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

Summary

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.

Abstract

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.

Introduction

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 neuropathy^1,2. 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 life^5,6,7,8. 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 mice^11,12. 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.

Protocol

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

1. Use male and female C57BL/6J mice (Janvier, 10 weeks old at arrival).
2. 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 ± 1 °C.
3. Provide water and food ad libitum.
4. Keep mice under a normal 12:12 h dark/light cycle (lights on at 08:00).

2. Surgery

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. Use a microscope for steps 2.7 to 2.14.
7. 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.
8. 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).
9. Using a rotating motion, slip the head of a hooked ligation aid under the IoN, taking care not to damage the nerve.
10. 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.
11. Tie a "slip knot" 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.
12. Cut the ends of the ligature leaving approximately 1.5 mm of free ends to prevent the knot from becoming undone.
13. Perform the sham surgery following steps 2.2-2.8.
14. 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.

3. Behavioral testing

1. Acclimate the mouse to the housing conditions for at least 8 days before pre-operative testing.
2. Before pre-operative testing, habituate the mouse to the test procedure at least once daily for 3 days.
3. Conduct testing in normal lighting conditions. If necessary, provide background noise to minimize disturbances from outside noises.
4. Observation of face grooming behavior
   1. 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.
   2. 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's face when its back is toward the camera.
   3. Record the mouse's behavior for 10 min. During recording, ensure that the experimenter is not present in the room.
   4. After recording the next animal, clean the observation cage.
   5. Have an observer who is blind to the experimental conditions of the mouse analyze the recorded behavior.
   6. 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.
   7. 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.
   8. 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.
5. Mechanical stimulation testing
   1. 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.
   2. Place the mice one at a time on a table.
      1. 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.
      2. 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).
   3. 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.
   4. Habituate the mice to the restrainer and reaching movements for 10 min. Every 30 s, make a reaching movement per animal.
   5. 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.
   6. Score the animal response to the stimulation to fit into one of the following response categories.
      1. Give a score of 0 when there is no response.
      2. Give a score of 1 for detection, i.e., the mouse turns its head toward the stimulating object and then explores the stimulus object.
      3. 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.
      4. Give a score of 3 for attacking, i.e., the mouse attacks the stimulus object, making biting and/or grabbing movements.
      5. 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.
   7. 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.
   8. 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.

Results

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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Figure 3: Postoperative changes in isolated face grooming behavior following DIoN-CCI. Data points denote the (A) amount of time spent (mean ± SEM; n =15 per group) on isolated face grooming and the (B) number (mean ± SEM; n = 15 per group) of isolated face grooming episodes 1 day before DIoN surgery (Pre-op) and on postoperative days 3-42. Please click here to view a larger version of this figure.

Figure 4: Time course of the effects of DIoN-CCI on responsiveness to mechanical stimulation. Data points denote the response score (mean ± 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. Please click here to view a larger version of this figure.

Discussion

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 benefits^13,14. 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 nerve^4,15,16. 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'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'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 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chromic catgut (6-0)	Dynek	CG602D	ligatures
Cotton applicator	Pharmacy
Digital video camera	Sony	HDR-CX330E
Dumont #5 forceps	Fine Science Tools	11251-10
Dumont forceps - Micro-blunted tips (#5/45)	Fine Science Tools	11253-25
Duratears	Alcon	0037-820	ophthalmic ointment
Hooked ligation aid	Fine Science Tools	18062-12
Ketalar	Pfizer		ketamine (50 mg/mL)
Operation microscope	Kaps	SOM 62
Precision cotton swab	Qosina	10225
Precision trimmer	Philips	HP6392/00
Rompun	Bayer		xylazine (2%)
Scissors - blunt tips	Fine Science Tools	14574-09
Semmes-Weinstein Von Frey Aesthesiometer kit	Stoelting	58011
Vicryl Rapide	Ethicon	MPVR489H	sutures