EFFECT OF D-RIBOSE- L-CYSTEINE ON REDOX BALANCE AND INFLAMMATION IN PARADOXICAL SLEEP-DEPRIVED PREGNANT RATS

TABLE OF CONTENTS

TITLE PAGE

CERTIFICATION

DEDICATION

ACKNOWLEDGEMENT

TABLE OF CONTENT

LIST OF TABLES

LIST OF FIGURES

ABSTRACT

CHAPTER ONE  

INTRODUCTION​​​​​​​​​​1

1.1 AIM OF THESTUDY​​​​​​​​​2

1.2 OBJECTIVES OF THESTUDY​​​​​​​​2

1.3 LIST OF ABBREVIATIONS​​​​​​​​3

CHAPTER TWO: LITERATURE REVIEW

2.1 STRESS​​​​​​​​​​​4

​2.1.1 TYPES OF STRESSORS​​​​​​​5

2.1.2 EFFECTS OF OXIDATIVE STRESS ON FEMALE REPRODUCTIVE SYSTEM​​​​​​​​​​6

2.2 THE MAMALIAN FEMALE REPRODUCTIVE SYSTEM

​2.2.1 FEMALE REPRODUCTIVE FUNCTIONS IN MAN​​​​7

​​2.2.1.1 OVARIES​​​​​​​​7

​​2.2.1.2 FALLOPIAN TUBES​​​​​​​7

​​2.2.1.3 UTERUS ​​​​​​​​7

​​2.2.1.4 VAGINA​​​​​​​​7

2.2.2 FEMALE REPRODUCTIVE SYSTEM IN RATS​​​​8

​​2.2.2.1 ESTROUS CYCLE​​​​​​​9

​​2.2.2.2 ESTROUS CYCLE PHASES​​​​​​9

2.2.2.3 ENVIRONMENTAL FACTORS AFFECTING THE ESTROUS CYCLE​​​​​​​​​10

​​2.2.2.4 MATING AND REPRODUCTIVE BEHAVIOR ​​​11

​​2.2.2.5 PREGNANCY DETECTION ​​​​​11

​​2.2.2.6 FERTILIZATION AND EARLY EMBRYONIC DEVELOPMENT 11

​​2.2.2.7 EMBRYO IMPLANTATION​​​​​12

​​2.2.2.8 GESTATION, PARTURITION AND WEANING​​​13

​2.2.3 HORMONAL CONTROL OF PREGNANCY IN RATS ​​​14

​​2.2.3.1 HORMONAL CHANGES​​​​​​15

2.3 SLEEP DEPRIVATION

​2.3.1 INTRODUCTION​​​​​​​​19

2.3.2 SLEEP LOSS AND ADRENOCORTICAL ACTIVITY​​​21

​2.3.3 SLEEP LOSS AND METABOLISM​​​​​​22

2.3.4 SLEEP DEPRIVATION AND ADVERSE MATERNAL AND FOETAL OUTCOMES​​​​​​​​​​24

​2.3.5 MECHANISMS​​​​​​​​​26

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2.4 OXIDATIVE STRESS AND PREGNANCY​​​​​​27

​2.4.1 ANTIOXIDANTS​​​​​​​​29

​2.4.2 REACTIVE OXYGEN SPECIES AND THEIR PHYSIOLOGICAL ACTIONS 29

​2.4.3 OXIDATIVE STRESS IN FEMALE REPRODUCTION​​​32

​2.4.4 OXIDATIVE STRESS AND PREGNANCY​​​​​33

2.5 OXIDATIVE STRESS ANDINFLAMMATION​​​​​33

2.5.1 C-REACTIVE PROTEIN​​​​​​​36

2.6 GLUTATHIONE(GSH)​​​​​​​​​37

​2.6.1 D-RIBOSE L-CYSTEINE​​​​​​​41

2.6.2 EFFECTS OF D-RIBOSE L-CYSTEINE ON GSH LEVEL​​​42

CHAPTER THREE: MATERIALS AND METHODOLOGY 

3.1 MATERIALS​​​​​​​​​​43

3.2 ANIMALS​​​​​​​​​​43

​3.2.1 INDUCTION OF PREGNANCY​​​​​​44

3.3 EXPERIMENTAL DESIGN​​​​​​​​44

3.4 D-RIBOSE L-CYSTEINE PREPARATION, DOSAGE AND ADMINISTRATION ​45

3.5 PARADOXICAL SLEEP DEPRIVATION PROTOCOL​​​​ 45

3.6 ANIMAL EUTHANASIA, BLOOD SAMPLING AND TISSUE COLLECTION​ 45

3.7 DETERMINATION OF OXIDATIVE PARAMETERS​​​​​47

​3.7.1 DETERMINATION OF SUPEROXIDE DISMUTASE (SOD)​​ 47

​3.7.2 DETERMINATION OF CATALASE (CAT)​​​​​ 48

​3.7.3 DETERMINATION OF GLUTATHIONE(GSH)​​​​ 48

​3.7.4 DETERMINATION OF MAIONDEALDEHYDE (MDA)​​​ 49

3.8 STATISTICAL ANALYSIS​​​​​​​​ 49

CHAPTER FOUR: RESULT​​​​​​​​ 50

CHAPTER FIVE:

