Effects of daily environmental enrichment on behavior and dendritic spine density in hippocampus following neonatal hypoxia–ischemia in the rat

Experimental Neurology 241 (2013) 25–33

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Effects of daily environmental enrichment on behavior and dendritic spine density in hippocampus following neonatal hypoxia–ischemia in the rat Joseane Jiménez Rojas a, b, Bruna Ferrary Deniz b, Patrícia Maidana Miguel b, c, Ramiro Diaz a, b, Érica do Espírito-Santo Hermel a, Matilde Achaval a, b, Carlos Alexandre Netto a, c, Lenir Orlandi Pereira a, b,⁎ a b c

Programa de Pós-graduação em Neurociências, ICBS, Universidade Federal do Rio Grande do Sul, Brazil Departamento de Ciências Morfológicas, ICBS, Universidade Federal do Rio Grande do Sul, Brazil Departamento de Bioquímica, ICBS, Universidade Federal do Rio Grande do Sul, Brazil

a r t i c l e

i n f o

Article history: Received 23 March 2012 Received in revised form 26 November 2012 Accepted 29 November 2012 Available online 6 December 2012 Keywords: Neonatal hypoxia–ischemia Environmental enrichment Dendritic spine density Behavior Neuroprotection

a b s t r a c t Hypoxia–ischemia (HI) is the main cause of mortality in the perinatal period and morbidity, in survivors, which is characterized by neurological disabilities. The immature brain is highly susceptible to hypoxic– ischemic insult and is responsive to environmental stimuli, such as environmental enrichment (EE). Previous results indicate that EE recovered memory deficits in adult rats without reversing hippocampal atrophy related to HI. The aim of this study was to investigate behavioral performance in the open field and rota-rod apparatuses, in object recognition and inhibitory avoidance tasks, as well as dendritic spine density in the hippocampus, in rats undergoing HI and exposed to EE. Seven-day old male rats were submitted to the HI procedure and divided into 4 groups: control maintained in standard environment (CTSE), controls submitted to EE (CTEE), HI in standard environment (HISE) and HI in EE (HIEE). Behavioral and morphological parameters were evaluated 9 weeks after the environmental stimulation. Results indicate impairment in the object recognition task after HI that was recovered by enrichment; however the aversive memory impairment in the inhibitory avoidance task shown by hypoxic–ischemic rats was independent of the environment condition. Hypoxic–ischemic groups showed more crossing responses during the first minute in the open field, when compared to controls, but no differences were found between experimental groups in the rotarod test. Dendritic spine density in the CA1 subfield of the right hippocampus (ipsilateral to the artery occlusion) was decreased after the HI insult, and increased in enriched controls; interestingly enriched HI rats did not differ from CTSE. In conclusion, EE was effective in recovering declarative memory impairment in object recognition and preserved hippocampal dendritic spine density loss after neonatal HI injury. © 2012 Elsevier Inc. All rights reserved.

Introduction Hypoxia–ischemia during gestation and perinatal periods are common causes of neonatal brain damage which are frequently associated with neurodevelopmental disabilities (Trollmann and Gassmann, 2009), including cerebral palsy, epilepsy and learning and memory and other cognitive impairments (Delsing et al., 2001; Koelfen et al., 1995). These sequelae result from injury to the hippocampus and some other brain structures (Kadam et al., 2009). The Levine and Rice method (Levine, 1960; Rice et al., 1981) is an animal model widely used to study neonatal encephalic hypoxia–ischemia (HI) in rodents. It is well established that neonatal HI causes significant long-term behavioral impairments of spatial memory in the water maze in adult rats (Arteni et al., 2003; Ikeda et al., 2001; Pereira et al., 2007), in aversive inhibitory avoidance memory (Arteni et al., 2003; Young ⁎ Corresponding author at: Rua Sarmento Leite, 500, ICBS, ZIP: 90050-170, Porto Alegre, RS, Brazil. Fax: +55 51 3308 3092. E-mail address: [email protected] (L.O. Pereira). 0014-4886/$ – see front matter © 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.expneurol.2012.11.026

