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Huanxing Su,* Yin Wu,† Qiuju Yuan,* Jiasong Guo,‡ Wenming Zhang,*§ and Wutian Wu*¶#**. *Department of Anatomy ..... ron death (21,43). For example, the ...
0963-6897/11 $90.00 + .00 DOI: 10.3727/096368910X522090 E-ISSN 1555-3892 www.cognizantcommunication.com

Cell Transplantation, Vol. 20, pp. 167–176, 2011 Printed in the USA. All rights reserved. Copyright  2011 Cognizant Comm. Corp.

Optimal Time Point for Neuronal Generation of Transplanted Neural Progenitor Cells in Injured Spinal Cord Following Root Avulsion Huanxing Su,* Yin Wu,† Qiuju Yuan,* Jiasong Guo,‡ Wenming Zhang,*§ and Wutian Wu*¶#** *Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong SAR, China †Molecular Immunology Unit, University College London Institute of Child Health, London, UK ‡Department of Histology and Embryology, Southern Medical University, Guangzhou, China §Department of Orthopaedics, The First Affiliated Hospital of Fujian Medical University, Fujian, China ¶State Key Laboratory of Brain and Cognitive Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China #Research Center of Reproduction, Development and Growth, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China **Joint Laboratory for Brain Function and Health (BFAH), Jinan University and The University of Hong Kong, Guangzhou, China

Root avulsion of the brachial plexus results in a progressive and pronounced loss of motoneurons. Cell replacement strategies have therapeutic potential in the treatment of motoneuron degenerative neurological disorders. Here, we transplanted spinal cord-derived neural progenitor cells (NPCs) into the cervical ventral horn of adult rats immediately, 2 weeks, or 6 weeks after root avulsion to determine an optimal time scale for the survival and differentiation of grafted cells. We showed that grafted NPCs survived robustly at all three time points and there was no statistical difference in survival rate. Interestingly, however, transplantation at 2 weeks postavulsion significantly increased the neuronal differentiation of transplanted NPCs compared to transplantation immediately or at 6 weeks postavulsion. Moreover, only NPCs transplanted at 2 weeks postavulsion were able to differentiate into choline acetyltransferase (ChAT)-positive neurons. Specific ELISAs and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) demonstrated that expression levels of BDNF and GDNF were significantly upregulated in the ventral cord at 2 weeks postavulsion compared to immediately or at 6 weeks postavulsion. Our study suggests that the cervical ventral horn at 2 weeks postavulsion both supports neuronal differentiation and induces region-specific neuronal generation possibly because of its higher expression of BDNF and GDNF. Key words: Spinal root avulsion; Neural progenitor cells; Motoneurons; Transplantation; Neuronal differentiation

INTRODUCTION

cular junctions with peripheral muscle targets after transplantation into the ventral horn, raising the possibility of cell replacement therapy for motoneuron loss (15). The adult spinal cord lacks appropriate promoting signals or contains inhibitory factors that make it a hostile environment for the survival and differentiation of grafted NPCs (6,9,35,40). It has been suggested that a time window exists for successful cell transplantation strategies for the treatment of CNS injuries and a delayed transplantation may optimize the survival and differentiation of grafted cells (23,29). Using the rat brachial plexus model, we have previously demonstrated that significant cell loss in the avulsed cervical spinal cord occurs starting at 2–3 weeks postinjury and ap-

Root avulsion of the brachial plexus is a devastating injury that results in massive motoneuron death and paralysis of the corresponding muscle groups (12,17,27). Transplantation of neural progenitor cells (NPCs) into the ventral horn could be a promising therapy to treat brachial plexus injury as NPCs have the capacity to differentiate into cell types appropriate to the structure into which they have been grafted. They can also secrete neurotrophic factors to protect injured neurons (2,4,14, 34,45). A recent study reported that human neural NPCderived motoneurons in vitro could send axons through the ventral root and peripheral nerve to form neuromus-

Received May 26, 2009; final acceptance June 25, 2010. Online prepub date: August 18, 2010. Address correspondence to Wutian Wu, Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong SAR, China. Tel: (852) 28199187; Fax: (852) 28170857; E-mail: [email protected]

