Induction of striatal neurogenesis and generation of region-specific functional mature neurons after ischemia by growth factors

Laboratory investigation

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The capacity to replace lost neurons after insults is retained by several regions of adult mammalian brains. However, it is unknown how many neurons actually replace and mature into region-specific functional neurons to restore lost brain function. In this paper, the authors asked whether neuronal regeneration could be achieved efficaciously by growth factor treatment using a global ischemia model in rats, and they analyzed neuronal long-term maturation processes.


Rat global ischemia using a modified 4-vessel occlusion model was used to induce consistent ischemic neuronal injury in the dorsolateral striatum. To potentiate the proliferative response of neural progenitors, epidermal growth factor and fibroblast growth factor–2 were infused intraventricularly for 7 days from Day 2 after ischemia. Six weeks after ischemia, the number of neurons was counted in the defined dorsolateral striatum. To label the proliferating neural progenitors for tracing studies, 5-bromo-2′-deoxyuridine (BrdU; 150 mg/kg, twice a day) was injected intraperitoneally from Days 5 to 7, and immunohistochemical studies were conducted to explore the maturation of these progenitors. Migration of the progenitors was further studied by enhanced green fluorescent protein retrovirus injection. The effect of an antimitotic drug (cytosine arabinoside) on the neuronal count was also evaluated for contribution to regeneration. To see electrophysiological changes, treated rats were subjected to slice studies by whole-cell recordings. Finally, the effect of neural regeneration was assessed by motor performance by using the staircase test.


Following epidermal growth factor and fibroblast growth factor–2 infusion into the lateral ventricles for 7 days beginning on Day 2, when severe neuronal loss in the adult striatum was confirmed (2.3% of normal controls), a significant increase of striatal neurons was observed at 6 weeks (~ 15% of normal controls) compared with vehicle controls (~ 5% of normal controls). Immunohistochemical studies by BrdU and enhanced green fluorescent protein retrovirus injection disclosed proliferation of neural progenitors in the subventricular zone and their migration to the ischemic striatum. By BrdU tracing study, NeuN- and BrdU-positive new neurons significantly increased at 6 and 12 weeks following the treatment. These accounted for 4.6 and 11.0% of the total neurons present, respectively. Antimitotic treatment demonstrated an approximately 66% reduction in neurons at 6 weeks. Further long-term studies showed dynamic changes of site-specific maturation among various neuronal subtypes even after 6 weeks. Electrophysiological properties of these newly appeared neurons underwent changes that conform to neonatal development. These regenerative changes were accompanied by a functional improvement of overall behavioral performance.


Treatment by growth factors significantly contributed to regeneration of mature striatal neurons after ischemia by endogenous neural progenitors, which was accompanied by electrophysiological maturation and improved motor performance. Recognition and improved understanding of these underlying dynamic processes will contribute to the development of novel and efficient regenerative therapies for brain injuries.

Abbreviations used in this paper: Ara-C = cytosine arabinoside; BrdU = 5-bromo-2′-deoxyuridine; ChAT = choline acetyltransferase; CNQX = 6-cyano-7-nitroquinoxaline-2,3-dione; DARPP-32 = dopamine and adenosine 3′:5′-monophosphate–regulated phosphoprotein with a molecular weight of 32 kD; DCX = doublecortin; EGF = epidermal growth factor; FGF-2 = fibroblast growth factor–2; GF = growth factor; GFAP = glial fibrillary acidic protein; GFP = green fluorescent protein; MCM2 = minichromosomal maintenance protein 2; NGF = nerve growth factor; NPY = neuropeptide Y; PARV = parvalbumin; SGZ = subgranular zone; SVZ = subventricular zone.

Article Information

Address correspondence to: Nobutaka Kawahara, M.D., Ph.D., Department of Neurosurgery, Graduate School of Medical Sciences, Yokohama City University, 3-9 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan. email: or

Please include this information when citing this paper: published online March 26, 2010; DOI: 10.3171/2010.2.JNS09989.

