Physiology & Behavior 138 (2015) 292–296

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Behavioral study of non-evoked orofacial pain following different types of infraorbital nerve injury in rats Kristof Deseure ⁎, Guy Hans a b

Laboratory of Pain Research, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium Multidisciplinary Pain Center, Antwerp University Hospital, Wilrijkstraat 10, 2650 Edegem, Belgium

H I G H L I G H T S • • • •

Face grooming after different types of infraorbital nerve ligation was compared. Tight ligation of the infraorbital nerve has long lasting effects on face grooming. Formalin grooming does not predict grooming after infraorbital nerve ligation. Repeated testing increases statistical power and reduces the number of animals.

a r t i c l e

i n f o

Article history: Received 3 October 2013 Received in revised form 1 July 2014 Accepted 9 October 2014 Available online 24 October 2014 Keywords: Neuropathic Trigeminal Orofacial Pain Infraorbital Nerve Grooming Behavior Formalin

a b s t r a c t Directed isolated face grooming following unilateral chronic constriction injury to the infraorbital nerve (IoN-CCI) is a unique measure of spontaneous neuropathic pain. Variability between rats and the limited duration of the increased face grooming behavior has hampered its usefulness. We studied three possible sources of variability: variations in surgery, pre-existing differences in nocifensive behavior between the rats and variation in time. Three different types of IoN lesion were performed: loose ligation (CCI), tight ligation (CCI-T) and partial tight ligation (PTL, Seltzer method); the latter two offer greater surgical standardization. Face grooming behavior following IoN injury, on the one hand, and during the orofacial formalin test, on the other hand, was analyzed and correlated. Significant differences in isolated face grooming behavior were found between the IoN groups. Interestingly, CCI-T rats continued to show significantly increased isolated face grooming for the duration of the experiment, i.e., up to 32 days post-operative, whereas CCI animals were no longer significantly different from sham animals after two weeks. Surprisingly, PTL operated rats only showed minor effects. Variability was not smaller in the CCI-T or PTL group. Face grooming behavior after IoN lesion was poorly correlated to that in the orofacial formalin test. It is therefore unclear if pre-existing behavioral differences between animals are a major cause of variability in the IoN-CCI model. Finally, repeated testing showed significant variability in time. It is concluded that tight ligation of the IoN nerve has long-lasting effects on face grooming behavior and that part of the variability in face grooming behavior may be reduced by performing repeated testing. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Neuropathic pain substantially reduces patients' quality of life [1,2]. It is a chronic condition that is challenging to treat [3]. Symptoms such as allodynia, hyperalgesia, paresthesia and dysesthesia vary considerably among patients and are largely resistant to treatments with commonly prescribed analgesics [4]. Animal models are used to examine the efficacy of existing and newly developed drugs and to identify the pathophysiological mechanisms involved in the development and

⁎ Corresponding author at: University of Antwerp, Laboratory of Pain Research S4.34, Universiteitsplein 1, 2610 Wilrijk, Belgium. Tel.: +32 3 265 2561. E-mail address: [email protected] (K. Deseure).

http://dx.doi.org/10.1016/j.physbeh.2014.10.009 0031-9384/© 2014 Elsevier Inc. All rights reserved.

maintenance of neuropathic pain. Most of these studies (90%; [5]) have focused exclusively on measuring hypersensitivity: mechanically (pinch/pressure) and thermally (heat/cold) evoked nocifensive behavior (e.g. vocalization, withdrawal responses). Only a very limited number of studies have attempted to measure spontaneous chronic pain. This is in contrast with the fact that the primary complaint of pain patients is ongoing pain, not hyperalgesia or allodynia [6]. Furthermore, based on existing evidence, the pathophysiology of hypersensitivity may be different from that of spontaneous chronic pain [5,7]. As a result, drug screening based on evoked pain will only have a limited predictive validity for efficacy in clinical neuropathic pain syndromes. To date, directed, isolated face grooming following infraorbital nerve (IoN) ligation is the best indicator available of spontaneous neuropathic pain in rats. Unfortunately, variability between rats and the limited duration

