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Chondroitinase ABC treatment opens a window of opportunity for task-specific rehabilitation

© 2009 Nature America, Inc. All rights reserved.

Guillermo Garcı´a-Alı´as, 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. 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 primarily 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 proteoglycans 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 investigated 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 anatomical sprouting, but there was only recovery of skilled paw function if ChABC treatment was combined with specific rehabilitation. General environmental enrichment rehabilitation enhanced locomotor function, 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 immunopositive 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. ([email protected]). Received 17 February; accepted 26 June; published online 9 August 2009; doi:10.1038/nn.2377

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Figure 1 ChABC or penicillinase (Pen) without rehabilitation. (a) Transverse 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 at the level of the injury. The ventral and lateral tracts cannot be seen at this magnification. (d) Following injury, rats were able to maintain their posture, locomote and hold objects with their forepaws. (e) The staircase device. Animals slide into the tunnel, gaining access to the left and right staircase. They grasp sugar pellets for 15 min. Supplementary Video 3 shows rats performing this task. (f) Following dorsal funiculus injury, there were severe impairments in the rats’ abilities to perform skilled reaching, as tested in the staircase test. The rats went from grasping 14 pellets prior 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 on the platform. To retrieve them, rats must extend their paw through a narrow window, grasp the pellet and pick it up. Pellets are replaced 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 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 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 with Pen did not (P o 0.001). Values are shown as mean ± s.e.m.

extent of maximal chondroitinase digestion, we killed four ChABCtreated 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).

Whishaw’s skilled paw reaching apparatus (Fig. 1g). In this task16, rats have to reach through a small window to pick up a sugar pellet on a shelf. In this test, ChABC-treated rats performed better than Pentreated rats (Fig. 1h,i); they showed a greater ability to extend their 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 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 different paw reaching task that they had not encountered before using

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 (Supplementary 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

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ARTICLES 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 6–8 rats placed in the general rehabilitation e cage actively participated for the full hour, running, climbing, exploring and eating with no differences in activity level between treatment groups (Supplementary Fig. 4). As in the previous groups, all of the rats had Chondroitinase treated – Neurocan 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 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 In contrast, in the ladder walking task, both area. Glycosaminoglycan (GAG) chain digestion affected both the gray and white matter, extending from of the groups of rats that received general the lower end of C2 to C5. (c) Immunostaining in alternate sections showed that the area digested with rehabilitation walked over the ladders faster ChABC did not stain for neurocan. Scale bar represents 1,000 mm. (d) Higher magnification image of 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 much smaller unlesioned lateral and ventral CST following BDA tary Fig. 3). At 42 dpo, in the Whishaw skilled paw-reaching apparatus, rats that injection bilaterally into the forepaw representation area of the sensorreceived 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 ChABC0 7 10 42 49 77 dpo treated and specific rehabilitation group.

© 2009 Nature America, Inc. All rights reserved.

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Effects of nonspecific rehabilitation combined with ChABC We next asked whether general environmental enrichment could achieve the same effects as specific forelimb rehabilitation (ChABC-general and Pen-general groups). We placed rats for 1 h per day in a large cage containing ladders, beams, hanging ropes and platforms and large

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Percentage of reaches

DISCUSSION We sought to test whether promoting spinal cord plasticity with ChABC makes it possible for rehabilitation to bring back useful

