Microscopic study of cell division in the cerebral cortex of adult brains after experimental focal cerebral ischemia

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1 Microscopic study of cell division in the cerebral cortex of adult brains after experimental focal cerebral ischemia Weigang Gu 1,2 and Per Wester 1 1 Umeå Stroke Center, Department of Public Health and Clinical Medicine, Medicine, University of Umeå 2 Department of Clinical Neuroscience and Neurology, University of Umeå, Umeå, Sweden It had long been a dogma in medical history that adult mammalian brains do not have the capacity to generate new neurons to replace the demised ones. This dogma was challenged in 1965 when Altman and Das reported their finding in postnatal rats that certain neurons became labeled by the radiolabelled DNA precursor [3H]-thymidine in their nuclei, suggesting that new-born cells had been generated, i.e., so called neurogenesis. With the same technique, Altman found postnatal neurogenesis in the olfactory bulb in With the advance of modern immunohistocytochemistry techniques, BrdUimmunolabeling has become one of the most commonly used techniques in cell regeneration research Cerebral cortex is one of the most important parts of the brain, which is often affected in the setting of ischemic stroke. After a stroke, a liquid cavity usually forms in the infarcted brain tissue. This phenomenon contributed largely to the historical assumption of the incapability of adult brains to generate new neurons. Nevertheless, most patients experience spontaneous neurological improvements to a certain degree after stroke. The discrepancy between the historical assumption and clinical experience aroused our curiosity of searching for any potential cell regeneration in the adult rat brains where remarkable morphological recovery was observed in the penumbral cerebral cortex after induction of thromboembolic ischemic stroke with spontaneous reperfusion. To detect cell regeneration and determine their phenotype in adult brains, two crucial criteria must be fulfilled. First, newborn cells need to be identified. Second, newborn cell types must be classified. To achieve this goal, exogenous BrdU was injected intraperitoneally to the poststroke rats. The exogenous BrdU is incorporated into the cell nuclei while proliferating mother cells duplicate their DNA strands, which could then be detected through BrdU-immunohistochemistry and visualized under microscopy. Surprisingly, widespread BrdU-immunolabeled cells were observed in the ischemic penumbral cortex at various times after ischemia, some of which were further recognized by the neuron-specific markers Map-2, beta-tubulin III and NeuN through the corresponding double immunohistochemistry, suggesting that they were newborn cortical neurons. To scrutinize a real colocalization of the BrdU with neuron-specific markers in the same cells, 3D confocal analysis of BrdU and NeuN double immunofluorescence was conducted, which confirmed this finding. The origin of the newborn neurons was further traced through in situ characterization of the proliferating cells at different phases of the cell cycle. At 16 hours post-stroke, cells in the penumbral cortex started to incorporate exogenous BrdU, marking their initial entrance into the S-phase. At 24h, these BrdU-immunolabelled nuclei expressed the M-phase specific marker Phos H3, indicating the start of the M-phase. Further, bipolar mitotic spindles immunolabeled by alpha-tubulin or gamma-tubulin appeared within the cells exhibiting Phos H3-immunolabelled M-phase nuclei, suggesting further progression of cell mitosis. Finally, Phos H3-immunolabelled doublet nuclei split into twin daughter cells recognized by Map-2, beta-tubulin III, hallmarking the completion of cell mitosis and thus generation of new neurons through in situ division of local cells inside the ischemic penumbral cortex in the adult brains. Keywords: BrdU; Phos H3; mitosis; stroke; infarct; reperfusion 1. Neurons in the brains Neurons are the basic functional units of our brains. It is estimated that an adult human brain contains up to 90 billion neurons. Neurons inside a brain gather together at different parts to form cerebrum, brain stem, and cerebellum. All the parts of the brain connect and communicate with each other through bundles of nerve fibers called axons or dendrites. Axons send out nerve signals to their next neurons while dendrites receive nerve signals from their previous neurons. Nerve signals conduct along the nerve fibers in a form of electrical impulses called action potentials. An action potential is initiated in the cell body of a neuron. It spreads itself along the axon to its ending where a membrane gap junction called synapse connects the first neuron (presynaptic) with the next (postsynaptic) neuron. The excitation of the presynaptic