Spontaneous pain following spinal nerve injury in mice

Experimental Neurology 206 (2007) 220 – 230 www.elsevier.com/locate/yexnr

Spontaneous pain following spinal nerve injury in mice Anne Minert a,1 , Eran Gabay a,1 , Cecilia Dominguez b , Zsuzsanna Wiesenfeld-Hallin b , Marshall Devor a,⁎ a

Department of Cell and Animal Biology, Institute of Life Sciences, and Center for Research on Pain, Hebrew University of Jerusalem, Jerusalem 91904, Israel b Department of Clinical Neuroscience, Section of Clinical Neurophysiology, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden Received 2 August 2006; revised 23 March 2007; accepted 26 April 2007 Available online 6 May 2007

Abstract Autotomy behavior is frequently observed in rats and mice in which the nerves of the hindlimb are severed, denervating the paw. This is the neuroma model of neuropathic pain. A large body of evidence suggests that this behavior reflects the presence of spontaneous dysesthesia and pain. In contrast, autotomy typically does not develop in partial nerve injury pain models, leading to the belief that these animals develop hypersensibility to applied stimuli (allodynia and hyperalgesia), but not spontaneous pain. We have modified the widely used Chung (spinal nerve ligation [SNL]) model of neuropathic pain in a way that retains the fundamental neural lesion, but eliminates nociceptive sensory cover of the paw. These animals performed autotomy. Moreover, the heritable across strains predisposition to spontaneous pain behavior in this new proximal denervation model (SNN) was highly correlated with pain phenotype in the neuroma model suggesting that the pain mechanism in the two models is the same. Relative reproducibility of strain predispositions across laboratories was verified. These data indicate that the neural substrate for spontaneous pain is present in the Chung-SNL model, and perhaps in the other partial nerve injury models as well, but that spontaneous pain is not expressed as autotomy in these models because there is protective nociceptive sensory cover. © 2007 Elsevier Inc. All rights reserved. Keywords: Autotomy; Chung model; Neuroma; SNL model; SNN model; Strain survey

Introduction Of all symptoms in patients with painful neuropathy, spontaneous pain is the most burdensome. Unfortunately, our ability to analyze underlying mechanisms is severely limited by the difficulty of recognizing spontaneous pain in experimental animals. Weight loss, gait abnormalities, sleep disturbance, reduced movement and spontaneous paw guarding, lifting or shaking have all been proposed to reflect spontaneous pain, but the inference in each case is tentative at best. For example, weight loss in animals with inflammation could be due to central effects of inflammatory mediators on appetite rather than ⁎ Corresponding author. Department of Cell and Animal Biology, Institute of Life Sciences, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel. Fax: +972 2 658 6027. E-mail address: [email protected] (M. Devor). 1 These two authors contributed equally to the study. 0014-4886/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2007.04.011

to pain per se (Barak et al., 2002). Arguably the best documented correlate of spontaneous pain is autotomy behavior in the neuroma model of neuropathic pain (Devor, 2006a; Wall et al., 1979). This behavior, in which animals tend to scratch and bite their denervated insensate limb, is thought to result from abnormal sensation (anesthesia dolorosa) associated with ectopic discharge, generated in afferent neurons and amplified centrally. Correspondingly, in patients with neuropathy, there is a close correlation between ectopia and spontaneous neuropathic pain (Devor, 2006b). There are many reasons for believing that autotomy in rodents reflects ongoing dysesthesia or pain rather than, say, simple numbness. Foremost among them is its suppression by drugs and procedures that reduce ectopia and provide pain relief in humans, e.g. anticonvulsants, tricyclic antidepressants, NMDA receptor antagonists, dorsal column stimulation and dorsal root entry zone lesions. Drugs such as NSAIDs, that are minimally effective against neuropathic pain in the clinic, do

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not affect autotomy (Coderre et al., 1986; Gao et al., 1996; Kauppilla, 1998; Levitt, 1985; Seltzer, 1995; WiesenfeldHallin, 1984; Zeltser et al., 2000). Correspondingly, autotomy is provoked by conditions that augment ectopia and pain such as palpating neuromas or spinal injection of irritants (Albe-Fessard and Lombard, 1983; Devor and Seltzer, 1999; Kryzhanovsky, 1976). Blockade of descending brainstem inhibition augments autotomy, while enhancing it suppresses autotomy (Coderre et al., 1986; Kauppila and Pertovaara, 1991; Saade et al., 1993). The time of onset of autotomy, which might reasonably be presumed to reflect the onset of spontaneous pain experience, matches the appearance of ectopic spontaneous activity in Cfibers, depleting C-fibers with neonatal capsaicin suppresses autotomy, and resecting neuromas delays autotomy until a new neuroma forms (Barbera et al., 1988; Devor and Seltzer, 1999; Devor et al., 1982; Seltzer, 1995; Zeltser et al., 2000). The autotomy phenotype is associated with a defined genetic locus on mouse chromosome 15 (Devor et al., 2005; Seltzer et al., 2001). Finally, rendering a rodent's limb numb for a prolonged period in the absence of ectopia by local anesthetic block rather than by nerve section does not trigger autotomy (Blumenkopf and Lipman, 1991). Unlike animals, powerful cognitive and social strictures mitigate against autotomy-like behavior in humans, with or without pain. Nonetheless, it does occur occasionally (Mailis, 1996; Stump et al., 2003; Vogel and Anderson, 2002). Complete limb denervation e.g. by brachial plexus avulsion causes chronic ongoing pain in humans (Wynn-Parry, 1980), but this is a relatively uncommon neuropathy. More common are partial nerve injuries where spontaneous pain coexists with residual, often enhanced, stimulation-evoked pain. In animals too, partial nerve injuries frequently induce allodynia. But do these animals have spontaneous pain like humans? Autotomy has been reported in the chronic constriction injury (CCI) model of neuropathic pain (Bennett and Xie, 1988), but it does not occur in other partial nerve injury models, notably the spinal nerve ligation (SNL) model of neuropathic pain (Chung model; Kim and Chung, 1992). In the Chung-SNL model hindlimb afferents are cut across far more proximally than in the neuroma and CCI models, just distal to the dorsal root ganglion (DRG). It is known that the response of DRG neurons to peripheral axotomy, and of neurons and glia in the dorsal horn of the spinal cord, are affected by the location of the nerve injury with respect to the DRG. For example, the extent of gene regulation, and both the prevalence and the patterning of ectopic afferent discharge, differ when hindlimb afferents are cut distally at midthigh (neuroma model) versus proximally (Chung-SNL model; Liu et al., 2000; Costigan et al., 2002; Wang et al., 2002). Does autotomy fail to occur in the Chung-SNL model because the changes in the peripheral and the central nervous system (PNS, CNS) that are evoked by this form of injury do not constitute an appropriate pathophysiological substrate for spontaneous pain? Or is spontaneous pain in fact present in this model but simply not expressed as autotomy behavior? In addition to the distal vs. proximal location of the nerve lesion, there is a second critical difference between the neuroma model and the Chung-SNL model. Specifically, in the neuroma

