Abstract
Aluminum is a ubiquitous metal that causes multiple brain pathologies such as, cognitive dysfunction and Alzheimer’s disease like symptoms. Exposure to aluminum through drinking water is responsible for hampering learning and memory. This study aimed to compare (1) the time-dependent effect of aluminum exposure (keeping total exposure of 5850 mg/kg same) in two durations, 30 and 45 days, and (2) to compare post-exposure self-recovery effect after 20 days in both (30 and 45 days exposure) groups. Rats were given 130 and 195 mg/kg of AlCl3·6H2O for 45 and 30 days respectively, to see the time-dependent exposure effect. At the end of exposure, rats were given distilled water and allowed to self-recover for 20 days to study the recovery. Expression levels of synaptic genes (Syp, SNAP25, Nrxn1/2, PSD95, Shank1/2, Homer1, CamkIV, Nrg1/2 and Kalrn) were measured using qPCR and compared in the exposure and recovery groups. Cellular morphology of the rat brain cortex and hippocampus was also investigated. Damage in lipid and protein profile was measured by employing FTIR. Results showed downregulation of mRNA expression of synaptic genes, plaques deposition, disorganization in lipid and protein profile by increasing membrane fluidity, and disorder and alteration of protein secondary structure after both exposure periods. However, better improvement/recovery in these parameters were observed in recovery group of 30 days aluminum exposure compared to 45 days aluminum exposure group. Taken together, these results suggested that short-term exposure resulted in better restoration of lipid and protein profile after time-dependent exposure of aluminum than prolonged exposure.
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Introduction
Aluminum (Al) is one of the most widely present metal on earth crust [1] and a known neurotoxicant even at low concentrations [2]. It can be consumed through foods as fruits and vegetables (estimated 10 mg/day), commercially processed foods (95 mg/day) [3] and drinking water (< 0.2 mg/L) [4] and also from sources as cooking utensils, pharmaceutical products and antiperspirants [5]. Al in drinking water contribute to only 1% of daily intake [6], but it is more bioavailable [7] owing to its uncomplexed nature and can cross blood brain barrier by replacing iron through iron transport protein transferrin [8]. Moreover, Al also binds to citrate through L-glutamate/cysteine exchanger transporter [9], thus leading to its accumulation in brain tissues. This accumulation has been associated with etiopathogenesis of neurodegenerative disorders [10, 11]. However, the exact mechanism by which Al causes these pathological changes is still unknown.
Synaptic physiology and plasticity are crucial for learning, memory, and higher cognitive functions [12]. Synaptic plasticity is regulated by various key components at presynaptic{synaptophysin (Syp), synaptosomal-associated protein (SNAP) 25 and neurexin (Nrxn)} [13,14,15] and post synaptic junction, where scaffolding molecules {post synaptic density (PSD) 95, Shank, and Homer} [16] and signaling molecules {calcium/calmodulin dependent protein kinase IV (CamkIV), neuregulin (Nrg) and kalirin (Kalrn)} [17,18,19,20] play crucial role in synaptic functions. Al modifies neurotrophins in synapse and promotes synaptic degeneration through adhesion molecules [21, 22]. This way Al can alter the complex link between the pre- and postsynaptic membranes and disrupt the cellular communication between synapses, necessary for synaptic signaling and strength. Keeping in view the abovementioned scenario, how Al exposure in time-dependent manner modify this complex process, still needs to be elaborated. Moreover, how the after-effects of Al exposure affect synapses was the prime question of this study and was investigated in recovery groups.
Neuronal membranes have a delicate composition of lipids and proteins making them crucial for normal cellular functioning, especially synaptic transmission in the brain [23]. Poor quality of these cell membrane lipids and proteins can trigger the formation of amyloid plaques, tangles, and other stressful conditions caused by metal neurotoxicity, which hampers normal synaptic transmission [24,25,26,27]. Little is known how Al can modify the balance between the functional groups in membranous macromolecules. Therefore, in this study, we aimed to look for such changes using appropriate method of Fourier transform infrared spectroscopy (FTIR) at the end of Al exposure and compared results with the recovery groups.
Chronic exposure to high doses of Al has been thoroughly investigated [21, 28]. However, no study has comprehensively reported the comparison of the same dose at different time points on synaptic markers. We aimed to understand and compare the adverse outcomes of Al exposure in a time-dependent manner and post-exposure recovery. Al was given through drinking water by keeping the total exposure same for two periods (30 and 45 days) and neurotoxicity was investigated through lipid and protein profile of membrane using FTIR spectroscopy, mRNA expression of synaptic genes and cellular histopathological changes. At the end of both exposure periods, a recovery period of 20 days was given to rats by providing distilled water and allowing them to self-recover. The same parameters were investigated and compared with the respective Al exposed groups.
Materials and Methods
Reagents and Chemicals
Aluminum chloride hexahydrate AlCl3·6H2O (AL0770) was purchased from Scharlau, Spain. AlCl3·6H2O had ≤ 0.005% impurities of heavy metals, such as Pb, and was stored in a ventilated area in an air-tight container. Potassium bromide (KBr) of FTIR grade, sodium hydroxide (NaOH), Congo red stain, silver nitrate (AgNO3), ammonium hydroxide, formaldehyde, citric acid, and sodium thiosulphate were purchased from Merck Germany. Deoxynucleotide (dNTP) 10 mM, 10 mM oligodT and RT enzyme and Maxima SYBR Green/ROX qPCR Master Mix (2 ×) were obtained from Thermo Scientific and TRI Reagent from Invitrogen®.
