JNS-13809; No of Pages 5 Journal of the Neurological Sciences xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of the Neurological Sciences journal homepage: www.elsevier.com/locate/jns

Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats Kei Kato a, Masao Koda a,⁎, Hiroshi Takahashi a, Tsuyoshi Sakuma a, Taigo Inada a, Koshiro Kamiya a, Mitsutoshi Ota a, Satoshi Maki a, Akihiko Okawa a, Kazuhisa Takahashi a, Masashi Yamazaki b, Masaaki Aramomi a, Masayuki Hashimoto a, Osamu Ikeda a, Chikato Mannoji c, Takeo Furuya a a b c

Department of Orthopedic Surgery, Chiba University Graduate School of Medicine, Japan Department of Orthopedic Surgery, University of Tsukuba, Japan Department of Orthopedic Surgery, Chiba Aoba Municipal Hospital, Japan

a r t i c l e

i n f o

Article history: Received 6 October 2014 Received in revised form 18 May 2015 Accepted 19 May 2015 Available online xxxx Keywords: Spinal cord injury Neuropathic pain G-CSF Allodynia Inflammatory cytokine Central pain

a b s t r a c t Spinal cord injury (SCI) can cause neuropathic pain (NeP), often reducing a patient's quality of life. We recently reported that granulocyte colony-stimulating factor (G-CSF) could attenuate NeP in several SCI patients. However, the mechanism of action underlying G-CSF-mediated attenuation of SCI-NeP remains to be elucidated. The purpose of the present study was to elucidate the therapeutic effect and mechanism of action of granulocyte colony-stimulating factor for SCI-induced NeP. T9 level contusive SCI was introduced to adult male Sprague Dawley rats. Three weeks after injury, rats received intraperitoneal recombinant human G-CSF (15.0 μg/kg) for 5 days. Mechanical allodynia was assessed using von Frey filaments. Immunohistochemistry and western blot analysis were performed in spinal cord lumbar enlargement samples. Testing with von Frey filaments showed significant increase in the paw withdrawal threshold in the G-CSF group compared with the vehicle group 4 weeks, 5 weeks, 6 weeks and 7 weeks after injury. Immunohistochemistry for CD11b (clone OX-42) revealed that the number of OX-42-positive activated microglia was significantly smaller in the G-CSF group than that in the vehicle rats. Western blot analysis indicated that phosphorylated-p38 mitogenactivated protein kinase (p38MAPK) and interleukin-1β expression in spinal cord lumbar enlargement were attenuated in the G-CSF-treated rats compared with that in the vehicle-treated rats. The present results demonstrate a therapeutic effect of G-CSF treatment for SCI-induced NeP, possibly through the inhibition of microglial activation and the suppression of p38MAPK phosphorylation and the upregulation of interleukin-1β. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Neuropathic pain (NeP) occurs as a result of alterations in neuronal activity in susceptible individuals produced by damage to the central or peripheral nervous system. Symptoms of NeP frequently include uncomfori, spontaneous sensations, dysesthesia, and exaggerated perception of non-noxious (allodynia) or noxious (hyperalgesia or hyperpathia) stimuli. Both abnormal sensations are described by patients as unrelenting, burning, biting, tingling, shooting and shock-like sensations. Currently, the precise pathomechanism of NeP remains unclear. Extensive laboratory and clinical exploration to establish novel therapeutics for NeP is ongoing worldwide [1]. ⁎ Corresponding author at: Department of Orthopedic Surgery, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-Ku, Chiba 2608670, Japan. E-mail address: [email protected] (M. Koda).

Spinal cord injury (SCI) is one of the major causes of central NeP. It is reported that the prevalence of NeP after SCI is 40% or more [2,3]. In SCI patients, NeP is one of the main causes of a patient's poor quality of life (QOL) [4]. Granulocyte colony-stimulating factor (G-CSF) is a 19.6 kDa glycoprotein initially identified as a serum factor that induces differentiation of a murine myelomonocytic leukemic cell line [5]. It is widely known as a hematopoietic cytokine that promotes survival, proliferation, and differentiation of cells of neutrophilic lineage [6]. G-CSF is used clinically for patients with leukocytopenia to increase white blood cell number and for donors of peripheral blood-derived hematopoietic progenitor cells prior to their collection for transplantation to mobilize bone marrow-derived hematopoietic progenitor cells into the peripheral blood [6]. Recently, nonhematopoietic effects of G-CSF have been reported, including effects on the central nervous system. G-CSF was found to protect neurons from ischemia-induced cell death and to promote neurogenesis in a rat model of brain ischemia [7,8]. Recently

http://dx.doi.org/10.1016/j.jns.2015.05.024 0022-510X/© 2015 Elsevier B.V. All rights reserved.

