Liver Growth Factor Promotes the Survival of ...

Current Stem Cell Research & Therapy, 2012, 7, 15-25

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Liver Growth Factor Promotes the Survival of Grafted Neural Stem Cells in a Rat Model of Parkinson’s Disease Diana Reimers1, Cristina Osuna1, Rafael Gonzalo-Gobernado1, Antonio S1. Herranz, Juan José Díaz-Gil2, Adriano Jiménez-Escrig3, Maria José Asensio1, Cristina Miranda1, Macarena Rodríguez-Serrano1 and Eulalia Bazán1,* 1

Servicio de Neurobiología, Hospital Ramón y Cajal, Madrid-28034, Spain; 2Servicio de Bioquímica Experimental, Hospital Universitario Puerta de Hierro-Mahadahonda, Madrid, Spain; 3Servicio de Neurología, Hospital Ramón y Cajal, Madrid, Spain Abstract: Neural stem cells (NSCs) with self-renewal and multilineage potential are considered good candidates for cell replacement of damaged nerve tissue. Several studies have focused on the ability of the neurotrophic factors coadministration to improve the efficiency of grafted NSCs. Liver growth factor (LGF) is an hepatic mitogen that promotes regeneration of damaged tissues, including brain tissue. It has neurogenic activity and has partially restored the nigrostriatal dopaminergic system in an experimental model of Parkinson’s disease. Present results demonstrate that in the dopamine-depleted striatum of 6-hydroxydopamine-lesioned rats, grafted NSCs retained their ability to differentiate into neurons, astrocytes, and oligodendrocytes. NSCs also differentiated into microglia/macrophages and endothelial cells. Thus, 23 ± 5.6% of them were inmunoreactive for isolectin IB4, and a small population integrated into blood vessels, showing an endothelial-like morphology. Intrastriatal infusion of LGF promoted the viability of the implants, and favored their differentiation to an endothelial-like phenotype. Moreover, LGF infusion raised the expression of the anti-apoptotic protein Bcl-2 by 3.9 ± 0.9 fold without affecting the levels of the pro-apoptotic protein Bax. Since LGF-treated rats also showed a significant reduction in apomorphine-induced rotational behavior, our results suggest that administration of this factor might be a convenient treatment for Parkinson’s disease cell replacement therapies based on NSCs transplantation.

Keywords: Neural stem cells, neuroregeneration, CNS transplantation, Parkinson’s disease, 6-hydroxydopamine, neurotrophic factors, liver growth factor. INTRODUCTION Parkinson’s disease (PD) is a neurodegenerative disorder involving a progressive loss of dopaminergic (DA) neurons projecting from the substantia nigra (SN) to the striatum [1]. The most widely used therapeutic approach is the administration of levodopa, but it loses effectiveness after several years of treatment. Cell replacement strategies have emerged as a promising approach for restoration of function in neurodegenerative diseases [2, 3]. Developing neural tissue has been used to replace damaged or dead DA neurons in PD [4, 5]. However, there is still no agreement regarding the ideal cell source for transplantation due to the inflammatory responses that compromise long-term graft survival, and the problems associated with the use of tissue from aborted fetuses [6, 7]. Neural stem cells (NSCs) are defined as clonogenic cells with self-renewal capacity and multilineage potential [8]. Cells with these characteristics have been isolated from the embryonic and adult Central Nervous System (CNS) [9-11]. Under specific conditions, these cells proliferate in culture as *Address correspondence to this autor at the Servicio de Neurobiología. Hospital Ramón y Cajal, Carretera de Colmenar Km. 9,1, 28034-Madrid, Spain; Tel.: 34-91-3368385; Fax: 34-91-3369016; E-mail: [email protected] (E. Bazan) 1574-888X/12 $58.00+.00

cell clusters, called neurospheres, and differentiate into neurons, glia, and non-neural cell types [12-15]. These cultures represent a potential source for cell replacement therapy [1619], but their use in PD remains unclear due to their low survival, and the lack of DA differentiation when grafted in the adult brain [20]. Recent studies suggest that the co-administration of trophic factors could improve the efficiency of grafted NSCs in PD [21, 22]. Neurotrophic factors are compounds that enhance the survival and differentiation of selected types of neurons [23, 24]. In addition, they are also critical for the expansion and differentiation of NSCs in vitro and in vivo [25]. Liver growth factor (LGF) is a hepatic mitogen purified by Díaz-Gil and colleagues some years ago [26]. Following an in-depth chemical and immunological study, they demonstrated that LGF is an albumin–bilirubin complex, the concentration of which is nearly undetectable in sera from healthy humans or rats, but dramatically increases in the presence of hepatobiliary disorders or liver injury [27, 28]. Recent studies show that LGF promotes proliferation of different cell types [29-31], and the regeneration of damaged tissues, including brain tissue. Thus, the intracerebral infusion of LGF stimulates the sprouting of DA terminals in the striatum of unilaterally 6-hydroxydopamine (6-OHDA)lesioned rats [32]. Moreover, LGF promotes the expansion © 2012 Bentham Science Publishers

16 Current Stem Cell Research & Therapy, 2012, Vol. 7, No. 1

of neural precursors located in the subventricular zone (SVZ) of the forebrain of 6-OHDA-lesioned rats, and the generation of new neurons [33, 34]. Considering the aforementioned neurogenic and neurotrophic activity of LGF observed in a known model of PD in rats, we questioned whether LGF could influence the survival of NSCs grafted in the striatum of 6-OHDA-lesioned rats and their differentiation to functional DA neurons. In the present study we report that intrastriatal (IS) infusion of LGF enhances cell viability of grafted NSCs and favors their differentiation to an endothelial-like phenotype, whose role in the synthesis of neurotrophines involved in neurogenesis and neuronal survival has recently been reported [35]. Since LGF-treated rats also showed a significant reduction in apomorphine-induced rotational behavior, we propose that the administration of the factor is a convenient treatment for PD cell replacement therapies based in NSCs transplantation. MATERIALS AND METHODS LGF Purification LGF was purified from serum of 5 weeks, bile ductligated rats following a previously reported procedure [26, 27]. LGF was quantitated by HPLC [36] and samples with the highest LGF serum concentration were selected to proceed with the purification process, three chromatography steps employing Sephadex G-150, DEAE-Cellulose and hydroxylapatite. Purity, that is, the absence of other growth factors and/or contaminants in the LGF preparation, was also assessed according to standard criteria [26, 29]. All LGF preparations showed a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). LGF preparations were lyophilized and kept at 4ºC until use, at which time aliquots were dissolved in saline for pump perfusion. Animals and 6-OHDA Lesion Surgery A total of 25 female Sprague Dawley rats weighing 220250g at the beginning of the experiments were used. The care and use of animals was carried out according to the European Union Council Directive (86/609/EEC). The animals were housed in a temperature-controlled environment with 12 h light/dark cycles and access to food and water ad libitum. Under fluothane anesthesia, the rats received two stereotaxic injections of 6-OHDA, one in left SN (pars compacta) and the other in the medial forebrain bundle [37]. Using a 10-l Hamilton syringe, 4.0 l of 6-OHDA (3.6 g/l in 0.2 mg/ml L-ascorbate-saline) were injected into the left mesostriatal pathway at AP -5.3, ML +2.1, DV -7.8 (in mm with respect to bregma and dura, tooth bar –3.0 mm) and at AP -4.3; ML +1.4; DV -8.7, according to the stereotaxic atlas of Paxinos and Watson [38]. The injection rate was 1 l/min, and the cannula was left in place for an additional 5 minutes before being slowly withdrawn. To minimize variability due to the degradation of the toxin, the 6-OHDA solutions were freshly prepared, kept on ice and protected from exposure to light. Rotational Behavior Apomorphine-induced rotational behavior was studied to determine whether the generation of the 6-OHDA lesions

