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Extract of deafferented hippocampus promotes in vitro radial glial cell differentiation into neurons
Neuroscience Letters 498 (2011) 93–98
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Extract of deafferented hippocampus promotes in vitro radial glial cell differentiation into neurons Heyan Zhao, Guohua Jin ∗ , Meiling Tian, Haoming Li, Xinhua Zhang Department of Anatomy and Neurobiology, the Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong 226001, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 2 March 2011 Received in revised form 27 April 2011 Accepted 27 April 2011 Keywords: Radial glial cell Differentiation Hippocampus Neuron
a b s t r a c t To explore the effects of deafferented hippocampal extracts on the differentiation of radial glial cells (RGCs), hippocampal RGCs of postnatal day 1 rats were isolated under adherent conditions in vitro. Protein extracts of deafferented hippocampus were prepared from adult rats following fimbria fornix lesion. RGCs were exposed to extracts of deafferented or normal hippocampus and the type and extent of proliferation and differentiation were evaluated. We report that extracts of deafferented hippocampus more effectively promoted RGC proliferation than extracts of normal hippocampus. Moreover, although RGC differentiation in vitro primarily generated cells of glial lineages, cells exposed to extracts of deafferented hippocampus, but not of normal hippocampus, showed a significantly increased trend towards the generation of cells of neuronal lineages. We conclude that extracts of deafferented hippocampus promote RGC proliferation and neurogenesis. © 2011 Elsevier Ireland Ltd. All rights reserved.
In central nervous system (CNS) RGCs constitute a special type of cell typically with single long and thin unbranched neuronal processes [3,10] and are widely distributed in the cerebral cortex, hippocampal dentate gyrus (DG) and cerebellum of vertebrates and in the CNS of newborns where they form RGC networks. These networks guide migrating neurons to target locations where they become mature cells, and RGCs themselves have been suggested to be neural stem cells (NSCs) [1,3,7,10,13,20,23,28]. It was previously reported that quiescent RGCs persist in the adult DG, and physiological changes taking place in the DG that lead to neurogenesis are accompanied by the proliferation and differentiation of RGCs [6,26,27,29]. A major nerve tract in the CNS, fimbria fornix (FF), contains multiple different fibers that include fibers connecting the hippocampus, a region centrally involved in learning and memory [16], to the anterior thalamic nucleus, medial mammilary nucleus, lateral septal nucleus, and the nucleus of diagonal band. The FF also contains cholinergic afferent fibers emanating from the medial septal nucleus and diagonal band and leading to the hippocampus [8,33]. Lesion of the FF therefore largely disconnects the hippocampus from external nuclei, and is generally considered to abolish
Abbreviations: RGCs, radial glial cells; CNS, central nervous system; DG, dentate gyrus; NSCs, neural stem cells; FF, fimbria fornix; BLBP, brain lipid-binding protein; GFAP, glial fibrillary acidic protein; GLAST, astrocyte-specific glutamate transporter; FACS, fluorescence-activated cell sorting; ES, embryonic stem. ∗ Corresponding author. Tel.: +86 513 85051718; fax: +86 513 85051718. E-mail address: [email protected] (G. Jin). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.04.070
major aspects of hippocampal function in vivo. Previous studies in our laboratory have revealed that major changes take place in the rat hippocampus following FF lesion. These changes in the internal environment of the hippocampus were found to promote the survival and migration of endogenous hippocampal and implanted NSCs and their differentiation into hippocampal neurons. Importantly, extracts of deafferented hippocampus could promote the in vitro differentiation of NSCs into neurons and their subsequent maturation, whereas extracts of normal hippocampus were largely inactive in this assay [31,32]. These results argue that deafferentation leads to changes in the hippocampal expression of molecules that modulate NSC differentiation, thereby promoting neuronal repair and regeneration. However, it is not known whether deafferentation of the hippocampus also promotes the differentiation and maturation of RGCs. In the present report we describe experiments in which extracts of normal and deafferented hippocampus were investigated for their ability to promote the in vitro proliferation and differentiation of primary adherent RGC cultures from neonatal hippocampus. Adult male and female Sprague-Dawley rats with a body weight of 220–250 g were supplied by Nantong University Laboratory Animal Center. All rats were maintained in a controlled temperature environment (23 ± 2 ◦ C) on a 12 h:12 h light:dark cycle and were housed in an approved facility with free access to food and water. Rats of different genders were maintained in separate cages. All animal experiments were conducted according to protocols approved by the US National Institutes of Health Guide for the Use and Care of Laboratory Animals. All efforts were made to minimize the number and suffering of animals used in this study.
