Combined delivery of Nogo-A antibody, neurotrophin-3 and the NMDA-NR2d subunit establishes a functional ‘detour’ in the hemisected spinal cord

Brain Research Institute, University and ETH of Zurich, Zurich, Switzerland.
European Journal of Neuroscience (Impact Factor: 3.18). 10/2011; 34(8):1256-67. DOI: 10.1111/j.1460-9568.2011.07862.x
Source: PubMed
ABSTRACT
To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by descending fibers after an intervening lateral thoracic (T10) hemisection (Hx), we treated adult rats with the following agents: (i) anti-Nogo-A antibodies to neutralize the growth-inhibitor Nogo-A; (ii) neurotrophin-3 (NT-3) via engineered fibroblasts to promote neuron survival and plasticity; and (iii) the NMDA-receptor 2d (NR2d) subunit via an HSV-1 amplicon vector to elevate NMDA receptor function by reversing the Mg(2+) block, thereby enhancing synaptic plasticity and promoting the effects of NT-3. Synaptic responses evoked by stimulation of the ventrolateral funiculus ipsilateral and rostral to the Hx were recorded intracellularly from ipsilateral lumbar motoneurons. In uninjured adult rats short-latency (1.7-ms) monosynaptic responses were observed. After Hx these monosynaptic responses were abolished. In the Nogo-Ab + NT-3 + NR2d group, long-latency (approximately 10 ms), probably polysynaptic, responses were recorded and these were not abolished by re-transection of the spinal cord through the Hx area. This suggests that these novel responses resulted from new connections established around the Hx. Anterograde anatomical tracing from the cervical grey matter ipsilateral to the Hx revealed increased numbers of axons re-crossing the midline below the lesion in the Nogo-Ab + NT-3 + NR2d group. The combined treatment resulted in slightly better motor function in the absence of adverse effects (e.g. pain). Together, these results suggest that the combination treatment with Nogo-Ab + NT-3 + NR2d can produce a functional 'detour' around the lesion in a laterally hemisected spinal cord. This novel combination treatment may help to improve function of the damaged spinal cord.

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NEUROSYSTEMS
Combined delivery of Nogo-A antibody,
neurotrophin-3 and the NMDA-NR2d subunit
establishes a functional ‘detour’ in the hemisected
spinal cord
Lisa Schnell,
1
Arsen S. Hunanyan,
2,3
William J. Bowers,
4
Philip J. Horner,
5
Howard J. Federoff,
6
Miriam Gullo,
1
Martin E. Schwab,
1
Lorne M. Mendell
3
and Victor L. Arvanian
2,3
1
Brain Research Institute, University and ETH of Zurich, Zurich, Switzerland
2
Northport Veterans Affairs Medical Center, 79 Middleville Road, Bld. 62, Northport, NY 11768, USA
3
Department of Neurobiology and Behavior SUNY at Stony Brook, Stony Brook, NY, USA
4
Center for Neural Development and Disease, Department of Neurology, University of Rochester Medical Center, Rochester, NY,
USA
5
Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA, USA
6
Department of Neurology, Georgetown University Medical Center, Washington, DC, USA
Keywords: motor neuron, spinal cord injury, spinal hemisection, sprouting, synaptic response, ventrolateral funiculus
Abstract
To encourage re-establishment of functional innervation of ipsilateral lumbar motoneurons by descending fibers after an intervening
lateral thoracic (T10) hemisection (Hx), we treated adult rats with the following agents: (i) anti-Nogo-A antibodies to neutralize the
growth-inhibitor Nogo-A; (ii) neurotrophin-3 (NT-3) via engineered fibroblasts to promote neuron survival and plasticity; and (iii) the
NMDA-receptor 2d (NR2d) subunit via an HSV-1 amplicon vector to elevate NMDA receptor function by reversing the Mg
2+
block,
thereby enhancing synaptic plasticity and promoting the effects of NT-3. Synaptic responses evoked by stimulation of the
ventrolateral funiculus ipsilateral and rostral to the Hx were recorded intracellularly from ipsilateral lumbar motoneurons. In uninjured
adult rats short-latency (1.7-ms) monosynaptic responses were observed. After Hx these monosynaptic responses were abolished.
In the Nogo-Ab + NT-3 + NR2d group, long-latency (approximately 10 ms), probably polysynaptic, responses were recorded and
these were not abolished by re-transection of the spinal cord through the Hx area. This suggests that these novel responses resulted
from new connections established around the Hx. Anterograde anatomical tracing from the cervical grey matter ipsilateral to the Hx
revealed increased numbers of axons re-crossing the midline below the lesion in the Nogo-Ab + NT-3 + NR2d group. The combined
treatment resulted in slightly better motor function in the absence of adverse effects (e.g. pain). Together, these results suggest that
the combination treatment with Nogo-Ab + NT-3 + NR2d can produce a functional ‘detour’ around the lesion in a laterally hemisected
spinal cord. This novel combination treatment may help to improve function of the damaged spinal cord.
Introduction
Several obstacles are known to prevent recovery of spinal cord
function after even less-than-complete transection of the adult
mammalian spinal cord. These include neurite growth-inhibiting
constituents of myelin, scar-associated inhibitory factors, and the lack
of sufficient neurotrophic support (Snow et al., 1990; Schnell et al.,
1994; Tuszynski & Gage, 1995; Fawcett, 2009). Whereas treatments
targeting these processes individually have proven somewhat effica-
cious in facilitating axon regeneration and functional recovery after
spinal cord injury, the effects are generally small. Therefore, it is
conceivable that developing combination treatments, e.g. neutralizing
the myelin-related inhibitory molecules, combined with growth- and
plasticity-enhancing factors, could be an important step in improving
repair of the damaged spinal cord. This is addressed in the present
study in an attempt to promote the re-establishment of a functional
detour around a hemisection (Hx).
The best-studied myelin-associated inhibitory molecule is Nogo-
A. Acute inactivation by function-blocking antibodies or Nogo
receptor antagonists, or blockade of the downstream signals RhoA
or ROCK, enhances both regeneration of lesioned fibers tracts and
compensatory sprouting of the spared corticospinal tract and other
fibers (Schwab, 2004; Yiu & He, 2006; Gonzenbach & Schwab,
2008). The effects of antibody-mediated neutralization of Nogo-A
on ventrolateral funiculus (VLF) axons have not been studied to
date.
Correspondence: Dr Victor L. Arvanian,
2
Northport Veterans Affairs Medical Center,
as above.
E-mail: vlarvanian@notes.cc.sunysb.edu
Re-use of this article is permitted in accordance with the Terms and Conditions set out at
http://wileyonlinelibrary.com/onli neopen#OnlineOpen_Terms
Received 24 May 2011, revised 5 August 2011, accepted 8 August 2011
European Journal of Neuroscience, Vol. 34, pp. 1256–1267, 2011 doi:10.1111/j.1460-9568.2011.07862.x
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience
Page 1
The neurotrophic factor neurotrophin-3 (NT-3) has been shown to
enhance regenerative sprouting of lesioned corticospinal, dorsal root and
reticulospinal fibers in the injured spinal cord (Schnell et al., 1994;
Tuszynski & Gage, 1995; Bregman et al., 2002; Alto et al., 2009).
