Spinal cord injury usually results in permanent paralysis because of lack of regrowth of damaged neurons. Here we demonstrate that
adult mice lacking EphA4 (?/?), a molecule essential for correct guidance of spinal cord axons during development, exhibit axonal
walk on and climb a grid, and the ability to grasp with the affected hindpaw within 1–3 months of injury. EphA4 expression was
EphA4?/? spinal cords. EphA4?/? astrocytes failed to respond to the inflammatory cytokines, interferon-? or leukemia inhibitory
factor, in vitro. Neurons grown on wild-type astrocytes extended shorter neurites than on EphA4?/? astrocytes, but longer neurites
Injury to the CNS usually results in very limited, if any, regener-
of function. Although some CNS neurons appear to lose the in-
trinsic ability to regenerate neurites postnatally (Goldberg et al.,
2002), many others, such as corticospinal tract (CST) neurons,
appear able to regenerate, but are inhibited from doing so by the
environment of the injury site. Major impediments to CNS re-
generation are the presence of myelin inhibitors and astrocytic
Myelin proteins that inhibit axonal regeneration include
Nogo (Caroni and Schwab, 1988; Schnell and Schwab, 1990,
1993; Bandtlow and Schwab, 2000), myelin-associated glycopro-
tein (MAG) (McKerracher et al., 1994; Mukhopadhyay et al.,
1994; DeBellard et al., 1996; Schafer et al., 1996), and
oligodendrocyte-myelin glycoprotein (OMgp) (Wang et al.,
2002b). Each of these proteins appears to inhibit regeneration by
the same mechanism. Nogo, MAG, and OMgp bind to the Nogo
receptor (Domeniconi et al., 2002; Liu et al., 2002; Wang et al.,
2002a), which interacts with the p75 neurotrophin receptor on
cord injury results in partial regeneration and improves func-
tional recovery, at best only a small percentage of axons regrow
(Bregman et al., 1995; GrandPre et al., 2002; Kim et al., 2003;
iments to regeneration still need to be overcome before robust
regeneration can occur.
The other major barrier to axonal regeneration is glial scar-
ring, the main component of which is astrocytic gliosis (Stichel
and Muller, 1998). Normally quiescent astrocytes in the adult
show a vigorous response to injury. They become hypertrophic,
proliferative, upregulate expression of glial fibrillary acidic pro-
tein (GFAP), and form a dense network of glial processes both at
and extending from the lesion site. Accompanying these mor-
phological changes are a range of physiological changes, includ-
ing secretion of a variety of cytokines and production of cell
ucts are inhibitory to regeneration, such as chondroitin sulfate
proteoglycan (CSPG) (McKeon et al., 1991) and collagen IV
(Stichel et al., 1999), and if their deposition is inhibited, axonal
regeneration is promoted (Stichel et al., 1999).
Other factors that may be implicated in inhibition of axonal
regeneration, but which have received little attention in this re-
This work was supported by The BHP Community Trust, the National Health and Medical Research Council of
of Melbourne, Melbourne, Victoria 3010, Australia, E-mail: email@example.com; or Perry Bartlett, The
Queensland Brain Institute, The University of Queensland, Brisbane, Queensland 4072, Australia. E-mail:
10064 • TheJournalofNeuroscience,November10,2004 • 24(45):10064–10073
during development. One such family of molecules is the Eph
receptor tyrosine kinase family, which, together with their li-
neural structures, including the corticospinal tract and anterior
commissure (Henkemeyer et al., 1996; Dottori et al., 1998;
Both the Eph receptors and the Ephrins are membrane-bound,
and cell–cell contact is required for signaling. In addition, some
of the Ephrins are able to transduce signals, so reverse signaling
can also occur (for review, see Kullander and Klein, 2002). Be-
cause Eph–Ephrin signaling appears to regulate axon guidance
through contact repulsion, inducing the collapse of neuronal
growth cones (Wahl et al., 2000; Kullander et al., 2001b), and
members of this family are upregulated in the adult after neural
injury (Moreno-Flores and Wandosell, 1999; Rodger et al., 2001;
Willson et al., 2002), the aberrant expression or absence of Eph
receptors could prove pivotal in determining the outcome of
injury in the adult CNS. This possibility was investigated in this
study by comparing neural regeneration after spinal cord he-
misection in wild-type and EphA4?/? mice.
as previously described (Coonan et al., 2001), were used in this study.
