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Orofacial neuropathic pain reduces spontaneous burrowing behavior in rats
Physiology & Behavior 191 (2018) 91–94
Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/physbeh
Orofacial neuropathic pain reduces spontaneous burrowing behavior in rats K. Deseure a b
a,b,⁎
, G. Hans
T
a,b
Laboratory for Pain Research, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium Multidisciplinary Pain Center, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium
A R T I C LE I N FO
A B S T R A C T
Keywords: Burrowing Behavior Neuropathic Pain Infraorbital Orofacial
It was recently reported that spontaneous burrowing behavior is decreased after tibial nerve transection, spinal nerve transection and partial sciatic nerve ligation. It was proposed that spontaneous burrowing could be used as a measure of the impact of neuropathic pain after peripheral nerve injury. It has remained unclear whether the reduction in burrowing behavior is caused directly by pain or hypersensitivity in the affected limbs, making it more difficult to perform burrowing, or by a pain induced decrease in the general wellbeing, thus reducing the motivation to burrow. We studied burrowing behavior after infraorbital nerve injury, a model of orofacial neuropathic pain that does not affect the limbs. Burrowing behavior was significantly reduced after infraorbital nerve injury. Isolated face grooming and responsiveness to mechanical von Frey stimulation of the infraorbital nerve territory were significantly increased after infraorbital nerve injury, indicative, respectively, of spontaneous pain and mechanical allodynia. It is concluded that spontaneous burrowing may provide a measure of the global impact of pain on the animal's wellbeing after peripheral nerve injury and incorporation of this behavioral assay in preclinical drug testing may improve the predictive validity of currently used pain models.
1. Introduction Neuropathic pain patients continue to suffer due to a lack of effective treatment options [1]. Unsurprisingly, pain research focuses on the development of new and more effective treatments. As a result, there is a need for valid and predictive animal models that require easy and reliable pain measures. Considering that ongoing pain is a common clinical symptom of neuropathic pain, there is a disproportionate number of studies that have used sensory reflex based pain testing paradigms with very few attempts to measure ongoing pain or evaluate the affective motivational component of pain [2,3]. These testing paradigms are furthermore subject to false positives necessitating appropriate control measurements. There is a need for pain models that are able to measure ongoing pain, evaluate the impact of pain on the animal and are able to discern between analgesia and sedation or motor impairment. Recently, some studies have proposed the innate burrowing behavior in rodents as a measure of general animal wellbeing and of the impact of pain thereon [4–8]. Unlike reflex based pain testing paradigms, sedation or motor impairment does not result in false positives, but would mask rather than falsely induce analgesic effects. Furthermore, because rats are not restrained, and assuming animals are sufficiently habituated to the testing conditions and are allowed to acclimate to the testing room, there is a much smaller risk for stress-induced
⁎
analgesia [9]. Finally, given that the experimenter is not present in the testing room, the potential for bias is also much smaller. Andrews et al. [5] showed that spontaneous burrowing behavior was reduced after nerve injury and intra-plantar injection of Complete Freund's Adjuvant. It was concluded that measuring burrowing behavior could be a successful approach to improving predictive validity and eliminating false positives from translational pain research. Unfortunately, because of the choice of the nerve injury in the latter study, as the rat uses its hind limbs to push the burrowing substrate out of the burrow, the plantar surface becomes stimulated. Therefore, and although no correlation was found between hind limb hypersensitivity and the level of burrowing, it cannot be excluded that evoked pain from the affected hind limb was at least partly responsible for the changes in burrowing behavior. It was suggested that in order to discern between the possibilities that either a pain induced change in wellbeing or evoked pain in the hind limbs is responsible for the change in burrowing behavior, a nerve injury model that does not affect the limbs should be used. The present study used the infraorbital nerve ligation model [10], which does not affect the limbs, to investigate the effect of spontaneous neuropathic orofacial pain on spontaneous burrowing behavior.
Corresponding author at: University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. E-mail address: [email protected] (K. Deseure).
https://doi.org/10.1016/j.physbeh.2018.04.020 Received 25 July 2017; Received in revised form 27 March 2018; Accepted 15 April 2018 Available online 17 April 2018 0031-9384/ © 2018 Elsevier Inc. All rights reserved.
Physiology & Behavior 191 (2018) 91–94
K. Deseure, G. Hans
2. Materials and methods
stopwatch. A distinction was made between isolated face grooming and face grooming during body grooming [14]. If a sequence was neither preceded nor followed by body grooming (i.e., movement patterns in which paws, tongue, or incisors are brought in contact with a body area other than the face or the forepaws, see [10]), the episode was categorized as isolated face grooming. The amount of time spent on isolated face grooming was determined as the sum of time spent on isolated face grooming episodes recorded during the observation period. If body grooming was present before or after a sequence of face grooming actions, the episode was categorized as face grooming during body grooming. The amount of time spent on face grooming during body grooming was calculated in the same way as for isolated face grooming.