et al., 1986), in working memory (Arteni et al., 2003; Chávez et al., 2008; De Paula et al., 2009; Pereira et al., 2007) and in sensorimotor tasks (Bona et al., 1997; Jansen and Low, 1996a,b). These behavioral findings are frequently associated to neural changes, mostly to the hippocampus. Pyramidal CA1 neuronal death (Kirino and Sano, 1984; Pulsinelli et al., 1982), decrease of dendritic spine density (Ruan et al., 2009) and neural atrophy in the hippocampus, sensorimotor cortex and striatum following neonatal HI have been already reported (De Paula et al., 2009; Jansen and Low, 1996a; Rodrigues et al., 2004). It is known that the immature brain is susceptible to environmental stimuli (Meaney and Aitken, 1985), therefore environmental enrichment (EE) has been recognized as a possible behavioral neuroprotective strategy. Enrichment involves a combination of social interaction, physical exercise and exposure to learning tasks (Harburger et al., 2007) and results in positive cognitive effects; for instance animals submitted to EE show improvement in learning and memory on spatial tasks, as compared to those maintained in the standard home cage environment (Kempermann et al., 1997; Leggio et al., 2005; Nilsson et al., 1999; Van Praag et al., 2000).

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Important morphological effects of environmental stimulation in the hippocampus have been reported (Van Praag et al., 2000), like increased neurogenesis (Kempermann, 2002; Kempermann et al., 1997), increased cortical thickness and dendritic branching in CA1 region pyramidal neurons (Greenough et al., 1973); enhanced synaptophysin levels in the hippocampus and the frontoparietal cortex in aged female rats (Frick and Fernadez, 2003) have been also shown. Dendritic spine growth and changes in spine morphology constitute relevant findings of enrichment studies (Diamond et al., 1976; Leggio et al., 2005; Nakamura et al., 1999). Moreover, EE was reported to improve neurological functions in animal models of Alzheimer's disease (AD), Parkinson's disease (PD) and traumatic brain injury (TBI). In addition to that, Jankowsky et al. (2005) demonstrated that in an AD animal model, learning and memory deficits on radial maze and Morris water maze tasks were reversed by enrichment exposure. Besides, Jadavji et al. (2006) reported that motor symptoms like tremor, rigidity and impairments on skilled limb movements in animals submitted to an animal model of PD were improved after an environmental stimulation; these data are in agreement with other studies showing that EE increases neurogenesis and neurotrophic factor expression (Bezard et al., 2003; Pham et al., 2002). Two recent papers demonstrate a relationship between TBI and EE. The first study, by De Witt et al. (2011), shows motor and cognitive benefits in TBI animals submitted to a protocol of 6 h per day EE, using Morris water-maze and beam-balance/beam-walk tasks, similar to continuous EE. The second study was performed by Matter et al. (2011), with the same TBI injury model, and demonstrates that early enrichment (starting 1 day after lesion) enhanced motor ability and delayed EE (starting 7 days after lesion) facilitated spatial learning in injured animals. Previous data from our laboratory with EE after neonatal hypoxia– ischemia evidenced that stimulation provided by late enrichment recovers spatial memory deficits, while having no effect on hippocampus or cerebral cortex atrophy (Pereira et al., 2007). Another study showed that early housing in enriched environment produced memory recovery in the object recognition task and an improvement in spatial memory in adolescent female and male rats after neonatal HI; however, enrichment did not affect hippocampus and striatum tissue atrophy due to hypoxia–ischemia-induced injury (Pereira et al., 2008). Considering that daily EE showed clear protective action on cognitive performance in rodents after a hypoxic–ischemic episode with no effects on studied hippocampal morphology aspects (Pereira et al., 2007, 2008), the present study was designed to investigate, in rats undergoing hypoxia–ischemia followed by a daily environmental enrichment protocol: 1) behavioral performance in the open field and rota-rod apparatuses, as well as in object recognition and inhibitory avoidance tasks, and 2) dendritic spine density in the dorsal hippocampus, using the Golgi technique. The working hypothesis is that both cognitive impairment and morphological damage caused by encephalic hypoxia–ischemia will be prevented, or alleviated, by the 1 hour environmental enrichment session administered 6 days per week during 9 weeks. This is the same procedure previously used (Pereira et al., 2007, 2009), where animals were stimulated until reaching adult age. With the spine density assessment we aimed to refine the study of morphological EE effects, since the gross hippocampal atrophy resulting from HI demonstrated not to be affected by.

