Effect of different doses of aluminum chloride on neurodegeneration in hippocampus region of the rat brain

Amit Massand; Mallika Basera; Sonal Grace; Reshma Kumarachandra; K Sudha; Rajalakshmi Rai; BV Murlimanju; K Sowndarya

doi:10.4103/jasi.jasi_39_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 71 | Issue : 4 | Page : 307-310

Effect of different doses of aluminum chloride on neurodegeneration in hippocampus region of the rat brain

Amit Massand¹, Mallika Basera², Sonal Grace², Reshma Kumarachandra², K Sudha², Rajalakshmi Rai¹, BV Murlimanju¹, K Sowndarya²
¹ Department of Anatomy, Kasturba Medical College Mangalore, Manipal Academy of Higher Education, Manipal, India
² Department of Biochemistry, Kasturba Medical College Mangalore, Manipal Academy of Higher Education, Manipal, India

Date of Submission	03-Mar-2022
Date of Decision	30-Jul-2022
Date of Acceptance	13-Sep-2022
Date of Web Publication	01-Dec-2022

Correspondence Address:
Dr. K Sudha
Department of Biochemistry, Kasturba Medical College Mangaluru, Manipal Academy of Higher Education, Mangaluru - 575 004, Karnataka
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jasi.jasi_39_22

Abstract

Introduction: Aluminum (AL) compounds are widely used as food additives, cosmetics, antacids, and buffered aspirins. Chronic consumption of AL may lead to its accumulation in tissues causing AL toxicity. The study aims to investigate the toxic effect of AlCl₃ on hippocampus region of rat brain by qualitative and quantitative analysis of neurons. Material and Methods: Adult male albino Wistar rats were divided into three groups with six rats in each group. Group 1 was the control, Group 2 rats received 100 mg/kg b. w, and Group 3 received 300 mg/kg b. w of AlCl₃ orally for 30 days. The neuronal count was done at the CA1, CA2, CA3, and CA4 regions of hippocampus by staining with cresyl violet stain. Neuronal damage in the AlCl₃ groups was compared with the control group. Results: A significant damage was observed in all the regions of hippocampus both in Groups 2 and 3 compared to the control group (P < 0.00001). Further higher dose of AL caused marked neuronal damage in CA1 (P < 0.03) and CA3 (P < 0.05) regions compared to the lower dose of AL. The neurons in the CA3 and CA1 regions were most vulnerable to AL toxicity and the CA2 region of the hippocampus had a maximum number of viable neurons indicative of resistance to AL toxicosis. Discussion and Conclusion: Consumption of higher dose of AL even for a short term could have variable degrees of deleterious effects on different regions of the rat brain. This study sets a background for an in-depth exploration on toxicology of AL compounds on human participants which could be of public health importance.

Keywords: Aluminum toxicity, CA2 region, CA3 region, hippocampus, neuronal degeneration

How to cite this article:
Massand A, Basera M, Grace S, Kumarachandra R, Sudha K, Rai R, Murlimanju B V, Sowndarya K. Effect of different doses of aluminum chloride on neurodegeneration in hippocampus region of the rat brain. J Anat Soc India 2022;71:307-10

How to cite this URL:
Massand A, Basera M, Grace S, Kumarachandra R, Sudha K, Rai R, Murlimanju B V, Sowndarya K. Effect of different doses of aluminum chloride on neurodegeneration in hippocampus region of the rat brain. J Anat Soc India [serial online] 2022 [cited 2023 Mar 27];71:307-10. Available from: https://www.jasi.org.in/text.asp?2022/71/4/307/362553

Introduction

Human beings are overexposed to aluminum (AL) these days as this metal exists as a component of food additives, cosmetics, antiperspirants, cooking utensils, paints, and buffered aspirin. According to the WHO, the tolerable intake is 1 mg/kg body weight/day.^[1] Chronic consumption of AL may lead to its accumulation in the bones, kidneys, and brain causing AL toxicity. Several researchers have detected elevated content of AL in the brains of patients with Alzheimer's disease (AD), pointing at its role in the pathogenesis of neurological disease. Since the hippocampus region of the brain is critical for learning and memory, it is most vulnerable to damage in AD.^[2] Al can cross blood–brain barrier and cause inflammation in the brain which may lead to loss of memory. Several neurodegenerative disorders such as Parkinson's disease and multiple sclerosis is attributed to AL accumulation in hippocampus and frontal cortex of the cerebrum.^[3],[4] Intestinal absorption of AL salts in rats is rapid through the oral route, and the bioavailability may go up to 0.21%.^[5] Administration in high dose leads to its accumulation in the brain, especially to a greater extent in cortex and hippocampus.^[6] Hence, more research is warranted to confirm the direct correlation between concentration and duration of exposure to AL with neurotoxicity. This study was undertaken to evaluate neurotoxicity of two different doses of AL chloride (100 and 300 mg/Kg body weight/day for 30 days) on four areas of hippocampus region of the rat brain by qualitative and quantitative analysis of neurons.

