Matt Feldman September 27, 2002 The adult brain after stroke: Neuronal replacement from endogenous precursors A. ARVIDSSON, T. COLLIN, D. KIRIK, Z. KOKAIA, O. LINDVALL. Nature Medicine, September, 2002

Overview Background: neurogenesis –Up to here: previous research –This work: what’s involved and why The system of study –Techniques and markers for measuring proliferation The question –Is true neurogenesis observed? The implications and the future

Background The adult brain: relevant anatomy 1. Ventricle and subventricular zone (SVZ) 2. Striatum Neurogenesis observed in the SVZ, dentate gyrus and olfactory bulb

Background: Magavi et al. Magavi et al. (Nature 405, 951–955 (2000)) –induced neuronal degradation and examined fate of dividing cells –chromophore-targeted apoptosis of pyramidal neurons of the cortex induced neurogenesis some reconstitution of damaged area –3D laser scanning confocal microscopy confirmed that new cells are not merely closely in close proximity to pre-existing neurons pyramidal morphology indicative of long distance projections –additional labeling was negative for GFAP and MBP (immature markers) new neurons had fully differentiated

Background: Magavi et al. Cell division can continue after an injury But, unlike a clinical stroke event, lesion only affected targeted neurons –Method makes damaged/destroyed neurons the source of the injury, rather than the pathological outcome –Ignores tissue complexity All the surounding cells (and precursors they express) are still intact –Relatively small lesion –Quiescent, but pre-determined survivors may differentiate with signals from adjacent cells

Approaching clinical relevancy Neurogenesis is observed in the adult brain After a more clinically relevant event (ischemic stroke – localized anemia following occlusion), is similar neurogenesis observed? –Can new neurons migrate to the site of an injury? –If so, are they appropriate? Long-lived? Are endogenous precursors sufficient to stimulate neurogenesis in adult rat striatum following stroke?

Methods: MCAO Injury model employed middle cerebral artery occlusion (MCAO) technique –monofilament inserted into common carotid artery and advanced to middle cerebral artery, held for 2 hours –Sham: filament placed into common carotid, no forward advancement

Methods: Markers of proliferation 5-bromo-2’-deoxyuridine (BrdU) Newly-injected BrdU is available for a few hours for incorporation Replaces tritiated thymidine and autoradiographic assays with immunological quantification Fluorescent Ab tagging in multiple excitation channels allows for simultaneous measurement of different probes DNA synthesis/cell proliferation measured by BrdU incorporation during S phase; detection using anti-BrdU monoclonal Antibody

Methods: Markers of neurogenesis Neuronal nuclear antigen (NeuN) Neuron-specific nuclear protein (vs cytoplasmic or cell-surface antigen) observed in invertebrates Recognized with a mAb in standard IHC Specifically reactive for post-migratory (late maturity) neurons No non-specific (ex. glial) reaction within NS; no non- neuronal detection Doesn’t detect all types of neurons, but most

Stroke leads to neurogenesis in damaged striatum NeuN BrdUNeuN/BrdU Individual neuron in X-Y plane Successive sections of neurons in the Z plane

BrdU injected 2x/day during days 4,5,6 post-stroke (n=9; 10) 31-fold increase in number of BrdU/NeuN-labeled cells Few observed BrdU/NeuN cells in contralateral striatum of MCAO; same in sham Massive inflammatory reaction, demonstrated in ischemic tissue by BrdU+/NeuN- cells Stroke leads to neurogenesis in damaged striatum “Intact” is uninjured striatum Cell numberCell density “Total” is entire striatum

Evidence for self-repair following stroke Neurogenesis is observed in the adult brain –Colocalization of BrdU and NeuN in lesion area But via what route?

Proliferation and recruitment of neuroblasts Where do new neurons originate? Examine ongoing cell proliferation in SVZ immediately following injury BrdU injected 2x/day for 2 weeks then rats were sacrificed LesionContralateralSham Sham- Contralateral Cell proliferation in SVZ Number of BrdU+ cells

Proliferation and recruitment of neuroblasts Confirmation that BrdU incorporation specifically results from SVZ proliferation Ara-C (cytosine-β-D-arabinofuranoside) –Antimitotic drug inhibits cell proliferation in mouse SVZ BrdU co-injected with Ara-C (saline controls) for 12 days after stroke Much lower BrdU in Ara-C-injected animals Cell proliferation in SVZ is responsible for BrdU immunopositivity Cell proliferation BrdU & Saline BrdU & Ara-C Number of BrdU+ cells