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had been successful. At two weeks post-lesion, the rats were placed in plastic rotometer bowls and connected to a mechanical counter. Counting of rotations began 5 minutes after the subcutaneous injection of apomorphine (0.5 mg/kg diluted in 0.9% saline), and rotational asymmetry was monitored over 15 minutes. Rats rotating at least 100 turns in 15 minutes were selected for intracerebral implants and infusions; after infusion began, rotation was monitored once a week until the end of the study period. Isolation of Neural Stem Cells from the Embryonic Rat Striatum Striatal primordia from E15 Sprague-Dawley rat embryos were dissected and mechanically dissociated. Cells were grown in suspension in a defined medium (DF12) composed of Dulbecco's modified Eagle's medium and Ham's F-12 (1:1), 2 mM L-glutamine, 1 mM sodium piruvate (all from Gibco BRL, Life Technologies Inc, Grand Island, NY), 0.6% glucose, 25 μg/ml insulin, 20 nM progesterone, 60 μM putrescine, and 30 nM sodium selenite (all from Sigma Chemical Co, St Louis, MO), 100 μg/ml human transferrin (Boehringer Mannheim GmbH, Germany) and 20 ng/ml human recombinant EGF (PreproTech EC Ltd., London, England). After 48-72 hours in vitro, the cells grew as free-floating neurospheres and were passaged by mechanical dissociation every 2-3 days [39]. After a minimum of 4 and a maximum of 5 passages, neurospheres were dissociated and 24 hours later 0.5 M 5-bromodeoxyuridine (BrdU), a marker of DNA synthesis, was added to cultures for an additional 48 hours. Previous to the implants, living cells were quantified by Tripan blue exclusion (saline/2 mM EDTA, 1:8). Implants of Neurosphere-Derived Cells and Intrastriatal Infusion of LGF in the Striatum of Healthy and 6OHDA-Lesioned Rats At 9 weeks post-lesion, healthy and 6-OHDA-lesioned rats were implanted with BrdU-labeled neurosphere-derived cells in the left striatum (Fig. 1). Each animal received a stereotaxic injection of 3-5 l of a cellular suspension containing 105 cells/l. The coordinates used were AP +0.5, ML +2.6, DV -5.5 (in mm with respect to bregma and dura). The neurospheres injection rate was 1 l/min, and the cannula was left in place for an additional 5 min. In the same surgical act, the rats were stereotaxically implanted with a 28-gauge infusion cannula (Alzet, brain infusion kit), connected to an osmotic minipump (Alzet, model 2002) via 5 cm of catheter tubing, for delivery of LGF and vehicle. The pumps were designed for infusion over 15 days at a rate of 0.5 l/h. The solution consisted of rat albumin at 100 g/ml in 0.9% saline in vehicle groups. LGF at a dose of 160 ng/day/rat was added to the vehicle solution. The minipumps were filled with 200 l of the corresponding solution and incubated overnight in normal saline at 37ºC. The 4.5-mm cannula attached to the minipump was stereotaxically implanted into the left striatum (AP +0.5; ML +3.5; DV -4.5) using bregma as a reference, and DV coordinates were calculated from of the skull surface. The tooth bar was set at –3.0 mm. The minipump was placed into a subcutaneous pocket slightly posterior to the scapula. Animals were divided in different groups according to the infusion solution

LGF and NSCs Grafts in Parkinson’s Disease

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Fig. (1). Representative scheme showing the methodological procedure followed for intracerebral grafts of NSCs and infusions of vehicle and LGF in the striatum of 6-OHDA-lesioned rats.

as follows: intrastriatal LGF infusion in 6-OHDA-lesioned rats (IS-LGF-6-OHDA) (n=5), intrastriatal vehicle infusion in 6-OHDA-lesioned rats (IS-vehicle-6-OHDA) (n=4), intrastriatal LGF infusion in naïve animals (IS-LGF-naïve) (n=3) and intrastriatal vehicle infusion in naïve animals (ISvehicle-naïve) (n=3). Animal Perfusion and Tissue Processing Six weeks after the neurosphere-derived cells were implanted (14 weeks after the creation of the 6-OHDA lesion), heavily anesthetized, animals were perfused via ascending aorta with 50 ml of heparanized saline (5 U of heparin per milliliter of 0.9% NaCl), followed by 250 ml of 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Brains were removed, postfixed in the same solution for 24 hours at 4ºC, transferred to sequential washes in 10%, 20% and 30% sucrose in PB, and frozen on dry ice before sectioning into 20 μm-thick coronal sections on a cryostat. Antibodies and Immunochemicals The primary monoclonal antibodies used in this study were: mouse anti-tyrosine hydroxylase (TH, 1:500; Chemicon International, Temecula, CA), mouse anti-BrdU (1:25; DakoCytomation, Denmark), rat anti-BrdU (1:25; AbD Serotec, Oxford, UK), mouse anti-neuronal nuclei (NeuN, 1:1000; Chemicon International Inc.), mouse anti-Reca (1:20; AbD Serotec), mouse anti-nestin (clone Rat 401, 1:20; Developmental Studies Hybridoma Bank, University of Iowa, Ames, IA), mouse anti-oligodendrocytes (RIP, 1:1000; Chemicon International Inc.). The polyclonal antibodies were: rabbit anti-TH (1:500, AbD Serotec), rabbit anti-tubulin III (1:2000; BabCO, Richmond, CA), rabbit anti-glial fibrillary acidic protein (GFAP, 1:200; DakoCytomation), guinea pig anti-doublecortin (Dcx, 1:3000; Chemicon International Inc.), and rabbit anti-laminin (1:25, Sigma Chemical Co; St. Louis, MO). The secondary antibodies used were: biotinylated goat anti-mouse IgG (Zymed Laboratories; South San Francisco, CA), streptavidin–biotin–peroxidase complex (DakoCytomation), diaminobenzidine (DAB) + substrate–chromogen system (both from DakoCytomation), Alexa Fluor-568 goat anti-mouse IgG, Alexa Fluor-488 donkey anti-rat IgG, and Alexa Fluor-488 goat anti-rabbit IgG (1:400; all from Molecular Probes; Eugene, OR), Fluorescein-conjugated goat

