sciatica

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Central administration of methotrexate reduces mechanical allodynia in an animal model of radiculopathy/sciatica q Hiroshi Hashizume a, Maria D. Rutkowski a, James N. Weinstein b,c, Joyce A. DeLeo a,d,* a

Department of Anesthesiology, Hinman Box 7125, Dartmouth -Hitchcock Medical Center, Lebanon, NH 03756-0001, USA b Department of Orthopaedic Surgery, Dartmouth -Hitchcock Medical Center, Lebanon, NH 03756-0001, USA c Department of Community and Family Medicine, Dartmouth Medical School, Hanover, NH 03755, USA d Department of Pharmacology, Dartmouth Medical School, Hanover, NH 03755, USA Received 9 June 1999; received in revised form 18 February 2000; accepted 28 February 2000

Abstract We have recently reported that injury to a lumbar root in a rat model of radiculopathy produces spinal glial activation associated with elevated proin¯ammatory cytokines. Based on our hypothesis that central neuroin¯ammatory processes may manifest clinically as radicular pain, we undertook pharmacological intervention using the immunosuppressive agent methotrexate (MTX). The L5 lumbar spinal root (central to the dorsal root ganglia) was exposed unilaterally and loosely constricted with chromic gut. In the prevention (phase I) study, MTX was administered intrathecally (1 mg/kg) and around the spinal root (1 mg/kg) at surgery and at days 2 and 4 postsurgery (group A). Saline injection was employed for the control group (group B). Sham operated animals were administered MTX to determine the potential for behavioral/neural side effects (group C). In the existing pain paradigm (phase II) study, the experiment was extended to day 14 with three additional groups. The same dose and method of delivery of MTX or saline was administered as in phase I in the ®rst week on days 0, 2, and 4 and in the second week on days 7, 9, and 11 postsurgery. To measure the effects of MTX on existing behaviors saline was administered in the ®rst week and MTX during the second (group D; Saline:MTX). The control group received saline during both weeks (group E; Saline:Saline). To examine the possible recurrence of radicular pain after MTX termination, MTX was given in the ®rst week and saline in the second (group F; MTX:Saline). Gait disturbance and mechanical allodynia (using von Frey ®laments) were assessed up to day 7 in the prevention study (Phase I) and day 14 in the existing pain paradigm (Phase II). The L5 spinal cord segments were harvested for assessment of immunohistochemical glial activation using the antibodies OX-42 (microglial marker) and glial ®brillary acidic protein (GFAP: astrocytic marker) and for the presence of Major Histocompatibility Complex (MHC) Class II expression. Group C (Sham 1 MTX) did not demonstrate any evidence of gait disturbance or mechanical allodynia after MTX administration. The rats in group B (Surgery 1 Saline) demonstrated mechanical allodynia from one day postsurgery to the time of euthanization. When allodynia was assessed using the 12 g von Frey ®lament, the MTX treated rats in group A showed signi®cantly decreased mechanical allodynia as compared to the saline treated rats (group B) (repeated measured ANOVA, P , 0:0001). In the phase II study, the rats in group D (Saline:MTX) and E (Saline:Saline) showed robust allodynia in the ®rst week after the surgery. In the second week, mechanical allodynia signi®cantly decreased in group D, while mechanical allodynia continued in the saline treated group (repeated measured ANOVA, P ˆ 0:0121). Allodynia was signi®cantly attenuated in group F (MTX: Saline) as compared to the response in groups D and E at day 7 (one-way ANOVA, P , 0:0001) and remained signi®cantly lower as compared to group E up to day 11 postsurgery (one-way ANOVA, P9 ˆ 0:0013: P11 ˆ 0:0048). OX-42 and GFAP expression were elevated in the gray matter of the L5 spinal section in all groups that underwent the root ligature with chromic gut (Groups A, B, D±F). There were no signi®cant differences in glial activation between the groups. However, spinal expression of MHC II was markedly reduced in the MTX treated group as compared with the saline treated group. The exact mechanism of action of MTX in attenuating mechanical allodynia has not yet been elucidated. The present results indicate that MTX administration may offer a new treatment modality for radicular pain with or without disc herniation as well as directing new research into the development of novel immunomodulators for the treatment of chronic neuropathic and radicular pain. q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. Keywords: Methotrexate; Nerve root; Radiculopathy; Chronic pain

1. Introduction q We dedicate this paper to Brianna Weinstein. * Corresponding author. Tel.: 11-603-650-6204; fax: 11-603-650-4928. E-mail address: [email protected] (J.A. DeLeo).

