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/pubmed?term=((The+clinical+picture+of+neuropathic+pain%5BTitle%5D)+AND+%22Eur+J+Pharmacol%22%5BJournal%5D)">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/pubmed?term=((Neuropathic+pain+a+multidimensional+burden+for+patients%5BTitle%5D)+AND+%22Eur+J+Pain%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+the+SF+36+and+Nottingham+Health+Profile+in+patients+with+chronic+neuropathic+pain%5BTitle%5D)+AND+%22Eur+J+Pain%22%5BJournal%5D)">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/pubmed?term=((Behavioral+evidence+of+trigeminal+neuropathic+pain+following+chronic+constriction+injury+to+the+rats+infraorbital+nerve%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">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/pubmed?term=((Application+of+the+formalin+test+to+the+study+of+orofacial+pain%5BTitle%5D)+AND+%22Neurosci+Lett%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">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/pubmed?term=((Comparison+between+two+types+of+behavioral+variables+of+non+evoked+facial+pain+after+chronic+constriction+injury+to+the+rat+infraorbital+nerve%5BTitle%5D)+AND+%22Comp+Med%22%5BJournal%5D)">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/pubmed?term=((Nonevoked+facial+pain+in+rats+following+infraorbital+nerve+injury+a+parametric+analysis%5BTitle%5D)+AND+%22Physiol+Behav%22%5BJournal%5D)">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/pubmed?term=((Behavioral+study+of+non+evoked+orofacial+pain+following+different+types+of+infraorbital+nerve+injury+in+rats%5BTitle%5D)+AND+%22Physiol+Behav%22%5BJournal%5D)">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/pubmed?term=((Continuous+administration+of+the+5+hydroxytryptamine1A+agonist+%283-Chloro-4-fluoro-phenyl%29-%5B4-fluoro-4-%5B%5B%285-methyl-pyridin-2-ylmethyl%29+-amino%5D-methyl%5Dpiperidin-1-yl%5D-methadone+%28F+13640%29+attenuates+allodynia+like+behavior+in+a+rat+model+of+trigeminal+neuropathic+pain%5BTitle%5D)+AND+%22J+Pharmacol+Exp+Ther%22%5BJournal%5D)">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/pubmed?term=((Ethical+Guidelines+for+investigations+of+experimental+pain+in+conscious+animals%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">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/pubmed?term=((What+should+we+be+measuring+in+behavioral+studies+of+chronic+pain+in+animals%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">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/pubmed?term=((Neuropathic+pain+symptoms+relative+to+overall+pain+rating%5BTitle%5D)+AND+%22J+Pain%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+heat+hyperalgesia+in+an+experimental+trigeminal+neuropathy+in+rats%5BTitle%5D)+AND+%22Exp+Brain+Res%22%5BJournal%5D)">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/pubmed?term=((Orofacial+cold+hyperalgesia+due+to+infraorbital+nerve+constriction+injury+in+rats+reversal+by+endothelin+receptor+antagonists+but+not+non+steroidal+anti+inflammatory+drugs%5BTitle%5D)+AND+%22Pain%22%5BJournal%5D)">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/pubmed?term=((Meal+Duration+as+a+Measure+of+Orofacial+Nociceptive+Responses+in+Rodents%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+bilateral+trigeminal+constriction+injury+using+an+operant+facial+pain+assay%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Use+of+the+Operant+Orofacial+Pain+Assessment+Device+%28OPAD%29+to+Measure+Changes+in+Nociceptive+Behavior%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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><tbody><tr><td>Chromic catgut</td><td>Dynek&amp;nbsp;</td><td>CG504</td><td></td></tr><tr><td>Nembutal&amp;nbsp; (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...

Watch this Scientific Journal Video about Chronic Constriction Injury of the Rat's Infraorbital Nerve IoN-CCI to Study Trigeminal Neuropathic Pain at JoVE.com

Chronic Constriction Injury of the Rat'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 ...

chronic-constriction-injury-rat-s-infraorbital-nerve-ion-cci-to-study

Research

JoVE Journal

Medicine

19.6K Views.  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

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

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 ...

Chronic Post-Ischemia Pain Model for Complex Regional Pain Syndrome Type-I in Rats

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical condition. However, the underlying mechanisms of CRPS-I have yet to be revealed. It is known that ischemia/reperfusion is one of the leading factors that causes CRPS-I. By means of prolonged ischemia and reperfusion of the hind limb, the rat chronic post-ischemia pain (CPIP) model has been established to mimic CRPS-I. The CPIP model has become a well-recognized animal model for studying the mechanisms of CRPS-I. This protocol describes the detailed procedures involved in the establishment of the rat model of CPIP, including anesthesia, followed by ischemia/reperfusion of the hind limb. Characteristics of the rat CPIP model are further evaluated by measuring the mechanical and thermal hypersensitivities of the hind limb as well as the nocifensive responses to acute capsaicin injection. The rat CPIP model exhibits several CRPS-I-like manifestations, including hind limb edema and hyperemia in the early stage after establishment, persistent thermal and mechanical hypersensitivities, and increased nocifensive responses to acute capsaicin injection. These characteristics render it a suitable animal model for further investigation of the mechanisms involved in CRPS-I.