anti-mouse IgG (1:25; Jackson ImmunoResearch Laboratories Inc, West Grove, PA), Cy3-conjugated donkey antiguinea pig IgG (1:500, Jackson ImmunoResearch Laboratories Inc.), and Rhodamine-conjugated goat anti-rabbit IgG (1:100, Chemicon International Inc.). Immunohistochemistry and Morphometric Analysis Sections were mounted on silane-coated slides, treated with sodium acetate 10 mM, pH 6.0, at 95ºC for 4 min, and preincubated with 5% normal goat serum (NGS) in Trisbuffered saline (0.15 M NaCl and 0.1 M Tris HCl, pH 7.4) / 0.1% Triton-X 100 for 30 min. Primary antibodies were applied for 24 hr at room temperature, and most of them were visualized using immunofluorescence procedures. The slides were coverslipped in a medium containing p-phenylenediamine and bisbenzimide (Hoechst 33342; Sigma) for detection of nuclei. Some series of sections were preincubated with 5% NGS and then processed for histochemical detection of isolectin IB4, a marker of microglia and macrophages, by incubating for 2 hr with isolectin IB4 conjugated to peroxidase (1:20; Sigma Chemical CO, St Louis, MO). Finally, the reaction product was detected with DAB chromogen. For double immunolabeling with BrdU, neural cell markers were detected prior to BrdU immunostaining. For detection of incorporated BrdU, sections were treated with 2N HCl at 37ºC for 30 min and rinsed in Tris-buffered saline, before blocking with NGS and primary antibody incubation. For quantitative estimation of TH immunostaining in the striatum, measurements were performed in several coronal sections from different rostrocaudal levels beginning at +1.00 mm relative to bregma (level 2), +0.2 mm (level 3), 0.3 mm (level 4), -0.92 mm (level 5), -1.4 mm (level 6) and 1.8 mm (level 7), using a 10x objective. The area occupied by TH-positive fibers was expressed as a percentage of the total striatal cross-sectional area. For quantitative estimation of BrdU-, neural antigen-, and isolectin IB4-positive cells, a systematic random sampling of 50 fields-of-view was carried out under fluorescence microscopy with the aid of the Computer Assisted Stereology Toolbox (CAST) grid® system (Olympus, Ballerup, Denmark). In each animal, measurements were performed in coronal sections containing the BrdU-positive grafted cells at two dif-


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Fig. (2). Distribution of grafted NSCs in the striatum of 6-OHDA-lesioned rats. BrdU-labeled neurosphere-derived cells were grafted in the DA depleted striatum of 6-OHDA-lesioned rats. In the same surgical act, the rats were implanted with an infusion cannula connected to an osmotic minipump designed for the infusion over 15 days of vehicle (IS-vehicle-6-OHDA). Two weeks after the end of IS-vehicle infusion BrdU inmunostaining was observed in the entire striatal surface (A and B, green) and in the subventricular zone (C, green), showing the highest density at the injection site (A, green). D, E and F, show the double inmunolabeling for BrdU (D, green, E and F, red), nestin (D, red), -tubulin III (E, green), or laminin (F, green). Note how in the striatal parenchyma some BrdU-positive cells are nestin-positive too (D, white dashed arrows), and how grafted cells are distributed upon the myelinated axonal bundles (B, white arrow heads), around the -tubulin III-positive neurons (E, arrows), and in the laminin-positive blood vessels (F). Lateral ventricle (LV), striatum (S), myelinated axonal bundles (MAB), subventricular zone (SVZ), (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper).

ferent levels of the striatum, the implantation site (levels 2 and 3), and at a posterior striatum site (levels 6 and 7). The number of TH-positive cells in the SN was assessed in coronal sections from 6 different levels 140 m apart, using a 20x objective. The first level being centered at -5.3 mm relative to bregma, and the last level at -6.00 mm relative to bregma. Terminal Deoxynucleotidyl Transferase-Mediated Biotinylated UTP Nick End Labeling Staining DNA fragmentation was evaluated using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated UTP nick end labeling (TUNEL) technique. Briefly, tissue sections mounted on silane-coated slides were permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate buffer (pH 7.4) for 2 min at 4ºC, and incubated with 5 units of TdT and 10 mM dUTP-biotin (Boehringer Mannheim) in Tris buffer saline (pH 7.6) for 1 hr at 37ºC. TUNEL detection was performed using a biotin-linked secondary antibody (1:200; Vector Laboratories, Inc., Burlingame, CA) followed by incubation with an avidin-biotinylated horseradish peroxidase complex (Vectastain Elite ABC Kit; Vector). Finally, 0.05% 3,3-diaminobenzidine in 0.05% H2O2 and 0.6% NiCl was used as chromogen. Western Blotting Protein Analysis A group of naïve (n=6) and 6-OHDA-lesioned rats (n=12) received in the left striatum IS-vehicle or IS-LGF for 24 hours. Their striata were removed and homogenized in

0.5 M Tris-HCl buffer (pH 7.4) containing 1 mM EDTA, 12 mM 2-mercaptoethanol, 1 mM benzamidine, 0.5% NP-40, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 10,000g for 30 min, and proteins were processed for Western blot analysis to determine the relative levels of Bcl-2 and Bax. Aliquots of 30g of protein were separated by electrophoresis on 10% SDS-polyacrylamide minigels and transferred to nitrocellulose filters. Membranes were soaked in blocking solution (0.2 M Tris-HCl, 137 mM NaCl, and 3–5% dry skimmed milk, pH 7.6) and incubated with primary antibodies diluted in the same blocking solution: mouse anti-Bcl-2 (1:400; Santa Cruz Biotechnology Inc., Burlingame, CA, USA), and rabbit anti-Bax (1:300; Santa Cruz Biotechnology Inc.). After extensive washing, membranes were incubated with the peroxidase-conjugated secondary antibodies diluted 1:1000 in blocking solution. The filters were developed with enhanced chemiluminescence Western blotting analysis, following the procedure described by the manufacturer (Amersham, Buckinghamshire, England). Membranes were immunolabeled for control charge using mouse anti- actine (1:5000; Sigma Aldrich). Autoradiograms were quantified by computer-assisted videodensitometry. Data Analysis Results are expressed as mean ± SEM from 3 to 5 independent animals. Statistical analyses were performed using Student’s t-test or ANOVA followed by the Newman-Keuls multiple comparison test, and the difference was considered significant when p 0.05.


