J Mol Neurosci (2015) 55:533–540 DOI 10.1007/s12031-014-0379-3
Intrathecal siRNA Against GPNMB Attenuates Nociception in a Rat Model of Neuropathic Pain Lili Hou & Yanfeng Zhang & Yong Yang & Kai Xiang & Qindong Tan & Qulian Guo
Received: 1 June 2014 / Accepted: 7 July 2014 / Published online: 17 July 2014 # Springer Science+Business Media New York 2014
Abstract Neuropathic pain is characterized by hyperalgesia, allodynia, and spontaneous pain. Recent studies have shown that glycoprotein nonmetastatic melanoma B (GPNMB) plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain remains unknown. The aim of the present study was to assess the role of GPNMB in neuropathic pain. In cultured spinal cord neurons, we used two small interfering RNAs (siRNAs) targeting the complementary DNA (cDNA) sequence of rat GPNMB that had potent inhibitory effects on GPNMB, and siRNA1GPNMB was selected for further in vivo study as it had the higher inhibitory effect. After sciatic nerve injury in rats, the endogenous level of GPNMB was increased in a timedependent manner in the spinal cord. Furthermore, the intrathecal injection of siRNA1-GPNMB inhibited the expression of GPNMB and pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia in the chronic constriction injury (CCI) model of rats. Taken together, our findings suggest that siRNA against GPNMB can alleviate the chronic neuropathic pain caused by CCI, and this effect may be mediated by attenuated expression of TNF-α, IL-1β, and IL-6 in the spinal cord of CCI rats. Therefore, inhibition of GPNMB may provide a novel strategy for the treatment of neuropathic pain. Keywords GPNMB . RNA interference . Neuropathic pain L. Hou : Y. Zhang : Y. Yang : K. Xiang : Q. Guo (*) Department of Anesthesiology, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha 410008, Hunan, People’s Republic of China e-mail: [email protected] Q. Tan Department of Anesthesiology, The Third Affiliated Hospital of Nanfang Medicine University, Guangzhou 510063, Guangdong, People’s Republic of China
Introduction Neuropathic pain is a severe health problem, which affects millions of people worldwide. Many disorders including diabetic polyneuropathy, radicular back pain, stroke, and spinal cord injury cause neuropathic pain by producing lesions in somatosensory pathways in the peripheral or central nervous system (Baron et al. 2010). Characteristic features of neuropathic pain are hyperalgesia, allodynia, and spontaneous pain. Despite the ever-increasing understanding of the neurobiology and molecular biology of neuropathic pain, it remains a prevalent and persistent clinical challenge. One reason is the absence of effective treatments: the drugs used to treat neuropathic pain tend to have only a modest effect and abundant side effects. The main reason is the inability to target underlying mechanisms of neuropathic pain (O’Connor and Dworkin 2009). Therefore, there is an urgent need to explore novel molecular mechanisms involved in neuropathic pain. Accumulating evidence demonstrates that injury to the peripheral or central nervous system results in maladaptive changes in neurons along the nociceptive pathway that can cause neuropathic pain. Neuropathic pain reflects complex remodeling of synaptic transmission and activation of neuroglial cells, astrocytes, and microglia. After nerve injury, neuroglial cells undergo structural and functional transformation; astrocytes, a type of neuroglial cell, release a lot of pronociceptive factors; and microglia in the resting state in the spinal dorsal horn are converted to an activated state with a series of morphological and molecular changes. As activation progresses, microglia stimulate the complement component of the immune system and release cytokines and chemokines, which contribute to maintenance of neuropathic pain (Zhuang et al. 2005). Glycoprotein nonmetastatic melanoma B (GPNMB) is a type I transmembrane protein and was initially cloned from
534
low-metastatic melanoma cells. It is involved in various biological processes, such as cell differentiation, tissue regeneration, inflammation, and invasion and metastasis of malignant tumors. A large body of evidence suggests that GPNMB expression is upregulated in various tumor cells, including gliomas (Loging et al. 2000), hepatomas (Onaga et al. 2003), and breast cancer (Rose and Siegel 2010), and in nontumor tissues, such as damaged skeletal muscles (Rose and Siegel 2010), injured liver (Haralanova-Ilieva et al. 2005), and fractured bones (Abdelmagid et al. 2010). However, little is known about its expression and roles in nontumorous neural tissues. Recently, it has been reported that the extracellular domain of GPNMB had protective effects against mutant superoxide dismutase 1 (SOD1)-induced neurotoxicity via extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) pathways (Tanaka et al. 2012). In addition, one report showed that GPNMB is upregulated in microglia after lipopolysaccharide treatment, and small interfering RNA (siRNA)-GPNMB inhibits the production of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines from activated microglia (Shi et al. 2014). These results suggested that GPNMB plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain is still unclear. Therefore, in this study, we investigated the role of GPNMB in neuropathic pain.