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model the hindlimb is denervated and entirely insensate. There is no response, for example, to applied pinch and pinprick stimuli. Thus, although spontaneous pain is present, the actual act of autotomy behavior is painless. In contrast, in the ChungSNL model the L4 spinal nerve is spared, the hindlimb has residual innervation and sensation is not only spared but exaggerated (allodynia and hyperalgesia). Thus, animals with a spinal nerve lesion may have ongoing pain just like animals with more distally placed lesions, but they may not express it with autotomy behavior because the very act of autotomy is painful. We addressed this possibility by modifying the Chung-SNL model in such a way that the location of the nerve injury was the same as in the conventional model, but with complete denervation of the hindlimb and hence elimination of protective sensory cover. Under these conditions spinal nerve injury did induce autotomy behavior. We call this new model the spinal nerve neuroma (SNN) model of neuropathic pain. We then went on to show in a range of mouse strains with heritable differences in pain behavior in the neuroma model that the same strain differences also occur in the SNN model. This is a strong indicator that autotomy scores in the two models derive from the same mechanism(s) and measure the same thing, vis. spontaneous dysesthesia and pain. Methods Subjects Four series of experiments were carried out. The first series used adult male mice of the inbred C3H/HeN strain, purchased from Harlan Laboratories (Jerusalem). This strain, which expresses high levels of autotomy in the neuroma model of neuropathic pain (Raber et al., 2006), was now tested in the SNN model. In a second series of experiments, we surveyed 11 other inbred mouse strains in the SNN model, for a total of 12 strains including C3H. Founder mice of the additional 11 strains were obtained from the Jackson Laboratory (Bar Harbor, ME) and bred in our Institute vivarium to provide stock for the study. The 11 strains were: 129/J, A/J, AKR/J, BALBc/J, C57BL/6J (also termed B6), C57BL/10J (also termed B10), C58/J, CBA/J, DBA/2J, RIIIS/J and SM/J. These strains were chosen so that new data obtained using the SNN model could be compared with observations that we obtained previously in the Jerusalem lab, in the same strains, using the neuroma model (Mogil et al., 1999a,b). In addition, new observations were made using the neuroma model in some of the strains, as indicated in the Results. Mice in the first two series of experiments, both of which were carried out in Jerusalem, were maintained in transparent plastic shoebox cages (23 × 17 × 12 cm) bedded with pine wood shavings, weaned from the dam at about 30 days of age, and reared in groups of 2–5, by sex. When they reached young adulthood (2–3 months of age, 25–35 g) males underwent surgery to induce pain phenotype in the neuroma or SNN models as described below. In a third series of experiments, we carried out a smaller strain survey of autotomy behavior in the neuroma model

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(only). These experiments were carried out in Stockholm under conditions intended to test whether the across strain differences observed in the Jerusalem laboratory are sufficiently robust to resist the variations often observed when behavioral experiments are carried out under different environmental conditions (Crabbe et al., 1999). This survey included adult males of a subset of five of the mouse strains tested in Jerusalem: AKR, C3H, C57BL/6, C58 and CBA. The mice used in these experiments were obtained directly from the Jackson Laboratory. Finally, a fourth series of experiments was carried out in Stockholm using these same five mouse strains. These were control experiments in which sham surgery was carried out, but no major nerves were cut. In all four series of experiments, solid pelleted food (in Jerusalem Koffolk, Petah Tikva, Israel, product #19520; in Stockholm Avelfoder, Lactamin AB, Sweden) and water were available ad libitum. The day–night cycle was 12:12 h, with lights on at 6:00 or 7:00 a.m. Experiments were approved by the Institutional Animal Care and Use Committee of the Hebrew University of Jerusalem and of the Karolinska Insitutet (Stockholm), and followed the ethical guidelines of the International Association for the Study of Pain (Zimmermann, 1983). Phenotyping Spinal nerve neuroma (SNN) model All of the experiments using the SNN model were carried out in Jerusalem. Briefly, animals were anesthetized with chloral hydrate (ca. 350 mg/kg i.p.) and skin of the lower back was shaved and cleaned. The lower lumbar spinal nerves were exposed unilaterally after removing the L6 transverse spinous process, and the L4, L5 and L6 spinal nerves were cut across using fine microsurgical scissors about 4 mm from the corresponding DRG (Fig. 1A). In pilot trials we discovered that because of variability in the architecture of the lumbosacral

