TABLE OF CONTENTS
Cover page …………………………………………………………………………………………………..i Fly leaf ………………………………………………………………………………………………………..ii Title page …………………………………………………………………………………………………….iii Declaration …………………………………………………………………………………………………..iv Certification …………………………………………………………………………………………………v Dedication ……………………………………………………………………………………………………vi Acknowledgements ……………………………………………………………………………………….vii Table of contents …………………………………………………………………………………………..viii List of figures ……………………………………………………………………………………………….xiii List of tables …………………………………………………………………………………………………xiv List of tables …………………………………………………………………………………………………xv Abstract ……………………………………………………………………………………………………….xviii CHAPTER ONE 1.0 INTRODUCTION………………………………………………………………………………….1 1.1 Background …………………………………………………………………………………………..1 1.2 Statement of Research Problem ……………………………………………………………..3 1.3 Justification of the Study ………………………………………………………………………..3 1.4 Significance of the Study ………………………………………………………………………..4 1.5 Scope of the Study ………………………………………………………………………………….4 1.6 Aim and Objectives of the Study ……………………………………………………………4 1.6.1 Aim of the Study …………………………………………………………………………………..4 1.6.2 Objectives of the Study ………………………………………………………………………….4 1.7 Study Hypothesis ……………………………………………………………………………………5
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CHAPTER TWO 2.0 LITERATURE REVIEW ………………………………………………………………………6 2.1 Forms in which Lead Exist……………………………………………………………………..6 2.2 Environmental Lead Exposure ……………………………………………………………….6 2.2.1 Sources of lead in the environment ………………………………………………………….6 2.2.2 Exposure to Lead Compounds ………………………………………………………………..8 2.3 Toxicokinetics of Lead Acetate ……………………………………………………………….9 2.3.1 Absorption of lead…………………………………………………………………………………9 3.3.2 Distribution of lead ……………………………………………………………………………….10 2.3.3 Excretion of lead …………………………………………………………………………………..11 2.4 Effects of Lead ……………………………………………………………………………………….11 2.4.1 Effects of lead on central nervous system …………………………………………………12 2.4.2 Effects of lead on genetic make up ………………………………………………………….13 2.4.3 Effects of lead on kidney ……………………………………………………………………….13 2.4.4 Effects of lead on reproductive system …………………………………………………….14 2.4.5 Toxicological effects of lead on blood ……………………………………………………..15 2.5 Psidium guajava ……………………………………………………………………………………..17 2.5.1 General description of Psidium guajava plants …………………………………………17 2.5.2 Scientific classification ………………………………………………………………………….20 2.5.3 Phytochemical constituents of Psidium guajava ……………………………………….20 2.5.4 Medicinal uses of Psidium guajava …………………………………………………………23 2.6 Standard Drug: Succimer (meso-2,3-dimercaptosuccinic acid) ………………..25 2.7 The Central Nervous System ………………………………………………………………….26 2.7.1 Hippocampus ……………………………………………………………………………………….26 2.7.2 Cerebellum …………………………………………………………………………………………..33 2.7.3 The cerebrum ……………………………………………………………………………………….38
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2.8 Memory …………………………………………………………………………………………………45 2.8.1 Types of memory ………………………………………………………………………………….46 CHAPTER THREE 3.0 MATERIALS AND METHOD ………………………………………………………………48 3.1 Materials ……………………………………………………………………………………………….48 3.1.1 Experimental Rats …………………………………………………………………………………48 3.1.2 Obtaining and identification of Psidium guajava leaves …………………………….48 3.1.3 Obtaining of lead acetate ……………………………………………………………………….48 3.2 Methods …………………………………………………………………………………………………49 3.2.1 Preparation of extract …………………………………………………………………………….49 3.3 Experimental Procedure ………………………………………………………………………..49 3.4 Experimental Design ………………………………………………………………………………50 3.5 Neurobehavioural Studies ………………………………………………………………………52 3.5.1 Morris water maze test …………………………………………………………………………..52 3.5.2 Balance Beam (Beam walking) test …………………………………………………………54 3.6 Wistar rats Sacrifice ………………………………………………………………………………56 3.7 Tissue Processing …………………………………………………………………………………..56 3.7.1 Haematoxylin and Eosin (H and E) staining method………………………………….56 3.7.2 Special stain using Cresyl Echt Violet Method …………………………………………57 3.8 Microscopic Cell count …………………………………………………………………………..57 3.9 Determination of Haematological Parameters …………………………………………60 3.10 Determination of Oxidative Stress Parameters ……………………………………..60 3.10.1 Determination of catalase activity …………………………………………………………60 3.10.2 Determination of superoxide dismutase activity ………………………………………61 3.10.3 Assessment of lipid peroxidation …………………………………………………………..61 3.10.4 Assay of reduced Glutathione concentration …………………………………………..62
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3.11 Data Analysis ……………………………………………………………………………………….62 CHAPTER FOUR 4.0 RESULTS ……………………………………………………………………………………………..63 4.1 General Observation ………………………………………………………………………………63 4.1.1 Physical changes in the Rats …………………………………………………………………..63 4.1.2 Movement of Rats on Balance Beam test …………………………………………………63 4.1.3 Spatial Learning and Memory of the Rats ………………………………………………..65 4.2 Analysis of Neurobehavioural Studies …………………………………………………….67 4.2.1 Motor coordination in the Rats ……………………………………………………………….67 4.2.2 Test for spatial learning and memory using Morris Water Maze test ……………69 4.3 Analysis of Oxidative Stress markers ……………………………………………………..71 4.4 Haematological Analysis ………………………………………………………………………..73 4.5 Histological Observation ………………………………………………………………………..75 4.5.1 Observation in number of cells in Hippocampus, Cerebellum and Cerebrum .75 4.5.2 Histopathology of cerebellum of the rats ………………………………………………….77 4.5.3 Histopathology of cerebrum …………………………………………………………………..92 4.5.4 Histopathology of the hippocampus ………………………………………………………..107 CHAPTER FIVE 5.0 DISCUSSION ………………………………………………………………………………………..122 5.1 Histopathology……………………………………………………………………………………….122 5.2 Neurobehavioural Studies ………………………………………………………………………124 5.3 Oxidative Stress Markers ……………………………………………………………………….126 5.4 Haematological Indices …………………………………………………………………………..127 CHAPTER SIX 6.0 CONCLUSION AND RECOMMENDATIONS ………………………………………130 6.1 Conclusions ……………………………………………………………………………………………130
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6.2 Recommendations ………………………………………………………………………………….130 REFERENCES ……………………………………………………………………………………………132
CHAPTER ONE
INTRODUCTION 1.1 BACKGROUND Lead poisoning has been a recurrent problem in the society for many centuries, and its deleterious effects on central nervous system (CNS) are known as lead encephalopathy or lead neuropathy (Flora et al., 2006). Neurobehavioural impairments, hyperactivity, alterations in brain structure, and learning and cognitive deficits in children have been observed even with low blood lead levels of 10–20μg/dl (Needleman, 1993). The metal (lead) is primarily found in leaded gasoline, (Tong et al., 2000). Automobile emissions have been an important source of lead exposure to urban residents, particularly in areas with congested traffic. The main source of adult human exposure is through contaminated food, which is believed to account for over 60% of blood levels; while air inhalation accounts for approximately 30% and water of 10% (John et al., 1991). Lead poisoning is also known as plumbism, colica Pictonum, saturnism, Devon colic, or painter‟s colic, which is a medical condition caused by increased levels of the heavy metal lead in the body (Rossi, 2008). Lead interferes with a variety of body processes and is toxic to many organs and tissues including the heart, bones, intestine, kidneys, reproductive and nervous systems. Interference with the development of the nervous system could lead to permanent learning and behaviour disorders. Symptoms of lead toxicity include abdominal pain, confusion, headache, anaemia, irritability, and in severe cases seizures, coma, and death (Barbosa et al., 2005).