Provided here is a protocol that details steps to establish an animal model of chronic post-ischemia pain (CPIP). This is a well-recognized model mimicking human complex regional pain syndrome type-I. Mechanical and thermal hypersensitivities are further evaluated, as well as capsaicin-induced nocifensive behaviors observed in the CPIP rat model.

Complex regional pain syndrome type-I (CRPS-I) is a neurological disease that causes severe pain among patients and remains an unresolved medical ...

Behavior

Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results by addressing the cognitive and motivational aspects of pain in rodents. This paper presents a protocol for assessing mechanical hypersensitivity following chronic constriction injury of the infraorbital nerves (CCI-ION) in rats using an orofacial operant pain system. Before CCI-ION surgery, rats were trained in an orofacial pain assessment device (OPAD) to drink sweetened condensed milk while making facial contact with the metal spiked bars and lick-tube.
In this assay, rats can choose between receiving milk as a positive reinforcer or escaping an aversive mechanical stimulus that is produced by a vertical row of small pyramid-shaped spikes on each side of the reward access hole. Following 2 weeks of training in the OPAD and before the CCI-ION surgery, baseline mechanical sensitivity data were recorded for 5 days for each rat during a 10 min testing session. During a session, the operant system automatically records the number of reward bottle activations (licks) and facial contacts, contact duration, and latency to the first lick, among other measures.
Following baseline measurements, rats underwent either CCI-ION or sham surgery. In this protocol, mechanical hypersensitivity was quantified by measuring the number of licks, latency to the first lick, the number of contacts, and the ratio of licks to facial contacts (L/F). The data showed that CCI-ION resulted in a significant decrease in the number of licks and the L/F ratio and an increase in the latency to the first lick, indicating mechanical hypersensitivity. These data support the use of operant-based pain assays to assess mechanical pain sensitivity in preclinical pain research.

This protocol describes the assessment of mechanical hypersensitivity in a rat model of neuropathic orofacial pain using an operant-based orofacial pain assessment device.

Pain has sensory and affective components. Unlike traditional, reflex-based pain assays, operant pain assays can produce more clinically relevant results ...

Facial Nerve Surgery in the Rat Model to Study Axonal Inhibition and Regeneration

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial nerve can be manipulated along its entire length, from its intracranial segment to its extratemporal course. There are three primary types of nerve injury used for the experimental study of regenerative properties: nerve crush, transection, and nerve gap. The range of possible interventions is vast, including surgical manipulation of the nerve, delivery of neuroactive reagents or cells, and either central or end-organ manipulations. Advantages of this model for studying nerve regeneration include simplicity, reproducibility, interspecies consistency, reliable survival rates of the rat, and an increased anatomic size relative to murine models. Its limitations involve a more limited genetic manipulation versus the mouse model and the superlative regenerative capability of the rat, such that the facial nerve scientist must carefully assess time points for recovery and whether to translate results to higher animals and human studies. The rat model for facial nerve injury allows for functional, electrophysiological, and histomorphometric parameters for the interpretation and comparison of nerve regeneration. It thereby boasts tremendous potential toward furthering the understanding and treatment of the devastating consequences of facial nerve injury in human patients.

This protocol describes a reproducible approach to facial nerve surgery in the rat model, including descriptions of various inducible patterns of injury.

This protocol describes consistent and reproducible methods to study axonal regeneration and inhibition in a rat facial nerve injury model. The facial ...

Neuroscience

In Vivo Visualization of the Rat Trigeminal Ganglion With Calcium Indicators

In-Vivo Calcium Imaging of Sensory Neurons in the Rat Trigeminal Ganglion

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their large signal-to-noise ratio makes GECIs a powerful tool for detecting stimulus-evoked activity in sensory neurons. GECIs facilitate population-level analysis of stimulus encoding with the number of neurons that can be studied simultaneously. This population encoding is most appropriately done in vivo. Dorsal root ganglia (DRG), which house the soma of sensory neurons innervating somatic and visceral structures below the neck, are used most extensively for in vivo imaging because these structures are accessed relatively easily. More recently, this technique was used in mice to study sensory neurons in the trigeminal ganglion (TG) that innervate oral and craniofacial structures. There are many reasons to study TG in addition to DRG, including the long list of pain syndromes specific to oral and craniofacial structures that appear to reflect changes in sensory neuron activity, such as trigeminal neuralgia. Mice are used most extensively in the study of DRG and TG neurons because of the availability of genetic tools. However, with differences in size, ease of handling, and potentially important species differences, there are reasons to study rat rather than mouse TG neurons. Thus, we developed an approach for imaging rat TG neurons in vivo. We injected neonatal pups (p2) intraperitoneally with an AAV encoding GCaMP6s, resulting in &gt;90% infection of both TG and DRG neurons. TG was visualized in the adult following craniotomy and decortication, and changes in GCaMP6s fluorescence were monitored in TG neurons following stimulation of mandibular and maxillary regions of the face. We confirmed that increases in fluorescence were stimulus-evoked with peripheral nerve block. While this approach has many potential uses, we are using it to characterize the subpopulation(s) of TG neurons changed following peripheral nerve injury.

