The authors have no acknowledgments.

Animals are treated and cared for according to the guidelines for pain research in conscious animals of the International Association for the Study of PAIN and in line with the Flemish and European regulations for animal research and the ARRIVE guidelines. The protocol is approved by the institutional Ethical Committee.
1. Animals
<ol>
	<li>Use male and female C57BL/6J mice (Janvier, 10 weeks old at arrival).</li>
	<li>House male and female mice separately in standard solid-bottom mice cages in a colony room with a humidity of 40%-60% and a room temperature (RT) of 21 &#177; 1 &#176;C.</li>
	<li>Provide water and food ad libitum.</li>
	<li>Keep mice under a normal 12:12 h dark/light cycle (lights on at 08:00).</li>
</ol>
2. Surgery
<ol>
	<li>Per mouse, prepare one piece of chromic gut ligature (6-0) approximately 6 cm long and place it in sterile saline to avoid drying and becoming stiff and brittle.</li>
	<li>Anesthetize the mouse with ketamine/xylazine (75/15 mg/kg, intraperitoneal, injection volume 10 mL/kg). Check the depth of anesthesia by pinching the skin between the toes. Ensure that the mouse does not flex its leg. If necessary, wait until the animal is fully anesthetized and/or administer additional ketamine/xylazine. 
	NOTE: Supplementary analgesics should not be administered to avoid pre-emptive analgesic effects that may interfere with the development of trigeminal neuropathic pain.</li>
	<li>Gently shave the buccal hair between the whisker pad and the eye to make an incision of approximately 4 mm just rostral to the infraorbital foramen. Take care not to damage the whiskers, as this may affect behavioral testing.</li>
	<li>Fix the head of the mouse in a stereotaxic frame or otherwise fixate the head. Place the mouse on a heated pad or take care to maintain body temperature otherwise.</li>
	<li>Apply ointment on both eyes to avoid drying. Scrub the shaved head area with alcohol, and then with betadine. Place a surgical drape exposing the shaved head area.</li>
	<li>Use a microscope for steps 2.7 to 2.14.</li>
	<li>Make a 4 mm skin incision perpendicular to the mid-line approximately halfway between the edge of the whisker pad and the eye, just rostral to the infraorbital foramen, and centered around the line between the center of the eye and the center of the whisker pad.</li>
	<li>Expose the IoN by bluntly separating the superficial connective tissue. Take care to minimize musculature damage and avoid the motor nerve fibers. Ensure that the trunk of the IoN (1-1.5 mm in diameter) is accessible approximately 3 mm deep between where it exits the skull and where it branches out to the whisker pad (Figure 1).</li>
	<li>Using a rotating motion, slip the head of a hooked ligation aid under the IoN, taking care not to damage the nerve.</li>
	<li>Place the chromic gut ligature through the hole on the tip of the ligation aid and retract the ligation aid so that the ligature remains under the IoN and both ends of the ligature are more or less equidistant from the IoN.</li>
	<li>Tie a &#34;slip knot&#34; from the two ends of the ligature and slide the knot against the IoN. Ensure that the slip knot allows a smooth action so that the degree of constriction can be accurately controlled. Slide the knot further and constrict the IoN. Reduce the diameter of the nerve by just a noticeable amount4. Place a normal knot on top of the slit knot to prevent it from slipping.</li>
	<li>Cut the ends of the ligature leaving approximately 1.5 mm of free ends to prevent the knot from becoming undone.</li>
	<li>Perform the sham surgery following steps 2.2-2.8.</li>
	<li>Close the skin incision using synthetic absorbable sutures (6-0) and allow the animal to recover on a heated pad or under an infrared heating lamp.</li>
</ol>
3. Behavioral testing
<ol>
	<li>Acclimate the mouse to the housing conditions for at least 8 days before pre-operative testing.</li>
	<li>Before pre-operative testing, habituate the mouse to the test procedure at least once daily for 3 days.</li>
	<li>Conduct testing in normal lighting conditions. If necessary, provide background noise to minimize disturbances from outside noises.</li>
	<li>Observation of face grooming behavior
	<ol>
