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
er o
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!