NDGA reduces secondary damage after spinal cord injury in rats via anti-inflammatory effects

brain research 1516 (2013) 83–92

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Research Report

NDGA reduces secondary damage after spinal cord injury in rats via anti-inflammatory effects Hui Xuea, Xiu-ying Zhangc, Jia-mei Liua,n, Yu Songd,a, Ting-ting Liua, Dong Chenb,nn a

Department of Histology & Embryology, Bethune School of Medical Science, Jilin University, Changchun 130021, China Department of Histology & Embryology, Guangdong Medical College, Dongguan 523808, China c Department of Fundamental Nursing, School of Nursing, Jilin University, Changchun 130021, China d Department of Anatomy, Changchun Medical College, Changchun 130021, China b

art i cle i nfo

ab st rac t

Article history:

After spinal cord injury (SCI), a series of complex pathophysiological processes follows the initial

Accepted 8 April 2013

injury. Because inflammation plays a key role in this secondary pathology damage, anti-

Available online 17 April 2013

inflammatory drug treatment may reduce secondary damage and protect neurons after SCI.

Keywords:

Though nordihydroguaiaretic acid (NDGA) can inhibit inflammatory responses, its potential

NDGA

roles in neuroprotection and anti- inflammation in an SCI model have not been studied. In this

Neuron protection

study, we investigated the anti-inflammatory effects of NDGA in SCI. First, histopathological

Secondary damage

alterations were evaluated with hematoxylin/eosin (HE) and Nissl staining, showing an

Rat

increased number of neurons after NDGA administration. Additionally, the extent of secondary

Spinal cord injury

damage was assessed by TUNEL assay and measurement of astrocyte proliferation. The data

Inflammation

showed that the numbers of apoptotic cells and the proliferative extent of astrocytes were significantly decreased by the use of NDGA. The anti-inflammatory effect of NDGA was evaluated by measuring myeloperoxidase (MPO) levels as an indicator of neutrophil activity, macrophage/microglia numbers, and expression of inflammatory cytokines including IL-1β and TNF-α. NDGA treatment significantly decreased the MPO level and the number of macrophages/ microglia. In addition, NDGA also suppressed the expression of IL-1β and TNF-α after SCI. These data suggest that anti-inflammatory action by NDGA can reduce secondary damage after SCI & 2013 Elsevier B.V. All rights reserved.

1.

Introduction

Spinal cord injury (SCI) is a traumatic event that has great physical, psychological, and social impacts on individuals, families, and society. After SCI, an inflammatory response is initiated, which plays an important role in the progression of secondary destructive phenomena (Fleming et al., 2006; n

Sinescu et al., 2010). During the immune response after SCI, inflammatory events in the first few days result in the most destructive activity (Leskovar et al., 2000; Sinescu et al., 2010). Those early inflammatory events also create a hostile microenvironment for various SCI treatments such as cell transplantation, thus creating obstacles for transplantation-based therapies (Coyne et al., 2006; Okano et al., 2003). The early

Corresponding author. Corresponding author. Fax: +86 769 22896 395. E-mail addresses: [email protected] (J.-m. Liu), [email protected] (D. Chen).

nn

0006-8993/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.brainres.2013.04.016

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brain research 1516 (2013) 83–92

immune response after SCI is induced by neutrophilic signaling and magnified by neutrophil-activated macrophages and microglia (Hirose et al., 2000; Liu et al., 2011; Taoka et al., 1998). Thus, anti-inflammatory treatment that prevents neutrophil and macrophage inflow, activation of microglia, or phagocytic and secretory activity of macrophages can be administered to improve the microenvironment for cellular transplantation after SCI (Sinescu et al., 2010). Nordihydroguaiaretic acid (NDGA) is a selective 5-LOX inhibitor from the creosote plant (Larrea tridentata) (Lu et al., 2010b). It has broad medicinal properties including inhibition of inflammation (Bhattacherjee et al., 1988; Salari et al., 1984), oxidation (Floriano-Sanchez et al., 2006; Lu et al., 2010a), virus activity (Craigo et al., 2000; Hwu et al., 2008), and tumor growth (Kubow et al., 2000; Nishimura et al., 2002; Park et al., 2004). Studies have shown that NDGA application in amyotrophic lateral sclerosis and chronic colitis can inhibit inflammation responses to promote tissue repair (Fitzpatrick et al., 1990; West et al., 2004). It has also been shown that NDGA can promote the survival of microencapsulated allogeneic islets by preventing the activation and chemotaxis of macrophages (Yang et al., 2005). In this study, we demonstrated that NDGA has a neuroprotective role through its anti-inflammatory effects after SCI.