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Fig. 1. Purity of isolated hippocampal RGCs. P1 RGCs were cultured under adherent conditions. (A) Cells positive for both BLBP and Nestin with characteristic morphological features were identified as RGCs. (B) RGCs incubated with specific antibodies against MAP2, DCX, or CNP; no cells were positive for these markers. Scale bar, 100 m.
Deafferented hippocampus model animals were prepared as previously reported by Hefti [15]. Briefly, rats underwent anesthesia by intraperitoneal injection of the composite anesthetic Chloropent (0.2 ml/100 g weight) and were immobilized on a stereotaxic apparatus (WPI, Sarasota, USA). In reference to the Paxinos anatomical map [24], two sites on the right side of the skull [A1 (anterior) = −1.4 plus L1 (lateral) = −1; also A2 = −1.4 plus L2 = −4] were located and a bony suture was prepared between the two sites. Acupotomy was performed in which a needle-knife was inserted into the brain at a depth of V (vertical) = +5.4, forming a return cut three times, and withdrawn. Four days after the operation the rats were anesthetized as before, chilled on ice, and deafferented hippocampus and contralateral normal hippocampus were excised, weighed, and transferred to precooled serum-free media at a concentration of 100 g tissue per ml. Tissues were homogenized (10 min) on ice; lysates were clarified by centrifugation (4 ◦ C, 10 000 rpm, 10 min) and the supernatants were aliquoted and stored at −70 ◦ C. Neonatal rat hippocampal tissues [postnatal day 1 (P1)] were dissected and primary cultures were prepared using standard protocols in the NSC expansion medium [Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12, 1:1) (Gibco, Invitrogen, USA) containing 2% B27 (GIBCO) and 20 ng/ml EGF and EGF-2 (Sigma, St. Louis, MO, USA)]. After 3–5 days, newly formed neurospheres were incubated in accutase (Sigma; 20 min, 37 ◦ C), triturated into singlecell suspensions, and replated onto poly-lysine-coated coverslips in 24-well plates (adherent conditions) [18]. To assess the differentiation ability of P1 RGCs, RGCs were transferred into DMEM/F12 medium supplemented with 1% fetal calf serum (FCS) without EGF or FGF-2 (differentiation medium; DM), then divided into three groups. (1) Deafferented extract group: RGCs were cultured in DM supplemented with 5% (v/v) deafferented hippocampal extract. (2) Normal extract group: RGCs were cultured in DM supplemented with 5% (v/v) normal hippocampal extract. (3) Control group: RGCs were cultured in DM without any extract. Seven days later cells were processed for immunocytochemistry. Cells were treated with 100% methanol (20 min) and then fixed with 4% formaldehyde in 0.01 M PBS (30 min). After rinsing in 0.01 M PBS (pH 7.4) three times and blocking with 10% goat serum at room temperature (RT) for 1 h, cells were separately incubated at RT overnight with primary antibodies (Chemicon, Billerica, USA): these were rabbit anti-BLBP (brain lipid-binding protein) antibody (1:600), mouse anti-BrdU (1:500), anti-nestin (1:100), anti-GFAP
(glial fibrillary acidic protein) (1:1000), anti-MAP2 (1:200), antiCNP (1:100) and guinea pig anti-DCX (1:500) antibodies. For double labeling, cells were incubated simultaneously with two primary antibodies of different species as above. Primary antibodies were removed and the cells were rinsed three times in PBS and incubated with the corresponding secondary antibodies (Invitrogen, California, USA) at RT for 4 h. Cells were washed three times with PBS and incubated with Hoechst 33342 (Sigma, St. Louis, USA) at a dilution of 1:1000 at 37 ◦ C for 30 min. Cells were washed three times with PBS and the coverslips were sealed with buffer containing glycerol. Labeled cells were visualized using fluorescence microscopy (Leica, Solms, Germany). Immunopositive cells were counted respectively in ten randomly selected microscopic visual fields; total cell numbers were counted using nuclear counterstaining with Hoechst and the lengths of cell processes were calculated using JD-801 image-analysis software (JEDA, Jiangsu, Nanjin, China). Data are shown as mean ± SD. Data processing employed GraphPad software (GraphPad Prism v4.0; GraphPad Software Company). Analysis of variance (ANOVA) was used for statistical analysis; P < 0.05 was taken to indicate statistical significance. RGCs were identified as double BLBP+ nestin+ cells showing typical morphological features of long, thin and unbranched neuronal processes [3,10] (Fig. 1A). Of the cells in the preparation, 93.76 ± 5.26% were bipolar double-positive cells. These were examined with specific antibodies against MAP2, DCX or CNP; no cells were positive for these antigens (Fig. 1B), confirming their identity as RGCs. To determine whether deafferented hippocampal extracts can promote RGC proliferation, RGCs were incubated for 7 days in the presence or absence of extracts of normal or deafferented hippocampus [FF lesions demonstrated by Nissl staining indicating that the deafferented hippocmapus models were successful (Fig. 2C)]. Dividing cells were labeled by incubation with BrdU (5 mol/L) for 12 h. The number of BrdU single-labeled cells versus the total cell number labeled by Hoechst (Fig. 2A) was 64.17 ± 9.20% in the deafferented extract group, 51.25 ± 7.17% in the normal extract group, and 24.92 ± 5.84% in the control group. Pairwise differences between groups achieved statistical significance in all cases (Fig. 2B, P < 0.01). This result indicates that although normal hippocampus extract can promote RGC proliferation, extract of deafferented hippocampus is significantly better at promoting the proliferation of hippocampal RGCs. When cultured in appropriate differentiation media the morphology of RGC was observed to change. Immunofluorescence
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Fig. 2. Proliferation of RGCs treated with extracts of deafferented hippocampus. (A) Immunofluorescence staining of cells after exposure to BrdU for 12 h. BrdU-positive cells were observed in all three groups. (B) The proportions of BrdU-labeled cells versus the total cell number (Hoechst) in three groups; intergroup differences were statistically significant in all cases; , P < 0.01 compared with control; N, P < 0.01 compared with the normal extract group. (C) Coronal section through FF was stained by cresyl violet 4 days after FF lesion. As shown (arrowed), the injured side FF was disconnected whereas the contralateral FF remains intact. Scale bar, 100 m.
