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Grafted neural stem cells migrate to substantia nigra and improve behavior in Parkinsonian rats
Neuroscience Letters 462 (2009) 213–218
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Grafted neural stem cells migrate to substantia nigra and improve behavior in Parkinsonian rats Zhu Qing feng, Ma Ji, Yu Ling li, Yuan Chong gang ∗ School of Life Science, East China Normal University, Shanghai 200062, China
a r t i c l e
i n f o
Article history: Received 19 March 2009 Received in revised form 11 May 2009 Accepted 6 July 2009 Keywords: Neural stem cells Parkinson’s disease Transplantation Migration
a b s t r a c t Neural stem cell (NSC) transplantation has the potential to treat neurodegenerative diseases such as Parkinson’s disease (PD). In this study, we investigated the effect of transplanted NSCs in a PD animal model. NSCs isolated from the subventricular zone (SVZ) of E14 rats were cultured in vitro to produce neurospheres, which were subsequently infected with recombinant adeno-associated virus (rAAV2 ) expressing enhanced green fluorescent protein (EGFP). The PD animal model was established by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) of Sprague–Dawley rats. Once the model was established, EGFP-expressing NSCs were transplanted into the substantia nigra pars compacta (SNc) or striatum of PD rats. We found that NSCs transplanted into either site significantly reduced apomorphine-induced circling behavior of PD rats. Pathological analysis revealed that the EGFP-expressing NSCs could be detected at both injection sites at 1, 2 and 4 months after transplantation. SNc transplanted cells dispersed within the SNc with a significant portion differentiated into tyrosine hydroxylase-positive neurons. Whereas cells transplanted into the striatum migrated ventrally and posteriorly towards the SNc. These results suggest that the 6-OHDA damaged brain area attracts grafted NSCs, which migrated from the striatum and survived for a long time in SNc, resulting in behavioral improvement of PD rats. © 2009 Elsevier Ireland Ltd. All rights reserved.
PD is characterized by a progressive loss of dopaminergic (DA) neurons in the SNc, leading to severe motor dysfunction [23,13]. Chemical replacement therapies are widely used to control behavioral symptoms of PD. However, the effectiveness of pharmacological treatments is limited [8,12], to the search for more effective therapies. The replacement of damaged neurons by cell transplantation has recently been explored as a potential treatment for many neurodegenerative diseases [4,20,19,16]. NSCs have attracted a great deal of interest, not only because of their importance in basic research on neural development, but also in terms of their therapeutic potential in neurological diseases including PD. Because of their abilities to proliferate and/or to migrate to injured areas, NSCs appear to be suitable candidates for cell replacement treatment. It has been shown that NSCs is able to migrate towards damaged areas, to produce trophic factors, and to replace lost cells in ways that might be beneficial in PD [2]. However, little information is available about the migration of NSCs in rat PD models. Moreover, the information about the functional effectiveness of transplanted stem cells is also limited [9,24,11]. In this study, we investigated the ability of NSCs to survive in 6-OHDA-injured areas,
∗ Corresponding author. Tel.: +86 21 62232729. E-mail address: [email protected] (C.g. Yuan). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.07.008
and their capacity to migrate to the lesioned SNc. More importantly, we monitored functional improvements of PD rats following NSCs transplantation into either SNc or striatum. We showed that natural cues available in the brain can constitutively direct the migration and differentiation of transplanted NSCs, resulting in their development into neuronal-like cells expressing DA traits. 105 male Sprague–Dawley (SD) rats weighing 180–220 g were received unilateral 6-OHDA (Sigma, USA) lesions using a 10-l Hamilton syringe inserted into the right MFB at the following coordinates (in mm, with reference to the bregma and dura): AP: −2.6; ML: 1.7; DV: −7.8. The concentration of 6-OHDA was at 10.5 g in 3.5 l of ascorbic-saline. Four weeks after injection, animals were tested for a period of 0.5 h for apomorphine-induced turning behavior (intraperitoneal injection of 0.25 mg/kg apomorphine in ascorbic-saline). Only those rats with proven rotational behavior (>7 rpm) were selected for the next phase of study. The evaluation was performed in the double blind manner. NSCs were obtained from the forebrain SVZ of 12 fetal E14 rat brains. The SVZ pieces were incubated in culture medium consisting of DMEM/F12 (GIBCO, USA), B27, 10 ng/ml FGF-2, 20 ng/ml EGF (Sigma, USA). The suspensions were centrifuged at 1000 rpm for 5 min. Pellets were resuspended in 1 ml of culture medium and cells were then seeded at a density of 2 × 105 cells/ml into 75-ml polystyrene plastic culture flasks. The cells were plated and cultured
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Fig. 1. Neural stem cells derived from SVZ cells. (A) Confocal microscope photograph of nestin-positive neurospheres. (B) NSCs expressing EGFP. Scale bar: 50 m.