Material and Methods

The present study was carried out on in-house bred male albino Wistar rats aged about 3–4 months, weighing between 200 and 250 gm. Rats were given ad libitum access to laboratory food and drinking water. The animals were housed in a polypropylene cage with a paddy husk bedding in controlled temperature, light and dark cycle (12:12 h), humidity (50% ±10%), and pathogen-free environment. The study was approved by the Institutional Animal Ethics Committee (KMC/MNG/IAEC/05-2019 on February 15, 2019). All procedures performed in the study were in accordance with the guidelines provided by the Indian government for the usage of laboratory animals.^[7]

The albino Wistar rats were divided into three groups with six rats in each group. The grouping details are as follows:

Group 1: Rats were given food and water for 30 days. This served as a control group
Group 2 (Al 100): Rats orally received 100 mg/kg body weight/day of AlCl₃ for 30 days
Group 3 (Al 300): Rats orally received 300 mg/kg body weight/day of AlCl₃ for 30 days.

AlCl₃ was procured from Sigma-Aldrich, USA, of the highest analytical grade. The solubility of AlCl₃ is 458 g/L at 20°C. The AlCl₃ was dissolved in distilled water at a final concentration of 300 mg/Kg b. w (1/10 LD50). Stock solution was prepared by dissolving 2 g of AlCl₃ in 20 ml of distilled water. This was administered orally at a dose of 0.5 ml/100 g b. w using gavage in one bolus.

Histological study

After 30 days of AlCl₃ administration, all the animals were sacrificed and the brain was perfused with cold phosphate buffer saline and 10% formalin and then stored in 10% formalin. Subsequently, paraffin blocks were prepared, 6–7 μm thick sections of the tissue were taken using a microtome, and the sections were processed with different grades of alcohol and xylene, and stained with 0.1% cresyl violet acetate solution. A minimum of six histological sections were taken from each region of each rat. The different regions of hippocampus were demarcated based on the topographical location of the dentate gyrus. The viable neurons were identified in CA1, CA2, CA3, and CA4 regions of both sides of the hippocampus by VUE software and counted using the imaging software NIS-Elements (Br version 4.30).^[8],[9] The cell count was expressed as the number of cells per unit length of the cell field (cells/300 μm length). A single person was involved in the counting of neurons at three different 300 μm areas of the same region to avoid interobserver bias. The average of the three readings was considered to prevent intraobserver bias. The neuronal cell count was compared between the three groups. The histopathological changes were examined using Nikon trinocular microscope (H600 L) under ×20 to evaluate the neuromorphological changes in the hippocampus. The percentage of neuronal loss was calculated as mean number of neurons in control sections minus the mean number of neurons in the treated section divided by the mean number of neurons in the control section multiplied by 100.^[10]

All the groups were statistically analyzed using the one-way analysis of variance followed by Tukey's post hoc test to compare between the different groups. The experimental data were represented as mean ± standard deviation with six rats in each group. The P < 0.05 was considered to be statistically significant.

Results

A significant decrease in healthy neurons was observed in rats fed with AL chloride compared to controls (P < 0.00001). The neurons in which the cell membrane was disrupted and those showing peripherally placed pyknotic nuclei or without nuclei were considered as degenerated neurons. Neuronal counts in four regions of hippocampus revealed a significant decline in healthy neurons in the CA3 followed by CA1, CA4, and CA2 in both Group 2 and Group 3 rats compared to the normal group [Table 1]. The histological study also depicts significant neurodegeneration in the CA3 and CA1 regions of hippocampus among all regions (CA1, CA2, CA3, and CA4) of the rat brains in the AL groups [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]. Neurodegeneration was almost double in CA1 and CA4 regions while it was four times higher in the CA3 region of hippocampus in rats fed with 300 mg/kg b. w of AL chloride compared to those fed with 100 mg/kg b. w. However, neurodegeneration was at the same level in these two groups in the CA2 region [Figure 1]. The CA3 region was most vulnerable to AL toxicity and a higher dose of AL was more effective in the induction of neurodegeneration. Compared to the control group, CA1 and CA3 regions were more sensitive to AL toxicity compared to other regions of the hippocampus.

