Primary cortical astrocytes cell cultures were prepared as described by Booher & Sensebrenner, (₂₃). Briefly, 2-3 days-old Wister rat pup decapitated and the heads were dipped into 96% ethanol and then into cold MEM-HEPE'S (10 µM). The brain was removed, dissected to obtain cerebral cortex and the cortecies were removed from the MEM-HEPE'S, cut into pieces, added to papain solution and incubated at 37 ⁰ C for 1 hr 15 min. After the incubation, papain inhibitor solution was added and cells were dissociated by passing the tissue through needles, filtration and centrifugation. Cell pellets were resuspended in primary medium DMEM containing 10% FCS v/v, 2 mM glutamine and 25µg/ml gentamycin (media). Cells were seeded onto 250 cm ² flask with 2.5 pups/flask which approximately containing 2.5 x10 ⁵ cells/cm ² and incubated at 37 ⁰ C in 5% CO2, 95% humidified atmospheric air. At 10 DIV flask were shaken and semi-pure culture of astrocytes were obtained.

Generally all the cells were continuously cultured and maintained in their respective media preparations until confluence was reached ( ≈5 x10 ⁵ cells/cm ² for astrocytes), with media changed every 3 days.

Three days before cell plating, the media was removed by aspiration from the cells followed by addition of 5 ml trypsin solution (0.25%) in order to detach the cells from the flask. The trypsinized cells were then incubated at 37 ⁰ C in 5% CO₂ humidified air incubator for 8 min. The trypsinization was stopped by the addition of an equal volume of FCS 10% v/v, the cells were centrifuged at 100 x g for 5 min and the resulting pellet suspended in 2 ml of the media. One ml of the suspension was then made up of to 25 ml of the media, swirled very well and 12.5 ml was transferred onto two separate 250 ml flasks and incubated until plating.

Cells were again trypsinized as mentioned above, but the resuspended pellet was made up to 10 ml with media and 25 µl from the suspension was used to count the cell number using a Neubauer double chambered haemocytometer. Cells were then diluted to 5,000 cell/100µl and plated onto either 96 well plate at 200 µl/well or 24 well plate at 500 µl/well. The plates were incubated at 37 ⁰ C in a humidified atmosphere of 5% CO₂ and 95% O₂ for 24 hr before further treatment.

Twenty four hours after plating, cells were treated with different concentrations of aluminium sulphate for different durations of exposure. In order to construct a dose response curve, cells were exposed to a wide range of concentration of aluminium (0-3000 µM) for up to five days.

Stringent precautions were taken to prevent Al contamination from the reagents and materials used. Double-deionized distilled water (DDW)[Al]< 1 ng/ml assay was used to prepare all solutions and aluminium levels were measured in most reagents and were < 4 ng/ml. Containers used for sample collection and during analysis were generally polypropylene rather than glass and if so usually made Al-free by acid-washing technique (₂₄).

Mitochondrial dehydrogenase activity was assessed based on the MTT colorimetric determination (₂₅,₂₆, ₂₇). MTT was dissolved in PBS at 5 mg/ml and filtered through a sterile filter (0.2µm) to remove any amount of insoluble residue. MTT stock solution (5 mg/ml) of which 11 µl per 100µl medium was added to all the wells of an assay 96-well plate and incubated at 37 ⁰ C in 95% O₂ and 5% CO₂ incubator for 1 hr. After one hour of formazan formation, the reaction was stopped by removing the MTT. The formazan crystals formed were subsequently dissolved in 150 µl DMSO/well, followed by addition of 25 µl glycine buffer (0.1 M glycine buffer, pH 10.5). To achieve complete solubilization of formazan crystals, plates were vigorously shaken on a microplate shaker at 200 rpm for 20 min. The absorbance was recorded directly, within 30 min, after the addition of DMSO using an iEMS microplate spectrophotometer (Labsystems Company) at a wavelength of 550 nm. Mitochondrial activity as a percentage of control (cytotoxicity or) was calculated relative to the calibration of the Al-free/untreated cells as follows:

Figure 1

These values were then used as index of cell viability.

Cell survival or active cells was determined using a fluorescence method employing fluoreseein diacetate and propidium iodide (₂₈). Cells were plated in a 24 well plate containing 5,000 cell/well in 500µl and exposured to 1 mM aluminium sulphate 24 hr after plating. The cells were exposed for a period of not more than five days, after which, the media was removed from the cell and replaced with equal volume of Kreb's-HEPE'S buffer pH 7.4. The Kreb's-HEPE'S buffer was removed from the wells and 150 µl FDA-PI mixture (60 µg/ml-20 µg/ml) was added, followed by incubation for 3 min at room temperature. After the incubation the cells were counted using a fluorescence microscope at an excitation of wavelength of 450-490 nm and emission wavelength of 510-520 nm.

