Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNOLOGICAL COMPOSITIONS AND METHODS OF USE TO TRANSIENTLY
DISRUPT MAM1VIALIAN CENTRAL NERVOUS SYSTEM MYELIN TO PROMOTE
NEURONAL REGENERATION
FIELD OF THE INVENTION
This invention relates to compositions and their methods of use in promoting
the growth and/or
regeneration of neurological tissue within the central nervous system (CNS).
BACKGROUND
CNS damage
Approximately 1,100 new spinal cord injuries occur eachyear in Canada; over
10,000 per year occur
in the United States. These numbers are five times higher if one also includes
patients suffering brain
damage involving inhibition to neural growth following traumatic brain injury.
The number of
patients with chronic spinal cord injuries in North America is in the order of
300,000. Again, this
number is five times higher if one includes patients suffering from brain
damage involving inhibition
to neural growth following traumatic brain injury.
Spinal cord injuries often result in a permanent loss of voluntary movement
below the site of
damage. Mostly young and otherwise healthy persons become paraplegic or
quadriplegic because
of spinal cord injuries. There are an estimated 200,000 quadriplegics in the
United States. Given
the amount of care required, it is not difficult to envision how health care
costs associated with
caring for patients with central nervous system (CNS) damage is well over $10
billion a year for
North America.
The CNS (the brain and the spinal cord) is comprised of neurons and glia, such
as astrocytes,
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microglia, and oligodendrocytes. Neurons typically have two types of
processes: dendrites, which
receive synaptic contact from the axons of other neurons; and axons, through
which each neuron
communicates with other neurons and effectors. The axon of a CNS neuron is
wrapped in a myelin
sheath.
In higher vertebrates, axons within the CNS possess a limited capacity for
repair after injury.
Axotomized neurons of the adult mammalian CNS fail to exhibit substantial
axonal regeneration,
in contrast to neurons within the embryonic or neonatal CNS or within the
adult peripheral nervous
system (PNS) (Saunders et al., (1992) Proc. R. Soc. Lond. B. Biol. 250:171-
180; Schwab and
Bartoldi (1996) Physiol. Rev. 76:319-370; Steeves et al., (1994) Prog. Brain
Res. 103:243-262). In
fact, complete CNS axonal disruption is likely to preclude recovery. Although
axotomized fibers
proximal to the neuronal cell body initiate regenerative growth, this is
subsequently aborted within
a short distance ( 1-2 mm) and is often followed by retrograde degeneration.
Although CNS axons
will not regrow in the environment of the adult spinal cord, peripheral nerve
grafts into the CNS
provide a favorable environment through which CNS axons will anatomically
regenerate (May et
al., Cajal 's Degeneration and Regeneration of the Nervous System, History of
Neuroscience Series
#S (NY and Oxford: Oxford Univ. Press, 1991) at 769). These findings indicate
that adult CNS
neurons retain intrinsic growth properties and, given favorable environmental
conditions, are capable
of successfully reactivating growth programs.
Current treatments ofspinal cord injuries
A number of currenttherapies exist for the treatment of spinal cord injuries.
Interventional therapies,
including opiate antagonists, thyrotropin-releasing hormone, local cord
cooling, dextran infusion,
adrenergic blockade, corticosteroids, and hyperbaric oxygen have been
utilized, but are of
questionable clinical value.
Peripheral nerve transplants have been suggested as bridges across CNS lesions
(David and Aguayo
(1981) Science 214:931-933; Houle (1991) Exp. Neurol. 113: i-9; Richardson et
al., (1984) J.
Neurocytol. 13:165-182; Richardson et al., (1980) Nature 284:264-265; Xu et
al., (1995) Exp.
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Neurol. 138:261-276; Ye and Houle (1997) Exp. Neurol. 143:70-81). Olfactory
ensheathing cell
transplants have been used recently to promote the regeneration of injured
corticospinal proj ections
in the rat (Li et al., (1997) Science 277:2000-2002). A recent study {Cheng et
al., (1996) Science
273:510-513) employed a combinatorial approach that extended earlier work
(Siegal et al., ( 1990)
Exp. Neurol. 109:90-97): after transection of the adult rat spinal cord,
peripheral grafts were used
to connect white matter tracts to central gray matter in such a way as to
direct regenerating fibers out
of an inhibitory environment and into the more permissive gray matter.
US Patents No. 5650148 and 5762926 describe amethod for treating damage to the
CNS by grafting
donor cells into the CNS that have been modified to produce molecules such as
neurotrophins.
The use of transplanted neural cells is also of limited clinical value:
although axons will be able to
grow into the transplanted tissue, they will not be able to grow out of the
transplanted tissue back
into the CNS due to inhibitory matter in the CNS.
This review of current methods of treating spinal cord injuries indicates that
a need remains for a
means of promoting regrowth, repair, and regeneration of neurons in the
mammalian CNS in both
the acute and chronic situations.
Myelin
It has been suggested that the failure of CNS axons to regenerate after injury
is associated with the
presence of myelin. The myelin sheath wrapping an axon is composed of
compacted plasma
membranes of Schwann cells and oligodendrocytes. Although its composition
resembles that of any
other plasma membrane in that it contains lipids, proteins, and water, the
relative proportions and
dispositions of these components are unique to myelin. Myelin in the CNS is
produced by
oligodendrocytes and is characterized by the expression of myelin basic
protein (MBP). MBP is
only associated with myelin and is one of the first proteins expressed at the
onset of myelination of
CNS axonal fibers. Galactocerebroside (GaIC) is the major sphingolipid
produced by
oligodendrocytes. GalC comprises approximately 15 percent of the total lipid
in human myelin and
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is highly conserved across species. Although GaIC is expressed on the surface
of oliogodendrocyte
cell bodies, it is expressed in greater concentration on the surface of myelin
membranes (Ranscht
et al., (1982) Proc. Natl. Acad. Sci. USA 79:2709-2713).
There is growing evidence that the presence of CNS myelin can retard or
inhibit the regenerative
growth of some severed CNS axons (Schwab and Bartoldi (I996) Physiol. Rev.
76:319-370),
including a number of examples from widespread vertebrate families {Schwegler
et al., (1995) J.
Neurosci. 15:2756-2767; Steeves et al., (1994) Prog. Brain Res. 103:243-262).
Both the lower
vertebrate CNS (e.g. lamprey) and the developing CNS of higher vertebrates
(e.g. birds and
mammals) exhibit substantial axonal regeneration after inj ury (Davis and
McClellan ( 1994) J. Comp.
Neurol. 344:65-82; Hasan et al., (1993) J. Neurosci. 13:492-507; Hasan et al.,
{1991) Restor.
Neurol. Neurosci. 2:137-154; Iwashita et al., (1994) Nature 367:167-170;
Saunders et al., (1992)
Proc. R. Soc. Lond. B. Biol. 250:171-180; Treherne etal., (1992) Proc. Natl.
Acad. Sci. USA 89:431-
434; Varga et al., {1995) Eur. J. Neurosci. 7:2119-2129). The common phenotype
for all these
positive examples of regeneration is either a CNS that lacks compact myelin
(lamprey) or incomplete
myelin development (embryonic chick, neonatal opossum and rat) at the time of
injury. The
developmental appearance of myelin temporally correlates with the loss of
regeneration by injured
CNS axons. In addition, the robust growth of transplanted fetal neurons in the
adult CNS (Bregman
et al., (1993) Exp. Neurol. 123:3-16; Li and Raisman (1993} Brain Res. 629:115-
127; Yakovleff et
al., (1995) Exp. Brain Res. 106:69-78) may be partially attributed to either a
lack of receptors for
myelin inhibitors at that stage of their differentiation and/or an ability to
override any inhibitory
signals from myelin.
Specific molecules associated with myelin have been identified as putative
mediators of this
inhibitory activity, including myelin-associated glycoprotein (MAG)
(McKerracher et al., (1994)
Neuron. 13:805-811; Mukhopadhyay et al., (1994) Neuron. 13:757-767)
andNI35/250, an as yet
unidentified myelin-derived protein (Bandtlow and Schwab (1991) Soc. Neurosci.
Abstr. 17:1495;
Carom and Schwab (1988) J. Cell Biol. 106:1281-1288; Caroni and Schwab (1988)
Neuron 1:85;
Crutcher (1989) Exp. Neurol. 104:39-54; Savio and Schwab (1989) J. Neurosci.
9:1126-1133;
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Schwab and Caroni (1988) J. Neurosci. 8:2381-2393); IN-1 (Brosamle, et al,
(1998) Abst. Soc
Neurosci., 24:1559; NI-35/250 (Huber et al., (1998) Abst. Soc Neurosci.,
24:1559; NI-220/250 (van
der Haar et al., ( 1998) Abst. Soc Neurosci., 24:1559; arretin, Janani et al.,
( 1998) Abst. Soc Neurosci.,
24:1560; and NOGO (Chen et al., (1998) Abst. Soc Neurosci., 24:1776.
Experimental attempts to functionally block myelin-associated~inhibition
involving NI35/250, by
using an anti-IVI35/250 antibody, IN-1, have facilitated some anatomical
regeneration of
corticospinal axons (Bregman et al., (1995) Nature 378:498-501; Caroni and
Schwab (1988)
Neuron. 1:85-96; Schnell and Schwab (1990) Nature 343:269-272).
The immunological disruption ofmature myelin within the avian spinal cord
(Keirstead et al., ( 1995)
J. Neurosci. 15:6963-6974), and the delay of onset of CN5 myelination during
normal avian or
mammalian neurodevelopment (Keirstead et al., ( 1992) Proc. Natl. Acad. Sci.
(USA) 89:11664-
11668; Keirstead et al., (1997) Brain. Res. Bull. 44:727-734; Varga et al.,
(1995) Eur. J. Neurosci.
7:2119-2129) have also facilitated CNS axonal re-growth and/or sprouting.
The presence of certain components located or embedded in myelin that are
inhibitory to the
regeneration of axonal growth after injury makes it desirable to transiently
remove myelin and its
inhibitory components to promote the repair of injured adult spinal cord.
Adult spinal cord can be
demyelinated in vivo via drugs (e.g. ethidium bromide); however, these drugs
have non-specific
deleterious effects on other cell types in the central nervous system (e.g.,
astrocytes). In addition,
myelin-deficient strains of mice and rats are readily available, but are of
limited experimental value
due to a shortened life span: most do not survive beyond a couple of weeks
after birth.
Consequently, there is a need for improved methods of disrupting myelin in
vivo in order to enhance
regeneration of neurological tissue. The present invention provides methods
that address this need.
Complement
The complement system is the primary humoral mediator of antigen-antibody
reactions. It consists
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of at least 20 chemical and immunologically distinct serum proteins capable of
interacting with one
another, with antibody, and with cell membranes (see, for example, J. Klein,
Immunology: The
Science of Self-NonseljDiscrimination (New York: John Wiley & Sons, 1982) at
310-346). The
principal actors in this system are 11 proteins, designated C1 to C9, B, and
D, which are present
normally among the plasmaproteins. These proteins are normally inactive, but
they can be activated
in two separate ways: the classical pathway or the alternate pathway.
The classical pathway is activated by an antigen-antibody reaction: when an
antibody binds with an
antigen, a specific reactive site on the constant portion of the antibody
becomes activated, which in
turn binds directly with the C 1 molecule of the complement system. This sets
into motion a cascade
of sequential reactions, beginning with the activation of the C1 proenzyme.
Only a few antigen-
antibody combinations are required to activate many molecules in this first
stage of the complement
system. The C 1 enzymes then activate successively increasing quantities of
enzymes in the later
stages of the complement system. Multiple end-products are formed, which cause
important effects
that help to prevent damage by an invading organism or toxin, including
opsonization and
phagocytosis, lysis, agglutination, neutralization of viruses, chemotaxis,
activation of mast cells and
basophils, and inflammatory effects.
The complement system can also be activated by an alternate pathway without
the intermediation
of an antigen-antibody reaction. Certain substances reactwith complement
factors B and D, forming
an activation product that activates factor C3, setting off the remainder of
the complement cascade;
thus, essentially all the same final products of the system are formed as in
the classical pathway,
causing the same effects. Since the alternate pathway does not involve an
antigen-antibody reaction,
it is one of the first lines of defense against invading microorganisms.