cell membrane by the action potential elicits a release of a kind of chemical molecules named neurotransmitter from the presynaptic neuron into the synaptic cleft. The neurotransmitter diffuses across the synaptic cleft and targets on the corresponding receptors anchored on the postsynaptic membrane and causes either excitation or inhibition of the postsynaptic neuron. A neuron that is presynaptic for one neuron can be postsynaptic for another and the eventual impact of neurotransmission on a neuron depends on the temporal summation of all neurotransmission from different neurons. Dispersed around the neurons are tremendous number of neuroglial cells, i.e., astrocytes and oligodendrocytes. They offer structural support, nutritional supply to neighboring neurons, the maintenance of local homeostasis and formation of myelin sheath around nerve fibers. Such a delicate complex network of the brains enables humans being not only the basic neurological functions such as feeling, hearing and moving but also the advanced 457

2 neurological functions such as thinking, talking, writing and intelligence. Any damage of this network may result in slight dysfunctions or severe disorders. 2. The history of adult neurogenesis One of the most striking differences between an adult brain and the most other organs in mammals is the limited capacity of self-regeneration. This concept can be dated back to the beginning of the 20 th century when Cajal and other neuroscientists agreed on this dogma of incapacity of regeneration in adult brains. Their conclusion was based on their microscopic observations of either normal and injured brains that were stained with Golgi s silver stain or Cajal s staining methods. Cajal remarked in his book that but the functional specialization of the brain imposed on the neurons two great lacunae; proliferative inability and irreversibility of intraprotoplasmatic differentiation. It is for this reason that, once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably``[1]. In 1965 this historical dogma was challenged by Altman and Das who introduced [3H]-thymidine autoradiography in detecting cell proliferation in postnatal animals of various species. As a DNA precursor, [3H]- thymidine was delivered in vivo that was incorporated into the cell DNA when proliferating cells duplicated their DNA strands, which was then detected through autoradiography. In their first report, they delivered [3H]-thymidine through intraperitoneal injection into postnatal rats [2]. Through histological examination of the brain sections, they found that certain differentiated granule cells with neuronal morphology in the dentate gyrus of the hippocampus were labeled by the [3H]-thymidine in their nuclei, suggesting that these cells were newly generated under the period of [3H]-thymidine delivery and therefore an occurrence of postnatal neurogenesis in this specific brain region [2]. Through the same cell labeling technique, they discovered postnatal neurogenesis in the olfactory bulks in the same year [3]. Further, they identified that the cells of the subependymal layer of the anterior lateral ventricle proliferated and migrated into the olfactory bulks, providing a constant cell resource of the continuous adult neurogenesis in this region [4]. In 1990 they proposed that the proliferation and migration of the cells in the subgranular zone into the granular zone contributed to the continuous adult neurogenesis in the dentate gyrus of the hippocampus [5]. Since then adult neurogenesis in the dentate gyrus of the hippocampus and olfactory bulbs have been extensively studied. Neurogenesis in these brain regions has been shown to be enhanced or inhibited by different physiological and pathological circumstances such as physical exercise, learning, stress, depression, drugs and cerebral ischemia [6-10]. 3. Clinical and experimental stroke Stroke is the second most common cause of mortality world-wide and the leading cause of disabiltiy during adult life in humans. In an ischemic stroke, a blood clot blocks an artery, which interrupts the blood flow into an area of the brain. When the local cerebral blood flow drops down to a critical threshold, neurons in the local brain tissue lose their function that results in acute neurological deficit symptoms such as aphasia, anopia, numbness and paralysis in the extremities, imbalance and sometimes loss of consciousness depending on which brain area that is involved. If stroke patients come in hospital within 4.5 hours after stroke onset, they may be treated with intravenous thrombolysis, which aims at dissolving the clot and recanalization of the occluded vessels and thus reliving the stroke symptoms [11]. Without prompt treatment, the local cerebral blood flow falls further and neurons and glial cells die eventually of a shortage of oxygen and energy supply, that