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Title: Spinal cord injury and repair I


1
Spinal cord injury and repair (I)
Dr Lawrence Moon
During the lecture, please interrupt with
questions when you have them. While you wait,
please fill in the questionnaire anonymously!
2
After these lectures and appropriate reading you
should be able to
  • Describe the gross anatomy of spinal cord
  • Describe the neuropathology of spinal cord injury
  • Describe possible mechanisms contributing to loss
    of function
  • Describe animal models of spinal cord injury
  • Critically evaluate current treatments
    (pharmacological, rehabilitative)
  • Critically evaluate potential therapies for
    spinal cord injury

Tips on answering exam questions
1. Answer the question, not just the part you
revised! 2. Read the reading list. 3. Read
beyond the reading list (esp. 2008, 2009) 4. Use
references (Smith et al., 2008) so pay attention
to names....
3
SFN video (Stop after successful life)
4
Billions of neurons in your brain!
Nervous system CNS, PNS, ANS CNS neurons
brain, spinal cord PNS nerves connects CNS
to outside world - sensory - motor
5
The CNS
  • Neurons
  • Cell body
  • Dendrites
  • Axon
  • Growth cone
  • Synapse
  • Glia
  • Microglia
  • Astrocytes
  • NG2 progenitors
  • Oligodendrocytes
  • Vasculature
  • Meninges
  • Ventricles
  • ependymal cells
  • CSF

6
Growth cones are dynamic!
Embryonic chick neurons. Time lapse in vitro
images courtesy of Dr Britta Eickholt.
7
Anatomy of a normal human vertebral column
Thuret, Moon, Gage, 2007
  • Corticospinal tract (dl)
  • Rubrospinal tract
  • Sensory axons in dorsal columns

8
Anatomy of a normal human spinal cord
9
Pathology of SCI depends on
  • anatomical level of injury
  • type of injury
  • magnitude of injury

10
  • Motor dysfunction below the injury site
  • Loss of sensation below the injury site
  • Pain
  • Bladder, bowel, sexual dysfunction

quadruplegia
Prevalence in USA 250,000 Incidence in
USA 11,000
Hand and finger Movement (C7-T1)
paraplegia
U Alabama
11
Pathology
  • Immediate
  • Axon interruption
  • Hours
  • Neurons die (necrosis)
  • Neutrophils invade
  • Macrophages invade
  • T cells invade
  • Days
  • Cyst / cavity forms
  • Wallerian degeneration
  • Demyelination, debris
  • Glial reactivity (3 types)
  • Apoptosis (ns, os)
  • Scarring (Fb, SCs, ECM)
  • Weeks, months, years
  • Ongoing cell death (syringo)

12
What can be done at present?
  • Critical care (Stoke Mandeville, Oswestry, etc)
  • Save life
  • Stabilise injury (decompress, fix vertebrae)
  • Few acute therapies
  • steroids (methylprednisolone) v contraversial
  • Hurlbert vs. NASCIS
  • Few chronic therapies
  • rehabilitation (locomotor)
  • adaptation (sexual, bladder, bowel)
  • None fully restorative

13
Spontaneous recovery is slight
  • Spinal shock
  • Respiration (Goshgarian, 2003)
  • ventilator weaning
  • Rehabilitation of limbs

14
Why only some spontaneous recovery?
  • Very few new neurons are born (neurogenesis)
  • Spontaneous failure of CNS axon regeneration
  • Limited endogenous repair (adult vs neonate)
  • Insufficient compensatory plasticity
  • Poor intrinsic axon growth
  • Pro-growth molecules down-regulated
  • Anti-growth pathways switched on
  • Inhospitable extrinsic environment
  • Cysts, cavities
  • Fibrotic scar
  • Growth-inhibitory molecules (intact injured)
  • Lack of growth factors, permissive substrates

15
Reactions of neurons to injury
16
Adult neurons grow very poorly
  • Goldberg et al., 2002

17
Inhibitors of growth
  • Development axon growth stops when
  • synapses form
  • myelin wraps axons
  • extracellular matrix nets accumulate
  • Critical window for regenerative success also
    closes
  • Partly a geometrical issue, partly a molecular
    signaling event
  • Pettigrew Crutcher, 1999

