Hello, I'm Dr.Jackie Vander Loot. Hi, and my name is Brian Room. Today we will be discussing a sensitive analysis of the cylinder test paw.Dragging.
The cylinder test is a test of four limb motor function in rodents. In this test, mice explore a plexiglass cylinder by rearing and using their four paws for postural support against the cylinder wall. Examining four paw use while the mouse explores the cylinder allows us to determine if mouse has motor deficits.
Specifically, we compare the use of each four paw before and after stroke. Following a stroke, the four paw contralateral to the injured hemisphere is used less often. This change in use of the affected four paw versus the unaffected four paw is a measure of four limb asymmetry.
Currently four limb asymmetry analysis is used in mice and rats after large ischemic injuries such as middle cerebral artery occlusion as a behavioral determinant of stroke. In contrast to large injuries, four limb asymmetry is not observed in mice with small focal ischemic injuries. This may be due to the fact that four limb asymmetry analysis of the cylinder test is sensitive to large injuries, but is not useful in detecting smaller injuries.
Small focal ischemic injuries such as photo thrombosis and endothelium one induced ischemia are becoming more popular in most models. This highlights need for functional behavioral assays to assess these smaller cortical injuries. In my lab, Brian Room has developed a novel analysis of the cylinder test we call paw dragging.
By examining how the mouse four paws contact the cylinder wall following an endothelium one ischemic injury, we observed a novel behavior with the affected paw. A paw drag uninjured mice will contact the cylinder wall with either one paw or both paws while rearing the mouse plants its four paws firmly against the wall for support while exploring and then uses their paws to push off from the wall. When dropping back to all fours, following an endy one ischemic injury, the affected paw does not provide firm support despite making contact with this cylinder wall.
Rather following contact the palm lifts off and the digits drag along the cylinder wall before releasing entirely. This paw dragging motion occurs either with the paw moving in a horizontal direction toward the body's midline or in a vertical direction toward the floor. We have previously quantified pod dragging behavior in mice following an inphi one ischemic injury and shown increased pod dragging behavior by the affected limb up to two weeks post ischemia.
Furthermore, our data showed that pod dragging behavior correlates with damage to the four motor cortex. In this video, we will show how to set up the cylinder test and analyze pod dragging behavior in recorded videos. The cylinder test is easy to set up and conduct and has low cost overhead.
It is necessary to have a table with a hollowed center, which is covered with a sheet of plexiglass. This allows us to film the most from below. The plexiglass cylinder is placed on the tabletop.
On the bottom of the tabletop, four black lines are drawn to indicate the position of the cylinder. This serves as a guide so that the cylinder can be removed and replaced between trials without affecting its position. In the frame of the video as well, the marks are placed on the underside of the table so that cleaning the table with 70%ethanol will not erase the ink.
In order to film the mouse from below using a camera and tripod, we include a mirror at approximately a 45 degree angle below the tabletop. This allows us to avoid the complications of building a separate apparatus to hold the camera in place below the tabletop. The mirror is held in place with support brackets as shown here and here.
The size of the brackets and the size of the mirror will depend on the dimensions of the space between the table legs. The camera and the tripod are placed at a distance such that all of the cylinder's wall can be seen in the field of view. This distance is typically no more than a few feet, but can vary due to the angle of the mirror, height of the tripod and the zoom capabilities of the camera.
To conduct the test, mice are loaded from the top of the cylinder before filming. Cue cards are used to indicate which mouse is performing. This can include the mouse number or a code name so that the person grading the cylinder videos will be blind to the treatment.
The date of filming and trial number can also be included on the card. Mice are filmed until they have reared 20 times rears. Counted in this way must involve both paws leaving the floor arrears not counted.
If the mouse touches the wall with one paw while the other remains on the ground, cylinder videos are analyzed using the VLC media player. Although any media player with playback speed modulation would be fine to use, pod drags can be counted using mechanical tallies or by writing a tally on paper. We record tallies on paper and later transferred the data to a spreadsheet.
Video playback at 0.5 times normal. Speed is recommended in order to be able to tally effectively. At times, pausing and replaying at a lower speed may be necessary.
Note that a mouse may also move its body around the cylinder during a rear making more than two contacts. These contacts are tallied, one for each left four pot touch and one for each. Right, four pot touch paw drags are counted as a percent of the total number of paw touches with the cylinder wall For each paw paw touches are assessed by counting the number of times the mouse makes contact with the cylinder wall with either one or both four paws.
Note that a mouse may also move its body around the cylinder during a rear making more than two contacts. These contacts are tallied, one for each left four po touch and one for each. Right four po touch quantifying paw dragging behavior is distinct from quantifying normal paw touches paw dragging can occur with a vertical drag or a horizontal drag paw.
Dragging often involves a slight tremor in the affected paw as it leaves the cylinder wall suggesting weakness and lack of motor control. Variations on the stereotypical paw drag are often seen with the affected paw. One example is an incomplete paw drag where the palm lifts away the digits drag against the cylinder, but both paws ultimately break contact with the wall at the same time while assessing paw dragging behavior, it is important not to mistake a mouse's lateral exploratory movement around the cylinder for a deficit in four limb motor function.
During lateral exploration, the mouse moves laterally around the cylinder and the trailing four paw is pulled following the direction of the mouse's torso. For instance, the mouse rears and touches the wall with both paws, repositions one of its paws, and then moves laterally toward the reposition of paw. When this occurs, the trailing paws then dragged along the wall until it is replanted for support.
Twisting of the mouse's torso is another cue of lateral exploratory behavior. This behavior is distinctly different from paw dragging as paw dragging does not involve lateral movement around the cylinder wall during or directly before the drag. Our studies have examined paw dragging after a small focal ischemic injury in the four limb motor cortex.
Here we show that paw dragging behavior with the affected four P peaks at one day post-stroke and declines to seven days post-stroke, where it plateaus and remains elevated up to four weeks post-stroke as the deficit appears to be prolonged. This will allow researchers to study the effects of chronic stroke after a small cortical injury. Moreover, stroke treatments can be studied in the short or long term in mice with small cortical injuries.
Our new PO dragging analysis of the cellular test has potential as a behavioral assay for focal ischemic stroke in mice such as those induced by photo thrombosis or endophilin. One in mice with injuries such as middle cerebral artery occlusion, where for lam assymetry occurs most of the time. POD dragging can be used to determine the extent of smaller injuries and such.
Experiments as pod dragging is elevated up to and including 28 days post-stroke. It has potential to be used in various short and long-term stroke treatment studies.
