Mechanical Allodynia Assessment in a Murine Neuropathic Pain Model

[Abstract] Experimental animal models are unique tools (i) to study pain transmission and pathophysiology of neuropathic pain, (ii) to identify novel molecular targets and (iii) to test the potential analgesic effect of specific molecules. The chronic constriction injury (CCI) model of neuropathic pain is the first model of post-traumatic painful peripheral neuropathy, originally developed by Bennett and Xie in the late 1980s. The chronic constriction is performed in the sciatic nerve and induces a partial denervation involving myelinated afferent axons and unmyelinated axons. Damage to unmyelinated axons is much more severe than myelinated afferents. As the model induces a partial denervation, it is very useful for the analysis of pain behaviours. Stimulation of the hind paw, a target of the sciatic nerve, induces pain which can be quantitated. Thus, mechanical allodynia is usually assessed 7, 14 and 21 days after CCI of the sciatic nerve by measuring the hind paw withdrawal response to von Frey filament stimulation. Here, we describe in detail the protocol allowing a reliable and reproducible CCI model in mice. Overall, researchers most commonly use this surgical model to discover more efficacious drugs for the pharmacological control of chronic pain states.

withdrawal response to von Frey filament stimulation. Mice are placed in a dark box with a wire grid bottom through which the von Frey filaments are applied by using the up-down paradigm previously described (Chaplan et al., 1994). Lack of response to a filament indicates the next filament with a higher bending force in the following stimulation, whereas a positive response indicated the next filament with a lower bending force. Each filament is applied and pressed perpendicularly to the plantar surface of the hind paw until it bends. The filament that evokes 3 paw withdrawals is assigned as the pain threshold in grams. Mice are treated with vehicle, novel potential analgesic drugs, or a classical analgesic drug, as positive analgesic control, and the mechanical thresholds are quantified. This protocol has been recently implemented by our research team (Font et al., 2017). Overall, CCI model and measurements of mechanical allodynia could be considered as an experimental approach to assess the analgesic activity of any potential drug. Thus, we provide here a complete description of the CCI models and assessment of mechanical allodynia aiming at facilitating its implementation by other scientists.      5. Move the two muscles to uncover the sciatic nerve.

Materials and Reagents
6. Using curved blunt-tipped forceps and micro-scissors, softly free the sciatic nerve (proximal to the sciatic trifurcation) from the surrounding connective tissue.
7. Under a dissecting microscope, make one ligature, proximal to the nerve trifurcation, while taking care to preserve epineural circulation, around the sciatic nerve with 6-0 silk (Figure 6).
The ligature has to be tied loosely around the nerve, until it elicits a brief twitch in the respective hind limb, which prevents over-tightening of the ligation.  Note: The amount (400-500 μl) of drug to be administered should be adjusted according to the dose to be tested (i.e., 10 mg/kg of raseglurant) and the mouse body weight (20-25 g). For example, for a mouse of 20 g of weight we will administer (i.p.) 400 μl of a 500 mg/L raseglurant solution in saline. In addition, all animal experimentation should be carried out by a researcher blind to drug treatments.
6. Place the mouse in a plastic cylinder placed on a wire mesh table. Habituate for 15 min in cylinders prior to testing to allowing mouse to stay calm and still. 7. After 20 min, assess mechanical pain thresholds by means of von Frey filaments ( Figure 3).
Thus, apply filament, bending force range from 0.008 to 3.5 g, to the mid-plantar surface of the hind paw (Figure 7).

Figure 7. Quantification of mechanical thresholds by the von Frey filaments
8. Apply each filament, starting from the one with a bending force of 0.008 g, and press it perpendicularly to the plantar surface of the hind paw until it bent for five times over a total period of 30 sec (approximately 2 sec per stimulus) and measure the mouse leg withdrawal after each application (Chaplan et al., 1994). Repeat this procedure 5 times with a 3-min interval.
Response in 3 out of 5 stimuli is considered as a positive reaction and the filament is assigned as the pain threshold in grams. Lack of response to a filament indicated the next filament with a higher bending force in the following stimulation, whereas a positive response indicated the next filament with a lower bending force. 9. Return the mouse to its home cage.

Data analysis
Representative example of data illustrating the type of results obtained is provided below.
2. The results are analyzed either by Student's t-test or by one-way analysis of variance (ANOVA) followed by a post hoc test, according to the experimental paradigm. P values < 0.05 are considered significant.