Methods: Markers of neurogenesis Doublecortin (Dcx) Specific for early post-mitotic neurons Microtubule-associated protein (366 a.a., 40kD) expressed exclusively in migrating and differentiating neurons (neuroblasts) Not expressed in mature neurons As Dcx expression declines, complex morphology (apical processes) increases –indicates increasing differentiation

Proliferation and recruitment of neuroblasts Dcx BrdU Saline Ara-C Dcx BrdU Dcx / BrdU Early-incorporated BrdU indicates production of migratory neuroblasts from SVZ

Evidence for self-repair following stroke Neurogenesis is observed in the adult brain Cells proliferating from SVZ –Stroke-generated migratory neuroblasts observed in SVZ (Dcx+) –Neuroblast production can be depressed by shutting down SVZ (Ara-C) –Some pre-existing (BrdU-) cells have neuroblast characteristics (Dcx+), but majority of Dcx+ cells are newly formed (BrdU+/Dcx+) But do new neurons move from SVZ to the lesion?

Neurons migrate from SVZ to lesion BrdU/Dcx neurons observed moving laterally and ventrally from SVZ to lesion (up to 2mm) in the 14 days following stroke –Controls: contralateral area and sham animals have Dcx confined solely to SVZ Observed morphologies: –Non-migratory symmetry, multidirectional processes –Migrating elongated, with leading processes Leading processes directed away from SVZ

Morphologies of migrating neurons Normal neuronal morphology is observed Dcx BrdUDcx/BrdU

Evidence for self-repair following stroke Neurogenesis is observed in the adult brain Cells proliferating from SVZ New stroke-generated neurons migrate from SVZ to the lesion –Neuroblasts with normal morphology observed to span a distance of up to 2mm What are the functional characteristics of these newly migrated neurons?

Cells express markers of striatal medium spiny neurons Meis2 Transcription factor normally expressed in proliferating striatal precursors Also expressed (to a lesser degree) in adult striatum Pbx Colocalized with Meis2 during neuronal development DARPP-32 Indicative of medium-sized spiny neurons

Markers of developing striatal neurons Striatal phenotype from neuroblasts Phenotype observed in BrdU+ neurons

Results: developmental markers BrdU injected at days 4-6 (to examine early cell proliferation) 2 weeks after injury: –96% of Dcx+ cells were Meis2+ –94% of Dcx+ cells were Pbx+ Early markers also seen in BrdU- cells (existing pre-injury, on lesion and control side), but stronger in BrdU+ cells –Consistent with prior observations of weaker mature expression 5 weeks after injury: –42% of BrdU+/NeuN+ cells were BrdU+/DARPP-32+

Evidence for self-repair following stroke Neurogenesis is observed in the adult brain Cells proliferating from SVZ New stroke-generated neurons migrate from SVZ to the lesion New neurons indicate phenotypic characteristics of the type within the lesion –Early markers (Meis2, Pbx) are expressed in new neurons –Markers of striatal medium spiny neurons (DARPP-32) are observed in mature stroke- generated cells Over what time frame does the maturation process occur?

Neurogenesis and maturation How fast is the maturation process? Sacrifice after 2 weeks of 2x/daily BrdU injection: 4 weeks after last BrdU injection, BrdU+/NeuN+ cells ~5- fold higher (~10x higher density) than measurements taken directly after last BrdU administration 6 weeks post-stroke represents a considerable loss of new neuroblast population Weeks after strokeNumber of cells/mm 3 BrdU/NeuN BrdU/Dcx 2 78 ± ± ± ± 214

Evidence for self-repair following stroke Neurogenesis is observed in the adult brain Cells proliferating from SVZ New stroke-generated neurons migrate from SVZ to the lesion New neurons indicate functional characteristics of the type within the lesion Neurogenesis leads to maturation which continues throughout survival Following stroke, endogenous precursors are sufficient to stimulate neurogenesis in the adult rat brain

Summary Neuronal replacement is observed, but critical determinations remain: –Nature of the signaling molecules involved –Long-term survival of neurons –Functionality of individual neurons –Are they sufficient functional replacement? (0.2%) If new neurons are functional, treatment might reinforce the processes at work

Thanks!