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Increased progenitor cell proliferation and astrogenesis in the partial progressive 6-hydroxydopamine model of Parkinson’s disease
Neuroscience 151 (2008) 1142–1153
INCREASED PROGENITOR CELL PROLIFERATION AND ASTROGENESIS IN THE PARTIAL PROGRESSIVE 6-HYDROXYDOPAMINE MODEL OF PARKINSON’S DISEASE P. M. APONSO,a R. L. M. FAULLa AND B. CONNORb*
progenitors undergo robust astrogenesis, newborn midbrain-derived progenitors remain in an undifferentiated state suggesting local environments differentially regulate endogenous progenitor cell populations in PD. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
a
Department of Anatomy with Radiology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
b
Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
Key words: adult neural progenitor cells, striatum, subventricular zone, substantia nigra, astrogenesis, Parkinson’s disease.
Abstract—The existence of endogenous progenitor cells in the adult mammalian brain presents an exciting and attractive alternative to existing therapeutic options for treating neurodegenerative diseases such as Parkinson’s disease (PD). However, prior to designing endogenous cell therapies, the effect of PD neuropathology on endogenous progenitor cell proliferation and their neurogenic potential must be investigated. This study examined the effect of dopaminergic cell loss on the proliferation and differentiation of subventricular zone- (SVZ) and midbrain-derived progenitor cells in the adult rodent brain, using the partial progressive 6-hydroxydopamine (6-OHDA) lesion model of PD. Cell proliferation and differentiation were assessed with 5-bromo-2=-deoxyuridine (BrdU) labeling and immunohistochemistry for cell type-specific markers. Tyrosine hydroxylase immunohistochemistry demonstrated a complete loss of nigrostriatal projections in the striatum and a subsequent progressive loss of dopamine (DA) cells in the SN. Quantification indicated that 6-OHDA lesion–induced cell degeneration produced a significant increase in BrdU immunoreactivity in the SVZ, ipsilateral to the lesioned hemisphere from 3 to 21 days post-lesion, compared with sham-lesioned animals. Similarly, in the striatum we observed a significant increase in the total number of BrdU positive cells in 6-OHDA-lesioned animals at all time points examined. More importantly, a significant increase in midbrain-derived BrdU positive cells was demonstrated in 6-OHDA-lesioned animals 28 days post-lesion. While we did not detect neurogenesis, BrdU labeled cells co-expressing the astrocytic marker glial fibrillary acidic protein (GFAP) were widely distributed throughout the 6-OHDA-lesioned striatum at all time points. In contrast, BrdU-labeled cells in the SN of 6-OHDA-lesioned animals did not co-express neural markers. These results demonstrate that DA-ergic neurodegeneration in the partial progressive 6-OHDA-lesioned rat brain increases SVZ- and midbrain-derived progenitor cell proliferation. While, newborn striatal
Parkinson’s disease (PD) is a common neurodegenerative disorder typically affecting the older population. It is pathologically hallmarked by the presence of intraneuronal Lewy bodies and the progressive neurodegeneration of the nigrostriatal dopaminergic neurons leading to debilitating motor dysfunction. The cardinal symptoms of idiopathic PD include resting tremor, rigidity and bradykinesia. The current treatment options available for PD primarily consist of pharmacological dopamine (DA) replacement (L-DOPA) and surgical procedures (e.g. deep brain stimulation, pallidotomy, thalamotomy) which partially alleviate motor symptoms. However, complications arising from chronic treatment of L-DOPA and the variable success of surgical procedures have led to the investigation of other novel treatment strategies including cell-based therapeutic options, such as restoring DA-ergic neurotransmission by exogenous cell replacement (e.g. transplantation of fetal mesencephalic tissue). The clinical success of these strategies has been variable with the inconsistent benefits for patients. Further, cell transplantation using aborted fetuses is surrounded by ethical and technical concerns. Therefore, the presence of endogenous progenitor cells in the adult mammalian brain presents as an exciting alternative to cell transplantation for the development of a novel therapeutic option for treating PD. Progenitor cells in the two main adult neurogenic regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the dentate gyrus, have been shown to proliferate and differentiate in response to neuronal cell loss in both the animal brain (Kuhn et al., 1996; Weinstein et al., 1996; Liu et al., 1998; Parent et al., 1998; Scott et al., 1998; Nait-Oumesmar et al., 1999; Takagi et al., 1999; Kay and Blum, 2000; Arvidsson et al., 2001; Mao et al., 2001; Yagita et al., 2001; Ferland et al., 2002; Nakatomi et al., 2002; Parent et al., 2002; Tattersfield et al., 2004) and the postmortem human brain (Curtis et al., 2003, 2005; Jin et al., 2004). Further, the presence of progenitor cells in non-neurogenic regions, such as the cortex (Palmer et al., 1999), septum (Palmer et al., 1995),
*Corresponding author. Tel: ⫹64-9-373-7599; fax: ⫹64-9-373-7556. E-mail address: [email protected] (B. Connor). Abbreviations: BDNF, brain-derived neurotrophic factor; BrdU, 5-bromo2=-deoxyuridine; CD68, cluster of differentiation 68; DA, dopamine; DAB, diaminobenzidine; DCx, doublecortin; GFAP, glial fibrillary acid protein; MAP2, microtubule-associated protein; MFB, medial forebrain bundle; NG2, chondroitin sulfate proteoglycan; PBS, phosphate-buffered saline; PD, Parkinson’s disease; PDGF-BB, platelet-derived growth factor; QA, quinolinic acid; SN, substantia nigra; SVZ, subventricular zone; TGF-␣, tumor growth factor-␣; TH, tyrosine hydroxylase; 6-OHDA, 6-hydroxydopamine.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.11.036
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spinal cord (Weiss et al., 1996), ventricular extension (Chouaf-Lakhdar et al., 2003) and the substantia nigra (SN) (Lie et al., 2002; Zhao et al., 2003), has also been demonstrated, but at a less appreciable level compared with the established neurogenic regions (Zhao et al., 2003). Currently, the neurogenic potential of the midbrain region is a highly debated topic. DA is reported to play a role in the regulation of both embryonic and adult neurogenesis (Borta and Hoglinger, 2007). Previous studies have demonstrated that a complete 6-hydroxydopamine (6-OHDA) lesion in the rat (Stromberg et al., 1986; Lie et al., 2002) or MPTP lesions in mice (Stromberg et al., 1986; Kay and Blum, 2000; Mao et al., 2001) results in increased cell proliferation in the striatum and the SN with no DA-ergic differentiation (Frielingsdorf et al., 2004). However, Zhao et al. (2003) postulated that DA-ergic differentiation occurs at a very low level in the SN of healthy mice and it is increased after a MPTP lesion. Transplantation of freshly isolated adult SN progenitor cells into the adult hippocampus showed that these cells have neuronal potential and can give rise to new neurons when exposed to the correct environment (Lie et al., 2002). Basal levels of neurogenesis, increased proliferation and a very low level of DAergic differentiation following MPTP treatment of LacZ transgenic mice were also demonstrated more recently (Shan et al., 2006). In contrast, experimental depletion of DA in rodents following complete 6-OHDA or MPTP lesioning has been shown to decrease progenitor cell proliferation in both the SVZ and SGZ (Baker et al., 2004; Hoglinger et al., 2004). The response of adult SVZ and, in particular, SN progenitor cells to DA depletion therefore requires further investigation. The controversy may be due to methodological differences between the reported studies. Furthermore, the previously reported studies have used standard 6-OHDA or MPTP rodent models of PD which have several disadvantages when examining interventions aimed at protecting or restoring the nigrostriatal system. In the standard 6-OHDA model of PD, 6-OHDA is injected to the medial forebrain bundle (MFB) or the SN which induces a near complete destruction of DA-ergic neurons (Jeon et al., 1995; Zuch et al., 2000). While identifiable nigral cell loss has been observed up to 31 days following complete 6-OHDA lesioning, the sudden and massive toxic insult near the cell bodies results in an acute rather than gradual and progressive loss of nigral cells, with the majority of cell death occurring within 12 h of nigral 6-OHDA injection (Jeon et al., 1995; Zuch et al., 2000). Second, the standard neurotoxic nigrostriatal lesion in rats is commonly used to achieve the maximum possible degeneration. Therefore, it makes it difficult to control when only a partial lesion is desired. In experiments investigating neuro-restoration strategies similar to this study, it is most ideal to use a partial progressive model where the initial lesion results in a partial loss of DA-ergic neurons followed by an ongoing progressive degeneration after injury (Sauer and Oertel, 1994; Kirik et al., 1998). Therefore, we have for the first time investigated the proliferation and differentiation poten-
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tial of endogenous SVZ-derived and midbrain progenitor cells by employing the partial progressive 6-OHDA lesion rat model. In contrast to the standard 6-OHDA models, the partial progressive 6-OHDA lesion model of PD allows for a retrograde degeneration of the nigrostriatal pathway, by first acting on the DA axon terminals within the striatum and exerting its degenerative effects along the axons toward the cell bodies within the SN (Sauer and Oertel, 1994). We report that a single intrastriatal injection of 6-OHDA induces a near complete loss of striatal DA and a subsequent progressive loss of DA-ergic neurons in the SN, which leads to a significant increase in progenitor cell proliferation in the SVZ, striatum and the midbrain. The newborn cells in the striatum readily differentiated into astrocytes and failed to acquire a neuronal phenotype. The midbrain-derived progenitor cells in contrast, did not acquire either a neuronal or a glial phenotype and appeared to remain in an undifferentiated state. The results of this study further suggest that endogenous progenitor cell populations in both neurogenic and non-neurogenic regions have the capacity to proliferate in response to DA-ergic neurodegeneration but may require region-specific environmental cues to undergo neurogenesis and DA-ergic differentiation.
EXPERIMENTAL PROCEDURES Surgical procedures Adult male Wistar rats weighing 240 –290 g (University of Auckland Animal Resources Unit, Auckland, New Zealand) were used in this study. The animals were housed in groups of three in a temperature- and humidity-controlled room that was kept on a 12-h light/dark cycle. Food and water were available ad libitum throughout the study. Experimentation was performed in strict compliance with the University of Auckland Animal Ethics Guidelines in accordance with the New Zealand Animal Welfare Act 1999 and conformed to named international guidelines on the ethical use of animals. All efforts were made to minimize the number of animals used and to minimize their suffering. All surgeries were performed following an i.p. injection of 60 mg sodium pentobarbital per kilogram of body weight. Rats received a unilateral intrastriatal infusion of either 6-OHDA (2.8 l) or vehicle solution (phosphate-buffered saline (PBS)) over a 5 min period at the following coordinates: 1.0 mm anterior–posterior, 2.8 mm medial–lateral and ⫺5.0 mm dorsal–ventral.
5-Bromo-2=-deoxyuridine (BrdU) labeling and tissue processing Following surgery, each animal received four BrdU (200 mg/kg i.p.) injections, over a period of 6 h (2 h interval between injections) on day 3, 7, 14, 21 or 28 after injection of 6-OHDA or PBS (n⫽3–5 per time point for each group) and were killed 2 h after the last BrdU injection. A concentration of 200 mg/kg BrdU per injection was chosen for this study as this concentration of BrdU has been demonstrated to produce optimal labeling of cells undergoing proliferation with little detection of cells exhibiting DNA repair (Cameron and McKay, 2001). Rats were killed by an overdose of sodium pentobarbital and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed, post-fixed in 4% paraformaldehyde overnight and cryoprotected for sectioning in a 30% sucrose solution. Coronal sections were cut through the striatum from frozen brains using a sliding microtome set at 40 m. Eight sets of
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sections were collected from each brain (distance of 320 m between consecutive sections in each set) and stored in a cryoprotective solution at ⫺20 °C.
