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American Journal of Biochemistry and Biotechnology 4 (2): 218-225,

2008

ISSN 1553-3468

© 2008 Science Publications

Corresponding Author: C. Wagner, Psychology, Busch Campus,

Rutgers University, New Brunswick, NJ 08854

Tel: 732-445-4660 Fax: 732-445-2263

218

Evidence of Oxidative Stress in Autism Derived from Animal Models

1Xue Ming, 2 A. Cheh and 2 L. Yochum,

3Alycia K. Halladay and 2 C. Wagner

1Pediatric Neuroscience, UMDNJ, Newark, NJ

2Psychology, Rutgers University, New Brunswick, NJ

3Autism Speaks, Princeton, NJ

Abstract: Autism is a pervasive neurodevelopmental disorder that

leads to deficits in social

interaction, communication and restricted, repetitive motor

movements. Autism is a highly heritable

disorder, however, there is mounting evidence to suggest that

toxicant-induced oxidative stress may

play a role. The focus of this article will be to review our animal

model of autism and discuss our

evidence that oxidative stress may be a common underlying mechanism

of neurodevelopmental

damage. We have shown that mice exposed to either methylmercury

(MeHg) or valproic acid (VPA) in

early postnatal life display aberrant social, cognitive and motor

behavior. Interestingly, early exposure

to both compounds has been clinically implicated in the development

of autism. We recently found

that Trolox, a water-soluble vitamin E derivative, is capable of

attenuating a number of

neurobehavioral alterations observed in mice postnatally exposed to

MeHg. In addition, a number of

other investigators have shown that oxidative stress plays a role in

neural injury following MeHg

exposure both in vitro and in vivo. New data presented here will show

that VPA-induced

neurobehavioral deficits are attenuated by vitamin E as well and that

the level of glial fibrillary acidic

protein (GFAP), a marker of astrocytic neural injury, is altered

following VPA exposure. Collectively,

these data indicate that vitamin E and its derivative are capable of

protecting against neurobehavioral

deficits induced by both MeHg and VPA. This antioxidant protection

suggests that oxidative stress

may be a common mechanism of injury leading to aberrant behavior in

both our animal model as well

as in the human disease state.

Key words: Vitamin E, trolox, valproic acid

INTRODUCTION

The core symptoms of autism include language

deficits, impaired social interactions and inappropriate,

stereotypic and sometimes self-injurious behaviors. The

etiology of autism remains unknown but may involve

early exposure to environmental toxicants acting upon

genetically-sensitive individuals. No single toxicant has

been identified; rather a broad range of toxicants

including drugs, metals, solvents, herbicides, pesticides,

etc. have been associated with autism[1-3]. A common

feature across this range of potential compounds is

toxicant-induced oxidative stress causing neuronal

damage leading to the behavioral phenotype of

autism[4-6]. Likewise, no single gene has been identified

but, rather, a constellation of as many as 15

polymorphisms may ultimately predispose the

individual to autism. Again, genetic alterations leading

to compromised handling of toxicant-induced reactive

oxygen species (ROS) has been a common theme.

Since the etiology of autism is unknown, it is

essential that animal models be developed. The

behavioral symptoms of autism have proven difficult to

model in other species. Accordingly, we have initiated

work on a novel strategy to model the behavioral

phenotype of autism in mice[1]. In this model, the

normal development of key behaviors is carefully

monitored from birth through adolescence. Once the

maturation of these key behaviors is understood in

terms of the postnatal day(s) of life in which subjects

are able to successfully perform the task or engage in

the behavior, the performance of mice with early

toxicant exposure and/or genetic modification can be

assessed.

