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,<sup class="...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chromic catgut (6-0)</td>
			<td>Dynek&#160;</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.

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

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

The overall goal of this procedure is to perform an infraorbital nerve injury in mice to study the development of trigeminal neuropathic pain and to test new treatment strategies. Isolated phase grooming behavior after infraorbital nerve ligation is one of the few animal models to study spontaneous neuropathic pain. But its use has long been hampered by the complexity of performing an infraorbital nerve ligation.Compared to sensory reflex based pain testing, such as from fray or withdrawal responses from heat or cold stimulation, measuring spontaneous or ongoing pain has not received sufficient attention, considering that this is an important clinical symptom affecting quality of life. The distal approach to the infraorbital nerve has the advantage of being a surgically less demanding procedure than the intraorbital approach. It is minimally invasive, and can be performed without using a stereotaxic frame.

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 stimul...

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

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 ...

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1.2K Views.  University of Antwerp. The overall goal of this procedure is to perform an infraorbital nerve injury in mice to study the development of trigeminal neuropathic pain and to test new treatment strategies. Isolated phase grooming behavior after infraorbital nerve ligation is one of the few animal models to study spontaneous neuropathic pain. But its use has long been hampered by the complexity of performing an infraorbital nerve ligation.Compared to sensory reflex based pain testing, such as from fray or withdr...

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

Infraorbital Nerve Surgery in Mouse

To begin, gently shave the buccal hair between the whisker pad and the eye of an anesthetized mouse. Fix the head of the mouse in a stereotaxic frame, or otherwise, fixate the head. Apply ointment on both eyes to avoid drying.Scrub the shaved head area with alcohol, and then with Betadine. Under a dissecting microscope, make a four millimeter skin incision perpendicular to the midline, 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. To expose the infraorbital nerve, bluntly separate the superficial connective tissue, minimizing musculature damage and avoiding the motor nerve fibers.Using a rotation motion, slip the head of a hooked ligation aid under the infraorbital nerve. 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 infraorbital nerve, with both ends of the ligature equidistant from the nerve. Tie a slip knot from both ends of the ligature and slide it against the infraorbital nerve.Slide the knot further to constrict the infraorbital nerve. Then place a normal knot on top of the slip knot. Cut the ends of the ligature, leaving approximately 1.5 millimeters of free ends to prevent the knot from becoming undone.Close the skin incision using 6-0 synthetic absorbable sutures. Allow the mouse to recover on a heated pad or under an infrared heating lamp.

Behavioral Testing and Mechanical Stimulation Assessment in Distal Infraorbital Nerve Chronic Constriction Injured Mouse

To begin, carry a single mouse from the housing to the test room in a covered plastic cage without bedding material. Place the cage in front of a video camera. Then place a mirror to view the mouse's face when its back is toward the camera.Record the mouse's behavior for 10 minutes. After recording, clean the observation cage before recording the next animal. Have an observer blind to the experimental conditions to analyze the recorded behavior of the mouse.Note each face grooming episode during the 10-minute recording. Make a distinction between isolated face grooming and face grooming behaviors during body grooming. Determine the number of face grooming episodes with a four-second cutoff criterion.The time between grooming actions of less than four seconds is defined as a pause within a single episode. A time greater than four seconds is defined as a full interruption of grooming actions between two episodes. Place the mouse tail in a soft silicone clamp and attach the clamp magnetically to a metal plate on the table.Place a three-walled plastic holder over the mouse so that only the head protrudes from the container. Place a weight on top of the holder to keep it in place. Use a graded series of four von Frey hairs to apply force to the mouse.Habituate the mouse to the restrainer and reaching movements for 10 minutes. When the mouse is relaxed, slowly apply the lightest von Frey hair within the infraorbital nerve territory near the center of the vibrissae until it bends. Score the mouse response to the stimulation into one of the following categories.Calculate the mean score for ipsilateral responses to the four von Frey hairs, then calculate mean scores for the contralateral sides. Distal infraorbital nerve chronic constriction injury mice exhibited a significant postoperative increase in isolated face grooming time and episodes, peaking in the first week and then gradually decreasing, remaining elevated for six weeks. Initially, distal infraorbital nerve chronic constriction injury mice showed almost complete unresponsiveness to mechanical stimulation, which, during subsequent weeks, transformed into hyperresponsiveness lasting for six weeks.A slight increase in responsiveness to contralateral mechanical stimulation was also observed.