ABSTRACT
Literature on the anatomy of the African Giant rats (AGR) is gradually building; there is paucity of information on the detailed morphometric, histologic analysis of the cerebrum and olfactory bulbs of the AGR. No stereologically study has been done on the olfactory bulb and cerebrum of the AGR. The quantification of cerebral layers and olfactory bulbs, their neuronal population and volume in AGRs, may shed light on neuroanatomical basis of their highly developed olfactory ability of the rodent and some of its behaviour. The general aim of the study was to investigate the cerebrum and olfactory bulbs of the AGR in order to elucidate the neuroanatomical basis for their special olfactory functions. Ten adult AGR (Cricetomys gambianus, Waterhouse-1840), consisting of five male and five females were used for this study and following standard procedures, the animals were captured alive around Zaria, using locally made rat traps, without any injury on them. The animals were anaesthetized with chloroform, sacrificed and transcardially perfused with a phosphate buffered solution of (pH 7.2, M = 0.12) 4% formaldehyde and 1% glutaraldehyde. The brain were removed and placed in the same fixative. The morphologic characteristics of the cerebrum and olfactory bulbs were observed with the naked eyes and electronic magnifying lens, and morphological features were recorded. Morphometric parameters of the cerebrum andolfactory bulb were taken, recorded and analyzed statistically using descriptive statistics and student‘s t- test. Histological sections were prepared after fixation and routine tissue processing, embedded in a low-gelling temperature agarose (6% agar), tissue sections of 60 um cut, perpendicular to the long axis (coronal section), were serially obtained with a calibrated vibratome and staining in thionin stain, stained tissues were viewed under the microscope and photomicrographs were taken. Designed-based stereological methods (optical fractionation and Cavalieri principle) were employed to estimate the neuronal number in olfactory bulb and volumes
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of the different layers in the olfactory bulb and cerebral cortex stained in 0.2% Thionin solution. The results showed that the adult AGR have lissencephalics cerebrum, a mean absolute cerebral weight of 3.44 ± 0.07g, relative cerebral weight of 0.29 ± 0.23, a globular shaped olfactory bulb, the olfactory bulb accounts for 8 % the total body weight in the AGR, cyto-architectural arrangement was similar to that of the olfactory bulb of dog, and other rodents, a mean neuronal number in the different layers of the olfactory bulbs irrespective of sex for the periglomerular cells, tufted cells, mitral cells, periglomerular cells and the granule cells account for 20.72%, 6.44%, 4.61%, 3.27% and 63.68% of the neurons in the five layers of the olfactory bulb in AGR respectively. Results of the mean volumes of the glomerular cell layers and the granule cell layer of the AGR, showed that Layers I, 11, 111, IV, V and VI accounted for 19.7%, 18.5%, 18.6%, 21% and 23.7% of the volume of the cerebral cortex of the AGR, respectively. This study for the first time reports on the volume of the different layers of the olfactory bulbs and cerebral cortex in the African giant rat (Cricetomys gambianus). The results obtained from this study can be used in comparative anatomy of rodents of similar species, comparative neurology of rodents and other mammals, and in elucidating the mechanism of olfaction and other brain functions in this macrosomatic rodent. The number and of neurons and the volumes of the individual layers in olfactory bulb and the cerebrum, may represent a more accurate indicator of the neural machinery and synaptic microcircuitry in these brain regions in rodents and other mammals.