The model strategy begins by characterizing

behavioral manifestations of developmental disorders

as retardations (a behavior fails to develop during a

critical period of maturation), regressions (a behavior

develops at about the right time but then is lost with

Am. J. Biochem. & Biotech., 4 (2): 218-225, 2008

219

later development, especially following toxicant

exposure), or intrusions (the appearance of behaviors

aberrant in form or frequency which mask normal

development). Most developmental disorders include

some combination of these conditions. In this

framework, the hypothesis that environmental toxicants

or genetic alterations are causally involved in autism

can be readily tested. That is, acute or repeated

exposure to a toxicant should disrupt neurobehavioral

development causing behavioral retardation, regression,

or intrusions and these toxicant-induced behavioral

deficits should occur at lower doses in the geneticallysensitive

mice. Traditionally, animal models of

developmental disorders have not examined these three

scenarios of retardations, regressions and/or intrusions

but, instead, focus on single aspects of neurobehavioral

development. The judicious use of toxicants associated

with autism or toxicants known to damage brain regions

associated with autism confers some selectivity of the

model for autism. Likewise, manipulation of genes

associated with autism also confers some selectivity of

this model for autism. Finally, administering a battery

of tests that assess social, cognitive and motor

maturation of the mice confers some selectivity for

autism. Ultimately, it is the possibility of combining

select toxicant exposure in genetically-sensitive mice

followed by thorough assessment of social, cognitive

and motor skill maturation that makes this a

comprehensive animal model of autism.

In our initial studies, we identified toxicant induced

retardation of motor and cognitive skills following pre-

or post-natal exposure to sodium valproate (VPA).

Likewise, we were able to demonstrate dramatic loss of

acquired skills, i.e. regressions, following post-natal

VPA administration[1]. Finally, we demonstrated

toxicant induced intrusions wherein toxicant-treated

subjects exhibited dramatic increases in stereotypic and

self-injurious behaviors akin to those seen in autism[7,8].

VPA was chosen as our first agent to test this

model following reports of an association between

autism and prenatal exposure to this teratogen[9-13].

Previous studies have also demonstrated impairment in

cognitive, motor, attention and social development in

rats administered pre- or post-natal VPA[14-17].

Accordingly, in our first studies[1] mice were exposed to

VPA either in utero or post-natally. The prenatal

exposure time reflected a period of cerebellar Purkinje

cell generation differentiation in the mouse[10,14,17,18].

The post-natal time of P14 was based on our

observation that critical cerebellar-mediated behaviors

of mid-air righting and negative geotaxis mature or first

appear on this day in the mouse[1] and because of

continued neuronal and glial development in other brain

regions[15,19,20]. Of importance, VPA administration

results in high levels of markers for oxidative stress and

lipid peroxidation including 15-F-isoprostane and

thiobarbituric acid reactive substances[21-23].

An organic mercury, MeHg, was selected as our

second compound for testing because it is an important,

widely distributed environmental toxicant. MeHg does

cross the placental barrier and, in humans exposed in

utero to acute high doses, was shown to cause

retardation in cognitive and locomotor development

along with numerous other neurological symptoms

including seizures and cerebral palsy[24]. Nonetheless, it

is important to note that autism was not found to be

associated with either pre- or neonatal exposure to

organic mercury.

The consequences of low dose, chronic exposure to

mercury through fish consumption are somewhat more

controversial with some studies showing deleterious

effects while others show no adverse consequences[2,24].

Early exposure to mercury has been shown to disrupt

the neurobehavioral development of other species

including rodents and primates[25]. The mechanism

through which MeHg exerts its toxicity is thought to be,

in part, mediated by disruption of neural cell adhesion

molecules[26]. In addition, oxidative stress is involved in

MeHg-induced neurotoxicity as demonstrated by

increased ROS and thiobarbituric acid reactive

substances and a reduction in GSH levels[27]. In

addition, the neurotoxicity of MeHg in cultured neurons

was blocked by the pretreatment with antioxidants[28].

Trolox, a water-soluble derivative of vitamin E,

protects against MeHg-induced neurotoxicity in rats[29].

Likewise, antioxidants produced protective effects

against MeHg toxicity in cultured human neurons and

astrocytes[30]. Indeed, ROS have been implicated in

MeHg-induced neurotoxicity in multiple experimental

models[27,31-34]. Finally, we have recently demonstrated

that pretreatment with Trolox protects mice against the

neurobehavioral deficits induced by postnatal MeHg[8].

Collectively, these data indicate that early exposure to

MeHg causes neurobehavioral deficits consequent, at

least in part, to the generation of ROS.

