This work was supported by the Centre National de la Recherche Scientifique (contract UPR3212), the University of Strasbourg and by a NARSAD Young Investigator Grant from the Brain &#38; Behavior Research Foundation (to I.Y). Publication costs are supported by the Neurex network (Program Interreg IV Upper Rhine).

Protocols have been approved by the &#34;comit&#233; d&#39;&#233;thique en mati&#232;re d&#39;exp&#233;rimentation animale de Strasbourg&#34; (CREMEAS).
1. Baseline Measurement of Paw Withdrawal Thresholds
<ol>
	<li>Allow the mice to habituate to the animal facilities for at least 10 days to 2 weeks before initiating the testing procedures.</li>
	<li>Habituate the mice to the von Frey testing set-up and to the von Frey procedure that are described in section 4.</li>
	<li>Before surgery, evaluate the mechanical paw withdrawal thresholds with von Frey filaments as described in the section 4.3. Note: Repeat the procedure on separate days until at least three stable consecutive values are obtained for paw withdrawal thresholds.</li>
	<li>Assign the mice to the different experimental groups so that these groups do not initially differ for paw withdrawal thresholds.</li>
</ol>
2. Surgery Procedure for Cuff Implantation
<ol>
	<li>Weigh the animal. Note: Mouse body weight should be over 20 g for the cuff insertion procedure described below.</li>
	<li>Anesthetize the animal with an intraperitoneal injection of 4 ml/kg of a mixture of ketamine (17 mg/ml) and xylazine (2.5 mg/ml) in 0.9% NaCl, which provides around 45 min&#160;of anesthesia.</li>
	<li>Check the absence of paw reflexes by pinching a hind paw with tweezers and check the absence of eye reflexes to make sure that the animal is fully anesthetized.</li>
	<li>Shave the right leg from the knee to the hip using an electrical shaver.</li>
	<li>Apply protective eye liquid gel to the eyes with a cotton-tipped swab.</li>
	<li>Place the animal on its left side and place the right hind limb on a small pillow and maintain the right hind limb to the pillow with adhesive tape.</li>
	<li>Disinfect the surgery field with chlorhexidine and&#160;70% ethanol using gauze pad or cotton-tipped swab.</li>
	<li>Find the femur using the forefinger and make an incision of approximately 0.5 cm, parallel to the femur and approximately 1.5 mm anterior to the femur.</li>
	<li>Separate the muscles close to the femur with two autoclaved sticks. Notes: Never cut the muscle. Normally, the muscle layers separate easily without any bleeding and the sciatic nerve is then visible. In case of bleeding, use a sterile cotton-tipped swab to absorb the blood.</li>
	<li>Insert two autoclaved sticks below the sciatic nerve to expose its main branch, and hydrate the nerve with a sterile physiological solution (0.9% NaCl).</li>
	<li>Hold the pre-prepared sterile 2 mm section of split PE-20 polyethylene tubing (cuff), 0.38 mm ID / 1.09 mm OD, with the help of a pointed steel stick and a bulldog clamp.
	<ol>
		<li>Insert the pointed steel stick into the cuff, which will slightly open it.</li>
		<li>Using the cuff lateral opening, insert the bulldog at one end of the cuff and parallel to the cuff. Rotate the bulldog (180&#176;) so that it will hold the cuff by the side that is opposite to the lateral opening. Close the bulldog and remove the pointed steel stick. Note: The rotation is done to allow holding-on the cuff in an optimized position for the insertion, the bulldog clamp is also helping to maintain the cuff partly open. The model and the size of the bulldog clamp are critical for this step of the procedure.</li>
	</ol>
	</li>
	<li>Have a second experimenter hold the two sticks under the nerve and gently separate the sticks to facilitate the access to a section of sciatic nerve that is around 4 mm long.</li>
	<li>Insert the 2 mm cuff around the main branch of the sciatic nerve, starting by inserting the part of the cuff that is distal to the bulldog around the part of the nerve that is proximal to the hip.</li>
	<li>Close the cuff gently by exerting pressure on its two distal sides with pliers, without squeezing or changing the form of the tube. Turn the cuff to ensure that it is closed correctly.</li>
	<li>Suture the shaved skin layer with surgical knots.</li>
	<li>Place the mouse on its left side in a clean home cage. Keep it under the heat lamp until the mouse is awake.</li>
	<li>Add extra water and place some chow directly in the home cage.</li>
</ol>
3. Surgery Procedure for Sham Controls
<ol>
	<li>Apply the same surgery procedure as described above from step 2.1 to step 2.9, then follow with steps 2.15 to 2.17. For sham controls, omit the steps 2.10 to 2.14 that only concern the cuff insertion.</li>
</ol>
4. von Frey Testing
<ol>
	<li>Place the mice in clear individual boxes (7 cm x 9 cm x 7 cm) with holes, on an elevated perforated plate of smooth stainless steel (1 m x 50 cm, 5 mm circle perforations with 2.5 mm between perforation borders). Note: Up to 12 mice can be concomitantly tested on this set-up. Operated animals can be tested the day after the surgery. However, 3 days of recovery are recommended to diminish the post-surgery hypersensitivity observed in sham controls.</li>
	<li>Allow the animals to habituate for 15 min prior to testing.</li>
	<li>Apply the von Frey filaments to the plantar surface of each hind paw in a series of ascending forces. Notes: The von Frey filaments are plastic hairs of calibrated diameters. They are 5 cm long and are fixed on hand-held applicators. The speed of filament application, the degree of bending and the duration of the application can influence the threshold values that are obtained with this test3. With the present procedure in mice, the filaments that are the most often used are the 0.16, 0.4, 0.6, 1, 1.4, 2, 4, 6, 8, and 10 g.
	<ol>
		<li>Apply the chosen filament to the plantar surface of the left paw until the filament just bends. Repeat the procedure three to five consecutive times, and then do the same to the right paw. Once the filament has been tested on both paws, test the next animal. Notes: Avoid paw lateral borders which can be more sensitive. The expected response is a paw withdrawal, sudden flinching or paw licking. Consider the response as positive if at least three expected responses are observed out of five trials. A given paw is always tested three times, but the fourth and the fifth trials are done only if 1 or 2 response(s) was (were) observed during the first three tests. In C57BL/6J mice, start the pre-surgery tests with the 1.4 g filament. After surgery, start the tests with the 0.4 g filament. If a positive response is observed with the first tested filament, test a filament of lower force (instead of greater) at step 4.3.2.</li>
		<li>Apply the same filament to the next animals according to the 4.3.1 procedure. Once all animals are tested, start again on the first animal with the next filament of greater force. Repeat the procedure until all mice give a positive response. Notes: Test each animal until two consecutive filaments give a positive response. Consider the gram value of the lower filament that gave a positive response as the paw withdrawal threshold for this animal.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