Fig. 2. Immunohistochemical analysis of TH neurons in the substantia nigra of PD rats at 4 weeks post 6-OHDA administration. (A) Coronal section showing TH-positive cells in both SNc regions. (B) Some TH-positive cells remained at ventral tegmental area in the lesioned side. (C) TH-positive cells in the non-lesioned side. (D) The loss of TH-positive cells in the 6-OHDA lesioned side. Scale bar: (B–D) 100 m.
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Fig. 3. (A) EGFP-labeled cells in the substantia nigra of the host brain 4 months after transplantation. (B) The same slide was stained for TH (brown). Scale bar: 50 m.
at 37 ◦ C in a humidified 5% CO2 and 95% air incubator. The neurospheres generated from this procedure were passaged weekly. Cells were cultured and expanded in flasks for 3 weeks, and then transferred to 24-well plates and cultured at a density of 5 × 105 cells/ml for 36 h. These cells were infected with rAAV2 containing the EGFP coding sequences (rAAV2 -EGFP, National Molecular Virology Lab of China) at 37 ◦ C for 4 h. The infected cells were harvested and resuspended, then cultured in the 75-ml flasks for 3–4 days before transplantation. A portion of cultured neurospheres was incubated with mouse anti-nestin antibody (1:500; Chemicon, USA) overnight at 4 ◦ C, followed by incubation for 1 h with the tetramethyl rhodamine isothiocyanate-IgG secondary antibody. To evaluate the expression of EGFP, some rAAV2-EGFP infected cells were cultured without FGF-2 and EGF in DMEM/F12 medium supplemented with 10% bovine serum for several days. All signals were detected using laser scanning confocal microscope (Leica, Germany). Five weeks after injection of 6-OHDA, EGFP-labeled cell suspensions were resuspended and grafted into the SNc (n = 20) or the striatum (n = 5). Using a glass capillary attached to a 10-l Hamilton syringe, 5 l of the cell suspension containing approximately 50,000 cells was injected into the right SNc or the right striatum at a rate of 1 l/min. Stereotaxic implantations were performed at the following coordinates: SNc AP: −5.2; ML: 1.8; DV: −7.8 and striatum AP: −0.2; ML: 3.5; DV: −5.5. PD rats and implanted rats were anesthetized and perfused transcardially with 4.0% PFA after final behavioral test. Brains were isolated and placed in the same solution for 4 h before saturation with 30% sucrose in PBS, and were then frozen at −80 ◦ C. Brains were sectioned at 30 m using a freezing microtome (Leica, Germany). To detect TH, sections were incubated with mouse antiTH antibody (1:300; Chemicon, USA) for 48 h at 4 ◦ C, followed by 1 h incubation of biotin-conjugated goat anti-mouse IgG, and then detected with an ABC kit (Chemicon, USA). Sigma-Plot Software version 10.0 was used for data analysis. Statistical differences between pre-transplantation and after transplantation were assessed using the Student’s t-test. The results were expressed as mean ± standard error. After 3–4 days, the NSCs obtained from the SVZ of E14 rats had proliferated and formed clusters. Most clusters grew to macroscopic size after 1 week. The number of cells increased from 2 × 105 seeded cells to about 6 × 105 cells within 1 week. During the expansion phase, most cells were positive for nestin, a marker of immature progenitor cells (Fig. 1A). To evaluate EGFP expression in NSCs, cells infected with rAAV2 -EGFP were allowed to differentiate and the EGFP expression was verified (Fig. 1B). The 6-OHDA lesion was confirmed by apomorphine-induced rotational behavior. A total of 57 rats were selected as successful PD models based on the presence of rotational behavior (>7 rpm).