Experimental Design and Animal Grouping
Experiments were performed according to Institute of Laboratory Animal Research, Division on Earth and Life Sciences, National Institute of Health, USA (Guide for the Care and Use of Laboratory Animals) rulings. Experimental protocol was permitted by the Internal Review Board (IRB) of Atta-ur-Rahman School of Applied Biosciences (ASAB), NUST (IRB No. 135). Male Wistar rats (total 40) aging 3–4 months, weighing 150–160 g were obtained from Laboratory Animal House, ASAB, and kept under specific environmental conditions of 25 ± 2 °C temperature, and natural light and dark cycle (14 h light and 10 h dark). Animals were provided water and standard diet ad libitum containing crude protein (30%), crude fat (9%), crude fiber (4%), and moisture (10%) [29]. The rats were classified into five groups, eight rats each group. The number of rats for mRNA expression analysis [30, 31], FTIR [30, 32] and histopathological experiments [33, 34] were carefully selected based on previously reported literature:
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(a)
control group received distilled water.
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(b)
30DAl group received 195 mg/kg AlCl3 through drinking water for 30 days.
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(c)
30DR group received 195 mg/kg AlCl3 through drinking water for 30 days. After Al exposure for 30 days, rats were given distilled water for 20 days for self-recovery.
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(d)
45DAl group received 130 mg/kg AlCl3 through drinking water for 45 days.
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(e)
45DR group received 130 mg/kg AlCl3 through drinking water for 45 days. After Al exposure for 45 days, rats were given distilled water for 20 days for self-recovery.
The primary objective of this study was to evaluate the time-dependent effects of Al exposure. Therefore, Al was given in time-dependent manner by keeping the total exposure of 5850 mg/kg at two time points categorized as short- and long-term exposure for 30 and 45 days, respectively. When the same dose of Al is given at different time points, it is presumed that it may have differential effects upon exposure. Humans are exposed to Al due to its omnipresence in daily life, that makes it difficult to set safe intake levels of Al. The limits set by the WHO are routinely exceeded by humans residing in different regions and circumstances [35]. Therefore, mentioned dose was selected to study Al neurotoxicity at the high end of human exposure.
After the completion of the trial, the animals were sacrificed (Fig. 1) through cervical dislocation, and the brain was rapidly isolated to collect the cortex and hippocampus through a scalpel in dry ice. Both the cortex and hippocampi from each cerebral hemisphere (two per brain sample) were collected and kept at − 80 °C till further use. From one hemisphere, the cortex and hippocampus were used for RNA extraction, while the other was used for FTIR analysis.
Body Weight and Formula of Brain Coefficients of Rats
The body weights of the animals were monitored twice a week and then averaged out. Brain, cortex, and hippocampus weights and respective coefficients (%) were measured according to formula described earlier [36]. Whole brains were collected after dissection and weighed. Whole cortex and whole hippocampus were extracted immediately from both hemispheres and weighed collectively after carefully removing all extra parts such as fat [37].
FTIR Spectroscopic Measurements of Membrane Lipids and Proteins Functional Group Changes
FTIR spectroscopy was performed as previously described method [32] for identification of functional groups and chemical structures [38] of biomolecules based on spectral differences. The tissue samples were lyophilized overnight to remove water and then grounded to get fine powder. The tissue powder was mixed with KBr at a ratio 1:100 to collect pellets by subjecting pressure of 1100 kg/cm2 through a hydraulic press (Perkin Elmer, Germany) [39]. The background energy from blank KBr pellet was also measured to give correction to the whole data. An attenuated total reflection (ATR) FTIR spectrometer by Bruker Alpha was used to record the absorption in the spectral region of 4000–500 cm−1 at a spectral resolution of 4 cm−1. The spectra were recorded at room temperature under similar conditions. Each sample was scanned thrice with different pellets and then averaged out. Each spectrum was baseline corrected and normalized to the absorbance of amide I band.
FTIR measurements were then followed by statistical method of Hierarchal cluster analysis (HCA), as described earlier [32]. The HCA is a clustering method that showed the organization of the tested groups in a hierarchy. The HCA results were presented in a dendrogram, which is a plot indicating the groups and their relationships in the form of a tree. There are two main approaches that are usually followed for grouping in HCA: agglomerative or divisive [40]. We followed the agglomerative method measured through suitable metric of Euclidean distance.