Table 1: Comparison of neuronal count at 300 μm area of different regions of hippocampus at the same topography between control and aluminum-treated groups

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Figure 1: Comparison of neurodegeneration in different regions of hippocampus with the two doses of aluminum

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Figure 2: The photomicrographs of CA1 subfield of hippocampus stained with cresyl violet. (Red arrows indicate normal neurons and black arrows indicate degenerated neurons)

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Figure 3: The photomicrographs of CA2 subfield of hippocampus stained with cresyl violet. (Red arrows indicate normal neurons and black arrows indicate degenerated neurons)

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Figure 4: The photomicrographs of CA3 subfield of hippocampus stained with cresyl violet. (Red arrows indicate normal neurons and black arrows indicate degenerated neurons)

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Figure 5: The photomicrographs of CA4 subfield of hippocampus stained with cresyl violet. (Red arrows indicate normal neurons and black arrows indicate degenerated neurons)

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Discussion

AL toxicity has multidimensional effects such as generation of free radicals, reduction in antioxidant defense, prevention of DNA repair, change in protein conformation, and inhibition of enzymes such as Na, K-ATPase, and inflammatory reactions.^[11] There is growing evidence to suggest that intraneuronal accumulation of metal ions such as AL, arsenic, lead, and nickel also leads to mitochondrial changes decreasing ATP production and cell death.^[12] In humans, the accumulation of AL in the brain has been associated with neurodegenerative diseases such as AD, Parkinson's disease, and multiple sclerosis.^[3],[4] AL intake promotes oxidative stress and amyloid deposition in nervous tissue which leads to neuronal necrosis and dysregulated neurogenesis.^[13] The memory loss in neurodegenerative disorder is attributed to the apoptosis of cortical neurons due to the accumulation of Al in the hippocampus and frontal cortex of the cerebrum.^[10] AL chloride administered in male Wistar rats for 1 month was found to increase the rate of protein and lipid damage in the brain.^[14] The histological changes observed in the present study prove that Al induces neuronal damage in the hippocampus region of the rat brain. Rats administered with AL (both doses) showed prominent morphological alterations in all four regions of hippocampus when compared to controls. The neurons in the CA3 subfield were most vulnerable to AL toxicity and a higher dose of AL was more effective in the induction of neurodegeneration. Compared to the control group, CA1 and CA3 regions were more sensitive to AL toxicity with the highest percentage of neurodegeneration compared to other regions of the hippocampus. Earlier studies have revealed that chronic administration of low dose of Al mimics natural aging process in rats.^[15] Chiroma et al.^[16] confirmed neurodegenerative changes in CA1 and CA3 subregions of hippocampus in rats administered with 200 mg/Kg/day of AlCl₃ for 70 days which is in agreement with the findings of our study. Morphologically, administration of 300 mg of caused major structural changes in the cytoarchitecture such as an increase in number of pyknotic cells and alteration of pyramidal cell arrangements. By the way of cresyl violet staining, a significant number of degenerative cells were observed in the CA3 region of hippocampus in rats administered with higher dose of AL compared to a lower dose (100 mg/kg/day), which only highlights the vulnerability of neurons of this region of the brain to AL toxicity which is consistent with previously published data. Kumar et al.^[17] reported that rats fed with 100 mg/kg/day of AlCl₃ for 60 days showed neuronal damage only in the CA3 region, whereas the same result was observed in our study within 30 days. Enas and Khalil^[18] demonstrated dementia in rats when (300 mg/kg/day) was administered for a month, which supports our study.

Conclusion

In the current investigation, animals exposed to 300 mg of AL have displayed a higher degree of neuronal necrosis in hippocampus which is the site for memory formation. Hence, the research outcome suggests that rats fed with 300 mg of AL/kg/day for 1 month could be the best choice for the study of AD-related pathologies. These findings set a background for an in-depth exploration on toxicology of AL compounds as it is of public health importance.