Low back pain is a common problem that will affect approximately two thirds of the adult population (Deyo

0304-3959/00/$20.00 q 2000 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved. PII: S 0304-395 9(00)00281-5

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and Tsui-Wu, 1987). It is the second leading reason for ambulatory care in the United States and direct medical costs are estimated at over $20 billion per year (Frymoyer and Cats-Baril, 1991). Of these, a small but signi®cant percentage will exhibit symptoms of radicular pain associated with a herniated disk(s). Current treatment for persistent lumbar radiculopathy includes, but is not limited to, invasive surgical procedures, pharmacology and physical therapy. The absence of an evidence-based approach to the diagnosis and treatment of low back pain has impeded increased clinical ef®cacy of its treatment. In addition, until recently, the absence of animal models for low back pain associated with radiculopathy have hindered the understanding of the pathophysiological mechanisms that produce radicular pain. Based on studies using various animal models to date, most researchers agree that two possible mechanisms produce radicular pain associated with herniated intervertebral discs (McCarron et al., 1987; Olmarker et al., 1989, 1993; Kawakami et al., 1994): a mechanical compression of the spinal root and a biological in¯ammatory effect on the root induced by the herniated nucleus pulposus. Recently, our laboratory has extended the development of animal models of chronic pain and theories of neuropathic hyperalgesia and allodynia from a peripheral nerve injury model to a spinal root injury model. We further characterized an animal model in which the L5 spinal root is loosely ligated with chromic gut (Hashizume et al., 2000). This model, which is a modi®cation of the Bennett chronic constriction injury model of the sciatic nerve (Bennett and Xie, 1988), produces behavioral changes suggestive of thermal hypersensitivity and mechanical allodynia by day one following surgery. We demonstrated a graded, mechanical allodynia dependent on the type of root injury, i.e. ligation with in¯ammatory chromic gut, tight ligation with silk suture, or loose ligation with silk suture. The combination of the chromic gut and ligation evoked the most pronounced mechanical allodynia followed by tight ligation of silk suture and then loose ligation of silk suture. Therefore, an important determinant to predict robust pain behaviors in this model is the chemical stimulation released from chromic gut combined with the mechanical compression of the spinal root. Of interest, the radiculopathic symptoms resolve within 10 days when the root is loosely ligated with hypotoxic silk suture. Therefore, the root injury model using chromic gut is useful for the study of pathophysiologic mechanisms of radicular pain since the chromic gut models both the in¯ammatory component of herniated nucleus pulposus and the ligation models root compression. We have put forth the hypothesis that central, neuroimmune mechanisms are an important factor for the development and maintenance of chronic neuropathic pain (Deleo and Colburn, 1996: DeLeo et al., 1996, 1997b). Supporting this hypothesis, we have demonstrated spinal glial cell (microglia and astrocytes) activation and increased cytokine (IL-1b) protein expression in the lumbar radiculopathy

model used in the present study (Hashizume et al., 2000). Our present working hypothesis is that injury to a lumbar root from a herniated disk causes a central, neuroimmune process associated with elevated spinal proin¯ammatory cytokines and associated membrane glycoproteins such as Major Histocompatibility Complex (MHC)Class II that may manifest clinically as radiculopathy. To examine this neuroimmune hypothesis and to develop a possible new treatment for chronic radicular pain, we undertook this study using the FDA approved pharmacological intervention, methotrexate (MTX). MTX is a folate antagonist originally developed for the treatment of malignancies and now widely used in the treatment of rheumatoid arthritis due to its potent, but selective, immunosuppresive actions. We designed a two phase study to address the effect of centrally administered MTX on both the prevention of mechanical allodynia as well as the ef®cacy of MTX on the reduction of existing mechanical allodynia following lumbar root injury. In order to begin to address possible mechanisms of action of MTX in this system, we also utilized immunohistochemistry to determine whether MTX altered spinal glial activation and MHC II expression. 2. Methods Experiments were performed using a total of 58 male Holtzman-strain rats, each weighing 200±250 g at the start of surgery, housed individually under USDA and AAALAC-approved condition with 12/12 h light-dark cycle and free access to food and water. All experimental procedures were approved by Dartmouth College Institutional Animal Care and Use Committee (IACUC). 2.1. Surgical procedure All surgical procedures were performed using aseptic technique as mandated by AAALAC and our IACUC. Rats were deeply anesthetized using inhalation anesthesia (induced at 4% and maintained at 2% halothane in 100% O2). The radiculopathy/sciatica model has been previously described (Hashizume et al., 2000). Brie¯y, the spinal root, dorsal root ganglia (DRG), and the adjacent dura mater on the left side at L5 were carefully exposed by hemilaminectomy in all rats using a surgical microscope. Five 0.3 cm pieces of 4-0 chromic gut ligature were laid adjacent to the root and secured by two loose ligatures of 5-0 chromic gut (Fig. 1). The muscle layers and incision were closed with 3-0 silk suture and staples, respectively. 2.1.1. Phase I protocol Three distinct combinations of treatments were investigated in phase I of the study. Methotrexate (4-Amino-10methylfolic acid, Sigma, St. Louis, MO) was reconstituted to 10 mg/ml in saline and pH adjusted to 7.4. Group A (n ˆ 10, root injury 1 MTX treatment): immediately following the surgical procedure and prior to incision