plexus, in some mouse strains there is a small afferent contribution of the L3 segment to hindlimb skin in some individuals, via the saphenous nerve. To insure that in all strains paw denervation was uniform and complete, we additionally exposed the saphenous nerve on the same side, tightly ligated it at mid-calf level with 6-0 silk, cut it just distal to the ligature and removed about 3–4 mm of the distal nerve stump (Fig. 1A). This was done in all 12 strains. In a small group of C3H control animals the saphenous nerve alone was cut, and in another the spinal nerves were cut without cutting the saphenous nerve. In all groups the side of the surgery was alternated from animal to animal so that in about half of the animals the hindpaw was denervated on the right, and in half on the left. Surgical exposures were closed in layers, a topical bacteriostatic powder (bismuth subgallate) was applied, and mice were given a prophylactic injection of penicillin (50 ku/kg i.m.). Following surgery the mice were returned to their original cages. Recovery was uneventful. Neuroma model Most of these data were collected in the Jerusalem laboratory. Some were reported previously in Mogil et al. (1999a,b), but new data were collected from males of three strains as indicated in the Results. Two of these three strains had been assayed previously (CBA and C57BL/6) and one was new (C57BL/10J). In addition, data were obtained in the Stockholm laboratory from males of five strains as noted above (AKR, C3H, C57BL/6, C58 and CBA). Anesthesia and preparation for surgery in the neuroma model was as described above for the SNN model. However, rather than exposing the lumbar spinal nerves, the sciatic nerve was exposed in the lower part of the popliteal fossa. The exposed nerve was tightly ligated with 5-0 or 6-0 silk near its point of trifurcation into tibial, peroneal and sural tributaries, cut just distal to the ligature, and 3–4 mm of the distal nerve stump were removed. In addition, the saphenous

Fig. 1. Description of surgery in the (A) SNN and (B) neuroma models of neuropathic pain. (C) Borders and shading illustrate the non-sensitive areas in 5 mice, 2 from the CBA strain, 2 from the SM strain, and 1 from the DBA strain, in the SNN model. (D) Borders and shading illustrate the non-sensitive areas in the neuroma model (mean of 5 C3H mice).

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Fig. 3. Behavioral phenotype in 12 inbred mouse strains in the SNN model of neuropathic pain, 35 days postoperative. Fig. 2. Development of autotomy in 15 male C3H/HeN mice in the SNN model of neuropathic pain. Each line represents an individual animal.

nerve was ligated and cut on the same side as described above. The relations of the nerves cut are shown in Fig. 1B. Sham surgery Mice were prepared as above and the lower lumbar spinal nerves were exposed in the same manner as for SNN surgery. However, no nerve injury was made. Rather, the incision was immediately re-closed. Postoperatively, the sham-operated mice were treated just as the experimentally operated groups. Behavioral observations In all animals, during the first week after surgery the entire surface of the denervated hindpaw was tested for residual sensation by pinching the skin in a distal-to-proximal pattern, using mouse-toothed forceps. Any animal that showed a withdrawal response to pinching anywhere distal to the ankle was excluded from the study on the grounds that surgical denervation had been incomplete. There was only one such exclusion 374 in the entire study. All mice included in the study had a completely anesthetic hindpaw. Table 1 Distribution of autotomy scores 35 dpo in 12 inbred mouse strains (males) in the spinal nerve neuroma (SNN) model of neuropathic pain Mouse strain

Mean ± SD n

129/J 0.1 ± 0.3 A/J 2.7 ± 3.9 AKR/J 0.0 ± 0.0 BALB/cJ 0.4 ± 0.5 C3H/HeN 10.3 ± 2.8 C57BL/6J 0.9 ± 0.6 C57BL/10J 1.8 ± 3.2 C58/J 0.5 ± 0.5 CBA/J 2.2 ± 3.9 DBA/2J 1.9 ± 3.2 RIIIS/J 1.1 ± 0.5 SM/J 3.5 ± 4.0

Score distribution, n mice (%) Low

15 15 15 15 15 15 15 15 23 15 15 15

Intermediate

High

(0–2)

(3–5)

(6–8)

(9–11)

15 (100%) 11 (73.3%) 15 (100%) 15 (100%) 1 (6.7%) 15 (100%) 13 (86.6%) 15 (100%) 17 (73.9%) 13 (86.6%) 15 (100%) 8 (53.4%)

0 1 0 0 0 0 0 0 2 0 0 2

0 (0%) 0 (0%) 1 (6.7%) 2 (13.3%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 14 (93.3%) 0 (0%) 0 (0%) 1 (6.7%) 1 (6.7%) 0 (0%) 0 (0%) 0 (0%) 4 (17.4%) 1 (6.7%) 1 (6.7%) 0 (0%) 0 (0%) 3 (20%) 2 (13.3%)

Data are from the Jerusalem laboratory.

