ARTICLES

Chondroitinase ABC treatment opens a window

of opportunity for task-specific rehabilitation
Guillermo Garcı́a-Alı́as, Stanley Barkhuysen, Miranda Buckle & James W Fawcett

Chondroitinase ABC treatment promotes spinal cord plasticity. We investigated whether chondroitinase-induced plasticity
combined with physical rehabilitation promotes recovery of manual dexterity in rats with cervical spinal cord injuries. Rats
received a C4 dorsal funiculus cut followed by chondroitinase ABC or penicillinase as a control. They were assigned to two
alternative rehabilitation procedures, the first reinforcing skilled reaching and the second reinforcing general locomotion.
© 2009 Nature America, Inc. All rights reserved.

Chondroitinase treatment enhanced sprouting of corticospinal axons independently of the rehabilitation regime. Only the rats
receiving the combination of chondroitinase and specific rehabilitation showed improved manual dexterity. Rats that received
general locomotor rehabilitation were better at ladder walking, but had worse skilled-reaching abilities than rats that received no
treatment. Our results indicate that chondroitinase treatment opens a window during which rehabilitation can promote recovery.
However, only the trained skills are improved and other functions may be negatively affected.

Following damage to the spinal cord and other parts of the CNS there is
a period of neurological recovery that is maximal in the first 3 months,
but continues for a year or more. Much of this recovery is probably a
result of the creation of new circuitry through sprouting and alterations
of synaptic strength, known collectively as plasticity1. In some
instances, the recovery process can be modified and enhanced through
the use of appropriate rehabilitation2, but in others, the advantages
over spontaneous recovery are unproven3. Following spinal cord injury
(SCI), tetraplegic patients indicated that improved arm and hand
function would most improve their quality of life4. In humans, hand
function and most other motor functions depend on the integrity of
the corticospinal tract (CST), whereas CST damage in rodents primar-
ily causes deficits in the fine control of the forearm muscles required
for grasping and holding objects5,6. Skilled paw use in rodents is
therefore a useful method for evaluating treatments relevant to the
CST and human SCI. Following SCI in rodents, there is limited
plasticity of the spared CST and other axons, which is associated with
some return of function7. However, in the injured spinal cord, there are
potent inhibitors of axon regeneration and plasticity, including several
molecules that are expressed on myelin8 extracellular matrix proteo-
glycans in the glial scar9 and various extracellular matrix molecules in
perineuronal nets surrounding neuronal somata and dendrites10.
Various therapies have been developed to overcome these inhibitory
influences and promote axon regeneration and plasticity11. Amongst
these, chondroitinase ABC (ChABC) has been shown to promote
regeneration, plasticity and functional recovery in various experimental
models12–14. In a previous study, however, treatment of rat cervical
spinal cord injuries with ChABC only produced a modest recovery in
CST function, as measured by skilled paw function15. We reasoned that
promoting plasticity by itself may not be sufficient to promote
functional recovery if it leads to random new connections. Instead, the
formation of appropriate connections in the spinal cord and brain may
need to be driven by appropriate rehabilitation. We therefore investi-
gated whether ChABC treatment combined with rehabilitation leads to
recovery of manual function following dorsal funiculus lesions in the
rat at level C4. We also asked whether the rehabilitation must be specific
to paw function or whether general environmental enrichment can be
equally effective. We found that ChABC treatment produced anatomi-
cal sprouting, but there was only recovery of skilled paw function if
ChABC treatment was combined with specific rehabilitation. General
environmental enrichment rehabilitation enhanced locomotor func-
tion, but extinguished skilled paw reaching.
RESULTS
Lesions and ChABC digestion
Cutting the dorsal funiculi of the C4 spinal segment led to the
formation of a cystic cavity in the spinal cord, which disrupted the
dorsal columns and the dorsal corticospinal tract axons and partially
compromised the gray matter of the dorsal and ventral horns (Fig. 1a
and Supplementary Fig. 1). Complete lesioning of the dorsal CST was
verified by staining spinal segments above and below the injury for
protein kinase C g (PKCg) and by observing the absence of immuno-
positive fibers below the injury (Fig. 1b,c) and by observing complete
transection of biotinylated dextran amine (BDA)-traced axons at the
site of injury. The cavity size and morphology were very similar in all of
the rats tested and we found no differences in the maximal width
between experimental groups (Supplementary Fig. 1).
Rats received intraparenchymal ChABC or control penicillinase
(Pen) above and below the lesion at the time of operation, followed
by five bolus intrathecal infusions on alternate days. To demonstrate the

Centre for Brain Repair, Department of Clinical Neuroscience, University of Cambridge, Cambridge, UK. Correspondence should be addressed to J.W.F. (jf108@cam.ac.uk).
Received 17 February; accepted 26 June; published online 9 August 2009; doi:10.1038/nn.2377

NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2009 1145

 ARTICLES
a b
d Paw use after SCI e Staircase pellet retrieval
Pellets eaten
g Reaching task in
Wishaw apparatus at 42 dpo
h i
30
Number of reaches
through window
20
10
© 2009 Nature America, Inc. All rights reserved.
0 0
Pen ChABC
no rehab no rehab
j Contact placing k with Pen did not (P o 0.001). Values are shown as mean ± s.e.m.
Number of responses
extent of maximal chondroitinase digestion, we killed four ChABC-
treated and two Pen-treated rats after the last ChABC intrathecal
injection and stained them with 1B5 anti-stub and neurocan antibodies
(Fig. 2). There was digestion throughout the cord within 5 mm rostral
and 1–3 mm caudal to the injury (Fig. 2b). In the digested area,
neurocan was completely removed and perineuronal net staining was
not seen with either antibody (Fig. 2c,e,f). Around the outside of the
cord, the meninges were strongly stained from C1 to the mid-thoracic
level and there was digestion of an outer rim of white matter
approximately 100 mm deep from level C1 to T1 or below (Fig. 2d).
Effects of ChABC treatment alone
Two groups of rats received a C4 lesion followed by ChABC or Pen
(ChABC alone and Pen alone groups). On the day after the injury, all of
the rats were able to stand and to use their forepaws and hindpaws to
locomote, with stable stance and raised tails. By 3 d, all rats were able to
flex and extend their forepaws and hold objects, and their general
locomotion in their cages was almost indistinguishable from normal
(Fig. 1d). However, major deficits were found in the rats’ abilities to
perform skilled reaching tasks. In the staircase test (Fig. 1e), rats were
trained before injury to retrieve at least 15 pellets, but rats in the Pen
and ChABC groups could only retrieve 0–4 pellets at 7 d after injury.
When observed eating small objects in their cage, they would first pick
them up in their mouth instead of with their paws, and then grasp them
with both paws.
In the weeks post injury, only modest recovery in skilled paw
reaching was observed in both the ChABC and Pen groups (Fig. 1f).
At the end of the experiment, at 6 weeks, we tested the rats in a differ-
ent paw reaching task that they had not encountered before using
1146
Figure 1 ChABC or penicillinase (Pen) without rehabilitation. (a) Transverse
c * section at the epicenter of the lesion immunostained for glial fibrillary
acidic protein (GFAP) showing a typical injury. Scale bar represents 500 mm.
(b,c) PKCg immunostaining at C2 (b) and T2 (c) spinal segments of an
injured spinal cord. Note the absence of dorsal CST staining in the T2 spinal
segment (star), indicating the disruption of the dorsal corticospinal tract
f at the level of the injury. The ventral and lateral tracts cannot be seen at
20
Pen no rehab
this magnification. (d) Following injury, rats were able to maintain their
posture, locomote and hold objects with their forepaws. (e) The staircase
15 ChABC no rehab
device. Animals slide into the tunnel, gaining access to the left and right
10
staircase. They grasp sugar pellets for 15 min. Supplementary Video 3
5 shows rats performing this task. (f) Following dorsal funiculus injury, there
were severe impairments in the rats’ abilities to perform skilled reaching,
PRE 7 14 21 28 35 42 as tested in the staircase test. The rats went from grasping 14 pellets prior
dpo
to injury to grasping 0–4 pellets afterwards, achieving only mild recovery
after 42 d. ChABC-treated rats performed no better than Pen-treated rats.
(g) The Whishaw skilled paw reaching apparatus. Sugar pellets are placed
30
* on the platform. To retrieve them, rats must extend their paw through
Pellets retrieved
a narrow window, grasp the pellet and pick it up. Pellets are replaced
20
for 5 min. Supplementary Video 1 shows rats performing this task.
(h,i) When tested at 42 d in the Whishaw apparatus, ChABC-treated rats
10 * were better able to extended their paws through the window and retrieve
pellets (P o 0.05). (j) In the contact placing response, which requires
CST function, rats spread their forepaw digits and place them on the
Pen ChABC
no rehab no rehab table when the dorsum of the paw touches the edge. (k) The rats treated
with ChABC recovered the ability to perform this task, but those treated
10.0
7.5
*** Whishaw’s skilled paw reaching apparatus (Fig. 1g). In this task16, rats
5.0
have to reach through a small window to pick up a sugar pellet on a
2.5 shelf. In this test, ChABC-treated rats performed better than Pen-
0.0 treated rats (Fig. 1h,i); they showed a greater ability to extend their
Pen ChABC
no rehab no rehab
forepaw through the window, after which they were able to grasp and
retrieve some pellets, whereas Pen-treated rats were usually unable to
extend their paws through the window (Supplementary Video 1).
Less-skilled forelimb motor tasks were also affected by C4 lesions.
CST function has been implicated in the contact placing response
(Fig. 1j), which was lost after lesioning, after which the ChABC-treated
rats recovered better than the control rats (Fig. 1k). The frequency with
which the rats missed the rungs during ladder walking was increased
for both groups at 7 days post operation (dpo), from which they
partly recovered (see below). Rats also showed a decrease in forelimb
grip strength, which largely recovered by 42 dpo. There was no
difference between the groups in the recovery of grip strength or
ladder walking.
Effects of task-specific rehabilitation combined with ChABC
Next, we investigated the effect of adding daily specific paw-reaching
rehabilitation to the ChABC or control Pen treatment described above
(ChABC-specific and Pen-specific groups). For this purpose, we
designed a task-specific rehabilitation therapy in which, starting at
1 week after injury, rats spent 1 h per d over a grid of deep wells in
which they could reach and grasp seeds and sugar pellets placed at the
bottom and use their fingers and mouth to open the seeds (Supple-
mentary Fig. 2 and Supplementary Video 2). The rats generally
engaged in this task for the full hour, leaving behind many seed shells
on the cage floor, which gave an indication of the involvement of the
rats in the training (Supplementary Fig. 2; the time line of the
experiment is shown in Fig. 3).
By 7 dpo, both rat groups had largely lost the ability to grasp pellets
in the staircase. During the following 42 d, Pen-treated rats that
received rehabilitation recovered no better than rats that received
no rehabilitation (Fig. 4a). In contrast, rats that received ChABC
with specific rehabilitation showed a large improvement in the task
VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2009 NATURE NEUROSCIENCE
 ARTICLES