© AANS, except where prohibited by US copyright law.



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    Region of quantification, neuronal degeneration, and subsequent recovery after ischemia. A: Overview of the distribution of neuronal degeneration following global ischemia on Day 2. Fluoro-Jade B staining depicts severe neuronal loss, mainly in the dorsolateral striatum. Bar = 300 μm. B: Boxed areas show the regions of interest for measurements in the dorsolateral striatum (500 × 500 μm, the center of Fluoro-Jade B–stained region) and the SVZ (4 squares of 250 × 250 μm). C–L: Cresyl violet staining (left column) and NeuN-immunostaining (right column) of coronal sections taken at the region of interest in the striatum in sham-operated (C and D), ischemic (E and F on Day 2; and G and H on Day 42), vehicle-infused (artificial CSF, I and J on Day 42), and GF-treated (K and L on Day 42) animals. Following GF treatment, neurons with clear pale nuclei and discrete nucleoli were visible (arrowheads in K), although they appeared smaller than those in sham controls. Bar = 10 μm. M: Neuronal count of rats subjected to ischemia, and then left untreated or treated with vehicle or GF on Day 42 (paraffin sections, 8 rats in each group with the exception of 4 in the sham-operated group). The numbers of neurons were corrected according to the changes of striatal area as described in the text. *p < 0.0001 compared with sham-operated group (t-test). **p < 0.0001 compared with ischemia-alone and vehicle-infused group on Day 42 in both cresyl violet staining and NeuN immunostaining (1-way ANOVA with Bonferroni/Dunn post hoc test).

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    Ischemic injury increases cell proliferation in the SVZ. A: Bar graph showing the number of BrdU-positive cells after ischemia alone or following GF treatment (4 rats in each group). The BrdU was intraperitoneally injected for 2 days before the rats were killed. Note that ischemia-induced cell proliferation peaked on Day 7, which was further increased by GF treatment. B and C: Overview of BrdU-positive cell distribution in the SVZ on Day 7 in ischemia alone (B) and GF-treated animals (C), showing an increased number of BrdU-labeled cells. D–F: Confocal images showing double-labeled cells in the SVZ with BrdU (D) and MCM2 (E) staining, and merged image (F) following GF treatment on Day 7, further indicating that BrdU-labeled cells are proliferating cells. Bar = 50 μm (B and C); 10 μm (D–F).

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    Expression of early neural progenitor markers and migration of SVZ cells into the lesioned striatum after ischemic insult. A–H: Distribution of BrdU- (A) and Mash 1– (B) labeled cells around the SVZ, and the merged image (C) following GF treatment on Day 7. Panel D shows high-magnification confocal images of these cells. Distribution of BrdU- (E) and Pax 6– (F) labeled cells around the SVZ, and the merged image (G) following GF treatment on Day 7. Panel H shows high-magnification confocal images of these cells. These observations indicate that proliferating cells in SVZ express early neural progenitor markers. I and J: Overview of the distribution of BrdU-positive (green) and DCX-positive (red) cells in the SVZ in animals treated with GF on Day 7 (I) and Day 14 (J), showing that BrdU-positive cells express DCX after cessation of GF treatment. K and L: Confocal high-magnification merged images of panel J. Note that BrdU/DCX double-positive cells were also observed at the distal SVZ (L, arrowheads). M–O: Dorsolateral striatum cells after 6 weeks following GF treatment. The NeuN-positive cells (M) double-labeled with DCX (N) were shown in the merged image (G). Note that these neurons appeared to be migrating young neurons with elongated cell bodies and 1 or 2 long leading processes directed away from the SVZ (O, arrowheads). P–S: At 12 weeks in the GF-treated animals, no DCX-positive cells (Q) colabeled with either NeuN (P) or DARPP-32 (R) were noted (merged image in S) in any part of the striatum. T and U: Five days after GFP-retrovirus injection, numerous GFP-positive cells are detected after GF treatment in the SVZ (T). Higher magnification image costained with DAPI showed cellular colocalization of GFP signal within the cell 5 days after the injection (U). V: At 12 weeks after ischemia and GF treatment, GFP-expressing cells (green) colabeled with NeuN (red) were noted in the lesioned striatum, demonstrating direct migration of neural progenitors from the subventricular zone. Bar = 50 μm (A–C, E–G, I, J, and T); 10 μm (D, H, K and L, M–S, U, and V). Arrowheads in M, N, P, R, S, and V depict double- or triple-labeled cells in the same row.