2.1. Subjects Male Sprague-Dawley rats (Charles River, N = 20, weighing 225250 g at arrival) were housed in solid-bottom polycarbonate cages in a colony room with a humidity of 40 ± 10% and a room temperature of 21 ± 1° C. Water and food were available ad libitum. Rats were kept under a reversed 12:12 h dark/light cycle (lights on at 20 h) and were allowed to acclimate for 9 days to the housing conditions before preoperative testing. Animals were treated and cared for according to the guidelines of the Committee for Research and Ethical Issues of IASP [11]. The protocol was approved by the Ethical Committee of the University of Antwerp (UA).
2.3.3. Mechanical stimulation A graded series of five von Frey hairs (Pressure Aesthesiometer®, Stoelting Co, Chicago, IL) were used. The force required to bend the filaments was 0.02 g, 0.16 g, 0.40 g, 1.00 g and 2.00 g, respectively. The stimuli were applied within the IoN territory, near the center of the vibrissal pad, on the hairy skin surrounding the mystacial vibrissae. Within each animal, stimuli were applied in an ascending order of intensity. After a stimulus intensity was applied to one side, it was applied to the other side before moving on to the next stimulus intensity. The order in which the ipsilateral and contralateral sides were stimulated, was randomized. The scoring system described by Vos et al. [10] was used to evaluate the response of the rats to the stimulation. The response was observed to belong to one of the following response categories: (score 0) no response; (score 1) detection = the rat turns the head toward the stimulating object and the stimulus object is then explored; (score 2) withdrawal reaction = the rat turns the head slowly away or pulls it briskly backward when the stimulation is applied, sometimes a single face wipe ipsilateral to the stimulated area occurs; (score 3) escape/attack = the rat avoids further contact with the stimulus object, either passively by moving its body away from the stimulating object to assume a crouching position against the cage wall, or actively by attacking the stimulus object, making biting and grabbing movements; (score 4) asymmetric face grooming = the rat displays an uninterrupted series of at least three face-wash strokes directed toward the stimulated facial area. For each rat, and at every designated time, a mean score for the five von Frey hairs was determined.
2.2. Surgery The unilateral ligation of the infraorbital nerve (IoN) was performed as described elsewhere [10]. Rats were anaesthetized with pentobarbital (60 mg/kg, i.p.) and treated with atropine (0.1 mg/kg, i.p.). Surgery was performed under direct visual control using a Kaps operation microscope (x10–25). The rat’s head was fixed in a stereotaxic frame and a mid-line scalp incision was made, exposing skull and nasal bone. The infraorbital part of the left IoN was exposed using a surgical procedure similar to that described earlier [12,13]. The edge of the orbit, formed by the maxillary, frontal, lacrimal and zygomatic bones, was dissected free. To give access to the IoN, the orbital contents were gently deflected with a cotton-tipped wooden rod. The IoN was dissected free and two chromic catgut ligatures (5–0) were loosely tied around the IoN (2 mm apart). The ligatures reduced the diameter of the nerve by a just noticeable amount and retarded, but did not interrupt the circulation through the superficial vasculature. The scalp incision was closed using polyester sutures (4–0). In sham operated rats, the IoN was exposed using the same procedure, but the exposed IoN was not ligated. Rats were randomly assigned to one of two experimental groups: 10 rats received an IoN ligation and 10 rats received a sham operation. 2.3. Behavioral testing Habituation/training and testing were conducted in a darkened room (light provided by a 60 W red light bulb suspended 1 m above the observation area) with a 45 dB background noise. Three habituation/ training sessions were performed before pre-operative testing. Burrowing and face grooming behavior were observed on pre-operative day −1 and on post-operative day +5. Mechanical stimulation testing was performed on pre-operative day −1 and on post-operative days, +5 and + 24. Behavior was analyzed by an experimenter who was blind to the experimental group of the rat.
2.4. Statistical analysis Data were analyzed using IBM SPSS Statistics 22 software. Data are expressed as mean ± S.E.M and were analyzed by means of a repeated measures ANOVA with time (rats were tested on different time points) as within-subjects factors, and surgery (two different types of IoN surgery) as between-subjects factor. This analysis was followed by independent sample t-tests per time point. The p-values for these t-tests were corrected for multiple testing using the Bonferroni method (i.e., the raw p-value was multiplied by the number of tests to account for multiple hypothesis testing).
