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Neurogenesis following brain ischemia
Developmental Brain Research 134 (2002) 23–30 www.bres-interactive.com
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Neurogenesis following brain ischemia Frank R. Sharp a , *, Jialing Liu b , Ramon Bernabeu a a
Department of Neurology and Neuroscience Program, Vontz Center Rm 2327, 3125 Eden Avenue, University of Cincinnati, Cincinnati, OH 45267 -0536, USA b Department of Neurosurgery, University of California, San Francisco, CA, USA Accepted 5 October 2001
Abstract Following 5 or 10 min of global ischemia in the adult gerbil there is a tenfold increase in the birth of new cells in the subgranular zone of dentate gyrus of the hippocampus as assessed using BrdU incorporation. This begins at 7 days, peaks at 11 days, and decreases thereafter. Over the next month approximately 25% of the newborn cells disappear. Of the remaining cells, 60% migrate into the granule cell layer where two-thirds become NeuN, calbindin and MAP-2 immunostained neurons. The remaining 40% of the cells migrate into the dentate hilus where 25% of these become GFAP labeled astrocytes. It is proposed that ischemia-induced neurogenesis contributes to the recovery of function, and specifically may serve to improve anterograde and retrograde recent memory function that is lost following global ischemia in animals and man. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Genesis of neurons and glia Keywords: Stem cell; Neurogenesis; Ischemia; Stroke; Memory
1. Introduction Stem cells that self-renew and that are multipotent are found in brain. Isolated cells from the adult mammalian forebrain form colonies of undifferentiated cells in vitro that can be dissociated to form many more secondary colonies, demonstrating renewal. Moreover, these cells are multipotent since they can be induced to differentiate into neurons, astrocytes and oligodendrocytes [49,57,15,44]. Stem cells can give rise to other stem cells as well as progenitor cells. Progenitor cells give rise to neurons, astrocytes, and oligodendrocytes. Microglia appear to be derived mainly from blood borne stem cells [15,44]. Though many stem cells are found in the adult subventricular zone (SVZ) which is adjacent to the lateral ventricles [11], stem cells can also be isolated from hippocampus [46], spinal cord [56], diencephalon [56] and other brain regions [33]. A number of recent reviews [57,15,44] have prompted this short review to focus on *Corresponding author. Tel.: 11-513-558-7087; fax: 11-513-5587009. E-mail address: [email protected] (F.R. Sharp).
ischemia-related neurogenesis in brain, and specifically on ischemia-induced cell proliferation and its possible relation to recovery of short-term memory following hippocampal damage produced by global ischemia.
2. Subventricular zone Proliferating subventricular zone (SVZ) cells are located along the lateral edge of the lateral ventricle, and are most numerous along the frontal horn. SVZ cells migrate via the rostral migratory stream (RMS) to the olfactory bulb. Once in the bulb they integrate into the granule and glomerular layers where they differentiate into local interneurons [36,40]. The identity of the stem cells in the SVZ remains uncertain. Electron microscopy shows ependymal cells lining the lateral ventricles, and three types of cells between the ependyma and the parenchyma of the striatum [11,17]. The majority of the cells in the SVZ are migrating type A neuroblast cells that continue to proliferate. Slowly proliferating type B cells (GFAP-positive, astrocyte-like cells) ensheath migrating type A cells. Type C cells, the
0165-3806 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00286-3
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least numerous but most actively proliferating cell in the SVZ, form small clusters throughout the SVZ. Following administration of an antimitotic drug (Ara-C), type B cells are the first to appear, and apparently give rise to type A and C cells [10]. Viral transfection studies also support the possibility that type B cells in the SVZ are a stem cell. It appears, therefore, that the slowly proliferating GFAPpositive type B cells represent at least one type of stem cell in brain [10]. There is also evidence that some ependymal cells might also be stem cells, since isolated dye-labeled ependymal cells appear to divide in vitro and develop into neurospheres [32]. Many factors can affect neural stem cells in vitro. BFGF, EGF, TGF, IGF-1 and monoamines increase proliferation [6]. Glutamate, GABA, and opioid peptides tend to decrease proliferation. PDGF, CNTF, VIP and other compounds can also affect proliferation and differentiation [6]. Infusions of EGF and bFGF into the lateral ventricle expand the SVZ progenitor population in vivo, bFGF increasing the numbers of neurons reaching the olfactory bulb and EGF enhancing astrocyte differentiation [37]. Following removal of the olfactory bulb, adult SVZ neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb [36], though this slows over time. This suggests that there is only modest feedback from the olfactory bulb to the SVZ. A recent study shows that focal cerebral ischemia increases the birth of cells in the SVZ, and that some of these cells form mature neurons and other cells form immature neurons or undifferentiated cells [27]. These changes of cell proliferation occur within days of the stroke [27].
3. Dentate subgranular zone Adult hippocampal stem and progenitor cells are located at the border between the dentate hilus and the inner margins of the upper and lower blades of the dentate granule cell layer (Fig. 1). The newborn cells in normal hippocampus migrate into the granule cell layer where the majority of the cells differentiate into neurons that assume a granule cell phenotype [2,16,7,20]. These cells are present in the brains of rodents [22], primates [21,23] and man [13]. Many factors up or down regulate the proliferation of the subgranular zone (SGZ) progenitor cells. NMDA receptor antagonists induce proliferation of dentate gyrus progenitor cells and increase newborn neurons in the granule cell layer. Enriched environments increase the total number of granule cell neurons by increasing the survival rate of the progeny from the dividing precursor cells [34]. Voluntary wheel running increases the proliferation of dentate gyrus stem cells and increases the survival of these cells [54]. Adrenal steroids, and stress on the other hand, decrease proliferation of dentate gyrus progenitor cells [5,18]. Similarly, differentiation of neurons within the
Fig. 1. BrdU labeled cells (green, left panels) and NeuN stained cells (red, right panels) in dentate gyrus from a control animal (upper two panels), from an animal 11 days after ischemia (middle two panels) and from an animal 30 days following ischemia (lower two panels). BrdU was given 24 h prior to sacrifice. Note the marked increase of BrdU labeled cells (middle, left panel) in the dentate subgranular zone compared to control (upper left) and and compared to 1 month following ischemia (lower left panel).
adult rat dentate gyrus is reduced in hypothyroid animals [41]. Cell birth rates are decreased in the SGZ of aged animals, and animals with higher rates of cell birth appear to have better memory function than those with lower rates of cell birth [35]. Since injury to granule cells promotes dentate neurogenesis [22], glutamate receptors modulate cell proliferation, and status epilepticus produces hippocampal injury and stimulates neurogensis in the SGZ [47], it was anticipated that cerebral ischemia would also modulate dentate cell birth rates as outlined below.
4. Injury induces stem cells in vertebrates Non-mammals, including fish, regenerate neurons and axons in the CNS after experimental injuries [1,33,52]. Acetylpyridine induced injury in the lizard medial cortex stimulates cell proliferation with neuroblasts and immature neurons gradually replacing dead neurons at the lesion site [45,14]. Lesions of the nucleus ectostriatum in the adult ring-dove increased the birth of newborn neurons over two-fold [62]. Therefore, in vertebrates at large, some injury-induced neurogenesis appears to be the rule. Injuryinduced neurogenesis also occurs in the brains of adult mammals, but it is much less robust and much more restricted.
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Fig. 2. Rate of cell birth (number of cells labeled with BrdU in a 24-h period) in the dentate SGZ (mm 2 ) at 6 days to 5 weeks following global cerebral ischemia in the adult gerbil brain compared to controls. Animals were administered BrdU 24 h prior to sacrifice. BrdU immunostained cells in the SGZ were counted and normalized to the area of the dentate hilus (mm 2 ).
5. Ischemia induces birth of cells in the rodent brain Cell birth was studied in the brain of the adult gerbil with the DNA analogue, bromodeoxyuridine (BrdU). BrdU is taken up into the DNA of dividing cells, and immunolocalized in the nuclei of these cells using an antibody specific for BrdU (see Figs. 1 and 3, green cells). There are a number of BrdU labeled cells in the SGZ of the dentate gyrus of the hippocampus in normal, adult rat brain (Fig. 1, control, upper left panel). BrdU incorporation into cells in the SGZ, given 24 h prior to sacrifice, did not change for the first 6 days following global ischemia (Fig. 2). Thereafter, cell proliferation increased markedly and was maximal at 11 days after ischemia (Fig. 1, middle panel) [39]. There was a twelve-fold increase in BrdU immunoreactive nuclei per mm 2 dentate gyrus compared to control animals (Fig. 2). Cell proliferation decreased by 14 days after ischemia but was still greater than control. The number of dividing cells appeared to return to control levels in most animals 3–5 weeks after the ischemia (Figs. 1 and 2). The explanation for the 1-week long delay between the ischemia and onset of neurogenesis is unknown.