RNA Extraction, Quantification, and Reverse Transcription
RNA was extracted from cortical and hippocampal tissues through Tri-reagent, as mentioned in manufacturer’s protocol [41]. mRNA quality was checked both qualitatively and quantitatively through agarose gel (1%) electrophoresis and spectrophotometry at 260 nm (Thermo Scientific Nanodrop), respectively and purity was estimated according to a 260/280 nm ratio of > 1.9. An equal amount of mRNA (3 µg) was reverse transcribed (RT) to cDNA through oligodT primer and MuLV RT.
qRT-PCR for Gene Expression Analysis
The experiment was performed according to method described earlier [42]. Synthesized cDNA from each group was taken as template for real-time PCR (qRT-PCR) amplification through AB QuantGene using Maxima SYBER green. Each reaction was performed with a master mix of 20 μl volume containing 1 µl cDNA, 4 µl 2 × Maxima SYBR green, 1 µl forward, and 1 µl reverse primers of the gene of interest (Table 6) diluted with H2O. The steps of PCR procedures followed were initial denaturation for 10 min at 95 °C, followed by 40 cycles for 15 s at 95 °C and 1 min at the annealing temperature, which varied for every gene (Table 6), followed by a final dissociation step of 45 s at 72 °C, proceeded by melt curve analysis for validation of specificity of the amplified products. The expression level was measured by 2−ΔΔCT method, as explained previously by Livak and Schmittgen [43]. The CT values were then normalized to the CT value of beta actin (Actb) gene in each sample. Actb was used as housekeeping gene, as it was considered as stable gene in cortex and hippocampus [44,45,46].
All primers were designed through NCBI primer BLAST according to rat mRNA sequences obtained from NCBI, except for Syp, PSD95, CamkIV, and Actb, which were selected from literature [42, 47]. Each primer was then tested for nonspecific products and correct amplicon size by 1% ethidium bromide (EtBr) agarose gel. Primer efficiency was also checked by cDNA serial dilutions.
Histopathological Examination
Congo Red Stain for Detection of Amyloid Beta Deposition
Congo red staining was performed as described earlier [33] for the detection of amyloid aggregates. The rat brain was fixed by transcardial perfusion with 4% paraformaldehyde according to a previously described method [41]. Coronal sections of 5 µm thickness were deparaffinized with xylene, and rehydrated in various concentrations of alcohol, washed with isopropanol (70%), and then with distilled H2O. Congo red stain 49.5 mL (0.5%) and 0.5 mL NaOH (1%) were poured on deparaffinized sections and retained for 20 min. The sections were then washed with distilled H2O and alcohol for 2 min and counterstained with hematoxylin for 30 s. After counterstaining sections were washed with 70% isopropanol (6–10 dips) and final washing was done with distilled H2O. The slides were mounted on coverslips after air-drying for 1 h.
Silver Staining for Detection of Amyloid Plaques Deposition
Silver staining was performed by modified method of Bielshowsky [48] to analyze the black colored amyloid plaque deposits. After deparaffinization of tissues in xylene and rehydration in various alcohol concentrations, slides were placed for 15 min in 10% AgNO3 solution until the sections became light brown. After washing with H2O thrice, concentrated ammonium hydroxide was mixed with AgNO3 till the precipitate became clear. The slides were incubated for 30 min at 40 °C and then kept in developer solution (20 ml of 40% formaldehyde, 0.5 g citric acid, 20 µl conc. nitric acid, and 100 ml distilled H2O) for 3 min. The slides were then placed in 1% ammonium hydroxide for 1 min to stop the silver reaction. After washing with H2O thrice, slides were placed for 5 min in 5% sodium thiosulphate. The sections were dehydrated and mounted on coverslips.
Quantitative Morphometric Analysis
Congo red and silver-stained sections from all groups were visualized through Optika microscope B-150 (Italy). Images were captured using the Optika Vision Lite 2.1 image analysis software. From each animal three slides were prepared and analyzed by calculating total number of cells from layer 2 and 3 of cortex and from dentate gyrus (DG), CA1, CA2 and CA3 regions of hippocampus at 40× resolution. Then number of Congo red and silver positive cells were calculated, and percentage was plotted.
Statistical Analysis
Data were represented as the mean ± standard error of mean (SEM) and n = number of animals used in the experiment. “Graph Pad Prism” software V8.0 was used for analyzing the statistical differences through one-way ANOVA followed by Bonferroni’s test for multiple comparisons for gene expression data and histopathological analysis. Two-way ANOVA was used for analyzing the body weight of rats to analyze the difference between initial and final body weights, and comparisons were made in all tested groups. Results with p < 0.05 was considered statistically significant. For FTIR data, Origin 2022 was used to calculate the band center and full width at half maximum (FWHM), and to perform HCA to study the similarity and differences in all tested rat brain tissues.
Results
Effect of Al Exposure and Post-Exposure Recovery on Body Weight
Initial and final body weights were plotted to examine the effect of Al exposure and post-exposure recovery on overall body weights of rats. There was no difference between control group, 30 days of Al exposed group and its respective recovery group (Fig. 2a). However, slight decrease in final body weight (257.67 ± 0.26 g, p < 0.01) of rats treated with Al for 45 days was observed in comparison to final body weight (266.00 ± 0.78 g) of animals of the control group, followed by increase in weight in recovery group (263.25 ± 0.30 g, p < 0.05; Fig. 2b). The brain, cortex and hippocampus weights and their coefficients were measured and compared (Table 1).
