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 JoVE Clinical and Translational Medicine

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

1, 2, 1, 1

1The Lundbeck Foundation Research Center MIND, Department of Biomedicine, Aarhus University, 2Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen

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    Summary

    The Spared Nerve Injury animal model is described here as a mouse model of peripheral neuropathic pain following partial denervation of the sciatic nerve by lesioning the tibial and common peroneal nerve branches, leaving the remaining sural nerve intact. Behavioral modification resulting from mechanical allodynia is quantified by von Frey filaments.

    Date Published: 8/18/2011, Issue 54; doi: 10.3791/3092

    Cite this Article

    Richner, M., Bjerrum, O. J., Nykjaer, A., Vaegter, C. B. The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice. J. Vis. Exp. (54), e3092, doi:10.3791/3092 (2011).

    Abstract

    Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1].

    The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn’t require expert microsurgical skills.

    The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold.

    As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.

    Protocol

    1. von Frey baseline measurements prior to surgery

    For von Frey testing procedure, please refer to section 5.

    2. Anesthesia/preparation

    1. Anesthetize the animals (intraperitoneal injection of a mixture of 100mg/kg ketamine and 15 mg/kg Xylazine).
    2. Place the animal in a quiet place until fully anesthetized (e.g. covered with a paper towel).
    3. Check reflexes by pinching the tip of the tail and the paws with a pair of tweezers. Be sure that the animals are unresponsive before proceeding.
    4. Subcutaneously inject (back shoulder area) 0.5ml isotonic saline with antibiotics, e.g. ampicillin (to avoid dehydration and prevent infection, although this is a minor surgical procedure).
    5. Using an electrical shaver, shave the operative field from slightly below the knee area to the hip area (for right-handed persons the left hind limb is recommended).
    6. Apply ophthalmic ointment to the eyes with a cotton-wool bud.
    7. Place the animal on its right side and place the left hindlimb on a small platform to keep it elevated. Secure the leg with adhesive tape.
    8. Disinfect the operative field with alternating scrubs of ethanol and betadine from surgical site out.

    3. Surgery

    1. Locate the knee with the thumb of your left hand and use a scalpel to make an app. 1 cm incision in the longitudinal direction proximal to the knee.
    2. Open the skin by blunt dissection using the tip of a pair of sterile scissors.
    3. Separate the muscle layer by blunt dissection right next to the clearly visible blood vessel, close to the thigh bone (femur). If done correctly, the muscle layers will easily separate without any bleeding, revealing the sciatic nerve right below the muscles.
      If bleeding occurs e.g. by damage to a blood vessel close to the knee, use sterile cotton-wool buds or pieces of gaze to absorb the blood. Press until the bleeding stops.
    4. Place the mouse under a stereo microscope and carefully separate the muscles with a pair of sterile tweezers (nr. 2) to visualize the sciatic nerve. Retractors may also be applied.
    5. Identify the area where the sural nerve branches from the sciatic nerve. The sural nerve is the smallest of the three branches, branching to the right in the left leg.
    6. Apply suture (6-0 suture) around the other two branches which are still running in parallel (the tibial and common peroneal nerves), being very careful not to touch the sural branch. This is a critical step as the sural nerve has to be left completely intact.
    7. Make a tight surgical knot. If the first knot is tight, contractions of the limb will be observed.
    8. Grab the nerves to be cut below the suture with a pair of tweezers (nr. 5) and cut the nerves above and below tweezers with a small pair of scissors. By first cutting above the pair of tweezers, pulling of the nerves is avoided.
    9. Cut off suture ends with a pair of micro scissors and gently close the muscle layer. Add a drop of lidocaine to the wound and suture with surgical knots.

    4. After surgery

    1. Check if eye ointment is still sufficient.
    2. Place the mouse in a clean cage under a paper towel in a comfortable posture. If the room is cold, place a heat pad under a part of the cage (only under part of the cage as the animal should be able to escape to a colder area if preferred).
    3. Ensure easily accessible water and chow.

