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. Author manuscript; available in PMC: 2014 Jun 8.
Published in final edited form as: J Neurochem. 2010 Jan 12;113(1):131–142. doi: 10.1111/j.1471-4159.2010.06579.x

Involvement of ERK2 in traumatic spinal cord injury

Chen-Guang Yu 1,*, Robert P Yezierski 2, Aashish Joshi 1, Kashif Raza 1, Yanzhang Li 1, James W Geddes 1
PMCID: PMC4048743  NIHMSID: NIHMS477095  PMID: 20067580

Abstract

Activation of extracellular signal-regulated protein kinase ½ (ERK1/2) are implicated in the pathophysiology of spinal cord injury (SCI). However, the specific functions of individual ERK isoforms in neurodegeneration are largely unknown. We investigated the hypothesis that ERK2 activation may contribute to pathological and functional deficits following SCI and that ERK2 knockdown using RNAi may provide a novel therapeutic strategy for SCI. Lentiviral ERK2 shRNA and siRNA were utilized to knockdown ERK2 expression in the spinal cord following SCI. Preinjury intrathecal administration of ERK2 siRNA significantly reduced excitotoxic injury-induced activation of ERK2 (p<0.001) and caspase 3 (p<0.01) in spinal cord. Intraspinal administration of lentiviral ERK2 shRNA significantly reduced ERK2 expression in the spinal cord (p<0.05), but did not alter ERK1 expression. Administration of the lentiviral ERK2 shRNA vector one week prior to severe spinal cord contusion injury resulted in a significant improvement in locomotor function (p<0.05), total tissue sparing (p<0.05), white matter sparing (p<0.05), and gray matter sparing (p<0.05) 6 weeks following severe contusive SCI. Our results suggest that ERK2 signaling is a novel target associated with the deleterious consequences of spinal injury.

Keywords: ERK2, tissue damage, spinal cord injury, locomotor function, RNAi

Introduction

Traumatic spinal cord injury (SCI) is a leading cause of permanent locomotor disability among young adults in the United States, with 11,000 new injures each year. At present, effective treatments for locomotor disability associated with SCI are inadequate (French et al., 2007). Understanding the complex biochemical mechanisms contributing to locomotor deficits after SCI and developing novel therapeutic strategies is of great clinical importance.

Extracellular signal-regulated kinase 1 and 2 (ERK1, a 44 kDa protein and ERK2, a 42 kDa protein) are mitogen-activated protein kinase (MAPK) family members (Chen et al., 2001). They are encoded by distinct genes, but share 83% homology at the amino acid level and are expressed in all tissues. In resting cells, the proteins are largely cytosolic and associated with the cytoskeleton. Upon activation, ERK1/2 can translocate to the nucleus or to membrane specializations. Activation of ERK1/2 by phosphorylation of threonine and tyrosine residues was originally identified as an effect of the growth factor signaling cascades that involve activation of Ras, Raf, MEK and ERK1/2 and are associated with neuronal survival and neuroprotection (Hetman and Gozdz, 2004).

However, recent reports have challenged this view and suggest a neurodegenerative role of ERK1/2 (Zhuang and Schnellmann, 2006; Yu and Yezierski, 2005; Genovese et al., 2008). For instance, blockade of ERK1/2 by MEK inhibitors (U0126 or PD98059) protects against neurodegeneration induced by excitotoxic SCI and spinal cord compression injury (Yu and Yezierski, 2005; Genovese et al., 2008); ERK2 knockdown with RNAi inhibits NK-1R or PKCα-mediated cell death (Matsumoto et al., 2005) and complete Freund's adjuvant-induced inflammatory pain (Xu, et al., 2008). However, the specific functions of the individual ERK isoforms in neurodegeneration after SCI are unknown. Isoform specific inhibitors of ERK 1 or ERK2 are not available. As a potent and specific inhibitor of gene expression, RNA interference (RNAi) has emerged as a promising tool to analyze individual gene function, using local delivery of sequence-specific small interfering RNA (siRNA) (Breckpot et al., 2007; Lu and Woodle, 2008). To test the hypothesis that ERK2 activation is involved in the injury-induced neurodegeneration of spinal tissue and locomotor dysfunction, we utilized microinjection of lentiviral-ERK2 shRNA or intrathecal administration of ERK2 siRNA to knockdown spinal ERK2 prior to contusive or excitotoxic SCI in rats.

Materials and Methods

Animals

For this study, thirty-eight female and twelve male Long-Evans rats (Charles River, Indianapolis, IN), weighing 200-250 g, were used. Animals were kept under standard housing conditions for at least 1 week following arrival. All experimental procedures were approved and carried out in accordance with the Guidelines of the US National Institutes of Health and Institutional Animal Care and Use Committee (IACUC) of the University of Kentucky and University of Florida.

ERK2 siRNA sequence

ERK2 siRNA sequences (Genebank accession number: M64300) were selected and chemically modified based on accepted criteria for the rational design of siRNA with off-target control using two web-based programs (Dharmacon and Invitrogen) (Reynolds et al., 2004). To minimize the potential off-target effects of siRNA, the ERK2 siRNA sequences (Sense, start 522, 5′pGCACCUCAGCAAUGAUCAUdTdT3′ and antisense 5′pAUGAUCAUUGCUGAGGUGCdTdT3′) were subjected to BLAST analysis and synthesized in the “ready-to-use in vivo” and modification option with off-target control by Thermo Scientific-Dharmacon Products (Lafayette, CO). ON-TARGET plus Non-Targeting siRNA was used as the negative control siRNA (Dharmacon).