Earlier electrophysiological studies have revealed that synaptic con-
nections from the VLF to individual motoneurons in uninjured young
(postnatal days 2–10) rats could be strengthened by administration of
NT-3 (Arvanian et al., 2003). However, this action of NT-3 required
reversing the developmental loss of NMDA receptor activity due to
Mg
2+
block by enhancing expression of the NMDA-receptor 2d (NR2d)
regulatory subunit in motoneurons using Herpes simplex virus (HSV-1)
amplicon-mediated delivery of NR2d (Arvanian et al., 2004).
In the current study we used a unilateral spinal cord Hx (corresponds
to Brown–Sequard lesion in humans) in adult rats as a model for partial
injuries because there is a clear lesion of one entire side of the cord with
intact fibers remaining on the contralateral side. We examined whether
intrathecal administration of a function-blocking anti-Nogo-A mono-
clonal antibody (Nogo-Ab) combined with long-term application of
NT-3 (via fibroblasts) and transient facilitation of NMDA-receptor
function (HSV-1-mediated viral delivery of the NMDA-nr2d subunit)
could enhance re-establishment of functional synaptic connections
from the transected lateral funiculi around the Hx lesion to ipsilateral
lumbar motoneurons. Under these conditions novel responses were
observed that differed from those in the uninjured cord in exhibiting
markedly longer latency and higher electrical threshold. These positive
electrophysiological findings were supplemented by anatomical studies
showing long propriospinal axons crossing to the opposite side of the
cord after the combination treatment. These changes were accompanied
by mild improvement in recovery of motor function.
Portions of these results have been published in abstract form
(Arvanian et al., 2006a; Schnell et al., 2007).
Materials and methods
Animals and experimental design
These studies were performed in accordance with protocols approved
by the Institutional Animal Care and Use Committees at SUNY SB,
University of Zurich, and Northport VAMC, USA. The design of
experiments is presented in Fig. 1. A total of 126 adult female
Sprague–Dawley rats (Charles River Laboratories, Wilmington, MA,
USA; approximately 200 g) were housed in groups of four to six
animals in standardized cages on a 12-h light–dark cycle with food
and water ad libitum. Because of the large number of experimental
groups (nine groups for the electrophysiological study, six groups for
the behavioral study and five groups for the fiber tracing studies; see
Results for details), the results were obtained in two separate
experimental studies performed by the same investigators at two
different times using the same suppliers for rats and the same
treatment agents. Generally rats were pre-trained for 4 weeks to obtain
baseline values in behavioral tests and then randomly divided into
experimental groups according to the treatment. The rats were coded
with random numbers and rats from the different groups were mixed in
the cages. The experimenters were blind with regard to treatment
throughout all phases of the experiment. Subsequent to surgery and
treatment and in most cases behavioral evaluation, rats were used for
electrophysiological recording or anatomical evaluation. However, not
all treatment groups could be evaluated using all analyses (see Results).
A general time line for these experiments is displayed in Fig. 1A.
Exclusion of animals
Seven animals were excluded from the study because evaluation of the
lesion post hoc (see Fig. 1B) revealed that it was too small or too big
in three animals we detected a portion of spared ipsilateral dorsal
white matter, while in four animals overhemisection extended beyond
the midline for > 10% of spared area of hemicord. Other rats were
eliminated either for general health problems, especially autophagia,
or because they expired during the in vivo electrophysiological
recordings (n = 16).
Surgical procedures and delivery of agents in combination
treatment
In this study we used a lateral hemisection spinal cord injury model.
This model allows electrophysiological evaluation of the the possi-
bility of establishing a functional detour around the lesion. Moreover,
unilateral injections of the anterograde tracer permit visualization of
midline-crossing fibers rostral to the lesion and recrossing fibers
caudal to the lesion (see below). Finally, transmission deficits in the
chronically hemisected spinal cord coincide with clear behavioral
impairments in challenging motor tasks, including irregular ladder and
narrowing beam, although rats exhibit a robust recovery of their ability
to walk in the open field (Arvanian et al., 2009).
After pre-training on the behavioral tasks for 4 weeks, rats were
deeply anesthetized and the lateral Hx was carried out at T10 as
previously described (Arvanian et al., 2009; Hunanyan et al., 2010).
Briefly, a dorsal laminectomy was performed to expose segment T10
of the spinal cord. A 1-mm slit was made in the dura at the midline at
T10. A complete Hx of the left hemicord at T10 was carried out with
the tip of an iridectomy scissor blade, as follows: first, a 32-gauge
needle was inserted through the midline from dorsal to ventral; then
one tip of the scissors was pushed along the needle through the entire
thickness of the spinal cord and the left dorsal and ventral columns
were cut; finally one tip of the scissors was guided along the lateral
surface of the spinal cord (down to the midline) and any uncut tissue in
the left lateral and ventral columns was cut.
A fine intrathecal catheter (32-gauge) was inserted from lumbar
level L2 L3 and pushed up to T10 to deliver the Nogo-Ab from an
osmotic minipump (Alzet
ª
2ML2; 5 lL h, 3.1 lg lL) for 2 weeks.
The tubing connecting the catheter with the minipump was sutured to
the back muscles for stabilization. Antibody treatment was started
immediately after the lesion by rinsing the wound with approximately
1 lL of the corresponding antibody. We used Nogo-Ab 11C7
(3.1 mg mL) and monoclonal mouse IgG directed against wheat
auxin as control antibody. Multiple studies have revealed the excellent
distribution and penetration of anti-Nogo antibodies infused intrathe-
cally throughout the spinal cord of adult rats and monkeys (Weinmann
et al., 2006). Function-blocking Nogo-Abs are also currently being
applied intrathecally to spinal cord-injured patients in an on-going
clinical trial (ATI-355 trial; Novartis, Basel, Switzerland).
Rat fibroblasts genetically modified to produce NT-3
(0.4 · 10
6
cells lL) or b-galactosidase (control) were suspended in
0.6% glucose-PBS and a cell volume of 2 lL, inserted into collagen
plugs (Kawaja & Gage, 1992; McTigue et al., 1998; Arvanian et al.,
2003) and placed on top of the lesion. These earlier studies
demonstrate that this procedure results in biological effects specific
to the released neurotrophin and elevation of neurotrophin levels in the
spinal cord days to weeks later.
HSV-1 amplicons encoding NR2d or control b-galactosidase were
administered in two injections of 1 lL each (approximately 10
4
viral
particles lL) into the left and right ventral horn at T11 caudal to the
injury region. We used a glass capillary with a tip of approximately
60 lm (calibrated for a volume of 1 lL) inserted into each side of the
cord dorsum 1 mm lateral to the midline. HSV-1 amplicons have a
Functional ‘detour’ in the hemisected spinal cord 1257
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 1256–1267
Page 2
transgene capacity sufficient to carry the NR-2d cDNA and the
co-expressed green-fluorescent protein (GFP) reporter gene (Arvanian
et al., 2004). Previous electrophysiological studies have revealed that
delivery of HSV-1 amplicon-based vectors themselves does not alter
synaptic function in hippocampus (Dumas et al., 1999) or spinal cord
(Arvanian et al., 2004, 2006b), thus supporting the use of the HSV-1
amplicon system as a safe method for delivering selected genes to the
central nervous system.