forms an infarct. Days after stroke, microglial cells start to clean up the dead cells. This process results in a cavity in the infarcted brain tissue several weeks after stroke. More than half of all stroke survivals suffer from certain forms of neurological deficit after stroke. Up to now, there is no established medication to regain the lost neuronal function. The macroscopic cavity formation in the post stroke brain tissue facilitates largely the historical postulation that adult brains do not have a capacity to regenerate new neurons in order to replace the dead ones. Nevertheless, most stroke patients experience spontaneous neurological improvement of various degrees, that begins days after stroke and lasts for weeks and even up to 18 months [12]. Thus, a discrepancy exists between the historical postulation and the clinical experience of post stroke spontaneous recovery. 4. Photothrombotic ring stroke model The complexity of stroke pathophysiology requires that experimental stroke should be studied in multiple animal models. We have been interested in a cortical photothrombotic stroke model in adult rodents. In this setting, reproducible thrombosis can be induced photochemically in the cortex of adult rodents, wherein the ischemic lesion may be placed in any desirable location [13]. In the photothrombotic ring version of this model [14], a large anatomically predefined cortical penumbra is induced in the somatosensory cortex of adult rats within the ischemic annulus of the ring. The interior of the ring annulus is concentrically encroached by the radially expanding ring annulus, thus simulating penumbral stroke-in-evolution in an inherently reproducible fashion (Fig. 1). Microvascular platelet thrombi appear in the cortical lesion [14, 15], reminiscent of clinical thromboembolic stroke. Delayed, but consistent, spontaneous reperfusion of the cortical penumbra can be presaged in the ring model by manipulation of the irradiating 458

3 laser beam thickness and intensity (Fig. 1) [15]. The local cerebral blood flow in the penumbra decreases to 59, 34, 26, and 33% of baseline values at 1, 2, 24, and 48h after ischemia, where neuronal necrosis and apoptosis prevail [16]. Meanwhile, a proliferation of microvessels, i.e., very early angiogenesis occurs in the same penumbra between 24-48h [17], which may contribute to the spontaneous reperfusion in the penumbra. Thus, the local cerebral blood flow recovers to 56 and 87% of the baseline values at 72 and 96h after stroke induction [16]. Astonishingly, a remarkable morphologic restoration of the nerve cells takes place in the reperfused cortical penumbra, which evolves into a chiefly unremarkable cytological appearance at 7 to 28 days after stroke [18]. Fig. 1 Representative photograph of the rat brains subjected to photothrombotic ring stroke or sham-operation and transcardial carbon-black perfusion. A ring-shaped cortical-perfusion deficit appears in the cortex at 4h (B) after the ischemic induction, which progressively increased at 10h (C), 24h (D), and maximizes at 48h (E). At 72h after ischemia (F), the distal MCA with its small branches in the centrally located region-at-risk becomes patent, representing reperfusion, which is more pronounced at 7d (G) and 28d (H) after ischemia (From Gu W, Jiang W, Wester P., Exp Brain Res 125: , With permission from Springer-Verlag Heidelberg). Fig. 2 Hematoxylin and Eosin (HE) staining of the coronal brain sections through the epicenter of the ring lesion. At 4h after ischemia (A), the neuropil is slightly pale and the nerve cells are mildly swollen. At 48h (B), the majority of the nerve cells are eosinophilic in their cytoplasm and pyknotic and hyperchromatic in their nuclei. The neuropil is pale and edematous. At 72h (C), only mild cytological changes, e.g., somal and nuclear swelling are observed in the penumbral cortex. At 28d after ischemia (D), tissue morphology becomes unremarkable. (From Gu el al., Exp Brain Res 125: , With permission from SpringerVerlag Heidelberg). 5. The finding of post stoke cortical neurogenesis With an observation of such a remarkable histological restoration in the reperfused penumbral cortex, we became curious if cells might have been regenerated in the cerebral cortex in this setting of focal cortical ischemia. To explore such a possibility, the DNA duplication marker 5-Bromo-2 -deoxyuridine (BrdU) was repeatedly injected intraperitoneally into the post stroke rats [19]. As a thymidine analogue, BrdU is incorporated into the cell nuclei when proliferating cells duplicate their DNA strands during the S-phase, which can be detected immunohistochemically [20, 21]. After each injection, the BrdU lasts for about two hours for cell uptake [22]. Repeated BrdU injections maximize 459

