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

Laboratory investigation

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Object

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.

Methods

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.

Results

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.

Conclusions

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: nkawa@yokohama-cu.ac.jp or kawahara-tky@umin.ac.jp.

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.

Headings

Figures

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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).

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