To confirm the ability of HSV-1 to infect cells at a distance from the
infection site, we measured GFP expression in identified motoneurons
at different segmental levels. Because HSV-1 gene expression is
highest 24–48 h after administration and decays to undetectable levels
by 2 weeks (Bowers et al., 2000), we could not use the same rats that
were studied electrophysiologically or anatomically 7–12 weeks after
HSV-1 administration. Therefore we used a separate control group of
three hemisected rats treated in an identical manner (Fig. 1C). The
A
B
C
Fig. 1. (A) Time schedule of experiments. Note that behavioral testing began before osmotic minipump removal. (B) Representative camera lucida drawing of a
cross-section from a representative cord in treatment groups used for behavioral testing. The maximal lesion area of each animal was reconstructed from at least 20
spinal cord cross-sections per animal, measured with the Image J program, and expressed as percentage of the area of the T10 segment of the intact cord.
Mean ± SEM value of lesion size as a percentage of an intact cord is shown for each treatment group. Arrows point to the midline. (C) Expression of GFP in
motoneurons at T12 and L5 7 days after GFP-expressing HSV-1 amplicons (HSV-NR2d-GFP) were injected intraspinally at the time of a T10 lateral hemisection:
GFP (as HSV-NR2d-GFP marker; red) and Peripherin (as motoneuron marker; green). Note infection of motoneurons bilaterally at T12 and L5. Further details in
text. Scale bars, 50 lm.
1258 L. Schnell et al.
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 1256–1267
Page 3
degree of motoneuron infection was determined as the percentace of
the total number of peripherin (green)-labeled cells also labeled with
GFP (red), i.e. that were yellow. The immunolabeling procedure and
analyses have previously been described (Arvanian et al., 2004). We
found a substantial level of infection of identified motoneurons in the
vicinity of the hemisection (79% in both the T5–T7 segment (rostral to
Hx) and the T11–T12 segments (caudal to Hx). Even as far caudal as
the L4–L6 segments infectivity of motoneurons was 67% 7 days after
injection of HSV-NR2d-GFP into the left and right ventral horn at T11
(Fig. 1C). Many other cells were also infected (GFP-labeled) at these
locations but their identity as neurons or glia was not verified. These
findings confirm the long-distance HSV-1 propagation within the CNS
and transfer to other neurons (Zemanick et al., 1991; Curanovic &
Enquist, 2009), in particular motoneurons upon which both ascending
and descending cells with axons in VLF have been shown to
terminate, using other tracing techniques as well as electrophysiolog-
ically (Petruska et al., 2007).
In order to reduce or prevent excitotoxicity that could be mediated
through activation of NMDA receptors, we delivered subanesthetic
doses of ketamine in all experiments (3 mg kg, i.m., twice per day)
during the initial 2 days post-injury, when transient elevation of
glutamate concentration following spinal cord injury occurs (Xu et al.,
2004). The rationale for using ketamine, an NMDA receptor blocker
known to be neuroprotective (Albers et al., 1989), was to minimize
glutamate-induced excitotoxicity during first 2 days after the initial
lesion. In this study we did not examine the effects of ketamine alone.
However, the comparisons of the effects of Nogo-Ab, HSV-NR2d and
NT-3 in the various combinations were performed using the same
surgical procedures and under the same recording conditions, and all
animals received the same post-surgery ketamine injections.
Behavior
The following tests were carried out. Motor tests: open-field
locomotion, ladder rung walk, narrowing beam, swim tests. Sensory
tests (withdrawal reflex): plantar heater, von Frey hairs. The perfor-
mance of each animal was normalized to its own pre-injury baseline.
Open-field locomotion
This was evaluated by using the 21-point Basso, Beattie, Bresnahan
(BBB) locomotor scale (Basso et al., 1995). The rats were placed in an
open field (diameter 150 cm) with a pasteboard-covered floor. In each
testing session the animals were monitored individually for 4 min.
Ladder rung walk
The animals were required to walk along a 1-m-long horizontal ladder
elevated to 30 cm above the ground. A defined stretch of 60 cm was
chosen for filming and analysis. To prevent habituation to a fixed bar
distance, the bars in this sector were placed irregularly (1–4 cm
spacing). The animals performed the ladder rung walk twice in the
same direction and once in the opposite direction. The number of
errors (any kind of foot slip or total miss) was divided by the total
number of steps in each crossing, yielding the percentage of missteps
(Kunkel-Bagden et al., 1993).
Narrowing beam
This paradigm assesses the ability of the rats to balance along a
tapered beam 20 cm above the ground. The beam is flanked by two
side boards and graded into 24 stretches of the same length but
different widths, starting with 5 cm and ending with 1.5 cm width,
and can be walked along easily by an intact animal. The maximum
possible score in this test is 24. Animals had to walk along the beam
three times.
Swim test
The setup for the swim test consisted of a rectangular Plexiglas basin
(150 · 40 · 13 cm) filled with water at 23 C. The water level was high
enough to prevent the rats from touching the bottom of the basin with the
tail. The animals’ task was to swim straight to the 60-cm-distant board
which they could climb to reach the home cage. A total of five runs per
rat was monitored using a mirror at 45 at the bottom of the pool to film
the rats from the side and the bottom simultaneously. Velocity, forelimb
stroke rate and inter-hindlimb coordination were analyzed.
Withdrawal reflex thermal stimulation
The thermal nociceptive threshold for both hind paws was evaluated
by performing a standardized plantar heater test (Hargreaves et al.,
1988) using a commercially available apparatus (Ugo Basile, Comerio,
Italy). Rats were placed in a Plexiglas box (17 · 23 cm) and were first
allowed to adjust to the new environment. When exploratory behavior
ceased, an infrared source producing a calibrated heating beam
(diameter 1 mm) was placed under the hind paw and triggered together
with a timer. After one initial trial, the time for the hind limb withdrawal
reflection was averaged from four successive measurements. A
minimum interval of 30 s was maintained between successive trials.
Withdrawal reflex mechanical stimulation
Von Frey hairs (Semmes-Weinstein Monofilaments; Stoelting Co.,
Wooddale, IL, USA) with target force ranging from 0.008 to 300 N
were used. Rats were placed in a Plexiglas box (17 · 23 cm) with a
fine grid bottom and were first allowed to adjust to the new
environment. The monofilament was pressed against the plantar
surface of the foot at a 90 angle until it bowed, and held in place for
1–2 s. This stimulation was repeated up to three times in the same
location. The test was performed by using increasing filament calibers
until the first withdrawal reflex was noted.
Tracing
We assessed the crossing of propriospinal fibers that would project
through the VLF as this was the tract that was activated electrophys-
iologically. Biotin dextran amine (BDA; 10%, MW 10 000) in a total
volume of 1.0
lL was injected unilaterally into four sites in the left
ventral horn at C4–C7 over a period of 10 min for anterograde tracing
of midline-recrossing fibers. Ten days later the rats were perfused and
spinal cords were removed and prepared for morphological evaluation.