Materials and Methods Spinal Cord Neuron Culture Primary cultures of embryonic spinal cord neurons were prepared as previously described (Ahlemeyer and Baumgart-Vogt 2005). Briefly, the spinal cords were dissected from embryonic day 16 rats. The spinal cord cells were dissociated with gentle trituration, grown in modified Eagle’s medium containing 10 % fetal bovine serum (Sigma, St. Louis, MO, USA), 100 U/ml penicillin (Sigma, St. Louis, MO, USA), and 100 μg/ml streptomycin (Gibco, Rockville, MD, USA). Animals Male Sprague-Dawley rats weighing 220–250 g were provided by the Center of Laboratory Animal Science of Xiangya Hospital of Central South University. The rats were housed under standardized conditions in a room on a 12-h light/dark cycle with food and water available ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Xiangya Hospital of Central South University. All efforts were undertaken to minimize the number of animals used and their discomfort.
J Mol Neurosci (2015) 55:533–540
Lumbar Subarachnoid Catheterization Lumbar subarachnoid catheters were implanted as previously described (Milligan et al. 1999). One week prior to chronic constriction injury (CCI), the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). A polyethylene-10 catheter was inserted into the lumbar subarachnoid space between the fifth and sixth lumbar vertebrae. Then, it was chronically implanted and the external portion of the catheter was protected. The Chronic Constriction Injury (CCI) Model The CCI model was established as previously described (Bennett and Xie 1988). In brief, the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The sciatic nerve was exposed and loosely ligated with 4-0 chromic gut thread at four sites with an interval of 1 mm. Meanwhile, a sham surgery was performed with the sciatic nerve exposed but not ligated. The animals were kept warm and allowed to recover from anesthesia. To determine GPNMB expression in the CCI model, injured and corresponding sham animals were sacrificed in a time course experiment at days 1, 3, 7, and 14 after CCI. Lentivirus Construction and siRNA Transfections For targeted knockdown of GPNMB, two siRNAs targeting the complementary DNA (cDNA) sequence of rat GPNMB were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Their nucleotide sequences were siRNA1: 5′-CCAUCUUGCUGUACAAAAAdTdT-3′ (sense) and 5′UUUUUGUACAGCAAGAUGGdTdT-3′ (antisense); siRNA2: 5′-GCACGGGUUUCUAUAAACAdTdT-3′ (sense) and 5′-UGUUUAUAGAAACCCGUGCdTdT-3′ (antisense). An additional scramble sequence was also designed as a negative control. The cDNAs corresponding to the two siRNAs and scramble were subcloned into a lentivirus vector, pLenti6/V5-D-TOPT (Invitrogen, Carlsbad, CA, USA), and the recombinant lentiviruses were propagated in 293T cells. For in vitro transfection, 5×104 spinal cord neurons were seeded in each cell of 24-well microplates, grown for 24 h to reach 30–50 % confluence, and then incubated with a mixture of siRNA and Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in 100 μl of serum-free medium (Abcam, Cambridge, UK) at 37 °C with 5 % CO2. After 24 h, the transfection efficiency was examined by realtime PCR and Western blotting. Quantitative Real-Time PCR (QRT-PCR) Total RNA was isolated from lumbar spinal cord (L4–L5) tissues and neurons using TRIzol (Takara, Dalian, China) according to the manufacturer’s protocol. Two micrograms