Immunohistochemistry and confocal microscopy Diaminobenzidine (DAB) peroxidase immunohistochemistry was performed on a complete set of free-floating coronal sections from each animal for tyrosine hydroxylase (TH) (mouse anti-TH; 1:1000; Chemicon, Temecula, CA, USA) immunoreactivity in order to confirm the selective neuronal loss induced by the 6-OHDA lesion. The presence of migrating neuroblasts was identified using an antibody to doublecortin (DCx; goat anti-DCx; 1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). To label proliferating cells, we used primary antibodies that recognize BrdU (mouse anti-BrdU, 1:250; Chemicon; rat anti-BrdU, 1:250; Accurate Chemicals, Westbury, NY, USA); both BrdU antibodies showed identical patterns of immunolabeling. For BrdU immunolabeling, standard BrdU immunohistochemical protocol was used (Tattersfield et al., 2004). Following incubation with the primary antibody, sections were incubated with the appropriate secondary biotinylated antibody (1:500; goat anti-mouse; Sigma-Aldrich, St. Louis, MO, USA; goat anti-rat, Sigma-Aldrich; donkey anti-goat; Jackson Immunoresearch Laboratories, West Grove, PA, USA) followed by incubation in ExtrAvidin peroxidase (1:1000; SigmaAldrich). Antibodies were visualized using 0.4 mg/ml DAB, 25 mg/ml nickel sulfate, 0.005% hydrogen peroxide in 0.2 M phosphate buffer. Brightfield images were taken using a digital camera on a light microscope. All the figures were compiled using Adobe Illustrator (Adobe Systems Inc., San Jose, CA, USA). Double-label immunofluorescence using rat anti-BrdU and a second primary antibody was performed as previously described (Tattersfield et al., 2004). Sections were serially incubated with the rat anti-BrdU antibody in conjunction with either one of the following mouse or rabbit second primary antibody; chondroitin sulfate proteoglycan (1:200; NG2; rabbit anti-NG2; Chemicon), microtubule-associated protein (1:200; MAP2; mouse anti-MAP2; Chemicon), cluster of differentiation 68 (1:200; CD68; mouse anti-CD68; Chemicon) and glial fibrillary acid protein (1:400; GFAP; mouse anti-GFAP; Dako, Glostrup, Denmark). Following incubation in the primary antibodies, the sections were incubated in pooled solutions of specific secondary fluorochromes (1:200; anti-rat Alexa Fluor 594; anti-mouse Alexa Fluor 488; anti-rabbit Alexa Fluor 488; Molecular Probes, Eugene, OR, USA) for 12 h at room temperature. Fluorescently labeled sections were imaged using a confocal laser-scanning microscope (Leica TCS SP2) equipped with UV, argon, argon/ krypton and helium/neon lasers (Biomedical Imaging Resource Unit, University of Auckland). Each fluorescent label was imaged serially to eliminate detection of bleed through and other artifactual fluorescence. The confocal images were captured in a z-series with an interslice gap of 1 m.
Quantification and statistical analysis Stereological counting of TH and BrdU positive cells. Non-biased stereological cell counts were carried out on every 8th section throughout the rostrocaudal extent of the brain using a digital camera (MicroFire 1.0) mounted on an upright microscope (Nikon ECLIPSE E800) equipped with a motorized stage driven in the X, Y and Z planes. TH positive cells in the SN of control and 6-OHDA-lesioned animals were counted at 20⫻ magnification using the optical fractionator stereological method (Stereo Investigator software Version 6, MicroBrightField Inc. USA) where 60 counting sites were examined (frame size: 75/75 m; spacing between frames: 250/250 m) (Connor et al., 1999) per section. TH positive cell counts in the control and 6-OHDA-lesioned animals were expressed as the total number of cells within the entire SN. BrdU positive cells in the striatum and the midbrain (SN and
PaG surrounding the cerebral aqueduct) of control and 6-OHDAlesioned animals were carried out exhaustively through every 8th section at 40⫻ magnification in order to estimate the total number of cells in each structure. Quantification of striatal denervation. The extent of the striatal denervation (lesion size) was evaluated on every 8th section throughout the rostrocaudal extent of the striatum. Both the nonlesioned and lesioned striatum was photographed at 2.5⫻ magnification using a digital camera mounted on a conventional transmitted light microscope. A standard level of grey-scale intensity was set to represent normal levels of TH immunoreactivity and from this level the area of reduced TH immunoreactivity was determined and quantified using ImageJ software (National Institutes of Health, USA). Quantification of BrdU positive cells in the SVZ. Since, BrdU positive cells in the SVZ occurred predominantly in clusters they could not be easily recognized as individual cells unless at very high magnification. Based on the fact that BrdU cells were unevenly distributed along the SVZ, it would be inappropriate or inaccurate to select a single or representative sample size in order to estimate the entire population. A quantification method was therefore employed to determine the total area of BrdU immunoreactivity in the SVZ lining the lateral ventricle adjacent to the striatum (Tattersfield et al., 2004; Henry et al., 2007). In order to clearly visualize BrdU positive cells in the SVZ high magnification images (40⫻) of the SVZ were acquired using an inverted microscope (Nikon TE2000E) and then automatically put together using automated montaging software (NIS Elements) to represent the entire length of the SVZ. The total area of BrdU positive cells was measured using the ImageJ 1.34s (National Institutes of Health) software. Area increase of BrdU positive cells in the SVZ in the ipsilateral, lesioned hemisphere was expressed as a percentage of the contralateral, non-lesioned hemisphere.
Statistical analysis All statistical analysis was performed using the Statistical Analysis Software (SAS) system version 9.1. All values and graphs are presented as mean⫾S.E.M. BrdU and TH immunoreactive cell counts from each brain region were analyzed using one-way ANOVA comparing control and 6-OHDA-lesioned groups. The striatal lesion area of 6-OHDA-lesioned animals was analyzed using one-way ANOVA comparing all time points combined. Results were considered statistically significant when the P-value was less than or equal to 0.05.
RESULTS Reduced TH immunolabeling in the striatum and SN following partial progressive 6-OHDA lesioning The extent of DA-ergic lesioning was determined from a set of coronal striatal and nigral sections stained for TH immunohistochemistry. A single unilateral injection of 6-OHDA into the striatum resulted in an almost complete loss of TH immunoreactivity in the striatum [Fig. 1A–B] and a subsequent loss of TH immunoreactivity in the SN [Fig. 1D–E] as previously described (Sauer and Oertel, 1994). Loss of TH-immunoreactivity does not necessarily indicate neuronal cell loss as it is well established that TH expression is down-regulated prior to cell loss (Sauer and Oertel, 1994; Bowenkamp et al., 1995, 1996; Choi-Lundberg et al., 1997; Connor et al., 1999, 2001; Rosenblad et al., 1999). Quantification demonstrated that loss of TH immunoreactivity in the lesioned striatum was completely
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Fig. 1. Reduced TH immunolabeling in the striatum and SN following partial progressive 6-OHDA lesioning. (A, B) Coronal sections of the striatum immunostained for TH in the 6-OHDA-lesioned animal at 28 days post-lesion. Extensive TH immunoreactivity is observed in the non-lesioned hemisphere (A) with complete loss of TH immunoreactivity observed in the 6-OHDA-lesioned hemisphere (B). (C) Graph demonstrating the mean area of reduced TH immunoreactivity (lesion size) in the lesioned hemisphere of 6-OHDA-treated animals. (D, E) Coronal sections of the SN immunostained for TH in the 6-OHDA-lesioned animal at 21 days post-lesion. Extensive TH immunoreactivity is observed in the non-lesioned hemisphere (D) with progressive loss of TH positive DA-ergic cells observed in the 6-OHDA-lesioned hemisphere (E). (F) Graph demonstrating the total number of TH positive DA-ergic cells in the SNpc in the ipsilateral hemisphere of 6-OHDA-lesioned and normal animals.
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formed by 3 days and remained fixed till 28 days postlesion, with no significant difference in lesion size between the time points [Fig. 1C]. In contrast, quantification of TH positive cells in the ipsilateral SN of 6-OHDA-lesioned animals indicated a significant progressive decrease in the total number of TH positive cells from 14 to 28 days postlesion compared with normal animals [Fig. 1F]. The observed temporal profile of TH expression in the striatum and SN following a partial progressive 6-OHDA lesion is in full agreement with previous observations (Sauer and Oertel, 1994). DA denervation in the 6-OHDA-lesioned striatum increases cell proliferation in the SVZ and the striatum We examined the effect of DA-ergic projection loss in the lesioned striatum on SVZ and striatal cell proliferation. Rats received multiple BrdU injections (four injections over 6 h; 200 mg/kg i.p. per injection) on day 3, 7, 14, 21 or 28 after an intrastriatal injection of 6-OHDA or PBS, and were killed 2 h after the last BrdU injection. In the contralateral and sham-lesioned hemispheres, BrdU labeling was confined to the SVZ [Fig. 2A] with very few BrdU positive cells seen in the adjacent striatum [Fig. 2D]. In contrast, in 6-OHDA-lesioned animals BrdU labeling was increased in the SVZ in the lesioned hemisphere [Fig. 2B] and a large number of BrdU positive cells were observed throughout the adjacent striatum [Fig. 2E] mainly accumulated around the lesion tract. BrdU positive cells were distributed singly or in nested clusters throughout the SVZ, while, in the striatum they were individually distinguishable and occurred predominantly as irregular and ellipsoid shaped homogenously stained nuclei. BrdU positive doublets were also observed in both the SVZ and the striatum indicating the occurrence of cell division [Fig. 2E]. Quantification revealed that 6-OHDA-induced DA denervation produced a significant increase in the area of BrdU positive cells in the SVZ from 3 to 21 days post-lesion compared with sham-lesioned animals [Fig. 2C; 3 day 6-OHDA 22⫾6% vs. sham 5⫾2%, Pⱕ0.004; 7 days 6-OHDA 32⫾10% vs. sham 4⫾2%, Pⱕ0.02; 14 days 6-OHDA 26⫾6% vs. sham 4⫾3%, Pⱕ0.006; 21 days 6-OHDA 18⫾4% vs. sham 5⫾2%, Pⱕ0.02]. At 28 days post-lesion, the area of BrdU immunoreactivity in the 6-OHDA lesioned hemisphere was reduced compared with earlier time points [Fig. 2C]. We also observed a small increase in the area of SVZ BrdU immunoreactivity in the ipsilateral hemisphere of sham-lesioned animals compared with the contralateral hemisphere at each time point [Fig. 2C], possibly reflecting a response in cell proliferation to mechanical injury induced by the intrastriatal vehicle injection. Stereological analysis also revealed a significant increase in BrdU positive cells in the striatum of 6-OHDA-lesioned animals from 3 to 28 days post-lesion, compared with shamlesioned animals [Fig. 2F; 3 day 6-OHDA 81392⫾4568 vs. sham 6405⫾2013, Pⱕ0.0005; 7 day 6-OHDA 3771⫾944 vs. sham 840⫾140, Pⱕ0.04; 14 day 6-OHDA 2570⫾653 vs. sham 552⫾169, Pⱕ0.05; 21 days 6-OHDA 1659⫾248 vs.
sham 53⫾19, Pⱕ0.003; 28 days 6-OHDA 520⫾55 vs. sham 48⫾17, Pⱕ0.002]. At 3 days post-lesion, 6-OHDAlesioned animals demonstrated the greatest significant increase in the number of BrdU positive cells which corresponded to a 12.7-fold increase compared with shamlesioned animals [Fig. 2F]. DA-ergic cell loss in the SN increases cell proliferation in the midbrain Multiple injections of BrdU resulted in successful labeling of proliferating cells in the midbrain following a partial progressive 6-OHDA lesion. BrdU positive cells were observed within the PaG [Fig. 3A–G], surrounding the cerebral aqueduct and in the SN [Fig. 3I–J] in both sham- [Fig. 3A] and 6-OHDA-lesioned [Fig. 3E and I–J] animals at all time points examined. In both treatment groups, BrdU positive cells were randomly dispersed within the PaG and SN with no clear pattern of labeling. Quantification indicated no significant difference in the number of BrdU-labeled cells from 3 to 21 days post-lesion in the PaG between sham- and 6-OHDAlesioned animals [Fig. 3H; 3 days 6-OHDA 904⫾536 vs. sham 483⫾101, Pⱕ0.4; 7 days 6-OHDA 411⫾183 vs. sham 388⫾20, Pⱕ1.0; 14 days 6-OHDA 570⫾129 vs. sham 387⫾154, Pⱕ0.4; 21 days 6-OHDA 506⫾53 vs. sham 280⫾134, Pⱕ0.2]. Similarly, no significant difference in the number of BrdU-labeled cells was observed in the SN between sham- and 6-OHDA-lesioned animals from 3 to 21 days post-lesion [Fig. 3K; 3 days 6-OHDA 4924⫾100 vs. sham 3493⫾415, Pⱕ0.2; 7 days 1029⫾292 vs. 624⫾ 0, Pⱕ0.6; 14 days 6-OHDA 910⫾142 vs. sham 928⫾100, Pⱕ0.2; 21 days 6-OHDA 584⫾103 vs. sham 977⫾288, Pⱕ0.9). However, we did observe a significant increase in the number of BrdU positive cells at 28 days post-lesion in both the PaG [Fig. 3H; 28 days 6-OHDA 264⫾52 vs. sham 51⫾31, Pⱕ0.03] and the SN [Fig. 3K; 28 days 6-OHDA 451⫾67 vs. sham 155⫾34, Pⱕ0.01] of 6-OHDA-lesioned animals compared with controls. This represents the first demonstration of progenitor cell proliferation in the adult rodent midbrain following a partial progressive 6-OHDA lesion of the nigrostriatal pathway. DA denervation induces striatal astrogenesis We next examined whether the newborn proliferating cells in the SVZ and the striatum resulted in the generation of new neurons and/or glial cells in the lesioned striatum. We first examined the expression of DCx (a microtubule-associated protein present in migrating neuronal precursors) to determine whether the increase of newborn cells in the SVZ is forming migrating neuroblasts which migrate toward the adjacent striatal lesion. No change in DCx positive labeling in the SVZ/RMS was noted between shamand 6-OHDA-lesioned animals [data not shown]. We also observed no DCx positive labeling in the ipsilateral and contralateral striatum of 6-OHDA-lesioned animals indicating that SVZ-derived progenitor cells are not migrating into the lesioned striatum as observed in other models of neuronal injury (Arvidsson et al., 2001; Tattersfield et al., 2004). Therefore, it is likely the increase in proliferating cells seen in the striatum is a result of locally derived
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Fig. 2. DA denervation in the 6-OHDA-lesioned striatum increases cell proliferation in the SVZ and the striatum. (A, B) Coronal sections of the SVZ adjacent to the striatum immunostained for BrdU in a 6-OHDA-lesioned animal at the 7-day time point. BrdU immunoreactivity was confined to the SVZ of the non-lesioned hemisphere (A) with increased BrdU immunoreactivity observed in the SVZ of the lesioned hemisphere (B). (C) Graph demonstrating the percentage mean area of BrdU immunoreactivity in the ipsilateral SVZ of sham- and 6-OHDA-lesioned animals. (D, E) Coronal sections of the striatum immunostained for BrdU in a 6-OHDA-lesioned animal at the 3-day time point. In the non-lesioned hemisphere, BrdU immunoreactivity was largely confined to the SVZ with a few BrdU positive cells observed in the striatum (D). Extensive labeling of BrdU positive cells was observed within the ipsilateral striatum of 6-OHDA-lesioned animals (E). (F) Graph demonstrating the total number of BrdU positive cells in the striatum of sham- and 6-OHDA-lesioned animals.