6. Ischemia induces new neurons To determine the fate of the BrdU labeled neurons, adult gerbils were subjected to 10 min of global ischemia and given BrdU twice a day on days 9–12 after ischemia to maximally label proliferating cells. At 15 days after ischemia, BrdU labeled cells were found mainly in the
SGZ (green cells, Fig. 3) and did not express either neuronal or astrocyte markers (Table 1). By 26 days after ischemia 27% of the BrdU immunoreactive cells expressed the neuronal marker NeuN (Table 1). By 40 days after
Fig. 3. Newborn cells in the dentate SGZ at 15 days following global ischemia. Gerbils subjected to 10 min of global ischemia were given BrdU on days 9–12 following ischemia. On day 15 following ischemia brain sections were immunostained for NeuN (red) and BrdU (green). BrdU labeled newborn cells (green) are shown in the dentate subgranular zone (SGZ) adjacent to the NeuN stained granule cell neurons (red) in the granule cell layer. Note that there are some BrdU and NeuN doublelabeled cells (yellow) that presumably represent newborn neurons.
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Table 1 Numbers of BrdU immunoreactive cells and their phenotypes in two regions of the dentate gyrus of hippocampus at 15, 26 and 40 days following global ischemia in the adult gerbil, GCL1SGZ5granule cell layer and subgranular zone Time after ischemia (days)
Treatment (n)
Regions of the dentate gyrus GCL1SGZ Total
a
Dentate hilus NeuN
b
GFAP
b
Total
NeuN
GFAP
15
Control (6) Ischemia (6)
4565 220657
0 0
0 0
261 1766
0 0
0 261
26
Control (6) Ischemia (6)
1663 113623
361 3065
0 0
361 69637
0 0
0 763.2
40
Control (6) Ischemia (6)
1163 9166
862 5664
0 0
361 78641
0 0
0 2066
GCL, granule cell layer; SGZ, subgranular zone; NeuN, a neuronal marker in the nucleus of mature mammalian neurons; GFAP, glial fibrillary acidic protein; the number in parentheses is the number (n) of animals in each control or ischemia group. This table has been previously published by Liu et al. [39]. a Total number of BrdU immunoreactive nuclei labeled by FITC detected by immunofluorescence microscopy. b Number of BrdU-labeled cells also immunoreactive for NeuN or GFAP, as detected by confocal microscopy.
ischemia 61% of the BrdU labeled cells expressed NeuN, and were therefore newborn, differentiated neurons (Table 1). The newborn neurons were no longer localized in the SGZ, but were distributed throughout the granule cell layer. Fig. 4 shows BrdU labeled cells in green and granule cell neurons in red. The BrdU and NeuN double-labeled neurons were yellow, and hence contained both labels as visualized by the confocal microscope (Fig. 4). These BrdU labeled neurons colocalized with calbindin-D28k and MAP-2 as well as with NeuN [39]. There could be some
Fig. 4. Newborn neurons in the granule cell layer 40 days following global ischemia. Gerbils subjected to 10 min of global ischemia were given BrdU on days 9–12 following ischemia. On day 40 following ischemia brain sections were immunostained for NeuN (red) and BrdU (green). Note that in this section through the granule cell layer that all of the BrdU labeled cells (which would be green) colocalize with NeuN. Hence, all of the yellow cells are BrdU and NeuN double-labeled cells that represent newborn neurons.
concern that BrdU would be taken up into cells that were undergoing DNA repair. However, we also showed the presence of PCNA (proliferating cell nuclear antigen) in NeuN labeled cells, confirming the birth of new neurons [39]. Moreover, we never observed BrdU uptake into dying neurons in CA1, or into any other injured cell. In addition, TUNEL staining following ischemia never shows any cells with fragmented DNA localized to the SGZ following global ischemia [24]. Lastly, because of the concern that newborn cells might undergo apoptosis, we also examined BrdU labeled cells many months following ischemia. Fig. 5 shows a number of BrdU and NeuN double labeled neurons (yellow cells) in the granule cell
Fig. 5. BrdU labeled, NeuN double labeled newborn neurons (yellow cells) survived for over 7 months in the granule cell layer (NeuN stained neuronal nuclei in the granule cell layer are red) following global ischemia in the adult gerbil brain.
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layer of one animal over 7 months following ischemia. This suggests that many of the newborn neurons can survive for very long periods following the ischemia.
7. Ischemia induces new glia To better address the fate of the BrdU labeled cells following global ischemia, cell counting was performed (Table 1) [39]. Beginning 15 days after ischemia roughly 220 cells (per mm 2 dentate657 cells) were detected in the SGZ of ischemic animals (Table 1). By 40 days after ischemia 91 cells (per mm 2 dentate16 cells) were found in the granule cell layer and 78 cells (per mm 2 dentate141 cells) were found in the dentate hilus (Table 1). Thus 75% of the cells that were present at 15 days after ischemia (3 days after labeling) were still present at 40 days after ischemia. Hence, a quarter of the newborn cells died or had sufficient dilution of the labeling that they could not be detected after the first month. When double labeling was performed, approximately 60% of the BrdU labeled cells in the granule cell layer were also NeuN double stained, and therefore had differentiated into neurons. None of the BrdU labeled cells in the granule cell layer appeared to differentiate into GFAP stained astrocytes. GFAP staining at 15 days after ischemia showed that none of the BrdU labeled cells in the SGZ had differentiated into astroyctes (Fig. 6). Indeed, most of the GFAP stained astrocytes in dentate are found in the dentate hilus (h) and not in the granule cell layer (Fig. 7). By 40
Fig. 6. Newborn cells in the dentate SGZ at 15 days following global ischemia. Gerbils subjected to 10 min of global ischemia were given BrdU on days 9–12 following ischemia. On day 15 following ischemia brain sections were immunostained for GFAP (red) and BrdU (green). BrdU labeled newborn cells (green) are shown in the dentate subgranular zone (SGZ) adjacent to the GFAP stained cells in the dentate hilus, and a few GFAP stained cells in the granule cell layer.
Fig. 7. Newborn astrocytes in the dentate hilus 40 days following global ischemia. Gerbils subjected to 10 min of global ischemia were given BrdU on days 9–12 following ischemia. Animals were allowed to survive 40 days following ischemia. Brain sections were immunostained for GFAP (red) and BrdU (green). Note that there are BrdU labeled cells (green), GFAP labeled astrocytes (red), and BrdU and GFAP double labeled cells (yellow). The yellow cells are newborn astrocytes that are presumably derived from cells in the SGZ that migrated into the hilus and differentiated into GFAP positive astrocytes.
days after ischemia a number of BrdU labeled cells (green) colocalized with GFAP (red) to produce double-labeled yellow cells in dentate hilus (Fig. 7). Only about 25% of the BrdU labeled, newborn cells in the dentate hilus, however, differentiated into GFAP stained astrocytes (Table 1). The 40% of the BrdU labeled cells that are not neurons or astrocytes in the granule cell layer could either be oligodendrocytes or regenerated stem or progenitor cells. The 75% of the BrdU labeled cells in the hilus that are not neurons or astrocytes similarly could be differentiated oligodendrocytes or regenerated stem or progenitor cells. They are not microglia, since newborn microglia are found between 1 and 6 days following ischemia [38], and the stem cells found in adult brain are not believed to develop into microglia [15,7]. In summary, 25% of the cells that are born in the SGZ following ischemia die in the first month. Of the surviving cells, 60% migrate into the SGZ and 40% migrate to the dentate hilus. In the granule cell layer 60% of the cells differentiate into neurons and none differentiate into astrocytes; this contrasts to the hilus where none of BrdU labeled cells differentiate into neurons whereas 25% of the cells differentiate into astrocytes. These data suggest that neuronal differentiation factors predominate in the granule cell layer, whereas astrocyte differentiation factors in the hilus are more prominent. The stimuli for this differential neuronal and glial differentiation are unclear since there is
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thought to be little neuronal cell death in the granule cell neuronal layer, while there is definite loss of somatostatin and other dentate hilar interneurons following global ischemia [53,28,26].
