[1] 12w Neuroprotective Effect of Vaccination with Autoantigen-Pulsed Dendritic Cells After Spinal Cord Injury

[1] 240w Background. Studies have shown that the development of a properly controlled autoreactive T cell response can serve as a therapeutic approach for spinal cord injury (SCI). Thus, vaccination with mature dendritic cells (DCs) pulsed with central nervous system (CNS) antigens that can prime autoreactive T cells have the potential for treating SCI.Materials and Methods. Mature DCs pulsed with spinal cord homogenate (SCH), nonpulsed mature DC or phosphate-buffer solution (PBS) were injected into spinal cord-injured mice peritoneally. The functional recovery of spinal cord was measured by Basso mouse scale and footprint analysis. Spinal cord specimen was preserved for immunohistochemical staining to detect T cell infiltration, differentiation of neural stem/progenitor cells, and tissue preservation. RT-PCR and enzyme linked immunosorbent assay (ELISA) was used to detect the expression of cytokines and neurotrophic factors.Results. Vaccination with DCs pulsed with SCH promoted pronounced functional recovery from SCI. The neuroprotection induced by SCH-pulsed DCs (SCH-DC) correlated to the accumulation of CD4 D T cells in the lesion site. SCH-DC markedly affected the production of interferon-g, interleukin-12, and granulocyte-macrophage colony stimulating factor. SCH-DC also promoted expression of neurotrophic factors in the injured spinal cord and spleen cells. Furthermore, vaccination with SCH-DC enhanced neuronal differentiation of neural stem/progenitor cells, and it led to better tissue preservation. Conclusion.The results of the present study suggest that DC-mediated immune regulation may be a potential therapeutic approach aimed at shifting the balance between immune and nerve cells in order to treat SCI.

[1] 108w Following primary mechanical spinal cord injury (SCI), secondary pathology processes contribute to further damage. The local immune response has been identified as a key component of secondary damage following SCI [1,2]. It is traditionally believed that the immune response in the damaged central nervous system (CNS) is harmful and should therefore be suppressed [3]. However, accumulating evidence indicates that the immune system can play both detrimental and beneficial roles in the nervous system [4]. Various immune cells including T cells, NK cells, macrophages, dendritic cells (DCs), and microglia participate in limiting damage to the nervous system during CNS trauma and in the process of repair after injury [4,5].

[2] 145w Previous studies have shown that self-reactive T cells under certain conditions can be harmful to CNS because the transfer of myelin basic protein (MBP) peptide-specific T cells to naive recipients causes experimental autoimmune encephalitis (EAE) [6,7]. However, recent studies have suggested that under some conditions, autoimmune T cells may convey ''protective autoimmunity'' to neurons and axons after mechanical nerve injury [5,[8][9][10]. In rodent models of optic nerve crush or SCI, passive or active immunization with T-cells specific to CNS-associated myelin antigens reduces neuronal loss and promotes immune neuroprotection [11][12][13][14]. Studies over the last few years have provided evidence indicating that a properly controlled T cell-mediated autoimmune response plays a key role in neurogenesis of the injured spinal cord and its protection from secondary degeneration [5]. Therefore, boosting the T cell response specific for CNS antigens could be considered a potential way of ameliorating SCI [8].

[3] 156w DCs have emerged as a key component of the immune balance and are critically involved in the T cell-mediated immune response. Current data indicate that DC functions are related to the stage of their maturation. After an antigenic stimulus, immature DCs become fully mature DCs, as shown by the up-regulation of major histocompatibility complex (MHC) and costimulatory molecules such as CD80 and CD86. These decisive signals from the DCs, concurrently with the release of proinflammatory cytokines, induce an antigen-specific T-cell immune response [15]. The capacity for the in vitro generation and manipulation of immunocompetent DCs promotes their use in immunotherapy approaches, particularly in cancer. Studies have shown that injection of DCs loaded with tumor-associated antigens leads to an anti-tumor immune response, and multiple clinical trials have been carried out using this method [16,17]. Moreover, in animal models of different diseases, vaccination with specific antigen-or peptide-pulsed DCs has prevented the development of arthritis, diabetes, and EAE [18][19][20].