Cell culture and transfection

Transfection was performed as described in the Invitrogen siRNA Transfection Manual using Lipofectamine 2000. To determine the efficacy of ERK2 siRNA sequences, PC-12 cells (3 × 105/well in 6-well plates) were transfected with 100 nM ERK2 siRNAs complexed with 6μl Lipofectamine 2000, based on the manufacturer's instructions (Invitrogen, Carlsbad, CA). ERK2 mRNA and protein levels in the cells were examined 72 h following transfection. Real-time PCR was employed to determine ERK2 mRNA levels. Western blot analysis was used to estimate protein levels. A 70% reduction in both mRNA and protein levels was considered an effective gene silencing effect. PC-12 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM supplemented with 5% fetal bovine serum, 10% heat-inactivated donor horse serum, 50μg/ml streptomycin, and 50 IU/ml penicillin in a humidified atmosphere at 37°C and 5% CO2 as described in the protocol provided by ATCC. Cells were maintained at a medium density (3 × 105/cm2).

Construction and production of lentiviral vectors

ERK2 shRNA was designed and synthesized based on the active ERK2 siRNA sequence using Invitrogen online software and cloned into BLOCK-iT U6 RNAi Entry Vector using U6 Entry Vector Kit (Invitrogen). The U6-ERK2 shRNA expression cassette was inserted between BamHI and XbaI sites in a lentiviral-CMV-RNAi vector (third generation lentiviral vector was kindly provided by Dr. George Smith, University of Kentucky). Expression of the shRNA is under control of a U6 promoter, while the eGFP reporter is linked to a CMV promoter. The lentiviral vector containing eGFP and ERK2 shRNA or the empty vector expressing only eGFP (LV-control) was cotransfected with packaging plasmid PAX2 and envelope plasmid pMD2G (obtained from Dr. Didier Trono, University of Geneva, Switzerland) into 293FT cells (Invitrogen) using calcium-phosphate precipitation. Viral supernatants were harvested and concentrated after 72 h. The titer of the lentiviral stocks was assessed using 10-fold serial dilutions to HT 1080 human fibrosarcoma cells (ATCC) using the eGFP reporter of the lenti-CMV-eGFP-RNAi vector to identify infected cells. The viral titer was 4 × 108 TU/ml for LV-ERK2 shRNA and 5 × 107 TU/ml for LV-eGFP control.

Intrathecal siRNA

The siRNA-transfectamine 2000 complexes were prepared by diluting ERK2 or control siRNA (2.5 μg) in opti-MEM (7.5 μl) and diluting lipofectamine 2000 (5 μg) in opti-MEM (7.5 μl) using lipofectamine 2000 kit (Invitrogen). The two mixtures were incubated for 5 min at room temperature, combined, and incubated for an additional 20 min. Complexes of ERK2 siRNA-lipofectamine 2000 or control siRNA-lipofectamine 2000 (one dose) were intrathecally delivered onto the surface of dorsal horn (QUIS injection site or L1 level) for a period of 72 hours prior to QUIS injections. The dose of ERK2 siRNA was chosen based on previous studies (Cai, et al., 2009).

Uptake of CY-3-labeled ERK2 siRNA in spinal cord

CY3-ERK2 siRNA (2.5μg) complexed with lipofectamine 2000 (5μg) were intrathecally delivered onto the surface of dorsal horn in uninjured rats. 24h after delivery, the rat was perfused and spinal cords dissected and processed for epifluorescent microscopy. The uptake of ERK2 siRNA was verified in spinal cord of rats by direct CY3 visualization with fluorescent microscopy.

Intraspinal microinjection

Lentiviral vectors encoding ERK2 shRNA or lentiviral control vectors were bilaterally injected into the gray and white matter of the spinal cord at T10 (four injections) in rats. Injections were made into the lateral funiculus 1.1 mm lateral to the midline at a depth of 1 mm. For gray matter, bilateral injections were made at 0.5 mm lateral to the midline at a depth of 1.3mm to target motor neurons in the ventral horn. Injections were made using a beveled glass micropipette (60μm tip diameter) and nano-injector (Stoelting Co., Wood Dale, IL). For each injection site, a volume of up to 1.0μl was slowly infused over 4 min (100nl/min). The injection protocol is similar to that described in detail by Ruitenbeng and colleagues for delivery of adeno-associated viral vectors to the spinal cord (Ruitenberg et al., 2002). Intraspinal injection of LV-ERK2 shRNA or LV-control was verified using Western blot, immunofluorescence staining, and fluorescent microscopy.

Excitotoxic Spinal Cord Injury

Excitotoxic injury was initiated by intraspinal injection of the glutamatergic AMPA/metabotropic receptor agonist quisqualic acid (QUIS) as previously described (Yu and Yezierski, 2005). The intraspinal QUIS injection has been used for study of glutamatergic excitotoxicity (Yu and Yezierski, 2005). Briefly, male Long-Evan rats weighing 200-250 g were anesthetized with a mixture of ketamine, acepromazine, and rompun (0.65 ml/kg, subcutaneously). Animals were placed in a stereotaxic unit and the spinal column immobilized with a vertebral clamp. One injection window for intraspinal injection was made by laminectomy between spinal segments T12-L2. QUIS (125 mmol/L) was injected unilaterally at 3 levels of the cord. Injections were made at a depth of 1000μm below the dura in segments between the dorsal root entry zone and the dorsal vein. These coordinates place injections in the center of the gray matter between spinal laminae IV-VI. The total volume of QUIS injected was 1.2 μl (0.4 μl each site, delivered over 60 seconds).