Alternating coronal sections were processed with Cresyl violet or were
stained for BDA using a nickel-enhanced diaminobenzidine protocol.
The number of fibers originating from the gray matter at C4 C7 and
traced with BDA was analyzed quantitatively using a light microscope
with bright-field illumination. We assessed the numbers of midline-
crossing fibers above and below the lesion in every fourth (30-lm-
thick) cross-section from the entire spinal cord (i.e. in approximately
500 sections per cord). For normalization, all midline-crossing fibers
were counted from T11 to S5 (below the lesion) and standardized to
the number of crossing fibers at T8 (above the lesion).
Electrophysiology
Experimenters were blinded as to the treatment of each rat. Rats were
deeply anesthetized using i.p. injection of ketamine (80 mg kg,
Functional ‘detour’ in the hemisected spinal cord 1259
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 1256–1267
Page 4
A
BF
G
H
I
C
D
E
J
Fig. 2. Intracellular recordings from L5 motoneurons demonstrating that Hx lesion abolished monosynaptic transmission and additive Nogo-Ab + NT-3 fibroblasts
+HSV-NR2d treatment established strong polysynaptic responses from ipsilateral VLF rostral to Hx. (A) Superimposed consecutive traces demonstrating
monosynaptic responses in non-injured cord with control treatments (n = 56 cells in seven rats). (B–I) Polysynaptic responses in Hx cords with following treatments:
(B) control-Ab+ control-FB+control-HSV (n = 35 cells in five rats; inset: response of same motoneuron, but from ipsilateral VLF caudal to Hx); see schematic
position of recording and stimulating electrodes for this particular configuration; (C) control-Ab+control-fibroblasts +HSV-NR2d (n = 31 cells in six rats); (D)
control-Ab + NT-3 fibroblasts +control-HSV (n = 39 cells in six rats); (E) control-Ab + NT-3 fibroblasts +HSV-NR2d (n = 65 cells in 10 rats); (F) Nogo-
Ab+control-fibroblasts+control-HSV (n = 41 cells in seven rats); (G) Nogo-Ab+control-fibroblasts +HSV-NR2d (n = 29 cells in six rats); (H) Nogo-Ab + NT-3
fibroblasts+control-HSV (n = 32 cells in six rats); (I) Nogo-Ab + NT-3 fibroblasts +HSV-NR2d (n = 76 cells 10 rats). (J) Summary of results for A–I groups,
respectively; pie charts represent the percentage of responding motoneurons in each group. Top drawing shows a schematic position of recording and stimulating
electrodes (except inset in B). *P < 0.05 for the effect of NT-3 + NR2d treatment compared to all groups, except full treatment group. **P < 0.01 for the effect of
full treatment group vs. all other groups.
1260 L. Schnell et al.
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 1256–1267
Page 5
0.5 mL) and xylazine (10 mg kg, 0.5 mL). Heart rate and expired
CO
2
were monitored continuously. Dorsal laminectomy of the spinal
cord was performed at T6–T8 for placement of the stimulation
electrode and L1–L6 for placement of the recording electrodes. L1–L6
ventral spinal segments were held tightly between custom-made bars,
and the dorsal surface of the cord was imbedded in a 3-mm-thick agar
layer to minimize movement of the cord during recordings. We
recorded responses from L5 motoneurons below the lesion (T10) on
the same side as the Hx. These responses were evoked by stimulation
of ventrolateral white matter tracts at T6 on the same side of the cord
(details in Arvanian et al., 2009). Motoneurons were identified by the
antidromic response to stimulation of the cut L5 ventral root. The resting
membrane potential of motoneurons used for analyses ranged from )55
to )65 mV. Peak excitatory postsynaptic potential amplitude was
measured from pre-stimulus baseline to peak. Latency was measured
from stimulus artifact to response onset. After completion of electro-
physiological recording, the rats were perfused and spinal cords
removed and prepared for morphological evaluation of the injury level.
Statistics
For the behavior experiments, two-way repeated-measures anova and
pairwise multiple comparison procedures (Holm–Sidak method) were
used to determine the statistical significance of the results (P < 0.05).
Data from the tracing experiments were subjected to one-way anova
followed by Bonferroni’s post hoc pairwise comparisons (*P < 0.05).
For the electrophysiological studies, the mean maximum response
from each motoneuron (50 consecutive responses per cell) was
averaged over all motoneurons recorded in each rat and these averages
were compared between treatment groups using one-way anova or
one-way anova on ranks (means are expressed ± SEM; n = number
of rats). If significant differences were observed between groups, a
Student–Newman–Keuls test or Dunn’s method were used for
pairwise comparisons as appropriate.
Results
Electrophysiology
The goal was to determine whether the combination treatment induced
the appearance of new functional connections spanning the hemisected
segment. We recorded intracellularly from motoneurons below the
lesion ipsilateral to the Hx. Responses were evoked by stimulation of
the ipsilateral VLF white matter above the lesion. This approach
improves detection of very weak functional connections across the
injury region and enables investigation of the impact of the various
treatments on these connections. For electrophysiology experiments
we used nine groups: one non-injured group that received all control
treatments, and eight groups that received a Hx lesion and no
treatment, or treatment with one, with two, or with all three
components of the combination treatment; appropriate controls were
administered in cases where only one or two active components were
delivered. The results are from experiments conducted 7–12 weeks
after the surgery with different treatment groups randomly assigned to
these times in order to minimize the variability of post-operation
recording time among the groups (Fig. 1).
Hx disrupted monosynaptic connections to motoneurons and additive
treatments established novel polysynaptic connections
In uninjured control rats that received laminectomy and treatments
with controls for all three agents in the combination treatment (Ringer-
filled catheter, control fibroblasts, and control HSV-1 virus), the
response in L5 motoneurons from ipsilateral T6 VLF exhibited the
following properties: large peak amplitude (6.2 ± 0.8 mV), short
latency (1.7 ± 0.1 ms), brief rise time and minimum fluctuation in both
amplitude and latency (Fig. 2A; n = 56 cells from seven rats). These
responses reached maximum amplitude at relatively low stimulus
current intensity (67.8 ± 11.5 lA, 50 ls), were similar to those
recorded in L5 motoneurons from ipsilateral VLF in untreated intact
adult rats (Arvanian et al., 2009), and were probably monosynaptic.
In Hx-lesioned rats that received either no treatment or control
treatment, the mean response was barely distinguishable from baseline
[no treatment: mean 0.2 ± 0.3 mV, n = 7, not shown; control
treatment (Fig. 2, B), mean 0.1 ± 0.2 mV; n = 5], even with VLF
stimuli as intense as 600 lAat50ls width. When we repositioned the
stimulation electrode caudal to the lesion, a typical monosynaptic
response was recorded from the same motoneuron at low stimulus
intensity (Fig. 2B inset). These results indicate that motoneurons
below the lesion remained viable and capable of receiving inputs from
surviving propriospinal fibers in the VLF below the lesion, and that
the lack of transmission from above the lesion was due to a disrupted
connection.
AB
C
Fig. 3. (A) Stimulus to VLF rostral to a hemisection evoked a response in an ipsilateral motoneuron caudal to the hemisection in a rat treated with Nogo-Ab + NT-
3 + NR2d. (B) Recording from the same motoneuron after a re-transection of the spinal cord through the Hx area. (C) Diagram to show position of the recording and
stimulating electrodes. Note that retransection did not eliminate the response to VLF stimulation.
