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Animal model of autism induced by prenatal exposure to valproate: Behavioral changes and liver parameters
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Research Report
Animal model of autism induced by prenatal exposure to valproate: Behavioral changes and liver parameters Victorio Bambini-Junior a, b, c,⁎, Leticia Rodrigues b , Guilherme Antônio Behr b , José Cláudio Fonseca Moreira b , Rudimar Riesgo a, c, d , Carmem Gottfried a, b, c a
Federal University of Rio Grande do Sul, Research Group in Neuroglial Plasticity, Porto Alegre, RS, Brazil Federal University of Rio Grande do Sul, Department of Biochemistry, Institute of Health's Basic Science, Porto Alegre, RS, Brazil c Federal University of Rio Grande do Sul, Pervasive Developmental Disorders Program (ProTID), Porto Alegre, RS, Brazil d Federal University of Rio Grande do Sul, Pediatric Neurology Center, Porto Alegre Clinical Hospital (HCPA), Porto Alegre, RS, Brazil b
A R T I C LE I N FO
AB S T R A C T
Article history:
Autism is characterized by behavioral impairments in three main domains: social interaction;
Accepted 6 June 2011
language, communication and imaginative play; and range of interests and activities.
Available online 12 June 2011
This syndrome has attracted social attention by its high prevalence. The animal model induced by prenatal exposure to valproic acid (VPA) has been proposed to study autism.
Keywords:
Several characteristics of behavioral abnormalities found in the VPA rats, such as
Autism spectrum disorder
repetitive/stereotypic-like activity and deficit in social interaction have been correlated
Sodium valproate
with autism. Features like flexibility to change strategy, social memory and metabolic
Animal model
status of the induced rats have not been examined. Thus, the main aim of this work
Behavioral impairment
was to investigate additional behavioral rodent similarities with autism, as well as, liver redox parameters after prenatal exposure to VPA. Young rats from the VPA group presented aberrant approach to a stranger rat, decreased conditioned place preference to conspecifics, normal spatial learning and a lack of flexibility to change their strategy. As adults, they presented inappropriate social approach to a stranger rat, decreased preference for social novelty, apparently normal social recognition and no spatial learning deficits. Examination of the liver from the VPA group presented significantly increased (12%) levels of catalase (CAT) activity, no alteration in superoxide dismutase (SOD) activity and a decrease in the SOD/CAT ratio. TBARS, sulfhydril and carbonyl contents, and serum levels of aminotransferases remained unchanged. In summary, rats prenatally exposed to VPA presented decreased flexibility to change strategy and social impairments similar to the autism symptoms, contributing to the understanding of neurodevelopmental symptoms and oxidative imbalance associated to the autism spectrum disorder. © 2011 Published by Elsevier B.V.
⁎ Corresponding author at: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, 90035-003, Porto Alegre, RS, Brazil. Fax: +55 51 3308 5540. E-mail address: [email protected] (V. Bambini-Junior). 0006-8993/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.brainres.2011.06.015
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1.
Introduction
Autism spectrum disorder (ASD), first described in 1943 by Leo Kanner (Kanner, 1943), is a complex, behaviorally defined disorder of the immature brain comprising autistic disorder, Asperger disorder and pervasive developmental disorder not otherwise specified (PDD-NOS) (Kogan et al., 2009). This syndrome has attracted public attention by its high prevalence (Stokstad, 2001). A recent study shows that the prevalence of parent-reported diagnosis of ASD among US children aged 3 to 17 years was estimated to be 110 in 10,000 (1 in 91) (Kogan et al., 2009). This data implies high impact on the family, and cost to society (DiCicco-Bloom et al., 2006). Autism is characterized by impairments in three main domains: 1) social interaction; 2) language, communication and imaginative play; and 3) range of interests and activities (Gadia et al., 2004; Rapin and Tuchman, 2008). Additionally, three to four times more males are affected by autism than females (Fombonne, 1999). Even though twin studies show a strong genetic component in ASD and multiple interacting genetic factors as the main causative determinants of autism (Muhle et al., 2004) the etiology remains unknown. However, in addition to the genetic predisposition, epidemiological studies indicate that it is necessary for these genetic factors to interact with exposure to environmental neurotoxicants (Currenti, 2010; Fombonne, 2003, 2009) e.g. prenatal exposure to xenobiotics, such as thalidomide and 2-propylpentanoate/ valproic acid (VPA) (Bello, 2007; Landrigan, 2010). Thalidomide has been shown to lead to a high incidence of autism when exposure occurs during the 20th to 24th days of gestation (Rodier et al., 1997). A clinical link between VPA and autism was recognized by examining the phenotypic abnormalities of the face and developmental disabilities in children who were exposed in utero to VPA (Christianson et al., 1994). There have been case studies and retrospective reports indicating that exposure to antiepileptic drugs may be associated with an increased risk of ASD (Bromley et al., 2008). Children exposed to these medications, particularly VPA, are more vulnerable to show poor cognitive development and may be monitored for more detailed cognitive assessment and early intervention (Bromley et al., 2010). These clinical studies gave evidence for, and a mechanism to develop a pharmacological animal model of autism by the prenatal exposure to neurotoxicants (Blaiss et al., 2009; Murcia et al., 2005; Rodier et al., 1997; Schneider and Przewlocki, 2005; Wagner et al., 2006). Therefore the investigation of potential metabolic dysfunctions in rodents prenatally exposed to VPA could reveal new affected patterns in the animal model (Aires et al., in press), opening a great range of possibilities, such as developmental, behavioral and immunological analyses which are not possible in humans (van der Worp et al., 2010). These studies include the evaluation of the teratogenic potential of VPA during pregnancy and increase the understanding about alterations in both human (Alsdorf and Wyszynski, 2005; Christianson et al., 1994) and rodent (Rodier et al., 1997) development. A single administration of VPA in utero leads to developmental delays and lifelong deficits in motor performance, social behavior, and anxiety-like behavior in rat offspring (Kolozsi et al., 2009; Schneider and Przewlocki, 2005), including develop-
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mental, anatomical and functional similarities with human autistic patients (Rodier et al., 1997; Schneider et al., 2006). Rats exposed to VPA showed reductions in neuron cell counts in the cranial nerve motor nuclei and also showed cerebellar anomalies like those reported in studies of autistic cases, supporting the idea that these animals may be a useful model of the developmental injury that initiates autism (Rodier et al., 1997). Although a significant number of studies show the consequences of prenatal exposure to VPA at the central nervous system level (Rodier et al., 1997; Tsujino et al., 2007), more investigations are needed to clarify the behavioral symptoms. Thus, the main aim of this work was to investigate additional behavioral rodent similarities with human autism, as well as, possible metabolic dysfunctions caused by the prenatal exposure to VPA, including variations in the redox state of the liver.
2.
Results
2.1.
Behavioral testing
2.1.1.
Young rats (35–50 days old)
2.1.1.1. Three-chamber sociability test.
These results are shown in Fig. 1. There was no difference between groups in the time (s) spent in the following chambers: in chamber 1, which contains the stranger rat; in chamber 2 which contains the object and in the empty central chamber. In addition, both groups spent less time in the central chamber than in other chambers. The control group spent more time in chamber 1 than in chamber 2 (p < 0.001) and the VPA group did not show this difference. A significant effect of group was observed on the time (s) spent exploring the stranger rat (180.5 ± 26.6 for the control and 66.1 ± 15.4 for the VPA) group. Mean comparisons revealed that VPA rats explored the stranger rat less than control males (p < 0.001). The time (s) exploring the object
Fig. 1 – Three-chamber sociability test in young rats (35–50 days old) prenatally exposed to VPA. Social approach was measured after 5 min of acclimation and 10 min of test, with the stranger rat and an object on each side. The stranger rat was positioned at chamber 1 and the object was placed at chamber 2. Data are expressed as mean±SEM (VPA group N=8, control group N=12). *different from control group, p < 0.001. #difference within groups, p< 0.001.