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Fig. 3. DA-ergic cell loss in the SN, increased cell proliferation in the midbrain. (A–K) Coronal sections of BrdU immunoreactivity in the PaG of a SHAM- (A) and 6-OHDA-treated (E) animal at 28 days post-lesion. (H) Total number of BrdU positive cells within the PaG. Quantification revealed a significant increase in the total number of BrdU positive cells in the PaG in 6-OHDA-treated animals was observed at 28 days post-lesion compared with SHAM controls. (I, J) Coronal section of BrdU immunoreactivity in the SN of a 6-OHDA-treated animal at 28 days post-lesion. (K) Total increased number of BrdU positive cells in the SNpc. Quantification revealed a significant increase in the total number of BrdU positive cells in the SNpc in the 6-OHDA-treated animals was observed at 28 days post-lesioned compared with SHAM controls.
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progenitors reacting to the lesion. Next, the phenotype of these newborn striatal progenitors was investigated using double immunofluorescent labeling using cell-type markers for neurons (MAP2, TH), astrocytes (GFAP), microglia (CD68) and oligodendrocyte precursors (NG2). BrdU positive cells in the striatum did not co-localize with any of the neuronal, microglial or oligodendrocyte precursor markers examined in any of the time points investigated [data not shown]. However, they readily co-localized with GFAP in all the time points examined demonstrating robust astrogenesis in the striatum [Fig. 4A–B]. High power inspection of the sections revealed that typically, GFAP positive newborn cells had triangular or irregular cell bodies with long radial processes [Fig. 4C–E]. A population of BrdU positive/GFAP negative cells was also present in the striatum [Fig. 4A–B]. Midbrain progenitor cells remain undifferentiated in the partial progressive 6-OHDA lesion Our results demonstrate for the first time an increase of midbrain progenitor cells in the partial progressive 6-OHDA lesion rat model of PD. This increase was observed both in the SVZ surrounding the cerebral aqueduct as well as in the SN, suggesting midbrain progenitor cells respond to progressive DA-ergic cell loss in the SN. Therefore, the differentiation potential of these newborn midbrain progenitor cells was also investigated using a range of
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neural cell markers. Since, a significant increase of BrdU cells was observed in both midbrain regions at 28 days post-lesion, the possibility of neuroblast migration from the PaG to the SN was investigated with DCx. We did not observe any DCx positive labeling in the PaG or the SN in sham or 6-OHDA-lesioned animals [data not shown] suggesting that these are locally derived progenitors. Next, the neurogenic and/or gliogenic potential of these midbrain progenitor cells was investigated using the same cell-type markers as previously described. We were unable to detect co-localization of BrdU positive cells in the PaG and the SN with any of the neuronal or glial cell markers examined indicating that they do not readily form neurons or glial cells in the midbrain [data not shown].
DISCUSSION The neurogenic potential of the midbrain region is a highly debated topic. Progenitor cell proliferation and differentiation in rodent animal models of PD has previously been investigated using either a systemic application of MPTP or an intracerebral application of 6-OHDA into either the MFB or the SN (Stromberg et al., 1986; Kay and Blum, 2000; Mao et al., 2001; Lie et al., 2002; Zhao et al., 2003). Due to the acute and near complete destruction of DAergic neurons, these models have several disadvantages when examining interventions aimed at protecting or re-
Fig. 4. BrdU labeled cells in the 6-OHDA-lesioned striatum co-express GFAP at all time points examined. (A, B) BrdU (red) and GFAP (green) double-immunolabeled cells (closed arrows) observed in the 6-OHDA-lesioned striatum at 3 days (A) and 28 days (B) post-lesion. BrdU negative/ GFAP positive (open arrows) cells were also observed. (C, D) Boxed areas in (A) and (B) indicate high magnification confocal images of BrdU-labeled cells (red) co-expressing GFAP (green).
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storing the nigrostriatal system. We therefore examined, for the first time, the neurogenic response of endogenous progenitor cells in the partial progressive 6-OHDA rat lesion model. This model results in a more delayed and partial DA-ergic cell loss than observed following standard 6-OHDA lesions due to retrograde degeneration of the nigrostriatal pathway, and we believe may provide an increased window of opportunity for a regenerative response (Sauer and Oertel, 1994; Lee et al., 1996; Kirik et al., 1998). In agreement with previous studies (Stromberg et al., 1986; Kay and Blum, 2000; Mao et al., 2001) we observed increased progenitor cell proliferation in the striatum of 6-OHDA-lesioned animals. The increase in cell proliferation was observed at all time points in the striatum with a decreasing gradient. This closely corresponded with the immediate complete loss of striatal DA and the secondary DA-ergic cell loss in the SN. First, the striatal DA is completely degenerated as early as 3 days post-lesion and remains fixed and complete till 28 days post-lesion. Therefore, the massive induction of proliferating cells seen in the striatum at 3 days post-lesion is a likely representation of initial reactive astrogliosis which is typically observed in a neurotoxic lesion of the DA-ergic system (Stromberg et al., 1986). The decreasing gradient of striatal progenitors from 3 to 28 days post-lesion indicates that the proliferating cells in the striatum are largely part of the injury-induced glial reaction in the striatum. Previous studies (Kay and Blum, 2000; Mao et al., 2001) using various animal models of PD have suggested that one of the noticeable responses to lesions of the nigrostriatal DA-ergic projection is reactive astrogliosis. Further confirming this, we found that the newborn BrdU positive cells in the striatum predominantly co-expressed the astrocytic marker GFAP and did not co-localize with neuronal markers. Experimental depletion of DA in rodents following complete 6-OHDA or MPTP lesioning has been shown to decrease progenitor cell proliferation in both the SVZ and SGZ (Baker et al., 2004; Hoglinger et al., 2004) with proliferation restored following delivery of a selective agonist of DA D2-like receptors (Hoglinger et al., 2004). Stimulation of the DA D3 receptor has been shown to increase SVZ and nigral cell proliferation (Van Kampen et al., 2004) and has also been shown to trigger neurogenesis in the SVZ and the SN of normal (Van Kampen et al., 2004; Van Kampen and Robertson, 2005) and 6-OHDA-lesioned rats (Van Kampen and Eckman, 2006). In contrast to these observations, we observed an increase in cell proliferation from 3 to 21 days post-lesion in the SVZ following a partial progressive 6-OHDA lesion of the nigrostriatal system. It is currently not clear how differences in the partial progressive lesion model may lead to stimulation of SVZ progenitor cell proliferation but this may reflect differences in the degenerative profile and expression of neurogenic cues between the partial and complete lesion models. Previous studies examining stroke- (Arvidsson et al., 2001) or quinolinic acid– (QA) induced (Tattersfield et al., 2004) striatal cell loss suggest the generation and migration of newly generated neurons into the damaged striatum from the SVZ/RMS. In contrast, we did not observe signif-
icant Dcx positive labeling in the partial 6-OHDA-lesioned striatum demonstrating a lack of progenitor cell migration from the SVZ to the damaged striatum. This observation may predominantly reflect a difference in the lesion models. Both the stroke and QA lesion models result in the degeneration of cell bodies predominantly within the striatum, which may produce environmental cues required to trigger neuronal migration and differentiation within the striatum. The partial progressive 6-OHDA lesion model in contrast exerts a retrograde type cell loss where predominant cell degeneration takes place within the SN away from the striatum and therefore, may provide a restrictive atmosphere with a lack of necessary environmental triggers to induce progenitor cell migration and neuronal differentiation in the lesioned striatum. Recent evidence suggests that the infusion of certain growth factors into forebrain structures can induce in vivo stimulation of endogenous progenitor cells in animal models of PD. However, studies examining the infusion of tumor growth factor-␣ (TGF-␣) into the striatum of rats with unilateral 6-OHDA lesions have provided differing results. Fallon et al. (2000) reported that TGF-␣ infusion into 6-OHDA-lesioned rats resulted in migration of progenitor cells into the striatum with an improvement in motor deficits, which was attributed to the appearance of a small number of new striatal neurons that expressed a DA-ergic phenotype, a cell type that is normally not observed in the striatum (Fallon et al., 2000). In contrast, while Cooper and Isacson (2004) also observed significant proliferation and migration of progenitor cells from the SVZ to the striatum in 6-OHDA-lesioned rats following intrastriatal infusion of TGF-␣, they did not observe neuronal differentiation of progenitor cells in either the striatum or the SN. Mohapel et al. (2005) investigated the effects of i.c.v.-infused plateletderived growth factor (PDGF-BB) and brain-derived neurotrophic factor (BDNF) on cell genesis, following BrdU incorporation in adult rats with unilateral 6-OHDA lesions. They found that both PDGF-BB and BDNF increased the number of newly formed cells in the striatum and SN to an equal extent following 10 days of treatment. At 3 weeks, after termination of growth factor treatment, co-localization of BrdU positive cells with the neuronal marker NeuN, revealed a significant increase in newly generated neurons in the striatum. In correspondence, many DCx-positive neuroblasts were also observed in the denervated striatum following growth factor infusion. Further, a subset of these new neurons expressed the early marker for striatal neurons Pbx, suggesting that administration of these growth factors is capable of recruiting new neurons into the striatum of hemiparkinsonian rats (Mohapel et al., 2005). Therefore, we propose that following a partial progressive 6-OHDA lesion in the rat striatum there is robust cell proliferation in the SVZ and the striatum in response to the ongoing degeneration of the nigrostriatal pathway generated in this model. However, the newborn striatal cells are only capable of extensive astrogenesis and not neurogenesis possibly due to the lack of stimulatory cues and restrictive microenvironment in the 6-OHDA-lesioned striatum.
P. M. Aponso et al. / Neuroscience 151 (2008) 1142–1153
The most significant finding to come out of this study is the demonstration of significantly increased midbrain-derived progenitor cells following the progressive DA-ergic denervation of the adult rodent SN. While the presence of proliferating progenitor cells in non-neurogenic regions has been previously demonstrated (Weiss et al., 1996; ChouafLakhdar et al., 2003), no study to date has examined the changing level of proliferating progenitor cells in the midbrain following a partial progressive 6-OHDA lesion. We demonstrate a significant increase in BrdU positive cells in the 6-OHDA-lesioned animals at 28 days post-lesion in both the PaG and the SN of the rodent midbrain. Interestingly, the degeneration profile of the partial progressive 6-OHDA lesion model indicates a significant decrease in the number of TH positive DA cells by 28 days post-lesion. Therefore, we initially hypothesized that progenitor cells located adjacent to the cerebral aqueduct may be responding to the degenerating process taking place in the adjacent SN by increased proliferation followed by potential migration to the site of degeneration. However, this was not found to be the case, as we were unable to detect the presence of newborn migrating neural precursors either adjacent to the cerebral aqueduct or in the SN in any of the time points examined. It was also interesting to note the presence of a large number of BrdU-labeled cells in the SN of both sham- and 6-OHDA-lesioned animals 3 days postlesion. We do not believe this reflects a response to mechanical damage as the lesion took place within the striatum, which is located more rostrally from the SN. However, we cannot rule out the possibility that BrdU-labeled cells in the midbrain may represent degenerating DA-ergic neurons that have lost phenotype (Taupin, 2007). Initially, we proposed this increase of BrdU-labeled cells may be an acute inflammatory response to the nigrostriatal denervation as demonstrated in previous studies (Kay and Blum, 2000; Mao et al., 2001). However, double immunolabeling was unable to demonstrate co-expression of midbrainderived BrdU positive cells with either microglial or astrocytic markers at any of the time points examined. The concept of cell differentiation and neurogenesis in the midbrain region still remains a controversial topic as previous studies have indicated contrasting findings. On the one hand, we and several other laboratories have demonstrated that 6-OHDA lesioning in rats or MPTP lesioning in mice results in cell proliferation in the SN without apparent DA-ergic differentiation (Kay and Blum, 2000; Mao et al., 2001; Lie et al., 2002; Steiner et al., 2006). On the other hand, DA-ergic differentiation has been shown to occur at a very low level in the SN of normal mice, and at an increased level after MPTP lesioning by Zhao et al. (2003). Further, basal levels of neurogenesis, increased proliferation and DA-ergic differentiation following MPTP have recently been shown in nestin-LacZ transgenic mice (Shan et al., 2006). In addition, functional DA-ergic differentiation has been demonstrated in vitro from neural progenitor cells isolated from the tegmental tissue of the midbrain and hindbrain, including the ependymal zone of the cerebral aqueduct and the fourth ventricle (Hermann et al., 2006). More interestingly, it has been shown that when
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midbrain-derived progenitor cells were isolated and implanted in a neurogenic environment, such as the dentate gyrus, a proportion of the transplanted cells differentiated into mature neurons (Lie et al., 2002). However, transplantation of midbrain precursor cells into SN of normal rats resulted only in the generation of glial cells, suggesting that local environmental factors may prevent the differentiation of precursor cells to distinct neuronal lineages in situ (Lie et al., 2002). Despite these contrasting findings from previous studies, we could not find any evidence of midbrain-derived proliferating cells co-expressing any of the neural markers examined, suggesting that they do not readily form neurons in the midbrain. Although, methodological differences between studies may also have contributed toward the disparity of the findings obtained with regard to midbrain progenitor cell differentiation, further detailed investigation of midbrain progenitor cell differentiation and neurogenesis remains a necessity.
