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Neuroscience 139 (2006) 1495–1506

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EHAVIORAL RECOVERY FROM UNILATERAL PHOTOTHROMBOTICNFARCTS OF THE FORELIMB SENSORIMOTOR CORTEX IN RATS:

OLE OF THE CONTRALATERAL CORTEX

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. V. SHANINA,a T. SCHALLERT,b O. W. WITTEa

ND C. REDECKERa*

Department of Neurology, Friedrich-Schiller-University, Erlangerllee 101, D-07747 Jena, Germany

Department of Psychology, University of Texas at Austin, Universitytation #A8000, 108 E Dean Keaton, Austin, TX 78712, USA

bstract—During sensorimotor recovery following strokepsi- and contralesional alterations in brain function haveeen characterized in patients as well as animal models ofocal ischemia, but the contribution of these bilateral pro-esses to the functional improvement is only poorly under-tood. Here we examined the role of the homotopic contralat-ral cortex for sensorimotor recovery after focal ischemicnfarcts at different time periods after the insult. One group ofnimals received a unilateral single photothrombotic infarctn the forelimb sensorimotor cortex, while four additionalroups received a second lesion in the contralateral homo-opic cortex either immediately or 2 days, 7 days, or 10 daysfter the first infarct. The time course of functional recoveryf the impaired forelimbs was assessed using different sen-orimotor scores: forelimb-activity during exploratory behav-or and frequency of forelimb-sliding in the glass cylinder asell as forelimb misplacement during grid walking. Focal

nfarcts in the forelimb sensorimotor cortex area significantlympaired the function of the contralateral forelimb in theseifferent behavioral tests. The subsequent damage of theontralateral homotopic forelimb sensorimotor cortex onlyffected the forelimb opposite to the new lesion but did noteinstate the original deficit. The time course of sensorimotorecovery after bilateral sequential cortical infarcts did notignificantly differ from animals with unilateral single lesions.hese data indicate that following small ischemic cortical

nfarcts in the forelimb sensorimotor cortex the contralateralortex homotopic to the lesion plays only a minor role forunctional recovery. © 2006 Published by Elsevier Ltd onehalf of IBRO.

ey words: forelimb, rat, recovery, sensorimotor cortex,troke.

ecovery of sensorimotor functions following stroke re-uires reorganization of central sensory and motor net-orks (Nudo and Friel, 1999). Several activation patternscross these networks have been characterized using

Corresponding author. Tel: �49-3641-9323430; fax: �49-3641-9323422.-mail address: redecker@med.uni-jena.de (C. Redecker).bbreviations: CTRL, sham-operated control animals; DL, double-

esioned animals with sequential bilateral infarcts; FLA, forelimb activ-ty; FL SMC, forelimb sensorimotor cortex; Fr1, primary motor cortex;

CAO, middle cerebral artery occlusion; MRI, magnetic resonance

imaging; PBS, phosphate-buffered saline; SL, single-lesioned animalsith a unilateral infarct.

306-4522/06$30.00�0.00 © 2006 Published by Elsevier Ltd on behalf of IBRO.oi:10.1016/j.neuroscience.2006.01.016

1495

unctional magnetic resonance imaging (MRI) (for reviewee Calautti and Baron, 2003), but very little is knownbout the contribution of these activated brain regions tohe recovery process. Better understanding of the topog-aphy of these adaptive mechanisms which the brain uti-izes to compensate the sensorimotor deficit will presum-bly lead to the development of more effective rehabilita-ive treatment strategies.

Several functional MRI studies in patients with hemi-aretic stroke clearly demonstrated bilateral activation pat-erns during movements of the affected hand involvingontralesional premotor or motor cortical areas (Johansen-erg et al., 2002; Seitz et al., 1998; Weiller et al., 1992). Itas therefore suggested that these alterations in the un-amaged hemisphere contribute to the motor recovery.xperimental findings support these observations demon-trating bilateral functional MRI activation patterns alsofter middle cerebral artery occlusion (MCAO) in rats (Di-

khuizen et al., 2001). Electrophysiological studies re-ealed bihemispheric hyperexcitability (Buchkremer-Ratz-ann et al., 1996) associated with widespread alterations

n excitatory and inhibitory receptor function and expres-ion following focal brain ischemia in rats (Que et al.,999a,b; Redecker et al., 2002). Morphological studiesurther showed an increase of axonal sprouting and den-ritic arborization in the contralesional cortex (Biernaskiend Corbett, 2001; Jones, 1999) that might be due toompensatory motor learning associated with over-reli-nce on the unimpaired forelimb (Jones and Schallert,994; Kozlowski and Schallert, 1998). A recent study byiernaskie et al. (2005) further supported the functional

elevance of the contralesional cortex for functional recov-ry. The authors observed reinstatement of the previousunctional deficit when they blocked neuronal activity bypplication of lidocaine into the contralateral cortex 3–4eeks after MCAO. In addition, a second injury to theomotopic contralesional cortex in the developing brainppeared to cause bilateral deficits consistent with take-ver of function (Barth et al., 1990; Barth and Stanfield,990).