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proximately 80% of adult rat spinal motoneurons die by 6 weeks following root avulsion (42,44). Although a cell transplantation strategy may prove to be a potential therapy for avulsion injury, the appropriate time point for engraftment to maximize survival and differentiation in the ventral horn remains unknown. To address this, we transplanted spinal cord-derived NPCs into the cervical ventral horn immediately, 2 weeks, or 6 weeks after root avulsion to investigate the survival and differentiation of grafted NPCs. MATERIALS AND METHODS All surgical interventions and subsequent care and treatment were approved by the Committee on the Use of Live Animals for Teaching and Research of the University of Hong Kong. Cell Isolation and Culture Under sterile conditions, spinal cords from embryonic day 13.5 wild-type or transgenic Sprague-Dawley rats expressing green fluorescent protein (GFP, “green rat” CZ-004; SLC, Shizuoka, Japan) were dissected out and prepared for stem cell culture following procedures described previously (18,37). Briefly, the spinal cord was separated from surrounding connective tissue before removal of the meninges. The cord was then transferred into a 15-ml centrifuge tube containing stem cell culture medium (described below) and dissociated to a singlecell suspension by gentle mechanical trituration through a fire-polished Pasteur pipette. The dissociated cells were filtered through a cell strainer (BD Falcon, San Jose, CA, USA) and plated onto poly-L-lysine (13.3 µg/ ml in distilled water; Sigma, St. Louis, MO, USA) and laminin (20 µg/ml; Sigma) coated dishes. Stem cell culture medium consisted of DMEM-F12 (Gibco, Grand Island, NY, USA), bovine serum albumin (BSA) (1 mg/ ml; Sigma), B27 (20 µl/ml; Invitrogen, Carlsbad, CA, USA), N2 (10 µl/ml; Invitrogen), basic fibroblast growth factor (bFGF) (20 ng/ml; Peprotech, Rocky Hill, NJ, USA), and penicilliin-streptomycin (100 IU/ml; Invitrogen). Cells were maintained in an incubator with a humidified atmosphere containing 5% CO2 at 37°C. The medium was changed every 2 days. At around 80% confluence, cells were passaged at a ratio of 1:4–6 after initial plating. These subcultured cells were designated as “first passage” (P1). All experiments were carried out using P1 NPCs in this study. To induce differentiation, dissociated cells in single cell suspensions were plated on cover slips coated with poly-L-lysine/laminin (1:1 ratio) at a density of 104/cm2 in a 24-well plate. Growth factors were removed from the culture medium and 1% fetal bovine serum (FBS; Gibco) was added. The cultures were allowed to differentiate for up to 5 days.

Animal Surgery and Cell Transplantation Forty-five adult female Sprague-Dawley rats (220– 250 g) were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (8 mg/kg). Brachial plexus avulsion was performed as described previously (25,46). Briefly, animals were placed in a supine position, and the right seventh cervical spinal nerve (C7) was avulsed by pulling the C7 spinal nerve out with a pair of microhemostatic forceps. Avulsed animals were first divided into two groups in a random manner. The first group (n = 27) was then subdivided evenly into three groups for cell transplantation immediately, 2 weeks, or 6 weeks after root avulsion according to procedures described previously (38,39). Briefly, a dorsal hemilaminectomy on the right side of the sixth cervical vertebra was carried out under aseptic conditions. Under the operating microscope, a small opening on dura of the right C7 spinal segment was made and a glass micropipette (100 µm outer diameter) connected to a Hamilton syringe was then stereotactically advanced into the avulsed ventral horn (ML: −0.8 mm; DV: −1.5 mm from dura) through the dura opening. NPCs in P1 derived from E13.5 day GFP rat spinal cords were dissociated and resuspended in Neurobasal medium (NB, Gibco) at a concentration of 1 × 105 cells/µl. Cell viability assessed by trypan blue exclusion at the end of transplantation was typically over 90%. One microliter of cell suspension was slowly injected over 5 min into the ventral horn and the micropipette was left in place for 2 min after the injection. The dura was closed with 10-0 sutures, muscles, and fascia were reapposed and sutured with 4-0 sutures, and the skin was closed with wound clips. After cell transplantation, animals were allowed to survive for 6 weeks. The second group (n = 18) was used to investigate the expression profiles of neurotrophic factors in the ventral horn of animals at the three time points (immediately, 2 weeks, and 6 weeks postavulsion; 3 animals for ELISA analysis and another 3 animals for quantitative RT-PCR studies at each time point). Tissue Processing At 6 weeks after cell transplantation, animals were sacrificed with a lethal dose of sodium pentobarbital and perfused intracardially with 0.01 M PBS (pH 7.4), followed by perfusion with 200–300 ml of fixative solution containing 4% paraformaldehyde in 0.1 M PB (pH 7.4). The C7 segments of spinal cords were harvested and postfixed in fresh fixative solution overnight and subsequently placed in 30% sucrose-0.1 M PB at 4°C for 2–3 days. The C7 segments were then cut into 30-µm-thick cross sections on a microtome (American Optical Company, NY, USA). The serial sections were collected in 0.01 M PBS and kept at 4°C for further study.