The extent of the 6-OHDA lesions in the MFB was also verified by immunohistochemistry. As shown in Fig. 2, a significant reduction in TH-positive cells was observed in the lesioned area (Fig. 2A, B and D), while TH-positive cells remained in the non-lesioned area (Fig. 2A and C). The NSCs grafted in the SNc were identified by EGFP fluorescence (Fig. 3A), and the same slide was used to for immunohistochemical staining of TH (Fig. 3B). We found that a significant number of grafted cells were TH-positive, indicating the conversion of NSCs to cells similar to dopaminergic neurons. The functional benefit of NSCs transplantation on PD rat behavior was evaluated at 1, 2 and 4 months after transplantation. Compared with the pretransplantation period, PD rats grafted with NSCs in the SNc showed significant reductions in rotational behavior after 2 and 4 months (Fig. 4), while there was a slight, but not statistically significant, reduction after 1 month. Notably, PD rats received NSCs transplantation in the striatum also showed a significant reduction in rotation after 4 months, but no significant reduction was observed after 1 or 2 months (Fig. 4). There was no improvement in rotational scores in PD rats without transplantation (Fig. 4).
Fig. 4. Behavioral effectiveness of grafted NSCs. The behavior of PD animals grafted with NSCs in the SNc (n = 20) or striatum (n = 5) was tested before transplantation (pre-TP) and at 1, 2 and 4 months postgrafting. The apomorphine-induced rotational response was monitored for 30 min. PD rats showed no change in rotation score over time (pre-TP, n = 13; 1 month, n = 13; 2 months, n = 11; 4 months, n = 9). Number of rotations at 4 months: 233.89 ± 70.43). Rats grafted with NSCs into the SNc showed a significant reduction in rotation at the second (n = 13) and fourth months (n = 10, number of rotations at 4 months: 189.3 ± 63.24), but a non-significant reduction after 1 month (n = 20). Animals grafted with NSCs into the striatum also showed a significant reduction in rotation after 4 months (n = 4, number of rotations at 4 months: 169 ± 47.28), but no significant reductions at 1 or 2 months (n = 4). Significantly different from pre-transplantation at *p < 0.05, **p < 0.01 and ***p < 0.001.
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Fig. 5. EGFP-labeled cells in the SNc of the host brain 1, 2 and 4 months after transplantation. (A) Cells migrating rostrally and caudally from the injection center, seen in a sagittal section after 1 month. (B) Cells migrating medially and laterally from the injection center, seen in a coronal section after 1 month. (C) Cells migrating rostrally and caudally from the injection center, seen in a sagittal section after 2 months. (D) Cells migrating medially and laterally from the injection center, seen in a coronal section after 2 months. (E) Cells migrating in a strap-like manner along most of the SNc area, seen in a coronal slice after 2 months. (F) Cells migrating rostrally and caudally from the injection center, seen in a sagittal section after 4 months. (G) Cells migrating medially and laterally from the injection site, seen in a coronal section after 4 months (AP: −5.2; ML: 1.8; DV: −7.8). (H) Magnification of the arrowed area in photomicrograph G. (I) Migrating cells in an area 0.8 mm ahead of the injection site(AP: −4.4). (J) Magnification of the arrowed area in photomicrograph (I). Scale bar: (A, B, H and J) 25 m; (C) 20 m; (D) 50 m; (E–G and I) 100 m.
EGFP-labeled cells were located around the transplantation site in SNc 1 month after transplantation. The cell bodies were round or oval with yellow–green fluorescence, and could easily be distinguished from the host cells. In the sagittal slices, the cells were
seen to have migrated about 90 m from the graft center (Fig. 5A). In the coronal slices, the migration distance was around 150 m (Fig. 5B). Two months after transplantation, large numbers of EGFPlabeled cells had migrated from the injection area to the periphery.