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showed no difference between the groups. There is also a significant difference within control groups in the exploration parameter (p < 0.001). They spent more time (s) exploring the stranger rat (180.5 ± 26.6) then the object (72.0 ± 12.5).
2.1.1.2. Y-maze. No statistical difference was found in the learning ability (Fig. 2A), expressed by the total trials until the criterion of learning was reached (10 right choices in a row) between VPA (59.13 ± 7.6) and control (63.75 ± 7.2) groups. The choice arm with food was the left arm. After the rat learned the task, the food was placed on the other choice arm. There was a significant increase in the number of trials the VPA subjects tried the new arm (Fig. 2B), showing persistence to sameness in the VPA (3.37 ± 0.9) group compared to controls (1.67 ± 0.12). The reversed Y-Maze was performed (with the food on the new right arm) and no statistical differences were observed (data not shown). 2.1.2.
Adult rats (80–115 days old)
2.1.2.1. Three-chamber sociability test. These results are shown in Fig. 3. The first phase of the experiment (Fig. 3A) showed a statistically significant decrease (22%) in the time spent exploring the stranger rat by VPA rats (116.8 ± 8.7 s) compared to the control group (150.8 ± 11.3 s, p < 0.05). Moreover, similarly to the young rats, there is a significant difference in
Fig. 2 – Learning ability and flexibility to change strategy measured in the Y-maze in young (35–50 days old) rats prenatally exposed to VPA. For the acquisition test the start arm was the left arm and for the reversion test, the start arm was always the right one. A. Learning ability, expressed as total trials until learn the task: 10 right response in 10 consecutive trials. B. Number of trials until change of the strategy. Data are expressed as mean ± SEM (VPA group N = 8, control group N = 12). *different from control group, p < 0.05.
Fig. 3 – Three-chamber sociability test in adult rats (80–115 days old) prenatally exposed to VPA. A. Phase 1: sociability measured after 5 min to acclimation and 10 min of test. B. Phase 2: social memory after 10 min of test. In the first phase of the task chamber 1 contains the stranger rat 1 and chamber 2 is the one which contains the object. In the second phase chamber 1 contains the “stranger 1” (present in phase 1) and chamber 2 contains a new rat, the “stranger 2”. The score was evaluated by the time spent in each chamber and by the time spent sniffing each wire cage. Data are expressed as mean ±SEM (VPA group N= 8, control group N= 8). *different from control group, p < 0.05. # difference within groups, p <0.05.
the adult rats within control groups in the exploration parameter (p< 0.001). They spent more time (s) exploring the stranger rat (150.8 ± 11.3) then the object (91.15±7.7). In the second phase (Fig. 3B), the time (s) spent in chamber 1, which contains the known rat was statistically different between groups (168.8 ± 13.1 for control and 224.5 ± 16.8 for VPA, p < 0.05). In addition, a difference in the time (s) spent exploring a new stranger rat was also found, showing a lower interest to begin a social approach in the VPA group (163.7 ± 18.2 for control and 105.6 ± 15.4 for VPA, p < 0.05). The analysis within groups showed also statistically significant differences. As expected, control group spent more time (s) in chamber 2 (299.81 ± 24.50) than in chamber 1 (168.65 ± 13.12). Moreover, control group demonstrated higher exploration of the new stranger rat (163.62 ± 18.15) when compared to the known rat (71.16 ± 9.39), p < 0.001. The same behavior was observed on the VPA group which spent more time exploring the new rat (105.62 ± 15.44) than the known (59.00 ± 7.07), p < 0.05.
2.1.2.2. Morris water maze. No statistical difference was found between the VPA and control groups in the Morris
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the activity of those enzymes in blood as compared to control group, suggesting that this teratogen did not affect hepatic cell integrity (Table 1). Moreover, the activity of two important antioxidant enzymes, SOD and CAT and oxidative damage, by damage on lipids (TBARS), damage on proteins (−SH and carbonyl content) had also been quantified in liver samples. The activity of SOD (U SOD.mg protein− 1) of control group (45.00±3.54) did not show a significant difference as compared to the VPA group (44.75 ±1.92) (Fig. 5A). However, VPA induced a significant increase (12%) in CAT activity (from 144.89 ± 7.82 to 169.02 ± 8.9 U CAT.mg protein − 1) in the VPA group (Fig. 5B). The SOD/CAT ratio (Fig. 5C) decreased in VPA rats (from 0.310±0.020 to 0.267±0.037). As showed in Fig. 6, no statistically significant differences were found on TBARS (from 0.0812± 0.0026 to 0.0755 ± 0.0065 nmol TMP.mg protein− 1), sulfhydryl (−SH) (from 36.7 ± 5.54 to 36.7 ± 2.28 μmol (−SH).mg protein− 1) and carbonyl content (from 2.80± 0.74 to 3.56 ± 0.39 μmol carbonyl.mg protein− 1).
3.
Discussion
The animal model of autism induced by VPA, has a significant number of characteristics observed in humans with autism.
Fig. 4 – Assessment of spatial learning ability determined by the Morris water maze (MWM) in adult rats (80–115 days old) prenatally exposed to VPA. Testing was conducted in 2 phases, acquisition and reversal. Each phase consisted of 4 trials/day to find the platform for 5 consecutive days. This was followed by one additional day when a single probe trial was given. The start positions used were: NW, N, E, and SE. A. Spatial learning ability. B. Ability to change the strategy to learning a new task on the reversed MWM (VPA group N = 8, control group N = 12).
Water Maze (MWM) and in the Reversed Morris Water Maze (RMWM) (Fig. 4).
2.1.2.3. Biochemical analysis. Liver cytotoxicity was investigated by the serum activity of the hepatic enzyme markers AST/GOT and ALT/GPT. Prenatal exposure to VPA did not modify
Table 1 – Activity of hepatic enzyme markers and urea content in serum from 120 days-old rats prenatally exposed to VPA.
Control VPA
AST/GOT (IU/L)
ALT/GPT (IU/L)
Urea (mg/dL)
95.36 ± 5.87 91.40 ± 4.45
46.5 ± 6.53 49.2 ± 5.6
186.14 ± 10.41 183.03 ± 9.79
AST/GOT, aspartate aminotransferase or glutamic-oxaloacetic transaminase; ALT/GPT, alanine aminotransferase or glutamicpyruvic transaminase. Values are means ± SEM, N = 4–6.
Fig. 5 – Liver antioxidant enzyme activity in adult rats prenatally exposed to VPA. At 120 days old, rats were sacrificed; the tissues were removed and kept in −70 °C until the assays were performed. A. SOD activity; B. CAT activity and C. SOD/CAT ratio. The experiments were performed in triplicate. Data are expressed as mean ± SEM (VPA group N = 6, control group N = 4). *different from control group, p < 0.05.
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Fig. 6 – Oxidative damage parameters in liver from adult rats prenatally exposed to VPA. At 120 days old, rats were sacrificed; the tissues were removed and kept in − 70 °C until the assays were performed. A. Lipid peroxidation assessed by TBARS (VPA group N = 4, control group N = 4). B. Protein damage by reduced sulfhydryl content (VPA group N = 6, control group N = 6). C. Protein damage by carbonyl content (VPA group N = 6, control group N = 6). Data are expressed as the mean ± SEM, and experiments were performed in quintuplicate.
