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. One half of the brain was homogenized for analysis of lead concentration via Atomic Absorption Spectrophotometry and oxidative stress markers such as Malondialdehyde (MDA), Superoxide Dismutase (SOD) and Reduced Glutathione (GSH). The hippocampus was isolated from the other half of the brain and were fixed in Bouin‘s fluid and paraformaldehyde fixatives for histological studies. The hippocampus was then processed routinely and stained using H and E for general cytoarchitecture, Cresyl violet stain for Nissl substance and Tomato lectin stain for microglia cells. The volume of the hippocampus and the number of activated microglia cells was then determined using stereology. 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
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(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. Neurotoxicity of lead was also evident as it caused weak staining for Nissl substanceand distortion in cytoarchitecture of the CA3 region of the hippocampus with presence of necrotic pyramidal cell, cell loss and pyknotic nucleus. In comparison with the controls, there was an insignificant (p>0.05) increase in the number of activated microglia cells in Wistar rat pups exposed to lead acetate via lactation from dams from PND 1-21. In conclusion, it can be inferred that exposure of Wistar rat pups to increasing doses of lead acetate from post-natal day 1-21 did not significantly trigger microglia activation but caused distortion in the cytoarchitecture of the CA3 region of the hippocampus of developing Wistar rats.
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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
1.0INTRODUCTION ……………………………………………………………………………………………….. 1
1.1 Background of Study …………………………………………………………………………………………… 1
1.2 Statement of Research Problem …………………………………………………………………………… 5
1.3 Justification of Study …………………………………………………………………………………………… 6
1.4 Aim of the Study …………………………………………………………………………………………………. 7
1.5 Objectives of the Study ………………………………………………………………………………………… 7
1.6 Hypothesis …………………………………………………………………………………………………………… 7
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CHAPTER TWO
2.0LITERATURE REVIEW …………………………………………………………………………………….. 8
2.1 Heavy Metals ………………………………………………………………………………………………………. 8
2.2 Lead ………………………………………………………………………………………………………………….. 11
2.2.1 Physical and chemical properties ………………………………………………………………………… 11
2.2.2 History of lead …………………………………………………………………………………………………. 12
2.2.3 Routes of exposure and absorption of Lead ………………………………………………………….. 14
2.2.4 Mechanism of toxicity ………………………………………………………………………………………. 17
2.2.5 Distribution of lead after absorption ……………………………………………………………………. 22
2.2.6 Excretion of lead ………………………………………………………………………………………………. 24
2.2.7 Effect of lead on body systems …………………………………………………………………………… 24
2.2.8 Prevention and treatment of lead poisoning …………………………………………………………. 30
2.3 Hippocampus …………………………………………………………………………………………………….. 32
2.3.1 History of hippocampus nomenclature ………………………………………………………………… 35
2.3.2 Development of the hippocampus ………………………………………………………………………. 35
2.3.3Structure of the hippocampus ……………………………………………………………………………… 37
2.3.4 Hippocampal formation …………………………………………………………………………………….. 38
2.3.5 Information flow in the hippocampal formation ……………………………………………………. 44
2.3.6 Functional implications of anatomical differences between hippocampal sub regions .. 48
2.4 Microglia …………………………………………………………………………………………………………… 50
2.4.1 Development of the microglia ……………………………………………………………………………. 51
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2.4.2 Physiological states of microglia ………………………………………………………………………… 55
2.4.3 Role of microglia in the brain …………………………………………………………………………….. 61
2.4.4 Role of microglia in immune regulation ………………………………………………………………. 65
2.4.5 Resident microglia versus infiltrating, blood-borne monocyte ……………………………….. 65
CHAPTER THREE
3.0 MATERIALS AND METHODS ………………………………………………………………………… 67
3.1 Materials …………………………………………………………………………………………………………… 67
3.1.1 Ethical approval ……………………………………………………………………………………………….. 67
3.1.2 Experimental animals ……………………………………………………………………………………….. 67
3.1.3 Chemical procurement ………………………………………………………………………………………. 68
3.1.4 Other materials …………………………………………………………………………………………………. 68
3.2 Methods …………………………………………………………………………………………………………….. 68
3.2.1 Determination of oestrous cycle in Wistar rats ……………………………………………………… 68
3.2.2 Mating of rats and confirmation of pregnancy ……………………………………………………… 69
3.2.3 Experimental design …………………………………………………………………………………………. 69
3.2.4 Drug administration ………………………………………………………………………………………….. 71
3.2.5 Body weight assessment ……………………………………………………………………………………. 71
3.2.6Euthanasia ………………………………………………………………………………………………………… 72
3.2.7 Brain weight and Brain somatic index estimation …………………………………………………. 72
3.2.8 Quantification of lead deposit in the brain ……………………………………………………………. 72
3.2.9 Oxidative stress markers ……………………………………………………………………………………. 74
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3.3 Histology and Histochemistry …………………………………………………………………………….. 77
3.3.1 Tomato lectin staining technique ………………………………………………………………………… 80