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Fig. 1. Schematic of the spinal root injury model: the spinal root, dorsal root ganglia (DRG), and the adjacent dura mater on the left side at L5 were exposed by hemilaminectomy. Five 0.3 cm pieces of 4-0 chromic gut ligature were laid adjacent to the root and secured by two loose ligatures of 5-0 chromic gut.

closure, 1 mg/kg of MTX (volume 100 ml/kg) was intrathecally injected by passing a PE-10 catheter through an incision in the exposed dura mater to the position 3 cm central to the incision. The PE-10 catheter was pulled out and the same dose and volume of MTX (1 mg/kg) was administrated around the injured roots (total dose 2 mg/kg) (DeLeo et al., 1997a). Drug administration was performed under direct visualization based on the need to verify an intrathecal site of delivery. Standard incision closing procedures followed. Under deep inhalation anesthesia, MTX was administered again at 2 and 4 days post surgery by opening the surgical wound and repeating the delivery method performed during surgery. Group B (n ˆ 10, root injury 1 saline treatment): 100 ml/kg of saline was intrathecally and perineurally administered in the same manner as group A (total volume 200 ml/kg) at surgery and at days 2 and 4 following surgery. Group C (n ˆ 10, sham operation 1 MTX treatment): the L5 nerve roots were exposed but not injured. MTX was administered using the same dose, protocol, and time points as in group A. Gait disturbance and mechanical allodynia were assessed in all rats daily until euthanization at day 7 postsurgery. 2.1.2. Phase II protocol The phase I protocol was extended to day 14 with the surgery, delivery method, volume and dose being identical to the phase I study. The rats were divided into three groups: Group D (n ˆ 10, saline: MTX): saline was administered at the time of surgery and at days 2 and 4 postsurgery. MTX was administered in the second week at days 7, 9, and 11 postsurgery. Group E (n ˆ 8, saline: saline): saline was administered throughout the 2 weeks at the same time points as in Group D. Group F (n ˆ 10, MTX: saline): MTX was administered during surgery and at days 2 and 4 postsurgery. Saline was administered on days 7, 9 and 11 postsurgery. Gait disturbance and mechanical allodynia were

assessed in all rats before the surgery and at days 2, 4, 7, 9, 11 and 14 until euthanization. 2.2. Behavioral tests The animals were tested for 3 days pre-operatively to acclimate them to the behavioral testing apparatus, the experimenter and to obtain baseline values. All behavioral testings were performed by an experimenter blinded to the treatment. 2.2.1. Gait disturbance and hind paw eversion Gait disturbance was scored using the following rating scale: normal gait (2), slight gait disturbance with motor weakness (1), severe gait disturbance with motor paresis of the ipsilateral hind paw (11). The presence or absence of hind paw eversion described as planter ¯exion of the toes and/or the presence of an inverted hind paw (the toes were held together or retro¯exed on the hind paw) was also recorded. 2.2.2. Hind paw withdrawal to mechanical stimulation Tactile sensitivity (mechanical allodynia) was measured as the frequency of foot-withdrawals elicited by a de®ned mechanical stimulus (DeLeo et al., 1996; Colburn et al., 1997, 1999). In each blinded testing session, rats were subjected to three sequential series of ten tactile stimulations to the plantar surface of the ipsilateral (nerve root injured) hind paw using 2 and 12 g von Frey ®laments (Stoelting, Wood Dale, IL). Baseline (pre-lesion) responsiveness was minimal as con®rmed from testing sessions prior to the surgery. Mechanical allodynia was assessed by recording the total number of responses elicited during three successive trials (ten stimulations/each ®lament) separated by at least 10 min for a total possible score of 30. The terms for the allodynic condition were de®ned retrospec-