FTIR Spectroscopic Measurement of Al Exposure and Post-Exposure Recovery
The average of normalized absorbance of FTIR spectra in range of 4000–500 cm −1, in all groups were recorded (Figs. 3a, 4a). The spectral differences were observed in the functional groups of the molecules as lipids, proteins, and carbohydrates among Al exposure and recovery groups. FTIR spectra of Al exposure after 45 days showed wavenumber shift towards higher wavenumbers in lipid and protein bands compared with control group and 30 days of Al exposure group in both cortex and hippocampus (Tables 2, 4). The decrease in wavenumber shift was only observed in recovery period of 30 days of Al exposure that was parallel to control group suggesting an alleviation in Al induced damage. The band center and FWHM were estimated for cortex and hippocampus through beginning and end of FTIR spectra (Fig. 5a, b). The spectral features of observed bands i.e., center, and FWHM were measured for cortex (Tables 2, 3) and hippocampus (Tables 4, 5).
The differences between IR spectra among groups were further evaluated statistically by HCA. The classification performed through cluster analysis is justified by the fact that there are two groups of Al exposure and their respective recovery groups in addition to control group. In this way, it was possible to substantiate whether the groups are distinct or similar. Normalized absorbance values of FTIR spectra were used for HCA (Figs. 3b, 4b). HCA recognizes every group; and have placed control and recovery group after 30 days of Al exposure in one branch, while Al exposure group in separate group. Moreover, Al exposure after 45 days and its respective recovery group were placed in separate branch confirming the difference in rate of recovery from control group.
Effect of Al Exposure and Post-Exposure Recovery on Synaptic Vesicle Genes
Syp
In the cortex, expression of Syp was not affected after 30 days of Al exposure (0.62 ± 0.15, p > 0.05) whereas expression was downregulated after 45 days of Al exposure (0.31 ± 0.14, p < 0.01) when compared with the control group (1.00 ± 0.01). In recovery group of 45 days Al exposure (0.39 ± 0.06, p > 0.99; Fig. 6a), no difference was observed when compared to its Al exposed group suggesting no improvement in recovery.
In the hippocampus, levels of Syp were downregulated after 30 days (0.46 ± 0.14, p < 0.05) and 45 days of Al exposure (0.02 ± 0.03, p < 0.001) relative to control group (1.00 ± 0.01). Upon completion of 20 days of recovery after 30 days exposure group, a trend of upregulation was observed but not significant (0.70 ± 0.08, p = 0.76) when compared with its Al exposed group. Syp expression was upregulated (0.47 ± 0.09, p = 0.04; Fig. 6c) in 45 days exposed recovery group when compared with its Al exposed group, and its gene expression recovery was better than 30 days exposed recovery group.
SNAP25
The expression of SNAP25 in the cortex was downregulated after 30 days (0.49 ± 0.09, p < 0.01) and 45 days of Al exposure (0.09 ± 0.02, p < 0.001) in relation to the control group (1.00 ± 0.01). The recovery after 30 days of Al exposure (0.71 ± 0.09, p = 0.43) showed no difference compared to its Al exposed group. Recovery after 45 days of Al exposure showed that SNAP25 expression was significantly upregulated (0.44 ± 0.05, p = 0.05; Fig. 6b) as compared to the Al exposed group.
When studied in hippocampus, SNAP25 expression was downregulated after 30 days (0.30 ± 0.12, p < 0.01) and 45 days (0.09 ± 0.03, p < 0.001) of exposure when compared with the control group (1.00 ± 0.01). No effect was observed in terms of recovery after both exposure groups (p > 0.99; Fig. 6d).
Effect of Al Exposure and Post-Exposure Recovery on Adhesion Molecule Genes
Nrxn1
The analysis of Nrxn1 in the cortex showed no difference in 30 days of Al exposure group (0.62 ± 0.12, p > 0.05) meanwhile expression was downregulated (0.34 ± 0.13, p < 0.01; Fig. 7a) after 45 days of Al exposure when compared with the control group (1.00 ± 0.04). No effect was observed in terms of recovery in after 30 (p = 0.25) and 45 days (p > 0.99) of exposure.
When studied in hippocampus, the mRNA expression was downregulated after 30 days (0.49 ± 0.12, p < 0.05) and 45 days of exposure (0.19 ± 0.11, p < 0.001; Fig. 7c) in comparison to the control group (1.00 ± 0.01). Upregulation in expression was observed in recovery period after 45 days of exposure (0.77 ± 0.06, p = 0.006) when compared with the respective Al exposed groups, whereas no difference was observed in recovery after 30 days of exposure (0.77 ± 0.07, p = 0.43).
Nrxn2
Expression of Nrxn2 in the cortex was downregulated after both, 30 days (0.42 ± 0.19, p < 0.05) and 45 days of exposure (0.29 ± 0.10, p < 0.05) relative to the control group (1.00 ± 0.01). The trend in downregulation was also observed in recovery after 30 days (0.86 ± 0.06, p = 0.21) and 45 days (0.36 ± 0.12, p > 0.99; Fig. 7b) of exposure when compared with Al exposed groups.
