DNA vaccine against NgR promotes functional recovery after spinal cord injury in adult rats

DNA vaccine against NgR promotes functional recovery after spinal cord injury in adult rats

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Research Report

DNA vaccine against NgR promotes functional recovery after spinal cord injury in adult rats Panpan Yu a , Lidong Huang a , Jian Zou a , Huiqing Zhu a , Xiaofei Wang a , Zhihua Yu a , Xiao-Ming Xu a,b,⁎, Pei-Hua Lu a,⁎ a

Department of Neurobiology, School of Medicine, Shanghai Jiaotong University, 280 South Chong Qing Road, Shanghai 200025, People's Republic of China b Kentucky Spinal Cord Injury Research Center, Departments of Neurological Surgery, and Anatomical Sciences and Neurobiology, University of Louisville, School of Medicine, Louisville, USA

A R T I C LE I N FO

AB S T R A C T

Article history:

NgR is a common receptor for three myelin-associated inhibitors and mediates their

Accepted 6 February 2007

inhibitory activities on neurite outgrowth. In the present study, we investigated whether a

Available online 17 February 2007

DNA vaccine targeting NgR could play a beneficial role in improving recovery from spinal cord injury (SCI). We demonstrated that a DNA vaccine against NgR was successfully

Keywords:

constructed and expressed efficiently in vitro and in vivo. After immunization with anti-NgR

Myelin-associated inhibitor

DNA vaccine, a low level of antibody response and a T cell-mediated immune response were

NgR

induced in the vaccinated rats. And the antisera taken from the anti-NgR DNA vaccinated

DNA vaccine

rats could partly reverse the inhibition of MAG on neurite outgrowth. When the rats were

Spinal cord injury

subjected to a contusive SCI, the vaccinated rats showed much better functional recovery

Functional recovery

than the controls. In those vaccinated rats that induced a T cell response and generated antibodies against NgR, functional improvements were even better. Histological assessments by three-dimensional reconstruction further demonstrated that the total lesion volume in the vaccinated rats was reduced by 30.8% compared to the controls. These results collectively suggest that DNA vaccine against NgR can significantly improve functional recovery in rats that received contusive SCI and that the vaccination approach may provide a promising strategy for promoting SCI repair. © 2007 Elsevier B.V. All rights reserved.

1.

Introduction

Axon regeneration in the adult mammalian central nervous system (CNS) is extremely limited and inhibitors associated with CNS myelin appear to play an important negative role. So far, at least three inhibitory components of myelin have been identified: Nogo-A (Chen et al., 2000; GrandPre et al., 2000), myelin-associated glycoprotein (MAG) (Mukhopadhyay

et al., 1994) and oligodendrocyte myelin glycoprotein (OMgp) (Kottis et al., 2002). In vitro, all three purified proteins have been identified to inhibit neurite outgrowth and induce growth cone collapse. Nogo-A is a member of the reticulon (RTN) family of proteins and has two distinct inhibitory domains: Nogo-66 and Amino-Nogo. MAG is a transmembrane protein with an extracellular segment comprised of five immunoglobulin-like domains and belongs to a subgroup

⁎ Corresponding authors. Department of Neurobiology School of Medicine, Shanghai Jiaotong University, 280 South Chong Qing Road, Shanghai 200025, People's Republic of China. Fax: +86 21 64453296. E-mail addresses: [email protected] (X.-M. Xu), [email protected] (P.-H. Lu). 0006-8993/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.02.013

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of sialic acid dependent adhesion molecule of the immunoglobulin superfamily. OMgp is a glycosyl phosphatidyl inositol (GPI)-anchored membrane protein that contains a leucine-rich repeat (LRR) domain followed by a C-terminal domain with serine/threonine repeats (Hunt et al., 2002). Though distinct in structure, all three molecules bind the same receptor NgR with high affinity and share a common set of downstream signaling pathway mediating growth inhibition (Liu et al., 2002; McGee and Strittmatter, 2003; Wang et al., 2002b). NgR is also a GPI-anchored membrane protein containing a signal sequence followed by eight LRR domains capped by N-terminal and C-terminal cysteine-rich modules (LRRNT and LRRCT) and a unique C-terminal region (CT) (Barton et al., 2003). LRRNT/LRR/LRRCT as a whole is the ligand binding domain and the CT domain is indispensable for interacting with its co-receptors that can transduce the inhibitory signal across the membrane(He et al., 2003). In the past 2 years, a soluble truncated version of NgR (Fournier et al., 2002), a Nogo-66 (1–40) antagonist peptide NEP1–40 (GrandPre et al., 2002) and IN-1, a neutralizing antibody against Nogo (Buffo et al., 2000; Fouad et al., 2001; Rubin et al., 1994) were examined