4 the chance to detect brain cells that proliferate at various times post stroke getting labeled by the exogenous BrdU, which thus enhances our detection sensitivity. To follow up a long term survival of the newborn cells in the post stroke adult brains, the animals were allowed to survive for various time periods after the last BrdU injections until sacrifice. Brain sections were processed for immunohistochemistry and analyzed under light microscope. Widespread BrdU-immunolabeled cells were observed in the ischemic penumbral cortex at 3, 7, 14, 30, 100 and 180 days after stroke induction (Fig. 3A) [19]. Some of these BrdU single-immunolabeled cells exhibited a large single nucleus with a distinct centered nucleolus (Figs. 3B and 3C), suggesting neuronal morphology. To identify the cell types of these BrdU-immunolabeled newborn cells, various neuron-specific markers and astrocyte-specific markers were used for double immunohistochemistry with BrdU. To our surprise, some of the cells that were BrdUimmunolabeled in their nuclei were doubly immunolabeled by the neuron-specific marker Map-2 in their cytoplasm, indicating their neuronal identity (Fig. 3E). To scrutinize a real cellular colocalization of BrdU with various neuronspecific markers in the same cells, double immunofluorescent labeling of BrdU with the neuron-specific marker NeuN was performed which was analyzed through 3-D confocal microscopy. At 3 days and 30 days after stroke induction (Fig. 4), BrdU was colocalized with NeuN in the same cells in the reperfused penumbral cortex, suggesting the occurrence of cortical neurogenesis in the post stroke adult brains [19]. In the ischemic penumbral cortex, the BrdU and Map-2 double-immunolabeled cells accounted for 3.3 ± 0.3% of the total BrdU single-immunolabeled cells at 7 days post stroke induction. This proportion was increased to 5.8 ± 1.4% at 100 days while the total number of the BrdU single-immunolabeled cells in the cortex was decreased. In the sham-operated animals, a few BrdU singleimmunolabeled cells were randomly scattered in the cortex, but none of which were recognized by the neuronal markers [19]. The generality of post stroke cortical neurogenesis was further studied in adult rats exposed to middle cerebral artery suture occlusion with reperfusion. Stroke induced neurogenesis was found in both the ischemic penumbral cortex and the post ischemic striatum in this stroke model [23]. Fig. 3 (A) Photograph of BrdU immunohistochemistry in a coronal brain section through the epicenter of the ischemic lesion at 7d after photothrombotic ring stroke. Widespread BrdU-immunolabeled cells (brown) appear in the ischemic penumbral cortex. (B) A cortical cell in the penumbral cortex exhibits a large round BrdU-immunolabeled cell nucleus with a single nucleolus in the center (arrow, brown) at 7d after stroke. (C) A BrdU-immunolabeled cell (arrow, brown) from the penumbral cortex exhibits a large round cell nucleus with a single nucleolus at 100d after stroke. (D) A newborn astrocyte in the penumbral cortex is BrdU-immunolabeled in the nucleus (arrow, red) and GFAP- immunopositive in the cytoplasm (brown) at 7d post ischemia. (E) A newborn neuron in cortical layer II in the penumbral cortex evinces a BrdU-immunolabeled cell nucleus (arrow left, red) and Map-2 immunopositive cytoplasm (brown) at 100d after stroke induction. (From Gu W, Brannstrom T, Wester P., J Cereb Blood Flow Metab 20: , With permission from Nature Publishing Group). 460

5 Fig. 4 Three-dimensional confocal analyses of BrdU (green) and NeuN (red) double immunofluorescence in the penumbral cortex at two adjacent Z-series planes at 30 days after photothrombotic ring stroke. The cell designated by the yellow arrows in square is both NeuN-immunopositive and BrdU-immunopositive. (From Gu W, Brannstrom T, Wester P., J Cereb Blood Flow Metab 20: , With permission from Nature Publishing Group). 6. Hypothesis of in situ cell division in the post ischemic cerebral cortex The concept of photothrombotic ring stroke with spontaneous reperfusion leads to penumbral inversion, in which the compromised tissue lies interior rather than exterior to the ischemic locus. This experimental set-up appears to facilitate the study of pathophysiological factors related to brain recovery. It provides progressive metabolic isolation owing to the radial encroachment of ischemia into the central zone, for which recovery probably is initiated by locally recruitable factors. It is the interaction of these factors that is believed to occur in a conventional penumbra [23], except the ring stroke model allows these events and the morphological recovery to occur in a highly reproducible localization of a cortical region. In this stroke model, the BrdU-immunolabeled cells began to accumulate initially inside the ischemic penumbral cortex rather than other regions of the brains shortly after stroke [19]. Accordingly, we assumed that the