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

Summary

To develop novel therapeutic interventions for the prevention and management of back pain, animal models are required to examine the mechanisms and effectiveness of these therapies from a translational perspective. The present protocol describes the BMS test, a standardized method to assess back mechanical sensitivity in the rat.

Abstract

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, animal models are needed to examine the mechanisms and effectiveness of novel therapies from a translational perspective. Several rodent models of back pain are used in current investigations. Surprisingly, however, no standardized behavioral test was validated to assess mechanical sensitivity in back pain models. This is critical to confirm that animals with presumed back pain present local hypersensitivity to nociceptive stimuli, and to monitor sensitivity during interventions designed to relieve back pain. The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. A test cage was fabricated specifically for this method; length x width x height: 50 x 20 x 7 cm, having a stainless-steel mesh on the top. This test cage allows the application of mechanical stimuli to the back. To perform the test, the back of the animal is shaved in the region of interest, and the test area is marked to repeat the test on different days, as needed. The mechanical threshold is determined with Von Frey filaments applied to the paraspinal muscles, utilizing the up-down method described previously. The positive responses include (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck (4) scratching or licking the back, and (5) escaping. This behavioral test (Back Mechanical Sensitivity (BMS) test) is useful for mechanistic research with rodent models of back pain for the development of therapeutic interventions for the prevention and management of back pain.

Introduction

Low back pain (LBP) is the leading cause of disability worldwide, which has dramatic personal, economic, and social consequences^1,2,3,4. Every year, approximately 37% of the population is affected by LBP⁵. LBP usually resolves within a few weeks but recurs in 24%-33% of individuals, becoming chronic in 5%-10% of cases². To understand the mechanisms and impacts of LBP as well as the effects of different therapeutic interventions, several animal models of LBP have been used, mimicking clinical conditions or some components of LBP⁶. These mouse and rat models can be classified in one or more of the following categories: (1) discogenic LBP^7,8,, (2) radicular LBP^8,9,10,11, (3) facet joint osteoarthritis¹², and (4) muscle-induced LBP^13,14. Since the pain cannot be measured directly in non-human species, numerous tests have been developed to quantify pain-like behaviors in these models⁸. These tests assess behaviors evoked by a noxious stimulus (mechanical force^15,16,17, thermal stimulation^{18,19,20,21,22,23,24,25}) or produced spontaneously^26,27,28,29.

The methods using mechanical stimuli include the Von Frey test^15,16 and the Randall-Selitto Test¹⁷. Methods using heat stimuli include the tail flick test¹⁸, hot plate test¹⁹, Hargreaves test²⁰, and thermal probe test²¹. Methods using cold stimuli include the cold plate test²², acetone evaporation test²³, and cold plantar assay²⁴. Methods for spontaneous behaviors include the grimace scales²⁶, burrowing²⁷, weight-bearing and gait analysis²⁸, as well as an automated behavioral analysis²⁹. Despite these numerous available tests, none of them is designed specifically for back pain models.

The objective of this study is to lay down a simple and accessible test to assess mechanical sensitivity in the back of rats. The technique is largely based on the Von Frey test applied to the plantar surface of the hind paw^15,16. The basic principle of the Von Frey test is to use a series of monofilaments to the region of interest, delivering constant pre-determined forces. A response is considered positive if the rat shows a nocifensive behavior. The mechanical threshold can then be calculated based on the filaments that evoked responses. In the present study, a simple and accessible method adapted from the Von Frey test is provided to determine mechanical sensitivity in the back of rats.

Protocol

The experimental protocol was approved by the animal care committee of Université du Québec à Trois-Rivières and conformed to the Guidelines of the Canadian Council on Animal Care and the Guidelines of the Committee for Research and Ethical Issues of the International Association for the Study of Pain (IASP). The present study used six male Wistar rats (body weight: 320-450 g; age: 18-22 weeks). The animals were obtained from a commercial source (see Table of Materials). Data from these rats are from the larger sample of a previous study³⁰.

1. Experimental preparation

1. House the animals in a temperature-controlled room in standard animal facilities with access to food and water ad libitum and a light-dark cycle of 14 h-10 h. Ensure that all animals are in good health on the day of the experiments.
2. Generate the chronic back pain animal model following the steps below.
   1. To induce chronic back pain, perform an intramuscular injection of Complete Freund Adjuvant (CFA) into the back muscles following the previous reports^14,30,31.
   2. Anesthetize the animal using isoflurane (4% for induction and 2%-2.5% for maintenance).
   3. Using a 27 G needle, inject 150 µL of a ready-to-use water-in-oil emulsion of CFA (see Table of Materials) into the paraspinal muscles unilaterally or bilaterally, depending on the protocol needs.
   4. Keep the injection needle in place for at least 3 min after completing the injection. For animals in the control group, use the same procedures³⁰, but inject a solution of sterile physiological saline solution (150 µL, 0.9%) instead of CFA.
3. Fabricate the test cage.
   1. Make a test cage for two animals that comprises one chamber for each animal.
      NOTE: For the present study, each chamber has the following dimensions: length x width x height: 50 x 20 x 7 cm (see Table of Materials).
   2. Mount the two contiguous chambers on four 33 cm long Plexiglass legs. Use transparent Plexiglass for the walls of the chambers, but use black Plexiglass to separate chambers to prevent animals from seeing each other.
   3. Use stainless-steel mesh made of 1 mm wire with an 8 mm inter-wire distance to make the floor and ceiling of the test cage (Figure 1).