The &#34;cuff&#34; model was initially developed in rats to obtain a standardized and reproducible chronic constriction injury with the implantation of multiple cuffs around the sciatic nerve6. It was then modified to implant a single cuff7,8, even though some research groups still use multiple cuff insertion19-22. It was then adapted to mice9,23, which opened the possibility to use transgenic animals. The cuff is usually 2 mm long, but other lengths have also been used in rats22. The polyethylene tubing depends on the species: PE-20 in mice9, and PE-6024,25 or PE-907,8,26,27 in rats.
The mechanical allodynia is measured with von Frey hairs. In this test, the absolute values for paw withdrawal thresholds may depend upon the surface on which the animal stands28 or upon the duration of filament bending3, but these factors do not affect the detection of the neuropathic allodynia.
The &#34;cuff&#34; model is of interest for the study of neuropathic pain mechanisms. It was used to study morphological changes in myelinated and unmyelinated fibers6,29, and functional changes in sensory neurons, primary afferents and spinal neurons19,21,22,30-35. It allowed demonstration that glial activation and a central shift in neuronal anion gradient participate in changes in the activity and in the responses of spinal nociceptive neurons and in neuropathic allodynia24,36-38. The influence of glutamate receptors7,39-41, of opioid receptors16,42-45 and of nicotinic receptors46 was also studied in this model.
Another interest of the model is its response to current treatments of neuropathic pain, i.e., gabapentinoids and antidepressants. Similar to clinical observations: gabapentinoids display both an acute short-lasting analgesic action at high dose and a delayed sustained relieving action that is observed after a few days of treatment, tricyclic antidepressants and selective serotonin and noradrenaline reuptake inhibitors have no acute analgesic effect at relevant dose but they display a delayed sustained relieving action that requires 1 to 2 weeks of treatment, and the selective serotonin reuptake inhibitor fluoxetine is ineffective16. The model is thus appropriate to study the molecular mechanism underlying these treatments16-18,44,45,47, which may reveal new therapeutic targets to test in patients48-51.
Lastly, the model also allows the study of the anxiodepressive consequences of neuropathic pain. Clinically, these consequences affect around a third of neuropathic pain patients but are preclinically less studied than the sensory aspects of pain. In this model, a time-dependent development of anxiety-like and depressive-like phenotypes is present52 and the related mechanism can thus be addressed.
The standardized cuffs and procedures in this mouse model of neuropathic pain result in low interindividual variability for the mechanical allodynia. The possibility to use genetically modified animals17,18,44-47,52, the long-lasting allodynia, the response to clinically used treatments and the time-dependent development of anxiodepressive symptoms make this model appropriate for the study of the various aspects and consequences of neuropathic pain and its treatments, which have already brought valuable information to this field of research.