    5. von Frey testing (baseline, and from the day after surgery)

    1. Place the mice in red colored plastic cylinders placed on a wire mesh table two days and one day prior to surgery. Habituate for 15 min in cylinders prior to testing. The red color of the cylinders helps the mice to quickly relax as they cannot see each other and are in a darker area.
    2. Verify that the mice are calm and apply von Frey filaments to the lateral part of the paw: Starting with the 0.02 g filament, first apply force to the left paw five times over a total period of 30 seconds (approximately 2 seconds per stimulus) and gauge the mouse's reaction after each application. Repeat with the same filament and the left paw of any other mice, and then start over again with the right paw of the first mouse before moving on to the next filament, e.g.:
      • Mouse 1: left paw 5 times with filament 0.02 g
      • Mouse 2: left paw 5 times with filament 0.02 g etc.
      • Mouse 1: right paw 5 times with filament 0.02 g
      • Mouse 2: right paw 5 times with filament 0.02 g etc.
      • Mouse 1: left paw 5 times with filament 0.04 g
      • Mouse 2: left paw 5 times with filament 0.04 g etc.
    3. Response in three out of five stimuli is regarded as a positive reaction. Filaments above threshold can be applied to verify the threshold level.
      Response = sudden paw withdrawal, sudden flinching, sudden paw licking.

    6. Representative results

    Figure 1
    Figure 1. Von Frey testing on C57BL/6 mice age 8-10 weeks old was performed 1 day prior to operation and repeatedly following surgery. 4-8 animals were included in each group. Von Frey testing on the operated side is represented by ipsilateral and the non-operated control side is represented by contralateral. The day after surgery the animals develop significant hypersensitivity on the operated paw while the non-operated is only slightly affected relative to sham operated mice. (* <0.05, **<0.01, ***<0.001, ipsilateral relative to contralateral).

    Discussion

    Critical steps

    Damage to the sural nerve should be avoided in order to study pathological changes in the intact sural nerve following injury to the tibial and common peroneal nerves. Collateral damage to the sural nerve may lead to paralysis and can thus be visualized as dragging of the operated hind limb.

    Only the lateral side of the paw is innervated by the spared sural nerve and, hence, only this area develops neuropathy. Testing other areas, innervated by the transected nerves, can strongly bias the evaluation of altered mechanical threshold.

    Possible modifications

    Other types of ligations may be supplementary in terms of studying pathological pain conditions following peripheral nerve injury, such as chronic constriction injury [5] or partial nerve ligation [6]. Each experimental procedure results in distinct phenotypic changes which should be considered prior to post-surgery testing.

    Furthermore, other sensory tests such as thermal hyperalgesia may be applied [7], although this phenotype is less robust following SNI.

    Future applications

    This technique can be used for testing of drugs altering the development or maintenance of mechanical allodynia [8]. Analysis of the sciatic nerve, the dorsal root ganglia (DRG) and/or the lumbar spinal cord allow research of the molecular mechanisms responsible for the induced phenotype.

    Animal experimentation was performed according to good laboratory practice in full compliance with Danish and European regulations. All experiments were approved by the Danish Animal Experiments Inspectorate under the Ministry of Justice (permission number 2006/561-1206).

    Disclosures

    No conflicts of interest declared.

    Acknowledgements

    This work was supported by the Lundbeck Foundation, the Carlsberg Foundation and Dagmar Marshall Foundation.

    References

    1. Costigan, M., Scholz, J., & Woolf, C.J. Neuropathic pain: a maladaptive response of the nervous system to damage. Annu. Rev. Neurosci. 32, 1-32 (2009).
    2. Decosterd, I. & Woolf, C.J. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain. 87 (2), 149-158 (2000).
    3. Bourquin, A.-F., Süveges, M., Pertin, M., Gilliar, N., Sardy, S., Davidson, A.C., Spahn, D.R., & Decosterd, I. Assessment and analysis of mechanical allodynia-like behavior induced by spared nerve injury (SNI) in the mouse. Pain. 122, 14.e1-14.e14 (2006).
    4. Chaplan, S.R., Bach, F.W., Pogrel, J.W., Chung, J.M., & Yaksh, T.L. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods. 53 (1), 55-63 (1994).
    5. Bennett, G.J., & Xie, Y.-K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain. 33 (1), 87-107 (1988).
    6. Seltzer, Z., Dubner, R., & Shir, Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain. 43 (2), 205-218 (1990).
    7. Hargreaves, K., Dubner, R., Brown, F., Flores, C., & Joris, J. A new and sensitive method for measuring thermal nociception in cutanous hyperalgesia. Pain. 32 (1), 77-88 (1988).
    8. Erichsen, H.K. & Blackburn-Munro, G. Pharmacological characterization of the spared nerve injury model of neuropathic pain, Pain. 98 (1-2), 151-161 (2002).