(0%) (6.7%) (0%) (0%) (0%) (0%) (0%) (0%) (8.7%) (0%) (0%) (13.3%)

In thee strains chosen at random, CBA, DBA/2 and SM, sensory testing was extended proximally up the leg in order to define the limits of the cutaneous denervation accomplished in the SNN surgery. Areas of cutaneous denervation in these mice are shown in Fig. 1C, and compared to the lesser area of denervation in the neuroma model (Fig. 1D). After both neuroma and SNN surgery, hindlimb paralysis results in limping and restricted use of the limb for weight bearing. This is more profound in the SNN model than the neuroma model as there is more denervation of proximal musculature. However, severing motor fibers could not be the proximate cause of autotomy because this behavior is not induced by extensive ventral rhizotomy. Mice, housed in groups of 2–5 per cage, were scored for autotomy behavior at weekly intervals from the day of surgery until 35 days postoperative (dpo) using the scale of Wall et al. (1979). One point was given for loss of one or more toenails, and an additional point was given for injury or loss of the proximal or distal half of each digit for a maximal possible score of 11. Animals were killed when their autotomy score reached 11, or after the final scoring at 35 dpo. Autotomy scores at 35 dpo are sometimes grouped in categories, where low = 0–2, intermediate = 3–5 or 6–8 and high = 9–11. The protocol used for evaluating pain phenotype in the Jerusalem and Stockholm laboratories was the same. The across strains predisposition for autotomy behavior in the SNN model was compared to heat sensitivity at baseline and in the (conventional) Chung-SNL model using Hargreave's test, and to tactile hypersensitivity using von Frey monofilaments in the Chung-SNL model. These tests are described in detail in Mogil et al. (1999a,b). Briefly, in the Hargreaves' test, a radiant heat source is focused from below on the plantar surface of the animal's hindpaw, and time to paw withdrawal is measured. In the von Frey test nylon monofilaments with calibrated bending forces are applied to the plantar hindpaw and the force just sufficient to evoke paw withdrawal is measured. Statistical evaluation Means are given either ± standard deviation (SD) or ± standard error of the mean (SEM) as indicated. Statistical evaluations are based on 2-tailed Fisher's exact probabilities

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tests, and linear regression tests (Pearson's product–moment correlation and Spearman's rank correlation) using SigmaStat 3.1 (Systat Software, Point Richmond, CA). Factoral ANOVA (analysis of variance) with repeated measures was also applied, followed by post hoc Fisher's PLSD (probable least-squares difference) tests. The criterion for significance was p = 0.05. Results Pain behavior in the spinal nerve neuroma (SNN) model of neuropathic pain Observations were made on 15 male C3H/HeN mice. All but one developed autotomy behavior during the first 35 dpo. In most autotomy developed rapidly once it began, with scores in

10 mice rising from the minimal to the maximal possible score (0 to 11) between one observation and the next, i.e. within a period of 7 days (Fig. 2). Three mice reached the maximal score by 7 dpo, and an additional five by 14 dpo. Autotomy behavior is attributable to the spinal nerve injury rather than the sectioned saphenous nerve as severing the saphenous nerve alone did not yield autotomy (5 C3H mice were tested and all scored 0 at 35 dpo). In contrast, sectioning the L4–6 spinal nerves while leaving the saphenous nerve intact yielded high levels of autotomy (7 of 7 C3H mice tested yielded scores of 9–11 at 35 dpo; Fisher's test, p = 0.001 compared to saphenous nerve cut). In fact, scores in the animals with spinal nerves cut but with an intact saphenous nerve were no different from animals with the full SNN surgery (Fisher's test, p N 0.2). Note that in C3H mice there was no patch of residual hindpaw

Fig. 4. The development of autotomy over 35 dpo in 12 inbred mouse strains (SNN model, all males, n = 15 except for CBA where n = 23).

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strains ( p b 0.001 compared to all lower-scoring strains). For other comparisons: SM scored NRII ( p b 0.05), B6 ( p b 0.01), C58 ( p b 0.01), BALBc ( p b 0.01), 129/J ( p b 0.001), and AKR ( p b 0.001); A scored NC58 ( p b 0.05), BALBc ( p b 0.05), 129 ( p b 0.01) and AKR ( p b 0.01); CBA scored NC58 ( p b 0.05), BALBc ( p b 0.05), 129 ( p b 0.05) and AKR ( p b 0.01); DBA scored N 129 ( p b 0.05) and AKR ( p b 0.05). Comparison to pain behavior in the neuroma model of neuropathic pain

Fig. 5. Comparison of autotomy behavior in the SNN model of neuropathic pain versus the neuroma model in 12 inbred mouse strains (data from Tables 1 and 2). Rank 1=lowest autotomy scores; rank 12=highest autotomy scores. Data points are inbred mouse strains. The Spearman rank correlation coefficient=0.79 (pb .01).

innervation following L4–6 spinal nerve section with a spared saphenous nerve, so these animals did not have protective nociceptive sensory cover. Strain survey, SNN model Autotomy was observed in male mice of an additional 11 strains for a total of 12 strains including C3H. Large differences were observed among the strains in the extent of pain behavior 35 dpo as shown in Table 1 and Fig. 3. Fig. 4 shows the development of autotomy behavior in each of the strains. Factoral ANOVA showed a significant effect of strain (F11,880 = 15.6, p b 0.001), and days postoperative (F5,880 = 61.6, p b 0.001). There was also a significant strain × days interaction (F55,880 = 10.5, p b 0.001). Post hoc Fisher's PLSD tests confirmed that C3H showed the highest level of autotomy behavior among the 12 Table 2 Distribution of autotomy scores 35 dpo in 12 inbred mouse strains (males) in the neuroma model of neuropathic pain Mouse strain

a

129/J A/J a AKR/J a BALB/cJ a C3H/HeN a C57BL/6J C57BL/10J C58/J a CBA/J a Replicate: Total: DBA/2J a RIIIS/J a SM/J a

Mean ± SD n

3.1 ± 3.6 3.9 ± 3.8 0.2 ± 0.4 3.1 ± 3.6 9.3 ± 3.7 1.2 ± 1.7 0.7 ± 0.5 0.1 ± 0.3 9.2 ± 1.9 9.8 ± 2.4 9.6 ± 2.2 3.6 ± 4.1 1.7 ± 1.6 5.1 ± 4.8