function. We addressed this question using a rat cervical dorsal column injury as the lesion and skilled forepaw reaching ability and other forepaw tasks as the outcome measures. Injury to the CST leads to a severe loss of manual dexterity in mice18, rats6,16, cats19, monkeys20 and humans21. The key functional role that the motor cortex and the CST has in human motor control makes it important to find treatments that enhance the recovery of CST function, and skilled paw reaching is the best test in rodents6,16. Dorsal funiculus lesions affect both the CST and sensory axons that convey fine touch and mechanoception. However, it is the CST lesion that is critical for the impairment of skilled paw function because rats with dorsal column lesions that preserve the CST fully recover skilled grasping22. In both our experiments and previous work, rats showed little spontaneous recovery in skilled paw reaching after a dorsal column lesion, although there was recovery in other tasks that require less skill. Promoting spinal cord plasticity by itself, as we did by injecting ChABC into the spinal cord, produced only modest recovery in skilled paw function in this and a previous study15, although there was an improvement in contact placing, a reflex that requires CST function. Reasoning that plasticity may not be useful unless appropriate connections are selected by training, we developed two rehabilitation regimens to drive plastic changes. A specific rehabilitation treatment in which rats reached down into wells to pick up small seeds was applied for an hour each day. This training specifically reinforces skilled reaching performance. Specific rehabilitation alone produced no improvement in the staircase reaching task, but did produce some improvement in the Whishaw skilled paw reaching apparatus17. However, when specific rehabilitation was combined with ChABC treatment, the rats showed markedly better manual dexterity, picking up more and dropping fewer pellets from the staircase. The combination of ChABC treatment and specific rehabilitation also allowed the rats to recover the ability to grasp pellets from above with a pronated paw in the Whishaw apparatus, whereas rats that received specific rehabilitation alone used an abnormal scooping movement. Optimal recovery therefore requires a combination of ChABC-induced sprouting and rehabilitation to mould the new connections. What might the combination of ChABC treatment and specific rehabilitation be achieving over and above either treatment alone? Our anatomical observations suggest that ChABC treatment

Percentage of reaches

© 2009 Nature America, Inc. All rights reserved.

e

14

Pellets retrieved

Pen

7

no P re en ha no ChA b re BC h Pe ab n sp ec C hA sp B ec C

0 PRE

Figure 4 Effects of task-specific rehabilitation. (a) After SCI, rats lost the ability to retrieve pellets in the staircase task. Only the rats treated with ChABC and specific rehabilitation showed substantial recovery. The results from Figure 1f (no rehabilitation) are shown in light gray. Statistical 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 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); ++, P o 0.01 versus ChABC no Rehab; *, P o 0.05 versus Pen treatment with specific rehabilitation (Pen spec); **, P o 0.01 versus Pen spec; ***, P o 0.001 versus Pen spec). (b) The rats treated with ChABC and specific rehabilitation showed improved dexterity in the staircase task, as demonstrated by increased accuracy and calculated as the number of pellets retrieved and eaten divided by the number of pellets removed from the wells (*, P o 0.05; **, P o 0.01). (c,d) After injury, the rats that received specific rehabilitation showed improved pellet retrieval in the Whishaw apparatus. 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; ***, P o 0.001). (e) Rats normally retrieve pellets in the Whishaw apparatus by grasping from above with a supinated paw. (f) After ChABC treatment and specific rehabilitation, rats usually regained the normal supinated grasping strategy. (g) Rats treated with Pen and specific rehabilitation regained the ability to retrieve pellets, but they used an abnormal scooping strategy. Values are shown as mean ± s.e.m.

crossing of axons into the gray matter. There was no increase in either sprouting measure in the undigested or partially digested segments two spinal levels further rostral or caudal to the injury. There was a modest amount of axon regeneration in all of the ChABC-treated groups. Examination of the lesion area and the absence of PKCg- and BDA-stained axons below the lesion indicated that all of the dorsal CST axons were lesioned. In ChABC-treated rats, there were fine tortuous axons close to the site of injury, around the cavity and below it (Supplementary Fig. 6). These may have been regenerated axons, as they could be seen coming from the cut end of the CST and took a trajectory around the cavity. In the C6 spinal segment below the injury, more CST axons and more arborizations in the gray matter were found in the ChABC-treated rats. This far from the cavity, it is not possible to differentiate between regenerated axons and processes that have sprouted in from the unlesioned lateral CST. No differences in numbers of axons were found that correlated with the type of rehabilitation treatment. We counted the density of serotonergic axons 1 mm above and below the lesion cavities. The density was higher in ChABC-treated rats, indicating increased sprouting (Supplementary Fig. 7). A summary of the behavioral and histological differences between groups is shown in Table 1.