Among those are repetitive and stereotypic behavior, decreased number of social behaviors (Schneider et al., 2006), impairment in the initiation of social interaction (DufourRainfray et al., 2010) and abnormality of circadian rhythm (Tsujino et al., 2007). The present work shows an evaluation of social memory, flexibility to change strategy and biochemical parameters. Behavioral tasks in young rats showed the following results: decreased time spent by the VPA rats exploring a stranger rat in a three chambered apparatus. The time spent exploring the stranger rat was significantly lower in the VPA group indicating reduced social approach to a stranger rat. This clearly shows a social dysfunction induced by the prenatal exposure to VPA that is a consistent characteristic of autism. In addition, control rats have shown a preference to conspecific. While control rats spent more time in chamber 1 containing the stranger rat, VPA rats did not show any preference. It is well known that mental retardation is a
characteristic often related with autism (Casanova, 2007). However, on the Y-maze, a spatial discrimination learning task, no difference between groups was found, indicating that the VPA rats have no spatial learning impairment. Moreover, increased latency of VPA rats to change their strategy was observed in the reverse Y-maze. These data are in agreement with previous work which shows enhanced repetitive behavior in the Y-maze (Markram et al., 2008). Once VPA rats learned the task normally, when challenged to try a new strategy, they showed a lack of flexibility, spending more trials on the first reward arm. It could be related with behavioral rigidity (South et al., 2007), which is very typical and present on autism. As adults, the tests performed were three-chambered apparatus (2 phases) and MWM (acquisition and reversion). The three-chamber apparatus tasks provided a good perspective about how social behavior is impaired in rats prenatally exposed to VPA. The first phase of the paradigm showed a statistically significant difference between groups; VPA group spends less time exploring the stranger rat. Therefore, it is a similar type of aberrant behavior prevailing at young ages on VPA rats. Interestingly, different from control groups, no difference was observed comparing the time exploring a stranger animal and an object within VPA groups, in both young and adult rats, suggesting that the VPA rats demonstrate the same interest exploring an object and a rat. Schneider et al. (2006) found that VPA young rats display lower number of pinnings, although the latency to pinning and duration of pinning did not differ between groups. Moreover, in the second phase of the test, where the preference for social novelty was evaluated, VPA rats showed less interest to make a new social approach. This was explained by the significantly lower time the VPA group spent exploring the new stranger rat, compared to the control group. Nevertheless, both groups decreased the time spent to explore the first stranger rat on the second phase when compared with the first phase, indicating that they showed the same degree of interest by the stranger rat already explored. In addition, within groups, both control and VPA rats explored the second (new) stranger rat more than the first on the second phase. Besides, between groups, the VPA group spent more time than the controls in chamber 1 (with the rat already explored). This could be a consequence of the fact that VPA rats were more willing to be in a chamber with a known rat than in a chamber in which they would be challenged to a new social approach, indicating apparently normal social recognition but decreased social interaction. Recent results obtained with VPA rodents on the threechambered apparatus showed increased time spent in the central chamber and increased number of crossing between the chambers by VPA compared to control rats (Dufour-Rainfray et al., 2010). In addition, similar results were found with mice in this task, where the VPA group decreased the number of nose pokes on social novelty test (Roullet et al., 2010). On the MWM task, the VPA rats showed the same behavior as the control group to both acquisition and reversion phases, indicating that spatial learning and memory references are still fully functional in the VPA group, at least in this task, in agreement with previous work that shows normal to slightly
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impaired spatial learning in the MWM (Markram et al., 2008). VPA rats showed a very good adaptation to the new task on the reversal MWM similar to the controls, which in part could be seen as a reversion of the behavior shown by young VPA rats, where the latency to try a new strategy was found. Moreover, this could be just a task sensitive limitation, in which reversed Y-maze findings were not reproduced by reversed MWM and deserve further studies to clarify this issue. It is of common knowledge that pharmacological tissue damage could lead to a huge spectrum of behavioral impairments (Delongeas et al., 2010). Accordingly, we must try to elucidate if those aberrant behaviors came selectively from a brain damage or if it came from, at least in part, other metabolic pathways. It possibly could happen, since the prenatal exposure to teratogenic by VPA is affecting the pups' body globally (Binkerd et al., 1988; Ingram et al., 2000). There is evidence that patients with autism may have serum markers for inflammation, including elevation of some cytokines, auto-antibodies, and lower levels of normal immunoglobulins for immune defense. Studies involving inflammatory response due to liver failure and metabolic impairment have shown correlation with brain and behavioral impairment (Chez and Guido-Estrada, 2010; Pardo et al., 2005). Moreover, children with autism were more likely to have mitochondrial dysfunction, mtDNA overreplication, and mtDNA deletions than typically developing children (Giulivi et al., 2010). Therefore, it seems proper to assume that the liver, an organ that plays a central role on metabolic status, is an important place to look at for possible damage or metabolic alterations. We evaluated urea and the enzyme markers of liver cytotoxicity AST/GOT and ALT/GPT, as a way to investigate if the VPA rodent impairments could be correlated to alterations in hepatic metabolism. However, these parameters were not changed between the VPA and control rats (Table 1), indicating absence of hepatic damage. Other pathological evidence of immunological reactions within the CNS is the presence of lymphocyte infiltration and microglial nodules and increases in pro-inflammatory cytokines in peripheral blood samples from patients with autism (Jyonouchi et al., 2001). Besides, we analyzed liver's oxidative damages which could compromise cellular function without causing a rupture of plasmatic membrane. No damage nor oxidative level alterations on proteins (by the evaluation of carbonyl and sulfhydryl groups, respectively) and no damage to lipid membranes (by TBARS) were observed in the VPA group. The enzyme SOD dismutates the \O−2 generating H2O2 which is converted in 2H2O + O2 by catalase. H2O2 can act as a signaling molecule or, with transition metals, yield the hydroxyl radical (UOH) (Fenton and Habber–Weiss reaction) (Halliwell, 2006). Thus, we have found an increased CAT activity and decreased SOD/CAT ratio; however, no oxidative damage was observed by the parameters studied. It is indicative that hepatic cells were able to handle the production of free radical and subproducts. It is also important to remember that reactive oxygen species also plays an important role as signaling molecules and that the imbalance between SOD and CAT activity (Fig. 5C) although apparently did not generate increased oxidative damage, may compromise long term cell function.
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In summary, the present data indicate that young VPA rats exhibited impaired approach to a stranger rat, decreased place preference to conspecifics, normal learning and a lack of flexibility on their strategy. As adults, they showed inappropriate social approach to a stranger rat, decreased preference for social novelty, apparently normal social recognition, no spatial learning deficits and normal resistance to change on MWM. It is an important contribution for an increased understanding of neurodevelopmental symptoms and oxidative imbalance associated to the autism spectrum disorder.
4.
Experimental procedures
4.1.
Animals
Experiments were performed according to the NIH Guide for the Care and Use of Laboratory Animals and approved by local authorities. Female Wistar rats coming from the local breeding colony (ICBS-Federal University of Rio Grande do Sul), with 12:12 light cycle (lights on at 7:00 and lights off at 19:00), controlled temperature (22 ± 1 °C), water and food ad libitum. They had their fertility cycle controlled, and, when on proestrus, mated overnight. In the morning, vaginal secretion was collected with a plastic pipette and placed on glass slides to be analyzed (Marcondes et al., 2002). If spermatozoa were found in the morning, it was designated as first day of pregnancy. Valproic acid (Acros Organics, New Jersey, USA) was purchased as the sodium salt and dissolved in 0.9% saline for a concentration of 250 mg/mL. Females received a single intraperitoneal injection of 600 mg/kg sodium valproate (VPA) or physiological saline (control) on E12.5 (Schneider and Przewlocki, 2005). The delivery of this dose can result in 900 μg/mL of total VPA in maternal plasma in less than 1 h, with a mean/average plasma elimination half life of 2.3 h (Binkerd et al., 1988). Females were kept separate and with free access to their own litters. Rats from both groups control and VPA were born healthy and the number of offspring was normal. Somatic aspects observed during the pups' development, included body weight, ear unfolding and eye opening which were unchanged between groups in agreement with previous work (Dufour-Rainfray et al., 2010). The offspring rats were weaned at 21 days old and were housed separately by sex. The experiments were performed twice, using 4–6 male rats from each litter. Rats had free access to food and water. All the experiments were performed between 12:00 and 17:00. In order to analyze the differences between young and adult rats, behavioral tests were performed at 35–50 and 80–115 days old, considered young and adult rats, respectively. At 120 days old, rats were sacrificed; the tissues were removed and kept in −70 °C until the assays were performed. 4.2.
Behavioral tests
4.2.1.