CONCLUSION Our findings provide the first quantitative demonstration of progenitor cell proliferation in the SVZ, striatum and the midbrain, following the partial progressive 6-OHDA lesion model of PD. In particular, we observed a significant increase of proliferating cells in the SVZ and the striatum which led to robust astrogenesis. More importantly, a significant increase of midbrain-derived proliferating cells was also demonstrated in the 6-OHDA-lesioned animals, however these did not differentiate into either new neurons or glia. We demonstrate that the endogenous neural progenitor cells in the striatum and the SN can provide potential repair for PD but require alteration or modulation of environmental cues to suppress astrogenesis in the striatum and enhance or direct neurogenesis or DA-ergic differentiation to replace the degenerating neurons in the SN following PD. Acknowledgments—This study was supported by the Health Research Council of New Zealand. P. Aponso was supported by a Neurological Foundation of New Zealand W. B. Miller Scholarship. The authors wish to thank Jacqui Ross, BIRU of University of Auckland and Dinusha Bandara, Institute of Environmental Science & Research Ltd. for their technical assistance with this research.
REFERENCES Arvidsson A, Kokaia Z, Lindvall O (2001) N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke. Eur J Neurosci 14:10 –18. Baker SA, Baker KA, Hagg T (2004) Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone. Eur J Neurosci 20:575–579. Borta A, Hoglinger GU (2007) Dopamine and adult neurogenesis. J Neurochem 100:587–595. Bowenkamp KE, David D, Lapchak PL, Henry MA, Granholm AC, Hoffer BJ, Mahalik TJ (1996) 6-Hydroxydopamine induced the loss of the dopaminergic phenotype in substantia nigra neurons of the rat: a possible mechanism for restoration of the nigrostriatal circuit mediated by glial cell line-derived neurotrophic factor. Exp Brain Res 111:1–7.
1152
P. M. Aponso et al. / Neuroscience 151 (2008) 1142–1153
Bowenkamp KE, Hoffman AF, Gerhardt GA, Henry MA, Biddle PT, Hoffer BJ, Granholm AE (1995) Glial cell line-derived neurotrophic factor supports survival of injured midbrain dopaminergic neurons. J Comp Neurol 355:479 – 489. Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:406 – 417. Choi-Lundberg DL, Lin Q, Chang Y-N, Chiang YL, Hay CM, Mohajeri H, Davidson BL, Bohn MC (1997) Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 275:838 – 841. Chouaf-Lakhdar L, Fevre-Montange M, Brisson C, Strazielle N, Gamrani H, Didier-Bazes M (2003) Proliferative activity and nestin expression in periventricular cells of the adult rat brain. Neuroreport 14:633– 636. Connor B, Kozlowski DA, Schallert T, Tillerson JL, Davidson BL, Bohn MC (1999) Differential effects of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the aged parkinsonian rat. Gene Ther 6:1936 –1951. Connor B, Kozlowski DA, Unnerstall JR, Elsworth JD, Tillerson JL, Schallert T, Bohn MC (2001) Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration. Exp Neurol 169:83–95. Cooper O, Isacson O (2004) Intrastriatal transforming growth factor a delivery to a model of Parkinson’s disease induces proliferation and migration of endogenous adult neural progenitor cells without differentiation into dopaminergic neurons. J Neurosci 24:8924– 8931. Curtis MA, Penney EB, Pearson AG, van Roon-Mom WM, Butterworth NJ, Dragunow M, Connor B, Faull RL (2003) Increased cell proliferation and neurogenesis in the adult human Huntington’s disease brain. Proc Natl Acad Sci U S A 100:9023–9027. Curtis MA, Penney EB, Pearson J, Dragunow M, Connor B, Faull RL (2005) The distribution of progenitor cells in the subependymal layer of the lateral ventricle in the normal and Huntington’s disease human brain. Neuroscience 132:777–788. Fallon J, Reid S, Kinyamu R, Opole I, Opole R, Baratta J, Korc M, Endo TL, Duong A, Nguyen G, Karkehabadhi M, Twardzik D, Loughlin S (2000) In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc Natl Acad Sci U S A 97:14686 –14691. Ferland RJ, Gross RA, Applegate CD (2002) Increased mitotic activity in the dentate gyrus of the hippocampus of adult C57BL/6J mice exposed to the flurothyl kindling model of epileptogenesis. Neuroscience 115:669 – 683. Frielingsdorf H, Schwarz K, Brundin P, Mohapel P (2004) No evidence for new dopaminergic neurons in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A 101:10177–10182. Henry RA, Hughes SM, Connor B (2007) AAV-mediated delivery of BDNF augments neurogenesis in the normal and quinolinic acidlesioned adult rat brain. Eur J Neurosci 25:3513–3525. Hermann A, Maisel M, Wegner F, Liebau S, Kim DW, Gerlach M, Schwarz J, Kim KS, Storch A (2006) Multipotent neural stem cells from the adult tegmentum with dopaminergic potential develop essential properties of functional neurons. Stem Cells 24:949 –964. Hoglinger GU, Rizk P, Muriel MP, Duyckaerts C, Oertel WH, Caille I, Hirsch EC (2004) Dopamine depletion impairs precursor cell proliferation in Parkinson disease. Nature Neurosci 7:726 –735. Jeon BS, Jackson-Lewis V, Burke RE (1995) 6-Hydroxydopamine lesion of the rat substantia nigra: time course and morphology of cell death. Neurodegeneration 4:131–137. Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC, Greenberg DA (2004) Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci U S A 101:343–347. Kay JN, Blum M (2000) Differential response of ventral midbrain and striatal progenitor cells to lesions of the nigrostriatal dopaminergic projection. Dev Neurosci 22:56 – 67. Kirik D, Rosenblad C, Bjorklund A (1998) Characterization of behavioral and neurodegenerative changes following partial lesions
of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat. Exp Neurol 152:259 –277. Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16:2027–2033. Lee CS, Sauer H, Bjorklund A (1996) Dopaminergic neuronal degeneration and motor impairments following axon terminal lesion by instrastriatal 6-hydroxydopamine in the rat. Neuroscience 72: 641– 653. Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage FH (2002) The adult substantia nigra contains progenitor cells with neurogenic potential. J Neurosci 22:6639 – 6649. Liu J, Solway K, Messing RO, Sharp FR (1998) Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J Neurosci 18:7768 –7778. Mao L, Lau YS, Petroske E, Wang JQ (2001) Profound astrogenesis in the striatum of adult mice following nigrostriatal dopaminergic lesion by repeated MPTP administration. Brain Res Dev Brain Res 131:57– 65. Mohapel P, Frielingsdorf H, Haggblad J, Zachrisson O, Brundin P (2005) Platelet-derived growth factor (PDGF-BB) and brain derived neurotrophic factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions. Neuroscience 132:767–776. Nait-Oumesmar B, Decker L, Lachapelle F, Avellana-Adalid V, Bachelin C, Van Evercooren AB (1999) Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination. Eur J Neurosci 11:4357– 4366. Nakatomi H, Kuriu T, Okabe S, Yamamoto S, Hatano O, Kawahara N, Tamura A, Kirino T, Nakafuku M (2002) Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110:429 – 441. Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH (1999) Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 19:8487– 8497. Palmer TD, Ray J, Gage FH (1995) FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci 6:474 – 486. Parent JM, Janumpalli S, McNamara JO, Lowenstein DH (1998) Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci Lett 247:9 –12. Parent JM, Valentin VV, Lowenstein DH (2002) Prolonged seizures increase proliferating neuroblasts in the adult rat subventricular zone-olfactory bulb pathway. J Neurosci 22:3174 –3188. Rosenblad C, Kirik D, Devaux B, Moffat B, Phillips HS, Bjorklund A (1999) Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson’s disease after administration into the striatum or the lateral ventricle. Eur J Neurosci 11:1554 –1566. Sauer H, Oertel WH (1994) Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neuroscience 59:401– 415. Scott BW, Wang S, Burnham WM, De Boni U, Wojtowicz JM (1998) Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci Lett 248:73–76. Shan X, Chi L, Bishop M, Luo C, Lien L, Zhang Z, Liu R (2006) Enhanced de novo neurogenesis and dopaminergic neurogenesis in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease-like mice. Stem Cells 24: 1280 –1287. Steiner B, Winter C, Hosman K, Siebert E, Kempermann G, Petrus DS, Kupsch A (2006) Enriched environment induces cellular plasticity in the adult substantia nigra and improves motor behavior function in the 6-OHDA rat model of Parkinson’s disease. Exp Neurol 199:291–300.
P. M. Aponso et al. / Neuroscience 151 (2008) 1142–1153 Stromberg I, Bjorklund H, Dahl D, Jonsson G, Sundstrom E, Olson L (1986) Astrocyte responses to dopaminergic denervations by 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as evidenced by glial fibrillary acidic protein immunohistochemistry. Brain Res Bull 17:225–236. Takagi Y, Nozaki K, Takahashi J, Yodoi J, Ishikawa M, Hashimoto N (1999) Proliferation of neuronal precursor cells in the dentate gyrus is accelerated after transient forebrain ischemia in mice. Brain Res 831:283–287. Tattersfield AS, Croon RJ, Liu YW, Kells AP, Faull RL, Connor B (2004) Neurogenesis in the striatum of the quinolinic acid lesion model of Huntington’s disease. Neuroscience 127:319 –332. Taupin P (2007) BrdU immunohistochemistry for studying adult neurogenesis: Paradigms, pitfalls, limitations, and validation. Brain Res Rev 53:198 –214. Van Kampen JM, Eckman CB (2006) Dopamine D3 receptor agonist delivery to a model of Parkinson’s disease restores the nigrostriatal pathway and improves locomotor behavior. J Neurosci 26: 7272–7280. Van Kampen JM, Hagg T, Robertson HA (2004) Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D receptor stimulation. Eur J Neurosci 19:2377–2387.
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Van Kampen JM, Robertson HA (2005) A possible role for dopamine D3 receptor stimulation in the induction of neurogenesis in the adult rat substantia nigra. Neuroscience 136:381–386. Weinstein DE, Burrola P, Kilpatrick TJ (1996) Increased proliferation of precursor cells in the adult rat brain after targeted lesioning. Brain Res 743:11–16. Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson AC, Reynolds BA (1996) Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16:7599 –7609. Yagita Y, Kitagawa K, Ohtsuki T, Takasawa K, Miyata T, Okano H, Hori M, Matsumoto M (2001) Neurogenesis by progenitor cells in the ischemic adult rat hippocampus. Stroke 32:1890 –1896. Zhao M, Momma S, Delfani K, Carlen M, Cassidy RM, Johansson CB, Brismar H, Shupliakov O, Frisen J, Janson AM (2003) Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A 100:7925–7930. Zuch C, Nordstroem V, Briedrick L, Hoernig G, Granholm A, Bickford P (2000) Time course of degenerative alterations in nigral dopaminergic neurons following a 6-hydroxydopamine lesion. J Comp Neurol 427:440 –545.
(Accepted 28 November 2007) (Available online 4 December 2007)
INCREASED PROGENITOR CELL PROLIFERATION AND ASTROGENESIS IN THE PARTIAL PROGRESSIVE 6-HYDROXYDOPAMINE MODEL OF PARKINSON’S DISEASE P. M. APONSO,a R. L. M. FAULLa AND B. CONNORb*
progenitors undergo robust astrogenesis, newborn midbrain-derived progenitors remain in an undifferentiated state suggesting local environments differentially regulate endogenous progenitor cell populations in PD. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved.
a
Department of Anatomy with Radiology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
b
Department of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
Key words: adult neural progenitor cells, striatum, subventricular zone, substantia nigra, astrogenesis, Parkinson’s disease.
