This video demonstrates a procedure to measure ground reaction forces and hind limb kinematics in freely behaving rats. Rats are first trained to consistently cross a runway for a food reward. Reflective markers then placed at key topographical anatomical landmarks for the hind limb.
Video recording and ground reaction force data are collected while the animal is locomoting for the food reward. The reflective markers are digitized and the data are processed retrospectively. Qualitative and quantitative differences in locomotor abilities are observed in rats of a variety of disease models.
Hi, my name is Aubrey Webb. I'm an assistant professor with the Faculty of Veterinary Medicine and with the Hotchkiss Brain Institute at the University of Calgary in Calgary, Alberta, Canada. The main advantage of this technique over existing methods like locomotive rating scales and other endpoint measures is that this technique provides sensitive, objective, quantitative data to help differentiate subtle differences in between groups of locomoting rats.
This method can help answer key questions in the neuroscience and orthopedic field, such as what are the effects of a potential therapeutic. The implications of this technique extend toward therapy for various models of disease because the likelihood of claiming no effect when there is one is limited. Using this technique though, this method provides insight into rat locomotion.
It can also be applied to other systems such as various models of neurological and orthopedic disease in a variety of species of animals. Generally, individuals new to this method will struggle because biomechanical analysis require substantial time and patience, and because advanced knowledge of biomechanics is required to troubleshoot data analysis, this methodology has been used in multiple species for several decades, though only recently has it been available for use in rodents because of commercially available Equipment. The first step in measuring rat locomotor abilities is to train each animal to cross a flat surface enclos runway.
Upon receiving rats from an appropriate animal supplier, animals should be acclimated to their new home. For one week. During this acclimation time, place several Cheerios into the rat's cage.
Daily animals are food restricted to their maintenance energy requirements to prevent obesity and ensure motivation to perform the task thereafter. Handle each animal for 10 to 15 minutes daily for one week during the same time period, place each animal into the runway with Cheerios located at either end. When the animals become familiar with their environment, they'll begin eating the Cheerios.
Once the animal is comfortable in eating Cheerios within the runway, it must be operantly conditioned to run the length of the runway for a food reward. This is accomplished by tossing one fourth of a cheerio to the opposite end of the runway where the rat is positioned. Once the rat eats this cheerio place another one fourth cheerio at the other end of the runway.
Repeat this until the rat consistently moves along the runway at a constant trotting velocity To eat the cheerio without galloping or bounding over conditioning of the animals to this task can lead to galloping and bounding gates, which biomechanically are more difficult to interpret. Now let's see how to conduct joint position. Marking four.
Limb kinematic analysis is unreliable due to the severely crouched posture and subsequent skin movement artifact imposed by placing skin markers on the four limbs. Instead, kinematics of the forelimbs must be achieved using x-ray cinematography or fluoroscopy. Therefore, this video will solely describe Hindin joint position marker placement.
Once the animal has been anesthetized, shave the hind limbs and the dorsum to the level of the iliac crests. Then place the animal external recumbent and place its hind limbs in an approximate standing position using firm packing foam to support it with a non-toxic permanent marker mark. The cranial most portions of the iliac crests, the greater trant of the femur, the lateral tibial protuberance, the tarsal joint, and the distal and lateral aspect of the fifth metatarsal.
Daily highlighting of the previously marked anatomical landmarks is required as rats will slowly remove the markers through natural grooming behavior. Now let's see how to record data. Place the calibration volume within the predetermined area of the runway.
A single frame of the calibration volume within the runway from each of the cameras is captured. Before initiating a recording session, examine the view from each camera. Ensure that the field of view is the same from all four cameras, and that it includes the force plate in the center, and a length of runway sufficient to capture two strides.
All calibrated marks along the length of each of the polls are digitized. This calibration step is critical prior to collecting data. If calibration is not performed accurately or does not occur immediately prior to a recording session, all resulting data will be inaccurate and unusable.
Only once a satisfactory error in digitizing is accomplished, proceed to collecting locomotive data immediately before placing the animal in the runway. Record its weight and adhere conical reflective skin markers to the predetermined felt marks made on the hind limb topographical landmarks. Once the markers are placed on the hind limbs, sit down at the computer and have in hand the event marker attached through the Vicon motor system.
Using the calibrated file as a template makes several files before recording. Typically 25 to 30 files are needed to ensure that sufficient numbers of runs, meaning the inclusion criter, and velocity are collected. Once the files are created, begin collecting data.
First, prompt the rat to shuttle within the runway by throwing one fourth pieces of Cheerios at either end of the runway with careful coordination and timing. The event marker is triggered at initiation and just prior to the rat successfully completing a pass along the runway. After examining the crude ground reaction, force tracing and after recording whether the left or right limbs, hit the force plate, save and close the file.
Equal numbers of left and right limb force plate hit should be recorded. The process of recording the data from a given run is repeated until sufficient numbers of runs have been recorded. Now let's see how to analyze the data from the runs upon completing data collection for kinematics and ground reaction forces, each run from every animal needs to be evaluated for speed using vicon modus software, two relatively fixed markers.
For example, the wings of the ileum can be used to evaluate a virtual point between the markers. The velocity of this virtual point in the horizontal direction can be calculated in doing so, only runs where the animal is moving between 60 to 90 centimeters per second are included. For analysis, a minimum of 10 runs are needed.
Five runs where the left limb makes contact with the force platform, and five runs where the right limb makes contact with the force platform. Once the acceptable runs are identified for each animal, digitization of the remaining skin markers must be completed to compensate for skin movement. Artifact over the knee estimation of the knee position is calculated using triangulation, hip, knee, and H joint angles.
Velocities and accelerations are determined stance and swing times can also be evaluated. The simultaneously collected ground reaction force data is analyzed using the same software, a variety of variables for forces in each of the three orthogonal directions can be calculated. The simultaneously collected ground reaction force data are analyzed Using the same software, a variety of variables for forces in each of the three orthogonal directions can be calculated.
Now we'll show some representative results to represent the utility of this form of locomotive analysis. Kinematics and ground reaction forces were determined for young, middle-aged, and geriatric female wister rats. From this analysis, age-related differences were found for female wister rats.
In particular, ground reaction force analysis demonstrates that geriatric rats have reduced four limb braking ability and tend to use their hind limbs more for lateral stabilization compared to the other groups of animals, kinematic analysis did not reveal any statistical differences between each group. Though demonstrates that kinematics can be readily recorded from virtually any age of rat While attempting to perform this procedure. It's important to weigh the animal immediately prior to or immediately after collecting data and to carefully calibrate the system prior to data collection following this procedure.
Other methods like whole animal clinical electrophysiology can be used to answer additional questions. For example, are the kinematic and ground reaction force findings related to neural regeneration or some other broader form of neuro anatomic plasticity in, for example, peripheral nerve regeneration? After watching this video, you should have a good understanding of how to collect ground reaction force and hind limb kinematic data from freely behaving rats using commercially available equipment.