		<li>Carry a single mouse from the housing to the test room in a covered plastic cage without any bedding material. Avoid external stimulation while transporting the animals.</li>
		<li>Place the mouse in a covered, transparent plastic cage without bedding (l x w x h: 12 cm x 12 cm x 17 cm) in front of a video camera. Place a mirror to view the animal&#39;s face when its back is toward the camera.</li>
		<li>Record the mouse&#39;s behavior for 10 min. During recording, ensure that the experimenter is not present in the room.</li>
		<li>After recording the next animal, clean the observation cage.</li>
		<li>Have an observer who is blind to the experimental conditions of the mouse analyze the recorded behavior.</li>
		<li>Note each face grooming episode while analyzing the 10 min recording. Face grooming is movement patterns in which the animal brings its forepaws in contact with facial areas.</li>
		<li>Make a distinction between isolated face grooming and face grooming behaviors during body grooming9. If a sequence is not preceded or followed by body grooming, the episode is labeled isolated face grooming. Body grooming is defined as patterns of movement that bring paws, tongue, or incisors in contact with a body area other than the face or the forepaws. If body grooming is present before or after a face grooming sequence, the episode is labeled as face grooming during body grooming.</li>
		<li>Determine the number of face grooming episodes by applying a 4 s cut-off criterion. A time period between grooming actions of less than 4 s is defined as a pause within a single episode. A time period greater than 4 s is defined as a full interruption of grooming actions between two episodes.</li>
	</ol>
	</li>
	<li>Mechanical stimulation testing
	<ol>
		<li>Carry mice in groups of up to 6 animals from the housing to the test room in a covered cage with bedding. Again, take care to avoid external stimulation.</li>
		<li>Place the mice one at a time on a table.
		<ol>
			<li>Place the tail of the mouse in a soft silicone clamp and attach the clamp magnetically to a metal plate on the table. The silicone material prevents the tail from slipping from the clamp while minimizing pressure on the tail.</li>
			<li>Place a three-walled plastic holder (65 mm x 25 mm x 23 mm) over the animal so that only the head of the mouse protrudes from the container. The size of the holder allows for head and forepaw movements but prevents the animal from turning around inside it. Finally, place a weight on top of the holder to keep the holder in place (Figure 2).</li>
		</ol>
		</li>
		<li>Use a graded series of four von Frey hairs. The force required to bend the hairs is 0.02 g, 0.16 g, 0.4 g and 1.0 g.</li>
		<li>Habituate the mice to the restrainer and reaching movements for 10 min. Every 30 s, make a reaching movement per animal.</li>
		<li>When the animal is in a relaxed state, slowly apply the lightest von Frey hair within the IoN territory near the center of the vibrissae until the von Frey hair bends. Ensure that the stimulation takes no more than 1 s.</li>
		<li>Score the animal response to the stimulation to fit into one of the following response categories.
		<ol>
			<li>Give a score of 0 when there is no response.</li>
			<li>Give a score of 1 for detection, i.e., the mouse turns its head toward the stimulating object and then explores the stimulus object.</li>
			<li>Give a score of 2 for withdrawal reaction, i.e., the mouse turns the head gently away or pulls it quickly backward when the stimulation is applied; sometimes, a single face wipe ipsilateral to the stimulated area occurs.</li>
			<li>Give a score of 3 for attacking, i.e., the mouse attacks the stimulus object, making biting and/or grabbing movements.</li>
			<li>Give a score of 4 for asymmetric face grooming, i.e., the mouse exhibits an uninterrupted series of at least three face-wash strokes directed toward the stimulated facial area.</li>
		</ol>
		</li>
		<li>For each mouse, apply von Frey hairs in an ascending order of intensity and randomly stimulate the ipsilateral and contralateral sides. Apply each stimulus intensity one time on each side.</li>
		<li>Calculate the mean score from the responses to the four von Frey hairs within each animal. Calculate separate scores for the ipsilateral and contralateral sides.</li>
	</ol>
	</li>
</ol>