Contusive SCI

Contusion SCI was produced following a T10 laminectomy using an Infinite Horizons (IH) spinal cord injury device (Precision Systems & Instrumentation, Lexington, KY) as described previously (Scheff et al., 2003; Yu and Geddes, 2007). Briefly, adult female Long-Evans rats, weighing 200-250 g, were anesthetized with ketamine (80 mg/kg, ip) and xylazine (10 mg/kg, ip), and a laminectomy was made to expose spinal segment T10. The exposed vertebral column was stabilized by clamping the rostral T9 and caudal T11 vertebral bodies with two spinal forceps. Spinal cord injury was then applied with the IH device using a 230 kdyn force setting, which resulted in severe contusion injury. Impact analyses, including actual force applied to the spinal cord, displacement of spinal cord, and velocity, were recorded. The impact tip was automatically retracted immediately, the wound irrigated with saline, and the muscle and skin openings were closed with sutures. The surgical procedure and postoperative care were similar to that described previously (Yu and Geddes, 2007).

Real-time PCR

Total RNA was isolated from PC-12 cell lyses sample using a modified guanidium thiocyanate-phenol-chloroform extraction method as described previously (Yu and Yezierski, 2005). First-strand cDNA was synthesized using 1.0μg of total RNA with the SuperScript First-strand Synthesis System (Invitrogen). The first-strand cDNA serves as a template for initial PCR and real time PCR analysis. Initial PCR using Taq DNA Polymerase PCR system (QIAGEN, Valencia, CA) was performed with GAPDH and ERK2 specific primers (see below) that resulted in 369-bp and 297-bp products, respectively, according to the protocol provided by the manufacturer. Agarose gels were used to evaluate the amplification product size. The use of DNase and no-RT control ensured lack of false-positive/negative data and genomic DNA contamination. The quantitative real-time PCR analysis was carried out using a Roche Light Cycler (Roche Diagnostics, Mannheim, Germany) as described previously (Yu and Yezierski, 2005). The quantitative assessment of PCR amplification was determined using the fluorescent dye SYBR Green I, which binds to the minor grove of the DNA double helix. Melting curve analysis of PCR products and gel electrophoresis confirmed the specificity of real-time PCR products. Two relative standard curves were obtained with five-tenfold dilutions from a PC-12 cell sample template: one for the target gene and another for the endogenous control GAPDH gene. The Light-Cycler program calculated a concentration value for each reaction. Calculated concentrations of RNA target and endogenous control (GAPDH) were determined by extrapolating from the relative standard curves. The target value was expressed relative to GAPDH value and normalized. All genes were amplified with high efficiency during real-time PCR.

ERK2 primers: Sense 5′-GCGCTACACTAATCTCTCGT-3′ Size: 297bp
Antisense 5′-CTGAGGTGCTGTGTCTTCAA-3′
GAPDH primers:  Sense 5′-GGTGAAGGTCGGTGTGAACGG-3′ Size: 369-bp
 Antisense 5′-GGCAGAAGGGGCGGAGATG-3′
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Western blotting

PC-12 cells were washed with 1 ×PBS, lysed by adding buffer, and sonicated for 15 sec to shear DNA and reduce sample viscosity. For spinal cord samples, animals were euthanatized by pentobarbital (100mg/kg, ip injection) and decapitated 2 weeks postinjury with treatment of LV-ERK2 shRNA/control vectors or 2 days postinjury with treatment of ERK2 siRNA/control siRNA. A 5-mm block of spinal cord centered on lesion site was removed and snap-frozen on dry ice, then stored at -80°C. The spinal cord samples were homogenized in a lysis buffer, sonicated, and centrifuged at 14,000 rpm for 10 min. Protein content of the supernatant was determined using the BCA method. Western blotting was performed as previously described (Yu and Yezierski, 2005) except that infrared-labeled secondary antibodies (IRDye Conjugates, Rockland Immunochemicals, Gilbertsville, PA) were used, designed for visualization with the LI-COR Odyssey infrared imaging system (Lincoln, NE). Briefly, PC-12 cell or spinal cord protein samples were loaded on SDS-PAGE gels and following electrophoresis were electrotransfered to nitrocellulose membranes. Blots were probed with a polyclonal antibody against ERK2 (1:1000; Cell Signaling Technology, Beverly, MA) and reprobed with a monoclonal antibody against GAPDH (1:1000, Chemicon, Temecula, CA), ERK1, pERK1, or pERK2 (1:1000, Cell Signaling). Blots were then incubated with infrared-labeled anti-rabbit or anti-mouse secondary antibodies (1:5000). All blots were visualized and analyzed on the LI-COR Odyssey infrared imaging system.

Immunofluorescence

Following anesthesia, the injured or naïve rats at 4 hours post-QUIS injection or 2 weeks postinjection with LV-ERK2 shRNA or control vectors were transcardially perfused with ice-cold saline followed by phosphate-buffered 4% paraformaldehyde. The spinal cord was removed, cut into blocks according to segments, and postfixed for 6 hours. The fixed spinal cords were serially cryosectioned at 20 μm. Spinal expression of eGFP in the lentiviral vector-infected sections and uptake of CY-3-labeled ERK2 siRNA in spinal sections were directly viewed using an epifluorescent microscopy system. Immunofluorescence staining was performed using Cell Signaling Technology protocols (Beverly, MA). Briefly, sections were immunolabeled with polyclonal antibodies against ERK2, pERK2 (1:200, Cell Signaling), cleaved caspase 3 (1:200, Cell Signaling) or ED-1 (1:200, Chemicon, Temecula, CA) followed by incubation with Alexa Fluor 594 or FITC-conjugated secondary antibodies. The fluorescent ERK2 or ED-1signal in the lentiviral vector injection epicenter were imaged/analyzed and the pERK2-positive or cleaved caspase 3-positive cells in the QUIS lesion epicenter were counted using an Olympus spinning disk fluorescent microscopy image system and Adobe Photoshop CS2 software (n=3 sections separated 60 μm apart per animal, n= 3-4 animals per group). For each animal, the calculated fluorescent density for expression of ERK2 and ED-1 or cell counts for activation of ERK2 and caspase 3 of 3 sections was averaged. Subsequently, the average density or cell counts per section of all animals in each group was averaged to obtain a mean value per section.