2. Back Mechanical Sensitivity (BMS) test

1. Familiarize the animal with the test cage 30 min/day for 5-7 consecutive days prior to the first test. Repeat the test as needed.
2. Anesthetize the animals using 2% isoflurane³¹ (see Table of Materials).
3. In a prone position under isoflurane anesthesia, shave the back hair of the area of interest (from T6 to L6 vertebral levels) using an animal hair trimmer (see Table of Materials). For repeated measures, shave the back hair every 3 days on a day without behavioral assessment to ensure that stimuli are always applied directly to the skin. Draw a black 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.
4. On the testing day, put the animals in the test cage for 15-30 min prior to the test until the animal is calm.
5. During the test, apply Von Frey filaments (0.07, 0.16, 0.4, 0.6, 1, 2, 4, 6, 10, 15, and 26 g) perpendicularly to the back, always beginning with the 2 g filament and using the up-down method¹⁵ (see Table of Materials). Approach the back of the animal slowly with the filament from behind the animal.
   1. Apply the filament only when the animal is awake, standing on its four paws, and not moving. Apply the filament for 2 s bilaterally to the area of interest, 10 mm from the spinous process (Figure 2), every 15-30 s.
      NOTE: A response is considered positive if the animal exhibits one or more of the following behaviors during or immediately after the filament is applied: (1) muscle twitching, (2) arching (back extension), (3) rotation of the neck to look at the back, (4) scratching or licking the back, and (5) escaping.
6. As described previously¹⁵, if no response is observed with the application of a filament, apply the next filament with a higher force in the series. If a response is observed, use the next filament with a lower force in the series. Continue this procedure until four readings are obtained after the first behavioral change (response after a series of "no response" or no response after a series of "response").
7. Once data collection is completed, calculate the value representing 50% of the mechanical threshold, as described by Chaplan et al.¹⁵, using this formula:
   50% threshold (g) = 10^(Xf+kδ)/10 000
   ​NOTE: In this formula, "Xf" is the Handle Mark of the last von Frey filament that was used. "k" is the tabular value based on the animal's response pattern¹⁵, and "δ" is the mean of Handle Mark's increments between Von Frey filaments. Depending on the experimental design and experimental needs, only one side of the spine may be assessed to report one threshold, or two sides may be evaluated, and thresholds are reported separately or as a mean. Refer to Supplementary Table 1 for the calculation template³².

3. Animal recovery

1. After the intramuscular injection is completed, discontinue anesthesia and place the animal alone in a standard housing cage for recovery.
2. During the recovery period, examine the animal's behavior and do not leave it unattended.
3. Confirm that the animal recovers from anesthesia and moves normally within 5 min. Then, return the animal to its usual housing cage with the other animals.
   NOTE: At the end of the experiment, the animal is perfused through the heart with a 10% formalin solution, under deep isoflurane anesthesia (5%). The back muscles in the injected area are then extracted for histology and confirmation of inflammatory changes.

Results

The method was used in a previous study, in which full data and statistics were presented to compare back mechanical sensitivity between CFA and control rats³⁰. Representative individual data (mean of left and right thresholds) from six rats included in the previous study are presented in Figure 3 and Table 1. At baseline, mechanical sensitivity was similar between groups. Intramuscular injection of CFA in the lumbar muscles caused a marked increase i...

Discussion

Critical steps
The BMS test is a simple method to assess mechanical sensitivity in the back of rats, either at one time point or repeatedly over days or weeks, when changes are expected to occur (pain models) or after pharmacological or non-pharmacological intervention. Critical issues of the method include the test cage, whose dimensions must ensure that the rat is comfortable but does not move too much. The animal's back must stay accessible through the mesh ceiling for reproducible mechanica...

Materials

Name	Company	Catalog Number	Comments
Aerrane (isoflurane, USP) - Veterinary Use Only	Baxter	NDC 10019-773-60	Inhalation Anaesthetic ; DIN 02225875, for inducing anasthesia
Complete Freund Adjuvant (CFA)	Fisher Scientific	#77140	Water-in-oil emulsion of Complete Freund Adjuvant (CFA) with killed cells of Mycobacterium butyricum.
Male Wistar Rats	Charles River Laboratories		body weight: 320–450 g; age: 18-22 weeks.
Penlon Sigma Delta Vaporizer	Penlon	990-VI5K-SVEEK	Penlon Sigma Delta Vaporizer used for anasthesia
Sharpie Permanent Marker	Sharpie	BC23636	Permanent Marker, Fine Point, Black
Test cage	Custom-made		Width: 20 cm;  Length: 50 cm; Height from the bottom to the top: 40 cm; Height from the bottom mesh to the top of the cage: 7 cm; Wall thickness: 5 mm; Mesh: 1 mm wire with an 8 mm inter-wire distance
Von Frey Filaments	Aesthesio, Precise Tactile Sensory Evaluator	514000-20C	Filaments from 0.07 g to 26 g
Wahl Professional Animal, ARCO Cordless Pet Clipper, Trimmer Grooming	Wahl	Kit #8786-1201	Animal hair trimmer, for shaving purposes, zero blade