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to promote axon regeneration and functional recovery after spinal cord injury (SCI) in rats. However, these reagents only targeted a single myelin inhibitor Nogo and there were still two other inhibitors in acting. Taking the advantage of NgR, the common receptor for three myelin inhibitors, a monoclonal anti-NgR antibody was developed recently that blocked all three ligands and promoted axon outgrowth in vitro (Li et al., 2004). Furthermore, a recombinant DNA vaccine encoding domains of myelin inhibitors was generated to promote axonal regeneration in a SCI model (Xu et al., 2004). The goal of this study was two-fold: (1) to generate a recombinant DNA vaccine against NgR, the common receptor for three myelin inhibitors, and (2) to test the ability of this DNA vaccine in promoting functional recovery in a rat contusive SCI model. Our results showed that anti-NgR DNA vaccination induced cellular immune responses mediated by CD8+ T cells in all immunized rats and promoted their functional recovery after contusive SCI. Furthermore, better functional recovery was achieved in those rats with both T cell response induced and low levels of antibodies against NgR generated.

Fig. 1 – Construction and expression of hNgR-Fc DNA vaccine. (A) Restriction enzyme analysis of the recombinant plasmid pcDNA3.1-hNgR-Fc. By digesting with restriction enzyme (HindIII/BamHI/XbaI), three bands respectively corresponding to the vector (4937 bp), hNgR (1419 bp) and Fc (677 bp) were obtained. (B) COS-7 cells transfected with plasmid pCDNA3.1-hNgR-Fc and vector were stained by NgR or Fc antibodies (red). The nuclei were stained in blue with Hoechst33258. Scale bar: 50 μm. (C) Western blot analysis of the secreted fusion protein hNgR-Fc in COS-7 cells culture supernatant. The culture medium of COS-7 cells transfected with vector or with pcDNA3.1-hNgR-Fc was incubated with protein A Sepharose respectively and then used for western blot analysis. A band (hNgR-Fc), with an estimated molecular weight of 78 kDa, reacted with the anti-NgR antibody in pcDNA3.1-hNgR-Fc group. (D) Immunohistochemical staining of rat quadriceps muscle with NgR or Fc antibody. NgR and Fc immunoreactivity was found in quadriceps muscles injected with pcDNA3.1-hNgR-Fc vaccine, while not found in vector injected controls. Scale bar: 100 μm.

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2.

Results

2.1.

Construction and expression of anti-NgR DNA vaccine

To improve the efficiency of antigen presentation, human NgR containing a leading peptide was fused in frame with the Fc fragment derived from human IgG1. The recombinant plasmid pcDNA3.1-hNgR-Fc was identified by restriction endonuclease digestion (Fig. 1A) and confirmed by DNA sequencing analysis. To test the expression efficiency in mammalian cells, the recombinant anti-NgR DNA vaccine was transiently transfected into COS-7 cells. Immunoflourescence labeling with anti-NgR and anti-Fc antibodies showed that the COS-7 cells transfected with the vaccine expressed the relevant fusion protein hNgR-Fc compared with those transfected with vector alone (Fig. 1B). And from the cultured medium, a protein about 80 kDa could be precipitated with protein A Sepharose and recognized by polyclonal anti-NgR antibody (Fig. 1C). These results demonstrated that a fusion protein encoded by the recombinant anti-NgR DNA vaccine could be expressed and secreted by mammalian cells in vitro and the fusion protein retained its binding ability to protein A. To further verify its expression in vivo, the recombinant anti-NgR DNA vaccine was injected intramuscularly into the right quadriceps of rats. Immunohistochemical staining with anti-NgR or anti-Fc antibodies indicated that the fusion protein hNgR-Fc was efficiently produced by rat muscles vaccinated with the recombinant DNA. While in the left quadriceps injected with vector only, there was no fusion protein detected (Fig. 1D).

2.2.

Identification of antisera from vaccinated rats

At 6 weeks and 8 weeks after the anti-NgR DNA vaccination, the sera from vaccinated rats were collected for antibody detection. Firstly, lysates of neuroblastoma cells that endogenously express NgR were used to react with the postvaccinated sera. The results showed that the sera from only three rats among total twenty vaccinated rats were observed a band at the same position as the positive control of NgR antibody. The pre-immune sera and saline vehicle sera showed no detectable band at this position (Fig. 2A). To further confirm the specificity of the antibody induced by vaccination, a recombinant NgR protein (about 50 kDa) was used to react with the antisera by Western blot assay. The results showed that the antisera from the three rats, that had the positive band in Fig. 2A, could react with the recombinant NgR protein (Fig. 2B). Furthermore, the results of ELISA revealed that the titers of antisera from the three rats were about 1:200 (Fig. 2C). And in both Western blot and ELISA analyses, the results of sera from saline vehicle and the other vaccinated rats, as well as the pre-immune sera were all negative.