Author Spotlight: Exploring Peripheral Mechanisms of Neuropathic Pain in Trigeminal Nerve Injury 

Genetically encoded calcium indicators (GECI) enable a robust, population-level analysis of sensory neuron signaling. Here, we have developed a novel approach that allows for in vivo GECI visualization of rat trigeminal ganglia neuron activity.

Genetically encoded calcium indicators (GECIs) enable imaging techniques to monitor changes in intracellular calcium in targeted cell populations. Their ...

Establishing the Mouse Model for Trigeminal Neuropathic Pain

Chronic Constriction Injury of the Distal Infraorbital Nerve (DIoN-CCI) in Mice to Study Trigeminal Neuropathic Pain

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury (DIoN-CCI) model to study trigeminal neuropathic pain in mice. This includes the surgical procedures to perform the chronic constriction injury and the postoperative behavioral tests to evaluate the changes in spontaneous and evoked behavior that are signs of ongoing pain and mechanical allodynia. The methods and behavioral readouts are similar to the infraorbital nerve chronic constriction injury (IoN-CCI) model in rats. However, important changes are necessary for the adaptation of the IoN-CCI model to mice. First, the intra-orbital approach is replaced by a more rostral approach with an incision between the eye and the whisker pad. The IoN is thus ligated distally outside the orbital cavity. Secondly, due to the higher locomotor activity in mice, allowing rats to move freely in small cages is replaced by placing mice in custom-designed and constructed restraining devices. After DIoN ligation, mice exhibit changes in spontaneous behavior and in response to von Frey hair stimulation that are similar to those in IoN-CCI rats, i.e., increased directed face grooming and hyperresponsiveness to von Frey hair stimulation of the IoN territory.

Author Spotlight: Utilizing Infraorbital Nerve Ligation in Mice for Investigating Trigeminal Neuropathic Pain and Treatment Strategies

Chronic constriction injury of the distal infraorbital nerve in mice induces changes in spontaneous behavior (increased face grooming activity) and nocifensive behavior in response to tactile stimulation (hyperresponsiveness to von Frey hair stimulation) that are signs of ongoing pain and allodynia and serves as a model for trigeminal neuropathic pain.

Animal models remain necessary tools to study neuropathic pain. This manuscript describes the distal infraorbital nerve chronic constriction injury ...

Transconjunctival Approach for Injection into the Rat Optic Nerve

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. Accessing the rat optic nerve for experimental manipulations and injections is challenging due to the small size of the orbit and the vascularity of the surrounding orbital tissues. Previous techniques have involved a cutaneous incision, which carries a higher risk of infection and requires wound closure. This study aims to describe a unique, less invasive, and potentially more efficient approach to accessing the rat optic nerve. A transconjunctival incision, along with a rat spinal cord hook, is used to isolate the optic nerve for experimental injections. In these experiments, an adenoviral vector is injected into the optic nerve with the goal of using this technique for direct gene therapy delivery in the future. This technique can also be applied by investigators requiring access to the rat optic nerve for other experimental studies, including drug delivery and optic nerve injury research.

This protocol describes a unique technique using a transconjunctival approach to access the rat optic nerve for injections.

The rat serves as an important model for studying disorders of the optic nerve, including heritable, traumatic, neoplastic, and autoimmune conditions. ...

Induction of Neuropathic Pain in a Rat Model by Constriction of the Infraorbital Nerve

<a href='https://app.jove.com/v/53167/chronic-constriction-injury-rat-s-infraorbital-nerve-ion-cci-to-study'>Source: Deseure, K., et al.&#160;Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain. J. Vis. Exp. (2015).</a>This video demonstrates a method to induce neuropathic pain in a rat model by constricting the infraorbital nerve. After exposing the infraorbital nerve and tying ligatures to constrict it, the immune response at the injury site increases the excitability of neighboring neurons, which transmit pain signals to the brain.

Source: Deseure, K., et al.&#160;Chronic Constriction Injury of the Rat's Infraorbital Nerve (IoN-CCI) to Study Trigeminal Neuropathic Pain. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Neuropathology

Neuromuscular Disorders

Inducing Orofacial Neuropathic Pain in a Rat Model via Infraorbital Nerve Ligations

Source: Donertas-Ayaz, B. et al., <a href="https://app.jove.com/v/64221">Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay.</a> J. Vis. Exp. (2022)
This video demonstrates the induction of orofacial neuropathic pain in a rat model by ligating the infraorbital nerve. This results in nerve damage and inflammation, causing abnormal pain signaling and mimicking chronic pain conditions, which can be used to study the orofacial pain assay.

Source: Donertas-Ayaz, B. et al., Assessment of Nerve Injury-Induced Mechanical Hypersensitivity in Rats Using an Orofacial Operant Pain Assay. J. Vis. ...