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tively based on the average responses to 12 g von Frey stimulation in each group as follows: minimal (0±5), mild (5±10), moderate (10±15), robust (15 and more). 2.3. Histological examination 2.3.1. Tissue preparation All animals were perfusion ®xed for immunohistochemistry. Under deep anesthesia (sodium pentobarbital, 50 mg/ kg, i.p.) rats were euthanized by transcardiac perfusion. Rats were then perfused with 300 ml phosphate buffered saline (PBS) followed by 500 ml of 4% paraformaldehyde in 0.1 M PBS. Following perfusion and laminectomy, the lesioned L5 roots were veri®ed and traced to their site of entry into the spinal cord. Appropriate L5 spinal cord segments were harvested and post ®xed for 4 h in ®xative and then cryoprotected 2±3 days in 30% sucrose/PBS at 48C. The segments were then freeze-mounted in OCT embedding medium on cork blocks for cryostat sectioning. 2.3.2. Immunohistochemistry Optimal dilutions and incubation time periods for each antibody and lot were determined prior to this study. Immunohistochemistry was performed by the avidin-biotin technique (Vector Labs, Burlingame, CA) on free ¯oating 20 mm sections. Elimination of the primary antibody was performed in each run as a negative control. Monoclonal antibodies OX-42 (labels complement receptor (CR3, CD11b)) and anti-MHC II were used as a microglial marker and for MHC II expression respectively, at a dilution of 1:2 (provided from Dr. William F. Hickey, Dartmouth Medical School, NH). A rabbit polyclonal antibody to glial ®brillary acidic protein (anti-GFAP, DAKO, Carpinteria, CA) was used as an astrocytic marker at the dilution of 1:20 000. Five or more sections per animal were prepared from the L5 segments in each run. Assessment of spinal glial activation and MHC II expression was performed by two experimenters blinded to the treatment groups. Following staining of lumbar spinal cord, all the sections from each animal were surveyed under low (10£) and medium (40£) magni®cation to arrive at a score based on a previously described scale for glial activation (Colburn et al., 1997). Brie¯y, the scale is as follows: baseline ( z ), mild response (1), moderate response (11), intense response (111). The ®nal score was based on the average reactivity of all sections per animal. The expression of MHC II-like immunoreactivity was based on the presence or absence of speci®c, cellular staining throughout the gray matter of the spinal cord. 2.4. Statistical analysis All data obtained from the observations of motor paresis and mechanical sensitivity were presented as the mean of eight or ten animals per treatment group ^ SEM. To compare the time-dependent curves among the groups, a repeated analysis of variance (ANOVA) with a Bonferroni

multiple comparison was used. The experimental period of Phase II study was separated into the ®rst 7 days and the second 7 days according to the changes of treatment. Additionally, the data from the tactile stimulation were analyzed by a one-way ANOVA at each time point. Differences between individual means were determined using a Sheffe's multiple comparison test. P , 0:05 for intergroup differences and P , 0:02 for post-hoc test were de®ned as significant. 3. Results 3.1. Phase I 3.1.1. Hind paw eversion and gait disturbance All rats in group A and B which underwent lumbar root injury demonstrated eversion of the ipsilateral hind paw. Forty percent of rats in these groups also showed a slight gait disturbance (scores of 1). The gait disturbance improved and all rats in both groups showed normal gait by day 7 postsurgery. There was no statistical difference in the postoperative change in hind paw eversion or gait disturbance between groups A and B (repeated measured ANOVA). The sham group treated with MTX (group C) did not demonstrate any hind paw eversion or gait disturbance. The postoperative changes in group C were signi®cantly different from groups A and B (P , 0:0001). 3.1.2. Hind paw withdrawal to mechanical stimulation The rats rarely demonstrated baseline stimulation responses with 2 or 12 g von Frey ®lament before the surgery. The rats in group B (L5 root injury 1 saline treatment) demonstrated mechanical allodynia from one day postsurgery to the time of euthanization (at day 7). In contrast, allodynia was reduced in group A (L5 root injury 1 MTX treatment) toward the baseline after the peak response at day 2 post surgery. The rats in group C (sham operation 1 MTX treatment) demonstrated only a minimal response after the surgery. The difference in allodynia was statistically signi®cant by repeated measures ANOVA followed by a post-hoc Bonferroni (2 g VF, PA±B ˆ 0:0138, PB±C ˆ N:S:, PC±A ˆ 0:0014; 12 g VF, PA±B ˆ 0:0034, PB±C ˆ 0:0045, PC±A , 0:0001; Fig. 2a). 3.2. Phase II 3.2.1. Hind paw eversion and gait disturbance The rats in all three groups demonstrated a similar time course in gait disturbance regardless of their postoperative treatments (repeated measured ANOVA). The slight gait disturbance observed at day 2 postsurgery gradually recovered and all the animals had normal gait at the time of euthanization (day 14 postsurgery). 3.2.2. Hind paw withdrawal to mechanical stimulation The rats in groups D and E, treated with intrathecal saline

