Intrathecal CGS-26303 Pretreatment Attenuates Spinal Nerve Ligation-Induced Neuropathic Pain in the Spinal Cord

Intrathecal CGS-26303 Pretreatment Attenuates Spinal Nerve Ligation-Induced Neuropathic Pain in the Spinal Cord

Accepted Manuscript Intrathecal CGS26303 Pre-treatment Attenuates Spinal Nerve Ligation-Induced Neuropathic Pain in the Spinal Cord Hung-Chen Wang, MD...

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Accepted Manuscript Intrathecal CGS26303 Pre-treatment Attenuates Spinal Nerve Ligation-Induced Neuropathic Pain in the Spinal Cord Hung-Chen Wang, MD, Kuang-I. Cheng, MD, PhD, Chao-Wen Chou, Aij-Lie Kwan, MD, PhD, Lin-Li Chang, PhD PII:

S1878-8750(16)00341-7

DOI:

10.1016/j.wneu.2016.02.093

Reference:

WNEU 3795

To appear in:

World Neurosurgery

Received Date: 23 December 2015 Revised Date:

22 February 2016

Accepted Date: 24 February 2016

Please cite this article as: Wang H-C, Cheng K-I, Chou C-W, Kwan A-L, Chang L-L, Intrathecal CGS26303 Pre-treatment Attenuates Spinal Nerve Ligation-Induced Neuropathic Pain in the Spinal Cord, World Neurosurgery (2016), doi: 10.1016/j.wneu.2016.02.093. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Intrathecal CGS26303 Pre-treatment Attenuates Spinal Nerve Ligation-Induced Neuropathic Pain in the Spinal Cord

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Hung-Chen Wang, MD1; Kuang-I Cheng, MD, PhD2,3; Chao-Wen Chou3;

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Aij-Lie Kwan, MD, PhD4; and Lin-Li Chang, PhD5

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Corresponding authors: ) Aij-Lie Kwan and Lin-Li Chang ( Department of Microbiology College of Medicine, Kaohsiung Medical University 100, Shih-Chuan 1st Road, Kaohsiung, 80708, Taiwan Tel: + 886-7-312-1101~9; Fax: + 886-7-321-2062

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E-mail: [email protected]

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Authorship: Drs. Aij-Lie Kwan and Lin-Li Chang contributed equally to this study.

_________________________________ 1 Departments of Neurosurgery, Kaohsiung Chang Gung Memorial Hospital, Chang

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Gung University College of Medicine, Kaohsiung, Taiwan Departments of 2Anesthesiology, 4Neurosurgery, and 5Microbiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan 3 Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

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ACCEPTED MANUSCRIPT ABSTRACT Background: Matrix metalloproteinase (MMPs) and endothelin-1 (ET-1) may prove

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to be important in the generation of pain induced by inflammation and nerve lesion. This study aimed to investigate the relationships between endothelin receptors and MMPs.

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Methods: Male Sprague-Dawley rats (250-300 gm) were divided into five groups: a

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normal (control) group; an L5 spinal nerve ligation (SNL) group; a CGS26303 IT + L5 SNL group; a BQ-123 IT + L5 SNL group; and a BQ-788 IT + L5 SNL group. The expression of GFAP, ETAR, ETBR, MMP2, and MMP9 in the ipsilateral L5 dorsal root ganglion (DRG) and the activation of microglia and astrocytes in the L5 spinal

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dorsal horn (SDH) were quantified by immunofluorescence and Western blotting. Results: Intrathecal pre-treatment with CGS26303 significantly attenuated the

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hyperalgesic and mechanical responses induced by SNL for 4 days, whereas BQ-123

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administration alleviated the hyperalgesia only for 3 hours and mechanical allodynia for only 1 hour. Pre-treatment with CGS26303 significantly down-regulated the GFAP, ET-A, MMP-2, and MMP-9 expressions in DRG and their effect lasted for 6 hours, 1 day, 7 days, and 1 day, respectively. By immunofluorescence and Western blotting, there was co-localization of ETAR and MMP‐9 in the DRG neurons, while MMP‐2 was expressed in DRG satellite cells. Furthermore, CGS26303 treatment also 2

ACCEPTED MANUSCRIPT reduced SNL-induced microglia and astrocyte activation on the SDH for 7 days. Conclusions: In this study, CGS26303 can attenuate SNL-induced neuropathic pain

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by down-regulating MMP-9, MMP-2, and ETAR expressions in the DRG and by glia cell activation in the SDH.