5.1 DISCUSSION AND FINDINGS​​​​​​​​61

5.2 CONCLUSION​​​​​​​​​​62

REFERENCES

LIST OF TABLES

TABLE 1: BEHAVIOUR AND VAGINAL CYTOLOGY WITH OESTROUS CYCLE PHASES​​​​​​​​​​10

TABLE 2: SOURCES AND FUCTIONS OF THE HORMONES OF THE FEMALE REPRODUCTIVE SYSTEM​​​​​​​​18

TABLE 3: EFFECT OF PSD AND DRLC ADMINISTRATION ON NUMBER AND WEIGHT OF IMPLANTATION IN RATS.​​​​​​49

TABLE 4: EFFECT OF PSD AND DRLC ADMINISTRATION ON FETAL PARAMETERS AND PREGNANCY OUTCOME IN RATS.​​​53

TABLE 5: OESTRADIOL CONCENTRATION​​​​​55

LIST OF FIGURES

FIGURE 1: HORMONAL CONTROL OF PREGNANCY IN RATS​​​14

FIGURE 2: EFFECTS OF OXIDATIVE STRESS ON FEMALE REPRODUCTIVE SYSTEM​​​​​​​​​​​30

FIGURE 3: EFFECT OF PSD AND DRLC ADMINISTRATION ON SERUM CONCENTRATION OF CORTICOSTERONE IN PREGNANT RATS SACRIFICED ON DIFFERENT DAYS OF GESTATION.​​​​​​​56

FIGURE 4: EFFECT OF PSD AND DRLCADMINISTRATION ON MDA CONCENTRATION INSERUM OF PREGNANT RATS.​​​​​57

FIGURE 5: EFFECT OF PSD AND DRLC ADMINISTRATION ON LEVEL OF SOD ACTIVITY INSERUM OF PREGNANTRATS.​​​​​​58

FIGURE 6: EFFECT OF PSD AND DRLC ADMINISTRATION ON LEVEL OF CAT ACTIVITY INSERUM OF PREGNANT RATS.

FIGURE 7: EFFECT OF PSD AND DRLC ADMINISTRATION ON GSH CONCENTRATION INSERUM OF PREGNANT RATS.

FIGURE 8: EFFECT OF PSD AND DRLC ADMINISTRATION ON CRP CONCENTRATION INSERUM OF PREGNANT RATS.

ABSTRACT

In a healthy body, ROS (reactive oxygen species) and antioxidants remain in balance. When the balance is disrupted towards an overabundance of ROS, oxidative stress (OS) occurs. OS influences the entire reproductive lifespan of a woman and even thereafter (i.e. menopause). OS results from an imbalance between pro-oxidants (free radical species) and the body's scavenging ability (antioxidants). ROS build up in the body can be aided by stress e.g. sleep deprivation. Sleep deprivation is becoming increasingly common in modern society, especially in undergraduate students. Sleep is crucial for optimal functioning of the human body. 

A modified multiple platform was used in this experiment, which uses a REM technique to manipulate sleep deprivation, to actuate sleep deprivation in pregnant rats. About 60 female Sprague-Dawley rats were used for this experiment, with 15 rats per group, 5 for each of 3 subgroups and four groups totalling to 60 rats. The rats were housed in a fully aerated standard cage at room temperature with a 12hour dark and light cycle. The rats were fed on daily basis in their respective cages with rat chow. Oestrous cycle was observed in the female rats and rats on proestrus were introduced to male rats on proestrus evening for mating. Mating was confirmed by the presence of sperm cells in the vaginal smear and was taken as day one of pregnancy. DRLC was administered to the GLU groups. A plastic cage (40 x 30 cm) with 7 columns (platforms, 5 x 3 cm) was filled with 1 cm water. Five mice, all from the same cage, were placed in each cage with water and food for twenty hours every day for seven days from the onset of pregnancy. The water is changed daily and the rats are removed from the stress cage to recuperate for four hours.The loss of muscle tone associated with sleep deprivation caused them to touch the water and wake up.

The result shows significant decrease in the number of foetusat term in the PSD group when compared to the control group (P