Stroke, which usually results from occlusion of a cerebral

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1 Evidene for stroke-indued neurogenesis in the human rain Kunlin Jin*t, Xiaomei Wangtt, Lin Xie*, Xiao Ou Mao*, Wei Zhu*, Yin Wang, Jianfeng Shen 1J, Ying Mao*, Surita Banwait*, and David A. Greenerg*1i *Buk Institute for Age Researh. Novato, CA 94945; Departments of *Neurosurgery and Neuropathology. Huashan Hospital, Fudan niversity, Shanghai China; and ~Department of Neurosurgery. First Affiliated Hospital, Zhejiang Medial niversity, Zhejiang , China Edited y Solomon H. Snyder, Johns Hopkins niversity Shool of Mediine. Baltimore, MD, and approved July (reeived for review May 9, 2006) Experimental stroke in rodents stimulates neurogenesis and migration of neworn neurons from their sites of origin into ishemi rain regions. We report that in patients with stroke, ells that express markers assoiated with neworn neurons are present in the ishemi penumra surrounding ereral ortial infarts, where these ells are preferentially loalized in the viinity of lood vessels. These findings suggest that stroke-indued ompensatory neurogenesis may our in the human rain, where it ould ontriute to postishemi reovery and represent a target for stroke therapy. ishemia I stem ells I penumra I infart I vasular nihe Stroke, whih usually results from olusion of a ereral artery leading to rain infartion, is among the ommonest auses of death and disaility in adulthood. However, many patients improve in the weeks to months following stroke, implying an innate apaity for rain repair. One means y whih repair might e ahieved is neurogenesis, whih ours in the adult human rain in situ (1), is inreased in animal models of rain injury inluding stroke (2-5), and is assoiated with migration of neworn neurons to injured rain regions (6-9). Whether the human rain responds to stroke in a similar manner has not een determined, ut it is important to examine this possiility eause of the prospet that therapeuti enhanement of injury-indued neurogenesis might improve linial outome. Results Setions were otained from human rain iopsies performed for the diagnosis of ereral lesions that proved to e ishemi strokes (Tale 1). The setions were stained with antiodies against markers of ell proliferation (proliferation-related Ki-67 antigen, Ki-67; Saharomyes eereviseae minihromosome maintenane 2 homolog, MCM2; and proliferating ell nulear antigen, PCNA) and neuronal lineage (douleortin, DCX; TOAD/lip/CRMP family protein 4, TC-4; emryoni nerve ell adhesion moleule, ENCAM; and 13m tuulin). Control speimens (n 6), from the ereral ortex of autopsied patients who died without rain pathology, ontained sl Ki-67-positive and no Ki-67/DCX-positive ells per mm 2 In stroke patients (n 6), however, the ortial region adjaent to the infart ore (ishemi penumra) ontained 63 ::':: 16 Ki-67-positive ells and 10::':: 2 Ki-67/DCX-positive ells per mm 2 (mean SEM) (Tale 1 and Fig. la). To onfirm that Ki-67 laeling refleted reent ell proliferation, some setions were also stained for MCM2 (Fig. I) or PCNA (Fig. Ie), whih were o-expressed in Ki-67-positive ells. Although dying neurons may aerrantly express ell-yle proteins, fewer than 3% of Ki-67 -positive ells in the ishemi penumra showed evidene of aspase-3 leavage, and these had distintly anormal nulear morphology (Fig. Id). Beause ell-yle markers do not disriminate ells of different lineages, some setions were stained with lineage-speifi markers. Markers expressed in new neurons, inluding DCX 2a) and 13m tuulin (Fig. 2), were loalized to the ishemi penumra. Moreover, DCX (Fig. 2), 13m tuulin (Fig. 2d), and TC4 (Fig. 2e) were deteted in ells that also expressed Ki-67, and multiple immature neuronal markers were expressed in a given ell (Fig. 2/), onfirming that these were neworn neurons. Consistent with this interpretation, som Ki-67/DCX-positive ells exhiited a migratory phenotype onsisting