maintained in standard environmental (CTSE); b) control exposed to environmental enrichment (CTEE); c) submitted to hypoxia–ischemia and maintained in standard environmental (HISE); and d) submitted to hypoxia–ischemia exposed to environmental enrichment (HIEE). Rats remained with their mothers until PND 21, even after the definition of experimental groups. All procedures were in accordance with the Guide for the Care and Use of Laboratory Animals adopted by national Institute of Health (USA) and with the Federation of Brazilian Societies for Experimental Biology. This project was approved by the Ethics Committee at the Universidade Federal do Rio Grande do Sul, under the number 2008247. The number of animals used was defined according to previous studies adopting similar methodology (Brown et al., 2008; Brusco, et al., 2008; Chen, et al., 2009; Marcuzzo et al., 2007). Thirty-six male rats were used for the behavioral study (CTSE, n = 10; CTEE, n = 10; HISE, n = 8; HIEE, n = 8). As for the morphological analysis, 20 rats were assessed (CTSE, n = 5; CTEE, n = 5; HISE, n = 5; HIEE, n = 5), making a total of 56 (36 + 20) animals utilized and randomly distributed among the four experimental groups at PND 7. A distinct set of rats was used for the morphological evaluation because it has been reported that performing cognitive tasks might increase dendritic spine density and neuronal activity in CA1 pyramidal cells in hippocampus (Berger et al., 1980; Leuner et al., 2003; Luine et al., 2011). However, considering that procedures (HI and EE) were the same in all animal groups, in the present study we sought to evaluate morphological and functional parameters, relating them. Only male rats were used in the present study, as above mentioned, since there is evidence in literature showing rapid changes in the number of dendritic spines during estrous cycle with consequences to hippocampal neuronal function (Brusco et al., 2008; Woolley et al., 1990). A time line containing all experimental events is showed (Fig. 1).

Materials and methods

Environmental enrichment procedure used in this study was previously described by Pereira et al. (2007, 2009). At 21st PND animals were weaned and separated from their mothers. Daily enrichment sessions began when rats reached 22-days-old and continued during 9 weeks, 6 days per week, 1 h per day, in groups of 7–10 animals. The enriched environment consisted of a large cage (40 × 60× 90 cm) with three floors, ramps, one running wheel and several objects with different shapes and textures modeled as previously described (Diamond, 2001; Wildman and Rosselini, 1990). Objects in the cage were changed once a week. Rats from CTSE and HISE groups (non-enriched) were

Animals Male Wistar rats were obtained from the Central Animal House of the Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Brazil. They were maintained in a temperaturecontrolled room (approximately 22 °C), on a 12/12 light/dark cycle and with food and water ad libitum. At postnatal day (PND) 7, animals were randomly divided into four experimental groups: a) control

Hypoxia–ischemia The Levine method (1960), as modified by Rice et al. (1981), was utilized in this study to produce unilateral brain injury to neonatal rats. At postnatal day 7, animals were anesthetized with halothane 2– 4% and submitted to surgical procedure. An incision was made to the ventral surface of the neck; the right common carotid artery was assessed, isolated from the adjacent structures and permanently occluded with 4.0 surgical silk threads. Animals were then maintained under controlled temperature to recover for 15 min, and then returned to their dams. After a period of 2.5 h, five pups at a time were exposed to 90 min to hypoxic atmosphere (8% oxygen and 92% nitrogen, 5 L/min flow) in a 1500 mL chamber partially immersed in a 37 °C water bath in order to maintain body temperature of lactating rats in the physiological limits. In the present work, control animals were sham-operated, i.e., they were submitted to manipulation, anesthesia and neck incision, but did not receive arterial occlusion or hypoxic atmosphere exposition. This procedure intends to prevent different maternal care and/or a rejection of the HI animals. Following the HI procedure, animals were returned to their respective home cages. Environmental enrichment

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Fig. 1. Time line of experimental procedures. HI: hypoxic–ischemic event.