18
Growth cones collapse dynamically!
Embryonic chick neurons. Time lapse in vitro
images courtesy of Dr Britta Eickholt.
19
Why is adult CNS inhibitory? What is mechanism?
What are the molecules? How do neurons recognise
them? Extracellular receptors and
co-receptors How does the signal reach the
intracellular region? How does it prevent axon
growth? - linking to intracellular signalling
pathways - linking to axon cytoskeleton -
growth cone collapse - slowing down / turning
away Thus, what pathways can be exploited to
boost axon regrowth? Translating basic science
to the clinic....
20
Inhibitors of growth
  • Purified myelin inhibits axon growth (Caroni
    Schwab, 1989)
  • Various myelin fractions contain
    growth-inhibitory molecules
  • NI 35, 250 (Caroni Schwab, 1989)
  • IN-1 antibodies raised against NI 250
  • promote spreading on myelin
  • boost neurite outgrowth (Schwab Caroni, 1989)
  • IN-1 enhances axon regeneration of corticospinal
    tract
  • Schnell et al 94 Bregman et al., 95
  • ? proper controls in early studies ?
  • confounded by spared axons, doesnt work in
    transection (2 papers)
  • CST growth after IN-1 treatment in 4 of 5
    marmosets (Fouad et al 04)
  • Need contusion studies, evaluation of pain

21
Strategy 2 targeting inhibitory factors present
in the CNS injury environment
Myelin-associated growth inhibition In vitro
observations of inhibitory factors present in
CNS white matter
22
Biochemical identification of neurite growth
inhibitors production of the IN-1 antibody
against inhibitory CNS myelin Schwab and Caroni
(1988), J, Neurosci., 8, 2381-2393.
23
Myelin-associated growth inhibition - summary
CNS myelin contains inhibitors that block the
growth of axons in culture and in vivo. IN-1
antibody blocks at least some of the inhibitory
activity of CNS myelin and leads to regeneration
of cut spinal cord axons functional recovery of
limb movements sprouting of injured and intact
pathways above and below the injury site (over a
decade of work from Martin Schwabs
lab). Identification of the antigen recognised
by the IN-1 antibody and further understanding of
the molecular determinants of myelin
inhibition 2000 Cloning and expression of a
novel protein revealed that Nogo-A is the
specific target for IN-1s actions (Prinjha et
al, 2000 Chen et al., 2000 Grand-Pre et al,
2000 Nature 403). Nogo-A is a component of CNS
myelin that inhibits axonal regeneration in the
adult mammalian CNS. 2001 The Nogo receptor
(NgR) was identified. The NgR binds with high
affinity to Nogo-66 (a 66 amino-acid peptide that
underlies the inhibitory activity of NogoA on
neurite outgrowth and growth cone collapse)
(Fournier et al., 2001, Nature 40, 9,
341-346). 2002 NgR was also found to be a
functional receptor for 2 other known
myelin-derived axonal outgrowth inhibitors, MAG
(myelin-associated glycoprotein) and Omgp
(oligodendrocyte myelin glycoprotein). Therefore,
NgR is a focal point for the convergence of three
myelin inhibitors (Liu et al, 2002, Science 297,
1190-1193). 2002 NEP1-40 (Nogo66 receptor
antagonist peptide) delivered in vivo to SCI rats
(GrandPre et al, 2002, Nature, 417, 547-511)
functional recovery and enhanced regeneration and
sprouting of the CST 2003 Nogo-A knockout mice.
Studied by 3 different groups who saw varying
degrees of regeneration after SCI (Kim et al,
2003 Simonen et al, 2003 Zheng et al, 2003
Neuron 38) 2004, 2005 NgR knockout mice. Again
different groups saw varying degrees of
regeneration in NgR-/- mice following SCI (Kim et
al, 2004 Neuron 44, 439-451 Zheng et al, 2005
PNAS, 102, 1205-1210).
24
Peptide against Nogo Receptor as a treatment for
SCI
Dorsal hemisection, thoracic, rat NEP 1-40
promotes axon regeneration (Grandpre et al.,
2002) NEP 1-40 subcutaneous and one week delayed
(Li Strittmatter, 2003) CST and 5HT growth Some
locomotor benefits NEP 1-40 intrathecal (Cao et
al., 2004 SfN) Rubrospinal axon growth Some
locomotor benefits
25
Nogo-A is a key inhibitor of axon growth in myelin
  • Publication of partial sequence of peptide
    recognised by IN-1
  • Spillmann et al., 1998
  • Race is on! Cloning of Nogo
  • rat nogo (Chen et al., 2000 GrandPre et al.,
    2000)
  • human nogo (Prinjha et al., 2000)
  • Three isoforms A,B,C. Nogo A is 200kDa, binds
    IN-1 IgM
  • New antibodies 7B12, 11C7 IgG
  • Three groups make Nogo-A knockout mice, variable
    results
  • Names to know
  • Stephen Strittmatter
  • Marc Tessier-Lavigne
  • Martin Schwab
  • Are there other inhibitors in myelin?