2.

Results

2.1. Reduction of secondary damage and neuroprotection by NDGA after SCI HE staining illustrated that at 1 week after operation, the injured area of spinal cord in two groups was filled with connective tissue or formed a large cavity. Surrounding the lesion, significantly fewer neurons survived in the injury group

compared with the NDGA group (Fig. 1). In line with the HE results, Nissl staining showed that the number of neurons in the NDGA group was significantly more than the injury group (Fig. 2). Neuron counting results showed that the difference between the two groups was statistically significant (Fig. 2), suggesting that NDGA promoted the survival of neurons after the injury. TUNEL staining showed that apoptotic cells were dramatically reduced in the NDGA group (Fig. 3), suggesting there was less neuronal apoptosis in the NDGA group. GFAP immunofluorescence staining 1 week post-operation showed that compared with the injury group, the percentage of GFAPpositive area in the NDGA group was significantly reduced (Fig. 4). This indicated that the application of NDGA reduced the proliferative extent of astrocytes after SCI, further supporting its role in limiting secondary damage.

2.2.

Anti-inflammation effects of NDGA in SCI

The MPO value measured results of postoperative day 3 demonstrated that the MPO values of the control group were significantly higher than those of the NDGA group (Fig. 5), suggesting that NDGA reduces neutrophil infiltration after SCI. The ED-1 immunofluorescence staining showed that the number of ED-1 positive cells in the NDGA group was significantly decreased compared with the injury group (Fig. 6). This result indicated that the NDGA application reduced the infiltration of macrophages/microglia after SCI. The results of Western blots for IL-1β (30 kD) and TNF-α (17 kD) showed a significantly decreased level in the NDGA group (Fig. 7), indicating that NDGA can efficiently decrease inflammatory factors in damaged spinal cord tissues. Taken together, the above data suggest that NDGA reduced the inflammatory response by inhibiting the inflammatory cells and molecules in SCI.

Fig. 1 – Coronal sections from each group at 1 week post-surgery stained with HE. Images of representative sections are from the injured control group ((A), (a)) and the NDGA-treated group ((B), (b)). Note that injured areas are filled with connective tissue. More neuronal survival is observed within the peripheral region of damage in the treated group than the injury group. Broken lines mark the lesion borders of injured spinal cords. Arrows indicate surviving neurons. Scale bars: (A) and (B), 625 μm; (a) and (b), 30 μm.

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Fig. 2 – Survival of neurons in the tissue surrounding the damage at postoperative week 1 shown by Nissl staining. (A) and (a) are from a representative section of the injured control group; (B) and (b) show a representative section of the NDGAtreated group. The neurons with clear Nissl bodies can only be observed in the region far from the injured area in the injury group, suggesting neuronal loss in tissue surrounding the damaged area. In contrast, Nissl stained neurons are located near the injured area in the treated group, indicating neuronal preservation. Broken lines mark the lesion borders. Arrows indicate Nissl positive neurons. The neurons within the areas between the triangles were counted. (C) the numbers of Nissl stained neurons of the two groups. nPo0.05 vs. the lesion control group. Scale bars: (A) and (B), 625 μm; (a) and (b), 30 μm.

3.

Discussion

After SCI, primary mechanical damage leads to secondary injuries that result in continuous neuronal damage and progressive loss of neurological function (Allen, 1914). The extent and scope of damage caused by secondary injury are usually far more than that caused by the primary injury. In the process of secondary injury, the inflammatory response, which involves a variety of inflammatory cells and molecules, is an important driving factor (Sinescu et al., 2010). In addition, astrocytes are activated in this process and form the glial scar, which is a physical and chemical barrier to axonal regeneration (Verma et al., 2008). Therefore, inhibition of the inflammatory response and astrocyte hyperplasia is an important measure for neuroprotection and for promoting the recovery of neurological function (Fitch et al., 1999). Nordihydroguaiaretic acid (NDGA) is a natural compound extracted from the creosote bush, with a wide range of biological