removed from their home cages to another standard cage during the enrichment period (Pereira et al., 2007, 2009). Behavioral tests All rats were submitted to a series of behavioral tests, in the following order: Open Field, Novel Object Recognition, Inhibitory Avoidance and the Rota-rod. This sequence was chosen after previous studies (Arteni et al., 2010; Schlager et al., 2011); the open field test was carried out 24 h after the end of environmental enrichment and remaining tests applied as depicted in Fig. 1. Open field The open field apparatus consisted of a wooden chamber (55 × 44 × 50 cm) with a dark gray floor divided into 12 fields. The box was placed in a quiet room, with the same illumination used in the other tests. Rats were placed in a corner square of the chamber and the latency to leave this first square was recorded. Subsequent rearing and crossing responses (in the 1st minute and total time) were recorded (Netto et al., 1985). Animals were observed individually for 5 min. Novel object recognition The novel object recognition task assesses declarative memory (Clark and Martin, 2005). In the first phase of the test, each animal was confronted with two different objects, placed in an open-field box, and the time of object exploration is registered in 5 min. Following this phase, the rodent was removed from the open-field box to another separate box for a period of 5 min. In the second phase, each animal was exposed to two objects placed in the same open-field box: one familiar object, used in the first phase, and one novel object. The time spent exploring the novel object and the familiar object was measured (Benice and Raber, 2005). A discrimination index was calculated in the test session (second), as follows: the difference in exploration time divided by the total time spent exploring the two objects (B− A/ B + A, where B is the new object and A is the familiar object) (Okuda et al., 2004). Inhibitory avoidance An acrylic box was used (50 × 25 × 25 cm), with the left-most 7 cm of the box floor was occupied by a 3 cm high platform. The box floor was a grid of parallel stainless steel bars (1.5 mm-diameter) spaced 1 cm apart. Animals were gently placed on the platform and their latencies to step down placing their four paws on the grid were measured with an automatic device. On stepping down, they received a 0.5 mA; 60 Hz scrambled foot shock for 2 s, and were withdrawn from the box. Animals were tested for retention 24 h later. Test session was procedurally similar to the training one except that foot shock was omitted; step-down latency in test was used as an index of retention (Arteni et al., 2003; Netto et al., 1985). Rota-rod test Motor coordination was assessed using a Rota-rod apparatus (InsightR, Brazil) (Capasso et al., 1996; Liu et al., 2007); this task was chosen especially because it does not involve a significant aversive stimulus. Animals were exposed to one habituation session during 3 min in the apparatus on slow velocity (20 rpm). In the test session, 24 h later,

animal's motor ability was evaluated. The rota-rod test was performed by placing rats on rotating drums (3 cm diameter) and measuring the time each animal was able to maintain its balance on the rod. The speed of the rota-rod accelerated from 16 to 40 rpm over a 5 min period. Variables recorded were: latency of the first downfall, number of falls (maximum 3) and time of permanence in the apparatus (Takao et al., 2010). Golgi staining Two days after the end of EE, animals were anesthetized with ketamine (80 mg/kg) and xylazine (10 mg/kg) i.p. and transcardially perfused with fixative solutions. The “single-section” Golgi method procedure followed the same methodology published in detail by De Castilhos et al. (2006). Brains were fixed with 4% paraformaldehyde and 1.5% picric acid in 0.1 M phosphate buffer (pH 7.4), coronally sectioned (200 μm thick) using a Vibratome (Leica, Germany) and impregnated in 1.5% silver nitrate following 3% potassium dichromate (Merck, Germany). After remaining in the dark for at least 48 h, the cover lips were removed and the sections were rinsed in distilled water, dehydrated, cleared with xylene, mounted on slides and covered with non acidic synthetic balsam and cover slips (Rasia-Filho et al., 2004). Microscopic analysis The microscopic analysis was performed using a camera lucida (1000×) coupled to an optic microscope. The hippocampal region CA1 (ipsilateral and contralateral to arterial occlusion) known as stratum lacunosum-moleculare was analyzed in all animals due its neuronal vulnerability to ischemic insults (Ito et al., 1975; Pulsinelli et al., 1982). The dorsal CA1 localization was based in the Paxinos and Watson Atlas of Stereotaxic Coordinates (2004). Selected pyramidal neurons must have the following characteristics: neuronal cell bodies undoubtedly located within the boundaries of the dorsal CA1, relatively away from its ultimate borders, and a typical pyramidal appearance, according to Lorente de Nó (1934); well-impregnated dendrites with defined borders that could be clearly distinguished from the background and a tapering appearance toward their endings throughout the section; and be relatively isolated from neighboring impregnated cells to avoid “tangled” dendrites with adjacent neurons (Brusco et al., 2008). One dendritic segment was studied per neuron and 10 different neurons per animal (5 per hemisphere); consequently, dendritic spine data were obtained from a total of 50 different dendrites in each experimental group. Spines were studied in the first 20 μm of the selected dendritic branches, which were all relatively thin and approximately with the same diameter. It is expected that only a small proportion of the spines along these segments could be hidden by the dendritic shafts in these Golgi-impregnated neurons (Woolley and McEwen, 1993). The most lateral tertiary dendrite on the apical tree of each CA1 pyramidal cell was selected, in order to have more regular sampling scheme. It is important to note that the draws were performed manually by only one of the authors who was blinded to the specimen origin. Drawings were scanned and had their lengths measured using an image analysis system (Image J 1.44, National Institutes of Health, USA) and spine density was defined as the number of spines divided per unit of dendritic length (Rasia-Filho et al., 2004).

