Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain

This protocol is useful to test mechanical sensitivity in the back. It is particularly interesting for longitudinal designs in rat models of chronic back pain, for which the goal is to examine effectiveness of an intervention to decrease back sensitivity to noxious stimuli. The method is simple, accessible, and yields reproducible results in trained experimenters.

It is largely based on the Von Frey test, which is commonly used to assess mechanical sensitivity at the hind paw. As for any other behavioral test, the experimenters should practice standardizing the application of the filaments, and until behaviors produced by mechanical stimuli are easily distinguished from other behaviors. The test is simple, otherwise, and there should be no major difficulty to use it.

Demonstrating the procedure will be Nasim Eskandari, a PhD student from my laboratory. To induce chronic back pain in the rat, perform an intramuscular injection of Complete Freund's Adjuvant, or CFA, into the back muscles. Using a 27 gauge needle, inject 150 microliters of a ready-to-use water-in-oil emulsion of CFA into the paraspinal muscles unilaterally or bilaterally.

Keep the needle in place for at least three minutes. Next, fabricate the test cage for two animals, comprising one chamber for each animal by mounting the two contiguous chambers on four 33-centimeter long plexiglass legs. Use transparent plexiglass for the walls of the chambers, and black plexiglass to separate chambers to prevent animals from seeing each other.

Then make the floor and ceiling of the test cage using stainless steel mesh made of one-millimeter wire with an eight-millimeter inter wire distance. Familiarize the rat with the test cage for 30 minutes per day for five to seven consecutive days before the first test. In a prone position under isoflurane anesthesia, shave the back hair in the area of interest over approximately 10 vertebral levels using an animal hair trimmer.

For repeated measures, shave the back hair every three days without behavioral assessment to ensure that stimuli are always applied directly to the skin. Draw a mark on the skin with a permanent marker to ensure that filaments are always applied to the same area when repeating the test on different days. On the testing day, place the rat in the test cage for 15 to 30 minutes before the test until the rat is calm.

Then apply Von Frey filaments perpendicular to the back, always beginning with the two-gram filament and using the up/down method. Apply the filament for two seconds bilaterally to the area of interest 10 millimeters from the spine is processed every 15 to 30 seconds when the rat is awake, standing on its four paws, and not moving. If no response is observed with the application of a filament, apply the next filament with a higher force in the series.

If a positive response such as arching the back, licking, muscle twitching, moving away from the filament, or rotation is observed use the next filament with a lower force in the series. Continue the procedure until four readings are obtained after the first behavioral change. In this study, back mechanical sensitivity between CFA and control rats was investigated.

At baseline, mechanical sensitivity was similar between groups. Intramuscular injection of CFA in the lumbar muscles caused a marked increase in mechanical sensitivity from seven to 28 days. In contrast, the control rats did not show any significant change.

Further, histological examination confirmed the presence of chronic inflammatory changes in the muscles injected with CFA with a clear leukocyte infiltration. Healthy back muscles from a controlled rat after intramuscular injection of saline is shown as a reference. The mechanical hypersensitivity was observed at the hind paw with a standard Von Frey test, in addition to the back muscles.

It is very important to make sure that the filament is always applied in the same way perpendicularly to the back, and that the animal is not moving when applying it. The experimenter should also feel confidence in distinguishing behavioral responses for modern movements.