Imidacloprid impacts on neurobehavioral performance, oxidative

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Food Safety and Toxicology

Imidacloprid impacts on neurobehavioral performance, oxidative stress, and apoptotic events in the brain of adolescent and adult rats yasmina Abd-Elhakim, Hesham H Mohammed, and Wafaa A Mohamed J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05793 • Publication Date (Web): 02 Dec 2018 Downloaded from http://pubs.acs.org on December 3, 2018

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Imidacloprid impacts on neurobehavioral performance, oxidative stress, and apoptotic

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events in the brain of adolescent and adult rats

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Yasmina M. Abd-Elhakim†,*, Hesham H. Mohammed ‡,Wafaa A.M. Mohamed §

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†

Department of Forensic Medicine and Toxicology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt.

7 ‡ Department

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of Veterinary Pubic Health, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt.

9 §

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Department of Clinical Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig 44519, Egypt

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*

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Email: [email protected]

Corresponding author: Yasmina Mohammed Abd-Elhakim Telefax number: +20552284283.

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ABSTRACT

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Currently, imidacloprid (IMI) is the first insecticide and the second agrochemical

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highly applied all over the world. Here we report on the impacts of IMI on neurobehavioral

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performance, oxidative stress, and apoptotic changes in the brain in either adult or adolescent

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rats. Forty male rats (adult and adolescent) were allocated to four groups. IMI groups were

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orally given 1 mg IMI/kg b.wt. dissolved in corn oil, whereas the controls were orally

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administered corn oil daily for 60 days. The obtained results demonstrated that IMI exposure

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resulted in less exploratory activity, deficit sensorimotor functions, and high depression.

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Levels of neurotransmitter including serotonin, gamma-aminobutyric acid, and dopamine

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were significantly reduced. Oxidative damage of brain tissues was evident following IMI

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exposure represented by the high levels of protein carbonyl, 8 hydroxyguanosine, and

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malondialdehyde, but total antioxidant capacity was reduced. Histopathological investigations

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of the brain tissues of IMI treated group revealed varying degrees of degeneration of the

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neuron. The immunohistochemical evaluation revealed a strong presence of glial fibrillary

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acidic protein (GFAP) and Bax positive cells, but a low expression of Bcl-2. These injurious

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impacts of IMI were very prominent in the adult rats than in the adolescent rats. Conclusively,

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exposure to IMI even at very low concentration could induce multiple neurobehavioral

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aberrations and neurotoxic impacts, especially in adults.

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Keywords: Imidacloprid; exploratory behaviors; neurotransmitters; Bcl2; Bax; oxidative

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stress.

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INTRODUCTION

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In non-targeted animal species, many of food contaminants and environmental pollutants can

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elicit inflammatory reactions and oxidative stress in brain of animals leading to

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neurobehavioral disorders

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is one of the highly growing insecticides in agriculture and veterinary fields 5. Imidacloprid

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(IMI) is the most known neonicotinoid used for crop safeguard and residential uses and for

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control of flea in pet animals all over the world

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commercial insecticide products 9.

1-4.

Neonicotinoid, a novel set of systemic neuro-active pesticides,

6-8.

It is the active ingredient in many

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In the global environment, IMI has been widely detected in soils and sediments. Because

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of its great solubility in water and fairly non-volatile nature, IMI persist in soil for a half-life

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of nearly 5 months 10. IMI was previously detected in soil samples from production fields in

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levels up to 8ppb11. Also, in a previous study to determine the leaching potential of IMI, it

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was identified at the greatest sampled soil at 105 cm depth at levels up to 120 ppb12. Also, it

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has a great runoff and releasing capacity to ground and surface water due to its extraordinary

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perseverance in the water bodies and its difficult biodegradation in the marine environments

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13.

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found up to 14 μg/L. Additionally, due to its persistence in crops, humans and non-target

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organisms could be widely exposed to it 15. For instance, there were several reported wild bird

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deaths cases because of the eating of seeds treated with IMI 16.

Based on the US Geological Survey 14 in the surface water, the concentration of IMI were

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IMI disturbs the stimuli synaptic transmission in the insect's central nervous system (CNS)

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producing systemic neurotoxic effects. It accomplishes this task via an agonistic action at the

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postsynaptic nicotinic acetylcholine receptor (nAChR) resulting in a block in the nicotinergic

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neuronal pathway and acetylcholine accumulation. But, nAChRs of mammals display a lesser

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binding affinity for IMI than insects. Hence, IMI has been categorized as “moderately toxic”

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compound

17.

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The occupational exposure of the human population to IMI is frequent all over the

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world18. Slight clinical signs such as hypertension, abnormally rapid heart rate, nausea,

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vomiting, and mydriasis occur but more severe consequences comprising seizures, respiratory

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failure, and death are documented19. A few numbers of lethal human poisoning cases have

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been documented due to consumption of IMI products20. However, its prolonged exposure

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may cause health hazards like oxidative stress, gastrointestinal disturbances, neurotoxic

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metabolic disturbances

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genotoxic, hepatotoxic, nephrotoxic, and reprotoxic effects

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found to affect early embryo development and elicit malformations

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worries on its supposed greater safety features rather than older compounds.

