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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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, the distal nerve stumps are anatomically separated from the proximal stumps. Indeed, anatomy dissection demonstrated that no nerve regeneration was observed 14 days after modified SNIt; (3) the procedure is much easier to perform with a much shorter procedure time.
It is critical to make skin incisions low around knee level, rather than high in the thigh near the hip, when performing the modified mouse SNIt. This is because the common peroneal, tibial, and sural nerves have not branched out from the sciatic nerve at the high thigh area, which makes it challenging to separate the three branches. In contrast, at the superior edge of the gastrocnemius muscle near the knee area, the three branches from the sciatic nerve traverse separately and are easily visualized and identified, as the tibial nerve passing under the gastrocnemius muscle, and the common peroneal nerve on the cranial side and sural nerve on the caudal side running above the gastrocnemius muscle11. Interestingly, although the common peroneal nerve is medial to the tibial nerve at the thigh area, it usually crosses the tibial nerve to get closer to the sural nerve at the superior edge of the gastrocnemius muscle at the knee area in the lateral position with knee flexion, which makes it easier for ligating common peroneal and sural nerves together. Importantly, the tibial nerve remains intact without contacting or stretching when the common peroneal and sural nerves are ligated above the gastrocnemius muscle. In addition, when dissecting BFM to expose underneath nerves, it is important to avoid damaging blood vessels (lateral proximal genicular artery, popliteal artery, distal caudal femoral artery, etc.) located above the gastrocnemius muscle16.
Although this modified SNIt model successfully develops neuropathic pain, some limitations are needed to be admitted. Because of damage to the common peroneal nerve, this could produce motor dysfunction with markedly extended hind leg17. Another, the mechanical hypersensitive area is innervated by intact nerve rather than injured nerve, but the neuropathic pain is often caused by the lesion or disease of the peripheral or central nervous system in clinical pain management18. Therefore, further study needs to be explored in the development of the neuropathic pain model.
In summary, this modified mouse SNIt method is a simplified SNI procedure with only one nerve-ligation and one nerve-cutting without any removal of nerve stumps. It is much easier to perform in producing nerve injury-induced mechanical hypersensitive, with reduced risk of sciatic or tibial nerves damage.

