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 proliferation5-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 BrdU NeuN/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

0.8

29

137

Cell number Cell 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

639

289

395

342

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Lesion Contralateral Sham Sham-ContralateralLesion Contralateral Sham Sham-

Contralateral

Cell proliferation in SVZ

Nu

mb

er o

f B

rdU

+ c

ells

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

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L e sio n + S a lin e L e sio n + A ra -C

Cell proliferation

BrdU &

SalineBrdU &

Ara-C

Nu

mb

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f B

rdU

+ c

ells

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 BrdU Dcx/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 stroke Number of cells/mm3

BrdU/NeuN BrdU/Dcx

2 78 ± 38 3900 ± 1000

5 137 ± 67

6 750 ± 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!