ABSTRACT
Alcohol is the most widely used and abused psychoactive drug globally. Ethanol is the main ingredient in the three classes of alcoholic beverages. Children of alcoholic pregnant women are prone to wide spectrum of disorder referred to as Foetal Alcoholic Spectrum Disorder (FASD). The present study aimed at evaluating the teratologic effects of ethanol on the histology, neurobehaviour and trace elements of cerebellar and cerebral cortices of the neonatal Wistar rats. Twenty eight (28) female Wistar rats were mated with matured males in ratio 2:1. Pregnant dams were then grouped into 7. Group A served as the control group that received distilled water, Groups B, C and D were administered 0.5ml 20% ethanol for 7, 14 and 20 days during pregnancy respectively. Groups E, F and G were given 0.5ml 30% ethanol in the above stated manner. Following parturition, morphometric indices of the litters were taken at birth. Neuro-behavioural assessments were done on postnatal days 5, 6 and 7. Cerebral and Cerebellar tissues were obtained and processed for paraffin embedding for histological evaluation using Haematoxylin and Eosin and histochemical study using Cresyl Fast Violet Stains. Atomic Absorption Spectrophotometry was used to quantify Iron, Zinc, Copper and Manganese in the cerebrum and cerebellum. The result showed effects on the weight and crown rump length of the pups by exhibiting lower weight and crown rump length values in the ethanol treated neonates. Intrauterine ethanol exposure was shown to affect the development of vestibular and postural reflexes, sensory and motor coordination with significance at p˂0.05.Histological evaluation of the cerebral cortex revealed histopathological presentations such as pyknosis, karyorhexis, clumping of cells and neural degeneration. Histology of the cerebellar cortex showed degeneration and disorientation of purkinje cells. Histochemical evaluation of the cerebral cortex showed degradation of nissl substance. Moreover, the histochemistry of the cerebellar
TABLE OF CONTENTS
Declaration ………………………………………………………………………………………………….. ii
Certification ………………………………………………………………………………………………… iii
Dedication …………………………………………………………………………………………………… iv
Acknowledgements …………………………………………………………………………………………v
Table of Contents ……………………………………………………………………………………….. vii
List of Figures …………………………………………………………………………………………….. xii
List of Tables ……………………………………………………………………………………………… xii
List of Plates ……………………………………………………………………………………………… xiii
Abstract ……………………………………………………………………………………………………… xv
1.0 Introduction ……………………………………………………………………………………………..1
1.1 Background ………………………………………………………………………………………………1
1.2 Statement of Research Problem ………………………………………………………………….5
1.3 Significance of the Study ……………………………………………………………………………5
1.4 Justification ………………………………………………………………………………………………5
1.5 Scope of the Study …………………………………………………………………………………….6
1.6 Aim and Objectives of the Study …………………………………………………………………6
1.6.1 Aim of the study ………………………………………………………………………………………6
1.6.2 Objectives of the study ……………………………………………………………………………..6
2.0 Literature Review ……………………………………………………………………………………..8
2.1 Review of Related Literature ……………………………………………………………………..8
2.2 Foetal Alcohol Spectrum Disorders (FASD) ………………………………………………. 10
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2.2.1 Clinical background on FASD …………………………………………………………………. 10
2.2.2 Foetal Alcoholic Spectrum Disorder (FASD) is a wide-spread and costly disorder …………………………………………………………………………………………………………………… 12
2.2.3 Current treatments for FASD excluding biological mechanisms …………………….. 12
2.2.4 Difficulty in diagnosing FASD based on phenotypes …………………………………… 14
2.3 Ethanol Exposure Can Lead to Adverse Effects in the Foetus …………………….. 16
2.3.1 Ethanol‟s mechanism of action ………………………………………………………………… 16
2.3.2 Ethanol toxicity in the developing foetus …………………………………………………… 16
2.3.3 Binge drinking and its adverse effects on offspring ……………………………………… 17
2.3.4 Timing and dose-dependent effects of alcohol on neurodevelopment ……………… 18
2.4 Cerebellum …………………………………………………………………………………………….. 19
2.4.1 Morphology of the cerebellum …………………………………………………………………. 19
2.4.2 Cellular layers of the cerebellum ………………………………………………………………. 20
2.4.3 Functions of the cerebellum …………………………………………………………………….. 21
2.5 The Cerebrum ……………………………………………………………………………………….. 22