Assessment of locomotor function

At 7 days postinjection with lentiviral-ERK2 shRNA or control vectors, the rats received severe contusion SCI (230 kdyn). Open-field locomotor function was assessed preinjury and 0, 3, 7, 14, 21, 28, 35, and 42 days postinjury using the Basso, Beattie, Bresnahan (BBB) locomotor rating scale as described previously (Basso et al., 1996; Yu and Geddes, 2007). The two examiners participating in the BBB evaluation were blinded to the experimental treatment received by each animal.

Assessment of tissue sparing

At 6 weeks postinjury, animals were euthanatized and transcardially perfused with ice-cold saline followed by phosphate-buffered 4% paraformaldehyde. The spinal cord was removed and prepared for histological assessment as previously described (Rabchevsky et al., 2002; Yu and Geddes, 2007). Spinal cords were serially cryosectioned at a thickness of 20μm. Every fifth section was mounted onto gelatin-coated slides and stored at -20°C. A modified eriochrome cyanine staining protocol for myelin that differentiates both white matter and cell bodies was used to visualize spared spinal tissue. Area measurements in lesion, gray matter, white matter, and total spinal tissue and calculation of lesion volume, total tissue sparing, white matter sparing, and gray matter sparing in transverse sections of the injured cords were performed as previously described (Rabchevsky et al., 2002; Yu and Geddes, 2007).

Experimental groups

For siRNA treatment in PC 12 cells: PC-12 cells were assigned to the following groups: for real-time PCR analysis, (A) ERK2 siRNA treatment and (B) control siRNA treatment, for Western blot analysis, (A) ERK2 siRNA treatment and (B) control siRNA treatment. Real-time PCR analysis was used to measure mRNA levels of ERK2 gene expression 72 h posttransfections (n=4 per group). Western blot analysis was used to evaluate the protein levels of ERK2 at 72 h posttransfections (n=4 per group).

For injury-induced ERK1/2 activation: Female Long-Evans rats were randomly assigned to the following groups: (A) sham control (laminectomy without injury) and (B) contusive SCI (n=4/group). Western blot analysis was used to determine whether injury causes ERK1/2 activation 2 days after contusion SCI.

For lentiviral shRNA pretreatment for contusion SCI: Female Long-Evans rats were randomly assigned to the following groups: (A) LV-ERK2 shRNA plus sham control (laminectomy without injury), (B) LV-Control plus sham control (laminectomy without injury), (C) LV-ERK2 shRNA plus SCI, and (D) LV-Control plus SCI. Fluorescent microscopy and immunofluorescent staining analysis were used to observe whether LV-ERK2 shRNA i.s. administration results in eGFP expression or ERK2 knockdown in spinal cord at 2 weeks post delivery of lentiviral vectors in uninjured rats (n=4). Western blot analysis was used to confirm whether LV-ERK2 shRNA i.s. administration results in ERK2 knockdown in spinal cord 2 weeks postinjury (n=4). Fourteen virus-infected animals in the SCI group were used to assess pathological and behavioral outcome 6 weeks following injury using histological and behavioral assessments (n=7). General evaluation observed in the present in vivo study includes daily observation of respiration, weight, hematuria, locomotor deficits, other abnormal behaviors, skin/spinal tissue damage, and inflammatory response (ED-1 marker for microglial activation) in virus-infected spinal tissue (sham animals).

For siRNA pretreatment for excitotoxic insult: Male Long-Evans rats were randomly assigned to the following groups: (A) CY-3-labeled ERK2 siRNA/lipofectamine 2000 with sham control (laminectomy without injury), (B) Control siRNA/lipofectamine 2000 with sham control (laminectomy without injury), (C) ERK2 siRNA/lipofectamine 2000 wtih excitotoxic insult, and (D) control siRNA/lipofectamine 2000 complexes with excitotoxic insult(n=3/group). Fluorescent microscopy was used to observe whether intrathecal delivery with CY3-labeled ERK2 siRNA results in CY-3 uptake in the spinal cord cells in uninjured rats (n=3). Immunofluorescent staining and cell counts were used to determine whether intrathecal delivery with ERK2 siRNA inhibits activation of ERK2 and caspase 3 in the spinal cord 4 h following excitotoxic insult using lipofectamine 2000 delivery system (3 sections separated 60 μm apart per animal, 3 rats per groups).

Statistical analysis was performed using StatView (SAS Institute, Cary, NC). Data are presented as mean ± S.E.M. Group differences were evaluated by t-test or repeated measures ANOVA and Bonferroni post hoc test (McTigue et al., 2007). Null hypotheses were rejected at the P < 0.05 level. Although the BBB scale is an ordinal scale, differences between the treatments were compared using parametric statistical methods recommended by Scheff and colleagues (Scheff et al., 2002).