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In order to evaluate the gross brain injury the percentage of rats with porencephalic cyst was recorded; porencephaly defined as focal cavitary lesions frequently communicating with lateral ventricles (Ten et al., 2004). Brain analysis revealed that rats subjected to neonatal HI and exposed to EE or standard environment had different degrees of cerebral injury. To evaluate this brain general injury, it was presented a percentage of rats with porencephalic cyst. Statistical analysis The Shapiro–Wilk test was run to determine whether data presented a normal distribution. So, data from the rota-rod, open field and recognition memory task, as long as those of dendritic spine density, were analyzed using parametrical statistics, conducted using two-way analysis of variance (ANOVA) with lesion and environment as independent variables, followed by post hoc Duncan's test for multiple comparisons whenever indicated. These results are expressed as mean ±S.E.M. Non-parametric data, i.e., latency to step-down platform in the inhibitory avoidance, are expressed as median latencies and interquartile ranges of each group and statistical differences were assessed using the Kruskall Wallis ANOVA followed by Mann Whitney test whenever indicated. The Bonferroni's correction was run to adjust for multiple comparisons, revealing that a p valueb 0.0125 is required to indicate statistical significance while maintaining an overall type-I error rate of 0.05. All statistics was performed using the Statistica® software package running on a compatible personal computer. Results Open-field Rats submitted to hypoxia–ischemia presented a greater number of crossings in the first minute of open-field exploration. There was effect of lesion (F(1,32)=21.95, pb 0.0125) but no effect of environment (F(1,32)=1.72, p>0.0125) nor factor interaction (F(1,32)=0.42, p> 0.0125); the Duncan post-hoc analysis indicated that HISE and HIEE groups presented more crossing responses compared to the control animals. Considering the total number of crossings ANOVA revealed effect of lesion (F(1,32)=1.50, pb 0.0125) and no effect on environment (F(1,32)=0.22, p>0.0125). Analysis of the latency to leave the first square also showed no significant effect on lesion (F(1,32)=1.97, p>0.0125), environment factors (F(1,32)=0.21, p>0.0125) nor interaction between factors (F(1,32)=0.008, p>0.0125); the same pattern emerged from the rearing response analysis, with no effects of lesion (F(1,32)=0.87 p>0.0125) or environment (F(1,32)=1.15, p>0.0125). Results are presented in Table 1. Novel object recognition Hypoxia–ischemia resulted in memory deficits in the novel-object recognition task. Two-way ANOVA of novel-object preference index indicated significant differences related to lesion (F(1,32)=15.45, pb 0.0125) and environment factors (F(1,32)=9.75, pb 0.0125), with a trend for lesion and environment interaction (F(1,32)=3.37). Duncan's post hoc test for multiple comparisons revealed that hypoxic–ischemic animals maintained in standard environment had lower preference index when compared to all other groups; interestingly, there was no difference between HI enriched and control groups (Fig. 2). ANOVA of first session indicated a difference in time exploration regarding the object A relative to lesion (F(1,32)= 9.76, p b 0.0125). No significant effect was found on environment (F(1,32) =1.55) and lesion and environment interaction (F(1,32) = 1.83). Duncan's test indicated that HISE and HIEE groups spent lower time exploring object A, as compared to CTSE group. Similarly, there was a lower exploration of object B by the HISE and HIEE groups when compared with CTEE group (data not shown).

Table 1 Open-field test. Mean ± S.E.M. of absolutes values. Group

Crossings 1st minute

Total crossings

Latency

Rearings

CTSE CTEE HISE HIEE

17.6 ± 2.1 19.5 ± 2.0 25.9 ± 2.3⁎ 30.7 ± 2.5⁎

75.7 ± 6.0 74.9 ± 5.8 85.6 ± 6.7 80.7 ± 7.2

7.2 ± 1.0 6.5 ± 0.9 6.1 ± 1.1 5.3 ± 1.2

47.8 ± 3.4 37.7 ± 3.3 37.6 ± 3.8 40.3 ± 4.1

Differences in relation to CTSE and CTEE. ⁎ p b 0.05, Duncan's post-hoc.

