Neuroprotective role of vitamin D in primary neuronal cortical culture

Abstract

Background: A role of Vitamin D in brain development and function has been gaining support over the last decade. There are compelling pieces of evidence that suggest vitamin D may have a neuroprotective role. The administration of vitamin D or its metabolites has been shown to reduce neurological injury and/or neurotoxicity in a variety of animal systems. The detail biochemical mechanism mediating neurons, to its ability to withstand greater oxidative stress in the presence of Vitamin D is unclear. This study was undertaken to study the biochemical effect of treatments of primary cortical neuronal cultures, with the active form of vitamin D(1,25(OH)₂D3), against the induced oxidative stress.

Methods: Primary neuronal cultures from cerebral cortex were set up from neonatal (from 6 to 7 days old) Wister Rat's brain. Different doses of [1,25(OH)₂D₃], ranges from 0 to 1 μg/ml, was added to the culture medium and the cells were cultured in its presence for 24 h to 120 h. The effect of induced extracellular oxidative stress was measured by subjecting these cultured cells with 0.5 mM H₂O₂ for 2 h, prior to collection of condition medium and the cell pellet for biochemical assay. The control and H₂O₂ treated cultures were maintained in similar culture conditions, for similar periods of time without any [1,25(OH)₂D₃] treatments.

Result: The optimum concentration of [1,25(OH)₂D₃] for treatment of primary cortical neuronal cultures was found to be 0.25 μg/ml by Trypan exclusion assay and MTT assay. Pre-treatments of cultured neuronal cells with 0.25 μg/ml of [1,25(OH)₂D₃] caused significantly increased levels of reduced glutathione, accompanied by a similar increase in the enzyme levels of GST, to neutralize the induced oxidative stress by H₂O₂. The level of Lipid peroxidation was significantly higher in the cells treated with H₂O₂ alone, but it was completely reversed in the neuronal cultures pre-treated with [1,25(OH)₂D₃]. The levels of Catalase enzyme also significantly reduced (≥0.05) in the [1,25(OH)₂D₃] pre-treated neuronal cultures.

Conclusion: We concluded that the systemic treatment of primary neuronal cultures with [1,25(OH)₂D₃] gave better protection to neurons against the induced oxidative stress, as shown by quantitative measurements of various biomarkers of oxidative stress. This study also suggested that Vitamin D is vital for the growth, survival, and proliferation of the neurons and hence it has a potential therapeutic role against various neurodegenerative diseases.

Fig. 1

Effect of Different doses of Vitamin D treatments on the viability of primary cortical neuronal cells after induced oxidative stress by 0.5mMH₂O₂ for 2 h after growing them in the presence and absence of [1,25(OH)₂D₃] for 24 h, 48 h and 72 h. The cell viability was measured either by MTT reduction for 24 h, 48 h and 72 h(a, c, e) or by Trypan blue exclusion assay (b, d, f). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Estimation of Lipid peroxidation in the cell pellet after culturing the primary cortical neuron cells in the presence and absence of 0.25 μg/ml of [1,25(OH)₂D₃] for 24 h, 48 h, 72 h and 120 h, after that they were induced oxidative stress by exposure to 0.5 mM H₂O₂ for 2 h. Cell pellet was isolated and of Lipid peroxidation was estimated as described in Materials and Methods. (*) represent the values of [1,25(OH)₂D₃] treated samples which were significantly different (≥0.05) from the H₂O₂ treated alone samples.(#) represent the significant different (≥0.05) values of H₂O₂ treated samples compared to the control samples.

Fig. 3

Estimation of reduced Glutathione from Cell pellet and conditioned medium derived from primary cortical neuron cultures grown in the presence and absence of 0.25 μg/ml,[1,25(OH)₂D₃] for 24 h, 48 h, 72 h and 120 h, after that they were exposed to 0.5 mM of H₂O₂ for 2 h. Conditioned medium and cell pellet was separated by centrifugation. Reduced GSH was estimated in both conditioned medium and the cell pellet as described in the methods section. (*) represent the values of Vitamin D treated groups which are significantly (≥0.05) different from the H₂O₂ treated alone samples. Each reading is an average of three readings from three independent sets of experiments. (#) represent the H₂O₂ treated samples significantly different to the control groups.

Fig. 4

Estimation of Catalase enzyme and GST enzyme activity as measured in the cell pellet derived from primary cortical neurons cultures grown in the presence and absence of 0.25 μg/ml of [1,25(OH)₂D₃] for 24 h, 48 h, 72 h and 120 h, followed by exposure to 0.5 mM of H₂O₂ for 2 h. (*) represents the P value as [1,25(OH)₂D₃] treated samples significantly different (≥0.05) from the H₂O₂ treated samples. (#) represent the H₂O₂ treated samples compared to the control samples as significant.