effects, such as anti-oxidant, anti-inflammatory, antineoplastic, and antiviral activities. It has been shown that NDGA has multiple promising applications in the treatment of various diseases such as cardiovascular diseases, neurological disorders, and cancers (Lu et al., 2010b). NDGA can selectively inhibit arachidonic acid 5-lipoxygenase (5-Lox) activity, resulting in the inhibition of leukotriene and prostaglandin synthesis, thus leading to a smaller inflammatory response (Lu et al., 2010b). Previous studies have shown that the inhibition or deletion of 5-Lox can exert anti-inflammatory and neuroprotective effects in a mouse model of SCI (Genovese et al., 2005, 2008). However, the anti-inflammatory and neuroprotective effects of NDGA after SCI remain unclear. After SCI, the inflammatory response becomes intense and is most destructive within the first few days (Leskovar et al., 2000; Sinescu et al., 2010). An anti-inflammatory treatment such as methylprednisolone should be given within a narrow time window in the acute phase to be effective after SCI (Amar and

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Fig. 3 – Apoptotic cells 7 days after surgery shown by TUNEL staining. (A) and (a): the injured control group; (B) and (b): the NDGAtreated group. Note that the apoptotic cells (brown color) are dramatically reduced in the treated group compared to the injury group. Statistical results are shown in (C). nPo0.05 vs. the control group. Scale bars: (A) and (B), 150 μm; (a) and (b), 75 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Levy, 1999; Bracken et al., 1997; Mabon et al., 2000). Additionally, to avoid an acute inflammatory response, the optimal timing for cell transplantation is 1–2 weeks after SCI (Okano et al., 2003). Thus, in the present study, we observed the anti- inflammatory and neuroprotective effects of NDGA in the acute phase of SCI (3 and 7 days post-injury). It was found that after SCI, NDGA application can inhibit the infiltration of neutrophils and macrophages/microglial cells, suppress expression of inflammatory cytokines including IL-1β and TNF-α, diminish the proliferation of astrocytes, and have a protective effect on damaged neurons. However, there was no apparent functional

recovery in the NDGA group compared with the control group (data not shown). These data suggest that NDGA significantly suppresses the secondary injury process with neuroprotective function after SCI, and these effects are related to its antiinflammatory function.

3.1. NDGA reduces secondary spinal cord injury and protects neurons through the inhibition of inflammatory cells Inhibition of inflammation helps to protect damaged neurons after SCI. Secondary spinal cord damage is a pathological

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Fig. 4 – GFAP positive astrocyte responses in each group 1 week after surgery. (A) and (a) GFAP-positive cells in the injury group. (B) and (b) GFAP-positive cells in the NDGA-treated group. The immunoreactivity of GFAP-positive cells in the NDGAtreated group decreased significantly compared to the injury group. (C) A comparison of the GFAP-positive area between each group (n ¼6). nPo0.05 vs. the injury group. Scale bars: (A) and (B), 250 μm; (a) and (b), 125 μm.

process involving a variety of factors, one of which is inflammation, a process in which neutrophils and macrophages/microglia are critical cellular components (Nguyen et al., 2012). Indeed, neutrophils are the first inflammatory cell type to reach the damaged tissue (Tjoa et al., 2003). The activated neutrophils play a role in promoting tissue repair through phagocytosis of necrotic tissue in the wound area, but simultaneously they produce large amounts of reactive oxygen species and promote release of elastase by the respiratory burst (Carlson et al., 1998; Taoka et al., 1998). On the one hand, the reactive oxygen attacks the polyunsaturated fatty acids on the biofilm, causing lipid peroxidation and disrupting the cell osmotic balance (Carlson et al., 1998). On the other hand, the reactive oxygen activates complement systems, resulting in the

formation of the complement – neutrophils – reactive oxygen positive feedback activation loop, thereby expanding the inflammatory response, and exacerbating secondary injury (Carlson et al., 1998). Elastase can damage the integrity of the vascular endothelium by increasing vascular permeability, which leads to spinal cord hemorrhage and microcirculation disorders, thus causing spinal cord ischemic injury (Taoka et al., 1998). Additionally, elastase can increase the expression of neutrophil surface CD18 molecules, further promoting neutrophil— endothelial cell interaction, resulting in a feedback loop that activates neutrophils and impairs endothelial cells (Taoka et al., 1998). The results of this study showed that NDGA could significantly inhibit the activity of neutrophils after spinal cord injury, which helped reduce secondary injury and provided a neuroprotective effect.