2.3. Antisera from vaccinated rats can promote the neurite outgrowth of neurons cultured on MAG To identify if the antisera, from the three vaccinated rats that anti-NgR antibody was detected in their sera, can neutralize

Fig. 2 – Detection of anti-NgR antibody in sera from vaccinated rats. (A) Lysates of SK-N-SH cells endogenously expressing NgR were blotted with post-vaccinated sera. The sera from three vaccinated rats (rat1–3) showed a band at the same position as the positive control of NgR antibody. And rat4–6 were three representative vaccinated rats without anti-NgR antibody induced. (B) Antisera from three vaccinated rats (rat1–3) that generated anti-NgR antibody could recognize recombinant 6*His-NgR protein. Sera from saline vehicle and pre-immune sera did not react with recombinant 6*His-NgR protein. (C) ELISA results showed the titer of anti-NgR antibody in sera of vaccinated rats with (rat1–3) or without (rat4 and 5) anti-NgR antibody production.

myelin associated inhibitors and promote neurite outgrowth in neurons cultured in inhibitory environment, the three antisera were added into the cultures of rat cerebellar neurons which had seeded on MAG, one of the most important myelin associated inhibitors. And a rabbit anti-NgR antibody was used as a positive control. The results (Fig. 3) showed that, compared with the neurite length (28.71 ± 1.62 μm) of neurons cultured on PLL, MAG-Fc could significantly inhibit the neurite outgrowth (reduced to 12.69 ± 1.25 μm, P < 0.01) from neurons. And the antisera from the three vaccinated rats could partly reverse the inhibition of MAG on neurite outgrowth (restored to 18.43 ± 1.32 μm, P < 0.05, vs. neurons on MAG). The rabbit antiNgR antibody (positive control) could reverse the inhibition of

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Fig. 3 – Antisera from vaccinated rats promoted neurite outgrowth of neurons cultured on MAG. (A) Representative images of cerebellar neurons cultured on PLL, MAG, MAG combined with antisera from vaccinated rats or MAG combined with rabbit anti-NgR antibody (positive control). Neurite outgrowth of cerebellar neurons was markedly inhibited by MAG, while antisera from vaccinated rats or anti-NgR antibody could partly reverse its inhibition. (B) Average neurite lengths of each group were measured and statistically analyzed. Data were presented as mean± SEM (*P < 0.05; **P < 0.01), scale bar: 50 μm. MAG more significantly (restored to 23.56 ± 1.32 μm, P < 0.01, vs. neurons on MAG). It is possible that the anti-NgR antibody in the three antisera from vaccinated rats was comparatively weak, which could not sufficiently block all the activity of NgR.

2.4. DNA vaccination promotes functional recovery in rats after SCI After the third vaccination, the rats were subjected to a contusive SCI and behavioral assessments were made before and after SCI up to 8 weeks. The Basso, Beatlie and Bresnahan (BBB) locomotor test showed that at 24 h after SCI, all rats obtained a minimal function with a BBB score of 0. From the first week on, the BBB scores in DNA vaccinated group were consistently higher than those in the control group and the differences between the two groups were statistically significant starting from week 3 and continued until week 8 (Fig. 4A). Furthermore, in the vaccinated rats that the anti-NgR antibody was detected in their sera (n = 3) showed much higher BBB scores than the other vaccinated rats whose anti-NgR antibody was not detected (n = 10) (Fig. 4B). Nonetheless, vaccinated rats with or without vaccine-induced antibodies showed significantly higher BBB scores than the control rats (Fig. 4B). The sham rats in BBB test all achieved maximal scores. In addition to BBB scoring, two other functional measures, grid walking and footprint analysis, were employed. Grid walking requires more sophisticate sensory and motor skills. In this test, the percent of missteps of hind paws was dramatically reduced in vaccinated rats compared to the saline controls (Fig. 5). In the footprint analysis at the eighth week after SCI, rats in the DNA vaccinated group had visible prints of all five toes. While in the saline vehicle group, the prints of toes were not clearly separated indicating signs of toe dragging (Fig. 6A). Furthermore, the base of support was

significantly reduced in the vaccinated group compared to the saline vehicle group (Fig. 6B).

2.5. DNA vaccination reduces the lesion volume of contused spinal cord Quantitative analyses of total lesion volume were performed in rats sacrificed at the eighth week after a contusive SCI. Stereological assessments by 3D reconstruction of individual lesion cavity indicated that the total lesion volume in the vaccinated rats was reduced by 30.8% compared to the controls (Fig. 7).

2.6. DNA vaccination induces accumulation of CD8+ T cells in the contused spinal cord Eight weeks after contusive SCI, rats were sacrificed and spinal cord sections including the epicenter of the lesion site were subjected to immunohistochemical staining with antiCD4 and anti-CD8 antibodies. Unexpectedly, in the samples from vaccinated rats, large numbers of CD8+ T cells were observed around the lesion cavity as well as the distant regions rostral and caudal to the lesion epicenter (Figs. 8A, C). However, only a few CD4+ T cells were found within the lesion site and were almost absent in the areas rostral and caudal to it (Fig. 8C). In the saline vehicle or sham-operated group, both CD4+ and CD8+ T cells were not observed at 8 weeks after SCI (Figs. 8B, C).