with antibodies directed against MAP2 (Fig. 3A) or DCX (Fig. 4A) revealed that 8.22 ± 1.93%, 8.99 ± 1.96% of RGCs differentiated into MAP2- and DCX-positive neurons respectively in the deafferented extract group, whereas the corresponding percentages in the normal extract group were 2.88 ± 1.08%, 3.56 ± 1.09%, and in the control group were 0.64 ± 0.45%, 1.48 ± 0.48%. Pairwise differences between groups achieved statistical significance in all cases (Figs. 3B and 4B, P < 0.01), indicating that deafferented hippocampal extracts promote RGCs differentiation into neurons, and significantly more effectively than do extracts of normal hippocampus. Moreover, the average lengths of neuronal processes in the deafferented extract group were 281.49 ± 83.63 m (Fig. 3C), 282.28 ± 71.30 m (Fig. 4C), markedly longer than
in either the normal extract group (67.19 ± 17.66 m, Fig. 3C; 68.85 ± 18.86 m, Fig. 4C) or the control group (60.19 ± 11.56 m, Fig. 3C; 61.02 ± 11.04 m, Fig. 4C) (P < 0.01). There was no statistically significant difference between the normal group and the control group (P > 0.05). Consistent with previous reports [18], a majority of RGCs (91.39 ± 2.01% in the deafferented extract group, 92.43 ± 2.08% in the normal extract group, and 93.01 ± 2.11% in the control group) differentiated into GFAP+ astrocytes including Type 1 (Fig. 3D white arrowheads) and Type 2 (Fig. 3D yellow arrowheads). They could be distinguished based on their morphology and there were no difference in the number of Type 1 and Type 2 in three groups (P > 0.05), Type 1 astrocytes had a fibroblast-like morphology, whereas Type
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Fig. 3. RGC differentiation. (A) Immunofluorescence staining for MAP2 (differentiation of RGCs to mature neurons). (B) The percentages of MAP2-positive neurons versus total cell number (Fig. 3A) in three groups; intergroup differences were statistically significant in all cases. (C) Average lengths of neuronal processes in three groups. The mean length in the deafferented extract group was markedly increased compared to both the normal extract group and the control group (P < 0.01 in both cases), whereas there was no statistically significant difference between the normal group and the control group (P > 0.05). , P < 0.01 compared with control, N, P < 0.01 compared with the normal extract group. (D) When cultured in differentiation medium, the majority of RGCs differentiated into GFAP+ astrocytes; two types of astrocytes could be distinguished based on their morphology, one resembled Type 1 astrocytes (white arrowheads) and the other resembled Type 2 astrocytes (yellow arrowheads); intergroup differences were not statistically significant (P > 0.05). (E) A minority of RGCs differentiated into oligodendrocytes, intergroup differences were also not statistically significant (P > 0.05). Scale bar, 50 m. (For interpretation of the references to color in text, the reader is referred to the web version of the article.)
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Fig. 4. Differentiation of RGC exposed to deafferented hippocampal extracts into immature neurons. (A) Immunofluorescence staining for DCX. (B) Percentages of DCXpositive neurons in three groups; intergroup differences were statistically significant in all cases. (C) Average lengths of neuronal processes in three groups. The mean length in the deafferented extract group was markedly higher than in either the normal extract group or in the control group (P < 0.01 in both cases); there was no statistically significant difference between the normal extract group and the control group (P > 0.05). , P < 0.01 compared to control, N, P < 0.01 compared with the normal extract group. Scale bar, 50 m.