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Fig. 5 – The mean MPO level in each group 3 days after surgery (n ¼ 6). The level of MPO was significantly decreased in the NDGA group compared to the injury group. nPo0.05 vs. the injury group.

Macrophages and microglia are important cellular components of the inflammatory response after SCI. Rat blood-derived monocytes or macrophages infiltrate the damaged area two days after SCI, with the highest density at 5–7 day post injury, and last a few weeks or a few months (Fleming et al., 2006). Microglial cells are activated within minutes to hours after SCI and form ED-1 positive macrophages (Fleming et al., 2006). Despite having the same origin, circulating monocytes/macrophages and microglial cells contain a wide range of different phenotypic and functional subsets (Soulet and Rivest, 2008). Studies have shown that the macrophage activation spectrum includes two composition subsets: the inflammatory response of macrophages (ED-1 positive) and alternatively activated macrophages (ED-2 positive) (Shechter and Schwartz, 2012). Each subgroup has different gene expression profiles, and accordingly, mediates different functions (Nahrendorf et al., 2007). The ED-1 positive macrophages express iNOS and TNFα, and have important proteolytic activity.

Fig. 6 – ED-1 immunoreactivity in each group 7 days after injury. (A) and (a): Images of a section from the lesion control group at 1 week post-surgery. (B) and (b): Images of a section from the NDGA-treated group at 1 week post-surgery. The pictures show that ED-1 positive cells are markedly reduced by the NDGA treatment. (C) A quantitative comparison of the ED-1 positive macrophages/microglia (n ¼ 6) nPo0.05 vs. the injury group. Scale bars: (A) and (B), 250 μm; (a) and (b), 125 μm.

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Fig. 7 – Expression levels of inflammatory factors in two groups at 3 days post-surgery. (A) Western blot was used to determine the protein levels of TNF-α and IL-1β in the injured control and NDGA-treated group. (B) Expression of these inflammatory factors significantly decreased in the NDGA-treated group compared with that of the injured control group. (*)Po0.05 vs. the injured control group. They can increase the inflammatory response and cause further damage to the injured cells and tissues. Conversely, ED-2 positive macrophages express IL-10 and are induced by IL-4 and glucocorticoids (Shechter and Schwartz, 2012). They can regulate the immune and inflammatory response by removing necrotic tissue fragments, thus promoting angiogenesis and tissue repair (Shechter and Schwartz, 2012). The present study found that NDGA application could suppress the number of ED-1 positive macrophages/microglia. Previous reports demonstrated that NDGA can reduce expression of TNF-α in macrophages (Dubois et al., 1989). Thus, the down-regulation of TNF-α in the NDGA treated group in our study suggested that NDGA could inhibit the activity of ED-1 positive macrophages/microglia after SCI. The decline in the number and activity of the ED-1 positive macrophages/microglia was conducive to reducing secondary injury of the spinal cord and helped protect neurons at the injury site. The increased number of surviving neurons and the decreased number of apoptotic cells after SCI in the NDGA group demonstrated that NDGA could reduce secondary injury of the spinal cord and provided a neuroprotective effect that correlates with the inhibition of inflammatory cells.

3.2. The reduction in the astrocytic response by NDGA is related to the inhibition of microglia after spinal cord injury After SCI, astrocytes sustain an excessively intense reaction leading to the formation of a glial scar that forms a physical and chemical barrier inhibiting the growth and regeneration of injured spinal cord axons (Verma et al., 2008). The dense structure of the glial scar combined with chemical inhibition

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within the scar creates an impenetrable barrier for regenerating axons to pass through. Therefore, inhibition of astrocytic activation is an important therapeutic strategy for SCI. Activation of astrocytes is mainly induced by IL-1 produced during the inflammatory response in SCI (Pineau et al., 2010). IL-1 can also induce astrocyte expression of MCP-1, which causes macrophage and neutrophil aggregation and leads to further tissue damage (Pineau et al., 2010). Previous studies showed that IL-1 is mainly secreted by microglia at early stages of SCI (Pineau et al., 2010). Our results showed that NDGA reduced the number of ED-1 positive cells. Combined with the result that IL-1 was also inhibited by NDGA, this suggested that the reduced response of astrocytes by NDGA might be related to the inhibition of microglia. Taking all of these observations together, NDGA could inhibit the inflammatory response after SCI, thereby limiting secondary damage, and may, thus, present a promising novel drug in the arsenal of neuroprotective agents that have already shown to be effective in experimental models of SCI (e.g. Iannotti et al., 2011; Koopmans et al., 2009; Arnold and Hagg, 2011). Although further studies are necessary to evaluate the long-term effects of NDGA treatment and completely clarify its mechanisms, we highlight the potential therapeutic interest of NDGA for the treatment of SCI due to its promising applications in the treatment of other diseases. Additionally, NDGA may provide a way to improve the microenvironment for cell transplantation therapy after SCI with its anti-inflammatory effect. In conclusion, our data set the initial stage to begin additional research on NDGA as a potential treatment for SCI.