In summary, wide ranges of toxicants and genetic

alterations have been associated with autism. The

toxicants are thought to have a common mechanism of

generating ROS[4-6] while the genetic alterations are

thought to result in enhanced sensitivity to the

deleterious effects of ROS. Accordingly, we now

hypothesize that autism may be the result multiple

exposures to any of a number of toxicants; the initial

exposure sensitizes the subject such that later exposures

to the same or different toxicants results in an enhanced

Am. J. Biochem. & Biotech., 4 (2): 218-225, 2008

220

oxidative stress response. Furthermore, we predict that

this sensitization will be exacerbated in individuals with

genetic alterations affecting their handling of ROS. In

previous studies we have demonstrated a sensitization

response to dopaminergic toxicants in adult mice

following prenatal administration of MeHg[7]. We have

also demonstrated that antioxidant pretreatment protects

mice against the behavioral deficits induced by early

exposure to MeHg[8]. Accordingly, the objective of this

study was to determine if antioxidants administered as a

pretreatment to VPA would protect the mice against the

VPA-induced behavioral regression. In addition, we

sought to determine if the early VPA administration

would alter levels of glial fibrillary acidic protein

(GFAP), a marker of astrocytic neural injury, thus

serving as a biological marker for the VPA-induced

behavioral deficits.

MATERIALS AND METHODS

Subjects: Male and female BALB/c mice (Taconic,

Germantown, NY) were housed together in plastic

cages with standard wood chip bedding and free access

to food and water. All mice were maintained in an

AAALAC-accredited facility under guidelines set forth

by the National Institutes of Health. Lights were set on

a 12 h on: 12 h off cycle and temperature was

maintained at 25 & #61616;C. Females were checked before 10

AM for presence of a vaginal plug which was recorded

as day 0 of embryonic development. Day of birth was

recorded as day 0 and all pups were labeled for

individual identification. Body weight was measured

daily. Female pups were removed from the cage on day

5. For the behavioral studies, the sodium valproate

(Sigma) dose was 400 mg kg & #61485;1 with a saline vehicle and

the vitamin E dose was also 400 mg kg & #61485;1 but with a

corn oil vehicle. All injections were s.c. in a volume of

1.0 mL 100 & #61485;1 g body weight.

Negative geotaxis: Negative geotropism was tested on

postnatal days P13-19 by placing the mouse facing

downward along a 45 & #61616;C incline. Latency to turn 180 & #61616;C

such that the head was facing upward along the incline

was recorded with a maximum of 30 seconds for each

trial.

Motor Activity: Motor activity was assessed on days

P14-19. The chamber consisted of a black

42 & #61605;22 & #61605;14 cm Plexiglass box. Six infrared sensors

placed approximately 7 cm apart and 2.5 cm above the

floor were used to measure activity over a 10 min

period.

Mid-air righting: When a mouse is dropped upside

down from a height of 45 cm onto a padded surface it

engages in a mid-air righting reflex with orderly, rostrocaudal

movements, initiated with head and concluded

with the hindlimbs such that the animal lands on its

paws. The behavior first appears on P13 and is fully

achieved by P17[35]. Mid-air righting has been linked to

cerebellum development[36]. For the mid-air righting

test, mice were elevated 45 cm above a foam pad,

dorsal side down. The animal was released and ability

to right in mid air assessed scored as the mouse landing

on its paws on two out of three trials each day. Mice

were tested on P13-20.

Protein determination: In order to determine changes

in protein expression following VPA treatment at

behaviorally significant time points, animals were

treated with VPA 600 mg kg & #61485;1 or saline on E13[1] and

assayed on days P4 and P5 with the cerebellum

removed and stored at -70 & #61616;C. Protein analysis via gel

electrophoresis, western blot and densiometry was

performed according to the methods of Dey et al.[26]