2 astrocytes had a neuron-like morphology [25]. Only a minority of RGCs (2.82 ± 0.76% in the deafferented extract group, 3.23 ± 0.60% in the normal extract group, and 3.41 ± 0.82% in the control group) differentiated into oligodendrocytes (Fig. 3E); there were no statistically significant differences between the three groups (P > 0.05). Brain injury can have profound consequences, and the development of techniques to promote neural regeneration in the injured CNS remains a major challenge. Replacement therapy with neural progenitor cells is a promising approach for the treatment of CNS injuries and degenerative diseases. In these studies researchers have focused primarily on the role of neurons in the nervous system and the important role of glial cells has tended to be overlooked. Although previous work has indicated that embryonic RGCs can act as progenitors for several types of neuron [14,19,23], these same cells can also develop into astrocytes and oligodendrocytes in vitro,
indeed RGCs were found to be far more effective in gliogenesis than in neurogenesis. Although no specific marker of RGCs has yet been identified, these cells express multiple growth and differentiation markers including GLAST (astrocyte-specific glutamate transporter), BLBP, GFAP, RC2, and Vimentin [2,5,19,21,22,30], and also express the neuronal stem-cell marker Nestin [14,17]. The identification of RGCs therefore relies on the conjunction of two or more such markers in combination with characteristic morphological features. Techniques used to isolate RGCs include fluorescence-activated cell sorting (FACS) [1,19], embryonic stem (ES) cell induction [4,11], and adherent culture. When cultured under adherent conditions and exposed to both EGF and FGF-2, NSCs can differentiae into RGCs and resemble ES-cell-derived RGCs [6,12]. Li [18] separately studied BLBP+ /Nestin+ , GFAP+ /Nestin+ and Vimentin+ /Nestin+ populations of RGCs and found that there were no significant differences
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between the three cell types. In the present work we cultured primary (postnatal day 1) adherent RGC in vitro; cells positive for both BLBP and Nestin that presented characteristic morphological features were identified as RGCs. Adult neurogenesis is affected by many factors and the internal environment of the CNS is thought to play a key role in the migration, localization and differentiation of NSCs [9]. Our previous studies revealed that the deafferented hippocampus provides a supportive microenvironment for the survival and neuronal differentiation of implanted neural progenitors. Furthermore, extracts of deafferented hippocampus showed enhanced ability to promote the differentiation of progenitor cells into cholinergic neurons [31]. In the present study we employed extracts of deafferented hippocampus to simulate the in vivo microenvironment of hippocampal nerve regeneration. We report that, although a majority of RGCs differentiated into radial glia–astrocyte lineages, and their capacity for gliogenesis far exceeded their capacity for neurogenesis, when stimulated in vitro by extracts of deafferented hippocampal extract a significantly increased number of RGCs differentiated into neurons. Furthermore, RGCs treated with extracts of deafferented hippocampus gave rise to neuronal processes that were markedly longer than those generated either by RGCs treated with normal extract or by control RGCs. We suggested that the primary role of RGCs under normal conditions is to act as glial progenitors to provide new cells of the glia–astrocyte lineages, but that when prompted by extracts of deafferented hippocampus a significant proportion can change specification to act as neural progenitors capable of differentiating into both glia and neurons. Together these results suggest that, in the damaged brain, selfrepair is promoted by an as-yet unknown signaling mechanism that is triggered by brain injury. It is possible that brain damage leads to upregulation of the production of secreted neurotropic factors that induce RGCs to proliferate and differentiate not only into glial cells, but increasingly into neuronal cells. Understanding the pathways of repair in the CNS and their regulation will be crucial for the development of new therapeutic approaches to brain damage; the identification of the pathways of induction and the roles of growthand differentiation-promoting factors induced by damage warrants further study. Acknowledgments This study was funded by the Jiangsu Province High-Technology Research Key Laboratory of Nerve Regeneration (SK2008-4), a Jiangsu Province Ordinary College Graduate Student Innovative Research Project grant (CX09S 023Z), the National Natural Science Foundation of China (31070937), a Nantong City Applied Research Project grant (K2009025), a Research Grant from Nantong University (08Z041), and by funding from the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] T.E. Anthony, C. Klein, G. Fishell, N. Heintz, Radial glia serve as neuronal progenitors in all regions of the central nervous system, Neuron 41 (2004) 881–890. [2] T.E. Anthony, H.A. Mason, T. Gridley, G. Fishell, N. Heintz, Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells, Genes Dev. 19 (2005) 1028–1033. [3] D. Barry, K. McDermott, Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord, Glia 50 (2005) 187–197.