Functional ‘detour’ in the hemisected spinal cord 1261
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The striking finding was that the additive treatment (Nogo-
Ab + NT-3 + NR2d) induced the appearance of large (4.7 ± 1.2
mV, n = 10 rats; Fig. 2I) responses in all (100%) injured rats.
However, in contrast to the short-latency monosynaptic responses in
uninjured rats, these responses exhibited a long latency
(9.8 ± 1.9 ms), showed greater fluctuation in both amplitude and
latency (Fig. 2, A vs. I), and required a markedly higher stimulus
intensity (415 ± 53 lA, 50 ls) to evoke a maximum response. These
results suggest that the functional connections established in the Hx-
lesioned spinal cords that received the combination treatment with
Nogo-Ab + NT-3 + NR2d probably involved conduction in smaller
axons than those responsible for the responses in intact preparations,
required more spatial summation on interneurons and were probably
multisynaptic. In rats treated with each agent alone or in pairs [with
corresponding controls for the missing agent(s)], the multisynaptic
responses evoked from segments above the Hx were either absent or
were much smaller than in rats with the full combination treatment,
even at the high stimulus currents (600 lA, 50 ls). Treatment with
Nogo-Ab alone (0.8 ± 0.5 mV; Fig. 3F), or in combination with either
NT-3 (1.1 ± 0.7; Fig. 3H) or NR2d (1.0 ± 0.7 mV; Fig. 3G), resulted
in weaker multisynaptic connections that could be recorded in over
half the rats (52–75%). Treatment with NT-3 + NR2d induced the
appearance of larger responses (peak amplitude 1.9 ± 0.7 mV;
Fig. 2E) but did so in only a few rats (in three rats out of 10 studied).
In rats that received NR2d alone (0.2 ± 0.1 mV; Fig. 2C) or NT-3
alone (0.4 ± 0.3 mV; Fig. 2D) very weak connections in a small
A
B
C
a1
a2
a1
a2
Fig. 4. Combination treatment enhanced ‘anatomical plasticity’ following Hx injury. (A) Representative photomicrographs of 30-lm cross-sections below the
lesion to demonstrate midline-recrossing fibers (MRCF) revealed by BDA anterograde transport from the gray matter at C4-C7. Further details in text. Left panel
shows example from animal treated with control-Ab and schematic depiction of the area shown. Only very few fibers could be found. Middle and right panels
showed MRCF in two different animals treated with Nogo-Ab + NT-3 + NR2d. (B) Bar graph of normalized number of re-crossing fibers counted from T11 L5.
The number of crossing fibers at the T8 level (above the lesion) was used to normalize these results for differences in staining intensity. Results of individual
experiments displayed as dots in line with the bars. Some points overlap. (C) Schematic drawing of the labeling in unlesioned animals (left panel), the effect of the
lesion on ipsilateral fibers (middle panel) and the regenerative sprouting effect (right panel) after the combination treatment with Nogo-Ab + NT-3 + NMDA2d.
Blue fibers cross the midline just once, and red fibers cross the midline twice (MRCF). Scale bar in A, 100 lm (does not apply to insets from a1 and a2, which are at
higher power for visualization of thin and thick fibers, respectively). * denotes treatments with more recrossing fibers than IgG control- treated preparations.
Fig. 5. Effects of treatment on behavioral outcome. (A) Open-field locomotion revealed similar scores in all six groups at 2 days post-operation, indicating
consistency of the lesion. However, there were no significant differences among the groups during up to 48 days post-injury. (B–D) In more challenging behavioral
tests, i.e. swim, narrow beam and ladder rung, the full triple-treatment group showed better recovery than animals of other groups (performance of each animal was
normalized to its own pre-injury baseline). As performance of all groups that received one or two treatment components was similar, the mean error bars are
displayed for the NR2d-alone and the triple combination groups. *denotes times at which the full treatment group was significantly different (P < 0.05) from the
partially treated groups.
Fig. 6. Sensory tests. (A) The von Frey filament test revealed no difference in mechanical threshold between the hind paws of the lesioned and unlesioned side in all
groups. (B) Withdrawal latency to noxious thermal stimulation was not different between hind paws on the intact and hemisected side.
1262 L. Schnell et al.
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European Journal of Neuroscience, 34, 1256–1267
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A
BC D
Fig. 5.
AB
Fig. 6.
Functional ‘detour’ in the hemisected spinal cord 1263
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European Journal of Neuroscience, 34, 1256–1267
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subset of rats were noted. Reconstruction of the Hx in each case
revealed no relation between the size of the lesion and the amplitude
of the responses (but see Discussion).
Novel polysynaptic responses were the result of a ‘functional detour’
around the Hx lesion
It was important to determine whether novel polysynaptic connections
established in the triple combination group travelled through the lesion
area or around the Hx. Therefore, in three rats treated with the full
combination treatment, the spinal cord was carefully retransected
through the existing scar after recording polysynaptic responses from
several motoneurons while maintaining penetration of a motoneuron.
We found that these responses persisted after this procedure (Fig. 3).
These results confirm that the novel responses recorded in Hx-lesioned
rats receiving the full combination treatment were the result of the
establishment of new connections around the hemisected cord rather
than regeneration through the lesion area.
Anatomical evaluation
The primary goal was to determine a possible anatomical substrate for
the novel polysynaptic responses around the Hx in the triple
combination treatment which gave the most robust change in the
electrophysiological studies. We were particularly interested in
determining whether the electrophysiological changes are related to
an increase in the number of midline crossings of fibers. Anterograde
BDA tracing from injections at C4 C7 ipsilateral to the Hx was
carried out in order to assess the number of fibers that crossed the
midline caudal to the Hx (Fig. 4A). This injection site allowed us to
follow the projection of interneurons (i.e. long propriospinal neurons;
Reed et al., 2008) to the side contralateral to the Hx and back to the
lesioned side more caudally. Above the lesion, fibers crossing the
midline to the contralesional side cannot be distinguished from fibers
that re-cross to the ipsilesional side but below the lesion only midline-
recrossing fibers are labeled (see Fig. 4C).
Because each group consisted of 500 sections from each of three to
eight cords, it was impractical to study all eight groups. Instead we
compared the results of animals treated with all three agents (Nogo-
Ab + NT-3 + NR2d; n = 8 rats after exclusion) to results obtained
from an untreated hemisected group (IgG control mAb; n = 3 rats after
exclusion) which displayed no electrophysiological evidence of a
functional detour. We made similar determinations using rats treated
with two agents (Nogo-Ab with either NT-3 or NR2d) that gave
intermediate electrophysiological evidence of a detour, or NR2d alone
that produced a minimal detour. In the unlesioned cord the number of
crossing fibers between T11–L5 was very high (approximately 5000;
n = 3). In Hx cords the number of crossing fibers above the lesion was
also high, and we used the number of fibers at T8 to normalize for
tracer injection quality. Counts of these crossing fibers (Fig. 4B)
revealed that the group of animals with the combined Nogo-Ab + NT-
3 + NR2d treatment was unique in having appreciable numbers of re-
crossing fibers (362 ± 90.1 SEM, n = 8 rats). This was significantly
higher (P < 0.05) than the number observed in the other treated Hx
groups. Administration of control-Ab alone yielded counts of
recrossing fibers that were uniformly very low. However, in individual
experiments in the three groups with intermediate treatments we
observed some recrossing fibers in all preparations treated with anti-
Nogo and NT-3 or NR2d and this might account for the electro-
physiological evidence of the detour (Fig. 4B). In preparations treated
with NR2d only we observed no recrossing fibers in three animals;
this is consistent with the lack of detour observed electrophysiolog-
ically. One preparation had an anomalously high number of
recrossing fibers.
