Repairing brain after stroke: A review on post-ischemic neurogenesis

Neurochemistry International 50 (2007) 1028–1041 www.elsevier.com/locate/neuint

Review

Repairing brain after stroke: A review on post-ischemic neurogenesis Charles Wiltrout a,1, Bradley Lang a,1, Yiping Yan a, Robert J. Dempsey a, Raghu Vemuganti a,b,c,d,* a

Department of Neurological Surgery, University of Wisconsin, Madison, WI, USA b Neuroscience Training Program, University of Wisconsin, Madison, WI, USA c Cardiovascular Research Center, University of Wisconsin, Madison, WI, USA d Regenerative Medicine Program, University of Wisconsin, Madison, WI, USA

Received 2 April 2007; received in revised form 13 April 2007; accepted 16 April 2007 Available online 4 May 2007

Abstract Stroke is devastating as currently no therapies are available that can prevent stroke-induced neurological dysfunction in humans. With the recent observations that acute insults to adult brain stimulate new neuronal formation in various species of animals, optimism is building for a possible regeneration of stroke-damaged brain. This article reviewed the advances in the understanding of the molecular mechanisms of the various steps of neurogenesis with an emphasis on the endogenous mediators and exogenous promoters of neural progenitor proliferation, migration and survival in the post-ischemic adult brain. # 2007 Elsevier Ltd. All rights reserved. Keywords: Cerebral ischemia; Neural progenitor proliferation; Stem cell migration; Growth factors

1. Introduction ‘‘In the adult centers the nerve paths are something fixed, ended and immutable. Everything may die, nothing may be regenerated.’’ Ramon y Cajal’s Degeneration and Regeneration of the Nervous System (1928). The above statement formed the basis for the scientific dogma ‘‘adult neurogenesis does not exist in humans’’ that dominated neuroscience for more than 70 years. A corollary to this is that for those who have sustained CNS damage, there is no hope of recovery. However, based on the new research on neurogenesis in adult brain this paradigm has been changed dramatically in the last decade. Joseph Altman made the initial discovery of new neural cell formation in adult rodent brain in 1962 which was subsequently confirmed by Michel Kaplan (Altman, 1962; Kaplan and Hinds, 1977). The process of adult neurogenesis was accepted beyond doubt when Fernando Nottebohm showed that canaries can learn new songs every

year as many new neurons will be formed in their hippocampi during the mating season (Nottebohm, 1981; Goldman and Nottebohm, 1983). Many studies now confirm that new neurons are continuously formed in adult brains in diverse species including primates and humans (Eriksson et al., 1998; Gould et al., 1999a). Furthermore, studies have shown that traumatic and ischemic injuries to adult brain stimulate neurogenesis, increasing hope for functional recovery after a CNS insult (Parent et al., 1997; Gould et al., 1997; Liu et al., 1998; Gu et al., 2000; Jiang et al., 2001; Dash et al., 2001; Dempsey et al., 2003; Yan et al., 2006; Rola et al., 2006; Ernst and Christie, 2006). In this article, we reviewed the characteristics, molecular mechanisms, putative endogenous mediators and exogenous stimulators of neurogenesis in adult brain following ischemic injury. 2. Neural stem cells (NSCs) 2.1. Embryonic NSCs

* Corresponding author at: Department of Neurological Surgery, University of Wisconsin, K4/8 (Mail Stop Code CSC-8660), 600 Highland Ave, Madison, WI 53792, USA. Tel.: +1 608 263 4055; fax: +1 608 263 1728. E-mail address: [email protected] (R. Vemuganti). 1 Both authors contributed equally. 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.04.011

In humans, formation of NSCs begins during the gastrulation. Interactions between the developing endoderm and mesoderm induce differentiation of neuroepithelium along the midline of the embryo, forming the neural plate which folds

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into the neural tube and the neuroepithelium differentiates into various CNS cell types (Clark and Chiu, 2003). In the embryonic brain, new neurons continuously proliferate and migrate from the ventricular zone. Post-parturition, the ventricular zone disappears, but some radial glia remains in the subventricular zone (SVZ) and retain the neural stem celllike properties (Clark and Chiu, 2003). Radial glia are known to act as neural and glial precursors in addition to guiding the migration of the newly formed cells. During development, mitotic precursor cells from the SVZ also migrate into the primordial dentate hilus of the hippocampus to form a germinal zone active for 2 weeks post-parturition. After this time, the precursor cells reside on the hilar side of the granule cell layer, forming the subgranular zone (SGZ) of the dentate gyrus (DG) in adult brain (Pleasure et al., 2000). 2.2. Adult NSCs The stem cells and/or neural progenitors that remained in the SVZ, SGZ and the posterior periventricular (PPv) area proliferate at a basal level to form new neurons and glia throughout the life of mammals (Lois and Alvarez-Buylla, 1993; Eriksson et al., 1998; Doetsch et al., 1999; Alvarez-Buylla et al., 2002; Gage, 2000). These are the three established neurogenic regions of the adult mammalian brain, but some studies also indicated the presence of minute amounts of progenitor cells in spinal cord, diencephalon, cerebral cortex and striatum as well (Weiss et al., 1996; Johe et al., 1996; Gould et al., 1999b; Nguyen et al., 2006). 2.3. Subventricular zone In the SVZ of the adult brain, NSCs are located adjacent to a layer of ependymal cells which line the lateral ventricles. SVZ precursor cells most likely arise from undifferentiated radial glial cells left over after embryonic neural development (Merkle et al., 2004). The adult SVZ contains four types of cells viz., A, B, C and E cells (Garcia-Verdugo et al., 1998). The astrocytes of the SVZ are the B cells which are considered to be the true adult neural stem cells. They form chains of young migratory neuroblasts known as the A cells which follow the rostral migratory stream (RMS) to the olfactory bulb and differentiate into interneurons (Altman, 1969; Lois and Alvarez-Buylla, 1994; Shapiro et al., 2006). The C cells are highly proliferative and are found at the base of the migratory chains of A cells. They are believed to be an intermediate population between the B cells and A cells. The neuroblast migration happens regardless of the presence of olfactory bulb, suggesting that the neural cell migration is an inherent property and is not mediated by the target (Kirschenbaum et al., 1999). These cells integrate into the odor discrimination and memory circuitry of the olfactory bulb (Gheusi et al., 2000; Petreanu and Alvarez-Buylla, 2002; Rochefort et al., 2002). 2.4. Dentate gyrus and the posterior periventricular area The NSCs in the hippocampal DG have a very limited ability to self-renew, and hence are considered neural