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Key words: MMP-9, MMP-2, ETAR, neuropathic pain, microglia, astrocyte

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ACCEPTED MANUSCRIPT INTRODUCTION Neuropathic pain, one of the most difficult clinical pain syndromes to treat,

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represents an unpleasant somatosensory experience due to injuries to the peripheral nervous system (PNS) or the central nervous system (CNS). Many common diseases involves PNS, such as post-herpetic neuralgia, trigeminal neuralgia, diabetic

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neuropathy, cancer and degenerative neurological diseases, and CNS, such as spinal

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cord injury and stroke, may produce neuropathic pain 1. Peripheral sensitization occurs when the primary afferent nociceptive neurons exhibit increased responsiveness to external mechanical or thermal stimuli at the original site of injury, as mediated by pro-inflammatory cytokines and other molecules 2, 3. Central

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sensitization refers to an increased activity of the dorsal horn neurons in response to input from primary afferents 4, 5.

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Endothelin-1 (ET-1) is expressed in neurons of the brain, spinal cord, and dorsal

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root ganglia 6-10. Two G-protein-linked transmembrane receptors for endothelin, endothelin-A receptor (ETAR) and endothelin-B receptor (ETBR), are present in neurons of the PNS and CNS 11-13. Previous studies suggest that endogenous ET-1 may be important in generating and maintaining persistent pain states 14-16. Moreover, ET-1 is generated from the post-translational processing of a precursor protein, pre-pro-ET-1, to a peptide precursor, big ET-1, which is further processed to 4

ACCEPTED MANUSCRIPT biologically active ET-1 by a specific phosphoramidon-sensitive metalloprotease called the endothelin converting enzyme (ECE) 17. The ECE inhibitor, (S)-2

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biphenyl-4yl-1-(1H-tetrazol-5-yl)-ethyl-amino-methyl phosphonic acid (CGS 26303), is a vasopeptidase inhibitor that simultaneously inhibits ECE and neutral

endopeptidase 18, which can inhibit pro-endothelin-1 conversion to ET-1. A previous

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spinal cord after ischemia-reperfusion injury 19.

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study has shown that CGS 26303 may have a promising neuroprotective effect in the

Matrix metalloproteinase (MMPs) may prove to be important in the generation of pain induced by inflammation and nerve lesion via their complex relationships with cytokines, chemokines, growth factors, and adhesion molecules, to which nociceptors

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are responsive 3. The up-regulation of MMP-9 and MMP-2 after acute peripheral nerve injury has been associated with both detrimental and beneficial effects on

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recovery 20-22, and mediate pain hypersensitivity by initiating IL-1β cleavage and

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microglial and astrocytic activation 23, 24. Several studies show that MMP activation is associated with endothelin receptors 25-27. The present study was designed to determine the effects of CGS26303 in acute

neuropathic pain, with focus on the expression of MMP2, MMP9, ETAR, ETBR, and glial cells in response to such pain. The study also aimed to investigate the relationship among ETAR, ETBR, and MMPs. 5

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MATERIALS AND METHODS

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The CGS 26303 (Novartis Pharmaceuticals Corp., East Hanover, NJ, USA) was dissolved in 0.25 M sodium bicarbonate at a concentration of 6mg/ml. Animals and Anesthesia

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The study used 223 male Sprague-Dawley rats weighing 250-300 gm. The rats

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were housed in plastic cages at room temperature in a 12-hour light-and-dark cycle, with free access to food and water. They rats were kept at least 7 days under these conditions before the study. The Kaohsiung Institutional Animal Care and Use

102049).

