MINISTRY OF DEFENCE
MINISTRY OF EDUCATION AND TRAINING VIETNAM MILITARY MEDICAL UNIVERSITY
DAO THU HONG
RESEARCH ON ESTABLISHING AN AUTISM MODEL USING
SODIUM VALPROATE AND AMELIORATIVE EFFECTS OF
ENRICHED ENVIRONMENT ON BEHAVIORS IN WHITE
MICE
Speciality: Physiology
Code: 62720107
SUMMARY OF MEDICAL DOCTORAL THESIS
HANOI - 2018
This research was carried out in
Vietnam Military Medical University
Supervisors:
1. Tran Hai Anh, M.D., Ph.D., Assoc.Prof.
2. Can Van Mao, M.D., Ph.D., Assoc.Prof.
Reviewer 1:
Reviewer 2:
Reviewer 3:
This thesis will be defended in doctoral examination council of
Vietnam Military Medical University at….on the date of
……………..
This thesis is available at:
1. National Library
2. Library of Vietnam Military Medical University
3. ………………………………………………….
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INTRODUCTION
1. Imperativeness
Autism is a neurodevelopmental disorder whose diagnosis is
based on three behavioral criteria: unusual reciprocal social
interactions, deficits in communication, and stereotyped repetitive
behaviors with restricted interests. The etiology and pathophysiology
of autism are unclear. Nowadays, autism is known as a modern-day phenomenon with the prevalence increase all over the world. The studies of CDC shown that the prevalence of autism was 0.5 per
1000 children in 1985, increased 12 per 1000 children in 2012.
Prenatal exposure to valproate (VPA) leads abnormal
neurodevelopment and increased risk of autism spectrum disorders.
In animal model, prenatal VPA-exposure also lead abnormal
behaviors and pathology similar to that observed in autism
individuals. For that reason, valproate is used to induce animal model
of autism in order to investigate the mechanism of biological changes
and to evaluate the effect of interventions for autism. In Vietnam,
there are several researches about some risk factors, some clinical
features in autistic patients. However, there is no research about
animal model of autism and pathophysiological mechanism for
autism. So, we conducted this research with two aims:
1. Establishing an animal model of autism using Swiss white
mice by prenatal exposure to sodium valproate.
2. Evaluating the effects of enriched environment on behaviors
in modelled white mice.
2. Scientific significance
The thesis has provided data about behavioral alterations of Swiss
mice prenatally exposed to sodium valproate (VPA) with dose 300-
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500mg/kg i.p., creating animal model of autism in Swiss mice by
using sodium valproate i.p. at dose 500mg/kg. Providing data about
the ameliorating of enriched environment on behaviors in VPA-
exposed mice. 3. Practical significance Using that VPA-exposed mice to determine neurobiological alterations related to the pathogenesis of autism and assess the effects of drugs or treatments on autism. 4. Structure of the thesis
The thesis consists 124 pages. 2 pages Introduction; Chapter 1 (Documentary Overview) 32 pages; Chapter 2 (Subject and methods) 14 pages; Chapter 3 (Results) 41 pages; Chapter 4 (Discussion) 33 pages; 1 page Conclusions and 1 page Recommendations. The thesis has 22 tables (2 appendix tables), 35 figuress (5 appendix figures) and 147 references (7 Vietnamese and 140 English references).
Chapter 1. DOCUMENTARY OVERVIEW
1.1. Terminology and brief history of autism
Autism was first mentioned from the 1900s, derived from the Greek "Autos" - "self", described patients with isolated, social withdrawal. Leo Kanner (1943) used the term "autism" to describe a group of children with abnormal development such as social interaction deficits, impairment in language development, strange and repetitive behaviors, early onset before 3 years of age. Hans Asperger (1944) described a milder form of autism called Asperger's syndrome. In 1960 – 1970s, several studies suggested that autism was caused by abnormal brain structures, or by biochemical and metabolic changes. In 1999, autism was classified as pervasive is a pervasive developmental disorder. Accordingly, autism
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developmental disorder, affecting many aspects of the development but the most impact on communication skills and social relations.