2.3.1. Burrowing The burrows were hollow plastic tubes (see [5]; 32 cm long × 10 cm diameter) sealed with an mdf plug on one end and open at the other. The open end of the tube was raised 65 mm above the ground to prevent the burrowing material from spilling onto the floor when the animals were not burrowing. Tubes were filled with 1 kg of food pellets. The test cage was a standard home cage used for housing groups of rats (56 cm long × 34 cm wide × 19 cm high) with wood chip bedding on the floor. Rats were placed individually in the test cages and were allowed to burrow for 4 h. The amount of food pellets that were removed from the burrow was weighed.
3. Results 3.1. Burrowing The amount of burrowing in IoN ligated animals was significantly decreased compared to that in sham operated animals [groups x time interaction: F(1,18) = 8.01, P < 0.05] (Fig. 1). Independent sample ttests, corrected for multiple testing using the Bonferroni method, showed a significant difference between the IoN-CCI and the Sham group on post-operative day +5 [t(18) = −3.75, P < 0.01], but not on pre-operative day −1 [t(18) = −0.79, NS]. Burrowing was reduced in IoN ligated animals whereas no change in burrowing behavior was observed in sham operated rats.
2.3.2. Face grooming Behavior was videotaped for 10 min and subsequently analyzed.The amount of time spent on face grooming (i.e., movement patterns in which paws contact facial areas; see [10]) was determined using a 92
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K. Deseure, G. Hans
P < 0.001] and + 24 [t(18) = 4.19, P < 0.01], but not on pre-operative day −1 [t(18) = 1.44, NS]. On day +5, ipsilateral response scores were significantly decreased in IoN ligated animals whereas no changes were observed in sham operated rats. On day +24, ipsilateral response scores were increased significantly more strongly in IoN ligated animals than in sham operated rats. Response scores to contralateral mechanical stimulation in IoN ligated animals were not significantly different from those in sham operated animals [groups x time interaction: F(2,36) = 0.60, NS] (Fig. 3B). 4. Discussion The present results confirm previous reports that pain, including neuropathic pain, impacts the innate burrowing behavior in rodents. Five days after infraorbital nerve ligation, i.e., at a time at which isolated face grooming behavior is most strongly increased, rats performed significantly less burrowing behavior and significantly more isolated face grooming [14–16]. A significant correlation was found between isolated face grooming and burrowing behavior (Pearson r = 0.45, P < 0.05 [17]). Face grooming behavior has been long used as a measure of non-evoked pain in the IoN-CCI (infraorbital nerve chronic constriction injury) model [10,14–16]. As such, burrowing constitutes an ethologically relevant measure in these animals of non-evoked pain [5,6,8]. Contrary to previous studies of burrowing behavior following nerve injury, in the infraorbital nerve ligation model, the limbs are not affected and can therefore be excluded as a potential cause of altered burrowing behavior. It should be pointed out that on post-operative day +5, i.e. the time point when burrowing and face grooming behavior were recorded, the animals were hyporesponsive to mechanical stimulation of the ipsilateral IoN territory. It is unclear to what extent intact vibrissal sensitivity and subsequent thigmotactic scanning is important to the burrowing behavior. To the best of our knowledge, the latter has not yet been documented. The hyporesponsiveness was complete (i.e., score 0) in all but one animal. As a result, restriction of range makes it impossible to correlate the burrowing data to the response scores. In order to investigate the effect of orofacial sensitivity on burrowing, it is proposed that a follow-up study examines the effects of sensory disturbances in the IoN territory, such as vibrissal clipping or local anesthetic blockade on burrowing behavior. Supressed burrowing is not a pain-specific measure. Burrowing is affected by many pathological conditions, including brain lesions (hippocampus and prefrontal cortex) and disorders (e.g. prion disease),
Fig. 1. Post-operative changes in burrowing behavior following IoN ligation. Data points represent the mean ( ± SEM; n = 10 per group) amount of burrowing material displaced (g) one day before IoN surgery (Pre-op) and on postoperative day +5. Asterisks indicate a significant difference compared to the sham group (**P < 0.01).