Neuropathic pain is usually chronic and arises as a consequence of a lesion or a disease affecting the somatosensory system. Maladaptive changes in injured sensory neurons and along the entire nociceptive pathway within the central nervous system participate in this complex syndrome. Various models have been developed in rodents for studying neuropathic pain and its treatments1-3.
Based on known etiologies, the models of neuropathic pain aim at mimicking the polyneuropathy observed in diabetes, the injuries to peripheral nerves, the central injuries, the trigeminal neuralgia, the neuropathies consecutive to chemotherapy, the post-herpetic neuralgia, etc.&#160;Different models of peripheral nerve injury in rodents focus on the sciatic nerve. These models depend on a compression and/or a section of this nerve. Indeed, the sciatic nerve affords relative easy surgery and allows for tests based on paw withdrawal reflexes. The models of chronic nerve compression include for example: the chronic constriction injury (CCI)4,5, the sciatic nerve cuffing6-9, the partial sciatic nerve ligation (PSL)10, the spinal nerve ligation (SNL)11, or the common peroneal nerve ligation12. Models referred to as &#34;spared nerve injury&#34; (SNI) are also widely used. They consist of a tight ligation and axotomy of two out of the three terminal branches of the sciatic nerve, while the third branch remains intact13-15. The various models of neuropathic pain, which target the sciatic nerve, result in a chronic mechanical allodynia (a nociceptive response to a normally non-nociceptive stimulus) on the injured hind paw.
Here, the detailed surgery procedure for the &#34;cuff model&#34; of neuropathic pain in mice is described. It consists in the implantation of a polyethylene cuff around the main branch of the sciatic nerve6-9. The use of von Frey filaments is also described. These filaments allow assessing the mechanical allodynia which is a long lasting nociceptive symptom present in this model.