The extent to which these contralesional alterationsontribute to functional recovery is further debatable be-ause many experimental and clinical studies clearly dem-nstrate that significant adaptive changes take place in theirect vicinity of the infarct (Butz et al., 2004; Chollet et al.,991; Loubinoux et al., 2003; Nudo et al., 1996). Perile-ional structural reorganization leading to an enlargementnd rearrangement of receptive fields has been described

n the somatosensory cortex of monkeys and rats (Jenkins

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E. V. Shanina et al. / Neuroscience 139 (2006) 1495–15061496

nd Merzenich, 1987; Schiene et al., 1999; Reinecke et al.,003), in the motor cortex of monkeys (Frost et al., 2003)nd the visual cortex of cats (Eysel and Schweigart, 1999;epeda et al., 2004). In these studies the cortical repre-entations shifted toward the vicinity of the lesion. Ablationf these perilesional areas in rats showing functional re-overy after a previous motor cortex lesion reinstated thenitial functional deficit (Castro-Alamancos and Borrel,995).

We here analyzed the role of the homotopic contralat-ral cortex for functional recovery using sequential bilateralortical infarcts in the forelimb sensorimotor cortex (FL SMC)nd behavioral analysis of sensorimotor performance. Inrder to elucidate time-dependent effects of the contrale-ional cortex, different groups of rats received sequentialhotothrombotic lesions at different delays between the

esions (immediately consecutive, 2, 7, or 10 days) and aattery of more recently developed behavioral tests (Bartht al., 1990; Jones and Schallert, 1994; Schallert et al.,000). Using this approach the present study demon-trates that the contralesional cortex is only of minor im-ortance for the functional improvement after circum-cribed cortical lesions.

EXPERIMENTAL PROCEDURES

nduction of photothrombotic infarcts

his study was performed in accordance with the guidelines of theational Institutes of Health (revised 1987) and all experimentalrocedures were approved by the German Animal Care and Useommittee. All efforts were made to minimize the number ofnimals and their suffering during the experiments. Focal cortical

nfarcts were induced in adult male Wistar rats (n�41, 280�30 g)sing the photothrombosis model as described by Watson et al.1985) with only minor modifications (Shanina et al., 2005). Briefly,nimals were anesthetized with 2.0% enflurane in oxygen/nitrousxide (1:2) and were placed in a stereotaxic frame. A midline

ncision was made through the scalp between the eyes and earsnd the skin was displaced to both sides to provide a clear skullurface. A fiberoptic bundle (2.4 mm diameter) connected to theold light source (Schott KL 1500, Mainz, Germany) was posi-ioned on the skull 0.5 mm anterior to Bregma and 3.7 mm lateralo the midline, aligned with stereotaxic coordinates correspondingo the FL SMC (Zilles, 1985). A light was then turned on for 20 minnd the dye Rose Bengal (1.3 mg/100 g body weight), dissolved

n saline (1 mg/100 �l), was injected into the tail vein during therst minute of illumination. Rectal temperature was kept constantt 37.0�0.2 °C. Sham procedure was performed identically with-ut illumination of the skull (n�6, 280�30 g).

In order to analyze the time-dependent effects of the contrale-ional cortex on functional recovery the following experimentalesign was used (see Fig. 1A). The animals were divided into fivexperimental groups: in one group of animals (n�8) a singlenilateral photothrombotic infarct (SL) was induced, while fourdditional groups received a second lesion on the contralateralide immediately (double-lesioned animals with sequential bilat-ral infarcts, DL0, n�6), or 2 days (DL2, n�10), 7 days (DL7,�8), and 10 days (DL10, n�9) after the first infarct. The secondhotothrombotic lesion was located in the homotopic cortex of theontralateral hemisphere. The side for the induction of the firsthotothrombotic infarct was varied in all groups. Sham-operated

nimals served as controls (CTRL, n�6). a

ime schedule of behavioral tests

n order to assess the baseline performance all animals receivedbattery of sensorimotor tests preoperatively (forelimb preferencend sliding test, foot-fault test). The same tests were used tonalyze the functional sensorimotor deficit at different postopera-ive time points: immediately at the first day after the infarct inrder to test the severity of the deficit and then weekly to assesshe time-course of functional recovery. Due to the different delaysetween the first and the second lesion, the intervals betweenhe behavioral tests slightly varied in the experimental groupsFig. 1A). The animals in the CTRL, SL, and DL0 groups wereested on days 1, 7, 14, and 21 after the surgery. In the DL2roup animals received postoperative tests on day 1 after therst lesion and day 1 after the second infarct corresponding toay 3 after the first one. The testing was then continued on day(9), day 12 (14), and day 19 (21) (Fig. 1A). For the DL7 group

he tests were performed on day 1 and day 5 after the firsturgery, and then again on day 1 after the second infarct asell as on days 7 (14), 14 (21), and 21 (28), respectively. TheL10-animals were tested on day 1 and day 7 after the first

esion and on days 1 (11), 14 (24), and 21 (31) after the secondesion (Fig. 1A).

