Our protocol proposes a new medial cerebral artery occlusion technique in rats to help study the mechanism of post-stroke depression development and to find a more efficient therapy. The main advantage of our technique is its ability to reduce the variability in weight changes, brain edema, and infarct volume, and to minimize procedure-related deaths. After confirming a lack of response to tail pinch in a male 300 to 350-gram Sprague Dawley rat, place the animal on a 37 degrees Celsius heating plate and use a rectal probe for monitoring the core body temperature.
Apply ointment to the animal's eyes and shave the hair from the neck. Then, disinfect the exposed skin with 70 percent alcohol chlorhexidine, and cover the rat with the sterile surgical drape before performing the occlusion according to standard techniques. To obtain the neurological severity score, place an occluded rat onto a flat surface and allow the animal to move about freely.
After one minute, gently pull the rat backward by the tail and grade the animal's gait response. To measure the infarct volume, scan the prepared stained brain slices at a 1, 600 by 1, 600 dots per inch resolution and crop and standardize the scale for all of the images. Then, load the standardized images into an appropriate image analysis software program and use a tracing tool to measure the area of marked pallor in six consecutive two-millimeter slices to determine the infarct size as a percentage of the whole brain slice.
30 to 33 days after the surgery, transfer the animals to a closed, quiet, light-controlled room in individual cages, and place a bottle containing 100 milliliters of 1%sucrose solution into each cage for 24 hours. The next day, remove the bottles as well as the food and drinking water for 12 hours. At the end of the deprivation period, place one bottle containing 100 milliliters of 1%sucrose solution and one bottle containing 100 milliliters of tap water into each cage for four hours.
Then, record the volume of the sucrose solution and water consumed to allow calculation of the affinity to sucrose preference for each animal. For Porsolt's forced-swim test, 30 to 33 days after the surgery, first place each rat in a vertical plexiglass cylinder containing 80 centimeters of 25 degrees Celsius water for 15 minutes. At the end of the habituation period, allow the rat to dry for 15 minutes in the heated enclosure at 32 degrees Celsius before returning the animal to its home cage.
The next day, return the rat to the cylinder for a final five minutes, recording the five-minute test to allow calculation of the total duration of immobility during that period. Histological analysis reveals a statistically significant infarct volume as a percentage of the total brain volume post-MCAO when compared to animals in the sham control group. A statistically-significant brain edema is also observed in the experimental animals compared to the sham control group.
MCAO animals demonstrate lower neurological performances compared to sham-operated animals at 50 minutes, 24 hours, and seven days post-surgery. MCAO rats also consume a significantly lower amount of sucrose and exhibit a longer immobility during Porsalt's forced-swim tests compared to the sham-operated animals. This is an important model for behavioral stroke studies and could serve as a tool for assessing future therapies or providing critical preclinical data on the efficacy of therapeutic substances.