Three-chamber sociability test
The social test was performed in a three chambered apparatus as described previously in a mice task, with modifications required to perform it in rats (Nadler et al., 2004). It is a wooden box no painted with partitions separating the box into three
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chambers with dimensions (length/width/height in cm) 120/40/50, being 60 cm length to the central chamber and 30 cm each side. The openings between compartments allowed free exploration to the different chambers. Time spent in each chamber; as well as the time spent exploring the stranger rat or an object in the chamber, was analyzed by two observers. The object was an empty identical cage used to enclose the stranger rat. Chambers were cleaned with 70% ethanol and water between tests. Animals used as “strangers” were Wistar males with the same age and no previous contact with the test rats. Animals were individually acclimated for 5 min into the apparatus on the day before the experiment. On the sociability test, rats were allowed to expend 5 min in the central chamber, and then the stranger rat was introduced into one of the side chambers. When performed with young rats (40–45 days old), the experiment was performed for up to 10 min, with the stranger rat and an object on each side. When performed with adult rats (80–115 days old), a 10-minute test to quantify preference for social novelty began immediately after the 10-minute test for sociability. In this test, the original stranger rat (stranger 1) remained in its wire cage on one side of the apparatus and a new unfamiliar rat (stranger 2) was placed in the wire cage on the opposite side, which was previously empty during the sociability test. The score was evaluated by the time spent in each chamber and by the time spent sniffing each wire cage. Stranger 1 and stranger 2 animals originated from different home cages had never been in physical contact with the subject rat. The three chambered apparatus was centered onto a lab bench to minimize lightgradients in, temperature, sound and other environmental conditions that could produce a side preference. 4.2.2. Y-maze Learning ability was tested as described (Aguilar et al., 2000; Piedrafita et al., 2008) in a wooden Y-shaped maze, which had three arms of equal size (60 cm long, 11.5 cm wide and 25 cm high). For the acquisition test the start arm was the left arm and for the reversion test, the start arm was always the right one. The other arms, each of which had a food cup located at the end, were considered the choice arms. Pre-training was carried out during 4 days with all rats to familiarize them with the maze. In each trial, rats were rewarded for choosing the left arm. The reward for the correct response consisted of half Froot-Loop® placed in the food cup at the end of the correct arm. If the rat made an incorrect choice, it was allowed to go to the empty food cup at the end of the incorrect arm and was removed after 5 s. Rats were trained for ten trials per day, with an inter-trial interval of approximately 5 min in their home cage until the completion of a criterion of ten correct responses in ten consecutive trials or until a maximum of 250 trials. After the subject reach the criterion, the reward was placed at the end of the reverse arm and the number of trials was measured until the rat changed its strategy, by choosing the other arm (the right side one). 4.2.3. Spatial learning in the Morris water maze (MWM) The MWM, an established test of spatial learning and reference memory, was conducted in a 210 cm diameter tank made of fiberglass and painted flat black (Vorhees et al., 2009). The 3 walls nearest to the maze (representing arbitrarily N, E,
and W walls of the test room) had large geometric figures mounted above the edge of the pool. The experimenter stood at the “S” position during testing and remained stationary. Testing was conducted in 2 phases, acquisition and reversal. Each phase consisted of 4 trials/day for 5 consecutive days; this was followed by one additional day when a single probe trial was given. Probe trials lasted for 30 s. The time limit on learning trials was 1 min and the inter-trial interval (ITI) was 5 min spent on the cage. Animals that did not find the platform within 1 min were placed on the platform again. Goal platforms (with diameters of 10 or 7 cm) were made of black acrylic with thin nylon screening attached to the surface to provide traction. The platform was positioned 1–2 cm below the surface of the water and was camouflaged by virtue of being transparent against a black background. Water temperature was 21 ± 1 °C. The acquisition phase occurred on P81–86. During acquisition, the 10 cm diameter platform was used and located in the SW quadrant halfway between the center and the side of the tank. Rats started at one of four positions located distal to the quadrant containing the platform in a random order with the restraint that they received one trial from each of the four starting positions per day. The start positions used were: NW, N, E, and SE. These positions were used to eliminate short paths to the goal, such as those that are possible if S or W starts are used. The day after the last acquisition trial, each rat was given a 30 s probe trial. For the probe trial, rats started from a position they had never started from before (NE). On P88–93 rats were tested in the reversal phase with a 7 cm diameter platform placed in the NE quadrant. The smaller platform was used to increase the spatial accuracy required to locate the platform. The same procedure was used as for acquisition (5 days, 4 trials/day). Start positions were SE, S, W, and NW. On the sixth day, the platform was removed and a 30 s reversal probe trial was administered with the start position at SW.
4.3.
Biochemical blood parameters and oxidative stress analysis
Blood sampling and analysis: at 120 days-old, overnightstarved animals were anesthetized by intramuscular injection of 75 mg/kg ketamine and 10 mg/kg of xylazine, respectively. Blood samples were obtained by intracardiac punction and animals were killed by decapitation. Blood samples were incubated at room temperature (25 °C) for 5 min and centrifuged at 3200 rpm for 5 min. Serum was stored at −70 °C until the day of analysis. Biochemical analyses of ALT/GPT (alanine aminotransferase or glutamic-pyruvic transaminase) and AST/GOT (aspartate aminotransferase or glutamic-oxaloacetic transaminase) activity were performed using kits from local supplier Human do Brasil. 4.3.1. Tiobarbituric acid reactive species (TBARS) assay As an index of lipid peroxidation, TBARS formation was measured using a hot acid reaction. The homogenates of rat liver slices were mixed with 0.6 mL of 10% trichloroacetic acid (TCA) and 0.5 mL of 0.67% thiobarbituric acid, and heated in boiling water for 25 min (Draper and Hadley, 1990). The levels of TBARS were spectrophotometrically determined at 532 nm. Results are expressed as nmol MDA equivalents/mg protein.
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4.3.2. Protein tiol (−SH) content A sample aliquot was diluted in 0.1% SDS and 10 mM 5,5dithiobis 2-nitrobenzoic acid (DTNB, Sigma). Ethanol was added to produce the intense yellowish color of the product of the reaction between the sulfhydryl (− SH) groups and DTNB. After 20 min, −SH levels were spectrophotometrically determined at 412 nm (Ellman, 1959). Results are expressed as μmol (−SH).mg protein− 1. 4.3.3. Determination of carbonyl groups Protein carbonyl formation was quantified as an index of protein oxidative damage (Levine et al., 1990). This method is based on the reaction of dinitrophenylhydrazine with protein carbonyl groups. The results are expressed as μmol.mg protein− 1. 4.3.4. Superoxid dismutase (SOD) activity assay SOD activity was quantified by the inhibition of superoxidedependent adrenaline auto-oxidation to adrenochrome using spectrophotometric measurements at 480 nm, as previously described (Misra and Fridovich, 1972). The results are expressed as absorbance/time (s). The area under the curve of the graph was used for statistical analysis and compared against the control values. Five units of CuZnSOD (E.C.: 1.15.1.1) were used to determine assay specificity. 4.3.5. Catalase (CAT) activity assay CAT (E.C.:1.11.1.6) activity was assayed as previously described (Aebi, 1984) by measuring the absorbance decrease at 240 nm in a reaction medium containing 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer, pH 7.0, and 50 μg protein. One unit (U) of the enzyme is defined as 1 μmol of H2O2 consumed per minute and the specific activity is reported as U/mg protein. 4.4.
Statistical analysis
Data are reported as mean±standard error mean (SEM) and were analyzed by Student's t-test. Analysis between the same groups was performed using repeated-measures ANOVA. Values of p<0.05 were considered significant. All analyses were performed using the SPSS program, Version 12.0 (SPSS, Chicago, IL).
Acknowledgments This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), FINEP/ Rede IBN 01.06.0842-00 and INCT-EN National Institute of Science and Technology for Excitotoxicity and Neuroprotection. Special thanks to Janine Mackenzie for the careful English review and to Cintia Zappe Fiori by the help with the statistical analysis. We are thankful to the reviewers and editor by their contributions which helped us to remarkably increase the quality of the present work and also improve our experimental protocols.