2.7. The expression of endogenous NgR in spinal cords of vaccinated rats To observe whether anti-NgR DNA vaccination would affect the expression of endogenous NgR, spinal cord sections from

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Fig. 4 – BBB test showed functional improvement in hNgR-Fc DNA vaccinated group after spinal cord contusion. (A) Comparison between DNA vaccinated group and saline vehicle group. (B) Comparison among DNA vaccinated groups with (Vaccine Ab+) or without (Vaccine Ab−) antibody production and saline vehicle group (*P < 0.05; **P < 0.01, compared with saline vehicle group; #, P < 0.01, compared with Vaccine Ab− group).

vaccinated rats and saline vehicle rats, 8 weeks after contusion, were stained with a rabbit anti-NgR antibody. The results showed that NgR immunoreactivity in the sections from the three vaccinated rats that anti-NgR anti-

Fig. 5 – Gridwalk testing 1–8 weeks after spinal cord contusion. Grid walking was significantly (*P < 0.01) improved in DNA vaccinated rats compared with saline vehicle controls.

Fig. 6 – Footprint analysis at 8 weeks after contusive SCI. (A) Fore paw footprints (red) and hind paw footprints (blue, arrow) from rats before contusion (pre-SCI), and from DNA vaccinated and saline vehicle rats 8 weeks after contusion. ‘d’ shows the vertical distance between the hind paws. (B) The distance of the base of support between the hind paws in DNA vaccinated group was significantly reduced comparing with saline vehicle group (**P < 0.01).

body was induced was much weaker than that from saline vehicle rats (P < 0.05, Fig. 9). However, the expression level of NgR in the sections from vaccinated rats that did not generate anti-NgR antibody showed no significant difference from that of saline vehicle rats, though there was a trend of decline (Fig. 9). These results suggested that anti-NgR DNA vaccination had not markedly affected the expression of endogenous NgR in spinal cord. The low immunoreactivity of NgR in the three vaccinated rats might be due to their endogenous anti-NgR antibody induced by DNA vaccination; i.e. the endogenous anti-NgR antibody could enter the injury spinal cord and block its target NgR, which consequently led to the decrease of NgR immunoreactivity by additional staining with a rabbit anti-NgR antibody.

3.

Discussion

Nogo-A, MAG and OMgp are the major myelin-associated inhibitors that play important roles in the failure of axon

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Fig. 7 – Stereological analysis of lesion volume 8 weeks after contusive SCI. (A) Left column, photomicrographs of representative spinal cord longitude-sections with the biggest boundary of lesion cavity among each set of sections. Right column, the corresponding three-dimensional (3D) images of lesion cavities reconstructed by neurolucida. (B) Quantitative data of lesion volumes from vaccinated group and saline vehicle group, respectively. It showed a significant reduction of total lesion volume in vaccinated rats compared to saline vehicle group (*P < 0.05).

regeneration in the adult mammalian CNS. Vaccines against these inhibitors have been generated to overcome the inhibitory activity associated with myelin in SCI animal models and are a promising strategy for clinical applications (Huang et al., 1999; Sicotte et al., 2003; Xu et al., 2004). The discovery that NgR is a common receptor for the three myelin inhibitors has provided a new potential target for vaccination therapy of CNS diseases. In this study, we reported the development of a DNA vaccine against NgR which resulted in a significant functional recovery in rats that received NgR DNA vaccination compared to the non-vaccinated control group. Our results revealed that rats induced antibodies against NgR showed the best functional recovery. This recovery was likely mediated mainly by the action of vaccine on blocking the interactions between NgR and the three myelin-associated inhibitors. However, the vaccinated rats that had no detectable anti-NgR antibodies also displayed a significant functional improvement compared to the controls. And NgR DNA vaccination could significantly reduce the spinal cord lesion volumes which might closely relate to the functional recovery after SCI. These results suggested that, in addition to blocking the actions of myelin-associated inhibitors, NgR vaccination might have a neuroprotective effect. While in our contusive SCI model, we could hardly distinguish whether the functional recovery resulted from axonal regeneration or neuroprotection.