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

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 approach took almost half the amount of procedure time (236.2 ± 28.6 s vs. 422.6 ± 53.8 s, p=0.0156; Figure 4).
The comparison of mechanical hypersensitivity between the two methods. 
No difference of von Frey assessment (manual) was observed in the two groups at baseline (1.05 ± 0.10 g vs. 0.96 ± 0.13 g, p=0.9405). Compared with the traditional method (n=9), the modified method (n=14) induced similar mechanical hypersensitivity on the ipsilateral side from post-operative day 1 to post-operative day 14 (0.33 ± 0.09 g vs. 0.32 ± 0.05 g, p&gt;0.9999, at post-operative day 1; 0.19 ± 0.02 g vs. 0.27 ± 0.06 g, p=0.9485 , at post-operative day 3; 0.20 ± 0.06 g vs. 0.15 ± 0.05 g, p=0.9979, at post-operative day 5; 0.13 ± 0.02 g vs. 0.15 ± 0.03 g, p&gt;0.9999, at post-operative day 7; and 0.14 ± 0.02 g vs. 0.19 ± 0.03 g, p=0.9937, at post-operative day 14; Figure 5A).
Compared with percentage response between traditional (n=5) and modified method (n=5), similar mechanical hypersensitivity was observed on the ipsilateral side from baseline (4.00% ± 2.45% vs. 6.00% ± 2.45%, p&gt;0.9999) to post-operative day 1 (20.00% ± 3.16% vs. 12.00% ± 3.74%, p=0.8987), day 3 (30.00% ± 5.48% vs. 32.00% ± 4.90%, p&gt;0.9999), day 5 (36.00% ± 9.27% vs. 30.00% ± 5.48%, p=0.9730), day 7 (32.00% ± 8.00% vs. 36.00% ± 4.00%, p=0.9968), and day 14 (38.00% ± 5.83% vs. 36.00% ± 7.48%, p&gt;0.9999; Figure 5B).
No neural reconnection between the distal and remaining stump at post-operative day 14. 
Because the axon regeneration proceeds at a rate of 1-3 mm/d15, whether there is nerve regeneration at post-operative day 14 should be taken into account. On the post-operative day 14, the modified SNI mice were perfused intracardially with 0.1 mol/L phosphate-buffered saline followed by a phosphate-buffered 10% formalin ﬁxative, and the nerve ligation site was dissected and exposed. No neural reconnection was observed between the distal and remaining stumps (Figure 3D).
<img alt="Surgical anatomy diagram of buccinator flap muscle (BFM) with labeled structures for educational use." class="xfigimg" src="/files/ftp_upload/63362/63362fig01.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Illustration of the 3 branches of the sciatic nerve that are separated at the superior edge of the gastrocnemius muscle (GM) around the knee area. The tibial nerve runs under the GM muscle, and the common peroneal nerve and sural nerve run above the GM muscle. 1: sciatic nerve; 2: tibial nerve; 3: common peroneal nerve; 4: sural nerve. Abbreviations: BFM = biceps femoris muscle; GM = gastrocnemius muscle. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63362/63362fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Surgical incision technique diagram for anatomical study; step-by-step process with surgical tools." class="xfigimg" src="/files/ftp_upload/63362/63362fig02v2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Illustration of surgical procedure in modified SNI mice. (A) In a lateral position, the left leg is upside with knee flexion. The yellow horizontal line (1) indicates the horizontal line crossing the knee level, and the yellow dashed arrow (2) indicates the incision. (B) Following a skin incision, two white lines can be visualized under the biceps femoris muscle (BFM). The femur is under the cephalad one (3), and the sciatic nerve is under the caudal one (4). (C) After careful dissection of BFM, the sciatic nerve (5) and its three branches can be visualized: the tibial (6), common peroneal (7), and sural (8) nerves. (D) The common peroneal (7) and sural (8) nerves were ligated together. 1: horizontal line crossing knee; 2: cut incision; 3: femur on the underneath; 4: sciatic nerve on the underneath; 5: sciatic nerve; 6: tibial nerve; 7: common peroneal nerve; 8: sural nerve; 9: white curve line indicates knee. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63362/63362fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Dissection process diagram; nerve anatomy mapping through tissue sections, labeled sequentially." class="xfigimg" src="/files/ftp_upload/63362/63362fig03.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Illustration of sciatic, tibial, sural, and common peroneal nerves with the ligation of modified SNI in intracardially perfused mice. (A) Sciatic nerve (1) and its three branches: the tibial nerve (2) passing under the gastrocnemius muscle (5), and the common peroneal (3) and the sural (4) nerves running above the gastrocnemius muscle. (B) Suture needle crossing under common peroneal and sural nerves together. (C) A 6-0 nylon suture was used for the ligation of common peroneal and sural nerves together. (D) No reconnection between distal and remaining nerve stump at post-operative day 14 was observed. 1: sciatic nerve; 2: common peroneal nerve 3: tibial nerve; 4: sural nerve; 5: gastrocnemius muscle; 6: ligation. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63362/63362fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bar chart comparing procedure time of traditional vs. modified methods; statistical analysis, p=0.0156." class="xfigimg" src="/files/ftp_upload/63362/63362fig04.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. The modified method is faster than the traditional SNI method to perform. Compared to the traditional SNI method (n=5), the modified method (n=5) requires much less procedure time to perform. Analyses were performed using an unpaired t-test, and data are presented as mean ± SEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63362/63362fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Mechanical threshold and response graph; data analysis comparing traditional vs modified method." class="xfigimg" src="/files/ftp_upload/63362/63362fig05.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. von Frey assessment of mechanical responsiveness in traditional and modified SNI models. (A) The modified SNI (n=9) and traditional SNI (n=14) models induced similar mechanical hypersensitivity on the ipsilateral side in 14-day follow-up. (B) The modified SNI (n=5) and traditional SNI (n=5) models acquired similar percentage responses on the ipsilateral side in a 14-day follow-up. Analyses were performed using two-way ANOVA with Sidak's multiple comparisons test. Data are presented as mean ± SEM. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/63362/63362fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice at JoVE.com

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

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 ...

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Universidad San Sebastian

Research

JoVE Journal

Neuroscience

4.5K Views.  Capital Medical University. 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.

Video: Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

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

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&#x2019;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.

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

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.

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared ...