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35

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Number of reaches through window

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21

28

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Reaching

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15 Pellets retrieved

c

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5

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no P re en ha no ChA b re BC ha Pe b n ge n C hA ge B n C

f Number of forepaw missteps

30

Pre-injury

7 dpo

42 dpo

** * *

25 20 15 10 5

0 ChABC – + – + – + – + – + – + – + – + – + Pen + – + – + – + – + – + – + – + – + –

g

h Max grip strength

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0

0

e

10

Figure 5 Effects of general rehabilitation. (a) ChABC- and Pen-treated rats with general rehabilitation lost their ability to retrieve pellets in the staircase test (results from the no rehabilitation groups in Fig. 1f are shown in gray for comparison). Both ChABC- and Pen-treated rats without rehabilitation showed mild recovery, but the rats that received general rehabilitation were significantly worse (*, P o 0.05, Pen treatment with general rehabilitation (Pen gen) + ChABC treatment with general rehabilitation (ChABC gen) versus Pen no rehab + ChABC no rehab). (b) Rats treated with general rehabilitation attempted the staircase task and were able to flick some pellets out of their 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 + 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 the Whishaw apparatus were similar to those from the staircase test. Rats with general rehabilitation had a limited ability to extend their paws through the window (*, P o 0.05), but were unable to successfully reach (c) and retrieve (d) the pellets. Again, their performance was worse than the rats with no rehabilitation in this task (*, P o 0.05). (e) The ladder walking test evaluates the abilities of the rats to place their forepaws correctly on the rungs while walking across the ladder. (f) After injury, the rats of each of the 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 strength, we had the rats grab a bar connected to a force transducer. The rats are pulled by the tail and held on to the bar until their grip failed. (h) The forepaw grip strength of all of the rats was decreased at 7 dpo and only the 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 groups). Values are shown as mean ± s.e.m.

600

Pre-injury

7 dpo

42 dpo

***

500 400 300 200 100

0 ChABC – + – + – + – + – + – + – + – + – + Pen + – + – + – + – + – + – + – + – + – Pen no rehab ChABC no rehab

Pen spec ChABC spec

Pen gen ChABC gen

promotes greater sprouting of the few unlesioned CST axons in the lateral and ventral tracts23, but only close to the lesion in the region of chondroitinase digestion. Two possibilities are that rehabilitation drives the formation of appropriate connections in this segment of the spinal cord or that cortical rearrangements caused by rehabilitation17,24 enable rats to make better use of the new spinal connections. Overall,

we conclude that promoting plasticity had opened a window during which rehabilitation becomes more effective. Similarly, lizards that retain regenerative ability into adulthood are able to regenerate their visual connections, but behavioral training is needed to make those connections functional25. Also, direct stimulation of the cortex or amphetamine combined with rehabilitation has also produced better recovery as a combined treatment after stroke26,27. There is evidence that general rehabilitation in which the level of physical activity increases can produce behavioral improvements in many conditions. Proposed mechanisms include the increase of neurotrophic factors in the spinal cord28, modified electrical properties of motoneurons29, alteration in neuronal energy balance30 and enhanced formation of new neuronal circuits17. It seemed probable that these mechanisms alone or combined with ChABC treatment to promote plasticity might lead to improved CST function after SCI. We therefore devised a nonspecific rehabilitation treatment in which rats spent an hour each day being very active in a large cage with ladders, climbing ropes, toys and large food pellets. As expected, this general rehabilitation was sufficient to improve the rats’ performance on ladder walking, which is driven mainly by the lateral and ventral spinal tracts, which

Table 1 Summary of behavioral and histological differences between groups

Pen

ChABC

Staircase reaching task

Reaching in Whishaw apparatus

Placing response

Ladder walking

Grip strength

Sensory threshold

CST sprouting

CST regeneration

No rehabilitation Specific rehabilitation

— —

— *

— —

— —

— —

— —

— —

— —

General rehabilitation 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.