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TABLE OF CONTENTS
Cover Page – – – – – – – – – i Title Page – – – – – – – – – ii Dedication – – – – – – – – – iii Declaration – – – – – – – – – iv Certification – – – – – – – – – v Acknowledgement – – – – – – – – vi Abstract – – – – – – – – – x Table of Contents – – – – – – – – xii List of Tables – – – – – – – – – xvii List of Figures – – – – – – – – – xviii List of Plates – – – – – – – – – xix List of Appendices – – – – – – – – xxi CHAPTER ONE 1.0 Introduction – – – – – – – – 1 1.1 Morphology – – – – – – – – 2 1.2 Morphometry – – – – – – – – 2 1.3 Stereology – – – – – – – – 3 1.4 Statement of the Problem – – – – – – – 6 1.5 Justification of the Study – – – – – – – 6 1.6 General Aim of the Study – – – – – – – 7 1.7 Specific Objectives of the Study – – – – – – 7 CHAPTER TWO 2.0 Literature Review – – – – – – – – 8 2.1 Geographic Range of African Giant Rats – – – – – 8
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2.2 Habitat of African Giant Rats – – – – – – 8 2.3 Physical Features of African Giant Rats – – – – – 8 2.4 Reproduction in African Giant Rats – – – – – 9 2.5 Brain Morphometry of African Giant Rats – – – – 11 2.6 Anatomy of the Olfactory Bulb – – – – – – 12 2.6.1 Development of the Olfactory Bulb – – – – – 13 2.6.2 Malformation of the Olfactory System – – – – – 16 2.6.3 Odour perception – – – – – – – 16 2.6.4 Main Olfactory Bulb – – – – – – – 19 2.6.5 Accessory Olfactory Bulb – – – – – – 21 2.6.6 Evolution of the Olfactory Bulb – – – – – 21 2.6.7 Olfactory Nerve Layer – – – – – – – 22 2.6.8 Olfactory Glomerular Layer – – – – – – 23 2.6.9 External Plexiform Layer – – – – – – 24 2.6.10 Mitral Cell Layer – – – – – – – 24 2.6.11 Granule Cell Layer – – – – – – – 26 2.7 Cerebral Cortex – – – – – – – – 29 2.8 Training of African Giant Rats for Mine Detection – – – 30 2.9 Use of African Giant Rats in Scent Detection of Tuberculosis – – 31 2.10 Cerebral Cortex – – – – – – – – 33 2.10.1 Embryology of the Cerebral Cortex – – – – – 34 2.10.2 Evolution of the Cerebral Cortex – – – – – 35 2.10.3 Layers of the Cerebral Cortex – – – – – – 36 2.10.4 Connections of the Cerebral Cortex – – – – – 40 2.10.5 Cortical Thickness – – – – – – – 41
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2.10.6 Comparative Anatomy of the Cerebral Cortex in Rodents and Primates- 41 CHAPTER THREE 3.0 Materials and Methods – – – – – – – 46 3.1 Experimental Design – – – – – – – 46 3.1.1 Capturing of Animal – – – – – – – 46 3.1.2 Housing and Feeding of Animals – – – – – 46 3.1.3 Sacrificing of Animals – – – – – – – 46 3.2 Extraction and Preparation of the Brain – – – – – 46 3.2.1 Extraction of the Brain – – – – – – – 46 3.2.2 Separation of the Olfactory Bulbs, Cerebrum and Cerebellum – – 47 3.3 Anatomical Study of the Olfactory Bulbs and Cerebrum of African Giant Rats 47 3.3.1 Morphologic (Gross Anatomical) Study – – – – 47 3.3.2 Morphometric Study – – – – – – – 48 3.3.3 Light Microscopy Study of the Olfactory Bulbs and Cerebrum of African Giant Rats – – – – – – – – 48 3.3.3.1 Fixation – – – – – – – – 48 3.3.3.2 Embedding – – – – – – – – 49 3.3.3.3 Mounting on metal block – – – – – – 49 3.3.3.4 Tissue Sectioning and staining – – – – – 49 3.3.3.5 Mounting on Chrome-Gelatine Slides – – – – 50 3.3.3.6 Dehydration and Thionin Staining – – – – – 50 3.3.3.7 Light Microscopy Study and Photomicrography of Slides – – 50 3.4 Stereological Study of the Olfactory Bulbs and Cerebrum of African Giant Rats – – – – – – – – – 50 3.4.1 Estimation of Neuronal Number in Olfactory Bulbs of African Giant Rats Using Optical Fractionator – – – – – – 51