orelimb preference and sliding test

orelimb use during spontaneous vertical exploration was ana-yzed based on the method described by Schallert et al. (2000).he rats were videotaped in a transparent glass cylinder for 5–7in depending on the degree of activity during the trial (Fig. 1B).wo mirrors combined at an angle of 90° were placed behind thelass cylinder allowing the recording of forelimb movements evenhen the animal turned away from the camera. Several behaviorallements were scored to determine the extent of forelimb impairmenturing spontaneous exploration of the glass cylinder. The indepen-ent or simultaneous use of the left or right forelimb was analyzed (a)t first contact with the wall and (b) during vertical and horizontalovements along the wall; and (c) also sliding movements of each

orelimb at the wall of the cylinder were scored (Fig. 1B). Forelimbctivity (FLA) was evaluated for each forelimb using the followingormula:

FLA�first contact � horizontal � vertical

number of rearings

In addition, the frequency of sliding movements (%) whichccurred during vertical activity at the wall of the glass cylinderas assessed for each forelimb using the following score:

sliding score (%)�sliding

first contact � horizontal � vertical�100

In order to illustrate the time course of alterations in theseehavioral tests data are in part given as percentage differenceetween preoperative baseline and different time points after the

nfarcts.

oot-fault test

s an additional sensorimotor behavioral test we used the foot-ault test which allowed the assessment of asymmetry in limb-useor fore- and hindlimbs on an elevated grid (Barth et al., 1990). Thenimals have to coordinate paw movements to gain footholds onhe wire frame. Inaccurate placement leads to a paw slippinghrough the openings in the grid (i.e. a foot-fault). Naive animalsarely show mistakes which are symmetric for both limbs. Allnimals had to walk five times from one end of the grid to theome cage at the other end. The movements were recorded using

videotape and analyzed in slow-motion offline for every limb

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E. V. Shanina et al. / Neuroscience 139 (2006) 1495–1506 1497

eparately. A foot-fault score (%) was calculated for every limbsing the following formula:

foot faults per limb

B

A

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1. infarct

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1.2. infarct

2. infarct

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ig. 1. (A) Schematic diagram of the experimental design. The gray hrrows indicate the induction of the photothrombotic infarcts in animalsL2, DL7, DL10). The white bars mark the time points of the behavioetween the experimental groups. (B) Series of video recordings of tharked with a black circle.

foot fault score�steps per limb

�100s

istological processing

or histological verification of lesion position animals were killed1 day after the surgery (SL and CTRL animals) or after the

(d)21 28

landing

t verticalmovement

sliding

arrows represent the different experimental groups. The black verticaland two DL lesions with different intervals between the lesions (DL0,(forelimb activity and sliding test, foot-fault-test) which slightly differ

b preference and sliding test. Characteristic behavioral elements are

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farct

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orizontalwith SL

ral testinge forelim

econd lesion, respectively (DL0, DL2, DL7 and DL10). Animals

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E. V. Shanina et al. / Neuroscience 139 (2006) 1495–15061498

ere deeply anesthetized (2% enflurane in N2O/O2, 2:1) andranscardially perfused with 100 ml phosphate-buffered salinePBS) followed by 450 ml of 4% paraformaldehyde fixative dis-olved in PBS. Brains were removed, postfixed for 24 h in theame fixative, and submerged in 10% sucrose in PBS for 24 h.he tissue was then transferred to a 30% sucrose/PBS solution for4–48 h. Sixty micrometer thick coronal sections were cut usingfreezing microtome and stained with Cresyl Violet. The extent of

he lesions was evaluated and compared with reference to theaxinos and Watson (1986) rat brain atlas and with Zilles’ (1985)tlas of rat cortex.

tatistical analysis

ll data are presented as mean�S.E.M. unless otherwiseoted. Behavioral alterations to the preinfarct baseline weressessed with nonparametric Wilcoxon matched pairs test us-

ng SPSS 11.5 for Windows (SPSS Inc., Chicago, IL, USA) andonferroni-Holm post hoc test for multiple comparisons (alpha-

evel: 0.05). Differences between the experimental groups weressessed using the Kruskal-Wallis test (nonparametric analysisf variance) followed by Dunn’s multiple comparisons post-test.level of P�0.05 was considered statistically significant.

RESULTS

orphology of the photothrombotic infarcts

nly animals with clear cortical infarcts were included inhis study. In the DL0 group two animals died due toomplications during the surgery and one animal was dis-

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ig. 2. Location and morphology of the photothrombotic infarcts in aocation of the lesions marked with dotted lines in animals from the SLocations on the modified rat brain atlas of Paxinos (1995). All lesion lo

ote that all infarcts affected the FL SMC and parts of the Fr1. Coronal Nissl-sta

nvolved in all cortical layers while the subcortical white matter remained intact

arded from the DL2 group because only a unilateral lesionas detected. The other experimental animals with singlenilateral photothrombotic lesion (SL, n�8) or two bilateralequential lesions (DL0, n�4; DL2, n�9; DL7, n�8; DL10,�9) recovered quickly without suffering from weight lossr any severe functional impairment. Histological process-

ng revealed well-defined cortical infarcts in all SL- or DL-nimals. There was only very little variability in lesion sizeetween the different experimental groups which was volu-etrically analyzed in detail in a recent study (Shaninat al., 2005). The lesions were consistently located in theL SMC with some extensions to the primary motor cortexFr1) and only little involvement of the somatosensoryortex (Par1) according to the rat brain atlas from Paxinosnd Watson (1986), Paxinos (1995) and Zilles (1985) (Fig.). The infarcts affected all cortical layers without damageo the underlying white matter (Fig. 2). In sham-operatedTRL no structural alterations were observed.