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progenitors as opposed to the multipotent and self-renewing stem cells of the SVZ. Within the DG, neural progenitors are located near the hilus in the SGZ (Kaplan and Hinds, 1977; Cameron and McKay, 2001). They continuously proliferate and the newly formed cells migrate to the granule cell layer (GCL). Similar to the SVZ, the DG precursor cells also express GFAP and could be remnants of radial glial cells (Seri et al., 2001; Namba et al., 2005). However, some studies suggest the presence of two distinct types of progenitors (neuronal progenitors that form neurons and the glial progenitors that form glia) in the SGZ (Seaberg and van der Kooy, 2002). There is an age-dependent decrease in the proliferation of cells in the DG (Kuhn et al., 1996), indicating that they cannot self-renew forever. It is also believed that type A-like neuroblasts migrate to the GCL to differentiate into granule cells (Bayer et al., 1982; Kuhn et al., 1996; Gage et al., 1998; Overstreet-Wadiche and Westbrook, 2006). Most of the newly proliferated DG cells die early, making it unlikely that these cells establish correct synaptic connections (Gould et al., 2001). Conversely, studies using green fluorescent protein (GFP) expressing retrovirus that preferentially labeled proliferating cells showed that the newly generated DG neurons can form synaptic connections and even become electrophysiologically active (Van Praag et al., 2002). The third germinal zone of brain, the subependymal layer of the PPv surrounding the hippocampus is thought to contain the true stem cells that replenish the hippocampal neurons (Seaberg and van der Kooy, 2002, 2003). Nakatomi et al. (2002) showed that the newly proliferated progenitors migrate and repopulate the hippocampal CA1 following ischemic cell death. 3. Functional significance of the neurogenesis in adult brain The major function of the neurogenesis in adult brain seems to be replacing the neurons that die regularly in certain brain areas. Granule neurons in DG continuously die and the progenitors might be proliferating at the same rate of mature neuronal death to maintain a constant DG cell number (Gage, 2000). Similarly, the newly proliferated cells from SVZ might be replenishing the dead olfactory bulb neurons. On the other hand, the resident neural progenitors might also be the emergency reserves that could be induced to replace neurons lost due to acute insults. It is interesting to note that adult brain neurogenesis is influenced by many conditions. For example, negative life experiences including stress, maternal separation of pups, chronic alcoholism, depression, drug abuse, irradiation, high-fat diet, diabetes and inflammation have all been linked to a down-regulation of neurogenesis (Herrera et al., 2003; Gould et al., 1997; Malberg et al., 2000; Dranovsky and Hen, 2006; Powrozek et al., 2004; Mirescu et al., 2004; Hauser et al., 2000; Raber et al., 2004; Ekdahl et al., 2003; Lindqvist et al., 2006; Beauquis et al., 2006). On the other hand, positive life experiences including exercise, learning, enriched environment, caloric restriction and induction of ischemic tolerance have been shown to upregulate neurogenesis (Van Praag et al., 1999; Gould et al., 1999c; Nakatomi et al., 2002; Lichtenwalner

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and Parent, 2006; Komitova et al., 2002; Lee et al., 2002; Naylor et al., 2005). 4. Neurogenesis following ischemia Many studies have shown that acute CNS insults such as seizures, oxidative damage, lesions, traumatic injury, global and focal ischemia significantly increase the progenitor cell proliferation in the adult brain (Parent et al., 1997; Gould et al., 1997; Liu et al., 1998; Gu et al., 2000; Jiang et al., 2001; Dash et al., 2001; Dempsey et al., 2003; Yan et al., 2006; Rola et al., 2006; Ernst and Christie, 2006). Furthermore, many studies also showed that the newly proliferated cells migrate to the damaged areas of the brain, particularly after cerebral ischemia (Magavi et al., 2000; Parent et al., 2002a,b; Arvidsson et al., 2002; Yan et al., in press; Sun et al., 2007; Hicks et al., 2007; Wang et al., 2007). 4.1. Neurogenesis following global cerebral ischemia In rodents, global ischemia induced by bilaterally occluding the common carotid arteries causes an almost complete neuronal death in the hippocampal CA1 region by 3–7 days after the insult (Imai et al., 2007). Several studies that global ischemia induces 10-fold enhanced progenitor proliferation in the SGZ of gerbils, rats, mice, monkeys as well as humans (Liu et al., 1998; Takagi et al., 1999; Iwai et al., 2001; Yagita et al., 2001; Kee et al., 2001; Schmidt and Reymann, 2002; Jin et al., 2006; Tonchev et al., 2007). Most studies reported an increased progenitor proliferation starting at 3–4 days, peaking at 7–10 days and returning to control levels by 3–5 weeks after global ischemia (Liu et al., 1998; Takagi et al., 1999; Kee et al., 2001; Yagita et al., 2001; Iwai et al., 2002; Tonchev et al., 2003). The level of progenitor proliferation in SGZ is known to be affected by the duration of global ischemia, but not by the intensity of CA1 cell death (Liu et al., 1998). While a 2 min occlusion had no effect, both 4 min and 10 min occlusions resulted in similar increases in the DG progenitor proliferation in adult gerbils (Liu et al., 2001). Following global ischemia, enhanced neurogenesis was also reported in the SVZ and PPv areas (Nakatomi et al., 2002; Iwai et al., 2003; Pforte et al., 2005). Following global ischemia, the newly generated progenitors in the SVZ express many immature neuronal makers including highly poly-sialated neural cell adhesion molecule (PSA-NCAM), doublecortin (DCX), nestin and bIII tubulin, and migrate along the RMS to the olfactory bulb (Iwai et al., 2003; Tonchev et al., 2005). Nakatomi et al. (2002) showed that 28 days following the induction of global ischemia in adult rats, several new neurons appear in the CA1 region. They demonstrated that the new neurons migrate from PPv into the CA1 to regenerate the damaged hippocampus. A later study by Bendel et al. (2005) showed that repopulation of the CA1 neurons after global ischemia leads to recovery of spatial learning and memory functions in adult rats. The phenomenon of increased neurogenesis in hippocampus following global ischemia was also demonstrated in the gerbil brain (Schmidt and Reymann, 2002).