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Committee approved all of the experimental procedures (Approval Nos. 97135 and

Experimental Groups

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The rats were divided into five groups: a normal (control) group; an L5 spinal

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nerve ligation (SNL) group; a CGS26303 IT + L5 SNL group;a BQ-123 IT + L5 SNL group, and a BQ-788 IT + L5 SNL group. Each group had 30-50 rats. Intrathecal injection of CGS26303, BQ123, or BQ788 Using a modification of the intrathecal injection technique described by De la Calle and Paino 28, rats were placed in the prone position and a 2 cm longitudinal skin incision was made on the midline just above L5 and L6 spinal process. The L5/L6 6

ACCEPTED MANUSCRIPT interspinous ligaments were incised, and half of the anterior L6 spinal process removed, allowing direct visualization of the L5/6 ligamentum flavum. A gauge 30

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needle was inserted between the ligamentum flavum at an angle of 15°-30° horizontal to the subarachnoid space of the cauda equina. A P-10 tube connected to a 50µl

Hamilton syringe with a gauge 30 needle (Hamilton, Reno, NV, USA) and inserted

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into the subarachnoid space of the cauda equina. A 50µl of CGS26303 (6mg/ml),

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BQ123 (3nmol), or BQ788 (3nmol) was administered by intrathecal injection through the L4-L5 intervertebral space. BQ-123 is a selective peptidic ETAR antagonist and BQ-788 is a selective peptidic ETBR receptor antagonist. BQ-123 and BQ-788 were used to selective inhibit ETAR and ETBR, respectively, and both of them were used to

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compare with the effects of CGS26303. Tail flick was another sign to identify successful intrathecal injection. After withdrawing the needle, gently check no fluid

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leaking out under microscope. The wound was approximated with surgical sutures.

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Placed the animal in a recovery cage to wake up and monitored until they resumed normal activity.

Surgical Procedures of L5 Spinal Nerve Ligation In the pathophysiology of neuropathic pain using a well characterized animal model on left L5 spinal nerve ligation (SNL), nerve injury was produced by surgical ligation of the L5 spinal nerve. Each mouse was anesthetized and placed in a prone 7

ACCEPTED MANUSCRIPT position. The left para-spinal muscles were separated from the spinous processes at the L5-L6 level and retracted. The left L6 transverse process was carefully removed

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with small forceps to visualize the L4 and L5 spinal nerves. The L5 spinal nerve was tightly ligated with a silk ligature proximal to the confluence of the spinal nerves and distal to the dorsal root ganglion (DRG).

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Behavioral Testing

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Allodynia (Mechanical Stimuli)

The rats were first acclimated to the environment for testing mechanical stimuli, which was a metal mesh floor covered by a transparent plastic dome (8 x 8 x 18 cm). A Dynamic Plantar Aesthesiometer (UgoBasil, Italy) filament underneath the metal

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mesh was applied perpendicular to the outer mid-plantar surface of the paw. An automated test machine was used to apply mechanical stimuli with a 2 mm diameter

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metal rod in increments of 2.5 g/s (to a maximum of 50 g) to either hind paw until an

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abrupt foot withdrawal was elicited. When rapid withdrawal of the paw was observed, duration and force intensity were recorded with approximately 0.1 g sensitivity. For each hind paw, measurements were repeated 5 times with 3-minute intervals.

The paw withdrawal force was the averaging measurements for each hind paw. Hyperalgesia (Noxious Heat Stimuli) The latency of foot withdrawal from noxious heat stimuli was measured using 8

ACCEPTED MANUSCRIPT the method described previously 29. Briefly, an infrared light beam emitted from a moveable light box was projected through a hole (2 × 5 mm) to heat the glass plate

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under one hind paw (Ugo Basile Model 7370, Italy). Abrupt lifting, withdrawal, licking of the hind paw, or guarding posture was considered a positive response. A

photocell was used to automatically turn off the light beam when the rat lifted its paw.

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The time from application of the light beam to the lifting of the hind paw was

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recorded and defined as foot withdrawal latency. Measurements were performed at 5-minute intervals and repeated five times on each hind paw, alternating between the two paws. The results were expressed as mean ± standard deviation of the 50%

Immunofluorescence

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withdrawal threshold.

As in a previous study 30, the rats were anaesthetized with 60 mg/kg thiopentone

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and perfused with 0.9% (w/v) saline followed by 4% (w/v) paraformaldehyde in 0.1

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mol/L phosphate buffer (pH 7.4). The L5 DRG and spinal cords were removed. The dissected tissues were then fixed in 4% (w/v) paraformaldehyde and then saturated in 10-30% (w/v) sucrose in 0.02 mol/L PBS (pH 7.4). After embedding the tissues in optimal cutting temperature (OCT) compound, L5 DRGs (10 µm) and L5 spinal cords (16 µm) were prepared for immunostaining. Activation of Microglia and Astrocytes in SDH 9

ACCEPTED MANUSCRIPT For the activation of microglia and astrocytes in spinal dorsal horn (SDH), OCT sections were incubated overnight with mouse monoclonal anti-OX-42 (mouse

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anti-rat-CD11b, 1:80, Serotec) or rabbit anti-glial fibrillary acidic protein (GFAP; 1:200, Millipore), followed by goat anti-mouse IgG Alexa Fluor 488 (1:100,

Invitrogen, Grand Island, NY, USA) or Cy3-conjugated goat anti-rabbit (1:100,

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Chemicon) secondary antibody.