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Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Extract of deafferented hippocampus promotes in vitro radial glial cell differentiation into neurons Heyan Zhao, Guohua Jin ∗ , Meiling Tian, Haoming Li, Xinhua Zhang Department of Anatomy and Neurobiology, the Jiangsu Key Laboratory of Neuroregeneration, Nantong University, 19 Qixiu Road, Nantong 226001, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 2 March 2011 Received in revised form 27 April 2011 Accepted 27 April 2011 Keywords: Radial glial cell Differentiation Hippocampus Neuron
a b s t r a c t To explore the effects of deafferented hippocampal extracts on the differentiation of radial glial cells (RGCs), hippocampal RGCs of postnatal day 1 rats were isolated under adherent conditions in vitro. Protein extracts of deafferented hippocampus were prepared from adult rats following fimbria fornix lesion. RGCs were exposed to extracts of deafferented or normal hippocampus and the type and extent of proliferation and differentiation were evaluated. We report that extracts of deafferented hippocampus more effectively promoted RGC proliferation than extracts of normal hippocampus. Moreover, although RGC differentiation in vitro primarily generated cells of glial lineages, cells exposed to extracts of deafferented hippocampus, but not of normal hippocampus, showed a significantly increased trend towards the generation of cells of neuronal lineages. We conclude that extracts of deafferented hippocampus promote RGC proliferation and neurogenesis. © 2011 Elsevier Ireland Ltd. All rights reserved.
In central nervous system (CNS) RGCs constitute a special type of cell typically with single long and thin unbranched neuronal processes [3,10] and are widely distributed in the cerebral cortex, hippocampal dentate gyrus (DG) and cerebellum of vertebrates and in the CNS of newborns where they form RGC networks. These networks guide migrating neurons to target locations where they become mature cells, and RGCs themselves have been suggested to be neural stem cells (NSCs) [1,3,7,10,13,20,23,28]. It was previously reported that quiescent RGCs persist in the adult DG, and physiological changes taking place in the DG that lead to neurogenesis are accompanied by the proliferation and differentiation of RGCs [6,26,27,29]. A major nerve tract in the CNS, fimbria fornix (FF), contains multiple different fibers that include fibers connecting the hippocampus, a region centrally involved in learning and memory [16], to the anterior thalamic nucleus, medial mammilary nucleus, lateral septal nucleus, and the nucleus of diagonal band. The FF also contains cholinergic afferent fibers emanating from the medial septal nucleus and diagonal band and leading to the hippocampus [8,33]. Lesion of the FF therefore largely disconnects the hippocampus from external nuclei, and is generally considered to abolish
Abbreviations: RGCs, radial glial cells; CNS, central nervous system; DG, dentate gyrus; NSCs, neural stem cells; FF, fimbria fornix; BLBP, brain lipid-binding protein; GFAP, glial fibrillary acidic protein; GLAST, astrocyte-specific glutamate transporter; FACS, fluorescence-activated cell sorting; ES, embryonic stem. ∗ Corresponding author. Tel.: +86 513 85051718; fax: +86 513 85051718. E-mail address: [email protected] (G. Jin). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.04.070
major aspects of hippocampal function in vivo. Previous studies in our laboratory have revealed that major changes take place in the rat hippocampus following FF lesion. These changes in the internal environment of the hippocampus were found to promote the survival and migration of endogenous hippocampal and implanted NSCs and their differentiation into hippocampal neurons. Importantly, extracts of deafferented hippocampus could promote the in vitro differentiation of NSCs into neurons and their subsequent maturation, whereas extracts of normal hippocampus were largely inactive in this assay [31,32]. These results argue that deafferentation leads to changes in the hippocampal expression of molecules that modulate NSC differentiation, thereby promoting neuronal repair and regeneration. However, it is not known whether deafferentation of the hippocampus also promotes the differentiation and maturation of RGCs. In the present report we describe experiments in which extracts of normal and deafferented hippocampus were investigated for their ability to promote the in vitro proliferation and differentiation of primary adherent RGC cultures from neonatal hippocampus. Adult male and female Sprague-Dawley rats with a body weight of 220–250 g were supplied by Nantong University Laboratory Animal Center. All rats were maintained in a controlled temperature environment (23 ± 2 ◦ C) on a 12 h:12 h light:dark cycle and were housed in an approved facility with free access to food and water. Rats of different genders were maintained in separate cages. All animal experiments were conducted according to protocols approved by the US National Institutes of Health Guide for the Use and Care of Laboratory Animals. All efforts were made to minimize the number and suffering of animals used in this study.
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Fig. 1. Purity of isolated hippocampal RGCs. P1 RGCs were cultured under adherent conditions. (A) Cells positive for both BLBP and Nestin with characteristic morphological features were identified as RGCs. (B) RGCs incubated with specific antibodies against MAP2, DCX, or CNP; no cells were positive for these markers. Scale bar, 100 m.
