Histology is the study of the tissue sectioned as a thin slice using a

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1 Histopathology Histology is the study of the tissue sectioned as a thin slice using a microtome. The photography of stained cell is called histography. The architectural dynamics of the tissue is very essential for maintaining the tissue integrity and for effective physiological, biochemical and metabolic functions. The knowledge of histopathology is useful to distinguish normal cells from abnormal or diseased one and helps in diagnosis of many diseases (Majumdar, 1980). Neuro-histopathology is an important tool in neuro-anatomical pathology of brain tissue since accurate diagnosis of nervous diseases usually requires histopathological examination of samples. Neurohistopathology is a critical part of the study involved in toxicologic and risk assessment of food, drugs, chemicals and medical devices which facilitates the sensitive and efficient evaluation of large amount of microscopic information (James et al., 2004). As high quality tissue specimens are necessary for histopathological evaluation, tissues must be promptly and appropriately fixed by immersion (commonly in ne utral buffered formalin) (Bancroft and Cook, 1994; Luna, 1968). Histopathology helps in diagnosing the tissue damages of an animal subjected to toxic stress (Jayantha Rao, 1982). Even though bio-chemical studies give an idea of the pathological state of the animal, a clear picture of cytoarchitectural changes produced during chemical intoxication can be traced by histopathological studies (Rajendra Prasad, 2007). Toxic Effect Aluminium on Nervous tissue Neurotoxicity refers to the ability of an agent to adversely affect the structural or functional integrity of the nervous system. Alterations in nervous 91

2 system may occur through toxicant interactions. Neurons have differentially vulnerability to certain neurotoxicants because of their functional characteristics. The neuropathology helps in understanding of chemical injury to the nervous system and also ensures the public safety (OECD, 2003). The adult brain has been able to consistently retain an ongoing ability to modify neural circuits in response to circumstances occurring during individual life span (Bertoni-Freddari et al., 1996). Aluminium is omnipresent and this nonessential metal exposure may lead to body burden. The use of aluminium in water purification, industry, food processing, cookwares and in medicine is a constant threat to human health, as the bioaccumulation of this metal may lead to metabolic interferences and many other deleterious effects (Eichhorn, 1993). Aluminium (Al) compounds are considered extremely neurotoxic (Griffioen et al., 2004). Animals overloaded with Al displayed a selective depletion in the hippocampal formation, especially in the CA1 field and also in the cerebral cortex (Exley, 1999). Animals supplemented with routine dietary aluminium have shown changes in behavioural responses such as impaired performance in learning and memory tasks, impaired concentration and behavioral changes including confusion and repetitive behaviors (Walton, 2007, 2009). Bishop et al. (1997) have shown that parenteral exposure to as little as 20 μg/kg b.wt of aluminium for >10 days may result in long- term detrimental outcomes in neurologic development in preterm infants. Melatonin role in neuroprotection The neuroprotective role of melatonin was demonstrated in animal models of neurodegeneration where the indole group in its structure acts as a potent antioxidant 92

3 protecting neurons and glia against the damage caused by free radicals (Jou et al., 2004). Melatonin is considered as a protector of cell and sub-cellular organelles because of its priming effects involved in the enhancement of antioxidant defense system (Reiter et al., 2001b). Melatonin is known to readily cross the blood-brain barrier and prevent the neuronal loss in subjects of Alzheimer s disease (AD). Several experimental evidences suggest the ability of melatonin in treating degenerative diseases related to CNS (Cheung, 2003). Central nervous system function analysis with the help of behavioral studies has shown that melatonin may also improve locomotor activity and reduce the severity in AD patients (Brusco et al., 1998; Cohen-Mansfield et al., 2000) by maintaining the healthy neuronal organization. The present study was conducted to examine the protective effects of melatonin against histological changes induced by aluminium acetate. Histological studies of nervous tissue in the present investigation were carried out, in an attempt to illustrate the cytoarchitectural changes in different brain regions viz. cerebral cortex, hippocampus, medulla oblongata and cerebellum of albino mice. These four regions of brain are considered as vital centers for the maintenance and coordination of various physical, psychological and physiological functions. Results A. LIGHT MICROSCOPY Histology of mice cerebral cortex The cerebral cortex is the largest part of the vertebrate and is the source of neural transactions that enhance memory, plasticity, cognition, speech and intellectual activity. The cerebral cortex consists of two major types of neurons, granule or stellate cells and pyramidal cells. Granule cells have small structure with 93

