The overall goal of this procedure is to quantify changes in tactile sensation at acute and chronic time points following spinal cord injury using two distinct Von Fry testing methods. This is accomplished by first acclimating rats to a plexiglass chamber for up down von fry testing and to being restrained in a towel for dorsal von fry testing. The second step of the procedure is to apply tactile stimulation to either the plantar or dorsal surface of the hind paw using von Fry monofilaments.
A positive response occurs when the rat quickly lifts the paw up away from the monofilament. The final step of the procedure is to determine the tactile sensory threshold of the hind paw by quantifying the number of positive responses or paw withdrawals to each force that was applied. Ultimately, results can be obtained that show both the up, down and dorsal von fry testing techniques are sensitive to declines in tactile thresholds that occur after moderate to severe spinal cord injury.
Today we'll be demonstrating two techniques to test the tactile sensation of the paw of rats with spinal cord injury. These techniques will help us determine future treatments for people with debilitating pain and spinal cord injury. At least two thirds of people with spinal cord injury go on to develop severe debilitating neuropathic pain.
For us as researchers, it's important to use accurate, sensitive, and valid behavioral testing techniques so that we can identify the cellular and molecular mechanisms for future therapeutics. Though this method can provide insight into sensory recovery after mid thoracic spinal cord contusion injury, it is also useful for other types of pathologies and injuries that affect tactile sensation. Visual demonstration of this test is critical because the in the assessment of sensation relies on the interpretation of a rat's Paul withdrawal response.
For new users, this can be difficult to determine whether or not the response is indeed due to a tactile stimulus rather than proprioceptive or visual cues. After watching this video, you'll be able to gentle and acclimate animals to the testing environment. Apply von fry monofilaments to the dorsal or planter surface of the hind P, interpret a A positive paw withdrawal response.
And finally, determine the tactile sensory threshold in animals. First, present the rat with fruit cereal in its home cage so that it recognizes and seeks the food reward. Once the rat is comfortable eating cereal in his home cage, place the rat in the testing chamber with three more pieces of fruit cereal.
Provide additional pieces of fruit cereal as they are eaten. A gentle rat that is ready for testing will eat nearly continuously for 10 minutes and will not be distracted by hand movements under the wire floor or subthreshold monofilament application to the paw. Place a gentle rat in the testing chamber along with two or three pieces of fruit cereal.
Roll a dye to determine which hind limb to start testing first with even numbers representing the right paw and odd numbers representing the left while the rat is eating. Begin the testing session using the 5.18 monofilament. If the rat freezes CROs or does not eat, wait for the rat to become calm.
Before proceeding with the testing, touch the monofilament to the L five planter dermatome of the hind paw between the foot pads until it bends. This smooth and steady application should take about one second before withdrawing the monofilament. A positive response occurs when the rat quickly lifts the paw up away from the monofilament.
Watch for and record additional evidence of awareness of the monofilament touch such as vocalization, looking at or licking the tested paw or twitching of the ears. If there is a positive response, apply the next lowest monofilament. If there is no response, move up to the next higher monofilament, allowing 30 seconds between each monofilament application for the sensory receptors to come back to baseline firing before retesting.
Be vigilant for invalid trials in which the hair physically displaces the paw. In such trials. The hair provides both tactile and proprioceptive feedback and such data must be excluded from further analysis.
Continue applying higher or lower monofilaments to the hind paw based on positive or negative responses. For a total of 10 trials loosely wrap a rat in a small towel so that the hindquarters are exposed. Position the rat over a table with the hind paws lightly resting on the surface.
Bracing your forearm on the table edge may reduce variability between rats, though a gentle rat that is ready for testing will not resist being held in the towel for several consecutive minutes, you may have to rewrap the rat during the testing of both hind paws. Again, roll a dye to determine which hind limb to start testing. First loosely wrap a gentle rat in the towel leaving the hindquarters exposed.
After the rat is calm for at least 10 seconds, apply the 4.56 von fry hair perpendicularly to the dorsal surface in between the first and second metatarsal about one centimeter proximal to the joint in a smooth, steady manner over a one second interval until it bends. A positive response occurs when the rat quickly moves the paw posteriorly or externally before flexing the hind limb. A negative response occurs when there is no movement of the hind paw in response to the monofilament or the movement is not directly related to von Fry hair application.
Apply the 4.56 monofilament twice more with at least 30 seconds between applications in order to prevent adaptations. If there are fewer than two positive responses to the 4.56 monofilament, apply the next largest in the same manner The test is completed when a monofilament elicits at least two positive responses out of three trials I here responses from both von Fry hair testing approaches are tallied to derive tactile thresholds for each hind paw. Tactile thresholds from dorsal von fry hair tests with acute SCI rats show excellent fidelity with up down von fry.
Hair thresholds derived at chronic times declines in tactile thresholds derived from plantar or dorsal tests occur for graded SCI with this hypersensitivity manifesting in the most severe SCI rats as early as seven days after SCI and lasting for the duration of the experiment. Importantly, the development of below level allodynia is an all or none response that occurs when there is a loss of more than 60%of the white matter at the lesion epicenter Once mastered. This technique can be done in about 15 to 20 minutes per rat when done properly.
To use these techniques accurately, it's important to control confounding variables in the environment, which can introduce variability in behavior factors such as testing at the same time of day and testing in the same temperature and humidity controlled room will help minimize variability. In addition, rats vocalize ultrasonically so it's important that we keep them outside of the testing room until it is their time to be tested. We also administer sugared cereal to stabilize sensory thresholds.
We find that by giving the cereal, the thresholds are stable, more stable than when we don't give the cereal. This behavior is likely due to anticipatory behaviors and the threat of pain, which activates fear and emotional centers and creates a hypervigilant and catastrophizing behavior in the rats.
