Induction of striatal neurogenesis and generation of region-specific functional mature neurons after ischemia by growth factors
Authors: Yoshikawa Gakushi , Momiyama Toshihiko , Oya Soichi , Takai Keisuke , Tanaka Jun-ichi , Higashiyama Shigeki , Saito Nobuhito , Kirino Takaaki , Kawahara Nobutaka 
Published: March 2010

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.