Assessment of the Effect of Post-Natal Lead Exposure on the Hippocampus of Developing Wistar Rats

Assessment of the Effect of Post-Natal Lead Exposure on the Hippocampus of Developing Wistar Rats.

ABSTRACT

Lead (Pb) is a highly toxic heavy metals found in every facet of environmental and biological systems. Evidence suggests that most of lead’s effects on a child’s central nervous system are irreversible. In this study we examined the effect of post-natal lead exposure on the hippocampus of developing Wistar rat.

Nine Pregnant rats were randomly distributed into three experimental groups of three rats each, consisting of a control group (1) and experimental groups (2 and 3).

After parturition, dams in Group 2 were administered 60mg/kg body weight (bwt) of lead acetate and Group 3 were administered 90mg/kg bwt of lead acetate.

The pups of the dams in the experimental groups (2 and 3) were exposed to lead acetate via lactation from dams‘ that were administered lead (Pb) acetate via oral gavage from post-natal day (PND) 1 – PND 21.The control group (1) was given distilled water (2ml/kg bwt) throughout the experimental period.

On PND 22, the pups were weighed and sacrificed after anaesthesia with 75mg/kg of ketamine. The brain tissue was excised and separated into halves along its longitudinal fissure.

The result from the present study showed a signicant decrease (p<0.05) in body weight,significant (p<0.05) increase in brain somatic index and an insignificant (p>0.05)decrease in brain weight of Wistar rat‘s pups exposed to lead acetate via lactation from PND 1-21 when compared with the control.

It also revealedsignificant (p<0.05)increase in accumulation of lead deposit in the brain of the Wistar rat pups exposed to increasing doses of lead acetate.

Lead exposure also induced oxidative stress in Wistar rat pups by causing an increase in free radicals production and a significant (p<0.05) decrease in the antioxidant enzymes.

TABLE OF CONTENTS

Title page……………….. i

Declaration……………… iv

Certification………………. v

Dedication…………….. vi

Acknowledgments………… vii

Abstract……………. ix

Table of Content…………… xi

List of Figures……………. xvi

List of Tables…………… xvii

List of Plates……………… xviii

List of Appendices…………… xx

List of Abbreviations………. xxi

CHAPTER ONE

• INTRODUCTION………………………………………………………………………………………….. 1
• Background of Study……………………………………………………………………………………… 1
• Statement of Research Problem……………………………………………………………………… 5
• Justification of Study……………………………………………………………………………………… 6
• Aim of the Study……………………………………………………………………………………………. 7
• Objectives of the Study…………………………………………………………………………………… 7
• Hypothesis…………………………………………………………………………………………………….. 7

CHAPTER TWO

• LITERATURE REVIEW…………………………………………………………………………………….. 8
• Heavy Metals……………………………………………………………………………………………………….. 8
• Lead………………………………………………………………………………………………………………….. 11
  • Physical and chemical properties………………………………………………………………………… 11
  • History of lead………………………………………………………………………………………………….. 12
  • Routes of exposure and absorption of Lead………………………………………………………….. 14
  • Mechanism of toxicity………………………………………………………………………………………. 17
  • Distribution of lead after absorption……………………………………………………………………. 22
  • Excretion of lead………………………………………………………………………………………………. 24
  • Effect of lead on body systems…………………………………………………………………………… 24
  • Prevention and treatment of lead poisoning………………………………………………………….. 30
• Hippocampus…………………………………………………………………………………………………….. 32
  • History of hippocampus nomenclature…………………………………………………………………. 35
  • Development of the hippocampus……………………………………………………………………….. 35
  • Structure of the hippocampus………………………………………………………………………………. 37
  • Hippocampal formation…………………………………………………………………………………….. 38
  • Information flow in the hippocampal formation……………………………………………………. 44
  • Functional implications of anatomical differences between hippocampal sub regions.. 48
• Microglia…………………………………………………………………………………………………………… 50
  • Development of the microglia…………………………………………………………………………….. 51

Physiological states of microglia 55

• Role of microglia in the brain…………………………………………………………………………….. 61
• Role of microglia in immune regulation………………………………………………………………. 65
• Resident microglia versus infiltrating, blood-borne monocyte………………………………… 65

