The overall goal of this procedure is to analyze mechanical allodynia of mice with a chronic constriction injury using an automated gait analysis system. This method can help answer key questions in the pain research field by measuring sensory function after peripheral nerve injury in rodents. The main advantage of this technique is that the analysis system is quick and easy to use and it also automatically scores various gait parameters.

For this study, house male ICR mice weighing 20 to 25 grams under standard conditions for at least one week. Select mice based on their appearance and behavior. Do not use mice that exhibit abnormal appearances or behavior such as hair loss, scarring and less movement.

Using the selected mice, starting five days before the surgery collect automated gait data on a daily basis to have a baseline measurement. One day before the surgery perform the Von Frey test with a one gram filament on a hind paw to get a baseline response. Balance the responses between the injury group and the sham group.

On each test day bring mice to the behavioral test room and acclimate the mice in their own home cage for at least 30 minutes before the test. Every other day after the surgery, measure mechanical allodynia using a Von Frey test. Put the mouse in a clean transparent acrylic box on a metal mesh floor and let it acclimate to the box for 30 minutes.

Next, gently apply a one gram Von Frey filament to the plantar surface of the ipsilateral hind paw until the filament bends. Repeat this pattern of stimulation 10 times observing for paw withdrawals. Wait at least 10 seconds between stimulations.

Record the number of withdrawals and score their frequency for analysis. Every other day, also perform the gait analysis test which must be done in the dark. Again, acclimate the mice to the test room in their home cage for at least 30 minutes.

To set up the gait analysis device go to the software control. Under the setup tab set the walkway length to 30 centimeters, set the maximum run duration to five seconds and set the maximum run variation to 50%Next, select a registered camera from the setup tab of the program menu and then select open acquisition from the acquire tab. Following the status message, click the snap background button to take a background picture of the empty walkway.

Now place a mouse onto the walkway and allow it to acclimate to the device for 10 minutes. The apparatus should always be washed with alcohol between mice. After the mouse has acclimated to the device, click the start acquisition button.

The recording starts automatically according to the movement of the mouse. When the mouse clearly walks across the walkway, the program will automatically classify the compliant runs with a green icon. If the software fails to detect the footprints, a red icon will light up.

After collecting at least five compliant runs select the classify runs option from the acquire tab of the program menu. Select which run should be analyzed and click the auto classify button. The results are automatically saved.

To view the results click on view run statistics from the analyze menu. The Von Frey test and automated gait analysis were used to assess mice that underwent the CCI surgery on 10 consecutive days post-operatively. The frequency of paw withdrawal increased two days after the surgery and was sustained for 10 days.

This effect could be significantly reduced by daily post-operative injections of gabapentin. Using the gait analysis software the size of the left and right hind paw prints could be compared. Normally the areas are the same which is indicated by a 50%value.

The CCI surgery had a significant effect on paw print and that effect could be significantly reduced using gabapentin. The gait analysis software is also able to pick up on the change of single stance durations when the mouse is weight-bearing on just one hind paw. This is calculated and shown as a percent change.

This metric shows the same effect for the CCI surgery and gabapentin treatments. After watching this video you should have a good understanding of how to operate and analyze automated gait analysis systems to investigate the pathophysiology of neuropathic pain in animal models like chronic constriction injury.