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PROJECT TOPIC AND MATERIAL ON EFFECT OF VITAMINS C AND E ON MEMORY IN ADULT MICE

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  • Name: EFFECT OF VITAMINS C AND E ON MEMORY IN ADULT MICE
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ABSTRACT

Vitamins C and E are naturally present in some foods and are available as dietary supplements. While vitamin C is also known as L-ascorbic acid, vitamin E is a generic name for tocopherols and tocotrienols each with α, β, γ and δ subunits. Neurobehavioural models have been used to study behaviour in animals with models specific for each animal species and behaviour. The aim of the study was to investigate the effect of vitamins C and E on memory and serum biochemical changes in adult mice. Twenty male and twenty female mice weighing 16-35 g were divided into 5 groups of eight mice each. The first group served as the control and received distilled water (1 ml/kg); mice in the second group served as the positive control and received soya oil (1 ml/kg), animals in group three received vitamin C (100 mg/kg), group four received vitamin E (100 mg/kg) and the last group received both vitamins C and E (100 mg/kg). The drugs were given once daily orally for a period of 21 days. Learning and memory were assessed using the elevated plus maze (EPM), object recognition and location models for memory (ORT and OLT) at the end of the experimental period. Memory index was calculated. The mice were sacrificed on day 22 and serum estrogen and testosterone levels and catalase, superoxide dismutase and glutathione peroxidase activities were also evaluated. Lipid peroxidation was determined by measuring the malondialdehyde concentration in the brain sample. The relationship between the gonadal hormone levels and the performance of animals in each of the neurobehavioural models for memory was positive. There was no significant difference in the malondialdehyde concentration (P < 0.05) of all groups in the males but in females between the control and vitamin C-treated group. The activities of superoxide dismutase showed no significant difference in the males but showed significance (P < 0.05) between control and treatment groups in the females, catalase showed no significant difference in the females but showed significant difference (P < 0.05) between control and all groups and glutathione peroxidase showed no significant difference between all groups (P < 0.05) of both males and females. The memory index for the EPM also showed significant difference (P < 0.05) between the vitamin E-treated group and the soya oil and vitamin C-treated groups on the first day and between vitamin E and C-treated group on the second day in the females. In the males the significant difference was observed between control and treatment groups on both days. In the ORT model, discriminatory and recognitive index showed a significant (P < 0.05) difference in the vitamin E and E+C treated groups. In the OLT model discriminatory and recognitive indices showed a significant difference (P < 0.05) in the vitamin E and vitamins C+E treated groups. In conclusion, administration of vitamin C and E improved memory indices and there was a positive relationship between endogenous gonadal hormones and recognition.

TABLE OF CONTENTS

Title page – – – – – – – – – i
Declaration – – – – – – – – – ii
Certification – – – – – – – – – iii
Dedication – – – – – – – – – iv
Acknowledgements – – – – – – – – v
Abstract – – – – – – – – – vii
Table of Contents – – – – – – – – viii
List of Tables – – – – – – – – xii
List of Figures – – – – – – – – xiii
List of Abbreviations – – – – – – – – xv
CHAPTER ONE
1.0 Introduction – – – – – – – – 1
1.1 Statement of Research Problem – – – – – 6
1.2 Justification of Study – – – – – – – 7
1.3 General Aim of the Study – – – – – – 7
1.3.1 Specific Objectives of the Study – – – – – 7
1.4 Research Hypothesis – – – – – – – 8
CHAPTER TWO
2.0 Literature Review – – – – – – – 9
2.1 Oxidative Stress and the Nervous System – – – – 9
2.2 Vitamin C in the Nervous System – – – – – 11
2.3 Vitamin E and Memory – – – – – – 12
2.4 Sex hormones and Memory – – – – – – 13
ix
2.4.1 Neurobiology of Oestrogen – – – – – – 14
2.4.2 Oestrogen and Cognition – – – – – – 16
2.4.3 Oestrogen and Learning and Memory – – – – – 17
2.4.4 Testosterone and Cognition – – – – – – 20
2.4.5 Gonadal Hormones and the Cholinergic system – – – 22
2.5 Cognition – – – – – – – – 22
2.6 Memory – – – – – – – – 22
2.6.1 Classification of Memory – – – – – – 23
2.7 Learning – – – – – – – – 26
2.8 Sex Differences in Cognitive functions – – – – – 27
CHAPTER THREE
3.0 Materials and Methods – – – – – – – 28
3.1 Site of experiment – – – – – – – 28
3.2.1 Experimental Animals – – – – – – – 28
3.2.2 Housing and grouping – – – – – – – 28
3.3 Drugs and soya oil preparation – – – – – – 29
3.3.1 Drugs and preparation – – – – – – – 29
3.4 Neurobehavioural Assessments – – – – – – 30
3.4.1 Elevated Plus Maze for Memory – – – – – 30
3.4.2 Object Recognition Test – – – – – – 30
3.4.3 Object Location Test – – – – – – – 33
3.5 Biochemical Tests – – – – – – – 37
3.5.1 Effect of Drugs on Brain Lipid Peroxidation – – – – 37
3.5.2 Evaluation of Brain Catalase Activity – – – – – 40
x
3.5.3 Evaluation of Brain Glutathione Peroxidase Activity – – – 40
3.5.4 Evaluation of Brain Superoxide Dismutase Activity – – – 41
3.6 Determination of Gonadal Hormone Levels – – – – 41
3.6.1 Determination of testosterone – – – – – – 41
3.6.2 Determination of Estrogen level – – – – – 43
3.7 Statistical Analysis – – – – – – – 44
CHAPTER FOUR
4.0 RESULTS – – – – – – – – 45
4.1 Neurobehavioural Assessments – – – – – 45
4.2 Elevated Plus Maze for Memory – – – – – – 48
4.3 Object Recognition Test – – – – – – – 51
4.4 Object Location Test – – – – – – – 51
4.5 Lipid Peroxidation Assessment – – – – – – 54
4.6 Gonadal Hormone Assay – – – – – – 57
4.7 Relationship between Serum Gonadal Hormones and Memory indices in mice 59
CHAPTER FIVE
5.1 Discussion – – – – – – – – 66
CHAPTER SIX
6.1 Summary – – – – – – – – 74
6.2 Conclusion – – – – – – – – 74
6.3 Recommendations – – – – – – – 74
References – – – – – – – – – 75