4.2. Neurogenesis following focal cerebral ischemia In contrast to global ischemia, during focal ischemia blood perfusion is only blocked to specific regions of brain. In rodents, focal ischemia can be induced by occluding the middle cerebral artery (MCA) either transiently or permanently. MCA occlusion (MCAO) in adult rodents results in an infarct encompassing the cerebral cortex and striatum on the occluded side of the brain. Many studies reported a significantly enhanced progenitor proliferation in both adult rats and mice in the SVZ as well as DG following MCAO (Jin et al., 2001; Zhang et al., 2001a,b; Komitova et al., 2002; Parent et al., 2002a; Dempsey et al., 2003; Yan et al., 2006, in press). Following transient focal ischemia, enhanced proliferation starts bilaterally in both SVZ and DG as early as 2 days, peaks at 1–2 weeks and returns to the sham level by 3–4 weeks of reperfusion (Jin et al., 2001; Zhang et al., 2001a,b; Takasawa et al., 2002; Dempsey et al., 2003; Zhu et al., 2003). Some studies also reported post-ischemic neurogenesis in the cerebral cortex following focal ischemia (Gu et al., 2000; Gould et al., 1999b; Jiang et al., 2001; Palmer et al., 1999; Rakic, 2002), but the origin of those cells is being debated. Gould et al. (1999a,b) reported cortical neurogenesis in the adult primates, but the phenomenon was not confirmed by others (Kornack and Rakic, 2001). Therefore, it is possible that the precursors which reside in the cortex alter their potential to generate neurons under pathological conditions such as ischemia, or extravasated blood cells might transdifferentiate to form some neural cells in the cortex (Hess et al., 2002). The magnitude of the post-ischemic progenitor proliferation was reported to be affected by the duration of MCAO. A 2 h MCA occlusion was shown to induce more proliferation than a 30 min MCAO (Arvidsson et al., 2001). However, neither infarction nor inflammation seems to be essential for the postischemic progenitor proliferation in rodents as a 15 min MCAO, which doesn’t induce any infarction or inflammation in adult rats, was also shown to induce progenitor proliferation (Naylor et al., 2005). 4.3. Fate of the newly generated cells in the ischemic brain Following focal ischemia, the newly proliferated progenitor cells will survive for at least 2–3 weeks in the DG, but disappear (either perish or migrate) within a week in the SVZ (Dempsey et al., 2003). The newborn cells express the marker DCX in the first week after focal ischemia, indicating an immature and migrating neuronal phenotype (Jin et al., 2001; Dempsey et al., 2003). In the DG, they either die or migrate into GCL. Most of the surviving newly formed DG cells differentiate into NeuN or calbindin positive mature neurons by 3–4 weeks after ischemia (Komitova et al., 2002; Dempsey et al., 2003; Jin et al., 2003; Zhu et al., 2003). About 10–20% of the newly generated cells differentiate into GFAP positive astrocytes in the GCL and the hippocampal hilus (Komitova et al., 2002; Zhu et al., 2003). In the normal brain, the progenitors proliferating in the SVZ migrate to the olfactory bulb by following the RMS. However, in the post-ischemic brain, many of the newly proliferated cells

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leave the RMS and migrate laterally from the ipsilateral SVZ into the striatal penumbral area (Parent et al., 2002a; Arvidsson et al., 2002; Jin et al., 2003; Yan et al., in press). Several of these survive and differentiate into mature striatal neurons and a few will differentiate into astrocytes (Parent et al., 2002a; Arvidsson et al., 2002). Some of the proliferating cells from the ipsilateral SVZ as well as PPv also migrate into the corpus callosum and the penumbral cortex (Jin et al., 2003), but almost no mature neurons differentiated from these migrated cells were observed in the cortex at the later time points (Parent et al., 2002a; Arvidsson et al., 2002). This may suggest that the injured cortex does not have a suitable environment for neuronal differentiation, either because of lack of instructive or survival cues or due to the presence of signals that inhibit neuronal survival or differentiation. 5. The mechanisms of ischemia-induced neurogenesis In the post-ischemic brain, neurogenesis can be controlled at three major steps—proliferation, migration and differentiation into neural phenotypes (Iwai et al., 2002). Although many studies evaluated these individual steps using various animal models, most focused on the first event, the neural progenitor proliferation after ischemia. 5.1. Factors that modulate post-ischemic progenitor proliferation As mentioned above, increased proliferation is reported not only in the ipsilateral DG and SVZ, but also contralaterally. This suggests that diffusible factors might play a major role in promoting post-ischemic progenitor proliferation. Transient focal ischemia is a strong stimulator of cerebral gene expression and several putative diffusible mitogens including growth factors, cytokines and cell division modulators are known to be upregulated in the ischemic cortex and striatum (Soriano et al., 2000; Vemuganti et al., 2002; Yan et al., 2006; Lu et al., 2003). 5.1.1. Epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) Of the various growth factors, EGF and FGF-2 are known to play a significant role in neurogenesis in vivo (Kuhn et al., 1997). They are also considered as essential for maintaining the pluripotency of the neural stem cell cultures in vitro (Reynolds et al., 1992; Richards et al., 1992). Importantly, FGF-2 expression significantly increases after cerebral ischemia (Endoh et al., 1994; Lin et al., 1997) and FGF-2 knockout mice show an attenuation of the focal-ischemia-induced progenitor proliferation (Yoshimura et al., 2001). Furthermore, FGF-2 expressing retrovirus significantly enhanced the postischemic progenitor cell proliferation in both wild-type mice and FGF-2 deficient mice (Yoshimura et al., 2001). A recent study showed that adenovirus programmed to deliver FGF-2 over longer time periods enhanced neurogenesis for 3 months after ischemia, suggesting its role in long-term neurological repair (Leker et al., 2007). EGF may have a similar effect as