with some small variations. In summary, whole

homogenate fractions were homogenized in 1:10 w/v of

a Tris extraction buffer [50 mM TrisHCl, pH 7.4,

0.32M sucrose, 1 mM EDTA, 1 vial to 100 ml protease

inhibitor (Sigma, St. Louis, MO). The supernatant was

removed following centrifugation for 10 min at 1000xG

and combined with equilibration buffer [0.125M

TrisHCl, pH 6.8, 4% SDS, 20% glycerol, 10%

mercapoethanol) and immediately heated for 30 min at

70 & #61616;C. Protein values were determined using the BCA

protein assay (Pierce, Rockford, IL) modified for a

BOBAS FARA II enzyme analyzer (Roche

Diagnostics, Nutley, NJ). Samples of 10 & #61549;g protein

were separated by SDS-PAGE on a 10%

polyacrylamide gradient gel using a Bio-Rad Mini-

Protean II System (Bio-Rad, Mellville, NY) for GFAP

and synaptophysin. Proteins were transferred to

nitrocellulose membranes and were washed twice for

10 min each in phosphate buffered saline (PBS) and

blocked with 5% non-fat dry milk in PBS for 1 h prior

to application of primary antibody. Immunoblotting for

GFAP and synaptophysin was performed overnight at

4 & #61616;C. All primary antibodies were obtained from Fisher

(Springfield, NJ). Anitgens were visualized following

1 h incubation with secondary peroxidase antibodies

(Southern Biotechnology Associates, Birmingham, AL)

and application of chemiluminescence ECL substrate

detection on Hyperfilm ECL autoradiographic film

(Amersham). For GFAP detection, this method was

verified in a separate study using a dose response of

trimethyltin treatment using a GFAP protein standard

Am. J. Biochem. & Biotech., 4 (2): 218-225, 2008

221

(Chemicon, Inc.). ECL images were scanned into an

IBM PC using a Hewlett Packard Scanner with a

transparency adapter. Densiometric analysis was

performed using Image Pro Analysis Software using

percent of saline treated controls as the standard.

Statistical analysis: All behavioral analysis were

performed using a repeated measures ANOVA

including both group, day and sex as main factors, with

the exception of the mid-air righting response which

was analyzed using Chi-Square and Fisher's Exact Test.

RESULTS AND DISCUSSION

Negative geotaxis: Control mice and those treated with

vitamin E alone were able to perform the reflexive

negative geotaxis response, reorienting their head to

point upward when placed on an inclined plane with

their head facing down. This reflex improved across

development, as the latency to re-orient improved

across testing (F (6, 312) = 5.4, p<0.001). VPA-treated

mice displayed an increased latency to perform this reorientation

response (F (1, 52) = 10.0, p<0.005). Posthoc

analysis revealed that following day 14 treatment

with VPA, there was a significant regression in the

performance of this response, which reached statistical

significance on days 16 and 17. Importantly, this VPAinduced

regression was blocked by vitamin E

pretreatment, such that pretreated mice were able to

perform this response with a similar latency as controls

on P16 and P17 (F (1, 52) = 5.3, p<0.05). Finally, VPAtreated

mice regained their ability to perform this

behavioral response similar to controls by the

completion of testing on P19 (Fig. 1).

Negative geotaxis

Day of testing

12 13 14 15 16 17 18 19 20

Latency to re-orient 180 & #61616;C

0

5

10

15

20

25

30 Corn oil/saline

Corn oil/VPA 400

Vit E/saline

Vit E/VPA 400

*

*

*

Fig. 1: Negative geotaxis: Latency to reorient from

head down to head up on a 45 & #61616;C incline for

groups of pups treated with VPA (400 mg kg & #61485;1)

or saline on P14. Some groups received vitamin

E pretreatment while others received corn oil.

*: p<0.05 compared to corn oil/saline

Mid-air righting

Day of testing

12 13 14 15 16 17 18 19 20

Percent mice able to right in mid-air

0

20

40

60

80

100

120 Corn oil/saline

Corn oil/VPA 400

Vit E/saline

Vit E/VPA 400

*

*

Fig. 2: Mid-air righting: Number of pups successfully

engaging in mid-air righting (expressed as a

percent of pups mid-air righting on 2 out of 3

trials/day) for groups of pups treated with VPA

(400 mg kg & #61485;1) or saline on P14. Some groups

received vitamin E pretreatment while others

received corn oil. *: p<0.05 compared to corn

oil/saline

Mid-air righting: Before any treatment was

administered, less than 20% of the pups were able to

engage in mid-air writing on P13 but this improved to

about 75% by P14. This observation is interpreted to

indicate that cerebellar and general muscular maturation

have matured by P14. & #61539;2 analysis revealed that

following VPA-treatment given after behavioral testing

on P14 caused a regression in mid-air righting on P15

[ & #61539;2 (3) = 39.8, p<0.0001] when compared to saline

controls. This regression was still observed on P16 in

the VPA-treated animals. However, the VPA-induced

regression was eliminated by pretreatment with vitamin

E (Fig. 2).

