Evaluations on the effects of mercury exposure on exploratory motor activity of Adult Wistar Rats

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Adamu. L.H.: Department of Human Anatomy, Faculty of Medicine, Bayero University, kano. Nigeria
A.A. Sadeeq; A.O. Ibegbu; S.P Akpulu;Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University. Zaria. Nigeria.
A.R. Hadiza I.S El-ladanDepartment of Human Anatomy, Faculty of Medicine, Kaduna State University. Nigeria.
H.O KwanashieDepartment of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences.Ahmadu Bello University, Zaria. Nigeria.

All correspondence to: A.A. Sadeeq Department of Human Anatomy, Faculty of Medicine, Ahmadu Bello University. Zaria. Nigeria.

ABSTRACT
Mercury is a widespread toxic environmental and industrial pollutant. The present study
was carried out to investigate the possible effect of mercury chloride (HgCl2) on exploratory motor activity (EMA) of adult wistar rats. Twenty four Wistar rats of both
sexes weighing (190-220 gm) were administered with HgCl2 orally for twenty one days at
concentrations of; 12.45mg/kg, 24.9mg/kg and 49.8mg/kg b.wt to group II, III, and IV respectively. While group I served as control which was administered normal saline. Exploratory motor activity (EMA) was evaluated using Montoya staircase test. Brain tissue specimens, representing all groups, were fixed in a Bouin’s fluid and taken for histopathological examination. Complex motor behavior (motor movements coordination) was significantly impaired due to mercury intoxication. Furthermore, histopathological evaluation revealed distinct neurodegenerative changes of nerve cells of cerebellar cortex. In conclusion, these results suggest that intoxication with mercury chloride has potentially deleterious effects on brain as reflected in exploratory motor activity and histopathological observations of the cerebellar cortex.
Keywords; Mercury, motor activity, wistar rats, cerebellar cortex, intoxication.

INTRODUCTION
Man in his environment is exposed to much potential hazards by heavy metals via bioaccumulation and biodegradation which is transferred in man via food chain due to anthropogenic activities (Wang et al., 2007). Mercury, a heavy metal is a highly deleterious environmental pollutant that can lead to many health problems in the world (WHO, 2003). Mercury can exist either as elemental, organic and inorganic mercury (WHO, 2003; ATDRS, 1995; Burger et al., 2011). Sources of Mercuric compounds are mostly from Industrial sources, gas, fumes, battery disposals, broken mercury thermometer, coal combustion (Akagi, 1995; Bjomberg et al., 2011). And majorly from Natural source such as Mercury chloride that is found in higher densities in rocks and volcanic activities which can give half of the HgCl 2 present in nature (Park, 2000; Booth, 2005). There are many routes of exposure to mercury which include: Oral exposure, inhalational exposure and dermal exposure (EHD, 2002; Vupputuri et al.,2005: WHO, 2005; Mohan et al., 2011; Berlin, 2006; Nardofy et al., 2000). Mercury and its compounds have been shown to also have effects on the growth, weight, renal system, liver, enzymes, memory and psychological disturbances to mention but a few (Kosan et al., 2001; WHO, 2003; Oliveri, 2000; Valera et al., 2008; Rao, 2001). Signs and symptoms of mercury poisoning include; Irritability, excitability, restlessness of the skin and eyes, headache, dizziness, difficulty in breathing and frequent urination (Amin- zaki et al., 1974; WHO, 2005; ATDRS, 2007 and 2011). Mercury has no known nutritional or biomedical importance but has various applications and uses such as preservation, employ by pharmaceutical company, agriculture and in cosmetic production (W.H.O, 2003 and 2005). The aim of the study is to evaluate the effects of Mercury exposure on exploratory Motor activity (EMA) of adult Wistar rats.