22-24,

1, 21,

behavioral deficits, teratogenic, mutagenic, immunotoxic, 15, 25-30.

Also, IMI exposure was 31, 32.

This raises critical

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Both acute, 10 µM i.v.for 2h, and subchronic, 1 mg/kg b.wt. /day orally for 30 consecutive

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days IMI exposure in rats was found to increase lipid peroxidation, xanthine oxidase, and

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myeloperoxidase activities and upregulate inflammatory cytokines IL-6, TNF-a, and IL-1b

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mRNA transcriptions in the brain tissue

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levels more than10 µM for less than 1 min can modulate the features and functions of

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membrane neurons containing nAChRs. Kara, et al. 35reported that IMI at the doses of 0.5, 2,

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and 8 mg/kg impaired cognition and learning behaviors in both infant and adult rats.

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However, the same authors reported that the expressions of learning related genes like

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synoptophysin, inotropic glutamate receptor, and growth-associated protein 43 were

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insignificantly changed. Notable reduction in spontaneous locomotors activity, pain threshold,

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acetylcholinesterase, ATPase and several serum biochemicals following IMI exposure (45

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and 90 mg/kg b.wt.; orally) for 4 weeks was previously reported

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neurobehavioral studies of pesticide exposure have dealt mainly with adults. However,

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pesticides are supposed to have a greater risk to adolescents than to adults, but the extent of

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health hazards of pesticides exposure on children and adolescents have paid little attention 37.

1, 33.

Bal, et al.

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documented that IMI exposure at

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36.

The majority of


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While certain work on the IMI neurotoxic potential has been adopted in rats

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1, 33, 36,

there

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is a vital need to further find out the impacts of age on the neurobehavioral response to IMI

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exposure in rats

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children to IMI via food, water, or direct contact with pets together with the earlier reports of

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the variation of the effects of some other pesticide with the age39, we hypothesized that the

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neurobehavioral response to IMI could differ between adolescent and adult. Hence, in the

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current study, both adolescent and adult rats were exposed to IMI for 60 days then subjected

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to

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neurobehavioral response. In particular, the effect of mutual interaction between IMI exposure

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and age on dopamine, serotonin, and gamma-aminobutyric acid (GABA) levels was examined

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for the first time.

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MATERIAL AND METHODS

behavioural,

38.

In view of the possibility of consistent exposure of adults as well as

biochemical,

and

neuropathological

evaluations

to

assess

their

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Tested compounds and chemicals. Technical grade (99.9% pure) IMI, CAS Number

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138261-41-3, was purchased (Sigma-Aldrich Co. St. Louis, MO, USA). The stock solution of

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the IMI was prepared by dissolving in corn oil (Arma food industries, 10th of Ramadan,

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Sharkia, Egypt). All other chemicals including dihydroxybenzylamine (DHBA), perchloric

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acid, and ascorbic acid were attained from Sigma-Aldrich Co. St. Louis, MO, USA.

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Animal grouping and experimental protocol. A total of 40 healthy male Sprague

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Dawley rats were got from the Laboratory Animal Housing Unit, Faculty of Veterinary

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Medicine, Zagazig University, Egypt. Half of the rats were adult (220 -250 g, 12 weeks of

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age), while the rest was adolescent (50-80 g, 3 weeks of age). The animals are housed in a

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well-ventilated stainless steel cage under a 12-h light/12-h dark cycle. Water and food were

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offered ad libitum. The experimental animals were adapted for two weeks before using in any

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trial herein. The guidelines of the National Institutes of Health, USA, were followed in all

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experimental steps involving animals and the protocol was accepted by the Ethics of the

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Animal Use Research Committee of Zagazig University, Egypt.

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The experimental animals were randomly distributed to four groups (10 rats/group).

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The control groups (adult and adolescent) were orally given corn oil (1 mL/kg b.wt). The

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IMI-exposed groups (adult and adolescent) orally dosed IMI (1 mg/kg b.wt.) for 60

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consecutive days

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observable effect level in rats (5–10 mg/kg bw/day) 35. This dose was equal to 1/450 of IMI

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LD5040. IMI was dissolved in corn oil (1 mg/mL) as a stock solution and the rats were orally

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administered the proper dose according to their weight. All rats were weighed once weekly to

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change the dose volumes consequently. The rats were carefully observed during the course of

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the experiment for signs of toxicity, sickness, and death.

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The IMI dose was selected to be lesser than the documented non-

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Behavioural analysis. All behavioural experiments were conducted in the same trial

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testing room. The identity of the animal groups was anonymous to the experimenter. On the

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test days, rats were transferred in their own cages to the testing room and allowed to

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acclimatize before testing procedures about 30 min. By the completion of each test, the

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animal was transferred to its household cage and then the device was cleaned to remove any

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trace of odour using a damp sponge.

