|Year : 2021 | Volume
| Issue : 6 | Page : 1228-1233
Aluminium induced neurodegeneration in rat cerebellum in the presence of ethanol coexposure
Buddhadeb Ghosh1, Ravi Kant Sharma1, Suman Yadav2
1 Department of Anatomy, Government Medical College, Amritsar, Punjab, India
2 Department of Anatomy, Dr Rajendra Prasad Government Medical College, Kangra, Himachal Pradesh, India
|Date of Submission||09-May-2021|
|Date of Decision||17-May-2021|
|Date of Acceptance||19-May-2021|
|Date of Web Publication||10-Nov-2021|
Department of Anatomy, Government Medical College, Amritsar, Punjab
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction: Both aluminium and ethanol are pro-oxidants and neurotoxic. Moderately intake of alcohol may favor the body in coronary heart disease and diabetes mellitus etc. Being cheaper aluminium and increasing consumption of alcohol in India mixed with each other and may induce neurotoxicity. The present study was planned to identify the level of aluminium induced neurodegeneration in presence of ethanol coexposure in the cerebellum. Materials and Methods: An experimental study was carried out at Dr. RP Government Medical College, Kangra, and Government Medical College, Amritsar, India after due approval from the Institute Animal Ethics Committee. Thirty-two Wistar rats were divided into one vehicle control and three experimental groups. Group I received the normal saline water as the vehicle control group. Group II received aluminium chloride 4.2 mg/kg body weight as the experimental group. Group III received ethanol 1 g/kg body weight as the experimental group. Group IV received both aluminium chloride 4.2 mg/kg body weight and ethanol 1 g/kg body weight as the experimental group. After 3 months of treatment, cerebellum was processed for histopathological observation under the microscope. Results: Experimental group treated with aluminium and ethanol separately showed reduction in the number of Purkinje cells, without a prominent nucleolus and well-defined nuclear membrane. Eosinophilic swelling adjacent to Purkinje cell bodies observed. The effects of combined administration of aluminium ethanol treated groups showed with acute neurodegeneration of Purkinje cell layer and granular layer. Pyknosis and neurofibrillary tangle seen in Purkinje cells. Conclusions: It has been suggested that the ethanol-induced the effects of aluminium on the cerebellum and plays a significant role in neurotoxicity.
Keywords: Aluminium, cerebellum, ethanol, neurodegeneration, neurotoxicity, Purkinje cells
|How to cite this article:|
Ghosh B, Sharma RK, Yadav S. Aluminium induced neurodegeneration in rat cerebellum in the presence of ethanol coexposure. J Pharm Bioall Sci 2021;13, Suppl S2:1228-33
|How to cite this URL:|
Ghosh B, Sharma RK, Yadav S. Aluminium induced neurodegeneration in rat cerebellum in the presence of ethanol coexposure. J Pharm Bioall Sci [serial online] 2021 [cited 2022 Aug 16];13, Suppl S2:1228-33. Available from: https://www.jpbsonline.org/text.asp?2021/13/6/1228/330114
| Introduction|| |
The brain is a susceptible organ for both aluminium (Al) toxicity and oxidative injury. Neurotoxicity caused by Al exposure is well evidenced and one of the suggested possible mechanisms of which is oxidative injury. In the 70s of the last century, the medical fraternity considered the toxicity of, so-called “biologically inert” metal, aluminium. A large volume of research, since then, is conducted to get insight and fight against the Al toxicity. While the awareness about the toxic effects is bringing down the daily and avoidable uses of the Al in developed countries, being the cheap, Al wares are the commonly used cooking utensils and containers in India. Neurotoxic effects of Al are well-established and specific effects on the human central nervous system such as memory loss and impaired coordination are documented. It is known to increase the permeability of blood-brain barrier and traverses it which results in the concentration of Al in the hippocampus, cortex, and corpus callosum.,, Aluminium neurotoxicity may play an important role in the development of anxiety disorders, depression, and memory deficit in mice, these alterations may also play an important role in the development of Alzheimer's diseases in the Al treated animals.