a C1 Penicillinase treated – Neurocan C8 d food pellets, the eating of which did not
require skilled paw function (Supplementary
Fig. 2 and Supplementary Video 2). Either
ChABC or Pen was given at the time of injury
and afterwards and rats received 1 h of general
rehabilitation per day starting at 7 dpo. The
b Chondroitinase treated-1B5
e 6–8 rats placed in the general rehabilitation
cage actively participated for the full hour,
running, climbing, exploring and eating with
no differences in activity level between treat-
ment groups (Supplementary Fig. 4).
Chondroitinase treated – Neurocan As in the previous groups, all of the rats had
c largely lost the ability to pick up pellets from
f
the staircase by 7 dpo. However, in contrast
with all of the other groups, which showed a
modest recovery over 6 weeks, rats receiving
general, nonspecific rehabilitation completely
lost the ability to pick up pellets from the
staircase (Fig. 5a,b). The same tendency was
Figure 2 To study CSPG digestion after ChABC treatment, we killed a set of rats by perfusion the day seen in the Whishaw apparatus, with few
© 2009 Nature America, Inc. All rights reserved.

after the last intrathecal injection. (a) Longitudinal section of the cervical enlargement of a Pen-treated successful reaches through the window and
rat stained with antibody to neurocan showing the distribution of this CSPG, prominently in the gray
hardly any pellets being retrieved (Fig. 5c,d).
spinal cord matter. (b) Spinal cords treated with ChABC were positive for 1B5 CS stub in the digested
area. Glycosaminoglycan (GAG) chain digestion affected both the gray and white matter, extending from In contrast, in the ladder walking task, both
the lower end of C2 to C5. (c) Immunostaining in alternate sections showed that the area digested with of the groups of rats that received general
ChABC did not stain for neurocan. Scale bar represents 1,000 mm. (d) Higher magnification image of rehabilitation walked over the ladders faster
the box in b showing GAG digestion in the periphery of the white matter. Scale bar represents 100 mm. and made fewer forepaw errors than rats that
(e,f) Higher magnification images of the boxes in c. In the area where there is no GAG digestion, received either no rehabilitation or specific
perineuronal structures were observable surrounding gray matter neurons (e). However, in areas in which
rehabilitation, with no difference occurring
GAGs were digested with ChABC, these structures were not present (f). Scale bars represent 100 mm.
between ChABC- and Pen-treated rats (Fig. 5f).
The ChABC-treated rats that were given
general rehabilitation showed greater forepaw
and had achieved about two thirds of their pre-lesion score by 42 dpo grip strength than the other rats (Fig. 5h). The rats of all the experi-
(Fig. 4a and Supplementary Video 3). This was accompanied by a mental groups showed sensory impairment when measured with the
marked improvement in manual dexterity, demonstrated by a greater plantar Von Frey filament test, but none showed abnormal sensory
proportion of pellets being picked up and eaten compared with the sensitivity indicative of allodynia (Supplementary Fig. 5).
number removed from the wells and dropped (Fig. 4b). Notably, these
differences were not the results of differences in motivation between Sprouting of corticospinal axons
the groups, because the time spent reaching was the same (Supplemen- We studied anatomical changes in the lesioned dorsal CST and in the
tary Fig. 3). much smaller unlesioned lateral and ventral CST following BDA
At 42 dpo, in the Whishaw skilled paw-reaching apparatus, rats that injection bilaterally into the forepaw representation area of the sensor-
received specific rehabilitation combined with either Pen or ChABC imotor cortex (Fig. 6). We quantified CST sprouting behavior by
treatment showed substantial recovery (Fig. 4c,d). The success of counting the number of branching axons in the lateral white matter
rehabilitated rats in this task was achieved in many cases by learning and by counting CST axons crossing the gray matter–white matter
an alternative reaching strategy, in which rats scooped up the pellets boundary in segments at the site of injury and above and below it,
rather than grasping them from above, which presumable required less similar to a previous study17 (Fig. 6c–h). In all ChABC-treated rats,
manual dexterity. Comparing reaching strategies between the two regardless of rehabilitation therapy, we observed an increase in the
groups of rehabilitated rats, the ChABC-treated rats had largely number of branching and crossing axons at the site of injury compared
recovered normal grasping from above with a pronated paw, whereas with control Pen-treated rats (Fig. 6d,g). Rehabilitation did not further
the Pen-treated rats tended to use the scooping strategy (Fig. 4e–g). increase sprouting over and above ChABC treatment. The increased
In the two specific rehabilitation groups, ChABC-treated rats showed sprouting was restricted to the enzyme-digested perilesional area,
an increased forepaw contact placing response, but there were no where there was increased branching of unlesioned CST axons and
differences in ladder walking or grip strength (see below). Grip strength ChABC or Pen
was no different to that of rats that received no rehabilitation, so this treatment
cannot explain the improvement in paw reaching in the ChABC- 0 7 10 42 49 77 dpo
treated and specific rehabilitation group.
SCI Last ChABC/Pen CST Perfusion
Effects of nonspecific rehabilitation combined with ChABC Begin staircase
Begin infusion tracing
training
We next asked whether general environmental enrichment could achieve rehabilitation
Pre-injury Last behavior
the same effects as specific forelimb rehabilitation (ChABC-general and behavior testing testing
7d

Pen-general groups). We placed rats for 1 h per day in a large cage

containing ladders, beams, hanging ropes and platforms and large Figure 3 Diagram showing the time line of the experiments.

NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2009 1147

 ARTICLES

Figure 4 Effects of task-specific rehabilitation. (a) After SCI, rats lost the
a 20
b 100 **
* ** ability to retrieve pellets in the staircase task. Only the rats treated with
ChABC and specific rehabilitation showed substantial recovery. The results
Pellets retrieved

15 75
xxx from Figure 1f (no rehabilitation) are shown in light gray. Statistical

Accuracy
++
x
10
x + *** 50 differences were found between the ChABC-treated with rehabilitation group
+
*** * and other groups at the later time points (x, P o 0.05 versus Pen treatment
**
5 25 without rehabilitation (Pen no rehab); xxx, P o 0.01 versus Pen no rehab; +,
P o 0.05 versus ChABC treatment without rehabilitation (ChABC no rehab);
0
++, P o 0.01 versus ChABC no Rehab; *, P o 0.05 versus Pen treatment
PRE 7 14 21 28 35 42

no ChA b

ec
re BC
re en

ec C
ha
with specific rehabilitation (Pen spec); **, P o 0.01 versus Pen spec;

Pe ab

sp B
sp
no P

hA
h
Pen ChABC Pen spec ChABC spec

C
***, P o 0.001 versus Pen spec). (b) The rats treated with ChABC and
specific rehabilitation showed improved dexterity in the staircase task, as
c 100 *** d 50
***
demonstrated by increased accuracy and calculated as the number of pellets
* retrieved and eaten divided by the number of pellets removed from the wells
Number of reaches

* ** 40 *
through window

Pellets retrieved

75 (*, P o 0.05; **, P o 0.01). (c,d) After injury, the rats that received specific
* **
30 rehabilitation showed improved pellet retrieval in the Whishaw apparatus.
50
20
They were better than controls in their ability to extend their paws through
the window and efficiently reach the pellets (*, P o 0.05; **, P o 0.01;
25
10 ***, P o 0.001). (e) Rats normally retrieve pellets in the Whishaw apparatus
0 0 by grasping from above with a supinated paw. (f) After ChABC treatment and
specific rehabilitation, rats usually regained the normal supinated grasping
n b

b
re en

re en

sp n
ec

re BC
re C

ec C

ec C
Pe ha

ha
Pe

C b
b
B

sp AB

ec
no P

sp B
no P
sp

ha
ha

no ChA
no ChA

hA

strategy. (g) Rats treated with Pen and specific rehabilitation regained the
h
C

ability to retrieve pellets, but they used an abnormal scooping strategy. Values
© 2009 Nature America, Inc. All rights reserved.

Pellet retrieval strategy - normal and learned compensation are shown as mean ± s.e.m.
Non-injured Reaching strategy
e 100
Percentage
of reaches

75 function. We addressed this question using a rat cervical dorsal column

50
injury as the lesion and skilled forepaw reaching ability and other
25
0 forepaw tasks as the outcome measures. Injury to the CST leads to a
Chondroitinase + specific rehabilitation
Pronation scooping severe loss of manual dexterity in mice18, rats6,16, cats19, monkeys20 and
100
f humans21. The key functional role that the motor cortex and the CST
Percentage
of reaches

75
50 has in human motor control makes it important to find treatments that
25 enhance the recovery of CST function, and skilled paw reaching is the
0
Pronation scooping best test in rodents6,16. Dorsal funiculus lesions affect both the CST and
Penicillinase + specific rehabilitation
100 sensory axons that convey fine touch and mechanoception. However,
g
Percentage
of reaches

75 it is the CST lesion that is critical for the impairment of skilled paw
50 function because rats with dorsal column lesions that preserve the CST
25
0
fully recover skilled grasping22.
Pronation scooping In both our experiments and previous work, rats showed little
spontaneous recovery in skilled paw reaching after a dorsal column
crossing of axons into the gray matter. There was no increase in either lesion, although there was recovery in other tasks that require less skill.
sprouting measure in the undigested or partially digested segments two Promoting spinal cord plasticity by itself, as we did by injecting ChABC
spinal levels further rostral or caudal to the injury. into the spinal cord, produced only modest recovery in skilled paw
There was a modest amount of axon regeneration in all of the function in this and a previous study15, although there was an
ChABC-treated groups. Examination of the lesion area and the absence improvement in contact placing, a reflex that requires CST function.
of PKCg- and BDA-stained axons below the lesion indicated that all Reasoning that plasticity may not be useful unless appropriate con-
of the dorsal CST axons were lesioned. In ChABC-treated rats, nections are selected by training, we developed two rehabilitation
there were fine tortuous axons close to the site of injury, around regimens to drive plastic changes.
the cavity and below it (Supplementary Fig. 6). These may have been A specific rehabilitation treatment in which rats reached down into
regenerated axons, as they could be seen coming from the cut end wells to pick up small seeds was applied for an hour each day. This
of the CST and took a trajectory around the cavity. In the C6 spinal training specifically reinforces skilled reaching performance. Specific
segment below the injury, more CST axons and more arborizations in rehabilitation alone produced no improvement in the staircase reach-
the gray matter were found in the ChABC-treated rats. This far from ing task, but did produce some improvement in the Whishaw skilled
the cavity, it is not possible to differentiate between regenerated axons paw reaching apparatus17. However, when specific rehabilitation was
and processes that have sprouted in from the unlesioned lateral CST. combined with ChABC treatment, the rats showed markedly better
No differences in numbers of axons were found that correlated with manual dexterity, picking up more and dropping fewer pellets from the
the type of rehabilitation treatment. We counted the density of sero- staircase. The combination of ChABC treatment and specific rehabi-
tonergic axons 1 mm above and below the lesion cavities. The density litation also allowed the rats to recover the ability to grasp pellets from
was higher in ChABC-treated rats, indicating increased sprouting above with a pronated paw in the Whishaw apparatus, whereas rats that
(Supplementary Fig. 7). A summary of the behavioral and histological received specific rehabilitation alone used an abnormal scooping
differences between groups is shown in Table 1. movement. Optimal recovery therefore requires a combination of
ChABC-induced sprouting and rehabilitation to mould the new con-
DISCUSSION nections. What might the combination of ChABC treatment and
We sought to test whether promoting spinal cord plasticity with specific rehabilitation be achieving over and above either treatment
ChABC makes it possible for rehabilitation to bring back useful alone? Our anatomical observations suggest that ChABC treatment