of an elongated ellular profile with a leading edge and trailing nuleus (Fig. 19). In the hippoampal dentate gyrus (whih, together with the suventriular zone, aounts for most neurogenesis in the adult rain), new neurons arise in proximity to lood vessels, whih are thought to provide a "vasular nihe" that supports their proliferation (10). A similar relationship was oserved in stroke rain speimens, in whih DCX-positive ells tended to luster near lood vessels laeled with antiodies against nestin (Fig. 3a), von Willerand fator (Fig. 3), or vasular smooth musle atin (Fig. 3). This finding may relate to oservations that vasular endothelial ells promote neurogenesis in vitro (11) and that vasular endothelial growth fator, whih stimulates the proliferation of neurons as well as endothelial ells (12), is indued in the rain after stroke (13). However, whether these new neurons arise loally or migrate from anonial neuropro \iferative regions suh as the suventriular zone annot e resolved on the asis of iopsies restrited to the ishemi rain area. Disussion Neurogenesis persists in the adult mammalian rain, where it an e stimulated y physiologial fators, suh as growth fators and environmental enrihment, and y pathologial proesses, inluding ishemia and neurodegeneration. Although these influenes are more diffiult to demonstrate in humans than in experimental animals, evidene for injuryindued human neurogenesis has een presented previously in Huntington's disease (14) and Alzheimer's disease (15). Here, we report similar evidene for stroke-indued neurogenesis in human rain. Standard methods used to detet neurogenesis in animals, suh as the administration of Brd or GFPexpressing viral vetors, annot e used in humans. Therefore, we relied on the expression of endogenous-ell-proliferation markers and neuronal-lineage markers to identify putative neworn neurons in the ortial ishemi penumra of patients with stroke. These markers have limitations in that their relationship to neurogenesis has een haraterized in most detail in rodents, and some of them may also e expressed in Conflit of interest statement: No onflits delared. This paper was sumitted diretly (frak II) to the PNAS offie. Areviations: Ki-67. proliferation-related Ki-67 antigen; MCM2. 5. (erevis""e minihromosome maintenane 2 homolog; PCNA. proliferating ell nulear antigen; DCX, douleortin; TC4, TOAD!lip!CRMP family protein 4; ENCAM, emryoni nerve ell adhesion moleule. tk.l. and X.W. ontriuted equally to this work. :To whom orrespondene should e addressed at: Buk Institute for Age Researh Redwood Boulevard, Novato. CA dgreenerg@ukinstitute.org y The National Aademy of Sienes of the SA I PNAS I August I vol.103 I no. 35

2 iii Tale 1. Clinial and immunohistologial features in patients with stroke Ki-67( +), l<i-67 / DCX( +), Patient Age, ells per mm 2 ells per mm 2 no. yr Sex Main symptoms Duration, d Stroke loation (mean.± SEM) (mean ± SEM) 65 M R hemianopia 40 L parietal 36 ± 7 6±6 R hemiparesis R hemisensory loss 2 45 M Headahe 14 L tempora-oipital 62 ± ± 7 Aphasia ~ 3 54 Headahe 38 R parietal 109 ± ± 3 L hemiparesis ~ 4 66 M L hemiparesis 42 R parietal 48 ± ± 3 L hem isensory loss 5 34 M Headahe 5 R oipital 11 ± 3 3 ± R hemiparesis 32 L parietal 112±12 17 ± 8 Cells were ounted in three X400 fieldsfrom the ortial ishemi penumra, per patient. Duration is the time from onset of symptoms to iopsy. M, male; F, female; R, right; L, left. present in the ishemi penumra ut not in the ishemi ore (where injured ell s would e expeted to e found), and we re situated preferentially in the viinity of patent (erythroyte o ntaining) lood vessels (whih is no t whe re ishemi e ll s injured e ll s. However, we oserved ells that stained for mltiple early neuronal markers, showed normal nu le ar morphology, did not stain for aspase-leavage produts, had features onsistent with a migratory ell phenotype, were a d w Z w '"o '" ~ z Fig. 1. Proliferative status of