CHAPTER ONE

1.0 INTRODUCTION
Vitamin C, also known as L-ascorbic acid, is a water-soluble vitamin that is naturally
present in some foods, added to others, and available as a dietary supplement and is
destroyed by heat or reduced by prolonged storage (Weinstein et al., 2001). Humans,
unlike most animals, are unable to synthesize vitamin C endogenously, so it is an essential
dietary component (Naidu, 2003; Li, 2007). Sex steroids are hormones produced mainly
by the reproductive glands, either the ovaries or testes, which share a similar basic
structure of three hexane rings and a pentane ring (Gasbarri, 2010). The primary role of
the gonadal steroid hormones in mammals is to regulate reproduction and related
behaviours; however, both androgens and estrogens are also integrally involved in
mediating higher brain function and processes, including cognition, neural development
and neural plasticity (Dohanich, 2002). The hippocampal system plays an important role
in memory function. Neurohormones like androgens and oestrogens are present in the
hippocampus and have important roles in learning and memory (Talebi et al., 2010).
Oestrogens act on the central nervous system (CNS) both through genomic mechanisms,
modulating synthesis, release and metabolism of neurotransmitters, neuropeptides and
neurosteroids, and through non-genomic mechanisms, influencing electrical excitability,
synaptic function and morphological features. Therefore, oestrogen’s neuroactive effects
are multifaceted and encompass a system that ranges from the chemical to the
biochemical to the genomic mechanisms, protecting against a wide range of neurotoxic
insults (Genazzani et al., 2007).
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Vitamin C, also known as ascorbic acid AA is required for the biosynthesis of collagen,
L-carnitine, and certain neurotransmitters; vitamin C is also involved in protein
metabolism (Carr and Frei, 1999). Collagen is an essential component of connective
tissue, which plays a vital role in wound healing. Vitamin C is also an important
physiological antioxidant (Carr and Frei, 1999) and has been shown to regenerate other
antioxidants within the body, including alpha-tocopherol (vitamin E) (Jacob, 2002). In
addition to its biosynthetic and antioxidant functions, vitamin C plays an important role in
immune function (Jacob, 2002) and improves the absorption of non-haeme iron, the form
of iron present in plant-based foods. Insufficient vitamin C intake causes scurvy, which is
characterized by fatigue or lassitude, widespread connective tissue weakness, and
capillary fragility (Weinstein et al., 2001; Wang, 2007).
Vitamin E is a generic name for tocopherols and tocotrienols. It is a family of α, β, γ and δ
tocopherols and corresponding tocotrienols. Tocopherol contains saturated phytol side
chains and tocotrienol have 3 double bonds in the side chain (Blatt et al., 2001; Dietrich et
al., 2006). The alpha-tocopherol form of vitamin E is an important lipid-soluble
antioxidant. In the brain and other tissues, alpha-tocopherol has a key role in preventing
oxidant-induced lipid destruction and is, therefore, vital in maintaining the integrity of
cell membranes (Blatt et al., 2001; Dietrich et al., 2006). Accordingly, vitamin E
deficiency causes lipid peroxidation in brain tissues. Severe vitamin E deficiency results
mainly in neurological symptoms, including impaired balance and coordination (ataxia),
injury to the sensory nerves (peripheral neuropathy), muscle weakness (myopathy), and
damage to the retina of the eye (pigmented retinopathy) (Traber et al., 2006). The
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developing nervous system appears to be especially vulnerable to vitamin E deficiency.
For instance, children who have severe vitamin E deficiency from birth and are not treated
with vitamin E rapidly develop neurological symptoms. In contrast, individuals who
develop malabsorption of vitamin E in adulthood may not develop neurological symptoms
for ten to 20 years (Drake, 2011).
Gender differences in cognitive function tests have been widely reported: men tend to
excel in fields of spatial cognition, whereas women show greater abilities in verbal
fluency (Halpern, 1992). Many factors influence the development of these abilities, such
as environment, education, cultural background and inherited factors, but sex hormones