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The hole-board test. In rodents, this test is frequently used to assess exploratory

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behavior and was done based on the protocol of File and Wardill 41. The test was conducted in

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a 60x60 cm open-field rectangular arena made of wood containing four identical 3 cm spaced

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holes in the floor. For the period of a 3-minutes, the number of head-dip (head of rat

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introduced into the hole) were calculated. A head-dip was counted if both eyes missing into

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the hole. 42

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Inclined plane. In line with the procedures followed by Abou-Donia, et al.

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were kept on a flat plane in the straight situation, with the head in front of the side of the 6


, rats


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board to be elevated. The inclined plane show was calculated by an ordinary protractor to the

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close 5 degrees. The angle at which the rat began to slip downward was recorded. A trial

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ended when the rat started to slip backward. The outcomes of the two trials with 1 h interval

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were averaged. The postural reflex. The postural reflex test was conducted to evaluate the

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43.

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sensorimotor function according to Bederson, et al.

The test includes suspending rats 20

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cm above the floor with their tails then estimate the degree of abnormal posture. Normal rats

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stretched both forelimbs toward the ground and a 0 score was recorded. If the unusual

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position as bending the contralateral limb to the trunk and/or circling the contralateral limb

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and shoulder medially was detected, the rat was kept on soft plastic-backed paper sheet that

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could be hold by its claws. A lateral compression was adopted from behind the shoulders

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where the forelimbs slipped smoothly to the left and formerly to the right. Animals that slided

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in both directions were classified as 1, animals had a reduced struggle to the adjacent push

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were recorded as 2, and those that encircled to the paretic side constantly were categorized as

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3.

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Tail suspension test (TST). TST, a behavior model to expect the antidepressant

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potential of chemicals in rodents, has been done according to the method of Chermat, et al. 44

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In this test, primarily, rats were completely isolated, then suspended 58 cm by adhesive tape

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on the floor kept about 1 cm from the tail tip. A plywood square platform was placed

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horizontally 15-20 cm under the bench so that the rat could hint the platform lightly. Rats

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were recorded immobile merely when they hung inactively and totally immobile. Throughout

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a 5-min phase, the total time of immobility was recorded.

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Sampling. By the end of the dosing, rats were euthanized and brain tissue samples

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were got, separated, washed with physiological saline and then distributed into two groups.

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The first set was homogenized and centrifuged for 15 at 4 °C min at 664 ×g to get the 7


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supernatants used for estimating the neurotransmitters levels and oxidative stress indicators.

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The second one was preserved in 10% neutral buffered formalin for histopathological and

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immunohistochemical examinations.

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Determination of brain neurotransmitters levels. For the estimation of

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neurotransmitter (serotonin, GABA, and dopamine) levels, the brain tissues specimens was

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homogenized in solution containing 10-7M ascorbic acid and 1.1 M Perchloric acid with 20

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ng DHBA/ml internal standard. Next to tissue homogenization, the samples were centrifuged

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and filtered. The neurotransmitter levels were accurately determined by reverse-phase high-

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performance liquid chromatography with an electrochemical detector and a C-18 column. The

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mobile phase contained phosphate buffer saline/methanol at a 1 ml/min flow rate. Detection

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was done at 270 NM with 20 µl injection volume. The neurotransmitters level was assessed

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by an external standard method by peak areas. Serial dilutions of standards were inoculated

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and their peak areas were recognized 45.

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Estimation of oxidative stress indicators in brain tissue. Malondialdehyde (MDA)

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was assessed following Ohkawa, et al. 46 method. Protein carbonyl was measured according to

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Levine, et al.

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(TAC)

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hydroxyguanosine (8-OHdG) was measured via ELISA kit (Cat. No. MBS 267513) along

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with manufacturer's instructions (My BioSource, San Diego, CA, United States).

was

47.

In line with the method of Koracevic, et al.

48,

total antioxidant capacity

estimated by kits reagent (Biodiagnostic Co. Dokki, Giza, Egypt). 8

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Histopathological and immunohistochemical investigations. The fixed brain

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specimens were stained with hematoxylin and eosin (HE) for histopathological investigation

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49.

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(15-35%), ++= Moderate alterations (40-55%), and +++= Severe alterations (55-75%).

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Another set of paraffinized sections was also ready for the immunodetection of GFAP, Bcl-2,

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and Bax positive cells by an avidin-biotin-peroxidase technique according to Abd-Elhakim, et

Lesion score was performed as the following: - = No alterations (0%), += Mild alterations

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50

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al.

For evaluating cellular GFAP, Bcl-2, and Bax expression, ten fields for each rat were

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examined at a ×400 magnification. The measurements were done in an anonymous way in

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line with Mustafa, et al.

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immunostaining were assessed by the following equation: OD = log (max intensity ÷ mean

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intensity), where max intensity equal to 255 for 8-bit images.

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protocol. The optical densities (OD) of GFAP, Bcl-2, and Bax

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Data Analysis. The computer program SPSS/PC+2001 was adopted for statistical

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analysis of the current study data. Two way ANOVA test, for the effects of age and toxicity,

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with Duncan’s multiple range test was adopted as the statistical method here. Data are shown

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as means plus or minus the standard error. p

Imidacloprid impacts on neurobehavioral performance, oxidative

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