J Mol Neurosci (2015) 55:533–540
of total RNA per sample was reverse transcribed using a HighCapacity RNA-to-cDNA Kit (Invitrogen, Carlsbad, CA, USA). The levels of gene mRNA transcripts were analyzed by using the specific primers and SYBR Green I reagent and the QRT-PCR kit, according to the manufacturer’s instructions, on Bio-Rad iQ5 Quantitative PCR System (Takara, Dalian, China). The specific primers for rat GPNMB were 5′-TCTGAACCGAGCCCTGACATC-3′ (sense), 5′-AGCA GTAGCGGCCATGTGAAG-3′ (antisense), and 5′-GGAG AAGCCAAGCCCATCA-3′ (antisense), and those for βactin were 5′-GATCATTGCTCCTCCTGAGC-3′ (sense) and 5′-ACTCCTGCTTGCTGATCCAC-3′ (antisense). These primers were all synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The PCR procedure was as follows: 95 °C for 3 min, followed by 40 cycles of 94 °C for 30 s, 59 °C for 20 s, and 72 °C for 40 s, and finally a single cycle at 72 °C for 5 min. For relative quantification, the levels of individual gene mRNA transcripts were firstly normalized to the control β-actin. Subsequently, the differential expression of these genes was analyzed according to the 2−ΔΔCt method.
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Enzyme-Linked Immunosorbent Assay (ELISA) The spinal cords were harvested 24 h after the last injection for pro-inflammatory cytokines analysis. Supernatants of spinal cord segments were assayed by the Rat TNF-α ELISA kit, IL1β, and IL-6 (Jingmei Biotech Co., Ltd., Shenzhen, China) according to the manufacturer’s instructions. Evaluation of the Pain Behavior The testing procedure was performed according to previously published protocols. An electronic automatic detector of sensor of touch was used to detect the paw withdrawal mechanical threshold (PWMT) (Chaplan et al. 1994), and the paw withdrawal thermal latency (PWTL) was determined using a pain detector (Hargreaves et al. 1988). Each rat was measured three times and the mean value was taken as the threshold value. Tests were performed 1 day before CCI surgery and 1, 3, 7, and 14 days after CCI surgery. Statistical Analysis
Western Blotting The proteins were extracted from lumbar spinal cord (L4–L5) tissues and cells using RIPA lysis buffer (Beyotime, Nantong, China). The protein concentration in the lysates was determined using a BCA protein assay kit (Beyotime, Nantong, China). Thirty micrograms of samples of total protein was separated by SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 10 % defatted milk in PBS at 4 °C overnight and incubated with the primary antibody against GPNMB (Invitrogen, Carlsbad, CA, USA) for 60 min. After washing with TBST buffer, the membranes were incubated with HRPconjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Immunodetection was by using the ECL reagents (Beyotime, Nantong, China). All experiments were performed in triplicate, and results were normalized according to β-actin. Intrathecal Delivery of siRNA1-GPNMB The rats were randomly divided into four groups with ten rats in each group: sham group, CCI group, CCI + siRNAscramble group, and CCI + siRNA1-GPNMB group. Prior to injection, the animals were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Ten micrograms of siRNA1GPNMB dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery. Seven days after intrathecal administration, the expression level of GPNMB in the spinal cord was measured by RTPCR and Western blot.