3.2. Face grooming The amount of time spent on isolated face grooming behavior in IoN ligated animals was significantly different from that in sham operated animals [groups x time interaction: F(1,18) = 16.51, P < 0.01] (Fig. 2A). Independent sample t-tests, corrected for multiple testing using the Bonferroni method, showed a significant difference between the IoN-CCI and the Sham group on post-operative day +5 [t (18) = 6.35, P < 0.001], but not on pre-operative day −1 [t (18) = 0.63, NS]. Isolated face grooming was increased in IoN ligated animals whereas no change was observed in sham operated rats. The amount of time spent on face grooming during body grooming in IoN ligated animals was not significantly different from that in sham operated animals [groups x time interaction: F(1,18) = 0.10, NS] (Fig. 2B). 3.3. Mechanical stimulation Response scores to ipsilateral mechanical stimulation in IoN ligated animals were significantly different from those in sham operated animals [groups x time interaction: F(2,36) = 160.10, P < 0.001] (Fig. 3A). Independent sample t-tests, corrected for multiple testing using the Bonferroni method, showed a significant difference between the surgical groups on post-operative day +5 [t(18) = −13.48,
Fig. 2. Post-operative changes in face grooming behavior following IoN ligation. Data points represent the mean ( ± SEM; n = 10 per group) amount of time spent on isolated face grooming (panel A) and face grooming during body grooming (panel B) one day before IoN surgery (Pre-op) and on post-operative day +5. Asterisks indicate a significant difference compared to the sham group (**P < 0.01). 93
Physiology & Behavior 191 (2018) 91–94
K. Deseure, G. Hans
Fig. 3. Post-operative changes in responsiveness to mechanical stimulation. Data points represent the mean ( ± SEM; n = 10 per group) response score to von Frey hair stimulation of the ipsilateral (panel A) and contralateral (panel B) territory of the ligated nerve one day before (Pre-op) and on post-operative days +5 and + 24. Asterisks indicate a significant difference compared to the sham group (**P < 0.01; ***P < 0.001).
peripheral and intestinal inflammation, viral and bacterial infections, but also in chronic stress, depression and under high fat dietary conditions [4,5,7,8,18,19]. Nevertheless, several studies have shown that in various pain models, analgesic drugs were able to reinstate burrowing behavior, suggesting that burrowing may have predictive validity as a measure of the global impact of pain. Particularly, the effect of ibuprofen, which arguably has no direct effects on wellbeing, on burrowing in rats with Complete Freund's Adjuvant induced inflammatory pain is compelling [8]. In this context, a follow-up study examining the effects of analgesics such as carbamazepine, baclofen or morphine on isolated face grooming and burrowing after IoN ligation would be of great value. The present results, ie the reduced burrowing behavior and simultaneous increase in isolated face grooming, also corroborates the validity of the IoN-CCI model as a model of trigeminal neuropathic pain. It has been long argued that the isolated face grooming behavior constitutes a measure of spontaneous unpleasant and probably painful sensations. The proposed reduction in wellbeing must be seen as a result of these painful sensations, although hyposensitivity may also be partly responsible (cf supra).
[3] J.S. Mogil, S.E. Crager, What should we be measuring in behavioral studies of chronic pain in animals? Pain 112 (2004) 12–15. [4] R.M.J. Deacon, Burrowing: a sensitive behavioural assay, tested in five species of laboratory rodents, Behav. Brain Res. 200 (2009) 128–133. [5] N. Andrews, E. Legg, D. Lisak, Y. Issop, D. Richardson, S. Harper, T. Pheby, W. Huang, G. Burgess, I. Machin, A.S. Rice, Spontaneous burrowing behaviour in the rat is reduced by peripheral nerve injury or inflammation associated pain, Eur. J. Pain 16 (2012) 485–495. [6] K. Rutten, K. Schiene, A. Robens, A. Leipelt, T. Pasqualon, S.J. Read, T. Christoph, Burrowing as a non-reflex behavioural readout for analgesic action in a rat model of sub-chronic knee joint inflammation, Eur. J. Pain 18 (2014) 204–212. [7] A.L. Whittaker, K.A. Lymn, A. Nicholson, G.S. Howarth, The assessment of general well-being using spontaneous burrowing behaviour in a short-term model of chemotherapy-induced mucositis in the rat, Lab. Anim. 49 (2015) 30–39. [8] S.A. Gould, H. Doods, T. Lamla, A. Pekcec, Pharmacological characterization of intraplantar complete Freund's adjuvant-induced burrowing deficits, Behav. Brain Res. 301 (2016) 142–151. [9] M.S. Fanselow, Conditioned fear-induced opiate analgesia: a competing motivational state theory of stress analgesia, Ann. N. Y. Acad. Sci. 467 (1986) 40–54. [10] B.P. Vos, A.M. Strassman, R.J. Maciewicz, Behavioral evidence of trigeminal neuropathic pain following chronic constriction injury to the rat's infraorbital nerve, J. Neurosci. 14 (1994) 2708–2723. [11] M. Zimmerman, Ethical guidelines for investigations of experimental pain in conscious animals, Pain 16 (1983) 109–110. [12] J.M. Gregg, A surgical approach to the ophthalmic-maxillary nerve trunks in the rat, J. Dent. Res. 52 (1973) 392. [13] M.F. Jacquin, H.P. Zeigler, Trigeminal orosensation and ingestive behavior in the rat, Behav. Neurosci. 97 (1983) 62–97. [14] K.R. Deseure, H.F. Adriaensen, Comparison between two types of behavioral variables of non-evoked facial pain after chronic constriction injury to the rat infraorbital nerve, Comp. Med. 52 (2002) 44–49. [15] K. Deseure, H. Adriaensen, Nonevoked facial pain in rats following infraorbital nerve injury: a parametric analysis, Physiol. Behav. 81 (2004) 595–604. [16] K. Deseure, G.H. Hans, Differential drug effects on spontaneous and evoked pain behavior in a model of trigeminal neuropathic pain, J. Pain Res. 10 (2017) 279–286. [17] R. Lowry, http://vassarstats.net/rsig.html. [18] A. Muralidharan, A. Kuo, M. Jacob, J.S. Lourdesamy, L.M. Carvalho, J.R. Nicholson, L. Corradini, M.T. Smith, Comparison of burrowing and stimuli-evoked pain behaviors as end-points in rat models of inflammatory pain and peripheral neuropathic pain, Front. Behav. Neurosci. 10 (2016) 88. [19] K. Luedtke, S.M. Bouchard, S.A. Woller, M.K. Funk, M. Aceves, M.A. Hook, Assessment of depression in a rodent model of spinal cord injury, J. Neurotrauma 15 (31) (2014) 1107–1121.