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    Confocal images of newly generated cells in the striatum showing site-specific phenotypic maturation on Day 42 and extension of a long axon following ischemia in the GF-treated animals. A–D: Newly generated neuron double-labeled with BrdU (green, A) and NeuN (red, B), and merged image (C). Panel D shows high-magnification confocal image of the double-labeled cell. E: The BrdU-positive cells (green) did not coexpress the astrocytic marker GFAP (red). F–I: Newly generated BrdU (green, F) and NeuN (red, G) double-positive neuron coexpressed DARPP-32 (blue, H), indicating maturation to medium spiny projection neuron (merged image, I). J–L: Confocal merged images of NeuN-positive (red) neurons double-labeled with PARV (green, J), NPY (green, K), and ChAT (green, L). M: Some ChAT-immunoreactive cells (green) also expressed the astrocytic marker, GFAP (red). N–Q: Confocal images of BrdU (blue, N), NeuN (red, O), and Fluoro-Gold (green, P) triple-labeled cells in the striatum at 12 weeks (merged image, Q). This cell retrogradely incorporated Fluoro-Gold injected into the globus pallidus. Bar = 50 μm (A–C, and E); and 10 μm (D, and F–Q). Arrowheads in A–C and N–Q indicate the cell.

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    Membrane and firing properties of neurons in the dorsolateral (A) and medial (B) striatum after ischemia. The resting membrane potential is indicated on the left of each trace. All the recordings were conducted in GF-treated animals, and the recording site was carefully selected to match the defined region for cell counting (see Fig. 1B) A: Membrane properties of recorded neurons in the dorsolateral striatum at 6, 16, and 25 weeks after ischemia. B: Membrane properties of recorded neurons in the medial striatum at 11 and 16 weeks after ischemia. The mean resting membrane potential was –67.0 ± 4.28 mV (5 rats) at 11 weeks and –67.5 ± 4.94 mV (4 rats) at 16–20 weeks, and the input resistance was 136.0 ± 16.9 MΩ (5 rats) at 11 weeks and 135.0 ± 30.7 MΩ (4 rats) at 16–20 weeks. C: Membrane properties of a glial cell recorded in the dorsolateral striatum 16 weeks after ischemia. D: The mean resting membrane potentials of neurons in the dorsolateral striatum at 6–10 weeks, 10–20 weeks, and more than 20 weeks after ischemia were –48.7 ± 1.04 mV (9 rats), –58.6 ± 2.98 mV (9 rats), and –63.7 ± 1.20 mV (3 rats), respectively. *p < 0.05 compared with 6–10 weeks. E: The mean input resistance of neurons in the dorsolateral striatum at 6–10 weeks, 10–20 weeks, and more than 20 weeks after ischemia were 255.6 ± 38.0 MΩ (9 rats), 132.7 ± 18.0 MΩ (9 rats), and 112.2 ± 27.4 MΩ (3 rats), respectively. *p < 0.05 compared with before 10 weeks. F–H: Voltage-clamp recording. Both outward synaptic currents with slow kinetics and inward currents with fast kinetics were recorded (F). Inward currents were blocked by CNQX (G). Remaining outward currents were blocked by additional bicuculline (H).