All results are reported as mean±S.E.M. Comparisons of means between two groups were carried out using t test and those between multiple groups with one-way analysis of variance (ANOVA). A value of P
Intrathecal siRNA Against GPNMB Attenuates Nociception in a Rat Model of Neuropathic Pain Lili Hou & Yanfeng Zhang & Yong Yang & Kai Xiang & Qindong Tan & Qulian Guo
Received: 1 June 2014 / Accepted: 7 July 2014 / Published online: 17 July 2014 # Springer Science+Business Media New York 2014
Abstract Neuropathic pain is characterized by hyperalgesia, allodynia, and spontaneous pain. Recent studies have shown that glycoprotein nonmetastatic melanoma B (GPNMB) plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain remains unknown. The aim of the present study was to assess the role of GPNMB in neuropathic pain. In cultured spinal cord neurons, we used two small interfering RNAs (siRNAs) targeting the complementary DNA (cDNA) sequence of rat GPNMB that had potent inhibitory effects on GPNMB, and siRNA1GPNMB was selected for further in vivo study as it had the higher inhibitory effect. After sciatic nerve injury in rats, the endogenous level of GPNMB was increased in a timedependent manner in the spinal cord. Furthermore, the intrathecal injection of siRNA1-GPNMB inhibited the expression of GPNMB and pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia in the chronic constriction injury (CCI) model of rats. Taken together, our findings suggest that siRNA against GPNMB can alleviate the chronic neuropathic pain caused by CCI, and this effect may be mediated by attenuated expression of TNF-α, IL-1β, and IL-6 in the spinal cord of CCI rats. Therefore, inhibition of GPNMB may provide a novel strategy for the treatment of neuropathic pain. Keywords GPNMB . RNA interference . Neuropathic pain L. Hou : Y. Zhang : Y. Yang : K. Xiang : Q. Guo (*) Department of Anesthesiology, Xiangya Hospital of Central South University, 87 Xiangya Road, Changsha 410008, Hunan, People’s Republic of China e-mail: [email protected] Q. Tan Department of Anesthesiology, The Third Affiliated Hospital of Nanfang Medicine University, Guangzhou 510063, Guangdong, People’s Republic of China
Introduction Neuropathic pain is a severe health problem, which affects millions of people worldwide. Many disorders including diabetic polyneuropathy, radicular back pain, stroke, and spinal cord injury cause neuropathic pain by producing lesions in somatosensory pathways in the peripheral or central nervous system (Baron et al. 2010). Characteristic features of neuropathic pain are hyperalgesia, allodynia, and spontaneous pain. Despite the ever-increasing understanding of the neurobiology and molecular biology of neuropathic pain, it remains a prevalent and persistent clinical challenge. One reason is the absence of effective treatments: the drugs used to treat neuropathic pain tend to have only a modest effect and abundant side effects. The main reason is the inability to target underlying mechanisms of neuropathic pain (O’Connor and Dworkin 2009). Therefore, there is an urgent need to explore novel molecular mechanisms involved in neuropathic pain. Accumulating evidence demonstrates that injury to the peripheral or central nervous system results in maladaptive changes in neurons along the nociceptive pathway that can cause neuropathic pain. Neuropathic pain reflects complex remodeling of synaptic transmission and activation of neuroglial cells, astrocytes, and microglia. After nerve injury, neuroglial cells undergo structural and functional transformation; astrocytes, a type of neuroglial cell, release a lot of pronociceptive factors; and microglia in the resting state in the spinal dorsal horn are converted to an activated state with a series of morphological and molecular changes. As activation progresses, microglia stimulate the complement component of the immune system and release cytokines and chemokines, which contribute to maintenance of neuropathic pain (Zhuang et al. 2005). Glycoprotein nonmetastatic melanoma B (GPNMB) is a type I transmembrane protein and was initially cloned from
534
low-metastatic melanoma cells. It is involved in various biological processes, such as cell differentiation, tissue regeneration, inflammation, and invasion and metastasis of malignant tumors. A large body of evidence suggests that GPNMB expression is upregulated in various tumor cells, including gliomas (Loging et al. 2000), hepatomas (Onaga et al. 2003), and breast cancer (Rose and Siegel 2010), and in nontumor tissues, such as damaged skeletal muscles (Rose and Siegel 2010), injured liver (Haralanova-Ilieva et al. 2005), and fractured bones (Abdelmagid et al. 2010). However, little is known about its expression and roles in nontumorous neural tissues. Recently, it has been reported that the extracellular domain of GPNMB had protective effects against mutant superoxide dismutase 1 (SOD1)-induced neurotoxicity via extracellular signal-regulated kinase 1/2 (ERK1/2) and protein kinase B (AKT) pathways (Tanaka et al. 2012). In addition, one report showed that GPNMB is upregulated in microglia after lipopolysaccharide treatment, and small interfering RNA (siRNA)-GPNMB inhibits the production of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines from activated microglia (Shi et al. 2014). These results suggested that GPNMB plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain is still unclear. Therefore, in this study, we investigated the role of GPNMB in neuropathic pain.
