Predicting sensorimotor and memory deficits after neonatal ischemic stroke with reperfusion in the rat

Behavioural Brain Research 212 (2010) 56–63

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Predicting sensorimotor and memory deficits after neonatal ischemic stroke with reperfusion in the rat Valentine Bouet a,b,∗ , Thomas Freret a,b , Steve Ankri c , Madeleine Bezault c , Sylvain Renolleau d,e , Michel Boulouard a , Etienne Jacotot c,f , David Chauvier c , Pascale Schumann-Bard a,b a

GMPc, EA4259 Memory and Behavioural Plasticity Group, University of Caen Basse-Normandie, Caen, France Hypoxia and Cerebrovascular Physiopathology, CI-NAPS, UMR 6232, University of Caen Basse-Normandie, Caen Cedex, France THERAPTOSIS Research Laboratory, THERAPTOSIS S.A., Romainville, France d UMR-CNRS 7102, Hypoxia and Developmental Cerebral Ischemia, University of Pierre and Marie Curie Paris VI, Paris, France e Pediatric Intensive Care Unit, Trousseau Children’s Hospital, Paris, France f Imperial College London, Laboratory of Fetal and Maternal Medicine, Institute of Reproductive and Developmental Biology, United Kingdom b c

a r t i c l e

i n f o

Article history: Received 1 March 2010 Accepted 19 March 2010 Available online 27 March 2010 Keywords: Development Gliosis Memory Perinatal ischemia Sensorimotor functions

a b s t r a c t Among experimental models of perinatal ischemic stroke, Renolleau’s model mimics selected types of stroke at birth, including ischemia and reperfusion. However, its behavioural consequences on development have been poorly described. Here, ischemia-reperfusion was performed in 7-day-old Wistar rats. Between the ages of 9 and 40 days, sensorimotor and memory functions were assessed. The infarcted area was analysed by immunohistochemistry at 40 days of age. The remaining lesion was in the parietal cortex, in the form of a cone-shaped area. This area contained glial cells but neither neurons nor macrophages. Transient focal neonatal ischemia led to sensorimotor alterations in early adulthood, such as postural asymmetry, motor coordination and somatosensory deficits, and hyperactivity, as well as cognitive impairments, such as spatial reference memory deficits. Based on these results, we propose here a selection of behavioural tests that should constitute meaningful tools for assessing sensory and cognitive functions after experimental neonatal ischemic stroke. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Perinatal diagnosed ischemic stroke occurs in 1 of 2300–5000 births [32]. It is defined as a heterogeneous condition in which there is a disruption of cerebral blood flow secondary to arterial or cerebral venous thrombosis or embolisation. Perinatal ischemic stroke appears between 20 weeks of foetal life and postnatal day 28, and it can be confirmed by neuroimaging or neuropathologic investigations [32]. In the case of cerebral blood flow disruption, which often occurs in the immediate antenatal or perinatal period, the left middle cerebral artery (MCA) is the most commonly involved vessel. The left cerebral hemisphere is therefore the most frequently affected, leading to right hemiparesia. Other situations of neonatal stroke are elicited by a global reduction of cerebral blood flow, thereby leading to cerebral hypoxia. The pathogenesis of perinatal stroke is not always obvious, or even known. Nevertheless, most strokes occurring in term births are associated with a vari-

∗ Corresponding author at: GMPc, EA4259 Memory and Behavioural Plasticity Group, University of Caen Basse-Normandie, 5 rue Vaubénard, 14000 Caen, France. Tel.: +33 231947255; fax: +33 231947255. E-mail address: [email protected] (V. Bouet). 0166-4328/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2010.03.043