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of the increased face grooming behavior has limited its usefulness in the study of neuropathic pain and the development of new drugs [8]. Lack of surgical standardization may be a major source of such variation. The criterion that is used for determining the amount of nerve constriction was first described by Bennet and Xie [9] and adopted by Vos et al. [10]. It states that the ligatures must reduce the diameter of the nerve by a just noticeable amount and should retard, but not interrupt the circulation through the superficial vasculature. Although this is a valuable guideline, it may still be the cause of variation, especially in the IoN model, considering the poor accessibility to the nerve. Several models of nerve ligation have attempted to overcome this problem, e.g. by tightly ligating (and cutting) one or more of several branches of a nerve [11], by tightly ligating one or more spinal nerves [12], or by tightly ligating part of the nerve [13]. Tightly ligating a nerve to the point where you cannot further apply force is expected to have greater standardization. In the present study, the effects of loosely ligating the IoN were compared to those of tightly ligating the entire or part of the IoN. Secondly, it is possible that differences in nocifensive behavior preexist between rats and are thus inducing variability [14,15]. In the present study, it is hypothesized that these differences may become apparent in the orofacial formalin test as well as in the IoN ligation model, i.e., if an animal prefers face grooming as nocifensive behavior in the IoN model, this may also be the case in the formalin test. Formalin injection in the upper lip has been shown to induce biphasic directed face grooming behavior in rats [16,17]; these data can thus be correlated to face grooming data in the IoN model. As a result, it may be possible to predict (and possibly select) which animals will be (un)responsive to IoN ligation based on data obtained in the orofacial formalin test. Finally, it is well known that trigeminal neuropathic pain is often characterized by paroxysmal pain episodes [18,19]. It is possible that also in the IoN model, spontaneous pain may be subject to variation in time. Therefore, in the present protocol, animals were tested twice daily instead of only once. 2. Materials and methods 2.1. Subjects Male Sprague–Dawley rats (Charles River, N = 48, weighing 275– 300 g at arrival) were housed in solid-bottom polycarbonate cages in a colony room with a humidity of 45 ± 5% 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). Animals were treated and cared for according to the guidelines of the Committee for Research and Ethical Issues of IASP [20]. The protocol was approved by the institutional Ethical Committee. Rats were allowed to acclimate for 8 days to the housing conditions before the formalin test. Rats were habituated to the formalin test procedure one and two days before the formalin test. A period of 7 days was respected between the formalin test and pre-operative observations. Rats were habituated to the test procedure every day for three days before pre-operative testing. Habituation 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. Rats were individually transported from the colony to the test room (15 s trip) in a covered plastic cage without bedding (l × w × h: 24 × 14 × 17 cm). 2.2. Surgery The unilateral ligation of the infraorbital nerve was performed as described elsewhere [10]. Rats were anesthetized 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 operating microscope (×10–25). The rat's head was fixed in a stereotaxic frame

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and a mid-line scalp incision was made, exposing the skull and nasal bone. The infraorbital part of the left IoN was exposed using a surgical procedure similar to that described earlier [21,22]. 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; Ethicon, Johnson & Johnson, Belgium). Tight ligation of the infraorbital nerve (CCI-T) was performed using the same procedure as used for the loose ligation, except that only a single catgut ligature (5-0) was tied around the IoN as tightly as possible. The same procedure was followed for the partial tight ligation (PTL), except that approximately one third to one-half the diameter of the nerve was tightly ligated with 6-0 Mersilk suture by passing the needle under the nerve and then up through the middle. In sham operated rats, the IoN was exposed using the same procedure, but the exposed IoN was not ligated. 2.3. Study design Rats were randomly assigned to one of four experimental groups: 12 rats received a loose IoN ligation (CCI), 12 rats received a tight IoN ligation (CCI-T), 12 rats received a partial tight ligation (PTL), and 12 rats received a sham operation. Eight days before IoN surgery, half the rats in each group (i.e., N = 6) received an injection with formalin in the snout, the other half received a saline injection in the snout. 2.4. Behavioral testing 2.4.1. Formalin test Rats were s.c. injected with 50 μl of formalin (1.5%) or saline near the center of the right – i.e. contralateral to the operated IoN – vibrissal pad. Face grooming behavior was recorded during a 35 min period. 2.4.2. IoN ligation Face grooming behavior was observed on pre-operative day −1 and on post-operative days +3, +5, +7, +10, +19, +25 and +32. Behavior was videotaped twice for 10 min (once in the morning between 9 h and 11 h; and once in the afternoon between 14 h and 16 h). Videotaped behavior was analyzed by an experimenter who was blind to the experimental group of the rat. The amount of time spent on face grooming (i.e., movement patterns in which paws contact facial areas; see [8]) was determined using a stopwatch. A distinction was made between isolated face grooming and face grooming during body grooming [23]. If a sequence was neither preceded nor followed by body grooming (i.e., movement patterns in which the 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. 2.5. Statistical analysis Data were analyzed using IBM SPSS Statistics 20 software. Data are expressed as mean ± S.E.M. and were analyzed by means of a repeated