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3.4.2 Estimation of Volume of Layer of Olfactory Bulbs in African Giant Rats Using the Cavalieri Principle – – – – – – 52 3.4.3 Point Counting – – – – – – – – 52 3.4.4 Z- Axis Analysis – – – – – – – 53 3.4.5 Counting Procedure for Neuronal Estimation of Olfactory Bulb Cells – 53 3.4.5.1 Counting Criteria for Cells of the Olfactory Bulb of African Giant Rats 54 3.4.5.2 Counting Criteria for Periglomerular Cells of olfactory bulb of African Giant Rats – – – – – – – – 54 3.4.5.3 Counting Criteria for Mitral Cells – – – – – 54 3.4.5.4 Counting Criteria for Granule Cells – – – – – 55 3.5 Sampling Fraction – – – – – – – – 55 3.6 Estimation of the Coefficient of Error – – – – – 56 3.7 Statistical Analysis – – – – – – – 56 CHAPTER FOUR 4.0 Results – – – – – – – – – 58 4.1. Physical Features of the Adult African Giant Rats – – – 58 4.2. Descriptive Statistics of the Morphometric Parameters of Adult African Giant Rats- – – – – – – – – 60 4.3. Sexual Dimorphism in the Body Morphometric Parameters in the Adult African Giant Rats – – – – – – – 62 4.4. Morphologic parameters of the Olfactory bulbs of Adult African Giant Rats 64 4.5. Morphological features of the cerebrum of adult African Giant Rats – 66 4.6. Morphometric Parameters of the Olfactory Bulbs of Adult African Giant Rats 73 4.7. Morphometric Parameters of the Olfactory Bulb in Male and Female Adult AGRs – – – – – – – – – 75 4.8. Morphometric Parameters of the Cerebrum of Adult African Giant Rats 77 4.9. Sexual dimorphism in the Morphometric Parameters in the Cerebrum of Adult African Giant Rats – – – – – – – 79 4.10. Cytoarchitecture of the Olfactory Bulb in the Adult African Giant Rats 81
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4.11. Cytoarchitecture of Adult African Giant Rat Cerebral Cortex – – 88 4.12. Designed-based estimation of neuronal number in Olfactory Bulbs of the Adult African giant rats – – – – – – – 95 4.13. Descriptive Statistics (Optical fractionator) Olfactory Bulbs of the male and female adult AGR – – – – – – – 97 4.14. Descriptive Statistics (Cavalieri Principle) Olfactory Bulbs of adult African Giant Rat – – – – – – – 99 4.15. Descriptive Statistics of Designed-Based (Cavalieri Principle) Estimation of Volume of the Layers of the Olfactory Bulb in Adult African Giant Rat 101 4.16. Volume (mm3) of the cerebral cortex layers of adult African Giant Rat, according to Cavalieri Principle- – – – – – 103 4.17. Descriptive Statistics of Designed-Based Stereological Estimation of Volume of Layers of Cerebral Cortex of Adult African Giant Rats by Cavalieri Principle – – – – – – – 105 CHAPTER FIVE 5.0 Discussion – – – – – – – – – 107 CHAPTER SIX 6.0 Summary, Conclusions and Recommendation – – – – 117 6.1 Summary – – – – – – – – – 117 6.2 Conclusion – – – – – – – – 118 6.3 Recommendation – – – – – – – – 119 References – – – – – – – – – 120
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CHAPTER ONE
1.0. INTRODUCTION
The African Giant rat (AGR) (Cricetomys gambianus, Waterhouse-1840), also known as the Gambian pouched rat, is a wild rodent that belongs to the order Rodentia, and is quite common in Nigeria. The rats are well distributed but more dominant in the Savannah areas of the country. The AGR has potentials as a laboratory animal, but it is better sought after as meat delicacy by the local people (Onyeanusi et al., 2007), the male AGR is called ―Burgu‖ and the female ‗gafiya‘ in Hausa language, ―Okete‖ in Yoruba language, ―Ewi‖ in Igbo and ―African rat geant‖ in French. An increasing amount of interest is presently being expressed on the biology of the AGR. The rats have already been successfully used to detect land mines in Mozambique because of their high acuity of odour perception (Magi, 2003). In Europe, AGR‘s imported from sub-Sahara Africa are trained to sniff out tuberculosis in humans. Attempts have been made to classify AGR as laboratory model (Ajayi, 1978; Olayemi and Adeshina, 2002). In United Kingdom, the AGR is increasingly becoming a popular pet (Copper, 2008). The brain, the organ of thought and cognizance perception, is composed of the cerebrum, cerebellum and brainstem (Moore et al., 2014; Nolte, 2007; Sinnatamby, 2011). The cerebrum is the largest part of the brain. In humans, it is the part of the brain, where activities, including reasoning, learning, sensory perception and emotional responses, take place (Moore et al., 2014; Singh, 2014). It consists of two partially connected cerebral hemispheres. The cerebral hemisphere has three different poles, namely: (i) the frontal pole, which lies anteriorly; (ii) the occipital pole, which is posteriorly placed; and (iii) the temporal pole, which lies between the frontal and occipital poles and points forward and same whole downwards (Drake et al., 2012; Sinnatamby, 2011; Moore et al., 2014).