3.3.2 Photomicrography …………………………………………………………………………………………….. 81
3.4 Stereology …………………………………………………………………………………………………………. 82
3.4.1 Volume estimation ……………………………………………………………………………………………. 82
3.4.2 Number of microglia cells …………………………………………………………………………………. 83
3.5 Data Analysis …………………………………………………………………………………………………….. 84
CHAPTER FOUR
4.0 RESULTS …………………………………………………………………………………………………………. 85
4.1 Body Weight, Brain Weight and Brain Somatic Index ………………………………………… 85
4.1.1 Body weight of Wistar rat pups from dams exposed lead acetate ……………………………. 85
4.1.2 Brain weight of Wistar rat pups from dams exposed to lead acetate ………………………… 86
4.1.3 Brain somatic index of Wistar rat pups from dams exposed to lead acetate ……………… 86
4.2 Lead Concentration in brain tissues of pups from dams exposed to lead acetate ….. 87
4.3 Oxidative Stress Biomarkers ……………………………………………………………………………… 90
4.3.1 Mean superoxide dismutase levels in Wistar rat pups from dams exposed to lead acetate postnatally ……………………………………………………………………………………………………………….. 90
4.3.2 Mean reduced glutathione levels in Wistar rat pups from dams exposed to lead acetate postnatally ……………………………………………………………………………………………………………….. 90
4.3.3 Mean malondialdehyde levels of Wistar rat pups from lead acetate exposed dams ……. 90
4.4 Histological Studies ……………………………………………………………………………………………. 94
4.4.1 Haematoxylin and eosin (H and E) stain ……………………………………………………………… 94
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4.4.2 Cresyl violet staining ………………………………………………………………………………………… 98
4.4.3 Tomato lectin stain for microglia ………………………………………………………………………. 102
4.5 Volume of Hippocampus and Number of Activated Microglia Cells ………………….. 106
4.5.1 Volume of hippocampus ………………………………………………………………………………….. 106
4.5.2 Number of Activated Microglia Cells ……………………………………………………………….. 106
CHAPTER FIVE
5.0 DISCUSSION ………………………………………………………………………………………………….. 109
5.1 Body Weight Assessment………………………………………………………………………………….. 109
5.2 Brain Weight and Brain Somatic Index ……………………………………………………………. 110
5.3 Lead Concentration …………………………………………………………………………………………. 112
5.4 Oxidative Stress Markers …………………………………………………………………………………. 113
5.5 Histological Studies ………………………………………………………………………………………….. 115
CHAPTER SIX
6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS …………………………… 122
6.1 Summary …………………………………………………………………………………………………………. 122
6.2 Conclusion ………………………………………………………………………………………………………. 123
6.3 Contribution to Knowledge ………………………………………………………………………………. 124
6.4 Recommendations ……………………………………………………………………………………………. 125
REFERENCES ……………………………………………………………………………………………………… 126
APPENDICES ………………………………………………………………………………………………………. 150
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CHAPTER ONE
1.0 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. Lead exists in three forms: metallic lead, inorganic lead and organic lead (Ahamed and Siddiqui, 2007).
Human activities such as mining, manufacturing and burning fossil fuels can result in lead exposure. Lead is used in production of car batteries, hair dye, farm equipment, gasoline,
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paints, ceramics, cosmetics, ammunition, water pipes, airplanes and shielding for x-ray machines (ATSDR, 2005). Despite the ban on using lead in paints or as a gasoline additive in the developed countries, human exposure to lead continues as lead does not degrade in the environment, remaining strongly absorbed by the soil. In humans, the routes of exposure include the ingestion of lead-contaminated food or drinking water containing lead leaching from older corroding pipes, inhalation in industrial settings, and dermal contact (ATSDR, 2005). Children can be exposed to peeling lead-based paint or weathered powdered paint when engaging in hobbies. Children suffering from pica (the compulsive, habitual consumption of non-food items) are particularly vulnerable (Bornschein et al., 1986). However, the magnitude of the toxic response depends on several factors, including the dose, the age of the person exposed, the life stage of a woman (childhood, lactating, menopausal), occupational exposure, duration of exposure, health and lifestyle, and nutritional status of the person exposed. Breast milk has been suggested as a significant potential source of lead exposure to nursing infants, maternal blood and bone lead levels are both important determinants of lead in breast milk (Silbergeld, 1991; Ettinger et al.. 2004). Lead is readily absorbed through the gastrointestinal tract and distributes into soft body tissues such as kidney, bone marrow, liver and brain, but accumulates in the blood and bone. It is also believed that the greater absorptive capacity of the gastrointestinal tracts of children puts them at a higher risk than adults (Ab Latif et al.,2015). The major mechanism of lead induced toxicity in most biological systems has been reported to be via oxidative stress (Flora et al., 2012). Excessive accumulation of Pb in human bodies leads to catastrophic effect on the haematopoietic, nervous, cardiovascular and renal tissues (Jacob et al., 2000; Anetor, 2002; Ekong et al., 2006; White et al., 2007; Sansar et al., 2012).