Experiments were approved by the Royal Melbourne Hospital and the
University of Melbourne Animal Ethics Committees in accordance with
the Australian Code of Practice for the Care and Use of Animals for
Spinal cord lesions
Mice were anesthetized with a mixture of ketamine and xylazine (100
mg/kg and 16 mg/kg, respectively). The spinal cord was exposed via a
laminectomy, in which two or three vertebral arches were removed at
levels T12–L1, corresponding to the level of the lumbar enlargement. A
spinal left hemisection at T12 was performed using a fine corneal blade
(cut twice in the same place to ensure complete section), and the overly-
ing muscle and skin were then sutured. A total of 71 wild-type and 83
of which 4% of wild-type mice and 12% of EphA4?/? mice died. Mice
of both genotypes that recovered from surgery showed good survival,
with no spontaneous deaths recorded, however three wild-type and four
EphA4?/? mice were killed because of infection, and two EphA4?/?
mice because of full left and right hindlimb paralysis. The success rate of
the hemisection model, as assessed by total paralysis of the left hindlimb
at 24 hr after surgery, was 96% for wild-type mice and 95% for
EphA4?/? mice. Mice that showed only partial paralysis at this time
point, indicative of incomplete lesion, were excluded from analysis. Spi-
nal cords of animals that were included in the analysis were also exam-
ined histochemically to ensure complete hemisection.
Five weeks after spinal cord lesion, tetramethylrhodamine dextran
(“Fluoro-Ruby”; molecular weight, 10,000 kDa) was injected as two 0.2
cervical enlargement, ipsilateral to the lesion, via a glass pipette attached
ways ipsilateral to the injection site (estimated to be 25%) but none
contralateral to the injection site (supplemental material, available at
The number of labeled axons running rostrally to caudally through a
250-?m-wide box placed in the white matter, at the border of the gray
matter, of all intact serial sections (8–10 per spinal cord) was counted at
400? by focusing up and down through the sections at 2.5 mm and
50–100 ?m proximal to the lesion site and 50–100 ?m, 1 and 5 mm
distal to the hemisection. Axons at the lateral edge of the white matter
and near the pial surface were therefore excluded from these counts, as
available. The lumbar site of the lesion precluded analysis of regrowth
longer than 5 mm because of termination of the fibers and commence-
Student’s t test.
The lumbar spinal cord below the lesion was exposed via a lower lumbar
GmBH, Gross-Umstadt, Germany], which labels the neuronal soma of
axons damaged by the injection, was injected into two sites of the spinal
cord ipsilateral to the lesion site with a glass micropipette attached to a
Hamilton syringe. After a 5 d survival period, the animals were perfused
with 4% paraformaldehyde in PBS. The brain and spinal cord were re-
moved and postfixed for 24 hr in 20% sucrose in fixative before being
serially sectioned at 50 ?m on a freezing microtome in the coronal–
transverse plane. Injections were considered successful by confirmation
of a unilateral injection site in the operated spinal cord longitudinal
sections. Qualitative and quantitative comparisons of labeled neurons
were made by mapping the locations of labeled cells in every fourth
section of a series using a computer-linked digitizing system (MD3 mi-
croscope digitizer and MD-plot software; Minnesota Datametrics
The hopping gait of the EphA4?/? mice precluded use of commonly
used behavioral assessments, such as the Basso, Beattie, Bresnahan scale.
The behavioral assessments chosen, and especially the grip strength test
and climbing on an angled grid, allow direct comparison of EphA4?/?
mice and wild-type mice, independent of hopping or reciprocal gait.
Stride length. Before and after hemisection, mice were footprinted by
painting their hindpaws with nontoxic ink and placing them in a tunnel
on blotting paper (wild type, n ? 7; EphA4?/?, n ? 9 mice). Stride
length was determined by measurement of multiple successive steps and
results were expressed as a percentage of each animal’s own baseline
mice to walk on a horizontal or angled (75° from horizontal) wire grid
their locomotion (Ma et al., 2001). The mice were tested 1, 2, and 3
months after the spinal cord hemisection and compared with nonle-
sioned mice from each group. On the horizontal grid each mouse was
allowed to walk freely around the grid for 5 min, during which a mini-
mum 2 min of walking time was required. On the angled grid, each
mouse was measured over 10 climbs. If the left hindpaw protruded en-
tirely through the grid, with all toes and heel extended below the wire
the left hindlimb was also counted. The results were expressed as the
percentage of accurate footsteps and significance was analyzed using the
Student’s t test.
Sensory and motor ability-grasp test
The ability of hemisected and nonlesioned wild-type (n ? 5) and
left hindlimb. The hindlimbs of the mice were lifted 2 cm from the table
top while allowing the forelimbs to remain in contact with the table.
Grasp ability was tested by lightly touching the left footpad with the rod
and assessing the response based on a scale from 0 to 4: 0, no movement
of paw and toes; 1, partial movement of the paw, no movement of the
not maintained with gentle rod movement; 4, strong grasp, maintained
with gentle rod movement. Mice were graded at least three times in
parallel with the grid tests described. Results were expressed as the
mean ? SEM of the score of each group, and significance was analyzed
using the Student’s t test.
Goldshmitetal.•AxonalRegenerationinEphA4-DeficientMice J.Neurosci.,November10,2004 • 24(45):10064–10073 • 10065
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