8. Mechanism of ischemia induced neurogenesis Neurogenesis in the developing brain is regulated at least in part by NMDA receptors, and blockade of NMDA receptors with MK801 increases the cell birth rate in adult dentate gyrus [19,20]. In addition, lesions of entorhinal cortex that decrease glutamate inputs to dentate and decrease NMDA receptor activation also stimulate dentate cell proliferation. However, neuronal cell death was not detected in entorhinal cortex following 5–10 min of global ischemia with either NeuN staining [39] or with TUNEL staining [24]. This suggests that loss of glutamatergic cells in the perforant pathway from entorhinal cortex to dentate is not the cause of ischemia-induced neurogenesis. It is possible that neuronal death within the hippocampus provided the stimulus for increased neurogenesis following ischemia. For example, limbic seizures that cause apoptosis of granule cells [3] increase dentate neurogenesis [47]. However, granule cell loss is not a significant feature of moderate durations of global ischemia [24,25]. To determine whether ischemic loss of CA1 neurons might be the stimulus for neurogenesis, gerbils were subjected to 2 min of preconditioning ischemia, followed 2 days later by 5 min of ischemia. There was no death of CA1 neurons using this preconditioning paradigm, but there was increased neurogenesis equal to that produced by 5 min of global ischemia alone [39]. However, it should be noted that there is death of dentate hilar interneurons using this ischemia preconditioning paradigm [28,53,25] that could result in altered dentate activity and alterations of NMDA receptor regulation that could stimulate neurogenesis. Growth and mitogenic factors could play a role in the ischemia-induced neurogenesis, but there is no direct evidence for this. The increases of basic FGF that occur in hippocampal glial cells after ischemia [12] might stimulate neurogenesis [39,47]. Infusions of bFGF into the ventricles increases the numbers of cells in the SVZ [37], though any effects on the SGZ were not reported. Lastly, changes of NMDA receptors may also mediate ischemia-induced neurogenesis. There is a 20% reduction in NMDA receptor binding in the rat dentate gyrus 1 week after transient forebrain ischemia [59,58]. The NMDA receptor binding returns to control levels 2 weeks after ischemia [58]. Ischemia-induced neurogenesis in gerbils reached a maximum at 9–11 days after ischemia and declined towards control levels 3 days later [39]. The similarity of these time courses raises the possibility that ischemia-induced decreases in NMDA receptor signaling contribute to neurogenesis in the dentate gyrus. We have
shown that administration of NMDA and AMPA-kainate antagonists directly into hippocampus prevents the cell death produced by global ischemia and also blocks the neurogenesis normally produced by global ischemia [4]. These data could mean that these glutamate receptors mediate neurogenesis via downstream signaling, or they could mean that hippocampal cell death is necessary to stimulate neurogenesis [4].
9. Neurogenesis in recovery of memory function following global ischemia The hippocampus plays a central role in learning and recent memory. Bilateral injury to CA3 neurons produced by status epilepticus and bilateral injury to granule cell neurons produced by hypoglycemia produces a disorder of recent memory similar to, if not identical to that produced by global ischemia. Following relatively short durations of global ischemia in rodents, primates and man [50,61] there is bilateral loss of some or all of the CA1 pyramidal neurons in hippocampus. This injury is associated with an anterograde and retrograde amnesia but with preservation of long-term memory. Animals and people lose memories for short periods prior to the ischemia, and also have difficulty forming new language or spatial memories [50]. Animals, for example, may perform well on a spatial task learned in the distant past, but will not be able to learn the location of a submerged platform in a new Morris water maze task [8,60]. It is tempting to speculate that the ischemia-induced neurogenesis observed in these studies could contribute to the recovery of memory function that has been documented in humans [9] and in experimental animals [8,60] following global ischemia. It is notable that rats housed in an enriched environment after global and focal ischemia showed improved cognitive functions compared to those housed in the standard environment [29–31,48]. Enriched environments increase the survival of newborn cells in normal adult mice [34,55]. The recovery of memory function following global ischemia and the improved cognitive function in enriched environments both correlate with dentate neurogenesis. A recent study also shows synapsin upregulation in the mossy fiber layer of CA3 following global ischemia [43]. This suggests that existing granule cell mossy fibers sprout in CA3, resulting in increased synaptogenesis. In addition, the newly formed granule cell neurons extend axons to CA3 [51], and form new synapses on CA3 target neurons [42]. These data suggest that following hippocampal injury produced by global ischemia, recovery of function could be mediated by at least two factors. Existing granule cell neurons may sprout and form more synapses on CA3 neurons, and the new neurons that are born in the granule cell layer may also form new synapses on CA3 neurons. The role of these new synapses in recovery of memory function and the
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signally pathways that set them in motion remain to be determined.
Acknowledgements Much of this material and the figures used in this review were previously published in The Neuroscientist, Neurogenesis and gliogenesis in the postischemic brain, 6(5): 362–370, 2000 which was authored by J. Liu, R. Bernabeu, A. Lu and F.R. Sharp.
References [1] M.J. Anderson, S.G. Waxman, Neurogenesis in adult vertebrate spinal cord in situ and in vitro: a new model system, Ann. NY Acad. Sci. 457 (1985) 213–233. [2] S.A. Bayer, J.W. Yackel, P.S. Puri, Neurons in the rat dentate gyrus granular layer substantially increase during juvenile and adult life, Science 216 (1982) 890–892. [3] J. Bengzon, Z. Kokaia, E. Elmer, A. Nanobashvili, M. Kokaia, O. Lindvall, Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures, Proc. Natl. Acad. Sci. USA 94 (1997) 10432–10437. [4] R. Bernabeu, F.R. Sharp, NMDA and AMPA / kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus, J. Cereb. Blood Flow. Metab. 20 (2000) 1669–1680. [5] H.A. Cameron, E. Gould, Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus, Neuroscience 61 (1994) 203–209. [6] H.A. Cameron, T.G. Hazel, R.D. McKay, Regulation of neurogenesis by growth factors and neurotransmitters, J. Neurobiol. 36 (1998) 287–306. [7] H.A. Cameron, R. McKay, Stem cells and neurogenesis in the adult brain, Curr. Opin. Neurobiol. 8 (1998) 677–680. [8] D. Corbett, S.J. Evans, S.M. Nurse, Impaired acquisition of the Morris water maze following global ischemic damage in the gerbil, NeuroReport 3 (1992) 204–206. [9] D.W. Desmond, J.T. Moroney, M. Sano, Y. Stern, Recovery of cognitive function after stroke, Stroke 27 (1996) 1798–1803. [10] F. Doetsch, I. Caille, D.A. Lim, J.M. Garcia-Verdugo, A. AlvarezBuylla, Subventricular zone astrocytes are neural stem cells in the adult mammalian brain, Cell 97 (1999) 703–716. [11] F. Doetsch, J.M. Garcia-Verdugo, A. Alvarez-Buylla, Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain, J. Neurosci. 17 (1997) 5046–5061. [12] M. Endoh, W.A. Pulsinelli, J.A. Wagner, Transient global ischemia induces dynamic changes in the expression of bFGF and the FGF receptor, Brain Res. Mol. Brain Res. 22 (1994) 76–88. [13] P.S. Eriksson, E. Perfilieva, T. Bjork-Eriksson, A.M. Alborn, C. Nordborg, D.A. Peterson, F.H. Gage, Neurogenesis in the adult human hippocampus [see comments], Nat. Med. 4 (1998) 1313– 1317. [14] E. Font, E. Desfilis, M. Perez-Canellas, S. Alcantara, J.M. GarciaVerdugo, 3-Acetylpyridine-induced degeneration and regeneration in the adult lizard brain: a qualitative and quantitative analysis, Brain Res. 754 (1997) 245–259. [15] F.H. Gage, Mammalian neural stem cells [in process citation], Science 287 (2000) 1433–1439. [16] F.H. Gage, G. Kempermann, T.D. Palmer, D.A. Peterson, J. Ray, Multipotent progenitor cells in the adult dentate gyrus, J. Neurobiol. 36 (1998) 249–266.