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Fig. (3). Effects of IS-LGF infusion in the survival and differentiation of grafted NSCs. BrdU-labeled neurosphere-derived cells were grafted in striatum of 6-OHDA-lesioned rats (A, C, and D-G) and naïve rats (B). In the same surgical act, the rats were implanted with an infusion cannula connected to an osmotic minipump designed for the infusion over 15 days of vehicle (IS-vehicle, white bars) or 160 ng/ml/day LGF (IS-LGF, black bars). At the implant injection level (zone 2, +1.0 mm from Bregma), IS-LGF infusion significantly increases the number of BrdU-positive grafted cells in the striatum of 6-OHDA-lesioned rats (A), and in the striatum of naïve rats (B). C, shows the percentage of BrdU-positive grafted cells that differentiate into NeuN-positive mature neurons (neurons), or showed an endothelial-like phenotype (endothelium) in the DA-depleted striatum of 6-OHDA-lesioned rats. Note how IS-LGF infusion significantly increases the number of endothelial-like cells. Panels D to G show the inmunolabeling for BrdU (D, green and F, red), NeuN (E, red), and laminin (G, green). Note how in the DA-depleted striatum of 6-OHDA-lesioned rats that received the infusion of vehicle, some of the BrdU-positive nuclei were NeuN-positive too (D and E, white arrowheads), and how grafted cells localized in the blood vessels show an endothelial-like morphology (G and F, white arrows). The results represent the mean ± SEM of 3 to 5 independent animals. *p 0.05 vs IS-vehicle infusion, (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper).

RESULTS Effects of IS-LGF Infusion in the Distribution and Survival of Grafted Neural Stem Cells Since several studies suggest that the co-administration of neurotrophic factors improves the efficiency of NSCs implants, the denervated striatum of 6-OHDA-lesioned rats received the implants of neurosphere-derived cells in combination with IS infusion of vehicle or 160 ng/rat/day LGF during 15 days. In the IS-vehicle-6-OHDA group of animals, most of the implanted cells, as analyzed by BrdU inmunostaining, were distributed in the entire striatal surface showing the highest density at the injection site and nearby striatal parenchyma (Fig. 2A). Moreover, implanted cells were able to migrate from the injection site about 2 mm caudally, occupying the most ventral area of the striatum, the globus pallidus, the internal capsula, and nearby thalamus.

In the striatal parenchyma, BrdU-positive implanted cells were localized inside myelinated axonal bundles (Fig. 2B), in close apposition to striatal -tubulin III-positive neurons where they seemed like satellite glial cells surrounding sensory neurons in dorsal ganglia (Fig. 2E), in the SVZ (Fig. 2C), and very frequently, they were associated to lamininepositive blood vessels (Fig. 2F). Intrastriatal infusion of LGF neither affected the distribution, nor the localization of the implanted NSCs within the striatum. However, at the implant injection level, the striatal parenchyma of the IS-LGF-6OHDA group showed a significantly higher number of BrdU-positive implanted cells as compared with the ISvehicle infused animals (Fig. 3A). This effect was not observed when BrdU-positive cells were analyzed in posterior regions of the striatum (102 ± 46 (n=3) and 80 ± 32 (n=4) BrdU-positive cells/mm2 in the IS-vehicle and IS-LGF treated rats, respectively).


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Fig. (4). Effects of IS-LGF infusion in Bcl-2 and Bax protein expression. 6-OHDA-lesioned rats were implanted in DA-depleted striatum with an infusion cannula connected to an osmotic minipump designed for the infusion of vehicle (IS-vehicle, white bars) or LGF (IS-LGF, black bars). Twenty four hours later, the animals were sacrificed and their striata were removed and prepared for Western blot analysis of Bcl-2 and Bax, as described in the methods section. Note how IS-LGF infusion significantly increases Bcl-2 expression (A) and the ratio Bcl-2/Bax (C) without affecting to the levels of the pro-apoptotic protein Bax (B). C, shows a representative Western blot for Bcl-2 and Bax. The results represent the mean ± SEM of 6 independent animals. *p 0.05 vs IS-vehicle infusion.

To determine whether or not LGF needs a DA depletion to increase the number of BrdU-positive implanted cells, a group of naïve animals received in the striatum an implant of neurosphere-derived cells in combination with the IS infusion of vehicle or LGF during 15 days. As shown in Fig. (3B), the number of BrdU-positive cells was significantly increased in the striatum of the IS-LGF-naïve group of animals. IS-LGF infusion could prevent apoptotic cell death of the implanted cells, but no TUNEL-positive cells were observed in the striatum of naïve or 6-OHDA-lesioned rats that received the infusion of vehicle or LGF for 15 days (data not shown). Programmed cell death is highly regulated by proteins of the Bcl-2 family, comprising members that have either anti-apoptotic (such as Bcl-2), or pro-apoptotic (such as Bax) effects [40]. To determine whether IS-LGF could regulate their expression, the DA-depleted striatum of 6OHDA-lesioned rats was infused with vehicle or LGF during

24 hours. Western blot analysis showed that IS-LGF significantly increased the levels of the anti-apoptotic protein Bcl-2 (Fig. 4A and 4D) without affecting the expression of the proapoptotic protein Bax (Fig. 4B and 4D). Moreover, the ratio Bcl-2/Bax was raised by 4.6 ± 1.1 fold in the infused striatum (Fig. 4C). Similarly, the infusion of LGF in the striatum of naïve rats potentiated the expression of Bcl-2 and increased the ratio Bcl-2/Bax by 4.2 ± 1.3 (n=3) and 4.1 ± 1.7 (n=3) fold, respectively. Altogether, these results strongly suggest that IS-LGF may exert a trophic effect on the implanted NSCs. Cellular Phenotypes Derived from Grafted Neural Stem Cells Nestin is a protein expressed by NSCs and undifferentiated neural precursors [8, 13, 39]. As shown in Fig. (2D), only a few of the BrdU-positive grafted cells double labeled for nestin in the DA depleted striatum of rats that received


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25 mm Fig. (5). Neural and no-neural cellular phenotypes derived from grafted NSCs in the striatum of 6-OHDA-lesioned rats. BrdU-labeled neurosphere-derived cells (A, green, C-F, red) were grafted in DA-depleted striatum of 6-OHDA-lesioned rats. In the same surgical act, the rats were implanted with an infusion cannula connected to an osmotic minipump designed for the infusion over 15 days of vehicle (ISvehicle-6-OHDA). Two weeks after the end of the infusion, grafted BrdU-positive cells co-expressed the neuronal markers Dcx (A, red) and -tubulina III (B, green). Moreover, they differentiated to GFAP-positive astrocytes (D, green, arrowheads) and RIP-positive oligodendrocytes (E, green, clear arrowheads). C and F show how a population of grafted cells co-labeled with isolectine IB4 and showed the morphology of activated macrophages (C, green) and microglia (F, green), (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper).