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measures ANOVA with time (post-operative days − 1 to + 32; postinjection 5-min blocks) as within-subjects factors, and surgery (four different types of IoN surgery) or injection group (saline/formalin) as between-subjects factor. Post-hoc comparisons were carried out using Dunnett pairwise multiple comparisons with sham as the control group; or two-tailed, unpaired (independent samples) Student's t-tests (saline versus formalin). Linear regression was performed to predict the amount of isolated face grooming following IoN ligation (dependent variable) based on the amount of isolated face grooming after formalin injection in the snout (independent variable). Coefficients of variation (Cv), which provide a relative measure of data dispersion, were calculated by dividing the standard deviation of a particular dataset by its corresponding mean (Cv = SD/mean). A smaller or larger Cv indicates that the scatter, compared to the mean, is respectively smaller or larger.

3.2. Repeated testing Average scores from observations in the morning and the afternoon were calculated per post-operative time point (Fig. 1, panels D–F). On most time points, greater significance was achieved as compared to the raw data (Fig. 1, panels A–C). Based on the observed standard deviations and true differences of means (between sham and IoN ligation groups), sample sizes were estimated to obtain statistical power of at least 0.8 (Table 1) [24]. Conventionally a test with a power greater than 0.8 is considered statistically powerful [25]. Sample sizes based on a single observation were systematically larger than based on average scores from two observations. Overall, there was a 38% reduction in the number of animals needed to obtain the same statistical power of 0.8.

3.3. Correlation of face grooming behavior following IoN ligation and after formalin injection 3. Results 3.1. Comparison between three types of IoN ligation The amount of time spent on isolated face grooming behavior in IoN ligated animals was significantly different from that in sham operated animals [surgery × time interaction: F(42,560) = 3.92, p b 0.001] (Fig. 1). Unifactorial ANOVA per time point, with surgery as betweensubjects factor, showed significant differences between the surgery groups on several time points [F(3,45) ≥2.99, p ≤ 0.042]. As indicated with asterisks in panels A–C of Fig. 1, post hoc comparisons between the different IoN ligated groups and sham operated rats showed significant differences between CCI and sham on days 3(1) to 10(1); CCI-T was significantly different from sham on all time points except 10(2) and 25(1). PTL was significantly different from sham on two time points, i.e. 5(2) and 10(1). 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 [surgery × time interaction: F(42,560) = 1.29, NS; between-subjects surgery effect: F(3,41) = 0.18, NS] (not shown).

The amount of time spent on isolated face grooming behavior in formalin treated animals was significantly different from that in saline treated animals [treatment × time interaction: F(6,186) = 2.47, p b 0.05] (Fig. 2). Unifactorial ANOVA per time point, with treatment as between-subjects factor, showed significant differences between the treatment groups on all time points [F(1,32) ≥ 8.39, p ≤ 0.007], except 5 to 10 min following injection [F(1,32) = 2.02, NS]. The amount of time spent on face grooming during body grooming in formalin treated animals was not significantly different from that in saline treated animals [treatment × time interaction: F(6,186) = 0.99, NS; between-subjects treatment effect: F(1,31) = 3.67, NS] (not shown). Using linear regression, it was found that the amount of isolated face grooming following IoN ligation could not be significantly predicted by the amount of isolated face grooming after formalin injection in the snout eight days before IoN ligation. This was true for grooming behavior both during the acute phase [i.e., 0–5 min after formalin injection; F(1,5) ≤7.25, NS, R2 ≤ 0.64] and the tonic phase [i.e., 10–35 min after formalin injection; F(1,5) ≤ 7.25, NS, R2 ≤ 0.64], and for all postoperative time points (3 to 32) and surgery groups.

