Name: Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice (Video) | JoVE Journal
Uploaded: 2022-01-25T14:00:00.000+00:00
Duration: 4 min 34 s
Description: Learn a modified spared nerve injury surgery in mice that reduces procedure time by half while maintaining efficacy. This streamlined technique ligates the common peroneal and sural nerves together to study neuropathic pain mechanisms.

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5 minutes

Z.G. is supported by NINDS R01NS100801.

Animal experiments were approved by UCSF Institutional Animal Care and Use Committee and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory animals. Adult C57BL/6 mice weighing 20-30 g was used in this study. The von Frey assessment was performed between 1:00 pm and 3:00 pm.
1. Anesthesia and mouse preparation
<ol>
	<li>Place a mouse in a plastic chamber filled with 2% isoflurane in O2 at a flow rate of 1.0 L/min until it is fully anesthetized.</li>
	<li>Cover the eyes with ophthalmic ointment using a cotton-wool applicator.</li>
	<li>Place the snout of the mouse into a flexible nose cone with 2% isoflurane flow throughout the surgical procedure.</li>
	<li>Place the mouse in a right lateral position. Keep the left leg up with knee flexion and secure it with adhesive tape.</li>
	<li>Remove the hair around the thigh and knee area with an electric shaver and hair removal cream. Disinfect the skin with 2% chlorhexidine and 70% alcohol.</li>
	<li>Ensure sufficient anesthesia depth before surgery by testing no response to pinch stimulus on the hind limb or tail with tweezers as the standard. Provide thermal support to the mouse. 
	NOTE: No local anesthetic or NSAID was used before and after performing the SNIt model because local anesthetic and NSAID significantly reduce the neuropathic pain behavior after SNIt12.</li>
</ol>
2. Modified SNIt surgery
<ol>
	<li>Cut a 1 cm incision starting at the first 1/3 of the horizontal line crossing the knee with an approximately 30&#176; angle from the vertical line with a scalpel (Figure 2A).</li>
	<li>Two white lines can be visualized under the biceps femoris muscle (BFM) after separating skin incision, with the medial (cephalad) thick line as the femur and lateral (caudal) thin line as sciatic nerve (Figure 2B).</li>
	<li>Blunt dissect BFM along the caudal white line with curved micro forceps and micro scissors to expose the sciatic nerve. Avoid blood vessel damage during blunt dissection. If accidental vessel damage occurs, use sterile cotton-wool swabs to absorb blood and apply proper pressure to stop bleeding.</li>
	<li>Differentiate the three branches from the sciatic nerve at the superior edge of the gastrocnemius muscle. The tibial nerve is the one with the biggest diameter passing under the gastrocnemius muscle, whereas the sural nerves (lateral, the smallest diameter) and the common peroneal nerve (medial) run above the gastrocnemius muscle (Figure 2C and Figure 3A).</li>
	<li>Depending on how BFM is dissected and opened, visualize the common peroneal nerve as lateral (Figure 2C) or medial (Figure 3A) to the tibial nerve.</li>
	<li>Separate the common peroneal and the sural nerves from the neighboring tissues using curve micro forceps.</li>
	<li>Ligate the common peroneal and sural nerves together with a 6-0 suture, as both nerves run above the gastrocnemius muscle, but the tibial nerve passes under the gastrocnemius muscle (Figure 2D and Figure 3B,C). Observe the limb for contraction following the tight ligature. For the traditional method, ligate the common peroneal and sural nerves separately.</li>
	<li>Cut nerve at a distal part within 2-4 mm of the ligation with a pair of micro scissors. Ensure that the tibial nerve remains untouched during the whole procedure. For the traditional method, cut the common peroneal and sural nerves at the distal part within 2-4 mm of the ligation and remove a 2 mm section, separately.</li>
	<li>Close the muscular layer with a 6-0 silk suture and the skin incision with wound clips.</li>
	<li>After surgery, return the mice to the animal room until full recovery from anesthesia. Check daily for intact incisions, normal food intake, water consumption, general body condition, regular movements, and grooming. Remove wound clips 7-14 days after surgery.</li>
</ol>
3. von Frey assessment for mechanical threshold
<ol>
	<li>Acclimatize the mice for 6 days to the testing room environment and testing materials to perform the von-Frey assessment. Place the mice in clear plastic cylinders on an elevated wire mesh grid for 1 h of habituation. Place white papers between each cylinder to prevent any visual cue from each testing animal.</li>
	<li>During this period, perform habituation every 2 days, and measure the baseline of von Frey monofilaments under the mid-plantar of the hind paw after the last habituation.</li>
	<li>Perform von Frey assessment with a blind tester. Stimulate the mid-plantar of the hind paw with von Frey filaments by using the up-down paradigm13. Apply the von Frey filament to the plantar surface perpendicularly with applied force to cause slight curling.</li>
	<li>Verify positive responses as sudden paw withdrawal, sudden flinching, or sudden paw licking. Exert the next stimulus at an interval of 5 s to avoid the influence of the previous stimulus.</li>
	<li>Confirm mechanical threshold per paw by taking an average of 3 sessions.</li>
	<li>Based on the 50% hind paw withdrawal threshold decided by the up-down method, use the percent response method with 0.16 g filament to assess the difference further.</li>
	<li>Record the percentage of the positive responses after 10 stimuli of 0.16 g filament applied to the mid-plantar of the hind paw regardless of the responses.</li>
	<li>Perform the von Frey assessments on pre-surgery day 1, and post-surgery on days 1, 3, 5, 7, and 14.</li>
</ol>
4. Statistical Analysis
<ol>
	<li>Report normally distributed continuous data as mean &#177; standard error of the mean (SEM). Analyze the continuous data with 2-tailed t-test or two-way repeated-measures ANOVA. Process all data using statistical analysis software, with statistical significance at the level of p &#60; 0.05.</li>
</ol>