Please cite this article as: K. Kato, et al., Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats, J Neurol Sci (2015), http://dx.doi.org/10.1016/j.jns.2015.05.024

2

K. Kato et al. / Journal of the Neurological Sciences xxx (2015) xxx–xxx

we reported that G-CSF protects neurons and oligodendrocytes from apoptosis in mouse and rat SCI models [9,10]. Moreover, it was reported that G-CSF attenuates peripheral NeP [11,12]. We recently conducted early-phase clinical trials to prove neuroprotective effects of G-CSF for SCI and acute aggravation of compressive myelopathy [13,14]. In the trial for compressive myelopathy, NeP was attenuated after G-CSF administration in 14/17 patients for several months. The ratio of pain reduction was 30–50% in average at 1 month after G-CSF treatment [15]. Therefore, we hypothesized that G-CSF can potentially attenuate neuropathic pain. However, the mechanism of action underlying G-CSF-mediated attenuation of SCI-NeP remains to be elucidated. In this study, we administered G-CSF after induction of NeP following SCI to elucidate the mechanism of G-CSF treatment for SCI-NeP.

vehicle and G-CSF groups was assessed using von Frey filaments, according to a previously described protocol [17] by blinded observer who did not know the experimental groups. In brief, von Frey filaments were applied to the central region of the plantar surface of a hind paw in ascending order of force (0.7, 1.2, 1.5, 2.0, 3.6, 5.5, 8.5, 11.7, 15.1, and 29 g). Each filament was applied 5 times. When the rats showed 1 withdrawal response to a given filament, the bending force of that filament was defined as the paw withdrawal threshold intensity. The median threshold intensity was calculated from the values following 1 descending and 2 ascending trials. The experimental conditions were identical for both groups of rats. Behavioral testing commenced one day after the operations and continued for 7 consecutive weeks. 2.4. Sample preparation

2. Materials and methods 2.1. Animals All animals were treated and cared for in accordance with the Chiba University School of Medicine guidelines pertaining to the treatment of experimental animals. The study was approved by the Animal Care and Use Committee of Chiba University Graduate School of Medicine (approval number 2572). We used a total of 46 (22 for locomotor and von Frey assessments, 12 for immunohistochemistry and 12 for western blot analysis) adult male Sprague Dawley rats (10 to 12 weeks old; 200 to 240 g; Japan SLC, Hamamatsu, Japan), which were housed in individual cages and allowed free access to food and water. Rats were anesthetized with 1.5% halothane inhaled with 0.5 L/min oxygen. The T9 spinal cord was exposed by a T8–T9 laminectomy, leaving the dura intact. Contusive SCI was introduced using a New York University Impactor (10 g weight was dropped from 6.25 mm height). Upon awakening, rats were evaluated neurologically and were monitored for food and water intake, and urine output. Manual bladder expression was performed twice a day until the rats regained their bladder reflex (usually one week after SCI). 2.2. Locomotor assessment The recovery of rat hind limb function in either group (n = 4 in sham-operated group, n = 10 in the G-CSF group and n = 8 in the vehicle group) was determined by measuring the hind limb motor function score with the Basso, Beattie and Bresnahan locomotor scale (BBB scale [16]). Rats were allowed to move freely on an open field with a rough surface for 5 min at each time tested. The hind limb movement of rats was videotaped and scored by two independent observers who were unaware of the treatment. If there were differences in the scores between observers, score was determined by discussion between observers. Measurement of motor function was performed weekly for six weeks after surgery.