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Some of cells had migrated in succession, forming a transitional zone along the SNc. In the sagittal slices, the migration distance was >150 m (Fig. 5C), while in the coronal slices, it was >300 m (Fig. 5D). In some rats, the grafted cells were seen in coronal slices to have migrated in a girdle shape along almost the whole SNc area (Fig. 5E). Four months after transplantation, the grafted cells had migrated and dispersed throughout the SNc. Sagittal slices showed that the density of grafted cells was reduced at the injection site, and many cells had migrated rostrally and caudally from the injection center along the SNc area (Fig. 5F). This phenomenon was also
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observed in the coronal slices that the cells migrated medially and laterally from the injection center (Fig. 5G and H). Many grafted cells had migrated more than 800 m from the center of the injection site and were also redistributed along the SNc (Fig. 5I and J). The grafted cells survived and were stable throughout the SNc, and many TH-positive cell bodies were present in the EGFP-labeled cell grafts (Fig. 3) by 4 months after transplantation. A few cells were also observed migrating away from the SNc region. EGFP-labeled NSCs were also distinguished along the injection tract 4 months after transplantation into the striatum. The cell bodies were similar to those found after injection into the SNc. In
Fig. 6. EGFP-labeled cells at striatum in the host brain 4 months after transplantation. (A) Upper arrow indicates the injection side (AP: −0.2; ML: 3.5; DV: −5.5), lower arrow shows the cells migrating from the injection site. (B) Magnification of the lower arrowed area in photomicrograph A. (C) Migrating cells 2.5 mm from the median sagittal plane. (D) Magnification of the arrowed area in photomicrograph (C). (E) Migrating cells 2.9 mm from the median sagittal plane. (F) Magnification of the arrowed area in photomicrograph (E). (G) Migrating cells 1.9 mm from the median sagittal plane. The right upper figure shows magnification of the arrowed area. (H) Distribution of many migrating cells within the SNc. The left upper figure shows magnification of the arrowed area. Scale bar: (A–C, E and H) 100 m; (D and F) 25 m; (G) 500 m.
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sagittal and coronal sections, the transplanted cells were seen to have migrated in specific directions. Most of the cells had migrated ventrally and caudally from the transplant sites (Fig. 6A and B). Sagittal sections showed that grafted cells had migrated in a cordlike arrangement from the striatum towards the ventral, medial and posterior site (Fig. 6C–G). Moreover, we found some EGFP-labeled cells migrating from the striatum over a long distance to part of the homolateral SNc, and some EGFP-labeled cells were dispersed within the SNc (Fig. 6H). There were very few EGFP-labeled cells in other brain regions. It is clear that stem cells have the capacity to migrate and integrate within the CNS [18,3]. NSCs have extensive capacities for self-renewal, and under certain conditions, their progeny can give rise to the principal cellular phenotypes comprising the mature CNS. It has been reported that many TH-positive cells were found at the injection sites in PD rats that showed functional improvements after implantation with human NSCs genetically transduced with the TH gene [10]. In our experiment, we used NSCs obtained from the SVZ of E14 rats without any prior genetic manipulation, and found that many grafted cells differentiated into TH-positive cells, even after migration. The pathology of PD is mainly due to the degeneration of dopaminergic neurons and their pathways. In order to achieve functional recovery in 6-OHDA induced lesions, some degree of dopaminergic re-innervation must occur. Studies in mice have indicated a threshold value of 1–2% of surviving TH neurons is adequate for the induction of behavioral changes [14]. We observed many TH-positive cell bodies among the EGFPlabeled cells 4 months after transplantation. Our data implied that, under certain conditions, stem cells possessed excellent capabilities to integrate and differentiate following transplantation, and could respond to environmental cues which determined their fate [1,5]. It is known that damaged cells or local tissue injury can lead to an increase in growth factor and cytokine production [7], including an upregulation of nerve growth factor production throughout the CNS by astrocytes and microglia [6,21]. NSCs have been shown to retain their ability to migrate towards injured or degenerating tissue, and then start to differentiate once they reach the target site [17,22]. In our 6-OHDA-lesioned rat model, the DA cells in the SNc were significantly depleted. This suggests that the pre-transplantation condition of the cells and the host microenvironment maybe the main factors determining the behavior of the grafted cells. We showed that transplanted NSCs could not only migrate to the injured brain area, but also differentiate into neuron-like cells. These results and those of others suggest that transplanted NSCs can survive well in vivo and possess the ability to migrate and differentiate, with a high degree of plasticity [15]. The migration and differentiation patterns of transplanted NSCs may be partially regulated by the injured brain. Acknowledgments We thank Bin Ni at the laboratory of material analysis and observation at ECNU for confocal technical assistance. This study was supported by the PhD Program Scholarship Fund of ECNU 2008 (No. 20080034). References [1] P. Akerud, J.M. Canals, E.Y. Snyder, E. Arenas, Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson’s disease, J. Neurosci. 21 (2001) 8108–8118.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Grafted neural stem cells migrate to substantia nigra and improve behavior in Parkinsonian rats Zhu Qing feng, Ma Ji, Yu Ling li, Yuan Chong gang ∗ School of Life Science, East China Normal University, Shanghai 200062, China