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<li>Decosterd, I., Woolf, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spared+nerve+injury%3A+an+animal+model+of+persistent+peripheral+neuropathic+pain%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">Spared nerve injury: an animal model of persistent peripheral neuropathic pain.</a> Pain. 87 (2), 149-158 (2000).</li>
<li>Cichon, J., Sun, L., Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spared+Nerve+Injury+Model+of+Neuropathic+Pain+in+Mice%5BTitle%5D)+AND+%22Bio-Protocol%22%5BJournal%5D)">Spared Nerve Injury Model of Neuropathic Pain in Mice.</a> Bio-Protocol. 8 (6), 2777(2018).</li>
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The authors declare no competing interests.

Compared to the traditional mouse SNIt method that ligates the common peroneal nerve and the sural nerve separately6,7,8,9, the modified SNIt model has three advantages: (1) it has less risk of contracting or stretching sciatic or tibial nerves; (2) there is no need to remove the distal nerve stumps after nerve-cutting because by ligating the common peroneal nerve and the sural nerve together, ...

Nerve injury-induced neuropathic pain following surgery or trauma has a significant economic burden that impairs quality of life. A host of nerve injury models, including spinal nerve ligation (SNL)1, chronic constriction injury (CCI)2 to the sciatic nerve, partial sciatic nerve ligation (pSNL)3, sciatic nerve transaction (SNT)4 and spared nerve injury (SNI)5,6,7,8, were successfully developed to mimic the cardinal symptoms of peripheral nerve injury in rats and mice for studying the molecular and cellular mechanism of neuropathic pain6,7,8,9,10. However, each surgical model has its benefits and limitations, therefore particular attention should be given to exploring and developing the surgery models10.
The rodent SNI model produces long-term hypersensitivity to mechanical stimulation. However, it is somewhat confusing because there are two different SNI models. The initial SNI model was developed in Woolf&#39;s lab, in which the common peroneal and the tibial nerves were injured, leaving the sural nerve intact5,6. The second SNI model was developed in Basbaum&#39;s lab, in which the common peroneal and the sural nerves were injured, leaving the tibial nerve intact7,8. The initial Woolf&#39;s model is defined as SNIs here because the sural nerve is left intact, and Basbaum&#39;s model is defined as SNIt here because the tibial nerve is left intact. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. However, unlike the SNIs model, mice in the SNIt model do not develop thermal hypersensitivity, but mechanical hypersensitivity develops in both models. Although the SNIt model is a relatively easy procedure, it requires the ligation of the sural and common peroneal nerves separately with the potential risk of stretching the sciatic or tibial nerves6,7,8,9.
The common peroneal, tibial, and sural nerves are three branches of the sciatic nerve and can be clearly identified at the superior edge of the gastrocnemius muscle (Figure 1): the tibial nerve goes under the gastrocnemius muscle, and the common peroneal (cephalad side) and sural nerve (caudal side) are above the gastrocnemius muscle11. Based on its anatomical features, a modified mouse SNIt surgery procedure was developed to ligate the common peroneal and sural nerves together with only one nerve-ligation and one nerve-cutting, which results in shortened procedure-time.