ety of risk factors such as birth asphyxia, cardiac diseases, blood disorders, trauma, or maternal disorders. Without a therapeutic intervention, multifocal lesions leading to functional deficits are expected. Long-term risks from perinatal ischemic stroke include seizures and neurodevelopmental impairments such as cerebral palsy, but also deficits of functions that are acquired later in life such as walking or language development. Nowadays, there is still no adequate treatment for perinatal ischemic stroke, and animal models are of prime importance to understand the physiopathology and functional consequences of early stroke and to set up efficient therapeutic interventions. Models of neonatal ischemic brain damage have been developed in the rodent (aged 7–18 days). Most of them mimic the clinical situation associated with a perinatal global reduction of cerebral blood flow and use intra- or extraluminal techniques [2]. Most studies investigating neonatal ischemia in rodents have used the hypoxiaischemia model of Rice-Vannuci [39], consisting of a permanent unilateral occlusion of the common carotid artery (CCA), followed by a hypoxic episode. This model induces a large hippocampal and cortical infarction [3,39], leading to massive sensorimotor and cognitive deficits [1,4,20,28]. Some models include reperfusion, which often occurs during perinatal intensive care. Renolleau’s model [36] consists of the permanent occlusion of the distal portion of the MCA

V. Bouet et al. / Behavioural Brain Research 212 (2010) 56–63

with a transient CCA occlusion. It involves the most often infarcted hemisphere in humans—the left hemisphere, and the most often occluded artery in humans—MCA [2]. This model mimics human neonatal ischemic stroke and is useful for evaluating the potential interest of new therapeutic strategies [12,33]. Consequently, we aimed to characterize the behavioural and histological consequences of Renolleau’s stroke model on postnatal development in rats. Our concern was to identify sensorimotor and memory that would serve for developing therapeutic strategies and to assess long-term brain damage in 7-day-old rats subjected to this ischemic-reperfusion stroke model. In particular, we adapted to developing animals tests previously used for adults. 2. Material and methods 2.1. Animals Animal experimentation was conducted according to the French and European Community guidelines for the care and use of experimental animals. Pregnant female Wistar rats, purchased from C.E.R.J. (Centre d’Elevage René Janvier, Le GenestSt-Isle, France) arrived in our animal facility 10 days before parturition. After birth (designated as P1—Postnatal day 1), litter size was reduced to 7–8 pups to ensure optimal body growth. When pups were 7 days old, they were randomly assigned to sham (n = 9) or MCAo (n = 10) groups. Housing conditions were as follows: food and water ad libitum, normal light/dark cycle (12 h/12 h-light on at 8:00), constant temperature (22 ± 1 ◦ C) and humidity (55 ± 10%). 2.2. Neonatal stroke model Ischemic arterial stroke was induced in 7 day-old (P7) Wistar rats (16–21 g) anesthetized by chloral hydrate (350 mg/Kg, i.p.), as previously described [36]. In brief, the left MCA was electro-coagulated at the inferior level of the cerebral vein and a clip was placed to occlude the left CCA. Sixty minutes later, the carotid blood flow was restored. Successful electro-coagulation was confirmed histologically by tissue invagination associated with MCAo disruption, found in all animals at P40. The same surgery was performed in sham-operated rats, but the left MCA and the CCA were not occluded. During the surgical procedure, body temperature was maintained at 37–38 ◦ C. Rats were then placed in an incubator (37 ◦ C) to avoid hypothermia until returned to their dam 12 h later. Due to surgery, anesthesia and/or mother cannibalism, mortality was 12%. 2.3. Histology and immunohistochemistry Coronal brain sections (50 ␮m) of fixed brains (4% paraformaldehyde) were collected from anterior striatum to posterior hippocampus. The residual lesion areas were measured on cresyl-violet stained sections using a SMZ680 stereomicroscope equipped with ACT-2U software (Nikon). Distances between sections (500 ␮m) were used to calculate the whole brain tissue loss, including infarction and atrophy, which was determined as follows: (contra minus healthy ipsilateral hemisphere volumes)/contralateral hemisphere volumes [36]. For immune-fluorescence staining of astrocytes, neuronal nuclei and preoligodendrocytes, coronal sections (25 ␮m) were incubated with mouse IgG primary antibodies, respectively diluted as follows: anti-GFAP (1:100, G3893, Sigma); antiNeuN (1:1000, MAB377, Chemicon); anti-NG2 (1:2000, MAB5384, Chemicon); anti-ED1 (1:500, MC341R, Serotec). Secondary antibody directly conjugated to FITC (1:500, F11021, Molecular Probes) was used before incubation with 2 ␮M Hoechst 33342. After washout, immunolabelled sections were mounted on cover glass in Vectashield (Vector) and were analysed on a Leica DM IRB inverted fluorescence microscope equipped with ×5 HCX PL FLUOTAR/×10 or ×20 N PLAN objectives. Cell counting was performed by two investigators in a blind manner on 3 fields per region and averaged. 2.4. Behaviour Sensorimotor and cognitive development was evaluated in a blind manner according to the experimental design illustrated in Fig. 1. Basic reflexes were assessed early in the rat’s development (grasp and tail reflexes, negative geotaxis, limb-placing reactions, and forepaw grip time). Contact righting was assessed until P14 (grey marks) and air-righting was assessed from P16 to P20 (dashed grey marks). More demanding sensorimototor and mnesic tasks were performed later, i.e. from P21 to the end of the protocol, P40. 2.4.1. Reflex development For the negative geotaxis test (from P9 to P20), rats were placed with the nose pointing downwards on a ramp tilted at 45◦ [11]. The time and the side for 180◦ turning and reaching a head-up position aligned with gravity were collected. Contact and air-righting were assessed as follows [11]: time to reach a prone from a supine position was video-recorded and measured, either after positioning the animal on a table