1.2. Diagnostic criteria for autism
and
Currently, there are two autism diagnostic criteria systems: "Diagnostic and Statistical manual of Mental disorders" (DSM) of The American Psychiatric Association "International Classification of Diseases" (ICD) of The World Health Organization. In which the DSM criteria are applied fairly common. 1.3. Pathophysiology of autism
The mechanisms that lead to autism are poorly understood, however scientists focus around the disruption of normal cerebral development and its subsequent implications on the functional brain unit. Others hypotheses are discussed the mechanisms of autism related to a role of environmental factors in association with genetic factors and epigenetics is a candidate biological mechanism for gene- environment interactions in autism. 1.4. Methods to model autistic features in animal
Animal models of autism is based on the mimics the causal factors in human disease. The same causal factors, obtain from animal manifestations and pathological behaviors similar to autism describe in human. The animal model of autism is divided to three main groups:
- Brain lesion models of autism: models obtain when damaging brain areas such as the cerebellum, amygdala, medial prefrontal cortex. - Environmental factors models of autism: exposure during pregnancy or early postnatal period with anticonvulsants and inflammatory factors.
- Genetic models of autism: mutagenic models of genes involved
in autism, genetic model diseases are associated with autism.
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1.5. Test for behavioral evaluation in autism modelled animals
Behavioral assays for animal model of autism are a battery of
tests based on cored symptoms and associated symptoms of autism.
The cored symptoms include abnormal reciprocal social interactions,
impaired communication, and repetitive behaviors, restricted
interests. Associated symptoms include seizures, anxiety, mental
retardation, hyper-reactivity and hypo-reactivity to sensory stimuli,
sleep disruption and gastrointestinal distress…
1.5.1. Tests for social interaction
To measure social interaction, we use three-chambered test or
maternal behaviors test.
1.5.2. Tests for communication
Responses to social, non-social odors, emitted ultrasonic
vocalizations in different contexts are means of assessing
communication in animals.
To count the number of repetitive behaviors such as rotating,
1.5.3. Tests for repetitive behaviors
digging, grooming or use T maze test, Y maze test.
1.5.4. Tests for associated symptoms
To measure seizures, sleep disorder using EEG recording.
Anxiety-related behaviors can be evaluated by elevated plus-maze
test or light–dark exploration. Assess learning and memory by using
Morris water maze test, object recognition and operant
discrimination tasks. Assess hypersensitivity to sensory stimuli by
acoustic startle. Assess motor clumsiness by using rotarod test.
1.6. Treatments for autism
Autism is a developmental disorder include abnormal behaviors,
impairment in communication and social interaction. To intervene
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effectively, we should have a specialist to assess impairments and
capacity of children with autism. The intervention areas include
psychology, language, education, medical and therapeutic
activities...which focuses on the skills deficit compare with the same
age children.
1.7. Effects of enriched environment on behaviors in animals
modelled for autism
Enriched environment (EE) is a laboratory condition in which the
environment is "riched" more than the standard conditions in the
laboratory. The enriched environments was first described by Donald
Hebb (1947) who noted qualitative differences in behavior between
the rats he brought home for his children to play with, and the rats
who were cage-kept in the laboratory. Until the 1960s, biochemical
and structural changes were found in the brains of rats exposed to
enriched environments. Autism is as a neurodevelopmental disorder,
using enriched environment having proven to improve the defects
caused by neurodevelopmental disorder.
1.8. Research about autism in Vietnam
Recent years in Vietnam, children with autism were examined
and treated increases quickly. Some study have focus on
epidemiology, clinical features, no study in animal model of
autism… The diagnosis and treatment for children with autism are
difficult because they have not been properly invested. So, many
children with autism have not diagnosed and treated, leading children
with autism difficult to integrate society.
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Chapter 2. SUBJECTS AND METHODS
2.1. Subjects
Swiss white offsprings of parent mice offer by National Institute
of Hygiene and Epidemiology. * Research aim 1: Establishing an animal model of autism in Swiss white mice. Mice were divided into control group (Control) n=35, prenatal VPA exposure groups at doses of 300, 400 and 500 mg/kg body weight (VPA300) n=31, (VPA400) n=32 and (VPA500) n=35. * Research aim 2: Evaluation effects of enriched environment on behaviors in modelled white mice. Mice were divided into: control group raised up in standard environment (Control-SE) n=69, control group raised up in enriched environment (Control-EE) n=72, VPA group raised up in standard environment (VPA-SE) n=66 and VPA group raised up in enriched environment (VPA-EE) n=65.
The care and use of laboratory animals were in accordance with the guidelines established by Laboratory Animal Production Center in Institute of Hygiene and Epidemiology and Laboratory Animal Board in Vietnam Military Medical University. 2.2. Methods 2.2.1. Research design
A prospective with intervention study.