Results

ERK2 siRNA in vitro

The silencing efficacy of the ERK2 siRNA sequence was validated on expression of the targeted mRNA and protein levels in PC-12 cells using real-time PCR and Western blots. Transfection of PC-12 cells with ERK2 siRNA or control siRNA showed that ERK2 siRNA (100nm) significantly decreased levels of ERK2 mRNA (Fig. 1A, P<0.001, t-test) and ERK2 protein (Fig. 1B and 1C, P<0.001, t-test) by 70% at 72 h after transfection in PC-12 cells compared with control siRNA transfection.

Figure 1.

Figure 1

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Silencing of ERK2 with siRNA in vitro. (A): ERK2 siRNA decreased ERK2 mRNA levels in PC-12 cells. PC-12 cells (3×105/well in 6-well plates) were transfected with 100 nM ERK2 siRNA complexed with 6μl lipofectamine 2000. The mRNA levels of ERK2 in the cells were measured at 72 h after transfection using real-time PCR. ***p<0.001, t-test, n=4 per group. Dark bar = control siRNA, Light bar=ERK2siRNA (ERK2 siRNA: Sense (start 522) 5′pGCACCUCAGCAAUGAUCAUdTdT3′). (B and C): ERK2 siRNA decreased ERK2 protein levels in vitro. PC-12 cells (3×105/well in 6-well plates) were transfected with ERK2 siRNA complexed with lipofectamine 2000 at 100nm of siRNA and liposome (6μl). The protein levels of ERK2 in the cells were measured at 72 h after transfection using Western blot analysis (***p<0.001, t-test, n=4 per group).

ERK1/2 activation after contusion SCI

Western blot analysis showed that contusion injury significantly induced activation of ERK1/2 at 2 days after contusive SCI (Fig. 2A, B & C, p<0.01, t test)

Figure 2.

Figure 2

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Contusive SCI induces upregulation of ERKI1/2 phosphorylation in the spinal cord 2 days after contusive SCI. Western blot analysis showed injury-induced ERK1/2 phosphorylation of serine residues of both ERK1 and ERK2 compared with sham-controls. The bottom panels in the Figure 2A show levels of total ERK1 and ERK2 protein expression, as loading controls. Antibodies on the top panels were specific for the phosphorylated form of the ERK proteins. **p<0.001, t-test.

LV-ERK2 shRNA

To evaluate effects of spinal ERK2 knockdown following SCI, we constructed ERK2 shRNA expression cassettes driven by the U6 promoter, cloned these into lentiviral-CMV-RNAi vectors and produced high-titer lentiviral ERK2 shRNA or control vectors as described in protocols provided by Invitrogen. As both vectors contain an eGFP reporter, expression of LV-ERK2 shRNA or LV-control vectors was verified in spinal cord of rats (Fig. 3A and 3B) by direct eGFP visualization with fluorescent microscopy. Intraspinal microinjection of LV-ERK2 shRNA resulted in eGFP expression throughout the gray matter and to a lesser extent in the white matter of spinal cord at injection epicenter (∼4 mm rostro-caudally, Fig. 3B). Intraspinal injection of LV-ERK2 shRNA reduced ERK2 protein levels, as compared to LV-control, as demonstrated by immunohistochemistry (Fig. 3C, D, and E) and Western blots (Fig. 4A and 4B). LV-ERK2 shRNA constructs did not decrease ERK1 levels (Fig. 4A top panel and C). These data indicate that intraspinal administration of LV-ERK2 shRNA selective reduces ERK2 expression in the rat spinal cord.

Figure 3.

Figure 3

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LV-ERK2 shRNA reduces ERK2 expression in rat spinal cord. (A and B): Intraspinal microinjection of LV-ERK2 shRNA (four injections in bilateral gray matter and gray matter at T10) resulted in expression throughout the spinal tissue (B), compared to uninjured rat (A), as visualized by the eGFP reporter. (C to E): Knockdown of ERK2 protein in the spinal cord with LV-ERK2 shRNA treatment was evident using Immunofluorescent staining (C. D, and E) with antibody specific for ERK2. Scale bar: 400 μm. ***p<0.001, t-test.

Figure 4.

Figure 4

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LV-ERK2 shRNA reduces ERK2 expression in rat spinal cord. Intraspinal microinjection of LV-ERK2 shRNA (four injections in bilateral gray matter and gray matter at T10) resulted in knockdown of ERK2 protein in the spinal cord two weeks postinjury using western blotting (A and B) with antibody specific for ERK2. These shRNA constructs do not alter ERK1 levels using Western blot with antibody against ERK1 (A, top panel and C). *p<0.05, t-test.

Following administration of the lentiviral particles in sham control rats, we did not observe pathological or behavioral alterations. Parameters examined included weight loss, hematuria, locomotor deficits, skin or spinal tissue damage, and inflammatory response in spinal tissue. Immunofluorescent staining revealed that no obvious ED-1 signal was seen in lentiviral vector-infected spinal tissue (data not shown).

ERK2 siRNA reduces excitotoxic injury

Direct epifluorescent visualization demonstrated uptake of CY-3-ERK2 siRNA throughout the spinal tissue twenty four hours following intrathecal delivery (Fig. 5). Intrathecal pretreatment for 72 h with ERK2 siRNA significantly reduced levels of pERK2 (Fig. 6A, B & C) and activated caspase 3 (Fig. 6D, E,& F) at 4 h following excitotoxic insult.

Figure 5.

