Download presentation source

The adult brain after stroke: Neuronal replacement
from endogenous precursors
A. ARVIDSSON, T. COLLIN, D. KIRIK, Z. KOKAIA, O. LINDVALL.
Nature Medicine, September, 2002
Matt Feldman
September 27, 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
Neurogenesis observed in the SVZ, dentate gyrus and olfactory bulb
1. Ventricle and subventricular
zone (SVZ)
2. Striatum
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)
• DNA synthesis/cell proliferation
measured by BrdU incorporation
during S phase; detection using
anti-BrdU monoclonal Antibody
• 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
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 nonneuronal detection
• Doesn’t detect all types of neurons, but most
Stroke leads to neurogenesis in damaged striatum
• Individual neuron in X-Y plane
NeuN
BrdU
NeuN/BrdU
• Successive sections of neurons in the Z plane
Stroke leads to neurogenesis in damaged striatum
• BrdU injected 2x/day during days 4,5,6 post-stroke (n=9; 10)
• 31-fold increase in number of BrdU/NeuN-labeled cells
Cell number
Cell density
137
“Intact” is
uninjured
striatum
29
0.8
“Total” is
entire striatum
• Few observed BrdU/NeuN cells in contralateral striatum of
MCAO; same in sham
• Massive inflammatory reaction, demonstrated in ischemic
tissue by BrdU+/NeuN- cells
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
Cell proliferation in SVZ
Number of BrdU+ cells
800
700
639
600
500
395
400
342
289
300
200
100
0
Lesion
Lesion
Contralateral
Contralateral
Sham
Sham
ShamSham-Contralateral
Contralateral
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
Number of BrdU+ cells
• BrdU co-injected with Ara-C (saline controls) for 12
days after stroke
Cell proliferation
1400
• Much lower BrdU in
1200
1027
Ara-C-injected animals
1000
• Cell proliferation in SVZ is
800
responsible for BrdU
600
400
immunopositivity
174
200
0
BrdU &
Saline
BrdU &
Ara-C
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
Dcx
Saline
BrdU
Ara-C
• Early-incorporated BrdU indicates production
of migratory neuroblasts from SVZ
Dcx /
BrdU
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 strokegenerated 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:
Weeks after stroke
Number of cells/mm3
BrdU/NeuN
BrdU/Dcx
2
78 ± 38
5
137 ± 67
6
750 ± 214
3900 ± 1000
• 4 weeks after last BrdU injection, BrdU+/NeuN+ cells ~5fold higher (~10x higher density) than measurements
taken directly after last BrdU administration
• 6 weeks post-stroke represents a considerable loss of
new neuroblast population
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!