4 clear nuclei and without any projections and act as intracortical neuronal cells. The pyramidal cells are large in structure with prominent nucleus, nucleolus, axon and many dendritic spines. The cytoarchitectural structure of cerebral cortex is characterized by the presence of six layered laminated pattern of cells. 1 st layer The molecular layer consists of mostly glial cells, axons of neurons of other layers and very few neurons. 2 nd /3 rd layer This layer is less distinct combination of two layers containing small pyramidal cells and granule cells 4 th layer Granular layer which is rich with stellate and granule cells which receive input to the cortex from thalamocortical fibers, association fibers and commissural fibers. 5 th layer Internal pyramidal layer containing cells known as giant pyramidal cells. 6 th layer Multiforme layer which consists of pleotrophic cells or martinotti. Microphotograph of control mouse cerebral cortex shows with neuroglial cells (NG), pyramidal neuron (PYNEU) with centrally placed nucleus (N) surrounded by cytoplasm triangular in shape with a prominent axon (AX) and associated dendrites and traversing neurofibrillary meshwork (NFM) which completely fill the background of the cortex of mice (Plate 4.1 and Fig.A). Aluminium treated mouse cortex showed degenerated pyramidal neuron (DGPYNEU), pyknosis of nuclei (PN) in the pyramidal neuronal cell, discontinuity in the neuro fibrillary network (DCNF), degenerated neuroglial cells (D GNG), loss of neuronal processes (LNP) and increased spaces with the vacuolation (V) (Plate 4.1 and Fig.B). 94

5 Under melatonin administration the cortex has pyramidal neuron (PYNEU) with centrally placed nucleus (N), axon (AX) associated as neuronal process, neuroglial cells (NG) and granule cell (GC) (Plate 4.1 and Fig. C). In aluminium plus melatonin treated mouse cortex the cytoarchitectural changes were remarkably more distinct, which include slight degeneration of pyramidal neuron (SDGPYNEU), neuroglia l cells (NG) and good architecture of nerve fibers(nf) (Plate 4.1 and Fig. D). Histology of mice Hippocampus The hippocampus is a brain region which lies under the medial temporal lobe one on each side of brain and is part of limbic system. Hippocampus plays significant role in the formation of long-term memories. Hippocampus is grouped with nearby structures including dentate gyrus and is called hippocampal formation. The hippocampal formation is bilateral structure sand- witched between the cortex and the thalamus. Hippocampal formation consists of hippocampus proper, the dentate gyrus, the subicular complex and the fornix. Hippocampus proper (Cornu Ammonis) is subdivided into four main cytoarchitectural fields namely CA 1, CA 2, CA 3 and CA 4 that are unidirectionally connected from CA 4 to CA 1. CA 2 and CA 4 are small and not well defined. The neurons of hippocampus have spatial firing fields called Place cells. Pyramidal cells are present in CA 3 and CA 1 regions. Some important anatomical features of hippocampus are as follows. 1) Dentate gyrus possesses 1.2 million granule cells, 4K basket cells 32 K hilar interneurons and 20 K mossy cells. 95

6 2) CA 3 subfield has16 x 10 3 Pyramidal cells and CA 1 consists of 250 x 10 3 Pyramidal cells. Together CA 3 / CA 1 have 330K / 420K pyramidal cells and various interneurons. 3) Subiculum possesses around 180 K cells. Hippocampal formation is formed of Enthorhinal cortex, dentate gyrus, CA 3, CA 1 and Subiculum. Microphotograph of control hippocampus shows characteristic curvature of hippocampus, Cornu Ammonis layer (CA 1 ) with compactly arranged pyramidal cells (PYNEU) in between stratum oriens (SO) and stratum radiatum (SR). Between the nerve cells a thick neuro fibrillary net work (NFN) known as neuropile is present (Plate 4.2 and Fig. E). Remarkable changes were viewed in aluminium administered mouse hippocampus with severe necrotic changes in neuro fibrillar network (SNCNFN), degenerative changes in pyramidal neuronal cells (DGCPYNEU), pyknosis of neuronal cells (PN) and vacuolated spaces (V) are observed (Plate 4.2 and Fig.F). Melatonin treated mouse hippocampus has a clear pyramidal neuronal cells (PYNEU), neurofibrillary network (NFN) with clear cytoarchitecture and inter - neuronal cells (INEU) (Plate 4.2 and Fig. G). The cellular changes were less prominent in hippocampus of mice coadministered with aluminium and melatonin. The changes include slight vacuolation (SV), slight congestion in blood vessel (SCBV), a layer of pyramidal neuron (PYNEU) and integrity of the neuronal cells was maintained with help of neurofibrillar network (NFN) (Plate 4.2 and Fig. H). 96

