The adult brain after stroke neuronal replacement from endogenous precursors
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The adult brain after stroke: Neuronal replacement from endogenous precursors. Matt Feldman September 27, 2002. A. ARVIDSSON, T. COLLIN, D. KIRIK, Z. KOKAIA, O. LINDVALL. Nature Medicine, September, 2002. Overview. Background: neurogenesis Up to here: previous research

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The adult brain after stroke neuronal replacement from endogenous precursors l.jpg

The adult brain after stroke: Neuronal replacement from endogenous precursors

Matt Feldman

September 27, 2002

A. ARVIDSSON, T. COLLIN, D. KIRIK, Z. KOKAIA, O. LINDVALL.

Nature Medicine, September, 2002


Overview l.jpg
Overview endogenous precursors

  • 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 l.jpg
Background endogenous precursors

  • 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 l.jpg
Background: Magavi endogenous precursors 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 al5 l.jpg
Background: Magavi endogenous precursorset 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 l.jpg
Approaching clinical relevancy endogenous precursors

  • 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 l.jpg
Methods: MCAO endogenous precursors

  • 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 l.jpg
Methods: Markers of proliferation endogenous precursors

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 l.jpg
Methods: Markers of neurogenesis endogenous precursors

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 l.jpg
Stroke leads to neurogenesis in damaged striatum endogenous precursors

Individual neuron in X-Y plane

NeuN

BrdU

NeuN/BrdU

Successive sections of neurons in the Z plane


Stroke leads to neurogenesis in damaged striatum12 l.jpg
Stroke leads to neurogenesis in damaged striatum endogenous precursors

137

Cell number

Cell density

29

0.8

  • 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

“Intact” is uninjured striatum

“Total” is

entire striatum


Evidence for self repair following stroke l.jpg
Evidence for self-repair following stroke endogenous precursors

  • Neurogenesis is observed in the adult brain

    • Colocalization of BrdU and NeuN in lesion area

  • But via what route?


Proliferation and recruitment of neuroblasts l.jpg
Proliferation and recruitment of neuroblasts endogenous precursors

Cell proliferation in SVZ

Number of BrdU+ cells

Sham-

Contralateral

Lesion

Contralateral

Sham

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


Proliferation and recruitment of neuroblasts15 l.jpg
Proliferation and recruitment of neuroblasts endogenous precursors

  • 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

Number of BrdU+ cells

BrdU &

Saline

BrdU &

Ara-C


Methods markers of neurogenesis16 l.jpg
Methods: Markers of neurogenesis endogenous precursors

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 neuroblasts17 l.jpg
Proliferation and recruitment of neuroblasts endogenous precursors

Dcx

BrdU

Dcx

Saline

BrdU

Ara-C

Dcx /

BrdU

  • Early-incorporated BrdU indicates production

    of migratory neuroblasts from SVZ


Evidence for self repair following stroke18 l.jpg
Evidence for self-repair following stroke endogenous precursors

  • 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 l.jpg
Neurons migrate from SVZ to lesion endogenous precursors

  • 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 l.jpg
Morphologies of migrating neurons endogenous precursors

Normal neuronal morphology is observed

Dcx

BrdU

Dcx/BrdU


Evidence for self repair following stroke21 l.jpg
Evidence for self-repair following stroke endogenous precursors

  • 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 l.jpg
Cells express markers of striatal medium spiny neurons endogenous precursors

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 l.jpg
Markers of developing striatal neurons endogenous precursors

Striatal phenotype from neuroblasts

Phenotype observed in BrdU+ neurons


Results developmental markers l.jpg
Results: developmental markers endogenous precursors

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 stroke25 l.jpg
Evidence for self-repair following stroke endogenous precursors

  • 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 l.jpg
Neurogenesis and maturation endogenous precursors

Weeks after strokeNumber of cells/mm3BrdU/NeuNBrdU/Dcx

2 78 ± 38 3900 ± 1000

5 137 ± 67

6 750 ± 214

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


Evidence for self repair following stroke27 l.jpg
Evidence for self-repair following stroke endogenous precursors

  • 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 l.jpg
Summary endogenous precursors

  • 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


Slide29 l.jpg

Thanks! endogenous precursors


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