Figure 5

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Uptake of CY-3-labeled ERK2 siRNA in dorsal horn (B) and ventral horn (C) in rat. CY3-ERK2 siRNA (2.5μg) complexed with lipofectamine 2000 (5μg) were intrathecally delivered onto the surface of dorsal horn in rat. 24h after delivery, the rat was perfused and spinal cords dissected and processed for epifluorescent microscopy. Intrathecal delivery of CY-3-labeled ERK2 siRNA resulted in uptake of CY-3-ERK2 siRNA throughout the spinal tissue (B and C), compared to naïve rat (A). Scale bar: 100 μm.

Figure 6.

Figure 6

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Silencing effects of pERK2 and caspase 3 with siRNA after excitotoxic SCI. (A, B, and C): Intrathecal pre-treatment for 72 h with ERK2 siRNA (B and C light bar, 2.5 μg) complexed with lipofectamine 2000 (5 μg) resulted in significant decrease in pERK2 protein expression 4 h after excitotoxic SCI, compared to control siRNA pretreatment (A and C dark bar, 2.5 μg). (D, E, and F): Intrathecal pre-treatment for 72 h with ERK2 siRNA (E and F light bar, 2.5 μg) complexed with lipofectamine 2000 (5 μg) resulted in significant decrease in caspase 3 activity (cleaved caspase 3) 4 h after excitotoxic SCI, compared to control siRNA pretreatment (D and F dark bar, 2.5 μg). The fluorescent labeled pERK2-positive or cleaved caspase 3-positive cells in the QUIS injection epicenter were counted using fliorescent microscopy image system (n=3 sections sparated 60 μm apart per animal, n=3 animals per group). Data were presented as mean ± S.E.M. and analyzed by t-test **p<0.01 and ***p<0.001, t test. Scale bar: 30 μm.

Lentiviral-ERK2 shRNA improves locomotor function 6 weeks following contusive spinal cord injury

To investigate the functional effects of LV-ERK2 shRNA on long-term locomotor deficits following traumatic SCI, fourteen lentiviral vector-infected rats received contusive injury using the IH impactor at the 230 kdyn force setting. No significant differences in actual force, displacement, or velocity were found between LV-ERK2 shRNA and LV-control-treated groups, indicating similar injuries to all animals (Table 1). Immediately following SCI, all animals exhibited complete bilateral hindlimb paralysis. Behavioral assessment demonstrated that administration of LV-ERK2 shRNA at 7 days preinjury resulted in significant improvement in locomotor function measured by BBB scores over 6 weeks after severe contusive SCI compared with the administration of LV-control (Fig. 7, p<0.05 at 2, 4, 5, and 6 weeks post injury, repeated measures ANOVA and Bonferroni post hoc test). At 6-weeks postinjury, most LV-ERK2 shRNA pretreated animals exhibited consistent weight-supported hindlimb movements and frequent or consistent forelimb-hindlimb coordination, whereas LV-control-pretreated animals exhibited limited hindlimb weight bearing. The 6-week BBB score in LV-ERK2 shRNA-pretreated animals was significantly higher than that in LV-control-pretreated animals (Fig. 7). These data suggest that intraspinal administration of LV-ERK2 shRNA significantly reduces locomotor impairment following traumatic SCI.

Table 1. Injury Parameters for LV-shRNA Treatment.

Groups Actual force (kdyn) Displacement (microns) Velocity (mm/sec)
LV-ERK2 shRNA 235 ± 9 1677 ± 73 124 ± 2
LV-Control 232 ± 13 1704 ± 91 120 ± 2
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Values are mean ± SEM. No significant differences in impact force, displacement, and velocity were found between LV-ERK2 shRNA and LV-control -treated groups.

Figure 7.

Figure 7

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Effects of the LV-ERK2 shRNA pretreatment on locomotor impairment measured by BBB scores over the 6 week testing interval following severe contusive SCI. Preinjury administration of LV-ERK2 shRNA (triangles) resulted in improved locomotor performance compared to LV-control -pretreated animals (circles). Contusive SCI was produced using the Infinite Horizons impactor, 230 kdyn setting, at T10. Data were presented as mean ± S.E.M. and analyzed with repeated measures ANOVA followed by Bonferroni post-hoc analysis, *p<0.05.

Lentiviral-ERK2 shRNA improves tissue sparing at 6 weeks following severe contusive spinal cord injury

Histological assessment of tissue sparing showed that i.s. injection of LV-ERK2 shRNA resulted in a significant increase in total tissue sparing (Fig 8, Fig. 9), gray matter sparing (Fig. 10), and white matter sparing (Fig. 11) at 6 weeks postinjury compared with animals receiving LV-control. These results suggest that intraspinal microinjection of LV-ERK2 shRNA produces a dramatic reduction in tissue damage following severe SCI.

Figure 8.

Figure 8

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Photomicrographs of representative transverse spinal cord sections taken from rats at 42 days following severe contusion SCI. A-E are photomicrographs of representative transverse spinal cord sections at the epicenter and 3-mm and 6-mm rostral and caudal to the lesion epicenter taken from a LV-ERK2 shRNA-pretreated rat. F-J are photomicrographs of representative transverse spinal cord sections at the epicenter and 3-mm and 6-mm rostral and caudal to the lesion epicenter taken from a LV-control-pretreated rat. The sections were stained with eriochrone cyanine for myelin. The treatment conditions are as described in Fig 4. The sections from the injured epicenter are indicated by an arrow. Scale bar: 500 μm.

Figure 9.