The mRNA expression analysis of Nrxn2 in hippocampus showed that expression was downregulated after both 30 days (0.34 ± 0.13, p < 0.01) and 45 days of Al exposure (0.14 ± 0.05, p < 0.001) as compared to the control group (1.00 ± 0.05). Recovery group in 30 (0.57 ± 0.10, p > 0.99) and 45 days (0.47 ± 0.08, p = 0.25; Fig. 7d) of Al exposure showed no difference when compared to the respective Al exposed groups suggesting no improvement in recovery.
Effect of Al Exposure and Post-Exposure Recovery on Post-synaptic Scaffolding Molecule Genes
PSD95
When studied in the cortex, no effect was observed (0.66 ± 0.06, p > 0.05) on expression of PSD95 in 30 days of Al exposure group however, after 45 days of exposure expression was downregulated (0.39 ± 0.13, p < 0.01) when compared to the control group (1.00 ± 0.02). No effect was observed in terms of recovery after both exposure groups (p > 0.99; Fig. 8a).
On the contrary, in hippocampus, expression was significantly downregulated in 30 days (0.41 ± 0.06, p < 0.01) and 45 days of Al exposure (0.02 ± 0.10, p < 0.001). In terms of recovery, expression was upregulated in recovery of 45 days Al exposure (0.49 ± 0.10, p = 0.01) while no significant change in expression was observed in recovery (0.56 ± 0.10, p > 0.99; Fig. 8e) of 30 days Al exposure group when compared with the respective Al exposed group.
Shank1
In the cortex, expression of shank1 was downregulated in both in 30 days (0.44 ± 0.08, p < 0.05) and 45 days of Al exposure groups (0.29 ± 0.07, p < 0.01) as compared to the control group (1.00 ± 0.01). No effect was observed in recovery period of both 30 days (0.73 ± 0.12, p = 0.39) and 45 days (0.54 ± 0.11, p = 0.70; Fig. 8b) of Al exposed groups compared with the respective exposure groups.
In hippocampus, expression was downregulated in 30 days (0.41 ± 0.09, p < 0.01) and 45 days (0.05 ± 0.02, p < 0.001) of Al exposure groups in comparison to the control group (1.00 ± 0.05). The expression was significantly upregulated (0.88 ± 0.06, p = 0.01; Fig. 8f) in recovery group of 30 days Al exposure as compared to its Al exposed group. Meanwhile, no recovery was observed in recovery of 45 days Al exposure (0.36 ± 0.11, p = 0.15) suggesting less recovery than 30 days of recovery period.
Shank2
When analyzed in the cortex no effect was observed in expression of shank2 in 30 days of Al exposure (0.74 ± 0.08, p > 0.05), whereas expression was downregulated (0.34 ± 0.14, p < 0.01; Fig. 8c) after 45 days of Al exposure when compared to the control group (1.00 ± 0.02). In terms of recovery no difference was observed in recovery of 45 days Al exposure (0.74 ± 0.15, p = 0.18) when compared with the respective exposure group.
In hippocampus, expression was downregulated after 30 days (0.56 ± 0.14, p < 0.05) and 45 days (0.24 ± 0.09, p < 0.001) of Al exposure when compared to the control group (1.00 ± 0.02). No upregulation (0.83 ± 0.06, p = 0.48) in expression was observed in recovery period of 30 days Al exposure compared with the respective exposure group, whereas expression was upregulated (0.79 ± 0.04, p = 0.009; Fig. 8g) in recovery after 45 days of Al exposure.
Homer1
In the cortex, the expression of Homer1 was downregulated (0.37 ± 0.12, p < 0.05) in 30 days and 45 days of Al exposure (0.10 ± 0.05, p < 0.001) when compared to control group (1.00 ± 0.05). Expression was upregulated in recovery of 30 days of Al exposure (0.92 ± 0.16, p = 0.03) suggesting an improved recovery as compared to its Al exposed group, however there was no recovery (0.33 ± 0.08, p > 0.99; Fig. 8d) after 45 days of Al exposure.
When analyzed in the hippocampus, downregulation in expression was observed in 30 days (0.67 ± 0.04, p < 0.05) and 45 days (0.36 ± 0.09, p < 0.001) of Al exposure groups when compared to control group (1.00 ± 0.05). No significant upregulation in expression was observed (0.81 ± 0.04, p > 0.99; Fig. 8h) in recovery group of 30 days Al exposure when compared with its exposure group, whereas expression was upregulated (0.68 ± 0.05, p = 0.03) in recovery group of 45 days Al exposure.
Effect of Al Exposure and Post-Exposure Recovery on Signaling Molecule Genes
CamkIV
In the cortex expression of CamkIV was downregulated after 30 days (0.66 ± 0.07, p < 0.05) and 45 days (0.53 ± 0.06, p < 0.01) of Al exposure groups when compared to the control group (1.00 ± 0.05). In terms of recovery, expression was not upregulated (0.84 ± 0.06, p = 0.05) in recovery group of 30 days Al exposure while this was upregulated (0.81 ± 0.05, p = 0.04; Fig. 9a) in recovery after 45 days of Al exposure when compared with the respective exposure groups.
In the hippocampus, expression of CamkIV was downregulated after 30 days (0.32 ± 0.09, p < 0.01) and 45 days (0.03 ± 0.02, p < 0.001; Fig. 9e) of Al exposure in comparison to the control group (1.00 ± 0.05). No change in expression was observed in both recovery groups (p > 0.99) compared with the respective exposure groups suggesting no improvement in gene expression.
