Materials and Reagents

1. Sterile gauze
   
2. Syringes. BD Micro-FineTM Demi, U-100 Insulin, 30 G x ½’’–0.33 x 8 mm (BD, catalog number: 324826 )
   
3. Eppendorf tubes (Eppendorf, catalog number: 0030120086 )
   
4. Scalpel blades (Sigma-Aldrich, catalog number: S2646 )
   
5. 6-0 silk suture (Ethicon, catalog number: MCP492G )
   
6. 5-0 Dermalon suture (Covidien, catalog number: 1756-21 )
   
7. Animals
   Adult C57BL/6J male mice (Charles River, Calco, Italy) weighing 20-25 g are used. Six-week-old male mice are allowed to habituate in the animal room for 2 weeks
   Note: All animals are housed in groups of five in standard cages with access to food and water ad libitum, and maintained under 12 h dark/light cycle (starting at 7:30 AM), 22 °C temperature, and 66% humidity. All manipulations are carried out between 9:00 and 16:00 h. Procedures were performed in accordance with relevant guidance from the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), the Guide for the Care and Use of Laboratory Animals (Clark et al., 1997) and European Union directives (2010/63/EU). The ethics committees of the IRCCS Neuromed Institute and the Italian Ministry of Health approved the protocol. All efforts were made to minimize suffering and reduce the number of animals used in the experiments. To reach statistical significance, at least 10 animals per group are highly recommended.
   
8. Isoflurane (Sigma-Aldrich, catalog number: Y0000858 )
   
9. Potential analgesic drug to be tested
   Note: In the example provided here we used raseglurant, a negative allosteric modulator of the metabotropic glutamate type 5 (mGlu5) receptor (Font et al., 2017).
   
10. Vehicle
    Sterile physiological saline (0.9% NaCl) solution (B. Braun Melsungen, catalog number: 3570380 )
    
11. Classical analgesic drugs for comparative purposes
    
12. Sterile cleaning solution
    Note: A 70% ethanol in H₂O was used.
    

Equipment

1. Surgical instruments, surgery bench, inhalation anaesthesia apparatus
   
2. Ugo Basile Gas Anaesthesia (Ugo Basile, catalog number: 22100 ), Ugo Basile Induction Box 7900 (Ugo Basile, catalog number: 7900/10 ) (Figure 1)
   
   
   Figure 1. Apparatus for inhalation anaesthesia in mice and rats with an induction chamber (25 x 13 x 13 cm)
   
   
3. Surgical Microscope Leica Wild M650 (Leica, model: Leica Wild M650) (Figure 2)
   
   
   Figure 2. Stereomicroscope with an anaesthetized mouse ready to undergo CCI
   
   
4. Fine watchmaker forceps (Sigma-Aldrich, catalog number: T4537-1EA )
   
5. Curved blunt-tipped forceps (Sigma-Aldrich, catalog number: T4787-1EA )
   
6. Micro-dissecting scissor (Sigma-Aldrich, catalog number: S3146-1EA )
   
7. Mathieu needle holder (Fine Science Tools, catalog number: 12510-14 )
   
8. Balances (Sartorius, models: CP124S and BL1500S )
   
9. von Frey filaments (North Coast Medical, Inc., San Jose, CA, USA) (Figure 3)
   
   
   Figure 3. von Frey filaments used to assess mechanical allodynia
   

Procedure

1. Surgical procedure
   1. Anaesthetize mice in an induction chamber by inhalation of 2.5% isoflurane in N₂O/O₂ (70:30), maintained at 2% by a facemask throughout surgery.
      
   2. Place mouse under a stereomicroscope, remove hair in the right or left hind leg and sterilize the area by the use of a sterile gauze with 70% alcohol.
      
   3. Place the mouse in horizontal position with the femur rotated by 90° with the aid of masking tape placed on the foot (Figure 4).
      
      
      Figure 4. Mouse placed in horizontal position with the right leg rotated at 90° with the aid of masking tape placed on the foot
      
      
   4. Make an incision in the skin and separate the muscles between the gluteus superficialis and the biceps femoris by cutting the connective tissue (Figure 5).
      
      
      Figure 5. Separation of muscles gluteus superficialis and biceps femoris by cutting the connective tissue
      
      
   5. Move the two muscles to uncover the sciatic nerve.
      
   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.
      
      
      Figure 6. Under the dissecting microscope, free the sciatic nerve by curved blunt-tipped forceps and make one ligature with 6-0 silk
      
      
   8. Stitch the muscle layer.
      
   9. Clean the incision and close the skin with 2-3 ligatures of 5-0 dermalon. Apply an iodine solution (Rodine, 10% solution) to disinfect the wound.
      
   10. Return the mouse to its home cage and check routinely for 72 h.
       
   11. Clean all the material used.
       
       
2. Mechanical allodynia measurement
   1. Handle mice daily in order to reduce stress.
      
   2. Prior to behavioural testing, mice need to be habituated to the testing procedure and the restricted area. The testing environment should be kept quiet and well controlled, with constant temperature and humidity levels. Testing sessions should be carried out at the same time (9:00-14:00).
      
   3. Reagents preparation (weight the analgesic drug and dissolve in vehicle).
      
   4. Remove the mouse from the house cage and measure its body weight using a balance.
      
   5. Administration of the vehicle or analgesic drug.
      Administer (i.e., intraperitoneally, i.p.) the analgesic drug to be tested (i.e., raseglurant) or the same volume of vehicle (physiological saline solution) using an insulin syringe.
      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.
      
   10. Clean all the material used.
       

Data analysis

Representative example of data illustrating the type of results obtained is provided below.

1. Administration of the analgesic drug (i.e., raseglurant) significantly increased pain thresholds in CCI mice (Figure 8).
   
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.
   
   
   Figure 8. Representative results. Antinociceptive effect of raseglurant (10 mg/kg) as analgesic drug using the CCI of the sciatic nerve animal model. Mechanical allodynia was measured in 21 days post-surgery CCI mice. Thus, animals were intraperitoneally injected with vehicle (Veh, saline) or raseglurant (Ras, 10 mg/kg) and 20 min later the mechanical thresholds were assessed using the von Frey filaments. Values are means ± SEM of 7-9 mice per group. **P < 0.001 Student’s t-test when compared to the vehicle (physiological saline solution) treated animals. Extracted from Font et al., 2017.
   

Acknowledgments

This work was supported by ERANET Neuron project ‘LIGHTPAIN’. This protocol was adapted from previous work: Font et al., 2017. The authors declare not conflict of interest.

References

1. Bennett, G. J. and Xie, Y. K. (1988). A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33(1): 87-107.
   
2. Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M. and Yaksh, T. L. (1994). Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53(1): 55-63.
   
3. Clark, J. D., Gebhart, G. F., Gonder, J. C., Keeling, M. E. and Kohn, D. F. (1997). Special report: The 1996 guide for the care and use of laboratory animals. ILAR J 38(1): 41-48.
   
4. Font, J., Lopez-Cano, M., Notartomaso, S., Scarselli, P., Di Pietro, P., Bresoli-Obach, R., Battaglia, G., Malhaire, F., Rovira, X., Catena, J., Giraldo, J., Pin, J. P., Fernandez-Duenas, V., Goudet, C., Nonell, S., Nicoletti, F., Llebaria, A. and Ciruela, F. (2017). Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator. Elife 6.