ABSTRACT
Trypanosomiasis, a diseases in the Sub-saharan Africa; its neurodegeneration phase of is yet to be understood. This study was designed to investigate the effect of Trypanosoma brucei brucei on spatial memory, histology and expression of calbindin-d28k in hippocampus of wistar rats. Thirty adult (male) Wistar rats were categorized into two groups, control(six rats) and infected (24 rats). Group 2 was divided into 4 subgroups (6 rats/ subgroup). Group 1 served as control, group 2 was inoculated with 1×106 innoculum dose of the parasite. Parasitaemia was studied using wet blood smear preparation. Morphological observations were made using weighting scale, Vanier caliper, and digimizer 2.2. Blood parameters were studied using automated hematological analyser. Histological and histomorphometrical studies were done using H and E, toluidine blue and. Expression of calbindin d28k was observed using the immunohistochemistry study, the antigen/antibody technique. Spartial memory and learning was studied using Morris water maze method and latency time to find the hidden platform was recorded. These were done after 3days for group 2a, 7days for 2b, 14days for group 2c, 21days for group 2d and the control group. Innoculation of the parasite, over time, significantly induced parasitaemia; significantly affected blood parameters(leucopenia, anemia, lymphopaenia and neutropaenia); significantly reduced body weight, neuronal cell count and diameter; it caused distortion of the laminated hippocampal arraingement; it reduced the expression of calbindin d28k. Affected learning and memory by decreasing latency time over the research period.In conclusion, Trypanosoma brucei brucei infection has deleterious effects on body, blood, hippocampus, and expression of calbindin d28k, which was reflected in the decrease in spartial memory and learning of the Wistar rats.
TABLE OF CONTENTS
Title Page ………………………………………………………………..…………………………i
Declaration ………………………………………..……………………………………………….iii
Certification ……………………………………………………….……………………..……….iv
Dedication ………. ……………………………………………………………………….…..……v
Acknowledgements ……………………………………………………….………………..……..vi
Abstract ………………………………………………………………….…………………..….viii
Table of contents ……………………………………………………………………….………….ix
List of Figures……………………………………………………………….………….…………xv
List of Tables……………………………………………………………….…………………….xvi
List of Plates……………………………………………………………….………….…………xvii
List of Appendices……………………………………………………………….………….…..xix
List of Abbreviations……………………………………………………………….……………xx
CHAPTER ONE
1.0 Introduction …………………………………………….……………….……………………..1
1.1 Background Information ……………………………….………………………….………….1
1.1.1 Incidence in Africa ……………..……………….…………………………………..…..1
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1.2 Symptoms of Trypanosomiasis……………………………………………..…………………2
1.3 Aims and Objectives of the Study………………..…………………..….…………………3
1.4 Significances of the Study.………………………………………………..…..……………4
1.5 Research Hypothesis.…………………………………………….………..…..……………4
CHAPTER TWO
2.0 Literature Review………………………………..…………………………………………5
2.1 Trypanosomiasis……………………….……………………..……………………………5
2.2 History …………………………………………………………………………..…….……5
2.3 Transmission.. …………………….……………………………………………….………6
2.3.1 Vector……………………….………………………….…………………………………..8
2.3.2 Morphology of Tsetse Fly. …………………….…………………….……………………9
2.3.3 Life cycle of Tsetse fly. …………………….…………………….………………………11
2.4 Parasites……………………….…………………………………………………………12
2.4.1 Life Cycle.. …………………….………………………….……………………………..14
2.5 Development of Trypanosomiasis.. ……………………….……….……………………15
2.6 Human African Trypanosomiasis……………………….………….……………………20
2.6.1 Diagnosis……………………….………………………..……………………………….21
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2.6.2 Prevention. …………………….…………………….…………………………………..21
2.6.3 Treatments.. …………………….…………………….………………………………….22
2.7 Animal trypanosomiasis. …………………………….….……………………………….24
2.8 Trypanosoma brucei brucei. ……………………………..….………………………………….25
2.9 Effect of Trypanosomiasis……………………………….….……………………………25
2.10 Calbindin D28k……………………….………………………………………………….29
2.12 The hippocampal formation………………………….……….…………………………..32
2.12.1 Hippocampus……………………….…………………………………………………….33
2.12.3 Hippocampal Histology……………………….………………………………………….35
2.12.6 Functions of the hippocampus……………………….……………………..……………40
2.12.7 Role in memory……………………….…………………………………………………..41
2.12.8 Role in spatial memory and navigation…………………………..…….………………..42
CHAPTER THREE
3.0 Material and Methods……………………….…………………..………………………45
3.1 Materials……………………….………………………………………………..………45
3.1.1 Experimental Animals…………………………..……….………………………………45
3.1.2 Equipment ………………………..……………………………………………..……….45
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3.1.3 Reagents……………………….………………………………………………..………..46
3.1.4 Parasites……………………….………………………………………………..………..46
3.2 Methodology…………………….………………….……………………………..…..…46
3.2.1 Screening of Animals……………………….…………………………………..………..46
3.2.2 Experimental Design…….…………………….………………………………………….46
3.2.5 Morris Water Maze Test……………………….…………………………………………53
3.2.6 Re-weighing of animals……………………….………………………………………….53
3.2.7 Infection of Animals……………………….…………………………………………….53