ehavioral performance in sham-operated CTRL

fter placing to the glass cylinder, sham-operated CTRLn�6) actively explored the cylinder, reared and supportedheir body against the walls with their forelimbs. Sham-perated animals did not show any asymmetry in forelimbse when they initiated a rearing movement. They equallysed either the right or the left forepaw to push off from theround. For vertical explorative activity, i.e. the first contact

2 m

m2

mm

r1

r1

ith SL (A) and DL lesions (B). The photographs on the left show thegroup. The schematic diagram in the middle demonstrates the lesionf the SL and DL2 group are transferred to the atlas and superposed.

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Fr1

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Fr1

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nimals wand DL2cations o

ined brain sections are displayed on the right showing that the infarcts. The borders of the infarcts were marked with dotted lines.

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E. V. Shanina et al. / Neuroscience 139 (2006) 1495–1506 1499

ith the cylinder wall after rearing and the following move-ents along the wall, all CTRL rats used both forelimbs

ymmetrically. Sliding of a forelimb occurred only in ap-roximately 20% of movements in sham-operated CTRLith no limb difference. The animals also did not show anyignificant asymmetry in forelimb use when they landedith their paws on the ground of the cylinder. In addition,ham-operated animals demonstrated only few misplace-ents before and after the surgery when they walkedlong the grid in the foot-fault test (Table 1).

able 1. Behavioral scores in the different experimental groups

roup Forelimb activity Sli

1st FL 2nd FL 1s

Mean�SEM Mean�SEM Me

TRL (n�8)Baseline 3.4�1.3 2.9�1.4 23Day 1 3.1�1.3 2.8�1.4 18Day 7 3.0�0.7 3.2�1.1 18

L (n�8)Baseline 3.4�0.8 3.4�1.4 22Day 1 1.5�0.7* 3.4�1.4 59Day 7 2.5�0.5 4.9�1.5 50Day 14 3.4�1.1 4.3�1.1 31Day 21 3.2�1.3 4.3�1.2 21L0 (n�4)Baseline 3.0�1.3 2.2�0.6 18Day 1 1.8�0.8 1.5�0.3 46Day 7 1.8�1.1 2.2�1.0 48Day 14 1.8�0.7 2.1�0.6 32Day 21 1.6�0.8 2.5�0.7 28L2 (n�9)Baseline 2.9�1.0 2.9�1.5 22Day 1 2.0�1.2 2.8�1.3 53Day 1(3) 2.0�0.7 1.6�0.6 54Day 7(7) 2.1�1.4 2.4�1.2 34Day 12(14) 2.1�0.7 2.5�0.8 23Day 19(21) 1.8�0.7 2.1�1.0 25L7 (n�8)Baseline 2.8�0.9 2.6�1.1 23Day 1 0.9�0.4** 2.8�1.5 65Day 5 2.0�0.7 3.3�1.2 41Day 1(8) 1.1�0.5** 1.2�0.7** 46Day 7(14) 2.2�0.9 2.3�1.5 37Day 14(21) 2.0�0.9* 2.5�0.9 23Day 21(28) 1.9�0.8 2.1�0.7 33

L10 (n�9)Baseline 3.1�1.4 3.0�1.1 21Day 1 2.1�1.0 3.1�1.9 54Day 7 2.9�1.1 3.9�1.6 37Day 1(11) 2.9�0.8 1.7�0.6 24Day 7(17) 2.7�0.4 1.8�1.0 29Day 14(24) 2.8�0.6 2.1�0.6 30Day 21(31) 2.4�0.2 1.8�1.0 29

Data are given as mean�SEM. For the forelimb activity (FLA) theovements a score is presented as percentage of numbers of forelimovements). The numbers given for the foot-fault test represent the pe

ignificant differences to baseline are indicated with asterisks * P �0.05

oc test).

unctional recovery after unilateral single andilateral sequential lesions

ll animals with SL or DL photothrombotic infarcts (DL0,L2, DL7, DL10) demonstrated transient functional deficitsf the affected forelimbs in the glass cylinder and foot-faultest. In animals with sequential cortical lesions the timeourse of functional recovery of the primarily impairedorelimb did not significantly differ from animals with onlyingle infarcts. Compared with unilaterally lesioned ani-als the progression of functional improvement in DL-