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FGF-2. Ischemia causes an upregulation of EGF-receptor on transient amplifying cells, a population that could be induced to proliferate by EGF infusion (Ninomiya et al., 2006). Heparin binding EGF (HB-EGF) was shown to be upregulated following ischemia (Tanaka et al., 1999; Jin et al., 2002a). HB-EGF is known to act through both EGF and non-EGF receptors (Raab and Klagsbrun, 1997), and was shown to increase progenitor proliferation under culture conditions as well as in vivo in rodent brain (Jin et al., 2002a, 2005). Furthermore, adenoviralmediated increase in HB-EGF levels was shown to increase the BrdU positive cell number and enhance neurological recovery following focal ischemia (Sugiura et al., 2005). Infusion of a cocktail of FGF-2 and EGF into the lateral ventricles of adult rats was shown to increase the progenitor proliferation in the PPv after global ischemia and in the DG and SVZ after focal ischemia (Nakatomi et al., 2002; Tureyen et al., 2005). Increased progenitor proliferation in rats infused with FGF-2 and EGF was also shown to be associated with enhanced postischemic memory formation and retention (Nakatomi et al., 2002). 5.1.2. Brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and the stem cell factor (SCF) Transient focal ischemia was shown to induce both BDNF and GDNF in adult rodent brain (Takami et al., 1992; Kokaia et al., 1995; Lin et al., 1997; Kitagawa et al., 1999). Infusion of GDNF into lateral ventricles was also shown to significantly enhance the progenitor proliferation in both SVZ and DG after transient MCAO in adult rats (Dempsey et al., 2003). On the other hand, the role of BDNF in post-ischemic neurogenesis is not clear. BDNF heterozygous knockout mice showed increased neurogenesis and enhanced motor function recovery compared to the wild-type mice following focal ischemia indicating an inhibitory role of BDNF (Nygren et al., 2006a). On the other hand, two mitogens, erythropoietin (EPO) and nitric oxide (NO) formed by the endothelial NO synthase (eNOS), were suggested to promote post-ischemic neurogenesis by increasing BDNF levels (Wang et al., 2004; Chen et al., 2005). SCF is known to be a major player in the embryonic neurogenesis. Jin et al. (2002b) showed that SCF mRNA and protein levels were elevated in cultures from hypoxic stem cells. SCF significantly stimulated BrdU uptake into cultured stem cells and when cultures were exposed to hypoxiaconditioned medium containing SCF antibodies, BrdU labeling was reduced (Jin et al., 2002a). Furthermore, SCF infusion enhanced the neurogenesis in ischemic rodent brain with many of the BrdU+ newly proliferated cells expressing NeuroD, a marker for immature neurons (Jin et al., 2002b). 5.1.3. Erythropoietin (EPO) and vascular endothelial growth factor (VEGF) EPO is an important mitogen that plays a significant role in post-ischemic neurogenesis. Treatment of rats with EPO 24 h after ischemia not only increased the number of proliferating cells in the SVZ, but also increased the levels of other growth factors such as VEGF and BDNF (Jin et al., 2002c; Wang et al.,

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2004). EPO and its upstream transcription factor hypoxia inducible factor-1 (HIF-1) are known to be stimulated after ischemia (Jiang et al., 2001). EPO is known to promote differentiation of type B stem cells into type C transient amplifying cells, reducing the true stem cell population and increasing the number of lineage protected neuronal precursors (Shingo et al., 2001). EPO knockout mice as well as EPO conditional neural knockout mice have shown deficiencies in proliferation in the SVZ and post-ischemic neurogenesis (Tsai et al., 2006). Both VEGF and EPO are known to be downstream to the transcription factor HIF1a. Hayashi et al. (1997) showed that cerebral ischemia induces VEGF expression and Jin et al. (2002c) showed that VEGF promotes neurogenesis both in vitro and in vivo. A recent study showed enhanced postischemic neurogenesis in VEGF-overexpressing transgenic mice (Wang et al., 2007). Furthermore, Tonchev et al. (2007) showed that following transient global ischemia in monkeys, the SVZ progenitors show significant expression of the VEGF receptor fms-like tyrosine kinase-1 supporting a role for VEGF in post-iscemic progenitor proliferation. 5.1.4. Insulin-like growth factor-1 (IGF-1) Focal ischemia is known to significantly induce IGF-1 expression (Gluckman et al., 1992). A recent study from our laboratory showed that transient MCAO induces a persistent increase in IGF-1 mRNA and protein expression in the reactive astrocytes throughout the penumbral cortex and striatum (Yan et al., 2006). In addition, focal ischemia also induces IGFbinding proteins that play a role in transporting IGF-1, as well as IGF-1 receptor in the proliferating neural progenitors of SVZ and DG in rat brain (Yan et al., in press). If IGF-1 was neutralized in the post-ischemic rat brain by infusing IGF-1 antibodies into lateral ventricles, there was a significant decrease in the ischemia-induced neural progenitor proliferation (Yan et al., 2006). Stimulation of cultured neural progenitor cells with IGF-I enhanced the phosphorylation of phosphatidylinositol 3-kinase (PI-3-kinase)/Akt but not MEK/ extracellular signal-regulated kinase (ERK) (Kalluri et al., 2007). PI-3-kinase inhibitor, but not the ERK inhibitor prevented the IGF-I-induced increase in the progenitor cell number (Kalluri et al., 2007). IGF-1 also increased the phosphorylation of glycogen synthase kinase, supporting its role in the survival of neural progenitor cells, while FGF-2 significantly enhanced the progenitor proliferation in the presence of IGF-1 (Kalluri et al., 2007). Thus, we could infer that IGF-1 promotes the survival of the newly formed cells so that other growth factors could induce the proliferation. Previous studies from our laboratory also showed that continuous i.c.v. infusion of IGF-1 into rat brain significantly enhances the transient MCAO-induced neural progenitor proliferation (Dempsey et al., 2003). 5.1.5. Nitric oxide (NO) NO, which is known to be formed in excess following cerebral ischemia is a putative modulator of stroke-induced neurogenesis. Zhang et al. (2001b) showed that administration of DETA/NONOate (a NO donor) significantly increases cell