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Research Report
Animal model of autism induced by prenatal exposure to valproate: Behavioral changes and liver parameters Victorio Bambini-Junior a, b, c,⁎, Leticia Rodrigues b , Guilherme Antônio Behr b , José Cláudio Fonseca Moreira b , Rudimar Riesgo a, c, d , Carmem Gottfried a, b, c a
Federal University of Rio Grande do Sul, Research Group in Neuroglial Plasticity, Porto Alegre, RS, Brazil Federal University of Rio Grande do Sul, Department of Biochemistry, Institute of Health's Basic Science, Porto Alegre, RS, Brazil c Federal University of Rio Grande do Sul, Pervasive Developmental Disorders Program (ProTID), Porto Alegre, RS, Brazil d Federal University of Rio Grande do Sul, Pediatric Neurology Center, Porto Alegre Clinical Hospital (HCPA), Porto Alegre, RS, Brazil b
A R T I C LE I N FO
AB S T R A C T
Article history:
Autism is characterized by behavioral impairments in three main domains: social interaction;
Accepted 6 June 2011
language, communication and imaginative play; and range of interests and activities.
Available online 12 June 2011
This syndrome has attracted social attention by its high prevalence. The animal model induced by prenatal exposure to valproic acid (VPA) has been proposed to study autism.
Keywords:
Several characteristics of behavioral abnormalities found in the VPA rats, such as
Autism spectrum disorder
repetitive/stereotypic-like activity and deficit in social interaction have been correlated
Sodium valproate
with autism. Features like flexibility to change strategy, social memory and metabolic
Animal model
status of the induced rats have not been examined. Thus, the main aim of this work
Behavioral impairment
was to investigate additional behavioral rodent similarities with autism, as well as, liver redox parameters after prenatal exposure to VPA. Young rats from the VPA group presented aberrant approach to a stranger rat, decreased conditioned place preference to conspecifics, normal spatial learning and a lack of flexibility to change their strategy. As adults, they presented inappropriate social approach to a stranger rat, decreased preference for social novelty, apparently normal social recognition and no spatial learning deficits. Examination of the liver from the VPA group presented significantly increased (12%) levels of catalase (CAT) activity, no alteration in superoxide dismutase (SOD) activity and a decrease in the SOD/CAT ratio. TBARS, sulfhydril and carbonyl contents, and serum levels of aminotransferases remained unchanged. In summary, rats prenatally exposed to VPA presented decreased flexibility to change strategy and social impairments similar to the autism symptoms, contributing to the understanding of neurodevelopmental symptoms and oxidative imbalance associated to the autism spectrum disorder. © 2011 Published by Elsevier B.V.
⁎ Corresponding author at: Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos 2600 anexo, 90035-003, Porto Alegre, RS, Brazil. Fax: +55 51 3308 5540. E-mail address: [email protected] (V. Bambini-Junior). 0006-8993/$ – see front matter © 2011 Published by Elsevier B.V. doi:10.1016/j.brainres.2011.06.015
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1.
Introduction
Autism spectrum disorder (ASD), first described in 1943 by Leo Kanner (Kanner, 1943), is a complex, behaviorally defined disorder of the immature brain comprising autistic disorder, Asperger disorder and pervasive developmental disorder not otherwise specified (PDD-NOS) (Kogan et al., 2009). This syndrome has attracted public attention by its high prevalence (Stokstad, 2001). A recent study shows that the prevalence of parent-reported diagnosis of ASD among US children aged 3 to 17 years was estimated to be 110 in 10,000 (1 in 91) (Kogan et al., 2009). This data implies high impact on the family, and cost to society (DiCicco-Bloom et al., 2006). Autism is characterized by impairments in three main domains: 1) social interaction; 2) language, communication and imaginative play; and 3) range of interests and activities (Gadia et al., 2004; Rapin and Tuchman, 2008). Additionally, three to four times more males are affected by autism than females (Fombonne, 1999). Even though twin studies show a strong genetic component in ASD and multiple interacting genetic factors as the main causative determinants of autism (Muhle et al., 2004) the etiology remains unknown. However, in addition to the genetic predisposition, epidemiological studies indicate that it is necessary for these genetic factors to interact with exposure to environmental neurotoxicants (Currenti, 2010; Fombonne, 2003, 2009) e.g. prenatal exposure to xenobiotics, such as thalidomide and 2-propylpentanoate/ valproic acid (VPA) (Bello, 2007; Landrigan, 2010). Thalidomide has been shown to lead to a high incidence of autism when exposure occurs during the 20th to 24th days of gestation (Rodier et al., 1997). A clinical link between VPA and autism was recognized by examining the phenotypic abnormalities of the face and developmental disabilities in children who were exposed in utero to VPA (Christianson et al., 1994). There have been case studies and retrospective reports indicating that exposure to antiepileptic drugs may be associated with an increased risk of ASD (Bromley et al., 2008). Children exposed to these medications, particularly VPA, are more vulnerable to show poor cognitive development and may be monitored for more detailed cognitive assessment and early intervention (Bromley et al., 2010). These clinical studies gave evidence for, and a mechanism to develop a pharmacological animal model of autism by the prenatal exposure to neurotoxicants (Blaiss et al., 2009; Murcia et al., 2005; Rodier et al., 1997; Schneider and Przewlocki, 2005; Wagner et al., 2006). Therefore the investigation of potential metabolic dysfunctions in rodents prenatally exposed to VPA could reveal new affected patterns in the animal model (Aires et al., in press), opening a great range of possibilities, such as developmental, behavioral and immunological analyses which are not possible in humans (van der Worp et al., 2010). These studies include the evaluation of the teratogenic potential of VPA during pregnancy and increase the understanding about alterations in both human (Alsdorf and Wyszynski, 2005; Christianson et al., 1994) and rodent (Rodier et al., 1997) development. A single administration of VPA in utero leads to developmental delays and lifelong deficits in motor performance, social behavior, and anxiety-like behavior in rat offspring (Kolozsi et al., 2009; Schneider and Przewlocki, 2005), including develop-
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mental, anatomical and functional similarities with human autistic patients (Rodier et al., 1997; Schneider et al., 2006). Rats exposed to VPA showed reductions in neuron cell counts in the cranial nerve motor nuclei and also showed cerebellar anomalies like those reported in studies of autistic cases, supporting the idea that these animals may be a useful model of the developmental injury that initiates autism (Rodier et al., 1997). Although a significant number of studies show the consequences of prenatal exposure to VPA at the central nervous system level (Rodier et al., 1997; Tsujino et al., 2007), more investigations are needed to clarify the behavioral symptoms. Thus, the main aim of this work was to investigate additional behavioral rodent similarities with human autism, as well as, possible metabolic dysfunctions caused by the prenatal exposure to VPA, including variations in the redox state of the liver.
2.
Results
2.1.
Behavioral testing
2.1.1.
Young rats (35–50 days old)
2.1.1.1. Three-chamber sociability test.
These results are shown in Fig. 1. There was no difference between groups in the time (s) spent in the following chambers: in chamber 1, which contains the stranger rat; in chamber 2 which contains the object and in the empty central chamber. In addition, both groups spent less time in the central chamber than in other chambers. The control group spent more time in chamber 1 than in chamber 2 (p < 0.001) and the VPA group did not show this difference. A significant effect of group was observed on the time (s) spent exploring the stranger rat (180.5 ± 26.6 for the control and 66.1 ± 15.4 for the VPA) group. Mean comparisons revealed that VPA rats explored the stranger rat less than control males (p < 0.001). The time (s) exploring the object
Fig. 1 – Three-chamber sociability test in young rats (35–50 days old) prenatally exposed to VPA. Social approach was measured after 5 min of acclimation and 10 min of test, with the stranger rat and an object on each side. The stranger rat was positioned at chamber 1 and the object was placed at chamber 2. Data are expressed as mean±SEM (VPA group N=8, control group N=12). *different from control group, p < 0.001. #difference within groups, p< 0.001.
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showed no difference between the groups. There is also a significant difference within control groups in the exploration parameter (p < 0.001). They spent more time (s) exploring the stranger rat (180.5 ± 26.6) then the object (72.0 ± 12.5).