7 Histology of mice Cerebellum One of the most impressive parts of the brain is cerebellum, located at the lower back of the brain and is more rapidly acting mechanism than any part of the brain. The cerebellum is not only involved in skilled motor performances but also involved in various sensory functions including sensory acquisition, discrimination, tracking, prediction etc. The three functional regions of cerebellum are vestibulocerebellum, spinocerebellum and cerebrocerebellum. The cerebellum can be divided into three cortical layers with the same basic neuronal circuitry everywhere which involve five main cell types as follows: 1) Outer Molecular layer neuroglial cells 2) Middle Purkinje layer- Purkinje cells(largest neurons) 3) Inner Granule layer - Granule cells Microphotograph of control mouse cerebellum shows outer molecular layer (OML), neuroglial cells (NG) also known as basket cells are present in OML, middle layer called as purkinje cell layer (MPKCL) containing purkinje cell (PKC), inner granule cell layer (IGCL) and white matter (WM) containing mossy fibers and climbing fibers (Plate 4.3 and Fig.I). The cytoarchitectural changes of aluminium treated mice cerebellum were necrosis of neuroglial cells (NNG) and diffuse gliosis (DFGLS) in molecular layer, loss of integrity among the granule cells (LIAGC) in the inner granular cell, degenerated purkinje cells (DGPKC) and marked loss of purkinje cells (LPKC) in the middle purkinje layer (Plate 4.3 and Fig.J). 97

8 Cerebellum of melatonin treated mice has shown clear cellular architecture consists of sparsely occupied neuroglial cells (NG) in outer molecular layer (OML), middle purkinje cell layer (MPCL), inner granular cell lay er (IGCL) and white matter (WM) (Plate 4.3 and Fig.K). The histopathological changes in aluminium plus melatonin treated mice cerebellum showed integrity among granule cells which is maintained within granular cell layer (GCL) inspite of Al inducing toxic effects, slight degeneration of purkinje cell (SDPKC) in middle purkinje cell layer, outer molecular layer (OML) containing neuroglial cells and white matter (Plate 4.3 and Fig.L) Histology of mice Medulla Oblongata Medulla oblongata is the lowermost portion of the vertebrate brainstem and functions as relay station for the crossing of motor tracts between the spinal cord and the brain. Medulla oblongata is responsible for autonomic functions such as respiration, blood pressure, heart rate, swallowing, vomiting, defecation, gagging, coughing etc. The cross section of control mouse medulla oblongata shows longitudinally arranged long axon pyramidal motor neuronal cells (MNEU) and nerve fibres (NF) between the nerve cells bodies (Plate 4.4 and Fig.M). In the mice group treated with aluminium, medulla oblongata showed severe hemorrhagic areas (SHA), congested blood vessel (CBV), numerous cystic spaces showing vacuolation (V), karyopyknosis of nucleus (KN) and loss of architectural details and degeneration of nerve cell bodies (LNEU) (Plate 4.4 and Fig. N). The cytoarchitectural changes in melatonin treated mice medulla oblongata included clear architectural details, motor neuron (MNEU), centrally placed nucleus (N) and nerve fibers (NF) (Plate 4.4 and Fig.O). 98

9 Under co-administered aluminium and melatonin treated mice medulla oblongata the neurohistopathological changes were less prominent showing only mild degenerative changes in neurofilament network (MDGCNFN), centrally placed nucleus (N) in motor neuron (MNEU) (Plate 4.4 and Fig.P). B. ELECTRON MICROSCOPY An electron microscope is scientific instrument that use a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield topography, morphology, composition and crystallographic information. The transmission electron microscope (TEM) was the first type of electron microscope to be developed and is patterned exactly as the light microscope except that a focused beam of electrons is used instead of light to See through the specimen. It was developed by Max knoll and Ernst Ruska in Germany in Transmission electron microscopy also has a well established role in the characterization of sub-cellular structural alterations in tissues which have been modified by the effects of xenobiotics. Electron microscopy provides a static morphological assessment of cells to characterize changes in sub-cellular organelles, can provide valuable information about any functional deficits. Despite the technological advances in transmission electron microscopy, it is highly selective and only small samples of tissues can be examined. So that appropriate and defined objectives are selected and examined within the context of the study. The electron microscope (EM) permits a direct study o f biological ultra structure. Its resolving power is much greater than that of the light microscope. In spite of the apparent similarities there are great differences between the light and the 99