Abstract—The existence of endogenous progenitor cells in the adult mammalian brain presents an exciting and attractive alternative to existing therapeutic options for treating neurodegenerative diseases such as Parkinson’s disease (PD). However, prior to designing endogenous cell therapies, the effect of PD neuropathology on endogenous progenitor cell proliferation and their neurogenic potential must be investigated. This study examined the effect of dopaminergic cell loss on the proliferation and differentiation of subventricular zone- (SVZ) and midbrain-derived progenitor cells in the adult rodent brain, using the partial progressive 6-hydroxydopamine (6-OHDA) lesion model of PD. Cell proliferation and differentiation were assessed with 5-bromo-2=-deoxyuridine (BrdU) labeling and immunohistochemistry for cell type-specific markers. Tyrosine hydroxylase immunohistochemistry demonstrated a complete loss of nigrostriatal projections in the striatum and a subsequent progressive loss of dopamine (DA) cells in the SN. Quantification indicated that 6-OHDA lesion–induced cell degeneration produced a significant increase in BrdU immunoreactivity in the SVZ, ipsilateral to the lesioned hemisphere from 3 to 21 days post-lesion, compared with sham-lesioned animals. Similarly, in the striatum we observed a significant increase in the total number of BrdU positive cells in 6-OHDA-lesioned animals at all time points examined. More importantly, a significant increase in midbrain-derived BrdU positive cells was demonstrated in 6-OHDA-lesioned animals 28 days post-lesion. While we did not detect neurogenesis, BrdU labeled cells co-expressing the astrocytic marker glial fibrillary acidic protein (GFAP) were widely distributed throughout the 6-OHDA-lesioned striatum at all time points. In contrast, BrdU-labeled cells in the SN of 6-OHDA-lesioned animals did not co-express neural markers. These results demonstrate that DA-ergic neurodegeneration in the partial progressive 6-OHDA-lesioned rat brain increases SVZ- and midbrain-derived progenitor cell proliferation. While, newborn striatal
Parkinson’s disease (PD) is a common neurodegenerative disorder typically affecting the older population. It is pathologically hallmarked by the presence of intraneuronal Lewy bodies and the progressive neurodegeneration of the nigrostriatal dopaminergic neurons leading to debilitating motor dysfunction. The cardinal symptoms of idiopathic PD include resting tremor, rigidity and bradykinesia. The current treatment options available for PD primarily consist of pharmacological dopamine (DA) replacement (L-DOPA) and surgical procedures (e.g. deep brain stimulation, pallidotomy, thalamotomy) which partially alleviate motor symptoms. However, complications arising from chronic treatment of L-DOPA and the variable success of surgical procedures have led to the investigation of other novel treatment strategies including cell-based therapeutic options, such as restoring DA-ergic neurotransmission by exogenous cell replacement (e.g. transplantation of fetal mesencephalic tissue). The clinical success of these strategies has been variable with the inconsistent benefits for patients. Further, cell transplantation using aborted fetuses is surrounded by ethical and technical concerns. Therefore, the presence of endogenous progenitor cells in the adult mammalian brain presents as an exciting alternative to cell transplantation for the development of a novel therapeutic option for treating PD. Progenitor cells in the two main adult neurogenic regions, the subventricular zone (SVZ) of the lateral ventricle and the subgranular zone of the dentate gyrus, have been shown to proliferate and differentiate in response to neuronal cell loss in both the animal brain (Kuhn et al., 1996; Weinstein et al., 1996; Liu et al., 1998; Parent et al., 1998; Scott et al., 1998; Nait-Oumesmar et al., 1999; Takagi et al., 1999; Kay and Blum, 2000; Arvidsson et al., 2001; Mao et al., 2001; Yagita et al., 2001; Ferland et al., 2002; Nakatomi et al., 2002; Parent et al., 2002; Tattersfield et al., 2004) and the postmortem human brain (Curtis et al., 2003, 2005; Jin et al., 2004). Further, the presence of progenitor cells in non-neurogenic regions, such as the cortex (Palmer et al., 1999), septum (Palmer et al., 1995),
*Corresponding author. Tel: ⫹64-9-373-7599; fax: ⫹64-9-373-7556. E-mail address: [email protected] (B. Connor). Abbreviations: BDNF, brain-derived neurotrophic factor; BrdU, 5-bromo2=-deoxyuridine; CD68, cluster of differentiation 68; DA, dopamine; DAB, diaminobenzidine; DCx, doublecortin; GFAP, glial fibrillary acid protein; MAP2, microtubule-associated protein; MFB, medial forebrain bundle; NG2, chondroitin sulfate proteoglycan; PBS, phosphate-buffered saline; PD, Parkinson’s disease; PDGF-BB, platelet-derived growth factor; QA, quinolinic acid; SN, substantia nigra; SVZ, subventricular zone; TGF-␣, tumor growth factor-␣; TH, tyrosine hydroxylase; 6-OHDA, 6-hydroxydopamine.
0306-4522/08$32.00⫹0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.11.036
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spinal cord (Weiss et al., 1996), ventricular extension (Chouaf-Lakhdar et al., 2003) and the substantia nigra (SN) (Lie et al., 2002; Zhao et al., 2003), has also been demonstrated, but at a less appreciable level compared with the established neurogenic regions (Zhao et al., 2003). Currently, the neurogenic potential of the midbrain region is a highly debated topic. DA is reported to play a role in the regulation of both embryonic and adult neurogenesis (Borta and Hoglinger, 2007). Previous studies have demonstrated that a complete 6-hydroxydopamine (6-OHDA) lesion in the rat (Stromberg et al., 1986; Lie et al., 2002) or MPTP lesions in mice (Stromberg et al., 1986; Kay and Blum, 2000; Mao et al., 2001) results in increased cell proliferation in the striatum and the SN with no DA-ergic differentiation (Frielingsdorf et al., 2004). However, Zhao et al. (2003) postulated that DA-ergic differentiation occurs at a very low level in the SN of healthy mice and it is increased after a MPTP lesion. Transplantation of freshly isolated adult SN progenitor cells into the adult hippocampus showed that these cells have neuronal potential and can give rise to new neurons when exposed to the correct environment (Lie et al., 2002). Basal levels of neurogenesis, increased proliferation and a very low level of DAergic differentiation following MPTP treatment of LacZ transgenic mice were also demonstrated more recently (Shan et al., 2006). In contrast, experimental depletion of DA in rodents following complete 6-OHDA or MPTP lesioning has been shown to decrease progenitor cell proliferation in both the SVZ and SGZ (Baker et al., 2004; Hoglinger et al., 2004). The response of adult SVZ and, in particular, SN progenitor cells to DA depletion therefore requires further investigation. The controversy may be due to methodological differences between the reported studies. Furthermore, the previously reported studies have used standard 6-OHDA or MPTP rodent models of PD which have several disadvantages when examining interventions aimed at protecting or restoring the nigrostriatal system. In the standard 6-OHDA model of PD, 6-OHDA is injected to the medial forebrain bundle (MFB) or the SN which induces a near complete destruction of DA-ergic neurons (Jeon et al., 1995; Zuch et al., 2000). While identifiable nigral cell loss has been observed up to 31 days following complete 6-OHDA lesioning, the sudden and massive toxic insult near the cell bodies results in an acute rather than gradual and progressive loss of nigral cells, with the majority of cell death occurring within 12 h of nigral 6-OHDA injection (Jeon et al., 1995; Zuch et al., 2000). Second, the standard neurotoxic nigrostriatal lesion in rats is commonly used to achieve the maximum possible degeneration. Therefore, it makes it difficult to control when only a partial lesion is desired. In experiments investigating neuro-restoration strategies similar to this study, it is most ideal to use a partial progressive model where the initial lesion results in a partial loss of DA-ergic neurons followed by an ongoing progressive degeneration after injury (Sauer and Oertel, 1994; Kirik et al., 1998). Therefore, we have for the first time investigated the proliferation and differentiation poten-
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tial of endogenous SVZ-derived and midbrain progenitor cells by employing the partial progressive 6-OHDA lesion rat model. In contrast to the standard 6-OHDA models, the partial progressive 6-OHDA lesion model of PD allows for a retrograde degeneration of the nigrostriatal pathway, by first acting on the DA axon terminals within the striatum and exerting its degenerative effects along the axons toward the cell bodies within the SN (Sauer and Oertel, 1994). We report that a single intrastriatal injection of 6-OHDA induces a near complete loss of striatal DA and a subsequent progressive loss of DA-ergic neurons in the SN, which leads to a significant increase in progenitor cell proliferation in the SVZ, striatum and the midbrain. The newborn cells in the striatum readily differentiated into astrocytes and failed to acquire a neuronal phenotype. The midbrain-derived progenitor cells in contrast, did not acquire either a neuronal or a glial phenotype and appeared to remain in an undifferentiated state. The results of this study further suggest that endogenous progenitor cell populations in both neurogenic and non-neurogenic regions have the capacity to proliferate in response to DA-ergic neurodegeneration but may require region-specific environmental cues to undergo neurogenesis and DA-ergic differentiation.
EXPERIMENTAL PROCEDURES Surgical procedures Adult male Wistar rats weighing 240 –290 g (University of Auckland Animal Resources Unit, Auckland, New Zealand) were used in this study. The animals were housed in groups of three in a temperature- and humidity-controlled room that was kept on a 12-h light/dark cycle. Food and water were available ad libitum throughout the study. Experimentation was performed in strict compliance with the University of Auckland Animal Ethics Guidelines in accordance with the New Zealand Animal Welfare Act 1999 and conformed to named international guidelines on the ethical use of animals. All efforts were made to minimize the number of animals used and to minimize their suffering. All surgeries were performed following an i.p. injection of 60 mg sodium pentobarbital per kilogram of body weight. Rats received a unilateral intrastriatal infusion of either 6-OHDA (2.8 l) or vehicle solution (phosphate-buffered saline (PBS)) over a 5 min period at the following coordinates: 1.0 mm anterior–posterior, 2.8 mm medial–lateral and ⫺5.0 mm dorsal–ventral.
5-Bromo-2=-deoxyuridine (BrdU) labeling and tissue processing Following surgery, each animal received four BrdU (200 mg/kg i.p.) injections, over a period of 6 h (2 h interval between injections) on day 3, 7, 14, 21 or 28 after injection of 6-OHDA or PBS (n⫽3–5 per time point for each group) and were killed 2 h after the last BrdU injection. A concentration of 200 mg/kg BrdU per injection was chosen for this study as this concentration of BrdU has been demonstrated to produce optimal labeling of cells undergoing proliferation with little detection of cells exhibiting DNA repair (Cameron and McKay, 2001). Rats were killed by an overdose of sodium pentobarbital and perfused transcardially with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed, post-fixed in 4% paraformaldehyde overnight and cryoprotected for sectioning in a 30% sucrose solution. Coronal sections were cut through the striatum from frozen brains using a sliding microtome set at 40 m. Eight sets of
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sections were collected from each brain (distance of 320 m between consecutive sections in each set) and stored in a cryoprotective solution at ⫺20 °C.
Immunohistochemistry and confocal microscopy Diaminobenzidine (DAB) peroxidase immunohistochemistry was performed on a complete set of free-floating coronal sections from each animal for tyrosine hydroxylase (TH) (mouse anti-TH; 1:1000; Chemicon, Temecula, CA, USA) immunoreactivity in order to confirm the selective neuronal loss induced by the 6-OHDA lesion. The presence of migrating neuroblasts was identified using an antibody to doublecortin (DCx; goat anti-DCx; 1:500; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). To label proliferating cells, we used primary antibodies that recognize BrdU (mouse anti-BrdU, 1:250; Chemicon; rat anti-BrdU, 1:250; Accurate Chemicals, Westbury, NY, USA); both BrdU antibodies showed identical patterns of immunolabeling. For BrdU immunolabeling, standard BrdU immunohistochemical protocol was used (Tattersfield et al., 2004). Following incubation with the primary antibody, sections were incubated with the appropriate secondary biotinylated antibody (1:500; goat anti-mouse; Sigma-Aldrich, St. Louis, MO, USA; goat anti-rat, Sigma-Aldrich; donkey anti-goat; Jackson Immunoresearch Laboratories, West Grove, PA, USA) followed by incubation in ExtrAvidin peroxidase (1:1000; SigmaAldrich). Antibodies were visualized using 0.4 mg/ml DAB, 25 mg/ml nickel sulfate, 0.005% hydrogen peroxide in 0.2 M phosphate buffer. Brightfield images were taken using a digital camera on a light microscope. All the figures were compiled using Adobe Illustrator (Adobe Systems Inc., San Jose, CA, USA). Double-label immunofluorescence using rat anti-BrdU and a second primary antibody was performed as previously described (Tattersfield et al., 2004). Sections were serially incubated with the rat anti-BrdU antibody in conjunction with either one of the following mouse or rabbit second primary antibody; chondroitin sulfate proteoglycan (1:200; NG2; rabbit anti-NG2; Chemicon), microtubule-associated protein (1:200; MAP2; mouse anti-MAP2; Chemicon), cluster of differentiation 68 (1:200; CD68; mouse anti-CD68; Chemicon) and glial fibrillary acid protein (1:400; GFAP; mouse anti-GFAP; Dako, Glostrup, Denmark). Following incubation in the primary antibodies, the sections were incubated in pooled solutions of specific secondary fluorochromes (1:200; anti-rat Alexa Fluor 594; anti-mouse Alexa Fluor 488; anti-rabbit Alexa Fluor 488; Molecular Probes, Eugene, OR, USA) for 12 h at room temperature. Fluorescently labeled sections were imaged using a confocal laser-scanning microscope (Leica TCS SP2) equipped with UV, argon, argon/ krypton and helium/neon lasers (Biomedical Imaging Resource Unit, University of Auckland). Each fluorescent label was imaged serially to eliminate detection of bleed through and other artifactual fluorescence. The confocal images were captured in a z-series with an interslice gap of 1 m.
