The overall goal of this procedure is to assess functional deficits and recovery after cervical spinal cord injury. This is accomplished by first training the animals to get comfortable during handling and within the testing environment. Next, the rats are assessed for their ability to alternate for limb stepping.
Then the animals are tested for their ability to respond to center of gravity changes and to adjust their posture appropriately. Finally, four paw and four limb function are analyzed during a fine motor handling task, which requires the animals to eat pasta. Ultimately, results can be obtained that show differences in deficit after cervical spinal cord injury, which allows animals to be grouped based on their functional deficits that correlate with their anatomical differences.
The main advantage of the methods described here over other existing methods, such as footprint gate analysis, is that it's much simpler to use and it doesn't require expensive equipment or software. These methods provide insight to functional recovery after spinal cord injury, but they can also be used for other disease models such as Parkinson's disease, stroke, or other neurological disorders that cause motor deficit Animals used in this study will have a C3 C four lateral spinal cord injury two to three weeks prior to surgery. Acclimate the animals to handling and the testing environment that is covered with sandpaper to prevent slipping, bracing, or dragging of four limbs.
The four limbs step alternation test determines whether the animal all donates the use of its forelimbs, and this test can be performed daily before beginning, ensure that the animal stops struggling and that its muscles are relaxed. Next, hold the animal on a tabletop in a forelimb only weight-bearing stance or a wheelbarrow position with its body at nearly 90 degrees to the table. Once the animal appears relaxed in your hands, push the animal forward to move along the surface of the tabletop.
Determine and record whether the rat alternates use of the forelimbs while moving across the surface. Repeat this test at least twice while holding the animal with one hand and then twice while holding it. In the other hand.
If the rat successfully demonstrated step alternation, perform the test again by introducing a five second wait after the first step to determine if the pattern of alternation remains after a delay. Note the results and record them in the score sheet. The postural instability test examines the distance it takes for the animal to take a step with the forelimb to regain its balance.
Similar to the alternation test, the animal should release tension in its muscles prior to beginning using a tabletop covered with sandpaper, allow the animal to adopt the wheelbarrow position with its body at a nearly 90 degree angle to the table, lightly restrained one four limb against the animal's torso and align its nose with the zero line as viewed from above. Next, move the rat forward. This will shift the animal center of gravity and stimulate it to step to regain its balance.
Determine the new position of the nose after the rat steps twice and use the average of these two steps as the distance needed to trigger a step. Record the results on the postural instability test score sheet provided in the text protocol. Test each limb five times.
The pasta handling test will measure the time it takes to eat a standard piece of pasta and the paw preference during each session, one to two weeks before surgery, give the rat five to six pieces of 4.0 centimeter strands of thin spaghetti every day in its home cage to prepare it for baseline measurements. Next, put the rat in a testing chamber and place pieces of pasta near the front of the chamber where the use of its for limbs can be easily observed. Then record the time it takes to eat each piece of pasta.
Allow the animal to eat at least three pieces per session. If the animal pauses while eating, then pause the timing. Also restarting the timing when the animal resumes eating.
Once the rat has shown a consistent pasta eating time for at least three days during training, the animal is ready for baseline recording to collect data place pieces of pasta on the floor near the front of the chamber using the pasta handling score sheet provided in the text protocol record the time the rat takes to eat a piece of pasta, which for limbs were used and the positions of the for limbs as shown here. Using the forelimb step alternation test. After surgical spinal cord injury, only 50%of the animals were able to alternate the use of their forelimbs and 50%of the successful alternators were able to alternate use of their limbs After a five second delay.
Correlating the findings with anatomical studies revealed that the alternators have a significantly higher area of spared cortical spinal tract and dorsal column on the contr lesional side, indicating that the for limb step alternation test can predict the severity of the lesion as well as the unilaterally of the lesions. The use of this test will allow for the separation of groups based on lesion severity. For example, four limb locomotor scale measurements can be separated based on lesion severity for clearer results.
Here, data collected from the postural instability test show changes in ips, lesional and contra lesional limbs after spinal cord injury. Unilateral lesions of the spinal cord can cause changes not only to the impaired and or affected limb, but also to the non impaired limb. Using the postural instability test after cervical spinal cord injury demonstrated that there was a significant effect from lesion status.
In addition for limb displacement was significantly longer in the IPS lesional side of injured animals compared to animals with sham surgery. While the displacement distance on the contral lesional for limb was much shorter compared to sham animals as shown in this figure at chronic time points after injury, both sham animals and those from the lesioned group took similar amounts of time to eat pasta. However, while both sham animals and lesioned alternators ate pasta using both paws during the test period, a smaller percentage of severely injured non alternators were able to use their ips lesional forelimbs to eat pasta.
These tests can be performed in less than five minutes per rat if performed correctly. The behavioral tests described here can be used in combination with other existing behavioral tests, such as foot fault cylinder paw preference tests, and for limb locomotive scale tests to fully assess the animal's Ipsy lesional, as well as contral lesional for limb function after cervical spinal cord injury.