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Fig. 2. Time course of tactile sensitivity using 12 g von Frey ®lament at the ipsilateral hind paw following L5 nerve root injuries. For each time point, all animals were exposed to a total of 30 stimulations. The average number of evoked foot withdrawal responses is recorded for each animal group. (a) Phase I study: arrows indicate the time points that MTX or saline was administered. In group A, MTX was administered intrathecally (1 mg/kg) and around the spinal root (1 mg/kg) at surgery and at days 2 and 4 postsurgery. Saline injection was employed as a control in group B. In group C, sham operated animals were administered MTX to determine the potential for behavioral /neural side effects. The difference in allodynia was statistically signi®cant between the three treatment groups by repeated measures ANOVA followed by a post-hoc Bonferroni (PA±B ˆ 0:0034, PB±C ˆ 0:0045, PC±A , 0:0001). (b) Phase II study: arrows (1) and (2) indicate the time points that the same numbered solution was administered in the ®rst week and the second week, respectively. In group D, the same dose and method of delivery of MTX was performed as in Phase I with the exception that MTX was administered at days 7, 9, and 11 postsurgery following saline administration in the ®rst week. Saline injection was continued in group E. For examining the recurrence of pain after MTX termination, saline was injected in the second week following MTX administration in group F. In the second week, mechanical allodynia signi®cantly decreased in the MTX treated group D, while mechanical allodynia continued in the saline treated group E (repeated ANOVA with Bonferroni, 12 g: P ˆ 0:0121). Allodynia was signi®cantly attenuated in group F as compared to the response in groups D and E at day 7 (one-way ANOVA, P , 0:0001) and remained signi®cantly lower as compared to group E up to day 11 postsurgery (one-way ANOVA, P9 ˆ 0:0013: P11 ˆ 0:0048).

injection for the ®rst week after the L5 nerve root injury (at the surgery, and days 2 and 4 after surgery), showed a robust allodynic response which developed by day 2 postsurgery and continued up to day 7 postsurgery. At day 7 postsurgery, there was not a signi®cant difference in the response scores between groups D and E (one-way ANOVA). The scores were: 2 g von Frey: 8.5 ^ 3.9, 12 g von Frey: 21.6 ^ 4.0 and

2 g von Frey: 8.0 ^ 4.4, 12 g von Frey: 20.7 ^ 6.0, groups D and E, respectively. In the second week, mechanical allodynia signi®cantly decreased in the MTX treated group D, while mechanical allodynia continued in the saline treated group E (repeated ANOVA with Bonferroni, 12 g: P ˆ 0:0121). The mean response scores at day 14 postsurgery in groups D and E were: 2 g von Frey: 3.9 ^ 4.2, 12 g

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Fig. 3. OX-42 immunoreactivity (microglial marker) in the ipsilateral spinal cord at day 7 following L5 spinal root injury: (a) rat in group A (administered MTX at surgery, at days 2 and 4 post injury); (b) rat in group B (administered saline at surgery, at days 2 and 4 postsurgery); (c) normal rat. Note the intensity of microglial reaction in the L5 root injured rats as compared to the normal rat.

von Frey: 10.7 ^ 6.4 and 2 g von Frey: 11.38 ^ 5.5, 12 g von Frey: 22.1 ^ 5.0, respectively. The animals in group F, which were treated with MTX after the L5 root injury in the ®rst week, demonstrated allodynia which was gradually decreased after the peak at day 2 postsurgery. At day 7 postsurgery, allodynia was signi®cantly attenuated in group F as compared to the response in the saline treated groups D and E (one-way ANOVA with Bonferroni, 2 g von Frey: P7D±F ˆ 0:0016, P7E±F ˆ 0:0020, 12 g von Frey: P7D±F , 0:0001, P7E±F , 0:0001). In the second week when the animals were treated with saline instead of MTX, the allodynic response remained signi®cantly lower as compared to group E up to day 11 postsurgery; 7 days from the last MTX administration (12 g von Frey: P9E±F ˆ 0:0013, P11E±F ˆ 0:0048; Fig. 2b).