29
[17] J.M. Garcia-Verdugo, F. Doetsch, H. Wichterle, D.A. Lim, A. Alvarez-Buylla, Architecture and cell types of the adult subventricular zone: in search of the stem cells, J. Neurobiol. 36 (1998) 234–248. [18] E. Gould, H.A. Cameron, D.C. Daniels, C.S. Woolley, B.S. McEwen, Adrenal hormones suppress cell division in the adult rat dentate gyrus, J. Neurosci. 12 (1992) 3642–3650. [19] E. Gould, H.A. Cameron, B.S. McEwen, Blockade of NMDA receptors increases cell death and birth in the developing rat dentate gyrus, J. Comp. Neurol. 340 (1994) 551–565. [20] E. Gould, B.S. McEwen, P. Tanapat, L.A. Galea, E. Fuchs, Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation, J. Neurosci. 17 (1997) 2492–2498. [21] E. Gould, A.J. Reeves, M. Fallah, P. Tanapat, C.G. Gross, E. Fuchs, Hippocampal neurogenesis in adult Old World primates, Proc. Natl. Acad. Sci. USA 96 (1999) 5263–5267. [22] E. Gould, P. Tanapat, Lesion-induced proliferation of neuronal progenitors in the dentate gyrus of the adult rat, Neuroscience 80 (1997) 427–436. [23] E. Gould, P. Tanapat, B.S. McEwen, G. Flugge, E. Fuchs, Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress, Proc. Natl. Acad. Sci. USA 95 (1998) 3168–3171. [24] J. Honkaniemi, S.M. Massa, M. Breckinridge, F.R. Sharp, Global ischemia induces apoptosis-associated genes in hippocampus, Brain Res. Mol. Brain Res. 42 (1996) 79–88. [25] M. Hsu, G. Buzsaki, Vulnerability of mossy fiber targets in the rat hippocampus to forebrain ischemia, J. Neurosci. 13 (1993) 3964– 3979. [26] M. Hsu, A. Sik, F. Gallyas, Z. Horvath, G. Buzsaki, Short-term and long-term changes in the postischemic hippocampus, Ann. NY Acad. Sci. 743 (1994) 121–139, discussion 139–140. [27] K. Jin, M. Minami, J.Q. Lan, X.O. Mao, S. Batteur, R.P. Simon, D.A. Greenberg, Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat, Proc. Natl. Acad. Sci. USA 98 (2001) 4710–4715. [28] F.F. Johansen, J. Zimmer, N.H. Diemer, Early loss of somatostatin neurons in dentate hilus after cerebral ischemia in the rat precedes CA-1 pyramidal cell loss, Acta Neuropathol. (Berl.) 73 (1987) 110–114. [29] B.B. Johansson, Brain plasticity and stroke rehabilitation. The Willis lecture, Stroke 31 (2000) 223–230. [30] B.B. Johansson, A.L. Ohlsson, Environment, social interaction, and physical activity as determinants of functional outcome after cerebral infarction in the rat, Exp. Neurol. 139 (1996) 322–327. [31] B.B. Johansson, L. Zhao, B. Mattsson, Environmental influence on gene expression and recovery from cerebral ischemia, Acta Neurochir. Suppl. (Wien) 73 (1999) 51–55. [32] C.B. Johansson, S. Momma, D.L. Clarke, M. Risling, U. Lendahl, J. Frisen, Identification of a neural stem cell in the adult mammalian central nervous system, Cell 96 (1999) 25–34. [33] K.K. Johe, T.G. Hazel, T. Muller, M.M. Dugich-Djordjevic, R.D. McKay, Single factors direct the differentiation of stem cells from the fetal and adult central nervous system, Genes Dev. 10 (1996) 3129–3140. [34] G. Kempermann, H.G. Kuhn, F.H. Gage, More hippocampal neurons in adult mice living in an enriched environment, Nature 386 (1997) 493–495. [35] G. Kempermann, H.G. Kuhn, F.H. Gage, Experience-induced neurogenesis in the senescent dentate gyrus, J. Neurosci. 18 (1998) 3206–3212. [36] B. Kirschenbaum, F. Doetsch, C. Lois, A. Alvarez-Buylla, Adult subventricular zone neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb, J. Neurosci. 19 (1999) 2171–2180. [37] H.G. Kuhn, J. Winkler, G. Kempermann, L.J. Thal, F.H. Gage,
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[38]
[39]
[40] [41]
[42]
[43]
[44] [45]
[46]
[47]
[48]
[49]
F.R. Sharp et al. / Developmental Brain Research 134 (2002) 23 – 30 Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain, J. Neurosci. 17 (1997) 5820–5829. J. Liu, M. Bartels, A. Lu, F.R. Sharp, Microglia / macrophages proliferate in striatum and neocortex but not in hippocampus after brief global ischemia that produces ischemic tolerance in gerbil brain, J. Cereb. Blood Flow. Metab. 21 (2001) 361–373. J. Liu, K. Solway, R.O. Messing, F.R. Sharp, Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils, J. Neurosci. 18 (1998) 7768–7778. M.B. Luskin, Neuroblasts of the postnatal mammalian forebrain: their phenotype and fate, J. Neurobiol. 36 (1998) 221–233. M.D. Madeira, A. Cadete-Leite, J.P. Andrade, M.M. Paula-Barbosa, Effects of hypothyroidism upon the granular layer of the dentate gyrus in male and female adult rats: a morphometric study, J. Comp. Neurol. 314 (1991) 171–186. E.A. Markakis, F.H. Gage, Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles, J. Comp. Neurol. 406 (1999) 449–460. E. Marti, I. Ferrer, J. Blasi, Transient increase of synapsin-I immunoreactivity in the mossy fiber layer of the hippocampus after transient forebrain ischemia in the mongolian gerbil, Brain. Res. 824 (1999) 153–160. R.D. McKay, Brain stem cells change their identity [news], Nat. Med. 5 (1999) 261–262. A. Molowny, J. Nacher, C. Lopez-Garcia, Reactive neurogenesis during regeneration of the lesioned medial cerebral cortex of lizards, Neuroscience 68 (1995) 823–836. T.D. Palmer, J. Takahashi, F.H. Gage, The adult rat hippocampus contains primordial neural stem cells, Mol. Cell. Neurosci. 8 (1997) 389–404. J.M. Parent, T.W. Yu, R.T. Leibowitz, D.H. Geschwind, R.S. Sloviter, D.H. Lowenstein, Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus, J. Neurosci. 17 (1997) 3727– 3738. K. Puurunen, J. Sirvio, J. Koistinaho, R. Miettinen, A. Haapalinna, P. Riekkinen Sr., J. Sivenius, Studies on the influence of enrichedenvironment housing combined with systemic administration of an a2-adrenergic antagonist on spatial learning and hyperactivity after global ischemia in rats, Stroke 28 (1997) 623–631. B.A. Reynolds, S. Weiss, Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system [see comments], Science 255 (1992) 1707–1710.
[50] L.R. Squire, S.M. Zola, Ischemic brain damage and memory impairment: a commentary, Hippocampus 6 (1996) 546–552. [51] B.B. Stanfield, J.E. Trice, Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections, Exp. Brain Res. 72 (1988) 399–406. [52] C.A. Stuermer, M. Bastmeyer, M. Bahr, G. Strobel, K. Paschke, Trying to understand axonal regeneration in the CNS of fish, J. Neurobiol. 23 (1992) 537–550. [53] A. Sugimoto, H. Shozuhara, K. Kogure, H. Onodera, Exposure to sub-lethal ischemia failed to prevent subsequent ischemic death of dentate hilar neurons, as estimated by laminin immunohistochemistry, Brain Res. 629 (1993) 159–162. [54] H. van Praag, B.R. Christie, T.J. Sejnowski, F.H. Gage, Running enhances neurogenesis, learning, and long-term potentiation in mice, Proc. Natl. Acad. Sci. USA 96 (1999) 13427–13431. [55] H. van Praag, G. Kempermann, F.H. Gage, Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus [see comments], Nat. Neurosci. 2 (1999) 266–270. [56] S. Weiss, C. Dunne, J. Hewson, C. Wohl, M. Wheatley, A.C. Peterson, B.A. Reynolds, Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis, J. Neurosci. 16 (1996) 7599–7609. [57] S. Weiss, D. van der Kooy, CNS stem cells: where’s the biology (a.k.a. beef)?, J. Neurobiol. 36 (1998) 307–314. [58] E. Westerberg, D.T. Monaghan, H. Kalimo, C.W. Cotman, T.W. Wieloch, Dynamic changes of excitatory amino acid receptors in the rat hippocampus following transient cerebral ischemia, J. Neurosci. 9 (1989) 798–805. [59] E. Westerberg, T.W. Wieloch, Changes in excitatory amino acid receptor binding in the intact and decorticated rat neostriatum following insulin-induced hypoglycemia, J. Neurochem. 52 (1989) 1340–1347. [60] R.P. Wiard, M.C. Dickerson, O. Beek, R. Norton, B.R. Cooper, Neuroprotective properties of the novel antiepileptic lamotrigine in a gerbil model of global cerebral ischemia, Stroke 26 (1995) 466– 472. [61] S. Zola-Morgan, L.R. Squire, N.L. Rempel, R.P. Clower, D.G. Amaral, Enduring memory impairment in monkeys after ischemic damage to the hippocampus, J. Neurosci. 12 (1992) 2582–2596. [62] M.X. Zuo, The studies on neurogenesis induced by brain injury in adult ring dove, Cell Res. 8 (1998) 151–158.