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a

C1–C3 spinal segments 3.0

d C4–C5 spinal segments eC6–C7 spinal segments 2.0

**

2.0

2.5 1.5

1.5

1.0

1.0

0.5

0.5

0.0

0.0

0.0

2.0

2.0

1.5

1.5

1.5

1.0

1.0

1.0

0.5

0.5

0.5

2.0 1.5 1.0 0.5

f Axon sproutings per cm per traced fibers

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Axon crossings per cm per traced fibers

c

Figure 6 Corticospinal axonal plasticity. We studied the anatomical changes 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.

b

g

h **

0.0

0.0

0.0 Pen no rehab ChABC no rehab

2.0

Pen spec ChABC spec

Pen gen ChABC gen

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, rehabilitation 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

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behavioral recovery. For human patients, it would be advantageous to start the treatment after 2–4 weeks to coincide with the start of rehabilitation. In addition, the variability of outcome following SCI becomes much less a month or more after injury, making it possible to conduct clinical trials with smaller patient numbers37. In previous experiments, we induced plasticity and behavioral recovery in completely uninjured spinal cords38, so it may be possible to extend the therapeutic window of ChABC treatment and rehabilitation to 1 month or more after injury. There is also a limited window during which rehabilitation can be effective in experimental stroke models, where rehabilitation started within 3 d may exacerbate motor cortex cell death39, but can be ineffective if delayed by as much as 30 d after injury40. We investigated anatomical changes in the CSTof our rats by tracing the axons at the end of the experiment. As in previous experiments with ChABC treatment, there appeared to be a modest amount of axon regeneration around the lesion15,41. More relevant to functional recovery was probably the sprouting of the small number of unlesioned 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 propriospinal 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.

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ARTICLES 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/.

© 2009 Nature America, Inc. All rights reserved.