The limitation of the study is that the tissue penetration of AlCl₃ could not be demonstrated due to the lack of facility involving fluorescein-based fluorescence detection.

Acknowledgment

The author would like to thank technical staff for their support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1.	Tietz T, Lenzner A, Kolbaum AE, Zellmer S, Riebeling C, Gürtler R, et al. Aggregated aluminium exposure: Risk assessment for the general population. Arch Toxicol 2019;93:3503-21.
2.	Gupta VB, Anitha S, Hegde ML, Zecca L, Garruto RM, Ravid R, et al. Aluminium in Alzheimer's disease: Are we still at a crossroad? Cell Mol Life Sci 2005;62:143-58.
3.	Nampoothiri M, John J, Kumar N, Mudgal J, Nampurath GK, Chamallamudi MR. Modulatory role of simvastatin against aluminium chloride-induced behavioural and biochemical changes in rats. Behav Neurol 2015;2015:210169.
4.	Abu-Taweel GM, Ajarem JS, Ahmad M. Neurobehavioral toxic effects of perinatal oral exposure to aluminum on the developmental motor reflexes, learning, memory and brain neurotransmitters of mice offspring. Pharmacol Biochem Behav 2012;101:49-56.
5.	European Food Safety Authority. Statement of EFSA on the evaluation of a new study related to the bioavailability of aluminium in food. EFSA J 2011;9:2157.
6.	Baydar T, Papp A, Aydin A, Nagymajtenyi L, Schulz H, Isimer A, et al. Accumulation of aluminum in rat brain: Does it lead to behavioral and electrophysiological changes? Biol Trace Elem Res 2003;92:231-44.
7.	Singh AP. Government of India notifies the rules for breeding of and conducting animal experiments. Indian J Pharmacol 1999;31:92-5.
8.	Joy T, Rao MS, Madhyastha S. N-Acetyl cysteine supplement minimize tau expression and neuronal loss in animal model of Alzheimer's disease. Brain Sci 2018;8:185.
9.	Standring S. Cerebral cortex. In: Gray's Anatomy. 39^th ed., Ch. 22. Spain: Elsevier, Churchill Livingstone; 2006. p. 407.
10.	Chiroma SM, Mohd Moklas MA, Mat Taib CN, Baharuldin MT, Amon Z. D-galactose and aluminium chloride induced rat model with cognitive impairments. Biomed Pharmacother 2018;103:1602-8.
11.	Maya S, Prakash T, Madhu KD, Goli D. Multifaceted effects of aluminium in neurodegenerative diseases: A review. Biomed Pharmacother 2016;83:746-54.
12.	Alasfar RH, Isaifan RJ. Aluminum environmental pollution: The silent killer. Environ Sci Pollut Res Int 2021;28:44587-97.
13.	Igbokwe IO, Igwenagu E, Igbokwe NA. Aluminium toxicosis: A review of toxic actions and effects. Interdiscip Toxicol 2019;12:45-70.
14.	Jyoti A, Sethi P, Sharma D. Bacopa monniera prevents from aluminium neurotoxicity in the cerebral cortex of rat brain. J Ethnopharmacol 2007;111:56-62.
15.	Roig JL, Fuentes S, Teresa Colomina M, Vicens P, Domingo JL. Aluminum, restraint stress and aging: Behavioral effects in rats after 1 and 2 years of aluminum exposure. Toxicology 2006;218:112-24.
16.	Chiroma SM, Hidayat Baharuldin MT, Mat Taib CN, Amom Z, Jagadeesan S, Adenan MI, et al. Protective effect of Centella asiatica against _D-galactose and aluminium chloride induced rats: Behavioral and ultrastructural approaches. Biomed Pharmacother 2019;109:853-64.
17.	Kumar P, Bairy KL, Nayak V, Reddy SK, Kiran A, Ballal A. Amelioration of aluminium chloride (AlCl3) induced neurotoxicity by combination of Rivastigmine and Memantine with artesunate in albino wistar rats. Biomed Pharmacol J 2019;12:703-11.
18.	Enas KA. Study of possible protective and therapeutic influence of coriander (Coriandrum sativum L.) against neurodegenerative disorders and Alzheimer's disease induced by aluminum chloride in cerebral cortex of male albino rats. Nat Sci 2010;8:202-13.