Establishing the Mouse Model for Trigeminal Neuropathic Pain

<ol>
	<li>Costigan, M., Scholz, J., Woolf, C. J. <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+maladaptive+response+of+the+nervous+system+to+damage.">Neuropathic pain: a maladaptive response of the nervous system to damage.</a> Annu Rev Neurosci. 32, 1-32 (2009).</li><li>Torrance, N., Smith, B. H., Bennett, M. I., Lee, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+chronic+pain+of+predominantly+neuropathic+origin.+Results+from+a+general+population+survey.">The epidemiology of chronic pain of predominantly neuropathic origin. Results from a general population survey.</a> J Pain. 7, 281-289 (2006).</li><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>Bennett, G. J., Xie, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+peripheral+mononeuropathy+in+rat+that+produces+disorders+of+pain+sensation+like+those+seen+in+man.">A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.</a> Pain. 33 (1), 87-107 (1988).</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, 491-497 (2004).</li><li>Basbaum, A. I., Campbell, J. N., 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=Measurrement+and+New+Technologies.">Measurrement and New Technologies.</a> Emerging Strategies for the Treatment of Neuropathic Pain. , (2006).</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, 12-15 (2004).</li><li>Vierck, C. J., Campbell, J. N., 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=Animal+Studies+of+Pain:+Lessons+for+Drug+Development.">Animal Studies of Pain: Lessons for Drug Development.</a> Emerging Strategies for the Treatment of Neuropathic Pain. , (2006).</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. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+constriction+injury+of+the+rat's+infraorbital+nerve+(IoN-CCI)+to+study+trigeminal+neuropathic+pain.">Chronic constriction injury of the rat's infraorbital nerve (IoN-CCI) to study trigeminal neuropathic pain.</a> J Vis Exp. (103), e53167 (2015).</li><li>Mogil, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+pain:+progress+and+challenges.">Animal models of pain: progress and challenges.</a> Nat Rev Neurosci. 10 (4), 283-294 (2009).</li><li>Wilson, S. G., Mogil, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+pain+in+the+(knockout)+mouse:+big+challenges+in+a+small+mammal.">Measuring pain in the (knockout) mouse: big challenges in a small mammal.</a> Behav Brain Res. 125 (1-2), 65-73 (2001).</li><li>Ding, W., 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=An+improved+rodent+model+of+trigeminal+neuropathic+pain+by+unilateral+chronic+constriction+injury+of+distal+infraorbital+nerve.">An improved rodent model of trigeminal neuropathic pain by unilateral chronic constriction injury of distal infraorbital nerve.</a> J Pain. 18 (8), 899-907 (2017).</li><li>Hardt, S., Fischer, C., Vogel, A., Wilken-Schmitz, A., Tegeder, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distal+infraorbital+nerve+injury:+a+model+for+persistent+facial+pain+in+mice.">Distal infraorbital nerve injury: a model for persistent facial pain in mice.</a> Pain. 160 (6), 1431-1447 (2019).</li><li>Krzyzanowska, A., Avenda&#241;o, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+testing+in+rodent+models+of+orofacial+neuropathic+and+inflammatory+pain.">Behavioral testing in rodent models of orofacial neuropathic and inflammatory pain.</a> Brain Behav. 2 (5), 678-697 (2012).</li><li>Martin, Y. B., Malmierca, E., Avenda&#241;o, C., Nu&#241;ez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+disinhibition+in+the+trigeminal+nucleus+caudalis+in+a+model+of+chronic+neuropathic+pain.">Neuronal disinhibition in the trigeminal nucleus caudalis in a model of chronic neuropathic pain.</a> Eur J Neurosci. 32 (3), 399-408 (2010).</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>Vuralli, D., Wattiez, A. S., Russo, A. F., Bolay, 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+and+cognitive+animal+models+in+headache+research.">Behavioral and cognitive animal models in headache research.</a> J Headache Pain. 20 (1), 11 (2019).</li><li>Krzyzanowska, A., 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=Assessing+nociceptive+sensitivity+in+mouse+models+of+inflammatory+and+neuropathic+trigeminal+pain.">Assessing nociceptive sensitivity in mouse models of inflammatory and neuropathic trigeminal pain.</a> J Neurosci Methods. 201 (1), 46-54 (2011).</li></ol>

The authors have no conflicts of interest to disclose.