The total protein content was determined according to the method of Lowry et al. (₂₉).

The media was removed from the wells and cell sample were washed with 150µl PBS, the removal of the PBS was followed by the addition of 50µl of 0.1M NaOH and the plate was frozen at -20 ⁰ C overnight to allow adequate solubilization.

Tissue supernatant were appropriately diluted and thereafter the amount of protein determined colorimetrically at 750 nm using BSA (0-250 µg/ml) as standard and amount in the unknown sample calculated from the standard curve of BSA. The concentrations of protein in the aliquots of samples were determined by employing the above method.

Methods Of Determinations

Determination Of Reduced Glutathione

Measurement Of Lipid Peroxidation Using The Thiobarbituric Acid Reaction (TBARS)

Superoxide dismutase was determined as described by Marklund (₃₄, ₃₅).

Data obtained in this research are expressed as mean ± standard error of the mean (SEM) and results tested for statistical significance (P<0.05 to P<0.001) of the differences between means by using simple student t-test or one way analysis of variance (ANOVA).

Mitochondrial Activity In Primary Cortical Astrocytes

Primary cortical astrocytes revealed a mitochondrial activity to both 1 and 3-days exposure of cell to aluminium sulphate (300-3000 µmol/L). There was however no significant effect at Al concentration up to 300 µM but a significant effect was observed at the highest dose for both day 1 and 3 as shown in figure 1.

Figure 2

Figure 1: Mitochondrial activity in astrocytes after a duration of exposure at different concentration (0-3000 µmol/L) of aluminium sulphate. Values are mean ± SEM (n=8) and values considered significance at *P<0.05 compared to control using paired ANOVA and post hoc t-tests.

Effects Of Aluminium On Astrocytes Viability Using Fda-Pi Staining

The number of surviving untreated primary cortical astrocytes cells was found to be greater than 95% regardless of the duration of experiment, while the number of Al treated (1000µM) cells were found to be 10% less than the untreated (Figure 2).

Figure 3

Figure 2: Effects to 1 mmol/L aluminium sulphate of primary rat cortical astrocytes . Values are means ± SEM (n=5).

Effects Of Aluminium Treatment On Cell Protein Content

protein content of astrocytes

The amount of protein in cells treated with 1 mmol/L aluminium sulphate showed an increase after 1-day exposure compared to non treated cells, with no significant change after 3-days exposure (Figure 3). This is either due to the increased number of cells per well or the protein content per well.

Figure 4

Figure 3: Protein content in astrocytes after 1 mmol/L aluminium sulphate exposure. Values are means ± SEM (n=5), and values considered significant at P

Effects Of Aluminium On Aluminium Content

Aluminium Content Of Astrocytes

Figure 4, shows the cell associated Al accumulation in astrocytes as determined by ETAAS following Al treatment of the cells. The results indicate a response with maximum cell uptake at a dose of 300 µmol/L irrespective of duration of exposure. At a high dose (1000 µmol/L), there were few cells/well. The results were found to be significant at 100-300 µmol/L using parametric ANOVA where P value <0.001. However, a comparison between the duration of exposure was not significantly different (P>0.05).

Figure 5

Figure 4: Aluminium content of cortical astrocytes after exposure to different concentration (0-1000 µmol/L) of aluminium sulphate. Values are means ± SEM (n=3) and values considered significance at *P<0.001 compared to control using paired ANOVA and post hoc t-tests.

Effects Of Aluminium Treatment On Cell Glutathione Content

Glutathione Content Of Astrocytes

The GSH content in astrocytes (Figure 5) revealed no significant changes at all levels of exposure to aluminium despite different duration of exposure.

Figure 6

Figure 5: Glutathione content in primary cortical astrocytes after 1 mmol/L aluminium sulphate exposure. Values are means ± SEM (n=5), and values considered not significant at P<0.05 compared to control using paired ANOVA and post hoc t-tests.

Effects Of Aluminium Treatment On Measures Of Cellular Lipid Peroxidation

Malondialdehyde Production In Astrocytes

Aluminium treatment was associated with significant increase in the production of MDA (Figure 6) a measure of lipid peroxidation.