1148 VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2009 NATURE NEUROSCIENCE

 ARTICLES

Figure 5 Effects of general rehabilitation. (a) ChABC- and Pen-treated rats

a 20 b 100
with general rehabilitation lost their ability to retrieve pellets in the staircase
Pen gen *
80
Pellets retrieved

15 ChABC gen ** test (results from the no rehabilitation groups in Fig. 1f are shown in gray

Accuracy
Pen no rehab 60 * for comparison). Both ChABC- and Pen-treated rats without rehabilitation
10 ChABC no rehab
40 showed mild recovery, but the rats that received general rehabilitation were
5 significantly worse (*, P o 0.05, Pen treatment with general rehabilitation
* * 20
(Pen gen) + ChABC treatment with general rehabilitation (ChABC gen) versus
0 Pen no rehab + ChABC no rehab). (b) Rats treated with general rehabilitation

no ChA b
PRE 7 14 21 28 35 42

n
re en

n C
re BC
ha

ge

ge B
attempted the staircase task and were able to flick some pellets out of their

Pe b
no P

ha

hA
n
C
wells, but could not retrieve them. This is reflected in a lower accuracy of
retrieval at 42 dpo (*, P o 0.05, Pen gen + ChABC gen versus Pen no rehab
c 50
Reaching
d 15 + ChABC no rehab; **, P o 0.01, ChABC gen versus ChABC no rehab;
*, P o 0.05, Pen gen versus Pen no rehab). (c,d) The results at 42 dpo in
Number of reaches

40 Pellets retrieved
through window

* * 10 * * the Whishaw apparatus were similar to those from the staircase test. Rats
30 with general rehabilitation had a limited ability to extend their paws through
20 the window (*, P o 0.05), but were unable to successfully reach (c) and
5
retrieve (d) the pellets. Again, their performance was worse than the rats with
10
no rehabilitation in this task (*, P o 0.05). (e) The ladder walking test
0 0 evaluates the abilities of the rats to place their forepaws correctly on the
no ChA b

n
re en

re en

n C
re BC

n C

re BC
ha

ge

ha

ge
rungs while walking across the ladder. (f) After injury, the rats of each of the
ge B
Pe b

Pe b
ge B
no P

no P
ha

ha

hA
hA

no hA
n

n
C
C

experimental groups made more mistakes. At 42 dpo, rats with general

rehabilitation performed better than rats with no rehabilitation or with
specific rehabilitation (*, P o 0.05; **, P o 0.01). (g) To test forepaw grip
© 2009 Nature America, Inc. All rights reserved.

e f 30 Pre-injury 7 dpo 42 dpo

strength, we had the rats grab a bar connected to a force transducer. The rats
25 ** are pulled by the tail and held on to the bar until their grip failed. (h) The
*
forepaw missteps

20 * forepaw grip strength of all of the rats was decreased at 7 dpo and only the
Number of

15
rats treated with ChABC and general rehabilitation showed greater strength
than the other experimental groups at 42 dpo (***, P o 0.01 versus all the
10 groups). Values are shown as mean ± s.e.m.
5

0
ChABC – + – + – + – + – + – + – + – + – +
Pen + – + – + – + – + – + – + – + – + – we conclude that promoting plasticity had opened a window during
which rehabilitation becomes more effective. Similarly, lizards that
g h 600 Pre-injury 7 dpo 42 dpo retain regenerative ability into adulthood are able to regenerate their
500 *** visual connections, but behavioral training is needed to make those
Max grip strength

400
connections functional25. Also, direct stimulation of the cortex or
amphetamine combined with rehabilitation has also produced better
300
recovery as a combined treatment after stroke26,27.
200
There is evidence that general rehabilitation in which the level of
100 physical activity increases can produce behavioral improvements in
0 many conditions. Proposed mechanisms include the increase of neuro-
ChABC – + – + – + – + – + – + – + – + – +
Pen + – + – + – + – + – + – + – + – + – trophic factors in the spinal cord28, modified electrical properties of
Pen no rehab Pen spec Pen gen
motoneurons29, alteration in neuronal energy balance30 and enhanced
ChABC no rehab ChABC spec ChABC gen formation of new neuronal circuits17. It seemed probable that these
mechanisms alone or combined with ChABC treatment to promote
promotes greater sprouting of the few unlesioned CST axons in the plasticity might lead to improved CST function after SCI. We therefore
lateral and ventral tracts23, but only close to the lesion in the region of devised a nonspecific rehabilitation treatment in which rats spent an
chondroitinase digestion. Two possibilities are that rehabilitation drives hour each day being very active in a large cage with ladders, climbing
the formation of appropriate connections in this segment of the spinal ropes, toys and large food pellets. As expected, this general rehabilitation
cord or that cortical rearrangements caused by rehabilitation17,24 was sufficient to improve the rats’ performance on ladder walking,
enable rats to make better use of the new spinal connections. Overall, which is driven mainly by the lateral and ventral spinal tracts, which