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DNA vaccine as the third generation of vaccine has a variety of applications and a major issue of DNA immunization today is to enhance its potency (Henke, 2002; Ulmer et al., 2006). In the present study, we introduced the Fc region of human IgG1 into the DNA vaccine to improve the efficiency of antigen presentation by targeting the antigen to antigen presenting cells (APCs) because most of APCs express Fc receptors (Liu et al., 2006; Qin et al., 2006; Regnault et al., 1999). Unexpectedly, after vaccination with the recombinant antiNgR DNA vaccine, only a small percentage of rats induced a low level of antibodies against NgR. In addition, all of the vaccinated rates showed a selective infiltration of CD8+T cells around and distal to the lesion site after SCI. It was reported that the inflammatory cell infiltration after CNS injury reached a peak on day 7, after which their numbers decreased. It was also shown that after CNS injury, T cells entered the lesion area with fewer numbers and were localized on a more restricted region around the injury site compared to the PNS injury (Moalem et al., 1999). Furthermore, it has been widely accepted that only activated T cells could migrate into the CNS and that only those who could recognize CNS antigens remained in the CNS (Hickey et al., 1991). In our study, the accumulation of CD8+ T cells remained at 8 weeks after SCI, either around or distal to the lesion, in all vaccinated rats compared to no CD8+ T cell infiltration in the Saline vehicle or sham-operated controls. We believe that these infiltrated CD8+ T cells in the spinal cord should be induced specifically by anti-NgR DNA vaccination. It indicates that in this study, the anti-NgR DNA vaccine has induced both humoral (generation of NgR antibodies) and cellular immune responses and that CD8+ T cells mediated the latter response in the NgR DNA vaccinated rats. The functional improvement along with the reduction of lesion size in vaccinated rats without detectable anti-NgR antibody, suggests that the CD8+ T cell-mediated cellular immune response induced by DNA vaccine is also beneficial for the repair of SCI. Although the role of infiltrated T cells in the injured spinal cord remains controversial, recent reports showed that CNS specific autoimmune T cells such as those specific for the myelin basic proteins could be protective from secondary injury (Hofstetter et al., 2003; Kipnis et al., 2002; Wolf et al., 2002). It has also been demonstrated that T cells produced neurotrophic factors such as NGF, BDNF and NT-3 and the secretion of these neurotrophins by T cells was antigen dependent (Hauben et al., 2000; Moalem et al., 2000). In addition, a recent study reported that vaccination with a Nogo-A derived peptide after SCI promoted recovery via a Tcell-mediated neuroprotective response (Hauben et al., 2001). Based on these findings, we suggest that the improvement of functional recovery in vaccinated rats that showed only cellular immune response could be mainly due to the CD8+ T cells infiltration. However, the actual function and the mechanism by which CD8+ T cells activation mediates by anti-NgR DNA vaccination remain to be investigated. There are mainly two pathways for antigen presentation. One is via the MHC class II molecules. It may prime the induction of antibody response as well as CD4+ T cell activation. And another way is via MHC class I molecules to activate CD8+ T cells. In general, the nature of immune response induced by DNA vaccine is dependent on the

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Fig. 8 – CD8+ T cells accumulated in spinal cord of vaccinated rats 8 weeks after contusion. (A) and (B) are the overviews of CD8+ T cell in the DNA vaccinated rat (A) and saline vehicle control (B) from the lesion site to the rostral area. Boxed areas in (A) and (B) are viewed in the right at higher power, respectively. (C) Quite a few CD8+ T cells were shown both at the lesion site and rostral or caudal to the lesion site in DNA vaccinated rats. Only several CD4+ T cells were found within the lesion site (arrow). Both CD8+ and CD4+ T cells were absent in Sham-operated and saline vehicle controls.

character of the protein encoded by plasmid DNA as well as the routes and methods of vaccine delivery. As the anti-NgR DNA vaccine, developed in this study, mainly induced a CD8+ T cell response, it indicated that the product of this DNA vaccine being taken up by myocytes and/or professional antigen-presenting cells (APCs) was subsequently presented by MHC class I molecules and thus recognized by CD8+ T cells. However, we could not explain the mechanisms behind it at present. The control of the nature and the extent of immune response induced by DNA vaccine is still a puzzle in DNA vaccination (Moore et al., 2002; Ulmer et al., 2006). Based on the fact that a subgroup of vaccinated rats initiated both cellular and humoral immunity and that these rats showed better functional recovery than those in which only T cell response was initiated, we suggest that antibodies induced by the anti-NgR DNA vaccine played an active role in mediating functional recovery following SCI in the present study. This was likely mediated by blocking the activity of NgR after SCI. It also suggests that the antibodies induced by the anti-NgR DNA vaccine were more effective than the T cell activation-mediated recovery of function. Further studies are needed to optimize the DNA vaccination strategy such as modifying the routes and improving methods of DNA delivery

to stimulate stronger antibody responses. Perhaps a peptide vaccine against NgR will be a promising alternative approach to induce a robust antibody response. In this study, we employed a vaccine approach targeting NgR for SCI repair. As expected, it could promote functional recovery in a rat contusive SCI model. It has been reported that a neutralizing anti-NgR monoclonal antibody could reverse the inhibition of neurite outgrowth by CNS myelin in vitro (Li et al., 2004). The other study also showed that siRNA-mediated knockdown of NgR resulted in disinhibition of neurite outgrowth of dorsal root ganglia neurons on CNS myelin (Ahmed et al., 2005). Our results together with these in vitro studies further demonstrated that NgR might be important in mediating the inhibitory actions of Nogo, MAG and OMgp. However, Zheng et al. (2005) recently reported that knockout of the NgR in mice failed to reduce neurite inhibition in vitro or promote corticospinal tract regeneration in vivo. Why does NgR gene knockout result in inferior regeneration compared to the treatment with neutralizing agents mentioned above? One possible explanation is that, besides blocking the action of NgR, vaccine as well as the other neutralizing agents targeting NgR may still have other indirectly pathway to promote regeneration such as by up-regulating some growth factors and growth-