2.2.2. Materials
The chemicals used in the study included sodium valproate (Sigma Aldrich, Germany) and 0.9% sodium chloride solution (B.BRAUN, Vietnam).
Mice were kept stable at 25 ± 1°C, humidity 60-70% and 12/12 hour dark light cycle. Control-SE and VPA-SE mice were housed in a standardized environment (SE), while the Control-EE and VPA-EE mice were housed in enriched environment (EE). SE was a cage with size 30x20x15 cm (L x W x H), freely accessed to pelleted food,
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water, and nesting material. EE was a large cage with size 50x30x30 cm (L x W x H), divided into two layers, including components: running wheels, ladder, vines, turtles, sleepers, tunnel, rolling ball, freely accessed to pelleted food, water and nesting material.
included:
A quiet laboratory room at a temperature of 25 ± 1°C had an experimental chamber for conducting behavioral assessments. The round cylindrical experimental chamber (2-m in diameter, 2-m in hight) was enclosed around the ceiling and made of thick black fabric and used 25 watt incandescent light bulbs. Equipments for behavior analysis incline surface, ultrasonic sound recording and analysis system, open field, rotarod, elevated plus maze, three-chamber apparatus, Morris water maze, and Any-maze behavioral recording and analysis system. 2.2.3. Research procedure * Research aim 1: Establishing an animal model of autism in Swiss white mice.
Female adult Swiss mice with controlled fertility cycle were mated overnight and the next morning when spermatozoa were found was designated as the first day of gestation. At day 12.5 of pregnancy, dams were randomly divided into groups. Control dams received a single intraperitoneal (i.p.) injection of physiological saline at dose 0.01 ml/g b.w., VPA dams received a single intraperitoneal injection of sodium valproate at dose 300 mg/kg, 400 mg/kg, 500 mg/kg and 600 mg/kg b.w. With these VPA doses, we conducted a dose/ toxicity test. Because the VPA 600 mg/kg dose was lethal/ toxic to the exposed pups during the first week postpartum, the exposed pups at VPA 600 mg/kg dose were evaluated behaviors limited. Therefore, the present study used doses of 300-500 mg/kg and experimental results corresponding to these doses.
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The offsprings were divided into control group or VPA300, VPA400, and VPA500 groups according to the dose exposure in utero. Behavioral examinations of mice were scheduled as follows: ultrasonic vocalization recording at 3 to 10 days of age; negative geotaxis test at 6 to 8 days of age; open field test, three-chambered test, elevated pluss maze test, rotarod test, Morris water maze test at 49 to 61 days of age. Results were compared amon groups of mice, then the most effective dose of VPA was chosen for modelling autism in mice. * Research aim 2: Evaluation effects of enriched environment on behaviors in modelled white mice.
Modelling autism in Swiss mice by injecting the dose of VPA 500 mg/kg b.w. The offspring were weaned on PND 21 and housed separately in a standard environmental cage (SE) or an enriched environmental cage (EE) for four weeks, with 3 to 6 animals per cage by gender.
Assess offspring’s behaviors before housing in the SE or EE: included ultrasonic vocalization recording at 3 to 10 days of age, and negative geotaxis test at 6 to 8 days of age.
Enriched environment housing procedure: EE cages were classified into samples labeled with ordinal numbers, each sample included five objects and changed every three days including a new replacement litter material, washed and dried the objects, a new replacement object and changed positions of two objects.
Assess offspring’s behaviors after housing in the SE or EE: using open field test, three-chambered test, elevated plus maze test, rotarod test, Morris water maze test at 49 to 61 days of age. Behavioral data were analyzed and compared among the groups to evaluate effects of enriched environment on behaviors in VPA-exposed mice.
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2.2.4. Data analysis
The results were represented by the mean, standard deviation, and percentage. The difference between percentages of groups were compared using Chi-square test, the difference between means of groups were analyzed by T-test/ANOVA (normal distribution) or Mann-Whitney test/ Kruskal-Walis test (non-standard distribution). The difference among means at different times were analyzed by Mix ANOVA. The data of research was processed by IBM SPSS software version 20.0. 2.2.5. Research location The research was conducted at Laboratory of Physiology in Vienam Military Medical University.
Chapter 3. RESULTS
3.1. Establishing an animal model of autism in Swiss white mice by prenatal exposure to sodium valproate
3.1.1. Effects of prenatal exposure to sodium valproate on motor coordination development in negative geotaxis test
+p<0.05, ++p<0.01 compared with that of the same group on PND6 The 1800 rotating time in VPA groups were higher than that in the control group, which was clearly shown in the VPA500 group.