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Immunocytochemistry For in vitro experiments, cultured cells on cover slips were fixed in 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (PB) for 20 min. After several washes with 0.01 M PBS, cells were processed for immunocytochemistry. For in vivo experiments, sections through the site of cell transplantation were collected for immunostaining. Cultured cells and sections were washed in 0.01 M PBS, blocked in 10% goat serum for 1 h, and incubated with primary antibodies at 4°C overnight. Astroglial cells were identified using rabbit anti-GFAP (1: 1000, Sigma); oligodendroglial cells were identified with mouse anti-adenomatus polyposis coli (APC, 1: 200; BD Biosciences Pharmingen, San Jose, CA, USA) for sections and mouse anti-Rip antibody (1:50, a generous gift from Dr. X. M. Xu, Indiana University School of Medicine, Indianapolis, IN, USA) for cultured cells; neurons were detected with mouse anti-NeuN (1:200; Chemicon) and goat anti-ChAT (1:400) for sections and mouse anti-βIII-tublin (1:500, Sigma) for cultured cells; undifferentiated cells were identified using monoclonal anti-nestin (1:200, Chemicon). Species-specific fluorescence-conjugated secondary antibodies conjugated to Alexa 568 (1:400, Molecular Probes) were applied for 2 h at room temperature. Sections and cell slides were then counterstained with DAPI to stain nuclei, and cover-slipped with antifade mounting media (FluorSave, Calbiochem). The images were taken with a laser confocal microscope (LSM510 META, Carl Zeiss Meditec, Jena, Germany). The total number of GFP-expressing cells, which were also labeled with DAPI in the ventral horn, was counted in every third section. To assess differentiation patterns of transplanted cells, we stained two sections per rat for each cell marker and then counted the number of GFP-expressing cells colocalized with the cell marker in 10 random fields per section at 40× magnification using the confocal microscope. All quantification was performed in an unbiased stereological manner (18). ELISA Animals were euthanized by decapitation and the ventral part of the right C7 segment was dissected out under a dissecting microscope. Tissue samples were homogenized in T-PER (Pierce, Rockford, IL) and total protein levels were measured with the Micro BCA protein assay reagent kit (Pierce). ELISA measurements were performed with the Promega BDNF, GDNF, NT3, or NGF Emax Immunoassay System following the recommendations of the manufacturer (Promega, Madison, WI, USA). A standard curve of pure BDNF, GDNF, NT-3, or NGF protein provided in the kit was used to quantify the concentration of these neurotrophic factors.

