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Axon Regeneration Why do injured adult CNS axons fail to regrow?
1) Axon regeneration is age-dependent
2) Severed adult axons can grow in vitro
3) Scar tissue prevents growth
Glial cells
ECM
cut axons
fibroblasts
reactive astrocytes
macrophages
ECM collagen
4) Permissive influence of peripheral glial cells
Traumatic brain injury statistics
Thurman et al., 1999
Gender Cause
Age (years)
30
20
10
0 0-4 20-24 40-44 60-64 80-84
Transportation
Firearms
Falls
40
female male
Hospitalization & death (7 states, 1994)
Rate (per 105)
Age (years)
Rate (per 105)
120
80
40
0 0-4 20-24 40-44 60-64 80-84
female male
http://www.cdc.gov/braininjuryinseniors/
Response Time: 45 mins Helicopter or Ambulance 69% of population
Level I/II Trauma Center Coverage (2008)
Response Time: 60 mins Ambulance 56% of population
http://www.spinalcord.uab.edu/show.asp?durki=21446
Spinal cord injury • 11,000 new cases per year in the U.S.
• Approx. 250,000 people alive
• Most injuries occur between 16 to 30 years
• Approx. 80% of injuries occur among males
• Car accidents account for about 50%
• Average cost for C1-4 injury: year one: $741,425 subsequent years: $132,807
What regenerates?
Cold-blooded vertebrate RGCs (rest of CNS is not good)
Mammalian peripheral nerves
Limited regeneration in neonatal mammals
Local sprouting (<500 µm) in adult mammals
Kalil and Reh, 1979
lesion
Adult lesion Infant lesion (PND1 to 7)
lesion
Axons can grow around lesion Control
Age-dependence
Age-dependent regeneration in vitro
Age-dependent regeneration in vitro
P12 control
P6 midline section
P12 midline section
Hafidi et al., 1995, 1999
Hafidi et al., 2004
Age-dependent regeneration in vitro
P6 P12
Club endings
Jorge Francisco Tello y Muñóz (Cajalʼs student)
Cajal in his lab
Kerschensteiner et al., 2005
In vivo imaging of cut axons
GFP-labeled axons in mouse spinal cord (Thy-1 promotor)
GFP-labeled DRG Axon is imaged
DRG
Dorsal view of mouse spinal cord
DRG axon: pre-lesion
DRG axon: post-lesion (200µm needle)
Kerschensteiner et al., 2005
In vivo imaging of cut axons
Proximal cut end: die back Distal cut end: Wallerian degeneration
GFP labeled DRG neuron axons running in the dorsal column of the spinal cord
Kerschensteiner et al., 2005
axonal fragmentation
retraction
bulb formation
In vivo imaging of cut axons
Attempted regrowth at 24 hours
In vivo imaging of cut axons
Adult axons can grow
Thanos et al., 1989
Adult retinal axons growing in vitro with BDNF
Scar tissue limits regeneration
Davies et al.1997
1 mm
MIDLINE 100 µm
No injury: regeneration
INJURY SITE
axons
Transplant DRG neurons in the corpus callosum
Injury: no regeneration
Rudge and Silver, 1990
Hippocampal axons don’t growth on adult scar material
Adult optic nerve Adult optic nerve + macrophages
Cortex
nitrocellulose
Neonatal scar stuff Adult scar stuff
Scar tissue limits regeneration
axon
axon
axon
cut axons
fibroblasts
reactive astrocytes
Macrophages
ECM
collagen
What’s in the scar?
myelin
Reactive astrocytes express EphA4 GFAP EphA4 Merged
Lesi
on s
ite
Goldshmit et al., 2004
… which is a poor growth substrate WT reactive astrocytes Eph4A-/- astrocytes
Better regeneration in EphA4-/- mice
Goldshmit et al., 2004
Eph
A4-
/-
Con
trol
ECM
chain of disaccharides covalently linked to a core protein
Core protein
Glycosaminoglycans (GAG)
“Average” Proteoglycan aggregate • one hyaluronic acid • 100 core proteins • 50 keratan sulfate chains (250 disaccharide units/chain) • 100 chondroitin sulfate chains (1000 disaccharide units/chain) • Molecular weight: tens of millions
Proteoglycan aggregate
Hyaluronic acid (also a GAG)
link protein
Scar tissue limits regeneration
Davies et al.1997
1 mm
MIDLINE 100 µm
No injury: regeneration
axons
Transplant DRG neurons in the corpus callosum
CSPG: no regeneration
laminin keratin sulfate &
chondroitin sulfate proteoglycan
laminin
50% laminin +
50% keratin sulfate & chondroitin sulfate
proteoglycan
laminin DRG
Neurocan: a proteoglycan expressed at injury site
Asher et al., 2000
Control cortex Injured cortex
Neurites do not grow on Neurocan
Asher et al., 2000
Cerebellum on L1
Cerebellum on L1 + Neurocan stripes
Neurocan L1
L1
(a chondroitin sulfate proteoglycan)
GC behavior on proteoglycan (Aggrecan)
Tom et al., 2004
Laminin
60x time lapse 60x time lapse
Laminin + Aggrecan
Growth cone behavior on Aggrecan
260x time lapse
Bradbury et al., 2002
Blockade of proteoglycans restores growth in spinal cord
labeled CST fibers no label
Control Lesioned
Control Lesioned _+ vehicle Lesioned _+ chondroitinase
Lesioned _+ chondroitinase: invasion of grey matter
Fibr
e co
unts
(%)
Distance from lesion (mm)
Sham Les+Veh Les+CHase
100
80
60
40
20
0
Moon et al., 2001
Blockade of proteoglycans restores growth in nigrostriatial pathway
chondroitinase
Tyrosine hydroxylase immunoreactivity
Chondroitinase
Control
cut axons
Macrophages
What’s in the scar?