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Expressions of MMP-2, MMP-9, and ETAR in DRG

By double immunofluorescence labelling, OCT sections were incubated for 24 h at 4°C with the combination of primary antibodies: rabbit anti-MMP-9 polyclonal antibody (1:800, AB19016, Millipore, USA) and mouse anti-NeuN monoclonal

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antibody (1:200, MAB377, Millipore, USA). The Cy3 conjugated goat anti-rabbit IgG or goat anti-mouse IgG Alexa Fluor 488 (1:100, Invitrogen, Grand Island, NY, USA)

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were used to visualize the primary antibody binding.

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To assess MMP-2 or ETAR co-localized in neuron or satellite cells in DRG, triple immunofluorescence was used. The OCT sections were incubated overnight simultaneously with primary antibody goat anti-MMP-2 polyclonal antibody (1:25, SC6838, Santa Cruz, USA) or sheep polyclonal to endothelin A receptor (1:200, ab30536, Abcam, USA), with mouse anti-NeuN monoclonal antibody (1:200, MAB377, Millipore, USA) and rabbit anti-Glial Fibrillary Acidic Protein (GFAP) 10

ACCEPTED MANUSCRIPT polyclonal antibody (1:200, AB5804, Millipore, USA). These were followed by incubation with secondary antibodies DyLight 405-

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conjugated Affinity Pure donkey anti-goat IgG (1:200, Jackson, USA), Alexa Fluor488-conjugated Affinity Pure Goat Anti-Mouse IgG (1:200, Jackson, USA), Cy3 conjugated goat anti-rabbit IgG antibody (1:200, AP132C, Millipore, USA), and Cy5

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conjugated donkey anti-sheep (1:200, AP184S, Chemicon, USA)

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The immunoreactivity of each section was examined. The images were captured using an Olympus FluoView 1000 confocal laser scanning microscope (Olympus, Tokyo, Japan). Western Blotting

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As in a previous study 30, the L5 DRG were removed to evaluate the ETAR, MMP-2, and MMP-9 expressions and GFAP (+) satellite cell activation in DRG. The

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L5 dorsal horn of the spinal cord was also sliced and removed for analysis of

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microglia and astrocyte activation. First, the tissues were homogenized in RIPA lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 0.1% (w/v) sodium dodecyl sulphate (SDS), 1% (v/v) NP-40, 0.5% (w/v) sodium deoxycholate] containing complete protease inhibitor mixture (Roche Diagnostics GmbH, Mannheim, Germany). Protein lysate (50 µg) from each sample was electrophoretically placed in 8% 11

ACCEPTED MANUSCRIPT SDS-polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (PVDF, Millipore, Bedford, MA, USA). The membranes were firstly blocked with 5%

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milk in phosphate-buffered saline (PBS) with 0.1% Tween-20 for 1 h at room temperature, and then probed overnight at 4°C with Rabbit anti-MMP-2 Polyclonal Antibody (1:200, AB19167, Millipore, USA), Rabbit anti-MMP-9 Polyclonal

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Antibody (1:800, AB19016, Millipore, USA), Sheep polyclonal to Endothelin A

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Receptor (1:500, Ab30536, Abcan, UK), Mouse Anti-Glial Fibrillary Acidic Protein Monoclonal Antibody (1:10000, MAB360, Millipore, USA), or mouse monoclonal anti-OX-42 antibody (1:100, CD11b, Serotec, Indianapolis, IN, USA) primary antibody to detect the expressions and activations of MMP-2, MMP-9, ETAR and

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GFAP (+) satellite cells in DRG and microglia and astrocyte activation in SDH. This was followed by reaction with a horseradish peroxidase-conjugated secondary

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antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