Table 1 Summary of behavioral and histological differences between groups

Staircase Reaching in Whishaw Placing Ladder Grip Sensory CST CST

reaching task apparatus response walking strength threshold sprouting regeneration

Pen No rehabilitation — — — — — — — —
Specific rehabilitation — * — — — — — —
General rehabilitation + — — ** — — — —
ChABC No rehabilitation — * ** — — — ** **
Specific rehabilitation ** ** * — — — ** **
General rehabilitation + + * ** ** — ** **

Improvements are indicated by an up arrow, larger improvements by two up arrows and decrements by down arrows. Overall, rats treated with ChABC showed greater behavioral
improvement over the range of tasks. Rehabilitation treatments affected the behaviors that were practiced; thus, paw reaching rehabilitation improved only paw reaching and
general rehabilitation improved locomotor tasks. There was competition between the rehabilitation effects, with paw reaching being extinguished by general rehabilitation.

NATURE NEUROSCIENCE VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2009 1149

 ARTICLES

Figure 6 Corticospinal axonal plasticity. We studied the anatomical changes

a b of the CST axons by counting the number of branching axons in the lateral
white matter and by counting CST axons crossing the gray matter-white
matter boundary in segments at the site of injury (C4–C5) and above (C1–C3)
and below (C6–C7) the site of injury. (a,b) Images of traced CST axons at the
level of the injury sending collateral branches (arrow), some of which crossed
the gray matter–white matter boundary (shown with dots and asterisks). Scale
bar represents 50 mm. (c–h) Quantification of axonal crossings (c–e) and
axonal sprouting (f–h) in the white matter at different spinal segments from
rats in the various treatment groups. Significant CST plasticity was observed
in the ChABC-treated rats only at the level of injury, with no effect of training
(P o 0.01). Values are shown as mean ± s.e.m.
c C1–C3 spinal segments
d C4–C5 spinal segments eC6–C7 spinal segments
3.0 2.0 ** 2.0 behavioral recovery. For human patients, it would be advantageous to
2.5 start the treatment after 2–4 weeks to coincide with the start of reha-
cm per traced fibers
Axon crossings per

1.5 1.5
2.0 bilitation. In addition, the variability of outcome following SCI becomes
1.5 1.0 1.0 much less a month or more after injury, making it possible to conduct
1.0
clinical trials with smaller patient numbers37. In previous experiments,
0.5 0.5 we induced plasticity and behavioral recovery in completely uninjured
0.5
spinal cords38, so it may be possible to extend the therapeutic window of
0.0 0.0 0.0
ChABC treatment and rehabilitation to 1 month or more after injury.
© 2009 Nature America, Inc. All rights reserved.

f g h There is also a limited window during which rehabilitation can be

2.0 2.0
** 2.0 effective in experimental stroke models, where rehabilitation started
within 3 d may exacerbate motor cortex cell death39, but can be
Axon sproutings per
cm per traced fibers

1.5 1.5 1.5

1.0 1.0
0.5 0.5
0.0 0.0
Pen no rehab Pen spec
ChABC no rehab ChABC spec
were not damaged31, and by serotonergic innervation32, which showed
increased sprouting following ChABC treatment. To our surprise,
this general environmental enrichment form of rehabilitation treatment,
either alone or combined with ChABC treatment, made rats worse at
skilled paw reaching in both the staircase and the Whishaw apparatus.
That training in one behavior can lead to the loss of another has been
suggested previously. Spinally transected cats could be trained to either
weight support or step, but successful stepping extinguished weight
support and vice versa33,34. Rodents with unilateral corticospinal and
rubrospinal injury that were trained for skilled reaching improved this
behavior, but made more missteps when running on a ladder17, and
training one paw can also reduce performance in the other following
cortical lesions in rodents35. Why might this interference have occurred
in our study? Two possibilities are that the limited number of new
connections that can be formed in the spinal cord become devoted to a
single behavior to the detriment of others following rehabilitation or
that rehabilitation-driven changes in the cortex may alter its ability to
use the small number of remaining CST connections. Taken together,
our results indicate that rehabilitation during a window of enhanced
plasticity can be much more effective than normal. However, rehabilita-
tion only enhances the functions that are practiced and may even worsen
behaviors that are not practiced.
The time window is critical for the successful therapeutic use of
treatments such as ChABC and rehabilitation. In our experiments, the
ChABC treatment was begun immediately after the lesion and continued
for 10 d, whereas rehabilitation began 7 d after the lesion. Active enzyme
remains for 10 d after injection and replacement of glycans in the
extracellular matrix takes up to 20 d36, so the matrix may have been
modified for around 30 d, which roughly corresponds to the timing of
ineffective if delayed by as much as 30 d after injury40.
1.0
We investigated anatomical changes in the CSTof our rats by tracing
the axons at the end of the experiment. As in previous experiments
0.5 with ChABC treatment, there appeared to be a modest amount
of axon regeneration around the lesion15,41. More relevant to functional
0.0 recovery was probably the sprouting of the small number of unlesioned
Pen gen
ChABC gen
CST axons, particularly those of the lateral CST (constituting
approximately 16% of the number of axons in the dorsal CST)23 into
the spinal cord gray matter23, which was increased in the digested
area close to the injury in all of the ChABC-treated groups, as in a
previous experiment42. These sprouts probably connected to pro-
priospinal circuits that relay down the cord43,44 and rubrospinal
sprouting may also have been substantial. Other treatments have
also been shown to enhance CST sprouting, including anti-NogoA,
inosine and neurotrophins45–47. ChABC digests chondroitin sulfate
proteoglycans (CSPGs) in perineuronal nets, structures that have
been associated with the restriction of plasticity at the end of
critical periods13,14.
Our experiments suggest that the ability of rehabilitation to promote
functional recovery after CNS damage can be greatly enhanced
by treatments that reactivate plasticity. However, the behavioral
improvements are restricted to the movements that are practiced and
successful reinforcement of one behavior may interfere with other
behaviors. The design and application of rehabilitation treatments will
be critical if they are applied during a window of plasticity.
METHODS
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/natureneuroscience/.
Note: Supplementary information is available on the Nature Neuroscience website.
ACKNOWLEDGMENTS
The authors acknowledge key advice and assistance from H. Steenson in animal
care and testing. This work was funded by grants from the Medical Research
Council, The Wellcome Trust, The Christopher and Dana Reeve Foundation
Consortium, The EU Framework 6 Network of Excellence NeuroNE, the Henry
Smith Charity and Action Medical Research.

