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 (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.
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.
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
2. Surgery
3. Behavioral testing
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.
In rats, it has been previously argued that an intra-orbital approach to the IoN is preferable, considering the importance of intact fine musculature controlling complex whisking patterns in vibrissotactile discrimination and the relative distance of the mid-line incision to the cutaneous infraorbital nerve territory10. Others have argued that a distal approach via an incision into the hairy skin caudal to the vibrissal pad has a number of benefits13,14. Surgically, it is an easier technique that is minimally invasive. It can be performed without using a stereotaxic frame and in a shorter amount of time. Both techniques result in comparable postoperative behavioral effects, both evoked and non-evoked pain behavior. Furthermore, the procedure avoids possible eye discomfort resulting from deflecting orbital contents during the procedure and potentially irritating contact between the nerve ligations and the eye. In mice, due to their smaller size, we found the intra-orbital approach too difficult to use as a standard procedure. Therefore, this procedure aims to use a distal approach to surgically induce an infraorbital nerve injury in a mouse, leading to the development of spontaneous and evoked pain behavior that can be used to study trigeminal neuropathic pain.
A crucial step in applying this model, as with other nerve ligation-induced chronic pain animal models, is placing the ligature with the correct amount of constriction around the infraorbital nerve4,15,16. Behavioral outcomes are vastly different in animals with varying degrees of nerve constriction17. The ratio of the size of the chrome gut (6-0) to the mouse infraorbital nerve diameter is not the same as that in rats where size 5-0 is used. In mice, it was found that two ligatures induced a level of nerve injury that was higher than that in rats. Because size 6-0 was the smallest chrome gut that could be found, in the present study, we chose to use a single ligature, which produced behavioral outcomes similar to those in rats. Two non-chrome gut ligatures size 7-0 could possibly also produce similar results.
Stimulation of the IoN territory with von Frey hairs requires animals to be relatively motionless. In rats, this can be achieved by habituating the animals to an observation cage. In mice, due to their high locomotor activity, using this method makes it difficult to stimulate the IoN territory with high precision. Holding the animal by hand is a stressful method that severely compromises the validity and reliability of the animal'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.
The authors have nothing to disclose.
The authors have no acknowledgments.
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 |