newborn neurons might have a cortical origin [24], in contrast to a possible tangential migration of stem cells from the subventricular zone (SVZ) in the setting of middle cerebral artery occlusion in rodents [25]. In order to test such a hypothesis, we searched for direct detection of possible cell division in the poststroke rat brains, which should theoretically have preceded the poststroke cortical neurogenesis. As quiescent cells start to proliferate, they first enter the interphase during which the cells grow and duplicate their nuclear DNA (S-phase). As soon as the DNA strands are duplicated, the cells enter the mitotic phase (M-phase) so that they build up mitotic spindles inside the cell cytoplasm, which split the mother cells into pairs of daughter cells. 7. Transition of proliferating cells from S-phase into M-phase In order to screen the initial entrance of brain cells into the S-phase, the DNA duplication marker BrdU was intraperitoneally injected every 4 hour up to 72 hours poststroke and then two times daily, which was ended at poststroke day 7 [24]. The rats were sacrificed at 4, 10, 16, 24, 48, and 72 hours and 7 and 14 days after stroke induction. To determine the initial transition of the S-phase into the M-phase and identify possible nuclear division, the M-phase-specific marker phosphorylated histone H3 (Phos H3) [26, 27] was examined through Phos H3 single immunocytochemistry and their double immunofluorescence with BrdU. At 4 and 10 hours after stroke, a few BrdUimmunolabeled cells were randomly scattered in the whole brain sections. At 16 hours poststroke, BrdUimmunolabeled cells started to accumulate in the ischemic penumbral cortex. These BrdU-immunolabeled cells did not show any specific spatial correlation with microvessels in the cortex. When consecutive sagittal brain sections were examined at this time, a few BrdU-immunolabeled cells were observed on the tangential migrating pathway from the SVZ toward the olfactory bulb, but generally not towards the penumbral cortex. The number of BrdU-immunolabeled cells increased steadily through 24, 48, 72 hours, 7 and 14 days poststroke in the penumbral cortex, during which Phos H3-immunolabeled cells were initially detected at 24h poststroke in the same cortical penumbra [24]. The cell density of the Phos H3-immunolabeled cells maximized quickly at 48h and 72h poststroke, and then declined at 7 days and disappeared at 14 days poststroke. During this time period, some of the BrdU-immunolabeled cell nuclei were Phos H3 461

6 doubly immunopositive (Fig. 5), suggesting their entrance from the S-phase into the M-phase. They appeared as a single nucleus (Fig. 5A, 6A), or as a nucleus in a partially duplicated shape (Fig. 5A, 7A), or as a pair of twin nuclei (Fig. 5A, 7B, 7C), indicating they were mitotic nuclei in metaphase, anaphase and telophase [24]. Fig. 5 Confocal analyses of Phos H3 and BrdU double immunofluorescence in the penumbral cortex at 48h after photothrombotic ring stroke. (A) In an optical section of Phos H3 single-channel image, three cell nuclei in different shapes are Phos H3- immunopositive (arrows, green). (B) In the Phos H3 and BrdU double-channel image from the same optical section as (A), all of the three Phos H3-immunopositive nuclei (green) are doubly immunopositive to BrdU (red), shown as yellow (arrows) in the doublelabeled nuclei. (From Gu el al., Stem Cell Res 2:68-77, With permission from Elsevier). 8. Mitotic spindles inside the M-phase cells To visualize mitotic spindles, the spindle components α-tubulin [28] and γ-tubulin [29] were detected through the corresponding immunocytochemistry and immunofluorescence. Mitotic spindles immunolabeled by α-tubulin and γ- tubulin were detected during the same time period in the same cortical tissue as that of Phos H3-immunolabeling [24]. In some cells, they appeared as bipolar spindles with their pole bodies anchored on the opposite ends of their singleshaped nuclei recognized by the M-phase marker Phos H3 (Fig. 6A). As mitosis proceeds, the microtubules were seen to stretch inside the spindles drawing the chromosomes towards the opposite poles (Fig. 6B, C, D). Fig. 6 (A) Phos H3 and γ-tubulin double immunofluorescence in the penumbral cortex at 48 h poststroke. In a cortical cell from layer II, a bipolar spindle immunolabeled by γ-tubulin (arrows, green) is built up around the Phos H3- immunopositive cell nucleus (red). (B) 3D confocal image of NeuN and γ-tubulin double immunofluorescence. In a cortical cell, the γ-tubulin immunolabeled spindle (green) draws the DNA (blue, DAPI) toward the opposite poles of the cell (arrows) that is expressing NeuN (red) in its cytosol. (C) An optical section confocal image of α-tubulin immunofluorescence in the penumbral cortex at 24h poststroke. In a large cortical cell in layer III (delineated), α-tubulin (green) appears as a bipolar spindle in which the chromosomes (blue, DAPI) are randomly scattered inside the cytosol. (D) The α-tubulin and NeuN double immunofluorescence from the same optical section as (C). NeuN (red) is detected inside the cell undergoing mitosis. (From Gu el al., Stem Cell Res 2:68-77, With permission from Elsevier). 