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The intensity of each band was visualized by ECL Western blotting detection reagents (Amersham Biosciences, Tokyo, Japan). Each protein expression was internal normalized using β-actin, while the expression level was normalized against the expression level of each protein in normal rats. Statistical Analysis Group comparisons for behavioral responses and microglia and astrocyte 12

ACCEPTED MANUSCRIPT activation in the SDH were performed using the Mann-Whitney U-test. Multiple comparisons of time-dependent differences in Western blots were determined by

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analysis of variance (ANOVA), followed by the least significant difference test for multiple post hoc analyses. The SPSS 18.0 (SPSS Inc., Chicago, IL, USA) software was used for all statistical analyses. Statistical significance was set at *p<0.05, p<0.001.



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**p<0.01, and

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ACCEPTED MANUSCRIPT RESULTS This study involved 223 rats. After surgery, none of the animals revealed

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autotomy and none exhibited permanent ventro-flexion or dragging of the hind paw during forward movement. Timeline and rats involved in the experiments was listed in Figure 1.

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Intrathecal pre‐‐treatment with CGS26303 was better than BQ-123

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administration

The left L5 SNL model produced immediate hyperalgesia and mechanical allodynia on the left hind paw 1 hour after nerve ligation and persisted for 7 days (Figs. 2A and 2B). In terms of hyperalgesic responses, compared to spinal nerve

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ligation, pre-treatment with CGS26303 and BQ-123 before nerve injury mitigated the hyperalgesic responses, and this effect lasted for 4 days and 3 hours, respectively (Fig.

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2A). In terms of mechanical responses, pre-treatment with CGS26303 and BQ-123

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palliated mechanical allodynia for 4 days and 1 hour, respectively (Fig. 2B). However, BQ-788 did not lessen both mechanical allodynia and thermal hyperalgesia. Intrathecal pre‐‐treatment with CGS26303 before SNL significantly decreased GFAP, ET‐‐A, MMP‐‐2, and MMP-9 protein expressions in DRG Peripheral nerve injury in the SNL model significantly increased GFAP, ET‐A, and MMP‐2 protein expressions in the ipsilateral L5 DRG at 6 hours and lasted up to 14

ACCEPTED MANUSCRIPT 7 days. The MMP‐9 expression increased significantly in the ipsilateral L5 DRG at 6 hours after SNL and lasted only for one day. Intrathecal pre‐treatment with

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CGS26303 before SNL significantly decreased MMP-2 protein expressions in DRG for 7 days, but this effect lasted for only 6 hours for GFAP and 1 day for ET‐A and MMP‐9 (Figs. 3A-3E).

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Confocal microscopic images showed the co-existence of ETAR and MMP‐9 in

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DRG neurons (Figs. 3F and 3G). The expression of MMP‐2 in glial cells was demonstrated by co‐staining with GFAP and NeuN (Fig 3H). In DRG, there was a short duration but significantly increased endothelin B (ET-B) receptor expression 1 day after SNL. Intrathecal pre-treatment with CGS-26303 significantly inhibited

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ET-B receptor expression after 1 day (Supplement 1). Intrathecal pre‐‐treatment with CGS26303 significantly alleviated SNL-induced

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activated glial cells in the spinal dorsal horn

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After SNL, the activated microglia and astrocytes significantly increased in the spinal dorsal horn from day 1 to day 7. There was a significant increase in OX-42 immunoreactivity in the ipsilateral spinal dorsal horn on day 1 (Normal vs. SNL groups, p≤0.01) (Fig. 4A). This reached a maximum on day 7 (Normal vs. SNL groups, p≤0.01) (Figs. 4B-4C). Intrathecal pre-treatment with CGS26303 attenuated the SNL-induced activated microglia on SDH from day 4 to day 7 (SNL vs. 15

ACCEPTED MANUSCRIPT SNL+CGS groups, p≤0.01 and p≤0.001, respectively) (Figs. 4D-4F). The quantification results were the same when Western blot hybridization was used (Figs.

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4G and 4H). The activated astrocytes significantly increased from day 1 to day 7 (Normal vs. SNL groups, p≤0.001, p≤0.01, and p≤0.01, respectively) (Figs. 5D-5F). Intrathecal

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pre-treatment with CGS26303 significantly reduced astrocyte activation caused by

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SNL in the spinal cord. The effect lasted for 7 days (SNL vs. SNL+CGS groups, p≤0.001, p≤0.05, and p≤0.05, respectively) (Figs. 5G-5I). The quantification results

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were the same when Western blot hybridization was used (Figs.5J and 5K).