12 9 12 8 20 39 12 23 10 15 25 9 10 11

Score distribution, n mice (%) Low

Intermediate

High

(0–2)

(3–5)

(6–8)

(9–11)

6 5 12 4 3 37 12 23 0 0 0 6 8 5

4 1 0 3 0 1 0 0 0 1 1 0 1 0

0 2 0 0 1 1 0 0 4 2 6 2 1 3

(50%) (55.6%) (100%) (50%) (15%) (94.9%) (100%) (100%) (0%) (0%) (0%) (66.7%) (80%) (45.4%)

(33.3%) (11.1%) (0%) (37.5%) (0%) (2.6%) (0%) (0%) (0%) (6.7%) (4%) (0%) (10%) (0%)

Data are from the Jerusalem laboratory. a Values reported previously in Mogil et al. (1999a,b).

(0%) 2 (16.7%) (22.2%) 1 (11.1%) (0%) 0 (0%) (0%) 1 (12.5%) (5%) 16 (80%) (2.6%) 1 (2.6%) (0%) 0 (0%) (0%) 0 (0%) (40%) 6 (60%) (13.3%) 12 (80%) (24%) 18 (72%) (22.2%) 1 (11.1%) (10%) 0 (0%) (27.3%) 3 (27.3%)

Fig. 5 plots mean autotomy scores 35 dpo in the SNN model of neuropathic pain in 12 inbred mouse strains against those obtained for the same strains in the neuroma model. For this plot the rank of each strain was used, from the lowest scoring strain (rank= 1) to the highest scoring (rank= 12). There was a significant correlation between strain ranks in the two models (Fig. 5; Spearman r = 0.79, p b 0.01). Interestingly, absolute scores were generally lower in the SNN model than in the neuroma model (Table 1 vs. Table 2, lower in 9 of 12 strains). The difference was sufficiently large in the case of CBA/J mice (3.2± 4.4 (n = 15) for the SNN model versus 9.2 ± 1.9 (n = 10) for neuroma model, Fisher's test, p b 0.01) that we decided to phenotype an additional 8 CBA/J animals using the SNN model (for a total n = 23). We also generated an independent replicate group in the neuroma model (n = 15). The contrasting scores in this strain persisted (Tables 1 and 2, Fisher's test, p b 0.001). Despite differences in absolute autotomy scores in the two models, the regression statistic based on absolute strain scores was statistically significant (Pearson r = 0.70, p b 0.02), as it was using rank scores. Autotomy behavior is relatively stable despite environmental variability Recent reports have indicated that strain difference is behavioral traits may be highly unstable, and fail to replicate from laboratory to laboratory despite heroic efforts to make environmental variables as uniform as possible (Crabbe et al., 1999). To assess the robustness of autotomy as a measure of spontaneous pain behavior, 5 of the 12 strains were evaluated in the neuroma model in a second laboratory setting (Karolinska Institutet, Stockholm; Table 3). We note also that several years separated the measurements made in the Jerusalem and Stockholm labs. These particular strains were

Table 3 Distribution of autotomy scores 35 dpo in 5 inbred mouse strains (males) in the neuroma model of neuropathic pain Mouse strain

Mean ± SD n

AKR/J C3H/HeJ C57BL/6J C58/J CBA/J

0.3 ± 0.5 8.6 ± 3.4 4.2 ± 3.0 4.7 ± 2.9 6.8 ± 3.9

Score distribution, n mice (%) Low

7 7 9 7 9

Intermediate

High

(0–2)

(3–5)

(6–8)

(9–11)

7 (100%) 1 (14%) 3 (33.3%) 2 (28.6%) 2 (22.2%)

0 1 4 3 1

0 (0%) 0 (0%) 1 (11.1%) 1 (14.3%) 3 (33.3%)

0 (0%) 5 (72%) 1 (11.1%) 1 (14.3%) 3 (33.3%)

Data are from the Stockholm laboratory.

(0%) (14%) (44.4%) (42.8%) (11.1%)

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chosen for the comparison because they cover a broad range of pain expression levels. The neuroma model was chosen rather than the SNN model because of its relative simplicity to execute, and its widespread use. Pain phenotype across strains was similar in the two laboratory settings, whether based on score rank (r =0.70) or absolute score (r= 0.83, Fig. 7). Differences appeared in the C57BL/6 and C58 strains with lower scores observed in Jerusalem than in Stockholm. However, this was not systematic as in C3H and CBA mice scores were somewhat higher in Jerusalem than in Stockholm (Fig. 6). Overall, the magnitude of the differences was similar to variability among replicate runs of a single strain within a single laboratory (Devor and Raber, 1990). The observed reproducibility of scores in spite of substantial environmental variability attests to the relative robustness of autotomy as a measure of spontaneous neuropathic pain. Sham surgery Sham surgery corresponding to the SNN model was carried out in 45 mice in 5 strains as follows: AKR (n = 9 mice), C3H (n = 10), C57BL/6 (n = 9), C58 (n = 8) and CBA (n = 9). All 45 mice retained withdrawal responses to pinch stimuli applied to the hindpaw, and all 45 had an autotomy score of zero at 35 dpo. Autotomy behavior in the neuroma model in C57BL/6J mice We have plotted data for C57BL/6 mice based on new observations rather than values published previously (Mogil et al.,1999a,b). In the first group of 11 male C57BL/6 mice that we phenotyped in 1993, autotomy scores were uniformly high (mean 11.0 ± 0.0, 0 low, 0 intermediate, 11 high). This is the value that appears in Mogil et al. (1999a,b). However, in light of the report by Seltzer et al. (2001) of low autotomy phenotype in this strain, we obtained additional breeding pairs of C57BL/6 from Jackson Labs