Figure 3.1. 1800 rotating time in control and VPA groups at PND 6 to 8. ** p<0.01, *** p<0.001 compared with that of control on the same day
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3.1.2. Effects of prenatal exposure to sodium valproate on social
communication by emitting ultrasonic vocalization (USV)
Figure 3.7. Number of calls in control and VPA groups at PND 3 to 10 in below 35 kHz bandwidth.
The number of calls in VPA groups were lower than that in the control group, which was clearly shown in the VPA500 group (p < 0.001). 3.1.3. Effects of prenatal exposure to sodium valproate on locomotor and exploratory activity in open field test
There were no significant difference between group (p > 0.05) for
Parameters
p
Number of entries in chamber 1
pa,b > 0.05 pa,c < 0.01 pa,d < 0.001
> 0.05
Time in chamber 1 (s)
Time in central chamber (s)
pa,b > 0.05 pa,c > 0.05 pa,d < 0.05
distance and speed travel. 3.1.4. Effects of prenatal exposure to sodium valproate on social interaction in three-chambered test Table 3.4. Number of entries and duration in chambers in session 1 in control and VPA groups. ± SD 14.51 ± 5.89 13.26 ± 7.06 9.45 ± 4.65 9.10 ± 6.69 295.61 ± 67.91 270.02 ± 55.48 291.33 ± 102.41 262.10 ± 122.78 91.61 ± 42.93 89.78 ± 30.05 118.71 ± 116.55 133.22 ± 99.27
Group a.Control b.VPA300 c.VPA400 d.VPA500 a.Control b.VPA300 c.VPA400 d.VPA500 a.Control b.VPA300 c.VPA400 d.VPA500
n 35 31 32 30 35 31 32 30 35 31 32 30
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Number of entries in strange mouse chamber in control group was higher than that in the VPA400 and VPA500 groups (p < 0.01 to p < 0.001). Duration in central chamber in control group was lower than that in the VPA500 group (p < 0.05).
3.1.5. Effects of prenatal exposure to sodium valproate on anxiety-
related behaviors in elevated-plus maze test
The results in Table 3.8 showed that number of entries in opened arms in control group was higher than that in the VPA300 and VPA400 groups (p < 0.05 and p < 0.01, respectively). Duration in opened arms in control group was higher than that in the VPA300, VPA400 and VPA500 groups (p < 0.05 and p <0.001, respectively). Duration in closed arms in control group was lower than that in the VPA400 group (p < 0.001), no significant difference between control group and VPA300 and VPA500 groups.
3.1.6. Effects of prenatal exposure to sodium valproate on motor
coordination, balance in rotarod test
Figure 3.12. Latency to fall off the rotating rod in control and VPA groups.
The latency to fall off the rotating rod in control group was longer
than that in the VPA groups (p < 0.01 to p < 0.001).
3.1.7. Effects of prenatal exposure to sodium valproate on spatial
learning and memory in Morris water maze test
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B
A
locomotor and
Figure 3.13. Distance (A) and duration (B) to find the hidden platform in control and VPA groups over six days of training. Distance to find the hidden platform in control group was shorter than that in the VPA300 and VPA500 groups (p < 0.05 to p < 0.01). Time to find the hidden platform in control group was shorter than that in the VPA500 group (p < 0.01). 3.2.2. Effects of enriched environment on behaviors in mice prenatally exposure to sodium valproate at dose of 500 mg/kg b.w. 3.2.2.1. Effects of enriched environment on exploratory activity of autism-modelled mice in the open field
Table 3.11. Activities in open field in control and VPA500 groups housed in the standard and enriched environment.
Parameters
p
Number of entries in central zone
pa,b > 0.05 pa,c < 0.05 pc,d < 0.001
Time in central zone (s)
pa,b > 0.05 pa,c > 0.05 pc,d < 0.01
Group a.Control-SE b.Control-EE c.VPA-SE d.VPA-EE a.Control-SE b.Control-EE c.VPA-SE d.VPA-EE
n 62 72 66 65 62 72 66 65
± SD 20.82 ± 11.72 18.72 ± 10.84 16.22 ± 9.22 22.82 ± 10.27 25.65 ± 22.18 30.08 ± 28.38 20.70 ± 13.20 29.79 ± 19.01
Number of entries and duration in central zone in VPA-SE group
were higher than those in the VPA-EE group (p < 0.01 to p < 0.001,
respectively).