Foot-fault test

2nd FL 1st FL 2nd FL

Mean�SEM Mean�SEM Mean�SEM

29.1�14.4 5.5�3.6 6.0�2.221.5�5.3 6.9�4.0 6.8�3.519.5�9.0 6.3�3.8 8.8�7.5

31.8�9.6 8.8�5.4 6.1�4.4* 23.0�10.2 25.7�7.0*** 10.1�5.7

21.3�12.3 15.1�6.1 6.8�3.124.1�8.4 7.3�5.0 5.9�3.814.9�8.8 10.1�6.1 10.1�2.0

28.4�10.3 6.8�5.2 6.9�3.642.0�23.5 28.6�6.8* 17.5�7.939.3�7.9 10.5�5.0 10.5�7.126.9�13.1 18.2�4.2 16.6�8.917.9�11.1 16.6�10.2 11.2�5.7

23.1�9.9 7.1�4.6 8.0�4.7* 20.5�11.6 25.6�5.7*** 6.2�4.0* 64.2�25.2* 19.2�5.6** 14.3�3.6**

26.7�17.6 13.5�4.9* 10.5�5.316.5�14.6 9.6�6.8 11.8�6.222.0�19.1 8.5�5.2 11.4�6.0

23.3�10.1 6.2�3.6 9.8�2.7** 18.9�13.2 22.4�6.0*** 7.9�2.9* 17.2�15.7 16.7�5.8*** 9.5�6.9* 63.6�16.9*** 13.1�4.7 20.8�4.1***

35.8�16.6 13.5�3.3** 15.2�7.922.1�13.0 10.7�6.1 15.8�6.028.8�13.2 8.9�2.0 8.8�2.5

22.7�8.5 6.5�4.8 6.9�2.4* 21.1�11.4 22.6�10.3** 5.1�4.2* 24.6�12.1 14.4�5.0 4.5�4.8

58.5�19.7*** 9.1�7.2 20.7�5.9**40.8�18.6 9.6�2.7 18.9�7.8**31.2�13.9 10.3�7.3 9.4�5.834.4�15.0 9.9�7.7 11.4�3.7

of forelimb movements on each side per rear is shown. For slidingper total number of movements (first touch, horizontal and vertical

of foot-faults of each forelimb to the total number of steps. Statistically.01, *** P �0.001; Wilcoxon matched-pairs test, Bonferroni-Holm post

ding

t FL

an�SEM

.0�14.3

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.6�18.8

.5�12.4

.3�13.0

.3�17.1

.3�12.1

.4�22.0*

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E. V. Shanina et al. / Neuroscience 139 (2006) 1495–15061500

nimals varied to some extent but reached similar scorest 2 weeks after the first infarct in the forelimb activity andliding as well as the foot-fault tests (Figs. 3-5). In allroups the functional deficits detected by the behavioralests nearly completely recovered to baseline levels within–4 weeks after the injury.

Statistical analysis of forelimb use during vertical ex-loration of the glass cylinder (Fig. 3A) revealed a signifi-ant decrease in use of the impaired forelimb in SL animalst day 1 after the photothrombotic infarct compared withhe preoperative level (P�0.016, Wilcoxon matched-pairsest, Bonferroni-Holm post hoc test) (Fig. 3B). This deficitompletely disappeared within 2 weeks. SL-animals in-reasingly used the unimpaired forelimb until day 21 afterhe lesion (Fig. 3B). All animals with DL (DL0, DL2, DL7,L10) demonstrated a similar deficit of the affected fore-

imb after the first lesion accompanied by an increasedctivity of the ipsilesional unimpaired paw as described innimals with SL (Table 1; Fig. 3B). After induction of the

first contact vertical or horizontal movements

days after lesion

*

10 5 20 25105

-50

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0

50

100

FL1 (impaired)

FL2 (unimpaired)

FL

A d

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ren

ce(%

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SLB

A

ig. 3. Forelimb-activity in the glass cylinder test in animals with SL aypical forelimb activity after rearing. The animal touched the wall of thovements of the same paw. (B) Diagrams of the relative alterations in

evels after cortical infarcts in the SL- (left) and DL10-animals (right). Tifferences to baseline (P�0.05, Wilcoxon matched-pairs test, BonferC) Diagram of the recovery of the primarily affected forelimb in differe

unctional recovery in SL-animals (gray zone reflects S.E.M.). Statistical analyseveal any significant difference between these groups. Data are given as mea

econd infarct in the contralateral cortex, the animalshowed a decreased use of the secondary affected fore-

imb which was previously unimpaired. The deficit of thisecondary impaired forepaw reached nearly the same ex-ent as observed for the primary affected forelimb (Table 1;ig. 3B). In all animals with sequential cortical lesionsDL0, DL2, DL7, DL10) the recovery of forelimb activity ofhe primarily affected paw was not significantly differentrom animals with SL (Fig. 3C), although the progression ofhe functional improvement varied to some extentP�0.05, Kruskal-Wallis test).