4.

Experimental procedures

4.1.

Animal and experimental groups

Male Wistar rats (230–280 g) were provided by the experimental animal center of Jilin University (Changchun, China). Rats were housed in groups of six animals per cage under standard laboratory conditions (12 h–12 h light/dark cycle; temperature of 2272 1C). The animal experiments were approved by the local Ethics Committee for Animal Research at Jilin University. The care and treatment of animals conformed to international standards for animal welfare. All animal experiments were performed under general anesthesia with 10% chloral hydrate to minimize pain or discomfort. Thirty rats were randomly assigned to two main groups: the NDGA-treated group and the injury control group. These two groups were randomly divided into two subgroups to be sacrificed on day 3 (n¼ 9 per group) or day 7 (n¼ 6 per group) after SCI. All rats underwent spinal cord lateral hemisection. After the injury, rats in the NDGA-treated group were intraperitoneally injected with NDGA (Sigma, 30 mg/kg) once per day until sacrifice. Rats in the injury group received an equal volume of saline each day until sacrifice. The rats were housed in separated cages with free access to food and water.

4.2.

Rat model of spinal cord injury

One purpose of this study was to determine if NDGA could inhibit inflammation after SCI, thus providing a way to

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improve the microenvironment for cellular treatment. We used a hemisection model because it has been used to assess the treatment effects of transplanted cells after SCI (Deumens et al., 2012; Xue et al., 2012a) and also leads to secondary degenerative damage (Dusart and Schwab, 1994). Hemisections were performed as previously described (Xue et al., 2012b; Zhang et al., 2012). Briefly, male Wistar rats were anesthetized with 10% chloral hydrate solution (3.5 ml/kg). The thoracic spinal cord was exposed by laminectomy, then hemisected (right-side) at the T10 level with iridectomy scissors. To ensure lesion completeness, the injured area was repeatedly scraped along the ventral surface of the vertebral canal with a fine surgical blade. After local bleeding stopped, the surgical wound was closed layer by layer in all animals. To prevent infection, rats received subcutaneous injections of ampicillin (100 mg/kg) and gentamicin (12 mg/kg) daily.

4.6.

4.3.

At 3 days post-surgery, 3 rats from each group were sacrificed for Western blot. The spinal cord tissue 1 mm rostral and caudal to the injury site was removed and placed on ice. Tissue was homogenized in radioimmunoprecipitation (RIPA) lysis buffer (Beyotime Institute of Biotechnology, China). Tissue lysates were sonicated three times for 5 s on ice and left to stand at 4 1C for 120 min. After centrifugation at 8000 g for 5 min, the supernatants were collected. Following the determination of protein concentration with the BCA protein assay kit (Beyotime Institute of Biotechnology, China), equivalent amounts of protein (40 μg) were separated by 12% (w/v) SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, USA). The membranes were blocked with 5% (w/v) skim milk in PBS for 1 h at room temperature then incubated with primary antibodies against β-actin (a mouse monoclonal antibody, Beijing Dingguo Changsheng Biotechnology Co., Ltd., China, 1:200 in PBST), rabbit polyclonal TNF-α (Beijing Dingguo Changsheng Biotechnology Co., Ltd., China, 1:400) or rabbit polyclonal IL-1β (Proteintech, USA, 1:100) in PBST overnight at 4 1C. After incubating with horseradish peroxidase-conjugated secondary antibodies (goat-anti-mouse or goat-anti-rabbit, Beijing Dingguo Changsheng Biotechnology Co., Ltd, China, 1:5000 in PBST), proteins were visualized using an enhanced chemiluminescence system (ECL, Beyotime Institute of Biotechnology, China). Images were acquired and analyzed with a Tanon GIS gel imager system. Protein expression levels were normalized to internal β-actin.