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Quantitative RT-PCR Analysis Animals were euthanized by decapitation and the ventral part of the right C7 segment was dissected out under a dissecting microscope and immediately frozen on dry ice. Total RNA was extracted using the TRIZOL reagent (Invitrogen) according to the manufacturer’s protocol. All samples were treated with DNaseI to remove residual genomic DNA in the RNA preparation. The integrity of the isolated total RNA was confirmed by gel electrophoresis with the presence of discrete 28S and 18S ribosomal RNA bands in each RNA preparation. Two micrograms of total RNA was reverse transcribed to cDNA using the SuperscriptTM first-strand synthesis system (Invitrogen) and used for RT-PCR according to the manufacturer’s protocol. The mRNA expression level of each sample was quantified by quantitative PCR using the iCycler iQTM (Bio-Rad) with SYBR Green (Molecular Probes). In brief, 1 µl of cDNA was added in a 24-µl reaction mixture containing 0.5× SYBR Green, Gibco PCR buffer (Invitrogen), 0.6 µM MgCl2?, 0.4 µM dNTP, 0.5 µM primer sets, and 0.5 U Gibco Taq polymerase (Invitrogen). Quantitative PCR was performed in triplicate and repeated three times. Primers were designed as described previously (38) and synthesized by Tech Dragon Limited (Shatin, N.T., Hong Kong, China): BDNF—forward, 5′-GAGCTGAGCGTGTGT GACAG and reverse, 5′-CGCCAGCCAATTCTC-TTT TTGC; GDNF—forward, 5′-GCGGTTCCTGTGAAG CGGCCGA and reverse, 5′-TAGATA-CATCCACAC CGTTTAGCGG; NT-3—forward, 5′-GAGCATAAGA GTCACCGAGG and reverse, 5′-AGTCAGTGCTCGG ACGTAGG; NGF—forward, 5′-CTTCAGCATTCCC TTG-ACAC and reverse, 5′-AGCCTTCCTGCTGAGC ACACA; GAPDH—forward, 5′-AACGACCCCTTCAT TGAC and reverse, 5′-TCCACGACATACTCAGCAC. PCR cycling conditions were 95°C for 5 min, 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 40 s. PCRamplified products were subjected to electrophoresis on a 1.0% agarose gel, stained with ethidium bromide, and visualized under ultraviolet light (White/Ultraviolet Transilluminator, Ultra Violet Products). Statistical Analyses Multiple group comparisons were made by one-way ANOVA and Tukey post hoc test. Data were presented as mean ± SEM. The significance level was set to 0.05 for all comparisons. RESULTS In Vitro Characterization of NPCs The majority of cells in bFGF-supplemented culture medium showed bipolar or multipolar morphology with small cell bodies and were immunopositive for nestin,

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a marker for NPCs (Fig. 1A–C), confirming that they remained at an immature stage. When bFGF was replaced with 1% FBS, NPCs began to differentiate. By the fifth day of culture in differentiating medium, around 13.7% of the NPCs differentiated into βIII-tublin-positive neurons (Fig. 1D), 62.5% into GFAP-positive astrocytes (Fig. 1E), and 8.3% into Rip-positive oligodendrocytes (Fig. 1F). There remained a small population of cells with the morphology of undifferentiated cells and positive for nestin immunoreactivity (data not shown). Transplantation of NPCs at 2 Weeks Postavulsion Increases Neuronal Differentiation Transplanted NPCs were detected based on the GFP expression. Cross sections from all three groups showed that the majority of grafted NPCs were located in the ventral horn although some were also found to be distributed along the injection tract (Fig. 2A1, B1, C1). Grafted NPCs survived robustly in all three transplantation groups. Approximately 13635 ± 2032 GFP-express-

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ing cells were detected in the ventral horn when NPCs were transplanted immediately after root avulsion. Similarly, 12756 ± 1829 and 13087 ± 2364 GFP-expressing cells were present in the ventral horn when NPCs were transplanted at 2 weeks and 6 weeks postavulsion, respectively. There was no significant difference in the survival rate of grafted NPCs among the three groups (Fig. 2D). Interestingly, neuronal differentiation of NPCs was significantly increased in the 2 week postavulsion group (Fig. 2B2–B4) compared to those transplanted immediately (Fig. 2A2–A4) or 6 weeks postavulsion (Fig. 2C2–C4). About 21.2 ± 4.7% grafted NPCs were differentiated into NeuN-positive cells when transplanted at 2 weeks postavulsion, which was significantly higher than 4.2 ± 1.3% neuronal differentiation seen in acute transplantation (immediately postavulsion) and 1.5 ± 0.4% in chronic transplantation (6 weeks postavulsion) (p < 0.001) (Fig. 2E). Notably, the intensity of GFP in NeuN-positive cells was much weaker compared with the NeuN-negative cells (Fig. 2A4, B4, C4). This