Quantitative evaluation of sliding movements (Fig. 4A)hich were observed during explorative activity on the wallf the glass cylinder revealed an immediate increase in theontralesional forelimb at day 1 after SL (P�0.016, Wil-oxon matched-pairs test, Bonferroni-Holm post hoc test)Table 1; Fig. 4B). The sliding score then decreased overhe next two weeks, reaching the preoperative level at day1 after the infarct. In the ipsilesional forelimb no signifi-

DL10

days after first lesion10 15 25205 35

FL1

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DL2

DL7

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farcts in the FL SMC. (A) Series of video recordings demonstrating ar with the left forelimb (black circle) followed by vertical and horizontalf the firstly (FL1) and the secondly affected forelimb (FL2) to baselinerrows mark the time-points of lesion induction. Statistically significantpost hoc test) are differentially indicated for the FL1 (*) and FL2 (#).of SL- and DL-animals. The fat line demonstrates the time course of

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E. V. Shanina et al. / Neuroscience 139 (2006) 1495–1506 1501

ant changes were detected. In animals with DL lesionsDL0, DL2, DL7, DL10) the functional deficit produced byhe first photothrombotic infarct was very similar comparedith the SL group (Table 1; Fig. 4C). The second lesion

hen caused a significant increase in the sliding score ofhe corresponding forelimb. Although the time course ofunctional improvement slightly varied between the firstlynd the secondly impaired forepaws in the different DLroups (DL0, DL2, DL7, DL10) no significant differencesave been detected (Kruskal-Wallis test) (Fig. 4B).

To assess deficits in coordinated limb placing we per-ormed the foot-fault test. All animals with single and se-uential infarcts demonstrated a significant deficit in fore-

imb placing with an increased number of foot-faults asompared with their preoperative level (Wilcoxon matched-airs test, Bonferroni-Holm post hoc test) (Table 1; Fig. 5).

days after lesion10 5 20 25105

FL1 (impaired)

FL2 (unimpaired)

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-100

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0

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sliding

ig. 4. Forelimb sliding in the glass cylinder test in animals with SL anliding-movement of the left forelimb (black circle) during vertical explornd the secondly affected forelimb (FL2) to baseline levels after corticime-points of lesion induction. Statistically significant differences to bre differentially indicated for the FL1 (*) and FL2 (#). (C) Diagram oL-animals. The fat line demonstrates the time course of function

Kruskal-Wallis test, Dunn’s post-test) did not reveal any significant d

n animals with SL lesions the foot-fault score of the con- d

ralateral forelimb significantly increased at day 1 after thenjury and then continuously recovered until day 14 (Fig.B). In animals with DL (DL0, DL2, DL7, DL10) similareficits were detected for both forelimbs after the first andecond lesion (Table 1; Fig. 5B and 5C). These ratshowed a significant increase in foot-fault score immedi-tely after lesion induction which recovered within 14–21ays. Interestingly, the functional deficit produced by theecond lesion was less pronounced in the DL2 group com-ared with the firstly impaired forelimb (P�0.021; Kruskal-allis test, Dunn’s multiple comparisons post-test) (Table 1).

nduction of the second lesion in the homotopic contrale-ional cortex did not reinstate the original functional deficit ofhe primarily impaired forelimb (Fig. 5B, C). Comparison ofhe time course of recovery between the different DLroups (DL0, DL2, DL7, DL10) did not reveal significant

0 5 10 15 20-50

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rcts in the FL SMC. (A) Series of video recordings illustrating a typicalvity. (B) Diagrams of the relative alterations in sliding of the firstly (FL1)

in the SL- (left) and DL10-animals (right). The gray arrows mark the�0.05, Wilcoxon matched-pairs test, Bonferroni-Holm post hoc test)very of the primarily affected forelimb in different groups of SL- andery in SL-animals (gray zone reflects S.E.M.). Statistical analysisbetween these groups. Data are given as mean�S.E.M.

C

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d DL infaative actial infarctsaseline (Pf the recoal recov

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ifferences (Kruskal-Wallis test) (Fig. 5B). Irrespective of

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E. V. Shanina et al. / Neuroscience 139 (2006) 1495–15061502

he interval between the lesions, the second infarct did notnfluence the functional performance of the initially im-aired forelimb.

DISCUSSION

ur study demonstrates that small ischemic infarcts in theL SMC produce significant sensorimotor deficits in con-

ralateral forelimb use and skilled walking. This functionalmpairment was transient and only detectable during therst two weeks after the infarct using the forelimb prefer-nce and coordinated limb placement tests. Induction of an

dentical infarct in the contralateral FL SMC at differentime points after the initial injury did not influence theunctional improvement of the primary impaired forepaw.hese findings suggest that the contralateral homotopicortex is only of minor relevance for the functional recovery

A

days after lesion0 15 20 25105

FL1 (impaired)

FL2 (unimpaired)

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300

ig. 5. Performance in the foot-fault test in animals with SL and DL infhrough the opening of the grid (black circle). (B) Diagrams of the relativorelimb (FL2) in SL- (left) and DL10-animals (right). The gray arrowsaseline (P�0.05, Wilcoxon matched-pairs test, Bonferroni-Holm posthe recovery of the primarily affected forelimb in different groups ofecovery in SL-animals (gray zone reflects S.E.M.). Statistical analysisetween these groups. Data are given as mean�S.E.M.