10 EM. In case of EM, molecules or supramolecular structures are now possible to obtain more detailed information ( Bozzola and Russell, 1999; Aughey and Frye, 2001). In the present investigation an attempt has been made to study the ultra structural changes in different brain regions and the analysis was carried to elucidate the alterations especially with respect to the cell bodies, its processes and their synaptic relations with other neural elements within the nucleus. Ultra structural study of mice cerebral cortex In control mice cerebral cortex showed a clear nucleus (N), heterochromatin along the nuclear membrane (HE), mitochondria (M) and golgi complex (GC) (Plate 4.5 and Figs. A& B). The ultrastructural alterations in aluminium acetate treated mice cerebral cortex revealed condensed chromatin within the nucleus (CC), vacuolation (V), discontinuity in cell membrane (DCM), distorted dendritic structure (DDS), discontinuity in nuclear membrane (DNM), degenerated neuronal cells, microglial cell (MG), foamy inclusions (FI), lipofuscin pigment (LP), swollen golgi (SG) and swollen mitochondria (SM) (Plate 4.6 and Figs. C& D). Melatonin treated mice cerebral cortex shown to consists of nucleus (N), cell membrane (CM), mitochondria (M), endoplasmic reticulcum (ER), neuroglial cell (NG), soma (SO), longitudinal section of axon (LSAX), node of ranvier (NR) and axodendritic synapse (ADS) (Plate 4.7 and Figs. E& F). Electron micro photograph of aluminium + melatonin treated mice Cerebral cortex showed nucleus (NU), nuclear membrane (NM) and cell membrane (CM), 100

11 astrocyte (AS), dendrite (DEN), neuron (NEU), microglial cell (MG) with golgi complex (GC) and dense cytoplasm (DC) (Plate 4.8 and Figs. G & H). Ultra structural study of mice Hippocampus Ultra-structural examination of control mice hippocampus shows a clear pyramidal neuron (PYNEU) with nucleus (N), mitochondria (M), synapse (S) and synaptic vesicles (SV) (Plate 4.9 and Figs. I & J). Aluminium acetate treated mice hippocampus showed condensed chromatin (CC), nucleus (N), vacuolation (V) and lipofuscin pigment (LP) (Pla te 4.10 and Figs. K & L). Electron micrograph of melatonin treated mice hippocampus showed synapse (S) and nucleus (N) (Plate 4.11 and Figs. M& N). Electron microscopic observation of aluminium + melatonin treated mice hippocampus showed mitochondria (M), nuclei (N), synapse (S), myelin sheath (MY) and clear cross section of pyramidal neuron (CSPYNEU) (Plate 4.12 and Figs. O & P). Ultra structural study of mice Cerebellum Conspicuous nuclei (N) within the neuron, cross section of granule cells (CSGC) and densely occupied supporting granular cell of cerebellum (DOGCCB) were observed in control mice cerebellum (Plate 4.13 and Figs. Q& R). Ultra structural observations of aluminium acetate treated mice cerebellum revealed condensed chromatin within the nucleus (CC), vacuolation (V), degenerated purkinje cell (DGPC), degenerated axon (DGAX) and degenerated granular cells(dgc) (Plate 4.14 and Figs. S & T). 101