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3.2.2.2. Effects of enriched environment on social interaction
Parameters
p
Number of entries in chamber 1
pa,b < 0.001 pa,c < 0.01 pc,d > 0.05
Time in chamber 1 (s)
pa,b < 0.05 pa,c > 0.05 pc,d > 0.05
Time in central chamber (s)
pa,b < 0.01 pa,c < 0.05 pc,d < 0.01
behaviours in autism-modelled mice Table 3.12. Number of entries and duration in chambers in session 1 in control and VPA500 groups housed in the standard and enriched environment. N 69 72 66 64 69 72 66 64 69 72 66 64
± SD 14.75 ± 9.98 8.76 ± 4.07 10.86 ± 8.66 9.34 ± 4.53 314.33 ± 95.94 360.03 ± 145.93 307.22 ± 113.21 342.63 ± 138.99 85.71 ± 51.68 80.95 ± 93.41 116.92 ± 91.61 83.48 ± 76.40
Group a.Control b.Control-EE c.VPA d.VPA-EE a.Control b.Control-EE c.VPA d.VPA-EE a.Control b.Control-EE c.VPA d.VPA-EE
Number of entries in chamber 1 in control-SE group was higher than that of the control-EE group (p < 0.001) and VPA-SE group (p < 0.01). Duration in chamber 1 in control-SE group was lower than that of the control-EE group (p < 0.05). Duration in central chamber of control-SE group was lower than that of the VPA-SE group (p < 0,05), in EE groups were lower than that of the SE groups (p < 0.01). 3.2.2.3. Effects of enriched environment on anxiety-related behaviours in autism-modelled mice
The results in Table 3.18 showed that number of entries in opened arms in control-SE group was higher than that of the control-EE group (p < 0.05) and VPA-SE groups (p < 0.01); in VPA-SE group was lower than that of the VPA-EE group (p < 0.05). Duration in opened arms in control-SE group was higher than that of the control- EE group (p < 0.01) and VPA-SE group (p < 0.01).
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3.2.2.4. Effects of enriched environment on motor coordination,
balance in rotarod test in autism-modelled mice
***: p < 0.001, compared with that of Control-SE; +++: p < 0.001,
compared with that of VPA-SE.
Figure 3.27. Latency to fall off the rotating rod in control and VPA500 groups house in SE and EE.
The latency to fall off the rotating rod in control-SE group was
shorter than that in the control-EE group; in VPA-SE group was
shorter than that in the VPA-EE group (p < 0.001).
3.2.2.5. Effects of enriched environment on spatial learning and
memory in autism-modelled mice
B
Figure 3.32. Distance (A) and duration to find the hidden platform (B) rod in control and VPA500 groups house in standard environment and enriched environment over six days of training.
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Distance to find the hidden platform in VPA-SE group was longer
than that of the VPA-EE group (p < 0.001). Duration to find the
hidden platform of control-SE group was longer than that of the
Control-EE group (p < 0.001), in VPA-SE group was longer than
that of the VPA-EE group (p < 0.01).
Chapter 4. DISCUSSION
4.1. Making animal model of autism in Swiss white mice by
prenatal injection of sodium valproate
There are three approaches to model autism in animal, as genetic
models, environmental factors models, and brain lesion models. Each
approach has its own advantages and disadvantages [9],[67],[68].
Researchers chose the method for modeling autism based on research
purposes and research facilities. In Viet Nam, there are currently no
technique to develop genetic models and/or brain lesion models for
autism. The present study chose modeling autism in mice by
exposure to sodium valproate (VPA) on day 12.5 of gestation based
on previous studies showed that VPA was an environmental factor
that altered neurodevelopment especially around the neural tube
closure and the completion of neural structures. Many studies used
the VPA dose of 100-800 mg/kg b.w, oral, subcutaneous,
intraperitoneal injection during day 9 to 15 of gestation. The
determination of day 12.5 was based on monitoring estrous cycle and
mating overnight at proestrus and estrus stage. The next morning,
determining the presence of sperm in the female mice vagina was the
first day of gestation (day 0.5 from the night before to the next
morning). Female mice after mating were separately care, monitored
the sign of pregnancy. In the morning of day 13 (day 12.5),
determined the pregnant mice for injection. The time for injection
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VPA was similar to that in the previous studies, which was
successful to create animal model of autism in mice. The choice for a
battery of tests to evaluate behaviors in mice based on behaviors in
autistic mice corresponding to autistic traits in human was suggested
by Crawley, Wöhr, etc.