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    Time course of motor performance in staircase test by the number of pellets taken at each session in GF- and vehicle (Veh)–treated groups and the number of striatal neurons. Left: The treated group exhibited a marked improvement at 6 and 12 weeks after ischemia. Statistical analysis by ANOVA showed overall significant effects of both group (F(1,18) = 5.34, p < 0.05) and time (F(2,36) = 10.74, p < 0.001), and the interaction of the 2 factors (F(2,36) = 3.695, p < 0.05). Post hoc Bonferroni-Dunn testing revealed a significantly better performance at 6 weeks compared with 2 and 12 weeks (p < 0.001). Values are means of 5 performance days in each stage on both paws. There were 11 rats in the GF-treated group and 9 in the vehicle-infused group. *p < 0.05 compared with vehicle group. Error bars = SEMs. Right: The corrected number of striatal neurons by NeuN immunohistochemistry exhibited statistically a significant difference between these groups at 12 weeks following the behavioral test (6 rats in the GF group and 5 in the vehicle group; p < 0.0001).


  • 1

    Aberg MAAberg NDHedbäcker HOscarsson JEriksson PS: Peripheral infusion of IGF-I selectively induces neurogenesis in the adult rat hippocampus. J Neurosci 20:289629032000

  • 2

    Alzheimer CWerner S: Fibroblast growth factors and neuroprotection. Adv Exp Med Biol 513:3353512002

  • 3

    Arvidsson ACollin TKirik DKokaia ZLindvall O: Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat Med 8:9639702002

  • 4

    Baldauf KReymann KG: Influence of EGF/bFGF treatment on proliferation, early neurogenesis and infarct volume after transient focal ischemia. Brain Res 1056:1581672005

  • 5

    Belluzzi OBenedusi MAckman JLoTurco JJ: Electrophysiological differentiation of new neurons in the olfactory bulb. J Neurosci 23:10411104182003

  • 6

    Benraiss AChmielnicki ELerner KRoh DGoldman SA: Adenoviral brain-derived neurotrophic factor induces both neostriatal and olfactory neuronal recruitment from endogenous progenitor cells in the adult forebrain. J Neurosci 21:671867312001

  • 7

    Bingham BLiu DWood ACho S: Ischemia-stimulated neurogenesis is regulated by proliferation, migration, differentiation and caspase activation of hippocampal precursor cells. Brain Res 1058:1671772005

  • 8

    Cameron HAMcKay RD: Adult neurogenesis produces a large pool of new granule cells in the dentate gyrus. J Comp Neurol 435:4064172001

  • 9

    Carleton APetreanu LTLansford RAlvarez-Buylla ALledo PM: Becoming a new neuron in the adult olfactory bulb. Nat Neurosci 6:5075182003

  • 10

    Collin TArvidsson AKokaia ZLindvall O: Quantitative analysis of the generation of different striatal neuronal subtypes in the adult brain following excitotoxic injury. Exp Neurol 195:71802005

  • 11

    Dayer AGFord AACleaver KMYassaee MCameron HA: Short-term and long-term survival of new neurons in the rat dentate gyrus. J Comp Neurol 460:5635722003

  • 12

    Doetsch FGarcía-Verdugo JMAlvarez-Buylla A: Regeneration of a germinal layer in the adult mammalian brain. Proc Natl Acad Sci U S A 96:11619116241999

  • 13

    Eriksson PSPerfilieva EBjörk-Eriksson TAlborn AMNordborg CPeterson DA: Neurogenesis in the adult human hippocampus. Nat Med 4:131313171998

  • 14

    Goldman JE: Lineage, migration, and fate determination of postnatal subventricular zone cells in the mammalian CNS. J Neurooncol 24:61641995

  • 15

    Grabowski MBrundin PJohansson BB: Paw-reaching, sensorimotor, and rotational behavior after brain infarction in rats. Stroke 24:8898951993

  • 16

    Jin KMinami MLan JQMao XOBatteur SSimon RP: Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat. Proc Natl Acad Sci U S A 98:471047152001