2.1.1.2. Y-maze. No statistical difference was found in the learning ability (Fig. 2A), expressed by the total trials until the criterion of learning was reached (10 right choices in a row) between VPA (59.13 ± 7.6) and control (63.75 ± 7.2) groups. The choice arm with food was the left arm. After the rat learned the task, the food was placed on the other choice arm. There was a significant increase in the number of trials the VPA subjects tried the new arm (Fig. 2B), showing persistence to sameness in the VPA (3.37 ± 0.9) group compared to controls (1.67 ± 0.12). The reversed Y-Maze was performed (with the food on the new right arm) and no statistical differences were observed (data not shown). 2.1.2.
Adult rats (80–115 days old)
2.1.2.1. Three-chamber sociability test. These results are shown in Fig. 3. The first phase of the experiment (Fig. 3A) showed a statistically significant decrease (22%) in the time spent exploring the stranger rat by VPA rats (116.8 ± 8.7 s) compared to the control group (150.8 ± 11.3 s, p < 0.05). Moreover, similarly to the young rats, there is a significant difference in
Fig. 2 – Learning ability and flexibility to change strategy measured in the Y-maze in young (35–50 days old) rats prenatally exposed to VPA. For the acquisition test the start arm was the left arm and for the reversion test, the start arm was always the right one. A. Learning ability, expressed as total trials until learn the task: 10 right response in 10 consecutive trials. B. Number of trials until change of the strategy. Data are expressed as mean ± SEM (VPA group N = 8, control group N = 12). *different from control group, p < 0.05.
Fig. 3 – Three-chamber sociability test in adult rats (80–115 days old) prenatally exposed to VPA. A. Phase 1: sociability measured after 5 min to acclimation and 10 min of test. B. Phase 2: social memory after 10 min of test. In the first phase of the task chamber 1 contains the stranger rat 1 and chamber 2 is the one which contains the object. In the second phase chamber 1 contains the “stranger 1” (present in phase 1) and chamber 2 contains a new rat, the “stranger 2”. The score was evaluated by the time spent in each chamber and by the time spent sniffing each wire cage. Data are expressed as mean ±SEM (VPA group N= 8, control group N= 8). *different from control group, p < 0.05. # difference within groups, p <0.05.
the adult rats within control groups in the exploration parameter (p< 0.001). They spent more time (s) exploring the stranger rat (150.8 ± 11.3) then the object (91.15±7.7). In the second phase (Fig. 3B), the time (s) spent in chamber 1, which contains the known rat was statistically different between groups (168.8 ± 13.1 for control and 224.5 ± 16.8 for VPA, p < 0.05). In addition, a difference in the time (s) spent exploring a new stranger rat was also found, showing a lower interest to begin a social approach in the VPA group (163.7 ± 18.2 for control and 105.6 ± 15.4 for VPA, p < 0.05). The analysis within groups showed also statistically significant differences. As expected, control group spent more time (s) in chamber 2 (299.81 ± 24.50) than in chamber 1 (168.65 ± 13.12). Moreover, control group demonstrated higher exploration of the new stranger rat (163.62 ± 18.15) when compared to the known rat (71.16 ± 9.39), p < 0.001. The same behavior was observed on the VPA group which spent more time exploring the new rat (105.62 ± 15.44) than the known (59.00 ± 7.07), p < 0.05.
2.1.2.2. Morris water maze. No statistical difference was found between the VPA and control groups in the Morris
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the activity of those enzymes in blood as compared to control group, suggesting that this teratogen did not affect hepatic cell integrity (Table 1). Moreover, the activity of two important antioxidant enzymes, SOD and CAT and oxidative damage, by damage on lipids (TBARS), damage on proteins (−SH and carbonyl content) had also been quantified in liver samples. The activity of SOD (U SOD.mg protein− 1) of control group (45.00±3.54) did not show a significant difference as compared to the VPA group (44.75 ±1.92) (Fig. 5A). However, VPA induced a significant increase (12%) in CAT activity (from 144.89 ± 7.82 to 169.02 ± 8.9 U CAT.mg protein − 1) in the VPA group (Fig. 5B). The SOD/CAT ratio (Fig. 5C) decreased in VPA rats (from 0.310±0.020 to 0.267±0.037). As showed in Fig. 6, no statistically significant differences were found on TBARS (from 0.0812± 0.0026 to 0.0755 ± 0.0065 nmol TMP.mg protein− 1), sulfhydryl (−SH) (from 36.7 ± 5.54 to 36.7 ± 2.28 μmol (−SH).mg protein− 1) and carbonyl content (from 2.80± 0.74 to 3.56 ± 0.39 μmol carbonyl.mg protein− 1).
3.
Discussion
The animal model of autism induced by VPA, has a significant number of characteristics observed in humans with autism.
Fig. 4 – Assessment of spatial learning ability determined by the Morris water maze (MWM) in adult rats (80–115 days old) prenatally exposed to VPA. Testing was conducted in 2 phases, acquisition and reversal. Each phase consisted of 4 trials/day to find the platform for 5 consecutive days. This was followed by one additional day when a single probe trial was given. The start positions used were: NW, N, E, and SE. A. Spatial learning ability. B. Ability to change the strategy to learning a new task on the reversed MWM (VPA group N = 8, control group N = 12).
Water Maze (MWM) and in the Reversed Morris Water Maze (RMWM) (Fig. 4).
2.1.2.3. Biochemical analysis. Liver cytotoxicity was investigated by the serum activity of the hepatic enzyme markers AST/GOT and ALT/GPT. Prenatal exposure to VPA did not modify
Table 1 – Activity of hepatic enzyme markers and urea content in serum from 120 days-old rats prenatally exposed to VPA.
Control VPA
AST/GOT (IU/L)
ALT/GPT (IU/L)
Urea (mg/dL)
95.36 ± 5.87 91.40 ± 4.45
46.5 ± 6.53 49.2 ± 5.6
186.14 ± 10.41 183.03 ± 9.79
AST/GOT, aspartate aminotransferase or glutamic-oxaloacetic transaminase; ALT/GPT, alanine aminotransferase or glutamicpyruvic transaminase. Values are means ± SEM, N = 4–6.
Fig. 5 – Liver antioxidant enzyme activity in adult rats prenatally exposed to VPA. At 120 days old, rats were sacrificed; the tissues were removed and kept in −70 °C until the assays were performed. A. SOD activity; B. CAT activity and C. SOD/CAT ratio. The experiments were performed in triplicate. Data are expressed as mean ± SEM (VPA group N = 6, control group N = 4). *different from control group, p < 0.05.
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Fig. 6 – Oxidative damage parameters in liver from adult rats prenatally exposed to VPA. At 120 days old, rats were sacrificed; the tissues were removed and kept in − 70 °C until the assays were performed. A. Lipid peroxidation assessed by TBARS (VPA group N = 4, control group N = 4). B. Protein damage by reduced sulfhydryl content (VPA group N = 6, control group N = 6). C. Protein damage by carbonyl content (VPA group N = 6, control group N = 6). Data are expressed as the mean ± SEM, and experiments were performed in quintuplicate.
