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Neural Regenerative Options in the Management of Ischemic Brain Injury ntil recently, it was widely believed † Shervin R. Dashti, MD, PhD that neuronal production in the † U Jason Wilson, MD mammalian brain occurred only during † Yin C. Hu, MD the prenatal period. Over the past 15 † Warren R. Selman, MD years, however, evidence has shown that Robert F. Spetzler, MD neurogenesis continues in certain areas of Peter Nakaji, MD the adult mammalian brain, namely, in the subventricular zone and hippocam- Ischemic brain injury is a difficult clinical scenario for which adequate treatment pal dentate gyrus (Fig. 1).20 An obvious paradigms are lacking. The formation of ischemic scar tissue in the cerebral question is whether adult neurogenesis cortex creates a barrier to regenerative efforts and restoration of function. Vari- has the potential to replace dying neurons ous agents that regulate the cell cycle hold promise for modulating the natural- in the setting of brain injury. New evi- ly progressive formation of this astrocytic scar tissue. Use of these agents will in- dence indicates that such neurogenesis is volve the development of strategies that influence the regenerative potential of indeed possible. The logical clinical im- neural progenitor cells. plication of this finding is the potential to Key Words: dentate gyrus, hippocampus, neural stem cells, neurogen- augment the production of mature neu- esis rons in the injured brain. This ability would be of considerable value in victims of large strokes who seldom regain significant function in distributions where neural tissue has died. In contrast to the recently discovered adult neurogenesis, hematopoietic stem cells have been studied extensively for a relatively long time. The hematopoietic system contains a pool of progenitor cells that differentiate into mature cells. Study- ing the molecular mechanisms govern- ing the proliferation, differentiation, and apoptosis of the hematopoietic stem cell population provides valuable clues about the biology of neural progenitor cells. This review examines neurogenesis in the normal brain and in response to ischemic insults. Factors that drive proliferation of stem cell populations into neu- Abbreviations Used: BrdU, bromodeoxyuridine; CNS, central nervous sys- ral progenitor cells and those that drive tem; DNA, deoxyribonucleic acid; G-CSF, granulocyte colony-stimulating factor; mRNA, messenger ribonucleic acid; NMDA, N-methyl-D-aspartate differentiation of neural progenitor cells into neurons and, ultimately, integration Division of Neurological Surgery, Barrow Neurological Institute, St. Joseph’s Hospital and Medical into neural networks are discussed. Such Center, Phoenix, Arizona therapeutic modalities can be broadly †Department of Neurosurgery, University Hospitals of Cleveland, Case Western Reserve University classified as using either an exogenous or School of Medicine, Cleveland, Ohio an endogenous approach. These basic is-

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This phenomenon peaks 7 to 10 days after ischemia is induced and returns to normal levels within a month.20