and repeated the phenotyping. Overall, three additional groups of males (39 mice), and two of females (24 mice) were phenotyped, independently. In all replicate groups autotomy scores were low (for males mean =1.0 ± 1.7, 35 low, 3 intermediate, 1 high; for females mean = 1.0 ± 0.4, 24 low, 0 intermediate, 0 high). Phenotype of most of the 9 C57BL/6 mice tested in the Stockholm laboratory was also relatively low (Table 3). Unlike C57BL/6, the other four strains from the original 11-strain survey (Mogil et al., 1999a,b) replicated the original results. Investigation of purchase and delivery orders of the original batch of C57BL/6 mice, and records made at the time of phenotyping, did not reveal an explanation of the divergent results obtained from the original batch of mice. Specifically, we found no evidence indicating that these mice were of a different strain and had been mislabeled, although this remains a possibility. We have no other explanation except for the possibility that an unidentified environmental factor might have skewed the results in the original batch of C57BL/6 mice (Dallman et al., 1999; Raber and Devor, 2002). In light of our multiple replications in Jerusalem, the results of the replication in Stockholm, and the results of Seltzer et al. (2001), all of which found autotomy phenotype of C57BL/6J mice in the neuroma model to be low, we now believe that this is the correct result, and not the value that we gave in Mogil et al. (1999a,b). That is, rather than the C57BL/6J strain being an instance of poor reproducibility, we suspect that an error occurred in the original assay. Comparison with other pain phenotypes Autotomy in the SNN model did not correlate significantly across strains with baseline thermal sensitivity as measured using the Hargreave's test (HT), or with heat (PNIHT) or von Frey tactile allodynia (PNIVF) in the (conventional) Chung-SNL model (Fig. 8; r = − 0.15 to 0.18, p N 0.2 for each comparison).

Fig. 6. Relative stability of pain phenotype. Plot shows the development of autotomy over 35 dpo in males of 5 inbred mouse strains in the neuroma model of neuropathic pain. Data from the Stockholm laboratory (Table 3) are compared to data from the Jerusalem laboratory (Table 2).

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triggered by sciatic nerve section, generate allodynia but not spontaneous pain, or (2) spontaneous pain is present after ChungSNL surgery, but not expressed in the form of autotomy behavior, presumably because sensory cover of the limb in these animals renders autotomy acutely painful and hence avoided. We have shown, by modifying the Chung-SNL model in a way that maintains the proximal location of the nerve surgery but eliminates sensory cover, that spinal nerve section induces the

Fig. 7. Autotomy behavior in the neuroma model of neuropathic pain in 5 inbred mouse strains, comparing data obtained in the Stockholm laboratory (Table 3) with data obtained in the Jerusalem laboratory (Table 2). Values from 35 dpo are plotted as ranks (upper, rank 1 = lowest autotomy scores; rank 5 = highest autotomy scores), and well as absolute scores (lower). Data points are inbred mouse strains.

The comparison data for the conventional Chung-SNL model were obtained from Mogil et al. (1999a,b), except for one data point, HT in the C57BL/10J strain, which was obtained from J. Mogil (unpublished, personal communication). Discussion Autotomy behavior after spinal nerve injury in the SNN model In the conventional Chung-SNL model of neuropathic pain, in which the L5 and 6 spinal nerves are severed leaving the hindlimb only partly denervated, prominent tactile allodynia develops within 24 h in many strains of rats and mice. However, these animals do not engage in autotomy behavior. We summarized in the Introduction the case for autotomy being an indicator of spontaneous dysesthesias and pain rather than a consequence of poor tending of a numb insensate, yet painless limb (Blumenkopf and Lipman, 1991; Devor, 2006a). The present study was not designed to revisit this case. Rather conclusions are predicated upon it. Thus, presuming that autotomy indeed reflects spontaneous pain, the observation that autotomy is absent in the conventional ChungSNL model has two alternative explanations. Either, (1) the PNS and CNS changes triggered by spinal nerve section, unlike those

Fig. 8. Comparison of pain phenotype in the SNN model of neuropathic pain (autotomy) with baseline thermal sensitivity (Hargreave's test, HT), thermal sensitivity (HT) in the (conventional) Chung-SNL model (PNIHT), and tactile sensitivity measured with von Frey filaments in the (conventional) Chung-SNL model (PNIVF). The tests are explained in the Methods. Data are plotted as ranks (rank 1 = lowest autotomy scores and least response to stimuli; rank 11 or 12 = highest autotomy scores and greatest response to stimuli). Data points are inbred mouse strains. HT and PNI data are from Mogil et al. (1999a,b).

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neural substrate required for the induction of autotomy behavior, spontaneous dysesthesia and pain. A corollary is that spontaneous pain is also present in the conventional Chung-SNL model, in segments L5 and 6 if not necessarily in L4, but that its expression in the form of autotomy is suppressed. This suppression is almost certainly due to residual L4 innervation of the paw. When residual innervation is present, the very act of autotomy is painful, and hence avoided despite the presence of spontaneous pain. When absent, in the SNN model, tissue damage during autotomy is not felt, only spontaneous pain. Autotomy behavior is therefore not suppressed. This situation is well know clinically where hand surgeons go to great lengths to provide nociceptive sensory cover in limbs with injured nerves, greatly reducing the risk of selfinduced tissue injury (Dellon, 1981; Sunderland, 1978). The autotomy reported in the original description of the CCI model of neuropathic pain (Bennett and Xie, 1988) was probably due to patches of denervated skin caused by excessive nerve constriction in some animals. It is likely that removal of sensory cover would also uncover autotomy behavior in many of the other partial nerve injury models commonly used, revealing the presence of spontaneous pain.