Since components of both the classical pathway and the alternative pathway of
the complement
system act locally to activate C3, this is the pivotal component of
complement.' C3 is a 195 kD
protein, which comprises two disulfide bridged chains of 105 and 75 kD. The
enzymatically active
C4-C2 complex, activated in the classical pathway by the binding of C 1 q to
an antigen-antibody
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complex, cleaves C3 into two fragments, C3a and C3b. The larger fragment, C3b,
binds covalently
to the surface of a target cell where it acts as a protease to catalyze the
subsequent steps in the
complement cascade. It is also recognized by specific receptor proteins on
macrophages and
neutrophils that enhance the ability of these cells to phagocytose the target
cell. In particular,
membrane-immobilized C3b triggers a further cascade of reactions that leads to
the assembly of
membrane attack complexes from the late components.
Complement fixation by cell-surface binding antibodies has been shown to
compromise the ionic
homeostasis of many different cells in vitro within minutes of activation
(Mayer ( 1972) Proc. Natl.
Acad. Sci. USA 69:2954-2958; Morgan (1989} Biochem. J. 264:1-14).
Use of Complement with myelin-specific Antibodies
After attachment of a specific complement-fixing antibody to a myelin surface
antigen, serum
complement forms a membrane attack complex through an enzymatic cascade
resulting in a rapid
influx of extracellular calcium (Dyer and Benjamins (1990) J. Cell Biol.
111:625-633) and
subsequent cytoskeletal re-arrangement (Dyer and Matthieu ( 1994) J.'
Neurochem. 62:777-787). In
vivo, this would make the disrupted myelin processes a target for phagocytosis
by subsequent
microglia, as well as by any invading macrophages.
The in vitro application of serum complement with myelin-specific antibodies
has been shown to
suppress myelin elaboration in purified oligodendrocyte cultures (Dorfman et
al., ( 1979) Brain Res.
177:105-114; Dubios-Dalcq et al., (1970) Pathol. Eur. 5:331-347; Dyer and
Benjamins (1990) J.
Cell Biol. 111:625-633; Fry et al., (1974) Science 183:540-542; Hruby et al.,
(1977) Science
195:173-175).
In vivo myelin disruption has been shown in the guinea pig optic nerve using
anti-GaIC serum and
complement (Sergott et al., (1984) J. Neurol. Sci. 64:297-303); myelin
disruption was observed
within 1 to 2 hours of treatment.
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The Chick Model
In the avian model, the onset of myelination in the embryonic chick spinal
cord at E 13 coincides
with the transition from a permissive to a restrictive period for the
functional repair of transected
spinal cord. The first appearance of chick oligodendrocytes on the tenth and
eleventh embryonic day
of development (E10-E11) precedes the initial formation of myelin by 2-3
embryonic days and is
characterized by the expression of galactocerebroside (GaIC), the major
sphingolipid produced by
oligodendrocytes.
In the mature avian spinal cord, after spinal cord transection, irnmunological
disruption of local
spinal cord myelin facilitated regeneration by brainstem-spinal neurons
(Keirstead et al., ( 1995) J.
Neurosci. 15:6963-6974; Keirstead et al., (1997) Brain Res. Bull., 44: 727-
734). The immunological
disruption of myelin was transient, produced by an intraspinal infusion of
both serum complement
and a myelin-specific, complement-fixing antibody (e.g. GaIC antibodies). Such
treatmentresulted
in the regeneration of up to 20% of mature brainstem-spinal axons.
This background information is provided for the purpose of making known
information believed by
the applicant to be of possible relevance to the present invention. No
admission is necessarily
intended, nor shouldbe construed, that any of the preceding information
constitutes prior art against
the present invention.
SUMMARY OF THE INVENTION
The purpose of the present invention is to provide a means of promoting
regrowth, repair, and
regeneration of neurons in the mammalian CNS. Accordingly, the invention
provides compositions
and methods of use for promoting regrowth, repair, and/or regeneration of
neurons in the CNS of
a mammalian subject, such as a human, in both chronic and acute disorders
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One embodiment of the present invention provides a composition comprising
therapeutically
effective amounts of the following:
(a) one or more complement-fixing antibodies or fragments thereof, which
specifically bind
to an epitope of myelin; and
(b) one or more complement proteins or fragments thereof;
wherein the binding of said antibodies to myelin causes transient disruption
and/or transient
demyelination of myelin. The antibodies may be monoclonal and/or polyclonal.
The complement
proteins or fragments thereof may be derived from a species different from
that species to which it
is administered. In a preferred embodiment, the complement proteins or
fragments thereof are
human. The complement component may be a physically distinct component from
the antibody
component, or it may be covalently or noncovalently attached directly to the
antibody component,
such that binding of the antibody to the surface of the myelin triggers the
endogenous immune
system attack. One or more growth factors may be added (in an appropriate
sequence) to facilitate
regrowth and regeneration.
In a specific embodiment, the epitope of myelin is a myelin sheath epitope,
such as
galactocerebroside (GaIC), 04, Myelin Oligodendrocyte Glycoprotein (MOG),
orMyelinAssociated
Glycoprotein (MAG), NOGO, NI22, NI-35/250, or arretin, or fragments thereof.
In a preferred
embodiment, the epitope of myelin is GaIC. Another preferred embodiment is
MOG.
In apreferred embodiment, the complement proteins or fragments thereof include
the C3 component
or a fragment, variant, analog, or chemical derivative thereof. In a preferred
embodiment, the
component C3b is used.
In another embodiment of the present invention, the composition further
comprises neurotrophins
and growth factors, such as NT-3, CNTF, FGF-1, BDNF, PDGF, GDNF, CT-1, or BNP.
The present invention also relates to the use of these compositions to promote
regrowth, repair,
and/or regeneration of neurons in a subj ect by the transient disruption
and/or transient demyelination
CA 02253078 2000-10-02
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of myelin.
In one embodiment of the present invention, the compositions are us ed in subj
ects requiring neuron
repair and/or regeneration due to neuron dysfunction. This neuron dysfunction
may be a result of
acute or chronic injury to the CNS. It may also be a result of degenerative
disease, such as
Alzheimer's or Parkinson's disease.
In another embodiment of the present invention, the compositions are used in
subjects to generate
an environment within the CNS that is relatively permissive to growth of
transplanted cells.
The present invention also relates to a method of promoting regrowth, repair,
and regeneration of
neurons in mammalian CNS, wherein the damage resulted from either a chronic or
acute disorder.
IO The method entails delivery of one or more complement-fixing antibodies or
fragments thereof,
which specifically bind to an epitope of myelin and delivery of one or more
complement proteins
or fragments thereof, delivered either together or separately to effect
transient disruption and/or
transient demyelination of myelin in the neuronal zone requiring regeneration.
Various other objects and advantages of the present invention will become
apparent from the
detailed description of the invention.
TABLES AND FIGURES
Table 1 presents rubrospinalneuronal cell counts obtainedfromindividual
control andexperirnental
TM
animals with retrograde Fluorogold labeling from the lumbar cord of an adult
rat.
Figure 1 presents (A) Photomicrograph of a transverse section of spinal cord
of an adult rat at the
level of T10 left side hemisection lesion, stained with cresyl violet. AlI
lesions were assessed and
always resulted in severing the funiculi through which the rubrospinal tract
traverses. The
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contralateral dorsal (dh) and ventral (vh) horns were always left undamaged;
the central canal (cc)
is labeled for orientation. (B) Assessment of visible Fluorogold diffusion in
the control treated and
immunologically disruptedhemisectedspinal cord. Diffusion of the retrograde
tracerwas measured
at the light microscope level at the time points indicated after injection
into the lumbar spinal cord
(see methods for details). Immunological demyelination did not significantly
affect the diffusion of
the tracer.
Figure 2 shows electron photomicrographs of transverse sections through the
dorsolateral funiculus
after continuous intraspinal infusion of immunological reagents for 7 days.
(A) Within one spinal
segment (<Zmm) of the infusion site, large regions of naked, demyelinated
axons were visible. Some
axons were observed to be associated with monocyte cells (M, e.g. infiltrating
macrophage) and or
endogenous microglia, some of which also contained myelin ovoids (arrow) or
myelin debris. (B)
On other grids, monocytes and invading polymolphonucleocytes (PMN) could also
be seen in close
association with demyelinated axons. Macrophages and/or microglia were
identified on the basis of
their high density endoplasmic reticulum (arrow-heads), and "finger-like"
processes. Some
monocytes have laid down basal lamina components such as collagen (Col), which
distinguishes
them from astrocytes. Multi-lobed nuclei are characteristic of PMNs and
facilitate their
identification. (C) Example of myelin-disruption. This is often observed 4-8mm
(1-2 spinal
segments) from the immunological infusion site where the axons were still
associated with myelin;
however, the myelin lamellae were disrupted (delaminated). Some regions of
coherence in the
myelin wrapping did persist (arrows). (D) Example of the appearance of axons
within the
dorsolateral funiculus after a control infusion of Guinea-pig complement
alone. Some non-specific
damage of myelin sheathes occurred, especially within one spinal segment of
the infusion site;
however, the compact nature of the myelin remained intact. Original
magiufication x 4000 (A, B,
D), x10000 (C).
Figure 3 presents demonstrations of regeneration of rubrospinal neurons after
left-side thoracic
hemisection and subsequent immunological myelin suppression treatment. Panels
A and B are
photomicrographs of rubrospinal neurons from the same experimentally-treated
animal (14 days
CA 02253078 1999-O1-28
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infusion of serum complement with anti-GaIC); A is from the uninjured Red
nucleus while B is from
the injured Red nucleus. Panels C and D are also from same control-treated
animal ( 14 days infusion
of serum complement only): C is the uninjured Red nucleus and D is the injured
Red nucleus.
Fluorogold injection within the rostral lumbar cord 28 days after injury
resulted in the retrograde
labeling of uninjured rubrospinal neurons (A and C) as well as those
rubrospinal neurons that had
regenerated from the injured Red nucleus (B and D). (E) and (Fj Axotomized
rubrospinal neurons
were retrograde labeled at the time of injury with the first Label RDA (solid
arrow heads) and
subsequently 28 days laterwith the secondlabel FG (open arrow heads). Double-
labeled (RDA+FG)
cells are indicated by an asterisk and represent those rubrospinal neurons
that had regenerated after
immunological myelin-suppression treatment. Scale bar = 100pm.
Figure 4 shows a relative quantitative assessment of regeneration of
rubrospinal neurons after
thoracic injury and immunological treatment. Regeneration was assessed by
counting FG-labeled
cells in alternating tissue sections: thosewith both multipolar neuronal
morphology and FG labeling
were deemed to be positive. Percentage regeneration was calculated by
comparison of the retrograde
labeled cell counts from the injured Red nucleus with the control uninjured
Red nucleus within the
same animal. For each animal, the degree of lesion was assessed. Filled bar:
myelin suppressed;
hatched bar: pooled control treated groups. Data shown t s.d.
Figure 5 demonstrates effects of removal of a single complement protein on
immunological
demyelination. (A) Controluninjuredspinal cord. Electronphotomicrographs
oftransversesections
through the dorsolateral funiculus indicating the ultrastructure of adult
myelin sheaths. (B) 7 day
infusion with myelin-specific antibody and human complement sera results in a
profound myelin
suppression. (C) The removal of the C3 component of complement results in a
lack of myelin-
removal, indicating the fundamental role of this protein in either (i)
opsonization, or (ii) the
propagation of the cascade to the lytic membrane attack complex (MAC), the
final lytic pathway
complex. It is believed that it is a fundamental and essential requirement of
a myelin specific cell
surface binding antibody to activate the classical complement pathway for
effective transient
demyelination.
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Figure 6 shows a relative quantitative assessment of regeneration of lateral
vestibulospinal neurons
after thoracic injury and delayed immunological treatment. Immunological
demyelination treatment
was delayed for 1 or 2 months after injury as indicated. Regeneration was
assessed by counting FG-
labeled cells in alternating tissue sections: those with both multipolar
neuronal morphology and FG
labeling were deemed to be positive. Percentage regeneration was calculated by
comparison of the
retrograde labeled cell counts from the injured lateral vestibulospinal
nucleus with the control
uninjured lateral vestibulospinal nucleus within the same animal. For each
animal, the degree of
lesion was assessed. Filled bar: myelin suppressed; open bar: pooled control
treated groups. Data
shown ~ s.d.