Materials And Method

Experimental Designed
Twenty four (24) adult Wistar rats of both sexes were obtained from the Faculty of Pharmaceutical Sciences, Ahmadu Bello University, Zaria. Nigeria. The weights of the animals were between 190 – 220g and were housed in polyester cages with wire gauze covering. The animals were allowed to acclimatize for two (2) weeks in the animal house of the Department of Human Anatomy, Ahmadu Bello University, Zaria. Nigeria. Animals were fed with Grower’s mesh brand of the Animal feed which was prepared in pellet form to reduce spillages, the animals were fed 3 times daily while clean water was provided in plastic

Administration of Mercury Chloride
The LD of Mercury chloride was adopted from (ATDRS, 50 (2011). as 166mg/kg body weight. The Concentration of Mercury chloride used was determined using 30%, 15% and 7.5% of the standard LD per kg body weight 50 according to the method. Animals in group I served as control and was given normal saline, while animals in group II, III and IV were administered with mercury chloride at 12.45 mg/kg, 24.9 mg/kg and 49.8 mg/kg respectively of the LD per kg body weight. 50
Animal Sacrifices
At the end of the 21 days of mercury chloride administration, animals were anasthesize using chloroform and the brain tissues were removed by opening through the sutures of the skull using a brain opener. The brain tissue were then transferred into specimen bottle, containing Bouin’s fluid.
Tissue Processing
The brain specimens were extracted placed in Bouin’s fluid for 48 hrs for proper fixation and was processed routinely by embedding in paraffin. Tissue sections (4-5 μm) were stained with haematoxylin-eosin for general features and examined under light microscope (Celestron).
Statistical analysis
Data obtained was expressed as Mean ± SEM (Standard error of Mean). One Way Analysis of Variance (ANOVA) was used to compare the Means difference between and within the groups and a P-value less than 0.05 was considered to be statistically significant. Statistical analysis was performed using EZanalyze v3.0 and a post hoc test of Bonferroni was applied. Chart was produce using Microsoft(R) Excel 2007 for windows.

NEUROBEHAVIORAL STUDIES

Montoya Staircase Test Method
The Montoya staircase test method was used to study the locomotor function and skills in motor co-ordination. This method helps in determining the effects of a test substance on motor function abilities (Montoya et al., 1990 and 1991). The tasks involved forepaw reaching for food pellets located at various steps along the staircase and the time taking to explore the activity cage (Abrous et
al.,1994; Whishaw et al.,, 1986 and 2000). The test was carried out as a measure of Motor coordination shown by muscle action in climbing and exploring the staircase and motor and locomotor function shown by the independent forelimb ability.
Training Time
At the outset of the test, animals were first trained to recognize that there are food pellets along the wells in Montoya staircase for three consecutive days. The animals were able to familiarize themselves with the Montoya staircase and then the animals were deprived of food for 18 hours in order to facilitate their search for food. During the test, each animal was placed on the foot of the staircase which will enable them to climb the stairs in order to reach out, grasp, or retrieve the food pellets and each test session lasted for 5 min, at the end of which the time taken to climb up and down from the basement was recorded as exploratory motor activity (EMA).

RESULT

Motor Activity Test Using Montoya Staircase Test
The results of exploratory motor activity test using Montoya staircase method were shown in Table 1. This showed that there was a non-significant decrease in the mean time taken for the animals to reach the top of the six stairs throughout the 3 weeks of administration. While the animals in group II that received the minimum concentration (12.45mg/kg b.w) of mercury chloride had the mean time taken to reach the stairs increased (P ≥0.05) between the 1st and 2nd week of administration. The increase in the mean time taken was statistically significant (P≤ 0.05) between the 1st and 3rd week, and between the 2nd and 3rd week, respectively as shown in Table 1. The results showed that the time taken for the animals to reach the top of the stairs was significantly increased in both groups III and IV animals. There was significant increase in time taken by the animals between week 1 and 2, between week 1 and 3 and between week 2 and 3, respectively in group III and IV animals.

Table 1: The mean time taken to explore Montoya staircase used for testing motor activity

Histological Observation
The cerebellum of the animals control group (group I) showed cytoarchitecture of the Molecular,
Granular and Purkinje cell layer appearing normal as shown in plate 1.While group II animals showed similar cellular architecture with cells and layers looking normal in plate 2. The histological observations for the group III animals as shown in plate 3 depicts normal molecular and granular layer but there was degeneration of the purkinje cells within the Purkinje cell layer. The same pattern of histological changes was observed in group IV for the purkinje cells with cells showing mark degeneration as shown in plate 4.