<table border="0" cellpadding="0" cellspacing="0" style="width:633px;" width="633">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:111px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PE-20 polyethylene tubing</td>
			<td>Harvard apparatus</td>
			<td>PY2-59-8323</td>
			<td>Splitted before surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ketamine</td>
			<td>Centravet</td>
			<td>IMA004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylazine HCl</td>
			<td>Sigma</td>
			<td>X1251</td>
			<td>Freshly prepared before surgery</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ocry-gel</td>
			<td>Centravet</td>
			<td></td>
			<td></td>
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			<td height="20" style="height:20px;">Plier</td>
			<td>FST</td>
			<td>11003-12</td>
			<td>52.5 mm straight</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Bulldog clamp</td>
			<td>FST</td>
			<td>p130 18038-45</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">Perforated plate</td>
			<td>CTTM</td>
			<td></td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">von Frey filaments</td>
			<td>Bioseb</td>
			<td>NC-12775</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The data are expressed as mean &#177; SEM. Statistical analyses were performed using multi-factor analysis of variance (ANOVA) or unpaired t-tests in accordance with the experimental design. For these analyses, the Sham and Cuff surgery groups as well as the saline vs. drug treatments were considered as between-group factors. When appropriate, repeated measure analyses were used for the time course data. The post-hoc comparisons were performed using the Duncan test. Statistical significance was considered at p&#60;0.05.
When using the procedures that are described above, the cuff implantation results in an ipsilateral allodynia as illustrated in Figure 1. Once the mouse is habituated to the testing procedure, the values for paw withdrawal thresholds in the von Frey test remain stable over time and are not affected by the surgical procedure per se, as illustrated in Sham animals. It should however be noted that a transitory post-surgical allodynia can usually be observed in Sham mice. When such allodynia is present, the paw withdrawal response returns to baseline after a few days post-surgery. In Cuff mice, the ipsilateral allodynia is already present on the first days post-surgery and is maintained for more than 2 months (see 9, and Figure 1; F8,344=29.5, p&#60;0.001). The cuff-induced allodynia remains ipsilateral in C57BL/6J mice when it is measured by the von Frey test as described above, but in other conditions a presence of allodynia on the contralateral paw can also be observed8. The absolute values for baseline are usually between 4 and 6 g in C57BL/6J mice, but the testing protocol may affect these values.
Tricyclic antidepressants are among clinical first-line treatments for neuropathic pain.&#160;In this model, the tricyclic antidepressant drug nortriptyline (5 mg/kg, intraperitoneal, twice a day) relieves the neuropathic allodynia after around 2 weeks of treatment, as illustrated in Figure 2 (F7,91=15.3, p&#60;0.001; post-hoc: (CuffNor=Sham)&#62;CuffSal at p&#60;0.001 on days 29 - 34). At this dose, no acute analgesic action of the antidepressant is observed16,17. To mimic the lasting pain relief that is present in patients taking such drugs, the mice can be tested before the morning drug administration rather than after. Such procedure allows assessment of a long-lasting effect primed by previous days of treatment. In this case, it requires 1 to 2 weeks of treatment to observe a lasting relief of the neuropathic allodynia. When the treatment is interrupted, a relapse is usually observed within 3 to 4 days18. Beside some antidepressants, gabapentinoids are the other first-choice treatments for neuropathic pain. Gabapentin has an acute and transitory analgesic action in this model16, but it also displays a delayed and long-lasting antiallodynic action when testing the animal each day before the drug administration (Figure 3; p&#60;0.001). This action is faster than with antidepressant drugs.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51608/51608fig1highres.jpg" src="/files/ftp_upload/51608/51608fig1.jpg" /> 
Figure 1. Mechanical paw withdrawal thresholds in the cuff model of neuropathic pain in mice. Adult male C57BL/6J mice were habituated to the von Frey procedure until a stable baseline was obtained (the baseline is represented at point 0 on the graph). Both paws were tested. The Cuff mice display ipsilateral mechanical allodynia as showed by the lowered paw withdrawal thresholds (n=10 per group).
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51608/51608fig2highres.jpg" src="/files/ftp_upload/51608/51608fig2.jpg" /> 
Figure 2. Delayed antiallodynic action of a tricyclic antidepressant. After two weeks post-surgery, mice received intraperitoneal treatment twice a day (morning and evening) with either 0.9% NaCl or 5 mg/kg nortriptyline hydrochloride (n=5 or 6 per group). The von Frey test was done before the morning treatment. With this procedure, a delayed antiallodynic action of nortriptyline is observed, which requires around 12 days of treatment.
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51608/51608fig3highres.jpg" src="/files/ftp_upload/51608/51608fig3.jpg" /> 
Figure 3. Antiallodynic action of a gabapentinoid. After three weeks post-surgery, mice received intraperitoneal treatment twice a day (morning and evening) with either 0.9% NaCl or 10 mg/kg gabapentin (n=5 per group). The von Frey test was done before the morning treatment. With this procedure, a delayed and lasting antiallodynic action of gabapentin is observed. Data are presented before starting the treatments and at the 6th day of treatments.

Watch this Scientific Journal Video about The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice at JoVE.com

The Sciatic Nerve Cuffing Model of Neuropathic Pain in Mice

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 ...

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47.9K Views.  Centre National de la Recherche Scientifique.  Невропатическая боль является следствием поражения или заболевания, влияющего на систему соматосенсорной. "Манжеты модель" невропатической боли у мышей состоит из имплантации полиэтиленовой манжеты вокруг главной ветви седалищного нерва. Механическ?...