In rats, it has been previously argued that an intra-orbital approach to the IoN is preferable, considering the importance of intact fine musculature controlling complex whisking patterns in vibrissotactile discrimination and the relative distance of the mid-line incision to the cutaneous infraorbital nerve territory10. Others have argued that a distal approach via an incision into the hairy skin caudal to the vibrissal pad has a number of benefits13,14. Surgically, it is an easier technique that is minimally invasive. It can be performed without using a stereotaxic frame and in a shorter amount of time. Both techniques result in comparable postoperative behavioral effects, both evoked and non-evoked pain behavior. Furthermore, the procedure avoids possible eye discomfort resulting from deflecting orbital contents during the procedure and potentially irritating contact between the nerve ligations and the eye. In mice, due to their smaller size, we found the intra-orbital approach too difficult to use as a standard procedure. Therefore, this procedure aims to use a distal approach to surgically induce an infraorbital nerve injury in a mouse, leading to the development of spontaneous and evoked pain behavior that can be used to study trigeminal neuropathic pain.
A crucial step in applying this model, as with other nerve ligation-induced chronic pain animal models, is placing the ligature with the correct amount of constriction around the infraorbital nerve4,15,16. Behavioral outcomes are vastly different in animals with varying degrees of nerve constriction17. The ratio of the size of the chrome gut (6-0) to the mouse infraorbital nerve diameter is not the same as that in rats where size 5-0 is used. In mice, it was found that two ligatures induced a level of nerve injury that was higher than that in rats. Because size 6-0 was the smallest chrome gut that could be found, in the present study, we chose to use a single ligature, which produced behavioral outcomes similar to those in rats. Two non-chrome gut ligatures size 7-0 could possibly also produce similar results.
Stimulation of the IoN territory with von Frey hairs requires animals to be relatively motionless. In rats, this can be achieved by habituating the animals to an observation cage. In mice, due to their high locomotor activity, using this method makes it difficult to stimulate the IoN territory with high precision. Holding the animal by hand is a stressful method that severely compromises the validity and reliability of the animal&#39;s response to a stimulus. Placing the animals on a small elevated platform has also been used in studies18. Although movements are more constrained, it was found that the platform allows the animals to move around more than is needed to stimulate the IoN territory with accuracy and a well-controlled bending force. A restraining method was devised and partly manufactured in our lab by the use of 3D printing. The three-walled plastic holder and weight are similar to that used by Krzyzanowska et al. (2011), but the method to keep the animal&#39;s tail in place is different19. Importantly, the device allows the animal to respond in a more natural way to the stimulus, including paw and head movements. The device does, however, prevent the animal from moving its body away to avoid further contact with the stimulus. In free-moving animals in an observation cage, the latter behavior is equivalent to grabbing or biting the stimulus (response score category 3).
The present article shows that DIoN-CCI in mice induces changes in spontaneous behavior in response to von Frey hair stimulation that are&#160;similar to those in IoN-CCI rats, i.e., increased directed face grooming and hyperresponsiveness to von Frey hair stimulation of the IoN territory10. The average isolated face grooming episode duration in mice was shorter than in rats (2 s vs. 10 s), but the average peak number of isolated face grooming episodes was higher in mice (13 vs. 5). It is unclear if this is purely a reflection of differences in innate grooming patterns or related to the nature of the spontaneous pain sensations.

Neuropathic pain arises from damage to the somatosensory nervous system, leading to abnormal transmission of sensory signals to the brain. Somatosensory nerve damage does not always lead to neuropathic pain, but the prevalence increases with the severity of clinical neuropathy1,2. Neuropathic pain patients experience specific symptoms such as spontaneous sensations (burning, pins and needles, electric sensations) and abnormally intense or prolonged pain to innocuous or noxious stimulation that tend to become chronic and resistant to treatment with conventional pain medication3. Significant progress in the field of neuropathic pain research stems from the discovery that loosely constricting ligatures around the sciatic nerve in rats leads to behaviors resembling human neuropathic pain conditions4. The animals display reduced thresholds to heat, cold, and mechanical stimulation, and exhibit nocifensive behaviors. Despite the inherent biological differences in pain processing between humans and rodents, animal models are a valuable tool for studying the underlying mechanisms in the development of neuropathic pain and testing newly proposed treatment strategies.
Sensory reflex-based pain testing paradigms have been extensively used in neuropathic pain models, but measuring ongoing pain or other frequently accompanied disturbances (sleeping disorder, depression, anxiety) has not received sufficient attention considering that these are common clinical symptoms affecting quality of life5,6,7,8. Face grooming behavior in rats has been documented as a measure of spontaneous neuropathic pain following chronic constriction injury (CCI) of the infraorbital nerve (IoN)9,10. In addition, rats also develop hyperresponsiveness to mild tactile stimulation of the IoN territory, which is indicative of mechanical allodynia.
Compared to mice, because of their larger size, rats are better suited for surgical injuries. However, mice offer cost and space efficiency and require smaller drug quantities. Also, the advent of transgenic technology has further boosted the use of mice11,12. Therefore, the overall goal of this procedure is to perform a surgical infraorbital nerve injury in mice, similar to that in rats, that induces changes in spontaneous and evoked behavior for the study of trigeminal neuropathic pain.