26
Be critical of the data you see
27
Myelin associated glycoprotein
  • Transmembrane and soluble forms
  • Purified / recombinant MAG usually (but not
    always) inhibits neurite growth (Mukhopadyay et
    al., 1994 McKerracher et al., 1994) and
    depleting / neutralizing MAG improves axon
    growth.
  • Overexpression of MAG in cells limits axon
    growth (Shen et al., 1998)
  • Name to know Marie Filbin
  • Caveat.
  • Axons do not regenerate appreciably better in
    MAG knockout mice relative to wildtypes (Bartsch
    et al., 1995 Li et al., 1996)

28
Oligodendrocyte myelin glycoprotein (OMgp)
  • GPi linked protein, 110 kDa
  • Found in myelin
  • Recombinant OMgp inhibits axon growth
  • Wang et al., 2002
  • Name to know - Zhigang He
  • To my knowledge, knockout has not yet been
    tested
  • All is in vitro

29
Chondroitin sulphate proteoglycans (CSPGs)
  • Family of proteins bearing CS glycosaminoglycan
    side chains
  • Neurocan, versican, brevican, phosphacan, etc.
  • CSPGs inhibit axon growth in vitro
  • Degrading CS using chondroitinase ABC boosts
    axon growth
  • in vitro McKeon et al., 1995 J Neurosci
  • after penetrating brain injury (Moon et al.,
    2001)
  • and improves outcome following spinal cord injury
  • (Bradbury et al., 2002)
  • Other names to know Jerry Silver, James Fawcett

30
How are neurons inhibited by these molecules?
CSPGs including versican Eph A4 EGF-like ligand?
Annexin as receptor for CSPGs? Ephrin B3 EGF
receptor EGF R kinase phosphorylates EGF
R Calcium increase
31
Remarkably, Nogo-A, MAG and OMgp all bind the
same receptor complex
  • Nogo receptor (NgR1) binds Nogo-A (Fournier et
    al,. 2001)
  • NgR1 binds OMgp (Wang et al., 2002a)
  • NgR1 binds MAG (Liu et al., 2002 Domeniconi et
    al., 2002)
  • GPi linked, lacks an intracellular domain, cant
    signal on its own
  • Nerve growth factor receptor (NGFR) interacts
    with NgR1 as a co-receptor for Nogo, MAG and OMgp
    (Wang et al., 2002b Wong et al., 2002)
  • p75
  • tumour necrosis factor (TNF) receptor
    superfamily, member 16
  • LINGO-1 (LRR and Ig domain containing, Nogo
    receptor interacting protein Mi et al., 2004)
  • Striking convergence of three anti-growth
    molecules with a pro-growth receptor (NGF R).
    Chao, 2003 Nat Rev Neurosci 4299-309

32
Raises more issues than it settles!
  • Do all inhibitory molecules signal through this
    complex?
  • all CSPGs?
  • semaphorins?
  • Are all parts of the complex necessary for all
    types of inhibitory signaling?
  • How does ligand / receptor complex binding
    transfer to an intracellular signal and thus to
    the cytoskeleton?

33
At least some CSPGs dont signal through p75, NgR
  • Versican V2 inhibits neurite growth independent
    of p75 and NgR (Schweigreiter et al., 2004)
  • Neurons derived from p75 knockouts are inhibited
    by V2
  • RhoA and rac1 are also modulated by V2
  • Neurons from p75 knockout mice are still largely
    inhibited by myelin how can this be?

34
More thorny issues for p75
  • Many adult mammalian neurons dont express p75
    yet they respond to myelin inhibitors (Park et
    al., 2005 Neuron 45345-351).
  • p75 is not detectable on P8 cerebellar granule
    neurons by immunolabeling (Moon, unpublished
    results).
  • Myelin from p75 knockout mice still contains
    inhibitors of axon growth in vitro and do not
    exhibit increased axon regeneration after spinal
    cord injury (Song et al., 2004 J Neurosci
    24542-546).
  • Is p75 really the key player? What else might
    act as a receptor for myelin inhibitors?