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ACCEPTED MANUSCRIPT DISCUSSION In the present study, SNL-induced hyperalgesic and mechanical responses were

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significantly attenuated by intrathecal pre-treatment with CGS26303. Its effect on ETA antagonist BQ-123 was for 3 hours, but it had no effect on ETB antagonist

BQ-788. Intrathecal pre‐treatment with CGS26303 before SNL also significantly

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decreased the expressions of these proteins in DRG, but the effect lasted for only 6

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hours for GFAP, 1 day for ET‐A, ET-B, and MMP‐9, and 7 days for MMP‐2. Compared to the SNL model, BQ-123 pre-treatment only significantly attenuated ET-A protein expression for one hour (data not shown). Based on the behavior and expressions of these proteins, the analgesic effects of intrathecal CGS26303

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pre-treatment before SNL might be due to ET-A and MMP9 inhibition for the first day and MMP2 inhibition from day 1 to day 4.

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Sensory neurons may be important sources of ET-1, which may act in an

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autocrine or paracrine manner to excite these neurons 7, 31. The spinal administration of ET-1 produces a dose-dependent anti-nociceptive effect in the tail flick test and the formalin test 32, 33. Another group of investigators has produced neuron-specific ET-1 knockout mice 9, which demonstrate significantly greater sensitivity to acute nociceptive pain, inflammatory pain, and neuropathic pain compared to control mice. These studies suggest that ET-1 in the CNS suppresses pain transmission and plays a 17

ACCEPTED MANUSCRIPT role in endogenous pain inhibitory control. However, these studies only reveal the difference of ET-1 spinal administration

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with normal mice, but not for pain treatment. The current SNL model produces significant nerve injury and profound painful behavior. The pain pathways can be multiple and not only via endothelin. Although intrathecal pre-treatment with

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CGS26303 only inhibited ETAR for one day compared to the SNL group, it

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significantly attenuated hyperalgesic and mechanical responses for 4 days. However, this response was shortened to only 3 hours after ETAR antagonist administration and was not eliminated after ETBR antagonist intrathecal pre-treatment. These data reveals that the analgesic effect of CGS26303 may not only be due to ETAR

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inhibition but also to another pathway.

The association between endothelin receptors and MMPs

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Several studies show that MMP activation is associated with endothelin receptors.

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One cardiac extracellular remodeling in rats with endothelin-dependent hypertension is associated with increased fibronectin, MMPs activity, and the up-regulation of inflammatory mediators, all of which are reduced by ETA receptor antagonism 25. In Ergul’s et al. study, total MMP activity and expression of MMP-2 and MMP-9 are increased in the stress group. In the stress group, ETA receptor antagonism prevents the increase in MMP expression and activation 26. Their results provide evidence that 18

ACCEPTED MANUSCRIPT the MMP system is activated before the development of hypertension and that ET-1 mediates these early events in vascular remodeling.

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One air pollution-induced exacerbation of cardiovascular disease animal model suggests that ET-1 may mediate MMP-9 expression, as BQ-123 treatment normalizes the expression and activity of MMP-9 27. The ET-1 regulation of MMP-9 may be

. In the present study, pre-treatment with CGS26303 significantly attenuates ETAR

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through mitogen activated protein kinase (MAPK) signaling pathways, via the ETAR

and MMP9 expressions and the effects last for 1 day. But MMP2 is attenuated from day 1 to day 7, suggesting that MMP9 activation, but not MMP2 activation, may be

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associated with ETAR.

Matrix Metalloproteinases (MMPs)

This MMP research is a new appreciation of the role played by these enzymes in

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nociception and hyperalgesia. In the current SNL model, MMP-9 shows a rapid and

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transient up-regulation in injured DRG primary sensory neurons, whereas MMP-2 shows a delayed response in DRG satellite cells. The same results are seen in the study by Kawasaki et al., whereby early and late phase neuropathic pain development after nerve injury required different matrix metalloproteinases (MMPs) 3. Confocal imaging shows that MMP-9 is expressed in DRG neurons and more MMP-9 positive neurons are found after SNL. Pre-treatment with CGS26303 can significantly 19

ACCEPTED MANUSCRIPT attenuate MMP9 expression in DRG neurons. The MMPs may prove important in the generation of pain induced by inflammation and nerve lesions via their complex relationship with cytokines, chemokines, growth factors, and adhesion molecules, to

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which nociceptors are responsive 24.