Inhibitory avoidance Hypoxic–ischemic animals demonstrated memory impairment independently of the environment. Kruskal–Wallis one way ANOVA showed no differences in the latencies to step down the platform the training session (H= 4.12, p > 0.0125), but a significant difference emerged on test session performance (H= 14.76, p b 0.0125). Mann– Whitney U-test demonstrated a lesion effect that was independent of the environment condition, since hypoxic–ischemic animals presented lower latencies to step down the platform than control animals (Fig. 3). Rota-rod There were no significant differences between the hypoxic–ischemic groups and control ones in the rota-rod test (Table 2). There were no effects, whatsoever, in any of the variables analyzed: latency of the first downfall, neither lesion (F(1,32) = 1.29, p > 0.0125) nor environment (F(1,32) = 1.04, p > 0.0125) effects; number of falls, neither lesion (F(1,32) = 1.27, p > 0.0125) nor environment (F(1,32) = 1.27, p > 0.0125) effects; and the maximum time of permanence in the apparatus, neither lesion (F(1,32) = 1.10, p > 0.0125) nor environment F(1,32) = 1.14, p >0.0125) effects. Dendritic spine density and morphological changes Analysis of gross brain injury revealed that rats subjected to neonatal HI and exposed to EE or standard environment did not differ in the degree of cerebral injury. Forty percent of animals in the HISE and HIEE groups presented porencephalic cyst. As expected, control groups had cysts (data not shown). Dendritic spine density was measured in pyramidal neurons of the stratum lacunosum-moleculare of hippocampal CA1 region (Fig. 4). Results demonstrate that HI caused a decrease of spine density in the hippocampus. Density of dendritic spines from the right hemisphere (ipsilateral to the lesion) suffered an effect of lesion (F(1,16) = 55.10, p b 0.0125) and of environment (F(1,16) = 20.68, p b 0.0125), along with a factor interaction (F(1,16)= 9.26, p b 0.0125). Duncan's post hoc test revealed that hypoxic–ischemic animals maintained in standard environment had lower dendritic spine densities when compared with CTSE and CTEE (pb 0.0125 in both groups). However, HIEE group had no significant difference when compared with the respective control, CTSE, group (p> 0.0125). Considering that HIEE spine density values did not differ from CTSE nor HISE ones, we suggest that environmental stimulation prevented the HI effect. Interestingly, dendritic spine density from left hemisphere (contralateral to the lesion) two-way ANOVA identified significant effect of lesion (F(1,16) = 39.34, p b 0.0125) and environment (F(1,16) = 13.25, p b 0.0125), with no Interaction between (Fig. 4). There was a clear reduction of spine density in HI groups, as compared to control groups in the left hippocampus. Discussion Present study investigated the effects of daily environmental enrichment (1 h/day for 9 weeks) in rats previously submitted to an early hypoxic–ischemic event and evaluated by means of open

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Fig. 2. Object exploration on novel-object recognition memory task, conducted 9 weeks after HI procedure. Bars represent the mean ± S.E.M. of preference index, calculated in the second exposition. *HISE group is significantly different from all other groups; ANOVA followed by Duncan's test, p b 0.0125.

field, object recognition, inhibitory avoidance and rota-rod tests, along with dendritic spine density in the hippocampus. Results partially confirmed the working hypothesis; since exposure to EE did recover from cognitive impairments associated to HI brain injury but did not reverse the decrease of dendritic spine density caused by HI. Morphological analysis indicate a trend towards a preventive effect of the enrichment protocol and provide a new avenue to explore in interpreting recent data showing cognitive recovery in rats submitted to neonatal hypoxia–ischemia and subjected to an enriched environment condition (Pereira et al., 2007, 2008). Hypoxic ischemic rats showed hyperactivity in the open field, as assessed by augmented number of crossing responses in the first minute compared to controls. It is well established that the hippocampal CA1 area is highly vulnerable to a hypoxic–ischemic event and several authors have reported that either complete, or partial, hippocampal injuries result in hyperactivity in an open-field test (Brotto et al., 2000; Cassel et al., 1998; Coutureau et al., 2000; Gray and McNaughton, 1983; Lipska et al., 1991; Shen et al., 1991; Valle and Gorzalka, 1980). Also, it has been described that hyperactivity could happen immediately after occlusion, in consequence to hippocampal CA1 neuronal death (Araki et al., 1999; Kuroiwa et al., 1991; Mileson and Schwartz, 1991; Wang and Corbett, 1990). However, the enrichment protocol here used was not able to reduce the hyperactivity typically found in HI animals. Memory in the object-recognition task was impaired in HI rats, an effect that was recovered after stimulation in enriched environment. Although daily enrichment has started two weeks after the HI event, an important memory deficit was prevented in rats submitted to neonatal insult. In another study we demonstrated that object-recognition memory was recovered in HI adolescent rats after an early permanent enrichment period (Pereira et al., 2008). Interestingly, present findings