<table><tbody><tr><td>6-0 suture</td><td>Henry Schein</td><td>9007482</td><td>Nerve ligation and close the muscular layer</td></tr><tr><td>Iris Scissors</td><td>Integra Miltex</td><td>12460598</td><td>Cut muscle and fascia</td></tr><tr><td>Mayo dissecting scissors</td><td>Fisherbrand</td><td>895120</td><td>Cut skin incision</td></tr><tr><td>Micro forcep</td><td>Fisherbrand</td><td>16100110</td><td>Blunt dissection biceps femoris muscle</td></tr><tr><td>Micro Scissors</td><td>Excelta</td><td>17467496</td><td>Cut nerve</td></tr><tr><td>Microdissection Forceps</td><td>Fisherbrand</td><td>16100123</td><td>Separate the common peroneal and the sural nerves from the neighboring tissues</td></tr><tr><td>Needle Holder</td><td>Fisherbrand</td><td>8966</td><td>Hold 6-0 needle</td></tr><tr><td>Prism software</td><td>Graph Pad</td><td>version 8.0</td><td>Statistical analysis software</td></tr><tr><td>Wound clips</td><td>Roboz Surgical</td><td>NC1878744</td><td>Close skin incision</td></tr></tbody></table>

modified spared nerve injury surgery model of neuropathic pain in mice

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The comparison of procedure time between modified and traditional methods. 
The procedure time from the beginning of cutting the skin to the end of closing skin was recorded in 5 mice with the modified approach and 5 mice with the traditional approach, respectively. A minimal number of animals was used to obtain results with statistical significance. Compared with the control of the traditional approach7,14 to perform SNIt, the modified approa...

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular ...

modified-spared-nerve-injury-surgery-model-of-neuropathic-pain-in-mice

Research

JoVE Journal

Neuroscience

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.

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

This video demonstrates testing sensory nerve function in a mouse model by surgically denervating one side and leaving the other as a control. The response to mechanical stimulation confirms the role of sensory nerves.

Testing Sensory Nerve Function in a Surgically Denervated Mouse Model

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of skeletal muscle atrophy. The model surgical protocol is described and demonstrated in C57Black6 mice.

Tibial Nerve Transection - A Standardized Model for Denervation-induced Skeletal Muscle Atrophy in Mice

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The “cuff model” of neuropathic pain in mice consists of the implantation of a polyethylene cuff around the main branch of the sciatic nerve. Mechanical allodynia is tested using von Frey filaments.

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

We present a surgical protocol detailing how to perform a cut or crush axotomy on the facial nerve in the mouse. The facial nerve axotomy can be employed to study the physiological response to nerve injury and test therapeutic techniques.

Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

This protocol describes the tibial neuroma transposition model, which entails a lesion of the tibial nerve with subsequent transposition of the proximal nerve end toward a subcutaneous pretibial or lateral position. Behavioral testing of neuroma pain and plantar hyperalgesia is quantified using Von Frey monofilaments.

Surgery and Behavioral Testing in the Tibial Neuroma Transposition Model in Rats

This protocol presents a reproducible rat model using sensory nerves to mimic human neuroma pain and assess surgical treatments, offering a valuable tool for translational neuroma research.

Novel Surgical Rodent Model for Studying Neuroma Pain Treatment Options using Targeted Muscle Reinnervation Through the Saphenous Nerve

Lateral fluid percussion (LFP), an established model of traumatic brain injury in mice, is demonstrated. LFP fulfills three major criteria for animal models: validity, reliability and clinical relevance. The procedure, consisting of surgical craniotomy, fixation of hub followed by induction of injury, resulting in focal and diffuse injuries, is described.

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Learn a modified spared nerve injury surgery in mice that reduces procedure time by half while maintaining efficacy. This streamlined technique ligates the common peroneal and sural nerves together to study neuropathic pain mechanisms.

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

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