[4] 152w It is now generally accepted that antigen-pulsed DCs are efficient at priming the proliferative response of CD4 þ T-cells and T-cell clones [21,22]. Immature DCs are poor antigen-presenting cells that are involved in the induction of peripheral T cell tolerance, while mature DCs are endowed with the capacity to initiate an antigen-specific T cell response [23,24]. Previously performed studies have reported the neuroprotective effect of vaccination with immature DCs [25,26], but a potential caveat to these studies is that once immature DCs are introduced into the host, immature DCs might cause tolerance instead of antigen-specific T cell proliferation. Therefore, to evoke T cell-mediated protective autoimmunity in the present study, we used mature DCs to investigate their therapeutic potentials for treating SCI. We show that vaccination with SCH-pulsed DCs (SCH-DC) leads to a protective effect from SCI in mice, and we explore the cellular basis and cytokine network in the injured spinal cord.

[1] 115w Motor recovery was assessed using the BMS openfield rating scale. Male BALB/c mice were subjected to a severe SCI as described in the Materials and Methods section. After 24 h, mice were injected peritoneally with PBS, DC, or SCH-DC. The mice treated with PBS were used as controls. As shown in Fig. 1A, during the entire experimental period, treatment with DC provided limited motor recovery that was not significantly different from that observed in the control group. However, injured mice treated with SCH-DC showed significantly improved recovery compared with other groups, which was detectable as early as 14 d after SCI. This suggests that vaccination with SCH-DC is effective at promoting functional recovery after SCI.

[2] 61w To further evaluate functional recovery in SCH-DCtreated mice, footprint analysis was performed. Footprint analysis at the eighth week after SCI revealed that mice in the SCH-DC group had relatively visible prints of plantar paws with short toe dragging, while in the control group, the plantar paw impressions were not detected or not clearly separated with the long-lasting toe dragging (Fig. 1B).

[3] 151w We first examined whether the observed differences in the extent of recovery could be correlated with local immunological changes. Because antigen-pulsed DCs are efficient at priming proliferative responses of CD4 þ T-cells [21], the CD4 þ cells in the injured spinal cords were examined using immunohistologic labeling. The spinal cords were taken on d 7 after injury from each experimental group. As shown in Fig. 2A, very few CD4 þ T cells were detected in the lesion site of the control group. This result implies that in the natural condition, the number of T cells infiltrating into the injured spinal cord within 1 wk is rather limited. In the DC-treated group, there were no statistically significant differences in the amounts of CD4 þ T cell infiltration compared with the control group. However, significantly higher numbers of CD4 þ T cells were observed in the SCH-DC group compared witih the control group.

[4] 197w To explore the protective mechanism underlying the SCH-DC-mediated immune response, the production of cytokines in injured spinal cords was analyzed at d 7 after injury. SCH-DC induced a significant increase in IFN-g production (Table 2) in the injured spinal cord compared with the other groups. However, there were no differences in the production of IL-4 and IL-10 among the control, DC, and SCH-DC groups. Moreover, the mRNA expression of IFN-g was significantly increased in SCH-DC group (Fig. 3), which is consistent with the protein expression pattern. These results suggest that SCH-DC induced a potential Th1 immune response in the injured spinal cord. Furthermore, a decrease of IL-17 and a significant increase of IL-12 and granulocyte-macrophage colony stimulating factor (GM-CSF) were detected after treatment with SCH-DC (Table 2). Next, to dissect whether the local immune response induced by SCH-DC could influence the production of proinflammatory cytokines, we determined the production of IL-1b, NO, and PGE 2 in the injured spinal cord (Table 2). Our results showed that no significant differences were observed in the production of IL-1b, NO, PGE 2 after SCH-DC administration in mice, suggesting that SCH-DC did not significantly alter the local production of proinflammatory cytokines.