Interactive report
Neurogenesis following brain ischemia Frank R. Sharp a , *, Jialing Liu b , Ramon Bernabeu a a
Department of Neurology and Neuroscience Program, Vontz Center Rm 2327, 3125 Eden Avenue, University of Cincinnati, Cincinnati, OH 45267 -0536, USA b Department of Neurosurgery, University of California, San Francisco, CA, USA Accepted 5 October 2001
Abstract Following 5 or 10 min of global ischemia in the adult gerbil there is a tenfold increase in the birth of new cells in the subgranular zone of dentate gyrus of the hippocampus as assessed using BrdU incorporation. This begins at 7 days, peaks at 11 days, and decreases thereafter. Over the next month approximately 25% of the newborn cells disappear. Of the remaining cells, 60% migrate into the granule cell layer where two-thirds become NeuN, calbindin and MAP-2 immunostained neurons. The remaining 40% of the cells migrate into the dentate hilus where 25% of these become GFAP labeled astrocytes. It is proposed that ischemia-induced neurogenesis contributes to the recovery of function, and specifically may serve to improve anterograde and retrograde recent memory function that is lost following global ischemia in animals and man. 2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Genesis of neurons and glia Keywords: Stem cell; Neurogenesis; Ischemia; Stroke; Memory
1. Introduction Stem cells that self-renew and that are multipotent are found in brain. Isolated cells from the adult mammalian forebrain form colonies of undifferentiated cells in vitro that can be dissociated to form many more secondary colonies, demonstrating renewal. Moreover, these cells are multipotent since they can be induced to differentiate into neurons, astrocytes and oligodendrocytes [49,57,15,44]. Stem cells can give rise to other stem cells as well as progenitor cells. Progenitor cells give rise to neurons, astrocytes, and oligodendrocytes. Microglia appear to be derived mainly from blood borne stem cells [15,44]. Though many stem cells are found in the adult subventricular zone (SVZ) which is adjacent to the lateral ventricles [11], stem cells can also be isolated from hippocampus [46], spinal cord [56], diencephalon [56] and other brain regions [33]. A number of recent reviews [57,15,44] have prompted this short review to focus on *Corresponding author. Tel.: 11-513-558-7087; fax: 11-513-5587009. E-mail address: [email protected] (F.R. Sharp).
ischemia-related neurogenesis in brain, and specifically on ischemia-induced cell proliferation and its possible relation to recovery of short-term memory following hippocampal damage produced by global ischemia.
2. Subventricular zone Proliferating subventricular zone (SVZ) cells are located along the lateral edge of the lateral ventricle, and are most numerous along the frontal horn. SVZ cells migrate via the rostral migratory stream (RMS) to the olfactory bulb. Once in the bulb they integrate into the granule and glomerular layers where they differentiate into local interneurons [36,40]. The identity of the stem cells in the SVZ remains uncertain. Electron microscopy shows ependymal cells lining the lateral ventricles, and three types of cells between the ependyma and the parenchyma of the striatum [11,17]. The majority of the cells in the SVZ are migrating type A neuroblast cells that continue to proliferate. Slowly proliferating type B cells (GFAP-positive, astrocyte-like cells) ensheath migrating type A cells. Type C cells, the
0165-3806 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 01 )00286-3
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F.R. Sharp et al. / Developmental Brain Research 134 (2002) 23 – 30
least numerous but most actively proliferating cell in the SVZ, form small clusters throughout the SVZ. Following administration of an antimitotic drug (Ara-C), type B cells are the first to appear, and apparently give rise to type A and C cells [10]. Viral transfection studies also support the possibility that type B cells in the SVZ are a stem cell. It appears, therefore, that the slowly proliferating GFAPpositive type B cells represent at least one type of stem cell in brain [10]. There is also evidence that some ependymal cells might also be stem cells, since isolated dye-labeled ependymal cells appear to divide in vitro and develop into neurospheres [32]. Many factors can affect neural stem cells in vitro. BFGF, EGF, TGF, IGF-1 and monoamines increase proliferation [6]. Glutamate, GABA, and opioid peptides tend to decrease proliferation. PDGF, CNTF, VIP and other compounds can also affect proliferation and differentiation [6]. Infusions of EGF and bFGF into the lateral ventricle expand the SVZ progenitor population in vivo, bFGF increasing the numbers of neurons reaching the olfactory bulb and EGF enhancing astrocyte differentiation [37]. Following removal of the olfactory bulb, adult SVZ neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb [36], though this slows over time. This suggests that there is only modest feedback from the olfactory bulb to the SVZ. A recent study shows that focal cerebral ischemia increases the birth of cells in the SVZ, and that some of these cells form mature neurons and other cells form immature neurons or undifferentiated cells [27]. These changes of cell proliferation occur within days of the stroke [27].
3. Dentate subgranular zone Adult hippocampal stem and progenitor cells are located at the border between the dentate hilus and the inner margins of the upper and lower blades of the dentate granule cell layer (Fig. 1). The newborn cells in normal hippocampus migrate into the granule cell layer where the majority of the cells differentiate into neurons that assume a granule cell phenotype [2,16,7,20]. These cells are present in the brains of rodents [22], primates [21,23] and man [13]. Many factors up or down regulate the proliferation of the subgranular zone (SGZ) progenitor cells. NMDA receptor antagonists induce proliferation of dentate gyrus progenitor cells and increase newborn neurons in the granule cell layer. Enriched environments increase the total number of granule cell neurons by increasing the survival rate of the progeny from the dividing precursor cells [34]. Voluntary wheel running increases the proliferation of dentate gyrus stem cells and increases the survival of these cells [54]. Adrenal steroids, and stress on the other hand, decrease proliferation of dentate gyrus progenitor cells [5,18]. Similarly, differentiation of neurons within the
Fig. 1. BrdU labeled cells (green, left panels) and NeuN stained cells (red, right panels) in dentate gyrus from a control animal (upper two panels), from an animal 11 days after ischemia (middle two panels) and from an animal 30 days following ischemia (lower two panels). BrdU was given 24 h prior to sacrifice. Note the marked increase of BrdU labeled cells (middle, left panel) in the dentate subgranular zone compared to control (upper left) and and compared to 1 month following ischemia (lower left panel).
adult rat dentate gyrus is reduced in hypothyroid animals [41]. Cell birth rates are decreased in the SGZ of aged animals, and animals with higher rates of cell birth appear to have better memory function than those with lower rates of cell birth [35]. Since injury to granule cells promotes dentate neurogenesis [22], glutamate receptors modulate cell proliferation, and status epilepticus produces hippocampal injury and stimulates neurogensis in the SGZ [47], it was anticipated that cerebral ischemia would also modulate dentate cell birth rates as outlined below.
4. Injury induces stem cells in vertebrates Non-mammals, including fish, regenerate neurons and axons in the CNS after experimental injuries [1,33,52]. Acetylpyridine induced injury in the lizard medial cortex stimulates cell proliferation with neuroblasts and immature neurons gradually replacing dead neurons at the lesion site [45,14]. Lesions of the nucleus ectostriatum in the adult ring-dove increased the birth of newborn neurons over two-fold [62]. Therefore, in vertebrates at large, some injury-induced neurogenesis appears to be the rule. Injuryinduced neurogenesis also occurs in the brains of adult mammals, but it is much less robust and much more restricted.
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Fig. 2. Rate of cell birth (number of cells labeled with BrdU in a 24-h period) in the dentate SGZ (mm 2 ) at 6 days to 5 weeks following global cerebral ischemia in the adult gerbil brain compared to controls. Animals were administered BrdU 24 h prior to sacrifice. BrdU immunostained cells in the SGZ were counted and normalized to the area of the dentate hilus (mm 2 ).
5. Ischemia induces birth of cells in the rodent brain Cell birth was studied in the brain of the adult gerbil with the DNA analogue, bromodeoxyuridine (BrdU). BrdU is taken up into the DNA of dividing cells, and immunolocalized in the nuclei of these cells using an antibody specific for BrdU (see Figs. 1 and 3, green cells). There are a number of BrdU labeled cells in the SGZ of the dentate gyrus of the hippocampus in normal, adult rat brain (Fig. 1, control, upper left panel). BrdU incorporation into cells in the SGZ, given 24 h prior to sacrifice, did not change for the first 6 days following global ischemia (Fig. 2). Thereafter, cell proliferation increased markedly and was maximal at 11 days after ischemia (Fig. 1, middle panel) [39]. There was a twelve-fold increase in BrdU immunoreactive nuclei per mm 2 dentate gyrus compared to control animals (Fig. 2). Cell proliferation decreased by 14 days after ischemia but was still greater than control. The number of dividing cells appeared to return to control levels in most animals 3–5 weeks after the ischemia (Figs. 1 and 2). The explanation for the 1-week long delay between the ischemia and onset of neurogenesis is unknown.