5. Conclusions It is concluded that spontaneous burrowing may provide a measure of the global impact of pain on the animal's wellbeing after peripheral nerve injury and incorporation of this behavioral assay in preclinical drug testing may improve the predictive validity of currently used pain models. References [1] J.H. Vranken, Elucidation of pathophysiology and treatment of neuropathic pain, Cent. Nerv. Syst. Agents Med. Chem. 12 (2012) 304–314. [2] M.M. Backonja, B. Stacey, Neuropathic pain symptoms relative to overall pain rating, J. Pain 5 (2004) 491–497.
94
Contents lists available at ScienceDirect
Physiology & Behavior journal homepage: www.elsevier.com/locate/physbeh
Orofacial neuropathic pain reduces spontaneous burrowing behavior in rats K. Deseure a b
a,b,⁎
, G. Hans
T
a,b
Laboratory for Pain Research, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium Multidisciplinary Pain Center, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium
A R T I C LE I N FO
A B S T R A C T
Keywords: Burrowing Behavior Neuropathic Pain Infraorbital Orofacial
It was recently reported that spontaneous burrowing behavior is decreased after tibial nerve transection, spinal nerve transection and partial sciatic nerve ligation. It was proposed that spontaneous burrowing could be used as a measure of the impact of neuropathic pain after peripheral nerve injury. It has remained unclear whether the reduction in burrowing behavior is caused directly by pain or hypersensitivity in the affected limbs, making it more difficult to perform burrowing, or by a pain induced decrease in the general wellbeing, thus reducing the motivation to burrow. We studied burrowing behavior after infraorbital nerve injury, a model of orofacial neuropathic pain that does not affect the limbs. Burrowing behavior was significantly reduced after infraorbital nerve injury. Isolated face grooming and responsiveness to mechanical von Frey stimulation of the infraorbital nerve territory were significantly increased after infraorbital nerve injury, indicative, respectively, of spontaneous pain and mechanical allodynia. It is concluded that spontaneous burrowing may provide a measure of the global impact of pain on the animal's wellbeing after peripheral nerve injury and incorporation of this behavioral assay in preclinical drug testing may improve the predictive validity of currently used pain models.
1. Introduction Neuropathic pain patients continue to suffer due to a lack of effective treatment options [1]. Unsurprisingly, pain research focuses on the development of new and more effective treatments. As a result, there is a need for valid and predictive animal models that require easy and reliable pain measures. Considering that ongoing pain is a common clinical symptom of neuropathic pain, there is a disproportionate number of studies that have used sensory reflex based pain testing paradigms with very few attempts to measure ongoing pain or evaluate the affective motivational component of pain [2,3]. These testing paradigms are furthermore subject to false positives necessitating appropriate control measurements. There is a need for pain models that are able to measure ongoing pain, evaluate the impact of pain on the animal and are able to discern between analgesia and sedation or motor impairment. Recently, some studies have proposed the innate burrowing behavior in rodents as a measure of general animal wellbeing and of the impact of pain thereon [4–8]. Unlike reflex based pain testing paradigms, sedation or motor impairment does not result in false positives, but would mask rather than falsely induce analgesic effects. Furthermore, because rats are not restrained, and assuming animals are sufficiently habituated to the testing conditions and are allowed to acclimate to the testing room, there is a much smaller risk for stress-induced
⁎
analgesia [9]. Finally, given that the experimenter is not present in the testing room, the potential for bias is also much smaller. Andrews et al. [5] showed that spontaneous burrowing behavior was reduced after nerve injury and intra-plantar injection of Complete Freund's Adjuvant. It was concluded that measuring burrowing behavior could be a successful approach to improving predictive validity and eliminating false positives from translational pain research. Unfortunately, because of the choice of the nerve injury in the latter study, as the rat uses its hind limbs to push the burrowing substrate out of the burrow, the plantar surface becomes stimulated. Therefore, and although no correlation was found between hind limb hypersensitivity and the level of burrowing, it cannot be excluded that evoked pain from the affected hind limb was at least partly responsible for the changes in burrowing behavior. It was suggested that in order to discern between the possibilities that either a pain induced change in wellbeing or evoked pain in the hind limbs is responsible for the change in burrowing behavior, a nerve injury model that does not affect the limbs should be used. The present study used the infraorbital nerve ligation model [10], which does not affect the limbs, to investigate the effect of spontaneous neuropathic orofacial pain on spontaneous burrowing behavior.