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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23. Steward, O., Zheng, B., Ho, C., Anderson, K. & Tessier-Lavigne, M. The dorsolateral corticospinal tract in mice: an alternative route for corticospinal input to caudal segments following dorsal column lesions. J. Comp. Neurol. 472, 463–477 (2004). 24. Ramanathan, D., Conner, J.M. & Tuszynski, M.H. A form of motor cortical plasticity that correlates with recovery of function after brain injury. Proc. Natl. Acad. Sci. USA 103, 11370–11375 (2006). 25. Beazley, L.D. et al. Training on a visual task improves the outcome of optic nerve regeneration. J. Neurotrauma 20, 1263–1270 (2003). 26. Adkins, D.L., Hsu, J.E. & Jones, T.A. Motor cortical stimulation promotes synaptic plasticity and behavioral improvements following sensorimotor cortex lesions. Exp. Neurol. 212, 14–28 (2008). 27. Hovda, D.A. & Fenney, D.M. Amphetamine with experience promotes recovery of locomotor function after unilateral frontal cortex injury in the cat. Brain Res. 298, 358–361 (1984). 28. Ying, Z., Roy, R.R., Edgerton, V.R. & Gomez-Pinilla, F. Exercise restores levels of neurotrophins and synaptic plasticity following spinal cord injury. Exp. Neurol. 193, 411–419 (2005). 29. Petruska, J.C. et al. Changes in motoneuron properties and synaptic inputs related to step training after spinal cord transection in rats. J. Neurosci. 27, 4460–4471 (2007). 30. Plunet, W.T. et al. Dietary restriction started after spinal cord injury improves functional recovery. Exp. Neurol. 213, 28–35 (2008). 31. Anderson, K.D., Gunawan, A. & Steward, O. Quantitative assessment of forelimb motor function after cervical spinal cord injury in rats: relationship to the corticospinal tract. Exp. Neurol. 194, 161–174 (2005). 32. Ribotta, M.G. et al. Activation of locomotion in adult chronic spinal rats is achieved by transplantation of embryonic raphe cells reinnervating a precise lumbar level. J. Neurosci. 20, 5144–5152 (2000). 33. de Leon, R.D., Hodgson, J.A., Roy, R.R. & Edgerton, V.R. Locomotor capacity attributable to step training versus spontaneous recovery after spinalization in adult cats. J. Neurophysiol. 79, 1329–1340 (1998). 34. De Leon, R.D., Hodgson, J.A., Roy, R.R. & Edgerton, V.R. Full weight–bearing hindlimb standing following stand training in the adult spinal cat. J. Neurophysiol. 80, 83–91 (1998). 35. Allred, R.P. & Jones, T.A. Maladaptive effects of learning with the less-affected forelimb after focal cortical infarcts in rats. Exp. Neurol. 210, 172–181 (2008). 36. Lin, R., Kwok, J.C., Crespo, D. & Fawcett, J.W. Chondroitinase ABC has a long-lasting effect on chondroitin sulphate glycosaminoglycan content in the injured rat brain. J. Neurochem. 104, 400–408 (2008). 37. Fawcett, J.W. et al. Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP panel: spontaneous recovery after spinal cord injury and statistical power needed for therapeutic clinical trials. Spinal Cord 45, 190–205 (2007). 38. Galtrey, C.M., Asher, R.A., Nothias, F. & Fawcett, J.W. Promoting plasticity in the spinal cord with chondroitinase improves functional recovery after peripheral nerve repair. Brain 130, 926–939 (2007). 39. Kozlowski, D.A., James, D.C. & Schallert, T. Use-dependent exaggeration of neuronal injury after unilateral sensorimotor cortex lesions. J. Neurosci. 16, 4776–4786 (1996). 40. Biernaskie, J., Chernenko, G. & Corbett, D. Efficacy of rehabilitative experience declines with time after focal ischemic brain injury. J. Neurosci. 24, 1245–1254 (2004). 41. Bradbury, E.J. et al. Chondroitinase ABC promotes functional recovery after spinal cord injury. Nature 416, 636–640 (2002). 42. Garcı´a-Alı´as, G. et al. Recovery of hindlimb motor function after a lateral spinal cord hemisection in adult rats is enhanced by combined administration of NT-3, NMDA-2D subunits and chondroitinase-ABC. Soc. Neurosci. Abstr. 646, 21 (2006). 43. Courtine, G. et al. Recovery of supraspinal control of stepping via indirect propriospinal relay connections after spinal cord injury. Nat. Med. 14, 69–74 (2008). 44. Lemon, R.N. & Griffiths, J. Comparing the function of the corticospinal system in different species: organizational differences for motor specialization? Muscle Nerve 32, 261–279 (2005). 45. Blo¨chlinger, S., Weinmann, O., Schwab, M.E. & Thallmair, M. Neuronal plasticity and formation of new synaptic contacts follow pyramidal lesions and neutralization of NogoA: a light and electron microscopic study in the pontine nuclei of adult rats. J. Comp. Neurol. 433, 426–436 (2001). 46. Benowitz, L.I., Goldberg, D.E., Madsen, J.R., Soni, D. & Irwin, N. Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury. Proc. Natl. Acad. Sci. USA 96, 13486–13490 (1999). 47. Grill, R., Murai, K., Blesch, A., Gage, F.H. & Tuszynski, M.H. Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J. Neurosci. 17, 5560–5572 (1997).

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ONLINE METHODS All procedures were performed in compliance with the UK Animals (Scientific Procedures) Act 1986 and institutional guidelines.

© 2009 Nature America, Inc. All rights reserved.

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. 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 rehabilitation, ChABC treatment with specific rehabilitation (n ¼ 13), Pen treatment 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.

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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 (Vectastain 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 phosphatebuffered 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