also play a role in development and maintenance of cognitive functions. Variation and
overlapping scores between genders show the limited extent of this impact; to a much
higher degree, an individual’s cognitive abilities are derived from experience, education
and inheritance (Zitzmann, 2001).
Oxidative stress is an imbalance between oxidants and antioxidants in favour of the
oxidants, potentially leading to damage (Sies, 1991). It occurs when the oxidative stress
related molecules, generated in the extracellular environment or within the cells exceed
the intracellular antioxidant defences (Vrba and Modriansky, 2002). Oxidative stress
involves a number of chemical reactions, leading to the production of free radicals (FRs)
and other reactive molecules that are able to induce cellular injury (Kennedy et al., 2005).
It results in loss of normal balance between free radical species production and the
antioxidant system (Himmelfab and Hakim, 2003) or interruption of the equilibrium
between pro- and antioxidant systems (Lazarova et al., 2004).
4
Free radicals are chemical species that have an unpaired delocalized electron in an outer
orbital (Campanella et al., 2007), hence they are molecules with an “open” or half bond
(McCord, 1985), and are capable of independent existence no matter how brief (Halliwell
and Gutteridge, 1999). This unstable configuration causes these chemical species to be
very aggressive and to have a short life span (Halliwell, 1987). The unpaired electron
confers on these molecules a strong propensity to react with target molecules, by
withdrawing one electron from target molecules to complete their own orbital (Halliwell
and Gutteridge, 1999; Younes, 1999), and thereby stabilizing themselves. Thus, FR
causes the molecule whose electron had been withdrawn to become unstable and even
become a FR itself (Halliwell and Gutteridge, 1999). The resulting FR can in turn react
with another molecule until the chain reaction is terminated; either by random collision of
two or three FRs to form a molecule with stable bond or by one of the cellular defence
mechanisms (McCord, 1985; Goldfarb, 1999). In view of their high reactivity and
instability, FRs can react with several chemical compounds such as proteins, lipids,
nucleic acids and carbohydrates, present in cellular compartments and other classes of
molecules like extracellular matrix glycosaminoglycans (hyaluronic acid) and damaging
them (Halliwell and Gutteridge, 1985; Schimdley, 1990; Campanella et al., 2007).
Biological systems have evolved endogenous defence mechanisms to protect against FR
induce cell damage (Singh et al., 2004). These are called antioxidants. Antioxidants were
originally defined as any substance that when present in low concentration, compared
with that of oxidizable substrate, significantly prevent the pro-oxidant-initiated oxidation
of that substrate (Halliwell and Gutteridge, 1985; Black, 2004). However, because of the
imperfection in the definition in relation to the concentration of antioxidants (Halliwell
5
and Gutteridge, 2007), Halliwell and Gutteridge (2007) redefined antioxidants as any
substance that delays, prevents or removes oxidative damage to a target molecule. Higher
organisms have developed a remarkably efficient antioxidants defense system over the
course of evolution. The antioxidants neutralize FR by donating an electron to stabilize
the FR (Chihuailaf et al., 2002), the antioxidant molecules “internalize” the loss of an
electron through resonance between carbon bonds. The antioxidant systems can prevent
oxidative damage by (Sen. 1995):
(a) Scavenging of reactive oxygen species (ROS) and other reactive derivatives;
(b) Decreasing the conversion of less reactive ROS to more reactive ROS
(c) Facilitating repair of damage caused by ROS; and
(d) Providing a favourable environment for the activity of other antioxidants
The antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase
(GPx), catalase (CAT) (Drewa et al., 1998; Schneider and De Oliveira, 2004), glutathione
reductase and transferases, thiol disulfide oxidoreductases and peroxiredoxins (Halliwell
and Gutteridge, 1990; Stocker and Keaney, 2004) are primary enzymes involved in the
direct elimination of toxic oxygen species. Other enzymes, including glucose-6-phosphate
dehydrogenase and cytosolic glutathione sulphur-transferase (GST), are secondary