Figure 7

Figure 6: Malondialdehyde production in astrocytes, after exposure to aluminium sulphate. Values are mean ± SEM (n=3), *P

Flow Cytometric Analysis Of Dcfh-Da Treated Cells

The results for astrocytes (Figure 7) interesting responses that utilises only DCFH-DA alone. The astrocytes revealed consistent fluorescence intensity similar to the data obtained from microscopic viability test that uses FDA-PI.

Figure 8

Figure 7: FACSan intensity of oxidative stress in astrocytes, after treatment with aluminium (1000 µmol/L) and iron (100 µmol/L). Values are mean ± SEM (n=5) and values are considered significance at *P<0.01 compared to control. Comparisons were assessed for significance using one way ANOVA and posthoc-test of Bonferroni multiple comparison.

In the present in vitro studies, isolated cell line of rat primary cortical astrocytes was utilized in order to assess the possible antioxidant/pro-oxidant effect to acute exposure to aluminium sulphate. The results of this study are in agreement with the previous report of Oteiza et al. (₄₂), where Al was found to exhibit a dual effect. From the MTT results (figures 1-3) it is seen that aluminium initially induces an increase in the mitochondrial dehydrogenase activity, which may be due to an increase in activity of viable cells. At higher concentration (3000 µM) there was simply a general trend of increased mitochondrial activity. This may be due to cell proliferation as compensatory responses to the toxic effect of Al. But what is not known is whether the increase in activity is due to proliferation of the cells or by an increase in protein synthesis. In order to answer this question, the amount of protein was determined in untreated and Al-treated (1000 µM) cells, and these did not change significantly. Thus, it was established that, the increase in mitochondrial activity observed was not due to change in protein content and this is similar to what was earlier observed by Campbell et al, (₄). Secondly, an attempt to assess clearly cell survival rate, investigations were carried out employing the simultaneous double staining method of FDA-PI (₂₉). The results of primary cortical astrocytes show that living viable cells (which are stained green by the method) were less than 10% different in Al-untreated and Al-treated. Hence, the characteristic increase in mitochodrial dehydrogenase activity observed in the different cell types showed their different susceptibility and also confirmed possible cell damage via radical species leading to the increase activity as a result of compensatory response to toxic agent. It should be noted that under normal conditions mitochondrial respiration results in about 3% of the oxygen consumed being converted to hydrogen peroxide (₄₃).

However, to determine whether the increased mitochondrial activity observed due to Al exposure is indeed as a result of reactive oxygen species generation, certain enzyme markers of oxidative stress were assessed. It is well established that electron leakage from the respiratory chain can lead to formation of excess reactive oxygen species (₃₈). Oxidative stress generated via ROS with the loss of neurones, occurs during the progression of neurodegenerative diseases particularly the ALS, AD and Parkinson's diseases (₄₅,₄₆,₄₇). Glutathione is one of the first natural intracellular defences against oxidant events. A biphasic effect of Al was observed on the GSH level primary cortical astrocytes. At lower Al concentration there seemed to be an increase in the GSH level and a decrease at high doses. One might argue on the observed effect, but it is not surprising as astrocytes have been reported to contain high levels of GSH with a range between 16 nmol/mg protein (₁₅) and 50 nmol/mg proteins (₄₈). Relatively, the cytosolic GSH level of astroglial cultures can even reach 8 mM (₁₇). These can however be modulated by a variety of condition and highly inhibited by the availability of glutamine (₁₇).

The effect of Al exposure on this cell type, mitochondrial dehydrogenases, GSH thiol status and lipid peroxidation as assessed by increase malondialdehyde production was also accompanied by about 10-15% increase of superoxide dismutase.

These studies show that the rat primary cortical astrocytes cells are able to accumulate Al against a concentration gradient. This is supported by the report of Guy et al. (₁).

Morphologically, using FDA-PI staining, the fluorescence photographs (Picture not shown), In Al treated, primary cortical astrocytes appeared less extended, disintegrated, and spread neurites were fewer and thinner, i.e. abnormal clustering and aggregation plus loose adhesion, but the number of live cells did not change (<10%) in comparison to untreated cells. The interesting point about this finding is that aggregation of cell bodies and fasciculation of processes have been previously reported (₄₉) in cultured cortical neurons following long-term exposures to Al. Moreover, both neuritic processes and aggregated cells could be confidently stained using an antibody to microtubule-associated protein tau, which is one of the main components of neurofibrillary tangles seen in Alzheimer's disease (₄₉).