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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
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
juan-carlosandaluz
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Retinal ganglion cells can regenerate;
Kalil and Reh, 1979
lesion
Adult lesion Infant lesion (PND1 to 7)
lesion
Axons can grow around lesion Control
Age-dependence
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In the adult, the cell will die if the axon is cut. In the infant, the cut axon will regrow around the lesion.
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
juan-carlosandaluz
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one way to test whether the environment is good enough to grow is to take an area of the brain that contains a ton of axons. Then transplant cells from another brain region into this new environment (without any injuries) What they found is that the axons grow normally
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On the other hand, when they transplant the cells into the new environment which contains an injury, there is no axonal regeneration. This shows that scar tissue limits axonal regeneration.
Rudge and Silver, 1990
Hippocampal axons don’t growth on adult scar material
Cerebellar neuron grows on Laminin but it does not grow in L1 + neurocan stripes. Neurocan is really good at repelling growth.
GC behavior on proteoglycan (Aggrecan)
Tom et al., 2004
Laminin
60x time lapse 60x time lapse
Laminin + Aggrecan
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Club ending forms in axon regrowing in 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
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Because myelin activates the RhoA/Rho kinase (ROCK)signaling pathway in PC12 cells and blocking of RhoA activityovercomes myelin inhibition
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
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RhoA activation has been shown to correlate with signals that induce growth cone collapse and axon guidance repulsion81. Evidence suggests that this pathway also mediates myelin inhibition
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myelin-based inhibitory signals might trigger the activation of RhoA and ROCK, leading to the phosphorylation of cofilin by LIM kinase to stabilize the growth cone cytoskeleton of damaged axons, restricting regenerative outgrowth
juan-carlosandaluz
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In the central nervous system (CNS), RHOA GTPase signaling through Rho kinase (ROK) promotes growth cone collapse and inhibits regrowth (Luo et al., 1997). Consequently RHOA activation has been associated with impaired regeneration of spinal cord axons
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http://people.ucalgary.ca/~mdnguyen/pdf/20081.pdf
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RHO is associated with the Rho kinase (ROK), in turn thought to activate LIM kinase that phosphorylates cofilin. Phosphorylated cofilin, in turn, has a loss of function that interferes with actin turnover and growth cone advance (Ng and Luo, 2004). RHOA is also linked to the inhibition of myosin phosphatase activity that in turn leads to increased myosin ATPase activity and growth cone retraction (Luo et al., 1997). An additional route to retract growth cones is through direct ROK phosphorylation of myosin (Giniger, 2002). Therefore, the net result of ROK activation is an increase in actin arc and central actin bundle contractility and stability with secondary actions on microtubular organization (Zhang et al., 2003). These actions collapse growth cones and mediate active repulsion
Inhibiting RhoA using C3 transferase or a dominant-negative approach also promotes axon outgrowth on inhibitory substrates
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y inhibiting the RHOA-ROK intracellular signaling pathway we observed an increase in axon outgrowth. HA- 1077 is a relatively specific inhibitor of ROK
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