In the current study, confocal microscopic images show the co-existence of

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ETAR and MMP‐9 in DRG neurons. The MMP-9 expression is significantly

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attenuated in CGS26303 pre-treated rats at 6 hours to 1 day, while the GFAP expression is significantly attenuated at 6 hours. The study by Berta et al. shows the same results, with confocal analysis revealing a close proximity of MMP-9 and GFAP immunostaining in DRG neurons and satellite cells, respectively 34. Furthermore,

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morphine-induced DRG up-regulation of GFAP expression activation is abolished after MMP-9 deletion. The up-regulation of GFAP expression in DRG is

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MMP-9-dependent 35.

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Increasing evidence indicates that the activation of microglia and astrocytes in the dorsal horn represents an essential amplification mechanism leading to neuropathic pain in the setting of spinal cord or nerve injury. Spinal or peripheral nerve ligation produces rapid and profound mechanical allodynia with intense glial responses 30, 36-39. The MMP-9 is expressed in DRG neurons and is transported to the dorsal horn of spinal cord to activate microglia 3. 20

ACCEPTED MANUSCRIPT In contrast to MMP9, MMP2 is induced and persists in DRG satellite cells and spinal cord astrocytes to activated astrocytes in the late phase of neuropathic pain.

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Activated glial cells are implicated in developing and potentiating neuropathic pain that is sensitized by ectopic discharge activities emitted from damaged afferent nerves 40, 41

, leading to central sensitization in dorsal horn through rendered light touch

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painful 36, 42, 43. Pre-treatment with CGS26303 provides a better and longer analgesic

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effect than BQ-123 in alleviating hypersensitive mechanical and thermal responses. This suggests that attenuation of neuropathic pain by CGS26303 may be due to the down-regulation of MMP2 and MMP9 expressions, except for ETAR and ETBR

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inhibition.

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ACCEPTED MANUSCRIPT CONCLUSIONS This study demonstrates that CGS26303 attenuates SNL-induced neuropathic

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pain by down-regulating MMP-9, MMP-2, and ETAR expressions in DRG and glia cell activation in SDH. Furthermore, MMP-9, but not MMP-2, is identified to be

partially regulated by ETAR. However, the mechanism of MMP inhibition of CGS

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26303 remains unclear. Develops drugs to inhibit ETAR or MMPs may have

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therapeutic potential in those patients had acute neuropathic pain.

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ACCEPTED MANUSCRIPT Acknowledgments This study was supported by grants from Chang Gung Memorial Hospital

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(Research Projects CMRPG8C0701 and CMRPG8C0702) and National Science Council (NSC Research Project 98-2314-B-182A-056-MY2). None of the authors

have any conflicts of interest associated with this study. The authors thank Dr. Gene

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Alzona Nisperos for editing and reviewing the manuscript for English language

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considerations.

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ACCEPTED MANUSCRIPT LEGEND Figure 1. Timeline and rats involved in the experiments. Each time point (*) had

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n=3~6 rats. I.T., intrathecal; h, hour(s); D, day(s) Figure 2. The attenuating effects of intrathecal pre-treatment with CGS26303 and endothelin (ET) receptor antagonist BQ-123, and BQ788, respectively, on

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SNL-induced nociceptive responses in rats. (A) In the CGS26303-infused group,

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the SNL-induced hyperalgesic response was significantly attenuated compared to the corresponding value of the SNL-operated group at each time point. The duration of pain alleviation by CGS26303 lasted for 4 days, while the alleviated effect by BQ-123 was for 3 hours only. (B) In mechanical responses, compared

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with spinal nerve ligation, pre-treatment with CGS26303 and BQ-123 before nerve injury palliated the mechanical allodynia for 4 days and 1 hour,

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respectively. The BQ-788 did not palliate either mechanical allodynia or thermal

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hyperalgesia. Significant differences of behavior hypersensitivity were as indicated. Each group had n=3~6 rats. Data represented the means of BQ-123 and SNL by the Mann-Whitney U-test, with *p<0.05, **p<0.01, and