indicate an extension of the therapeutic window for functional prevention in consequence to neonatal HI event, in agreement with our previous results with spatial memory in water maze task (Pereira et al., 2007). In accordance with our findings, other studies demonstrate that pharmacologic and non-pharmacologic approaches require relatively early initiation for therapeutic efficacy (Dixon et al., 2003; Kline et al., 2001, 2004; Kokiko and Hamm, 2007). On the other hand, Matter et al. (2011) found that cognitive performance was enhanced in animals submitted to enriched environment starting 7 days after traumatic brain injury. Additionally, our results revealed impaired performance of HI animals (groups HISE and HIEE) as regards to the exploration of objects in the first session of EE. Since hyperactivity was observed in HI animals, as commented above, we suggest that this finding is not consequent to a motor deficit but may indicate a motivational effect of brain hypoxia. Aversive memory impairment in HI animals, independent of the environment condition, was demonstrated in the inhibitory avoidance task. This result could be due to the fact that HI injury has damaged other brain areas implicated in acquisition and expression of aversive memory (Arteni et al., 2003; Baker and Kim, 2004; Coleman-Mesches and McGaugh, 1995). The HI rat model is known to cause cerebral atrophy but, unlike human neonates, rats that underwent neonatal HI do not show gross motor deficits: they apparently move like control animals and do not display evident postural and locomotor abnormalities (Jansen and Low, 1996a). We found no gross motor impairment, assessed in the rota-rod, in adult HI rats; this is supported by results from Balduini et al. (2000) and Lubics et al. (2005), who found no differences between control and HI adult animal performance in the rota-rod test. However, conflicting studies provided evidence for long term deficits in motor coordination tasks after HI (Bona et al., 1997; Jansen and

Fig. 3. Latencies to step down the platform, in training and test sessions on inhibitory avoidance task. Bars represent the median ± interquartile range of latencies. *HI groups are significantly different from CT groups in the test session; Kruskal–Wallis analysis followed by Mann–Whitney, p b 0.0125.

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Table 2 Rota-rod test. Mean ± S.E.M. of values. Group

Number of falls

Latency of the 1st downfall

Maximum time of permanence

CTSE CTEE HISE HIEE

3 3 3 2.87 ± 1.12

119.1 ± 12.9 108.8 ± 17.8 106.7 ± 18.3 81.9 ± 20.1

199.1 ± 11.9 173.5 ± 20.3 188.2 ± 17.7 171.7 ± 27.7

Low, 1996a,b; Spandou et al., 2005). Im et al. (2010) also identified motor impairment in animals submitted to neonatal HI at 5–6 weeks of age, compared to control group, on rota-rod. Additionally, Ten et al.

(2004) demonstrated that mice which had severe brain damage (with porencephalic cyst formation), observed 7 to 9 weeks after hypoxic–ischemic insult, also presented a significant dysfunction in the rota-rod test. Although we have collected data on the presence porencephalic cysts in HI rats, no statistical correlation with rota-rod performance was run since animals subjected to morphological analysis were not the same that performed behavioral tasks (see Animals). Karasev et al. (2010), demonstrated severe motor dysfunction in HI animals during the 5th to 10th weeks; however a spontaneous recovery was found in the 11th and 12th weeks after the event. Since the rota-rod test is useful to evaluate balance and coordination, we suggest that gross locomotor activity in adult animals is not impaired by neonatal HI. In addition, it is known that the neonatal brain has an impressive level of plasticity

Fig. 4. Dendritic spine density at CA1 region from right and left hippocampus. In A coronal slice of rat brain; in B hippocampal CA1 region indicating the stratum lacunosum-moleculare; in C a drawing of a pyramidal neuron showing in the box an example of studied dendritic branch (HIEE group); in D a dendritic branch; E–H: representative photomicrographs of tertiary branches from CA1 pyramidal neurons; E — CTSE group; F — CTEE group; G — HISE group and H — HIEE group; I: Graphic representation of dendritic spine density. Bars represent mean ± S.E.M. of dendritic spine density per hemisphere (spines/μm); all analyses were performed comparing each hemisphere separately. #CTEE is significantly different from other groups in right hemisphere; *HISE is significantly different from control groups considering right hemisphere; ANOVA followed by Duncan's test, p b 0.0125.