proliferation in the SVZ and DG of normal rats as well as those subjected to transient MCAO. Zhu et al. (2003) showed that inhibiting the inducible isoform of NOS (iNOS) prevents postischemic neurogenesis and iNOS knockouts failed to show MCAO-induced neurogenesis. A recent study suggested that increased iNOS modulates the astrocytic differentiation from the SVZ-generated progenitors after transient MCAO (Sehara et al., 2006). In addition, knockout mice lacking endothelial isoform of NOS (eNOS) showed curtailed neurogenesis as well as angiogenesis following focal ischemia (Chen et al., 2005). The effects of eNOS seem to be mediated by the growth factors BDNF and VEGF which were lowered in the eNOS knockout mice following focal ischemia (Chen et al., 2005). On the other hand, Sun et al. (2005) showed that lack of neuronal isoform of NOS (nNOS) as well as treatment with nNOS inhibitor 7nitroindazone induces post-ischemic neurogenesis. Hence, it seems that the isoform forming NO is very important in determining the stimulation or prevention of ischemia-induced neurogenesis. It was also shown that increasing production of NO and the down-stream cGMP by treating ischemic rats with sildenafil significantly enhances the neurogenesis (Zhang et al., 2006b). 5.1.6. Neurotransmitters Disrupted glutamatergic neurotransmission, in particularly altered receptor expression and function, is known to play a significant role in post-ischemic neuronal death. In addition, recent studies indicated a role for the glutamate system in postischemic neurogenesis. Activation of the NMDA subtype of glutamate receptors has been shown to repress progenitor proliferation, while NMDA receptor antagonists induce it (Cameron et al., 1995, 1998; Nacher et al., 2003). Bernabeu and Sharp (2000) observed that both NMDA and AMPA antagonists prevent neurogenesis in the DG following global ischemia. Kluska et al. (2005) also observed an increase in neurogenesis in the DG of rats when an NMDA antagonist was given during a focal ischemic insult induced by photothrombosis. Arvidsson et al. (2001) reported that after focal ischemia, NMDA, but not AMPA antagonists block neurogenesis. Other neurotransmitters are also thought to modulate neurogenesis in adult brain. Serotonin reuptake inhibitors, which are common antidepressants, have been shown to increase neurogenesis, providing a link between increased exposure to serotonin and increased proliferation (Malberg et al., 2000). Dopamine (which is the neurotransmitter that controls mood and motivation) is recently suggested to play a significant role in neurogenesis in adult brain (Borta and Hoglinger, 2007). Pharmacological manipulation of acetylcholine was also shown to alter adult brain neurogenesis (Kotani et al., 2006). However, the significance of these neurotransmitters in modulating post-ischemic neurogenesis still needs to be evaluated. 5.1.7. Hormones, inflammation and other factors A recent study showed that estradiol promotes post-ischemic neurogenesis in ovariectomized female mice (Suzuki et al., 2007). This effect was observed to be a receptor-mediated event as estrogen receptor knockout mice failed to respond to

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estradiol. In song birds, a causal relationship to the levels of testosterone and adult brain neurogenesis was demonstrated (Louissaint et al., 2002). On the other hand, 19-nortestosterone was shown to negatively effect the neural progenitor proliferation in adult rodent brain (Brannvall et al., 2005). The vasodilatory hormone adrenomedullin was recently shown to promote post-ischemic neurogenesis (Miyashita et al., 2006). While these studies indicate that hormones affect neurogenesis, no in-depth studies have evaluated their role in modulating post-ischemic neurogenesis. Inflammation seems to be another important modulator of neurogenesis. Focal ischemia is known to induce chronic inflammation in adult rodent brain which inhibits neurogenesis (Ekdahl et al., 2003; Monje et al., 2003). A recent study showed that preventing inflammation by treating rats with minocycline enhances post-ischemic neurogenesis (Liu et al., 2007). On the other hand, the complement system which plays a significant role in post-ischemic inflammatory reactions was recently shown to be a promoter of neurogenesis. Mice lacking complement factor C3 or C3a receptor as well as those treated with a C3a receptor antagonist showed significantly decreased SVZ neurogenesis following focal ischemia (Rahpeymai et al., 2006). Prevention of inflammation by the non-steroidal antiinflammatory drug indomethacin was also found to increase the number of BrdU+ cells co-labeled with DCX, nestin, GFAP and NG2 in the striatum and cortex (probably migrated from SVZ) at 14 and 28 days after focal ischemia (Hoehn et al., 2005). Cyclooxygense-2 (COX2) is a known promoter of inflammation in the post-ischemic brain and treatment with COX2 inhibitors was shown to significantly lower the newly proliferated cells after global ischemia in adult mice (Sasaki et al., 2003). This study also showed significantly lower neurogenesis in the COX2 knockout mice as compared to wildtype littermates following global ischemia. Tissue kallikrein is a serine protease that cleaves kininogen substrate to form kinin which is a potent vasodialator. Infusion of kallikrein protein into post-ischemic brain was shown to prevent inflammation and oxidative stress leading to neuroprotection (Chao and Chao, 2006). Furthermore, induction of kallikrein gene 8 h after transient MCAO was shown to significantly alleviate the neurological deficits at 2 as well as 7 days (Xia et al., 2006). This protective effect of kinin was thought to be mediated in part by its ability to promote angiogenesis and neurogenesis in addition to prevention of apoptosis. By virtue of its capability to induce arteriogenesis, granulocyte colony stimulating factor (G-CSF) infusion into the post-ischemic brain was shown to significantly induce neural progenitor proliferation (Schneider et al., 2005; Kawada et al., 2006). A recent study showed that treating rats with Notch ligands Delta-like4 and Jagged1 following permanent MCAO leads to enhanced neurogenesis (Androutsellis-Theotokis et al., 2006). These authors showed that activation of Notch induces PI-3kinase/AKT and the down-stream transcriptional mechanisms signal transducer and activator of transcription-3 (STAT3) and mammalian target of rapamycin (mTOR), and the further down-stream hairy and enhancer of split 3 (Hes3) and Sonic Hedgehog (Shh) leading to enhanced neurogenesis and