ells in the ishemi penumra of human ereral ortex after stroke. (a) Nulei stained for the ell proliferation marker Ki-67 (green) are present in the ishemi penumra, where TOTO-3 (red) has een used to ounterstain all nulei. (Sale ar, 150 /Lm.) (Inset) Ki-67-stained nuleus shown at higher magnifiation. () Ki -67 (red) (pper Left) is oloalized with the ell proliferation marker MCM2 (green) (pper Right) in the nuleus of a ell in the ishemi penumra. (Sale ar, 10 /Lm.) () Ki-67 (red) (pper Left) is oloalized with the ell proliferation marker PCNA (green) (pperright) in the nuleus of a ell in the ishemi penumra. (Sale ar, 5Ikm.) (d) Ki-67 (red) (pper Left) oloalizes with a ell-death marker, the 17- to 20-kDa leavage produt of aspase-3 (green) (pper Right) in ells with misshapen nulei (top right orner of eah panel and Right Inset), ut not in ells with normal-appearing nulei (ottom left orner of eah panel and Left Inset). (Sale ar, 10 /Lm.) (-d) Bottom Left and Right panels show DAPI-stained nulei and merged images, respetively. lin et al. PNAS I August 29, 2006 I vol. 103 I no. 35 I 13199

3 a e 9 f Fig. 2. Neuronal harater of ells in the ishemi penumra of human ereral ortex after stroke. (a) The early neuronal marker DCX (green) is expressed in the ytoplasm of numerous ells in the ishemi penumra, where its expression does not overlap with that of the astroglial marker GFAP (red). (Sale ar, 150 ",m.) () The neuronal marker J3111-tuulin (red) is highly expressed in the ishemi penumra (enter of panel), is less highly expressed in adjaent normal ortex (top of panel), and is asent in the ishemi ore (ottom of panel). (Sale ar, 200 ",m.) (e) DCX (green) is expressed in the ytoplasm of a ell with Ki -67-positive (red) nuleus. (Sale ar, 5 ILm.) (d) J3111 -tuulin (red) is expressed in the ytoplasm of a ell with Ki -67-positive (green) nuleus. (Sale ar, 7 ILm.) (e) TC-4 (red) is expressed in the ytoplasm of a ell w ith Ki-67-positive (green) nuleus. (Sale ar, 5 ILm.) (I) ENCAM (red), DCX (green), and TC-4 (purple) are oloalized; the nul eus is ounterstained with DAPI (lue). (Sale ar, 10 ILm.) (g) A ell with a Ki-67-stained nuleus (red) and DCX-positive ytoplasm (green) exhiits harateristi migratory morphology, w ith a leading proess and trailing nuleus; nulei are ounterstained with DAPI (lue) I gi/ doi/ / pnas Jin et al.

4 a... u ẓ.. o'" ii! "" z Fig. 3. Vasular nihe for stroke indued neurogenesis in human ereral ortex. (a) A setion stained for DCX (green). TC-4 (purple), and nestin (red) and ounterstained with DAPI (lue) shows DCX- and TC-4-positive ells, some with the elongated morphology oserved in migrating neurons (Inset), in lusters in the viinity of a apillary that ontains erythroytes. (Sale ar, 20 /Lm.) () DCX-positive e lls (green) are in the viinity of, ut distint from, von Willerand fator-positive endothelial ells (red); nulei areounterstained with DAPI (lue). " vessel lumen. (Sale ar, 15 /Lm.) (e) DCX-positive ells (g reen) are in the viinity of, ut distint from, (l',-aetin-positive vasular smooth musle ells (red); nulei are ounterstained with DAPI (lue). " vessel lumen. (Sale ar, 15 /Lm.) would e antiipated). It is possile th a t all of these attriutes are assoiated with ishemi injury rather than neurogenesis, ut it seems unlikely. In rodent models of stroke, the soure of new neurons that migrate to ishemi rain areas appears to e the suventriular zone (6-9). In human iopsy speimens, the soure of these ells is unlear, ut their perivasular loation may provide lues. One possiility is that neworn neurons migrate from elsewhere (suh as the suventriular zone), using lood vessels as saffolds for migration or as destination markers. Alternatively, these neurons ould arise loally, although whether neurogenesis an our in situ in primate ereral neoortex is ontroversial (16, 17). The extent to whih stroke-indued neurogenesis in human rain results in the prodution of funtional neurons with the