Fig. 1. Post-operative changes in isolated face grooming behavior following loose (CCI), tight (CCI-T) or partial tight (PTL) IoN ligation. Data points represent the mean (±SEM; n = 12 per group) amount of time spent on isolated face grooming one day before IoN surgery (pre) and on post-operative days +3 to +32. Panels A–C represent raw data from observations in the morning (1) and in the afternoon (2). Panels D–F represent average scores from (1) and (2). Asterisks indicate a significant difference compared to sham operated control rats (Dunnett's test, *p b 0.05, **p b 0.01, ***p b 0.001).

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Table 1 Differences in sample size between one or two observations.

CCI CCI-T PTL

3(1/2)

3

5(1/2)

5

7(1/2)

7

10(1/2)

10

19(1/2)

19

25(1/2)

25

32(1/2)

32

6 17 49

4 14 18

5 17 24

4 11 17

7 13 36

4 11 27

13 17 39

7 13 15

52 11 3255

14 7 212

95 28 187

58 8 243

205 16 39

80 13 28

Sample sizes refer to the number of animals needed to include to obtain statistical power of 0.8 based on observed standard deviations and true differences in means between the sham group and the respective IoN ligation groups [24]. 3–32(1/2) refers to average sample sizes from scores obtained on post-operative days 3–32(1) and 3–32(2); 3–32 refers to sample sizes from average scores on post-operative days 3–32.

4. Discussion 4.1. IoN ligation During the first post-operative week, isolated face grooming behavior was significantly higher in CCI rats compared to the other IoN ligation groups. From day 19 onward, it was significantly higher in CCI-T rats. It is clear that different types of IoN ligation induce different time courses in isolated face grooming behavior. This finding may help to explain how even small surgical differences may be partially responsible for differences in time course in face grooming behavior between Vos et al. [10], where IoN rats were significantly different from control rats up to 4 months after IoN-CCI, and other studies that showed a much more limited duration [26 and unpublished data]. The present results indicate that nerve constriction may have been more extensive in Vos et al. [10]. IoN-CCI is characterized by substantial axonal degeneration of mainly thickly myelinated fibers, rapid regeneration and neuroma formation. It has been hypothesized that spontaneous discharges in surviving myelinated primary afferents in the early postoperative period and in C-fibers later post-injury underlie the recurrent paresthetic or dysesthetic sensations evoking directed face grooming [10]. It is therefore possible that, in as much as CCI induces a less intense nerve injury than CCI-T, more surviving afferents induced a greater amount of painful sensations in the early postoperative period and a more rapid return to preoperative neurophysiological conditions was achieved. Unfortunately, in the present study, behavioral testing was not continued beyond post-operative day 32. In a future study it would be interesting to see how long isolated face grooming behavior would remain significantly increased in CCI-T rats. Surprisingly, compared to CCI and CCI-T, PTL did not strongly affect isolated face grooming behavior. This is in contrast with Seltzer et al. [13] where it was concluded that PTL of the sciatic nerve (SN) induces spontaneous burning pain, as evidenced by intense, repeated licking of

the affected hind paw. Hyperesthesia to repetitive fine touch is a prominent feature in the SN-PTL model, but does not appear after SN-CCI [9]. It is possible that this hyperesthesia suppressed face grooming behavior. Alternatively, functional differences between the IoN and the SN may also underlie this finding. Finally, it should be considered that surgical differences between Seltzer et al. [13] and the present study may also have contributed to these effects. Indeed, the relative difficulty to reach the IoN significantly hampered the PTL approach to ligate the nerve in the present study. Despite the greater standardization that the present authors were able to apply in tightly ligating the IoN, coefficients of variation (Table 2) were not systematically smaller in the CCI-T group compared to CCI and PTL. It should therefore be considered that even very minor differences in IoN ligation in CCI-T and PTL may induce variability between rats that is comparable to that seen in CCI animals. Finally, it cannot be excluded that minor variations in surgery before IoN ligation, such as when dissecting the IoN free, are partially responsible for the observed variability between rats. 4.2. IoN ligation versus orofacial formalin injection Differences between isolated face grooming and face grooming during body grooming that have been extensively documented in the IoN model of neuropathic pain [8,23] were corroborated by the present findings in the orofacial formalin model. Isolated face grooming is specifically linked to nocifensive behavior whereas face grooming during body grooming is part of the rat's normal behavior. Also, the biphasic time course in the present data matches those in previous studies [17, 27]. Isolated face grooming data after formalin injection did not correlate very well with isolated face grooming data after IoN ligation. Hence, it was not possible to predict (and possibly select) which animals are likely to be unresponsive to IoN ligation based on data obtained in the orofacial formalin test. Therefore, it is concluded that the above theorized pre-existing differences in nocifensive behavior either do not exist, or cannot be measured by means of the orofacial formalin test. 4.3. Repeated testing A substantial amount of variation was observed between the two observations on a particular day, especially during the first postoperative week. A significant reduction in variability was obtained by averaging scores from these observations. From an ethical point of view, it is interesting to note that a 38% reduction in the number of animals needed to reach the same statistical power of 0.8 could be Table 2 Coefficients of variation (Cv) for the amount of time spent on isolated face grooming.