Figure 9

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Effects of the LV-ERK2 shRNA pretreatment on tissue sparing 6 weeks following severe contusive SCI. Preinjury administration of LV-ERK2 shRNA resulted in a significant increase in total tissue sparing (A) and tissue sparing at 3 to 6 mm caudal to the injury epicenter (B) following contusion injury to the spinal cord. Injury conditions and treatment groups are as described in Fig 7. Data were presented as mean ± S.E.M. and analyzed by t-test (A) or repeated measures ANOVA followed by Bonferroni post-hoc analysis (B). *p<0.05, **p<0.01.

Figure 10.

Figure 10

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Effects of the LV-ERK2 shRNA pretreatment on gray matter sparing 6 weeks following severe contusive SCI. Preinjury administration of LV-ERK2 shRNA resulted in a significant increase in total gray matter sparing (A) and gray matter sparing at 3 to 6 mm caudal to the injury epicenter (B) following contusion injury to the spinal cord. Injury conditions and treatment groups are as described in Fig 7. Data were presented as mean ± S.E.M. and analyzed by t-test (A) or repeated measures ANOVA followed by Bonferroni post-hoc analysis (B). *p<0.05, **p<0.01, ***p<0.001.

Figure 11.

Figure 11

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Effects of the LV-ERK2 shRNA pretreatment on white matter sparing 6 weeks following severe contusive SCI. Preinjury administration of LV-ERK2 shRNA resulted in a significant increase in white matter tissue sparing at 3 to 6 mm caudal to the injury epicenter (B, 300% increase at 3 mm, 100% increase at 4 mm, 89% increase at 5 mm, and 64% increase at 6 mm caudal to the epicenter) and non-significant increase in total white matter sparing (A) following contusion injury to the spinal cord. Injury conditions and treatment groups are as described in Fig 7. Data were presented as mean ± S.E.M. and analyzed by t-test (A) or repeated measures ANOVA and Bonferroni post-hoc analysis (B). *p<0.05.

Discussion

In the present study, we report the development of the RNAi strategy to knockdown ERK2 expression in spinal cord and the involvement of ERK2 expression in the glutamate excitotoxicity, progressive tissue damage and locomotor dysfunction following SCI. To our knowledge, this is the first report demonstrating a detrimental role of ERK2 in spinal cord injury using either shRNA or siRNA. The major findings of the present study include: 1) ERK2 siRNA intrathecal pretreatment significantly reduces early excitotoxic injury-induced caspase 3 activation and 2) preinjury intraspinal administration of lentiviral-vectors expressing ERK2 shRNA significantly improves long-term locomotor function and tissue sparing over 6 weeks following contusive SCI; Traumatic SCI results in secondary damage and locomotor deficits as well as sustained activation of ERK1/2 (Crown et al., 2006; Zhao et al., 2007). Whether an increase in ERK1/2 activity is neuroprotective or detrimental is unclear (Hetman and Gozdz, 2004; Zhuang and Schnellmann, 2006). Most studies examining the role of ERK1/2 signaling in neuroprotection and neurodegeneration have relied on MEK inhibitors which inhibit activity of both ERK1 and ERK2. Relatively little data concerning the roles of individual ERK isoforms has been reported previously (Matsumoto et al., 2005; Xu, et al., 2008; Agrawal, et al., 2006; Nakazawa, et al., 2008). In the present study, we examined the hypothesis that ERK2 may contribute to neurodegeneration following spinal cord injury and selective inhibition of ERK2 may provide a novel therapeutic strategy for spinal injury.

Selective inhibitor for ERK2 is not available. To test the hypothesis that ERK2 expression is involved in the pathogenesis of SCI, we utilized intraspinal microinjection of lentiviral-ERK2 shRNA vectors to knockdown ERK2 expression in the spinal cord prior to contusive SCI in rats. We initially evaluated ERK2 siRNA efficacy in PC12 cells, then examined intrathecal delivery of ERK2 siRNA into the spinal cord in vivo using CY-3 labeled ERK2 siRNA. Although intravenous administration of lipid-based transfection reagents is problematic due to rapid liver metabolism, such agents have been used successfully for intrathecal siRNA administration (Luo et al., 2005; Aigner, 2006). Intrathecal ERK2 siRNA reduced activation of ERK2 and caspase3 at 4 hours after excitotoxic insult. These results provided support for the hypothesis that ERK2 inhibition may protect against neurodegeneration following SCI.

For subsequent studies, we utilized Lentiviral-shRNA vectors which have the ability to infect both dividing and nondividing cells and are an effective method for RNAi in postmitotic cells, with a longer time course of gene knockdown and less inflammatory/immune response (Rubinson et al., 2003; Breckpot et al., 2007; Meunier et al., 2008). Lentiviral vector-based RNAi has been approved in clinical trials for gene therapy in HIV, human beta-thalassemia, and sickle-cell anemia with no obvious side effects (Chen et al., 2007). However, the use of lentiviral vector expressing shRNA for analysis of individual gene function and gene therapy in SCI has not been reported. This study developed lentiviral-mediated delivery of ERK2 shRNA in vivo and verified the efficacy of this approach in locally reducing ERK2 expression in the spinal cord. In the present study, no side effects including spinal tissue damage, weight loss, hematuria, locomotor deficits, other abnormal behaviors, or inflammatory response in spinal tissue were observed after lentiviral-ERK2 shRNA intraspinal administration in uninjured controls. The important finding of the present study is that selective ERK2 knockdown by intraspinal injection of lentiviral-ERK2 shRNA (pre-injury) resulted in significant improvement in locomotor function and tissue sparing over 6 weeks following severe contusive SCI using IH SCI impact device.