Corresponding author at: University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. E-mail address: [email protected] (K. Deseure).
https://doi.org/10.1016/j.physbeh.2018.04.020 Received 25 July 2017; Received in revised form 27 March 2018; Accepted 15 April 2018 Available online 17 April 2018 0031-9384/ © 2018 Elsevier Inc. All rights reserved.
Physiology & Behavior 191 (2018) 91–94
K. Deseure, G. Hans
2. Materials and methods
stopwatch. A distinction was made between isolated face grooming and face grooming during body grooming [14]. If a sequence was neither preceded nor followed by body grooming (i.e., movement patterns in which paws, tongue, or incisors are brought in contact with a body area other than the face or the forepaws, see [10]), the episode was categorized as isolated face grooming. The amount of time spent on isolated face grooming was determined as the sum of time spent on isolated face grooming episodes recorded during the observation period. If body grooming was present before or after a sequence of face grooming actions, the episode was categorized as face grooming during body grooming. The amount of time spent on face grooming during body grooming was calculated in the same way as for isolated face grooming.
2.1. Subjects Male Sprague-Dawley rats (Charles River, N = 20, weighing 225250 g at arrival) were housed in solid-bottom polycarbonate cages in a colony room with a humidity of 40 ± 10% and a room temperature of 21 ± 1° C. Water and food were available ad libitum. Rats were kept under a reversed 12:12 h dark/light cycle (lights on at 20 h) and were allowed to acclimate for 9 days to the housing conditions before preoperative testing. Animals were treated and cared for according to the guidelines of the Committee for Research and Ethical Issues of IASP [11]. The protocol was approved by the Ethical Committee of the University of Antwerp (UA).
2.3.3. Mechanical stimulation A graded series of five von Frey hairs (Pressure Aesthesiometer®, Stoelting Co, Chicago, IL) were used. The force required to bend the filaments was 0.02 g, 0.16 g, 0.40 g, 1.00 g and 2.00 g, respectively. The stimuli were applied within the IoN territory, near the center of the vibrissal pad, on the hairy skin surrounding the mystacial vibrissae. Within each animal, stimuli were applied in an ascending order of intensity. After a stimulus intensity was applied to one side, it was applied to the other side before moving on to the next stimulus intensity. The order in which the ipsilateral and contralateral sides were stimulated, was randomized. The scoring system described by Vos et al. [10] was used to evaluate the response of the rats to the stimulation. The response was observed to belong to one of the following response categories: (score 0) no response; (score 1) detection = the rat turns the head toward the stimulating object and the stimulus object is then explored; (score 2) withdrawal reaction = the rat turns the head slowly away or pulls it briskly backward when the stimulation is applied, sometimes a single face wipe ipsilateral to the stimulated area occurs; (score 3) escape/attack = the rat avoids further contact with the stimulus object, either passively by moving its body away from the stimulating object to assume a crouching position against the cage wall, or actively by attacking the stimulus object, making biting and grabbing movements; (score 4) asymmetric face grooming = the rat displays an uninterrupted series of at least three face-wash strokes directed toward the stimulated facial area. For each rat, and at every designated time, a mean score for the five von Frey hairs was determined.