Video: The Sciatic Nerve Cuffing 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 ...

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie's (1988) unilateral sciatic nerve chronic constriction injury (CCI)3 (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.
CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.
For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs4. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.

Due to the simplicity of surgery and the robust behavioural outcome, chronic constriction of the sciatic nerve is one of the pre-eminent animal models of neuropathic pain. Within 24 hrs following surgery, pain hypersensitivity is established and can be quantitatively measured using a von Frey aesthesiometer (mechanical test) and plantar analgesia meter (thermal test).

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of ...

Breathing-controlled Electrical Stimulation (BreEStim) for Management of Neuropathic Pain and Spasticity

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. It has been extensively used in various clinical settings. Based upon recent discoveries related to the systemic effects of voluntary breathing and intrinsic physiological interactions among systems during voluntary breathing, a new EStim protocol, Breathing-controlled Electrical Stimulation (BreEStim), has been developed to augment the effects of electrical stimulation. In BreEStim, a single-pulse electrical stimulus is triggered and delivered to the target area when the airflow rate of an isolated voluntary inspiration reaches the threshold. BreEStim integrates intrinsic physiological interactions that are activated during voluntary breathing and has demonstrated excellent clinical efficacy. Two representative applications of BreEStim are reported with detailed protocols: management of post-stroke finger flexor spasticity and neuropathic pain in spinal cord injury.

Breathing-controlled Electrical Stimulation BreEStim for Management of Neuropathic Pain and Spasticity

The purpose is to present a new method, breathing-control electrical stimulation (BreEStim) for management of neuropathic pain and spasticity.

Electrical stimulation (EStim) refers to the application of electrical current to muscles or nerves in order to achieve functional and therapeutic goals. ...

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. ...

Urinary Bladder Distention Evoked Visceromotor Responses as a Model for Bladder Pain in Mice

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized by increased urgency and frequency of urination, as well as nocturia and general pelvic pain, especially upon bladder filling or voiding. Despite years of research, the cause of IC/BPS remains elusive and treatment strategies are unable to provide complete relief to patients. In order to study nervous system contributions to the condition, many animal models have been developed to mimic the pain and symptoms associated with IC/BPS. One such murine model is urinary bladder distension (UBD). In this model, compressed air of a specific pressure is delivered to the bladder of a lightly anesthetized animal over a set period of time. Throughout the procedure, wires in the superior oblique abdominal muscles record electrical activity from the muscle. This activity is known as the visceromotor response (VMR) and is a reliable and reproducible measure of nociception. Here, we describe the steps necessary to perform this technique in mice including surgical manipulations, physiological recording, and data analysis. With the use of this model, the coordination between primary sensory neurons, spinal cord secondary afferents, and higher central nervous system areas involved in bladder pain can be unraveled. This basic science knowledge can then be clinically translated to treat patients suffering from IC/BPS.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition characterized in part by pelvic pain. In order to study nervous system contributions to the condition, a physiological model of urinary bladder pain is used in mice and rats.

Approximately 3-8 million people in the United States suffer from interstitial cystitis/bladder pain syndrome (IC/BPS), a debilitating condition ...

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

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.

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.

The goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and ...

Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral procedures to study trigeminal neuropathic pain in rats. To meet the specificity of trigeminal neuropathic pain syndromes, the infraorbital nerve (IoN) is subjected to a chronic constriction injury (CCI) by loosely ligating the nerve. An intra-orbital approach is presented here to expose and ligate the IoN in the orbital cavity. After IoN ligation, rats exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are indicative of persistent pain and mechanical allodynia. Two phases can be defined in the development of the behavioral changes. During the first week following IoN-CCI (phase 1), rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. A distinction is made between face grooming behavior that is part of a more general body grooming behavior, which remains largely unaffected by IoN-CCI, and face grooming that is neither preceded nor followed by body grooming, which is significantly increased after IoN-CCI. During this period, responsiveness to mechanical stimulation of the IoN territory is reduced. This hyporesponsiveness is abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior (phase 2). The phenomenological similarities between these behavioral alterations and reported signs of facial pain (i.e., responses to noxious stimulation of the face) suggest the presence of dysesthesia/paresthesia and mechanical allodynia in the ligated IoN territory.