Figure 7 presents A) Drawing of a dorsal view of the rat central nervous
system, indicating the
relative origins and course of the rubrospinal tract (RN) and lateral
vestibular tract (LVe). Also
illustrated (solid line) is the left-side thoracic hemisection lesion (~ T10,
line), the immunological
infusion site (~ T11, vertical hatching), and the site of the Fluorogold
injection (~L1, diagonal
hatching). B) composite photomicrograph of parasagittal sections through the
lower thoracic and
rostral lumbar spinal cord (T9- L1, rostral is up). Some Fluorogold diffusion
can be clearly
emanating from the inj ection site as an intense white "halo", however, this
staining rapidly decreased
with distance from the site of injection and none was ever visible rostral to
Tl 1, the immunological
infusion site (i. e. no diffusion to or above the lesion at T 10, thus no
evidence for any "false" positive
retrograde labeling of brainstem-spinal proj ections). C) photomicrograph of a
transverse section of
spinal cord at the level of T10 left side hemisection lesion, stained with
cresyl violet. All lesions
were assessed and always resulted in severing the funiculi through which the
rubrospinal and lateral
vestibulospinal tracts traverse. The contralateral dorsal (dh) and ventral
(vh) horns were always left
undamaged; the central canal (cc) is labeled for orientation. D and E) Non-
specific fluorescence
associated with blood cells within the lesion and pump implantation sites
indicating the degree of
damage associated with the lesion and cannula implantation, respectively.
Specific Fluorogold
fluorescence labeling was never observed at the level of the cannula
implantation or hemisection
injury.
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Figure 8 shows regeneration of lateral vestibulospinal neurons after left-side
thoracic hemisection
and subsequent immunological myelin suppression treatment. Panels A and B are
photomicrographs
of lateral vestibulospinal neurons from the s ame experimentally-treated
animal ( 14 days infusion of
serum complement with anti-GalC); A is of the injured lateral vestibular
nucleus and B is from the
uninjured lateral vestibular nucleus and. Panels C and D are also from same
control-treated animal
( 14 days infusion of serum complement only); where C is the inj~ed lateral
vestibulospinal nucleus
and D is the uninjured lateral vestibulospinal nucleus. Fluorogold inj ection
within the rostral lumbar
cord 28 days after injury resulted in the retrograde labeling of uninjured
lateral vestibulospinal
neurons (B and D) as well as those lateral vestibulospinal neurons that had
regenerated from the
injured lateral vestibulospinal nucleus (A and C), please see results for
further details. Panel E is a
drawing of a transverse section through the midbrain indicating the location
of the lateral vestibular
nucleus (LVe), SpVe = spinal vestibular nucleus, MVe = medial vestibular
nucleus, 4V = 4~'
ventricle, FN = facial nerve tract, 7 = 7'" cranial (facial) nucleus, PFl =
paraflocculus. Scale bar =
100 ~.m
Figure 9 shows relative quantitative assessment of regeneration of rubrospinal
and lateral
vestibulospinal neurons after thoracic injury and immunological treatment.
Regeneration was
assessed by counting FG-labeled cells in alternating tissue sections; those
with both multipolar
neuronal morphology and FG labeling, were deemed to be positive. Percentage
regeneration was
calculatedby comparison of the injurednucleus with the contralateral
(uninjured) nucleus within the
same animal. For each animal the degree of lesion was assessed. Filled bars,
experimental; open
bars, pooled control groups.
Figure 10 shows a quantitative assessment of regeneration of descending
brainstem-spinal axons
after chronic lateral hemisection & delayed immunological treatment.
Percentages of retrogradely
labeled red nucleus (red) and lateral vestibular (green) neurons vs.
Contralateral uninjured, after
control (PBS, Ab, GpC) treatment (open bars) or immunological
disruption/demyelination (filled
bars). Expressed as percentage labeled cells in the injured nucleus vs.
Uninjured contralateral.
CA 02253078 1999-O1-28
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DETAILED DESCRIPTION OF THE INVENTION
The following terms and abbreviations are used throughout the specification
and in the claims:
The term "antibodies or fragments thereof' includes recombinant, chimeric, and
affinity modified
forms made by techniques of molecular biology well known in~the art;
"CNS" refers to the central nervous system;
The term "complement protein or fragment thereof' (C) refers to any of 13
whole serum proteins or
any of more than 20 intermediates and complexes of the complement system, the
primary humoral
mediator of antigen-antibody reactions, and includes variants, analogs, and
chemical derivatives
thereof;
The term "composition" is used to indicate more than one component. The
elements of the
composition can be mixed together, however, it is not necessary that they be
combined in the same
solution. In an alternative embodiment, they do not need to be packaged,
stored or even mixed
together. The elements (antibody-type and complement-type) can be delivered to
the site of nerve
damage sequentially, oratthesametime.
Theneedforatherapeuticallyeffectivetemporalsequence
is understood by one skilled in the art. The concept of at least one
complement fixing antibody or
fragment thereof, plus at least complement protein or active fragment thereof
equates with the
concept of the composition. These elements are delivered to the site of damage
to form a complex
with an appropriate epitope present in myelin to be transiently demyelinated.
Thus, the first two
types of elements (of which there can be more than one member of each type of
element, for
example, two or more antibody or component proteins or fragments) are
delivered to the site targeted
for transient demyelination to form a complex in situ, in vivo with the
epitope(s) on myelin.
The term "demyelination" refers to the removal or breakdown of myelin in
neurological tissue.
CA 02253078 1999-O1-28
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Demyelination consists of the removal of the myelin sheath, such as that
surrounding neurons or
neuronal projections (e.g., the axons). This process may be chemical or
immunological in both the
experimental and pathological states. This invention effects transient
demyelination in order to
promote repair and regrowth.
The term "disruption" refers to delamination or disruption of the three-
dimensional conformation
of myelin;
The term "dysfunction" when used to describe the therapeutic use of the
invention encompasses any
type of trauma to the nervous system and resulting loss of function. Such
trauma can arise from
either physical injury or disease;
The term "Fab" means an antibody fragment that is obtained by cleaving an
antibody in the hinge
region yielding two Fab fragments, each having the heavy and light chain
domains of the antibody,
along with an Fc region;
The term "Fc" means the constant region of the antibody, which may activate
complement;
The term "Fv fragment" means a heterodimer of the heavy and light chain
variable domain of an
antibody. These variable domains may be joined by a peptide linker or by an
engineered disulphide
bond;
Growth factors are extracellular polypeptide signaling molecules that
stimulate a cell to grow or
proliferate. Examples are epidermal growth factor (EGF) and platelet=derived
growth factor
(PDGF). Most growth factors have other actions besides the induction of cell
growth or proliferation.
Growth factors can be divided into broad- and narrow-specificy classes. The
broad-specificity
factors, like PDGF and EGF affect any classes of cells. At the opposite
extreme lie narrow-
specificity factors. In intact animals proliferation of mot cell types depends
on a specific
combination of growth factors rather than a single growth factor. Thus a
fairly smal number of
CA 02253078 1999-O1-28
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growth factor families may serve, in different combinations, to regulate
selectively the proliferation
of each of the many types of cells in a higher animal.
Fibroblast Growth Factor (FGF) is any one of a group of proteins, usually
intracellular, that have
important angiogenic function and enhance would healing and tissue repair.
Over-activity of these
factors has been associate with neoplasia.
Neurotrophic factors are a family of substances that promote growth and
regeneration of neurons.
While growth factors elsewhere in the body promote and support cell division,
neurons cannot
divide; but they can regenerate after injury and neurotrophic factors promote
this regeneration. They
also promote the growth of axons and dendrites, the neuron branches that form
connections with
other neurons.
"GaIC" refers to galactocerebroside;
"MAG" refers to myelin-associated glycoprotein;
"MBP" refers to myelin basic protein;
"MOG" refers to myelin oligodendrocyte glycoprotein;
The term "neurological tissue" refers to neurons and other cells typically
situated in the region of the
nervous system, such as the spinal cord of the CNS;
"PNS" refers to the peripheral nervous system;
The term "recombinant antibodies or fragments thereof' collectively includes
chimeric or
recombinant forms of the antibodies or fragments thereof wherein the Fc domain
is substituted for
an Fc domain of another species or isotype, affinity modified forms of the
antibodies or fragments
CA 02253078 1999-O1-28
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thereof wherein the binding sites are altered, avidity modified forms of the
antibodies or fragments
thereof wherein the hinge regions are altered, immunoreactive fragments
thereof, and combinations
thereof; and
The term "regeneration of neurological tissue" includes the regrowth of
neurons that results in the
reformation of neuronal connections, both anatomically and/or~functionally.
The present invention resides in the unexpected discovery that a combination
of both antibody,
which binds an epitope on a myelin-producing glial cell, and complement can be
used for disruption
and demyelination of the myelin sheath, such that repair and regeneration of
mammalian
neurological tissue is enhanced. The composition of this invention is valuable
as a therapeutic agent
in cases in which there is injury or disease of the mammalian nervous system
such that there is a
need to facilitate neuronal plasticity and the regrowth of neural connections.
The neurological tissue
is exposed to the myelin disrupting composition, according to the invention,
as soon as possible
following the injury, trauma, or disease. The nature of the protocol to effect
transient demyelination
can be determined from Kierstead and Blakemore, 1997, J.
Neuropath.Expt.Neurol. 56:1 I91-1201;
Kierstead et al., 1998, Glia, 22:161-170.
The present invention provides compositions and methods of their use for
promoting regeneration
of neurological tissue in a mammalian subject, such as a human, with a nervous
system dysfunction
by contacting the neurological tissue with a therapeutically effective amount
of a composition
comprising a complement fixing antibody, which binds to myelin, and
complement. Uses of the
composition in the field of veterinary medicine are also an embodiment of the
present invention.
The compositions of the present invention are comprised of one or more
antibodies or fragments
thereof, which bind myelin, and one or more serum complement proteins or
fragments thereof.
Antibodies
The antibodies used in this invention can be any antibodies or fragments
thereof that specifically
CA 02253078 1999-O1-28
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bind to myelin, wherein said antibodies activate the complement system. The
preferred antibodies
ofthe present invention specifically bind amyelin sheath epitope, such as
galactocerebroside (GalC),
04, Myelin Oligodendrocyte Glycoprotein (MOG), or Myelin Associated
Glycoprotein (MAG).
Other preferred epitopes are NOGO (formerly NI 35/250) and NI220 and arretin.
Generation ofAntibodier
The antibodies of the present invention, or fragments thereof, can be:
a) naturally occurring;
b) antibodies obtained from disease states such as B-cells from multiple-
sclerosis patients;
b) produced by recombinant DNA technology;
c) produced by biochemical or enzymatic fragmentation of larger molecules;
d) produced by methods resulting from a combination of a) to c); or
e) produced by any other means for producing antibodies.
Human antibodies can be generated by a number of techniques known to those
skilled in the art,
including the use of insect cells and transgenic plants such as tobacco or
corn seed (framer, C.L.,
CropTech Development Corp; Reno, J., NeoRx - IVC's IV Annual Conference: Sept
9-12, S.F.,
U.S.A.)
The antibodies of the present invention can also be made by traditional
technqiues such as
monoclonal or polyclonal, althoughmonoclonal antibodies are preferred. In
general, antibodies may
be obtained by inj ecting the desired immunogen into a wide variety of
vertebrates or invertebrates
in accordance with conventional techniques. While rodents, particularly mice,
are preferred, other
species may be employed, such as members of the bovine, ovine, equine,
porcine, or avian families.
Immunization of these animals can be readily performed and their lymphocytes,
particularly
splenocytes, may be obtained for fusions.
Immunization protocols are well known and can vary considerably yet remain
effective (Goding,
CA 02253078 2000-10-02
-21-
MonoclonalAntibodies.~ Principles and Practice(2nded.) {Academic Press,1986).
Isolatedproteins,
synthetic peptides, and bacterial fusion proteins which contain antigenic
fragments of the myelin
molecule may be used as immunogens. Preferably the immunogen of peptides or
recombinant
proteins will be enriched for proteins or fragments thereof containing the
epitopes to which antibody-
producing B cells or splenacytes are desired.
Once the proteins or peptides thereof have been purified to the extent
desired, they may be
suspended or diluted in an appropriate physiological carrier for immunization,
or may be coupled
to an adjuvant. Immunogenic amounts of antigenic preparations enriched in
myelin, or antigenic
portions thereof, are injected, generally at concentrations in the range of 1
ug to 100 mg/kg ofhost.