IS-vehicle or IS-LGF during 15 days. NSCs in culture differentiate into neurons, glia, and non-neural cell types [12, 25]. To determine if grafted NSCs can differentiate into different cell types, double inmunolabeling for BrdU and neural- and non-neural markers was performed. Animals receiving IS-vehicle infusion, as well as those that received LGF, showed BrdU-positive cells that co-expressed Dcx (Fig. 5A) or -tubulin III (Fig. 5B). Moreover, about 12% of the implanted cells were NeuN-positive in both experimental groups (Fig. 3C, D, and E). These results indicate that the denervated striatum of 6-OHDA-lesioned rats allows the differentiation of NSCs to a neuronal phenotype.

they were localized in laminin-positive blood vessel walls (Fig. 3F), we may argue that grafted NSCs differentiate to an endothelial-like phenotype. Interestingly, they could be found in big blood vessels that were probably already present in the striatal parenchyma prior to the implant, and also in smaller vessels that could be newly generated due to their location near the injection site of the implant. The striatal parenchyma of the IS-vehicle as well as the IS-LGF group of animals showed BrdU-positive cells within the blood vessels, but the number of cells showing the morphology of endothelial cells was significantly increased in the LGF infused animals (Fig. 3C).

Besides neuronal differentiation, BrdU-positive implanted cells were able to differentiate into different glial phenotypes present in the adult CNS. Thus, in the striatal parenchyma of the IS-vehicle and IS-LGF infused animals some BrdU-positive cells co-stained with anti-GFAP (Fig. 5D), and others with anti-RIP (Fig. 5E). On the other hand, 23 ± 5.6% (n=6) of the BrdU-positive cells expressed also isolectin IB4, and showed the morphology of microglia (Fig. 5F). At the cannula level, some of the BrdU-/isolectin IB4positive cells looked like activated macrophages with abundant lysosomes in their cytoplasm (Fig. 5C). Taken together, these results point out the capability of implanted NSCs to differentiate into hematopoietic derived cell phenotypes.

Effects of Grafted NSCs in the Apomorphine-Induced Rotational Behavior and the TH-Positive Innervation

As mentioned above, BrdU-positive cells were frequently located near the blood vessels. A significant number of these cells was integrated into the blood vessels and showed a similar morphology to endothelial cells (Fig. 3E). Because

In rats receiving unilateral injections of 6-OHDA in the SN apomorphine-induced rotational behavior was progressively increased to reach a plateau at 9 weeks post-lesion, which was maintained for several additional weeks. Intrastriatal infusion of vehicle, in combination with implants of NSCs in the striatum, significantly decreased apomorphineinduced rotational behavior 3 weeks after beginning the infusion, as compared with those animals that only received ISvehicle infusion without cell implants (lesion-control group) (Fig. 6A). Notably, rotational behavior was more reduced in 6-OHDA-lesioned rats receiving NSCs implants and IS-LGF infusion, as compared with the lesion-control group, and with those animals that received intrastriatal infusion of vehicle and cells (Fig. 6A).


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Fig. (6). Effects of grafted NSCs and LGF infusion in rotational behavior and TH expression in the striatum of 6-OHDA-lesioned rats. BrdU-labeled neurosphere-derived cells were grafted in the DA depleted striatum of 6-OHDA-lesioned rats. In the same surgical act, the rats were implanted with an infusion cannula connected to an osmotic minipump designed for the infusion over 15 days of vehicle (ISvehicle-6-OHDA) or with 160 ng/ml/day of LGF (IS-LGF-6-OHDA). A, shows apomorphine-induced rotational behavior of IS-vehicle-6OHDA rats (open circles) and IS-LGF-6-OHDA rats (black circles) as compared with a group of rats receiving unilateral injections of 6OHDA in the SN and IS- vehicle infusion (lesion-control group, black squares). Note how rotational behavior was significantly improved in the IS-LGF-6-OHDA group of rats. B, shows how TH-positive innervation in the DA-depleted striatum of IS-vehicle-6-OHDA (B, white bars) and IS-LGF-6-OHDA rats (B, black bars) differed of that observed in the lesion-control group (B, horizontal lined bars). C, shows how TH-positive innervation in the striatum of IS-vehicle-6-OHDA rats depends on the zone where NSCs were grafted (C, black arrows). Thus, TH-positive innervation through the striatum was higher in those animals receiving the implants in zone 2 (+1.0 mm from Bregma) than in those animals were grafts were applied in zone 3 (+0.2 mm from Bregma). D to F show TH inmunostaining in the striatum of naïve rats (D), in the lesion-control group (E), and in the striatum of IS-vehicle-6-OHDA rats (F). Note how the IS-vehicle-6-OHDA group shows higher density of TH-positive terminals than the lesion-control group. The results represent the mean ± SEM of 3 to 5 independent animals. *p 0.05,**p 0.01 vs lesion control group of animals. +p 0.05 vs IS-vehicle-6-OHDA group of animals, (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper).

To determine if the improvement in rotational behavior was due to an increase in dopaminergic innervation, and/or to the differentiation of implanted NSCs into cathecolaminergic neurons, double immunostaining for TH and BrdU was performed in the striatum of 6-OHDA-lesioned rats that received implants of NSCs in combination with IS-vehicle or IS-LGF infusions.

As previously reported [32], striata from unilaterally 6OHDA-lesioned rats that have not received a NSCs graft nor an infusion, contain no TH-positive neurons, and only 5.5 ± 1.9% of the structure, corresponding to the most ventral region of the striatum, shows TH-positive innervation, Similar results were observed in the striatum of the lesion-control group, where only 4.0 ± 1.3% of the structure showed TH-