Motor activity: Mice engaged in a similar amount of

locomotor activity at the start of testing. This activity

significantly increased across post-natal development

[F (1, 38) = 80.2, p<0.001]. Interestingly, there was a

trend for mice treated with VPA to engage in intrusive

behaviors following P14 treatment. This was evidenced

by increased levels of activity across testing, beginning

on P16 through P18. However, this hyperactivity did

not reach statistical significance. In addition, this

hyperactive behavior was blocked by pre-treatment

with vitamin E (Fig. 3).

GFAP and synaptophysin: Previous work in our lab

revealed retarded neurobehavioral development in mice

treated with 600 mg kg & #61485;1 VPA on embryonic day 13[1].

In order to determine whether we could detect a

Am. J. Biochem. & Biotech., 4 (2): 218-225, 2008

222

Motor activity:

Day of testing

13 14 15 16 17 18 19 20

Number of horizontal beam breaks

0

200

400

600

800

1000

Corn oil+saline

Corn oil+VPA

Vitamin E+VPA

Fig. 3: Motor activity: Horizontal beam breaks for

groups of pups treated with VPA (400 mg kg & #61485;1)

or saline on P14. Some groups received vitamin

E pretreatment while others received corn oil.

*: p<0.05 compared to corn oil/saline

Prenatal saline

Sacrificed day 4

Prenatal saline

Sacrificed day 5

Pprenatal VPA

Sacrificed day 5

Glia-fibrillary acidic protein

0

1000

2000

3000

4000

5000

6000

7000

SALINE (PND4) SALINE (PND5) VPA (PND5)

Protein level (arbitrary units)

*

Animals treated with VPA 600 mg kg & #61485;1 or Saline on E13

Fig. 4: GFAP: Pups received either VPA (600 mg kg & #61485;1)

or saline in utero on E13 and were sacrificed on

either P4 or P5. *: p<0.05 compared to corn

oil/saline

biological marker of VPA-induced neurobehavioral

retardation, we examined early postnatal levels of

GFAP and synaptophysin in the cerebellum following

in utero exposure to VPA. Mice treated with

600 mg kg & #61485;1 VPA on E13 and sacrificed on P5 showed

decreased concentrations of GFAP in the cerebellum

compared to saline treated controls sacrificed on both

P4 and P5 (F (2, 8) = 12.3, p<0.01) (Fig. 4). Likewise,

the concentration of synaptophysin was significantly

decreased in the cerebellum of E13 VPA-treated mice

compared to both P4 and P5 saline controls

Prenatal saline

Sacrificed day 4

Prenatal saline

Sacrificed day 5

Prenatal VPA

Sacrificed day 5

0

1000

2000

3000

4000

5000

6000

7000

8000

SALINE (PND4) SALINE (PND5) VPA (PND5)

Protein level (arbitrary units)

*

Synaptophysin

Animals treated with VPA 600 mg kg & #61485;1 or Saline on

E13

Fig. 5: Synaptophysin: Pups received either VPA

(600 mg kg & #61485;1) or saline in utero on E13 and

were sacrificed on either P4 or P5. *: p<0.05

compared to corn oil/saline

(F (2, 6) = 10.0, p<0.01) (Fig. 5). During this period of

postnatal development, GFAP and synaptophysin levels

increase to promote normal astrocytes-neuron

interactions and synaptogensis, respectively[37].

Therefore, mice treated with VPA in utero show

immature neural development, since the levels of GFAP

and synaptophysin observed on P5 are much lower than

those found in mice from an earlier postnatal period

(P4). This suggests that the behavioral retardations seen

in our previous study are influenced by retarded neural

development.

The etiology of autism is thought to involve early

exposure to ROS-generating toxicants acting upon

genetically-sensitive individuals. We have developed a

new strategy to assess the detrimental effects of early

toxicant exposure on neurobehavioral development,

classifying the behavioral deficits as retardations,

regressions or intrusions. In previous studies we

demonstrated that early exposure to VPA or MeHg

results in behavioral deficits in the maturation of social,

cognitive and motor skills[1,8]. Furthermore, we

demonstrated that our behavioral model was useful in

demonstrating that pretreatment with an antioxidant

protected mice against the behavioral deficits induced

by early exposure to MeHg[8]. In the present study, we

demonstrated that vitamin E was capable of protecting

mice against VPA-induced regression in negative

geotaxis and mid-air righting as well as against

intrusive VPA-induced hyperactivity. Collectively, the

present data together with our previous MeHg study

indicate that the generation of ROS may be a common

Am. J. Biochem. & Biotech., 4 (2): 218-225, 2008

223

factor mediating toxicant-induced neuronal damage

associated with autism and that neurobehavioral

assessments provide an important functional measure of

the potential benefits of antioxidants.