Tissue processing

At 7 days post-surgery, 6 rats in each group were transcardially perfused with 0.9% sodium chloride and then with 4% paraformaldehyde (PFA) in PBS. The region of the spinal cord containing the injury site as well as neighboring tissue was dissected and post-fixed in 4% PFA overnight. Tissue was soaked overnight in 15% sucrose followed by 30% sucrose, and serial coronal sections with a thickness of 16 μm were obtained using a cryostat. Two out of every ten sections were taken for HE staining and Nissl staining.

4.4.

Immunofluorescence

Sections were immersed in PBS containing 0.1% Triton X-100 for 30 min. Nonspecific antibody binding was blocked with 5% bovine serum albumin (BSA) for 30 min followed by incubation with 10% normal goat serum in PBS for 30 min. The primary antibodies mouse monoclonal ED-1 (Serotec, 1:100) and mouse monoclonal glial fibrillary acidic protein (GFAP, Labvision, 1:100) were applied for 12 h in 5% BSA in PBS at 4 1C to label microglia/macrophages and astrocytes, respectively. For primary antibody detection, Cy3-conjugated secondary antibodies (goat anti-mouse, Jackson Immunoresearch Labs, 1:400) were applied for an hour at room temperature in 5% BSA in PBS. In negative control slides, the primary antibody was replaced with PBS. After staining, preparations were visualized, and digitized images were taken under a fluorescence microscope using standard fluorescent filters.

On postoperative day 3, 6 rats from each group were sacrificed by chloral hydrate overdose. Spinal cord tissue 1 mm rostral and caudal to the injury site was obtained and placed on ice. The MPO activity of tissue from each group was measured with an MPO activity assay kit (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer's protocol. Briefly, samples were homogenized and sonicated (10 s) in MPO Buffer with a ratio of 1:19 (W/V). After that, 0.9 ml of homogenate was mixed with a series of reaction solutions and incubated at 37 1C for 15 min. The absorbance at 460 nm was then measured, and MPO activity was calculated with the following formula: MPO unit/g¼OD value (sample)−OD value(control)/11.3  sample(g).

4.7.

4.8. 4.5.

MPO activity

Western blot assays

Quantification and statistical analysis

TUNEL staining

Apoptotic cells were analyzed using an In Situ Cell Death Detection kit (Roche, Shanghai, China). According to the kit's protocol, sections were incubated with permeabilization solution (0.1% Triton X-100, 0.1% sodium citrate) for 1 h at 4 1C. After washing with PBS, TUNEL reaction mixture was added to the samples for 1 h at 37 1C in a humidified and dark atmosphere. Apoptotic cells were visualized using the ConverterPOD and DAB Substrate. Sections were then counter-stained with hematoxylin (Beijing Dingguo Changsheng Biotechnology Co., Ltd., China,).

For neuronal counts, five Nissl stained sections from each animal were randomly selected that were approximately 200 mm apart from each other and running across the gray matter. All neurons within 200 mm rostral to the lesion edge in each section were counted, with counting restricted to neurons having a well-defined nucleolus and a soma rich in Nissl bodies. The quantification of macrophages/microglia and apoptotic cells was performed in the gray matter and white matter adjacent to the lesion edge as previously described (Zhang et al., 2012). Five sections from each spinal cord 200 mm apart

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from each other were randomly chosen. The cells of six microscopic fields at a magnification of 400  in each section were counted. For macrophages/microglia, only the cells with a diameter larger than 5 μm were counted no matter if it contains a nucleus. During quantification, overlapping cells were defined only if a clear outline of each cell was discernible. The mean number of all six animals in each group was calculated. For astrocytes, the data were obtained by determining GFAP positive area as a percentage of the total image area per microscopy field with Image-Pro Plus, v. 6.0, and were calculated in a similar way as that described above. All data were acquired by two people who are blind to the group assignments and expressed as the mean7SD, and a statistical analysis of the data was assessed by two-tailed Student's t-test, with P values of o0.05 considered statistically significant.

Acknowledgment This work was funded in part by a grant to Dong Chen from the Chinese National Natural Science Foundation (30970739), and by grants to J.M. Liu provided by the Jilin Province Science Foundation (20090726, 201115116). All authors are in agreement with the content and have no conflict of interest to declare.

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