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(daily from P9 to P16–contact righting) or after dropping the animal 50 cm above a soft pad (daily from P16 to P20—air-righting). To assess the grasp reflex, rats (until P20) were held by the experimenter, and a thin rod (diameter: 1 mm) was placed to the palmar surface of fore- or hind-paws. Scoring was performed as follows: digit flexion: 2, delayed response (more than 2 s): 1, digit extension: 0. To assess the postural tail reflex, rats (until P20) were held by the tail, suspended one meter above the floor, and scored: extension of fore and hind limbs: 2, delayed response or only fore- or hind limb extension: 1, no extension: 0). A global score was then obtained for each rat. Forepaw grip time was used to evaluate force and fatigability from P9 to P20. After grasping a rod (diameter, 2 mm), the rat was suspended above a cotton pad. Time before falling was recorded. The limb-placing reaction (P9–P20) was triggered by visual, tactile, and vibrissae stimulation and scored: no response: 0, delayed response (i.e., more than 2 s): 1, immediate reaction: 2. 2.4.2. Actimetry (P20) Spontaneous activity was measured using a photoelectronic actimeter (APELAB® ). The apparatus was a Perspex box (25.5 cm × 20.5 cm × 9 cm) crossed in the centre by two infrared beams. Beam interruptions were counted by an automated system. 2.4.3. Corner test (P22 and P37) Each rat was placed at the entry of a corner made by two vertical boards (30 cm × 20 cm × 1 cm) attached on one side at an angle of 30◦ . Over 10 trials, the side chosen for turning after rearing was noted. The laterality index (LI) was calculated for each rat, according to the formula: LI = (number of right turns − number of left turns)/total number of turns [10]. 2.4.4. Accelerated rotarod (P22, P23, P29, P30, P36 and P37) After a habituation period to the apparatus (LETICA LE 8500, Bioseb® , France), each rat pup was tested three times (with an inter-trial interval of 5 min) on the accelerating rotarod (speed from 4 to 40 rpm over 2 min) [10]. Time before falling was collected (maximum: 120 s). 2.4.5. Adhesive removal test (from P20 to P24) [9,17,41] After a habituation period to the box (20 cm × 25 cm × 32 cm, duration: 60 s), two adhesive tapes (0.5 cm × 0.5 cm) were applied with equal pressure on each rat’s fore-paws. Each animal was tested once a day. Time to contact and to remove the adhesive was measured, with a maximum of 120 s. 2.4.6. Passive avoidance (P21 and P22) A step-through type passive avoidance box designed for mice (Bioseb® , France) was used [10]. The first day of the acquisition phase, each rat was placed in the illuminated compartment. Thirty seconds later, the door opened, and as soon as the animal entered the dark compartment, it received an electric foot shock (0.4 mA, 3 s). The animal was thereafter placed back in its home cage for 45 s before the next trial. The acquisition phase was achieved when the pup did not enter the dark compartment within 120 s during two consecutive trials [38]. If this criterion was reached before the last, 10th, trial, then the value of 120 s was attributed to all the remaining trials. The retention phase, performed 24 h later, consisted of a single trial in which the pup was placed in the illuminated compartment. The time before entering the dark compartment was noted. No electric shock was given during the retention session. 2.4.7. Morris water maze [31] (from P20 to P24) The water maze procedure consisted of three phases: learning (hidden platform), retention phase (no platform), and visual testing (visible platform) [27]. The pool (diameter: 136 cm) was virtually divided into four equally spaced quadrants. Data (swim speed, path length) were collected with a video computer-based storing system. For learning, a circular platform (13 cm × 13 cm) was submerged 0.5 cm below the surface in the centre of the target quadrant of the pool. The animals were trained over 4 days (from P19 to P23), 4 trials a day (60 s per trial, alternated starting positions, 60 s inter-trial, 20 s on the platform). For the long-term memory testing performed 24 h after the last learning session, the rats were placed in the tank during 60 s without platform. Visual testing was performed at P26: 3 trials with a visible platform (trial duration: 60 s). 2.4.8. Spontaneous alternation (P30 and P40) [29] The rat was placed in one of the three arms of a Y-maze (arm length: 50 cm, height: 24 cm, width: 15 cm) and the number of entries (four paw criterion), the order of entries, and the number of rearing were collected over 5 min. 2.5. Statistical analysis Results, presented as mean ± standard deviation (SD), were analysed by twoway analyses of variance (ANOVA) with repeated measurements, followed by post hoc multiple comparisons tests (Fischer’s PLSD) (Statview® ). Univariate t-test was used to compare the behavioural performances to a reference value when appropriate. Relationships between functional impairment and histological outcome were investigated using Pearson’s correlation test for the adhesive removal, Morris water

