The authors acknowledge the work that was done by the first author of the IoN-CCI model, Bart Vos&#8224;.

Ethics statement: Animals are treated and cared for according to the guidelines of the Committee for Research and Ethical Issues of IASP11. The protocol is approved by the institutional Ethical Committee.
1. Subjects
<ol>
	<li>Use male Sprague-Dawley rats (250-300 g at arrival).</li>
	<li>House rats in solid-bottom cages in a colony room with a humidity of 40-60% and a RT of 21 &#177; 1 &#176;C.</li>
	<li>Make water and food available at libitum.</li>
	<li>Keep rats under a reversed 12:12 hr dark/light cycle (lights on at 20 hr).</li>
</ol>
2. Surgery
<ol>
	<li>Per rat, prepare two pieces of chromic gut ligature (5-0) of approximately 6 cm long and place them in sterile saline to avoid drying and becoming stiff and brittle.</li>
	<li>Anesthetize the animal with pentobarbital (60 mg/kg, i.p.) and treat with atropine (0.1 mg/kg, i.p.). Monitor the level of anesthesia by extending one leg and pinching the web of skin between the toes with a fingernail to ensure adequate pressure is applied. Ensure that the animal does not flex its limb. If necessary, administer additional pentobarbital.</li>
	<li>Shave the head of the rat so that a mid-line scalp incision can be made of approximately 25 mm with equal distances anterior and posterior to the center of the eyes.</li>
	<li>Fix the rat&#8217;s head in a stereotaxic frame or otherwise fixate the rat&#8217;s head. Place the rat on a heated pad to maintain body temperature.</li>
	<li>Apply ophthalmic ointment on both eyes to prevent damage from drying.</li>
	<li>Scrub the shaved head area with alcohol, then betadine.</li>
	<li>Make a mid-line scalp incision, exposing skull and nasal bone.</li>
	<li>Use an operation microscope to perform steps 2.9 to 2.16.</li>
	<li>Dissect the edge of the orbit free, formed by the maxillary, frontal, lacrimal and zygomatic bones, using a cotton-tipped swab and a pair of Dumont forceps. Use the swab to soak up blood from possible bleeding.</li>
	<li>To give access to the IoN, gently deflect the orbital contents with a precision cotton swab, taking care not to overstretch the anterior ethmoidal nerve which crosses superior to the IoN. Dissect the IoN free from the surrounding connective tissue, using a precision cotton-tipped swab and a pair of Dumont forceps using a spreading motion. Use the swab to soak up blood from possible bleeding.</li>
	<li>To perform a ligation, push one end of a single piece of chromic gut downward along the frontal bone, medial to the IoN and advance a few millimeters parallel and inferior to the IoN. The ligature is now held in place between the IoN and the frontal bone.</li>
	<li>Slip the tip of a 45 degree angled Dumont forceps under the IoN to place it in close proximity to the ligature. Gently retract the IoN laterally to reveal the ligature and tip of the forceps. Grip the ligature with the forceps and withdraw the forceps laterally from under the IoN.</li>
	<li>Pull out the ligature from the orbital cavity until both ends of the ligature are more or less equidistant from the IoN.</li>
	<li>Make a &#8220;slip knot&#8221; from the two ends of the ligature to allow perfect control over the degree of constriction and slide the knot against the IoN. Slide the knot further to constrict the IoN so that the diameter of the nerve is reduced by a just noticeable amount. Make a normal knot on top of the slip knot so that the amount of constriction remains constant (i.e., to prevent the slit knot from slipping).</li>
	<li>Cut the ligatures to leave approximately 2 mm of free ends from the knot.</li>
	<li>Repeat the same procedure (steps 2.11 to 2.15) to make a second ligation 2 mm apart from the first.</li>
	<li>For sham surgery, perform steps 2.2 to 2.10.</li>