12 Melatonin treated mice cerebellum displayed the nucleus (N), axon (AX), purkinje cell (PC) and densely occupied supporting granule cells (DOGCCB) (Plate 4.15 and Figs. U& V). In aluminium + melatonin treated mice cytoarchitectural changes revealed intact nucleus (N) and nucleolus (NU) associated with mild degeneration in granular layer of cells (MDGCCB) and purkinje cell (PC) (Plate 4.16 and Figs. W& X). Ultra structural study of mice Medulla Oblongata In control mice medulla oblongata the electron microscopic observations showed the cell with clear nucleus (N), blood vessel (BV), cross section of motor cell neuron (CSMNEU) and neuroglial cell (NGC) (Plate 4.17 and Figs. Y& Z). Aluminium acetate treated mice medulla oblongata showed degenerated nerve fibers (DGNF) and complete loss of motor cell neurons (LMCNEU) (Plate 4.18 and Figs. Z 1 & Z 2 ). The observations of melatonin treated mice medulla oblongata showed nerve fibers (NF), clear cross section of neuron (CSNEU) and axon (AX) (Plate 4.19 and Figs. Z 3 & Z 4 ). Electron micro photograph of aluminium + melatonin treated mice medulla oblongata showed neuroglial cells (NGC) and cross section of nerve fibers (NF) (Plate 4.20 and Figs. Z 5 & Z 6 ). Discussion Extensive cell death in the central nervous system is present in all neurodegenerative diseases. The type of nerve cell loss and the particular part of the brain affected describes the symptoms associated with an individual disease (Waters, 1994). Chronic exposure to aluminium has been reported to cause 102

13 behavioural, neuropathological and neurochemical changes (Kumar and Gill, 2009; Ribes et al., 2010; Sethi et al., 2008). The present study has clearly revealed the cytoarchitectural changes in different regions of mice brain administered with aluminium, melatonin and aluminium plus melatonin. The neuro-histopathological changes were more pronounced in mice brain regions treated with aluminium acetate than those of melatonin and aluminium plus melatonin administered mice. This may be due to the toxic effects of aluminium and its ability to bind structural proteins and enzymes in neurons. As a result of this binding the normal structure and activities of the neuron become impossible and the cell body undergoes changes from its original histological structure. There have been many experimental studies on animals and on isolated cells showing that aluminium has toxic effects on the nervous system. In 1991, Guy et al. showed that the aluminium uptake in human neuroblastoma cells displayed an epitope similar to Alzheimer's diseases. However, several studies document that pharmacological treatment with melatonin could significantly prevent premature ageing and delays the onset of various degenerative disorders of central nervous system (Poeggeler et al., 1993). The changes in cell dynamics of aluminium treated mice cortex showed changes such as degeneration of pyramidal neuron, pyknosis of nuclei in the pyramidal neuronal cell, discontinuity in the neuro fibrillary network, degenerated neuroglial cells, loss of neuronal processes and increased spaces showing the vacuolation were observed in light microscopic studies (Plate 4.1: F ig.b). In the electron microscopic observations aluminium treated mice group showed condensed chromatin with in the nucleus, vacuolation, discontinuity in cell membrane, distorted dendritic structure, discontinuity in nuclear membrane, degenerated neuronal cells, 103

14 microglial cell, foamy inclusions, lipofuscin pigment, swollen golgi and swollen mitochondria (Plate 4.5: Figs A&B). This is in agreement with Deloncle et al. (2001) who demonstrated that chronic administration of young rats with Al displayed cell vacuolation, mitochondrial swelling demyelination and increased accumulation of lipofuscin. The neurotoxic property of aluminium also contributes to degeneration of neurons similar to ageing (Suarez Fernandez et al., 1999). Phosphorylation induced damage of neurofilaments in cerebral cortex was observed in rats exposed to aluminium (Kaur et al., 2006). Bihaqi et al. (2009) has shown that AlCl 3 produces cerebral lesions associated with degenerative changes such as vacuolated cytoplasm, hemorrhage, ghost cell and gliosis. Flaten (2001) found that aluminium has a relationship with cerebral diseases and syndromes of dialysis dementia. Melatonin normalized the severity of damage to the brain cerebral cortex in aluminium induced melatonin treated mice group the cytoarchitectural changes were remarkably more distinct under light microscope, which include slight degeneration of pyramidal neuron, neuroglial cells and good architecture of nerve fibers were clearly visible (Plate 4.1: Fig D). The degree damage resultin g from aluminium intoxication to the cerebral cortex was attenuated by melatonin is also observed in electron microscopy and labeled as nucleus, nuclear membrane and cell membrane, astrocyte, dendrite, neuron, microglial cell with golgi complex and dense cytoplasm (Plate 4.8: Figs. G&H). Pei et al. (2003) have found that a single dose of melatonin was able to reduced brain infarction up to 2 hr after the onset of transient focal cerebral ischemia. Regrigny et al. (2001) demonstrated that there was a marked reduction in 104