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Fig. 1. Experimental design of the study. Sensorimotor and mnesic tests were performed from P9 to P40. Black marks indicate the date/period at which the behaviour was analysed. Basic reflexes were assessed early in development (grasp and tail reflexes, negative geotaxis, limb-placing reactions, and forepaw grip time). Contact righting was assessed until P14 (grey marks) and air-righting was assessed from P16 to P40 (dashed grey marks). More demanding sensorimototor and mnesic tasks were performed later, i.e. from P21 to P40. maze, rotarod, actimetry and passive avoidance tasks while Spearman’s test was used for scoring-based tests.

3. Results Focal ischemia in the neonatal rat led to a significant body weight decrease until P13, to functional impairments during the early phase of development (from P9 to P40) and to brain tissue alterations as assessed at the end of this period, i.e. P40.

estingly, oligodendrocyte progenitor cells (NG2+ ) were found in the radial connecting structures. Moreover, a drastic lack of neuronal nuclei (NeuN) immuno-staining was observed in the core of astrocyte-rich cone-shaped areas (˛ and ˇ, for instance) in MCAo animals (Fig. 4A). Note that margins were clearly NeuN+ and that high magnification confirmed that most of the NeuN− cells had smaller nuclei than those of sham animals (Fig. 4B). Moreover, in the ␥ area, NeuN immune-staining density was similar to that of sham animals (Fig. 4B).