Tissues from a subset of rats (n = 4/group) were prepared for histological evaluation 3 weeks plus 6 days after surgery (the next day of last G-CSF administration). Animals were euthanized by pentobarbital overdose and perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4). Tissue blocks of the spinal lumbar enlargement were removed, postfixed overnight in 4% paraformaldehyde, stored for two days at 4 °C in 20% sucrose in PBS, and then embedded in Tissue-Tek O.C.T. Compound (optimum cutting temperature formulation; SAKURA Finetechnical Co., Ltd. Tokyo, Japan). The cryoprotected samples were frozen and stored at − 80 °C until use. The samples were cut into serial 20 μm transverse sections. 2.5. Immunohistochemistry For immunofluorescent labeling, sections were permeated with 0.3% Triton X-100 in PBS and treated for 1 h in blocking solution containing 1% bovine serum albumin and Block Ace (Dainippon Pharma, Japan). Sections were then incubated with the following primary antibodies: mouse monoclonal anti-glial fibrillary acidic protein antibody (GFAP, 1:400, Sigma, St Louis, MO) for astrocytes; or anti-CD11b mouse monoclonal antibody (clone OX-42, 1: 400, Serotec AbD, Kidlington, UK) for activated microglia. The sections were incubated with primary antibodies overnight at 4 °C, after which they were washed in PBS and then incubated for 1 h at room temperature with secondary antibodies: Alexa 488-labeled anti-rabbit or anti-mouse IgG (1:800, Invitrogen, Eugene, OR). Finally, the sections were washed twice in PBS and protected with coverslips. Positive labeling was observed using fluorescence microscopy (Eclipse E600; Nikon, Tokyo, Japan). To determine the specificity of staining, procedures were performed on control sections with the omission of primary or secondary antibodies. Positive immunofluorescent signals were counted for every fifth 20-μm transverse section (i.e., at intervals of 100 μm) from the spinal lumbar enlargement using Scion Image computer analysis software (version beta 4.0.3, Scion Corporation, Frederick, MA). At least ten sections from each animal were counted, covering a 1-mm length of spinal cord.

2.3. G-CSF treatment and assessment for mechanical allodynia 2.6. Western blotting Three weeks after SCI, 40% of rats showed mechanical allodynia as revealed by hypersensitivity to von Frey filament stimulation. Rats exhibiting no mechanical allodynia were euthanized and excluded from further experiments. The rats showing mechanical allodynia were randomized to one of two groups. Those in the G-CSF group received intraperitoneal recombinant human G-CSF dissolved in normal saline (15.0 μg/kg; Kyowa Kirin Pharma, Tokyo, Japan) for 5 consecutive days. Rats in the vehicle group received an equivalent volume of normal saline at the same time points. We followed the drug-administration regimen described in our previous report of our rat model of SCI [10]. On the day following the final administration of G-CSF, peripheral blood samples were collected for leukocyte counts. Blood leukocyte counts for rats in the vehicle and G-CSF groups were 3800 ± 500/mm3 and 9700 ± 700/mm3 respectively. Mechanical allodynia in rats from the

Three weeks plus 6 days after SCI (the day following the last administration of G-CSF), 10-mm sections of the spinal lumbar enlargement were removed from rats in both the vehicle and G-CSF-treated groups (n = 4/group). The tissues were homogenized in homogenization buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl and 1% Triton X-100) containing a protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland). The homogenates were centrifuged at 100,000 g for 10 min at 4 °C to remove cellular debris. Protein concentrations of the supernatants were measured using the Bradford method (Bio-Rad Laboratories, Hercules, CA) and were adjusted to 1 mg/mL by dilution with homogenization buffer. Protein samples were mixed with an equal volume of concentrated (2×) sample buffer: 250 mM Tris–HCl, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 0.02% bromophenol

Please cite this article as: K. Kato, et al., Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats, J Neurol Sci (2015), http://dx.doi.org/10.1016/j.jns.2015.05.024

K. Kato et al. / Journal of the Neurological Sciences xxx (2015) xxx–xxx

3

2.7. Statistics Mechanical allodynia data from von Frey filaments testing were analyzed using a repeated measures ANOVA followed by a post hoc Fisher's protected least significant difference (PSLD) test. Results of immunohistochemistry and western blotting were analyzed using Student's t test. Results are presented as mean values ± S.E. Values of p b 0.05 were considered statistically significant. 3. Results 3.1. G-CSF attenuated SCI-induced mechanical allodynia

Fig. 1. Mechanical allodynia data from von Frey hair testing. Three weeks after the surgery, both the G-CSF (triangle) and the vehicle groups (circle, dashed line) showed decreased paw withdrawal threshold indicating mechanical allodynia compared with that in the sham group (square, dotted line). G-CSF-treated rats showed significant attenuation of paw withdrawal threshold compared with that of the vehicle rats. * indicates p b 0.05. Error bar indicates standard error.