	<li>Close the scalp incision using polyester sutures (4-0) and allow the rat to recover on a heated pad. Do not leave the animal unattended until it has regained sufficient consciousness to maintain sternal recumbency and do not return the animal to the company of other animals until it is fully recovered.</li>
	<li>To avoid pre-emptive analgesic effects on the development of neuropathic pain, do not administer analgesia for post-surgical pain.</li>
</ol>
3. Behavioral Test Procedure
<ol>
	<li>Allow rats to acclimate for at least 8 days to the housing conditions before pre-operative testing.</li>
	<li>Habituate rats to the test procedure at least once daily for three days before pre-operative testing.</li>
	<li>Conduct testing in a darkened room with light provided by a 60 W red light bulb suspended 1 m above the center of the test area lighting the center but leaving the circumference dim and the remainder of the room dark.</li>
	<li>Make sure there is sufficient background noise to decrease interference from sudden auditory stimulation. If necessary, provide white noise.</li>
	<li>Observation of face grooming behavior
	<ol>
		<li>Transport a single rat from the colony to the test room in a covered plastic cage without bedding. Note: Ideally, this should be a short trip with minimal external stimuli.</li>
		<li>Place the rat in a covered, transparent plastic cage without bedding (l x w x h: 24 cm x 14 cm x 17 cm) with a mirrored back in front of a video camera.</li>
		<li>Make a 10 min recording of the animal&#8217;s behavior. During recording, make sure the experimenter is not present in the test room.</li>
		<li>Clean the observation cage before placing the next animal.</li>
		<li>Make sure the recorded behavior is analyzed by an experimenter who is blind to the experimental condition of the rat.</li>
		<li>Make a record of each face grooming episode during the 10 min recording. Note: Face grooming is defined as movement patterns in which forepaws are brought in contact with facial areas.</li>
		<li>Make a distinction between two types of face grooming behavior. If a sequence is neither preceded nor followed by body grooming (i.e., grooming of a body area other than the face), categorize the episode as isolated face grooming. If body grooming is performed immediately before or after face grooming, categorize the episode as face grooming during body grooming.</li>
		<li>Determine the number of face grooming episodes.
		<ol>
			<li>Categorize a time period between face grooming actions that does not exceed 4 sec as an intra-episode space (i.e., a pause between face grooming actions within a single episode).</li>
			<li>Categorize a time period between face grooming actions that exceeds 4 sec as an inter-episode space (i.e., a complete interruption of face grooming actions between two episodes).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Mechanical stimulation testing
	<ol>
		<li>Transport rats in groups of up to 6 animals from the colony to the test room in a covered cage with bedding. Ensure that this is a short trip with minimal external stimuli.</li>
		<li>Place the rats individually in transparent plastic cages with bedding (l x w x h: 24 cm x 14 cm x 17 cm) with a hinged lid so that it can be easily opened and closed.</li>
		<li>Use a graded series of five von Frey hairs. Note: The force required to bend the hairs is 0.015 g, 0.127 g, 0.217 g, 0.745 g and 2.150 g.</li>
		<li>Habituate the rats to the observation cage and reaching movements for 10 min. Every 30 sec, open the lid and gently touch the wall of the cage with a plastic rod.</li>
		<li>When the animal is in a sniffing/ no locomotion state (i.e.,&#160;with four paws placed on the ground, neither moving nor freezing, but exhibiting sniffing behavior), apply the first von Frey hair within the IoN territory near the center of the vibrissal pad, on the hairy skin surrounding the mystacial vibrissae.</li>