astrocytic activation (111). There was no signi®cant difference in the glial activation between the MTX-treated groups (A, D, and F) and the saline treated groups (B and E). (Figs. 3 and 4) MHC II-like immunoreactivity was markedly increased in 5/5 rats in the saline treated group from the Phase II study (see Fig. 5). The morphology of MHC II staining was identical to microglia. In contrast, 5/6 rats demonstrated no detectable MHC II staining in Group D (Saline:MTX group); see Table 1.

3.3. Gross pathology

These data demonstrate a statistically signi®cant attenuation of the incidence of withdrawal to von Frey stimulation in a radicular pain model following central administration of low dose MTX, a recognized immunosuppressive agent.

Gross anatomical inspection following perfusion demonstrated no evidence of suppurative in¯ammation around the nerve roots or DRGs. In all groups except the sham operated (group C), the treated nerve roots were encapsulated with dense granulation tissue and ®brous adhesions. Of interest, granulation and adhesions around the injured nerve root were markedly decreased in the MTX treated groups (A, D, and F) as compared to the saline treated groups (B and E). 3.4. Immunohistochemistry OX-42 and GFAP expression were elevated in the gray matter of the L5 spinal section in all groups that underwent the root ligature with chromic gut (Groups A, B, D±F). All rats in these groups demonstrated intense microglial and

4. Discussion 4.1. Summary

Table 1 Presence of MHC II-like immunoreactivity in the gray matter of the dorsal horn following root injury in either saline treated rats or saline:MTX treated rats Rat number Treatment MHC II 1 Rat number Treatment MHC II 1 5 10 11 22 23

Sal:Sal Sal:Sal Sal:Sal Sal:Sal Sal:Sal

1 1 1 1 1

8 19 21 27 28 29

Sal:MTX Sal:MTX Sal:MTX Sal:MTX Sal:MTX Sal:MTX

2 2 1 2 2 2

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165

Fig. 4. Spinal astrocytic responses as illustrated by GFAP immunoreactivity. (a) Spinal cord GFAP immunoreactivity in a normal rat. The rectangle indicates the area of the ipsilateral dorsal horn which was magni®ed for (b±d). (b) Ipsilateral spinal dorsal horn GFAP immunoreactivity in the rat of group A (MTX was administered intrathecally (1 mg/kg) and around the spinal root (1 mg/kg) at surgery and at days 2 and 4 postsurgery), (c) rat in group B (saline treatment), (d) rat in group C (MTX administration following sham operation).

The possibility that the increased response of withdrawal to von Frey stimulation is due to a confounding factor such as weight bearing as discussed by Kauppila et al. (1998) is very unlikely. Previous studies have con®rmed mechanical allodynia is present in restrained rats after the identical root injury (Kawakami et al., 1994). Although the presence of mechanical allodynia in humans with radiculopathy is observed, the incidence is relatively rare. This raises the possibility that the rats are responding to ongoing pain, not pain evoked by tactile stimulation. This factor is very dif®cult to ascertain in any rat model of persistent pain. However, it is highly relevant to consider for the clinical translation of drug studies performed in animal models. The precise site of action (nerve root versus spinal cord) was not determined in the present study. Since MTX was effective both as a preventative agent (Phase I) as well as demonstrating ef®cacy on existing radicular pain (Phase II), these behavioral results support a neuroimmune/in¯ammatory component in the generation and maintenance of persistent pain associated with root injury. 4.2. Potential mechanisms of action We are con®dent that effective levels of MTX reach spinal nociceptive regions because of the report by Burch et al. (1988). In their autoradiographic study using rabbits, 67±99% of the total area of the spinal cord sections were

shown to be exposed to intrathecally administered MTX 1 h after administration. They also reported that high drug levels were seen in the area of the substantia gelatinosa and peripheral white matter. 4.3. Action as an immunomodulator The exact mechanism of action of MTX in attenuating mechanical allodynia in this study has not yet been fully elucidated. In our initial investigation, we focused on the role of glial cells as a potential drug target. MTX is a folic acid antagonist that inhibits dihydrofolate reductase and, thereby blocks thymidilate synthesis, arresting cells in late S phase (Jolivet et al., 1983). Billingsley et al. (1982) reported that intraperitoneal pretreatment of MTX (10 mg/ kg per day £ 5 days) depressed trauma-induced glial proliferation in the rat brain. The astrocytic depression was also reported in MTX administered cell culture (Gregorios and Soucy, 1990). Previously, we have demonstrated activation of glial cells (microglia and astrocytes) in the relevant spinal segments using the same animal model as this study (Hashizume et al., 2000). In the present study, using immunohistochemistry, there was no signi®cant difference in the activation of glial cells between the MTX treated and the saline-injected groups. This may not be surprising if one recognizes that both OX-42 and GFAP are sensitive but not speci®c markers of CNS injury. In addition, it is unlikely

