Calcium Supplementation Ameliorates Cerebellar Oxidative Stress in Lactational Aluminum-induced Neurotoxicity in Rats

Abstract

Introduction: The neurotoxic effects of aluminum exposure during the critical period of neurodevelopment have been well documented. This study investigated the known protective effects of calcium supplementation on the cerebellum of juvenile Wistar rats following aluminum-induced neurotoxicity during lactation.

Methods: Four groups of juvenile rats were exposed via lactation to distilled water (control group), aluminum (40 mg/kg/d), calcium supplement (50 mg/kg/d), and a combination of both aluminum and calcium from postnatal day 4 to day 28. The cerebella of the animals were excised to access the levels of antioxidant enzymes (superoxide dismutase [SOD], glutathione peroxidase [GPx]), lipid peroxidation (malondialdehyde), histomorphological alterations (hematoxylin and eosin staining), Nissl profile (cresyl fast violet staining), and glial activation (glial fibrillary acidic protein immunohistochemistry).

Results: Lactational aluminum significantly decreased the activities of superoxide dismutase and glutathione peroxidase while exacerbating lipid peroxidation and reactive astrocyte in cerebellar lysates. Lactational calcium supplementation normalized the activities of SOD and GPx, thereby preventing excessive lipid peroxidation and glial activation. Despite no apparent changes in the general histology of the cerebellum, aluminum-induced chromatolysis changes in the Purkinje cell layer, which was counteracted by the antioxidant propensities of calcium supplementation.

Conclusion: These findings support that calcium supplementation significantly protects the cerebellum against aluminum-induced oxidative stress, chromatolysis, and neuroinflammation.

Keywords: Aluminum; Astroglia; Calcium; Cerebellum; Glial activation; Lactation; Oxidative stress.

Figure 1.

Superoxide dismutase activity The activity of superoxide dismutase (SOD) in the cerebellum of juvenile rats shows a significant reduction in SOD level in group B compared to other groups, while group C shows an increase in SOD level compared to group B (P<0.05). A, control group; B, aluminum chloride group; C, calcium supplementation group; D, aluminum chloride + calcium group. ^* and ^** are significant values at P<0.05 and P<0.01, respectively.

Figure 2.

Glutathione peroxidise activity The cerebellar level of glutathione peroxidase (GPx) enzyme in juvenile rats. A, control group; B, aluminum chloride group; C, calcium group; D, aluminum chloride + calcium group. GPx activity was significantly reduced in group B relative to other experimental groups (P<0.05), with the highest level of GPx seen in the calcium-treated group (C); the difference was significant between groups C and B (P<0.05), and between groups C and A (P<0.05), but not group D. ^* is the significant value at P<0.05.

Figure 3.

Lipid peroxidation Cerebellar levels of lipid peroxidation in rats as shown by the level of malondialdehyde (MDA). A, control group; B, aluminum chloride group; C, calcium group; D, aluminum chloride + calcium group. MDA level was highest in group B with significant differences compared to the control and other treated groups, while the level was least in the calcium-treated group (C) with significant differences compared with the control and other treated groups. ^* and ^** are significant values at P<0.05 and P<0.01, respectively.

Figure 4.

Histology and nissl profile of the cerebellar cortex Representative photomicrographs of the general histology (H&E: A-D) and Nissl staining (CFV: E-L) of the cerebellar cortex show the molecular layer (ML), Purkinje cell (yellow circles), Purkinje cell layer (PCL) and Granule layer (GL). A, E, and I, control; B, F, and J, AlCl3-induced; C, G, and K, calcium supplementation group; D, H, and L: AlCl3+calcium group.

Figure 5.

Immunoreactivity of astrocytes Immunohistochemical demonstration of astroglia using anti-rat-glial fibrillary acidic protein (GFAP) across the layers of the cerebellar cortex in rats. The molecular cell layer (ML), Purkinje cell layer (black arrows), and granule cell layer (GL) were demonstrated across the study groups. GFAP immunopositive cells (red arrows) in the control (A) appeared sparse around neurons and between layers, with regular processes, distribution, and sizes within the neuropil. However, increased astrocytic densities with reactive astroglia within the granule cell layer and hypertrophic cells appeared within the cerebellar layers in aluminum-treated rats (B). The cerebellar cortex of rats co-treated with aluminum and calcium (D) had close similarities with those in the control (A) and calcium supplementation (C) groups. In groups A and C, astrocytic processes, cellular distribution, and size appeared normal (GFAP x400).

Figure 6.

Immunoreactive astrocytes count Immunopositivity for anti-glial fibrillary acidic protein (GFAP). A, Control group; B, Aluminum chloride group; C, Calcium supplementation group; ;D, Aluminum chloride + Calcium group. ^** is significant at P<0.01.