35
TROY can substitute for p75
  • Only one other TNFR superfamily member, TROY,
    binds NgR1 and forms a complex with LINGO-1 (and
    does so better than p75)
  • Park et al., 2005 Neuron 45345-351
  • Shao et al., 2005 Neuron 45353-359
  • Overexpressing TROY in neurons retards axon
    growth on myelin
  • Axon growth can be increased on myelin by
    interfering with TROY (by providing truncated or
    soluble variants)

36
Given that Nogo-A binds this receptor complex,
how does it signal intracellularly?
  • p75 and TROY both activate RhoA, a small GTPase
    (Park, Shao)
  • p75 is needed to activate RhoA, at least for
    MAG, Nogo-66 and OMgp (Yamashita et al,. 2002
    Wang et al., 2002)
  • Rho kinase (Fournier et al., 2003 J Neurosci 23
    1416-1423) activates rho which in turn rigidifies
    the actin cytoskeleton, causing growth cone
    collapse (Yamashita Tohyama, 2003 Nat Neurosci
    6461-467).
  • Inhibitors of rhoA and (downstream) rho kinase
    boost axon growth and enhance axon sprouting and
    functional recovery after spinal cord injury
    (Dergham et al., 2002 Fournier et al., 2003).
  • ? sprouting of collaterals ?

37
MAG binding to p75 causes cleavage
First alpha then gamma. Blocking secretases
reduces inhibition. Intracellular fragment may be
growth inhibitory Domeniconi et al., 2005 How
does this signal no-grow?
38
Rho No Grow PKC Grow Free
  • Activation of small GTPase RhoA inhibits neurite
    growth (Niederost et al,. 2002)
  • After dorsal hemisection of thoracic spinal cord
    in adult rats,
  • inhibiting Rho using C3 botulinum toxin promotes
    axon regeneration in vivo (Dubrueil et al,. 2003)
  • inhibiting Rho kinase also promotes axon
    regeneration in vivo (Fournier et al,. 2003)
  • Protein kinase C (PKC) activation is required
    for MAG and Nogo to activate Rho and inhibit
    growth (Sivasankaran et al., 2004).

39
How does rho no grow ?
RhoGDP to RhoGTP
  • MAG binds to p75 and causes activation of Rho
    (Yamashita et al 2002)
  • Gamma secretase requires protein kinase C
    activation (Domeniconi)
  • Cytoplasmic p75 activates RhoA and results in
    axon growth inhibition

40
Summary of mechanisms for axon growth
  • Some neurotrophins signal through p75
  • Some inhibitors in myelin signal through p75
  • some convergence on p75
  • Does p75 balance or integrate Go and No-go
    signals?
  • How?
  • MAG-induced cleavage of p75 increases ratio of
    intracellular fragment
  • Other mechanisms less well understood
  • EGF receptor and EGF receptor kinases
  • role of p75-like receptors
  • role of cyclic AMP

41
Ephrin B3 in myelin inhibits axon growth
ephrin b3 signals to CST neurons via binding to
EphA4 receptor In vitro study needs in vivo
42
EGF receptor phosphorylation...
Screened 400 compounds Two inhibitors of EGF R
kinases boosted neurite growth of DRGs and CGNs
43
Summary
CSPG Eph A4 EGF-like ligand?
Annexin as receptor for CSPGs? Ephrin B3 EGF
receptor EGF R kinase phosphorylates EGF
R Calcium increase
44
Reading list (I)
Anatomy of human spinal cord Kandel, Schwartz
Jessell, Principles of Neural Science Spinal cord
injury statistics http//www.spinalcord.uab.edu/s
how.asp?durki21446 Critical reviews on animal
models for spinal cord injury Moon Bunge, 2005.
From animal models to humans. Journal of
Neurological Physical Therapy 2955-70. Jeffery
et al., 2006. Clinical canine spinal cord injury
provides an opportunity to examine the issues in
translating laboratory techniques into practical
therapy. Spinal Cord. 44584-593. Critical
reviews of therapies Hurlbert, 2006. Strategies
of medical intervention in the management of
acute spinal cord injury. Spine.
15S16-21. Thuret, Moon Gage, 2007.
45
End of session I
Please be back at 1 pm sharp!
Before you go, please fill in feedback form I
anonymously and leave in a pile at the front !
46
Spinal cord injury and repair (II)
Dr Lawrence Moon
During the lecture, please interrupt with
questions when you have them. While you wait,
please fill in the questionnaire....
47
After these lectures and appropriate reading you
should be able to
  • Describe the gross anatomy of spinal cord
    v
  • Describe the neuropathology of spinal cord
    injury v
  • Describe possible mechanisms contributing to loss
    of function v
  • Describe animal models of spinal cord injury
  • Critically evaluate current treatments
    (pharmacological, rehabilitative)
  • Critically evaluate potential therapies for
    spinal cord injury