Figure 1. NPCs culture and differentiation pattern in vitro. (A–C) NPCs were isolated from E-13.5 spinal cords of transgenic SD rats expressing GFP and plated as a monolayer on poly-L-lysine/laminin-coated culture dishes in bFGF-supplemented culture medium. These GFP-positive cells had the morphology of round-shaped cell bodies with either bipolar or multipolar processes (A). Immunocytochemistry on these cells revealed efficient expression of the progenitor cell marker nestin (B). The majority of these GFP-expressing cells were nestin positive (C). (D–F) When bFGF was removed from the culture and replaced with 1% FBS for 5 days, a number of NPCs (13.7%) differentiated into βIII-tublin (TUJ1)-positive neurons (D). The majority of NPCs (62.5%) differentiated into GFAP-positive astrocytes (E) and a small population of NPCs (8.3%) into Rip-positive oligodendrocytes (F). Scale bar: 50 µm in (A–C); 20 µm in (D–F).

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Figure 2. Transplantation of NPCs into the ventral horn of adult rats at 2 weeks postavulsion increased neuronal differentiation as revealed by anti-NeuN immunostaining. (A) NPCs were transplanted immediately postavulsion. (B) NPCs were transplanted at 2 weeks postavulsion. (C) NPCs were transplanted at 6 weeks postavulsion. (A4, B4, C4) Higher magnification images showing colocalization of GFP-positive cells with NeuN (arrows). The intensity of GFP in NeuN-positive cells was much weaker compared with the NeuN-negative cells. (D) The total number of GFP-expressing cells in the ventral horn was counted as described in Materials and Methods. There were no statistically significant differences in the survival rate of grafted cells among three groups. (E) The percentage of both NeuN- and GFP-positive cells in the injured ventral horn was counted and represented as the mean ± SEM. Transplantation at 2 weeks postavulsion significantly increased the neuronal differentiation of NPCs compared to transplantation immediately or at 6 weeks postavulsion. #p < 0.01 compared with transplantation at 6 weeks postavulsion. *p < 0.001 compared with the other two transplantation models. Scale bar: 250 µm (A1–A3, B1–B3, C1–C3); 15 µm (A4, B4, C4).