The cerebellum is a part of hindbrain plays an important role to perform the sensory and motor function. It is coordinate all the muscle tone and motor activities with its connection with the cerebrum. Its main function is to maintain equilibrium and balance. The cerebellum contains the large number of neuron cells, but it takes up only ten percent of brain volume. The cortex of the cerebellum having three layers outer molecular layer contains stellate and basket cell and the dendrons of Purkinje cells and middle Purkinje layer having Purkinje cells and inner granular layer contain granule cells and Golgi cells and axons of the Purkinje cells. Stellate and basket cells form GABAergic synapses onto Purkinje cell dendrites.
Ethanol has many effects in the nervous system and reproductive organs. It is also noted that moderate alcohol consumption increase the HDL level and reduces the cholesterol level in the blood and reduces the risk of stroke and stress, anxiety and tension, Alzheimer's disease. Moderate alcohol consumption and risk of coronary heart disease among women is documented. It is also noted that moderate alcohol consumption lowers the risk of type 2 diabetes mellitus. Consumes large amounts of alcohol can result in acute and delayed impairments in cognitive and executive functions, spatial learning, and memory impairment. These impairments lead to medical and social problems including dementia, violence, and decreased work productivity. General neurotoxicity produced by Al is not modified by ethanol. However, the Al load caused by Al exposure may be influenced by ethanol coexposure., Consumption of alcohol is suggested to increase the susceptibility of rats to certain effects of Al but it is also noted that moderate beer consumption, possibly affording a protective factor for the toxic effect of Al.,,
Davis hypothesized about the role of Al on the alcohol-related neurodegeneration and suggested the altered pharmacokinetics of Al when coexposed with ethanol. However, it is found that the available reports are inconclusive to evidently suggest any alteration in terms of pharmacokinetics and toxicokinetic. However, co-occurrence of Al and alcohol exposure could compromise the compensatory changes in the brain in addition to the enhanced toxicity. The dependency of neuropathological risk factors on the local oxidant status for their effectuality has been reviewed. While Dlugos CA suggested that ethanol might be a positive inducer of age-related deterioration of neuronal cells and Tripathi et al. showed aging could markedly influence the Al-induced oxidative stress, neuropathological and neurobehavioral toxicity., Thus, the oxidant status of the brain has been evaluated in Al exposure along with graded doses of ethanol coexposure. Although the brain is the primary organ affected by Al toxicity, only a few studies have described its effect on the structure of the cerebellum. Furthermore, the use of ethanol against Al toxicity needs to be investigated.
The aim of the study was to identify the effects of Al administration on the microscopic structure of the cerebellum with the concomitant administration of ethanol.
| Materials and Methods|| |
An experimental study was carried out at Dr. RP Government Medical College, Kangra and Government Medical College, Amritsar, India after due approval from the Institute Animal Ethics Committee, Registration no 668/02/C/CPCSEA. Thirty-two Wistar rats include the equal number of males and females of an average age of 120 days and the average weight of 200 g used in this study. After 1 week of acclimatization, rats were randomly divided into four groups containing 8 animals (4 male and 4 female) with the help of Random Allocation software version 1.0, May 2004. Animals were kept in the air-conditioned animal house and fed with diet pellets and filtered tap water.
- Group I received the normal saline water as the vehicle control group
- Group II received aluminium chloride 4.2 mg/kg body weight as the experimental group
- Group III received ethanol 1 g/kg body weight as the experimental group
- Group IV received both aluminium chloride 4.2 mg/kg body weight and also ethanol 1 g/kg body weight as experimental group.
The doses were calculated according to the body weight and given 10 ml/kg body weight through oral feeding (gavage) once daily for periods of 90 days. Their weights were recorded daily. After 3 months, the animals were anesthetized with chloroform and intracardiac perfusion of normal saline followed by 10% formaldehyde was performed. The brains of all groups of animals were extracted on an ice-chilled tray. After sectioning, the cerebellum of all animals was processed for routine paraffin embedding.