In agreement with the electrophysiological findings above, where
no monosynaptic response in motoneurons was found in response to
stimulating VLF rostral to the Hx and where a re-transection of the
spinal cord did not alter the polysynaptic response, we did not find any
evidence for axons that could have crossed through the lesion. We
therefore consider it likely that the conduction path involves newly
formed re-crossing fibers from long propriospinal axons, which
connect via interneurons to the L5 motoneuron pool in the ventral horn
of the ipsilesional side (Fig. 4D; see Discussion).
Assessment of lesion size
After completion of physiological recordings or tracing experiments
the lesion site was reconstructed from cross-sections and measured as
a percentage of the area of the intact cord (Fig. 1B). Camera lucida
drawings of six representative lesion sites from rats used for the
behavioral studies are shown in Fig. 1B. It can be seen that the mean
lesion size was virtually identical for all treatments. There were
differences in the tissue that was spared from rat to rat but these were
not systematic in the different treatments (see Discussion).
Behavioral evaluation
In order to minimize the role of extraneous factors, it was important to
carry out behavioral testing on animals that arrived at the institutional
animal facility from the same vendor at the same time and received
treatment using the same lot of compounds. The need to do surgery
and behavioral testing on a single group of animals placed a limit on
the number of animals that could be studied. Thus we limited this
experiment to six groups (vs. nine groups in the electrophysiology
experiment). The groups were chosen to cover the range of results
obtained in the initial electrophysiology experiments. All rats were
pre-trained for 4 weeks, then received injury and treatment within
4 days and behavioral testing for the following 6 weeks using four
motor and two sensory tests.
Two days after the operation, the rats were scored with the BBB test
(Basso et al., 2002) to assess the extent of the lesion. The left leg did
not score > 3 in any group while the right hindlimb was mostly able to
support the body weight and perform plantar stepping, which is
equivalent to a score of 8 or higher. In the course of a 3-week recovery
the different groups improved their performance gradually by
approximately 3–4 points and reached a plateau of 12 points (which
is just below the score for coordinated forelimb–hindlimb stepping),
with no significant difference between the groups (Fig. 5A). Although
the quasi-quantitative protocol of BBB scoring is useful for evaluating
the loss of function and recovery following injury, it has a major
disadvantage in assessing the subtle improvements that result from
treatments after thoracic Hx in rodents because of the robust
spontaneous recovery of locomotor function that takes place after
this type of injury (Courtine et al., 2008; Arvanian et al., 2009).
Therefore all animals were also tested in more challenging tests such
as the symmetry of swimming, narrowing beam and horizontal ladder
paradigms (Fig. 5B and C; see below).
In the swim test there was no difference in swimming speed
between the groups (P
> 0.05), as the Hx lesion allowed a relatively
fast recovery of performance in this weight-supported test. We
therefore focused on inter-hindlimb rhythm and measured the
difference in beat duration (time for a complete stroke) between the
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right and the left hindlimb. In healthy, uninjured animals this
difference is close to zero as the legs beat in a very regular pattern.
After a lateral Hx lesion the animals exhibited ‘limping’, which is
better observed and measured during swimming than in overground
locomotion (BBB test). This interhindlimb coordination remained
disturbed over the entire 42-day period following the lesion, but the
difference in beat duration was smallest in the triple-combination
treatment group (Fig. 5B). While the performance among the groups
with one or two treatment components was similar (P > 0.05), the
performance of the rats from the triple combination treatment group
was significantly better than these other groups (P < 0.05).
In the narrow beam test, unlesioned animals normally reach the
narrow end of the scaled bar without missteps. After the lesion, this
capability was greatly reduced. Normalized values revealed that the
triple combination treatment group performed significantly better than
groups with one or two treatment components (P < 0.05). The
performance among groups with one or two treatment components
was similar (P > 0.05).
In the horizontal ladder rung test, the ability of the animals to place
their hindpaws on the same rung as the forepaws was greatly reduced
on the ipsilesional side, where hardly any successful hindpaw
placements were performed (Fig. 5D). Significantly better perfor-
mance was evident in the triple combination treatment group
compared to the other groups tested at post-operative day 28; this
difference was maintained at day 42, the last time point tested
(P < 0.05). The performance among groups with one or two treatment
components was similar (P > 0.05).
In order to evaluate the effects of the treatment on the sensitivity to
nociceptive stimuli, we performed standardized von Frey filament and
plantar heater tests. Over the 7, 21 and 42 days post-operation time
points tested, treatment groups were indistinguishable from each other
(Fig. 6).
Discussion
This study has revealed that a functional ‘detour’ can be established
around a Hx, from the lesioned ventrolateral white matter above to
ipsilateral motoneurons below, using a novel combination treatment
with the following components: an antibody to the major inhibitory
molecule Nogo-A, the neurotrophin NT-3, and the NR2d regulatory
subunits to enable NT-3-induced plasticity. Combining neurotrophins
with other agents to improve their effectiveness in restoring function is
consistent with the results of recent studies adopting this approach (Lu
et al., 2004; Nothias et al., 2005; Arvanian et al., 2006b; Chen et al.,
2008; Massey et al., 2008). Furthermore, the VLF contains reticu-
lospinal and long propriospinal fibers (Reed et al., 2008) known to
participate in the recovery of locomotor function in rats following
thoracic injuries (Basso et al., 2002; Schucht et al., 2002; Arvanian
et al., 2009). Therefore, development of a treatment aimed at the
restoration of VLF projections is an important strategy in promoting
recovery of function after thoracic injuries.
Electrophysiological experiments revealed the appearance of novel
long-latency responses in L5 motoneurons from the ipsilateral VLF
rostral to Hx in rats receiving the combination treatment (Fig. 2). The
fact that these responses were preserved after re-transection of the
spinal cord through the pre-existing lesion strongly suggests that these
novel responses were not due to axons regenerating through the lesion,
but were the result of the establishment of novel functional
connections around the Hx. Although the increase in branching from
white-matter fibers observed in the tracing studies is suggestive of new
connections being responsible for the detour, we cannot rule out a
contribution from already existing subliminal connections or ‘silent’
synapses (Kerchner & Nicoll, 2008) that were strengthened and
became visible after the treatment (Wall, 1988).
Previous double-labeling studies in intact adult rats using tracers
injected into the lumbar cord and VLF have identified a population of
cervical neurons that cross to the contralateral VLF and recross to
terminate in the ipsilateral upper lumbar cord (Reed et al., 2008).