<table><tbody><tr><td>Chromic catgut (6-0)</td><td>Dynek&amp;nbsp;</td><td>CG602D</td><td>ligatures</td></tr><tr><td>Cotton applicator</td><td>Pharmacy</td><td></td><td></td></tr><tr><td>Digital video camera</td><td>Sony</td><td>HDR-CX330E</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>Duratears</td><td>Alcon</td><td>0037-820</td><td>ophthalmic ointment</td></tr><tr><td>Hooked ligation aid</td><td>Fine Science Tools</td><td>18062-12</td><td></td></tr><tr><td>Ketalar</td><td>Pfizer</td><td></td><td>ketamine (50 mg/mL)</td></tr><tr><td>Operation microscope</td><td>Kaps</td><td>SOM 62</td><td></td></tr><tr><td>Precision cotton swab</td><td>Qosina</td><td>10225</td><td></td></tr><tr><td>Precision trimmer</td><td>Philips</td><td>HP6392/00</td><td></td></tr><tr><td>Rompun</td><td>Bayer</td><td></td><td>xylazine (2%)</td></tr><tr><td>Scissors - blunt tips</td><td>Fine Science Tools</td><td>14574-09</td><td></td></tr><tr><td>Semmes-Weinstein Von Frey Aesthesiometer kit</td><td>Stoelting</td><td>58011</td><td></td></tr><tr><td>Vicryl Rapide</td><td>Ethicon</td><td>MPVR489H</td><td>sutures</td></tr></tbody></table>

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.

DIoN-CCI mice show a strong postoperative increase in time spent on isolated face grooming and the number of isolated face grooming episodes (Figure 3). The strongest increase occurs during the first postoperative week and then becomes smaller during the following weeks but is significantly increased for at least 6 weeks. Face grooming during body grooming is more or less unaffected.
DIoN-CCI mice are almost completely unresponsive to ipsilateral mechanical stimulation of the ipsilateral IoN territory during the first week after DioN-CCI (Figure 4). During the next weeks, this hyporesponsiveness is replaced by hyperresponsiveness that persists for at least 6 weeks. There may also be a small increase in responsiveness to contralateral mechanical stimulation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66420/66420fig01.jpg" /> 
Figure 1: Location of the distal infraorbital nerve ligation. The location of the distal infraorbital nerve ligation is rostral to its exit from the skull but caudal to where it branches out to the whisker pad. (A) Schematic drawing of the right IoN. (B) Surgical view of a ligated IoN on the left side. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66420/66420fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66420/66420fig02.jpg" /> 
Figure 2: Restraining device used for mechanical stimulation testing. The tail of the mouse is held in place by a (A) soft silicone clamp that is magnetically attached to a (B) metal plate on the table. (C) A plastic holder allows for head and forepaw movements but prevents the animal from turning around inside it. A metal weight keeps the holder in place. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66420/66420fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66420/66420fig03.jpg" /> 
Figure 3: Postoperative changes in isolated face grooming behavior following DIoN-CCI. Data points denote the (A) amount of time spent (mean &#177; SEM; n =15 per group) on isolated face grooming and the (B) number (mean &#177; SEM; n = 15 per group) of isolated face grooming episodes 1 day before DIoN surgery (Pre-op) and on postoperative days 3-42. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66420/66420fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66420/66420fig04.jpg" /> 
Figure 4: Time course of the effects of DIoN-CCI on responsiveness to mechanical stimulation. Data points denote the response score (mean &#177; SEM; n = 15 per group) to von Frey hair stimulation of the territory of the (A) ligated nerve and of the (B) contralateral side 1 day before (Pre-op) and 3-42 days after DIoN surgery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66420/66420fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Utilizing Infraorbital Nerve Ligation in Mice for Investigating Trigeminal Neuropathic Pain and Treatment Strategies at JoVE.com

