Effect of Yi-Nao-Jie-Yu Prescription on Post-Stroke Depression in Rats using Middle Cerebral Artery Occlusion Combined with Behavioral Restraint

We developed a RED model of post-stroke depression and evaluated whether the Chinese medicine alleviates depression-like behaviors. Existing models inadequately combined stroke and chronic stress. This protocol integrates artery blockage, prolonged restraint, and isolation to better replicate post-stroke depression.

To begin, perform behavioral restraint and isolation housing on post middle cerebral artery occlusion day seven to avoid the acute stroke phase. Prepare the custom T-shaped restraint platform. Ensure the restraint area contains slots for fixing the rat's head and limbs and three adjustable straps to fit different rat sizes.

At any random two-hour window each day, secure each rat to the platform using the slots and strap. Then adjust the tightness to prevent struggling without causing respiratory suppression. At the start of behavioral restraint, transfer rats in the post-stroke depression, conventional medicine, and traditional Chinese medicine groups from group housing with five animals per cage to individual housing with one animal per cage.

Maintain individual housing until behavioral restraint is completed on post middle cerebral artery occlusion day 14. For the sucrose consumption test, randomly select six rats from the groups at two, four, and eight weeks post middle cerebral artery occlusion surgery. Fast and deprive the rats of water for 24 hours before the sucrose consumption test.

Ensure the testing room temperature is maintained at 23 plus or minus two degrees Celsius and the lighting matches the housing environment. Then place two 150-milliliter bottles in each cage, one containing tap water and the other containing 1%sucrose solution. Swap the positions of the two bottles halfway through the drinking period to eliminate position bias.

After one hour of free drinking, record the volumes of sucrose solution and water consumed. To calculate sucrose preference, use the given formula. For the open-field test, set up a black square box.

Keep the testing room quiet and dimly lit. Place each rat in the center of the open field box and start a five-minute timer. Record the rat's horizontal activity movement within the box and vertical activity as the rearing with the four paws off the floor or climbing against the wall.

Optionally, record grooming and other typical behaviors for additional observation. To conduct the forced swim test, first fill a glass cylinder with water to a level that prevents the rat from touching the bottom with its feet. Adjust the water temperature to 25 plus or minus one degree Celsius.

Place each rat in the cylinder and let it swim for five minutes. Record the total immobility time during the five minutes. Define immobility as passive floating with only minimal movements to keep the nose above water and activity as limb paddling or wall scratching.

After the test is complete, remove the rat from the cylinder, dry it with a towel, and place it under a heating lamp set to 30 plus or minus two degrees Celsius for 30 minutes to prevent hypothermia. At four weeks, the immobility time of post-stroke depression rats in the forced swim test was significantly higher than that of stroke group rats. At both four and eight weeks, the sucrose preference of PSD rats in the sucrose preference test was significantly lower than that of the stroke group rats.

The YNJYP treatment significantly reduced the immobility time of PSD rats at both four and eight weeks. YNJYP treatment also significantly increased their sucrose preference at both time points. At eight weeks, the rearing frequency of PSD rats was significantly lower than that of the sham-operated group in the open-field test.

At two weeks, the locomotor activity of PSD rats was significantly lower than that of the stroke group in the open-field test. At eight weeks, the rearing frequency in both the traditional Chinese medicine and conventional medicine groups was significantly higher than that in the PSD group. In addition, the locomotor activity of both treatment groups was also significantly higher than that of the PSD group at both two and eight weeks.

This protocol let us measure behaviors linked to depression. We record time spent floating, sugar water intake, and activity in open field. The key challenge is ensuring precise humane execution, requiring accurate suture placement and secure restraint without compromising respiration or animal welfare.

Future world can study how this Chinese medicine works in the brain. We can also test if combining aids with other treatments like rehabilitation or western tracks is more effective.