Administration may be by injection, such as intramuscularly, peritoneally,
subcutaneously, or
intravenously. Administration may be one or a plurality of times, usually at
one to four week
intervals.
Immunized animals are monitored for production of antibody to the desired
antigens, then the
spleens are removed and splenic B-lymphocytes isolated and fused with a
myeloma cell line or
transformed. The B-lympocytes can also be isolated from the blood. The
transformation or fusion
can be carried out in conventional ways, the fusion technique being described
in an extensive
number of patents, such as U.S. Patent Nos. 4,172,124; 4,350,683; 4,363,799;
4,381,292; and
4,423,147. The manner of immortalization is not critical, but the most common
method is fusion
with a myeloma fusion partner. Other techniques of immortalization include EBV
transformation,
transformationwithbare DNA, such as oncogenes orretroviruses, or any
othermethodthatprovides
for stable maintenance of the cell line and production of monoclonal
antibodies. The general process
for obtaining monoclonal antibodies has been described (Kohler and Milstein
(I975) Nature
256:495-497). Human monoclonal antibodies may be obtained by fusion of the
spleen cells with an
appropriate human fusion partner, such as WI-L2, described in European Patent
No. 0062409. A
detailed technique for producing mouse X-mouse monoclonal antibodies has been
taught (Oi and Herzenberg (1980) in Mishell and Shiigi (eds.) Selected Methods
in Cellular
Immunology 35I-372). The resulting hybridomas are screened to isolate
individual clones, each of
CA 02253078 1999-O1-28
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which secretes a single antibody species to the antigen.
The immortalized cell lines may be cloned and screened in accordance with
conventional
techniques, and antibodies in the cell supernatants detected that are capable
of binding to myelin.
The appropriate immortalized cell lines may then be grown in vitro or injected
into the peritoneal
cavity of an appropriate host for production of ascites fluid. Immortalized
hybridoma cell lines can
be readily produced from a variety of sources. Alternatively, these cell lines
rnay be fused with other
neoplastic B-cells, where such other B-cells may serve as recipients for
genomic DNA coding for
the antibody.
The monoclonal antibody secreted by the transformed or hybrid cell lines may
be of any of the
classes or subclasses of immunoglobulins, such as IgM, IgD, IgA, IgGi_4, or
IgE. As IgG is the most
common isotype utilized in diagnostic assays, it is often preferred.
To circumvent the possible antigenicity in a human host of a monoclonal
antibody derived from an
animal other than human, chimeric antibodies may be constructed. For example,
the antigen binding
fragment of an immunoglobulin molecule (variable region) may be connected by
peptide linkage to
at least part of another protein not recognized as foreign by humans, such as
the constant portion of
a human immunoglobulin molecule. This can be accomplished by fusing the animal
variable region
exons with human kappa or gamma constant region exons. Various techniques are
known to the
skilled artisan, such as those described in PCT 86/01533, EP171496, and
EP173494.
As an alternative method of producing antibodies, US Patent No. 5627052
describes methods of
producing proteins that replicate the binding characteristics and desired
function of particular
antibodies. An example of application of this method includes the isolation
and chaxacterization of
a human B-lymphocyte cell, producing a specific anti-myelin antibody, for
example from the blook
of a patient with Multiple Sclerosis.
CA 02253078 1999-O1-28
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Antibody Engineering
The antibodies may be used intact, or as fragments, such as Fv, Fab, and
F{ab')2 as long as there is
an Fc region present to bind complement. Such antibody fragments provide
better diffusion
characteristics in vivo than the whole antibody, due to their smaller size.
The means for engineering
antibodies by recombinant DNA and chemical modification methods are considered
well-known in
the axt.
The antibodies may be fragmented to obtain highly immunoreactive F(ab')2,
F(ab'), and Fab
fragments using the enzyme pepsin by methods well known in the art (see
Colcher et al., (1983)
Cancer Res. 43:736-742).
Due to the development of molecular cloning technqiues, it is now possible to
produce human
monoclonal antibody fragments quickly by paring phage display libraries
against predefined
antigenic specificities. For exemplary techniques see: Pistillo et al., Human
Immunology, 57( I):19-
26, 1997 Sep 15).
Antibodies or fragments thereof are also made into recombinant forms by
techniques of molecular
biology well known in the art (see Rice et al., (1982) Proc. Natl. Acad. Sci.
USA 79:7862-7865;
Kurokawa et al., (1983) Nucleic Acids Res. 11:3077-3085; Oi et al., (1983)
Proc. Natl. Acad. Sci.
USA 80:825-829; Boss et al., (1984) Nucleic Acids Res. 12:3791-3806; Boulianne
et al., (1984)
Nature (London) 312:643-646; Cabily et al., ( 1984) Proc. Natl. Acad. Sci. USA
81:3273-3277;
Kenten et al., ( 1984) Proc. Natl. Acad. Sci. USA 81:2955-2959; Liu et al., (
1984) Proc. Natl. Acad.
Sci. USA 81:5369-5373; Morrison et al., (1984) Proc. Natl. Acad. Sci. USA
81:6851-6855;
Neuberger et al., {1984) Nature (London) 312:604-608; Potter et al., (1984)
Proc. Natl. Acad. Sci.
USA 81:7161-7165; Neuberger et al., (1985) Nature (London) 314:268-270; Jones
et al., (1986)
Nature (London) 321:522-525; Oi et al., (1986) BioTechniques 4:214-221;
Sahagan et al., (1986)
J. Immunol. 137:1066-1074; Sun et al., (1986) Hybridoma 5 (Supp. 1):517-520;
and Sun et al.,
CA 02253078 1999-O1-28
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(1987) Proc. Natl. Acad. Sci. USA 84:214-218).
More specifically, the antibodies and fragments thereof may be altered to a
chimeric form by
substituting antibody fragments of another species, e.g., human constant
regions (Fc domains) for
mouse constant regions by recombinant DNA techniques known in the art as
described in the above
cited references. These Fc domains can be of various human isotypes, i.e.,
IgGI, IgG2, IgG3, IgG4,
or IgM.
In addition, the antibodies and fragments thereof may be altered to an
affinity modified form, avidity
modified form, or both, by altering binding sites or altering the hinge region
using recombinant DNA
techniques well known in the art as described in the above cited references.
The recombinant antibody forms may also be fragmented to produce
immunoreactive fragments
F{ab')2, F(ab'), and Fab in the same manner as described.
Antibody fragments may also include Fv fragments, the smallest functional
modules of antibodies
required to maintain the binding and specificity of the whole antibody. Fv
fragments are
heterodimers composed of a variable heavy chain and a variable light chain
domain. Proteolytic
digestion of antibodies can yield isolated Fv fragments, but the preferred
method of obtaining Fvs
is by recombinant technology (See Skerra and Pluckthun (1988) Science 240:1038-
1041).
Fvs can be noncovalently-associated VH and VL domains, although these tend to
dissociate from one
another. Stable Fvs can be produced by making recombinant molecules in which
the VH and VL
domains are connected by a peptide linker so that the antigen-combining site
is regenerated in a
single protein. These recombinant molecules are termed single chain Fvs
(scFvs). The means for
preparing scFvs are known in the art (S ee: Raag and Whitlow ( 1995) FASEB
9:73; Bird et al., ( 1988)
Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-
5883).
Alternatively, the two variable domains may be joined and stabilized by an
engineered disulphide
bond; these are termed disulfide Fvs (dsFvs} (Reiter and Pastan (1996) Clin.
Cancer Res. 2:245-
CA 02253078 1999-O1-28
-25-
252).
The Fc domain of an antibody is required for the activation of complement. Fv
fragments, which
lack the Fc domain, cannot activate complement. In order for Fv fragments to
be useful in the
present invention, they would have to be designedwith a novel activator of the
complement cascade.
As an example, the Fv fragment could be designed to include the CH2 domain of
an IgG antibody.
As an alternative example, awholly synthetic molecule may be linked to the Fv
fragment to activate
complement, or an activator of complement familiar to those in the field may
be linked to the Fv
fragment.
The antibody may also be modified by the addition of such molecules as
polyethylene glycol (as
described in U. S. Patent 5766897) as to prolong its biological half life,
potency, or the diffusion of
the molecule in situ (U.S. Patent 5747446, Chinol et al., 98 Brit. J. Cancer,
78:189-197; Francis et
al., 98, Intl J. Hematol. 68:1-18).
Labeling ofAntibodies or Fragments:
The antibodies of this invention, or fragments thereof, may be us ed without
modification or may be
modified in a variety of ways, for example, by labeling. Labeling is intended
to mean j oining, either
covalently or non-covalently, a label which directly or indirectly provides
for a means of detection
of the antibody to enable monitoring of the progress of therapeutic treatment
using the composition.
A label can comprise any material possessing a detectable chemical or physical
property. A wide
variety of labels is known, including radionuclides, enzymes, enzyme
substrates, enzyme cofactors,
enzyme inhibitors, ligands (particularly haptens), fluorescers, chromophores,
luminescers, and
magnetic particles. These labels are detectable on the basis of either their
own physical properties
(eg., fluorescers, chromophores and radioisotopes), or their reactive or
binding properties (eg.,
enzymes, substrates, cofactors and inhibitors). These materials are well known
to one skilled in the
art. U.S. Patent 4,671,958 teaches methods that can be used for labelling
antibodies or attaching
complement to antibodies.
CA 02253078 1999-O1-28
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Complement
The complement portion of the composition may be comprised of one or more
complement proteins,
fragments, variants, analogs, and/or chemical derivatives.
A fragment of a complement protein refers to any subset of the C ~rnolecule.
For example, fragments
of C3 include C3b, iC3b, C3a, C3c, C3dg, and C3d.
A "variant" of a complement protein or fragments thereof refers to a molecule
substantially similar
to either the entire protein or a fragment thereof, which possesses biological
activity that is
substantially similar to a biological activity of the complement protein or
fragments thereof. A
molecule is said to be "substantially similar" to another molecule if both
molecules have
substantially similar structures or if both molecules possess a similar
biological activity.
Variants of C3b, for example, include C3b dimers, and higher oligomers. When C
activation occurs
at the cell-surface, multiple cycles of enzyme reactions result in the
deposition on the surface of C3b
in multimeric form. C3b dimers or higher oligomers indeed have higher affinity
for the cell than do
C3b monomers.
Variants of complement protein or fragments thereof are produced by chemical
or recombinant
means well-known in the art. Such variants include, for example, deletions
from, or insertions or
substitutions of, amino acid residues within the amino acid sequence. For
example, at least one
amino acid residue may be removed and a different residue inserted in its
place. Substantial changes
in functional or immunological properties are made by selecting substitutions
that are less
conservative, ie. that differ more significantly in their effect on
maintaining (a) the structure of the
peptide backbone in the area of the substitution, for example, as a sheet or
helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or (c) the
bulk of the side chain. The
substitutions that in general are expected to induce greater changes are those
in which (a) glycine
and/or proline is substituted by another amino acid or is deleted or inserted;
(b) a hydrophilic
CA 02253078 1999-O1-28
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residue, e.g., seryl orthreonyl, is substituted for Corby)
ahydrophobicresidue, e.g., leucyl, isoleucyl,
phenylalanyl, valyl, or alanyl; (c) a cysteine residue is substituted for (or
by) any other residue; (d)
a residue having an electropositive side chain, e.g., lysyl, arginyl, or
histidyl, is substituted for (or
by) a residue having an electronegative charge, e.g., glutamyl or aspartyl; or
(e) a residue having a
bulky side chain, e.g., phenylalanine, is substituted for (or by) one not
having such a side chain, e.g.,
glycine.
Most deletions, insertions, and substitutions are not expected to produce
radical changes in the
characteristics of the protein molecule; however, when it is difficult to
predict the exact effect of the
substitution, deletion, or insertion in advance of doing so, one skilled in
the art will appreciate that
the effectwill be evaluatedby routine screening assays. For example, a change
in the immunological
character of the protein molecule, such as binding to a given antibody, is
measured by an
immunoassay such as a competitive type immunoassay.
An "analog" of a complement protein or fragment thereof refers to a non-
natural molecule
substantially similar to either the entire protein or a fragment thereof.