Deafferented hippocampus model animals were prepared as previously reported by Hefti [15]. Briefly, rats underwent anesthesia by intraperitoneal injection of the composite anesthetic Chloropent (0.2 ml/100 g weight) and were immobilized on a stereotaxic apparatus (WPI, Sarasota, USA). In reference to the Paxinos anatomical map [24], two sites on the right side of the skull [A1 (anterior) = −1.4 plus L1 (lateral) = −1; also A2 = −1.4 plus L2 = −4] were located and a bony suture was prepared between the two sites. Acupotomy was performed in which a needle-knife was inserted into the brain at a depth of V (vertical) = +5.4, forming a return cut three times, and withdrawn. Four days after the operation the rats were anesthetized as before, chilled on ice, and deafferented hippocampus and contralateral normal hippocampus were excised, weighed, and transferred to precooled serum-free media at a concentration of 100 g tissue per ml. Tissues were homogenized (10 min) on ice; lysates were clarified by centrifugation (4 ◦ C, 10 000 rpm, 10 min) and the supernatants were aliquoted and stored at −70 ◦ C. Neonatal rat hippocampal tissues [postnatal day 1 (P1)] were dissected and primary cultures were prepared using standard protocols in the NSC expansion medium [Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12, 1:1) (Gibco, Invitrogen, USA) containing 2% B27 (GIBCO) and 20 ng/ml EGF and EGF-2 (Sigma, St. Louis, MO, USA)]. After 3–5 days, newly formed neurospheres were incubated in accutase (Sigma; 20 min, 37 ◦ C), triturated into singlecell suspensions, and replated onto poly-lysine-coated coverslips in 24-well plates (adherent conditions) [18]. To assess the differentiation ability of P1 RGCs, RGCs were transferred into DMEM/F12 medium supplemented with 1% fetal calf serum (FCS) without EGF or FGF-2 (differentiation medium; DM), then divided into three groups. (1) Deafferented extract group: RGCs were cultured in DM supplemented with 5% (v/v) deafferented hippocampal extract. (2) Normal extract group: RGCs were cultured in DM supplemented with 5% (v/v) normal hippocampal extract. (3) Control group: RGCs were cultured in DM without any extract. Seven days later cells were processed for immunocytochemistry. Cells were treated with 100% methanol (20 min) and then fixed with 4% formaldehyde in 0.01 M PBS (30 min). After rinsing in 0.01 M PBS (pH 7.4) three times and blocking with 10% goat serum at room temperature (RT) for 1 h, cells were separately incubated at RT overnight with primary antibodies (Chemicon, Billerica, USA): these were rabbit anti-BLBP (brain lipid-binding protein) antibody (1:600), mouse anti-BrdU (1:500), anti-nestin (1:100), anti-GFAP
(glial fibrillary acidic protein) (1:1000), anti-MAP2 (1:200), antiCNP (1:100) and guinea pig anti-DCX (1:500) antibodies. For double labeling, cells were incubated simultaneously with two primary antibodies of different species as above. Primary antibodies were removed and the cells were rinsed three times in PBS and incubated with the corresponding secondary antibodies (Invitrogen, California, USA) at RT for 4 h. Cells were washed three times with PBS and incubated with Hoechst 33342 (Sigma, St. Louis, USA) at a dilution of 1:1000 at 37 ◦ C for 30 min. Cells were washed three times with PBS and the coverslips were sealed with buffer containing glycerol. Labeled cells were visualized using fluorescence microscopy (Leica, Solms, Germany). Immunopositive cells were counted respectively in ten randomly selected microscopic visual fields; total cell numbers were counted using nuclear counterstaining with Hoechst and the lengths of cell processes were calculated using JD-801 image-analysis software (JEDA, Jiangsu, Nanjin, China). Data are shown as mean ± SD. Data processing employed GraphPad software (GraphPad Prism v4.0; GraphPad Software Company). Analysis of variance (ANOVA) was used for statistical analysis; P < 0.05 was taken to indicate statistical significance. RGCs were identified as double BLBP+ nestin+ cells showing typical morphological features of long, thin and unbranched neuronal processes [3,10] (Fig. 1A). Of the cells in the preparation, 93.76 ± 5.26% were bipolar double-positive cells. These were examined with specific antibodies against MAP2, DCX or CNP; no cells were positive for these antigens (Fig. 1B), confirming their identity as RGCs. To determine whether deafferented hippocampal extracts can promote RGC proliferation, RGCs were incubated for 7 days in the presence or absence of extracts of normal or deafferented hippocampus [FF lesions demonstrated by Nissl staining indicating that the deafferented hippocmapus models were successful (Fig. 2C)]. Dividing cells were labeled by incubation with BrdU (5 mol/L) for 12 h. The number of BrdU single-labeled cells versus the total cell number labeled by Hoechst (Fig. 2A) was 64.17 ± 9.20% in the deafferented extract group, 51.25 ± 7.17% in the normal extract group, and 24.92 ± 5.84% in the control group. Pairwise differences between groups achieved statistical significance in all cases (Fig. 2B, P < 0.01). This result indicates that although normal hippocampus extract can promote RGC proliferation, extract of deafferented hippocampus is significantly better at promoting the proliferation of hippocampal RGCs. When cultured in appropriate differentiation media the morphology of RGC was observed to change. Immunofluorescence
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Fig. 2. Proliferation of RGCs treated with extracts of deafferented hippocampus. (A) Immunofluorescence staining of cells after exposure to BrdU for 12 h. BrdU-positive cells were observed in all three groups. (B) The proportions of BrdU-labeled cells versus the total cell number (Hoechst) in three groups; intergroup differences were statistically significant in all cases; , P < 0.01 compared with control; N, P < 0.01 compared with the normal extract group. (C) Coronal section through FF was stained by cresyl violet 4 days after FF lesion. As shown (arrowed), the injured side FF was disconnected whereas the contralateral FF remains intact. Scale bar, 100 m.