2.2. Surgery The unilateral ligation of the infraorbital nerve (IoN) was performed as described elsewhere [10]. Rats were anaesthetized with pentobarbital (60 mg/kg, i.p.) and treated with atropine (0.1 mg/kg, i.p.). Surgery was performed under direct visual control using a Kaps operation microscope (x10–25). The rat’s head was fixed in a stereotaxic frame and a mid-line scalp incision was made, exposing skull and nasal bone. The infraorbital part of the left IoN was exposed using a surgical procedure similar to that described earlier [12,13]. The edge of the orbit, formed by the maxillary, frontal, lacrimal and zygomatic bones, was dissected free. To give access to the IoN, the orbital contents were gently deflected with a cotton-tipped wooden rod. The IoN was dissected free and two chromic catgut ligatures (5–0) were loosely tied around the IoN (2 mm apart). The ligatures reduced the diameter of the nerve by a just noticeable amount and retarded, but did not interrupt the circulation through the superficial vasculature. The scalp incision was closed using polyester sutures (4–0). In sham operated rats, the IoN was exposed using the same procedure, but the exposed IoN was not ligated. Rats were randomly assigned to one of two experimental groups: 10 rats received an IoN ligation and 10 rats received a sham operation. 2.3. Behavioral testing Habituation/training and testing were conducted in a darkened room (light provided by a 60 W red light bulb suspended 1 m above the observation area) with a 45 dB background noise. Three habituation/ training sessions were performed before pre-operative testing. Burrowing and face grooming behavior were observed on pre-operative day −1 and on post-operative day +5. Mechanical stimulation testing was performed on pre-operative day −1 and on post-operative days, +5 and + 24. Behavior was analyzed by an experimenter who was blind to the experimental group of the rat.
2.4. Statistical analysis Data were analyzed using IBM SPSS Statistics 22 software. Data are expressed as mean ± S.E.M and were analyzed by means of a repeated measures ANOVA with time (rats were tested on different time points) as within-subjects factors, and surgery (two different types of IoN surgery) as between-subjects factor. This analysis was followed by independent sample t-tests per time point. The p-values for these t-tests were corrected for multiple testing using the Bonferroni method (i.e., the raw p-value was multiplied by the number of tests to account for multiple hypothesis testing).
2.3.1. Burrowing The burrows were hollow plastic tubes (see [5]; 32 cm long × 10 cm diameter) sealed with an mdf plug on one end and open at the other. The open end of the tube was raised 65 mm above the ground to prevent the burrowing material from spilling onto the floor when the animals were not burrowing. Tubes were filled with 1 kg of food pellets. The test cage was a standard home cage used for housing groups of rats (56 cm long × 34 cm wide × 19 cm high) with wood chip bedding on the floor. Rats were placed individually in the test cages and were allowed to burrow for 4 h. The amount of food pellets that were removed from the burrow was weighed.
3. Results 3.1. Burrowing The amount of burrowing in IoN ligated animals was significantly decreased compared to that in sham operated animals [groups x time interaction: F(1,18) = 8.01, P < 0.05] (Fig. 1). Independent sample ttests, corrected for multiple testing using the Bonferroni method, showed a significant difference between the IoN-CCI and the Sham group on post-operative day +5 [t(18) = −3.75, P < 0.01], but not on pre-operative day −1 [t(18) = −0.79, NS]. Burrowing was reduced in IoN ligated animals whereas no change in burrowing behavior was observed in sham operated rats.
2.3.2. Face grooming Behavior was videotaped for 10 min and subsequently analyzed.The amount of time spent on face grooming (i.e., movement patterns in which paws contact facial areas; see [10]) was determined using a 92
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K. Deseure, G. Hans
P < 0.001] and + 24 [t(18) = 4.19, P < 0.01], but not on pre-operative day −1 [t(18) = 1.44, NS]. On day +5, ipsilateral response scores were significantly decreased in IoN ligated animals whereas no changes were observed in sham operated rats. On day +24, ipsilateral response scores were increased significantly more strongly in IoN ligated animals than in sham operated rats. Response scores to contralateral mechanical stimulation in IoN ligated animals were not significantly different from those in sham operated animals [groups x time interaction: F(2,36) = 0.60, NS] (Fig. 3B). 4. Discussion The present results confirm previous reports that pain, including neuropathic pain, impacts the innate burrowing behavior in rodents. Five days after infraorbital nerve ligation, i.e., at a time at which isolated face grooming behavior is most strongly increased, rats performed significantly less burrowing behavior and significantly more isolated face grooming [14–16]. A significant correlation was found between isolated face grooming and burrowing behavior (Pearson r = 0.45, P < 0.05 [17]). Face grooming behavior has been long used as a measure of non-evoked pain in the IoN-CCI (infraorbital nerve chronic constriction injury) model [10,14–16]. As such, burrowing constitutes an ethologically relevant measure in these animals of non-evoked pain [5,6,8]. Contrary to previous studies of burrowing behavior following nerve injury, in the infraorbital nerve ligation model, the limbs are not affected and can therefore be excluded as a potential cause of altered burrowing behavior. It should be pointed out that on post-operative day +5, i.e. the time point when burrowing and face grooming behavior were recorded, the animals were hyporesponsive to mechanical stimulation of the ipsilateral IoN territory. It is unclear to what extent intact vibrissal sensitivity and subsequent thigmotactic scanning is important to the burrowing behavior. To the best of our knowledge, the latter has not yet been documented. The hyporesponsiveness was complete (i.e., score 0) in all but one animal. As a result, restriction of range makes it impossible to correlate the burrowing data to the response scores. In order to investigate the effect of orofacial sensitivity on burrowing, it is proposed that a follow-up study examines the effects of sensory disturbances in the IoN territory, such as vibrissal clipping or local anesthetic blockade on burrowing behavior. Supressed burrowing is not a pain-specific measure. Burrowing is affected by many pathological conditions, including brain lesions (hippocampus and prefrontal cortex) and disorders (e.g. prion disease),
Fig. 1. Post-operative changes in burrowing behavior following IoN ligation. Data points represent the mean ( ± SEM; n = 10 per group) amount of burrowing material displaced (g) one day before IoN surgery (Pre-op) and on postoperative day +5. Asterisks indicate a significant difference compared to the sham group (**P < 0.01).
