The overall goal of these behavioral tests is the assess motor function, as well as spacial learning and memory performance in a novel comorbid rat model of Alzheimer's disease and stroke. This method can help answer key questions in the dementia field, such as how stroke and Alzheimer's disease pathology interact, producing a phenotype of potential enhanced motor deficit and accelerated cognitive decline. The main advantage of these techniques is that they test for a range of brain functions while being easy to conduct, cost efficient, and suitable for repeated testing at later time points.
The implications of this technique extend toward prevention of dementia after stroke because novel preventative compounds can be tested for their potential to ameliorate the comorid behavioral phenotype. To begin, place a mirror at a 45 degree angle beneath a previous mounted cylinder apparatus. Then affix a video camera to a tripod and position it at a distance of approximately 50 centimeters from the apparatus.
Check that the entire diameter of the cylindar in the mirror can be visualized on the camera. Next, choose an acclimatized rat and on a white board write down the animal's number and additional trial information. Place this board in front of the mirror and then press record on the video camera.
Continue by picking up the rat by the base of the tail and placing it in the cylinder. Then secure the lid of the apparatus and remove the white board so that it no longer blocks the mirror. Once the white board has been removed, proceed to film the animal for five minutes, a period that constitutes a single trial.
At the end of this time, stop the recording, return the rat to it's home cage and clean the cylinder with paper towels and water. Then repeat the previously described steps twice more to achieve a total of three trials per rat. Once data has been collected for all rats, import the camera files into a video editing program and proceed to quantify the number of four limb contacts as described in the text protocol.
First, position two shelving units approximately 100 centimeters apart from one another in front of a black wall. Then, use tape to anchor the ends of a previously acquired wooden beam to the shelves so that there is approximately one meter of unsupported wood between them. Next place a video camera on a stand and position it so that the camera captures the entire length of the beam.
Proceed to choose a rat that has undergone a beam walking training session and record it's number and additional trial information on a white board. When finished, tape the white board to the wall behind the beam. Continue by placing the animal's home cage and an environmental enrichment tube which serve as a motivating queue which the rat can immediately enter after crossing plank, at one end of the beam.
Then press record on the video camera. Immediately afterwards, pick up the rat by the base of the tail and place it on the opposite end of the beam without the cage and tube. Record the animal until it sucessfully traverses the full length of the elevated wood, which marks the end of the trial.
If the rat pauses midway across the beam, gently touch it's tail to promote movement. Once the animal completes the task, move it's home cage to the other end of the beam. The change the trial number on the white board and repeat the procedure as previously described.
When all animal's trials have been recorded, import the camera files into a video editing program and analyze the relevant step, limb slips, and fall data as describe in the text protocol. Start by securing a video camera above the center of an empty, circular pool. Then, designate the four quadrants of the tank by placing tape along the rim of the pool and align them properly with the outline of the tank in the tracking software program.
Next, fill the tank with water to a depth of 36 centimeters and then add black, non-toxic paint until the liquid if opaque in order to ensure the platform is not visible below the surface of the water and to better visualize white rats. Once the pool has been prepared, surround it with blank wall surfaces. Then prepare spatial cues by cutting four shapes from different colors of poster board.
Afterwards, attach one shape to each of the walls associated with the north, south, east, and west pool locations. To perform a spatial learning experiment, continue by placing a circular platform in the southwest quadrant of the pool. In the accompanying computer program, check that this structure is aligned with a designated platform area in the quadrant.
Proceed by grasping a rat by the base of it's tail and gently placing it at a designated start site in the water along the pool wall. Once the animal is in the tank, quickly move our of it's sight and signal another experimenter to start the tracking software. Allow the rat to swim until it locates and climbs onto the platform.
At which point, the other researcher should stop the trial on the computer. Let the animal remain on the platform for 30 seconds. If the rat fails to find the platform within the allotted trial time, here 90 seconds, guide the animal to the structure and let it stay there for 30 seconds.
Afterwards, lift the rat by the base of the tail and remove it from the maze. Placing it on a towel draped over an arm. Repeat this procedure three additional times per animal with an inter-trial interval of 20 minutes, so that each rat completes of total of four trials.
Once the spatial learning experiment has been completed, remove the platform from the pool to commence the probe trials. In the software, check that the previous platform position, in the southwest quadrant remains defined. Then introduce a rat into the pool.
Placing it in the water along the wall of the tank. As before, immediately move out of the animal's sight and instruct another experimenter to start the tracking software. At the end of the 30 second probe trial, grasp the animal by the base of the tail, remove it from the tank and then place it one a towel covered arm.
Repeat this procedure one week later. Once spatial learning and probe trials have been performed for all animals, analyze the data as described in the text protocol. Four limb use data from the cylinder apparatus was collected from APP21 transgenic rats three days before and seven and 20 days after stroke induction.
When standardized and compared to the value representing equal four limb use, the red line here, these results indicate that comorbid rats do not statistically demonstrate a four limb deficit. Similar results were obtained for wild type animals with or without stroke and APP21 transgenic rats alone. However, comorbid animals showed differences in locomotion associated motor function.
Beam task data collected for these rats seven days prior to and 21 days after stroke induction reveals a significant increase in the steps needed to traverse the plank, which was not observed in other groups. Morris water maze results also reveal memory deficits in comorbid animals, as evidenced by this graph showing the percentage change in latency, the time animals take to reach the quadrant of the pool where the platform once was between the first and second probe trials. Here APP21 rats with stroke take significantly longer in the second trial to enter the target quadrant.
A difference that was not demonstrated by the transgenic rats alone or wild type animals with stroke. This indicates that comorbid animals have a long term memory deficit. While attempting this procedure, it's important to keep all variables as consistent as possible throughout and across testing sessions and create a stress free environment for the animals.
After watching this video, you should have a good understanding of how to assess motor and cognitive performance in rat models of post-stroke dementia.