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Fig. 5. Representative photomicrograph of MHC II-like immunoreactivity in the ipsilateral dorsal horn from a rat in the saline treated group (rat #23 in Phase II).

that, in the doses given in the present study, methotrexate diminished proliferation of glial cells by inhibiting de novo purine and pyrimidine synthesis. It has been recognized, however, that a subpopulation of microglia (the macrophages of the central nervous system)

become immunocompetent in response to infection or injury. One way in which this occurs is through the expression of MHC class II. In normal CNS, both MHC class I and MHC class II expression is minimal compared to other tissues (Hickey and Kimura, 1988; Kreutzberg, 1996).

H. Hashizume et al. / Pain 87 (2000) 159±169

This low expression of MHC and reduced immune surveillance are believed to contribute to the immune-privileged status of non-renewable CNS neurons. Part of the complex peripheral immune response involves CD4 1 T cells recognizing foreign antigens bound to self class II MHC molecules. MHC class II is expressed on antigen presenting cells (APC) which present antigen to T cells. Microglia are the antigen presenting cells of the CNS (Wekerle et al., 1986). Cytokines are key modulators of MHC class I and II genes in a wide variety of cells. Of relevance to the discussion of cytokines and pain processing, proin¯ammatory cytokines such as Tumor Necrosis Factor (TNF) enhance MHC class II expression. Glia do not normally express MHC class II but its expression can be induced by cytokines (Roitt et al., 1998). The mediators which alter the expression of the MHC class II antigens are tissue speci®c. This has implications for possible selective immunomodulation of MHC class II in tissues in which it is over expressed without affecting MHC class II expression in other tissues. We have previously demonstrated an increase in spinal MHC class II expression as compared with a sham surgery group or normal rats in the same radiculopathy model as used in the present study (Hunt et al., 1999). The presence of spinal cord MHC class II-like immunoreactivity in the lumbar radiculopathy model in cells which morphologically resemble activated microglia (see Fig. 5) further supports the notion that central neuroin¯ammatory processes involving immune recognition are involved in root injury. Genes encoding MHC class II are immensely polymorphic. Speci®c alleles for MHC class II has been associated with a variety of autoimmune diseases like multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus (Roitt et al., 1998). A genetic component of chronic pain has recently emerged to help explain why all individuals with a similar injury or disease may not experience the same type of pain (Mogil et al., 1999). Similarly, we have put forth a provocative theory that speci®c polymorphisms in membrane glycoproteins such as MHC class II may render an individual more susceptible to persistent pain after a root or peripheral nerve injury (DeLeo, 1999). It is well known that immune cells (macrophages, monocytes and lymphocytes) are commonly observed in the herniated discs and that various cytokines (IL-1, TNF-a etc.) are produced by the disc materials (Kang et al., 1996; Kawakami et al., 1996). Several reports note reduction in immunoglobulin (IgG) synthesis with signi®cantly lowered levels of all IgG isotypes after 3 months of MTX treatment (Roitt et al., 1998). Administration of low-doses of MTX (450 mg/kg per week £ 4 weeks) inhibits LPSinduced TNF-production by macrophages (Durez et al., 1998). In addition, it has been demonstrated that immunosuppressive therapy with MTX modulates cytokine production by T cells and macrophages (Becker et al., 1998; Neurath et al., 1999). It has been reported that MTX inhibited macrophage activation, PGE2 and IL-1 production, cyanine dye accumulation as well as the in¯ux of Ia positive