		<li>Score the response of the animal to the stimulation to belong to one of the following response categories4.
		<ol>
			<li>Categorize a complete lack of response as score 0.</li>
			<li>Categorize a stimulus detection (i.e., the rat turns the head toward the stimulating object and the stimulus object is then explored) as score 1.</li>
			<li>Categorize a withdrawal reaction (i.e., the rat turns the head slowly away or pulls it briskly backward when the stimulation is applied, sometimes a single face wipe ipsilateral to the stimulated area occurs) as score 2.</li>
			<li>Categorize an escape/attack response (i.e., the rat avoids further contact with the stimulus object, either passively by moving its body away from the stimulating object to assume a crouching position against the cage wall, or actively by attacking the stimulus object, making biting and grabbing movements) as score 3.</li>
			<li>Categorize asymmetric face grooming (i.e., the rat displays an uninterrupted series of at least three face-wash strokes directed toward the stimulated facial area) as score 4.</li>
			<li>Within each animal, apply stimuli in an ascending order of intensity. After a stimulus intensity has been applied to one side, apply it to the other side before moving on to the next stimulus intensity. Randomize the order in which the ipsilateral and contralateral sides are stimulated. Note that the animal must be in a sniffing/ no locomotion state before each stimulation.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Jensen, T. S., Gottrup, H., Sindrup, S. H., Bach, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+clinical+picture+of+neuropathic+pain.">The clinical picture of neuropathic pain.</a> Eur J Pharmacol. 429 (1-3), 1-11 (2001).</li><li>Meyer Rosberg, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathic+pain+a+multidimensional+burden+for+patients.">Neuropathic pain a multidimensional burden for patients.</a> Eur J Pain. 5 (4), 379-389 (2001).</li><li>Meyer Rosberg, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+SF+36+and+Nottingham+Health+Profile+in+patients+with+chronic+neuropathic+pain.">Comparison of the SF 36 and Nottingham Health Profile in patients with chronic neuropathic pain.</a> Eur J Pain. 5 (4), 391-403 (2001).</li><li>Vos, B. P., Strassman, A. M., Maciewicz, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+evidence+of+trigeminal+neuropathic+pain+following+chronic+constriction+injury+to+the+rats+infraorbital+nerve.">Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rats infraorbital nerve.</a> J Neurosci. 14, 2708-2723 (1994).</li><li>Clavelou, P., Pajot, J., Dallel, R., Raboisson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+formalin+test+to+the+study+of+orofacial+pain.">Application of the formalin test to the study of orofacial pain.</a> Neurosci Lett. 103, 349-353 (1989).</li><li>Vos, B. P., Hans, G., Adriaensen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+assessment+of+facial+pain+in+rats+face+grooming+patterns+after+painful+and+non-painful+sensory+disturbances+in+the+territory+of+the+rats+infraorbital+nerve.">Behavioral assessment of facial pain in rats face grooming patterns after painful and non-painful sensory disturbances in the territory of the rats infraorbital nerve.</a> Pain. 76 (1-2), 173-178 (1998).</li><li>Deseure, K. R., Adriaensen, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+between+two+types+of+behavioral+variables+of+non+evoked+facial+pain+after+chronic+constriction+injury+to+the+rat+infraorbital+nerve.">Comparison between two types of behavioral variables of non evoked facial pain after chronic constriction injury to the rat infraorbital nerve.