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The olfactory bulb (Bulbus olfactorious) is a structure of the vertebrate forebrain involved in olfaction, the perception of odours (Nolte, 2007). In most vertebrates, the olfactory bulb is the most rostral (forward) part of the brain; while in humans, the olfactory bulb is on the inferior (bottom) side of the brain. The olfactory bulb is supported and protected by the cribriform plate of the ethmoid bone, which in mammals separates it from the olfactory epithelium, and which is perforated by olfactory nerve axons (Sinnatamby, 2011). The olfactory bulb is a highly organized structure, composed of several distinct layers and synaptic specializations. The layers (from outside toward the centre of the bulb) are differentiated into the following: glomerular layer, external plexiform layer, mitral cell layer, internal plexiform layer, granule cell layer (Mori et al., 2006; Wei et al., 2008). 1.1. Morphology
Morphology is the study of the form and structure of organisms and their specific structural features. This includes aspects of the outward appearance (shape, structure, colour, and pattern) (Hongtu et al., 2007). Shape descriptors offer a more informative representation of morphology, adding additional information over volumetric measurements (Wachinger et al., 2014). Morphology offers advantages for studying the shape variability within and across populations because additional information is available for the statistical analysis. 1.2. Morphometry
Morphometry as the measurement of morphology, in three dimension (3D), such measurements can be done directly; for example through determination of the height of a person using a measuring tape. However in biomedical research, morphometry is most frequently performed on thin sections using microscopy. These sections can often be
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regarded as nearly two dimensions and, what is seen is not the whole three dimensions objects themselves, but only a fraction of their two dimensions representation. The goal of morphometry is the estimation of quantitative parameters such as volume, surface area, length or number of the 3D objects, and it has to take into account the loss of one dimension during the cutting process (Singleton et al., 2011; Serag et al., 2012). Brain morphometric is a subfield of both morphometry and the brain sciences, concerned with the measurement of brain structures and changes there of during development aging, learning, disease and evolution. Since autopsy-like dissection is generally impossible on living brains, brain morphometry starts with noninvasive neuroimaging data, typically obtained from magnetic resonance imaging (MRI). These data are born digital, which allows researchers to analyze the brain images further by using advanced mathematical and statistical methods such as shape quantification or multivariate analysis. This allows researchers to quantify anatomical features of the brain in terms of shape, mass, volume (e.g. of the cerebellum, or of the primary versus secondary visual cortex), and to derive more specific information, such as the encephalization quotient, grey matter density and white matter connectivity, gyrification, cortical thickness, or the amount of cerebrospinal fluid. These variables can then be mapped within the brain volume or on the brain surface, providing a convenient way to assess their pattern and extent over time, across individuals or even between different biological species (Fred, 2001). 1.3. Stereology
Stereology provides efficient tools for estimation of geometric quantities such as volume, surface area, length, or number of objects for 3-D tissue structures, contained within an organ from measurements made on a set of 2-D sections (West, 1993; Gundersen, 1999). Stereological analyses involve a two-step process (West, 2002). Statistical sampling principles are used to obtain statistically valid histological sections from an organ to
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reduce the amount of tissue for analysis without reducing the precision of the estimate (Gundersen, 1999; Nyengaard, 1999). Design-based stereology provides estimates of total volume, surface area, length, or cell number in an organ, making no assumptions about the structures in an organ. Strict adherence to the principles of stereology guarantees accuracy; that is, the mean of the estimates comes closer and closer to the true value with replication. Accurate estimation of particle numbers from tissue sections became possible only in 1984 with the invention of the physical dissector (Sterio, 1984; Boyce, 2010). Unbiased stereology has been considered by modern neuroscientists as the most preferred method to measure or quantify morphological properties of the central nervous system (CNS) (Grady et al., 2003). This is particularly important in identification of subtle, yet important alterations in the morphology of the brains, based on disturbances during development or in neuropsychiatric and neurodegenerative diseases (Boyce et al., 2010). Some of the probes employed in unbiased stereology include optical fractionator and Cavalieri principle (Glaser et al., 2007). The optical fractionator is a designed-based stereological method using a two-stage systematic sampling method to estimate the number of objects in a specified region of an organ (West, 2002). This method combines the optical disector methods with fractionator sampling method. It is the most commonly used stereological probe in the life sciences. It can be used to estimate the total number of objects in any three-dimensional volume regardless of that volume‘s shape (Glaser et al., 2007). The optical fractionator is appropriate for the quantification of such objects as cells, synapses, glomeruli, and any discrete objects within biological tissues (Gundersen et al., 1999; Hosseini-Sharifabad and Nyengaard, 2007). The optical fractionator uses thick sections and estimates the total number of cells from the number of cells sampled with a systematic uniform, randomly sampled set of unbiased virtual counting spaces, covering the entire region of interest with uniform distance between unbiased virtual