  • 17

    Jin KSun YXie LChilds JMao XOGreenberg DA: Postischemic administration of heparin-binding epidermal growth factor-like growth factor (HB-EGF) reduces infarct size and modifies neurogenesis after focal cerebral ischemia in the rat. J Cereb Blood Flow Metab 24:3994082004

  • 18

    Kawaguchi Y: Neostriatal cell subtypes and their functional roles. Neurosci Res 27:181997

  • 19

    Kimpinski KMearow K: Neurite growth promotion by nerve growth factor and insulin-like growth factor-1 in cultured adult sensory neurons: role of phosphoinositide 3-kinase and mitogen activated protein kinase. J Neurosci Res 63:4864992001

  • 20

    Kirino T: Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239:57691982

  • 21

    Kobayashi TAhlenius HThored PKobayashi RKokaia ZLindvall O: Intracerebral infusion of glial cell line-derived neurotrophic factor promotes striatal neurogenesis after stroke in adult rats. Stroke 37:236123672006

  • 22

    Kuhn HGWinkler JKempermann GThal LJGage FH: Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain. J Neurosci 17:582058291997

  • 23

    Larsson ELindvall OKokaia Z: Stereological assessment of vulnerability of immunocytochemically identified striatal and hippocampal neurons after global cerebral ischemia in rats. Brain Res 913:1171322001

  • 24

    Liu JSolway KMessing ROSharp FR: Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. J Neurosci 18:776877781998

  • 25

    Marin OAnderson SARubenstein JL: Origin and molecular specification of striatal interneurons. J Neurosci 20:606360762000

  • 26

    Martens DJSeaberg RMvan der Kooy D: In vivo infusions of exogenous growth factors into the fourth ventricle of the adult mouse brain increase the proliferation of neural progenitors around the fourth ventricle and the central canal of the spinal cord. Eur J Neurosci 16:104510572002

  • 27

    Maslov AYBarone TAPlunkett RJPruitt SC: Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice. J Neurosci 24:172617332004

  • 28

    Meade CAFigueredo-Cardenas GFusco FNowak TS JrPulsinelli WAReiner A: Transient global ischemia in rats yields striatal projection neuron and interneuron loss resembling that in Huntington's disease. Exp Neurol 166:3073232000

  • 29

    Momiyama T: Parallel decrease in omega-conotoxin-sensitive transmission and dopamine-induced inhibition at the striatal synapse of developing rats. J Physiol 546:4834902003

  • 30

    Nakatomi HKuriu TOkabe SYamamoto SHatano OKawahara N: Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors. Cell 110:4294412002

  • 31

    Nieto MSchuurmans CBritz OGuillemot F: Neural bHLH genes control the neuronal versus glial fate decision in cortical progenitors. Neuron 29:4014132001

  • 32

    Ouimet CCGreengard P: Distribution of DARPP-32 in the basal ganglia: an electron microscopic study. J Neurocytol 19:39521990

  • 33

    Ouimet CCLangley-Gullion KCGreengard P: Quantitative immunocytochemistry of DARPP-32-expressing neurons in the rat caudatoputamen. Brain Res 808:8121998

  • 34

    Oya SYoshikawa GTakai KTanaka JHigashiyama SSaito N: Region-specific proliferative response of neural progenitors to exogenous stimulation by growth factors following ischemia. Neuroreport 19:8058092008

  • 35

    Palmer TDRay JGage FH: FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci 6:4744861995

  • 36

    Parent JMVexler ZSGong CDerugin NFerriero DM: Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann Neurol 52:8028132002

  • 37

    Pencea VBingaman KDWiegand SJLuskin MB: Infusion of brain-derived neurotrophic factor into the lateral ventricle of the adult rat leads to new neurons in the parenchyma of the striatum, septum, thalamus, and hypothalamus. J Neurosci 21:670667172001