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functional recovery following focal ischemia. However, the role of Notch seems to be confusing as Notch activation during the acute phase of ischemia promotes neuronal death (Arumugam et al., 2006). A recent study showed that the Down syndrome cell adhesion molecule, which is known to play a significant role in neuronal development, was observed to be upregulated in the SGZ of monkey brain at 9–15 days following global ischemia indicating its putative significance in post-ischemic neurogenesis (Yamashima et al., 2006). 5.1.8. Enriched environment and preconditioning Owing to its stimulatory effect on many growth factors, exposing animals to an enriched environment following stroke was shown to enhance the post-ischemic neurogenesis (Komitova et al., 2005; Nygren et al., 2006b; Matsumori et al., 2006). In a similar fashion preconditioning (a short duration ischemia) which induces ischemic tolerance was also shown to promote growth factor expression and neurogenesis in rat brain (Naylor et al., 2005). 6. Migration and maturation of precursor cells following ischemia Progenitor cells proliferating in the DG, SVZ and PPv could help replace the neurons lost after ischemia if they could migrate to the areas of damage. In a normal adult brain, the cells proliferating in the SGZ migrate to the GCL and this pattern is not altered even after ischemia. On the other hand, the cells proliferating in the SVZ follow the RMS to migrate to olfactory bulb in the normal adult brain. Ischemia induces these newly generated neural precursors of SVZ to revoke their normal migratory pattern to migrate towards the injured areas of brain (Sun et al., 2004; Thored et al., 2006; Yan et al., in press). Arvidsson et al. (2002) showed that in the post-ischemic brain, many BrdU+/NeuN+ cells and a subset of BrdU+/DCX+ cells with the morphology of migrating neurons could be seen at the site of the infarct. These BrdU+/DCX+ cells extensively colabeled with Hu and Meis2, markers of proliferating progenitors and striatal neurons, showing that the cells might have migrated from the SVZ to the damaged area and assumed the phenotype of the neurons in the ‘‘target’’ area (Arvidsson et al., 2002). Using co-labeling with BrdU and DCX, as well as labeling SVZ cells with the fluorescent tracer DiI, Jin et al. (2003) showed that progenitor cells directly migrate from the SVZ to the striatum in the post-ischemic rat brain. The migrating cells become elongated, and express DCX and PSANCAM (the glycoprotein regulating cell adhesion and recognition) (Iwai et al., 2002; Tanaka et al., 2004). They migrate via chain migration to the infarcted region, reminiscent of migration to the olfactory bulb in the rostral migratory stream (Luskin, 1993; Shapiro et al., 2006). These newly generated cells can survive in the ipsilateral striatum and corpus callosum for >3 months Gu et al., 2000; Jiang et al., 2001). 3– 10% of the surviving cells were shown to express the immature neuronal marker MAP-2 and mature neuronal markers such as bIII Tubulin and NeuN at 2–3 months after ischemia (Gu et al.,

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2000; Jiang et al., 2001). The proliferation and survival of the newly generated neurons in the post-ischemic brain diminishes with age (Yagita et al., 2001; Rao et al., 2006). Interestingly, the migration of the newly proliferated progenitors to the area of damage was also shown following global ischemia (Nakatomi et al., 2002). Following global ischemia, the CA1 neurons will be completely lost by 1 week. Surprisingly, 1 month after ischemia some neurons reappear in the CA1 that are the newly proliferated progenitors from PPv that migrate to CA1 to repopulate that area (Nakatomi et al., 2002). While ischemia acts as a major stimulant for the SVZ-derived progenitors to go to the damaged areas of the brain instead of following RMS, many newly formed cells were also found in the olfactory bulb after focal ischemia in primates indicating that some cells still follow their natural route (Koketsu et al., 2006). 6.1. Factors that modulate migration of the newly proliferated progenitors in the ischemic brain Focal ischemia induces macrophage/leukocyte infiltration, astrocytosis and microglial activation in the ischemic areas in the infarct core as well as penumbral areas. These cells produce several factors including cytokines and chemokines which might be the putative chemoattractants that induce migration of proliferating progenitors to the areas of damage (Aarum et al., 2003; Dempsey et al., 2003; Imitola et al., 2004; Robin et al., 2006; Yan et al., 2006). 6.1.1. Monocyte chemoattractant protein-1 (MCP-1) MCP-1 is a chemokine of the CC family that binds to the Gprotein-coupled receptor CCR2 on its target cells (Rossi and Zlotnik, 2000). MCP-1 is a known chemoattractant for monocytes/macrophages, T lymphocytes, basophils, and NK cells (Rossi and Zlotnik, 2000). Moreover, MCP-1 increases the cultured neural progenitor migration across a chemotaxis chamber (Widera et al., 2004), with those progenitor cells expressing diverse chemokine receptors (Tran et al., 2004; Ji et al., 2004). MCP-1 was previously shown to increase the migration of bone marrow stromal cells and neural progenitors in vitro (Wang et al., 2002; Widera et al., 2004). We recently reported that transient MCAO in adult rats induces a sustained induction of MCP-1 expression in the activated microglia and astrocytes lasting for more than 3 days of reperfusion (Yan et al., in press). We observed that MCP-1 is a strong attractant of neurospheres in cultures as well as when transplanted into adult rat brain. Furthermore, the migrating neuroblasts in the ischemic brain express the MCP-1 receptor CCR2, and MCP-1 and CCR2 knockout mice display significantly lower progenitor migration after transient MCAO (Yan et al., in press). 6.1.2. Stromal cell derived factor-1a (SDF1-a) and SCF The migration of the newly formed cells has been shown to last for at least 4 months following ischemia, with SDF1-a as the proposed major regulator possibly formed by reactive astrocytes (Thored et al., 2006; Miller et al., 2005). Blocking SDF-1a by a neutralizing antibody against its receptor CXCR4