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Animals were obtained from the Animal Breeding Center of Chinese Academy of Sciences and housed in a light- and temperature-controlled room. All surgical interventions and postoperative animal care were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996, USA) and were approved by the Animal Use and Care Committee of School of Medicine, Shanghai Jiaotong University.

4.2. Construction of expression plasmid pcDNA3.1-hNgR-Fc The full-length human NgR and human IgG Fc cDNA were amplified by PCR using the following forward and reverse primers respectively: 5-TTAAGCTTACCATGAAGAGGGCTCCGCTGG-3′ (HindIII) and 5-TTGGATCCGCAGGGCCCAAGCACTGTC-3′ (BamHI) for hNgR; 5-TTGGATCCAAAACTCACACATGCCCACC-3′ (BamHI) and 5-TTTCTAGATCATTTACC CGGAGACAGGGAGA-3′ (XbaI) for Fc. The forward primer of hNgR contained an ATG-start code and the reverse primer of Fc contained a stop code. Then these two fragments were sequentially inserted into pcDNA3.1/HisA vector (Invitrogen, Carlsbad, California). The resultant vector was identified by restriction enzyme analysis and confirmed by DNA sequencing.

4.3.

Fig. 9 – Immunohistochemical staining for NgR in spinal cord of vaccinated rats 8 weeks after contusion. (A) Sections from saline vehicle rats, DNA vaccinated rats without generating anti-NgR antibody (vaccine Ab−) or DNA vaccinated rats that generated anti-NgR antibody (vaccine Ab+) were stained with a rabbit anti-NgR antibody. Representative images showed a decrease of NgR immunoreactivity in vaccine Ab (+) group compared with saline vehicle. Micrographs were taken from longitude sections at corresponding areas of grey matter (GM) or white matter (WM) 2–4 mm rostral to the lesion site. Scale bar: 50 μm (B) The Quantifications of NgR immunoreactivity (*P < 0.05, compared with saline vehicle).

related proteins (Bareyre et al., 2002; Teng and Tang, 2005). It is also likely that NgR is not the only receptor to mediate the inhibitory action. And once the NgR gene was deleted, the other receptors might be up-regulated compensatively. However, this compensation might not appear or was fairly weak in the models treated with neutralizing approaches as well as the siRNA-mediated-knockdown of NgR.

4.

Experimental procedures

4.1.

Animals

A total of 35 adult female Sprague–Dawley (SD) rats, weighing 180–200 g, were used for DNA or 0.9% saline vaccination.

hNgR-Fc fusion protein expression in vitro and in vivo

For in vitro assay, COS-7 cells grown in 100-mm dishes or on coverslips were transiently transfected with recombinant plasmid or mock transfected with vector pcDNA3.1 using Lipofectamin™ 2000 reagent (GibcoBRL, Life technologies, USA). After 48 h, the cells on coverslips were rinsed in 0.01 M PBS and fixed with 4% paraformaldehyde (PFA) in PBS for 20 min at room temperature (RT), after three rinses in PBS, the cells were incubated with 10% normal goat serum (NGS) in PBS containing 0.3% Triton X-100 for 1 h at RT and then incubated with rabbit anti-NgR antibody (1:1000, prepared by our lab) or rabbit anti-Fc antibody (1:1000; Rockland) overnight at 4 °C. On the following day, rhodamine-conjugated goat anti-rabbit IgG (1:60; Sigma) was applied to the cells for 1 h at 37 °C, the coverslips were then rinsed and mounted with Gel/Mount aqueous mounting media (Biomeda Corp., Foster City, CA) containing Hoechst 33342 (0.5 M; Sigma, St. Louis, MO). The results were examined using an Olympus BX60 microscope. The culture medium in 100-mm dishes was harvested and incubated with Protein A Sepharose CL-4B (Amersham Pharmacia Biotech, Uppsala, Sweden). The eluted proteins were fractionated on SDS-PAGE, then transferred to a nitrocellulose membrane (Schleicher & Schuell, Dassel, Germany) and detected by Western blot analysis. Briefly, the membrane was blocked with 2% bovine serum albumin (BSA, Sigma) in Tris-buffered saline containing 0.1% (v/v) Tween20 (TBST-2%BSA) for 30 min at 37 °C, and then incubated with rabbit anti-NgR antibody diluted in TBST-2%BSA (1:3000) for 1 h at 37 °C. After washing with TBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (SABC, 1:10,000) at 37 °C for 1 h. After the final wash, the membrane was visualized with the super ECL kit (Pierce, Rockford, IL) following the manufacture's instructions.

