ABSTRACT
Lead is a widespread environmental and industrial pollutant which is used in food preservation, cosmetics, pharmaceutical companies and laboratories. Despite many efforts to minimize lead poisoning, it continues to be a major health concern in many developing and developed countries.The present study was aimed at evaluating the effects of aqueous garlic extract on lead induced changes on the hippocampus and cerebellum of Wistar rats.Forty fiveWistar rats (both sexes) were used in the study and were randomly divided into nine groups with five rats in each group. The rats in Group one (G1) served as the Control and received distilled water. The rats in Groups 2 and 3 received lead only at low (60 mg/kg) and high (120 mg/kg) respectively. The rats in Group 4 (G4) received lead at low dose (60 mg/kg) and aqueous garlic extract at low dose (300 mg/kg) while the rats in Group 5 (G5) received lead at high dose (120 mg/kg) and aqueous garlic extract at low dose (300 mg/kg). The rats in Group 6 (G6) received lead at low dose (60 mg/kg) and aqueous garlic extract at high dose (500 mg/kg) while the rats in Group 7 (G7) received lead at high dose (120 mg/kg) and aqueous garlic extract at high dose (500mg/kg). The rats in Group 8 (G8) received lead at low dose (60 mg/kg) and Succimer (DMSA) at 30mg/kg b w while the rats in Group 9 (G9) received lead at high dose (120 mg/kg) and Succimer (DMSA) at 30mg/kg. All the administration were carried out orally for twenty one (21) days. The rats were trained for both spartial learning and memory and beam walking before the administration and tests performed after the administration. At the end of the administration, rats were sacrificed and histological specimens of the cerebellum and hippocampus of the rats across the groups were prepared using the routine haematoxyline and eosin and cresyl violet staining
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techniques.Sections of the lead treated groups revealed that lead produced deleterious effects on tissues of thehippocampal formation and cerebellum, disrupting the general histoarchitecture and cellular integrity while sections of lead and aqueous garlic extract administration revealed that aqueous garlic extract had promising positive effects on lead induced changes on the histological changes of the hippocampus and cerebellum. The result of Morris water maze and beam walking test showed that the rats in lead acetate treated Group experienced an increase in the meantime taken to complete task which when compared with the rats in Group one (Control) was found to be statistically significant (P˂0.01), while the rats in the lead and aqueous garlic extract or Succimer (DMSA) treated Groups experienced a marked decrease in the meantime taken to complete task which when compared with the rats in Group one (Control) was found to be non-significant (P˂0.05). The result of oxidative stress analyses of lead treated Groups rats showed decrease of superoxide dismutase, catalase and glutathione activities and increased level of malondialdehyde while there was increased superoxide dismutase, catalase and glutathione activities and decreased malondialdehyde levels in the aqueous garlic extract Groups. On the basis of the present study, it can be concluded that the promising potentials of aqueous garlic extracton lead induced toxicity were considered and it was found to be a good candidate for the therapeutic intervention of lead poisoning.
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TABLE OF CONTENTS
Tittle Page …………………………………………………………………………….……………………………………….I
Declaration …………………………………………………………………………………….…………………………….II
Certification ………………………………………………………………………………………………………………..III
Dedication ……………………………………………………………………………………….……………….…………IV
Acknowledgements ………………………………………………………………………………………………………V
Table of Contents ………………………………………………………………………………………………………..VI
List of Figures …………………………………………………………………………………….………………………XI
List of Tables …………………………………………………………………………………….………………………XII
List of Plates …………………………………………………………………………………….………………………XIII
Abstract ……………………………………………………………………………………………….………………….XVII
CHAPTER ONE
1.0 Introduction……………………………………………………………………………….1
1.1 Significant of the Study ……………………………………………………………………………………..3
1.2 Aim and objectives ………………………………………………………………………….………………..3
1.2.1 Aim of the Study ………………………………………………………………………………………………3
1.2.2 Objectives of the Study…………………………………………………………………….……………….3
1.3 Scope of the Study ……………………………………………………………………………..……………..4
1.4 Study hypothesis ……………………………….…………………………………………….……………….4
CHAPTER TWO
2.0 Literature review …………………………………………………..………………5
2.1 Background of the Lead Poisoning …………………………………………………………………….5
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2.2 Absorption of lead and its Distribution …………………………..…………………………………6
2.3 Toxic effects and Mechanism of Lead Toxicity ………………………………………………..8
2.4.1 Oxidative stress ………………………………………….…………………………………………………..11
2.4.2 Reactive oxygen species and oxidative stress ………………………………………………..…14
2.4.2.1 Superoxide anion.………………………………………………………………………….…………………14
2.4.2.2 Hydrogen peroxide ………………………………………………………………………….………………15
2.4.2.3 Hydroxyl radical ………………………………………………………….…………………………………..16
2.4.2.4 Nitric oxide ……………………………………………………………………………………..….…………..16