Nrg1
When studied in the cortex, Nrg1 expression was downregulated after 30 days (0.49 ± 0.07, p < 0.05) and 45 days (0.02 ± 0.04, p < 0.001) of Al exposure in relation to the control group (1.00 ± 0.04). Expression was not upregulated (0.82 ± 0.13, p = 0.26) in recovery period after 30 and 45 days (0.43 ± 0.12, p = 0.09; Fig. 9b) of Al exposure when compared with Al exposed groups.
In the hippocampus, there was no difference in expression after 30 days of Al exposure (0.60 ± 0.09, p > 0.05) but the expression was significantly downregulated in 45 days of Al exposure group (0.03 ± 0.02, p < 0.001) in comparison to the control group (1.00 ± 0.02). No effect was observed in terms of upregulation of expression in recovery groups of both Al exposed groups (p > 0.99; Fig. 9f).
Nrg2
The expression analysis of Nrg2 in the cortex was downregulated after 30 days (0.45 ± 0.21, p < 0.05) and 45 days (0.02 ± 0.04, p < 0.001) of Al exposure when compared to the control group (1.00 ± 0.04). In the recovery group after 30 days Al exposure no difference was observed (0.83 ± 0.07, p = 0.34) when compared with respective Al exposure group. Expression was not upregulated (0.36 ± 0.08, p = 0.51; Fig. 9c) in recovery group of 45 days Al exposure.
When analyzed in the hippocampus, expression of Nrg2 was significantly downregulated in both 30 days (0.46 ± 0.06, p < 0.01) and 45 days of Al exposure groups (0.01 ± 0.03, p < 0.001) when compared to control group (1.00 ± 0.03). The expression was not upregulated (0.77 ± 0.10, p = 0.36) in recovery after 30 and 45 days (0.39 ± 0.15, p = 0.12; Fig. 9g) of Al exposure when compared with respective Al exposed groups.
Kalrn
The expression of Kalrn in the cortex was downregulated in both 30 days (0.31 ± 0.04, p < 0.05) and 45 days of Al exposure (0.22 ± 0.12, p < 0.01) when compared to control group (1.00 ± 0.05). In recovery group after 30 days (0.69 ± 0.18, p = 0.37; Fig. 9d) and 45 days (0.42 ± 0.11, p > 0.99) of Al exposure no difference was observed when compared with respective Al exposure groups.
On the contrary, in hippocampus no prominent change in expression was observed in both 30 days (0.65 ± 0.07 p > 0.05) and 45 days of Al exposure groups (0.79 ± 0.08, p > 0.05; Fig. 9h) in comparison to control group (1.00 ± 0.05). No effect on recovery groups were also observed (p > 0.99).
Effect of Al Exposure and Post-Exposure Recovery on Brain Histopathology
Cortex
In the cortical layer 2 and 3, percentage of Congo red positive cells significantly increased after both 30 days (42.20 ± 1.20, p < 0.05) and 45 days of Al exposure (57.30 ± 2.19, p < 0.001) as compared to the control group (26.5 ± 0.76). However, in 30 days exposure recovery group no notable increase in Congo red positive cells (38.10 ± 2.37, p > 0.05) were observed when compared to the control group suggesting an improvement in recovery. Meanwhile in recovery period given after 45 days of Al exposure, there was a significant increase in Congo red positive cells (50.90 ± 5.22, p < 0.01; Fig. 10b), relative to control group showing no improvement in recovery.
Al exposure after both 30 days (60.40 ± 3.78, p < 0.05) and 45 days (81.31 ± 2.66, p < 0.001) have resulted in significant increase in silver deposits when compared to the control group (37.21 ± 1.59). The percentage of silver deposits was significantly decreased in recovery period given after 30 days of Al exposure (43.91 ± 5.10, p > 0.05) whereas number was slightly decreased in recovery group of 45 days of Al exposure (63.81 ± 7.01, p < 0.05; Fig. 11b).
Hippocampus
Congo red staining in both 30 and 45 days of Al exposure resulted in significant increase in Congo red positive cells in DG (57.20 ± 3.01, p < 0.01 and 82.41 ± 0.30, p < 0.001, respectively), CA1 (58.71 ± 4.37, p < 0.01 and 85.70 ± 4.96, p < 0.001, respectively), CA2 (47.91 ± 4.11, p < 0.01 and 87.30 ± 5.26, p < 0.001, respectively) and CA3 (72.10 ± 1.89, p < 0.01 and 82.71 ± 4.37, p < 0.001, respectively) when compared to the percentage of cells in the control group in DG (37.10 ± 2.92; Fig. 12b), CA1 (31.91 ± 2.34; Fig. 12c), CA2 (24.50 ± 3.78; Fig. 12d) and CA3 (42.81 ± 5.21; Fig. 12e). In terms of recovery, for 30 days exposure group, percentage of Congo red cells were significantly decreased that showed an improved recovery in DG, CA1, CA2 and CA3 (43.81 ± 3.36; 37.90 ± 3.01; 35.30 ± 1.83 and 45.42 ± 5.73, p > 0.05, respectively). In recovery after 45 days exposure group percentage of cells were only improved in CA1 (45.62 ± 4.46, p > 0.05) while the percentage of Congo red cells were increased in DG, CA2 and CA3 (56.20 ± 1.58, p < 0.01, 60.12 ± 0.97, p < 0.001 and 67.32 ± 2.41, p < 0.05, respectively).