Formalin-fixed whole brain was carefully dissected to isolate the cerebellum. Cerebellum was stained with hematoxylin and eosin (H and E) staining, Periodic acid Schiff (PAS) staining and Bielchowsky silver staining according to John D Bancroft Theory and Practice of Histological Techniques. The stained slides were labeled properly and placed under light microscope obtained with a digital camera attached to the microscope for observation.
| Results|| |
The cerebellum of vehicle control (Group I) showed that the normal architecture with H and E staining in [Figure 1]a and [Figure 1]b and PAS staining in [Figure 2]a and [Figure 2]b and modified Bielchowsky Silver staining in [Figure 3]a. The outer molecular layer contains small scattered stellate and basket cells. The Purkinje cell layer contains Purkinje cells, are arranged in one row which have a pyriform-shaped cell body with prominent nucleoli and cytoplasm with their dendrites towards the molecular layer. The granular layer shows tightly packed small rounded cells.
|Figure 1: Cerebellum of control group in (a: ×100), (b: ×400) and aluminium group in (c: ×100) and (d: ×400) and ethanol group in (e: ×100) and (f: ×400) and aluminium ethanol group in (g: ×100) and (h: ×400). ML: Molecular layer, PL: Purkinje layer, GL: Granular layer. Normal Purkinje cell (arrow) in (b). Necrotic Purkinje cells (arrow) in (d, f and h). Eosinophilic swelling (arrow) in (f). Scale bar = 100 μm and 20 μm. H and E stain|
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|Figure 2: Cerebellum of the control group in (a: ×100), (b: ×400) and aluminium group in (c: ×100) and (d: ×400) and ethanol group in (e: ×100) and (f: ×400) and aluminium ethanol group in (g: ×100) and (h: ×400). ML: Molecular layer, PL: Purkinje layer, GL: Granular layer. Normal Purkinje cell (arrow) in (b). Degenerated Purkinje cells (arrow) in (d, f and h). Eosinophilic swelling (green arrow) in (f). Scale bar = 100 μm and 20 μm. PAS stain|
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|Figure 3: Photomicrograph of the cerebellum from the control group in (a: ×400) and aluminium treated group in (b: ×400) and ethanol-treated group in (c: ×400) and aluminum ethanol treated group in (d: ×400). ML: Molecular layer, PL: Purkinje layer, GL: Granular layer. Normal Purkinje cell (arrow) in (a). Necrotic Purkinje cells (arrow) in (b and c). Neurofibrillary tangle (arrow) in (d). Scale bar = 20 μm. Bielchowsky silver staining|
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The aluminium treated (Group II) showed neurodegeneration of the cerebellum. Purkinje cells were observed without a prominent nucleolus and well-defined nuclear membrane in [Figure 1]c and d with H and E staining. Pyknosis of Purkinje cells were observed in [Figure 2]c and [Figure 2]d with PAS staining. The outer molecular layer showed few scatter cells and morphological changes and reduced number of Purkinje cells noted in [Figure 3]b with silver staining. The ethanol-treated (Group III) showed with neurodegeneration of Purkinje cells. Purkinje cells were observed without a prominent nucleolus or well-defined nuclear membrane. Eosinophilic swelling adjacent to Purkinje cell bodies observed in [Figure 1]e and [Figure 1]f with H and E staining and [Figure 2]e and [Figure 2]f with PAS staining. Apoptotic cell death of Purkinje cells seen in [Figure 3]c with Bielchowsky silver staining.
The aluminium ethanol combined treated (Group IV) showed acute neurodegeneration and necrosis of the cerebellum. Reduced number of Purkinje cells observed without nucleus and many dead cells noted in the molecular layer, pyramidal layer and granular layer in [Figure 1]g and [Figure 1]h with H and E staining. Purkinje cells degeneration seen in [Figure 2]g and [Figure 1]h with PAS staining. Pyknosis and neurofibrillary tangle of Purkinje cell seen in [Figure 3]d with Bielchowsky silver staining.
| Discussions|| |
In this study, Al exposure resulted in acute neurodegeneration of Purkinje cells. Eosinophilic swelling seen adjacent to Purkinje cell bodies. Purkinje cells were observed without a prominent nucleolus and well-defined nuclear membrane. Pyknosis of Purkinje cells was also observed. Morphological changes of the Purkinje cells and deposition of neuritic plaques seen. Disorganization of the Purkinje cell layer with loss of the Purkinje cells was previously reported with Al exposure. The few observed Purkinje cells in this study showed a darkly stained cytoplasm and dark (pyknotic) nuclei. Pyknosis was described as irreversible condensation of chromatin in the nucleus of the cell undergoing programmed cell death or apoptosis. Histological alterations in the cerebellum found in this study after Al administration could be explained by increased oxidative stress. This is based on the fact that Al is known to possess pro-oxidant activity. Al induces oxidative stress. All types of brain cells have been reported to be affected by the oxidative stress induced by Al. On exposure to oxidative stress a variety of oxidative products released in the neurons, which include malondialdehyde, carbonyls, peroxynitrites, nitrotyrosines, and enzymes like superoxide dismutase, hemoxygenase-I.