  • 38

    Peng HWen TCTanaka JMaeda NMatsuda SDesaki J: Epidermal growth factor protects neuronal cells in vivo and in vitro against transient forebrain ischemia- and free radical-induced injuries. J Cereb Blood Flow Metab 18:3493601998

  • 39

    Pisa M: Motor functions of the striatum in the rat: critical role of the lateral region in tongue and forelimb reaching. Neuroscience 24:4534631988

  • 40

    Pulsinelli WABrierley JBPlum F: Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 11:4914981982

  • 41

    Reynolds BAWeiss S: Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:170717101992

  • 42

    Richards LJKilpatrick TJBartlett PF: De novo generation of neuronal cells from the adult mouse brain. Proc Natl Acad Sci U S A 89:859185951992

  • 43

    Schmidt-Hieber CJonas PBischofberger J: Enhanced synaptic plasticity in newly generated granule cells of the adult hippocampus. Nature 429:1841872004

  • 44

    Schmitz CHof PR: Design-based stereology in neuroscience. Neuroscience 130:8138312005

  • 45

    Schmued LCAlbertson CSlikker W Jr: Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Res 751:37461997

  • 46

    Semba KVincent SRFibiger HC: Different times of origin of choline acetyltransferase- and somatostatin-immunoreactive neurons in the rat striatum. J Neurosci 8:393739441988

  • 47

    Shingo TSorokan STShimazaki TWeiss S: Erythropoietin regulates the in vitro and in vivo production of neuronal progenitors by mammalian forebrain neural stem cells. J Neurosci 21:973397432001

  • 48

    Smith MLAuer RNSiesjö BK: The density and distribution of ischemic brain injury in the rat following 2-10 min of forebrain ischemia. Acta Neuropathol 64:3193321984

  • 49

    Takahashi MOsumi N: Pax6 regulates specification of ventral neurone subtypes in the hindbrain by establishing progenitor domains. Development 129:132713382002

  • 50

    Tandé DHöglinger GDebeir TFreundlieb NHirsch ECFrançois C: New striatal dopamine neurons in MPTP-treated macaques result from a phenotypic shift and not neurogenesis. Brain 129:119412002006

  • 51

    Tattersfield ASCroon RJLiu YWKells APFaull RLConnor B: Neurogenesis in the striatum of the quinolinic acid lesion model of Huntington's disease. Neuroscience 127:3193322004

  • 52

    Tepper JMSharpe NAKoós TZTrent F: Postnatal development of the rat neostriatum: electrophysiological, light- and electron-microscopic studies. Dev Neurosci 20:1251451998

  • 53

    Teramoto TQiu JPlumier JCMoskowitz MA: EGF amplifies the replacement of parvalbumin-expressing striatal interneurons after ischemia. J Clin Invest 111:112511322003

  • 54

    Uchida KMomiyama TOkano HYuzaki MKoizumi AMine Y: Potential functional neural repair with grafted neural stem cells of early embryonic neuroepithelial origin. Neurosci Res 52:2762862005

  • 55

    van Praag HSchinder AFChristie BRToni NPalmer TDGage FH: Functional neurogenesis in the adult hippocampus. Nature 415:103010342002

  • 56

    Wu JSun ZSun HSWu JWeisel RDKeating A: Intravenously administered bone marrow cells migrate to damaged brain tissue and improve neural function in ischemic rats. Cell Transplant 16:99310052008

  • 57

    Xu ZC: Neurophysiological changes of spiny neurons in rat neostriatum after transient forebrain ischemia: an in vivo intracellular recording and staining study. Neuroscience 67:8238361995

  • 58

    Yamamoto SNagao MSugimori MKosako HNakatomi HYamamoto N: Transcription factor expression and Notch-dependent regulation of neural progenitors in the adult rat spinal cord. J Neurosci 21:981498232001

  • 59

    Yamanaka S: Strategies and new developments in the generation of patient-specific pluripotent stem cells. Cell Stem Cell 1:39492007




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