Differentiation of Stem Cells to Mature Neurons. Neural stem cells from adult rats co-cultured with primary neurons and astrocytes from neonatal hippocampus differentiate into electrically ac- tive neurons and integrate into neural networks with functional synaptic trans- mission (Fig. 2).29 Preliminary evidence suggests that these cells play a role in hippocampal-dependent memory formation.29 At 35 days after stroke, the chain Figure1. Flow chart of the pathways involved in the differentiation of endogenous neu- of neuroblasts that migrates from the ral stem cells. subventricular zone to the peri-infarct striatum also expresses neuronal mark- sues must be understood before strategies grate tangentially toward the olfactory ers.25 As with the hippocampal dentate can be devised to improve outcomes bulb and striatum. In the olfactory bulb, gyrus, the persistent neurogenesis in the based on repopulating neurons and these neurons climb radially into the gran- subventricular zone has been implicat- achieving functional integration in the ule and periglomerular cell layers. There, ed in olfactory memory and odor dis- setting of acute ischemic injury. they assume the nuclear morphology of crimination.1 granule cells and express neuron-specific markers.11 Most of these adult-generated Migration of Neural Precursors. Under- Hippocampal Dentate granule neurons persist within the olfac- standing the movement of neural pre- Gyrus tory bulbs for at least 4 months.10 Chains cursors generated in the adult mam- Neural stem cells are located in the of neuroblasts also extend from the sub- malian brain to their final destination has subgranular zone of the adult mammalian ventricular zone to the peri-infarct stria- important implications for functional re- hippocampal dentate gyrus.15,19 The pri- tum. Many of these newly generated cells covery after ischemic injury. Recall that mary neural progenitor cells, bipotent ra- persist in the striatum and cortex adjacent normal neural stem cells in the hip- dial glia-like stem cells with astrocytic to infarcts. However, 35 days after stroke, pocampal dentate gyrus differentiate into properties, differentiate into lineage-de- only the cells in the neostriatum express progenitor cells and then to mature neu- termined progenitor cells and then to ma- neuronal markers.25 As with the hip- rons.6,16 The latter migrate a short dis- ture neurons.6,16 The mature neurons mi- pocampal dentate gyrus, the persistent tance to populate the granular cell layer of grate a short distance and populate the neurogenesis in the subventricular zone the hippocampal dentate gyrus. Newly granular cell layer of the hippocampal has been implicated in olfactory memo- divided neuronal precursor cells also mi- dentate gyrus. The S-phase marker bro- ry and odor discrimination.1 grate from the subgranular zone into the modeoxyuridine (BrdU) has been used granule cell layer and differentiate into to track dividing cells in the adult rat den- neurons.21 Migrating neuroblasts move tate gyrus. Based on such studies, young as chains through a well-defined path- Response to Ischemic adult rats generate 9,000 new cells each way, the rostral migratory stream. These Insults day or more than 250,000 per month. chains contain only closely apposed neu- The new granule neurons generated each Stroke-Induced Neurogenesis roblasts. Another astrocytic cell type en- month constitute 6% of the total size of Recent evidence suggests that ischemia sheathes the chains of migrating neurob- the granular cell population as well as 30 significantly induces neurogenesis in the lasts. Hence during chain migration, to 60% of afferent and efferent cells.5 brain of adult rodents.20 Liu et al. first neural precursors move in association demonstrated a marked increase in cell with each other; they are not guided by birth in the dentate subgranular zone of radial glial or axonal fibers.22 Forebrain Subventricular gerbils in response to transient global is- Parent et al. induced focal stroke in Zone chemia.21 Subsequent studies confirmed adult rats and assessed cellular prolifera- Adult neurogenesis also occurs in the that focal9,12,31,32 and global30 cortical is- tion and neurogenesis with BrdU label- mammalian subventricular zone. Neu- chemia significantly increases the prolif- ing and immunostaining.25 Brains exam- rons are generated in the subependymal eration of neural stem cells in both the ined 10 to 21 days after stroke showed layer of the lateral ventricles and then mi- dentate gyrus and subventricular zone. chains of neuroblasts extending from the

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subventricular zone to the peri-infarct Neurotransmitters glutamate negatively affects adult neu- striatum. These findings combined with Glutamate, the main excitatory neurogenesis. those from several other studies indicate rotransmitter in the brain, appears to Serotonin is another neurotransmit- that focal and global ischemic injury di- play a role in adult neurogenesis. Acti- ter implicated in adult neurogenesis. rects the migration of the neuroblasts to vation of NMDA-glutamate receptors Chronic antidepressant therapy with the site of the infarct.2,13 rapidly decreases proliferation of neu- serotonin-reuptake inhibitors signifi- ronal stem cells in rats.4 In contrast, ad- cantly increases proliferation of adult neu- Long-Term Survival of New Adult Neurons. ministration of NMDA-receptor an- ronal precursor cells in the dentate An important question is how long the tagonists rapidly increases the number gyrus.23 Conversely, inhibiting the sero- newly generated neurons in the ischemic of cells during the S phase.4 Lesioning toninergic pathway decreases neuronal adult brain persist. It is estimated that of the entorhinal cortex, the main exci- proliferation in the dentate gyrus and fewer than 1% of the ischemic and dying tatory (glutamate-mediated) afferent subventricular zones.3 However, the ef- striatal neurons are replaced through input to the hippocampal granule neu- fect of serotonin on ischemia-induced adult neurogenesis.2 This estimate sug- rons, also increases neurogenesis in the proliferation of neural progenitor cells has gests that most neurons generated in the dentate gyrus. This finding suggests that not yet been established. adult brain in response to injury do not survive for a significant length of time. Understanding the mechanism of cell death in adult-born neurons is the first step in developing therapies intended to maximize functional recovery after stroke.