enzymes, which help in the detoxification of ROS by decreasing peroxide levels or
maintaining a steady supply of metabolic intermediates like glutathione or NADPH
necessary for optimum function of the antioxidant enzymes (Singh et al., 2003). The
antioxidant enzymes are primarily intracellular and, thus, extracellular FRs either
produced extracellularly, or from the environment must be inactivated by circulating
antioxidants such as vitamins and ceruloplasmins (Machlin and Bendich, 1987). Their
6
effectiveness varies with the stage of development and other physiological aspect of the
organism (Halliwell and Gutteridge, 1999; Livingstone et al., 2001). The antioxidants
require micronutrients such as selenium, zinc, iron, copper, and manganese as co-factors
for optimum catalytic activity and effective defence mechanisms (Halliwell and
Gutteridge, 1992; Halliwell, 2001).
1.1 Statement of Research Problem
The need to preserve cognitive functioning is present at every stage in life, whether at
childhood when the focus is cognitive development or at adulthood when the need is
protection or alleviation of short-term or long-term cognitive decline. This has led to
promotion of the use of prescription drugs, herbal supplements, among others, in order to
promote or preserve cognitive performance levels (Schmitt, 2010). Starting from
adulthood, there is an age-related decline in the level of gonadal hormones, which has
been correlated with impairment in some cognitive tasks (Leonard, 2011). Hence, the
need for a substance that can promote cognitive development as well as protect against
cognitive decline (Schmitt, 2010). Micronutrient supplementation has been shown to be
associated with less cognitive decline (Morris, et al., 2002). It is reported that treatment
with ascorbic acid significantly improved cognitive function in rats (Shahidi et al., 2008)
and it ccould enhance learning and memory processes (Cho, et al., 2003). While a lot of
work has been done on the effects of antioxidant supplementation in improving cognitive
functions in aged rodents and humans (Durga, et al., 2007; Saleem, et al., 2012) there is
paucity of information on effects of vitamins C and E supplementation on serum
testosterone and estrogen levels as well as on learning and memory in adult mice of both
sexes. Thus, the question this study seeks to answer is whether vitamins C and E affect
7
the level of the testosterone and estrogen which in turn affect learning and memory, and
whether endogenous levels of these hormones can be correlated to the optimum cognitive
capacity observed in adulthood.
1.2 Justification
The maintenance of brain health underpinning intact cognition is a key factor to
maintaining a positive, engaged, and productive lifestyle (Stough et al., 2012). There is an
age-related decline in the level of gonadal hormones, which has been correlated with
impairment in some cognitive tasks such as learning and memory (Leonard, 2011).
It is reported that treatment with ascorbic acid significantly improved cognitive function
in aged rats (Shahidi, et al., 2008), and it can enhance learning and memory processes
(Cho, et al., 2003). Vitamin E intake, from foods or supplements, was also shown to be
associated with less cognitive decline with age (Morris et al., 2002). The combined
effects of multiple antioxidant nutrients might be more influential on cognition than a
single antioxidant as reported by Devore et al. (2010).
1.3 General Aim
The general aim of this study was to investigate the effect of vitamins C and E on learning
and memory in healthy adult mice.
1.3.1 Specific objectives
The specific objectives of this work were as follows:
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1. To assess the effect of administration of vitamins C and E on learning and
memory using the elevated plus maze for memory (EPM), object recognition test
(ORT) and object location test (OLT) in mice.
2. To determine the effect of vitamins C and E on lipid peroxidation;
malondialdehyde (MDA), and enzymatic activities; superoxide dismutase (SOD),
glutathione peroxidase (GPx) and catalase (CAT) in mice.
3. To relate the effect of endogenous testosterone and estrogen with animals
performance in the EPM, ORT and OLT models for memory.
1.4 Hypothesis
Ho: Vitamins C and E have no effect on learning and memory in adult mice.

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