Medicine

Lateral Fluid Percussion: Model of Traumatic Brain Injury in Mice

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and mortality. Based on the nature of primary injury following TBI, complex and heterogeneous secondary consequences result, which are followed by regenerative processes 1,2. Primary injury can be induced by a direct contusion to the brain from skull fracture or from shearing and stretching of tissue causing displacement of brain due to movement 3,4. The resulting hematomas and lacerations cause a vascular response 3,5, and the morphological and functional damage of the white matter leads to diffuse axonal injury 6-8. Additional secondary changes commonly seen in the brain are edema and increased intracranial pressure 9. Following TBI there are microscopic alterations in biochemical and physiological pathways involving the release of excitotoxic neurotransmitters, immune mediators and oxygen radicals 10-12, which ultimately result in long-term neurological disabilities 13,14. Thus choosing appropriate animal models of TBI that present similar cellular and molecular events in human and rodent TBI is critical for studying the mechanisms underlying injury and repair.
Various experimental models of TBI have been developed to reproduce aspects of TBI observed in humans, among them three specific models are widely adapted for rodents: fluid percussion, cortical impact and weight drop/impact acceleration 1. The fluid percussion device produces an injury through a craniectomy by applying a brief fluid pressure pulse on to the intact dura. The pulse is created by a pendulum striking the piston of a reservoir of fluid. The percussion produces brief displacement and deformation of neural tissue 1,15. Conversely, cortical impact injury delivers mechanical energy to the intact dura via a rigid impactor under pneumatic pressure 16,17. The weight drop/impact model is characterized by the fall of a rod with a specific mass on the closed skull 18. Among the TBI models, LFP is the most established and commonly used model to evaluate mixed focal and diffuse brain injury 19. It is reproducible and is standardized to allow for the manipulation of injury parameters. LFP recapitulates injuries observed in humans, thus rendering it clinically relevant, and allows for exploration of novel therapeutics for clinical translation 20.
We describe the detailed protocol to perform LFP procedure in mice. The injury inflicted is mild to moderate, with brain regions such as cortex, hippocampus and corpus callosum being most vulnerable. Hippocampal and motor learning tasks are explored following LFP.

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.

Traumatic brain injury (TBI) research has attained renewed momentum due to the increasing awareness of head injuries, which result in morbidity and ...

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

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. Although originally developed and used extensively in the rat due to its larger size, the tibial nerve in mice is big enough that it can be easily manipulated with either crush or transection, leaving the peroneal and sural nerve branches of the sciatic nerve intact and thereby preserving their target muscles. Thus, this model offers the advantages of inducing less morbidity and impediment of ambulation than the sciatic nerve transection model and also allows investigators to study the physiologic, cellular and molecular biologic mechanisms regulating the process of muscle atrophy in genetically engineered mice. The tibial nerve supplies the gastrocnemius, soleus and plantaris muscles, so its transection permits the study of denervated skeletal muscle composed of fast twitch type II fibers and/or slow twitch type I fibers. Here we demonstrate the tibial nerve transection model in the C57Black6 mouse. We assess the atrophy of the gastrocnemius muscle, as a representative muscle, at 1, 2, and 4 weeks post-denervation by measuring muscle weights and fiber type specific cross-sectional area on paraffin-embedded histologic sections immunostained for fast twitch myosin.

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. &#160;

The tibial nerve transection model is a well-tolerated, validated, and reproducible model of denervation-induced skeletal muscle atrophy in rodents. ...

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system. It is usually chronic and challenging to treat. In order to study neuropathic pain and its treatments, different models have been developed in rodents. These models derive from known etiologies, thus reproducing peripheral nerve injuries, central injuries, and metabolic-, infectious- or chemotherapy-related neuropathies. Murine models of peripheral nerve injury often target the sciatic nerve which is easy to access and allows nociceptive tests on the hind paw. These models rely on a compression and/or a section. Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. In this model, a cuff of PE-20 polyethylene tubing of standardized length (2 mm) is unilaterally implanted around the main branch of the sciatic nerve. It induces a long-lasting mechanical allodynia, i.e., a nociceptive response to a normally non-nociceptive stimulus that can be evaluated by using von Frey filaments. Besides the detailed surgery and testing procedures, the interest of this model for the study of neuropathic pain mechanism, for the study of neuropathic pain sensory and anxiodepressive aspects, and for the study of neuropathic pain treatments are also discussed.