A "chemical derivative" of a complement protein or fragment thereof contains
additional chemical
moieties that are notnormally part of the protein or fragment. Covalent
modifications of the peptides
are included within the scope of this invention. Such modifications may be
introduced into the
molecule by reacting targeted amino acid residues of the peptide with organic
derivatizing agents
that are capable of reacting with selected side chains or terminal residues,
as is well-known in the
art (T. E. Creighton Proteins: Structure and Molecule Properties (San
Francisco: W. H. Freeman,
1983) at 70-86 ). '
The complement portion of the composition may be a physically distinct
component from the
antibody component. Alternatively, the complement proteins or fragments
thereof, may be
covalently or noncovalently attached directly to the antibody component, such
that binding of the
antibody to the surface of the myelin triggers the endogenous immune system
attack.
CA 02253078 1999-O1-28
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The complement components may be fractions that have been purified as well as
those that have
been enriched in the proteins which comprise the complement system. Such
preparations should
take into account the relative lability of complement and provide a sufficient
combination of factors
to allow complete activation of the complement cascade to allow transient
demyelination to occur.
The complement portion ofthe composition may be comprisedof one ormore
complementproteins,
fragments, variants, analogs, and/or chemical derivatives. It should be noted,
however, that the C3
component of complement plays a fundamental role either in opsonization or in
the propagation of
the cascade to the lytic MAC. In apreferred embodiment, the C3 component or a
fragment, variant,
analog, or chemical derivative thereof should be included in the complement
portion of the
composition. In situations targeted for demyelination, the C3 component should
certaintly be
present for optimal results. In situations targeted for regeneration, it is
less certaintly required.
The complement portion of the composition may be derived from a subject's own
serum, from the
serum of a donor, or from the pooled sera of a number of donors, such as those
available
commercially, which are produced to consistent, approved standards.
The complement components may be derived from species different from that
species to which it
is administered due to the fact that the compositions are introduced directly
to the neural tissue (e.g.,
intrathecally).
Other Factors
The composition may optionally include other chemicals or drugs such as growth
factors and
neurotrophins. It is known that the beneficial effects of blocking CNS myelin-
associated inhibitors
on axonal regeneration can be augmented by the concomitant application of
neurotrophins, such as
NT-3 (Bregman et al., (1995) Nature 378:498-501; Schnell et al., (1994) Nature
367:170-173).
FGF-1 can also be used (Chang et al., 1996, supra).
CA 02253078 1999-O1-28
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In a preferred embodiment, the composition is comprised of a GaIC-specific
monoclonal antibody
and human serum complement.
In another preferred embodiment, the composition is comprised of a MOG-
specific monoclonal
antibody and human serum complement.
Uses
The compositions of the present invention can be used to promote regrowth,
repair, and/or
regeneration of neurons in the CNS of a subject by stimulating transient
immunological disruption
of myelin or transient demyelination of axons. Preferably, the transient
demyelination process of
the present invention occurs in the CNS, most preferably in the spinal cord.
The subject may be any mammal. In a preferred embodiment, the subject is
human.
The compositions of the present invention can be used to promote regrowth,
repair, and/or
regeneration of dysfunctional neurons in the CNS that have been damaged as a
result of injury, such
as a spinal cord injury. The method can be used following immediate or chronic
injury.
The compositions of the present invention can also be used to promote
regrowth, repair, and/or
regeneration of dysfunctional neurons in the CNS that have been damaged as a
result of disease,
such as degenerative diseases including Alzheimer's and Parkinson's disease.
The compositions of the present invention can also be used to generate an
environment within the
mammalian CNS that is relatively permissive to growth of transplanted cells.
For example, if PNS
cells are transplanted into a site in the CNS that has been damaged, axons
will be able to grow into
the transplanted tissue but will be unable to grow out of this tissue back
into the CNS due to the
inhibitory effects of myelin. The compositions of the present invention can be
used to disrupt the
myelin in the CNS to allow the axons to extend into this area.
CA 02253078 1999-O1-28
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Preparations and Administration
Methods of using the compositions of the present invention comprise
administering a therapeutically
effective amount of such a composition to the subject. As used herein, the
term "therapeutically
effective amount" refers to an amount of composition sufficientto effectively
and transiently disrupt
and/or demyelinate the CNS so that repair and regeneration of neurological
tissue and neuronal
connections is enhanced. Generally, the therapeutic composition is
administered at a range from
about 0.03 mg antibody to about 0.6 mg antibody in a 20% to 30% complement
solution per kg body
weight. Preferably, the range is from 0.05 mg antibody to 0.4 mg antibody in a
20% to 30%
complement solution per kg body weight. Most preferably, the range is from 0.1
mg antibody to 0.3
mg antibody in a 20% to 30% complement solution per kg body weight. The
exactratio of antibody
to complementwill vary depending on the circumstances; however, since the
amount of complement
activated is directly proportional to the number of bound antibody molecules,
it is possible to
administer relatively high concentrations of complement in excess of the
relative concentration of
antibody. In addition, the particular concentration of antibody administered
will vary with the
particular dysfunction and its severity, as well as with such factors as the
age, sex, and medical
history of the patient. Those of skill in the clinical arts will know of such
factors and how to
compensate the dosage ranges of the composition accordingly.
The majority of spinal cord injuries result from damage to the vertebral
column surrounding the
spinal cord. This damage includes fractures, dislocations, or both. Much of
the damage to the spinal
cord is due to secondary phenomena that occur within hours following the
injury. At this point, the
resultant damage may be reversible; consequently, a critical factor for
recoverable CNS function is
the amount of time that evolves between injury and the institution of therapy.
Most preferably,
when the nervous system dysfunction is a result of injury, administration of
the composition to the
subject will be as close in time to the time of the injury as possible.
A composition according to the method ofthe invention can be administered to a
subj ect parenterally
by injection or by gradual infusion over time. For example, the composition
can be administered
intrathecally or inj ected directly into the spinal cord.
CA 02253078 2000-10-02
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Preparations forparenteral administration are containedin apharmaceutically
acceptable carrier that
is compatible with both the components of the composition and the patient.
Such carriers include
sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples
of non-aqueous
solvents include propylene glycol, polyethylene glycol, metabolizable oils
such as olive oil or
squalane, and injectable organic esters such as ethyl oleate. ~~Aqueous
carriers include water,
alcoholic/acqueous solutions, and emulsions or suspensions, including saline
and buffered media.
Parenteral vehicles include sodium chloride solution, R.in.ger's dextrose,
dextrose and sodium
chloride, lactated Ringer's, or fixed oils. Preservatives and other additives
may also be present such
as, for example, antimicrobials, anti-oxidants, chelating agents, and inert
gases and the like. A
preferred carrier is artificial cerebrospinal fluid.
Kit
The materials for use in the method of the invention are ideally suited for
the preparation of a kit.
Such a kit may comprise a carrier means compartmentalized to receive in close
confinement one or
more container means, such as vials, tubes, andthe like, each of the container
means comprising one
of the separate elements to be used in the method. For example, one of the
container means may
comprise a GalC-specific antibody. Alternatively, the antibody and complement
may be present in
the same container. The constituents may be present in liquid or lyophilized
form, as desired.
Needles and/or other equipment that facilitates delivery of the complement and
antibody to the site
of damage may include:
a) silastic, Polyethylene, Tygon (Norton Performance Plastics) tubing;
b) subcutaneous pumps, (such as the Medtronic pump system known for the
administration
of baclofen intrathecally);
c) spinal needle for direct intraspinal administration, or for short-term
intrathecal
administration.
One example of a method using such a kit can be described as, a 14-gauge Tuohy
needle is ins erted
into the lumbar subarachnoid space. A 5-F catheter is coaxially placed with
the tip at L10 and
CA 02253078 2000-10-02
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tunneled to the flank (or appropriate location). This type of instruction
would be understood by one
familiar with the technique. Tubing is placed intrathecally, and connected to
the pump. The pump,
containing a finite folume of the reagents is placed under the skin this can
be refilled in the Doctor's
TM
office, via a needle inserted into a septa in the pump. Or the Infusaid pump
may be used in the
alternative.
Advantages over Current Methods
The compositions and theiruses of the present invention have a number of
advantages over methods
currently available for the regeneration of neuronal growth in the CNS.
Interventional therapies, including opiate antagonists, thyrotropin-releasing
hormone, local cord
cooling, dextran infusion, adrenergic blockade, corticosteroids, andhyperbaric
oxygen, are targeted
at reducing secondary inflammatory damage after a traumatic injury to the
spinal cord in order to
prevent the spread of damage to uninjuredneurons. Unlike the present
invention, however, they do
not promote regeneration of the damaged neurons.
Peripheral nerve transplants andthe grafting of donor cells into the CNS are
useful in that axons can
grow into them; however, the axons cannot grow out of them into the
surrounding CNS due to the
inhibitory myelin present. In contrast, the present invention disrupts the
inhibitory myelin to allow
regrowth of neurons in the CNS.
The present invention is des cribed in further detail in the following non-
limiting examples. It is to
be understood that the examples described below are not meant to limit the
scope of the present
invention. It is expected that numerous variants will be obvious to the person
skilled in the art to
which the present invention pertains, without any departure from the spirit of
the present inv'~ntion.
The appended claims, properly construed, form the only limitation upon the
scope of the present
invention.
EXAMPLE I
CA 02253078 2000-10-02
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Regeneration of Brainstem-Spinal Axons
The following example illustrates thatthe transient developmental suppression
of myelination orthe
disruption of mature myelin by local intraspinal infusion of s erum complement
proteins along with
a complement-fixing, myelin-specific antibody facilitates brainstern-spinal
axonal regeneration after
spinal transection in a mammalian subject.
Materials and Methods:
Surgical Spinal Transection and Transient Immunological Myelin Disruption:
Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g in
weight, were
anaesthetized with Ketamine/Xyiazine (60mglkg and 7.5mg/kg, respectively).
After a limited
dorsolateral laminectomy at T10, a left-side spinal cord hemisection lesion
was made with micro-
scissors. The extent of the lesion was then confirmed by passing a sharp
scalpel through the lesion
site three times (Fig. IA). Immediately after the lesion, an intraspinal
cannulawas implanted at T 11
(n=22 total) and connected to an Alzet osmotic pump ( 14 day) to subsequently
deliver a continuous
intraspinal infusion (@ 0.5 ~1/hr) of serum complement (GIBCO BRL, # I 9195-O
15, 33 % v/v) along
with a complement-fixing IgG antibody to galactocerebroside (either our own
polyclonal antibody
or Chemicon Intl. Ltd., #AB 142, 25% v/v). Cannulae were held in place by
means of dental acrylic
applied to the vertebral bone. Muscle layers were then sutured over the dental
acrylic, and the
superficial tissue and skin were closed. The extent of the hemisection lesion
was always confirmed
histologically at the end of the 5-week treatment and recovery period.
All control animals received an identical hemisection lesion andwere then
intraspinally infused via
an osmotic pump, for the same time period, with either vehicle alone (0.1 M
phosphate buffered
saline, PBS, n=5), antibody alone (25% v/v, n=2), or serum complement alone
(33% v/v, n=6). All
surgical procedures and subsequent animal care protocols were in accordance
with Canadian and
University of British Columbia Animal Care Committee guidelines.
CA 02253078 1999-O1-28
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Electron Microscopy:
Tissue for ultrastructural analysis was obtained from 10-12 week old adult
female Sprague-Dawley
rats sacrificed 7 days after infusion of serum complement along with a
complement-fixing IgG
antibody to GaIC (see above for details) via an osmotic pump. Animals were
lethally anaesthetised
with Ketamine/Xylazine ( 120mg/kg and 1 Smg/kg, respectively), then perfused
intracardially with
200 ml of 0. I M PBS (pH 7.4) followed by 100 ml of 4% glutaraldyhyde in 0.1 M
PB, (pH 7.3) and
subsequently postfixed overnight in the same fixative. The infusion site and
surrounding cord was
cut into lmm transverse blocks and processed to preserve rostral-caudal
sequence. Blocks were
washed in O.IM sodium cacodylate buffer (24 hours), postfixed in 2% Os04,
dehydrated through
ascending alcohols, and embedded in Spurrs' resin according to standard
protocols. Tissue blocks
from experimental and untreated-control animals were processed in parallel.
Thin sections (lp,ln)
were cut from each block, stained with alkaline Toluidine Blue, and examined
under a light
microscope. For electron microscopic examination, blocks were trimmed then
sections were cut at
80-1 OOnm, mounted on copper grids, stained with uranyl acetate and lead
citrate, and viewed under
a Ziess EM l OC electron microscope (at 80kV).