Fig. 2. Time course of isolated face grooming behavior after s.c. injection of saline or formalin (1.5%) in the snout. Data points represent the mean (±SEM; n = 18 for formalin, n = 15 for saline) amount of time spent on isolated face grooming for each 5-min block during the 35 min post-injection observation period. Asterisks indicate a significant difference (Student's t-test, *p b 0.05, ** p b0.01, *** p b0.001).

CCI CCI-T PTL Sham

Pre

3

5

7

10

19

25

32

0.79 1.10 0.63 1.66

0.34 0.64 0.55 0.52

0.32 0.62 0.67 0.80

0.33 0.62 0.76 0.80

0.45 0.64 0.61 0.74

0.54 0.48 0.65 0.55

0.66 0.43 1.03 0.85

0.95 0.73 0.82 1.22

CCI = chronic constriction injury (loose ligation); CCI-T = chronic constriction injury — tight (tight ligation); PTL = partial tight ligation. Pre = pre-operative and 3–32 refer to days post-operative relative to IoN surgery.

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obtained by adding the second observation. It remains unclear, however, if fluctuations in ongoing pain, as typically seen in trigeminal neuralgia patients [18,19], on the one hand, or confounding variables (such as time of day, activity pattern), on the other hand, are the cause of this variation in time. 5. Conclusions The present study shows that differences in IoN ligation have distinctly different effects on isolated face grooming behavior, both in intensity and time course. It remains however unclear how much variation can be attributed to lack of surgical standardization. It would be interesting to further study the differential effects of a tight versus loose ligation. Lack of correlation between isolated face grooming behavior following IoN ligation and after formalin injection in the snout makes it unlikely that differences in nocifensive behavior are a major cause of variability in the IoN model of neuropathic pain. Finally, a significant reduction in the number of animals needed to include in IoN-CCI studies can be obtained by doing more than one observation per time point. References [1] Meyer-Rosberg K, Kvarnström A, Kinnman E, Gordh T, Nordfors LO, Kristofferson A. Peripheral neuropathic pain—a multidimensional burden for patients. Eur J Pain 2001;5:379–89. [2] Meyer-Rosberg K, Burckhardt CS, Huizar K, Kvarnström A, Nordfors LO, Kristofferson A. A comparison of the SF-36 and Nottingham Health Profile in patients with chronic neuropathic pain. Eur J Pain 2001;5:391–403. [3] Jensen TS, Gottrup H, Sindrup SH, Bach FW. The clinical picture of neuropathic pain. Eur J Pharmacol Oct 19 2001;429:1–11. [4] Orza F, Boswell MV, Rosenberg SK. Neuropathic pain: review of mechanisms and pharmacologic management. NeuroRehabilitation 2000;14:15–23. [5] Mogil JS, Crager SE. What should we be measuring in behavioral studies of chronic pain in animals? Pain Nov 2004;112:12–5. [6] Backonja MM, Stacey B. Neuropathic pain symptoms relative to overall pain rating. J Pain Nov 2004;5:491–7. [7] Gottrup H, Bach FW, Juhl G, Jensen TS. Differential effect of ketamine and lidocaine on spontaneous and mechanical evoked pain in patients with nerve injury pain. Anesthesiology 2006 Mar;104:527–36. [8] Deseure K, Adriaensen H. Nonevoked facial pain in rats following infraorbital nerve injury: a parametric analysis. Physiol Behav 2004 Jun;81:595–604.

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Behavioral study of non-evoked orofacial pain following different types of infraorbital nerve injury in rats.

Directed isolated face grooming following unilateral chronic constriction injury to the infraorbital nerve (IoN-CCI) is a unique measure of spontaneou...
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