[5] 191w To investigate whether the local immune response induced by SCH-DC could influence the synthesis of neurotrophic factors, protein production and mRNA expression of BDNF and NT-3 were assessed in the injured spinal cord on d 7 after injury. The production and mRNA expression of BDNF and NT-3 were significantly increased in the SCH-DC group compared with the control and DC groups (Fig. 4A). We next asked whether administration of SCH-DC could also affect the levels of neurotrophic factors in the spleen. The production of BDNF and NT-3 was analyzed in spleen cells that had been cultured for 48 h, and mRNA expression of BDNF and NT-3 was measured in freshly isolated spleen cells. The secretion of BDNF from splenocytes obtained from mice treated with SCH-DC was higher than that in mice treated with PBS (Fig. 4B). An increase in BDNF mRNA expression was observed in freshly isolated spleen cells from SCH-DC-treated mice compared with control and DC-treated mice (Fig. 4B). NT-3 production was not detected in the supernatants of spleen cell cultures in any group, and no significant difference in NT-3 mRNA expression was observed among any groups (data not shown).

[6] 13w The Effect of SCH-DC on the Differentiation of Neural Stem/ Progenitor Cells (NSPCs)

[7] 127w It is generally accepted that proliferating NSPCs in the injured spinal cord differentiate mainly into astrocytes, which are likely to be nestin þ GFAP þ . Therefore, we focused on the effects of SCH-DC treatment by evaluating the number of nestin þ GFAP À NSPCs. We observed a great number of nestin þ GFAP À cells in the SCH-DC group, while the number of nestin þ GFAP À cells in the control and DC groups were limited (Fig. 5A). This result prompted us to examine whether SCH-DC treatment affects neuronal differentiation of NSPCs in the injured spinal cord. Staining for nestin and b-tubulin III showed significantly more nestin/btubulin III double-positive cells in mice that had received treatment with SCH-DC compared with control and DC-treated mice (Fig. 5B).

[8] 113w To determine the effect of SCH-DC on the size of the injury site, we stained serial longitudinal sections of the spinal cord with anti-GFAP antibodies at the eighth wk post-injury and measured the unstained area. As shown in Fig. 7, the GFAP-delineated area was significantly smaller in mice treated with SCH-DC than in control and DC-treated mice. No statistically significant difference in the size of the injury site was found between DC-treated mice and control mice after SCI. Quantitative assessment of the size of the lesion site (three sections per spinal cord) showed that SCH-DC treatment led to a significant reduction in lesion area following SCI compared with the control group (Fig. 7D).

[1] 71w In the present study, we demonstrated that mature DCs pulsed with SCH induced significant infiltration of CD4 þ T cells in the lesion site and activated local microglia/macrophages, which may lead to a beneficial immune response in the injured spinal cord. The microenviron-ment constituted by the local immune response and the up-regulation of growth factors leads to better tissue preservation, enhanced neuronal differentiation of neural stem/progenitor cells, and improved functional recovery.

[2] 129w Three types of ''immunotherapy'' DCs have been described: immature, semi-mature, and fully mature. Mature DCs were used in the present study because they FIG. 5. SCH-DC promotes neuronal differentiation of NSPCs in the injured spinal cord. (A) NSPCs stained with nestin (green) and GFAP (red) were analyzed in the injured spinal cord on d 7. Arrows indicate typical nestin þ GFAP À cells. Nuclei were stained with DAPI (blue). Scale bar, 20 mm. (B) Number of double-labeled cells with nestin (green), and b-tubulin III (red) in areas 0.5 mm rostral and caudal to the injury epicenter at d 7 after injury. Scale bar, 20 mm. Data are given as means 6 SEM, three animals per group, *P < 0.05 versus control (ANOVA). (Color version of figure is available online.)