a r t i c l e
i n f o
Article history: Received 19 March 2009 Received in revised form 11 May 2009 Accepted 6 July 2009 Keywords: Neural stem cells Parkinson’s disease Transplantation Migration
a b s t r a c t Neural stem cell (NSC) transplantation has the potential to treat neurodegenerative diseases such as Parkinson’s disease (PD). In this study, we investigated the effect of transplanted NSCs in a PD animal model. NSCs isolated from the subventricular zone (SVZ) of E14 rats were cultured in vitro to produce neurospheres, which were subsequently infected with recombinant adeno-associated virus (rAAV2 ) expressing enhanced green fluorescent protein (EGFP). The PD animal model was established by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) of Sprague–Dawley rats. Once the model was established, EGFP-expressing NSCs were transplanted into the substantia nigra pars compacta (SNc) or striatum of PD rats. We found that NSCs transplanted into either site significantly reduced apomorphine-induced circling behavior of PD rats. Pathological analysis revealed that the EGFP-expressing NSCs could be detected at both injection sites at 1, 2 and 4 months after transplantation. SNc transplanted cells dispersed within the SNc with a significant portion differentiated into tyrosine hydroxylase-positive neurons. Whereas cells transplanted into the striatum migrated ventrally and posteriorly towards the SNc. These results suggest that the 6-OHDA damaged brain area attracts grafted NSCs, which migrated from the striatum and survived for a long time in SNc, resulting in behavioral improvement of PD rats. © 2009 Elsevier Ireland Ltd. All rights reserved.
PD is characterized by a progressive loss of dopaminergic (DA) neurons in the SNc, leading to severe motor dysfunction [23,13]. Chemical replacement therapies are widely used to control behavioral symptoms of PD. However, the effectiveness of pharmacological treatments is limited [8,12], to the search for more effective therapies. The replacement of damaged neurons by cell transplantation has recently been explored as a potential treatment for many neurodegenerative diseases [4,20,19,16]. NSCs have attracted a great deal of interest, not only because of their importance in basic research on neural development, but also in terms of their therapeutic potential in neurological diseases including PD. Because of their abilities to proliferate and/or to migrate to injured areas, NSCs appear to be suitable candidates for cell replacement treatment. It has been shown that NSCs is able to migrate towards damaged areas, to produce trophic factors, and to replace lost cells in ways that might be beneficial in PD [2]. However, little information is available about the migration of NSCs in rat PD models. Moreover, the information about the functional effectiveness of transplanted stem cells is also limited [9,24,11]. In this study, we investigated the ability of NSCs to survive in 6-OHDA-injured areas,
∗ Corresponding author. Tel.: +86 21 62232729. E-mail address: [email protected] (C.g. Yuan). 0304-3940/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2009.07.008
and their capacity to migrate to the lesioned SNc. More importantly, we monitored functional improvements of PD rats following NSCs transplantation into either SNc or striatum. We showed that natural cues available in the brain can constitutively direct the migration and differentiation of transplanted NSCs, resulting in their development into neuronal-like cells expressing DA traits. 105 male Sprague–Dawley (SD) rats weighing 180–220 g were received unilateral 6-OHDA (Sigma, USA) lesions using a 10-l Hamilton syringe inserted into the right MFB at the following coordinates (in mm, with reference to the bregma and dura): AP: −2.6; ML: 1.7; DV: −7.8. The concentration of 6-OHDA was at 10.5 g in 3.5 l of ascorbic-saline. Four weeks after injection, animals were tested for a period of 0.5 h for apomorphine-induced turning behavior (intraperitoneal injection of 0.25 mg/kg apomorphine in ascorbic-saline). Only those rats with proven rotational behavior (>7 rpm) were selected for the next phase of study. The evaluation was performed in the double blind manner. NSCs were obtained from the forebrain SVZ of 12 fetal E14 rat brains. The SVZ pieces were incubated in culture medium consisting of DMEM/F12 (GIBCO, USA), B27, 10 ng/ml FGF-2, 20 ng/ml EGF (Sigma, USA). The suspensions were centrifuged at 1000 rpm for 5 min. Pellets were resuspended in 1 ml of culture medium and cells were then seeded at a density of 2 × 105 cells/ml into 75-ml polystyrene plastic culture flasks. The cells were plated and cultured
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Fig. 1. Neural stem cells derived from SVZ cells. (A) Confocal microscope photograph of nestin-positive neurospheres. (B) NSCs expressing EGFP. Scale bar: 50 m.
