This work was supported by a grant from the Fondation Chiropratique du Qu&#233;bec and the Natural Sciences and Engineering Research Council of Canada (MP: grant #06659). The contribution of HK was supported by the Universit&#233; du Qu&#233;bec &#224; Trois-Rivi&#232;res (PAIR program). The contribution of BP was supported by the Fonds de recherche du Qu&#233;bec en Sant&#233; (FRQS) and the Fondation Chiropratique du Qu&#233;bec. The contribution of TP was supported by the Natural Sciences and Engineering Research Council of Canada. The contribution of NE and EK was supported by the Fondation Chiropratique du Qu&#233;bec. The contribution of MP was supported by the FRQS.

The experimental protocol was approved by the animal care committee of Universit&#233; du Qu&#233;bec &#224; Trois-Rivi&#232;res 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 the...

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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&#39;s back must stay accessible through the mesh ceiling for reproducible mechanica...

Low back pain (LBP) is the leading cause of disability worldwide, which has dramatic personal, economic, and social consequences1,2,3,4. 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 LBP7,8,, (2) radicular LBP8,9,10,11, (3) facet joint osteoarthritis12, and (4) muscle-induced LBP13,14. 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 force15,16,17, thermal stimulation18,19,20,21,22,23,24,25) or produced spontaneously26,27,28,29.
The methods using mechanical stimuli include the Von Frey test15,16 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 paw15,16. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aerrane (isoflurane, USP) - Veterinary Use Only</td>
			<td>Baxter</td>
			<td>NDC 10019-773-60</td>
			<td>Inhalation Anaesthetic ; DIN 02225875, for inducing anasthesia</td>
		</tr>
		<tr>
			<td>Complete Freund Adjuvant (CFA)</td>
			<td>Fisher Scientific</td>
			<td>#77140</td>
			<td>Water-in-oil emulsion of Complete Freund Adjuvant (CFA) with killed cells of Mycobacterium butyricum.</td>
		</tr>
		<tr>
			<td>Male Wistar Rats</td>
			<td>Charles River Laboratories</td>
			<td></td>
			<td>body weight: 320&#8211;450 g; age: 18-22 weeks.</td>
		</tr>
		<tr>
			<td>Penlon Sigma Delta Vaporizer</td>
			<td>Penlon</td>
			<td>990-VI5K-SVEEK</td>
			<td>Penlon Sigma Delta Vaporizer used for anasthesia</td>
		</tr>
		<tr>
			<td>Sharpie Permanent Marker</td>
			<td>Sharpie</td>
			<td>BC23636</td>
			<td>Permanent Marker, Fine Point, Black</td>
		</tr>
		<tr>
			<td>Test cage</td>
			<td>Custom-made</td>
			<td></td>
			<td>Width: 20 cm;&#160; 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&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Von Frey Filaments</td>
			<td>Aesthesio, Precise Tactile Sensory Evaluator</td>
			<td>514000-20C</td>
			<td>Filaments from 0.07 g to 26 g</td>
		</tr>
		<tr>
			<td>Wahl Professional Animal, ARCO Cordless Pet Clipper, Trimmer Grooming&#160;</td>
			<td>Wahl</td>
			<td>Kit #8786-1201</td>
			<td>Animal hair trimmer, for shaving purposes, zero blade&#160;</td>
		</tr>
	</tbody>
</table>

back mechanical sensitivity assessment in the rat for mechanistic investigation of chronic back pain

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.

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...

Watch this Scientific Journal Video about Back Mechanical Sensitivity Assessment in the Rat for Mechanistic Investigation of Chronic Back Pain at JoVE.com

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

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.

Low back pain is the leading cause of disability worldwide, with dramatic personal, economic, and social consequences. To develop novel therapeutics, ...

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.

back-mechanical-sensitivity-assessment-rat-for-mechanistic

Research

JoVE Journal

Behavior

2.4K vistas.  Université du Québec à Trois-Rivières. Este protocolo es útil para probar la sensibilidad mecánica en la espalda. Es particularmente interesante para diseños longitudinales en modelos de ratas de dolor de espalda crónico, cuyo objetivo es examinar la efectividad de una intervención para disminuir la sensibilidad de la espalda a estímulos nocivos. El método es simple, accesible y produce resultados reproducibles en experimentadores entrenados.Se basa en gran medida en la prueba de Von Frey, que se usa comúnmente para...