was shown to significantly attenuate neural progenitor migration in the post-ischemic brain (Robin et al., 2006). In addition, SCF which was shown above to increase proliferation also induces migration and survival of the newly formed cells (Sun et al., 2004). Ohab et al. (2006) demonstrated that postischemic neuroblast migration and behavioral recovery is mediated by SDF1 and the down-stream angiopoietin-1. 6.1.3. Matrix metallloproteinases (MMPs) Previous studies have shown that MMP induction during the acute phase after focal ischemia is detrimental as MMPs damage the neurovascular unit to precipitate the edema, BBB breakdown and hemorrhaging leading to neuronal death (Asahi et al., 2001; Kamada et al., 2007; Yang et al., 2007; Gu et al., 2005; Tejima et al., 2007). However, recent studies showed that MMP induction plays a vital role in promoting progenitor proliferation during the chronic phase after focal ischemia. MMP9, which is upregulated in the infarcted cortex at 7–14 days in rats (Zhao et al., 2006) has been shown to colocalize with DCX and BrdU positive cells migrating from the SVZ, while blocking the MMP activation can severely diminish striatal migration (Lee et al., 2006). Wang et al. (2006) showed that if mouse SVZ neural progenitor cells are co-cultured with epithelial cells and treated with recombinant EPO, MMP2 and MMP9 expression increases significantly. Furthermore, it was shown that conditioned medium from the EPO-treated epithelial cell cultures significantly increased the neural progenitor migration which was prevented by an MMP inhibitor (Wang et al., 2006). 7. Functional integration of the newly formed cells in the post-ischemic brain Even with the increased progenitor proliferation and subsequent migration, the question is whether these cells are capable of functionally integrating into the adherent architecture and reconnecting to the correct pathways. They need to regrow long axonal projections to the correct target to adequately augment the injury, and axons do not grow well in the adult brain. After migration to the damaged penumbral area of the striatum, some of the newly generated cells differentiate into mature striatal neurons (Arvidsson et al., 2002; Yamashita et al., 2006) and to a lesser degree to astrocytes (Parent et al., 2002a). The newly proliferated cells also migrate to the damaged cortex (laterally from the RMS, or along the interface of the corpus callosum), but they rarely express mature neuronal markers at later time points (Parent et al., 2002a; Arvidsson et al., 2002; Jin et al., 2003). This suggests that the ischemic areas have cues to attract neuroblasts, but not to efficiently orchestrate their differentiation and integration. Nakatomi et al. (2002) most convincingly showed the functional integration of the new neurons in the adult brain following transient global ischemia. Their studies have shown that following complete loss of the CA1 neurons, the newly proliferated cells remain in the germinal zones at 10–12 days, but many of these from the PPv migrate to CA1 by 28 days and survive for at least 2 months (Liu et al., 1998; Nakatomi et al.,

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2002). These studies also showed that the dendritic structures were restored in the CA1, the Schaffer collaterals elicited correct field excitatory potentials and the animals performed significantly better in the Morris Water-Maze tasks by 84–120 days if the animals were treated with EGF and FGF-2 following global ischemia (Nakatomi et al., 2002). However, Arvidsson et al. (2002) cast doubt on the functional significance of postischemic neurogenesis as <0.2% of neurons destroyed after MCAO were observed to be replaced in the infarcted striatum and an even smaller percentage in the infarcted cortex. As most newly formed cells undergo apoptosis, prevention of apoptosis by overexpressing B-cell leukemia/lymphoma 2 (Bcl2) gene was shown to improve the survival of the newly proliferated cells in the post-ischemic brain (Zhang et al., 2006a; Sasaki et al., 2006). Magavi et al. (2000) showed that SVZ neuroblasts migrated to the injury site and differentiated into NeuN+ mature neurons which survived for at least 28 weeks, and developed corticothalamic connections following targeted induction of apoptosis of the corticothalamic neurons in layer VI of the adult mouse anterior cortex. Using a GFP retrovirus injected into the dentate gyrus of gerbils 48 h prior to ischemia, Tanaka et al. (2004) showed that the newly formed cells coexpress GFP and adult neuronal markers with increasing percentages at 5, 10, and 30 days after global ischemia. They also showed that with this increased time, the dendritic lengths of these cells increase. There is some controversy on the role of growth factors in the functional integration of the newly formed cells after ischemia. Bendel et al. (2005) showed that EGF and FGF2 are not needed for the functional integration. However, growth factors may increase the incorporation of new neurons into the DG at 3 weeks after focal ischemia (Tureyen et al., 2005). Conversely, Fluorogold injection into the ipsilateral substantia nigra pars reticulata after focal ischemia has failed to show any striatal colocalization 6 weeks after ischemia (Lichtenwalner and Parent, 2006). A recent study showed that infusion of bone morphogenic protein-7 (BMP7) into lateral ventricles of adult rats following transient MCAO promotes progenitor proliferation in the SVZ and many of the newly formed cells (BrdU/ NeuN colabeled) migrate to the ischemic striatum and cortex and promote significant functional recovery at 1 month after stroke (Chou et al., 2006). As many new cells formed in the ischemic brain perish, increasing their survival promotes their maturation and functional integration. In addition to EGF and FGF-2 which were shown to promote survival of the newly proliferated progenitors (Nakatomi et al., 2002; Tureyen et al., 2005), BDNF was shown to improve the functional outcome and striatal survival in the ischemic brain (Andsberg et al., 2002). Treating ischemic animals with tumor necrosis factor-a (TNF-a) antibody led to the formation of much fewer BrdU+ striatal and hippocampal neurons indicating that TNF-a formed in excess in the ischemic brain might promote the survival of the newly formed cells (Heldmann et al., 2005). Other studies showed that infusion of GDNF (Kitagawa et al., 1999; Tsai et al., 2000), EGF alone (Teramoto et al., 2003) and VEGF (Zhang et al., 2000) also decreases post-ischemic neuron death and/or increase functional recovery. Particularly VEGF may be