The IH impact device-induced contusive SCI at T10 is a clinically relevant model of thoracic traumatic SCI (Scheff et al., 2003). The 230 kdyn force setting used in the present study results in severe secondary damage and locomotor dysfunction (Scheff et al., 2003). The final outcome of SCI directly depends on the degree of the initial traumatic insults and the secondary damage (Scheff et al., 2003). In the severe contusion injury model, the BBB score assessment and histological assessment demonstrated that LV-ERK2 shRNA pretreatment resulted in significant improvement in locomotor function and tissue sparing over six weeks postinjury. These data suggest that ERK2 knockdown with LV-ERK2 shRNA i.s. administration is a highly potent approach for long-term recovery of locomotor dysfunction and tissue damage after SCI.

Secondary tissue damage after SCI spreads both rostral and caudal to the lesion epicenter with increasing functional deficits (Wells et al., 2003). Tissue loss, including loss of neurons (gray matter) and/or axons (white matter), is the underlying cause of permanent disability following cervical and/or thoracic SCI (el-Bohy et al., 1998; Magnuson et al., 1999; Reier et al., 2002). In the T10 contusive injury model, it is likely that only white matter sparing contributes to the functional improvement because the locomotor deficits at this level of injury are largely due to demyelination and loss of white matter axons (Magnuson et al., 1999; Reier et al., 2002; Teng and Wrathal, 1997; Blight 1983). Gray matter damage at the thoracic level has relatively minor consequences, including loss of sensory input from one or two dermatomes and deficits in motor output to some axial musculature (Magnuson et al., 1999). Preservation of as few as 5% to 10% of myelinated axons in white mater can confer locomotor recovery (Azari et al., 2006). Thus, strategies aimed at improving white matter sparing by ERK2 knockdown are likely to be of therapeutic significance. On the other hand, very small sparing of tissue at the lesion epicenter or rostral/caudal to the lesion site has profound effects on locomotor recovery (Basso et al., 1996; Wrathall et al., 1996; Wrathall et al., 1997; Lankhorst et al., 1999; Rabchevsky et al., 2000; Hillard et al., 2004). In the present study, although total white matter sparing was not statistically different between the groups, 300% to 64% increase in white matter sparing at 3 to 6 mm caudal to the lesion epicenter was observed with intraspinal administration of LV-ERK2 shRNA. The improvement in locomotor function obtained with intraspinal LV-ERK2 shRNA is presumably associated with white matter sparing caudal to the lesion epicenter. These findings are similar to those observed with other pharmacological interventions in studies of spinal cord contusion injury by other investigators and may be responsible for the functional recovery (Wrathall et al., 1996; Teng and Wrathall, 1997; Wrathall et al., 1997; Madsen et al., 1998; Lankhorst et al., 1999; Rabchevsky et al., 2000).

The tissue sparing achieved with intraspinal administration of LV-ERK2 shRNA was primarily caudal to the injury. Although the reason for this marked rostral and caudal differences is uncertain, it may be related to the following factors: 1) an unequal local gene knockdown in different cells (Meunier et al., 2008), 2) an unequal local expression/activation of ERK2 downstream targeted genes (Zhao et al., 2007), and/or 3) an unequal local gain in blood perfusion (Oudega et al., 1999). Further studies in the future are necessary to examine the local pattern in eGFP expression, cell type-selective ERK2 knockdown, and downstream targeted gene expression/activation with LV-ERK2 shRNA vectors in the spinal cord after SCI.

Although the mechanisms of pathological and behavioral dysfunction after SCI are not fully understood, it is known that SCI activates ERK1/2 that targets multiple secondary injury events including glutamate excitotoxicity, inflammation, apoptosis, pain hypersensitivity, and mitochondrial dysfunction (Klussmann and Martin-Villalba, 2005; Yu and Yezierski, 2005; Barrett et al., 2006; Genovese et al., 2008). Glutamatergic excitotoxicity is believed to be a major mechanism in neurotrauma (Park et al., 2004). Strategies aimed at blocking the deleterious effects of ERK signaling leading to inhibition of glutamatergic cascades may be of therapeutic significance. In the present study, we showed that selective ERK2 knockdown with ERK2 siRNA significantly reduced glutamatergic signaling-mediated caspase 3 activation after excitotoxic insult (Yu, et al., 2009). The present findings suggest that ERK2 activation after SCI contributes to activation of glutamatergic cascades, resulting in secondary damage and locomotor deficits.

The major objective of the present study was to investigate the pathological contributions of ERK2 in the secondary damage following contusive SCI. Although pretreatment with lentiviral vectors before SCI is not a viable therapeutic strategy, the strategy enabled examination of the role of ERK2 isoform, which is not possible using current pharmacologic inhibitors. The lentiviral vectors were administered preinjury to allow sufficient time for genomic integration of the retroviral cDNA and the decreased gene and protein expression.

Treating SCI remains a medical challenge and developing new therapeutic strategies is clinically important. Our study provides a proof-of-concept that ERK2 knockdown can reduce tissue damage and locomotor impairment, thereby revealing a detrimental role for ERK2 pathway in secondary injury and paving the way for further investigation with postinjury administration of lentiviral shRNA or siRNA targeting ERK2 in the pathophysiology and treatment of SCI.

Acknowledgments

This research was supported by the Paralysis Project of America, the Kentucky Spinal Cord and Head Injury Research Trust, and a University of Florida Seed grant. The authors wish to thank Dr. George Smith for lentiviral-CMV-RNAi vectors, and Paula Thomason for manuscript editing.

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