Fig. 2. Immunohistochemical analysis of TH neurons in the substantia nigra of PD rats at 4 weeks post 6-OHDA administration. (A) Coronal section showing TH-positive cells in both SNc regions. (B) Some TH-positive cells remained at ventral tegmental area in the lesioned side. (C) TH-positive cells in the non-lesioned side. (D) The loss of TH-positive cells in the 6-OHDA lesioned side. Scale bar: (B–D) 100 m.
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Fig. 3. (A) EGFP-labeled cells in the substantia nigra of the host brain 4 months after transplantation. (B) The same slide was stained for TH (brown). Scale bar: 50 m.
at 37 ◦ C in a humidified 5% CO2 and 95% air incubator. The neurospheres generated from this procedure were passaged weekly. Cells were cultured and expanded in flasks for 3 weeks, and then transferred to 24-well plates and cultured at a density of 5 × 105 cells/ml for 36 h. These cells were infected with rAAV2 containing the EGFP coding sequences (rAAV2 -EGFP, National Molecular Virology Lab of China) at 37 ◦ C for 4 h. The infected cells were harvested and resuspended, then cultured in the 75-ml flasks for 3–4 days before transplantation. A portion of cultured neurospheres was incubated with mouse anti-nestin antibody (1:500; Chemicon, USA) overnight at 4 ◦ C, followed by incubation for 1 h with the tetramethyl rhodamine isothiocyanate-IgG secondary antibody. To evaluate the expression of EGFP, some rAAV2-EGFP infected cells were cultured without FGF-2 and EGF in DMEM/F12 medium supplemented with 10% bovine serum for several days. All signals were detected using laser scanning confocal microscope (Leica, Germany). Five weeks after injection of 6-OHDA, EGFP-labeled cell suspensions were resuspended and grafted into the SNc (n = 20) or the striatum (n = 5). Using a glass capillary attached to a 10-l Hamilton syringe, 5 l of the cell suspension containing approximately 50,000 cells was injected into the right SNc or the right striatum at a rate of 1 l/min. Stereotaxic implantations were performed at the following coordinates: SNc AP: −5.2; ML: 1.8; DV: −7.8 and striatum AP: −0.2; ML: 3.5; DV: −5.5. PD rats and implanted rats were anesthetized and perfused transcardially with 4.0% PFA after final behavioral test. Brains were isolated and placed in the same solution for 4 h before saturation with 30% sucrose in PBS, and were then frozen at −80 ◦ C. Brains were sectioned at 30 m using a freezing microtome (Leica, Germany). To detect TH, sections were incubated with mouse antiTH antibody (1:300; Chemicon, USA) for 48 h at 4 ◦ C, followed by 1 h incubation of biotin-conjugated goat anti-mouse IgG, and then detected with an ABC kit (Chemicon, USA). Sigma-Plot Software version 10.0 was used for data analysis. Statistical differences between pre-transplantation and after transplantation were assessed using the Student’s t-test. The results were expressed as mean ± standard error. After 3–4 days, the NSCs obtained from the SVZ of E14 rats had proliferated and formed clusters. Most clusters grew to macroscopic size after 1 week. The number of cells increased from 2 × 105 seeded cells to about 6 × 105 cells within 1 week. During the expansion phase, most cells were positive for nestin, a marker of immature progenitor cells (Fig. 1A). To evaluate EGFP expression in NSCs, cells infected with rAAV2 -EGFP were allowed to differentiate and the EGFP expression was verified (Fig. 1B). The 6-OHDA lesion was confirmed by apomorphine-induced rotational behavior. A total of 57 rats were selected as successful PD models based on the presence of rotational behavior (>7 rpm).