CHAPTER THREE

• MATERIALS AND METHODS………………………………………………………………………… 67
• Materials…………………………………………………………………………………………………………… 67
  • Ethical approval……………………………………………………………………………………………….. 67
  • Experimental animals………………………………………………………………………………………… 67
  • Chemical procurement………………………………………………………………………………………. 68
  • Other materials…………………………………………………………………………………………………. 68
• Methods…………………………………………………………………………………………………………….. 68
  • Determination of oestrous cycle in Wistar rats……………………………………………………… 68
  • Mating of rats and confirmation of pregnancy……………………………………………………… 69
  • Experimental design………………………………………………………………………………………….. 69
  • Drug administration………………………………………………………………………………………….. 71
  • Body weight assessment……………………………………………………………………………………. 71
  • Euthanasia………………………………………………………………………………………………………… 72
  • Brain weight and Brain somatic index estimation…………………………………………………. 72
  • Quantification of lead deposit in the brain……………………………………………………………. 72
  • Oxidative stress markers……………………………………………………………………………………. 74
• Histology and Histochemistry……………………………………………………………………………… 77
  • Tomato lectin staining technique………………………………………………………………………… 80

• Photomicrography…………………………………………………………………………………………….. 81

• Stereology………………………………………………………………………………………………………….. 82
  • Volume estimation……………………………………………………………………………………………. 82
  • Number of microglia cells………………………………………………………………………………….. 83
• Data Analysis…………………………………………………………………………………………………….. 84

CHAPTER FOUR

• RESULTS…………………………………………………………………………………………………………. 85
• Body Weight, Brain Weight and Brain Somatic Index………………………………………… 85
  • Body weight of Wistar rat pups from dams exposed lead acetate……………………………. 85
  • Brain weight of Wistar rat pups from dams exposed to lead acetate………………………… 86
  • Brain somatic index of Wistar rat pups from dams exposed to lead acetate……………… 86
• Lead Concentration in brain tissues of pups from dams exposed to lead acetate…… 87
• Oxidative Stress Biomarkers………………………………………………………………………………. 90
  • Mean superoxide dismutase levels in Wistar rat pups from dams exposed to lead acetate postnatally……………………………………………………………………………………………………………….. 90
  • Mean reduced glutathione levels in Wistar rat pups from dams exposed to lead acetate postnatally……………………………………………………………………………………………………………….. 90
  • Mean malondialdehyde levels of Wistar rat pups from lead acetate exposed dams……. 90
• Histological Studies……………………………………………………………………………………………. 94
  • Haematoxylin and eosin (H and E) stain……………………………………………………………… 94
  • Cresyl violet staining…………………………………………………………………………………………. 98
  • Tomato lectin stain for microglia………………………………………………………………………. 102

• Volume of Hippocampus and Number of Activated Microglia Cells…………………… 106
  • Volume of hippocampus………………………………………………………………………………….. 106
  • Number of Activated Microglia Cells………………………………………………………………… 106

CHAPTER FIVE

• DISCUSSION………………………………………………………………………………………………….. 109
• Body Weight Assessment………………………………………………………………………………….. 109
• Brain Weight and Brain Somatic Index…………………………………………………………….. 110
• Lead Concentration…………………………………………………………………………………………. 112
• Oxidative Stress Markers…………………………………………………………………………………. 113
• Histological Studies………………………………………………………………………………………….. 115

CHAPTER SIX

• SUMMARY, CONCLUSION AND RECOMMENDATIONS……………………… 122
• Summary…………………………………………………………………………………………………………. 122
• Conclusion……………………………………………………………………………………………………….. 123
• Contribution to Knowledge………………………………………………………………………………. 124
• Recommendations……………………………………………………………………………………………. 125

REFERENCES…………….. 126

APPENDICES…………………… 150

INTRODUCTION

1.1 Background of Study

Human child development is regulated by the interactions of both endogenous and exogenous factors. One of the exogenous factors affecting early development of neurobehavior in children is the exposure to heavy metals (Wieslawet al., 2008).

Human and animal populations are in constant interaction with their environment and as such are exposed to a range of chemicals and heavy metals such as lead, mercury, cadmium. Interestingly, these interactions with the environment occur through food, air and water (Burger et al., 2011).

Heavy metal poisoning has become an increasingly major health problem by nature of their environmental persistence, especially since the industrial revolution(Abbaset al., 2002).

Almost all organ systems are involved in heavy metal toxicity; however, the most susceptible systems include the nervous, renal, haematopoietic, and cardiovascular system (McDowell, 2003).

Lead (Pb) as one of the highly toxic heavy metals has been detected in every facet of environmental and biological systems (Payne, 2008; Bilandzˇic et al., 2009; Clark et al., 2009), particularly in  industrialized   cities.

Lead is one of the oldest harmful agents known to mankind and has been reported to be toxic in both human and experimental animals (Goswami and Bhattacharya, 2000;Ahmad et al., 2003; Loumbourdis et al., 2003; Reza et al., 2008) since historic times of the Greeks, Romans, Arabs and even the Egyptians.

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