48
Human pathology Animal models
  • Contusion Weight drop
  • Compression / Maceration Clip, balloon
  • Solid core injuries n/a
  • Laceration Complete transection
  • Partial section
  • Dorsal hemisection
  • Lateral hemisection
  • Pyramidotomy
  • Animals used as models Comments
  • Mouse transgenics, cheap (Zhang et al)
  • Rat larger, still cheap
  • Cat classical physiology
  • Dog spontaneous (Jefferys et al)
  • Non-human primate not great apes (Freund et al)

49
Animal models pros and cons
  • bipeds vs quadrupeds
  • differences in nervous system anatomy,
    physiology other - ologies

Lemon Griffiths, 2005. Muscle Nerve 32261-279.
50
Overview of potential therapies
  • Rehabilitation / prosthetics / robots
  • Cellular therapies
  • Molecular therapies

51
Prosthetics / robotsSFN video (re-start after
successful life)
52
Rehabilitation research
Ronaldo Ichiyama (now Leeds), Reggie Edgerton
(UCLA)
Four videos showing treadmill and Neonatally
transected rat, no training rare
stepping Neonatally transected rat, treadmill
training amazing stepping Adult transected rat,
no training no stepping Adult transected rat,
epidural stimulation stepping Ichiyama et al.,
2005 Neuroscience Letters 383339-44 Gerasimenko
et al., 2007 J Neurophysiol epub ahead of print
53
Rehabilitation research
Susan Harkema, Reggie Edgerton, David
Reinkensmeyer
  • Video of PAM/POGO (no sound) showing
  • Incomplete paraplegic patient (ASIA B or C)
  • on treadmill
  • partial body weight support (harness)
  • PAM/POGO robot can control pelvis and legs
  • first, physiotherapists teach the robot two
    handed
  • second, the robot takes over physios now one
    handed

54
Prosthetics / robotsParastep video showingT4
complete paraplegicNo treadmilllElectrical
stimulators on leg musclesPartial weight bearing
on frame using armsPalm pilot
55
Ethics
  • A need to establish reasonable expectations
  • hope v depression
  • Therapies about five years from now ???

56
Other goals for spinal cord repair
  • Reduce cell death (neuroprotection)
  • Promote regrowth of injured axons (regeneration)
  • Promote compensatory regrowth by uninjured axons
    (collateral sprouting)
  • Demyelination (?)

57
Primary sensory neurons in the dorsal root
ganglia provide a unique system for studying
regeneration (Bradbury et al (2000) Trends
Pharmacol Sci 21 389-394.)
58
The three branches of DRG neurons have different
regenerative capacities
59
The ascending dorsal column projection a
myelinated tract which ascends in the dorsal
columns of the spinal cord and terminates in the
brainstem
Formed by collaterals of centrally projecting DRG
axons Important for fine discriminative touch
and proprioception Can be selectively labelled
with CTB
60
NT-3 promotes regeneration of dorsal column
axons (Bradbury et al, Eur J Neurosci 1999
113873-83.)
In NT3-treated rats there is less fibre
retraction, abundant sprouting at the crush site,
and regeneration of fibres through lesioned tissue
61
The death of corticospinal neurons after axotomy
is prevented by neurotrophins (Giehl Tetzlaff
1996 Eur J Neurosci 8, 1167-75)
62
Most corticospinal tract neurons express trkB and
trkC almost none express trkA (Giehl
Tetzlaff, 1996)
trkC - 73
trkB - 88
trkA
63
Neurotrophins promote axon growth
  • Nerve growth factor
  • Brain derived neurotrophic factor
  • NT-3
  • NT-4/5
  • Deliver to cell body (Kwon et al,. 2002)
  • or to injury site by
  • Direct injection (Bradbury et al,. 1999)
  • Osmotic minipump (Xu et al., 1995)
  • Ex vivo genetically modified cells (Grill et
    al,. 1997)
  • Viral vectors in vivo (Blits et al., 2004)
  • No studies in injured primates
  • Some studies in Alzheimers disease
  • Side effects (Apfel, 2002)