15 size of cerebral arteriolar wall thickness in melatonin deficient rats, suggesting a prominence of melatonin in protecting the arterial wall mass. Administration of melatonin alone shown to preserve the cytoarchitecture of the cerebral cortex the histological section consists of pyramidal neuron with centrally placed nucleus, axon as neuronal process, neuroglial cells and granule cell (Plate 4.1: Fig.C). Ultra structure is shown to consist of nucleus, cell membrane, mitochondria, endoplasmic reticulcum, neuroglial cell, cytosoma of the adjacent cell, longitudinal section of axon, nodes of ranvier and axodendritic synapse (Plate 4.7: Figs. E&F). Exposure to Al resulted in marked histopathological alterations both at light and electron microscopic levels in the hippocampus which were represented by necrotic changes in neuro fibrillar network, degenerative changes in pyramidal neuronal cells, pyknosis of neuronal cells and vacuolated spaces at light microscopic level (Plate 4.2: Fig.F). Electron mic roscopic observation revealed condensed chromatin, vacuolation and lipofuscin pigment accumulation (Plate 4.9: Figs. I&J). Sun et al. (1999) have demonstrated the presence of granulovacuolar degenerations (GVD) in hippocampus. However, the incidence of GVD per 300 nerve cells was observed with increase in dosage of aluminium. Matyja, (2000) noticed alterations within the hippocampus such as neuronal degeneration, perikaryal cytoplasm and gliosis following aluminium administration. Rodella et al. (2001) found that cortical nitroxidergic neurons and granule cells are very sensitive to the toxic effects of Al. Chronic aluminium intoxication in rats resulted in dose-dependent morphological changes. In brain, the appreciable neurodegenerative changes were observed in hippocampus. The changes were seen as spongioform changes in 105

16 pyramidal layer, nuclear deformity and presence of vacuoles in the nuclei. The effects of Al induced neurofibrillary degeneration are quite similar to those in patients with Alzheimer's disease (Somova et al., 1997). Melatonin was observed to play a beneficial role in reducing the neuropathology in hippocampus of mice brain that arises from aluminium induced damage. Under light microscopy, cellular changes were less prominent and the changes include slight vacuolation, slight congestion in blood vessel, a layer of pyramidal neuron and integrity of the neuronal cells was maintained with help of neurofibrillar network inspite of aluminium toxicity (Plate 4.2: Fig H). The ultrastuctural sections consisted of mitochondria, nuclei, synapse, myelin sheath and clear cross section of pyramidal neuron(plate 4.12: Figs. O&P). Uz et al. (1996) reported that melatonin can protect hippocampal neurons in adult rats against kainite induced neuronal damage. Ozdemir et al. (2005) reported that melatonin plays an important role in protecting the morphological integrity of the pyramidal neuronal cells of hippocampus and also found to restore the spatial memory deficits in animals after induction of head trauma. Melatonin is found to prevent the nerve cell loss in both CA1 and CA3 regions of hippocampus. Melatonin treated mouse showed that hippocampus has a clear pyramidal neuronal cells, neurofibrillary network with clear cytoarchitecture and inter-neuronal cells (Plate 4.2: Fig.G). Electron micrograph of melatonin treated mice hippocampus is illustrated in Plate 4.11: Figs. M &N and labeled as synapse and nucleus. Animals exposed to aluminium acetate was shown to cause changes in the cellular structures of cerebellum such as necrosis of neuroglial cells and diffuse gliosis in molecular layer, loss of integrity among the granule cells in the inner 106