positive innervation, in comparison with the striatum of naïve rats (Fig. 6D and 6E). None of the different experimental groups of animals analyzed in this study showed TH-/BrdUpositive neurons in the striatum, suggesting that our NSCs do not have the ability to differentiate into DA neurons when implanted in the striatum of 6-OHDA-lesioned rats. However, in comparison with the lesion-control group, the striatum of rats receiving IS-vehicle infusion for 15 days in combination with implants of NSCs, showed a 23 ± 7% of the structure with TH-positive innervation at the infusion site (Fig. 6B and 6F). 6-OHDA-lesioned rats receiving IS-LGF infusion and cells also showed increased TH-positive innervation, but no significant differences were observed when compared with the lesion-control group, and with those animals that received intrastriatal infusion of vehicle and cells (Fig. 6B). Irrespective of the IS-vehicle or IS-LGF infusion, THpositive innervation through the striatum was dependent on the zone where NSCs were implanted. Thus, 6-OHDAlesioned rats receiving cells at +1.0 mm from Bregma showed higher TH-positive innervation than those animals where NSCs were implanted more caudally, at +0.2 mm from Bregma (Fig. 6C). Unilaterally 6-OHDA-lesioned rats showed a significant loss in the number of TH-positive neurons in the SN [32]. Similarly, almost no TH-positive neurons were observed in the lesioned SN of rats receiving IS infusion of vehicle or LGF in combination with grafted NSCs (1 ± 0.6 (n=3) and 1.3 ± 1.3 (n=3) TH-positive cells/section in the IS-vehicle and IS-LGF treated rats, respectively). DISCUSSION In the present study we show that NSCs are able to survive in the denervated striatum of 6-OHDA-lesioned rats. Implanted NSCs keep their pluripotentiality. Thus, they differentiate into neurons and glial cells, as well as endothelium and cells from the hematopoietic lineage system, such as macrophages and microglia. Present results also show the ability of implanted NSCs to stimulate TH-positive neurite outgrowth in the striatum. This effect was accompanied by a significant improvement in apomorphine-induced rotational behavior, and was favored by the administration of LGF in the denervated striatum of 6-OHDA-lesioned rats. The ability of grafted NSCs to survive in the mammalian adult brain has been widely reported [16, 17, 41]. The present study clearly shows that the adult rat brain contains the signals required for the survival of NSCs, since BrdUpositive implanted cells were observed both in the naïve, and in the denervated striatum. In vitro, NSCs differentiate into neurons, astrocytes and oligodendrocytes [39, 42]. The adult rat striatum also allows migration and differentiation of the implanted cells. Thus, BrdU-positive cells that co-expressed neuronal and glial specific markers were widely distributed in the striatal parenchyma, including the most ventral zones. In addition, a population of the implanted cells was located in the SVZ and in the proximity of the blood vessels, two favorable environments for the maintenance and renewal of NSCs [43-45]. In agreement with other studies [46, 47], a small proportion of these cells showed endothelial cells-like


23

morphology, and was marked with anti-laminin antibodies. These results indicate the capability of grafted NSCs to generate new blood vessels that may be needed for tissue regeneration. In vitro and in vivo studies suggest that NSCs may also generate cells from the hematopoietic lineage [15, 48]. Although these results have been refuted by other authors [49], our data clearly show the hematopoietic potential of NSCs because activated macrophages as well as abundant microglia derived from the grafted cells were observed in the denervated striatum of 6-OHDA-lesioned rats. As reported here, IS-LGF infusion significantly increased the number of implanted cells in the striatum of naïve and 6OHDA-lesioned rats. This increase could be due to an antiapoptotic effect of LGF, as occurs in the liver of bile ductligated rats [50]. Under our experimental conditions, no TUNEL-positive cells were observed in animals receiving vehicle or LGF for 15 days. Moreover, TUNEL analysis at early time points of infusion (24 to 72 hours) was not very accurate due to the tissue damage promoted by the insertion of cannula. Bcl-2 is an anti-apoptotic protein involved in the survival of neural cells [51, 52]. Interestingly, IS-LGF significantly raised its expression in the striatum. Since the infusion of the factor did not affect the levels of the proapoptotic protein Bax, we may argue that LGF probably strengthens the mechanisms that regulate the viability of the grafted NSCs. However, we cannot discard the possibility that LGF promotes the proliferation of the implanted cells, because intracerebral LGF infusion significantly increases PCNA expression [32], and stimulates the proliferation of neural precursors located in the SVZ of the adult rat brain [33, 34]. We have previously reported that LGF has neurogenic activity and promotes the activation of microglia in the striatal parenchyma of 6-OHDA-lesioned rats [32, 34]. IS infusion of LGF did not affect the number of neurons or microglia derived from the implanted cells. However, LGF increased the number of BrdU-positive cells showing an endothelial-like morphology. In the liver, LGF locally stimulates the secretion of tumor necrosis factor alpha (TNF-alpha) [53], whose role in stimulating angiogenesis has been reported [54, 55]. Our preliminary data show that 48 hours after IS-LGF infusion has begun, TNF-alpha levels are increased by 1.8 ± 0.16 fold (n = 3) in the DA-depleted striatum of 6-OHDA-lesioned rats. Similar results are observed after the infusion of vehicle, so we may argue that this effect is probably due to the tissue damage promoted by the insertion of the osmotic minipum. However, the fact that one single intraperitoneal injection of LGF also raises TNF-alpha levels by 1.6 fold in the striatum of naïve rats (our unpublished observations), seems to suggest that this cytokine might be responsible for the increase in the number of BrdUpositive cells showing an endothelial-like morphology, as observed in the IS-LGF infused animals. Grafted NSCs differentiated into neurons, but no THpositive cells derived from them were observed at any of the different experimental conditions analyzed in this study. Being that under some in vitro specific conditions our NSCs are able to differentiate into TH-positive neurons [25, 56], pre-


sent results suggest that the dopamine-depleted striatum is devoid of the signals required for the differentiation of NSCs into DA neurons. A curious finding was that TH-positive innervation in the striatum was increased when NSCs were grafted in anterior levels (zone 2, +1.0 mm from Bregma), as compared to more posterior grafts (zone 3, + 0.2 mm from Bregma). As already mentioned in this study, grafted NSCs differentiate into microglia/macrophages, which are able to synthesize trophic factors involved in the dopamine fibers sprouting in the injured striatum [57, 58]. Their presence in the anterior zones of the striatum could explain the increase in TH-positive innervation observed, because most of the dopaminergic neurons in the SN project to these areas [59, 60]. In agreement with other studies [19], our results show that apomorphine-induced rotational behavior was improved in 6-OHDA-lesioned rats with grafted NSCs. Some of the implanted cells were localized in the proximity of striatal neurons, and showed a similar location to the satellite glial cells of the Peripheral Nervous System. Since satellite glial cells are able to synthesize neurotrophins [61], we may argue that implanted NSCs represent a source of trophic factors for striatal neurons, and therefore are able to improve their function. Alternatively, newly generated neurons may partially replace a small proportion of striatal neurons affected by the lack of DA. IS-LGF infusion significantly reduced the rotational behavior of 6-OHDA-lesioned rats with grafted NSCs. As previously commented, LGF drives the differentiation of NSCs to an endothelial phenotype. This angiogenic effect could derive in a better irrigation of the host tissue, and a higher supply of neurotrophic factors [35]. On the other hand, LGF could improve the functionality of the remaining DA neurons by stimulating the synthesis of factors involved in their survival and/or function [62-64]. Note worthily, LGF stimulates the synthesis of vascular endothelial growth factor in testis [30], and increases the levels of the dopamine transporter (DAT) in the striatum of 6-OHDA-lesioned rats [65]. In conclusion, our results show that grafted NSCs survive, migrate, and keep their pluripotentiality in the dopamine depleted striatum of 6-OHDA-lesioned rats. As IS-LGF infusion elicits their survival, and improves apomorphineinduced rotational behavior, we propose that the administration of the factor might be a convenient treatment for Parkinson’s disease cell replacement therapies based in NSCs transplantation.