fter cortical brain infarcts. g

ensorimotor deficits after focal ischemicortical lesions

ollowing lesions in the FL SMC functional impairment ofhe corresponding forelimb has been previously describedn different injury models and behavioral tests. One sensi-ive parameter for functional impairment is the measure-ent of forelimb preference during exploratory behavior in

he glass cylinder (Jones and Schallert, 1992; Kozlowski etl., 1996; Metz et al., 2005; Schallert et al., 2000). Animalsreferentially use the unimpaired ipsilesional forepaw forearing and support during exploratory activity in the glassylinder (Schallert et al., 2000). In accordance with theserevious reports we observed asymmetric use of the fore-

imbs with significant impairment of the paw contralateral tohe lesion. Furthermore, animals with photothrombotic le-ions showed significantly increased sliding movements ofhe impaired forelimb during vertical exploration of the

0 5 10 15 20-100

0

300

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e FL SMC. (A) Photograph illustrating a typical slip of the left forelimbions in the foot-fault score of the firstly (FL1) and the secondly affected

time-points of lesion induction. Statistically significant differences toare differentially indicated for the FL1 (*) and FL2 (#). (C) Diagram of

DL-animals. The fat line demonstrates the time course of functional-Wallis test, Dunn’s post-test) did not reveal any significant difference

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lass cylinder. Sham-operated animals usually placed

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E. V. Shanina et al. / Neuroscience 139 (2006) 1495–1506 1503

heir forelimbs accurately on the wall of the glass cylindernd held it on the same position until the next movement.n contrast animals with infarcts in the FL SMC were notble to keep this position gliding down with their impairedorelimb. Increased sliding movements were only observedn the affected forelimb indicating that they were notaused by unspecific effects of the anesthesia and/or sur-ical procedure. Moreover, an increase in the sliding scoreid not occur after sham surgery. It is conceivable, that theliding movements were primarily caused by the paresisollowing corticospinal tract damage but also sensory im-airment might contribute to this dysfunction. Therefore,

ncreased sliding movements were a sensitive marker formpaired forelimb function. However, this increase in slid-ng movements was transient and declined to baseline atwo weeks after the infarct.

Animals with infarcts in the FL SMC additionallyhowed functional deficits in the foot-fault test duringkilled walking on a regular grid. This finding is in accor-ance with previous studies demonstrating that rats withMC lesions increasingly misplace the contralateral fore-

imb (Barth et al., 1990; Jones et al., 2003; Napieralski etl., 1998). Placing deficits were also caused by corticospi-al tract injury and impairment of sensory function. Inarticular, deficits in proprioception might occur and thenimals might lose the necessary feedback from the limbshen they slip too deep into the openings of the grid (Bartht al., 1990).

The tests used in our study focus on sensorimotorunction during exploratory behavior and skilled walkingnd allow the assessment of both forelimbs. A very sensi-

ive test for fine sensorimotor forelimb function is the singleellet reaching test (Whishaw et al., 1993) which has beenhown to detect even slight deficits in grasping after differ-nt brain lesions (Metz et al., 2005; Gharbawie et al., 2005;’Graggen et al., 2000) However, increasing evidence in-icates that the complex grasping movements learned inhis test require the integrity of both hemispheres (Bier-askie et al., 2005; Gonzalez et al., 2004). Moreover, theellet reaching task additionally necessitates a trainingeriod prior to the lesion and only analyzes the sensori-otor performance of a single (“trained”) forelimb. Since

here are currently no additional sensitive tests availablehich allow the assessment of fine sensorimotor functionsf both forelimbs we were not able to evaluate theseapabilities in the present study.

ole of the contralateral cortex for postlesionalunctional reorganization

e here provide further evidence that the contralesionalortex did not significantly contribute to the functional re-overy of the impaired forelimb. Lesions to the contralat-ral cortex homotopic to the initial infarct did not signifi-antly influence the functional improvement of the primarilyffected forelimb. This was a surprising finding since sev-ral experimental (Biernaskie and Corbett, 2001; Jonesnd Schallert, 1992; Jones, 1999) and clinical studiesButefisch et al., 2005; Cramer, 1999; Fisher, 1992; Fries

t al., 1993; Johansen-Berg et al., 2002) indicate that o

lterations contralateral to the damaged hemisphere pos-ibly facilitate sensorimotor recovery after brain lesions.urther support for this hypothesis came from a recenttudy by Biernaskie and colleagues (2005) analyzing sen-orimotor function after endothelin-1 induced MCAO in ratsnd single intracortical application of the anesthetic lido-aine into the sensorimotor contralesional cortex 3–4eeks later. Using this experimental approach they ob-erved a reinstatement of the initial forelimb deficit in theellet reaching task suggesting that the contralesional cor-ex is recruited for functional recovery after focal brainschemia. These findings are in contrast to a study byndersen et al. (1991) who analyzed the sensorimotorerformance in animals with a second MCAO inducedontralaterally 28 days after the first one. Following theecond infarct the animals showed a deficit in removing andhesive tape of the contralesional forelimb only, while the

nitially impaired limb was not (re-)affected. Using sequen-ial bilateral electrolytic lesions of the FL SMC and behav-oral analysis of coordinated forelimb placing Barth andolleagues (1990) demonstrated no reinstatement of the

nitial deficit too. Our findings are in accordance with theserevious reports (Barth et al., 1990; Andersen et al., 1991).