3.2. Face grooming The amount of time spent on isolated face grooming behavior in IoN ligated animals was significantly different from that in sham operated animals [groups x time interaction: F(1,18) = 16.51, P < 0.01] (Fig. 2A). Independent sample t-tests, corrected for multiple testing using the Bonferroni method, showed a significant difference between the IoN-CCI and the Sham group on post-operative day +5 [t (18) = 6.35, P < 0.001], but not on pre-operative day −1 [t (18) = 0.63, NS]. Isolated face grooming was increased in IoN ligated animals whereas no change was observed in sham operated rats. The amount of time spent on face grooming during body grooming in IoN ligated animals was not significantly different from that in sham operated animals [groups x time interaction: F(1,18) = 0.10, NS] (Fig. 2B). 3.3. Mechanical stimulation Response scores to ipsilateral mechanical stimulation in IoN ligated animals were significantly different from those in sham operated animals [groups x time interaction: F(2,36) = 160.10, P < 0.001] (Fig. 3A). Independent sample t-tests, corrected for multiple testing using the Bonferroni method, showed a significant difference between the surgical groups on post-operative day +5 [t(18) = −13.48,
Fig. 2. Post-operative changes in face grooming behavior following IoN ligation. Data points represent the mean ( ± SEM; n = 10 per group) amount of time spent on isolated face grooming (panel A) and face grooming during body grooming (panel B) one day before IoN surgery (Pre-op) and on post-operative day +5. Asterisks indicate a significant difference compared to the sham group (**P < 0.01). 93
Physiology & Behavior 191 (2018) 91–94
K. Deseure, G. Hans
Fig. 3. Post-operative changes in responsiveness to mechanical stimulation. Data points represent the mean ( ± SEM; n = 10 per group) response score to von Frey hair stimulation of the ipsilateral (panel A) and contralateral (panel B) territory of the ligated nerve one day before (Pre-op) and on post-operative days +5 and + 24. Asterisks indicate a significant difference compared to the sham group (**P < 0.01; ***P < 0.001).
peripheral and intestinal inflammation, viral and bacterial infections, but also in chronic stress, depression and under high fat dietary conditions [4,5,7,8,18,19]. Nevertheless, several studies have shown that in various pain models, analgesic drugs were able to reinstate burrowing behavior, suggesting that burrowing may have predictive validity as a measure of the global impact of pain. Particularly, the effect of ibuprofen, which arguably has no direct effects on wellbeing, on burrowing in rats with Complete Freund's Adjuvant induced inflammatory pain is compelling [8]. In this context, a follow-up study examining the effects of analgesics such as carbamazepine, baclofen or morphine on isolated face grooming and burrowing after IoN ligation would be of great value. The present results, ie the reduced burrowing behavior and simultaneous increase in isolated face grooming, also corroborates the validity of the IoN-CCI model as a model of trigeminal neuropathic pain. It has been long argued that the isolated face grooming behavior constitutes a measure of spontaneous unpleasant and probably painful sensations. The proposed reduction in wellbeing must be seen as a result of these painful sensations, although hyposensitivity may also be partly responsible (cf supra).
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5. Conclusions It is concluded that spontaneous burrowing may provide a measure of the global impact of pain on the animal's wellbeing after peripheral nerve injury and incorporation of this behavioral assay in preclinical drug testing may improve the predictive validity of currently used pain models. References [1] J.H. Vranken, Elucidation of pathophysiology and treatment of neuropathic pain, Cent. Nerv. Syst. Agents Med. Chem. 12 (2012) 304–314. [2] M.M. Backonja, B. Stacey, Neuropathic pain symptoms relative to overall pain rating, J. Pain 5 (2004) 491–497.
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