[3] 169w can initiate a significant antigen-specific T cell response. These CNS autoantigen-specific T cells can directly migrate into the spinal cord and exert protective effects in the local lesion site. Treatment with nonpulsed DCs had no effect on the functional recovery of the hindlimbs of SCI mice, further suggesting that the neuroprotective effect of SCH-DC is achieved through primed antigenspecific T cells. To acquire an effective DC immune intervention therapy, antigens should be carefully selected in order to avoid pathogenicity. The SCH contains multiple autoantigen components that are rich in myelin and contain some inhibitory proteoglycans. Several studies have reported that SCH immunization can lead to a neuropro-tective effect after nerve injury but not to the development of EAE [31]. In fact, induction of EAE in most rodents is related to the dose of adjuvant and EAE susceptibility of the experimental animal [32]. The mice in the present study did not show any evidence of cellular or proinflammatory cytokine changes in the spinal cord that would be suggestive of EAE.

[4] 287w The reconstitution of the mediated immune response by local T cells is important for neuroprotection and neurogenesis after CNS damage [8]. Following an insult in the CNS, T cells can cross the blood-brain barrier and be recruited to the damaged tissue [33]. In the lesion site, T cells can be a source of neurotrophic factors and can promote the expression of neurotrophic factors by neurons and microglia [8]. However, the T cell response to CNS injury was relatively limited and was not sufficient to produce neuroprotective effects. Therefore, augmentation of the T cell response at the site of CNS injury might affect the outcome of the secondary degeneration. CD4 þ T cells seem to play an important role in protecting motoneurons after SCI [34]. Severe combined immunodeficient mice that lack functional B and T cells display impaired recovery after facial nerve transfection, which could be restored by passive transfer of wild-type CD4 þ T cells but not B cells or CD8 þ T cells [35,36]. Injection of MBP-specific T cells into thymectomized Lewis rats led to significant neuroprotection to damaged neurons only when they were transferred along with an enriched population of CD4 þ T cells [37]. Therefore, augmentation of the CD4 þ T cell response induced by SCH-DC in lesion sites, as shown in this study, may constitute a favorable condition for the protection of motoneurons and the promotion of neuronal differentiation of NSPCs [14,38,39]. Our results also showed that vaccination with SCH-DC does not significantly affect NO and IL-1b production. Autoimmune T cells were recently found to be able to mediate the recruitment of myeloid cells from the peripheral blood, and these infiltrating blood-borne cells help to promote termination of the local immune response [8].

[5] 132w In this study, we found that systemic injection of SCH-DC up-regulated the production of IFN-g, GM-CSF, and IL-12 in injured spinal cords compared with control mice. IFN-g and IL-12 are representative Th1 cytokines. The significantly up-regulated levels of IFN-g and IL-12 in the injured spinal cords suggest that vaccination with SCH-DC evoked a T cell response with a T helper 1 (Th1/Th0) bias. Although both Th1 and Th2 cells were reported to be neuroprotective, Th1 cells may play unique regulatory and functional roles in neuroprotection [37,40,41]. The beneficial effect of IL-12 on the injured spinal cord is thought to act mainly through the activation of local microglia/ macrophages and DCs [42]. The upregulated production of GM-CSF helps to activate neural stem cells and enhances neurotrophic factor expression via activation of microglia/macrophages [43].