3.2.8 Parasitemia Estimation…………………….…………………………………………….54
3.2.9 Weighing of animals……………………….…………………………………………….54
3.2.10 Morris Water Maze Test……………………….…………………………………………54
3.2.11 Hematological Analysis……………………….………………………………………….54
3.2.12 Histological Studies……………………….……………………………………………..55
3.2.13 Histomorphometric Study….…………………….………………………………………56
CHAPTER FOUR
4.0 Results……………………….………………………………………………..…………57
4.1 Morphological Studies……………………….………..…………………………………57
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4.2 Degree of Parasitaemia…………………………….…………………………………….57
4.3 Haematological Studies……………………..………………………….………………..57
4.4 Morris Water Maze Training and Test Result ………….…………..……………………58
4.5 MorphometricStudies…….…………………….…………………………………………67
4.6 Histological Study….…………………….………………………………………………70
4.6.1 Histological Features of Hippocampus in both the Control (Uninfected) and Infected Experimental Animals……………………….…………………………………………….…70
4.7 Histomorphometrical Studies…………………………………………………………….79
4.8 Immunohistochemical Studies……………………………………………………………79
CHAPTER FIVE
5.0 Discussion……………………….………………………………………………………..85
5.1 Mophorlogical Studies……………………….…….……………………………………..85
5.2 Parasitaemia Observation….…………………….……………………………………….86
5.3 Hematological Study…..…………………….…………………………………………..86
5.4 Neurobehavioural Study……………………….…………………………………………87
5.5 Histological Study……………………….……………………………………………….88
5.6 Histomophormetrical Studies…………………………………………………………….89
5.7 Immunohistochemical Studies………….……………………………………………….89
CHAPTER SIX
6.0 Summary, Conclusion and Recommendation………………………………..……..…….90
xiv
6.1 Summary…….……………………..……………………………………..…………….….90
6.2 Conclusion….……………………..……………………………………..…………….….90
6.3 Recommendations……..………………………………………………..……………..….90
APPENDICES……………………….…………………………………………………………..91
REFERENCES ……………………………………………………………………………….….98
CHAPTER ONE
1.0 Introduction
1.1 Background Information
Trypanosomiasis is a parasitic disease of people and animals, caused by protozoa of the Trypanosoma genus and transmitted by the tsetse fly (WHO,2006). It ranks high in importance amongst protozoan infections of animals and man. It occurs in tropical and subtropical regions of the world and affects different species of animals like horse, camel, dog, cattle, sheep and goats (Boid, 2011). The disease is also called sleeping sickness, Human African Trypanosomiasis (HAT) or Congo trypanosomiasis (Robinson,2009).
1.1.1 Incidence in Africa
162,877 cases have been reported in africa between 2001 to 2011, according to WHO in 2012, with the distribution shown in the table below:
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Table 1.1: Incidence of trypanosomiasis in Africa from 2001 to 2011.
Country
Percentage(%)
DRC
58.27
Angola
11.18
Sudan
8.85
Uganda
1.68
Chad
2.04
Cameroon
0.12
Nigeria
0.08
Benin, Burkina faso, Ghana and Mali
0
1.2 Symptoms
African trypanosomiasis symptoms occurs in two stages:
Haemolymphatic Phase
Invasion of the circulatory and lymphatic systems by the parasite is associated with severe swelling of the lymph nodes, often to treamendous sizes. This is characterised by: Fever Headaches Joint pains and Itching.
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If left untretad, the disease overcomes the host‘s defences and causes more extensive damage, broadening symptoms to include: Anemia Endocrine disfunction Cardiac disfunction and Kidney disfunction.
Neurologic Phase
The neurological phase begins when the parasite invades the CNS by passing through the blood-brain barrier. The term ‗sleeping sickness‘ comes from the symptoms of the neurological phase. The symptoms include confusion, reduced coordination and disruption of the sleep cycle, with bouts of fatigue punctuated with manic periods, leading to daytime slumber and night-time insomnia. Without treatment, the disease is invariably fatal, with progressive mental detoriation leading to coma and death. Damage caused in the neurological phase is irreversible (New Vision,2008).
1.3 Aims and Objectives of the Study
Aims:
To investigate the effect of T. brucei brucei infection on spartial memory, histology and expression of calbindin-D28k neurons of hippocampus in Wistar rats.
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Objectives:
i. To determine the effect of T. brucei brucei on the spartial memory and learning using Morris water maze test.
ii. To determine the extent of neurodegenerative damage caused by T. brucei brucei in hippocampus using routine histological stains (H & E) and special stains and histomorphometric analysis.
iii. To compare the expression of calbindin-D28k in neurons of hippocampus in rats infected or uninfected with Trypanosoma brucei brucei using immunohistochemistry.
1.4 Significances of the Study
Results of this study could help in the understanding of the mechanism involved in the neurologic phase of trypanosoma infections and exploited in the development of drugs.
1.5 Research Hypothesis
T. brucei brucei infection will affect the spartial memory, histology, and expression of calbindin d28k in the hippocampus of Wistar rats.
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