2.5.1 The morphology of the cerebrum ……………………………………………………………… 22
2.5.2 Cellular layers of the cerebral cortex …………………………………………………………. 23
2.5.3 Variations of cortical structure …………………………………………………………………. 26
2.5.4 Functions associated with cerebrum ………………………………………………………….. 27
2.6 Development of the Central Nervous System …………………………………………….. 28
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2.6.1 Pre-differentiation stage ………………………………………………………………………….. 28
2.6.2 Embryonic stage ……………………………………………………………………………………. 28
2.6.3 Development of the neural tube ……………………………………………………………….. 29
2.6.4 Start of neurogenesis………………………………………………………………………………. 29
2.6.5 Foetal stage…………………………………………………………………………………………… 30
2.6.6 Pre and post natal Stage ………………………………………………………………………….. 31
2.7 Teratogenicity ………………………………………………………………………………………… 31
2.7.1 Background on teratology ……………………………………………………………………….. 31
2.7.2 Principle of teratogenicity ……………………………………………………………………….. 32
2.7.3 Mechanisms of teratogenicity ………………………………………………………………….. 33
2.7.4 Transfer of teratogens across the placenta ………………………………………………….. 34
2.7.5 Site of action of teratogens ………………………………………………………………………. 35
2.7.6 Nutritional role in teratogenesis ……………………………………………………………….. 36
2.7.7 Genetic role in teratogenesis ……………………………………………………………………. 37
2.7.8 Effective dosage of teratogens …………………………………………………………………. 39
2.7.9 Effects of teratogens on DNA synthesis …………………………………………………….. 40
3.0 Materials and Methods ……………………………………………………………………………. 41
3.1 Materials ……………………………………………………………………………………………….. 41
3.2 Experimental Animals …………………………………………………………………………….. 41
3.3 Experimental Design ………………………………………………………………………………. 41
3.3.1 Route of ethanol administration and basis for the choice of the concentration 42
3.3.2 Cycling of the animals ……………………………………………………………………………. 43
3.3.3 Giemsa stain preparation …………………………………………………………………………. 46
3.3.4 Confirmation of pregnancy ……………………………………………………………………… 46
3.3.4 Ethanol Preparation ……………………………………………………………………………….. 46
3.3.5 Animal sacrifice…………………………………………………………………………………….. 47
3.3.6 Tissue processing …………………………………………………………………………………… 47
3.3.7 Routine paraffin sectioning ……………………………………………………………………… 47
3.4 Staining Methods ……………………………………………………………………………………. 48
3.4.1 Haematoxylin and Eosin (H & E) staining methods …………………………………….. 48
3.4.2 Cresyl fast violet staining (Nissl Stain) ……………………………………………………… 48
3.5 Atomic Absorption Spectrophotometry …………………………………………………….. 49
3.6 Morphometric Analysis …………………………………………………………………………… 50
3.7 Photomicrography ………………………………………………………………………………….. 50
3.8 Neuro-Behavioural Studies ……………………………………………………………………… 50
3.8.1 Surface righting reflex on postnatal day 5 (PND 5) ……………………………………… 51
3.8.2 Cliff avoidance (PND 6) …………………………………………………………………………. 53
3.8.3 Negative geotaxis (PND 7) ……………………………………………………………………… 55
3.9 Statistical Analysis ………………………………………………………………………………….. 57
4.0 Results …………………………………………………………………………………………………… 58
4.1 Physical Observation ………………………………………………………………………………. 58
4.2 Morphometric Analysis …………………………………………………………………………… 58
4.3Pre-weaning Neuro-Behavioural Battery Tests …………………………………………… 62
4.3.2Surface righting reflex …………………………………………………………………………….. 62
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4.3.3Cliff Avoidance ……………………………………………………………………………………… 62
4.3.3Negative geotaxis …………………………………………………………………………………… 62
4.5 Histologic Findings …………………………………………………………………………….. 64
4.5.1 Histology of the cerebral cortex ……………………………………………………………….. 64
4.5.2 Histology of cerebellar cortex ………………………………………………………………….. 65
4.5.3 Histochemistry of cerebral cortex …………………………………………………………….. 66
4.5.4 Histochemistry of the cerebellar cortex ……………………………………………………… 67
4.4 Atomic Absorption Spectrometry (AAS) ………………………………………………. 97
5.0 Discussion …………………………………………………………………………………………….. 101
5.1 Morphometric Analysis …………………………………………………………………………. 101
5.2 Histological Analysis ……………………………………………………………………………… 101
5.3 Trace Element Analysis Using Atomic Absorption Spectrophotometry (AAS) …………………………………………………………………………………………………………………. 104
5.4 Neuro-Behavioural Study ………………………………………………………………………. 108
6.0 Conclusion and Recommendations …………………………………………………………. 111
6.1 Conclusion……………………………………………………………………………………………. 111
6.2 Recommendations for Further Studies ……………………………………………………. 112
References …………………………………………………………………………………………………. 114