6. Ischemia induces new neurons To determine the fate of the BrdU labeled neurons, adult gerbils were subjected to 10 min of global ischemia and given BrdU twice a day on days 9–12 after ischemia to maximally label proliferating cells. At 15 days after ischemia, BrdU labeled cells were found mainly in the
SGZ (green cells, Fig. 3) and did not express either neuronal or astrocyte markers (Table 1). By 26 days after ischemia 27% of the BrdU immunoreactive cells expressed the neuronal marker NeuN (Table 1). By 40 days after
Fig. 3. Newborn cells in the dentate SGZ at 15 days following global ischemia. Gerbils subjected to 10 min of global ischemia were given BrdU on days 9–12 following ischemia. On day 15 following ischemia brain sections were immunostained for NeuN (red) and BrdU (green). BrdU labeled newborn cells (green) are shown in the dentate subgranular zone (SGZ) adjacent to the NeuN stained granule cell neurons (red) in the granule cell layer. Note that there are some BrdU and NeuN doublelabeled cells (yellow) that presumably represent newborn neurons.
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Table 1 Numbers of BrdU immunoreactive cells and their phenotypes in two regions of the dentate gyrus of hippocampus at 15, 26 and 40 days following global ischemia in the adult gerbil, GCL1SGZ5granule cell layer and subgranular zone Time after ischemia (days)
Treatment (n)
Regions of the dentate gyrus GCL1SGZ Total
a
Dentate hilus NeuN
b
GFAP
b
Total
NeuN
GFAP
15
Control (6) Ischemia (6)
4565 220657
0 0
0 0
261 1766
0 0
0 261
26
Control (6) Ischemia (6)
1663 113623
361 3065
0 0
361 69637
0 0
0 763.2
40
Control (6) Ischemia (6)
1163 9166
862 5664
0 0
361 78641
0 0
0 2066
GCL, granule cell layer; SGZ, subgranular zone; NeuN, a neuronal marker in the nucleus of mature mammalian neurons; GFAP, glial fibrillary acidic protein; the number in parentheses is the number (n) of animals in each control or ischemia group. This table has been previously published by Liu et al. [39]. a Total number of BrdU immunoreactive nuclei labeled by FITC detected by immunofluorescence microscopy. b Number of BrdU-labeled cells also immunoreactive for NeuN or GFAP, as detected by confocal microscopy.
ischemia 61% of the BrdU labeled cells expressed NeuN, and were therefore newborn, differentiated neurons (Table 1). The newborn neurons were no longer localized in the SGZ, but were distributed throughout the granule cell layer. Fig. 4 shows BrdU labeled cells in green and granule cell neurons in red. The BrdU and NeuN double-labeled neurons were yellow, and hence contained both labels as visualized by the confocal microscope (Fig. 4). These BrdU labeled neurons colocalized with calbindin-D28k and MAP-2 as well as with NeuN [39]. There could be some
Fig. 4. Newborn neurons in the granule cell layer 40 days following global ischemia. Gerbils subjected to 10 min of global ischemia were given BrdU on days 9–12 following ischemia. On day 40 following ischemia brain sections were immunostained for NeuN (red) and BrdU (green). Note that in this section through the granule cell layer that all of the BrdU labeled cells (which would be green) colocalize with NeuN. Hence, all of the yellow cells are BrdU and NeuN double-labeled cells that represent newborn neurons.
concern that BrdU would be taken up into cells that were undergoing DNA repair. However, we also showed the presence of PCNA (proliferating cell nuclear antigen) in NeuN labeled cells, confirming the birth of new neurons [39]. Moreover, we never observed BrdU uptake into dying neurons in CA1, or into any other injured cell. In addition, TUNEL staining following ischemia never shows any cells with fragmented DNA localized to the SGZ following global ischemia [24]. Lastly, because of the concern that newborn cells might undergo apoptosis, we also examined BrdU labeled cells many months following ischemia. Fig. 5 shows a number of BrdU and NeuN double labeled neurons (yellow cells) in the granule cell
Fig. 5. BrdU labeled, NeuN double labeled newborn neurons (yellow cells) survived for over 7 months in the granule cell layer (NeuN stained neuronal nuclei in the granule cell layer are red) following global ischemia in the adult gerbil brain.
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layer of one animal over 7 months following ischemia. This suggests that many of the newborn neurons can survive for very long periods following the ischemia.
7. Ischemia induces new glia To better address the fate of the BrdU labeled cells following global ischemia, cell counting was performed (Table 1) [39]. Beginning 15 days after ischemia roughly 220 cells (per mm 2 dentate657 cells) were detected in the SGZ of ischemic animals (Table 1). By 40 days after ischemia 91 cells (per mm 2 dentate16 cells) were found in the granule cell layer and 78 cells (per mm 2 dentate141 cells) were found in the dentate hilus (Table 1). Thus 75% of the cells that were present at 15 days after ischemia (3 days after labeling) were still present at 40 days after ischemia. Hence, a quarter of the newborn cells died or had sufficient dilution of the labeling that they could not be detected after the first month. When double labeling was performed, approximately 60% of the BrdU labeled cells in the granule cell layer were also NeuN double stained, and therefore had differentiated into neurons. None of the BrdU labeled cells in the granule cell layer appeared to differentiate into GFAP stained astrocytes. GFAP staining at 15 days after ischemia showed that none of the BrdU labeled cells in the SGZ had differentiated into astroyctes (Fig. 6). Indeed, most of the GFAP stained astrocytes in dentate are found in the dentate hilus (h) and not in the granule cell layer (Fig. 7). By 40
Fig. 6. Newborn cells in the dentate SGZ at 15 days following global ischemia. Gerbils subjected to 10 min of global ischemia were given BrdU on days 9–12 following ischemia. On day 15 following ischemia brain sections were immunostained for GFAP (red) and BrdU (green). BrdU labeled newborn cells (green) are shown in the dentate subgranular zone (SGZ) adjacent to the GFAP stained cells in the dentate hilus, and a few GFAP stained cells in the granule cell layer.
Fig. 7. Newborn astrocytes in the dentate hilus 40 days following global ischemia. Gerbils subjected to 10 min of global ischemia were given BrdU on days 9–12 following ischemia. Animals were allowed to survive 40 days following ischemia. Brain sections were immunostained for GFAP (red) and BrdU (green). Note that there are BrdU labeled cells (green), GFAP labeled astrocytes (red), and BrdU and GFAP double labeled cells (yellow). The yellow cells are newborn astrocytes that are presumably derived from cells in the SGZ that migrated into the hilus and differentiated into GFAP positive astrocytes.
days after ischemia a number of BrdU labeled cells (green) colocalized with GFAP (red) to produce double-labeled yellow cells in dentate hilus (Fig. 7). Only about 25% of the BrdU labeled, newborn cells in the dentate hilus, however, differentiated into GFAP stained astrocytes (Table 1). The 40% of the BrdU labeled cells that are not neurons or astrocytes in the granule cell layer could either be oligodendrocytes or regenerated stem or progenitor cells. The 75% of the BrdU labeled cells in the hilus that are not neurons or astrocytes similarly could be differentiated oligodendrocytes or regenerated stem or progenitor cells. They are not microglia, since newborn microglia are found between 1 and 6 days following ischemia [38], and the stem cells found in adult brain are not believed to develop into microglia [15,7]. In summary, 25% of the cells that are born in the SGZ following ischemia die in the first month. Of the surviving cells, 60% migrate into the SGZ and 40% migrate to the dentate hilus. In the granule cell layer 60% of the cells differentiate into neurons and none differentiate into astrocytes; this contrasts to the hilus where none of BrdU labeled cells differentiate into neurons whereas 25% of the cells differentiate into astrocytes. These data suggest that neuronal differentiation factors predominate in the granule cell layer, whereas astrocyte differentiation factors in the hilus are more prominent. The stimuli for this differential neuronal and glial differentiation are unclear since there is
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thought to be little neuronal cell death in the granule cell neuronal layer, while there is definite loss of somatostatin and other dentate hilar interneurons following global ischemia [53,28,26].