blue, and 10% β-mercaptoethanol. After boiling for 5 min, equal volumes of samples were subjected to 10% SDS-polyacrylamide gel electrophoresis under reducing conditions, and the proteins were transferred to a polyvinylidene difluoride membrane (Immobilon-P; Millipore Corporation, Billerica, MA, USA). After blocking the membrane with PBS containing 0.3% skim milk and 0.05% Tween 20, the membrane was reacted with an anti-IL-1β (BD Biosciences, Franklin lakes, NJ), antip-p38 MAPK (Cell Signaling Technology), and an anti-β-actin antibody as a loading control (Santa Cruz Biotechnology, Santa Cruz, CA). For detection, a horse radish peroxidase-conjugated secondary antibody (Cell Signaling Technology) and an ECL chemiluminescence system (GE Healthcare, Piscataway, NJ) were used. Western blot analysis was performed in triplicate for each sample. Protein bands were quantified using image J software.

Results of von Frey filament testing showed that G-CSF attenuates mechanical allodynia induced by SCI in the contusive injury model. Three weeks after the injury, there were no significant differences between the average paw withdrawal threshold for rats in the vehicle and G-CSF groups (vehicle: 1.85 ± 1.45 g; G-CSF: 1.10 ± 0.62 g, Fig. 1). The administration of G-CSF caused a marked attenuation of mechanical allodynia (i.e., increase in paw-withdrawal threshold) relative to that seen in the vehicle group (Fig. 1). Post hoc analysis with Fisher's PSLD revealed a significant increase in the paw withdrawal threshold in the G-CSF group compared with the threshold in the vehicle group 4 weeks after injury (G-CSF 4.68 ± 3.10 g; vehicle 2.52 ± 1.97 g, p b 0.05), 5 weeks after injury (G-CSF 5.00 ± 3.02 g; vehicle 1.85 ± 1.42 g, p b 0.05), 6 weeks after injury (G-CSF 5.00 ± 3.38 g; vehicle 2.38 ± 2.05 g, p b 0.05) and 7 weeks after injury (G-CSF 5.43 ± 3.60 g; vehicle 2.18 ± 1.57 g, p b 0.05). 3.2. G-CSF reduced the number of microglia in spinal cord lumbar enlargement Immunohistochemistry for OX-42 (a marker for activated microglia) in rats from the vehicle group revealed that the number of OX-42positive cells was greater in the dorsal horn from the spinal cord lumbar enlargement compared to that in the naive rat (not shown). In the dorsal horn of rats from the G-CSF group, the number of

Fig. 2. Immunohistochemistry for glial markers in dorsal horn of spinal cord lumbar enlargement. The photomicrographs (A–C) depict immunohistochemistry for glial fibrillary acidic protein (GFAP, a marker for astrocyte) and E–G depict immunohistochemistry for CD11b (clone OX-42, a marker for activated microglia). Graphic representations of the immunocytochemical data are illustrated in panels D and H. There was no significant difference in GFAP-positive cell number in both groups (A–C and D). G-CSF significantly reduced the number of OX-42-positive activated microglia (E–G and H). Bar = 100 μm. * indicates p b 0.05. Error bar indicates standard error.

Please cite this article as: K. Kato, et al., Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats, J Neurol Sci (2015), http://dx.doi.org/10.1016/j.jns.2015.05.024

4

K. Kato et al. / Journal of the Neurological Sciences xxx (2015) xxx–xxx

Fig. 3. Western blot analysis for phosphorylated-p38 mitogen activated protein kinase (p-p38MAPK) and interleukin-1β (IL-1β). Spinal cord injury-induced allodynia promoted upregulation of both p-p38MAPK and IL-1β (A, closed and hatched column in graph B) compared with sham control rats (A, open column in graph B). G-CSF reduced protein expression level of p-p38MAPK and IL-1β compared with that in the vehicle group (A, hatched column in graph B, p b 0.05). β-actin was served as a loading control. * indicates p b 0.05. Error bar indicates standard error.