Silver staining in both 30 and 45 days of Al exposure resulted in significant increase in silver deposits in DG (64.31 ± 2.33, p < 0.05 and 77.00 ± 2.08, p < 0.001, respectively), CA1 (58.80 ± 1.95, p < 0.01 and 80.71 ± 2.89, p < 0.001, respectively), CA2 (81.23 ± 2.30 and 86.84 ± 3.45, p < 0.001, respectively) and CA3 (65.45 ± 3.08, p < 0.01 and 81.00 ± 2.01, p < 0.001, respectively) as compared to silver deposits in control group in DG (50.32 ± 2.91; Fig. 13b), CA1 (35.12 ± 2.51; Fig. 13c), CA2 (41.90 ± 3.97; Fig. 13d) and CA3 (44.12 ± 3.67; Fig. 13e). In recovery group after 30 days of exposure, percentage of silver deposits decreased in DG, CA2 and CA3 (53.31 ± 3.38, 41.10 ± 0.34 and 55.51 ± 0.356, p > 0.05, respectively), except for CA1 where no recovery was observed (52.32 ± 1.31, p < 0.05). In recovery group after 45 days of Al exposure, improvement was observed only in DG (59.71 ± 2.60, p > 0.05) while percentage of silver deposits remained high in CA1, CA2 and CA3 (64.43 ± 4.47, 65.71 ± 4.83 and 77.72 ± 3.14, p < 0.001, respectively).
Discussion
Studies on Al neurotoxicity are almost a century old [49]. Humans are exposed to Al through different routes, and it is absorbed through gastrointestinal tract and excreted in urine [50]. During normal functioning of kidneys, Al elimination occurs from body almost completely with very little accumulation, specifically in the brain. However, when accumulated, Al is responsible for cognitive deficits [51]. Animal model studies have also suggested that Al is responsible for hampering synaptic plasticity [31]. Synaptic membranes hold many receptors and channels; therefore, any alteration in lipid and protein content of synaptic membranes due to Al exposure can affect neurotransmitter release, thus altering synapse function [24, 27].
This study was designed to investigate the toxic effects of Al on synaptic elements in terms of critical gene expression studies and lipid and protein content of cortical and hippocampal tissues through FTIR. High doses of Al were selected to ensure the Al accumulation in brain with aim to understand the next objective of our study which is post-exposure recovery effect after Al exposure. The administered doses of Al to rats per day in our study does not exceed from doses taken by individuals consuming antacids and buffered aspirin chronically that contribute to thousands of mg/kg of Al [50]. Furthermore, a longitudinal study from China revealed the high amount of Al intake from daily food items [52]. For example, pancakes provide 180 mg Al per serving [53]. Another study suggested that 5% of the American population ingested more than 95 mg/kg/day through food items [54]. Previous studies also reported that administration of 260 mg/kg Al for 5 weeks refers to maximum estimated human intake [2, 55]. Additionally, the LD50 for AlCl3 given through oral route is 200–1000 mg/kg in mice and rats [56]. According to these studies, as well as previous studies on Al neurotoxicity by our research group [28, 29], the above-mentioned doses were selected.
In this study, we assessed the body weights of animals to analyze the progress of model development for Al neurotoxicity. Body and brain weights are one of the key indexes that initially indicate the toxicity influence on animal's health. In our study, Al exposure for 45 days resulted in reduced body weight compared to Al exposure for 30 days, where no change was observed in relation to the control group. This decrease in body weight is consistent with previous research [57, 58]. The decrease in body weight was recovered after 20 days, whereas in previous studies, no recovery was made in terms of body weight [59] which could be due to the recovery period of one week after exposure to 200 mg/kg of Al for 21 days. Furthermore, the cortex and hippocampus tissues were weighed, and the respective coefficients were measured. Brain coefficients provide important information in toxicological studies in which target organs are tested for toxic chemicals [60] and it is expressed by dividing the weight of each animal by the weight of corresponding organ of an animal [61]. The decrease in hippocampal weight and coefficient was observed after 45 days of Al exposure. These findings were according to previous studies [31], which further confirmed the establishment of an animal model by indicating the improvement in hippocampal weight and coefficient after recovery group. However, no effect was observed on the cortex weight. Moreover, no animal died during the trial.
FTIR spectroscopy was recorded for both Al exposure and post-exposure recovery group that provides information about biomolecules, i.e., proteins, lipids and carbohydrates and in-depth analysis about protein secondary structure, membrane ordering, fluidity, and information as hydrocarbon chain length can also be obtained [32]. In the cortex, decrease in wavenumber of amide A band was observed only in 45DAl group that represent the N–H stretching in proteins. This may be because of Al induced free radicals that can damage the N–H bond in proteins [62]. This was restored in its respective recovery group, while FWHM was significantly altered in both Al exposed groups, which was fully recovered in 30DR group and slightly restored in 45DR group. However, amide A band was not noticed in hippocampal spectra.