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It has been reported that lead exposure produces free radicals, such as, active oxygen species (O2•-, •OH or lipid peroxyl radical, LOO•), cause oxidative damage to lipids, proteins, and nucleic acids and may lead to oxidative stress, biological carcinogenesis, mutagenesis, aging, atherosclerosis, neuro-degenerative diseases and stress-induced depression (Sapolsky, 2000; Bilici et al., 2001). Oxidative stress may adversely affect neuronal cells in which they may die when oxygen supply is reduced or eliminated (Ibegbu et al., 2012). Oxidative stress has been implicated in nerve cell death that occurs in a variety of neurodegenerative disorders like dementias, multiple sclerosis, Alzheimer‟s disease and Parkinson‟s disease (Maher, 2001; Prentice et al., 2011). Oxidative stress leads to metabolic cellular processes in which oxidative species such as super oxide radical anions, hydrogen peroxide and lipid peroxides are generated intracellularly (Chen and Buck, 2000; Lee et al., 2012). These reactive species, if not eliminated, may damage DNA, proteins or membrane lipids and cause oxidative cell death. Endogenous antioxidative enzymes as well as antioxidants are required for cells to survive (Semenza, 2005), while exogenous antioxidants have been shown to effectively prevent oxidative cell death in cultured cells (Du et al., 2012).
It is generally known that the consumption of a variety of local herbs and vegetables by man contributes significantly to the improvement of human health, in terms of prevention, and or treatment of diseases because, plants have long served as a useful and natural source of therapeutic agents (Chevellier, 1996). About 80% of the World population depends on herbal based alternative systems of medicine. The activities of these curative plants are evaluated by their chemical components. Indian Ayurveda utilizes about 2000 plants to cure different ailments (Daniel, 2006). Some of these curative plants have natural antioxidants, which neutralize free radicals, are receiving more attention from
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nutritionists and medical researchers for their potential effects in the prevention of chronic and degenerative changes, such as cancer, cardiovascular disease and aging (Young and Woodside, 2001). Psidium guajava leaf is an important part of guava tree which is useful in treating many health problems. Guava leaves have anti-oxidant, anti-cancer, antibacterial, and anti ulcer properties needed in treatment of many diseases (Vibha et al., 2012). 1.2 STATEMENT OF RESEARCH PROBLEM Lead remains a considerable occupational and public health problem, which is known to cause a number of adverse effects like brain damage, mental impairment and severe behavioural anomalies, neuromuscular weakness, decreased hearing, impaired cognitive functions in experimental animals and coma (Liuji et al., 2002; Verina et al., 2007; Flora et al., 2007). The toxic effects of lead are treated by chelation therapy which also depletes the body store of essential cations (Ruff et al., 1996) and as such there is need to look for alternative solution to lead poisoning. There are reports on lead induced toxicity in experimental studies but there are scanty works on medicinal plants as treatment options for lead induced brain damage in our environment. 1.3 JUSTIFICATION OF THE STUDY
The recent increase in lead exposure in our environment, especially the cases of Zamfara (Elaine, 2010) and Niger States (Lucia, 2015) has placed a heavy burden on health risk in both adults and children which may result to oxidative stress and severe behavioural anomalies. This incessant exposure to lead and related products require investigation on locally available modes of treatment. Psidium guajava has been reported to elicit strong
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antioxidative activity of which the present study intends to evaluate, following lead exposure. 1.4 SIGNIFICANCE OF THE STUDY The present study would be of importance in the identification and evaluation of available natural remedy that may possibly be used as alternatives to the presently used chelation therapy to treat lead poisoning, which are not always free from adverse effects. Therefore, from the result of the present study, Psidium guajava leaves could be of help in the management of lead poisoning. 1.5 SCOPE OF THE STUDY This study was be limited to the study of the general histomorphological and degenerative changes in the cerebellum, cerebrum and hippocampus and the study of spatial learning and memory; motor coordination; oxidative stress markers; and haematological analysis. 1.6 AIM AND OBJECTIVES OF THE STUDY 1.6.1 Aim of the study The aim of the study was to evaluate the effects of aqueous extract of Psidium guajava leaves on lead induced changes in brain tissues mainly cerebellum, cerebrum and hippocampus, behaviour, biochemical and haematological parameters of adult Wistar rats. 1.6.2 Objectives of the study The objectives of the study were to:
i. Evaluate the effects of Psidium guajava aqueous leaves extract on lead induced changes in spatial learning, memory and motor coordination in adult Wistar rats.
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ii. Study the effects of the extract on lead induced alterations in haematological and biochemical parameters of adult Wistar rats.
iii. Determine the effects of the extract on lead induced changes in brain tissues mainly cerebellum, cerebrum and hippocampus of adult Wistar rats.
1.7 STUDY HYPOTHESIS Aqueous extract of Psidium guajava leaves has ameliorative effects on lead acetate induced changes on the brain tissues, behaviour, haematological and biochemical parameters of adult Wistar rats.
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