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counting spaces in directions X, Y, and Z (Nyengaard, 1999; Schmitz and Hof, 2005; Glaser et al., 2007). The optical fractionator is immune to tissue shrinkage due to histological processing (Dorph-Petersen et al., 2004; Boyce et al., 2010). Additionally, it is easier to estimate the precision of the number of estimate; that is, the coefficient of error (CE) generated using the optical fractionator, making it the method of choice (Gundersen et al., 1999; Nyengaard, 1999; Schmitz and Hof. 2005). The Cavalieri is used to estimate volume (Grady et al., 2003). This instrument/method is often used in conjunction with other estimators that approximate density such as number density, length density, or surface density. The sections for Cavalieri principle are sampled using the fractionator principle. A rule of thumb is to use 10 to 15 sections. Suppose that the tissue is sectioned into 80 sections; If every 6th section is used, then around 13 sections are used. This is usually an adequate number of sections. The fractions are used in the sampling fraction (ssf). The Cavalieri estimator is usually performed using a point grid. The points need to be appropriately spaced. The points are usually spaced equally both across and down. A rule of thumb is to count 200 to 250 points. Some experimentation is usually required to determine an adequate spacing of the point grid. Regions that have complicated profiles or profiles that are thin require closer points than simple shapes. The distance between the points can be used to compute the intensity of the probe (Nielsen et al., 2001). The number of points that hit each section is counted and it is used to compute the volume and CE (Gundersen et al., 1986a). The formula for calculating volume in the Cavalieri Principle is given as: V= t* a/p *ΣP Formula for volume
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Where V is the volume, t is the thickness of the tissue section, a/p is the area of points and ΣP is the sum of points (Gundersen et al., 1986b). 1.4. Statement of the Problem The National Wildlife Conservation Committee has encouraged the domestication of the African Giant rats because of its numerous usefulness (Ikede and Ajayi, 1976, NRC, 1991). Therefore, there is a need to understand the biology of this rodent in order to elucidate the neuroanatomical basis of the olfactory behaviour of AGR. It is because of this need that the study of the cerebrum and olfactory bulbs of the AGR was carried out. Although literature on the anatomy of the AGR is gradually building up (Ogwuegbu et al, 1983; Oke and Aire, 1989, 1990; Oke et al., 1996; Nzalak et al., 2005; Ibe et al, 2012), there is paucity of information on the detailed morphometric, histologic analysis of the olfactory bulbs and cerebrum of the AGR. There is also no information on the stereologic study of the olfactory bulb and cerebrum of the AGR. Quantitative morphology is important in neurobiology, developmental and clinical human biology. The quantification of cerebral layers and olfactory bulbs, their neuronal population and volume in AGRs, may shed light on the neuroanatomical basis of the highly-developed olfactory ability of the rodent and some of its behaviour. 1.5. Justification of the Study
There is need to study the olfactory bulb cerebrum and of this rodent. This may serve as a fulcrum for future clinical and research applications, involving this macrosomatic animal. The findings from the anatomical studies of structures of the cerebrum and olfactory bulbs in the rodent may contribute to our current understanding of some of its unique behaviour, such as the ability to detect explosives in mines and tuberculosis bacteria in saliva. A detailed stereological study of the neuronal assortment of olfactory bulbs of the
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AGR may also provide information on the behavioural trait, especially the strong olfactory ability of the AGR. 1.6. General Aim of the Study The general aim of the study was to investigate the olfactory bulbs and cerebrum of the AGR in order to elucidate the neuroanatomical basis for their special olfactory acquity and visual attention. 1.7. Specific Objectives of the Study The specific objectives of the study were to:
i. Determine the gross morphology of the olfactory bulbs cerebrum and of the African Giant rat.
ii. Evaluate the morphometric features of the olfactory bulbs cerebrum and of the African Giant rat.
iii. Investigate the histological structures of the olfactory bulbs cerebrum and of the African Giant rat.
iv. Describe some stereological parameters such as the Optical fractionation and Cavalieri principle of the olfactory bulbs cerebrum and of the African Giant rat.
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