2.4.3 Ionic mechanism of lead toxicity ……………………………………………………………..………17
2.5 Natural Antioxidant ………………………………………………………………………………….……..18
2.6 Role of antioxidant in lead induced oxidative stress …………………………………………19
2.6.1 Superoxide dismutase …………………………………………………….………………………………..19
2.6.2 Glutathione Peroxidases …………………………………………………………………………….…….20
2.6.3 Catalase …………………………………………………………………………………………………………..21
2.6.4 Vitamins ………………………………………………………………………………………………………..21
2.6.4.1 Ascorbic acid (Vitamins C) ……………………………………………………………………………..22
2.6.4.2 Vitamin E (α-tocopherol) …………………………………………………………………………….22
2.6.5 Garlic (Allium Sativum) ……………………………………………………………………….…………23
2.6.5.1 Origin and history ……………………………………………………………………………………..….…23
2.6.5.2 Chemical composition of garlic ………………………………………………………………..……..26
2.7 Nervous System ………………………………………………………………………………….……………26
2.7.1 Cerebellum ………………………………………………………………………………………………………27
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2.7.1.1 Morphology ……………………………………………………………………………………….……………27
2.7.1.2 Cellular layer of the cerebellum ………………………………………………..…………………….28
2.7.1.3 Function of the cerebellum ………………………………………….…………………………………..30
2.7.2 Hippocampus ……………………………………………………………………………………………..……30
2.7.2.1 Morphology and hippocampal formation ……………………………………..……….…………30
2.7.2.2 The cells and layers of hippocampus ……………………………………………………………….32
2.7.2.3 The dentate gyrus……………………..……………………………………………………………..………33
2.7.2.4 Subiculum……………………………………………………………………………………………………….33
2.7.2.5 Function of the hippocampus …………………………………………………………………..………34
CHAPTER THREE
3.0 Materials and Method …………………………………………………………….35
3.1 Animals ………………………………………………………………………………………….……………….35
3.2 Lead Acetate and its Preparation ……………………………………………………………………..38
3.3 Source of Garlic and Preparation of the Extract ……………………………………………….38
3.4 Neurobehavioral Study …………………………………………………………………………………….39
3.4.1 Assessment of spatial memory ….…………………………………………………………..…………39
3.4.2 Beam walking assay …………………….…………………………………………………………….……41
2.6 Biochemical Analysis ………………………………………………………………………………………43
2.6.1 Measurement of lipid peroxidation level ………………………………………….………………43
2.6.2 Measurement of antioxidant enzymes activities ……………………………………………….43
3.5 Animals sacrifice and tissues processing ……………..……………………………………..……43
3.5.1 Sacrifice ………………………………………………………………………………………….………………43
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3.5.2 Tissues processing ……………………………………………………………………………………………44
3.5.3 Routine paraffin section …………….…………………………………………….………………………44
3.5.4 Staining ……………………………………………………………………………………………..…………….44
3.5.4.1 Haematoxylic and eosin staining …………………………………………..……………………….. 44
3.5.4.2 Cresyl violet staining ……………………………………………………………………………………….45
2.7 Data analysis …………………………………………………………………………………….……………..45
CHAPTER FOUR
4.0 Results ……………………………………………………………………………46
4.1 Body weight changes of the animals ………………………………………………………………..46
4.2 Effect of lead acetate on the neurobehavioral Changes ……………………….……………48
4.2.1 Effect of lead acetate on spatial learning and memory using morris water maze..48
4.2.2 Effect of lead acetate on motor coordination using beam walking assay……………50
4.3 Effect of lead acetate on oxidative stress biomarkers…………………………………………52
4.4 Effect of lead acetate on histological changes………………………………….……………… 54
CHAPTER FIVE
5.0 Discussion ……………………………………………………………………….92
5.1 Effect of Lead Acetate on Body Weight………………………..………….……………..………92
5.2 Effect of Lead Acetate on the Neurobehavioral Changes …………………………….……93
5.3 Effect of Lead Acetate on Oxidative Stress Biomarker …………………………………….94
5.4 Histological studies ………………………………………………………………………………………..96
CHAPTER SIX
6.0 Conclusion, and Recommendations …………………………..……………………………….……98
6.2 Conclusion ……………………………………………………………………………………………………….98
6.3 Recommendations ……………………………………………………………………………………………99
References ………………………………………………………………………………………………………………100
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LIST OF FIGURES
Figure 2.1 Garlic (Allium Sativum) ……………………………………………………………….…………….25
Figure 2.2 Cellular layers of cerebellum ………………………………………………………………………29
Figure 2.3 cells and layers of the hippocampus………….………………………………………………. 31
CHAPTER ONE
1.0 INTRODUCTION
1.1 Background of the study
Exposure of the body organs and systems to heavy metals including lead has been found to cause adverse effects which affect their functionality (Owolabiet al.,2012). Although lead is a useful metal in life and is used in modern industries and agriculture, it is one of the most toxic heavy metals in the body and its poisoning is known as an important public health hazard (Patrick, 2006). General researches have demonstrated that lead can cause neurological, haematological, gastrointestinal, reproductive, circulatory and immunological disorders (Al Saleh et al., 2009). Despite the strong evidences indicating the association between lead exposure and behavioral and cognitive impairments, but the mechanism by which lead causes these impairments remain poorly understood (Alirezaet al., 2013). Lead can enter the body mainly via eating, drinking or inhalation and transported to many tissues such as kidney, liver, bones and brain. As estimated by the World Health Organisation (WHO), the total lead intake from food by an adult is in the range of 26-28μg/dl/day in various Countries (Al-Saleh et al., 2009).