Plate 1; A section of the cerebellum of the control group (G1) showing normal orientation of the Molecular layer (ML), Granular layer (GCL), and Purkinje cell layer (PCL) with normal Purkinje cells (PC) (H&E Stain, ×250)

Plate 2; A section of the cerebellum of the group II showing normal orientation of the Molecular layer (ML), Granular layer (GL), and the Purkinje cell layer (PCL) with normal Purkinje cells (PC) (H&E Stain, ×250)

Plate 3; A section of the cerebellum of the Animals in group III showing the Molecular layer (ML), Purkinje cell layer (PCL) with degenerating Purkinje cells (DPC) and some densely populated areas in granular layer (DPA) in the Granular layer (GL) (H&E Stain,×250).

Plate 4; A section of the cerebellum of the animals in group IV showing the normal Molecular layer (ML), with degeneration of the Purkinje cell layer (DPL) and degeneration of the Purkinje cells (DPC) while the Granular layer (GL) showed sparsely granular cells (SGC) (H&E Stain, ×250)

Discussion

The results in the present study showed a significant decrease in motor activity which was shown by the increase in the time taken to reach the maximum attainable distance in the Montoya staircase method. This could be attributed to various concentrations mercury administered. Mercury intoxication was reported to cause a decrease in essentials trace elements copper; that modulates the activity of the neurotransmitter, dopamine which is important in motor function activity. (Gagelli et al., 2011) had reported an association between copper and dopamine concentration that was positive in patients with Parkinson’s disease which was characterized by motor function disorders. The decrease in the level of dopamine was observed in patients with Parkinson’s disease, in which there was loss of smooth and controlled movements (Rasia, 2005; Madsen, 2007). Histomorphological changes in the cerebellum were observed, which can in turn interfere with the motor ability of animals in the experimental group. It has been shown that ingestion of mercury of 220ml affected muscle coordination and physical movement which caused fatigue and tremor. (Dolbec, 2000). It has been shown that neurobehavioral manifestation, affected motor functions which was associated with high and medium dose of mercury exposure especially in movements, grip and balance (Mutter, 2010). Studies also shown that exposure to high concentration of mercury vapor causes tremor initially affecting the hand and then spread to the other part of the body (Dolbec et al.,2000; Yoshida et al.,2011). The cerebellum showed evidence of mercury chloride exposure by manifesting some histopathological changes and these changes include the destruction of the Purkinje cell layer, infiltration of cells in the granular cell layer and the loss of the architecture of the Purkinje cell layers. There was sparse distribution of the Purkinje cells of the Purkinje cell layer. These could result in interference with the motor activity and other motor functions such as loss of fine movement, lost of grasping, maintenance of equilibrium and loss of regulation of muscle tone which are modulated by the spinal cord and brain stem mechanisms involved in postural control. Moreover, it has been shown that neuronal degeneration in the cerebellum can affect the level of copper concentration in the cerebellum and this can affect the action of the neurotransmitter dopamine which is very crucial in motor activity. This is because copper serve as a modulator for the neurotransmitter, dopamine activity. Mutter et al., 2010 had reported decrease in dopamine concentration in the cerebellum of mice ingested with methy mercury chloride for a week which resulted in motor function disorder. It has been shown that mercury intoxication does not show any significant changes in the granular layer of chicken treated with mercury chloride in their food (Wolf et al., 2009; Quirino et al., 2012). This is not in agreement with the findings of the present study which showed infiltration of granular cells of the granular layer into the molecular layer.

Conclusion

Mercury chloride intoxication causes cytoalterations of the cerebellar cortex and also impaired motor activity (exploratory motor activity) in adult Wistar rats.

ACKNOWLEDGMENT

I appreciate Mal. Ado Garba of Ahmadu Bello
University,Zaria. Nigeria for his financial support and to
the technical assistance of the laboratory staff of the
Department of Pathology and Morbid Anatomy, Ahmadu
Bello University Teaching Hospital, Shika, Zaria, Nigeria.

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