with antibodies directed against MAP2 (Fig. 3A) or DCX (Fig. 4A) revealed that 8.22 ± 1.93%, 8.99 ± 1.96% of RGCs differentiated into MAP2- and DCX-positive neurons respectively in the deafferented extract group, whereas the corresponding percentages in the normal extract group were 2.88 ± 1.08%, 3.56 ± 1.09%, and in the control group were 0.64 ± 0.45%, 1.48 ± 0.48%. Pairwise differences between groups achieved statistical significance in all cases (Figs. 3B and 4B, P < 0.01), indicating that deafferented hippocampal extracts promote RGCs differentiation into neurons, and significantly more effectively than do extracts of normal hippocampus. Moreover, the average lengths of neuronal processes in the deafferented extract group were 281.49 ± 83.63 m (Fig. 3C), 282.28 ± 71.30 m (Fig. 4C), markedly longer than
in either the normal extract group (67.19 ± 17.66 m, Fig. 3C; 68.85 ± 18.86 m, Fig. 4C) or the control group (60.19 ± 11.56 m, Fig. 3C; 61.02 ± 11.04 m, Fig. 4C) (P < 0.01). There was no statistically significant difference between the normal group and the control group (P > 0.05). Consistent with previous reports [18], a majority of RGCs (91.39 ± 2.01% in the deafferented extract group, 92.43 ± 2.08% in the normal extract group, and 93.01 ± 2.11% in the control group) differentiated into GFAP+ astrocytes including Type 1 (Fig. 3D white arrowheads) and Type 2 (Fig. 3D yellow arrowheads). They could be distinguished based on their morphology and there were no difference in the number of Type 1 and Type 2 in three groups (P > 0.05), Type 1 astrocytes had a fibroblast-like morphology, whereas Type
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Fig. 3. RGC differentiation. (A) Immunofluorescence staining for MAP2 (differentiation of RGCs to mature neurons). (B) The percentages of MAP2-positive neurons versus total cell number (Fig. 3A) in three groups; intergroup differences were statistically significant in all cases. (C) Average lengths of neuronal processes in three groups. The mean length in the deafferented extract group was markedly increased compared to both the normal extract group and the control group (P < 0.01 in both cases), whereas there was no statistically significant difference between the normal group and the control group (P > 0.05). , P < 0.01 compared with control, N, P < 0.01 compared with the normal extract group. (D) When cultured in differentiation medium, the majority of RGCs differentiated into GFAP+ astrocytes; two types of astrocytes could be distinguished based on their morphology, one resembled Type 1 astrocytes (white arrowheads) and the other resembled Type 2 astrocytes (yellow arrowheads); intergroup differences were not statistically significant (P > 0.05). (E) A minority of RGCs differentiated into oligodendrocytes, intergroup differences were also not statistically significant (P > 0.05). Scale bar, 50 m. (For interpretation of the references to color in text, the reader is referred to the web version of the article.)
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Fig. 4. Differentiation of RGC exposed to deafferented hippocampal extracts into immature neurons. (A) Immunofluorescence staining for DCX. (B) Percentages of DCXpositive neurons in three groups; intergroup differences were statistically significant in all cases. (C) Average lengths of neuronal processes in three groups. The mean length in the deafferented extract group was markedly higher than in either the normal extract group or in the control group (P < 0.01 in both cases); there was no statistically significant difference between the normal extract group and the control group (P > 0.05). , P < 0.01 compared to control, N, P < 0.01 compared with the normal extract group. Scale bar, 50 m.