Neuropathic pain is a consequence of a lesion or disease affecting the somatosensory system. The &#8220;cuff model&#8221; 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.

Neuropathic pain arises as a consequence of a lesion or a disease affecting the somatosensory system. This syndrome results from maladaptive changes in ...

Traumatic Peripheral Nerve Injury in Mice

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve and denervating muscle have long been a goal in musculoskeletal research. Many traumatically injured limbs have nerve trauma that defines the long-term patient outcome. Over several years, precise methods of producing microsurgical nerve injuries have been developed, including crush, lacerations, and nerve-gap grafting, allowing for reproducible outcome assessments. Moreover, newer methods are created for calibrated crush injuries that offer clinically relevant correlations with outcomes used to assess human patients. The principles of minimal manipulation to ensure low variability in nerve injury allow for adding still more associated tissue injuries into these models. This includes direct muscle crush and other components of limb injury. Finally, atrophy assessment and precise analysis of behavioral outcomes make these methods a complete package for studying musculoskeletal trauma that realistically incorporates all the elements of human traumatic limb injury.

The present protocol describes traumatic peripheral nerve injuries (TPNIs), including precisely calibrated crush, strictly aligned and misaligned laceration, as well as grafted and non-grafted gaps of the sciatic nerve in mice. Custom-designed sensors are developed to gauge nerve trauma, induced with commonly available tools to ensure reproducible post-TPNI outcomes.

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity following orthopedic trauma. Reproducible and precise methods of injuring nerve ...

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

The tibial neuroma transposition (TNT) is a rat model in which allodynia at the neuroma site (tibial nerve) can be independently evaluated from allodynia at the plantar surface of the hind paw innervated by the intact sural nerve. This TNT model is suitable to test therapies for neuroma pain, such as the potential superiority of certain surgical therapies that are already used in the clinic, or to evaluate new drugs and their effect on both pain modalities in the same animal. In this model, a distal lesion (neurotmesis) is made in the tibial nerve, and the proximal nerve end is transposed and fixed subcutaneously and pretibially to enable assessments of the neuroma site with a 15 g Von Frey monofilament. To assess allodynia over the sural nerve, Von Frey monofilaments can be used via the up-down method on the plantar lateral region of the hind paw. After cutting the tibial nerve, mechanical hypersensitivity develops at the neuroma site within 1 week after surgery and persists at least until 12 weeks after surgery. Allodynia at the sural innervated plantar surface develops within 3 weeks after surgery compared to the contralateral limb. At 12 weeks, a neuroma forms on the proximal end of the severed tibial nerve, indicated by dispersion and swirling of axons. For the TNT model surgery, multiple critical (micro)surgical steps need to be followed, and some surgery practice under terminal anesthesia is advised. Compared to other neuropathic pain models, such as the spared nerve injury model, allodynia over the neuroma site can be independently tested from sural nerve hypersensitivity in the TNT model. However, the neuroma site can be tested only in rats, not in mice. The tips and directions provided in this protocol can help research groups working on pain successfully implement the TNT model in their facility.

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.

The tibial neuroma transposition (TNT) is a rat model in which allodynia at the neuroma site (tibial nerve) can be independently evaluated from allodynia ...

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

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

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.

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...

Targeting Mouse Sympathetic Neurons: A Lumbar Sympathectomy Approach

Surgical Lumbar Sympathectomy in Mice

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays many critical roles in maintaining bodily homeostasis, but it has rarely been studied in the context of peripheral nerve injury. The extent of postganglionic sympathetic neuronal functions in distal targets in the periphery is currently unclear. To better explore the role of sympathetic innervation of peripheral targets, a surgical &#34;knock-out&#34; model provides an alternative approach. Although this can be achieved chemically, chemical destruction of postganglionic sympathetic neurons can be nonspecific and dose-dependent. The use of a surgical lumbar sympathectomy in mice, once thought to be &#34;virtually not practicable&#34; in small animals, allows for specific targeting of postganglionic sympathetic neurons that innervate the hind limbs. This manuscript describes how to surgically remove the L2-L5 lumbar sympathetic ganglia from a mouse as a survival surgery, which reliably decreases the hind paw sweat response and the number of sympathetic axons in the sciatic nerve.