Chronic Constriction Injury of the Rat&#039;s Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain

This manuscript describes a rodent model to study trigeminal neuropathic pain. The methods described include surgical procedures to perform a chronic constriction injury of the rat&#8217;s infraorbital nerve and the post-surgical behavioral tests to measure the changes in spontaneous and evoked behavior that are indicative of persistent pain and allodynia.

Animal models are important tools to study the pathophysiology and pharmacology of neuropathic pain. This manuscript describes the surgical and behavioral ...

The Monoiodoacetate Model of Osteoarthritis Pain in the Mouse

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The current treatments for OA pain such as NSAIDS or opiates are neither sufficiently effective nor devoid of detrimental side effects. Animal models of OA are being developed to improve our understanding of OA-related pain mechanisms and define novel pharmacological targets for therapy. Currently available models of OA in rodents include surgical and chemical interventions into one knee joint. The monoiodoacetate (MIA) model has become a standard for modelling joint disruption in OA in both rats and mice. The model, which is easier to perform in the rat, involves injection of MIA into a knee joint that induces rapid pain-like responses in the ipsilateral limb, the level of which can be controlled by injection of different doses. Intra-articular injection of MIA disrupts chondrocyte glycolysis by inhibiting glyceraldehyde-3-phosphatase dehydrogenase and results in chondrocyte death, neovascularization, subchondral bone necrosis and collapse, as well as inflammation. The morphological changes of the articular cartilage and bone disruption are reflective of some aspects of patient pathology. Along with joint damage, MIA injection induces referred mechanical sensitivity in the ipsilateral hind paw and weight bearing deficits that are measurable and quantifiable. These behavioral changes resemble some of the symptoms reported by the patient population, thereby validating the MIA injection in the knee as a useful and relevant pre-clinical model of OA pain.
The aim of this article is to describe the methodology of intra-articular injections of MIA and the behavioral recordings of the associated development of hypersensitivity with a mind to highlight the necessary steps to give consistent and reliable recordings.

Osteoarthritis (OA), or degenerative joint disease, is a debilitating condition associated with pain that remains only partially controlled by available analgesics. Animal models are being developed to improve our understanding of OA-related pain mechanisms. Here we describe the methodology for the monoiodoacetate model of OA pain in the mouse.

A major symptom of patients with osteoarthritis (OA) is pain that&#160;is triggered by peripheral as well as central changes within the pain pathways. The ...

Subcutaneous Trigeminal Nerve Field Stimulation for Refractory Facial Pain

Chronic or neuropathic trigeminal facial pain can be challenging to treat. Neurosurgical procedures should be applied when conservative treatment fails. Neuromodulation techniques for chronic facial pain include deep brain stimulation and motor cortex stimulation, which are complex to perform. Subcutaneous nerve field stimulation is certified for chronic back pain and is the least invasive form of neuromodulation. We applied this technique to treat chronic and neuropathic trigeminal pain as an individual therapy concept. First, trial stimulation is performed. Subcutaneous leads are placed in the painful trigeminal dermatome under local anesthesia. The leads are connected to an external neurostimulator that applies constant stimulation. Patients undergo a 12 day outpatient trial to assess the effect of the stimulation. Electrodes are removed after the trial. If the patient reports pain reduction of at least 50% in intensity and/or attack frequency, a reduction in medication or increase in quality of life, permanent implantation is scheduled. New electrodes are implanted under general anesthesia and are subcutaneously tunneled to an infraclavicular internal pulse generator. Patients are able to turn stimulation on and off and to increase or decrease the stimulation amplitude as needed.
This technique represents a minimal invasive alternative to other more invasive means of neuromodulation for trigeminal pain such as motor cortex stimulation or deep brain stimulation.

Treating chronic or neuropathic facial pain can be challenging when medical or standard treatment fails. Subcutaneous nerve field stimulation is the least invasive form of neuromodulation and is used for chronic back pain. We applied this technology to treat chronic and neuropathic trigeminal facial pain.

Chronic or neuropathic trigeminal facial pain can be challenging to treat. Neurosurgical procedures should be applied when conservative treatment fails. ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...