Chen et al., 2000
Macrophage migrate to axon injury site in leech
5 minutes post-injury 3 hours post-injury E
xten
t of n
erve
cru
sh
Davalos et al., 2005
Microglial response to injury in Cortex
Lesion with laser ATP
David et al.
Macrophages accumulate at optic nerve lesion
Lesion site
Cut optic nerve
Obtain cryostat section of optic nerve lesion
lesion site
Do macrophages have an effect in CNS injury?
Cut optic nerve
Obtain cryostat section of optic nerve lesion
lesion site
DRG growth near lesion site
DRG growth distal to lesion site
Adult optic nerve Adult optic nerve + macrophages
Cortex
nitrocellulose
Adult optic nerve Adult optic nerve + macrophages
Injuring lens induces macrophages in retina
Do macrophages have an effect in CNS injury?
Leon et al., 2000; Lorber et al.,2005
nerve crush nerve crush + lens injury
brown = macrophage stain
injuring lens induces regeneration
injure lens
crush
lens retina
crush
regeneration Leon et al., 2000
100 µm
Do macrophages have an effect in CNS injury?
Macrophage activation mimics lens injury
Zymosan injection increases macrophages in the retina (Zymosan = a yeast cell wall suspension)
Leon et al., 2000; Lorber et al.,2005
Macrophages in retina Regeneration in Zymogen-treated retina
Macrophages produce oncomodulin, a Ca+2 binding protein
Yin et al., 2006
control
lens injury
macrophages oncomodulin
Oncomodulin enhances axon regeneration
control
oncomodulin
cut axons
What’s in the scar?
myelin
Bandtlow et al.
tubulin = yellow actin = red
GC collapse after contact with oligodendrocyte
Contact Collapse
ethidium bromide knife cut
CNS regeneration following depletion of glia
Moon et al., 2000
myelin stain
Saline
Ethidium bromide
Dopaminergic fibers regenerate past lesion (but not into striatum)
anterior posterior
lesion
Antibody to oligodendrocyte protein: Nogo-A
vehicle-treated
anterior posterior
lesion
Antibody-treated
Yiu & He, 2006
RhoA activity leads to growth cone collapse
Gaillard et al., 2007
Blocking Rho permits RGC axon regeneration Control Rho inhibitor (C3) Protocol
Sciatic nerve graft in cortex
Tello, F (1911) La influencia del neurotropismo en la regeneration de los centros nerviosos. Trab Lab Invest Univ Madrid 9:123-159
David and Aguayo, 1981
Sciatic nerve bridge in spinal cord
David and Aguayo, 1981
Fibers entering medulla Fibers entering spinal cord
200 µm
Sciatic nerve bridge in spinal cord
Olfactory bulb ensheathing cells promote regeneration
Thuret et al., 2006
Olfactory bulb ensheathing cells promote regeneration
Li et al., 1997
Lesion of corticospinal tract
0.5 mm
Green = axons Red = ensheathing cells
rost
ral
caud
al
200 µm
Spinal cord injury patients: olfactory mucosa autografts
Lima et al., 2006
7 patients, ages 18-32, treated 6 mos to 6 yrs after injury
scar tissue removed from injury site before graft nerve bundle
collagen is stained green
GFAP is stained brown
*
Lima et al., 2006
Spinal cord injury patients: olfactory mucosa autografts
Lima et al., 2006
Spinal cord injury patients: olfactory mucosa autografts
Lima et al., 2006
Spinal cord injury patients: olfactory mucosa autografts
Gaillard et al., 2007
Growth of embryonic rat cortex in lesioned adult
E14 tissue
GFP+ fibers were found in:
Busch and Silver, 2007