Author Spotlight: Exploring Plasticity of Sympathetic Neurons

This manuscript presents a protocol for surgically removing the postganglionic lumbar sympathetic neurons from a mouse. This procedure will facilitate a multitude of studies aimed at investigating the role of sympathetic innervation in distal tissue targets.

Peripheral nerve injuries are common, and full functional recovery after injury is achieved in only 10% of patients. The sympathetic nervous system plays ...

Dual Hind Paw Injection for Enhanced Analgesic Testing

A Modified Inflammatory Pain Model to Study the Analgesic Effect in Mice

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with an intraplantar injection of carrageenan in one hind paw. However, the findings from our laboratory showed that mice with a single-hind-paw injection of carrageenan lifted their paws to avoid thermal nociception during the hot plate test. Because of this response,&#160;previous injection method cannot accurately reflect the thermal pain threshold. Thus, we investigated a new method to avoid this issue. In the present study, we modified the previous method by injecting carrageenan into both hind paws to establish the model of inflammatory pain. The results demonstrated that both-hind-paw injection with carrageenan was sensitive and a better method to induce inflammatory pain when using the hot plate test than single-hind-paw injection. On the basis of these findings, we designed further experiments in which mice with either both-hind-paw or single-hind-paw injection of carrageenan were treated intragastrically with celecoxib (30 mg/kg). The results of the hot plate test showed that celecoxib augmented the thermal pain threshold in mice with both-hind-paw injection of carrageenan but not in mice with single-hind-paw injection of carrageenan. In summary, we developed a superior method to induce a model of inflammatory pain to evaluate analgesic effect.

Author Spotlight: Enhanced Method for Evaluating Analgesic Effects &#8212; Dual Hind Paw Carrageenan Injection in Mice

Here, we present a modified inflammatory pain model with the both-hind-paw carrageenan injection to evaluate the analgesic effect.

The hot plate test is widely used to evaluate analgesic effects on inflammatory pain in mice. A commonly used model of inflammatory pain was induced with ...

Testing Sensory Nerve Function in a Surgically Denervated Mouse Model

Anesthetize the animal. Shave the entire dorsal skin and clean the area similar to the biopsy procedure. Now, make an incision using a sterile scalpel along the dorsal midline from the base of the neck, to roughly half a centimeter above the tail. Using sterile blunt forceps, gently reflect the skin on the left side away from the flank, to visualize the underlying tissue from the scapular fat pads near the neck to just above the hind limb.
Now, identify the dorsal cutaneous nerves under a dissecting light microscope. These appear as white strands that travel caudally through the translucent fascia of the trunk wall before making sharp bends and entering the loose connective tissue underneath the skin. Next, using ultra-fine forceps, remove the nerves exclusively from the left side of the animal located at anatomical sites T3 to T12 by plucking them from where their segments bend at the trunk wall to their entry sites into the skin.
Orient the forceps vertically and grasp the nerves about half a centimeter below their bend sites. Once grasped, pull upwards to cause the nerve to stretch and separate from the surrounding tissue to remove them. Avoid damaging the adjacent blood vessels. Be sure to keep the tissue moist throughout the procedure by periodically applying drops of 0.9% sterile saline solution.
Continue removing all nerves extending from the trunk wall to the skin, but do not disrupt the nerves within the dense fascia of the trunk wall. Next, remove any nerves from the exposed skin flap. These fibers comprise the distal branches of the dorsal cutaneous nerves, and appear as white branching strands located sporadically within the connective tissue on the dermal side of the skin flap.
To remove these fine branches, position the forceps roughly parallel to the dermal surface, grasp the nerves, and pluck upwards. Remove all the visible nerves in this fashion. Do not puncture the blood vessels and skin.
During this step, it&#39;s important to avoid rupturing neighboring blood vessels. If you find a nerve with an adjacent blood vessel, follow it ventrally to an area where there is no adjacent vessel, and remove it by plucking.
Now, bluntly reflect the skin on the right side of the dorsal midline incision, but do not remove any nerves. This will serve as the contralateral sham-operated control. Finally, suture up the animal and monitor postoperatively as before. Later, to confirm the stable loss of nerves weeks after surgery, gently prick the denervated area using a sterile hypodermic needle. Note whether the animal responds typically by shuddering or turning its head. If the skin area has been stably denervated, the animal will exhibit little or no response.