4.1.1. Effects of prenatal exposure to sodium valproate on motor
coordination development in negative geotaxis test
Negative geotaxis test evaluates function of the vestibular and
motor coordinative development. Cheaha et al. studied on autistic
Swiss mice by using subcutaneous VPA dose of 600mg/kg on day 13 of pregnancy found that the 1800 rotating time in negative geotaxis test at PND3-10 in VPA group was longer than that in the control
group. Wöhr et al. studied on Shank1 gene mutant mice (autism-
related genes), coding for shelving postsynaptic protein, also pointed out in the period 2-12 days old, Shank1-/- mice was prolonger the 1800 rotating time in the negative geotaxis test than Shank1+/- mice and control mice (Shank1+/+). The results in the preset study showed that the 1800 rotating time in VPA group was longer than that in control group, which was clearly in VPA500 group, suggesting the
development of motor function of VPA500 group was slower than of
the control group. Our study is similar to previous studies in rats and
mice on motor function.
4.1.2. Effects of prenatal exposure to sodium valproate on
communication by emitted ultrasonic vocalization (USV)
Mice emit USV in different contexts during development and
maturation. Research on USV is a tool for behavioural phenotyping
of mouse models of neurodevelopmental disorders, including autism.
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Cheaha et al. studied on autistic Swiss mice by using VPA found
that number of calls per minute of VPA group was lower than control
group in the period 3-10 days old. Wöhr et al. studied on Shank1 gene mutant mice showed that at PND8, in Shank1-/- mice number of calls was lower, total call time was shorter, peak frequency was
higher, and frequency modulation was lower than those of the Shank1+/- and Shank1+/+ groups, but no difference in characteristics of calls by gender. In our study, the VPA mice (particularly
VPA500) reduced emitting USV ratio of above 35 kHz bandwidth,
reducing number of calls, duration of call, and frequency of call
below 35 kHz bandwidth. So, prenatal exposure to VPA leads to
reduce social communication on 3-10 days old mice.
4.1.3. Effects of prenatal exposure to sodium valproate on
locomotor and exploratory activity in open field
Open field test use to evaluate locomotor activity and exploratory behavior. In this study, there were no difference in distance and speed travelled in open field test among groups, indicating that VPA had no effect on locomotor activity. Number of entries and time in central zone in the VPA500 group tended lower than those in the control group showed a partly impairment exploration and restrict interest in the VPA500 group. Roullet et al. studied on mice exposed to VPA dose of 800 mg/kg b.w orally on day 11 of pregnancy found that no difference in locomotor activity in open field between VPA and control group. Mehta et al. studied on mice exposed to VPA dosed 600 mg/kg b.w subcutaneously on day 13 of pregnancy also found that VPA had no effect on spontaneous motor activity, there were no difference in distance and speed in open field between VPA and control group, but number of entries and time in central zone in
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VPA500 groups was lower than those of the control group. Thus, VPA group increased anxiety-related behaviours and reduced exploratory. These changes may relate to a decrease in number of Purkinje cells in cerebellum or abnormal in prefrontal cortex and amygdala. The results of activity in open field test in our study were similar to these hypotheses.
4.1.4. Effects of prenatal exposure to sodium valproate on social
interaction behaviors in the three-chambered tests
In session 1, VPA500 group decreased number of entries to
novelty mouse chamber, decreased number of contact with novelty
mouse, increased time in the central chamber; VPA400 group
decreased the number of entries to novelty mouse chamber; VPA300
group decreased number and time contact with novelty mouse. In
session 2, VPA500 group decreased number of entries to novelty
mouse chamber and number of contact with novelty mouse,
increased time in the central chamber; VPA300 group decreased
number of contact with novelty mouse. These results expressed
impairment in social interaction and social recognition in mice
prenatally exposed to VPA. Cheaha et al. studied on Swiss mice
model autism by using VPA also found that time contact with
strange mouse in VPA group was lower than control group in session
1 of three-chambered test. Markram et al. studied on rats model
autism by using VPA dose 500 mg/kg on day 12.5 of pregnancy
found that VPA group exhibited less play behavior, explored each
other less, as indicated by sniffing and touching each other and
avoided interaction by hiding more than control group. The results of
in vitro slice electrophysiology showed that the basolateral amygdala
network of VPA group was hyperreactive to stimulation and
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hyperplastic to intense synaptic stimulation and inhibition was
impaired. Abnormal in the amygdala might lead to excessive anxiety,
fears and phobias. Fears could lead to isolation, avoidance, social
withdrawal, and loss of social behaviors.