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macrophages into synovial tissue (Johnson et al., 1988). Finally, MTX is capable of modulating the cytokine network by increasing Th2 anti-in¯ammatory cytokines and decreasing Th1 proin¯ammatory cytokines (Constantin et al., 1998). This mechanism has been proposed to explain the in vivo action of MTX as a potent anti-in¯ammatory and immunoregulatory agent. We speculate that MTX may act as an anti-in¯ammatory agent or as an immunomodulator in the spinal cord and/or the injury site based on the ®nding that central administration of MTX reduced or eliminated MHC II expression on microglia after root injury. In addition, our observation that granulation and adhesion around the injured nerve root was much less in the MTX treated group as compared to the saline treated group demonstrated that MTX may reduce in¯ammation around the nerve root caused by the in¯ammatory chromic gut. Due to the semi-quantitative nature of immunocytochemistry, analysis of cytokine expression was not attempted in this study. Experiments in progress and future studies planned include an assessment of cytokine expression after MTX treatment utilizing quanti®able methods like enzyme linked immunosorbent assays for protein detection (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) for mRNA detection. Given the multifaceted dimensions of nociceptive processing after nerve or root injury and the complex pharmacology of MTX, it may be dif®cult to assess the speci®c mechanism of action. Indeed, MTX may have multiple actions which may partly explain its enhanced ef®cacy over other agents. 4.4. Enhanced adenosine Unlike its use in the treatment of malignancies (pulses of 20±250 mg/kg), MTX is administered weekly in low doses (0.1±0.3 mg/kg) to treat rheumatoid arthritis and other in¯ammatory diseases (Furst and Kremer, 1988). Cronstein et al., 1993 demonstrated that low-dose weekly methotrexate therapy leads to intracellular accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), which promotes increased adenosine release at sites of in¯ammation. Several studies have shown the antinociceptive effects of adenosine and adenosine analogs when administered intrathecally to rodents in models of acute and chronic pain (Holmgren et al., 1983; Reeve and Dickenson, 1995; Cui et al., 1997). Moreover, adenosine, acting at an A3 receptor, interrupts the endotoxin CD14 receptor signal transduction pathway and blocks induction of cytokine TNF in macrophages (McWhinney et al., 1996). 4.5. Potential spinal toxicity The dosage of MTX in this study was 1 mg/kg for intrathecal injection and 1 mg/kg for the nerve root administration. Clinically, MTX can be administered orally, intravenously, intramusculary, or intrathecally. MTX diffuses from the periphery into the cerebrospinal ¯uid with dif®-

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culty and peak CSF levels are less than 0.1% of corresponding plasma levels following intravenous injection of a single 50 mg dose (Shapiro et al., 1975). For this reason, we performed intrathecal administration for MTX delivery to the spinal cord. Of particular clinical interest, intrathecal MTX has been associated with a variety of neurotoxic effects ranging from acute chemical arrachnoiditis to motor dysfunction, seizures, and coma (Gagliano and Costanzi, 1976; Kaplan and Wiernik, 1982). The MTX dosage in this study, 1 mg/kg, was a quarter of the previously reported dose that was maximally tolerated by rats without acute neurotoxic effects (Morris et al., 1992). The rats in group C, which underwent a sham operation and MTX administration, showed neither evidence of motor paresis nor allodynia in response to von Frey stimulation. Intrathecal injections of MTX or saline were performed intermittently without catheterization because we have previously reported that intrathecal catheterization itself induces neuroimmune activation (enhanced glial activation and cytokine expression) in the rat and may alter nociceptive processing (Serpell et al., 1993; DeLeo et al., 1997a). In conclusion, we have demonstrated that MTX administered intrathecally and around the spinal root had an antinociceptive effect in this combined chemical/mechanical lumbar root injury model. MTX administration, thus, may offer a new treatment modality for radicular pain with or without disc herniation as well as directing new research into the development of novel immunomodulators for the treatment of chronic pain. In addition, these data also support the use of this model as a tool to study new therapies for chronic low back pain model associated with radiculopathy. Acknowledgements We would like to thank Dr William F. Hickey for providing the antibodies, OX-42 and anti-MHC II, Janice Arruda and Tracy Wynkoop for editorial assistance; and the following for grant support: National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR44757); Orthopaedic Research and Education Foundation; and Bristol-Myers Squibb/Zimmer Orthopaedic Foundation. References Becker C, Barbulescu K, Kildner K, Meyter zum Buschenfelde KH, Neurath ME. Activation and methotrexate-mediated suppression of the TNF alpha promoter in T cells and macrophages. Ann NY Acad Sci 1998;859:311±314. Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87± 107. Billingsley ML, Hall N, Mandel HG. Trauma-induced glial proliferation: possible involvement of the immune system. Immunopharmacology 1982;5:95±101. Burch PA, Grossman SA, Reinhard CS. Spinal cord penetration of intrathe-

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