Materials and Methods Spinal Cord Neuron Culture Primary cultures of embryonic spinal cord neurons were prepared as previously described (Ahlemeyer and Baumgart-Vogt 2005). Briefly, the spinal cords were dissected from embryonic day 16 rats. The spinal cord cells were dissociated with gentle trituration, grown in modified Eagle’s medium containing 10 % fetal bovine serum (Sigma, St. Louis, MO, USA), 100 U/ml penicillin (Sigma, St. Louis, MO, USA), and 100 μg/ml streptomycin (Gibco, Rockville, MD, USA). Animals Male Sprague-Dawley rats weighing 220–250 g were provided by the Center of Laboratory Animal Science of Xiangya Hospital of Central South University. The rats were housed under standardized conditions in a room on a 12-h light/dark cycle with food and water available ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Xiangya Hospital of Central South University. All efforts were undertaken to minimize the number of animals used and their discomfort.
J Mol Neurosci (2015) 55:533–540
Lumbar Subarachnoid Catheterization Lumbar subarachnoid catheters were implanted as previously described (Milligan et al. 1999). One week prior to chronic constriction injury (CCI), the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). A polyethylene-10 catheter was inserted into the lumbar subarachnoid space between the fifth and sixth lumbar vertebrae. Then, it was chronically implanted and the external portion of the catheter was protected. The Chronic Constriction Injury (CCI) Model The CCI model was established as previously described (Bennett and Xie 1988). In brief, the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The sciatic nerve was exposed and loosely ligated with 4-0 chromic gut thread at four sites with an interval of 1 mm. Meanwhile, a sham surgery was performed with the sciatic nerve exposed but not ligated. The animals were kept warm and allowed to recover from anesthesia. To determine GPNMB expression in the CCI model, injured and corresponding sham animals were sacrificed in a time course experiment at days 1, 3, 7, and 14 after CCI. Lentivirus Construction and siRNA Transfections For targeted knockdown of GPNMB, two siRNAs targeting the complementary DNA (cDNA) sequence of rat GPNMB were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). Their nucleotide sequences were siRNA1: 5′-CCAUCUUGCUGUACAAAAAdTdT-3′ (sense) and 5′UUUUUGUACAGCAAGAUGGdTdT-3′ (antisense); siRNA2: 5′-GCACGGGUUUCUAUAAACAdTdT-3′ (sense) and 5′-UGUUUAUAGAAACCCGUGCdTdT-3′ (antisense). An additional scramble sequence was also designed as a negative control. The cDNAs corresponding to the two siRNAs and scramble were subcloned into a lentivirus vector, pLenti6/V5-D-TOPT (Invitrogen, Carlsbad, CA, USA), and the recombinant lentiviruses were propagated in 293T cells. For in vitro transfection, 5×104 spinal cord neurons were seeded in each cell of 24-well microplates, grown for 24 h to reach 30–50 % confluence, and then incubated with a mixture of siRNA and Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) in 100 μl of serum-free medium (Abcam, Cambridge, UK) at 37 °C with 5 % CO2. After 24 h, the transfection efficiency was examined by realtime PCR and Western blotting. Quantitative Real-Time PCR (QRT-PCR) Total RNA was isolated from lumbar spinal cord (L4–L5) tissues and neurons using TRIzol (Takara, Dalian, China) according to the manufacturer’s protocol. Two micrograms
J Mol Neurosci (2015) 55:533–540