In this study, prominent eosinophilic swelling was observed in the Purkinje layer around the granule cells. To our knowledge, no previous study reported the presence of such swelling. However, vacuoles were described earlier around pyramidal cells in the cerebral cortex but not in the cerebellum after Al exposure. Granule cells showed increased condensation of nuclear chromatin inside their nuclei and areas of vacuolated cytoplasm, which might be a feature of apoptosis. Some previous studies agree with the present findings. These studies showed that the granule cells are specific targets for Al neurotoxicity. They explained that Al toxicity may occur because of impairment of the glutamate nitric oxide cyclic GMP pathway in neurons. It was suggested that the degenerated Purkinje cells fail to establish contact with the granule cells, leading to the lack of normal synchronism between them, impairing their regulatory role. It was concluded that Al adversely affected the cerebellum by a triad of gross morphological, histological, and histomorphometric changes. These alterations are dangerous due to heavy human exposure to Al in the environment.
The present study demonstrated that consumption of moderate dose of ethanol for 3 months decreased the count of Purkinje cells in most of the lobules of the cerebellum. In a previous study, it was observed that there was 20%–25% reduction in the total number of Purkinje cells per midline section in rats exposed to ethanol for 20 weeks. Chronic intake of ethanol alters cell membrane fluidity by complex neurochemical adaptive changes, and neurochemical alteration of the cell is important for ethanol dependence. Purkinje cells use gamma-aminobutyric acid (GABA) as a neurotransmitter. It has been also noted that GABA receptor structure and function change during ethanol exposure. In Purkinje cells, the toxicity is related to alcohol-induced mitochondrial damage with cytochrome release from mitochondria, caspage 3 activation and apoptosis of targeted neuronal cells. In my previous study, it was also observed that early chronic ethanol exposure reduces the number of neuronal cells in the cerebellum.
Most previous studies focused on the individual toxic effects of a single chemical; however, there is a possibility that humans and animals can be exposed to a mixture of toxic agents.
In co-exposed rats, cerebellum damages were significantly increased when compared to the individual toxic effects of aluminium or acrylamide. These toxicants exhibited synergism which was also supported by histopathological studies. Alteration in the Purkinje cell layer after Al exposure may cause changes in motor coordination and changes or loss of motor behavioral activities. It is established that Purkinje cells send inhibitory projections to the deep cerebellar nuclei, and they constitute the sole output of motor coordination in the cerebellar cortex.
However, when the animals were coexposed to ethanol and Al, the brain tissue became more vulnerable, especially with the higher doses of ethanol. Thus, the chances of neurotoxicity increase when the subjects are being exposed to both Al and ethanol than either one of these neurotoxicants alone. It has been already proven that ethanol is a causative agent for brain oxidative stress while aluminium is not directly involve in the oxidative stress or response coexposure of Al with pro-oxidant might favor the development of Al-induced oxidative stress in the cerebrum., The chances of neurotoxicity increase when the subjects are being exposed to both Al and ethanol than either one of these neurotoxicant alone. Ultra-structurally, mitochondria of some Purkinje cells appeared damaged with destroyed cristae; other mitochondria showed dilated cristae. Some authors reported that myelin figure formation is a sign of degeneration. Moreover, the present data demonstrated that chronic ethanol exposure induces Al toxicity in the cerebellum.
| Conclusions|| |
Present results showed that aluminium and ethanol both are neurotoxicant. Most importantly, acute neurodegeneration of Purkinje cells, pyknosis, and neurofibrillary tangle seen in combined exposure of aluminium ethanol treatment. It has been suggested that the ethanol induced the effects of aluminium on the cerebellum and plays a significant role in neurotoxicity.
The authors would like to thank the Asst Director, I/C Central Lab Animal House and Sr. Lab Technician, Department of Anatomy, Dr. RP Government Medical College, Kangra India to carry out this project in the Institute. The authors wish to thankfully acknowledge the support received from Department of Pharmacology and department of Pathology, Government Medical College, Amritsar, India to carry out the work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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