2 astrocytes had a neuron-like morphology [25]. Only a minority of RGCs (2.82 ± 0.76% in the deafferented extract group, 3.23 ± 0.60% in the normal extract group, and 3.41 ± 0.82% in the control group) differentiated into oligodendrocytes (Fig. 3E); there were no statistically significant differences between the three groups (P > 0.05). Brain injury can have profound consequences, and the development of techniques to promote neural regeneration in the injured CNS remains a major challenge. Replacement therapy with neural progenitor cells is a promising approach for the treatment of CNS injuries and degenerative diseases. In these studies researchers have focused primarily on the role of neurons in the nervous system and the important role of glial cells has tended to be overlooked. Although previous work has indicated that embryonic RGCs can act as progenitors for several types of neuron [14,19,23], these same cells can also develop into astrocytes and oligodendrocytes in vitro,
indeed RGCs were found to be far more effective in gliogenesis than in neurogenesis. Although no specific marker of RGCs has yet been identified, these cells express multiple growth and differentiation markers including GLAST (astrocyte-specific glutamate transporter), BLBP, GFAP, RC2, and Vimentin [2,5,19,21,22,30], and also express the neuronal stem-cell marker Nestin [14,17]. The identification of RGCs therefore relies on the conjunction of two or more such markers in combination with characteristic morphological features. Techniques used to isolate RGCs include fluorescence-activated cell sorting (FACS) [1,19], embryonic stem (ES) cell induction [4,11], and adherent culture. When cultured under adherent conditions and exposed to both EGF and FGF-2, NSCs can differentiae into RGCs and resemble ES-cell-derived RGCs [6,12]. Li [18] separately studied BLBP+ /Nestin+ , GFAP+ /Nestin+ and Vimentin+ /Nestin+ populations of RGCs and found that there were no significant differences
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between the three cell types. In the present work we cultured primary (postnatal day 1) adherent RGC in vitro; cells positive for both BLBP and Nestin that presented characteristic morphological features were identified as RGCs. Adult neurogenesis is affected by many factors and the internal environment of the CNS is thought to play a key role in the migration, localization and differentiation of NSCs [9]. Our previous studies revealed that the deafferented hippocampus provides a supportive microenvironment for the survival and neuronal differentiation of implanted neural progenitors. Furthermore, extracts of deafferented hippocampus showed enhanced ability to promote the differentiation of progenitor cells into cholinergic neurons [31]. In the present study we employed extracts of deafferented hippocampus to simulate the in vivo microenvironment of hippocampal nerve regeneration. We report that, although a majority of RGCs differentiated into radial glia–astrocyte lineages, and their capacity for gliogenesis far exceeded their capacity for neurogenesis, when stimulated in vitro by extracts of deafferented hippocampal extract a significantly increased number of RGCs differentiated into neurons. Furthermore, RGCs treated with extracts of deafferented hippocampus gave rise to neuronal processes that were markedly longer than those generated either by RGCs treated with normal extract or by control RGCs. We suggested that the primary role of RGCs under normal conditions is to act as glial progenitors to provide new cells of the glia–astrocyte lineages, but that when prompted by extracts of deafferented hippocampus a significant proportion can change specification to act as neural progenitors capable of differentiating into both glia and neurons. Together these results suggest that, in the damaged brain, selfrepair is promoted by an as-yet unknown signaling mechanism that is triggered by brain injury. It is possible that brain damage leads to upregulation of the production of secreted neurotropic factors that induce RGCs to proliferate and differentiate not only into glial cells, but increasingly into neuronal cells. Understanding the pathways of repair in the CNS and their regulation will be crucial for the development of new therapeutic approaches to brain damage; the identification of the pathways of induction and the roles of growthand differentiation-promoting factors induced by damage warrants further study. Acknowledgments This study was funded by the Jiangsu Province High-Technology Research Key Laboratory of Nerve Regeneration (SK2008-4), a Jiangsu Province Ordinary College Graduate Student Innovative Research Project grant (CX09S 023Z), the National Natural Science Foundation of China (31070937), a Nantong City Applied Research Project grant (K2009025), a Research Grant from Nantong University (08Z041), and by funding from the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References [1] T.E. Anthony, C. Klein, G. Fishell, N. Heintz, Radial glia serve as neuronal progenitors in all regions of the central nervous system, Neuron 41 (2004) 881–890. [2] T.E. Anthony, H.A. Mason, T. Gridley, G. Fishell, N. Heintz, Brain lipid-binding protein is a direct target of Notch signaling in radial glial cells, Genes Dev. 19 (2005) 1028–1033. [3] D. Barry, K. McDermott, Differentiation of radial glia from radial precursor cells and transformation into astrocytes in the developing rat spinal cord, Glia 50 (2005) 187–197.
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