64
Other growth factors
Glial-derived neurotrophic factor
(GDNF) Fibroblast growth factor (FGF) LIF, CNTF,
others...
65
How do neurotrophins signal?
  • Neurotrophins are 12kDa
  • They form dimers
  • p75 binds all four plus -gt
  • trkA binds NGF
  • trkB binds BDNF and NT4
  • trkC binds NT3
  • Classically trks considered high affinity
    whereas actually
  • NGF to trkA low affinity
  • BDNF to trkB low affinity
  • although co-expression of p75 increases trkA
    affinity for NGF
  • On binding, receptors dimerise and signal
    intracellularly.
  • Chao, MV, 2003 Nat Neurosci Rev

66
How do neurotrophin receptors signal?
Dominant negative rhoA activity boosts neurite
growth Constitutively active rhoA blocks
neurotrophin-induced growth Gehler et al., 2004 J
Neurosci ---- well return to RhoA later....
67
Growth inhibitors
injured, some spontaneous changes
combination therapies
68
IN-I treatment permits the growth of
corticospinal axons around the injury site
Recovery from spinal cord injury mediated by
antibodies to neurite growth inhibitors Bregman
et al (1995), Nature 378, 498-501
IN-I treatment increases the growth of brain
stem- spinal axons
Contact-placing is abolished after injury and
recovers after IN-1 treatment
69
Blockade of myelin associated neurite growth
inhibitors induces functional recovery and
enhanced sprouting and plasticity following
lesions of the corticospinal tract Thallmair et
al (1998) Nat Neurosci 1, 124-131
In the spinal cord (below the lesion) sprouting
of intact CST fibres into the lesioned side In
the brainstem (above the lesion) sprouting from
lesioned side into the intact side
70
Corticospinal tract lesions lead to impairments
in food-pellet reaching tasks IN-1 treatment
reverses these impairments
71
Corticospinal tract lesions lead to impairments
in rope-climbing and sticky-paper tests IN-1
treatment reverses these impairments
72
The glial scar as a barrier to regeneration
Physical barrier blocks growth cone
advancement Molecular barrier scar associated
extracellular matrix contains inhibitory
molecules such as chondroitin sulphate
proteoglycans (CSPGs)
73
Proteoglycans Consist of a protein core with
attached sugar moieties called glycosaminoglycans
(GAGs). They are characterised by their GAG
compositions as chondroitin sulphate, heparin
sulphate, keratin sulphate and dermatin
sulphate. The chondroitin sulphate proteoglycans
(CSPGs) broadly fall into 3 classes The
Lecticans Aggrecan Versican Neurocan
Brevican Matrix-associated proteoglycans Phosph
acan Cell surface proteoglycans Neuroglycan C NG2
74
(No Transcript)
75
  • CSPGs are important growth inhibitors in the CNS
  • In vitro evidence
  • In astrocyte cell lines inhibition of neurite
    outgrowth was correlated with expression of CSPGs
    (Fawcett)
  • Degrading CSPG side chains using the enzyme
    chondroitinase ABC can make inhibitory substrates
    permissive to growth (Silver Muir)
  • In vivo evidence
  • CSPGs are up-regulated at CNS injury sites
    (Levine Fawcett Tuszynski)
  • CSPGs are present where axons stop regenerating
    (Davies)
  • Degrading CSPG side chains in vivo promotes
    regeneration of CNS axons (Moon Yick Bradbury)
  • The GAG side chain component of CSPGs is
    inhibitory