Quantification and statistical analysis Stereological counting of TH and BrdU positive cells. Non-biased stereological cell counts were carried out on every 8th section throughout the rostrocaudal extent of the brain using a digital camera (MicroFire 1.0) mounted on an upright microscope (Nikon ECLIPSE E800) equipped with a motorized stage driven in the X, Y and Z planes. TH positive cells in the SN of control and 6-OHDA-lesioned animals were counted at 20⫻ magnification using the optical fractionator stereological method (Stereo Investigator software Version 6, MicroBrightField Inc. USA) where 60 counting sites were examined (frame size: 75/75 m; spacing between frames: 250/250 m) (Connor et al., 1999) per section. TH positive cell counts in the control and 6-OHDA-lesioned animals were expressed as the total number of cells within the entire SN. BrdU positive cells in the striatum and the midbrain (SN and
PaG surrounding the cerebral aqueduct) of control and 6-OHDAlesioned animals were carried out exhaustively through every 8th section at 40⫻ magnification in order to estimate the total number of cells in each structure. Quantification of striatal denervation. The extent of the striatal denervation (lesion size) was evaluated on every 8th section throughout the rostrocaudal extent of the striatum. Both the nonlesioned and lesioned striatum was photographed at 2.5⫻ magnification using a digital camera mounted on a conventional transmitted light microscope. A standard level of grey-scale intensity was set to represent normal levels of TH immunoreactivity and from this level the area of reduced TH immunoreactivity was determined and quantified using ImageJ software (National Institutes of Health, USA). Quantification of BrdU positive cells in the SVZ. Since, BrdU positive cells in the SVZ occurred predominantly in clusters they could not be easily recognized as individual cells unless at very high magnification. Based on the fact that BrdU cells were unevenly distributed along the SVZ, it would be inappropriate or inaccurate to select a single or representative sample size in order to estimate the entire population. A quantification method was therefore employed to determine the total area of BrdU immunoreactivity in the SVZ lining the lateral ventricle adjacent to the striatum (Tattersfield et al., 2004; Henry et al., 2007). In order to clearly visualize BrdU positive cells in the SVZ high magnification images (40⫻) of the SVZ were acquired using an inverted microscope (Nikon TE2000E) and then automatically put together using automated montaging software (NIS Elements) to represent the entire length of the SVZ. The total area of BrdU positive cells was measured using the ImageJ 1.34s (National Institutes of Health) software. Area increase of BrdU positive cells in the SVZ in the ipsilateral, lesioned hemisphere was expressed as a percentage of the contralateral, non-lesioned hemisphere.
Statistical analysis All statistical analysis was performed using the Statistical Analysis Software (SAS) system version 9.1. All values and graphs are presented as mean⫾S.E.M. BrdU and TH immunoreactive cell counts from each brain region were analyzed using one-way ANOVA comparing control and 6-OHDA-lesioned groups. The striatal lesion area of 6-OHDA-lesioned animals was analyzed using one-way ANOVA comparing all time points combined. Results were considered statistically significant when the P-value was less than or equal to 0.05.
RESULTS Reduced TH immunolabeling in the striatum and SN following partial progressive 6-OHDA lesioning The extent of DA-ergic lesioning was determined from a set of coronal striatal and nigral sections stained for TH immunohistochemistry. A single unilateral injection of 6-OHDA into the striatum resulted in an almost complete loss of TH immunoreactivity in the striatum [Fig. 1A–B] and a subsequent loss of TH immunoreactivity in the SN [Fig. 1D–E] as previously described (Sauer and Oertel, 1994). Loss of TH-immunoreactivity does not necessarily indicate neuronal cell loss as it is well established that TH expression is down-regulated prior to cell loss (Sauer and Oertel, 1994; Bowenkamp et al., 1995, 1996; Choi-Lundberg et al., 1997; Connor et al., 1999, 2001; Rosenblad et al., 1999). Quantification demonstrated that loss of TH immunoreactivity in the lesioned striatum was completely
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Fig. 1. Reduced TH immunolabeling in the striatum and SN following partial progressive 6-OHDA lesioning. (A, B) Coronal sections of the striatum immunostained for TH in the 6-OHDA-lesioned animal at 28 days post-lesion. Extensive TH immunoreactivity is observed in the non-lesioned hemisphere (A) with complete loss of TH immunoreactivity observed in the 6-OHDA-lesioned hemisphere (B). (C) Graph demonstrating the mean area of reduced TH immunoreactivity (lesion size) in the lesioned hemisphere of 6-OHDA-treated animals. (D, E) Coronal sections of the SN immunostained for TH in the 6-OHDA-lesioned animal at 21 days post-lesion. Extensive TH immunoreactivity is observed in the non-lesioned hemisphere (D) with progressive loss of TH positive DA-ergic cells observed in the 6-OHDA-lesioned hemisphere (E). (F) Graph demonstrating the total number of TH positive DA-ergic cells in the SNpc in the ipsilateral hemisphere of 6-OHDA-lesioned and normal animals.
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formed by 3 days and remained fixed till 28 days postlesion, with no significant difference in lesion size between the time points [Fig. 1C]. In contrast, quantification of TH positive cells in the ipsilateral SN of 6-OHDA-lesioned animals indicated a significant progressive decrease in the total number of TH positive cells from 14 to 28 days postlesion compared with normal animals [Fig. 1F]. The observed temporal profile of TH expression in the striatum and SN following a partial progressive 6-OHDA lesion is in full agreement with previous observations (Sauer and Oertel, 1994). DA denervation in the 6-OHDA-lesioned striatum increases cell proliferation in the SVZ and the striatum We examined the effect of DA-ergic projection loss in the lesioned striatum on SVZ and striatal cell proliferation. Rats received multiple BrdU injections (four injections over 6 h; 200 mg/kg i.p. per injection) on day 3, 7, 14, 21 or 28 after an intrastriatal injection of 6-OHDA or PBS, and were killed 2 h after the last BrdU injection. In the contralateral and sham-lesioned hemispheres, BrdU labeling was confined to the SVZ [Fig. 2A] with very few BrdU positive cells seen in the adjacent striatum [Fig. 2D]. In contrast, in 6-OHDA-lesioned animals BrdU labeling was increased in the SVZ in the lesioned hemisphere [Fig. 2B] and a large number of BrdU positive cells were observed throughout the adjacent striatum [Fig. 2E] mainly accumulated around the lesion tract. BrdU positive cells were distributed singly or in nested clusters throughout the SVZ, while, in the striatum they were individually distinguishable and occurred predominantly as irregular and ellipsoid shaped homogenously stained nuclei. BrdU positive doublets were also observed in both the SVZ and the striatum indicating the occurrence of cell division [Fig. 2E]. Quantification revealed that 6-OHDA-induced DA denervation produced a significant increase in the area of BrdU positive cells in the SVZ from 3 to 21 days post-lesion compared with sham-lesioned animals [Fig. 2C; 3 day 6-OHDA 22⫾6% vs. sham 5⫾2%, Pⱕ0.004; 7 days 6-OHDA 32⫾10% vs. sham 4⫾2%, Pⱕ0.02; 14 days 6-OHDA 26⫾6% vs. sham 4⫾3%, Pⱕ0.006; 21 days 6-OHDA 18⫾4% vs. sham 5⫾2%, Pⱕ0.02]. At 28 days post-lesion, the area of BrdU immunoreactivity in the 6-OHDA lesioned hemisphere was reduced compared with earlier time points [Fig. 2C]. We also observed a small increase in the area of SVZ BrdU immunoreactivity in the ipsilateral hemisphere of sham-lesioned animals compared with the contralateral hemisphere at each time point [Fig. 2C], possibly reflecting a response in cell proliferation to mechanical injury induced by the intrastriatal vehicle injection. Stereological analysis also revealed a significant increase in BrdU positive cells in the striatum of 6-OHDA-lesioned animals from 3 to 28 days post-lesion, compared with shamlesioned animals [Fig. 2F; 3 day 6-OHDA 81392⫾4568 vs. sham 6405⫾2013, Pⱕ0.0005; 7 day 6-OHDA 3771⫾944 vs. sham 840⫾140, Pⱕ0.04; 14 day 6-OHDA 2570⫾653 vs. sham 552⫾169, Pⱕ0.05; 21 days 6-OHDA 1659⫾248 vs.
sham 53⫾19, Pⱕ0.003; 28 days 6-OHDA 520⫾55 vs. sham 48⫾17, Pⱕ0.002]. At 3 days post-lesion, 6-OHDAlesioned animals demonstrated the greatest significant increase in the number of BrdU positive cells which corresponded to a 12.7-fold increase compared with shamlesioned animals [Fig. 2F]. DA-ergic cell loss in the SN increases cell proliferation in the midbrain Multiple injections of BrdU resulted in successful labeling of proliferating cells in the midbrain following a partial progressive 6-OHDA lesion. BrdU positive cells were observed within the PaG [Fig. 3A–G], surrounding the cerebral aqueduct and in the SN [Fig. 3I–J] in both sham- [Fig. 3A] and 6-OHDA-lesioned [Fig. 3E and I–J] animals at all time points examined. In both treatment groups, BrdU positive cells were randomly dispersed within the PaG and SN with no clear pattern of labeling. Quantification indicated no significant difference in the number of BrdU-labeled cells from 3 to 21 days post-lesion in the PaG between sham- and 6-OHDAlesioned animals [Fig. 3H; 3 days 6-OHDA 904⫾536 vs. sham 483⫾101, Pⱕ0.4; 7 days 6-OHDA 411⫾183 vs. sham 388⫾20, Pⱕ1.0; 14 days 6-OHDA 570⫾129 vs. sham 387⫾154, Pⱕ0.4; 21 days 6-OHDA 506⫾53 vs. sham 280⫾134, Pⱕ0.2]. Similarly, no significant difference in the number of BrdU-labeled cells was observed in the SN between sham- and 6-OHDA-lesioned animals from 3 to 21 days post-lesion [Fig. 3K; 3 days 6-OHDA 4924⫾100 vs. sham 3493⫾415, Pⱕ0.2; 7 days 1029⫾292 vs. 624⫾ 0, Pⱕ0.6; 14 days 6-OHDA 910⫾142 vs. sham 928⫾100, Pⱕ0.2; 21 days 6-OHDA 584⫾103 vs. sham 977⫾288, Pⱕ0.9). However, we did observe a significant increase in the number of BrdU positive cells at 28 days post-lesion in both the PaG [Fig. 3H; 28 days 6-OHDA 264⫾52 vs. sham 51⫾31, Pⱕ0.03] and the SN [Fig. 3K; 28 days 6-OHDA 451⫾67 vs. sham 155⫾34, Pⱕ0.01] of 6-OHDA-lesioned animals compared with controls. This represents the first demonstration of progenitor cell proliferation in the adult rodent midbrain following a partial progressive 6-OHDA lesion of the nigrostriatal pathway. DA denervation induces striatal astrogenesis We next examined whether the newborn proliferating cells in the SVZ and the striatum resulted in the generation of new neurons and/or glial cells in the lesioned striatum. We first examined the expression of DCx (a microtubule-associated protein present in migrating neuronal precursors) to determine whether the increase of newborn cells in the SVZ is forming migrating neuroblasts which migrate toward the adjacent striatal lesion. No change in DCx positive labeling in the SVZ/RMS was noted between shamand 6-OHDA-lesioned animals [data not shown]. We also observed no DCx positive labeling in the ipsilateral and contralateral striatum of 6-OHDA-lesioned animals indicating that SVZ-derived progenitor cells are not migrating into the lesioned striatum as observed in other models of neuronal injury (Arvidsson et al., 2001; Tattersfield et al., 2004). Therefore, it is likely the increase in proliferating cells seen in the striatum is a result of locally derived
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Fig. 2. DA denervation in the 6-OHDA-lesioned striatum increases cell proliferation in the SVZ and the striatum. (A, B) Coronal sections of the SVZ adjacent to the striatum immunostained for BrdU in a 6-OHDA-lesioned animal at the 7-day time point. BrdU immunoreactivity was confined to the SVZ of the non-lesioned hemisphere (A) with increased BrdU immunoreactivity observed in the SVZ of the lesioned hemisphere (B). (C) Graph demonstrating the percentage mean area of BrdU immunoreactivity in the ipsilateral SVZ of sham- and 6-OHDA-lesioned animals. (D, E) Coronal sections of the striatum immunostained for BrdU in a 6-OHDA-lesioned animal at the 3-day time point. In the non-lesioned hemisphere, BrdU immunoreactivity was largely confined to the SVZ with a few BrdU positive cells observed in the striatum (D). Extensive labeling of BrdU positive cells was observed within the ipsilateral striatum of 6-OHDA-lesioned animals (E). (F) Graph demonstrating the total number of BrdU positive cells in the striatum of sham- and 6-OHDA-lesioned animals.