of total RNA per sample was reverse transcribed using a HighCapacity RNA-to-cDNA Kit (Invitrogen, Carlsbad, CA, USA). The levels of gene mRNA transcripts were analyzed by using the specific primers and SYBR Green I reagent and the QRT-PCR kit, according to the manufacturer’s instructions, on Bio-Rad iQ5 Quantitative PCR System (Takara, Dalian, China). The specific primers for rat GPNMB were 5′-TCTGAACCGAGCCCTGACATC-3′ (sense), 5′-AGCA GTAGCGGCCATGTGAAG-3′ (antisense), and 5′-GGAG AAGCCAAGCCCATCA-3′ (antisense), and those for βactin were 5′-GATCATTGCTCCTCCTGAGC-3′ (sense) and 5′-ACTCCTGCTTGCTGATCCAC-3′ (antisense). These primers were all synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The PCR procedure was as follows: 95 °C for 3 min, followed by 40 cycles of 94 °C for 30 s, 59 °C for 20 s, and 72 °C for 40 s, and finally a single cycle at 72 °C for 5 min. For relative quantification, the levels of individual gene mRNA transcripts were firstly normalized to the control β-actin. Subsequently, the differential expression of these genes was analyzed according to the 2−ΔΔCt method.
535
Enzyme-Linked Immunosorbent Assay (ELISA) The spinal cords were harvested 24 h after the last injection for pro-inflammatory cytokines analysis. Supernatants of spinal cord segments were assayed by the Rat TNF-α ELISA kit, IL1β, and IL-6 (Jingmei Biotech Co., Ltd., Shenzhen, China) according to the manufacturer’s instructions. Evaluation of the Pain Behavior The testing procedure was performed according to previously published protocols. An electronic automatic detector of sensor of touch was used to detect the paw withdrawal mechanical threshold (PWMT) (Chaplan et al. 1994), and the paw withdrawal thermal latency (PWTL) was determined using a pain detector (Hargreaves et al. 1988). Each rat was measured three times and the mean value was taken as the threshold value. Tests were performed 1 day before CCI surgery and 1, 3, 7, and 14 days after CCI surgery. Statistical Analysis
Western Blotting The proteins were extracted from lumbar spinal cord (L4–L5) tissues and cells using RIPA lysis buffer (Beyotime, Nantong, China). The protein concentration in the lysates was determined using a BCA protein assay kit (Beyotime, Nantong, China). Thirty micrograms of samples of total protein was separated by SDS-PAGE and then transferred onto a polyvinylidene difluoride membrane. Membranes were blocked with 10 % defatted milk in PBS at 4 °C overnight and incubated with the primary antibody against GPNMB (Invitrogen, Carlsbad, CA, USA) for 60 min. After washing with TBST buffer, the membranes were incubated with HRPconjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Immunodetection was by using the ECL reagents (Beyotime, Nantong, China). All experiments were performed in triplicate, and results were normalized according to β-actin. Intrathecal Delivery of siRNA1-GPNMB The rats were randomly divided into four groups with ten rats in each group: sham group, CCI group, CCI + siRNAscramble group, and CCI + siRNA1-GPNMB group. Prior to injection, the animals were anesthetized with pentobarbital sodium (40 mg/kg, i.p.). Ten micrograms of siRNA1GPNMB dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery. Seven days after intrathecal administration, the expression level of GPNMB in the spinal cord was measured by RTPCR and Western blot.
All results are reported as mean±S.E.M. Comparisons of means between two groups were carried out using t test and those between multiple groups with one-way analysis of variance (ANOVA). A value of P