Among those are repetitive and stereotypic behavior, decreased number of social behaviors (Schneider et al., 2006), impairment in the initiation of social interaction (DufourRainfray et al., 2010) and abnormality of circadian rhythm (Tsujino et al., 2007). The present work shows an evaluation of social memory, flexibility to change strategy and biochemical parameters. Behavioral tasks in young rats showed the following results: decreased time spent by the VPA rats exploring a stranger rat in a three chambered apparatus. The time spent exploring the stranger rat was significantly lower in the VPA group indicating reduced social approach to a stranger rat. This clearly shows a social dysfunction induced by the prenatal exposure to VPA that is a consistent characteristic of autism. In addition, control rats have shown a preference to conspecific. While control rats spent more time in chamber 1 containing the stranger rat, VPA rats did not show any preference. It is well known that mental retardation is a
characteristic often related with autism (Casanova, 2007). However, on the Y-maze, a spatial discrimination learning task, no difference between groups was found, indicating that the VPA rats have no spatial learning impairment. Moreover, increased latency of VPA rats to change their strategy was observed in the reverse Y-maze. These data are in agreement with previous work which shows enhanced repetitive behavior in the Y-maze (Markram et al., 2008). Once VPA rats learned the task normally, when challenged to try a new strategy, they showed a lack of flexibility, spending more trials on the first reward arm. It could be related with behavioral rigidity (South et al., 2007), which is very typical and present on autism. As adults, the tests performed were three-chambered apparatus (2 phases) and MWM (acquisition and reversion). The three-chamber apparatus tasks provided a good perspective about how social behavior is impaired in rats prenatally exposed to VPA. The first phase of the paradigm showed a statistically significant difference between groups; VPA group spends less time exploring the stranger rat. Therefore, it is a similar type of aberrant behavior prevailing at young ages on VPA rats. Interestingly, different from control groups, no difference was observed comparing the time exploring a stranger animal and an object within VPA groups, in both young and adult rats, suggesting that the VPA rats demonstrate the same interest exploring an object and a rat. Schneider et al. (2006) found that VPA young rats display lower number of pinnings, although the latency to pinning and duration of pinning did not differ between groups. Moreover, in the second phase of the test, where the preference for social novelty was evaluated, VPA rats showed less interest to make a new social approach. This was explained by the significantly lower time the VPA group spent exploring the new stranger rat, compared to the control group. Nevertheless, both groups decreased the time spent to explore the first stranger rat on the second phase when compared with the first phase, indicating that they showed the same degree of interest by the stranger rat already explored. In addition, within groups, both control and VPA rats explored the second (new) stranger rat more than the first on the second phase. Besides, between groups, the VPA group spent more time than the controls in chamber 1 (with the rat already explored). This could be a consequence of the fact that VPA rats were more willing to be in a chamber with a known rat than in a chamber in which they would be challenged to a new social approach, indicating apparently normal social recognition but decreased social interaction. Recent results obtained with VPA rodents on the threechambered apparatus showed increased time spent in the central chamber and increased number of crossing between the chambers by VPA compared to control rats (Dufour-Rainfray et al., 2010). In addition, similar results were found with mice in this task, where the VPA group decreased the number of nose pokes on social novelty test (Roullet et al., 2010). On the MWM task, the VPA rats showed the same behavior as the control group to both acquisition and reversion phases, indicating that spatial learning and memory references are still fully functional in the VPA group, at least in this task, in agreement with previous work that shows normal to slightly
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impaired spatial learning in the MWM (Markram et al., 2008). VPA rats showed a very good adaptation to the new task on the reversal MWM similar to the controls, which in part could be seen as a reversion of the behavior shown by young VPA rats, where the latency to try a new strategy was found. Moreover, this could be just a task sensitive limitation, in which reversed Y-maze findings were not reproduced by reversed MWM and deserve further studies to clarify this issue. It is of common knowledge that pharmacological tissue damage could lead to a huge spectrum of behavioral impairments (Delongeas et al., 2010). Accordingly, we must try to elucidate if those aberrant behaviors came selectively from a brain damage or if it came from, at least in part, other metabolic pathways. It possibly could happen, since the prenatal exposure to teratogenic by VPA is affecting the pups' body globally (Binkerd et al., 1988; Ingram et al., 2000). There is evidence that patients with autism may have serum markers for inflammation, including elevation of some cytokines, auto-antibodies, and lower levels of normal immunoglobulins for immune defense. Studies involving inflammatory response due to liver failure and metabolic impairment have shown correlation with brain and behavioral impairment (Chez and Guido-Estrada, 2010; Pardo et al., 2005). Moreover, children with autism were more likely to have mitochondrial dysfunction, mtDNA overreplication, and mtDNA deletions than typically developing children (Giulivi et al., 2010). Therefore, it seems proper to assume that the liver, an organ that plays a central role on metabolic status, is an important place to look at for possible damage or metabolic alterations. We evaluated urea and the enzyme markers of liver cytotoxicity AST/GOT and ALT/GPT, as a way to investigate if the VPA rodent impairments could be correlated to alterations in hepatic metabolism. However, these parameters were not changed between the VPA and control rats (Table 1), indicating absence of hepatic damage. Other pathological evidence of immunological reactions within the CNS is the presence of lymphocyte infiltration and microglial nodules and increases in pro-inflammatory cytokines in peripheral blood samples from patients with autism (Jyonouchi et al., 2001). Besides, we analyzed liver's oxidative damages which could compromise cellular function without causing a rupture of plasmatic membrane. No damage nor oxidative level alterations on proteins (by the evaluation of carbonyl and sulfhydryl groups, respectively) and no damage to lipid membranes (by TBARS) were observed in the VPA group. The enzyme SOD dismutates the \O−2 generating H2O2 which is converted in 2H2O + O2 by catalase. H2O2 can act as a signaling molecule or, with transition metals, yield the hydroxyl radical (UOH) (Fenton and Habber–Weiss reaction) (Halliwell, 2006). Thus, we have found an increased CAT activity and decreased SOD/CAT ratio; however, no oxidative damage was observed by the parameters studied. It is indicative that hepatic cells were able to handle the production of free radical and subproducts. It is also important to remember that reactive oxygen species also plays an important role as signaling molecules and that the imbalance between SOD and CAT activity (Fig. 5C) although apparently did not generate increased oxidative damage, may compromise long term cell function.
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In summary, the present data indicate that young VPA rats exhibited impaired approach to a stranger rat, decreased place preference to conspecifics, normal learning and a lack of flexibility on their strategy. As adults, they showed inappropriate social approach to a stranger rat, decreased preference for social novelty, apparently normal social recognition, no spatial learning deficits and normal resistance to change on MWM. It is an important contribution for an increased understanding of neurodevelopmental symptoms and oxidative imbalance associated to the autism spectrum disorder.
4.
Experimental procedures
4.1.
Animals
Experiments were performed according to the NIH Guide for the Care and Use of Laboratory Animals and approved by local authorities. Female Wistar rats coming from the local breeding colony (ICBS-Federal University of Rio Grande do Sul), with 12:12 light cycle (lights on at 7:00 and lights off at 19:00), controlled temperature (22 ± 1 °C), water and food ad libitum. They had their fertility cycle controlled, and, when on proestrus, mated overnight. In the morning, vaginal secretion was collected with a plastic pipette and placed on glass slides to be analyzed (Marcondes et al., 2002). If spermatozoa were found in the morning, it was designated as first day of pregnancy. Valproic acid (Acros Organics, New Jersey, USA) was purchased as the sodium salt and dissolved in 0.9% saline for a concentration of 250 mg/mL. Females received a single intraperitoneal injection of 600 mg/kg sodium valproate (VPA) or physiological saline (control) on E12.5 (Schneider and Przewlocki, 2005). The delivery of this dose can result in 900 μg/mL of total VPA in maternal plasma in less than 1 h, with a mean/average plasma elimination half life of 2.3 h (Binkerd et al., 1988). Females were kept separate and with free access to their own litters. Rats from both groups control and VPA were born healthy and the number of offspring was normal. Somatic aspects observed during the pups' development, included body weight, ear unfolding and eye opening which were unchanged between groups in agreement with previous work (Dufour-Rainfray et al., 2010). The offspring rats were weaned at 21 days old and were housed separately by sex. The experiments were performed twice, using 4–6 male rats from each litter. Rats had free access to food and water. All the experiments were performed between 12:00 and 17:00. In order to analyze the differences between young and adult rats, behavioral tests were performed at 35–50 and 80–115 days old, considered young and adult rats, respectively. At 120 days old, rats were sacrificed; the tissues were removed and kept in −70 °C until the assays were performed. 4.2.
Behavioral tests
4.2.1.