1150 VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2009 NATURE NEUROSCIENCE


AUTHOR CONTRIBUTIONS
G.G.-A. and J.W.F. designed the experiments. G.G.-A., S.B. and M.B. performed
the experiments. G.G.-A. and J.W.F. analyzed the data and wrote the paper.
COMPETING INTERESTS STATEMENT
The authors declare competing financial interests: details accompany the full-text
HTML version of the paper at http://www.nature.com/natureneuroscience/.
Published online at http://www.nature.com/natureneuroscience/.
Reprints and permissions information is available online at http://www.nature.com/
reprintsandpermissions/.
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1151
 ONLINE METHODS
All procedures were performed in compliance with the UK Animals (Scientific
Procedures) Act 1986 and institutional guidelines.
Spinal cord injury and drug delivery. Male Lister Hooded rats (250–300 g)
were anaesthetized with halothane/nitrous oxide. We performed a C4 dorsal
laminectomy, inserted the tips of sharpened forceps (2 mm in depth and 2 mm
apart) between the dorsal roots and held them there for 20 s, leading to a cut
extending between the dorsal roots15. Immediately after, a glass micropipette
(outer diameter, 30 mm) was introduced into the gray matter 1 mm rostral and
then 1 mm caudal to the injury, lowered 1 mm and 1 ml of ChABC at 100 U ml–1
(Seikagaku) or Pen (Sigma) was injected at a rate of 6 ml h–1. Through an
opening at the cisterna magna, a 32 gauge catheter (ReCathCo) was inserted
intrathecally, with the tip lying on top of the injury. The rats received five
injections of ChABC (Acorda Therapeutics) or Pen (3 ml, 100 U ml–1 per
injection), one every 2 d following the first injection.
Rehabilitation. For specific rehabilitation, the rats were placed from 7 d after
injury for an hour a day in a cage, the floor of which was a plastic grid with
square (1.7
1.7 cm) openings 2.2 cm deep. In the grid bottom were seeds that
the rats could retrieve by extending and grasping with their forepaws.
© 2009 Nature America, Inc. All rights reserved.
For general rehabilitation, rats were placed in an enriched environment cage,
with ladders, beams, ropes and ramps. Food pellets were positioned at the top
of the cage to encourage the rats to explore the cage and to use the devices. The
training started 7 d after the injury and was performed for 1 h, 5 d per week.
Some sessions were recorded and the positions of that rats were recorded every
10 min to ensure that all animals and groups participated equally in the activity.
Experimental groups. The rats were divided into six experimental groups. All
of the rats received a dorsal funiculi cut in the C4 spinal segment, received
ChABC or Pen infusion and no rehabilitation, specific rehabilitation or general
rehabilitation: ChABC treatment without rehabilitation (n ¼ 6), Pen treatment
without rehabilitation (n ¼ 10), reaching and grasping task-specific rehabi-
litation, ChABC treatment with specific rehabilitation (n ¼ 13), Pen treat-
ment with specific rehabilitation (n ¼ 17), or performed general motor
rehabilitation with ChABC treatment (n ¼ 6) or Pen treatment (n ¼ 8).
Because of the large numbers, the experiment was performed in two stages.
Each stage contained control rats that received ChABC or Pen without
rehabilitation. There was no difference in behavioral recovery between the
controls in the two stages.
Behavioral assessment. For the forepaw reaching tasks, rats were trained to
grasp and eat sugar pellets from a staircase device over 15 min prior to injury15.
In the staircase reaching task, we scored the number of pellets displaced from
the wells, the number dropped in the apparatus and, from this, the number of
pellets retrieved and eaten. We calculated the percentage of displaced pellets
that were successfully retrieved (accuracy). We also monitored the time spent
attempting to retrieve pellets.
For the Whishaw apparatus, the rats, without any prior training at the
last time point of the behavioral evaluation, were placed for 5 min in a
chamber as described previously48 and sugar pellets were placed on the
platform and replaced when eaten. We scored the number of times the rats
extended their paw through the narrow opening and the number of pellets
they retrieved.
To test the forelimb placing response, we held the rats horizontally with the
forelimbs suspended. The rats were advanced slowly until the dorsum of the
forepaw touched the edge of a table. Normally, rats show a placing response by
extending the forepaw digits and putting them on the table46. The task was
repeated five times and the number of responses elicited was counted.
To test grip strength, we used a lateralized grip strength meter (Linton
Instruments). Rats were allowed to grip a left and right horizontal bar with each
forepaw and were then pulled away until the grip was released. The force