[6] 214w Neurotrophic factors are essential for neuronal survival and maintenance during development and for the regulation of neurotransmitter release and dendritic growth [44]. Several studies have shown that administration of neurotrophic factors can rescue injured or degenerating neurons and induce axonal outgrowth and regeneration [45]. In this study, vaccination with SCH-DC led to increased production of BDNF and NT-3, and these abundant neurotrophic factors are known to be expressed by CD4 þ T cells, microglia/macrophages, and astrocytes. The increased levels of BDNF and NT-3 after SCH-DC treatment play important roles in the protection of CNS neurons from the potentially detrimental effects of IFN-g and other proinflammatory cytokines [40]. Increased levels of BDNF and NT-3 in the spinal cord also contribute to tolerance against EAE. For stimulation of an immune response, DCs migrate to the spleen or draining lymph nodes. There, DCs prime the immune response to antigens and may also prime self-antigen-specific responses in autoimmunity. Because neurotrophic factors can be produced by lymphocytes outside of the nervous system, the spleen is considered to be an important source of neurotrophic factors [46,47]. As we expected, vaccination with SCH-DC increased neurotrophic factor production in spleen cells. This is an important protective mechanism induced by SCH-DC that constitutes a rich neurotrophic pool for supporting the injured spinal cord.

[7] 84w In summary, our results show a neuroprotective effect of SCH-DC on recovery of mice from SCI. SCH-DC changed the local immune response in injured spinal cord and promoted the secretion of neurotrophic factors in the spleen. The up-regulation of growth factors and the alteration of cytokine networks induced by SCH-DC constitute a microenvironment that is favorable for neuroprotection in the injured spinal cord. The results of the present study support the concept that DC-mediated immune regulation may be a novel therapeutic approach for SCI.

[1] 80w A homogenate of the spinal cord (SCH) was harvested from BALB/c mice (aged 4-6 weeks, weighing 18-23 g). The process is described in brief as follows: BALB/c mice were euthanized by anesthesia overdose, and T 7-10 were sectioned. The spinal cord segments were ground and filtered (200 mm), then ground by an ultrasonic homogenizer and finally centrifuged at 15,000 rpm for 20 min. The supernatant was collected, and the protein concentration was measured using the bicinchoninic acid assay (BCA) method.

[2] 89w Motor function of the hindlimbs was evaluated by the locomotor rating test on the Basso mouse scale (BMS) [29]. Blind scoring ensured that the observers were not aware of the treatment received by each mouse. The locomotor activities of the trunk, tail, and hindlimbs were evaluated in an open field for 4 min. Before each evaluation, the mouse was examined carefully for perineal infection, wounds in the hindlimbs, and tail and foot autophagia. Behavioral analysis was performed on d 0, 7, 14, 21, 28, 42, and 56 after injury.

[3] 98w To examine the step patterns of the forelimbs and hindlimbs during forward locomotion, mice with consistent plantar steppings were required to traverse a straight, well-lit runway (2 in. width) to receive a food treat in a darkened box at the far end. The forelimb and hindlimb plantar surfaces were inked with nontoxic red and blue dyes, respectively. A minimum of two nonstop passes were required. The forelimb stride lengths and toe drags were analyzed from a minimum of five step cycles per trial. Footprint analysis was performed on the day prior to surgery and 56 d after injury.

[4] 265w After transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS), the spinal cord and spleens were removed and then sectioned in the appropriate plane at 20 mm. The sections were blocked with 5% bovine albumin diluted in 0.1% Triton X-100/0.1M TBS for 60 min. For immunocytochemical analysis, the following primary antibodies were used for immunofluorescent staining: rat anti-CD4 (1:200; Abcam, Cambridge, UK), rabbit anti-b-tubulin III (1:300; Abcam), goat anti-glial fibrillary acidic protein (GFAP) (1:200; Abcam), and rat anti-nestin (1:200; Abcam). All of the antibodies were applied to the corresponding sections overnight at 4 C. On the following day, the sections were incubated for 60 min at 37 C with the corresponding secondary antibody, which was either FITC-labeled goat anti-rat IgG, FITC-labeled goat anti-rabbit IgG, Cy3-labeled goat anti-goat IgG, or Cy3-labeled goat anti-rat IgG (all of the secondary antibodies were purchased from Beyotime Institute of Biotechnology, Jiangsu, PR China). Slides were washed, and some were incubated with diamidino-phenyl-indole (DAPI) for 2 min at 37 C, then washed and coverslipped. All of the samples were examined using a laser scanning confocal fluorescence microscope (Zeiss 510 LSM; Jena, Germany). A single image was compiled by stacking four confocal images taken at 1 mm intervals. Twenty sections were inspected from each spinal cord, of which the fifth, tenth, and fifteenth sections (representing bilateral and midsagittal areas of interest) were selected for further quantitative analysis. CD4 þ cells were analyzed in the areas that were 0.5 mm rostral and caudal to the lesion epicenter. CD4 þ cells and the GFAP-delineated area were quantified using Image-Pro Plus 5.1 software.