Retrograde Neuronal Labeling:
If a retrograde tracer (single label) is inj ected into the rostral lumbar
cord ( 1 cm caudal to the injury
site), it should be incorporated and transported back to the cell bodies of
origin by both intact axons,
as well as regeneratedproj ections. Consequently, it is essential that the
retrograde tracer reliably and
extensively label most, if not all, descending spinal projection neurons. An
equally important
parameter is that the tracer must be inj ected in a controlled and
reproducible manner at a distance
sufficiently caudal to the spinal injury to prevent any direct diffusion of
the tracer to the level of the
hemisection injury. The retrograde label that best satisfies all these
conditions is Fluorogold
{Sahibzada, et al., (1987) Brain Res. 415:242-256). Fluorescent dextran
amines, such as RDA,
require a recent axonal injury to facilitate axonal uptake (Heimer and
Zaborszky Neuroanatomical
Tract-tracing Methods 2: Recent Progress (New York: Plenum, 1989)), and are
therefore better
suited for use in the double label retrograde-tracing studies.
CA 02253078 2000-10-02
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Single Label Studies:
Twenty-eight days after the hemisection lesion and, consequently, 14 days
after completion of the
intraspinal infusion of the immunological reagents, each adult rat was
anaesthetized with
TM
Ketarriine/Xylazine(60mglkgand7.5mg/kg,respectively). Fluorogold(FG,100-
150nltotalvolume,
5% w/v in sterile dH20; Fluorochrome Inc. Englewood, C0, USA) was inj ected
(50-75n1) bilaterally
into the middle of the spinal tissue at the L1 level, approximately 1cm caudal
to the lesion site.
The specific effect of the demyelinating protocol on the extent of diffusion
of FGwas also assessed.
Rats (n=8) were experimentally treated as described above; however, animals
were killed at 12, 24,
72, and 120 hours after injection of FG into the LI cord. Eight other rats
served as controls, where
the pump contained vehicle only, and were processed in parallel with the
experimentally treated
animals. Cryostat sections (25pm thick) were analyzed for the extent of FG
diffusion from each
injection site (Fig. 1B). There were no significant differences in the extent
of visible FG diffusion,
as detected atthe light microscope level, between experimentally treated and
control treated animals.
In all cases, the range of FG diffusion was 4-6mm (1-1.5 spinal segments) from
the injection site or
at least 1.5 spinal segments caudal to the lesion site.
Double Label Studies:
At the time of lesion, the hemisection site was packed with gel-foam soaked
with 12% (w/v in sterile
TM
dHzO) rhodamine-conjugated dextran amine (RDA,10,000MW FluoroRuby, Molecular
Probes) for
30 minutes. The gel-foamwas thenremoved, andthe remaining surgical procedures
were completed
(as outlined above). After 28 days survival, all animals were
anaesthetizedwith KetamineIXyIazine
(60mglkg and 7.5mglkg, respectively). FG (100-150n1 total volume, 5% w/v in
sterile dHzO) was
injected (50-75n1) bilaterally into the spinal parenchyma at the L1 level of
the cord (n=6).
Analysis of Axonal Regeneration:
Seven days following the injection of the FG tracer into the lumbar cord,
animals were lethally
anaesthetised with KetaminelXylazine (120mg/kg and l5mg/kg, respectively) and
then perfused
intracardially with 200m1 of O.1M PBS (pH 7.4) followed by 100 mI of 4%
paraformaldehyde in
CA 02253078 2000-10-02
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0.1 M PBS, {pH 7.3). The brain and spinal cord were then removed and postfixed
overnight in the
same fixative. Subsequently, each brain and spinal cord was cleared of
fixative and cryo-preserved
by placing the tissue in a series of sucrose solutions (15% followedby 21 %).
Coronal or parasagital
sections were cut at 25~n thickness on a cryostat. The brainstem and spinal
cord tissue sections
were examined under a Zeiss Axioskop vaith a 100W mercury bulb
(excitation/emission
wavelengths: FG, 365/420nm; RDA, 546/590nm).
The brainstem-spinal nucleus used to assess the axonal regenerative abilities
of experimentally
treated auima.Is was the Red Nucleus (RN, origin is contralateral to the
hemisection). Spinal-
projecting axons from each RN cross to the opposite side of the midbrain and
descend throughout
the spinal cord within the contralateral dorsolateral funiculus. This
contralateral spinal projection
pathway is known to be a completely lateralized tract with the possible
exception of 2-5 % of the
axons, which may project to the cord via an ipsilatera~i route (Brown (1974)
J. Comp. Neurol.
154:169-188; Huisman et al., (1981) Brain Res. 209:217-286; Shieh et al.,
(1983) J. Comp. Neurol.
214:79-86; Waldron and Gwyn (1969) J. Comp. Neurol. 137:143-154).
Using a single-blind protocol, the number of retrograde labeled neurons within
the Red Nucleus
(RN) were counted in every other tissue section throughout the nucleus to
avoid counting the same
neuron twice. Only those cells exhibiting a nucleus and a neuronal morphology
(i. e. mufti-polar in
appearance), and that were specifically labeled with FG (i.e. not visible
using other fluorescent
filters; see above) extending into the proximal processes, were deemed to be
positively labeled
spinal-proj ecting neurons. The percentage of regenerating neurons was then
determined in
comparison to the number of labeled neurons within the contralateral
(uninjured) control nucleus
within the same animal.
Results:
Extent of Spinal Cord Demyelination and Myelin Disruption after Immunological
Treatment
Direct intraspinal infusion of 33% heterologous (guinea pig) serum complement
along with
CA 02253078 1999-O1-28
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polyclonal antibodies to GalC (25%) in PBS over 7 days (@ 0.5~,1/hr) resulted
in extensive
demyelination up to 2mm away from the infusion cannula (total rostrocaudal
distance of 4mm or
approximately 1 spinal segment (Fig. 2A). This zone of demyelination was
bounded on either side
by a further 8mm or 2 segments of spinal cord characterized by disrupted
myelin (i.e. myelin that
is extensively de-laminated, having an unraveled appearance, Fig. 2C). As
shown in previous studies
(Keirstead et al., ( 1995) J. Neurosci. 15:6963-6974; Keirstead et al., (
1992) Proc. Natl. Acad.
Sci. (USA) 89:11664-11668; Keirstead et al., ( 1997) Brain. Res. Bull. 44:727-
734), control infusions
of heterologous serum complement alone, myelin-specific antibody alone, or PBS
alone resulted in
only limited non-specific damage immediately centered around the cannula site.
There was no
surrounding zone of demyelination or myelin disruption (Fig. 2D).
The immunological demyelination and disruption of myelin within the
experimentally-treated adult
rat spinal cord is an active process extending throughout the entire cross-
sectional profile of the cord.
Immunological myelin disruption commences within 1 day and is associated with
an invasion of
macrophages or resident microglia and polymorphonuclear cells (e.g. leukocytes
such neutrophils,
eosinophils and basophils). Many macrophages/microglia contain myelin
fragments and complete
their phagocytic activity within 7 days (Fig. 2B). This pattern of
demyelination and myelin
disruption can be maintained for as long as the serum complement and myelin-
specific antibody are
infused. Recent evidence suggests that afterthe immunological infusion is
terminated, remyelination
begins within 2 weeks (Keirstead and Blakemore ( 1997) GI is (In Press); Dyer,
B ourque, and Steeves
(unpublished observations)); the new myelin originates from differentiating
oligodendrocyte
progenitors, although invading Schwann cells and surviving "mature"
oligodendrocytes may also
contribute to remyelination.
Choice of Retrograde Tracer and Its D~usion Distance from the Injection Site
In this study, the major anatomical evidence for axonal regeneration within
the hemisected and
immunologically myelin-suppressed spinal cord of adult rats depends on a
comparison between the
number of retrogradely-labeled neurons within a homologous pair of brainstem-
spinal nuclei. For
these comparisons to be valid, the candidate brainstem spinal nuclei must have
highly unilateral
CA 02253078 1999-O1-28
-3 8-
projections that are confined to one side of the spinal cord at all levels. A
left thoracic hemisection
(Fig. lA) severedthe contralaterally-projectingmagnocellularneurons
oftherightrednucleus (RN),
but left the projections from the left RN undamaged (as they project through
the intact right
dorsolateral funiculus of the thoracic cord).
In all cases, the Fluorogold label ( 100-1 SOnI) was inj ected bilaterally
within the rostral lumbar cord
(lcm or 3 spinal segments caudal to the hemisection injury site). We assessed
the time course and
degree of rostrocaudal diffusion of Fluorogold within the lumbar and thoracic
spinal cord of
normally myelinated (control) animals and experimentally treated rats (i. e.
under demyelinated and
myelin disrupted conditions). Random 25~,m sections of experimental and
control-treated spinal
cords {extending from L2 to T8) were examined under a fluorescent microscope
using the highest
intensity s etting of the 1 OOW mercury lamp. Spinal tissue was examined for
the extent of Fluorogold
diffusion at varying survival intervals after injection, including: l2hr
(n=4), 24hr (n=4), 3d {n=4),
and 5d (n=4). The maximum rostral diffusion distance observed was 4-6mm (or 1-
1.5 spinal
segments) and occurred within a time span of 24h. The degree of Fluorogold
diffusion within the
lumbar cord did not change over the subsequent time points examined (Fig. 1
B).
In summary, no animal (experimental or control) showed any evidence of the
Fluorogold label
within the spinal cord at the level of the hemis ection lesion {T 10); thus,
by this criteria, no animals
had to be excluded from this study. The available evidence indicates that the
retrograde label was
restricted to labeling intact and regenerating.brainstem-spinal neurons having
axonal projections
caudal to the T10 injury site.
Evidence for Brainstem-spinal Axonal Regeneration by Retrograde Neuronal
Labeling
28 animals ( 12 experimental (9 retrogradely single-labeled, 3 double-labeled)
and 16 control ( 13
retrogradely single-labeled, 3 double-labeled)) were subjected to a left-side
lateral hemisection of
the T 10 spinal cord. Immediately after hemisection, an infusion cannula
(connected to a 14d osmotic
pump) was inserted directly into the spinal cord 4-5 mm ( 1 spinal segment)
caudal to the injury site.
The osmotic pump contained one of a number of 3 different control solutions or
the experimental
CA 02253078 1999-O1-28
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treatment (i.e. PBS vehicle alone, serum complement alone, anti-
galactocerebroside antibody alone,
or serum complement with anti-GaIC antibodies, respectively). Animals were
then allowed to
recover for 28 days before the Fluorogold was injected into the rostral
lumbar, lcm {i.e. 3 spinal
segments) caudal to the lesion site. After a further 7 days survival, each
animal was killed, and the
brain and spinal cord were removed for examination and analysis (see Materials
and Methods for
criteria used to determine a labeled neuron).
The extent of the hemisection lesion was assessed in every animal. In all but
one experimental and
one control-treated animal, the leftthoracic spinal cordwas hemisected (Fig.
lA). Most importantly,
the region of the rubrospinal tract (dorsolateral funiculus) was severed. The
right side white matter
tracts were always remained intact and undamaged; usually the gray matter of
the contralateral side
was also undamaged.
Comparing "blind" counts of the number of labeled neurons within each RN (Fig.
3A-B, Table 1 ),
the data indicated that 31.8% t 13.38% (n=9, range 10-50%) of the injured
rnagnocellular RN
neurons had regenerated a sufficient distance into the caudal lumbar cord to
incorporate and
retrogradely transport the Fluorogold (Fig. 4). In contrast, control treated
animals, receiving either
the PBS vehicle alone, GaIC antibody alone, or serum complement alone, did not
exhibit a
significant amount of RN labeling: 1.49% t 0.84%, (Fig. 3C-D; Fig. 4, n=13,
range 0-3, Table 1).
The labeling of some neurons within the injured right RN nucleus may represent
the small number
of RN that do not project to the opposite side of the midbrain and descend
within the ipsilateral
(uninjured) cord (Shieh et al., (1983) J. Comp. Neurol. 214:79-86). No
retrograde-labeling of cells
was observed within the parvocellular region of the RN. This was expected
since this RN region
predominantly proj ects only as far as the cervical region of the cord.