Lead passes through the blood brain barrier (BBB) rapidly and causes neurotoxicity via oxidative stress molecular mechanism (El Sokkaryet al., 2003). The damage inflicted is usually a consequence of the ability of the lead to induce free radical generation within the cells. The elevated production of oxygen and nitrogen based radicals and related non radical products leads to the oxidation of essential macromolecules including lipids, proteins and DNA (Reiter et al.,2010). The resultant damage referred to as
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oxidativestress and when the molecular damage is sufficiently severe, it can cause apoptosis or necrosis of neurons and glia. The brain and spinal cord are highly metabolically active organs which, even at rest, utilize an estimated 20% of the total oxygen taken by the lungs. This percentage increases substantially when the brain is active (Shuklaet al., 2011). Depriving the brain of its rich oxygen supply even for short intervals often has severe and irreversible detrimental morphological and physiological consequences for both neurons and glia (Shuklaet al., 2011). This high utilization of oxygen, however, comes at a heavy biological price. As important as oxygen is for the survival of neurons and glia, it also indirectly contributes to their destruction and death over time. The reason for this is that a small percentage of oxygen that enters the cells is metabolized to derivatives that gradually erode and destroy essential molecules(Reiteret al., 2010). These destructive derivatives of oxygen are often referred to as free radicals or reactive oxygen species (ROS). Some of the notably destructive oxygen metabolites include the superoxide anion radical, hydrogen peroxide and hydroxyl radical; this latter agent is especially toxic to cells and indiscriminately harms or destroys any molecules in the vicinity of where the radical is generated (Volkaet al.,2005).
Garlic (alliumsativum) as a phytomedicine is a specie in the onion genus, allium. Garlic is one of the most researched plants, with long history of medicinal use (Omotosoet al., 2009). Garlic containssulphur, phosphorus, potassium and zinc ions, moderate amounts of selenium, vitamin A, vitamin C and smaller amounts of calcium, magnesium, sodium, iron, B complex vitamins and allicin, a compound to trap free radicals (El Demerdashet al., 2005). Recently, it was found that the sulphur containing compounds of garlic have
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anti mutagenic and anti-carcinogenic effects. Aqueous garlic extract exerts its antioxidant action by scavenging enzymes superoxidase dismutase, catalase and glutathione peroxidise and inhibits lipid peroxidation (Ataeiet al., 2014).
1.2 Significance of the Study
Lead poisoning can cause irreversible genetic and reproductive toxicity, haematological effects, neurological damage and cardiovascular effects. Despite many efforts to minimize lead poisoning, it continues to be a major health concern in many developing and developed countries (Shaiket al.,2014) and as such this study seeks to investigate the effects of aqueous garlic extract on lead poisoning particularly on the central nervous system.
1.3 Aim and Objectives of the study
1.3.1 Aim of the study
This study is aimed at determining the effects of aqueous garlic extract on lead induced damages on the hippocampus and cerebellum of adult Wistar rats.
1.3.2 Objectives
This study seeks to:
i. Evaluate the neurobehavioral effects of aqueous Allium sativumextract on lead induced changes on the hippocampus and cerebellum of adult Wistar rats using Morris water maze and beam walking methods respectively.
ii. Biochemically evaluate the effect of aqueous allium sativum extract on lead induced changes on hippocampus and cerebellum of adult Wistar rats using
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oxidative stress markers namely; catalase (CAT), superoxide dismutase (SOD), glutathione (GSH) and lipid peroxidation (LPO).
iii. Evaluate the possible histological and histochemical effects of aqueous Allium sativum extract on lead induced changes on the hippocampus and cerebellum of adult Wistar rats using routine Hand E staining and cresylviolet methods.
1.4 Scope of the Study
This study was limited to the general histochemical, histomorphological, behavioural and cognitive changes in the hippocampus and cerebellum of the adult Wistar rats as a result of exposure to lead acetate.
1.5Study Hypothesis
Aqueous garlic extract has protective effects on lead induced changes on the antioxidants system, spatial learning and memory and cytoarchitectureof the hippocampus and cerebellum of the Wistar rats.
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