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.

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

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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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

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 ...

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory system is characterized by its ability to give rise to new neurons even in adult animals. This particular ability is partly due to the presence of OECs which create a favorable microenvironment for neurogenesis. This property of OECs has been used for cellular transplantation such as in spinal cord injury models. Although the peripheral nervous system has a greater capacity to regenerate after nerve injury than the central nervous system, complete sections induce misrouting during axonal regrowth in particular after facial of laryngeal nerve transection. Specifically, full sectioning of the recurrent laryngeal nerve (RLN) induces aberrant axonal regrowth resulting in synkinesis of the vocal cords. In this specific model, we showed that OECs transplantation efficiently increases axonal regrowth.
OECs are constituted of several subpopulations present in both the olfactory mucosa (OM-OECs) and the olfactory bulbs (OB-OECs). We present here a model of cellular transplantation based on the use of these different subpopulations of OECs in a RLN injury model. Using this paradigm, primary cultures of OB-OECs and OM-OECs were transplanted in Matrigel after section and anastomosis of the RLN. Two months after surgery, we evaluated transplanted animals by complementary analyses based on videolaryngoscopy, electromyography (EMG), and histological studies. First, videolaryngoscopy allowed us to evaluate laryngeal functions, in particular muscular cocontractions phenomena. Then, EMG analyses demonstrated richness and synchronization of muscular activities. Finally, histological studies based on toluidine blue staining allowed the quantification of the number and profile of myelinated fibers.
All together, we describe here how to isolate, culture, identify and transplant OECs from OM and OB after RLN section-anastomosis and how to evaluate and analyze the efficiency of these transplanted cells on axonal regrowth and laryngeal functions.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth of the primary olfactory neurons. This specific property can be used for cellular transplantation. We present here a model of cellular transplantation based on the use of OECs in a laryngeal nerve injury model.

Olfactory ensheathing cells (OECs) are neural crest cells which allow growth and regrowth of the primary olfactory neurons. Indeed, the primary olfactory ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also demonstrate that traumatic brain injuries cause changes to the gut microbiota. However, mechanisms underlying the bidirectional regulation of the brain-gut axis remain unknown. Currently, few models exist for studying the changes in gut microbiota after traumatic brain injury. Therefore, the presented study combines protocols for inducing traumatic brain injury using a lateral fluid percussion device and analysis of caecum samples following injury for investigating alterations in the gut microbiome. Alterations of the gut microbiota composition after traumatic brain injury are determined using 16S-rDNA sequencing. This protocol provides an effective method for studying the relationships between enteric microorganisms and traumatic brain injury.

Presented here is a protocol to induce diffuse traumatic brain injury using a lateral fluid percussion device followed by the collection of the caecum content for gut microbiome analysis.

Increasing evidence shows that the microbiota-gut-brain axis plays an important role in the pathogenesis of brain diseases. Several studies also ...

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and motor phenotypes involved in different patterns of neurotrophic factor gene expression and other biological processes, affecting nerve regeneration. The present study has established a protocol to obtain highly purified rat sensory and motor SCs and fibroblasts more rapidly. The ventral root (motor nerve) and the dorsal root (sensory nerve) of neonatal rats (7-days-old) were dissociated and the cells were cultured for 4-5 days, followed by isolation of sensory and motor fibroblasts and SCs by combining differential digestion and differential adherence methods sequentially. The results of immunocytochemistry and flow cytometry analyses showed that the purity of the sensory and motor SCs and fibroblasts were &#62;90%. This protocol can be used to obtain a large number of sensory and motor fibroblasts/SCs more rapidly, contributing to the exploration of sensory and motor nerve regeneration.

Here, we present a method to purify fibroblasts and Schwann cells from sensory and motor nerves in vitro.

The principal cells in the peripheral nervous system are the Schwann cells (SCs) and the fibroblasts. Both these cells distinctly express the sensory and ...

Burn Injury-Induced Pain and Depression-Like Behavior in Mice

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the high mortality and sequelae in low- and middle-income countries. Burns frequently induce intense spontaneous pain and persistent allodynia as well as life-threatening problem. More importantly, excessive pain is often accompanied by depression, which may significantly decrease the quality of life. This article shows how to develop an animal model for the study of burn-induced pain and depression-like behavior. After anesthesia, burn injury was induced by dipping one hind paw of the mouse into hot water (65 &#176;C &plusmn; 0.5 &#176;C) for 3 s. The von Frey test and automated gait analysis were performed every 2 days after burn injury. In addition, depression-like behavior was examined using the forced swimming test, and the rota-rod test was performed to differentiate the abnormal motor function after burn injury. The main purpose of this study is to describe the development of an animal model for the study of burn injury-induced pain and depression-like behavior in mice.

A transient scald injury (65 &#176;C &plusmn; 0.5 &#176;C, 3 s) of one hind paw decreases the threshold (g) to von Frey filament stimulation of the ipsilateral side and alters gait pattern. Besides, burn injury induces depression-like behavior in the forced swimming test.

Scalding water is the most common cause of burn injury in both elderly and young populations. It is one of the major clinical challenges because of the ...

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice

Spared nerve injury (SNI) is an animal model that mimics the cardinal symptoms of peripheral nerve injury for studying the molecular and cellular mechanism of neuropathic pain in mice and rats. Currently, there are two types of SNI model, one to cut and ligate the common peroneal and the tibial nerves with intact sural nerve, which is defined as SNIs in this study, and another to cut and ligate the common peroneal and the sural nerves with intact tibial nerve, which is defined as SNIt in this study. Because the sural nerve is purely sensory whereas the tibial nerve contains both motor and sensory fibers, the SNIt model has much less motor deficit than the SNIs model. In the traditional SNIt mouse model, the common peroneal and the sural nerves are cut and ligated separately. Here a modified SNIt surgery method is described to damage both common peroneal and sural nerves with only one ligation and one cut with a shorter procedure time, which is easier to perform and reduces the potential risk of stretching the sciatic or tibial nerves, and produces similar mechanical hypersensitivity as the traditional SNIt model.

The modified surgery is a simplified method for mouse or rat spared nerve injury model that requires only one ligation and one cut to injure both common peroneal and sural nerves.

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

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

Transplantation of Olfactory Ensheathing Cells to Evaluate Functional Recovery after Peripheral Nerve Injury

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

Automated Gait Analysis in Mice with Chronic Constriction Injury

Investigating Alterations in Caecum Microbiota After Traumatic Brain Injury in Mice

Purification of Fibroblasts and Schwann Cells from Sensory and Motor Nerves in Vitro

Burn Injury-Induced Pain and Depression-Like Behavior in Mice

Modified Spared Nerve Injury Surgery Model of Neuropathic Pain in Mice