</a> Comp Med. 52 (1), 44-49 (2002).</li><li>Deseure, K., Adriaensen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonevoked+facial+pain+in+rats+following+infraorbital+nerve+injury+a+parametric+analysis.">Nonevoked facial pain in rats following infraorbital nerve injury a parametric analysis.</a> Physiol Behav. 81 (4), 595-604 (2004).</li><li>Deseure, K., Hans, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+study+of+non+evoked+orofacial+pain+following+different+types+of+infraorbital+nerve+injury+in+rats.">Behavioral study of non evoked orofacial pain following different types of infraorbital nerve injury in rats.</a> Physiol Behav. 138, 292-296 (2015).</li><li>Deseure, K., Koek, W., Adriaensen, H., Colpaert, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+administration+of+the+5+hydroxytryptamine1A+agonist+(3-Chloro-4-fluoro-phenyl)-[4-fluoro-4-[[(5-methyl-pyridin-2-ylmethyl)+-amino]-methyl]piperidin-1-yl]-methadone+(F+13640)+attenuates+allodynia+like+behavior+in+a+rat+model+of+trigeminal+neuropathic+pain.">Continuous administration of the 5 hydroxytryptamine1A agonist (3-Chloro-4-fluoro-phenyl)-[4-fluoro-4-[[(5-methyl-pyridin-2-ylmethyl) -amino]-methyl]piperidin-1-yl]-methadone (F 13640) attenuates allodynia like behavior in a rat model of trigeminal neuropathic pain.</a> J Pharmacol Exp Ther. 306 (2), 505-514 (2003).</li><li>Zimmerman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethical+Guidelines+for+investigations+of+experimental+pain+in+conscious+animals.">Ethical Guidelines for investigations of experimental pain in conscious animals.</a> Pain. 16, 109-110 (1983).</li><li>Mogil, J. S., Crager, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+should+we+be+measuring+in+behavioral+studies+of+chronic+pain+in+animals.">What should we be measuring in behavioral studies of chronic pain in animals.</a> Pain. 112 (1-2), 12-15 (2004).</li><li>Backonja, M. M., Stacey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathic+pain+symptoms+relative+to+overall+pain+rating.">Neuropathic pain symptoms relative to overall pain rating.</a> J Pain. 5 (9), 491-497 (2004).</li><li>Imamura, Y., Kawamoto, H., Nakanishi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+heat+hyperalgesia+in+an+experimental+trigeminal+neuropathy+in+rats.">Characterization of heat hyperalgesia in an experimental trigeminal neuropathy in rats.</a> Exp Brain Res. 116, 97-103 (1997).</li><li>Chichorro, J. G., Zampronio, A. R., Souza, G. E., Rae, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orofacial+cold+hyperalgesia+due+to+infraorbital+nerve+constriction+injury+in+rats+reversal+by+endothelin+receptor+antagonists+but+not+non+steroidal+anti+inflammatory+drugs.">Orofacial cold hyperalgesia due to infraorbital nerve constriction injury in rats reversal by endothelin receptor antagonists but not non steroidal anti inflammatory drugs.</a> Pain. 123 (1-2), 64-74 (2006).</li><li>Kramer, P. R., Bellinger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meal+Duration+as+a+Measure+of+Orofacial+Nociceptive+Responses+in+Rodents.">Meal Duration as a Measure of Orofacial Nociceptive Responses in Rodents.</a> J Vis Exp. (83), e50745 (2014).</li><li>Rossi, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+bilateral+trigeminal+constriction+injury+using+an+operant+facial+pain+assay.">Characterization of bilateral trigeminal constriction injury using an operant facial pain assay.</a> Neuroscience. 224, 294-306 (2012).</li><li>Anderson, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+Operant+Orofacial+Pain+Assessment+Device+(OPAD)+to+Measure+Changes+in+Nociceptive+Behavior.">Use of the Operant Orofacial Pain Assessment Device (OPAD) to Measure Changes in Nociceptive Behavior.</a> J Vis Exp. (76), 50336 (2013).</li></ol>