A second objective of the present study was to

develop a biological marker of the VPA-induced

damage. Toward this end we used our initial model,

delivering the VPA prenatally on E13[1]. We had

demonstrated that this prenatal VPA treatment resulted

in later behavioral deficits as assessed in the surface

and mid-air righting tests, negative geotaxis and in

water maze. Furthermore, this prenatal VPA treatment

resulted in sex-dependent differences in these

behavioral deficits with males more affected than

females. In the present study, we found that both

cerebellar GFAP and cerebellar synaptophysin were

reduced postnatally following the prenatal VPA

administration. GFAP is a marker of astroglia in the

brain and is involved in astrocyte-neuron interactions.

GFAP mutant mice have abnormal structure and exhibit

deficient long-term depression in cerebellar Purkinje

cell synapses[37]. Therefore, major alterations in GFAP

may alter Purkinje cell communication that, in turn,

may alter behavior. Synaptophysin is a widely used

marker for nerve terminals and can indicate

synaptogenesis. Therefore, a reduction in

synaptophysin in the cerebellum could signify a

reduction in synatpogenesis in that region. More

generally, it is intriguing that these biological markers

may reflect the behavioral deficits of cognitive and

motor retardation caused by the early VPA exposure.

Future studies are designed to determine if the

antioxidant pretreatment also protects the mice against

these neurological changes induced by the VPA.

There is ample evidence that ROS are involved in

human autism. Free oxygen radicals could result from

ingested or inhaled environmental toxins, food or food

additives, inflammation or infection (overt or occult).

The interaction of free oxygen radicals and

polymorphic oxidative genes during gestation or

postnatally could disrupt neurogenesis in developing

brain at multiple time windows, eliciting immediate

stage-dependent effects in specific systems that

influence subsequent ontogenetic processes, leading to

the phenotype of autism. Indeed an exacerbated

oxidative stress response has been implicated in autism.

Specifically lower glutathione peroxidase (GPX) and

superoxide dismutase (SOD) activity were found in

children with autism[38-40]. An increase in body burden

of various toxins was reported in autism[41,42]. In

addition, provoked urinary mercury excretion is found

to be higher in autism[43]. These toxins could generate

oxidative stress in children with autism. Elevated nitrite

and nitrate in plasma[44] and red cells[38] have been

reported in children with autism. This elevation

indicates excess generation of nitric oxide free radicals.

In addition, two independent double blind placebo

controlled clinical trials of antioxidants (vitamin C or

carnosine) showed beneficial effects in autism[45,46].

Finally, we conducted a study of oxidative stress

biomarkers in children with autism and age matched

healthy controls. Our results showed that urinary

excretion of 8 isoprostane F2_ was significantly higher

in children with autism as compared to healthy

controls[47]. There was also a trend of increased 8-

OHdG urinary excretion in autistic subjects. These

results suggest that oxidative stress is exacerbated in

autism and are consistent with the present results of

antioxidant protective effects against VPA-induced

behavioral deficits in mice.

In summary, we have developed a comprehensive

neurobehavioral model in which mice are exposed to

candidate toxicants during critical periods of neural

development. The mice may have altered expression of

genes thought to be associated with autism and/or to

confer increased sensitivity to the toxicants. The mice

are then assessed in a battery of tests designed to assess

behavioral maturation of skills in the social, cognitive

and motor domains. Toxicant or genetic-induced

deficits in the behavioral maturation are classified as

retardations, regressions or intrusions. In the present

studies, we further demonstrate that pretreatment with

an antioxidant protects the mice against the toxicantinduced

behavioral deficits. We conclude that our

model is useful for evaluation of the theory that

oxidative stress may play a role in the etiology of

autism.

ACKNOWLEDGEMENT

Supported by ES11256, EPAR829391, New Jersey

Governor's Council on Autism and Autism Speaks.

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