[5] 95w In addition, transverse sections from some samples of spinal cord were analyzed by immunohistochemical staining of b-tubulin III to detect neurons. After incubation with primary antibody, secondary antibodies labeled with biotin for DAB detection were used. The biotinylated antibodies were visualized using the ABC kit (Botster, Wuhan, PR China). The b-tubulin III-stained sections at the lesion epicenter and 0.5, 1.5, and 2.5 mm rostral and caudal to the epicenter were analyzed using light microscopy, and the number of surviving ventral horn (VH) neurons were quantified. All of the calculations were performed in a blinded manner.

[6] 68w After sacrificing the animals, spleens were aseptically removed, and cells were suspended at a concentration of 5 3 10 6 /mL in RPMI-1640 supplemented with 10% fetal calf serum and 1% l-glutamine; the cells were then cultured in flat-bottomed, 24-well plates. Cells were cultured in triplicate (1 mL/well) for 24 h at 37 C and 5% CO 2 . Supernatants of spleen cell cultures were collected and assayed.

[7] 231w Total RNA from injured spinal cord (1 cm spinal cord segment containing the injury epicenter) or freshly isolated spleen cells was extracted with TRIzol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. cDNA was synthesized from the mRNA using the Reverse Transcription System (Promega Corporation, Madison, WI) according to the manufacturer's instructions. RT-PCR for detection of IFN-g, IL-4, IL-10, BDNF, and NT-3 mRNA expression was performed using a Lightcycler instrument and a Lightcycler FastStart DNA Master Hybridization Probes Kit (Roche Diagnostics, Mannheim, Germany). Primers and hybridization probes were purchased from and designed by Invitrogen. The primers used for PCR are given in Table 1. Each primer pair was tested for the optimal Mg 2þ concentration. RT-PCR reactions were performed in a 20 mL volume containing 1 mL of cDNA sample. The reaction was initiated by activating the polymerase with a 10 min pre-incubation at 95 C. Amplification was performed for 45 cycles with a 15 s denaturation at 95 C, a 10 s annealing at 65 C, and a 10 s extension at 72 C in each cycle. b-Actin was used as an internal control and analyzed together with the cytokines using dual color detection. Color compensation was performed using the Lightcycler Color Compensation Set (Roche). Negative controls (reactions without cDNA or cDNA reactions without mRNA) were included in each run. Cycle threshold (CT) values were calculated as previously described [30].

[8] 48w The data were analyzed by one way ANOVA followed by Tukey tests of multiple comparisons to determine whether there were significant differences between individual groups. For BMS scores, repeated measure ANOVA was used to determine statistical significance. Statistical significances were set at *P 0.05 and **P 0. 01.

[1] 198w Adult BALB/c mice (weighing 24 to 26 g) were purchased from the Animal Breeding Center of Harbin Medical University (Harbin, PR China). All surgical procedures 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 was approved by the Animal Use and Care Committee of School of Medicine, Harbin Medical University. In accordance with the clip compression model described previously [27], all mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (5 mL/kg) and then positioned on a cork platform. The skin was incised along the midline of the back, and the paravertebral muscles of the thoracic-level (T8-T10) vertebrae were dissected out. Laminectomy was performed at the T9 level under visual guidance using an operating microscope. A 9-se-long extradural compression with a vascular clip (with 8 g force) was performed around the exposed spinal cord in order to cause an acute compression injury. Animals were left to recover on a warm pad until thermoregulation and an alert state were reestablished. Animals received manual bladder expression twice daily until the return of bladder function; they also received appropriate veterinary care when needed.