The extent of the 6-OHDA lesions in the MFB was also verified by immunohistochemistry. As shown in Fig. 2, a significant reduction in TH-positive cells was observed in the lesioned area (Fig. 2A, B and D), while TH-positive cells remained in the non-lesioned area (Fig. 2A and C). The NSCs grafted in the SNc were identified by EGFP fluorescence (Fig. 3A), and the same slide was used to for immunohistochemical staining of TH (Fig. 3B). We found that a significant number of grafted cells were TH-positive, indicating the conversion of NSCs to cells similar to dopaminergic neurons. The functional benefit of NSCs transplantation on PD rat behavior was evaluated at 1, 2 and 4 months after transplantation. Compared with the pretransplantation period, PD rats grafted with NSCs in the SNc showed significant reductions in rotational behavior after 2 and 4 months (Fig. 4), while there was a slight, but not statistically significant, reduction after 1 month. Notably, PD rats received NSCs transplantation in the striatum also showed a significant reduction in rotation after 4 months, but no significant reduction was observed after 1 or 2 months (Fig. 4). There was no improvement in rotational scores in PD rats without transplantation (Fig. 4).
Fig. 4. Behavioral effectiveness of grafted NSCs. The behavior of PD animals grafted with NSCs in the SNc (n = 20) or striatum (n = 5) was tested before transplantation (pre-TP) and at 1, 2 and 4 months postgrafting. The apomorphine-induced rotational response was monitored for 30 min. PD rats showed no change in rotation score over time (pre-TP, n = 13; 1 month, n = 13; 2 months, n = 11; 4 months, n = 9). Number of rotations at 4 months: 233.89 ± 70.43). Rats grafted with NSCs into the SNc showed a significant reduction in rotation at the second (n = 13) and fourth months (n = 10, number of rotations at 4 months: 189.3 ± 63.24), but a non-significant reduction after 1 month (n = 20). Animals grafted with NSCs into the striatum also showed a significant reduction in rotation after 4 months (n = 4, number of rotations at 4 months: 169 ± 47.28), but no significant reductions at 1 or 2 months (n = 4). Significantly different from pre-transplantation at *p < 0.05, **p < 0.01 and ***p < 0.001.
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Fig. 5. EGFP-labeled cells in the SNc of the host brain 1, 2 and 4 months after transplantation. (A) Cells migrating rostrally and caudally from the injection center, seen in a sagittal section after 1 month. (B) Cells migrating medially and laterally from the injection center, seen in a coronal section after 1 month. (C) Cells migrating rostrally and caudally from the injection center, seen in a sagittal section after 2 months. (D) Cells migrating medially and laterally from the injection center, seen in a coronal section after 2 months. (E) Cells migrating in a strap-like manner along most of the SNc area, seen in a coronal slice after 2 months. (F) Cells migrating rostrally and caudally from the injection center, seen in a sagittal section after 4 months. (G) Cells migrating medially and laterally from the injection site, seen in a coronal section after 4 months (AP: −5.2; ML: 1.8; DV: −7.8). (H) Magnification of the arrowed area in photomicrograph G. (I) Migrating cells in an area 0.8 mm ahead of the injection site(AP: −4.4). (J) Magnification of the arrowed area in photomicrograph (I). Scale bar: (A, B, H and J) 25 m; (C) 20 m; (D) 50 m; (E–G and I) 100 m.
EGFP-labeled cells were located around the transplantation site in SNc 1 month after transplantation. The cell bodies were round or oval with yellow–green fluorescence, and could easily be distinguished from the host cells. In the sagittal slices, the cells were
seen to have migrated about 90 m from the graft center (Fig. 5A). In the coronal slices, the migration distance was around 150 m (Fig. 5B). Two months after transplantation, large numbers of EGFPlabeled cells had migrated from the injection area to the periphery.
