e.g., the following day. Always choose an identical time period for control and experimental conditions.
2. Immobility time
   1. Proof-of-concept
      1. To use immobility time as a read-out method, evaluate each day of the induction phase and the test day to provide a proof-of-concept (see Figure 1). For further experimental series, reduce the assessments to day 1, day 5, and the test day (see Figure 1C).
      2. Videotape each experiment. Allow two trained observers who are blinded to the experimental conditions to perform the analysis independently. Video analysis enables the experimenter to observe the behavior from a different room, therefore minimizing the interference with the test (for example, see the video file in the supplementary material).
   2. Conditions: Observe and identify the three different behavioral conditions during the swim test: struggling, swimming, and immobility. Most researchers focus on immobility; a further differentiation between struggling and swimming is rarely useful and dramatically increases the complexity and duration of the analysis.
      1. Struggling: The animal actively tries to escape from the threatening situation. This involves pawing the side of the cylinder with the head oriented toward the wall and movements of all limbs. The water surface is typically slightly turbulent.
      2. Swimming: The animal moves at least both hind paws and travels a distance throughout the water. It actively searches for a way out but does not try to overcome the glass wall of the vessel. Swimming does not involve lifting the paws above the water surface, and the body is usually oriented parallel to the walls of the cylinder. In this condition, animals frequently turn around or move in circles.
      3. Immobility: The animal keeps still, in a freezing-like position, and does not move at all or only moves the tail, or the forepaws to keep its head above the water surface. No distance is actively traveled except for passive floating, and no directed movement of the front paws is observed.
   3. Tracking
      1. Perform the assessment using offline video recordings. Use blinded ratings by two independent and experienced examiners and calculate averages between the two ratings.
      2. Repeat the ratings if the results of the two raters differ above a previously determined range. Continuously observe the mice as the different conditions frequently change between struggling, swimming, and immobility.
      3. Use a stopwatch to measure the total time spent in a focused stage (usually immobility) over the 10 min the mouse stays in the water. Consider a short latency of about a second before changing the ongoing time measurement (e.g., if an animal remains for 20 s in immobility and only moves once for less than a second and returns to immobility for another 10 s, the total immobility time is 30 s).
   4. Statistics: Due to the relatively high inter-individual standard deviation (probably caused by a transfer of hierarchy-depending behavior from the cage to the swim test), mark or label the animals to perform paired (instead of unpaired) parametric tests afterward. Evaluate the normality distribution and, depending on the specific question, perform analysis of variance (ANOVA) with post-hoc t-tests or paired t-tests to compare the different groups. Perform the analysis using absolute values of immobility time (s) or as normalized values.
      1. Absolute values: Give mean values of the immobility time from day 1 to day 5 and for the test day ± SEM (see Figure 1A). Compare the averaged values for day 1 and day 5, preferably using a paired t-test to validate the induction of a depressed state. If there is a significant difference between day 1 and 5, compare the mean values of day 5 to the averaged results of the test day. Ensure that a typical group size in one experiment is between 6 and 10 animals and expect significant differences between baseline and post-induction immobility times in wild-type animals. Comparing different groups with an unpaired t-test is difficult if absolute values are used because of baseline differences; therefore, use normalized values.
      2. Relative/Normalized values: Compare the different treatment effects by normalization to the individual result on day 5, and then express the values as a percentage of day 5 (see Figure 1B).
   5. Control experiments
      NOTE: The swimming performance might be correlated with locomotion. Substances that cause a hyper-locomotion could induce false-positive results (namely, a decrease of immobility time); as well as sedative agents could artificially increase immobility time.
      1. Evaluate the changes in locomotion for unknown substances before performing the swim analysis. Use Open Field Test (OFT) in a separate group of animals for at least 10 min.
      2. Choose the same observation time (10 min) in the OFT as in the CDM to detect unspecific hyper-locomotive effects of the tested compound that might influence CDM read-out via measurement of immobility-time with high validity.
      3. In case of significant hyper-locomotive effects, do not evaluate the swim session to assess the anti-depressive potency but use different read-out methods (for instance, sucrose preference, tail suspension test, etc.).

4. Evaluation of the development of a depressive-like state

1. To evaluate the development of a depressive disorder, assess each day of the induction phase to measure the immobility-time.
   NOTE: In this case, a minor increase of immobility-time between each day describes resilience, whereas a stronger and earlier increase compared to untreated or wild-type animals represents an enhanced vulnerability to stress-induced despair. By treating mice before the swimming event, the preventive intervention or transgenic mouse lines could be assessed concerning the development of behavioral despair.

Wyniki

In the first swim session of the induction phase of CDM, mice usually show a mean immobility time between 190 s and 230 s, which constantly rises with every additional swim session (Figure 1A). This increase is more pronounced in the first 3 days and reaches a plateau-like phase during the last 2-3 days. The immobility-time measured on day 5 remains stable over up to 4 weeks, indicating stable behavioral despair. The antidepressant potency of an intervention ...

Dyskusje

The CDM model represents a relevant and established model for testing the anti-depressive potency of new interventions and opens an extended time window for molecular or electrophysiological experiments to elucidate the pathophysiology of depression. Especially when combined with other tests to assess a depression-like state, CDM has a high face and concept validity. It combines subchronic stress and acquired helplessness for induction and produces a long-lasting depressive-like state. It is insensitive to the single app...

Materiały

Name	Company	Catalog Number	Comments
Beaker, 2000 mL	Kimble Kimax	14000-2000	any vessel >2000ml and diameter of 24-26 cm possible
Digital Thermometer	Hanna Instruments	846-4708	any digital thermometer possible
Digitalwaage 200 g Dipse	DIPSE	tp200	any digital scale possible
Lenovo ThinkCentre V50a-24IMB AiO 11FJ00DVGE - 60,5 cm	Lenovo	A 908278	any standard Personalcomputer possible
Logitech PTZ Pro	Logitech	1000005246	any high resolution camera possible
Stopwatch ROTILABO	Carl Roth	L423.1	any stopwatch possible
Timer ROTILABO	Carl Roth	A802.1	any timer possible