    Comments

    7 Comments

    Dear Prof. Dr. Christian B. Vaegter,

    It stated in your kind honor that, currently, I'm working on anti-inflammatory and analgesic effect of naturally occuring compound in vitro
    and in vivo models using macrophages and randall selitto apparatus for analgesic activity.

    I watched your video article in Journal of visualized experiments "The Spared Nerve Injury (SNI) Model of Induced
    Mechanical Allodynia in Mice"
    It is really very nice and impressive animal model for anti-nociceptive effect.
    I'm very interested to follow your animal model for evaluation of anti-nociceptive effect of single component isolated
    from natural products.

    I have some question about this article, would you mind to answer me?

    1. What if I use drug to evaluate the analgesic or anti-hyperalgesic effect,so what rout of administration will you
    suggest (i.p, s.c or orally?
    ². What if I use randall selitto apparatus to measure the withdraw effect?
    3. Why you used the ointment in the eye?

    Your answers will be highly appreciated.
    I'll be very thankful to you.

    Regards with best wishes

    S. Khan
    Reply

    Posted by: salman k.January 4, 2012, 2:33 AM

    Dear Salman

    I'm happy to hear that you could benefit from your JoVE protocol!

    Regarding your questions:

    1) I believe this would depend solely on the drug used .. whether this needs to be administered orally or i.p depends on the specific properties of the drug. But in general, injection is preferable as this allows accurate dosage and consistent delivery.
    ²) We haven't used the randall selitto apparatus on the SNI model so I don't know if this works well. In the SNI model we ligate the main branches of the sciatic nerve and leave the sural nerve intact, this only the lateral part of the paw displays hypersensitivity whereas the rest of the paw is numb. Von frey filaments allow us to test only the hypersensitive area .. I'm not sure if this also is true with the paw pressure test?
    3) We use eye ointment during surgery to prevent that the eye dry out during the procedure. The blink reflex is impaired during anesthesia thus we need to protect the eye (especially during longer procedures).

    Best regards,
    Christian
    Reply

    Posted by: Christian V.January 4, 2012, 2:44 AM

    Dear Prof. Dr. Christian B. Vaegter,
    Thanks for you video article in Journal of Visualized Experiments, It's really very useful for me to assess compouds for neuropathic pain. I have one question about the surgey step 6 and 7 'Apply suture (6-0 suture) around the other two branches which are still running in parallel ...',and after that 'Make a tight surgical knot.' Since we need cut off the nevers in step 8, can I just cut off the tibial and common peroneal nerves without ligation the nerves?

    Your kind and early reply will be highly appreciated.
    Regards with best wishes,
    Longhe Yang
    Reply

    Posted by: Longhe Y.June 28, 2013, 2:12 AM

    Dear Prof. Dr. Christian B. Vaegter,
    Thanks for you video article in Journal of Visualized Experiments, It's really very useful for me to assess compouds for neuropathic pain. I have one question about the surgey step 6 and 7 'Apply suture (6-0 suture) around the other two branches which are still running in parallel ...',and after that 'Make a tight surgical knot.' Since we need cut off the nevers in step 8, can I just cut off the tibial and common peroneal nerves without ligation the nerves?

    Your kind and early reply will be highly appreciated.
    Regards with best wishes,
    Longhe Yang
    Reply

    Posted by: Longhe Y.June 28, 2013, 2:28 AM

    Dear Longhe Yang,
    thanks for your question regarding our Jove video.
    We apply a suture, i.e. a mechanical barrier, in addition to cutting off a piece of the nerves to ensure that there is absolutely no nerve regeneration occuring that may affect our von Frey analysis.

    Kind regards,
    Mette
    Reply

    Posted by: Mette R.September 10, 2013, 5:36 AM

    Hello,

    Thanks for this video! We do electronic von Frey on our SNI mice and one of the problems is that they move so much. The red cylinders look promising though. What are the dimensions of the cylinder? And did you buy them from a commercial vendor or did you just get some red plexiglass tubing and cut your own?

    Many thanks.
    -Alex
    Reply

    Posted by: Alexander C.September 5, 2013, 3:10 PM

    Hi Alex,
    thanks for your questions.
    1) The dimensions of the cylinders are:
    plastic thickness: 3 mm; diameter: 8 cm; hight: 7.5 cm
    2) They are home made from red plexiglass tubings and the lids consist of standard petri dishes with drilled holes.

    Kind regards,
    Mette
    Reply

    Posted by: Mette R.September 10, 2013, 6:24 AM

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