The overall goal of this procedure is to assess the progressive neurological impairment in mice with chronic demyelinating diseases. We accomplish this by offering recommendations of testing parameters suitable for studying long term neurological disability in the Theilers virus model of progressive multiple sclerosis, using the Rotarod test. This procedure provides a baseline against which to assess the relevance of mouse model to progressive demyelination and it's usefulness for testing therapies aimed at treating progressive neurological condition such as multiple sclerosis.
The advantage of this optimized test over the traditional visual scoring system is that it generates an objective variable to quantify the long term effect of therapies and experimental procedures on motor functions. Demonstrating this procedure will be Darlene Royce, a technician from the laboratory of neuroimmunology at Dartmouth. In order to assess baseline balance coordination and motor control, start the adaption protocol five days prior to TMEV infection by first allowing the mice to acclimate to the testing room for at least 30 minutes.
While the mice are acclimating, preset the Rotarod with the minus five DPI training parameters. Then, pickup a mouse by the tail and place it on the rod facing away from the operator. If the mouse falls or jumps, place it back in its lane on the Rotarod until all mice are in position.
After loading all four mice, press the enter button to start the experiment. Ensure that the timers start automatically, and observe the rotations per minutes on display for each lane. Then, as each animal falls from the rod, record the speed of the rod at the time of the fall, as well as the duration of time the animal remained on the rod.
After all the mice have fallen, use a tissue to remove any fecal boli and urine from the rod as the presence of urine and fecal material may affect the ability of mice to properly grip the rod. After a three minute rest give the mice a second and then a third trial in which the maximum time per single trial is 240 seconds. Administer a total of three trials during each testing day.
Finally, return the mice to their home cage. On days negative four, negative three, negative two, and negative one PI, preset the Rotarod with the appropriate training protocol parameters and repeat the Rotarod experiment. Begin by moving cages containing four to six week old female SJL mice from the rack to a comfortable working space.
Use ear punches to mark the mice to allow for individual evaluation of clinical and histological disease. Next, draw 30 microliters of TMEV infecting stock in PBS into a 29 gauge insulin syringe needle. Prepare the anesthesia gas machine by ensuring the presence of adequate amounts of oxygen and isoflurane for the duration of the procedure.
Place the animal into the induction chamber and seal the top. After a few minutes, remove the animal from the chamber, and test the mouse by pinching the foot pad to ensure adequate anesthesia. Next, clean the injection site with 70%isopropyl alcohol and inject the 30 microliters of TMEV infecting stock into the right cerebral hemisphere by freehand injection.
Finally, return the mouse to its holding cage once it is fully alert and mobile. On day seven post infection, preset the Rotarod with the appropriate experimental parameters, and repeat the complete Rotarod training excercise with the appropriate experimental protocol parameters. After the third trial on each day, weigh each mouse and make a note of the body weight on the data sheet.
Test the mice twice a week for the following six weeks in the same manner. After six weeks, test the mice once a week with the same experimental protocol for an average of 150 experimental days. Next, analyze the raw data, and express as a neurological functional index, or NFI.
To determine the individual NFI value, calculate the baseline performance threshold of each mouse as the mean of all running times from 15 to 45 days post infection. In order to allow for the evaluation of motor performance as a result of progressive demyelination and exclude any contributing deficits due to early encephalitis. Then, calculate the NFI value as the mean of the three most recent average running times divided by the baseline performance threshold of the mouse.
Finally, calculate an adjusted NFI by dividing the NFI value by the average NFI value obtained by the sham treated group on that specific day. This figure shows the TMEV infected mice display significantly increased neurological deficits over time when compared with control mice. Chronic infection with two different strains of TMEV negatively affected the running times of the mice.
Both groups of TMEV infected mice had significantly lower NFI values than those of sham mice. Furthermore, there was no difference comparing disability progression in mice infected with the BeAn strain, and those infected with the DA strain at all time points. Following this procedure are the traditional visual scoring methods, like the righting reflex test, can be performed in order to rapidly assess general health in mice.
After its development, this technique paved the way for researchers in the field of multiple sclerosis and neurodegenerative diseases to test the effectiveness of therapies and other intervention in delaying progressive disability. After watching this video, you should have a good understanding of how to evaluate the long term progressive disability in mice. This procedure is not only useful in detecting the progressive neurological disability in the Theiler virus model, but is also useful in uncovering impairments in other mouse models of neurodegenerative diseases.