76
Comparing astrocyte cell lines for their ability
to promote growth (Smith-Thomas et al, 1994 and
Fidler et al, 1999)
Growth inhibition was correlated with the
production of CSPGs
A function blocking antibody against the
proteoglycan NG2 neutralised Neu7 matrix
inhibition
DRG neurons grown on inhibitory Neu7 matrix
DRG neurons grown on permissive A7 matrix
77
Structure of CSPGs before and after treatment
with the enzyme chondroitinase ABC (ChABC)
Protein core
ChABC degrades CS-GAG chains
CS- GAG chains
Stub region
Addition of ChABC
78
Chondroitinase ABC can make an inhibitory
substrate more permissive to growth (McKeon et
al, 1995)
ChABC treated
Control treated
Treatment with ChABC promotes neurite outgrowth
on inhibitory glial scar explants
79
Chondroitinase ABC can make an inhibitory
substrate more permissive to growth (Zuo et al,
1998)
Untreated
ChABC 24 hr
ChABC 48 hr
Normal spinal cord
Injured spinal cord
Treatment with ChABC promotes neurite outgrowth
on inhibitory spinal cord tissue
80
The CSPG Neurocan is up-regulated in injured
cortex (Asher et al, 2000)
81
The CSPG versican is up-regulated in injured
cortex (Asher et al, 2002)
82
The CSPG NG2 is up-regulated at a spinal cord
injury site (Jones et al, 2002)
NG2 expression is up-regulated within 24 hr of
injury, peaks at 1 week, and remains elevated for
at least 8 weeks
83
Regeneration of adult axons in degenerating white
matter tracts (Davies et al, 1999)
Axon regeneration of transplanted cells (green)
in degenerated dorsal columns Axons grow in
degenerating white matter tracts but stop when
they reach the lesion scar, labelled with GFAP
(red) and CSPG (blue)
84
Regeneration in the nigrostriatal tract after
treatment with ChABC (Moon et al, 2001 Nat Rev
Neurosci 4, 465-466 )
ChABC treated
Vehicle control
85
Chondroitinase ABC promotes functional recovery
after spinal cord injury (Bradbury et al
(2002), Nature, 636-640)
ChABC promotes functional recovery in a number of
behavioural tests
ChABC promotes regeneration of descending CST
axons regenerating axons sent collaterals from
white matter to gray matter, indicating terminal
arborisation.
Electrophysiological analysis confirms that ChABC
restores functional connectivity of CST axons
below the lesion
86
CSPG-associated growth inhibition summary In
vitro and in vivo evidence indicates that CSPGs
are potent growth inhibitors in the adult CNS.
The GAG side-chains of CSPG molecules are
inhibitory to growth in vitro, the enzyme ChABC
can make inhibitory substrates become permissive
to neurite growth. In vivo, delivery of ChABC can
promote regeneration of injured axons, recovery
of motor function and increased connectivity of
descending motor pathways. Combining ChABC
treatment with other treatment interventions can
lead to enhanced axon regeneration (Chau et al
2004, FASEB J 18, 194-196 Fouad et al 2005 J
Neurosci 25, 1169-1178). ChABC can also promote
plasticity ChABC can restore synaptic
plasticity in the visual cortex of adult rats to
a level normally seen only during development
(Pizzorusso et al Science 2002 298,
1248-1251. ChABC can promote functional
plasticity of ascending spinal projections within
the brainstem (Massey et al 2006 J Neurosci 26,
4406-4414). ChABC can promote plasticity of
injured and intact spinal systems following
spinal injury (Bradbury et al 2006 J Neurosci 26,
10856-67).
87
Cellular transplantation
  • Peripheral nerve
  • Schwann cells
  • Olfactory ensheathing glia
  • Macrophages
  • Stem cells (aaaaaaaaaregh)
  • Embryonic
  • Adult
  • Progenitor cells
  • Many have been tried in various models of injury

88
Olfactory ensheathing glia
89
Olfactory ensheathing glia
  • Transection, OEG (Ramon-Cueto et al., 2000)
  • Improved climbing
  • Serotonergic growth distally
  • Not reproduced
  • Cervical lateral hemisection, acute or delayed
    transplant of OEG (Li et al 2003 inc Raisman)
  • Improved respiration and climbing
  • Incomplete lesions?
  • Autologous transplants in dogs (Franklin)
  • Naturally occurring injury
  • Feasible, safe

90
Olfactory ensheathing glia
  • 500 humans (Huang)
  • Fetal cells, largely uncharacterised OEG?
  • No controls
  • Few follow-ups for safety or efficacy
  • Guest et al., (in press)