3.1. Long-term brain morphology and tissue recovery after neonatal stroke Ipsilateral (IL) brain hemispheres from ischemic animals exhibited an invagination at P40, associated with a MCA disruption (Fig. 2). Cresyl-violet staining revealed tissue alterations in IL hemisphere of MCAo animals, i.e. a cone-shaped area along cortical layers. By contrast, no damage was detected in sham-operated animals. The whole brain tissue loss in ischemic animals was 6.4 ± 1.6% of the contralateral hemisphere. 3.2. Astrocytic and neuronal pattern in P40 brain after neonatal stroke Tissue alterations were found in the cingulum and the external capsule at the basis of cortical cone-shaped area as sequels of previous tissue loss (Fig. 3A). Other residual thinner cone-shaped projections were observed in the cortical tissue (Fig. 4A), whereas glial fibrillary acidic protein (GFAP) positive cells were observed in the cingulum-alveus in sham animals, this labelling was strongly relocated in the cortical cone-shaped area and in its projections in ischemic animals (Fig. 3B and C, areas ˛, ˇ and  represent the narrow cone area, the cone projection area, and the adjacent cortex, respectively). These animals showed strong GFAP staining connecting the cingulum to the cortical layer I (Fig. 3C and D). In these projections (˛,ˇ and ), high magnification (Fig. 3D and E) showed numerous astrocytes (GFAP+ cells) with small nuclei. A transition in astrocytic pattern was clearly observed from the core to the margin of the cone-shaped area; astrocytes were less present cortical layers adjacent to the lesioned site (Fig. 3E). Inter-

Fig. 2. Morphological brain modifications in P40 rats after MCAo at P7. Illustrative brains of sham-operated (A) and MCAo animals (B). That electro-coagulation was successful was checked by MCAo disruption (black arrow: invagination in ipsilateral hemisphere). Bars: 5 mm. (C) Cresyl-violet stained sections in sham and MCAo animals. A cone-shaped area was observed after MCAo. Cortical layers (I–VIb), external capsule (ec), alveus (alv), cingulum (cg), and CA1 and CA2 hippocampus fields are indicated.

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Fig. 3. Reorganization of astrocytes in the P40 cortex after MCAo at P7. (A) Cresyl-violet stained coronal section (˛ = cone-shaped area; ˇ = cone projection area;  = cortical tissue adjacent to the lesion) (B–F). Hoechst, anti-GFAP staining, and merge in MCAo and sham animals. (B) Astrocyte radial projections emerging from the cingulum (cg) towards cortex layers. (C) Astrocytic pattern in areas ˛ and ˇ in MCAo and sham animals. (D) Enlarged micrographs of areas ˛ and ˇ. Note that astrocytes were mainly located in cortex I layer in sham animals. (E) Astrocytic pattern in area  in MCAo animals. (F) Infarcted area contained both GFAP+ (upper image) and NG2+ (lower image) cell located within a radial connection between the cingulum (cg) and the cone-shaped cortical area.

3.3. Reflex development Postural reflexes (negative geotaxis, contact and air-righting) were not affected by MCAo. Concerning the grasp and tail reflexes,

ANOVA did not reveal any group differences; all animals acquired the maximal score between P11 and P15. However, while sham animals displayed a score of 3.7 ± 0.7 at P9 (not significantly different from the maximal value of 4—univariate t-test), a deficit

Fig. 4. (A) Neuronal pattern in the P40 cortex after stroke at P7 showed by Hoechst, anti-NeuN staining, and merge in MCAo and sham animals in ˛ and ˇ areas. Note the lack of NeuN staining within infracted area in MCAo animals. (B) High magnification images depicting NeuN+ cells in intact and ischemic brain (˛ and  areas).

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Fig. 5. Effect of neonatal ischemia on sensorimotor performances. (A) Light beam interruptions during 30 min in the actimetry box in sham and MCAo animals at P20 (mean ± SD). ANOVA revealed a global significant difference between groups (p = 0.0234). (B) Corner test performances expressed by the laterality index in MCAo and shamoperated rats at P22 and P37 (mean ± SD). A negative laterality index indicates preference for ispsilateral turns. *Difference between groups (ANOVA, repeated measurements); # different from 0 (univariate t-test). (C) Accelerated rotarod performances expressed by the mean time (±SD) before falling (speed increased from 4 to 40 rpm over 2 min) at P22–P23, P29–P30, and P36–P37. *Inter-group difference (ANOVA with repeated measurements, Fisher’s PLSD, p < 0.05). (D) Adhesive removal performances expressed by the time-to-remove the contralateral adhesive tape (mean ± SD). *Difference between groups (ANOVA with repeated measurements, p < 0.05).