4.1.5. Effects of prenatal exposure to sodium valproate on anxiety-
related behaviors in the elevated plus maze test
Elevated plus maze test is used by many previous experiments to
evaluate anxiety-related behavior. In our study, the results showed
that number of entries and time in closed arms in all VPA groups
tended higher than those of the control group, which was clearly
shown in VPA400 group. Thus, prenatally exposure to VPA leads to
anxiety-related behaviours. Markram et al. studied on autistic rats by
using VPA found that VPA exposed rats spent less time in the opened arms and more time in the closed arms than in the control group. VPA exposed rats also enhanced fear memories, fear
generalization, and impaired fear extinction in a fear conditioning
test. The behavioral manifestations such as increased anxiety, fear or
impaired social interaction were mentioned above may be due to
decreased inhibition and increased excitation in the basolateral
amygdala.
4.1.6. Effects of prenatal exposure to sodium valproate on motor
coordination and balance in the rotarod test
In this study, the latency to fall off the rotating rod in VPA300,
VPA400 and VPA500 groups were shorter than that in the control
group, demonstrating the ability to coordinate movement and balance
in VPA injected mice was less than that of the control group.
Morakotsriwan et al. studied on VPA rats model autism showed that
VPA group decreased the latency to fall off the rotating rod
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compared to control group in the rotarod test. The therapy for VPA
mice by using combined extract of purple rice and silkworm pupae
increased latency to fall off the rotating rod.
Until now, the cause of motor impairments in autism spectrum
disorders are poorly understood. Piochon et al. found that
impairments in synaptic plasticity related motor problems. Others
hypothesis supposed that motor impairments in autism spectrum
disorders related decreases in motor nuclei cells and Purkinje cells in
the cerebellar vermis.
4.1.7. Effects of prenatal exposure to sodium valproate on spatial
learning and memory in the water maze
In this study, the results showed that VPA500 injected mice had
lower ratio, longer swimming distance and time to find the hidden
platform in acquisition phase, which expressed an impairment in learning and spatial memory. However, in the probe test, there were no difference among groups in swimming time in target quadrant.
Kataoka et al. studied on ICR mice exposed to VPA dosed 500
mg/kg b.w on day 12.5 of pregnancy showed that at 8 weeks old
VPA500 injected mice had a longer time to find the hidden platform
in acquisition phase and a lower time in target quadrant in probe test.
Thus, Swiss mice prenatally exposed to VPA doses of 300 to 500
mg/kg b.w. leaded to autistic-like behaviours, which was clearly
shown in the VPA500 group as impairment in communication, social interaction, and motor coordination, a tendency to reduce exploratory
activity, an increased anxiety-related behaviour; and a decreased
spacial learning and memory. Compared with the standard model of
autism in animals proposed by Crawley, VPA intraperitoneal
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injection at the dose of 500 mg/kg could be used for modelling
autism in mice.
4.2. Effects of enriched environment on behaviours in mice
prenatally exposed to sodium valproate dosed 500 mg/kg b.w.
4.2.2.1. Effects of the enriched environment on locomotor and
exploratory activity in the open field in autism-modelled mice
The results in open field test showed that VPA mice raised up in
enriched environment (EE) increased numbers of entries and
duration in central zone compared with those in VPA mice raised up
in the standard environment (SE), while control mice raised up in the
EE were not higher than those of control mice raised up in the SE. So
enriched environment enhances locomotor and exploratory activity in VPA injected mice but not in the control mice. 4.2.2.2. Effects of the enriched environment on social interaction
behaviors in autism-modelled mice
In both two sessions of this study, VPA mice and control mice
raised up in the EE decreased time in central chamber in which
without the partner mouse. So EE tendly improved social interaction
behaviours. Renner et al. found that rats raised up in EE had
significantly higher weight of several brain regions but no reliable
differences in the social interactions compared with rats raised up in
SE. Schneider et al. showed that VPA rats raised up in EE enhanced
number of social interactions with partner rat compared with VPA
rats raised up in SE. Favre et al. also showed that VPA rats raised up
in predictable EE enhanced social interaction time with partner rat
compared with control group, while VPA rats raised up in
unpredictable EE had no effect, and control rats raised up in predictable EE with control group raised up in SE.