17 granular cell and marked loss of purkinje cells (Plate 4.3: Figs. I &J). Electron microscope studies have shown condensed chromatin within the nucleus, vacuolation, degenerated purkinje cell, degenerated axon and degenerated granular cells (Plate 4.19: Figs. S&T). These observations are in agreement with Nehru et al. (2007). The histological alterations induced by high tissue aluminium levels of brain are demyelination, brain ventricle dilatation and thinning of the corpus callosum (Lapresle et al., 1975), marked neuronal injury, depletion in density of neuronal cell bodies, loss of neuronal integrity, degenerative changes like pyknosis, vacuolization, chromatin condensation (Varner et al., 1998), degeneration of neurons with striking similarity to that observed in AD, clusters of neural filaments in neuron from the spinal cord and brainstem (Terry and Pena, 1965), degenerating neuronal cytoskeleton, damage to axons (Troncoso et al., 1982), degenerating granule cells, degeneration of the Purkinje cells, proliferation of glial cells, nerve cell bodies with shrunken nuclei and dark cytoplasm (Ghetti et al., 1985; Yokel, 1994). Under light microscopic observation, the histological changes in aluminium plus melatonin treated mice cerebellum showed integrity among granules cells which was maintained within granular cell layer inspite of Al inducing toxic effects, slight degeneration of purkinje cell of middle purkinje cell layer, inner layer of white matter which was placed below granular cell layer and outer molecular layer consisting of neuroglial cells (Plate 4.3: Fig. L). Under transmission electron microscopic studies, cytoarchitectural changes showed intact nucleus and nucleolus associated with mild degeneration in granular layer of cells and purkinje cell (Plate 4.16: Figs. W&X). 107

18 Giusti et al. (1995) have shown that co-treatment of melatonin with kainite protected primary cultured cerebellar granule neuronal cells effectively which is shown by the reduced death of neurons from undergoing death against exicitotoxicity induced by kainite. Cagnoli et al. (1995) showed that melatonin provides protection of cerebellar neurons in vitro against photosensitive dye rose bengal induced oxidative stress. Cerebellum of melatonin only treated mice has shown clear cellular architecture consists of sparsely occupied neuroglial cells in outer molecular layer, middle purkinje cell layer, inner granular cell layer and white matter (Plate 4.3: Fig.K). Ultra semi-thin sections of melatonin treated mice cerebellum displayed the nucleus, axon, purkinje cell and densely occupied supporting granule cells (Plate 4.15: Figs. U&V). Morphological changes were much prominent in aluminium treated mice medulla oblongata in both light and electron microscopic analysis. From the results by light microscopy the pathological changes such as severe hemorrhagic areas, congested blood vessel, numerous cystic spaces showing vacuolation, karyopyknosis of nucleus, loss of architectural details and degeneration of nerve cell bodies were observed (Plate 4.4: Fig. N). The results obtained from ultrastructural observation of medulla oblongata were shown degenerated nerve fibers and complete loss of motor cell neurons (Plate 4.17: Figs. Y&Z). This may be due to Al accumulation in the medulla oblongata of brain. Nehru and Bhalla, (2006) reported that aluminium induces the cellular damage in different brain regions including medulla oblongata after the administration of aluminium orally at a dose of 40mg/kg b.wt/day for a period of six weeks. 108

19 Light photomicrograph of medulla oblongata in mice were co-administered with aluminium and melatonin, the neurohistopathological alterations were less prominent showing only mild degenerative changes in neurofibrillary network, centrally placed nucleus in motor neuron (Plate 4.4: Fig. P). Electron micrograph has shown the structures such as neuroglial cells and cross section of nerve fibers (Plate 4.20: Figs. Z 5 & Z 6 ). Rao et al. (2011) demonstrated that melatonin ameliorated the pathological changes in medulla oblongata of rat brain from mercury induced toxicity. Kaur et al. (2011) showed that administration of the melatonin (10mg/kg) before and after the hypoxia improved ultrastructural abnormalities in medulla oblongata induced by hypoxia, suggesting its therapeutic efficacy in reducing hypoxia-associated brainstem damage. Histomorphology of melatonin only treated mice medulla oblongata is represented with clear architectural details such as motor neuron, centrally placed nucleus with in the neuron and nerve fibers (Plate 4.4; Fig.O). The observations of ultra structure of melatonin treated mice medulla oblongata show nerve fibers, clear cross section of neuron and axon (Plate 4.19: Figs. Z 3 & Z 4 ). In conclusion, the analysis by both light and electron microscopy reveals that aluminium administration can result in Al accumulation in cells and different tissues of brain. Thus from the above observations, supplementation of melatonin can aid in repair of damaged neurons by restoration of neuronal synthesis, synaptic activity, prevents mitochondrial changes and nuclear alterations. These observations reveal that Al induces neurotoxicity and melatonin has a potential to prevent Al induced effects. This study suggests that melatonin s antioxidant functions presumably help in preserving neurons from mutilation and death. 109