OX-42-positive cells was significantly smaller than that in the vehicle rats (Fig. 2D,E,F). By contrast, there was no statistical difference in the number of GFAP-positive cells in the dorsal horn of spinal cord lumbar enlargement in either group (Fig. 2A,B,C). 3.3. G-CSF suppressed expression of p38MAPK and IL-1β Western blot analysis indicated that the expression level of p-p38MAPK and IL-1β protein in spinal cord lumbar enlargement was lower in the G-CSF-treated rats compared to that in the vehicle-treated rats (Fig. 3A,B). 4. Discussion The present study revealed that G-CSF can attenuate mechanical allodynia induced by contusive SCI in an adult rat model. When G-CSF is administered 3 weeks after SCI, a time when mechanical allodynia is apparent, the expression of p38MAPK and IL-1β is suppressed in the lumbar enlargement and the number of activated microglia is reduced. Many studies have reported that microglia in the spinal cord can respond to nerve injury and that activation of microglia may be responsible for the initiation of allodynia [18,19]. Thus inhibition of microglial activation is thought to be one of therapeutic strategies for NeP. This suggests that SCI induces microglial activation at a site remote from the injury epicenter. In addition, our results show that midthoracic SCI induces allodynia and causes an increase in the number of microglia in the dorsal horn of the spinal cord lumbar enlargement. p38MAPK is widely known as a key signal mediator related to the inflammatory cytokine network. Activation of p38MAPK leads to the upregulation of several downstream inflammatory cytokines, including IL-1β [20]. Previous reports have indicated that IL-1β can exacerbate neuropathic pain in various animal models [21,22]. Our results show that G-CSF suppresses the phosphorylation of p38MAPK and the upregulation of IL-1β. Thus, the suppression of IL-1β expression by G-CSF is a possible explanation for G-CSF-mediated attenuation of neuropathic pain. We previously reported, G-CSF suppressed phosphorylation of p38MAPK and upregulation of IL-1β, resulting in attenuation of mechanical allodynia in the chronic constriction injury of the sciatic nerve model in adult rats [12]. Despite the difference of experimental models, the present results show a similar mechanism of action of G-CSF for SCI-induced neuropathic pain. The possible limitations of the present study are as follows. Firstly, the difference in pharmacokinetics between intraperitoneal injection, the method used to administer G-CSF in the present study, and intravenous injection, the method used in clinics, remains unclear [23]. A clinical dose–response study is needed to establish the appropriate dose regimen of G-CSF therapy for SCI-induced neuropathic pain. Secondly, the therapeutic time window of G-CSF therapy for SCI-induced

neuropathic pain is unclear. We cannot conclude that G-CSF is effective for chronic stage SCI-induced neuropathic pain because we have only proven the efficacy of G-CSF for SCI-induced neuropathic pain in the subacute phase. This limits the clinical implications of this study because most patients needing treatment in the clinic are at the chronic stage of neuropathic pain. Finally, we assessed mechanical allodynia as an indicator of neuropathic pain. Spontaneous pain known as dysesthesia, a characteristic of neuropathic pain in clinical settings, cannot be evaluated by assessment of allodynia. Therefore we cannot make any conclusions regarding the effects of G-CSF for dysesthesia, limiting the clinical relevance of the present result. In conclusion, the present results demonstrate a therapeutic effect of G-CSF treatment for SCI-induced mechanical allodynia, a marker for neuropathic pain. The results of this study provide evidence that further work is warranted to determine whether G-CSF may be a viable option for managing SCI-induced neuropathic pain. Acknowledgment The present work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (22591626). References [1] M.M. Backonja, Neuropathic pain therapy: from bench to bedside, Semin. Neurol. 32 (2012) 264–268. [2] P.J. Siddall, J.D. Loeser, Pain following spinal cord injury, Spinal Cord 39 (2001) 63–73. [3] P.J. Siddall, J.M. McClelland, S.B. Rutkowski, et al., A longitudinal study of the prevalence and characteristics of pain in the first 5 years following spinal cord injury, Pain 103 (2003) 249–257. [4] V.K. Noonan, J.A. Kopec, H. Zhang, et al., Impact of associated conditions resulting from spinal cord injury on health status and quality of life in people with traumatic central cord syndrome, Arch. Phys. Med. Rehabil. 89 (2008) 1074–1082. [5] N.A. Nicola, D. Metcalf, M. Matsumoto, et al., Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor, J. Biol. Chem. 258 (1983) 9017–9023. [6] A.W. Roberts, G-CSF: a key regulator of neutrophil production, but that's not all! Growth Factors 23 (2005) 33–41. [7] W.R. Shäbitz, R. Kollmar, M. Schwaninger, et al., Neuroprotective effect of granulocyte colony-stimulating factor after focal cerebral ischemia, Stroke 4 (2003) 745–751. [8] A. Schneider, C. Krüger, T. Steigleder, et al., The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis, J. Clin. Invest. 115 (2005) 2083–2098. [9] Y. Nishio, M. Koda, T. Kamada, et al., Granulocyte colony-stimulating factor (G-CSF) attenuates neuronal death and promotes functional recovery after spinal cord injury in mice, J. Neuropathol. Exp. Neurol. 66 (2007) 724–731. [10] R. Kadota, M. Koda, J. Kawabe, et al., Granulocyte colony-stimulating factor (G-CSF) protects oligodendrocyte and promotes hindlimb functional recovery after spinal cord injury in rats, PLoS One 7 (2012) e50391, http://dx.doi.org/10.1371/journal. pone.0050391. [11] P.K. Chao, K.T. Lu, Y.L. Lee, et al., Early systemic granulocyte-colony stimulating factor treatment attenuates neuropathic pain after peripheral nerve injury, PLoS One 7 (2012) e43680, http://dx.doi.org/10.1371/journal.pone.