91
Olfactory ensheathing cells
OECs - provide a substrate for axons to grow
from the nasal epithelium into the CNS - a unique
cell type that accompanies axons from PNS into
CNS Astrocyte-like properties - express GFAP -
form the glia limitans within the olfactory
bulb Schwann cell-like properties - axonal
ensheathment - myelinating capability - support
of axonal regrowth Advantages over Schwann cells
for CNS repair - greater propensity to exist
within a CNS environment - can migrate within the
CNS following transplantation - secrete
neurotrophic factors - remyelinate damaged axons
92
Repair of adult rat corticospinal tract by
transplants of olfactory ensheathing cells Li et
al (1997), Science 277, 2000-2002
Corticospinal tract lesion
OEC transplants promoted CST regeneration and
recovery of directed forepaw reaching
Regenerating CST axons (green) are aligned with
OECs (red)
93
Conclusion
Transection any therapy Weight bearing
stepping on hindlimbs is the exception Contusion
any therapy Few studies have been reproduced
independently Things to think about Very few
safety or efficacy studies in primates Is going
straight to humans sensible? Does it have to be
100 safe? How much do we need to know?
94
Next steps
... using this new understanding of mechanism,
test new therapeutics for SCI and stroke
95
include Barrette on where the receptors are
tract dependent repaircafferty
strittmatterngr1 kos and nogo kos show
collateral sprouting even if CST regn is not
always greatSteward et al 2007 on BDA tracing
artefactCafferty et al BDA retort with 8 v 14
96
Embryonic chick neurons. Growth cone collapse in
response to soluble sema3A. Time lapse in vitro
images courtesy of Dr Britta Eickholt.
97
videos of transected rats from Ronaldo, including
his N Letts paper as reference
2_L2_No Stim is a completely transected adult
rat with no stim. This is two months after
transection. 2_L2_40Hz_11v is the same rat
under epidural stimulation. The specifications
are on the title. They do step better when we
combine stim with Quipazine and after we train
them. But those videos are HUGE. Nonetheless, I
think you can make the point that the epidural
stimulation alone can have an enormous effect
acutely, just by engaging the Lumbar CPG. Those
videos are from the Ichiyama et al 2005, Neurosci
Letters, short paper. Let me know if you can use
them.
98
for future.cAMP / PKAmore on actin cytoskeleton
and collapse!oncomodulinpolyaminesinclude
prosthesesgo through Nogo etc more quickly
maybe a bit more matter of facthighlight debate
on gamma secretase have two papers in reading
listsema3a- fred de winter, new nature medicine
paper. check NEW REVIEW folderLIF CNTFJAK
STATgp130ATF3?conditioning lesion
99
Future directions
  • Maximise spared function
  • Develop a neuroprotective therapy that works
    better than MP
  • Figure out what rehab works best
  • MAKE SURE WE DONT MAKE PATIENTS WORSE

100
BE CRITICAL WHEN READING
  • Does the paper state it was performed blind and
    randomised?
  • Were the controls suitable? With cellular
    therapies, did they use another cell type rather
    than just medium?
  • Has the work been independently validated?
  • Was the animal model suitable?
  • Have they tested for adverse consequence?
  • Was the therapy given in a clinically relevant
    time frame?
  • Can the therapy be commercialised / rolled-out
    across clinics?

101
Reading list (II)
Reviews of therapies for SCI Thuret, Moon Gage,
2006. Therapeutic interventions after spinal cord
injury. Nature Reviews Neuroscience.
7628-643. Reviews on animal models for
developing therapies Zheng et al., 2006. Genetic
models for studying inhibitors of spinal axon
regeneration. TINS 29640-6. Steward et al.,
2003. False resurrections. J Comparative Neurol.
4591-8. Key papers to read critically Jeffery
et al., 2006. Autologous Olfactory Glial Cell
Transplantation Is Reliable and Safe in Naturally
Occurring Canine Spinal Cord Injury. J
Neurotrauma 221282-1293 Freund et al., 2006.
Nogo-A-specific antibody treatment enhances
sprouting and functional recovery after cervical
lesion in adult primates. Nature Medicine.
12790-2 and pages 1220, 1231-1233. Include
Supplementary Materials.
102
Dr Lawrence Moon Download some of the reading
list at www.lawrencemoon.co.uk
103
End of session II
Thanks to Drs Britta Eickholt, Liz Bradbury and
Ronaldo Ichiyama
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