The authors have nothing to disclose.

The most interesting aspect of the IoN-CCI model is that it provides a unique measure of spontaneous, non-evoked (neuropathic) pain. Most animal studies of neuropathic pain, using spinal nerve models, exclusively address nocifensive behaviors (e.g., vocalization and withdrawal responses), reflecting hypersensitivity to mechanical or thermal stimulations12. However, the chief clinical complaint in patients is ongoing pain, not hyperalgesia or allodynia13.
The pres...

Damage to somatosensory nerves most often leads to loss of sensation. It is not very well understood why and how, in some cases, peripheral nerve damage leads to chronic pain. As a result, painful neuropathies remain a challenging condition to treat1 and result in substantial reductions in patients&#8217; quality of life2-3. Animal models are an important tool to examine the efficacy of existing and newly developed drugs and to identify the pathophysiological mechanisms involved in the development and maintenance of neuropathic pain. Among the key challenges for translational pain research is identifying and measuring spontaneous pain in animals (i.e., physiological, behavioral or other manifestations that indicate the presence of pain in animals that are not being challenged with probing stimulations that are acutely superimposed on the subject&#8217;s basal condition). The overall goal of this procedure is to surgically induce an infraorbital nerve injury in a rat that leads to the development of both spontaneous and evoked nocifensive behavior that can used to study the mechanisms involved in trigeminal neuropathic pain and its treatment.
Directed, isolated face grooming is a unique measure of spontaneous neuropathic pain in rats. Following chronic constriction injury (CCI) of the infraorbital nerve (IoN), rats exhibit changes in spontaneous behavior and in response to tactile stimulation that are indicative of persistent pain and mechanical allodynia4. Following IoN-CCI, rats show an increased and asymmetric face grooming activity, i.e., with face wash strokes primarily directed to the nerve-injured IoN territory. In contrast to noxious stimulation of the face (e.g., formalin injection)5, non-painful sensory disturbances (i.e., unilateral vibrissae clipping, anesthetic infraorbital nerve blockade, application of mineral oil on vibrissae) did not or only very briefly (i.e., the first minutes after onset) induce a significant change in face grooming behavior6. It was concluded that this abnormal grooming after IoN-CCI is a behavioral manifestation of spontaneous, strongly aversive sensations in the injured nerve territory. This behavior in IoN-CCI rats bears strong resemblance to spontaneous pain observed in patients. Further studies have identified an important difference between face grooming behavior that is part of a more general body grooming behavior (i.e., &#8220;face grooming during body grooming&#8221;), which remains largely unaffected by IoN-CCI, and nocifensive face grooming that is neither preceded nor followed by body grooming (i.e., &#8220;isolated face grooming&#8221;), which is significantly increased after IoN-CCI7-9. Pharmacologically, face grooming during body grooming constitutes an excellent control measurement for non-specific drug effects (e.g., sedation) that may impair motor function. Movements of the forelimbs during the two types of face grooming are exactly the same.
Following IoN-CCI, rats also exhibit changes in response to tactile stimulation that are indicative of mechanical allodynia4, 10. During the first week(s) after IoN-CCI, rats no longer respond to mild or moderate tactile (i.e., von Frey) stimuli. This hyporesponsiveness is then abruptly replaced by an extreme hyperresponsiveness whereby even very weak stimulus intensities provoke nocifensive behavior. The onset of hyperresponsiveness may vary between rats, but most rats have become hyperresponsive after three to four weeks post-operative.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Chromic catgut</td>
			<td>Dynek&#160;</td>
			<td>CG504</td>
			<td></td>
		</tr>
		<tr>
			<td>Nembutal&#160; (pentobarbital)</td>
			<td>Pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine</td>
			<td>Pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clippers for small animals - Favorita II</td>
			<td>Aesculap</td>
			<td>GT104</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic instrument</td>
			<td>Stoelting</td>
			<td>51600</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophthalmic ointment (Liposic)</td>
			<td>Pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic monitor system</td>
			<td>Harvard Apparatus</td>
			<td>PY8 50-7221-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton applicator</td>
			<td>Pharmacy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Precision cotton swab</td>
			<td>Qosina</td>
			<td>10225</td>
			<td></td>
		</tr>
		<tr>
			<td>Operation microscope</td>
			<td>Kaps</td>
			<td>SOM 62</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps - Micro-blunted tips (#5/45)</td>
			<td>Fine Science Tools</td>
			<td>11253-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors - blunt tips</td>
			<td>Fine Science Tools</td>
			<td>14574-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyester sutures - Mersilene (4-0)</td>
			<td>Ethicon</td>
			<td>R871H</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital video camera</td>
			<td>Sony</td>
			<td>HDR-CX330E</td>
			<td></td>
		</tr>
		<tr>
			<td>Semmes-Weinstein Von Frey Aesthesiometer kit</td>
			<td>Stoelting</td>
			<td>58011</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

IoN-CCI rats show a strong post-operative increase in the amount of time spent on isolated face grooming (Figure 1). The increase peaks at the end of the first post-operative week and then declines during the following weeks. Most recent studies have found face grooming to be significantly increased for up to three weeks. Face grooming behavior in sham operated rats is more or less unaffected.
IoN-CCI rats also show an almost complete lack of responsiveness to ipsilateral mech...

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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.

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 ...

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19.3K vistas.  University of Antwerp. 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’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. 

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

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. ...

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 ...

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 ...