exerted on the bar at the time of release was measured. The rats were given
three trials per session and the mean average of the right and left forepaw
strength was calculated15.
For ladder walking, the rats walked over a ladder with unequally spaced rungs.
The number of times that a forepaw slipped between the rungs was counted.
NATURE NEUROSCIENCE
Corticospinal tract tracing and histological assessment. At the end of the
functional evaluation, the rats received a stereotaxic injection of 0.5 ml of
10% BDA (wt/vol) at coordinates (+2, ±3), (+1, ±3,5), (1, ±2) and
(0, ±2) (anterior/posterior, medial/lateral). We killed the rats by perfusion
4 weeks later. Each spinal cord was cut into five blocks compromising
C1, C1–C3, C3–C5, C5–T1 and T2 spinal segments, frozen and processed
for immunohistochemistry.
To verify the injury to the corticospinal tract, we stained transverse sections
of the C1 and T2 spinal segments with antibody to PKCg (1:250, Chemicon)
and Alexa Fluor 488 (1:500, Jackson Immunoresearch). For BDA visualization,
alternate longitudinal sections were stained with Alexa Fluor 568–conjugated
streptavidin (1:500) and GFAP-conjugated Cy3 (1:200, Sigma) or processed by
the avidin-biotin amplification method using peroxide as a substrate (Vectas-
tain ABC Elite Kit, Vector) and stained with diaminobenzidine and NiCl2.
To confirm CSPG GAG digestion, we killed by perfusion four rats that
received ChABC and two rats that received Pen intrathecal infusion the day
after the last infusion. A spinal block from C1 to C8 was removed and
processed. Alternate sections were immunostained with monoclonal antibody
to chondroitin sulfate DDi-0S, which recognizes unsulfated CSPG stubs
produced following chondroitinase digestion (1.200, Seikagaku) and mouse
monoclonal antibody to neurocan (1:3, DSHB), followed by biotinylated goat
antibody to mouse (1:500, Vector), and were amplified by the avidin-biotin
method using peroxide as a substrate (Vectastain ABC Elite Kit, Vector) and
stained with diaminobenzidine and NiCl2.
Staining and quantification of serotonergic processes. Longitudinal sections
from the C3–C5 spinal segments were washed in free-floating in phosphate-
buffered saline with 0.3% Triton X-100 (wt/vol, Fluka) and 5% goat serum
(vol/vol, Biological Industries) for 1 h. The spinal sections were incubated
overnight at 4 1C with rabbit polyclonal antibodies to 5-HT (Immunostar) in
Triton X phosphate-buffered saline (PBST) and 1% goat serum (vol/vol). After
several washes, the sections were incubated with secondary antibodies, goat
antibody to rabbit (1:500, Alexa Fluor conjugated, Invitrogen), overnight at
4 1C. Following additional washes, sections were mounted on gelatin-coated
slides and visualized under the microscope using appropriate filters. Two lines
were drawn transversely between the lateral edges of the gray matters at 1 mm
above and 1 mm below the lesion cavity. All 5-HT–positive fibers crossing these
lines were counted.
Lesion size and CST axon quantification. From the C3–C5 spinal block, one of
nine sections was stained with cresyl violet and the width and area of the cavity
was measured using ImageJ (US National Institutes of Health), showing no
difference in lesion width or area between groups. We used width as the best
indicator of lesion extent because the functional deficit after dorsal spinal cord
lesions depends on the lateral extent and damage to the lateral gray matter49.
Corticospinal BDA traced axons were quantified from the C1–C3, C3–C5 and
C5–T1 spinal blocks.
Corticospinal axonal sprouting and crossing between the white matter of the
lateral funiculi and gray matter was quantified in three serial sections from the
C1–C3, C3–C5 and C5–T1 spinal blocks. For sprouting, we counted every
instance in each area of analysis of a CST axon branching to send a
process medially or laterally. For crossing, we counted every instance of a
stained process crossing the boundary between the lateral white matter and
the dorsal gray matter. From this, an overall figure for crossing axons was
calculated for each rat by dividing the sum of CST crossings by the number
of total CST fibers traced and the length of the section studied, as
described previously22.
Statistical analysis. Data are shown as mean ± s.e.m. The behavioral data was
analyzed by two-way ANOVA and Bonferroni post hoc analysis. Histological
data was analyzed by t test when there were two groups, and one-way ANOVA
with post hoc analysis for multiple groups.
48. Whishaw, I.Q., Pellis, S.M., Gorny, B., Kolb, B. & Tetzlaff, W. Proximal and distal
impairments in rat forelimb use in reaching follow unilateral pyramidal tract lesions.
Behav. Brain Res. 56, 59–76 (1993).
49. Yamamoto, M., Raisman, G. & Li, Y. Loss of directed fore-limb reaching after destruction
of spinal grey matter. Brain Res. 1265, 47–52 (2009).
doi:10.1038/nn.2377