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.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Neuroscience

1.3K Views.  University of Antwerp. 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.

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

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 ...

Medicine

An Optic Nerve Crush Injury Murine Model to Study Retinal Ganglion Cell Survival

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible vision loss. Examples of such diseases in human include traumatic optic neuropathy and optic nerve degeneration in glaucoma. It is characterized by typical changes in the optic nerve head, progressive optic nerve degeneration, and loss of retinal ganglion cells, if uncontrolled, leading to vision loss and blindness.
The optic nerve crush (ONC) injury mouse model is an important experimental disease model for traumatic optic neuropathy, glaucoma, etc. In this model, the crush injury to the optic nerve leads to gradual retinal ganglion cells apoptosis. This disease model can be used to study the general processes and mechanisms of neuronal death and survival, which is essential for the development of therapeutic measures. In addition, pharmacological and molecular approaches can be used in this model to identify and test potential therapeutic reagents to treat different types of optic neuropathy.
Here, we provide a step by step demonstration of (I) Baseline retrograde labeling of retinal ganglion cells (RGCs) at day 1, (II) Optic nerve crush injury at day 4, (III) Harvest the retinae and analyze RGC survival at day 11, and (IV) Representative result.

This protocol shows how to retrogradely label retinal ganglion cells, and how to subsequently make an optic nerve crush injury in order to analyze retinal ganglion cell survival and apoptosis. It is an experimental disease model for different types of optic neuropathy, including glaucoma.

Injury to the optic nerve can lead to axonal degeneration, followed by a gradual death of retinal ganglion cells (RGCs), which results in irreversible ...

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 ...

Behavior

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

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

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

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

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

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

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

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

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

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 ...

Dural Stimulation and Periorbital von Frey Testing in Mice As a Preclinical Model of Headache

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system. For example, they protect the brain from the skull and anchor/organize the vascular and neuronal supply of the cortex. However, the meninges are also implicated in nervous system disorders such as migraine, where the pain experienced during a migraine is attributed to local sterile inflammation and subsequent activation of local nociceptive afferents. Of the layers in the meninges, the dura mater is of particular interest in the pathophysiology of migraines. It is highly vascularized, harbors local nociceptive neurons, and is home to a diverse array of resident cells such as immune cells. Subtle changes in the local meningeal microenvironment may lead to activation and sensitization of dural perivascular nociceptors, thus leading to migraine pain. Studies have sought to address how dural afferents become activated/sensitized by using either in vivo electrophysiology, imaging techniques, or behavioral models, but these commonly require very invasive surgeries. This protocol presents a method for comparatively non-invasive application of compounds on the dura mater in mice and a suitable method for measuring headache-like tactile sensitivity using periorbital von Frey testing following dural stimulation. This method maintains the integrity of the dura and skull and reduces confounding effects from invasive techniques by injecting substances through a 0.65 mm modified cannula at the junction of unfused sagittal and lambdoid sutures. This preclinical model will allow researchers to investigate a wide range of dural stimuli and their role in the pathological progression of migraine, such as nociceptor activation, immune cell activation, vascular changes, and pain behaviors, all while maintaining injury-free conditions to the skull and meninges.

The most notable symptom of migraine is severe head pain, and it is hypothesized that this is mediated by sensory neurons innervating the meninges. Here, we present a method to locally apply substances to the dura in a minimally invasive manner while using facial hypersensitivity as an output.

The cranial meninges, comprised of the dura mater, arachnoid, and pia mater, are thought to primarily serve structural functions for the nervous system.  ...

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. ...

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

<a href='https://app-jove-com.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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. ...