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Furthermore, increasing prevalence of various abnormalities of the female reproductive system has been associated to lead exposure. Blood lead level above 10 μg/dl may adversely affect pregnancy outcome, resulting in increased risk of gestational hypertension, reduced length of gestation, miscarriage, spontaneous abortion, and preterm delivery (Torres-Sanchez et al., 1999; Sowers et al., 2002; Vigeh et al., 2006). Indeed, no blood-lead levels appeared to be safe, because blood lead levels as low as 2.5 μg/dl in children with no observable peripheral symptoms of Pb exposure (asymptomatic) have been associated with, for example, reduced academic achievement, lower IQ, poorer memory, attention, motor dexterity and problem- solving, suggestive of altered brain development (Canfield et al., 2003; Chiodo et al., 2004; Lanphear et al., 2005). Additionally, in mouse and rodent models, early chronic exposure to Pb resulted in abnormal motor, decreased memory and exploratory behavior (Leasure et al., 2008; Azzaoui et al., 2009; Kasten-Jolly et al., 2012). In vivo and in vitro studies have suggested that neurotoxicity may be promoted by disruption of neuroimmune system function via reduced numbers of microglia, difference in microglial cell body volume by lead exposure (Kraft and Harry, 2011; Sobin et al., 2013). The neuroimmune system is comprised of microglial cells. Microglia cells are highly motile resident macrophages of the central nervous system with important roles in development, homeostasis, disease, and injury (Aguzzi et al., 2013). They account for 5–20% of the total glial cell population within the central nervous system (CNS) parenchyma (Perry, 1998) and protect it through constant surveillance and scavenging of debris and foreign substances from the local environment and interacts functionally with astrocytes (Schwartz et al., 2006).
Microglia keenly surveys the surrounding parenchyma via dynamic movement of their processes while the soma remains static (Nimmerjahn et al., 2005) while also facilitating neuronal activity (Aloisi, 2001). They are the only cells in the CNS that originate outside the
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brain. Although the origin of microglia and its cell lineage still remains highly contentious and debatable. It has been suggested that the haematopoietic stem cells in developing and adult brain transform into microglia (Alliot et al., 1991; Hickey et al., 1992) and the bone marrow-derived progenitors or monocytes are recruited for supplementing the microglial population. In contrast, it has also been reported that microglia also existed before brain vascularization and production of monocytes in hematopoietic tissues indicating thereby that all microglia are not haematopoietic in origin (Shepard and Zon, 2000; Takahashi 2001). Saijo and Glass (2011) reported that they arise early during development from neuroectodermal matrix cells and yolk sac cells that kernel the brain of the embryo and appear to persist throughout life. During development, activated microglia support and protect neurite development, guide synaptic pruning, and sculpt neural circuits (Paolicelli et al., 2011; Schafer et al., 2012). The neuroprotective role of microglia during early development is suggested by the acute sensitivity of these cells to CNS changes, as indicated by extremely rapid activation and proliferation response times (Dissing-Olesen et al., 2007). Microglia cells are activated by various agents that trigger a sequence of unique morphologic changes, including cell body enlargement. In response to these various agents, microglia activates a number of surface proteins, (CD40, MHC II), cytokines (IL-18, IL-1β, TNF-α, IL-6) and neurotoxic mediators, such as nitric oxide (NO), prostaglandin (PG) and superoxide anions (Kierdorf and Prinz, 2013). These factors initiate both repair and cytotoxic processes via interactions with other brain cells, e.g., astrocytes and neurons.