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(Balduini et al., 2000; De Paula et al., 2009) and that could be responsible by spontaneous recovery of sensory motor deficits. Previous environmental enrichment reports from our group demonstrated cognitive improvements in HI rats; however it failed to show any effect on the extension of damage to hippocampus and striatum (Pereira et al., 2007, 2008). Several authors ratify that EE promotes neural protection, i.e., increases neurogenesis, dendritic branching, and dendritic spine number and enhances cell survival (Leggio et al., 2005; Van Praag et al., 2000). Since EE may cause changes to dendritic spine morphology (Alvarez and Sabatini, 2007), the present study evaluated spine density in tertiary dendrites of pyramidal neurons of hippocampal CA1. Confirming the working hypothesis, a decrease of spine density in the hippocampus after the HI insult was shown. Dendritic spine density from right hemisphere (ipsilateral to arterial occlusion) indicated a partial recovery effect of the environmental enrichment on hypoxic–ischemic damage. As regards the left hippocampus (contralateral to arterial occlusion), results confirmed a lower spine density in rats submitted to HI, in agreement with previous studies showing that brain unilateral insults induce neuronal changes both in ipsilateral and contralateral hemispheres (Cheng et al., 1997; Nicolelis, 1997; Shimada et al., 1997). Interestingly, EE increased dendritic spine density in hippocampal CA1 in control animals (group CTEE). This finding corroborates previous results of maintained enriched housing (Bindu et al., 2007; Leggio et al., 2005) and. is the first demonstration of spine density increase after a daily enrichment protocol (only 1 h/day). To our knowledge, this is the first report showing an effect of environmental enrichment initiated 2 weeks after injury on morphological brain damage after a neonatal HI event. In studies evaluating effects of enrichment in adult rats submitted to brain ischemia, some data demonstrated plastic effects on neural tissue such as: increase in number and size and changes in shape of dendritic spines (Johansson and Belichenko, 2002; Leggio et al., 2005), decrease infarct volume and increase number of astrocytes and oligodendrocyte progenitors (Leggio et al., 2005). Certainly, there is a complex group of molecular events associated to neuroprotective effect following EE stimulation. It has been proposed that the development of dendritic spines occurs concurrently with the growth of the pre-synaptic elements, suggesting that cell–cell interaction and extrinsic cues likely induce the formation of dendritic spines (Lippman and Dunaevsky, 2005). The increase in dendritic spine density could be explained by spinogenesis and a consequent synaptogenesis. Benson and Huntley (2012) suggest that some spines form in concert with their synapses, while others arise from previously established shaft synapses, and that different stages of development or neuronal states might favor one or the other. During early stages of synaptogenesis, dendrites are covered with filopodia, which contain signaling molecules sufficient to establish transient communication between pre and postsynaptic elements; they are quiet transient (Ziv and Smith, 1996). Results using images techniques demonstrated that dendritic filopodia are actively seeking synaptic partners (Ziv and Smith, 1996). These data could indicate that the first step in synapse formation, can occur on a rather simple and transient postsynaptic structure and is probably mediated by broadly distributed, lightly adhering cell surface molecules. In addition, it has been demonstrated that spines move and change shape with a surprisingly high frequency in young neurons, but substantially lower in mature neurons (Dunaevsky et al., 1999; Fischer et al., 1998), confirming that this mobility can be important for aspects of synapse formation and functional maturation. Moreover, it should be considered that spine density is just one of morphological variables which can be studied aiming to understand EE effects on neurons of the hypoxic–ischemic animals. Arborization, length and morphology of dendritic spines deserve further investigation. Concluding, environmental enrichment was effective in recovering behavioral impairment and preserving dendritic spine density in

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the hippocampus consequent to neonatal hypoxia–ischemia in the rat. These findings suggest that changes in dendritic spines may play a role in the functional effects of EE after neonatal HI and support the potential of environmental enrichment as a non-invasive rehabilitation strategy to ameliorate cognitive deficits. Acknowledgments We would like to thank the technical assistance given by Ana Paula Maciel Ornaghi. This work was supported by Brazilian funding agencies: CNPq, CAPES and FAPERGS. References Alvarez, V.A., Sabatini, B.L., 2007. Anatomical and physiological plasticity of dendritic spines. Annu. Rev. Neurosci. 30, 79–97. Araki, H., Hino, N., Karasawa, Y., Kawasaki, H., Gomita, Y., 1999. Effect of dopamine blockers on cerebral ischemia-induced hyperactivity in gerbils. Physiol. Behav. 66 (2), 263–268. Arteni, N.S., Salgueiro, J., Torres, I., Achaval, M., Netto, C.A., 2003. Neonatal cerebral hypoxia–ischemia causes lateralized memory impairments in the adult rat. 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