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important due to its ability to increase the neovascularization (Sun et al., 2003; Mabuchi et al., 2005). With increased capillary number, the chances for survival of the newly formed cells increases. It was shown that neurons may only be able to survive and differentiate and in very close proximity to a blood nutrient source in monkey brain (Yamashima et al., 2004). In addition, environmental enrichment which is known to improve several motor and cognitive tasks, might act by increasing proliferation as well as survival of the newly formed neurons under normal as well as ischemic conditions (Ohlsson and Johansson, 1995; Dahlqvist et al., 2004; Gobbo and O’Mara, 2004; Pereira et al., 2007). 8. Neurogenesis after traumatic brain injury (TBI) and other neurological insults Several other acute and chronic insults to CNS also modulate the neurogenesis in adult brain. Any injury to CNS induces stress, and stress-related hormones were shown to downregulate neurogenesis in the DG (Cameron and Gould, 1994). On the other hand, excitotoxic and mechanical lesions can stimulate DG proliferation (Gould et al., 1997). Interestingly, increased progenitor proliferation following TBI in mouse brain was shown 32 years ago (Reznikov, 1975). TBI-induced neurogenesis in rodents was confirmed by many later studies that used controlled cortical impact (CCI) and fluid percussion (FP) injuries as well as subarachnoid hemorrhage models (Kernie et al., 2001; Dash et al., 2001; Chirumamilla et al., 2002; Mino et al., 2003). Chen et al. (2003) showed that increased proliferation persists for almost a year after FP injury in adult rats. Similar to ischemia, FGF-2 was shown to play a significant role in post-TBI neurogenesis in rodents (Yoshimura et al., 2003). Furthermore, infusion of the astrocytic mitogenic protein S100B significantly enhances the post-TBI neurogenesis associated with cognitive improvement (Kleindienst et al., 2005). In addition to neurogenesis, the proliferating progenitors also form glial cells in mouse brain after TBI (Rola et al., 2006). The gliogenic response following TBI seems to be mediated by the induction of Notch signaling pathway mediated by its activators Jagged1 and Delta 1 (Givogri et al., 2006). Studies also have shown that environmental enrichment increases the DG progenitor proliferation following FP injury (Gaulke et al., 2005). Similar to ischemia, the newly proliferated progenitors tends to migrate from SVZ to the site of injury in the cortex after TBI (Sundholm-Peters et al., 2005). Recent studies showed that the newly generated neurons in the SGZ extend axons towards CA3 region as well as integrate into the DG to promote cognitive recovery following TBI (Emery et al., 2005; Sun et al., 2007). TBI can lead to epileptic seizures which are known to increase the progenitor proliferation in both SVZ and SGZ of rodents (Parent et al., 2002b; Smith et al., 2006; Gray et al., 2002). 9. Conclusions Ischemia causes seemingly irresolvable damage to brain. The discovery of the neural stem cells and the subsequent observations of increased neurogenesis in adult brain following

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ischemia has led to optimism that the post-ischemic brain can be repaired at least to a certain extent. Many endogenous molecular mechanisms synergistically promote the postischemic progenitor cell proliferation, survival, migration to the areas of damage and functional integration. In addition, several exogenous agents including a variety of growth factors promote the post-ischemic neurogenesis. At this point, the major challenges seem to be inducing sufficient proliferation to repair the devastating neuronal damage following ischemia, promoting the survival of the newly formed cells in the hostile environment that is skewed towards cell death, and more importantly to induce proper connectivity of the newly formed cells with the existing circuitry. Acknowledgments The authors’ laboratories were funded by grants from the United States National Institutes of Health, the American Heart Association and the University of Wisconsin Medical School. References Aarum, J., Sandberg, K., Haeberlein, S.L., Persson, M.A., 2003. Migration and differentiation of neural precursor cells can be directed by microglia. Proc. Natl. Acad. Sci. U.S.A. 100, 15983–15988. Altman, J., 1962. Are new neurons formed in the brains of adult mammals? Science 135, 1127–1128. Altman, J., 1969. Autoradiographic and histological studies of postnatal neurogenesis. IV. Cell proliferation and migration in the anterior forebrain, with special reference to persisting neurogenesis in the olfactory bulb. J. Comp. Neurol. 137, 433–435. Alvarez-Buylla, A., Seri, B., Doetsch, F., 2002. Identification of neural stem cells in the adult vertebrate brain. Brain Res. Bull. 6, 751–758. Androutsellis-Theotokis, A., Leker, R.R., Soldner, F., Hoeppner, D.J., Ravin, R., Poser, S.W., Rueger, M.A., Bae, S.K., Kittappa, R., McKay, R.D., 2006. Notch signalling regulates stem cell numbers in vitro and in vivo. Nature 442, 823–826. Andsberg, G., Kokaia, Z., Klein, R.L., Muzyczka, N., Lindvall, O., Mandel, R.J., 2002. Neuropathological and behavioral consequences of adenoassociated viral vector-mediated continuous intrastriatal neurotrophin delivery in a focal ischemia model in rats. Neurobiol. Dis. 9, 187–204. Arumugam, T.V., Chan, S.L., Jo, D.G., Yilmaz, G., Tang, S.C., Cheng, A., Gleichmann, M., Okun, E., Dixit, V.D., Chigurupati, S., Mughal, M.R., Ouyang, X., Miele, L., Magnus, T., Poosala, S., Granger, D.N., Mattson, M.P., 2006. Gamma secretase-mediated Notch signaling worsens brain damage and functional outcome in ischemic stroke. Nat. Med. 12, 621–623. Arvidsson, A., Kokaia, Z., Lindvall, O., 2001. N-methyl-D-aspartate receptormediated increase of neurogenesis in adult rat dentate gyrus following stroke. Eur. J. Neurosci. 14, 10–18. Arvidsson, A., Collin, T., Kirik, D., Kokaia, Z., Lindvall, O., 2002. Neuronal replacement from endogenous precursors in the adult brain after stroke. Nat. Med. 8, 963–970. Asahi, M., Wang, X., Mori, T., Sumii, T., Jung, J.C., Moskowitz, M.A., Fini, M.E., Lo, E.H., 2001. Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia. J. Neurosci. 21, 7724–7732. Bayer, S.A., Yackel, J.W., Puri, P.S., 1982. Neurons in the rat dentate gyrus granular layer substantially increase during juvenile and adult life. Science 216, 890–892. Beauquis, J., Roig, P., Homo-Delarche, F., De Nicola, A., Saravia, F., 2006. Reduced hippocampal neurogenesis and number of hilar neurones in streptozotocin-induced diabetic mice: reversion by antidepressant treatment. Eur. J. Neurosci. 23, 1539–1546.

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