Environmental stability of the autotomy phenotype Surgical procedures and the protocol for evaluating pain phenotype were matched in Jerusalem and Stockholm. However, there was a myriad of subtle known and unknown differences between the two laboratory settings. Some such parameters, the constituents of food pellets used, for example, are known to affect pain behavior (Shir et al., 1997). Others, such as water quality, particulates in the air and shelf position of neighbors in the animal colony room, are of unknown functional importance. Despite these differences the autotomy phenotype displayed a remarkable robustness over time and between the laboratories. Across-strain rankings of other behavioral phenotypes, including other pain measures, are much less robust (Crabbe et al., 1999). For example, we failed to replicate the across-strain rankings reported by Mogil et al. (1999a,b) for tactile allodynia in the conventional Chung-SNL model (Z. Wiesenfeld-Hallin, unpublished). Robustness in the face of environmental variation is an essential advantage in the effort to identify the genes and neurobiological processes that underlie strain differences. Neural mechanisms

Across strain correlation of pain behavior A second major observation was the significant across-strain correlation between spontaneous pain behavior in the SNN and the neuroma models of neuropathic pain. Since all animals of an inbred mouse strain are genetically identical, phenotypic variability within a strain is necessarily due to environmental factors, while variability across strains reflects genetic polymorphisms and genetic– environmental interactions (Mogil et al., 2004). Gene expression, in turn, controls pain phenotype through numerous constitutive and/or injury-evoked neural mechanisms. Complex behavioral phenotypes are typically determined by the coordinated action of numerous genes. Hence, heritable predisposition to one pain modality, say thermal sensibility, does not necessarily imply heritable predisposition to another pain modality, say spontaneous neuropathic pain. A mouse strain high in one may well be low in the other. By the same token, if a pair of strains is high and low, respectively, in one parameter, they may be high and low, respectively, in a second parameter strictly by chance, with no assurance that the two parameters are necessarily controlled by a common pattern of gene expression or by the same neural mechanisms (Valder et al., 2003). However, as the number of strains increases the chances of an across strain correlation occurring by chance alone diminishes rapidly. For two independent pain phenotypes, the likelihood of two strains showing the same rank order is p = 2−(n−1) = 0.5, where n is the number of strains tested. However, for 12 strains, the likelihood of identical rank order occurring by chance is 2− 11. The significant correlation in spontaneous pain phenotype that we found comparing the SNN and neuroma models across 12 inbred mouse strains supports the twin inferences that autotomy behavior is measuring the same thing in the two models, vis. spontaneous dysesthesia and pain, and that these models share the same underlying neural mechanism(s).

The evidence that autotomy indeed reflects neuropathic dysesthesias and pain rather than, say, simple numbness of the limb (Blumenkopf and Lipman, 1991) is strong (Introduction, Devor, 2006a). The underlying neural mechanism, however, is less certain. The significant correlation between autotomy in the SNN and the neuroma models indicates that these models share a constellation of expressed genes and neural mechanism(s). Considerable evidence links ongoing pain and autotomy behavior to ongoing neuropathic discharge and its cellular and molecular substrates (Abdulla and Smith, 2001a; Devor and Seltzer, 1999; Liu et al., 2001; Lombard et al., 1984). However, many details of this relationship still need to be resolved. For example, in both rats and mice the time of onset of ectopic firing in afferent A-fibers (b 24 h post-axotomy) closely matches that of tactile allodynia (Liu et al., 2000; Tal et al., 2006) but autotomy usually begins days or weeks later. We have previously proposed that autotomy is associated with ongoing burning pain due to ectopia in C-fibers on the grounds that: (1) there is a match between autotomy onset and the onset of ectopia in afferent C-fibers in Sabra strain rats, (2) autotomy in rats is suppressed following depletion of C-fibers, and (3) autotomy behavior correlates across-strains with various measures of heat sensibility (Devor et al., 1982; Devor and Seltzer, 1999; Mogil et al. 1999a,b). A similar association was reported recently for spontaneous foot lifting, a proposed marker of spontaneous pain (Djouhri et al., 2006; Yoon et al., 1999). If spontaneous pain as reflected in autotomy behavior depends on C-fiber ectopia, and tactile allodynia depends on Afiber ectopia, this could account for the poor association of these pain phenotypes across-strains and within individual animals (Fig. 8; Kauppila and Xu, 1996; Mogil et al. 1999a,b). But other parameters cast a shadow on this conclusion. For example, comparing individual animals Abdulla and Smith (2001a,b) found a relation between the extent of autotomy and the amount

