The overall goal of the behavioral test presented here is to detect functional deficits in rats after traumatic brain injury. Four different tests will be presented that detect deficits in either neurological or cognitive function. These methods can help answer key questions in the field of brain injury research, such as which areas of the brain are damaged after an injury or whether or not an experimental treatment is effective.
The main advantages of these techniques is that they are well established in many laboratories and proven to be reproducible, as well as sensitive to different types of brain injuries, such as blast injury and stroke. These techniques contribute towards further understanding of how traumatic brain injury affects neurological and cognitive functions, as well as testing of experimental treatments in rats. Generally, individuals new to the method will struggle because of inadequate rat handling experience and not allowing enough time to acclimate the rats prior to training and testing.
This is needed to calm the rat in order to make testing time more effective. Begin the training with the forelimb flexion test by lifting the rat by the tail and holding it about six to 12 inches above the table surface. Observe whether the rat extends or flexes forelimbs.
Score the presence of flexion with a one or the absence of flexion with a zero on the score sheet. Next, for the hindlimb flexion test, lift the rat by the tail again and hold about six to 12 inches above the table surface. Observe whether the rat extends or flexes its hindlimbs, and score the presence of flexion with a one or the absence of flexion with a zero.
For the next test, lift the rat by the tail and slowly lower it toward the edge of the table, until the nose is about 10 centimeters from the edge. Move the rat slowly toward the edge without letting the whiskers touch the edge. Observe whether the rat reaches and extends forepaws towards the table.
Score the presence or absence of extending forepaws. Next, for the contact triggered placing test, hold the rat with body and hand parallel to the table edge and forelegs free. Slowly lower the rat toward the edge of the table until the whiskers on one side touch the edge of the table.
Observe whether the rat extends the forelimb on the same side as the whiskers that are touching the table toward the table edge, as soon as the whiskers touch. Score the presence or absence of reaching toward the table. Finally, for the hindpaw grasping reflex test, hold the rat in one hand with the thumb and index finger around the chest under the forelimbs.
Gently touch the palm of each hindpaw with a forefinger. Observe whether the rat grasps the forefinger and score the presence or absence of grasping. 24 hours after surgery, begin the beam-balance testing by placing the rat on the beam and starting the timer.
Observe the rat closely for 60 seconds and record the score on the worksheet. Allow the rat to rest briefly between trials, either on the experimenter's arm or in the home cage. Begin by placing the rat in the goal box for one minute.
After one minute, remove the rat and start the trial by turning on the light and white noise. Place the rat on the beam at the location of the peg hole closest to the goal box, and allow the rat to enter the goal box. When the rat's front feet cross the threshold of the goal box, immediately turn off the light and noise sources to note the end of the one trial.
Allow the rat to rest in the goal box for 30 seconds between each trial. Repeat the beam-walk training once at each peg location and from the starting position. Insert the pegs in the holes and run one complete beam-walk with the pegs in place.
Then, using a stopwatch, time each trial and repeat until the rat has completed three trials under five seconds. 24 hours after surgery, test the rat on the beam-walk using the complete beam with pegs in place. Visual cues are located around the room to provide landmarks for the rat to learn and remember the location of the hidden platform.
Begin the first trial by opening the video tracking software and loading the correct protocol, including the water maze map with platform locations, length of swim, number of trials, number of animals, and treatment groups. Place the platform in the assigned location and check that it matches the map in the software. Then, place the rat in the tank facing the wall at the assigned location and immediately start the timer of the tracking program and/or stopwatch.
Allow the rat 120 seconds to find the platform. When the rat finds the platform, stop the stopwatch and record the time on the worksheet. Note, if the rat fails to find the platform, lead it to the platform by hand and record 120 seconds.
Allow the rat to remain on the platform for 15 seconds between trials. After trial two, place the rat in the heated enclosure for four minutes. Finally, move the platform to the second location and check that it matches the map in the software.
Repeat trial one and two procedures until all four starting location and platform pairings are completed. Results of the neuroscore procedure demonstrate both the potential for false positive at day zero, which is prior to surgery, and the sensitivity of this test to detect small differences in the TBI group, such that day zero is different from days one, two, and three. The beam-balance results demonstrate the sensitivity of the beam-balance test to deficits shortly after injury and at a time point longer after injury.
Results from the beam-walk test indicated deficits early after injury. However, by six months after injury, there are no significant differences between the groups, suggesting healing occurred in the injured group. Further, deficits in working memory are shown at three months after injury, while both working and reference memory showed deficits at 12 months after injury.
It's important to acclimate the rats to handling and to pre-train for neuroscore, beam-walk, and beam-balance tests. Once mastered, the short neurological assessment can be done quickly and without any specialized equipment. After the neuroscore, beam-balance, and beam-walk procedures, different versions of the water maze may be used, as well as other behavioral paradigms, like the novel object recognition test, to answer additional questions pertaining to learning and memory.
After watching this video, you should have a good understanding of how to detect neurological and cognitive deficits using behavioral measures. Acclimation to handling, pre-training, and consistency when conducting behavioral experiments are all critical.