9. Generation of daughter neurons Cortical cells exhibiting nuclear division were observed to generate daughter neurons in the penumbral cortex at 24, 48, and 72 hours after stroke induction [24]. Thus, large cortical cells containing Phos H3-immunopositive nuclei in duplicated shape expressed the neuron-specific marker β-tubulin III (Fig. 7A), NeuN or Hu in the cytoplasm [24]. Further, Phos H3-immunopositive twin nuclei committing cytokinesis were recognized by the neuron-specific marker β-tubulin III in the cytoplasm (Fig. 7B). Finally, we observed daughter neurons generated through the cytoplasmic 462

7 division of the pairs of cells exhibiting Phos H3-immunopositive nuclei and Map-2-immunopositive cytoplasm (Fig. 7C). The cell density of the Map-2 and Phos H3-double-immunopositive cells was 1±5 cells/mm 3 (mean±sd) at 24h post stroke. It maximized at 770±33 cells/mm 3 at 48h and 320±35 cells/mm 3 at 72 hours post stroke, and then decreased at 3±2 cells/mm 3 at 7 days. Therefore, division of the cortical cells gave birth to daughter neurons in situ in the post ischemic penumbral cortex [24]. Fig. 7 (A) Phos H3 and β-tubulin III double immunohistochemistry in the penumbral cortex at 48h after stroke. A cortical cell containing a Phos H3-immunolabeled nucleus in a partially duplicated shape (arrows, blue) expresses β-tubulin III in the cytoplasm (purple). The arrowhead points to a cortical neuron that is singly immunolabeled by β-tubulin III. (B) A cortical cell containing a pair of Phos H3 immunolabeled nuclei (arrows, blue) undergoes cytokinesis in which a cleavage furrow is forming at the center on either side of the cell pinching through the β-tubulin III-immunopositive cytoplasm at 48h after stroke. (C) A pair of daughter neurons immunopositive to Phos H3 in their nuclei (blue) is splitting the Map-2 immunopositive cytoplasm (purple) connecting in between (From Gu el al., Stem Cell Res 2:68-77, With permission from Elsevier). 10. Conclusion Photothrombotic ring stroke model in rats features a large anatomically predefined cortical penumbra that undergoes critical hypoperfusion followed by late spontaneous reperfusion and a remarkable morphological restoration. As early as at 16h after stroke induction, cortical cells in the penumbra begin to enter the S-phase to duplicate their nuclear DNA during which the exogenous BrdU become incorporated which could be visualized through immunohistochemistry and immunofluorescence. At 24 hours poststroke, the S-phase cortical cells labeled by BrdU in the nuclei start to transit into the M-phase while expressing the mitosis nuclear marker Phos H3 and building up spindles in their cytoplasm while dividing. Cortical cells express the neuron-specific markers NeuN, Hu, Map-2 and beta-tubulin III while committing nuclear division and cytokinesis, that generates daughter neurons in the ischemic penumbral cortex of the adult brains. Future studies need to be directed at the presumptive function of the new-born cells in the ischemic penumbra and whether the observed neurogenesis after ischemic stroke is related to the clinically observed functional brain recovery. References [1] Cajal SRY. Degeneration and Regeneration of the Nervous System. New York: Oxford University Press, American Branch; [2] Altman J, Das GD. Autoradiographic and histological evidence of postnatal hippocampal neurogenesis in rats. The Journal of comparative neurology. 1965;124(3): Epub 1965/06/01. [3] Altman J, Das GD. Post-natal origin of microneurones in the rat brain. Nature. 1965;207(5000): [4] Altman J. 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. The Journal of comparative neurology. 1969;137(4): [5] Altman J, Bayer SA. Migration and distribution of two populations of hippocampal granule cell precursors during the perinatal and postnatal periods. The Journal of comparative neurology. 1990;301(3): Epub 1990/11/15. [6] Brown J, Cooper-Kuhn CM, Kempermann G, Van Praag H, Winkler J, Gage FH, et al. Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis. The European journal of neuroscience. 2003;17(10): Epub 2003/06/06. [7] Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, et al. Neurogenesis in the adult human hippocampus [see comments]. Nat Med. 1998;4(11): [8] Liu J, Solway K, Messing RO, Sharp FR. Increased neurogenesis in the dentate gyrus after transient global ischemia in gerbils. The Journal of neuroscience : the official journal of the Society for Neuroscience. 1998;18(19):

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