Reimers et al.

observer.Z1 ZEISS microscope coupled to the ApoTome system. REFERENCES [1]

[2] [3]

[4] [5]

[6] [7] [8]

[9] [10] [11]

[12] [13] [14]

[15] [16] [17]

[18] [19]

ACKNOWLEDGEMENTS

[20]

The authors wish to acknowledge the editorial for its invitation to contribute with a scientific article in CSCRT. This work was supported by the Fondo de Investigaciones Sanitarias (FISS PI060315). RG-G was the recipient of a FiBio Hospital Ramón y Cajal fellowship, and MRS was the recipient of a Contrato de Personal de Apoyo a la Investigación (FISS). We thank Bárbara Luna for technical help and Kerry Davis for manuscript review. We also thank Dr. Myriam Escobar and Mr. Manel Roldan for obtaining the microphotographies showed in Fig. 3D and 3E with an Axio

[21] [22]

[23] [24]

Ehringer H, Hornykiewicz O. Distribution of noradrenaline and dopamine (3-hydroxytyramine) in the human brain and their behavior in diseases of the extrapyramidal system. Klin Wochenschr 1960; 38: 1236-9. Björklund A, Lindvall O. Cell replacement strategies for central nervous system disorders. Nature Neuroscience 2000; 3 (6): 53744. Hermann A, Gerlach M, Schwarz J, Storch A. Neurorestoration in Parkinson's disease by cell replacement and endogenous regeneration. Expert Opin Biol Ther 2004; 4(2): 131-43. Freed CR, Greene PE, Breeze RE, et al. Transplantation of embryonic dopamine neurons for severe Parkinson's disease. N Engl J Med 2001; 344 (10): 710-9. Olanow CW, Kordower JH, Freeman TB. Fetal nigral transplantation as a therapy for Parkinson's disease. Trends Neurosci 1996; 19 (3): 102-9. Björklund A. Cell therapy for Parkinson's disease: problems and prospects. Novartis Found Symp 2005; 265: 174-86. Meyer AK, Maisel M, Hermann A, Stirl K, Storch A. Restorative approaches in Parkinson's Disease: Which cell type wins the race? J Neurol Sci 2010; 289(1-2): 93-103 Bazán E, Alonso FJ, Redondo C, et al. In vitro and in vivo characterization of neural stem cells. Histol Histopathol 2004; 19 (4): 1261-75. Gil-Perotín S, Alvarez-Buylla A, García-Verdugo JM. Identification and characterization of neural progenitor cells in the adult mammalian brain. Adv Anat Embryol Cell Biol 2009; 203: 1-01. Merkle FT, Alvarez-Buylla A. Neural stem cells in mammalian development. Curr Opin Cell Biol 2006; 18(6): 704-9. Weiss S, Reynolds BA, Vescovi AL, Morshead C, Craig CG, van der Kooy D. Is there a neural stem cell in the mammalian forebrain? Trends Neurosci 1996; 19(9): 387-93. Kennea NL, Mehmet H. Neural stem cells. J Pathol 2002; 197 (4): 536-50. Lobo MV, Alonso FJ, Redondo C, et al. Cellular characterization of epidermal growth factor- expanded free-floating neurospheres. J Histochem Cytochem 2003; 51(1): 89-103. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992; 255(5052): 1707-10. Vescovi AL, Rietze R, Magli MC, Bjornson C. Hematopoietic potential of neural stem cells. Nat Med 2002; 8(6): 535; 536-7. Arias-Carrión O, Yuan TF. Autologous neural stem cell transplantation: A new treatment option for Parkinson's disease? Med Hypotheses 2009; 73(5): 757-9. Bjugstad KB, Teng YD, Redmond DE Jr, et al. Human neural stem cells migrate along the nigrostriatal pathway in a primate model of Parkinson's disease. Exp Neurol 2008; 211(2): 362-9. Pluchino S, Zanotti L, Deleidi M, Martino G. Neural stem cells and their use as therapeutic tool in neurological disorders. Brain Res Brain Res Rev 2005; 48(2): 211-9. Zhu Q, Ma J, Yu L, Yuan C. Grafted neural stem cells migrate to substantia nigra and improve behavior in Parkinsonian rats. Neurosci Lett 2009; 462(3): 213-8. Sharp J, Keirstead HS. Stem cell-based cell replacement strategies for the central nervous system. Neurosci Lett 2009; 456(3): 107-11. Liu J, Huang HY. How to improve the survival of the fetal ventral mesencephalic cell transplanted in Parkinson's disease? Neurosci Bull 2007; 23(6): 377-82. Sharma R, McMillan CR, Niles LP. Neural stem cell transplantation and melatonin treatment in a 6-hydroxydopamine model of Parkinson's disease. J Pineal Res 2007; 43(3): 245-54. Fernández-Espejo E. Pathogenesis of Parkinson's disease: prospects of neuroprotective and restorative therapies. Mol Neurobiol 2004; 29(1): 15-30. Krieglstein K. http://www.ncbi.nlm.nih.gov/pubmed/15300492?ordinalpos=72&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSumFactors promoting survival of mesencephalic dopaminergic neurons. Cell Tissue Res 2004; 318(1): 73-80.

LGF and NSCs Grafts in Parkinson’s Disease [25]

[26] [27]

[28] [29] [30]

[31]

[32]

[33]

[34]

[35]

[36] [37] [38] [39]

[40]

[41]

[42]

[43] [44] [45] [46]