Hippocampus Neurogenesis in the Aging Brain Dentate Most of the new neurons generated gyrus in response to stroke in adult rats do not survive, and stroke most often occurs in older populations. Therefore, an important question is whether aging in- fluences the survival of adult-born neurons. Yagita et al. used a four-vessel transient ischemia model in rats to in- A vestigate the role of aging on proliferation of neural progenitor cells.34 In both young and old rats, neurogenesis signif- Cerebrum icantly increased in the subventricular zone in response to transient global is- Olfactory chemia. Advanced age, however, pro- bulb foundly influenced the survival of the SVZ newly generated neural precursor cells. RMS One month after the ischemia was in- Cerebellum duced, the number of surviving neural Midbrain progenitor cells decreased seven-fold in the older rats. To develop strategies to maximize functional recovery after stroke in the aging population, we must first understand the molecular mechanism by which senescence decreases the B survival of neurons. Is it through programmed cell death or tissue necrosis? Figure 2. The two niches containing neural stem cells in the CNS of the adult mammal. (A) adult hippocampus. (B) Longitudinal section of the adult mouse brain in which the sub- What signaling pathways are involved? ventricular zone (SVZ) and the rostral migratory stream (RMS) are indicated. Neuronal How can this cell death mechanism be precursors migrate tangetially along the rostral migratory stream from the SVZ to the ol- down-regulated? factory bulb.

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Environmental Enrichment bral ischemia, suggesting an autocrine prevents quiescent progenitor cells in the Environmental enrichment, consist- neuroprotective signaling cascade. brain from entering the cell cycle.26 Qiu ing of social interactions, various phys- In myeloid lineage cells, G-CSF in- and colleagues demonstrated that is- ical activities, and exposure to novel ob- duces enhanced survival and differentia- chemic brain injury induces proliferation jects, enhances functional recovery after tion into mature neutrophilic granulo- of quiescent neural progenitor cells in the stroke.17,33 At the cellular level, environ- cytes.28 It appears that G-CSF exerts a hippocampus and in the subventricular mental enrichment has increased den- similar effect on survival and differentia- zone of p21 knockout mice.26 The in- dritic branching and dendritic spines in tion of neural stem cells.28 Adult neural creased proliferation of stem cells, how- pyramidal neurons of layers II-III in the stem cells isolated from the subventricu- ever, did not occur in nonlesioned p21 contralateral cortex.14 At the molecular lar zone and hippocampus of rats express knockout mice. level, environmental enrichment in- G-CSF in vitro.28 G-CSF greatly induces duces differential gene regulation in the promoter activity of the mature neuronal injured brain. marker gene III-tubulin. In culture it also Conclusion Recent evidence suggests that postis- induces dose-dependent increases in lev- Future efforts aimed at investigating chemic environmental enrichment also els of the mRNA of several differentia- other regulators of the cell cycle, includ- enhances neurogenesis in the adult ger- tion markers. It does not, however, de- ing the other members of the cyclin-de- minal zones. Komitova et al. induced plete the pool of undifferentiated cells. pendent kinase-inhibitor family, will im- stroke by ligating the middle cerebral ar- prove understanding of the relative tery of spontaneously hypertensive rats, quiescence of neural progenitor cells. which were then exposed to a standard Endogenous Suppression Eventually, such studies may lead to im- or enriched environmental condition.18 of Neural Regeneration proved functional recovery of the CNS One and 7 days after stroke, rats exposed (p21 Cyclin-Dependent in response to ischemic injury by to the enriched environment performed Kinase Inhibitor) spurring the development of strategies to significantly better than rats exposed to The efficacy of neural stem cell ex- enhance proliferative potential of neural the standard environment. Five weeks pansion in response to injury depends on progenitor cells. after stroke, there was less cellular prolif- the proliferative capacity of the cells. Un- eration in the subventricular zone of the derstanding the molecular kinetics of the stroke-lesioned rats in the standard envi- cell cycle is the key to inducting the pro- ronment compared to intact animals. liferation of stem cells for therapeutic pur- References Postischemic environmental enrich- poses. In the hematopoietic stem cell 1. Alvarez-Buylla A, Garcia-Verdugo JM: Neuroge- nesis in adult subventricular zone. J Neurosci ment counteracts the decreased level of model, there are two types of cellular pro- 22:629-634, 2002 cellular proliferation in the subventric- liferation: high-capacity proliferation of 2. Arvidsson A, Collin T, Kirik D, et al: Neuronal re- ular zone while increasing the pool of progenitor cells in response to cytokines placement from endogenous precursors in the neural progenitor cells in the subven- and low-proliferation capacity quiescent adult brain after stroke. Nat Med 8:963-970, 2002 3. Brezun JM, Daszuta A: Depletion in serotonin de- tricular zone. 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