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CHAPTER ONE
1.0 Introduction
1.1 Background
Alcohol is the most widely used and abused psychoactive drug in many regions of Nigeria (WHO, 2004). Common slangs used include booze, bubbly, firewater, joy juice, sauce, liquid courage, and many others (NIAAA, 2000). Legal for those aged 18years and above in Nigeria (WHO, 2014) and the National legal Blood Alcohol Concentration (BAC) while driving is 0.05% (WHO, 2014). While there are many types of alcohol which is (an entire class of chemicals), the type that is found in drinks and medicines is known as „ethyl alcohol‟ or „ethanol.‟ A yeast enzyme changes the simple sugars that are found in grapes, potatoes, or corn into ethanol – the alcohol found in beer, malt liquor, wine, liquors such as vodka and whiskey, wine coolers, and liqueurs like Irish cream (NIAAA, 2000). One of the ways ethanol is thought to effect its toxicity is by the inhibition of folic acid uptake by the intestinal bacteria, and its metabolism in the liver. Folic acid is a well-known essential co-factor in the synthesis of purine and pyrimidine components of DNA and RNA, which are important in the formation of protein for normal development, growth, and repair of tissues (Adebisi, 2003a). Ethanol is the main ingredient in the three classes of alcoholic beverages: distilled spirit, wine and beer. Other forms such as methanol have immediate toxic effects that make them unsuitable for drinking (Adebisi, 2004). The ethanol concentration for common types of alcoholic drinks is as follows:
Beer: 4-6%, Malt liquor: 5-8%, Wine: 7-15%, Wine coolers: 5-10%, Champagne: 8-14%, Hard liquor (Distilled spirits – vodka, rum, whiskey, etc.): 40-95%, and Grain Alcohol: 95-97.5%. (NIAAA, 2000). A standard drink (wine and champagne) contains
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18 grams of pure ethanol – approximately the amount found in; Beer 12 Oz. (1 Can or Bottle), Wine 5 Oz. (1 Glass), Hard Liquor 1.5 Oz. (1 Shot) (NIAAA, 2014). Alcohol is widely recognized as a neuroteratogen (Allam and Abdulhamid, 2013; Moutard et al., 2012; Ohrtman, 2006; Maier and West, 2003; Sandra and Michael, 2003; and Thomas et al., 1998). Some women who drink heavily during pregnancy may have their children affected with alcohol-related deficits, such as neuroanatomical malformations (Alex and Feldmann, 2012; Clarren et al., 1978), cognitive dysfunction (Coles et al., 1991), or other behavioral disorders (Thanabhorn, 2006; Coles et al., 1985; Ernhart et al., 1985). Other reported observations include absence of skull vault, under-ossification and asymmetry of the constituent skull bones resulting in severe reduction of the cranial volume in rats (Adebisi, 2002a; Adebisi 2002b). Foetal alcohol syndrome (FAS) results from maternal consumption of alcohol during pregnancy and represents the extreme end of a continuum of foetal alcohol spectrum disorders (FASD). A diagnosis of FAS is made on the basis of three defining characteristics: central nervous system (CNS) damage or dysfunction, pre- and/or post-natal growth retardation with height or weight at/or below the 10th percentile, and distinct dysmorphic facial anomalies including a smooth philtrum, thin upper lip and small palpebral fissures (Jones and Smith, 1973). Growth impairment either antenatal, postnatal or both is a feature of FAS and a common finding in the mild effect called foetal alcoholic effects (FAE). Severe growth retarded children do not show significant accelerated growth following rehabilitation (Adebisi, 2002a; Adebisi 2002b).
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The most severe end of the spectrum is foetal alcohol syndrome (FAS) (Warren et al., 2001; Warren and Bast, 1988; Abel, 1984; Streissguth et al., 1980), for which facial abnormalities, growth deficits and central nervous system (CNS) abnormalities are the defining diagnostic features. Despite efforts to educate the public about FASD, the prevalence of ethanol consumption in women of child-bearing age has remained essentially the same (Caetano et al., 2006; CDC, 2004; NIAAA, 2000). Moreover, diminution in cranial and limb bone dimensions, body emaciation, low skeletal weights and decrease in size of ethanol-treated animals had been observed (Adebisi, 2004; Adebisi, 2003a; Adebisi, 2003b; Adebisi, 2000; Adebisi, 1995).