Reimers D, Gonzalo-Gobernado R, Herranz AS, et al. Driving Neural Stem Cells Towards a Desired Phenotype. Curr Stem Cell Res Ther 2008; 3(4): 247-53. Díaz-Gil JJ, Escartín P, García-Cañero R, et al. Purification of a liver DNA synthesis promoter from plasma of partially hepatectomized rats. Biochem J 1986; 234: 49-55. Díaz-Gil JJ, Gavilanes JG, Sánchez G, et al. Identification of a liver growth factor as an albumin-bilirubin complex. Biochem J 1987; 243: 443-8. Díaz-Gil JJ, Sánchez G, Trilla C, Escartín P. Identification of biliprotein as a liver growth factor. Hepatology 1988; 8 (3): 484-6. Díaz Gil JJ, Rúa C, Machin C, et al. Hepatic growth induced by injection of the liver growth factor into normal rats. Growth Regul 1994; 4(3): 113-22. Martín-Hidalgo A, Arenas MI, Sacristán S, et al. A. Rat testis localization of VEGFs and VEGF receptors in control and testicular regeneration stimulated by the liver growth factor (LGF). The FEBS Journal 2007; 274 (Suppl.1): 296. Somoza B, Abderrahim F, González JM, et al. Short-term treatment of spontaneously hypertensive rats with liver growth factor reduces carotid artery fibrosis, improves vascular function, and lowers blood pressure. Cardiovasc Res 2006; 69(3): 764-71. Reimers D, Herranz AS, Díaz-Gil JJ, et al. Intrastriatal infusion of liver growth factor stimulates dopamine terminal sprouting and partially restores motor function in 6-hydroxydopamine-lesioned rats. J Histochem Cytochem 2006; 54(4): 457-65. Bazán E, Herranz AS, Reimers D, et al. LGF “Liver Growth Factor” as a factor involved in the proliferation, migration and differentiation of neural stem cells: Potential use in Parkinson’s disease. Mapfre Medicina 2005; 16 (2): 1-11. Gonzalo-Gobernado R, Reimers D, Herranz AS, et al. Mobilization of neural stem cells and generation of new neurons in 6-OHDAlesioned rats by intracerebroventricular infusion of liver growth factor. J Histochem Cytochem 2009; 57(5): 491-502. Dong Z, Su L, Mino J. Effects of endothelial cells on renewal and differentiation of neural stem cells. Sheng Wu Yi Xue Gong Cheng Xue Za Zhi 2007; 24(5): 1184-6. Singh J, Bowers LD. Quantitative fraction of serum bilirubin species by reversed-phase high-performance liquid chromatography. J Chromatogr 1986; 380: 321-30. Ungersted U. Stereotaxic mapping of the monoamine pathways in the rat brain. Acta Physiol Scand 1971; 367: 1-48. Paxinos G, Watson CH. The Rat Brain in Stereotaxic Coordinates, 3rd ed., San Diego, CA, Academic Press 1997. Reimers D, López-Toledano MA, Mason I, et al. Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment. Neurol Res 2001; 23(6): 612-21. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999; 13(15): 1899911. Eriksson C, Björklund A, Wictorin K. Neuronal differentiation following transplantation of expanded mouse neurosphere cultures derived from different embryonic forebrain regions. Exp Neurol 2003; 184(2): 615-35. Redondo C, López-Toledano MA, Lobo MV, et al. Kainic acid triggers oligodendrocyte precursor cell proliferation and neuronal differentiation from striatal neural stem cells. J Neurosci Res 2007; 85(6): 1170-82. Barami K. Relationship of neural stem cells with their vascular niche: Implications in the malignant progression of gliomas. J Clin Neurosci 2008; 15(11): 1193-7. Doetsch F. A niche for adult neural stem cells. Curr Opin Genet Dev 2003; 13(5): 543-50. Palmer TD, Willhoite AR, Gage FH. Vascular niche for adult hippocampal neurogenesis. J Comp Neurol 2000; 425: 479-94. Song YS, Lee HJ, Park IH, Lim IS, Ku JH, Kim SU. Human neural crest stem cells transplanted in rat penile corpus cavernosum to repair erectile dysfunction. BJU Int 2008; 102(2): 220-4.

Received: May 26, 2010

[47]

[48]

[49]

[50] [51]

[52]

[53] [54]

[55] [56]

[57]

[58]

[59] [60] [61]

[62]

[63]

[64]

[65]

25

Ii M, Nishimura H, Sekiguchi H, et al. Concurrent vasculogenesis and neurogenesis from adult neural stem cells. Circ Res 2009; 105(9): 860-8. Papavasiliou AK, Mehler MF, Dobrenis K, Marmur R, Mabie PC, Kessler JA. Microglial lineage species are expressed in mammalian epidermal growth factor-generated embryonic neurospheres. J Neurosci Res 1996; 46(1): 49-57. Levison SW, Druckman SK, Young GM, Basu A. Neural stem cells in the subventricular zone are a source of astrocytes and oligodendrocytes, but not microglia. Dev Neurosci 2003; 25(2-4): 184-96. Díaz-Gil JJ, García-Monzón C, Rúa C, et al. Liver growth factor antifibrotic activity in vivo is associated with a decrease in activation of hepatic stellate cells. Histol Histopathol 2009; 24(4): 473-9. Frebel K, Wiese S. Signalling molecules essential for neuronal survival and differentiation. Biochem Soc Trans 2006; 34(Pt 6): 1287-90. Lee SI, Kim BG, Hwang DH, Kim HM, Kim SU. Overexpression of Bcl-XL in human neural stem cells promotes graft survival and functional recovery following transplantation in spinal cord injury. J Neurosci Res 2009; 87(14): 3186-97. Díaz-Gil JJ, Majano PL, López-Cabrera M, et al. The mitogenic activity of the liver growth factor is mediated by tumor necrosis factor alpha in rat liver. J Hepatol 2003; 38: 598-604. Otrock ZK, Mahfouz RA, Makarem JA, Shamseddine AI. Understanding the biology of angiogenesis: review of the most important molecular mechanisms. Blood Cells Mol Dis 2007; 39(2): 212-20. Sainson RC, Johnston DA, Chu HC, et al. TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype. Blood 2008; 111(10): 4997-5007. López-Toledano MA, Redondo C, Lobo MV, et al. Tyrosine hydroxylase induction by basic fibroblast growth factor and cyclic AMP analogs in striatal neural stem cells: role of ERK1/ERK2 mitogen-activated protein kinase and protein kinase C. J Histochem Cytochem 2004; 52(9): 1177-89. Batchelor PE, Liberatore GT, Wong JY, et al. Activated macrophages and microglia induce dopaminergic sprouting in the injured striatum and express brain-derived neurotrophic factor and glial cell line derived neurotrophic factor. J Neurosci1999; 19: 1708-16. Batchelor PE, Porritt MJ, Martinello P, et al. Macrophages and microglia produce local trophic gradients that stimulate axonal sprouting toward but not beyond the wound edge. Mol Cell Neurosci 2002; 21: 436-53. Gerfen CR, Wilson CJ. The basal ganglia. 1996. Handbook of Chemical Anatomy, Vol. 12: Integrated Systems of the CNS, Part III. Chapter II. Merello M, Cammarota A. Functional anatomy of the basal ganglia. Rev Neurol 2000; 30(11): 1055-60. Wetmore C, Olson L. Neuronal and nonneuronal expression of neurotrophins and their receptors in sensory and sympathetic ganglia suggest new intercellular trophic interactions. J Comp Neurol 1995; 353(1): 143-59. Jiang Q, Yan Z, Feng J. Neurotrophic factors stabilize microtubules and protect against rotenone toxicity on dopaminergic neurons. J Biol Chem 2006; 281(39): 29391-400. Singh S, Ahmad R, Mathur D, Sagar RK, Krishana B. Neuroprotective effect of BDNF in young and aged 6-OHDA treated rat model of Parkinson disease. Indian J Exp Biol 2006; 44(9): 699704. Yasuhara T, Shingo T, Kobayashi K, et al. Neuroprotective effects of vascular endothelial growth factor (VEGF) upon dopaminergic neurons in a rat model of Parkinson’s disease. Eur J Neurosci 2004; 19: 1494-504. Gonzalo-Gobernado R, Bazán E, Reimers D, et al. Neurotrophic activity of Liver Growth Factor in a Parkinson´s disease experimental model. Neuroscience 2009, Chicago, 2009-S-7516-SfN.

Revised: September 28, 2010

Accepted: November 06, 2010