In many models of neurodegeneration and neurotoxicity, early events of synaptic degeneration and neuronal loss are accompanied by an inflammatory response including activation of microglia, perivascular monocytes, and recruitment of leukocytes and systemic macrophages (Harry and Kraft, 2012). In culture, microglia have been shown to be capable of
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releasing several potentially cytotoxic substances, such as reactive oxygen intermediates, nitric oxide, proteases, arachidonic acid derivatives, excitatory amino acids, and cytokines; however, they also produce various neurotrophic factors. Microglia cells induce long-term changes within the brain because they have the capacity to become and remain sensitized (Town et al., 2005; Branchi et al., 2014). The sensitized state of ‗‗primed‘‘ microglia may be prolonged following the initial activation by stimuli, such as injury, heavy metals or stress and therefore affecting neuronal function. Findings from in vivo and in vitro studies show that Pb exposure alters cellular functions in ways that might be expected to promote chronic microglial activation. Extensive studies have revealed that Pb exposure at low doses is extremely dangerous and can cause learning and memory impairments which is sub served by the hippocampus (Sobin et al., 2013). Long term potentiation (LTP) of the hippocampal excitatory synaptic transmission is thought to be a pattern of manifestation for synaptic plasticity (Bliss and Collingridge, 1993) which is believed to be the underlying mechanisms for hippocampus-dependent learning and memory (Morris and Frey, 1997). Microglia activation can impair the learning and memory via induction of high-level expression of many factors. Either alone or in combination, these inflammatory factors may cause hippocampal neuronal injury. This neurotoxic damage may affect long term potentiation (LTP), thus leading to learning and memory deficits.
1.2 Statement of Research Problem
Lead exposure accounts for about 0.2% of all deaths and 0.6% of disabilities globally. Since the 1970s, regulation reducing lead in products has greatly reduced exposure in the developed world, however lead is still allowed in products in many developing countries (Pokras and Kneeland, 2008). Children tend to have the highest blood lead levels, possibly because at this age they breathe faster, they begin to walk and explore their environment, and they use their
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mouths in their exploration (Ab Latif et al., 2015). Substantial progress has been made in reducing the numbers of children exposed to higher levels of environmental lead. However, early chronic low-level lead exposure remains an unresolved child public health problem. As at October 5, 2010 at least 400 children have died from the effects of lead poisoning in Zamfara State, Nigeria (Lo et al., 2010). Illegal mining activities in Niger state, Nigeria also led to disabilities and death of over 28 children.No blood-lead level is safe, because several studies have reported that blood lead levels (BLLs) as low as 2 μg/dl are associated with lower measured intelligence, poorer memory, attention andreduced neurocognitive function in children (Chiodo et al., 2004; Lanphear et al., 2005).While peripheral effects in adults often go away when lead exposure ceases, evidence suggests that most of lead’s effects on a child’s central nervous system are irreversible (Bellinger, 2004).
1.3 Justification of Study
The continuous use of lead in pesticides, water pipes, cosmetics and gasoline in developing countries necessitates a detailed investigation on its resultant effects on the brain.The activity of the hippocampus is distorted particularly in children by exposure to lead. Lead damages the cells within the hippocampus and also interferes with the release of neurotransmitters causing the disruption of communication between cells. Most studies have reported that the mechanism of lead induced neurotoxicity is via ionic mechanism and that of oxidative stress. Hence there is a need to investigate other mechanisms that may be responsible for lead induced neurotoxicity in the hippocampus. Therefore, this study was conducted to investigate the effect of post-natal lead exposure on the hippocampus of developing Wistar rats. The results from this study could help elucidate the mechanism of post-natal lead neurotoxicity in the hippocampus as well as if the effect of post-natal lead exposure on microglia activation is dose dependent, thus its effect on learning and memory.
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1.4Aim of the Study
The aim of the study was to assess the effect of post-natal lead exposure on the hippocampus of developing Wistar rats.
1.5Objectives of the Study
The research objectives are to assess the effect of post-natal lead exposure on the:
i. body weight, brain weight and brain somatic index of the Wistar rat pups.
ii. concentration of lead deposit in the brain using Atomic Absorption Spectrophotometer-lamp standard.
iii. changes in oxidative stress markers such assuperoxide dismutase activity (SOD), reduced glutathione (GSH) and malondialdehyde (MDA) concentration using chemical method.
iv. CA3 regions of the hippocampus of the pups using H and E and Cresyl fast violet stains.
v. cytoarchitecture of microglia in the hippocampus of the pups using Tomato lectin stain.
vi. volume of the hippocampus of the pups and number of activated microglia in the hippocampus of the pups using Cavalieri estimator and physical fractionator probe respectively.
1.6Hypothesis
Post-natal lead exposure will trigger microglia activation and cause distortion in the cytoarchitecture of the hippocampus of developing Wistar rats.
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