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suggests that NPCs downregulated the GFP expression after differentiation into neuron-like cells. Transplantation of NPCs at 2 Weeks Postavulsion Induced ChAT-Positive Neuron Generation We then investigated whether NPCs could differentiate into region-specific neurons after transplantation into the injured ventral horn. NPCs failed to differentiate into ChAT-positive neurons when transplanted immediately or at 6 weeks after root avulsion (data not shown), which was consistent with our previous reports (38,39). However, NPCs transplanted at 2 weeks postavulsion were able to generate ChAT-positive neurons with approximately 5.8 ± 0.7% grafted NPCs in the injured ventral horn ChAT-positive (Fig. 3A, D). The majority of NPCs differentiated into glial cells after transplantation at 2 weeks postavulsion with 61.6 ± 7.2% GFAP-positive astrocytes (Figs. 3B, D) and 9.5 ± 3.3% APC-positive oligodendrocytes (Figs. 3C, D). Higher Expression Levels of BDNF and GDNF Were Observed in the Ventral Cord at 2 Weeks Postavulsion Compared With Immediately or at 6 Weeks Postavulsion To explore the potential mechanisms of the ventral cord after avulsion on grafted NPCs differentiation, specific ELISAs for BDNF, GDNF, NT-3, and NGF protein levels and quantitative RT-PCR for gene expression were performed on tissue samples of avulsed ventral cords at the three time points. ELISAs showed that the average concentration of BDNF in the ventral cord at 2 weeks postavulsion was 634 ± 32 pg/mg of tissue, which was significantly higher than the 267 ± 26 pg/mg of tissue in the ventral cord immediately postavulsion and 281 ± 29 pg/mg of tissue at 6 weeks postavulsion (p < 0.001) (Fig. 4A). Increased expression of GDNF in the ventral horn at 2 weeks postavulsion (432 ± 36 pg/ mg of tissue) was also observed compared to immediately postavulsion (250 ± 17 pg/mg of tissue) and at 6 weeks postavulsion (224 ± 21 pg/mg of tissue) (p < 0.001) (Fig. 4B). No differences in the concentration of NT-3 and NGF were detected among the three time points in that the average concentration of NT-3 was 548 ± 50, 576 ± 56, and 521 ± 38 pg/mg of tissue, respectively (Fig. 4C) and the average concentration of NGF was 649 ± 69, 696 ± 45, and 667 ± 76 pg/mg of tissue, respectively (Fig. 4D). Quantitative RT-PCR confirmed that normalized mRNA expression levels of BDNF and GDNF in the ventral cord at 2 weeks postavulsion were significantly increased by fivefold and twofold, respectively, compared to the other two time points (p < 0.001) (Figs. 4E, F). No changes in mRNA expression of NT-3 and NGF were found among the three time points in the study (Figs. 4E, F).

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DISCUSSION Recently cell replacement strategies have been applied to treat motoneuron degeneration (10,19,20,28,41). NPCs are of particular interest for neurological repair as they are already committed to a neural fate and may be easier to differentiate into neural phenotypes (5,36). However, the injured adult spinal cord milieu does not appear to favor differentiation of grafted NPCs towards a neuronal phenotype. Several studies provide evidence that grafted NPCs differentiate predominantly into glial phenotypes in the injured adult spinal cord (6,9,35). Here we present novel evidence that grafted NPCs can produce a large number of NeuN-positive neurons after transplantation into the ventral horn at 2 weeks postavulsion. Moreover, some of these graft-derived neurons express ChAT. These findings suggest that the ventral horn at 2 weeks after avulsion is supportive for neuronal differentiation and even conducive for region-specific neuronal generation. Several reports have previously shown that there is only a narrow time window for successful cell transplantation strategies for the treatment of spinal cord injury (29,32). It has been demonstrated that severe acute inflammation occurs immediately following spinal cord transection and many inflammatory cytokines with neurotoxic or astrocyte-inducing effects, such as IL-1, IL-6, and TNF-α, are highly expressed and then decline sharply within 24 h (26). In light of this acute inflammatory response it is not surprising that the microenvironment of the injured spinal cord during the acute phase is unfavorable for neuronal differentiation of grafted cells. The chronic phase of spinal cord injury is also considered to be inappropriate for therapeutic transplantation due to the development of glial scars, the presence of inhibitory molecules and the lack of appropriate trophic factors (30,31). Furthermore, subacute transplantation of NPCs (2 weeks postinjury) leads to not only a substantial survival and differentiation of engrafed cells but also functional recovery whereas grafted cells survive poorly and exert no effects on the functional recovery in chronic transplantation models (6–8 weeks postinjury) after spinal cord contusion injury (23,24). The exact mechanisms underlying the benefits of subacute transplantation for grafted cells remain unknown. An interesting finding in our present study is that the expression levels of BDNF and GDNF in the 2-week avulsed ventral cord are significantly elevated compared to acutely or chronically avulsed ventral cords. It has been previously reported that BDNF plays a critical role in regulating neuronal development, survival, and plasticity (22). NPCs in culture increase neuronal generation after exposure to BDNF (1) and the induction of BDNF in the striatum promotes the neu-

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Figure 3. Generation of ChAT-positive neurons and glial differentiation of NPCs transplanted at 2 weeks postavulsion. (A) Confocal images showing ChAT immunostaining of NPCs. Arrows indicate colabeling of ChAT and GFP. (B) Confocal images showing GFAP immunostaining of NPCs. Arrows indicate colabeling of GFAP and GFP. (C) Confocal images showing APC immunostaining of NPCs. Arrows indicate colabeling of APC and GFP. The intensity of GFP in ChATpositive cells (A) was much weaker compared with GFAP-positive cells (B) and APC-positive cells (C). (D) The percentage of ChAT-positive, GFAP-positive, and APC-positive cells differentiated from transplanted GFP-positive cells was counted and represented as the mean ± SEM. Scale bar: 30 µm.