was observed in MCAo animals, score of 2.9 ± 0.9 (p < 0.01, different from 4, univariate t-test). At P10, MCAo animals reached a score of 3.5 ± 0.8 (not different from 4). Forepaw grip time and limb-placing reactions were similar between groups. 3.4. Global locomotor activity At P20, MCAo rats displayed significantly more beam interruptions than sham-operated animals (p < 0.05) (Fig. 5A). 3.5. Sensorimotor performances 3.5.1. Corner test A postural asymmetry was observed in the MCAo animals (Fig. 5B, ANOVA p < 0.05). The laterality index was significantly different from the theoretical value of 0 in MCAo animals at P37 (univariate t-test, p < 0.01). 3.5.2. Accelerated rotarod MCAo animals displayed motor coordination deficits that persisted until P40 (Fig. 5C, ANOVA group effect p < 0.05). A significant time effect (p < 0.001) showed, however, a global improvement of performances with time, which was similar in the two groups (no group × time interaction). 3.5.3. Adhesive removal test Time to contact the ispsi- or contro-lateral adhesive did not differ between MCAo and sham animals (from P20 to P24). By contrast, the time to remove the contralateral adhesive was higher

in MCAo than in sham animals (p < 0.05) (Fig. 5D). Furthermore, contrary to sham-operated animals (ANOVA with repeated measurements, time effect, p < 0.05), MCAo animals did not improve their performances with time (Fig. 5D). 3.6. Memory performances 3.6.1. Passive avoidance The number of trials to reach the learning criterion at the acquisition phase did not differ between groups (4.9 ± 1.3 for sham and 4.5 ± 1.0 for MCAo animals). Likewise, the mean latency to enter the dark compartment was not different between groups at the retention phase (92.7 ± 41.1 for sham and 77.9 ± 49.4 for MCAo animals) 3.6.2. Morris water maze The mean latency to find the platform during the acquisition phase (from P20 to P23) was not significantly different between MCAo and sham animals (Fig. 6A). Both groups improved their performances across trials (time effect, p < 0.0001). Considering the retention phase (P24), there was no difference between groups for the latencies to reach the target quadrant or the previous platform location and for the number of platform crossings. However, MCAo animals tended to spend less time than sham animals in the target quadrant (p = 0.0803). Moreover, as opposed to sham animals, MCAo animals did not significantly stay more than 15 s in the target quadrant (equal exploration time of each quadrant for a 60 s session) (Fig. 6B).

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Fig. 6. Morris water maze performances in sham and MCAo rats. (A) Learning phase performances expressed by the latency to reach the platform between P20 and P23. Both groups learned the platform location in the learning phase (ANOVA with repeated measurements, p < 0.0001). (B) Retention session performances expressed by the time spent in the target quadrant. # Different from the reference value of 15 s (univariate t-test). Data expressed as mean (±SD).