8. Mechanism of ischemia induced neurogenesis Neurogenesis in the developing brain is regulated at least in part by NMDA receptors, and blockade of NMDA receptors with MK801 increases the cell birth rate in adult dentate gyrus [19,20]. In addition, lesions of entorhinal cortex that decrease glutamate inputs to dentate and decrease NMDA receptor activation also stimulate dentate cell proliferation. However, neuronal cell death was not detected in entorhinal cortex following 5–10 min of global ischemia with either NeuN staining [39] or with TUNEL staining [24]. This suggests that loss of glutamatergic cells in the perforant pathway from entorhinal cortex to dentate is not the cause of ischemia-induced neurogenesis. It is possible that neuronal death within the hippocampus provided the stimulus for increased neurogenesis following ischemia. For example, limbic seizures that cause apoptosis of granule cells [3] increase dentate neurogenesis [47]. However, granule cell loss is not a significant feature of moderate durations of global ischemia [24,25]. To determine whether ischemic loss of CA1 neurons might be the stimulus for neurogenesis, gerbils were subjected to 2 min of preconditioning ischemia, followed 2 days later by 5 min of ischemia. There was no death of CA1 neurons using this preconditioning paradigm, but there was increased neurogenesis equal to that produced by 5 min of global ischemia alone [39]. However, it should be noted that there is death of dentate hilar interneurons using this ischemia preconditioning paradigm [28,53,25] that could result in altered dentate activity and alterations of NMDA receptor regulation that could stimulate neurogenesis. Growth and mitogenic factors could play a role in the ischemia-induced neurogenesis, but there is no direct evidence for this. The increases of basic FGF that occur in hippocampal glial cells after ischemia [12] might stimulate neurogenesis [39,47]. Infusions of bFGF into the ventricles increases the numbers of cells in the SVZ [37], though any effects on the SGZ were not reported. Lastly, changes of NMDA receptors may also mediate ischemia-induced neurogenesis. There is a 20% reduction in NMDA receptor binding in the rat dentate gyrus 1 week after transient forebrain ischemia [59,58]. The NMDA receptor binding returns to control levels 2 weeks after ischemia [58]. Ischemia-induced neurogenesis in gerbils reached a maximum at 9–11 days after ischemia and declined towards control levels 3 days later [39]. The similarity of these time courses raises the possibility that ischemia-induced decreases in NMDA receptor signaling contribute to neurogenesis in the dentate gyrus. We have
shown that administration of NMDA and AMPA-kainate antagonists directly into hippocampus prevents the cell death produced by global ischemia and also blocks the neurogenesis normally produced by global ischemia [4]. These data could mean that these glutamate receptors mediate neurogenesis via downstream signaling, or they could mean that hippocampal cell death is necessary to stimulate neurogenesis [4].
9. Neurogenesis in recovery of memory function following global ischemia The hippocampus plays a central role in learning and recent memory. Bilateral injury to CA3 neurons produced by status epilepticus and bilateral injury to granule cell neurons produced by hypoglycemia produces a disorder of recent memory similar to, if not identical to that produced by global ischemia. Following relatively short durations of global ischemia in rodents, primates and man [50,61] there is bilateral loss of some or all of the CA1 pyramidal neurons in hippocampus. This injury is associated with an anterograde and retrograde amnesia but with preservation of long-term memory. Animals and people lose memories for short periods prior to the ischemia, and also have difficulty forming new language or spatial memories [50]. Animals, for example, may perform well on a spatial task learned in the distant past, but will not be able to learn the location of a submerged platform in a new Morris water maze task [8,60]. It is tempting to speculate that the ischemia-induced neurogenesis observed in these studies could contribute to the recovery of memory function that has been documented in humans [9] and in experimental animals [8,60] following global ischemia. It is notable that rats housed in an enriched environment after global and focal ischemia showed improved cognitive functions compared to those housed in the standard environment [29–31,48]. Enriched environments increase the survival of newborn cells in normal adult mice [34,55]. The recovery of memory function following global ischemia and the improved cognitive function in enriched environments both correlate with dentate neurogenesis. A recent study also shows synapsin upregulation in the mossy fiber layer of CA3 following global ischemia [43]. This suggests that existing granule cell mossy fibers sprout in CA3, resulting in increased synaptogenesis. In addition, the newly formed granule cell neurons extend axons to CA3 [51], and form new synapses on CA3 target neurons [42]. These data suggest that following hippocampal injury produced by global ischemia, recovery of function could be mediated by at least two factors. Existing granule cell neurons may sprout and form more synapses on CA3 neurons, and the new neurons that are born in the granule cell layer may also form new synapses on CA3 neurons. The role of these new synapses in recovery of memory function and the
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signally pathways that set them in motion remain to be determined.
Acknowledgements Much of this material and the figures used in this review were previously published in The Neuroscientist, Neurogenesis and gliogenesis in the postischemic brain, 6(5): 362–370, 2000 which was authored by J. Liu, R. Bernabeu, A. Lu and F.R. Sharp.
References [1] M.J. Anderson, S.G. Waxman, Neurogenesis in adult vertebrate spinal cord in situ and in vitro: a new model system, Ann. NY Acad. Sci. 457 (1985) 213–233. [2] S.A. Bayer, J.W. Yackel, P.S. Puri, Neurons in the rat dentate gyrus granular layer substantially increase during juvenile and adult life, Science 216 (1982) 890–892. [3] J. Bengzon, Z. Kokaia, E. Elmer, A. Nanobashvili, M. Kokaia, O. Lindvall, Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures, Proc. Natl. Acad. Sci. USA 94 (1997) 10432–10437. [4] R. Bernabeu, F.R. Sharp, NMDA and AMPA / kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus, J. Cereb. Blood Flow. Metab. 20 (2000) 1669–1680. [5] H.A. Cameron, E. Gould, Adult neurogenesis is regulated by adrenal steroids in the dentate gyrus, Neuroscience 61 (1994) 203–209. [6] H.A. Cameron, T.G. Hazel, R.D. McKay, Regulation of neurogenesis by growth factors and neurotransmitters, J. Neurobiol. 36 (1998) 287–306. [7] H.A. Cameron, R. McKay, Stem cells and neurogenesis in the adult brain, Curr. Opin. Neurobiol. 8 (1998) 677–680. [8] D. Corbett, S.J. Evans, S.M. Nurse, Impaired acquisition of the Morris water maze following global ischemic damage in the gerbil, NeuroReport 3 (1992) 204–206. [9] D.W. Desmond, J.T. Moroney, M. Sano, Y. Stern, Recovery of cognitive function after stroke, Stroke 27 (1996) 1798–1803. [10] F. Doetsch, I. Caille, D.A. Lim, J.M. Garcia-Verdugo, A. AlvarezBuylla, Subventricular zone astrocytes are neural stem cells in the adult mammalian brain, Cell 97 (1999) 703–716. [11] F. Doetsch, J.M. Garcia-Verdugo, A. Alvarez-Buylla, Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain, J. Neurosci. 17 (1997) 5046–5061. [12] M. Endoh, W.A. Pulsinelli, J.A. Wagner, Transient global ischemia induces dynamic changes in the expression of bFGF and the FGF receptor, Brain Res. Mol. Brain Res. 22 (1994) 76–88. [13] P.S. Eriksson, E. Perfilieva, T. Bjork-Eriksson, A.M. Alborn, C. Nordborg, D.A. Peterson, F.H. Gage, Neurogenesis in the adult human hippocampus [see comments], Nat. Med. 4 (1998) 1313– 1317. [14] E. Font, E. Desfilis, M. Perez-Canellas, S. Alcantara, J.M. GarciaVerdugo, 3-Acetylpyridine-induced degeneration and regeneration in the adult lizard brain: a qualitative and quantitative analysis, Brain Res. 754 (1997) 245–259. [15] F.H. Gage, Mammalian neural stem cells [in process citation], Science 287 (2000) 1433–1439. [16] F.H. Gage, G. Kempermann, T.D. Palmer, D.A. Peterson, J. Ray, Multipotent progenitor cells in the adult dentate gyrus, J. Neurobiol. 36 (1998) 249–266.