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Fig. 3. DA-ergic cell loss in the SN, increased cell proliferation in the midbrain. (A–K) Coronal sections of BrdU immunoreactivity in the PaG of a SHAM- (A) and 6-OHDA-treated (E) animal at 28 days post-lesion. (H) Total number of BrdU positive cells within the PaG. Quantification revealed a significant increase in the total number of BrdU positive cells in the PaG in 6-OHDA-treated animals was observed at 28 days post-lesion compared with SHAM controls. (I, J) Coronal section of BrdU immunoreactivity in the SN of a 6-OHDA-treated animal at 28 days post-lesion. (K) Total increased number of BrdU positive cells in the SNpc. Quantification revealed a significant increase in the total number of BrdU positive cells in the SNpc in the 6-OHDA-treated animals was observed at 28 days post-lesioned compared with SHAM controls.
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progenitors reacting to the lesion. Next, the phenotype of these newborn striatal progenitors was investigated using double immunofluorescent labeling using cell-type markers for neurons (MAP2, TH), astrocytes (GFAP), microglia (CD68) and oligodendrocyte precursors (NG2). BrdU positive cells in the striatum did not co-localize with any of the neuronal, microglial or oligodendrocyte precursor markers examined in any of the time points investigated [data not shown]. However, they readily co-localized with GFAP in all the time points examined demonstrating robust astrogenesis in the striatum [Fig. 4A–B]. High power inspection of the sections revealed that typically, GFAP positive newborn cells had triangular or irregular cell bodies with long radial processes [Fig. 4C–E]. A population of BrdU positive/GFAP negative cells was also present in the striatum [Fig. 4A–B]. Midbrain progenitor cells remain undifferentiated in the partial progressive 6-OHDA lesion Our results demonstrate for the first time an increase of midbrain progenitor cells in the partial progressive 6-OHDA lesion rat model of PD. This increase was observed both in the SVZ surrounding the cerebral aqueduct as well as in the SN, suggesting midbrain progenitor cells respond to progressive DA-ergic cell loss in the SN. Therefore, the differentiation potential of these newborn midbrain progenitor cells was also investigated using a range of
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neural cell markers. Since, a significant increase of BrdU cells was observed in both midbrain regions at 28 days post-lesion, the possibility of neuroblast migration from the PaG to the SN was investigated with DCx. We did not observe any DCx positive labeling in the PaG or the SN in sham or 6-OHDA-lesioned animals [data not shown] suggesting that these are locally derived progenitors. Next, the neurogenic and/or gliogenic potential of these midbrain progenitor cells was investigated using the same cell-type markers as previously described. We were unable to detect co-localization of BrdU positive cells in the PaG and the SN with any of the neuronal or glial cell markers examined indicating that they do not readily form neurons or glial cells in the midbrain [data not shown].
DISCUSSION The neurogenic potential of the midbrain region is a highly debated topic. Progenitor cell proliferation and differentiation in rodent animal models of PD has previously been investigated using either a systemic application of MPTP or an intracerebral application of 6-OHDA into either the MFB or the SN (Stromberg et al., 1986; Kay and Blum, 2000; Mao et al., 2001; Lie et al., 2002; Zhao et al., 2003). Due to the acute and near complete destruction of DAergic neurons, these models have several disadvantages when examining interventions aimed at protecting or re-
Fig. 4. BrdU labeled cells in the 6-OHDA-lesioned striatum co-express GFAP at all time points examined. (A, B) BrdU (red) and GFAP (green) double-immunolabeled cells (closed arrows) observed in the 6-OHDA-lesioned striatum at 3 days (A) and 28 days (B) post-lesion. BrdU negative/ GFAP positive (open arrows) cells were also observed. (C, D) Boxed areas in (A) and (B) indicate high magnification confocal images of BrdU-labeled cells (red) co-expressing GFAP (green).
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storing the nigrostriatal system. We therefore examined, for the first time, the neurogenic response of endogenous progenitor cells in the partial progressive 6-OHDA rat lesion model. This model results in a more delayed and partial DA-ergic cell loss than observed following standard 6-OHDA lesions due to retrograde degeneration of the nigrostriatal pathway, and we believe may provide an increased window of opportunity for a regenerative response (Sauer and Oertel, 1994; Lee et al., 1996; Kirik et al., 1998). In agreement with previous studies (Stromberg et al., 1986; Kay and Blum, 2000; Mao et al., 2001) we observed increased progenitor cell proliferation in the striatum of 6-OHDA-lesioned animals. The increase in cell proliferation was observed at all time points in the striatum with a decreasing gradient. This closely corresponded with the immediate complete loss of striatal DA and the secondary DA-ergic cell loss in the SN. First, the striatal DA is completely degenerated as early as 3 days post-lesion and remains fixed and complete till 28 days post-lesion. Therefore, the massive induction of proliferating cells seen in the striatum at 3 days post-lesion is a likely representation of initial reactive astrogliosis which is typically observed in a neurotoxic lesion of the DA-ergic system (Stromberg et al., 1986). The decreasing gradient of striatal progenitors from 3 to 28 days post-lesion indicates that the proliferating cells in the striatum are largely part of the injury-induced glial reaction in the striatum. Previous studies (Kay and Blum, 2000; Mao et al., 2001) using various animal models of PD have suggested that one of the noticeable responses to lesions of the nigrostriatal DA-ergic projection is reactive astrogliosis. Further confirming this, we found that the newborn BrdU positive cells in the striatum predominantly co-expressed the astrocytic marker GFAP and did not co-localize with neuronal markers. Experimental depletion of DA in rodents following complete 6-OHDA or MPTP lesioning has been shown to decrease progenitor cell proliferation in both the SVZ and SGZ (Baker et al., 2004; Hoglinger et al., 2004) with proliferation restored following delivery of a selective agonist of DA D2-like receptors (Hoglinger et al., 2004). Stimulation of the DA D3 receptor has been shown to increase SVZ and nigral cell proliferation (Van Kampen et al., 2004) and has also been shown to trigger neurogenesis in the SVZ and the SN of normal (Van Kampen et al., 2004; Van Kampen and Robertson, 2005) and 6-OHDA-lesioned rats (Van Kampen and Eckman, 2006). In contrast to these observations, we observed an increase in cell proliferation from 3 to 21 days post-lesion in the SVZ following a partial progressive 6-OHDA lesion of the nigrostriatal system. It is currently not clear how differences in the partial progressive lesion model may lead to stimulation of SVZ progenitor cell proliferation but this may reflect differences in the degenerative profile and expression of neurogenic cues between the partial and complete lesion models. Previous studies examining stroke- (Arvidsson et al., 2001) or quinolinic acid– (QA) induced (Tattersfield et al., 2004) striatal cell loss suggest the generation and migration of newly generated neurons into the damaged striatum from the SVZ/RMS. In contrast, we did not observe signif-
icant Dcx positive labeling in the partial 6-OHDA-lesioned striatum demonstrating a lack of progenitor cell migration from the SVZ to the damaged striatum. This observation may predominantly reflect a difference in the lesion models. Both the stroke and QA lesion models result in the degeneration of cell bodies predominantly within the striatum, which may produce environmental cues required to trigger neuronal migration and differentiation within the striatum. The partial progressive 6-OHDA lesion model in contrast exerts a retrograde type cell loss where predominant cell degeneration takes place within the SN away from the striatum and therefore, may provide a restrictive atmosphere with a lack of necessary environmental triggers to induce progenitor cell migration and neuronal differentiation in the lesioned striatum. Recent evidence suggests that the infusion of certain growth factors into forebrain structures can induce in vivo stimulation of endogenous progenitor cells in animal models of PD. However, studies examining the infusion of tumor growth factor-␣ (TGF-␣) into the striatum of rats with unilateral 6-OHDA lesions have provided differing results. Fallon et al. (2000) reported that TGF-␣ infusion into 6-OHDA-lesioned rats resulted in migration of progenitor cells into the striatum with an improvement in motor deficits, which was attributed to the appearance of a small number of new striatal neurons that expressed a DA-ergic phenotype, a cell type that is normally not observed in the striatum (Fallon et al., 2000). In contrast, while Cooper and Isacson (2004) also observed significant proliferation and migration of progenitor cells from the SVZ to the striatum in 6-OHDA-lesioned rats following intrastriatal infusion of TGF-␣, they did not observe neuronal differentiation of progenitor cells in either the striatum or the SN. Mohapel et al. (2005) investigated the effects of i.c.v.-infused plateletderived growth factor (PDGF-BB) and brain-derived neurotrophic factor (BDNF) on cell genesis, following BrdU incorporation in adult rats with unilateral 6-OHDA lesions. They found that both PDGF-BB and BDNF increased the number of newly formed cells in the striatum and SN to an equal extent following 10 days of treatment. At 3 weeks, after termination of growth factor treatment, co-localization of BrdU positive cells with the neuronal marker NeuN, revealed a significant increase in newly generated neurons in the striatum. In correspondence, many DCx-positive neuroblasts were also observed in the denervated striatum following growth factor infusion. Further, a subset of these new neurons expressed the early marker for striatal neurons Pbx, suggesting that administration of these growth factors is capable of recruiting new neurons into the striatum of hemiparkinsonian rats (Mohapel et al., 2005). Therefore, we propose that following a partial progressive 6-OHDA lesion in the rat striatum there is robust cell proliferation in the SVZ and the striatum in response to the ongoing degeneration of the nigrostriatal pathway generated in this model. However, the newborn striatal cells are only capable of extensive astrogenesis and not neurogenesis possibly due to the lack of stimulatory cues and restrictive microenvironment in the 6-OHDA-lesioned striatum.
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The most significant finding to come out of this study is the demonstration of significantly increased midbrain-derived progenitor cells following the progressive DA-ergic denervation of the adult rodent SN. While the presence of proliferating progenitor cells in non-neurogenic regions has been previously demonstrated (Weiss et al., 1996; ChouafLakhdar et al., 2003), no study to date has examined the changing level of proliferating progenitor cells in the midbrain following a partial progressive 6-OHDA lesion. We demonstrate a significant increase in BrdU positive cells in the 6-OHDA-lesioned animals at 28 days post-lesion in both the PaG and the SN of the rodent midbrain. Interestingly, the degeneration profile of the partial progressive 6-OHDA lesion model indicates a significant decrease in the number of TH positive DA cells by 28 days post-lesion. Therefore, we initially hypothesized that progenitor cells located adjacent to the cerebral aqueduct may be responding to the degenerating process taking place in the adjacent SN by increased proliferation followed by potential migration to the site of degeneration. However, this was not found to be the case, as we were unable to detect the presence of newborn migrating neural precursors either adjacent to the cerebral aqueduct or in the SN in any of the time points examined. It was also interesting to note the presence of a large number of BrdU-labeled cells in the SN of both sham- and 6-OHDA-lesioned animals 3 days postlesion. We do not believe this reflects a response to mechanical damage as the lesion took place within the striatum, which is located more rostrally from the SN. However, we cannot rule out the possibility that BrdU-labeled cells in the midbrain may represent degenerating DA-ergic neurons that have lost phenotype (Taupin, 2007). Initially, we proposed this increase of BrdU-labeled cells may be an acute inflammatory response to the nigrostriatal denervation as demonstrated in previous studies (Kay and Blum, 2000; Mao et al., 2001). However, double immunolabeling was unable to demonstrate co-expression of midbrainderived BrdU positive cells with either microglial or astrocytic markers at any of the time points examined. The concept of cell differentiation and neurogenesis in the midbrain region still remains a controversial topic as previous studies have indicated contrasting findings. On the one hand, we and several other laboratories have demonstrated that 6-OHDA lesioning in rats or MPTP lesioning in mice results in cell proliferation in the SN without apparent DA-ergic differentiation (Kay and Blum, 2000; Mao et al., 2001; Lie et al., 2002; Steiner et al., 2006). On the other hand, DA-ergic differentiation has been shown to occur at a very low level in the SN of normal mice, and at an increased level after MPTP lesioning by Zhao et al. (2003). Further, basal levels of neurogenesis, increased proliferation and DA-ergic differentiation following MPTP have recently been shown in nestin-LacZ transgenic mice (Shan et al., 2006). In addition, functional DA-ergic differentiation has been demonstrated in vitro from neural progenitor cells isolated from the tegmental tissue of the midbrain and hindbrain, including the ependymal zone of the cerebral aqueduct and the fourth ventricle (Hermann et al., 2006). More interestingly, it has been shown that when
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midbrain-derived progenitor cells were isolated and implanted in a neurogenic environment, such as the dentate gyrus, a proportion of the transplanted cells differentiated into mature neurons (Lie et al., 2002). However, transplantation of midbrain precursor cells into SN of normal rats resulted only in the generation of glial cells, suggesting that local environmental factors may prevent the differentiation of precursor cells to distinct neuronal lineages in situ (Lie et al., 2002). Despite these contrasting findings from previous studies, we could not find any evidence of midbrain-derived proliferating cells co-expressing any of the neural markers examined, suggesting that they do not readily form neurons in the midbrain. Although, methodological differences between studies may also have contributed toward the disparity of the findings obtained with regard to midbrain progenitor cell differentiation, further detailed investigation of midbrain progenitor cell differentiation and neurogenesis remains a necessity.