3.6.3. Spontaneous alternation Sham-operated and MCAo animals displayed similar exploratory behaviour (arm entries and rearing) in the Y-maze. The alternation percentage did not significantly differ between MCAo and sham-operated animals. Although sham animals performed above 50% (chance level) at P30 and P40 (p < 0.05 and p < 0.001, respectively), this was not the case for MCAo at both ages (p = 0.85 and p = 0.27, respectively). 3.7. Correlations between histological and behavioural outcome No significant correlations were found between brain lesion volumes at P40 and performances of MCAo animals for most behavioural tests (actimetry at P20, r = −0.16; corner test at P37, r = −0.05; rotarod, r = −0.29; passive avoidance at P22, r = −0.06; spontaneous alternation at P40, r = −0.06; adhesive removal test at P24, p = 0.39), except for the Morris Water Maze during the retention phase at P24. Indeed, in this task, the number of platform location crossings at P24 was significantly inversely correlated with the lesion volume at P40 (r = −0.64, p < 0.05, Pearson test). 4. Discussion A few hours after ischemia-reperfusion onset, cortical injury occurs through apoptotic features in the penumbra, whereas necrotic features rather occur in the ischemic core [5,13,24,36,37]. A previous study by Renolleau et al. [36] showed a well-delineated cortical infarct 48 h post-reperfusion (20% of IL hemisphere), and we observed a large cavity in the full thickness of the frontoparietal cortex 14 days after the ischemic onset [36]. Surprisingly, we did not find here any histological evidence of a cavitation at a later stage, i.e. P40. Increased neurogenesis after cerebral ischemia has been demonstrated in the adult rodent [26] as well as in HI (hypoxia-ischemia) model [14]. Interestingly, previous works with Renolleau’s model evidenced that proliferating cell number increased in the border of the cortical infarct 7 days after perinatal stroke [6], coming possibly from the dorsolateral subventricular zone (SVZ) [7]. It has been reported that after ischemia-hypoxia, brains keep the potential to generate new oligodendrocytes up to 4 weeks within and surrounding the infarct [45]. GFAP+ astrocytes are located in the infarct prior to surrounding the cavity, and they show signs of degradation afterwards [6,18]. Our data suggest that strong GFAP immuno-reactivity persisted at P40 only within coneshaped lesioned areas and radial projections. Therefore at P40, the remaining cortical lesion was restricted to a small cone-shape area containing glial cells, but neither neurons nor macrophages. Behaviourally, in contrast to what was observed with the RiceVannucci model [28,30], Renolleau’s neonatal stroke model did not induce deficits in negative geotaxis, contact and air-righting,

forepaw grip time, and limb-placing tests. Postural reaction and developmental reflexes were not affected. Regarding the neurological score, our results contrast with those of a previous study [37], which reported a neurological deficit at P28. This discrepancy is probably due to differences in the items used for neurological scoring as well as the extensive training of our animals, related to the number of tests we used. We observed, however, a 1-day delay in the acquisition of a normal neurological score (not significantly different from 4), which could be related to the stroke and/or to the weight loss, larger after surgery in MCAo than in sham animals. As frequently reported after HI [4,28,30], but contrary to Pabello et al. [33], we found spontaneous locomotor hyperactivity in MCAo rats. In adult rats, hyperactivity has been reported after focal ischemia [8,44], but mostly when the lesion involves the striatum. Slight damage to the head of the caudate-putamen is encountered in a small fraction of animals (20% approximately) [36], and could therefore partly account for this increased activity. Ischemia-induced hyperactivity could also possibly be related to cognitive dysfunctions, such as increased anxiety [44], which was unfortunately not measured in our experiment. Our data show that motor coordination and equilibrium were altered in MCAo animals, which is also the case with Rice-Vanucci model [4,28] or with adult rat models of focal ischemia [46]. However, note that adult animals generally show transient motor coordination deficits, while young animals display long-lasting deficits (about 50 days) [28]. Disturbances of brain development in the early stage of life could therefore lastingly affect some aspects of motor coordination capacities. The timetable of cerebral development displayed so-called critical periods, and a modification in the sensorimotor cortex mapping at early age could modify for a long period the integration of somatosensory afferents and therefore related motor functions. Early histological measurements (i.e. 24 or 48 h after ischemia) indeed showed that the parietal cortex was massively infarcted in this model, including S1 (barrel fields, jaw, forelimb, hindleg cortical representation) and S2 somatosensory cortices [37]. The notion that early-life disturbances have lasting effects could be applied to the asymmetries in postural and forelimb use evidenced on the corner and cylinder tests, which were also longlasting herein, as reported in adults [10,12,20,22,47]. The adhesive removal test is relevant to detect long-term deficits after stroke in adult rodents [9,10,16,47]. We show here for the first time that this test can be adapted to juvenile rats for assessing deficits after neonatal stroke. A contralateral deficit was indeed highlighted by the time-to-remove the adhesive, indicating a deficit in sensorimotor function, and a possible impairment in motor control (the latter is doubtful since the motor cortex was not comprised in the ischemic area). Given the fronto-parietal cortical distribution of the lesion [36], this impairment might reflect an alteration in face-related somatososensory perception (i.e. a dif-