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For in vivo assay, recombinant DNA vaccine and vector were injected intramuscularly into the right and the left quadriceps of rat, respectively. After 48 h, the corresponding muscles were taken out for frozen section preparation. Then the sections were subjected to immunohistochemical staining with anti-NgR (1:1000) or anti-Fc (1:1000) antibody using the avidin-biotinylated peroxidase complex (ABC) immunoperoxidase-DAB method (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) according to the manufacturer's recommendation.

4.4.

DNA vaccination

The recombinant plasmid was purified with the EndoFree Plasmid Mega Kit (Qiagen, Hilden, Germany) according to the manufacturer's instruction. DNA plasmid was adjusted to a final concentration of 1.5 mg/ml for injection. The quantity and purity of isolated plasmid DNA were assessed spectrophotometrically and the ratio of OD260/OD280 for immunization was >1.8. Rats were first injected with 100 μl of 0.25% tetracaine hydrochloride via intramuscular route into the quadriceps of both hind legs. After 24 h, DNA was injected intramuscularly at the same position. Lipofectamine2000 was used as an adjuvant. Briefly, Lipofectamine2000 was combined with plasmid DNA at a ratio of 1:2 and incubated 20 min at room temperature to form complexes. Every rat was injected with 300 μg plasmid once and immunized three times at a 3week interval. Sera were collected at 4, 6 and 8 weeks after the first immunization.

4.5.

Antibody detection by Western blot and ELISA

At 6 weeks and 8 weeks after the anti-NgR DNA vaccination, sera from vaccinated rats were collected for antibody detection. (1) The lysates of a human cell line SK-N-SH which endogenously express NgR were fractionated on SDSPAGE and reacted with pre-immune sera, saline vehicle sera and post-vaccinated sera (1:50) by Western blot analysis. A rabbit anti-NgR antibody (1:5000, prepared by our lab) was used as a positive control. (2) A recombinant NgR protein tagged with 6*His expressed in E. coli was used to react with pre-immune sera, saline vehicle sera and post-vaccinated sera by Western blot assay. (3) The titers of the postvaccinated sera were assessed by ELISA. Briefly, microtiter plates coated with 100 μl purified NgR (1 μg/ml), overnight at 4 °C, were incubated with sera from vaccinated rats, which had serially diluted with PBS, for 2 h at 37 °C. Bound antibody was reacted with HRP-conjugated goat anti-rat IgG (SABC, 1:5000) for 1 h at 37 °C. The optical density (OD) at 490 nm was measured with an ELISA reader. Sera that produced net OD values greater than the mean OD plus 3 SD obtained with a panel of pre-immune sera were considered to have significant antibody responses.

4.6.

Neurite outgrowth assay

Neurite outgrowth assay was performed with cerebellar neurons from P7–9 rats as previously described (Wang et al., 2002a; Mi et al., 2004). Glass coverslips in 24-well plate were coated with 100 μg/ml poly-L-lysine (PLL, sigma) and washed, and then 8 μl drops PBS containing 0 or 100 ng MAG-

Fc (R&D systems) were spotted and dried. Cerebellar neurons were dissected, dissociated and resuspended with medium in the presence or absence of antisera from vaccinated rats or sera from saline vehicle rats (1:100), and then plated respectively at a density of 1 × 105 cells per ml onto corresponding coveslips pre-coated with immobilized substrates (PLL or PLL + MAG-Fc). A rabbit anti-NgR polyclonal antibody (1:1000) was used as a positive control. Cells were cultured 24 h before fixed with 4% paraformaldehyde and the neurite lengths were measured, with a Neurolucida system (MicroBrightField Inc., Colchester VT), from at least 100 neurons per condition, and from three independent experiments in duplicate wells. Average neurite length was used for statistic analysis.

4.7.

Spinal cord contusion injury model

After the third vaccination, rats were subjected to a contusive SCI. Animals for contusion included 3 groups: DNA vaccination group (n = 13), saline vehicle (0.9%; n = 7) group and shamoperated group (n = 5) which contained both DNA vaccinated and saline vehicle rats. Contusive SCI was performed as reported previously (Wang et al., 2006a; Yan et al., 2003). Briefly, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg, i.p.) and their spinal cords were exposed by laminectomy at the level of T9. Body temperature was maintained at 37 °C during the period of anesthesia with a temperature-controlled heating pad. After the spinous processes of T8 and T11 were clamped to stabilize the spine, a 10 gm rod (2.5 mm in diameter) was dropped onto the exposed cord from a height of 12.5 mm, using an NYU impactor. After contusion, the muscles and skin were closed in layers, and rats were placed in a temperature- and humidity-controlled chamber. Manual bladder emptying was performed three times daily until reflex bladder emptying was established. Sham-operated rats were laminectomized but receiving no contusion.