Please cite this article as: K. Kato, et al., Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats, J Neurol Sci (2015), http://dx.doi.org/10.1016/j.jns.2015.05.024

K. Kato et al. / Journal of the Neurological Sciences xxx (2015) xxx–xxx [12] M. Koda, T. Furuya, K. Kato, et al., Delayed granulocyte colony-stimulating factor treatment in rats attenuates mechanical allodynia induced by chronic constriction injury of the sciatic nerve, Spine 39 (2014) 192–197. [13] H. Takahashi, M. Yamazaki, A. Okawa, et al., Neuroprotective therapy using granulocyte colony-stimulating factor for acute spinal cord injury: a phase I/IIa clinical trial, Eur. Spine J. 21 (2012) 2580–2587. [14] T. Sakuma, M. Yamazaki, A. Okawa, et al., Neuroprotective therapy using granulocyte colony-stimulating factor for patients with worsening symptoms of thoracic myelopathy: a multicenter prospective controlled trial, Spine 37 (2012) 1475–1478. [15] K. Kato, M. Yamazaki, A. Okawa, et al., Intravenous administration of granulocyte colony-stimulating factor for treating neuropathic pain associated with compression myelopathy: a phase I and IIa clinical trial, Eur. Spine J. 22 (2013) 197–204. [16] D.M. Basso, M.S. Beattie, J.C. Bresnahan, A sensitive and reliable locomotor rating scale for open field testing in rats, J. Neurotrauma 12 (1995) 1–21. [17] Y.Q. Zhang, N. Guo, G. Peng, et al., Role of SIP30 in the development and maintenance of peripheral nerve injury-induced neuropathic pain, Pain 146 (2009) 130–140.

5

[18] H. Cao, Y.Q. Zhang, Spinal glial activation contributes to pathological pain states, Neurosci. Biobehav. Rev. 32 (2008) 972–983. [19] Y.S. Gwak, J. Kang, G.C. Unabia, et al., Spatial and temporal activation of spinal glial cells: role of gliopathy in central neuropathic pain following spinal cord injury in rats, Exp. Neurol. 234 (2012) 362–372. [20] R.R. Ji, M.R. Suter, p38 MAPK, microglial signaling, and neuropathic pain, Mol. Pain 3 (2007) 33, http://dx.doi.org/10.1186/1744-8069-3-33. [21] M. Zelenka, M. Schafers, C. Sommer, Intraneural injection of interleukin-1beta and tumor necrosis factor-alpha into rat sciatic nerve at physiological doses induces signs of neuropathic pain, Pain 116 (2005) 257–263. [22] K. Ren, R. Torres, Role of interleukin-1β during pain and inflammation, Brain Res. Rev. 60 (2009) 57.64, http://dx.doi.org/10.1016/S0929-6646(11)60074-0. [23] H. Tanaka, T. Kaneko, Pharmacokinetics of recombinant human granulocyte colony-stimulating factor in the rat. Single and multiple dosing studies, Drug Metab. Dispos. 19 (1991) 200–204.

Please cite this article as: K. Kato, et al., Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats, J Neurol Sci (2015), http://dx.doi.org/10.1016/j.jns.2015.05.024

Granulocyte colony-stimulating factor attenuates spinal cord injury-induced mechanical allodynia in adult rats.

Spinal cord injury (SCI) can cause neuropathic pain (NeP), often reducing a patient's quality of life. We recently reported that granulocyte colony-st...
648KB Sizes 0 Downloads 12 Views