29
[17] J.M. Garcia-Verdugo, F. Doetsch, H. Wichterle, D.A. Lim, A. Alvarez-Buylla, Architecture and cell types of the adult subventricular zone: in search of the stem cells, J. Neurobiol. 36 (1998) 234–248. [18] E. Gould, H.A. Cameron, D.C. Daniels, C.S. Woolley, B.S. McEwen, Adrenal hormones suppress cell division in the adult rat dentate gyrus, J. Neurosci. 12 (1992) 3642–3650. [19] E. Gould, H.A. Cameron, B.S. McEwen, Blockade of NMDA receptors increases cell death and birth in the developing rat dentate gyrus, J. Comp. Neurol. 340 (1994) 551–565. [20] E. Gould, B.S. McEwen, P. Tanapat, L.A. Galea, E. Fuchs, Neurogenesis in the dentate gyrus of the adult tree shrew is regulated by psychosocial stress and NMDA receptor activation, J. Neurosci. 17 (1997) 2492–2498. [21] E. Gould, A.J. Reeves, M. Fallah, P. Tanapat, C.G. Gross, E. Fuchs, Hippocampal neurogenesis in adult Old World primates, Proc. Natl. Acad. Sci. USA 96 (1999) 5263–5267. [22] E. Gould, P. Tanapat, Lesion-induced proliferation of neuronal progenitors in the dentate gyrus of the adult rat, Neuroscience 80 (1997) 427–436. [23] E. Gould, P. Tanapat, B.S. McEwen, G. Flugge, E. Fuchs, Proliferation of granule cell precursors in the dentate gyrus of adult monkeys is diminished by stress, Proc. Natl. Acad. Sci. USA 95 (1998) 3168–3171. [24] J. Honkaniemi, S.M. Massa, M. Breckinridge, F.R. Sharp, Global ischemia induces apoptosis-associated genes in hippocampus, Brain Res. Mol. Brain Res. 42 (1996) 79–88. [25] M. Hsu, G. Buzsaki, Vulnerability of mossy fiber targets in the rat hippocampus to forebrain ischemia, J. Neurosci. 13 (1993) 3964– 3979. [26] M. Hsu, A. Sik, F. Gallyas, Z. Horvath, G. Buzsaki, Short-term and long-term changes in the postischemic hippocampus, Ann. NY Acad. Sci. 743 (1994) 121–139, discussion 139–140. [27] K. Jin, M. Minami, J.Q. Lan, X.O. Mao, S. Batteur, R.P. Simon, D.A. Greenberg, Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat, Proc. Natl. Acad. Sci. USA 98 (2001) 4710–4715. [28] F.F. Johansen, J. Zimmer, N.H. Diemer, Early loss of somatostatin neurons in dentate hilus after cerebral ischemia in the rat precedes CA-1 pyramidal cell loss, Acta Neuropathol. (Berl.) 73 (1987) 110–114. [29] B.B. Johansson, Brain plasticity and stroke rehabilitation. The Willis lecture, Stroke 31 (2000) 223–230. [30] B.B. Johansson, A.L. Ohlsson, Environment, social interaction, and physical activity as determinants of functional outcome after cerebral infarction in the rat, Exp. Neurol. 139 (1996) 322–327. [31] B.B. Johansson, L. Zhao, B. Mattsson, Environmental influence on gene expression and recovery from cerebral ischemia, Acta Neurochir. Suppl. (Wien) 73 (1999) 51–55. [32] C.B. Johansson, S. Momma, D.L. Clarke, M. Risling, U. Lendahl, J. Frisen, Identification of a neural stem cell in the adult mammalian central nervous system, Cell 96 (1999) 25–34. [33] K.K. Johe, T.G. Hazel, T. Muller, M.M. Dugich-Djordjevic, R.D. McKay, Single factors direct the differentiation of stem cells from the fetal and adult central nervous system, Genes Dev. 10 (1996) 3129–3140. [34] G. Kempermann, H.G. Kuhn, F.H. Gage, More hippocampal neurons in adult mice living in an enriched environment, Nature 386 (1997) 493–495. [35] G. Kempermann, H.G. Kuhn, F.H. Gage, Experience-induced neurogenesis in the senescent dentate gyrus, J. Neurosci. 18 (1998) 3206–3212. [36] B. Kirschenbaum, F. Doetsch, C. Lois, A. Alvarez-Buylla, Adult subventricular zone neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb, J. Neurosci. 19 (1999) 2171–2180. [37] H.G. Kuhn, J. Winkler, G. Kempermann, L.J. Thal, F.H. Gage,
30
[38]
[39]
[40] [41]
[42]
[43]
[44] [45]
[46]
[47]
[48]
[49]
F.R. Sharp et al. / Developmental Brain Research 134 (2002) 23 – 30 Epidermal growth factor and fibroblast growth factor-2 have different effects on neural progenitors in the adult rat brain, J. Neurosci. 17 (1997) 5820–5829. J. Liu, M. Bartels, A. Lu, F.R. Sharp, Microglia / macrophages proliferate in striatum and neocortex but not in hippocampus after brief global ischemia that produces ischemic tolerance in gerbil brain, J. Cereb. Blood Flow. Metab. 21 (2001) 361–373. J. Liu, K. Solway, R.O. Messing, F.R. Sharp, Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils, J. Neurosci. 18 (1998) 7768–7778. M.B. Luskin, Neuroblasts of the postnatal mammalian forebrain: their phenotype and fate, J. Neurobiol. 36 (1998) 221–233. M.D. Madeira, A. Cadete-Leite, J.P. Andrade, M.M. Paula-Barbosa, Effects of hypothyroidism upon the granular layer of the dentate gyrus in male and female adult rats: a morphometric study, J. Comp. Neurol. 314 (1991) 171–186. E.A. Markakis, F.H. Gage, Adult-generated neurons in the dentate gyrus send axonal projections to field CA3 and are surrounded by synaptic vesicles, J. Comp. Neurol. 406 (1999) 449–460. E. Marti, I. Ferrer, J. Blasi, Transient increase of synapsin-I immunoreactivity in the mossy fiber layer of the hippocampus after transient forebrain ischemia in the mongolian gerbil, Brain. Res. 824 (1999) 153–160. R.D. McKay, Brain stem cells change their identity [news], Nat. Med. 5 (1999) 261–262. A. Molowny, J. Nacher, C. Lopez-Garcia, Reactive neurogenesis during regeneration of the lesioned medial cerebral cortex of lizards, Neuroscience 68 (1995) 823–836. T.D. Palmer, J. Takahashi, F.H. Gage, The adult rat hippocampus contains primordial neural stem cells, Mol. Cell. Neurosci. 8 (1997) 389–404. J.M. Parent, T.W. Yu, R.T. Leibowitz, D.H. Geschwind, R.S. Sloviter, D.H. Lowenstein, Dentate granule cell neurogenesis is increased by seizures and contributes to aberrant network reorganization in the adult rat hippocampus, J. Neurosci. 17 (1997) 3727– 3738. K. Puurunen, J. Sirvio, J. Koistinaho, R. Miettinen, A. Haapalinna, P. Riekkinen Sr., J. Sivenius, Studies on the influence of enrichedenvironment housing combined with systemic administration of an a2-adrenergic antagonist on spatial learning and hyperactivity after global ischemia in rats, Stroke 28 (1997) 623–631. B.A. Reynolds, S. Weiss, Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system [see comments], Science 255 (1992) 1707–1710.
[50] L.R. Squire, S.M. Zola, Ischemic brain damage and memory impairment: a commentary, Hippocampus 6 (1996) 546–552. [51] B.B. Stanfield, J.E. Trice, Evidence that granule cells generated in the dentate gyrus of adult rats extend axonal projections, Exp. Brain Res. 72 (1988) 399–406. [52] C.A. Stuermer, M. Bastmeyer, M. Bahr, G. Strobel, K. Paschke, Trying to understand axonal regeneration in the CNS of fish, J. Neurobiol. 23 (1992) 537–550. [53] A. Sugimoto, H. Shozuhara, K. Kogure, H. Onodera, Exposure to sub-lethal ischemia failed to prevent subsequent ischemic death of dentate hilar neurons, as estimated by laminin immunohistochemistry, Brain Res. 629 (1993) 159–162. [54] H. van Praag, B.R. Christie, T.J. Sejnowski, F.H. Gage, Running enhances neurogenesis, learning, and long-term potentiation in mice, Proc. Natl. Acad. Sci. USA 96 (1999) 13427–13431. [55] H. van Praag, G. Kempermann, F.H. Gage, Running increases cell proliferation and neurogenesis in the adult mouse dentate gyrus [see comments], Nat. Neurosci. 2 (1999) 266–270. [56] S. Weiss, C. Dunne, J. Hewson, C. Wohl, M. Wheatley, A.C. Peterson, B.A. Reynolds, Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis, J. Neurosci. 16 (1996) 7599–7609. [57] S. Weiss, D. van der Kooy, CNS stem cells: where’s the biology (a.k.a. beef)?, J. Neurobiol. 36 (1998) 307–314. [58] E. Westerberg, D.T. Monaghan, H. Kalimo, C.W. Cotman, T.W. Wieloch, Dynamic changes of excitatory amino acid receptors in the rat hippocampus following transient cerebral ischemia, J. Neurosci. 9 (1989) 798–805. [59] E. Westerberg, T.W. Wieloch, Changes in excitatory amino acid receptor binding in the intact and decorticated rat neostriatum following insulin-induced hypoglycemia, J. Neurochem. 52 (1989) 1340–1347. [60] R.P. Wiard, M.C. Dickerson, O. Beek, R. Norton, B.R. Cooper, Neuroprotective properties of the novel antiepileptic lamotrigine in a gerbil model of global cerebral ischemia, Stroke 26 (1995) 466– 472. [61] S. Zola-Morgan, L.R. Squire, N.L. Rempel, R.P. Clower, D.G. Amaral, Enduring memory impairment in monkeys after ischemic damage to the hippocampus, J. Neurosci. 12 (1992) 2582–2596. [62] M.X. Zuo, The studies on neurogenesis induced by brain injury in adult ring dove, Cell Res. 8 (1998) 151–158.