Several differences in the experimental design such ashe behavioral tests as well as permanent versus functionallockade of the contralesional cortex might explain the con-

roversial findings of Biernaskie et al. (2005). Furthermore,esion type and size as well as the precise location are crucialactors for the sensorimotor deficits of the corresponding limbut also for functional alterations on the contralesionalemisphere (Napieralski et al., 1998; Voorhies and Jones,002; Gonzalez and Kolb, 2003). Functional MRI studiesn chronic stroke patients clearly demonstrated a bilateralctivation when subcortical brain regions were involvedhile in patients with cortical lesions predominantly ipsile-ional activation was observed (Feydy et al., 2002; Luft etl., 2004). These data allow the hypothesis that the mech-nisms of reorganization are dependent on lesion location.onsiderable methodological differences complicate theirect transfer of functional activation patterns in humansoward behavioral findings after brain lesions in animals.owever, the animal data strongly support the importancef the lesion site for functional recovery. The endothelin--induced MCAO model used by Biernaskie et al. (2005)amaged the primary somatosensory cortex including thearrel fields and subcortical areas whereas a considerableart of the FL SMC located in the vicinity remained intact.he subcortical damage in this model causing bilateral

unctional alterations might contribute to the reinstatementf the previous sensorimotor deficit after contralesional

idocaine application. In our study as well as in the study byarth et al. (1990), ischemic injury was restricted to theensorimotor cortex leaving subcortical structures intact.ne possibility is that the reduced function in pellet reach-

ng by the forelimb ipsilateral to the first infarct after theecond infarct was due in part to impaired capacity in thepposite limb to support body weight essential to properse of the reaching forelimb. Pellet reaching requires use

f both limbs. The animals must aim the reaching forelimb

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recisely without visual guidance, a movement that de-ends heavily on motor function in the non-reaching fore-

imb (Metz et al., 2005; Whishaw et al., 2002).Functional MRI studies further indicate that contrale-

ional activation patterns are temporally limited (Cao et al.,998; Cramer et al., 1997; Luft et al., 2005). Dijkhuizen andolleagues (2003) demonstrated a bihemispheric activa-ion of FL SMC following sensory stimulation of the im-aired forelimb only 1 and 3 days after transient MCAO inats. At 2 weeks after the infarct, when functional recoveryn gross locomotor activity tests was completed, the stim-lation response only occurred in the ipsilesional hemi-phere. These findings indicate that the contralesional acti-ation patterns do not significantly correlate with functionalecovery. The transient activation of the contralesional cortex,hich was increasing with the size of the infarcts, might

eflect postischemic cortical hyperexcitability which definitelynvolves the undamaged hemisphere but probably also con-ributes to dysfunction and diaschisis (Buchkremer-Ratz-ann et al., 1996; Reinecke et al., 1999; von Monakow,914; Witte et al., 2000).

Several experimental and clinical studies suggest thateorganization of lost sensorimotor functions after corticalesions predominantly occurs in the direct vicinity of theamaged cortex (for review see Calautti and Baron, 2003;udo and Friel, 1999). Ablation of perilesional areas in ratshich showed functional recovery after a primary forelimbotor cortex lesion reinstated the initial functional impair-ent, while the same lesion in CTRL did not affect motor

orelimb function (Castro-Alamancos and Borrel, 1995).udo and colleagues further demonstrated in primates thatortical representations shift toward adjacent areas afterotor cortex lesions and that these processes are trainingependent (Frost et al., 2003; Nudo et al., 1996; Xerri etl., 1998). Similar processes occur after lesions in theisual cortex of kittens (Zepeda et al., 2004). Transcranialagnetic stimulation studies in humans further support the

nding that perilesional cortical areas are predominantlynvolved in functional reorganization after stroke (Cicinellit al., 1997; Liepert et al., 2000).

Our data indicate that following small ischemic corticalnfarcts in the FL SMC the contralateral cortex homotopico the lesion did not significantly influence sensorimotorecovery of the contralesional forelimb. Even when thentervals between the infarcts varied, no effect of the sec-nd infarct on functional recovery of the initially impaired

orelimb was observed. These results suggest that there iso critical period for the involvement of contralateral ho-otopic cortex in functional recovery during the first 10ays after the initial infarct. Together with the literature

hese findings might support the hypothesis that ipsile-ional cortical reorganization predominantly facilitatesunctional recovery after ischemic cortical infarcts, and thatontralesional cortex may contribute to learn motor com-ensation in the better-functioning forelimb. However, it islso conceivable that the functional compensation aftermall cortical lesions may rely upon ipsilateral processes,hereas that of larger lesions might require plastic

hanges on the contralateral side. Additional studies are

eeded to elucidate these probably different reorganiza-ion patterns.

cknowledgments—This work was supported by grants of theMBF 0311578, 01GI9905 and 01GZ0306. The authors thankr. A. Kastrup for stimulating discussions and helpful commentsn the manuscript.

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(Accepted 23 January 2006)(Available online 3 March 2006)