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of ectopic hyperexcitability in injured afferent A-fibers, and Wu et al. (2001) reported early onset of spontaneous firing in “uninjured” C-fibers in the Chung-SNL model in rats, with timing that corresponds to the onset of tactile allodynia. Counter to simple intuition, absolute autotomy scores were mostly lower in the SNN model than in the neuroma model. This was so despite the facts that the amount of tissue denervated was greater in the former, and that axotomy was closer to the DRG and hence expected to trigger a more profound retrograde cell reaction. This observation further excludes the hypothesis that autotomy behavior reflects simple numbness (Blumenkopf and Lipman, 1991) as if anything, SNN surgery produces more numbness than neuroma surgery. We also note the variable onset time of autotomy, ranging over several weeks, even though the animals were inbred and genetically identical. Perhaps autotomy is affected by important environmental effects such as random exacerbations of ectopia as a result of mechanical forces applied to the injured nerve (neuroma and DRG) during movement. This factor could also explain the higher level of autotomy in the neuroma model, where the nerve injury site is relatively exposed to stresses and strains during movement compared to the SNN model, where the cut nerve end is near the spine and relatively protected (Dorsi et al., 2003; Devor and Seltzer, 1999). Another potential factor is retrograde cell loss in the DRG after axotomy. Proximal lesions, such as in the ChungSNL and SNN models, result in much more rapid and severe loss of DRG neurons than more distal lesions (Tandrup et al., 2000; Vestergaard et al., 1997). Loss of DRG neurons is expected to reduce the overall ectopic afferent barrage, that originating in the neuroma and that originating in the DRG itself, and hence to reduce spontaneous pain sensation. The observation that once begun autotomy tends to advance rapidly to maximal levels suggests that the underlying cause is paroxysmal and is triggered at different times in different animals. Anecdotal observations of sudden, brief pain attacks resulting in autotomy behavior support this idea (Devor and Seltzer, 1999; Duckrow and Taub, 1977). Paroxysms could be caused by mechanical stimuli (Tinel sign), sympathetic activity, or positive feedback (Rappaport and Devor, 1994). Long-term recordings of ectopic neural activity in vivo will be needed to detect occasional intense paroxysmal impulse storms. Finally, in animals, as in humans, it is essential to distinguish between the first-person experience of pain and the expression of pain in the form of behaviors that can be seen by an outside observer. Spontaneous pain may be present in our mice soon after spinal nerve injury despite the fact that it's expression in the form of autotomy behavior is variably delayed. The delay may not be due to the fundamental pain signal itself, but to complex intervening variables such as the animal equivalent of the psychosocial factors that affect the expression of pain behavior in humans (Raber and Devor, 2002; Langford et al., 2006). Ethical considerations The conflicting imperatives of developing more effective means of pain relief for patients, for example based on human homologs of mouse pain genes, while minimizing pain inflicted on experimental animals, sometimes raises ethical dilemmas. The

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costs in potential suffering need to be balanced against the benefits of the knowledge obtained (Zimmermann, 1983). The neuroma model has been criticized on the grounds that it generates spontaneous, inescapable pain, while the various partial nerve injury models produce hypersensibility that the animal can minimize by avoiding cutaneous stimuli. Our data indicate that this difference is illusory, at least for the Chung-SNL model of neuropathic pain. Both types of nerve injury evoke spontaneous pain, and for the same reasons. In the Chung model, however, pain expression is suppressed because of the presence of sensory cover. While this might have esthetic benefits, and parry criticism by the non-initiated, the ethical dilemma posed by the presence of spontaneous pain is the same in both types of models. Acknowledgments We thank Ira Dor and Pnina Raber for their contributions to this work, and Jeff Mogil for sharing unpublished strain data. Supported by grants from the German–Israel Foundation for Scientific Research (GIF), the Hebrew University Center for Research on Pain, and the European Community's 6th Framework Program (project LSHM-CT-2004-502800 PainGenes). The manuscript reflects only the authors' views. The European Community is not liable for any use that may be made of the information contained therein. References Abdulla, F.A., Smith, P.A., 2001a. Axotomy- and autotomy-induced changes in the excitability of rat dorsal root ganglion neurons. J. Neurophysiol. 85, 630–643. Abdulla, F.A., Smith, P.A., 2001b. Changes in Na+ channel currents of rat dorsal root ganglion neurons following axotomy and axotomy-induced autotomy. J. Neurophysiol. 88, 2518–2529. Albe-Fessard, D., Lombard, M.C., 1983. Use of an animal model to evaluate the origin of and the protection against deafferentation pain. Adv. Pain Res. 5, 691–700. Barak, O., Weidenfeld, J., Goshen, I., Ben-Hur, T., Taylor, A.N., Yirmiya, R., 2002. Intracerebral HIV-1 glycoprotein 120 produces sickness behavior and pituitary-adrenal activation in rats: role of prostaglandins. Brain Behav. Immun. 16, 720–735. Barbera, J., Garcia, G., Lopez-Orta, A., Gil-Salu, J., 1988. The role of the neuroma in autotomy following sciatic nerve section in rats. Pain 33, 373–378. Bennett, G., Xie, Y.-K., 1988. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33, 87–107. Blumenkopf, B., Lipman, J.J., 1991. Studies in autotomy, its pathophysiology and usefulness as a model of chronic pain. Pain 45, 203–210. Coderre, T.J., Grimes, R.W., Melzack, R., 1986. Deafferentiation and chronic pain in animals, an evaluation of evidence suggesting autotomy is related to pain. Pain 26, 61–84. Costigan, M., Befort, K., Karchewski, L., Griffin, R.S., Da'Urso, D., Allchorne, A., Sitarski, J., Mannion, J., Pratt, R., Woolf, C., 2002. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury. BMC Neurosci. 3, 16. Crabbe, J.C., Wahlsten, D., Dudek, B.C., 1999. Genetics of mouse behavior, interactions with laboratory environment. Science 284, 1670–1672. Dallman, M.F., Akana, S.F., Bell, M.E., Bhatnagar, S., Choi, S., Chu, A., Gomez, F., Laugero, K., Soriano, L., Viau, V., 1999. Warning! Nearby construction can profoundly affect your experiments. Endocrine 11, 111–113. Dellon, A.L., 1981. Evaluation of Sensibility and Re-Education of Sensation in the Hand. Williams and Wilkins, Baltimore, MD. 263 pp. Devor, M., 2006a. Anesthesia dolorosa model, autotomy. In: Schmidt, R.F., Willis, W. (Eds.), Encyclopedia of Pain. Springer-Verlag, Berlin.

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