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4.2.2.3. Effects of the enriched environment on anxiety-related
behaviors in autism-modelled mice
The results of elevated-plus maze showed that VPA mice
increased time in closed arms, decreased number of entries and time
in opened arms expressed anxiety-related behaviours, and VPA mice
raised up in enriched environment increased number of entries to
opened arms expressed tendency to reduce anxiety-related
behaviours. Schneider et al. studied on rats model autism by using
VPA found that VPA injected mice decreased number of entries and
time in opened arms compared with control rats and VPA mice
raised up in EE increased number of entries and time in opened arms
compared with VPA mice raised up in SE. Favre et al. studied on
VPA injected mice showed that VPA rats raised up in predictable EE
increased time in opened arms compared with control rats, while
VPA rats raised up in unpredictable EE had no effect, and control
rats raised up in predictable or unpredictable EE had no increase time
in opened arms compared with control rats raised up in SE. These
results suggest a study on effects of environmental adjustments on
animal behaviors.
4.2.2.4. Effects of the enriched environment on motor coordination
and balance in autism-modelled mice
The latency to fall off the rotating rod of VPA mice and control
mice raised up in EE were longer than VPA mice and control mice
raised up in SE expressed that enriched environment enhance motor
coordination and balance in both VPA injected mice and control
mice. Kondo et al. also found that in rats model Rett Syndrome, EE
ameliorated motor coordination and motor learning. These resullts
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related alteration excitatory, inhibitory synaptic density and increase
Brain-derived neurotrophic factor in cerebellum.
4.2.2.5. Effects of the enriched environment on learning and spatial
memory in autism-modelled mice
The results of Morris water maze test showed that the EE
ameliorated learning and spatial memory in VPA injected mice by
decreased swimming distance and latency to find out the hidden
platform. Learning and spatial memory may relate to brain regions
affected in autism, such as hippocampus. Improvements in the
performance of learning tasks also relate to these brain structures.
CONCLUSIONS
1. Establishing an animal model of autism in Swiss white mice by
prenatal exposure to sodium valproate at day 12.5 of gestation at
some doses had results: The VPA 300 mg/kg (b.w.) did not affect on
motor coordination development; decreased communication and
social interaction; increased anxiety-related behavior; reduced motor
coordination and balance; reduced the spatial learning and memory.
The VPA 400 mg/kg (b.w.) induced delay motor coordination
development; reduced communication and social interaction;
increased anxiety-related behavior; reduced motor coordination and
balance; did not affect spatial learning and memory. The VPA 500
mg/kg (b.w.) induced delay motor coordination development;
reduced communication and social interaction; increased anxiety-
related behavior; reduced motor coordination and balance; reduced
spatial learning and memory.
From above results, we found that VPA at the dose of 500 mg/kg
(b.w.) is the most appropriate dose for modelling autism in white
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mice with behavioral disturbances that replicated human autistic
features.
2. VPA-exposed mice raised up in enriched environment had
improved behaviors, such as increase in exploratory activity in open
field; enhancement of motor coordination and balance; increase in
spatial learning and memory; slight improvement on social
interaction and reduction of anxiety-related behavior compared with mice raised in standard environment.
PERSPECTIVES
Using model of mice prenatally exposed to VPA at dose of
500mg/kg b.w. to determine alterations of brain structures,
neurotransmitters, and of genetics traits to elucidate etiology of
autism and evaluate to find drugs that most effective for treatment of
the disease.
ARTICLES PUBLISHED THE RESULTS OF THE THESIS
1. Dao Thu Hong, Nguyen Le Chien, Can Van Mao, Tran Hai
Anh (2016) Modelling autism in mouse using valproic acid.
Vietnam Medical Journal, 446, 311-321.
2. Dao Thu Hong, Tran Thi Quynh Trang, Nguyen Le Chien,
Can Van Mao, Nguyen Duy Bac, Tran Hai Anh (2016)
Enriched environment ameliorates behaviours in autism-
modelled mice. Vietnam Medical Journal, 446, 322-333.
3. Dao Thu Hong, Nguyen Le Chien, Can Van Mao, Tran Hai
Anh (2016) Characteristics of ultrasonic vocalization in 3 to 10
PNDs mice. Vietnam Journal of Physiology, 20(4), 136-143.