However, the absence of electrophysiological responses observed in
control hemisected preparations suggests very little functional con-
nectivity to L5 motoneurons mediated by propriospinal fibers crossing
above and below the Hx (Fig. 4). The propriospinal fibers studied here
apparently did not sprout below the lesion spontaneously, as indicated
by the virtual absence of midline-recrossing fibers below the Hx in the
absence of treatments. However, after the full combination treatment
the number of fibers recrossing caudal to Hx increased substantially
(Fig. 4).
The mild behavioral effects of the combination treatment occurred
on a background of robust spontaneous recovery of locomotor
function observed after thoracic Hx in rodents (Courtine et al., 2008;
Arvanian et al., 2009); this spontaneous recovery makes further
improvements difficult to detect. More challenging tests such as
narrowing beam, horizontal ladder and the swimming symmetry
revealed minor yet significant improvement of motor function in rats
with the full combination treatment (Fig. 5), with no change in
nociceptive function (Fig. 6). More robust recovery may depend on
strengthening the synaptic connectivity from the descending fiber
systems on the hemisected side to neurons responsible for the detour.
Together these studies suggest that the combination treatment
produced larger effects electrophysiologically, anatomically and
behaviorally than components tested separately or in pairs. In the
electrophysiology where all possible combinations were tested with
corresponding controls, the ability of these agents to produce a detour
is clear. In the case of the anatomy and behavior, the full combination
treatment elicited more sprouting or functional recovery than any of
the treatments tested. However, because not all combinations were
studied anatomically and behaviorally, and because the behavioral
recovery may not parallel the electrophysiological recovery, we
remain cautious about making conclusions concerning the ability of
these treatments to promote recovery of behavior. Another caveat is
the possibility of small differences in tissue sparing among the
different preparations (Fig. 1B). However, the uniform differences in
plasticity at all levels between the treatment groups, and the uniformity
of the lesion size, make it very unlikely that systematic differences in
tissue sparing were a major factor determining the findings reported
here.
How does the combination treatment produce the detour? The
requirement for Nogo-A specific antibody and NT-3 and NR2d
suggests that detour formation required sprouting or growth of axons
as well as an increase in synaptic efficacy. Although our present
results do not reveal the location of the novel connections, we believe
that they are distributed throughout the cord but are probably most
numerous or strongest close to the lesion site where the concentration
of the exogenous agents is highest.
NMDA receptors on motoneurons become functional in the prenatal
period (Ziskind-Conhaim, 1990; Kalb & Hockfield, 1992), but they
suffer a decline in function during the second postnatal week due to
Mg
2+
blockade (Arvanian et al., 2004). We previously found that
restoring NMDA receptor function by adding back the NR2d subunit
of the NMDA receptor using an HSV viral construct enabled NT-3 to
induce NMDA receptor-dependent potentiation of VLF synaptic
transmission (Arvanian et al., 2004). When combined with NT-3, the
NR2d subunit induced the appearance of synaptic responses in
motoneurons from damaged VLF axons (Arvanian et al., 2006c).
Functional ‘detour’ in the hemisected spinal cord 1265
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European Journal of Neuroscience, 34, 1256–1267
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These results suggest that activity of NMDA receptors in the target
neurons might be an essential factor required for growing axons to
establish glutamatergic synaptic contacts upon them. Although our
studies were in motoneurons, it seems likely that novel connections on
interneurons are important for establishing the connections to
motoneurons described here.
Our current results demonstrate that combinatorial treatment with
NT-3 and NR2d resulted in VLF connections in approximately 33% of
the motoneurons in injured adult rats. Similar treatments with NT-3
and NR2d in contused or staggered double-hemisected neonatal rats
resulted in recovery of some connectivity to virtually all motoneu-
rons (Arvanian et al., 2006c). One possible explanation for the
limited efficacy of the treatment with NT-3 and NR2d in adult rats is
the age-related development of myelin-associated neurite growth
inhibition in the spinal cord. The localization of Nogo-A in
oligodendrocytes, where expression starts at a relatively late
developmental stage (Huber et al., 2002; Taketomi et al., 2002), fits
well with its role as an age-dependent myelin-associated inhibitor of
regenerative fiber growth in adult mammals. Here we demonstrate
that additive treatment with NT-3 and HSV–NR2d in adult rats was
sufficient to form connections via conduction around the Hx only
when combined with anti-Nogo-A antibody. Considering that HSV-
1-mediated NR2d expression lasts 1–2 weeks and Nogo-Ab delivery
lasts 2 weeks, we hypothesize that they play a role in initiating the
establishment of polysynaptic connections observed 7–12 weeks
post-injury.
The establishment of connections to motoneurons via the detour is
supported by anatomical experiments that indicate an increase in the
number of branches given off by propriospinal or supraspinal axons
(either ascending or descending) in the contralateral white matter
caudal to Hx. Growth of these branches to the ipsilesional side of the
cord could provide access to the stimulating electrode above the
lesion; similarly, the recrossing between the hemisection and L5 could
provide access to ipsilesional motoneurons, perhaps via strengthening
of polysynaptic connections. Branches of fibers ipsilateral to the Hx
may also send branches to contact propriospinal neurons on the
contralateral side which then recross below the Hx to contact L5
motoneurons either directly or via relays from short propriospinal
interneurons (Courtine et al., 2008; Etlin et al., 2010). Axotomized
fibers descending from supraspinal centers (Reed et al., 2008)
including the corticospinal tract and serotonergic raphe spinal fibers
known to be influenced by anti-Nogo (Liebscher et al., 2005; Mu
¨
llner
et al., 2008) could also play a role in re-establishing the connectivity
observed in these experiments.
A further consideration is the recent finding that thoracic Hx can
reduce conduction through the uninjured contralateral white matter
beginning 1–2 weeks after Hx as well as a decline in conduction
velocity for axon segments across from the Hx (Arvanian et al., 2009).
These changes were associated with decreased excitability in these
axons (manifested by an increased rheobase), partial demyelination of
the VLF and rubrospinal tract axons contralateral to the Hx (Hunanyan
et al., 2011) and accumulation of chondroitin sulfate proteoglycans
(CSPGs) in tissue surrounding the Hx (Hunanyan et al., 2010). Such
changes undoubtedly contributed to the absence of any response
through the region of injury in the controls; the treatments given at the
time of the injury could have either prevented this decline in
conduction or reversed it. In this context, NT-3 has been found to
induce oligodendrocyte proliferation and myelination of regenerating
axons in the contused adult rat spinal cord (McTigue et al., 1998), and
the presence of Nogo has complex effects on oligodendrocyte
differentiation which could affect myelination and impulse conduction
(Pernet et al., 2008). The effects of NT-3 and anti-Nogo on
myelination of regenerating fibers and conduction through the region
contralateral to Hx, as well as the combination of this treatment with
intraspinal digestion of CSPGs (Fawcett, 2009), remain to be
determined in the current Hx model.
In conclusion, these results demonstrate that combination treatments
using anti-Nogo, NT-3 and the NR2d subunit promote the establish-
ment of a synaptic detour around a Hx. This pathway involves
sprouting of white-matter fibers to the opposite side and may
contribute to behavioral improvement. Future experiments should
explore this combination approach to studying other spinal injuries,
e.g. contusion, to determine whether recovery of function is improved
under these experimental conditions.