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Figure 4. Differential expression levels of neurotrophic factors in the cervical ventral cord after avulsion at different time points. (A–D) Specific ELISAs showed that the expression levels of BDNF (A) and GDNF (B) in the ventral cord at 2 weeks postavulsion were significantly higher than those expressed in the ventral cord immediately and at 6 weeks postavulsion, respectively; no differences in the production of NT-3 (C) and NGF (D) were found in the avulsed ventral cord among the three time points. (E) Ethidium bromide-stained gels demonstrated RT-PCR products of BDNF, GDNF, NT-3, and NGF from the avulsed ventral cord at the three time points. GAPDH was used as an internal control for equal amounts of template cDNA. (F) Densitometric comparison of the average amount of cDNA product of BDNF, GDNF, NT-3, and NGF among the three time points. The data showed that BDNF and GDNF mRNA in the ventral cord were significantly upregulated at 2 weeks postavulsion compared with immediately and at 6 weeks postavulsion, whereas NT-3 and NGF mRNA expression did not differ among the three time points. All results represented mean values of three animals at each time point; *p < 0.001 when compared with other two time points.

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ronal differentiation of transplanted NPCs (7). GDNF is a potent factor for neuronal survival and axonal elongation (3,33). Therefore, an increase in BDNF and GDNF expression may account for the neurogenic induction of the 2-week avulsed ventral horn. An interesting recent study reported that BDNF and GDNF levels in the peripheral nerve were significantly upregulated at 1 or 2 weeks after ventral root avulsion and decreased gradually towards 6–8 weeks (11), which are similar to their expression profiles in the ventral cord after avulsion as shown in our present study. It is still not known which cell types in the avulsed segment are responsible for the distinct temporal expression profiles of neurotrophic factors after injury. Activated astrocytes, remaining avulsed spinal roots, which are rich in Schwann cells, or even injured motoneurons in the ventral horn are possible candidate cell types responsible for the elevated expression of BDNF and GDNF. It is also possible that some as yet unknown molecules expressed in the 2-week avulsed ventral horn may contribute to converting the host milieu from nonneurogenesis to neurogenesis. It has been reported that specific genes are induced in the ventral horn after avulsion injury, some of which are correlated with motoneuron death (21,43). For example, the expression of neuronal nitric oxide synthase (nNOS) is first observed by 3 days, increases from then, and reaches a maximal level by 2 weeks following root avulsion injury. Some studies have demonstrated that NO, a gaseous intercellular messenger synthesized by NOS-positive cells in CNS, plays a role in both embryonic nervous tissue formation and adult neurogenesis (13,16). Exposure of NO supports neuronal differentiation of NPCs not only in vitro but also in vivo (8). The high expression of nNOS in motoneurons at 2 weeks postinjury may be involved in regulating the plasticity of avulsed ventral horns for grafted NPCs differentiation. Future studies using NOS knockout animals as hosts to investigate the survival and differentiation of transplanted NPCs in vivo would help to clarify the role of NO. Clinically, root avulsion most frequently occurs in multitraumatized events where it can be difficult to determine the exact location and degree of the injury. Therefore, chronic transplantation is more clinically relevant due to the delayed diagnosis of avulsion injury. However, our present study suggests that subacute transplantation within 2–3 weeks after injury should be considered in NPCs-based transplantation therapy because of the optimal neuronal regeneration and higher expression levels of some specific neurotrophic factors. ACKNOWLEDGMENTS: This study was supported by HKU Spinal Cord Injury Foundation and National key basic research support foundation (973 project: 2011CB504402).

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