4.8.

Behavior analysis

Gross locomotor recovery after SCI was scored in an open field in accordance with BBB locomotor rating scale of 0 (complete paralysis) to 21 (normal locomotion) (Basso et al., 1996). One week before surgery, rats were acclimated to the testing environment. At 24 h after contusion and weekly thereafter up to 8 weeks post-contusion, each rat was observed for 4 min. Each hind limb was scored by two observers blinded to the treatment. For each animal, the scores for both hind limbs were averaged to yield one score per test session. For grid walking (Merkler et al., 2001; Metz et al., 2000), the ability of rats to walk on a horizontal wire grid was determined to assess their locomotion. Rats were tested 1–8 weeks after the contusive SCI. Each rat was allowed to walk freely around for 4 min. If the hind paw of one side protruded entirely through the grid, with all toes and heel extended below the wire surface, it was counted as a misstep. The total number of steps made by the hind limb of the same side was also counted. The result was expressed as the percentage of missteps which is calculated as: the number of missteps / (the number of missteps + the number of normal steps).

BR A IN RE S E A RCH 1 1 47 ( 20 0 7 ) 6 6 –7 6

For footprint assay (De et al., 1982; Merkler et al., 2001), the animal's hind paws were inked and footprints were made on paper covering a narrow runway of 100 cm long and 7 cm wide. The base of support was determined by measuring the core to core distance of the central pads of the hind paws. Each rat tested its footprints before and 8 weeks after spinal contusion, respectively. Blind scoring ensured that observers were not aware of the treatments received by each rat.

4.9. Histological and three-dimensional (3D) reconstruction of lesion volume After the final behavioral assessments, rats were deeply anesthetized by intraperitoneal injections of sodium pentobarbital (80 mg/kg) and sacrificed by transcardial perfusion with 100 ml of 0.9% saline followed by 400 ml of 4% paraformaldehyde in cold 0.1 M phosphate buffered saline (PBS, pH 7.4). After perfusion, the spinal cord containing the injury epicenter was carefully removed and post-fixed for an additional 2 h in the same fixation solution. The specimen was transferred to a solution containing 30% sucrose in 0.1 M PBS (pH 7.4) and stored at 4 °C. For frozen section preparation, the spinal cord segment was embedded in tissue freezing medium (Tissue-Tek, Miles, Elkart, IN), serial 20-μm-thick longitudinal sections containing the entire injury site were obtained using a cryostat (Leica CM1900, Bannockburn, IL) and thaw-mounted on gelatin-coated slides. A set of slides containing serial sections spaced 40 μm apart were used to assessment of the lesion volume. The lesion cavity was three-dimensionally reconstructed with an Olympus BX60 microscope attached to a Neurolucida system (MicroBrightField Inc., Colchester VT). The lesion volume was used for statistical analyses.

4.10.

Immunohistochemistry

To identify the infiltrated lymphocytes, sections from each group were immunohistochemically stained for CD4 or CD8. Briefly, after being blocked with 10% normal goat serum in PBS for 1 h at RT, sections were incubated with a mouse anti-CD4 (1:100, Serotec) or a mouse anti-CD8 antibody (1:50, Serotec) in PBS containing 1% BSA overnight at 4 °C. After several rinses in 0.01 M PBS, the sections were reacted with Cy3-conjugated goat anti-mouse IgG (1:250, Jackson ImmunoResearch Lab, West Grove, PA) for 1 h at 37 °C. Sections were washed and coverslipped. Slides were examined using an Olympus BX60 microscope. To detect the expression of endogenous NgR, sections of spinal cord from each group were stained with a rabbit antiNgR polyclonal antibody (1:1000) using the avidin-biotinylated peroxidase complex (ABC) immunoperoxidase-DAB method (Santa Cruz Biotechnology, Inc. Santa Cruz, CA) according to the manufacturer's recommendation and as previously described (Wang et al., 2006b).

4.11.

Statistical analysis

Behavioral scores from BBB analysis, grid walking and mean values of lesion volume were compared using a two-tailed Student's t-test. Average neurite lengths, base of support analysis and quantitative of the density of NgR immunohis-

75

tochemical staining were compared using a one-way analysis of variance (ANOVA). Differences were considered to be statistically significant when P < 0.05.

Acknowledgments We thank Zhen Guan, Research Associate from the Spinal Cord Injury Research Center, Ohio State University, for his technical assistance for inducing spinal cord contusion and behavioral testing. This work was supported by the grant from Major State Basic Research Development Program of China (973 Project) (NO, 2003CB515302) and the Science and Technology Development Foundation of Shanghai (NO, 02JC14014). REFERENCES

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