Three-chamber sociability test
The social test was performed in a three chambered apparatus as described previously in a mice task, with modifications required to perform it in rats (Nadler et al., 2004). It is a wooden box no painted with partitions separating the box into three
14
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chambers with dimensions (length/width/height in cm) 120/40/50, being 60 cm length to the central chamber and 30 cm each side. The openings between compartments allowed free exploration to the different chambers. Time spent in each chamber; as well as the time spent exploring the stranger rat or an object in the chamber, was analyzed by two observers. The object was an empty identical cage used to enclose the stranger rat. Chambers were cleaned with 70% ethanol and water between tests. Animals used as “strangers” were Wistar males with the same age and no previous contact with the test rats. Animals were individually acclimated for 5 min into the apparatus on the day before the experiment. On the sociability test, rats were allowed to expend 5 min in the central chamber, and then the stranger rat was introduced into one of the side chambers. When performed with young rats (40–45 days old), the experiment was performed for up to 10 min, with the stranger rat and an object on each side. When performed with adult rats (80–115 days old), a 10-minute test to quantify preference for social novelty began immediately after the 10-minute test for sociability. In this test, the original stranger rat (stranger 1) remained in its wire cage on one side of the apparatus and a new unfamiliar rat (stranger 2) was placed in the wire cage on the opposite side, which was previously empty during the sociability test. The score was evaluated by the time spent in each chamber and by the time spent sniffing each wire cage. Stranger 1 and stranger 2 animals originated from different home cages had never been in physical contact with the subject rat. The three chambered apparatus was centered onto a lab bench to minimize lightgradients in, temperature, sound and other environmental conditions that could produce a side preference. 4.2.2. Y-maze Learning ability was tested as described (Aguilar et al., 2000; Piedrafita et al., 2008) in a wooden Y-shaped maze, which had three arms of equal size (60 cm long, 11.5 cm wide and 25 cm high). For the acquisition test the start arm was the left arm and for the reversion test, the start arm was always the right one. The other arms, each of which had a food cup located at the end, were considered the choice arms. Pre-training was carried out during 4 days with all rats to familiarize them with the maze. In each trial, rats were rewarded for choosing the left arm. The reward for the correct response consisted of half Froot-Loop® placed in the food cup at the end of the correct arm. If the rat made an incorrect choice, it was allowed to go to the empty food cup at the end of the incorrect arm and was removed after 5 s. Rats were trained for ten trials per day, with an inter-trial interval of approximately 5 min in their home cage until the completion of a criterion of ten correct responses in ten consecutive trials or until a maximum of 250 trials. After the subject reach the criterion, the reward was placed at the end of the reverse arm and the number of trials was measured until the rat changed its strategy, by choosing the other arm (the right side one). 4.2.3. Spatial learning in the Morris water maze (MWM) The MWM, an established test of spatial learning and reference memory, was conducted in a 210 cm diameter tank made of fiberglass and painted flat black (Vorhees et al., 2009). The 3 walls nearest to the maze (representing arbitrarily N, E,
and W walls of the test room) had large geometric figures mounted above the edge of the pool. The experimenter stood at the “S” position during testing and remained stationary. Testing was conducted in 2 phases, acquisition and reversal. Each phase consisted of 4 trials/day for 5 consecutive days; this was followed by one additional day when a single probe trial was given. Probe trials lasted for 30 s. The time limit on learning trials was 1 min and the inter-trial interval (ITI) was 5 min spent on the cage. Animals that did not find the platform within 1 min were placed on the platform again. Goal platforms (with diameters of 10 or 7 cm) were made of black acrylic with thin nylon screening attached to the surface to provide traction. The platform was positioned 1–2 cm below the surface of the water and was camouflaged by virtue of being transparent against a black background. Water temperature was 21 ± 1 °C. The acquisition phase occurred on P81–86. During acquisition, the 10 cm diameter platform was used and located in the SW quadrant halfway between the center and the side of the tank. Rats started at one of four positions located distal to the quadrant containing the platform in a random order with the restraint that they received one trial from each of the four starting positions per day. The start positions used were: NW, N, E, and SE. These positions were used to eliminate short paths to the goal, such as those that are possible if S or W starts are used. The day after the last acquisition trial, each rat was given a 30 s probe trial. For the probe trial, rats started from a position they had never started from before (NE). On P88–93 rats were tested in the reversal phase with a 7 cm diameter platform placed in the NE quadrant. The smaller platform was used to increase the spatial accuracy required to locate the platform. The same procedure was used as for acquisition (5 days, 4 trials/day). Start positions were SE, S, W, and NW. On the sixth day, the platform was removed and a 30 s reversal probe trial was administered with the start position at SW.
4.3.
Biochemical blood parameters and oxidative stress analysis
Blood sampling and analysis: at 120 days-old, overnightstarved animals were anesthetized by intramuscular injection of 75 mg/kg ketamine and 10 mg/kg of xylazine, respectively. Blood samples were obtained by intracardiac punction and animals were killed by decapitation. Blood samples were incubated at room temperature (25 °C) for 5 min and centrifuged at 3200 rpm for 5 min. Serum was stored at −70 °C until the day of analysis. Biochemical analyses of ALT/GPT (alanine aminotransferase or glutamic-pyruvic transaminase) and AST/GOT (aspartate aminotransferase or glutamic-oxaloacetic transaminase) activity were performed using kits from local supplier Human do Brasil. 4.3.1. Tiobarbituric acid reactive species (TBARS) assay As an index of lipid peroxidation, TBARS formation was measured using a hot acid reaction. The homogenates of rat liver slices were mixed with 0.6 mL of 10% trichloroacetic acid (TCA) and 0.5 mL of 0.67% thiobarbituric acid, and heated in boiling water for 25 min (Draper and Hadley, 1990). The levels of TBARS were spectrophotometrically determined at 532 nm. Results are expressed as nmol MDA equivalents/mg protein.
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4.3.2. Protein tiol (−SH) content A sample aliquot was diluted in 0.1% SDS and 10 mM 5,5dithiobis 2-nitrobenzoic acid (DTNB, Sigma). Ethanol was added to produce the intense yellowish color of the product of the reaction between the sulfhydryl (− SH) groups and DTNB. After 20 min, −SH levels were spectrophotometrically determined at 412 nm (Ellman, 1959). Results are expressed as μmol (−SH).mg protein− 1. 4.3.3. Determination of carbonyl groups Protein carbonyl formation was quantified as an index of protein oxidative damage (Levine et al., 1990). This method is based on the reaction of dinitrophenylhydrazine with protein carbonyl groups. The results are expressed as μmol.mg protein− 1. 4.3.4. Superoxid dismutase (SOD) activity assay SOD activity was quantified by the inhibition of superoxidedependent adrenaline auto-oxidation to adrenochrome using spectrophotometric measurements at 480 nm, as previously described (Misra and Fridovich, 1972). The results are expressed as absorbance/time (s). The area under the curve of the graph was used for statistical analysis and compared against the control values. Five units of CuZnSOD (E.C.: 1.15.1.1) were used to determine assay specificity. 4.3.5. Catalase (CAT) activity assay CAT (E.C.:1.11.1.6) activity was assayed as previously described (Aebi, 1984) by measuring the absorbance decrease at 240 nm in a reaction medium containing 20 mM H2O2, 0.1% Triton X-100, 10 mM potassium phosphate buffer, pH 7.0, and 50 μg protein. One unit (U) of the enzyme is defined as 1 μmol of H2O2 consumed per minute and the specific activity is reported as U/mg protein. 4.4.
Statistical analysis
Data are reported as mean±standard error mean (SEM) and were analyzed by Student's t-test. Analysis between the same groups was performed using repeated-measures ANOVA. Values of p<0.05 were considered significant. All analyses were performed using the SPSS program, Version 12.0 (SPSS, Chicago, IL).
Acknowledgments This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), FINEP/ Rede IBN 01.06.0842-00 and INCT-EN National Institute of Science and Technology for Excitotoxicity and Neuroprotection. Special thanks to Janine Mackenzie for the careful English review and to Cintia Zappe Fiori by the help with the statistical analysis. We are thankful to the reviewers and editor by their contributions which helped us to remarkably increase the quality of the present work and also improve our experimental protocols.
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