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ficulty for the animal to sense the adhesive with its whiskers and/or with its tongue) and/or a forelimb motor and/or sensory alteration (since the corresponding forelimb sensory cortex is affected by ischemia). Regarding memory functions, ischemia did not alter spatial learning abilities in the Morris water maze. This finding agrees with results reported in adult models of focal ischemia [15,21]. Acquisition of this task is known to rely on the dorsal hippocampus. Given that this brain structure is spared in 80–85% of the animals subjected to the present neonatal model [42], no deficit was expected. Besides, the spatial reference memory (retention session) was significantly impaired in the MCAo animals. In addition, this deficit was significantly correlated with the lesion volume. The parietal and frontal cortices, the medial septal area and the dorsal hippocampus play an important role in mediating reference memory [25]. As discussed above, the parietal cortex was massively affected, while the dorsal hippocampus was only partially altered in some animals (15–20%) [36]. It is therefore conceivable that ischemiainduced alteration of the neuronal network, implicating at least those two structures, may lead to the observed impairment. In addition, spatial working memory assessed by spontaneous alternation performances was altered by focal ischemia. This finding could be related to the implication of the frontal cortex in this task [34] and of the parietal cortex in processing topological spatial information [19]. No deficit in long-term memory was evidenced in the passive avoidance test. This task is known to strongly involve the hippocampus and the lateral amygdala, especially during the memory consolidation phase [23]. The retention phase involves both structures in addition to the entorhinal and parietal cortices. None of these structures, except the parietal cortex and, in a small fraction of the animals (15–20% [42]), part of the CA2 and CA3 fields of the hippocampus, was included in the ischemic lesion. The relative integrity of the hippocampus and amygdala, associated to possible plastic adaptations of the neonate brain towards an aversive stimulus, may therefore explain the memory impairment. Except for the spatial reference memory impairment, no significant correlation was found between the lesion size and the various behavioural deficits. However, the number of MCAo animals used in the study is rather small, rendering the correlation analysis difficult to interpret. It nevertheless suggests that the Morris water maze could be a particularly sensitive test of neonatal ischemia-induced deficits. However, one must keep in mind that the absence of a significant linear relation between behaviour and histology does not mean the absence of functional relationship between both parameters. In that sense, such a result does not argue against the use of those behavioural tests in studies where the functional impact of neuroprotective strategies is prospected. The presence, or absence, of a correlation between functional deficit and lesion size is rather contradictory in the literature, even in adult models of ischemia, when brain plasticity is lower than in pups [35,40,43]. In the neonate, one study reported a correlation between the extent of ischemiainduced sensorimotor asymmetry measured on the cylinder test and the volume of brain lesion [34]. In conclusion, we show here that neonatal focal ischemia, according to Renolleau’s model, leads to sensorimotor alterations in the early adulthood, such as postural asymmetry and hyperactivity. Additionally, we adapted and performed for the first time the adhesive removal test in juvenile rats, and highlighted its relevance in the field of neonatal stroke models. Besides, we showed a deficit in spatial reference memory, never described so far in this model. Therefore, corner test, adhesive removal, rotarod test, and spontaneous alternation should constitute meaningful tools for assessing the efficacy of neuroprotective and/or regenerative therapies on sensorimotor and cognitive functions in preclinical neonatal stroke studies.

Acknowledgments We thank Dr. C. Charriault-Marlangue for stimulating discussions at the initiation of this work, Prof. C. Mallard, and Drs. M. Bernaudin, and C. Rousset for critical reading and suggestions.

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