Double retrograde labeling of the injured and myelin-suppressed rubrospinal
tract was also
qualitatively assessed (Fig. 3E and F). Large numbers of RDA-positive (first
label) magnocellular
RN neurons were observed after direct labeling of the lesion site at the time
of hemisection injury
to the thoracic spinal cord. After intraspinal myelin-suppression and
subsequent injection of
CA 02253078 1999-O1-28
-40-
Fluorogold caudal to the lesion site, a small overlapping population of FG-
positive neurons was
observed (i.e. some neurons were labeledwith both R.DA and FG). Cells labeled
exclusively by the
first or the second tracer were also present in every brainstem analysed. The
low number of double
labeled brainstem-spinal neurons may in part be due to the failure of a
severed axon to take up RDA
prior to the sealing of the cut end, i.e. must be freshly injured (Heimer and
Zaborszky
Neuroanatomical tract-tracing methods 2: Recent Progress (New York: Plenum,
1989)). Also the
population of rubrospinal neurons that do not cross the brainstem will also
appear as FG-positive
cells in the "injured" nucleus. Due to the small number of animals that were
assessed, we did not
attempt to quantify these results. Nevertheless, they probably represent an
under-estimate of the
axonal regeneration facilitated by immunological demyelination and myelin
disruption, but
definitely not an over-estimate of the degree of brainstem-spinal regeneration
after myelin
suppression.
As compared with prior art using spinal transection (Keirstead et al., ( 1995)
J. Neurosci. 15:6963-
6974; Keirstead et al., (1992) Proc. Natl. Acad. Sci. (USA) 89:11664-11668),
the present invention
is demonstrated using a hemisection model for this study so that each animal
could serve as its own
internal control (i. e. axonal regeneration from injured brainstem-spinal proj
ections could be readily
compared to the uninjured contralateral homologue). In addition, the present
invention strove to
minimize the degree of cyst cavity formation that often occurs with larger
spinal lesions, as well as
the amount of animal discomfort over the relatively long recovery periods
required for this study.
Examinations for any functional or behavioral differences during the 5 week
recovery period after
experimental treatment indicated no notable differences in locomotor patterns
between injured
animals and uninj cared control animals (i. e. all animals walked and all
animals were comparable with
respect to basic reflex functions}. These observations were true regardless of
the treatment infused
intraspinally after a hemisection injury (e.g. PBS alone, GaIC antibody alone,
serum complement
alone, or serum complement plus GaIC antibody).
These findings indicate that the immunological suppression of myelin
(demyelination and myelin
CA 02253078 1999-O1-28
-41-
disruption) facilitate anatomical regeneration of brainstem-spinal axons
within the injured adult rat
spinal cord.
EXAMPLE II
Effects of Removal of a Single Complement Protein on Immunological
Demyelination
Materials and Methods:
Surgical Spinal Transection and Transient Immunological Myelin Disruption:
Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g in
weight, were
anaesthetizedwith Ketamine/Xylazine (60mg/kg and 7.5mg/kg, respectively). A
limited dorsolateral
laminectomy was performed at T 10, and connected to an Alzet osmotic pump ( 14
day) to
subsequently deliver a continuous intraspinal infusion (@ 0.5~.1/hr) of C3-
depleted serum
complement (Sigma 58788, 33% v/v) along with a complement-fixing IgG antibody
to
galactocerebroside (either our own polyclonal antibody or Chemicon Intl. Ltd.,
#AB 142, 25% v/v).
Cannulae were held in place by means of dental acrylic applied to the
vertebral bone. Muscle layers
were then sutured over the dental acrylic, and the superficial tissue and skin
were closed.
All control animals were intraspinally infused via an osmotic pump, for the
same time period, with
whole human serum complement (Sigma 51764, 33% v/v) along with a complement-
fixing IgG
antibody to galactocerebroside (either our own polyclonal antibody or Chemicon
Intl. Ltd., #AB 142,
25% v/v). All surgical procedures and subsequent animal care protocols were in
accordance with
Canadian and University of British Columbia Animal Care Committee guidelines.
Electron microscopy was performed as described in Example I.
Results:
As seen in Figure 5, the removal of the C3 component of complement results in
a lack of myelin-
removal. This indicates that this protein has a fundamental role in either (i)
opsonization, or (ii) the
CA 02253078 1999-O1-28
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propagation of the cascade to the lytic membrane attack complex (MAC), the
final lytic pathway
complex.
EXAMPLE III
Regeneration of Chronically Injured Neurons
Materials and Methods:
11 animals (6 experimental and 5 control ) were subjected to a left-side
lateral hemisection of the
T 10 spinal cord as follows: 10 to 12 week old adult female rats (Sprague-
Dawley), approximately
200g inweight, were anaesthetizedwith Ketamine/Xylazine (60mg/kg and 7.Smg/kg,
respectively).
After a limited dorsolateral laminectomy at T 10, a left-side spinal cord
hemisection lesion was made
with micro-scissors. The extent of the lesion was then confirmed by passing a
sharp scalpel through
the lesion site three times.
One month (S animals) or 2 months (6 animals) after hemisection, an infusion
cannula (connected
to a 14d osmotic pump) was inserted directly into the spinal cord 4-5 mm ( 1
spinal segment) caudal
to the injury site. Cannulae were held in place by means of dental acrylic
applied to the vertebral
bone. Muscle layers were then sutured over the dental acrylic, and the
superficial tissue and skin
were closed. The osmotic pump delivered a continuous intraspinal infusion
(0.5~.1/hr) of guinea-pig
serum complement (33% v/v) along with a complement-fixing IgG antibody to
galactocerebroside
(either our own polyclonal antibody or Chemicon Intl. Ltd., #AB 142, 0.25
mg/mL).
All control animals received an identical hemisection lesion and were then
intraspinally infused via
an osmotic pump for the same time period with whole guinea-pig serum
complement (33% v/v}
alone.
Animals were then allowed to recover for 28 days before Fluorogold was
injected into the rostral
lumbar, lcm (i.e. 3 spinal segments) caudal to the lesion site, as described
in Example I. After a
further 7 days survival, each animal was killed, and the brain and spinal cord
were removed for
CA 02253078 1999-O1-28
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examination and analysis as described in Example I.
The extent of the hemisection lesion was confirmed histologically at the end
of both the 5-week
treatment and the recovery period. All surgical procedures and subsequent
animal care protocols
were in accordance with Canadian and University of British Columbia Animal
Care Committee
guidelines.
Results:
The extent of the hemisection lesion was assessed in every animal. In all
animals the region of the
vestibulospinal tract was severed. The right side white matter tracts always
remained intact and
undamaged while the gray matter of the contralateral side usually remained
undamaged.
Comparing "blind" counts of the number of labeled neurons within each LVe
(Fig. 6), the data
indicated that in the 1 month chronically injured animals, 31.5% ~ 5% (n=3) of
the injured lateral
vestibulospinal neurons had regenerated a sufficient distance into the caudal
lumbar cord to
incorporate andretrogradely transportthe Fluorogold. In contrast, control
treated animals, receiving
serum complement alone, did not exhibit a significant amount of LVe labeling:
3.6% ~ 2.7%, (n=2).
Of those animals in which treatment was delayed for 2 months before treatment
commenced, 26.8%
~ 13% (n=3) of the injured lateral vestibulospinal neurons had regenerated a
sufficient distance into
the caudal lumbar cord to incorporate and retrogradely transport the
Fluorogold. In contrast, control
treated animals, receiving serum complement alone, did not exhibit a
significant amount of LVe
labeling: 5.4% ~ 1.8%, (n=2). These results indicate that the compositions of
the present invention
are useful for promoting regrowth, repair, and regeneration of chronically
injured neurons in the
CN5 of a mammalian subject.
EXAMPLE IV
Surgical Spinal Transection and Transient Immunologicc~l Myelin Disruption
Ten to 12 week old adult female rats (Sprague-Dawley), approximately 200g in
weight, were
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anaesthetized with Ketamine/Xylazine (60mg/kg, 7.Smg/kg respectively). After a
limited
laminectomy at T10, a left-side spinal cord hemisection lesion was made with
micro-scissors and
the extent of the lesion was then confirmed by passing a sharp scalpel through
the lesion site (Fig.
7). Immediately after the lesion, an intraspinal cannula was implanted at T11
(n=22 total) and
connected to an Alzet osmotic pump (14 day) to subsequently deliver a
continuous intraspinal
infusion (@ O.Sp.I/hr) of serum complement (GIBCO BRL, #1'9195-015, 33% v/v)
along with a
complement-fixing IgG antibody to galactocerebroside (either our own
polyclonal antibody or
Chernicon Intl. Ltd., #AB142, 25% v/v). Cannulae were held in place by means
of dental acrylic
applied to the vertebral bone. Muscle layers were then sutured over the dental
acrylic, and the
superficial tissue and skin closed. The extent of the hemisection lesion was
always confirmed
histologically at the end of the 5-week treatment and recovery period.
All control animals received an identical hemisection lesion and were then
intraspinally infused via
an osmotic pump, for the same time period, with either vehicle alone (0.1 M
phosphate buffered
saline, PBS, n=5), antibody alone (25% v/v, n=2), or serum complement alone
(33% v/v, n=6). All
surgical procedures and subsequent animal care protocols were in accordance
with Canadian and
UBC Animal Care Committee guidelines.
Electron Microscopy:
Tissue for ultrastructural analysis was obtained from 10-12 week old adult
female Sprague-Dawley
rats sacrificed 7 days after infusion of serum complement along with a
complement-fixing IgG
antibody to GaIC (see above for details) via an osmotic pump. Animals were
lethally anaesthetised
with Ketamine/Xylazine (120mg/kg, l5mg/kg respectively), then perfused
intracardially with 200
ml of O.1M PBS (pH 7.4) followed by 100 ml of 4% glutaraldyhyde in O.IM PB,
(pH 7.3) and
subsequently postfixed overnight in the same fixative. The infusion site and
surrounding cord was
cut into lmm transverse blocks and processed to preserve rostral-caudal
sequence. Blocks were
washed in O.1M sodium cacodylate buffer (24 hours), post fixed in 2% Os04,
dehydrated through
ascending alcohols and embedded in Spurns' resin according to standard
protocols. Tissue blocks
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from experimental and untreated-control animals were processed in parallel.
Thin sections (1 pm)
were cut from each block, stained with alkaline Toluidine Blue and examined
under a light
microscope. For electron microscopic examination blocks were trimmed and
sections cut at 80
100nm, mounted on copper grids, stained with uranyl acetate and lead citrate
and viewed under a
Ziess EM lOC electron microscope (at 80kV).
Retrograde Neuronal Labeling:
Sidle label Studies-
Twenty-eight days after the hemisection lesion and consequently 14 days after
completion of the
intraspinal infusion of the immunological reagents, each adult rat was
anaesthetized with
Ketamine/Xylazine (60mg/kg, 7.Smg/kg respectively). Fluorogold (FG,100-1 SOnl
total volume, 5%
w/v in sterile dH20; Fluorochrome Inc. Englewood, CO, USA) was injected (50-
75n1) bilaterally
into the middle of the spinal tissue at the L 1 level, approximately 1 cm
caudal to the lesion site (Fig.
7).
Double Label Studies-
At the time of lesion, the hemisection site was packed with gel-foam soaked
with 12% (w/v in sterile
dH20) rhodamine-conj ugated dextran amine (RDA,1 O,OOOMW FluoroRuby, Molecular
Probes) for
30 minutes. The gel-foam was then removed and the remaining surgical
procedures were completed
(as outlined above). After 28 days survival, all animals were
anaesthetizedwith Ketamine/Xylazine
(60mg/kg, 7.Smg/kg respectively) and FG (100-150n1 total volume, 5% w/v in
sterile dHzO) was
injected (50-75n1) bilaterally into the spinal parenchyma at the L1 level of
the cord (n=6).
Analysis o, f'Regeneration:
Seven days following the injection of the FG tracer into the lumbar cord,
animals were lethally
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anaesthetised with Ketamine/Xylazine (120mg/kg, l5mg/kg respectively) and then
perfused
intracardially with 200 ml of O.1M PBS (pH 7.4) followed by 100 ml of 4%
paraformaldehyde in
0.1 M PBS, (pH 7.3). The brain and spinal cord were then removed and postfixed
overnight in the
same fixative. Subsequently, each brain and spinal cord was cleared of
fixative and cryo-preserved
S by placing the tissue in a series of sucrose solutions (15% followed by 21
%). Coronal or parasagital
sections were cut at 25~.m thickness on a cryostat. The brainstem and spinal
cord tissue sections
were examined under a Zeiss Axioskop with a 100 W mercury bulb
(excitation/emission wavelength
at: FG, 365/420nm; RDA, 546/590nm; fluorescein, 490/5lSnm)
The two brainstem-spinal nuclei used to assess the axonal regenerative
abilities of experimentally
treated animals were the Red Nucleus (RN) (origin is contralateral to the
hemisection) and the
Lateral Vestibular (LVe) Nucleus (origin is ipsilateral to the hemisection).