Acknowledgements
The research was supported by Merit Review Funding from the Department of
Veterans Affairs (V.L.A.), Christopher and Dana Reeve Foundation (L.M.M.
and M.E.S.), the Department of Defense Award and the New York State Spinal
Cord Injury Research Board (V.L.A.), the NIH 5R01 NS 16996 (L.M.M.) and
the William J. Heiser Foundation (L.M.M.). We would like to thank Mr Clark
Burris and Mr Lou Lotta (University of Rochester) for HSV amplicon vector
packaging.
Abbreviations
BDA, biotin dextran amine; GFP, green-fluorescent protein; HSV-1, Herpes
simplex virus; Hx, hemisection; Nogo-Ab, anti-Nogo-A monoclonal antibody;
NR2d, NR2d subunit of NMDA receptor; NT-3, neurotrophin-3; VLF,
ventrolateral funiculus.
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Functional ‘detour’ in the hemisected spinal cord 1267
ª 2011 The Authors. European Journal of Neuroscience ª 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd
European Journal of Neuroscience, 34, 1256–1267
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    • "Lateral hemisection of the spinal cord at the low thoracic level is one of the models frequently used for testing new therapeutic strategies, which can be applied after spinal cord injury123. Although there are numerous inventive studies designed to improve locomotion via a variety of rehabilitation techniques or pharmacological agents after spinal cord hemisection, there is still a lack of precise data describing spontaneous locomotor recovery after such injury. "
    [Show abstract] [Hide abstract] ABSTRACT: Lateral thoracic hemisection of the rodent spinal cord is a popular model of spinal cord injury, in which the effects of various treatments, designed to encourage locomotor recovery, are tested. Nevertheless, there are still inconsistencies in the literature concerning the details of spontaneous locomotor recovery after such lesions, and there is a lack of data concerning the quality of locomotion over a long time span after the lesion. In this study, we aimed to address some of these issues. In our experiments, locomotor recovery was assessed using EMG and CatWalk recordings and analysis. Our results showed that after hemisection there was paralysis in both hindlimbs, followed by a substantial recovery of locomotor movements, but even at the peak of recovery, which occurred about 4 weeks after the lesion, some deficits of locomotion remained present. The parameters that were abnormal included abduction, interlimb coordination and speed of locomotion. Locomotor performance was stable for several weeks, but about 3-4 months after hemisection secondary locomotor impairment was observed with changes in parameters, such as speed of locomotion, interlimb coordination, base of hindlimb support, hindlimb abduction and relative foot print distance. Histological analysis of serotonergic innervation at the lumbar ventral horn below hemisection revealed a limited restoration of serotonergic fibers on the ipsilateral side of the spinal cord, while on the contralateral side of the spinal cord it returned to normal. In addition, the length of these fibers on both sides of the spinal cord correlated with inter- and intralimb coordination. In contrast to data reported in the literature, our results show there is not full locomotor recovery after spinal cord hemisection. Secondary deterioration of certain locomotor functions occurs with time in hemisected rats, and locomotor recovery appears partly associated with reinnervation of spinal circuitry by serotonergic fibers.
    Full-text · Article · Nov 2015 · PLoS ONE
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    • "The behavioral data were correlated with the highest number of sprouting axons in the spinal cord and multisynaptic responses in the motor-neurons. Similar results could be found if anti-Nogo-A antibodies were combined with NT-3 and the NMDA-NR2D subunit (Schnell et al., 2011). Furthermore, the combinatorial treatment of acutely applied anti-Nogo-A antibody followed by delayed Chondroitinase ABC treatment starting 3 weeks after spinal cord injury, and forelimb grasp training starting at 4 weeks was much more effective in terms of functional recovery , sprouting and axonal regeneration than the single treatments (Rehme et al., 2011). "
    [Show abstract] [Hide abstract] ABSTRACT: After stroke the central nervous system reveals a spectrum of intrinsic capacities to react as a highly dynamic system which can change the properties of its circuits, form new contacts, erase others, and remap related cortical and spinal cord regions. This plasticity can lead to a surprising degree of spontaneous recovery. It includes the activation of neuronal molecular mechanisms of growth and of extrinsic growth promoting factors and guidance signals in the tissue. Rehabilitative training and pharmacological interventions may modify and boost these neuronal processes, but almost nothing is known on the optimal timing of the different processes and therapeutic interventions and on their detailed interactions. Finding optimal rehabilitation paradigms requires an optimal orchestration of the internal processes of re-organization and the therapeutic interventions in accordance with defined plastic time windows. In this review we summarize the mechanisms of spontaneous plasticity after stroke and experimental interventions to enhance growth and plasticity, with an emphasis on anti-Nogo-A immunotherapy. We highlight critical time windows of growth and of rehabilitative training and consider different approaches of combinatorial rehabilitative schedules. Finally, we discuss potential future strategies for designing repair and rehabilitation paradigms by introducing a "3 step model": determination of the metabolic and plastic status of the brain, pharmacological enhancement of its plastic mechanisms, and stabilization of newly formed functional connections by rehabilitative training.
    Full-text · Article · Jun 2014 · Frontiers in Human Neuroscience
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    • "Enhancement of the proliferation and orientation differentiation ability of stem cells by gene modification has been widely studied in tissue engineering. Vascular endothelial growth factor 165 (VEGF 165 ) (Widenfalk et al., 2003), neurotrophin-3 (NT-3) (Schnell et al., 2011), brain-derived neutrophic factor, or the derived scaffolds or vectors constructed based on these factors have been widely used in experimental studies and gradually applied in the clinical setting. The goal of this study was to construct a bicistronic eukaryotic expression vector, pIRES 2 -VEGF 165 -NT-3, using a simple and efficient method to enable further study on the functions of the VEGF 165 and NT-3 genes. "
    [Show abstract] [Hide abstract] ABSTRACT: We used a simple and efficient method to construct the bicistronic eukaryotic expression vector pIRES2-VEGF165-NT-3. The neurotrophin-3 (NT-3) gene was obtained from the genomic DNA of human peripheral blood mononuclear cells by polymerase chain reaction. The NT-3 cDNA fragment was cloned into the pIRES2-VEGF165-EGFP vector in place of enhanced green fluorescent protein (EGFP) to create the plasmid pIRES2-VEGF165-NT-3. Next, pIRES2-VEGF165-NT-3 was transfected into HEK293 cells, and reverse transcription-polymerase chain reaction and Western blotting were used to test co-expression of the double genes. The vascular endothelial growth factor 165 (VEGF165) and NT-3 genes were cloned; DNA sequencing analysis demonstrated that the VEGF165 and NT-3 sequences were the same as those recorded in GenBank. Restriction analysis indicated that the VEGF165 and NT-3 genes were correctly inserted into the expression vector pIRES2-EGFP. The double gene was expressed at both the mRNA and protein levels. The VEGF165 and NT-3 co-expression plasmid was successfully constructed, providing a novel expression system for further study of the functions of the VEGF165 and NT-3 genes.
    Preview · Article · Jun 2014 · Genetics and molecular research: GMR
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