CONCLUSION Our findings provide the first quantitative demonstration of progenitor cell proliferation in the SVZ, striatum and the midbrain, following the partial progressive 6-OHDA lesion model of PD. In particular, we observed a significant increase of proliferating cells in the SVZ and the striatum which led to robust astrogenesis. More importantly, a significant increase of midbrain-derived proliferating cells was also demonstrated in the 6-OHDA-lesioned animals, however these did not differentiate into either new neurons or glia. We demonstrate that the endogenous neural progenitor cells in the striatum and the SN can provide potential repair for PD but require alteration or modulation of environmental cues to suppress astrogenesis in the striatum and enhance or direct neurogenesis or DA-ergic differentiation to replace the degenerating neurons in the SN following PD. Acknowledgments—This study was supported by the Health Research Council of New Zealand. P. Aponso was supported by a Neurological Foundation of New Zealand W. B. Miller Scholarship. The authors wish to thank Jacqui Ross, BIRU of University of Auckland and Dinusha Bandara, Institute of Environmental Science & Research Ltd. for their technical assistance with this research.
REFERENCES Arvidsson A, Kokaia Z, Lindvall O (2001) N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke. Eur J Neurosci 14:10 –18. Baker SA, Baker KA, Hagg T (2004) Dopaminergic nigrostriatal projections regulate neural precursor proliferation in the adult mouse subventricular zone. Eur J Neurosci 20:575–579. Borta A, Hoglinger GU (2007) Dopamine and adult neurogenesis. J Neurochem 100:587–595. Bowenkamp KE, David D, Lapchak PL, Henry MA, Granholm AC, Hoffer BJ, Mahalik TJ (1996) 6-Hydroxydopamine induced the loss of the dopaminergic phenotype in substantia nigra neurons of the rat: a possible mechanism for restoration of the nigrostriatal circuit mediated by glial cell line-derived neurotrophic factor. Exp Brain Res 111:1–7.
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Bowenkamp KE, Hoffman AF, Gerhardt GA, Henry MA, Biddle PT, Hoffer BJ, Granholm AE (1995) Glial cell line-derived neurotrophic factor supports survival of injured midbrain dopaminergic neurons. J Comp Neurol 355:479 – 489. Cameron HA, McKay RD (2001) Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:406 – 417. Choi-Lundberg DL, Lin Q, Chang Y-N, Chiang YL, Hay CM, Mohajeri H, Davidson BL, Bohn MC (1997) Dopaminergic neurons protected from degeneration by GDNF gene therapy. Science 275:838 – 841. Chouaf-Lakhdar L, Fevre-Montange M, Brisson C, Strazielle N, Gamrani H, Didier-Bazes M (2003) Proliferative activity and nestin expression in periventricular cells of the adult rat brain. Neuroreport 14:633– 636. Connor B, Kozlowski DA, Schallert T, Tillerson JL, Davidson BL, Bohn MC (1999) Differential effects of glial cell line-derived neurotrophic factor (GDNF) in the striatum and substantia nigra of the aged parkinsonian rat. Gene Ther 6:1936 –1951. Connor B, Kozlowski DA, Unnerstall JR, Elsworth JD, Tillerson JL, Schallert T, Bohn MC (2001) Glial cell line-derived neurotrophic factor (GDNF) gene delivery protects dopaminergic terminals from degeneration. Exp Neurol 169:83–95. Cooper O, Isacson O (2004) Intrastriatal transforming growth factor a delivery to a model of Parkinson’s disease induces proliferation and migration of endogenous adult neural progenitor cells without differentiation into dopaminergic neurons. J Neurosci 24:8924– 8931. Curtis MA, Penney EB, Pearson AG, van Roon-Mom WM, Butterworth NJ, Dragunow M, Connor B, Faull RL (2003) Increased cell proliferation and neurogenesis in the adult human Huntington’s disease brain. Proc Natl Acad Sci U S A 100:9023–9027. Curtis MA, Penney EB, Pearson J, Dragunow M, Connor B, Faull RL (2005) The distribution of progenitor cells in the subependymal layer of the lateral ventricle in the normal and Huntington’s disease human brain. Neuroscience 132:777–788. Fallon J, Reid S, Kinyamu R, Opole I, Opole R, Baratta J, Korc M, Endo TL, Duong A, Nguyen G, Karkehabadhi M, Twardzik D, Loughlin S (2000) In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc Natl Acad Sci U S A 97:14686 –14691. Ferland RJ, Gross RA, Applegate CD (2002) Increased mitotic activity in the dentate gyrus of the hippocampus of adult C57BL/6J mice exposed to the flurothyl kindling model of epileptogenesis. Neuroscience 115:669 – 683. Frielingsdorf H, Schwarz K, Brundin P, Mohapel P (2004) No evidence for new dopaminergic neurons in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A 101:10177–10182. Henry RA, Hughes SM, Connor B (2007) AAV-mediated delivery of BDNF augments neurogenesis in the normal and quinolinic acidlesioned adult rat brain. Eur J Neurosci 25:3513–3525. Hermann A, Maisel M, Wegner F, Liebau S, Kim DW, Gerlach M, Schwarz J, Kim KS, Storch A (2006) Multipotent neural stem cells from the adult tegmentum with dopaminergic potential develop essential properties of functional neurons. Stem Cells 24:949 –964. Hoglinger GU, Rizk P, Muriel MP, Duyckaerts C, Oertel WH, Caille I, Hirsch EC (2004) Dopamine depletion impairs precursor cell proliferation in Parkinson disease. Nature Neurosci 7:726 –735. Jeon BS, Jackson-Lewis V, Burke RE (1995) 6-Hydroxydopamine lesion of the rat substantia nigra: time course and morphology of cell death. Neurodegeneration 4:131–137. Jin K, Peel AL, Mao XO, Xie L, Cottrell BA, Henshall DC, Greenberg DA (2004) Increased hippocampal neurogenesis in Alzheimer’s disease. Proc Natl Acad Sci U S A 101:343–347. Kay JN, Blum M (2000) Differential response of ventral midbrain and striatal progenitor cells to lesions of the nigrostriatal dopaminergic projection. Dev Neurosci 22:56 – 67. Kirik D, Rosenblad C, Bjorklund A (1998) Characterization of behavioral and neurodegenerative changes following partial lesions
of the nigrostriatal dopamine system induced by intrastriatal 6-hydroxydopamine in the rat. Exp Neurol 152:259 –277. Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J Neurosci 16:2027–2033. Lee CS, Sauer H, Bjorklund A (1996) Dopaminergic neuronal degeneration and motor impairments following axon terminal lesion by instrastriatal 6-hydroxydopamine in the rat. Neuroscience 72: 641– 653. Lie DC, Dziewczapolski G, Willhoite AR, Kaspar BK, Shults CW, Gage FH (2002) The adult substantia nigra contains progenitor cells with neurogenic potential. J Neurosci 22:6639 – 6649. Liu J, Solway K, Messing RO, Sharp FR (1998) Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J Neurosci 18:7768 –7778. Mao L, Lau YS, Petroske E, Wang JQ (2001) Profound astrogenesis in the striatum of adult mice following nigrostriatal dopaminergic lesion by repeated MPTP administration. Brain Res Dev Brain Res 131:57– 65. Mohapel P, Frielingsdorf H, Haggblad J, Zachrisson O, Brundin P (2005) Platelet-derived growth factor (PDGF-BB) and brain derived neurotrophic factor (BDNF) induce striatal neurogenesis in adult rats with 6-hydroxydopamine lesions. Neuroscience 132:767–776. Nait-Oumesmar B, Decker L, Lachapelle F, Avellana-Adalid V, Bachelin C, Van Evercooren AB (1999) Progenitor cells of the adult mouse subventricular zone proliferate, migrate and differentiate into oligodendrocytes after demyelination. Eur J Neurosci 11:4357– 4366. Nakatomi H, Kuriu T, Okabe S, Yamamoto S, Hatano O, Kawahara N, Tamura A, Kirino T, Nakafuku M (2002) Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110:429 – 441. Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH (1999) Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 19:8487– 8497. Palmer TD, Ray J, Gage FH (1995) FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci 6:474 – 486. Parent JM, Janumpalli S, McNamara JO, Lowenstein DH (1998) Increased dentate granule cell neurogenesis following amygdala kindling in the adult rat. Neurosci Lett 247:9 –12. Parent JM, Valentin VV, Lowenstein DH (2002) Prolonged seizures increase proliferating neuroblasts in the adult rat subventricular zone-olfactory bulb pathway. J Neurosci 22:3174 –3188. Rosenblad C, Kirik D, Devaux B, Moffat B, Phillips HS, Bjorklund A (1999) Protection and regeneration of nigral dopaminergic neurons by neurturin or GDNF in a partial lesion model of Parkinson’s disease after administration into the striatum or the lateral ventricle. Eur J Neurosci 11:1554 –1566. Sauer H, Oertel WH (1994) Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat. Neuroscience 59:401– 415. Scott BW, Wang S, Burnham WM, De Boni U, Wojtowicz JM (1998) Kindling-induced neurogenesis in the dentate gyrus of the rat. Neurosci Lett 248:73–76. Shan X, Chi L, Bishop M, Luo C, Lien L, Zhang Z, Liu R (2006) Enhanced de novo neurogenesis and dopaminergic neurogenesis in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson’s disease-like mice. Stem Cells 24: 1280 –1287. Steiner B, Winter C, Hosman K, Siebert E, Kempermann G, Petrus DS, Kupsch A (2006) Enriched environment induces cellular plasticity in the adult substantia nigra and improves motor behavior function in the 6-OHDA rat model of Parkinson’s disease. Exp Neurol 199:291–300.
P. M. Aponso et al. / Neuroscience 151 (2008) 1142–1153 Stromberg I, Bjorklund H, Dahl D, Jonsson G, Sundstrom E, Olson L (1986) Astrocyte responses to dopaminergic denervations by 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as evidenced by glial fibrillary acidic protein immunohistochemistry. Brain Res Bull 17:225–236. Takagi Y, Nozaki K, Takahashi J, Yodoi J, Ishikawa M, Hashimoto N (1999) Proliferation of neuronal precursor cells in the dentate gyrus is accelerated after transient forebrain ischemia in mice. Brain Res 831:283–287. Tattersfield AS, Croon RJ, Liu YW, Kells AP, Faull RL, Connor B (2004) Neurogenesis in the striatum of the quinolinic acid lesion model of Huntington’s disease. Neuroscience 127:319 –332. Taupin P (2007) BrdU immunohistochemistry for studying adult neurogenesis: Paradigms, pitfalls, limitations, and validation. Brain Res Rev 53:198 –214. Van Kampen JM, Eckman CB (2006) Dopamine D3 receptor agonist delivery to a model of Parkinson’s disease restores the nigrostriatal pathway and improves locomotor behavior. J Neurosci 26: 7272–7280. Van Kampen JM, Hagg T, Robertson HA (2004) Induction of neurogenesis in the adult rat subventricular zone and neostriatum following dopamine D receptor stimulation. Eur J Neurosci 19:2377–2387.
1153
Van Kampen JM, Robertson HA (2005) A possible role for dopamine D3 receptor stimulation in the induction of neurogenesis in the adult rat substantia nigra. Neuroscience 136:381–386. Weinstein DE, Burrola P, Kilpatrick TJ (1996) Increased proliferation of precursor cells in the adult rat brain after targeted lesioning. Brain Res 743:11–16. Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson AC, Reynolds BA (1996) Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16:7599 –7609. Yagita Y, Kitagawa K, Ohtsuki T, Takasawa K, Miyata T, Okano H, Hori M, Matsumoto M (2001) Neurogenesis by progenitor cells in the ischemic adult rat hippocampus. Stroke 32:1890 –1896. Zhao M, Momma S, Delfani K, Carlen M, Cassidy RM, Johansson CB, Brismar H, Shupliakov O, Frisen J, Janson AM (2003) Evidence for neurogenesis in the adult mammalian substantia nigra. Proc Natl Acad Sci U S A 100:7925–7930. Zuch C, Nordstroem V, Briedrick L, Hoernig G, Granholm A, Bickford P (2000) Time course of degenerative alterations in nigral dopaminergic neurons following a 6-hydroxydopamine lesion. J Comp Neurol 427:440 –545.
(Accepted 28 November 2007) (Available online 4 December 2007)