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Some of cells had migrated in succession, forming a transitional zone along the SNc. In the sagittal slices, the migration distance was >150 m (Fig. 5C), while in the coronal slices, it was >300 m (Fig. 5D). In some rats, the grafted cells were seen in coronal slices to have migrated in a girdle shape along almost the whole SNc area (Fig. 5E). Four months after transplantation, the grafted cells had migrated and dispersed throughout the SNc. Sagittal slices showed that the density of grafted cells was reduced at the injection site, and many cells had migrated rostrally and caudally from the injection center along the SNc area (Fig. 5F). This phenomenon was also
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observed in the coronal slices that the cells migrated medially and laterally from the injection center (Fig. 5G and H). Many grafted cells had migrated more than 800 m from the center of the injection site and were also redistributed along the SNc (Fig. 5I and J). The grafted cells survived and were stable throughout the SNc, and many TH-positive cell bodies were present in the EGFP-labeled cell grafts (Fig. 3) by 4 months after transplantation. A few cells were also observed migrating away from the SNc region. EGFP-labeled NSCs were also distinguished along the injection tract 4 months after transplantation into the striatum. The cell bodies were similar to those found after injection into the SNc. In
Fig. 6. EGFP-labeled cells at striatum in the host brain 4 months after transplantation. (A) Upper arrow indicates the injection side (AP: −0.2; ML: 3.5; DV: −5.5), lower arrow shows the cells migrating from the injection site. (B) Magnification of the lower arrowed area in photomicrograph A. (C) Migrating cells 2.5 mm from the median sagittal plane. (D) Magnification of the arrowed area in photomicrograph (C). (E) Migrating cells 2.9 mm from the median sagittal plane. (F) Magnification of the arrowed area in photomicrograph (E). (G) Migrating cells 1.9 mm from the median sagittal plane. The right upper figure shows magnification of the arrowed area. (H) Distribution of many migrating cells within the SNc. The left upper figure shows magnification of the arrowed area. Scale bar: (A–C, E and H) 100 m; (D and F) 25 m; (G) 500 m.
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sagittal and coronal sections, the transplanted cells were seen to have migrated in specific directions. Most of the cells had migrated ventrally and caudally from the transplant sites (Fig. 6A and B). Sagittal sections showed that grafted cells had migrated in a cordlike arrangement from the striatum towards the ventral, medial and posterior site (Fig. 6C–G). Moreover, we found some EGFP-labeled cells migrating from the striatum over a long distance to part of the homolateral SNc, and some EGFP-labeled cells were dispersed within the SNc (Fig. 6H). There were very few EGFP-labeled cells in other brain regions. It is clear that stem cells have the capacity to migrate and integrate within the CNS [18,3]. NSCs have extensive capacities for self-renewal, and under certain conditions, their progeny can give rise to the principal cellular phenotypes comprising the mature CNS. It has been reported that many TH-positive cells were found at the injection sites in PD rats that showed functional improvements after implantation with human NSCs genetically transduced with the TH gene [10]. In our experiment, we used NSCs obtained from the SVZ of E14 rats without any prior genetic manipulation, and found that many grafted cells differentiated into TH-positive cells, even after migration. The pathology of PD is mainly due to the degeneration of dopaminergic neurons and their pathways. In order to achieve functional recovery in 6-OHDA induced lesions, some degree of dopaminergic re-innervation must occur. Studies in mice have indicated a threshold value of 1–2% of surviving TH neurons is adequate for the induction of behavioral changes [14]. We observed many TH-positive cell bodies among the EGFPlabeled cells 4 months after transplantation. Our data implied that, under certain conditions, stem cells possessed excellent capabilities to integrate and differentiate following transplantation, and could respond to environmental cues which determined their fate [1,5]. It is known that damaged cells or local tissue injury can lead to an increase in growth factor and cytokine production [7], including an upregulation of nerve growth factor production throughout the CNS by astrocytes and microglia [6,21]. NSCs have been shown to retain their ability to migrate towards injured or degenerating tissue, and then start to differentiate once they reach the target site [17,22]. In our 6-OHDA-lesioned rat model, the DA cells in the SNc were significantly depleted. This suggests that the pre-transplantation condition of the cells and the host microenvironment maybe the main factors determining the behavior of the grafted cells. We showed that transplanted NSCs could not only migrate to the injured brain area, but also differentiate into neuron-like cells. These results and those of others suggest that transplanted NSCs can survive well in vivo and possess the ability to migrate and differentiate, with a high degree of plasticity [15]. The migration and differentiation patterns of transplanted NSCs may be partially regulated by the injured brain. Acknowledgments We thank Bin Ni at the laboratory of material analysis and observation at ECNU for confocal technical assistance. This study was supported by the PhD Program Scholarship Fund of ECNU 2008 (No. 20080034). References [1] P. Akerud, J.M. Canals, E.Y. Snyder, E. Arenas, Neuroprotection through delivery of glial cell line-derived neurotrophic factor by neural stem cells in a mouse model of Parkinson’s disease, J. Neurosci. 21 (2001) 8108–8118.
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