[2] 377w DCs were obtained from bone marrow using a method described previously [28]. The brief process is described as follows: femurs and tibias were removed from the dead mature male BALB/c mice, the muscles and connective tissues were stripped, and bones were placed in 70% ethanol for 5 min for disinfection and then washed with phosphate-buffer solution (PBS). Both ends of the bones were cut with scissors, and the marrow was flushed out with calcium-free and magnesium-free PBS using a syringe with a 23-gauge needle. Cell aggregates were broken down by vigorous pipetting. Red blood cells were lysed with ACK buffer (150 mM NH 4 Cl, 10 mM KHCO 3 , 0.1 mM Na 2 EDTA, pH 7.2-7.4). At d 0, bone marrow cells were counted and plated at a density of 0.5 3 10 6 cells/mL in a 2.5 mL flask (total 5 mL). The cells were cultured in RPMI-1640 medium (Gibco Invitrogen, Beijing, PR China) supplemented with 100 mg/mL penicillin and streptomycin, 2 mM L-glutamine, 50 mM b-mercaptoethanol, 1 mM pyruvate, 1:100 nonessential amino acids, and 10% heatinactivated and filtered fetal calf serum (Gibco Invitrogen, Grand Island, NY) (referred to hereafter as DC medium). Cytokine recombinant murine granulocyte macrophage colony-stimulating factor (rmGM-CSF, PeproTech, Rocky Hill, NJ) at a concentration of 200 U/mL was added at d 0. On d 3, an additional 2.5 mL of RPMI-1640 medium containing 200 U/mL rmGM-CSF was added to the plates. On d 6 and 8, half of the culture supernatant was collected and centrifuged, and the cell pellet was resuspended in 2.5 mL of fresh RPMI-1640 containing 200 U/mL rmGM-CSF and returned to the original plate. On d 10, cells were ready for use. Some cells were resuspended in fresh DC medium (without additional cytokines; 2 3 10 6 cells/mL) containing SCH (100 mg/mL) or not. DCs that were pulsed with SCH were stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 24 h to obtain SCH-pulsed mature DCs. Unpulsed mature DCs were stimulated with LPS in the absence of SCH as naive DCs. All of the cells were kept on ice until they were injected. Just before injection, the cells were centrifuged and resuspended in PBS three times (1 3 10 6 cells in 0.3 mL of PBS for intraperitoneal injection).

[3] 122w The proteins in SCI mice were extracted from the injured spinal cord at the T9 level using a lysis buffer containing 1% NP-40, 10% glycerol, 137 mM Tris-HCl (pH 8.0), 1 mM PMSF, 10 mg/mL aprotinin, 1 mg/mL leupeptin, and 0.5 mM sodium vanadate. Spinal cord extracts were centrifuged for 10 min at 13,000 g at 4 C, and the supernatants were then subjected to detection using corresponding ELISA kits according to the manufacturer's instructions. Prostaglandin E 2 (PGE 2 ) and Nitric oxide (NO) ELISA kits were purchased from R&D Systems, Minneapolis, MN. All other ELISA kits were purchased from Wuhan Boster, Wuhan, PR. China. The absorbance was read at 450 nm by an automated ELISA plate reader (Bio-Tech, Winooski, VT).

[4] 99w To confirm the effects of SCH-DC on neuronal survival, the number of VH motoneurons at the injury epicenter as wells as at different distances further away were counted at the eighth wk after injury. There were no motoneurons left at the epicenter at the 8th week after SCI in each experimental group (not shown). As shown in Fig. 6, the number of motoneurons in the control and DC groups showed no statistically significant difference. However, more residual motoneurons were found in SCH-DC-treated mice at 1.5 or 2.5 mm rostral and caudal to the lesion epicenter compared with control mice.