This document is presented in theiler'Experimental design and results in a model of demyelinating disease caused by S murine encephalomyelitis virus (TMEV). Following TMEV infection, susceptible SJL mice develop chronic progressive demyelinating disease similar to progressive multiple sclerosis (MS). These mice exhibit progressive disability, accompanied by loss of motor and sensory function, which can be assessed with a variety of devices and protocols. Among them, the Rotarod performance test is a commonly used behavioral test that is favored because it provides objective measurements. The test generates an objective, measurable, continuous variable that allows for almost perfect inter-rater agreement. However, to achieve inter-laboratory reliability, a variety of test parameters need to be replicated. Therefore, the paper provides recommendations for specific test parameters for the RotoRod test, including the size, velocity, and acceleration of the rod, as well as the amount of training and data processing performed on the animals.
1. Mouse model (TMEV-induced demyelinating disease).
Move the cage containing the 4- to 6-week-old female SJL Jhan mouse from the stand to a comfortable workspace. Label mice (e.g., with ear tags or ear piercings) for individual assessment of clinical and tissue lesions.
Aspirate 30 L of TMEV infection depot (2 x 10 6 plaque-forming units) with a 29-gauge insulin syringe and needlePFU) in PBS.
Prepare the anesthetic gas machine: check the system to ensure that there is enough oxygen and isoflurane** throughout the process.
Turn on the flow meter to 1 L min. Place the animal in the induction chamber and close the top. Turn on the vaporizer to 35% and monitor the animal to lie down.
Remove the animal from the chamber and test the mouse by pinching the paws to ensure adequate anesthesia. Lack of response to strong pinching indicates adequate anesthesia.
Clean the injection site with 70% isopropanol.
Inject 30 L of TMEV infection reservoir into the right cerebral hemisphere by hand-held injection (Figure 1). The injection site is about between the eye and the ear line and away from the midline.
Once the mouse is fully awake and active (usually 3-5 min), return it to the holding cage.
At 3 to 6 months after TMEV infection, mice are euthanized by excretion or cardiac perfusion, depending on the rate of disease progression.
Second, the experimental process
The RotoRod device is used to test mice prior to TMEV infection to familiarize them with the device and assess their normal basal balance, coordination, and motor control abilities.
5 dpi (i.e., 5 days before TMEV infection), start the acclimatization protocol. Allow mice to acclimatize to the environment for at least 30 min in the testing room before performing the Rotorod test.
Make sure both the Rotorod unit and the computer are plugged in and connected to each other (see Figure 1). Preset Rotarod according to the -5 dpi training protocol parameters described in Table 1. Save working documents with date and identification information.
Move the cage of the team to be tested from the shelf to the table next to Rotarod. Typically 6 mice are tested at a time. Pick up the mouse with its tail and place it on the runner with its back to the operator. Repeat this step until the fourth mouse is in place. If there are mice falling or jumping, place them back on the wheel until all mice are in place. Ignore any mice turning around to face the operator.
Once all mice are in place, press the "Enter" button to start the experiment. The observation timer automatically starts timing and displays the revolutions per minute (rpm) for each road.
Record the rotational speed of each mouse when it falls off the runner and the time it stays on the runner. The runner will continue to rotate until the last mouse drops.
After the mouse drops, use a paper towel to remove feces and urine from the runner. Urine and feces may affect the ability of mice to grasp the runner.
After a 3-min break, mice are given a second and third trial. The maximum time for a single trial is 240 seconds. A total of 3 tests were performed on each test day.
Return the mouse to its home cage and return to the shelf. At the end of the experiment, clean the rotarod with soap and water to remove all feces. Wipe the bottom plate with 70% ethanol. Disinfect by spraying the entire machine with chlorine dioxide.
On the days of -4, -3, -2, and -1 pi, preset Rotrod according to the appropriate training protocol parameters in Table 1 and repeat step 21.2 to 21.12。
After baseline data is obtained, mice are subjected to TMEV infection. A 6-day post-infection recovery period is allowed.
The specific parameters of the training and experimental phases are listed in the table, including the starting speed, maximum velocity, acceleration, number of trials, and trial intervals for each trial.