Some of the common traditional alcoholic beverages in Nigeria are: Burukutu which is a popular alcoholic beverage of a vinegar-like flavour prepared from sorghum grains and fermented guinea corn and consumed in the Northern Guinea savannah region of Nigeria (Haard et al., 1999). It is also typically consumed in the Ibadan region and ranges in alcohol content from 3–6% (Bennet et al., 1998). Burukutu is the most popular alcoholic beverage in the rural areas of northern Nigeria and in poor urban neighbourhoods because it is more affordable than commercially brewed beer. It is often consumed as food because it is thick and heavy. The producers of burukutu are overwhelmingly women (Obot, 2000). Palm wine is to Southern Nigerians what burukutu is to the Northerners. Unlike burukutu, it is not synthesised but obtained as a natural whitish sap collected in vessels attached to the base of the tree from where some leaves have been removed. Fresh wine from these sources is sweet and contains little alcohol but, with fermentation, the alcohol content increases with time. Unbottled palm wine has a lower alcohol content of around 3% than bottled palm wine which has around 4% (Stanley and Odejide, 2002). In general, palm wine, which has an alcohol
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content of 3–6%, is also widely consumed in the Ibadan region of Nigeria (Bennet,1998). The main alcoholic beverages produced and consumed by the Tiv people of Central Nigeria are tashi and ityo, also known as palm wine. Both alcoholic beverages contain nutrients rich in vitamins such as B and C found in ityo and complex carbohydrates in tashi. Akpetashi, a native gin to some Ghanians community, is distilled from tashi (Gire and Dimah, 2001). Pito is the traditional beverage of the Binis in the mid-western part of Nigeria. It is now very popularly consumed throughout Nigeria owing to its low price. Prepared from cereal grains mainly maize, sorghum or a combination of both, pito is a dark brown liquid which varies in taste from sweet to bitter. It contains lactic acid, sugars, amino acids and has an alcohol content of 3% (Haard, 1999). Emu common among the Yorubas in the Southwest part of Nigeria is produced from sugary palm saps. The most frequently tapped palms are raphia palms and the oil palm. It has an alcoholic content of around 5% (SCTD, 2004). Ogogoro (also known as kinkana and apetesi) is a gin-like drink distilled from oil or raffia palm wine. In Nigeria, distillation takes place in small sheds dotted along the coastal areas and in villages across the South. The end product is a clear liquid with alcohol content often higher than 40% (Obot, 2000). In the rural town of Igbo-Ora, guinea corn is malted and fermented to produce oti baba or oti’ka, with baba and ka being local names for the corn. There is also agadangidi, a fermented beverage made from mashed ripe plantain, fresh chili peppers and water (Mamman et al., 2002).
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1.2 Statement of Research Problem
Despite the fact that ethanol is known to have numerous toxic effects on humans, the level of alcohol abuse and alcoholism remain a very pressing issue throughout the world (WHO, 2014b). There are evident data on billions of Dollars being spent by developed countries on Foetal Alcohol Spectrum Disorder (FASD). In Canada, the annual cost of FASD amounts to 5.3billion Dollars (Stade et al., 2009). There are reports by numerous researchers on the use and misuse of alcohol by women in different parts of Nigeria (WHO, 2014b). Alcohol is also known to have toxic effects on nervous system and ethanol is known to have potency in inducing teratogenicity (Allam and Abdulhamid, 2013). However, some proportion of pregnant women still take alcohol either in moderate or in binge drinking pattern (Allam and Abdulhamid, 2013).
1.3 Significance of the Study
Children of alcoholic women are exposed to the potential risk of ethanol-induced disorders as a result of ethanol ingestion during pregnancy, as such, this study will create awareness on the risks associated with alcohol intake during pregnancy with emphasis on the nervous system. The result of the study will provide scientific data on the pattern of teratologic effects of ethanol as a function of timing and dosage on nervous tissues.
1.4 Justification
There are evidences that ethanol has toxic effects on the nervous system of adult mice and rats, but little is known of its prenatal effects on the nervous system. The study seeks to provide information on prenatally administered effects of ethanol on the
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developing cerebrum and cerebellum. The study outcome could serve as the basis on which further studies could be carried out.
1.5 Scope of the Study
The study was on the histological and histochemical changes in the developing cerebrum and cerebellum as a result of prenatal ethanol exposure. Haematoxylin and Eosin (H and E) and Cresyl Fast Violet stains were employed in the study of the tissues. Atomic Absorption Spectrophotomery was also conducted to quantify multi-elements in the tissues of the experimental animals.
1.6 Aim and Objectives of the Study
1.6.1 Aim of the study
The aim of the study is to evaluate the histology, histochemistry, neuro-behaviour and quantify the amount of trace elements on the cerebellar and cerebral cortices of noenatal Wistar rats following intrauterine ethanol exposure.
1.6.2 The objectives of the study are to:
i- Determine the morphometric variables of the pups by measuring the crown rump length and weight of the pups.
ii- Assess the neurobehavioral effects of ethanol on the pups prior to weaning by using cliff avoidance, surface righting and negative geotaxis reflexes methods.
iii- Study the teratologic effects of ethanol on the histology of cerebral and cerebellar cortices of the pups using the routine Haematoxylin and Eosin stain.
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iv- Investigate the histochemical changes in the cerebral and cerebellar cortices of the pups using Cresyl Fast Violet to demonstrate nissl substance.
v- Study the effects of intra-uterine ethanol exposure on the concentration of trace elements (Zn, Cu, Fe and Mn) present in the noenatal brain tissue using Atomic Absorption Spectrophotometry.
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