Spinal-projecting axons
from each RN cross to the opposite side of the midbrain and descend throughout
the spinal cord
within the contralateral dorsolateral funiculus. This contralateral spinal
proj ection pathway is known
to be a completely lateralized tract with the possible exception of 2-5 % of
the axons which may
project to the cord via an ipsilateral route (Waldron and Gwyn 1969; Brown,
1974; Huisman et al.,
1981; Shieh et al., 1983). The LVe tract projects from the dorsolateral
pontine hindbrain,
maintaining an exclusive ipsilateral course throughout the brainstem and the
ventrolateral white
matter of the spinal cord (Zemlan et al., 1979; Shamboul, 1980).
Using a single-blind protocol, the number of retrograde labeled neurons within
the Red Nucleus
(RN) (contralateral to the hemisection) and the Lateral Vestibular (LVe)
Nucleus (ipsilateral to the
hemisection) were counted in every othertissue section (throughout these
brainstemnuclei) to avoid
counting the same neuron twice. Only those cells exhibiting a nucleus, a
neuronal morphology (i. e.
multi-polar in appearance) and specifically labeledwith FG (i.e. not visible
using other fluorescent
filters; see above) extending into the proximal processes, were deemed to be
positively labeled
spinal-projecting neurons. The percentage of regenerating neurons for each
brainstem-spinal
projection was then detelxrlined in comparison to the number of labeled
neurons within the
contralateral (uninjured) control nucleus within the same animal.
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Extent of Spinal Cord Demyelination and Myelin Disruption after Immunological
Treatment
Direct intraspinal infusion over 7 days (@ 0.5u1/hr) of 33% heterologous
(guinea pig) serum
complement along with polyclonal antibodies to GaIC (25%) in PBS resulted in
extensive
demyelination up to 2mm away from the infusion cannula (total rostrocaudal
distance of 4mm or
approximately 1 spinal segment (Fig. 2A). This zone of demyelination was
bounded on either side
by a further 8mm or 2 segments of spinal cord characterized by disrupted
myelin (i.e. myelin that
is extensively de-laminated, having an unraveled appearance, Fig. 2B). As
shown in previous studies
(Keirstead et al., 1992, 1995), control infusions of heterologous serum
complement alone, myelin-
specific antibody alone, or PBS alone resulted in only limited non-specific
damage immediately
centered around the cannula site. There was no surrounding zone of
demyelination or myelin
disruption (Fig. 2C).
The immunological demyelination and disruption of myelin within the
experimentally-treated adult
rat spinal cord was an active process extending throughout the entire cross-
sectional profile of the
cord. Immunological myelin disruption commenced within 1 day and was
associated with an
invasion of macrophages or resident microglia and polymorphonuclear cells
(e.g. leukocytes such
neutrophils, eosinophils and basophils). Many macrophageslmicroglia contained
myelin fragments
and completed their phagocytic activity within 7 days (Fig. 2D). This pattern
of demyelination and
myelin disruption could be maintained for as long as the serum complement and
myelin-specific
antibody were infus ed. Recent evidence suggests that after the immunological
infusion is terminated
remyelination begins within 2 weeks (Keirstead and Blakemore, 1997; Dyer,
Bourque and Steeves
unpublished observations) and the new myelin originates from differentiating
oligodendrocyte
progenitors, although invading Schwann cells and surviving "mature"
oligodendrocytes may also
contribute to remyelination.
Choice of Retrograde Tracer and Its Diffusion Distance from the Injection Site
In this study, the major anatomical evidence for axonal regeneration within
the hemisected and
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immunologically myelin-suppressed spinal cord of adult rats depends on a
comparison between the
number of retrogradely-labeled neurons within a homologous pair of brainstem-
spinal nuclei. For
these comparisons to be valid, the candidate brainstem spinal nuclei must have
highly unilateral
proj ections that are confined to one side of the spinal cord at all levels.
As summarized in Fig. 7A,
a left thoracic hernisection severed the contralaterally-proj ecting
magnocellular neurons of the right
red nucleus (RIB, but left the projections from the left RN undariiaged (as
they project through the
intact right dorsolateral funiculus of the thoracic cord). Likewis e, a left
thoracic hemis ection s evered
the ipsilateral proj ecting neurons of the left lateral vestibulospinal
nucleus (LVe), but left the axons
from the right LVe nucleus undamaged (as they also project through the intact
right side of the
thoracic cord via the ventrolateral white matter).
If a retrograde tracer (single label) is inj ected into the rostral lumbar
cord ( 1 cm caudal to the injury
site), it should be incorporated and transported back to the cell bodies of
origin by both intact axons,
as well as regenerated proj ections. Consequently, it is essential that the
retrograde tracer reliably and
extensively label most, if not all, descending spinal projection neurons. An
equally important
parameter is the tracer must be injected in a controlled and reproducible
manner at a distance
sufficiently caudal to the spinal injury to prevent any direct diffusion of
the tracer to the level of the
hemisection injury. The retrograde label that best satisfied all these
conditions was Fluorogold
(Sahibzada, et al., 1987). Fluorescent dextran amines, such as RDA, require a
recent axonal injury
to facilitate axonal uptake (c.f. Heimer and Zaborszky, 1989), and were
therefore better suited for
use in the double label retrograde-tracing studies (see description below).
In all cases, the Fluorogold label ( 100-I SOnI) was inj ected bilaterally
within the rostral lumbar cord
(1 cm or 2-3 spinal segments caudal to the hemisection injury site, Fig. 7).
We assessed the time
course and degree ofrostrocaudal diffusion of Fluorogoldwithin the lumbar and
thoracic spinal cord
of normally myelinated (control) animals and experimentally treated rats (i.e.
under demyelinated
and myelin disrupted conditions). Random 25 ~,m sections of experimental and
control-treated spinal
cords (extending from L2 to T8) were examined under a fluorescent microscope
using the highest
intensity setting of the 100W mercury lamp. Spinal tissue was examined for the
extent of Fluorgold
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diffusion at varying survival intervals after injection, including: l2hr
(n=6), 24hr (n=6), 3d (n=6),
5d (n=6) and 7d (n=22). The maximum rostral diffusion distance observed was 4-
6 mm (or 1- 1.5
spinal segments) and occurred within a time span of 24h. The degree of
Fluorogold diffusion within
the lumbar cord did not change over the subsequent time points examined (Fig.
7).
Evidence, f'or Braintem-spinal Axonal Regeneration by Retrograde Neuronal
Labeling
Inbrief, 28 animals;12 experimental (9 retrogradely single-labeled, 3 double-
labeled) and 16 control
(13 retrogradely single-labeled, 3 double-labeled) were subjected to a left-
side lateral hemisection
of the T10 spinal cord. Immediately after hemisection, an infusion cannula
(connected to a 14d
osmotic pump) was inserted directly into the spinal cord 4-5 mm (1 spinal
segment) caudal to the
injury site. The osmotic pump contained one of a number of 3 different control
solutions or the
experimental treatment (i.e. PBS vehicle alone, serum complement alone, anti-
galactocerebroside
antibody alone, or serum complement with anti-GaIC antibodies, respectively).
Animals were then
allowed to recover for 28 days before the Fluorogold was inj ected into the
rostral lumbar, l cm (i. e.
at least 2 spinal segments) caudal to the lesion site. After a further 7 days
survival, each animal was
killed and the brain and spinal cord were removed for examination and analysis
(see above for
criteria used to determine a labeled neuron).
The extent of the hemisection lesion was assessed in every animal. In all but
one experimental and
one control-treated animal, the left thoracic spinal cordwas hemisected (Fig.
7). Most importantly,
the regions of the rubrospinal tract (dorsolateral funiculus) and the lateral
vestibulospinal tract
{ventrolateral funiculus) were severed. The right side white mattertracts were
always remainedintact
and undamaged and usually the gray matter of the uninjured side was also
undamaged.
As discussed above, the 2 pairs of brainstem-spinal nuclei examined for
evidence of retrograde
labeling (after spinal cord hemisection and immunological myelin suppression)
were the RN and the
LVe. These brainstem-spinal nuclei were chosen for their unilateral projection
patterns within the
thoracic and lumbar cord, enabling comparisons to be made between the
retrograde-labeling within
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an injured nucleus and the uninjured contralateral homologue. Comparing
"blind" counts of the
number of labeled neurons within each RN (Fig. 3A-B), the data indicated that
31.8% ~ 4.7% {n=8,
range 10-50%) of the injured magnocellular RN neurons had regenerated a
sufficient distance into
the caudal lumbar cord to incorporate and retrogradely transport the
Fluorogold (Fig. 9). In contrast,
control treated animals, receiving either the PBS vehicle alone, GaIC antibody
alone, or serum
complement alone did not exhibit a significant amount of RN labeling; 1.49% ~
0.23 %, (Fig. 3 C-D;
Fig. 9, n=13, range 0-3). The labeling of some neurons within the injured
right RN nucleus may
represent the small number of RN that do not project to the opposite side of
the midbrain and
descend within the ipsilateral (uninjured) cord (Shieh et al., 1983). No
retrograde-labeling of cells
was observed within the parvocellular region of the R~.'~t.
Retrograde-1'abeling of regenerating LVe neurons was also observed, but only
after experimental
demyelination and disruption of spinal cord myelin (Fig. 8). In 8 experimental
animals, the mean
percentage of regenerating LVe labeling, in comparison to the uninjured
contralateral control
nucleus, was 41.8% ~ 3.1 % (n=8, range 33-49%). In control-treated animals
(see above) the percent
LVe labeling was 2.24% ~ 0.55% (Fig. 5, n=13, range 0-6).
Double retrograde labeling of the injured and myelin-suppressed rubrospinal
tract was also
qualitatively assessed (Fig. 9E and F). Large numbers of RDA-positive (first
label) magnocellular
RN neurons were observed after direct labeling of the lesion site at the time
of hemisection injury
to the thoracic spinal cord. After intraspinal myelin-suppression and
subsequent injection of
Fluorogold caudal to the lesion site (see above for details) a small
overlapping population of FG-
positive neurons was observed (i.e. some neurons were labeled with both RDA
and FG). Cells
labeled exclusively by the first or the second tracer were also present in
every brainstem analysed.
Examinations for any functional or behavioral differences during the 5 week
recovery period after
experimental treatment indicated no notable differences in locomotor patterns
between injured
animals and uninjured control animals (i. e. all animals walked and all
animals were comparable with
respect to basic reflex functions). These occurred regardless of the treatment
infused intraspinally
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after ahemisection injury (e.g. PBS alone, GaIC antibody alone, serum
complement alone, or serum
complement plus GaIC antibody). Thus, subtle differences were very difficult
to observe or quantify
and 'gross' motor patterns were essentially the same.
As compared with prior art using spinal transection (Keirstead et al., 1992,
1995), the present
invention is demonstrated using a hemisection model so that each animal could
serve as its own
internal control (i.e. axonal regeneration from injured brainstem-spinal proj
ections could be readily
compared to the uninjured contralateral homologue). In addition, the present
invention strove to
minimize the degree of cyst cavity formation that often occurs with larger
spinal lesions, as well as
the amount of animal discomfort over the relatively long recovery periods
required.
The present invention also illustrates that the demyelination produced by the
intraspinal infusion of
serum complement and a myelin-specific antibody (e.g. GaIC) produced a rapid
and active
demyelination over 1-2 segments of the cord with myelin disruption extending a
further 2 segments,
either side of the infusion site. Resident microglia and/or invading
macrophages were observed to
contain myelin debris. The immunological suppression of spinal cord myelin
surrounding the
thoracic hemisection facilitated significant axonal regeneration by 2
unilaterally projecting
brainstem-spinal pathways, the rubrospinal and lateral vestibulospinal (RN and
LVe, respectively)
tracts. Control treated animals (hemisection injury plus local intraspinal
infusion of PBS alone, GaIC
antibody alone, or serum complement alone) showed little or no retrograde
labeling within the
injured RN or LVe.