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(between the CGS26303 and SNL groups) and a p<0.05, bp<0.01, and cp<0.001 (between the BQ-123 and SNL groups); s, second; g, gram Figure 3. (A) Western blot and quantitative analysis of (B) GFAP, (C) ET‐A receptor, 28

ACCEPTED MANUSCRIPT (D) MMP‐2, and (E) MMP‐9 protein expressions in L5 DRG at 6 hour, 1 day, 4 days, and 7 days after SNL and CGS26303 pre‐treatment. The GFAP, ET‐A, and

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MMP‐2 protein expressions were significantly increased in the SNL group at 6 hours and this lasted up to 7 days. The MMP‐9 expression increased significantly at 6 h after SNL and lasted only for 1 d. Intrathecal pre‐treatment with

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CGS26303 before SNL significantly decreased the expressions of these proteins

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in DRG, but the effect only lasted 6 h for GFAP, 1 d for ET‐A and MMP‐9, and 7 d for MMP‐2. (F and G) Confocal microscopic imaging showed co-localization ETAR and MMP‐9 in DRG neurons. (H) Expression of MMP‐2 was demonstrated by co‐localization with GFAP and NeuN. Each group had n=3~6



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rats. Scale Bars: 20 µm for ETAR and 50 µm for MMPs; *p<0.05, **p<0.01, and p<0.001, by one‐way ANOVA.

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Figure 4. Photomicrographs showing OX-42 positive microglia expression in the

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ipsilateral SDH at (A) day 1 day, (B) day 4, and (C) day 7 day post-SNL. There was low OX-42 immunoreactivity in the SDH of normal control rats. Quantification of OX-42 immunoreactivity revealed significantly increased levels of OX-42 in the ipsilateral SDH from day 1 to day 7 after SNL. (D-F) Photomicrographs revealed OX-42 immunoreactivity in the ipsilateral SDH after SNL in rats pre-treated with CGS26303. (G and H) The quantification results 29

ACCEPTED MANUSCRIPT were the same when Western blot hybridization was used. Compared to SNL, intrathecal pre-treatment with CGS26303 before nerve injury inhibited microglia

n=3~6 rats per group; *p<0.05, **p<0.01, and



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activation on SDH for 7 days. Scale bars=200 µm, by Mann-Whitney U test; p<0.001; NS; non-significantly

different; Data shown as mean 0 in the ipsilateral SDH from day 1 to day 7

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Figure 5. Intrathecal pre-treatment CGS26303 reduced astrocyte activation caused by

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SNL in the spinal cord. (A-C) By immunofluorescence, the normal control group had a low expression level of GFAP in astrocytes. (D-F) SNL caused a significantly increased GFAP expression of spinal cord ipsilateral to nerve injury.

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(G-I) Pre-treatment with CGS26303 via intrathecal significant reduced spinal GFAP expression after SNL. The effect lasted for 7 days. (J and K) The quantification results were the same when Western blot hybridization was used.

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p<0.001, NS; non-significantly different; Data was shown as

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**p<0.01, and

mean±SEM, with n=3~6 rats /each treatment in each time point

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ACCEPTED MANUSCRIPT SUPPLEMENT Supplement 1. In DRG, there was short duration but significantly increased

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endothelin B (ET-B) receptor expression at 1 day after SNL. Intrathecal pre-treatment with CGS-26303 significantly inhibited ET-B receptor expression, although the effect lasted only for 1 day. n=3~6 rats per group; *p<0.05, ☆

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ACCEPTED MANUSCRIPT Abbreviations list: PNS, peripheral nervous system; CNS, central nervous system; ET-1, endothelin-1; ETAR, endothelin-A receptor; ETBR, endothelin-B receptor; ECE,

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endothelin converting enzyme; MMPs, Matrix metalloproteinase; SNL, spinal nerve ligation; DRG, dorsal root ganglion; OCT, optimal cutting temperature; SDH, spinal dorsal horn; GFAP, glial fibrillary acidic protein; PBS, phosphate-buffered saline;

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ANOVA, analysis of variance

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patients had acute neuropathic pain.

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neuropathic pain by down-regulating MMP-9, MMP-2, and ETAR expressions in DRG and glia cell activation in SDH.  MMP-9, but not MMP-2, is identified to be partially regulated by ETAR.  Develops drugs to inhibit ETAR or MMPs may have therapeutic potential in those

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