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 ...

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. ...

Development of Recombinant Proteins to Treat Chronic Pain

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates for treating debilitating pain conditions due to their capacity to regulate aberrant neuro-immune interactions. However, physiologically they work in a network of various cytokines, and therefore their therapeutic effect may not be optimal when used as stand-alone drugs. To overcome this limitation, we developed a fusion protein of the anti-inflammatory cytokines IL4 and IL10. Here, we describe the methods for production and quality control of IL4-10 recombinant fusion protein and we test the effectiveness of the IL4-10 fusion protein to resolve pain in a mouse model of persistent inflammatory pain.

In this paper we provide details for the methods of production and quality control of an IL4-10 recombinant fusion protein. We also show how to test the effectiveness of this protein to resolve pain in a mouse model of inflammatory pain.

Chronic pain is difficult to treat and new approaches to resolve persistent pain are urgently needed. Anti-inflammatory cytokines are promising candidates ...

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. ...

Improving FSN Therapy in the Rat Model&amp;#58; Minimizing Discomfort and Maximizing Efficiency

Efficacy of Fu's Subcutaneous Needling on Sciatic Nerve Pain: Behavioral and Electrophysiological Changes in a Chronic Constriction Injury Rat Model

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the mechanisms of action are still not fully understood. During FSN treatment, the FSN needle is inserted and retained in the subcutaneous tissues for a long duration with a swaying movement. However, challenges arise from maintaining a posture while manipulating FSN in animal models (e.g., rats) for researchers. Uncomfortable treatment can lead to fear and resistance to FSN needles, increasing the risk of injury and may even affect research data. Anesthesia may also affect the study results too. Hence, there is a need for strategies in FSN therapy on animals that minimize injury during the intervention. This study employs a chronic constriction injury model in Sprague-Dawley rats to induce neuropathic pain. This model replicates the pain induced by nerve injury observed in humans through surgical constriction of a peripheral nerve, mimicking the compression or entrapment seen in conditions such as nerve compression syndromes and peripheral neuropathies. We introduce an appropriate manipulation for easily inserting an FSN needle into the subcutaneous layer of the animal&#39;s body, including needle insertion and direction, needle retention, and swaying movement. Minimizing the rat&#39;s discomfort prevents the rat from being tense, which causes the muscle to contract and hinder the entry of the needle and improves the study efficiency.

Author Spotlight: Unveiling the Therapeutic Effects of FSN Treatment &amp;#8211; Bridging Research and Clinical Applications in Neuropathic Pain

We present a protocol for using Fu's subcutaneous needling in a chronic constriction injury model to induce sciatic nerve pain in rats.

Fu&#39;s subcutaneous needling (FSN), an invented acupuncture technique from traditional Chinese medicine, is used worldwide for pain relief. However, the ...

Standardized Tuina Protocol for Rat DRG Compression Model

Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar disc herniation, spinal canal stenosis, and intervertebral foramen stenosis. Although Tuina, a traditional Chinese manual therapy, has shown analgesic effects in clinical practice for the treatment of neuropathic pain, its underlying neurobiological mechanisms remain unclear. Animal models are essential for elucidating the basic principles of Tuina. In this study, we propose a standardized Tuina protocol for rats with compression of the dorsal root ganglion (DRG), which involves inducing DRG compression by inserting a stainless steel rod into the intervertebral foramen, performing Tuina manipulation with specific parameters of location, intensity, and frequency in a controlled environment, and assessing the behavioral and histopathological outcomes of Tuina treatment. This article also discusses the potential clinical implications and limitations of the study and suggests directions for future research on Tuina.

Author Spotlight: Analgesic Effect of Tuina on Rat Models with Compression of the Dorsal Root Ganglion Pain  

This article presents a manipulation for treating chronic compression of the dorsal root ganglion in rats using Tuina therapy, along with a method for evaluating its effectiveness based on pain behavior and histopathological results.

Neuropathic pain is a prevalent condition that affects 6.9%-10% of the population and results from nerve damage due to various etiologies, such as lumbar ...