apaity to integrate into rain iruitry is also unertain and is not addressed y our findings. Several drugs an stimulate neurogenesis in the dentate gyrus or suventriular zone of ishemi rodent rain, inluding statins (18) and mediations used to treat eretile dysfuntion (19). Beause none of the patients we studied was known to have taken any of these drugs, the inreased neurogenesis we oserved is unlikely to have een drug-indued. On the other hand, the aility of drugs and environmental alterations to promote nem'ogenesis suggests linial interventjons that might e used to enhane this proess. Although its funtional impat is as yet unknown, postishemi neurogenesis in the human rain may represent oth an e ndogenous repair mehanism and a therapeuti target for stroke. Materials and Methods Human Brain Tissue. Brain speimens from patients aged years and without linial or postmortem evidene of neurolog- Jin et al. PNAS I August 29, 2006 I vol. 103 I no. 35 I 13201

5 ial disease (n = 6) were otained at autopsy, and human stroke speimens (/7 = 9) were otained at iopsy performed for diagnosti purposes, with informed onsent and in aordane with protools approved y the Institutional Researh Review Board at Huashan Hospital of Fudan niversity, China. Cereral ortial infarts were onfirmed y histopathology in six of nine patients, who were inluded in the prese nt study; the rem ai ning three of nine iposied patients were exluded eause of other diagnoses (one eah with ereellar infart, intraereral hemorrhage, and intraventriular hemorrhage). Immunohistohemistry. Tissues were postfixed in paraformaldehyde for 24 h, inuated with 30% surose for 3 days, and emedded in paraffin; 6-p,m setions were ut on a mirotome and stored at room temperature. Setions were deparaffinized with xylene and rehydrated with ethanol. Peroxidase ativity was loked with 1% H20 2, and setions were treated with itrate uffer (ph 6.0) (Zymed, Carlsad, CA) at 96 C for 30 min, ooled for 25 min, and inuated in loking uffer (2% horse serum/0.2% Triton X-100/0.1% BSA in PBS) for 1 h at room temperature. Primary antiodies used were mouse monolonal anti-ki-67 antigen (1:50; Novoastra, Newastle upon Tyne,.K.), rait anti-ki-67 antigen (1:100; Zymed), goat anti MCM2 (1:100; Santa Cruz Biotehnology, Santa Cruz, CA), mouse monolonal anti-pcna (1:200; Chemion, Temeula, CA), rait anti-leaved aspase 3 (1:200; BD PharMingen, San Diego, CA), affinity-purified goat anti-dcx (1:200; Santa Cruz Biotehnology), rait anti-gfap (1:1,000; Sigma, St. Louis, MO), mouse monolonal anti-/3ili-tuulin (TJl, 1:250; Covane, Berkeley, CA), rait polylonal anti-tc-4 (1:10,000; Chemion), mouse monolonal anti-encam (1:500; Chemi- on), mouse monolonal anti-human-speifi nestin (1:200; Chemion), rait anti-von Willerand fator (1:500; Chemion), and mouse monolonal anti-a2- (vasular smooth musle) atin (1:100; Maine Biotehnology, Portland, ME). Primary antiodies were added in loking uffer and inuated with setions at 4 C overnight. The seondary antiodies were Alexa Fluor 488-, 594-, or 647-onjugated donkey anti-mouse, anti-goat, or anti-rait IgG (1: ; Moleular Proes, Carlsad, CA). Nulei were ounterstained with DAPI using prolong Gold antifade reagent or with TOTO-3 (all from Moleular Proes), and fluoresene signals were deteted with Nikon (Melville, NY) PCM-2000 lasersanning onfoal mirosopy a t exitation/emission wavelengths of 650/668 nm (Alexa Fluor 647, far red), 590/617 nm (Alexa Fluor 594, red), 495/519 nm (Alexa Fluor 488, green), or 360/400 nm (DAPI, lue). Slides were examined with an LSM 510 NLO onfoal sanning system mounted on an Axiovert 200 inverted mirosope (Carl Zeiss, Oerkohen, Germany) equipped with a two-photon Chameleon laser (Coherent, Santa Clara, CA). Images were aquired using LSM 510 imaging software (Carl Zeiss). Four-olor images were sanned using argon, 543 HeNe, 633 HeNe, and Chameleon ( nm for DAPI) lasers. 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