Band assignment 2955, 2920 and 2850 cm−1 represents the lipid profile. In cortex no change was noticed in asymmetric stretching of CH2, however prominent shift was observed in spectral features of both 30DAl and 45DAl group with no recovery in 45DR group in hippocampus, which indicates the increase in membrane fluidity and disoriented acyl chains [26, 63]. Membrane fluidity is vital for regulating axonal information, receptors, and ion channels [64] and any alterations in fluidity affect signaling pathways. Previous studies have suggested that free radicals can increase the membrane fluidity by disrupting acyl chain [65, 66]. Studies have also reported that Al causes increase in fluidity [67, 68]. Reports have also shown that amyloid beta peptide 1–40 increases neuronal membrane fluidity in brain tissues [69, 70]. On the contrary, decrease in fluidity in brain was taken as positive factor due to decreased cholesterol levels [71]. These alterations in fluidity are dependent on many factors and cholesterol level is an important factor, but it should be taken with caution as alteration in cholesterol have also varying implications, as studies have reported that reduced cholesterol levels causes decrease in fluidity, accompanied by reduction in amyloid beta clustering and causes amyloid induced neurotoxicity [72]. However, our studies are limited only to FTIR analysis of lipid and protein profile, in future it can be validated by separately collecting the membranes.
In the cortex, spectra of asymmetric stretching of CH3 were not noticed. However, in the hippocampus for both 30DAl and 45DAl groups, spectral characters of asymmetric stretching of CH3 were significantly changed with recovery in 30DR, showing an increase in unsaturated lipids in hippocampal membrane, resulting in degradation of fatty acid to shorter chains because of Al induced oxidative stress [65]. Brain is susceptible to oxidative stress due to unsaturated lipid content specially in axonal layer of hippocampus than the cortical region, and low levels of antioxidant enzymes. Previous studies from our lab have confirmed that prolonged exposure to Al results in irreversible oxidative stress [73]. Spectral data from symmetric stretching of CH2 in cortex have shifted significantly in 45DAl group with recovery in 45DR group while in hippocampus shift was prominent in both Al exposure groups with no recovery in 45DR group that indicates structural loss of acyl chain which happens during trans-to-gauche transition [74]. Increase in wavenumber shift means higher disordering in membrane and unsaturation in gauche rotomers forms [69].
In the cortex, spectra of amide, I and II was changed in both Al exposed groups with no recovery in 45DR group that suggests change in secondary structure of proteins. In the hippocampus, amide I and II was changed only in 45DAl group, with no recovery in 45DR group. Furthermore, amide III spectra were only shifted in 45DAl group with no restoration of protein structure in 45DR group. It is an established fact that aggregation and misfolding of proteins in body can cause pathologies like, Alzheimer’s, Parkinson's disease, and Diabetes type II [75]. The protein misfolding is associated with changes in secondary structure of protein. In the cortex, spectral data of symmetric stretch of carbohydrates was modified in both 30DAl and 45DAl groups with only recovery in 30DR group, while in hippocampus, shift was seen in 45DAl group which was restored in 45DR group. The alteration in carbohydrate vibrations may indicate the modifications in sugar backbone of molecules [76]. The results of our study coincided with previous studies where Al-induced model of Alzheimer’s disease altered the lipid profile of cortex [67]. Similarly in a separate study, Al induced toxicity resulted in modification of hippocampal membrane lipids that was alleviated through CoQ [32]. Moreover, recent studies on rat brain tissues, through Al-lactate resulted in alterations in both lipid and protein profile of membrane. In the same study, recovery period of 2 weeks were given after Al exposure, but no improvement was observed which may be because of higher Al doses [33].
The exact mechanism by which Al hampers synaptic transmission is not well understood, as there is lack of enough evidence about Al toxicity studies at molecular expression level of critical genes which were studied here. Synaptic changes are accompanied by neurotransmitter’s release and exocytosis of synaptic vesicles which alters the signaling cascade in post synaptic membrane [77]. Results of mRNA expression of genes studied here showed that order of genes affected the most during Al exposure in the cortex were SANP25, Homer1, Nrg1/2, Shank1, CamkIV, Kalrn and Nrxn2, and in the hippocampus were SNAP25, PSD95, Shank1, CamkIV, Syp, Nrxn1, Shank2, Homer1 and Nrg2. Similarly, in recovery groups, the order of improved recovery in the cortex were SNAP25, CamkIV and Homer1 while in the hippocampus were Nrxn1, PSD95, Shank1, Syp, Shank2 and Homer1.
Earlier studies have also reported decreased expression of Syp in hippocampus, after induction of amyloid beta and iron [47, 78, 79]. Moreover, cadmium, lead and manganese exposure have also resulted in downregulation of Nrxn in zebra fish [80]. Although fluoride exposure does not change SNAP25 levels in the rat hippocampus [81], protein expression studies on lead exposure in the hippocampus have resulted in decreased expression of SNAP25 [82]. The irreversibility in damage studied here may be because of possibility of Al binding to ionotropic nicotinic receptor that modulates transmitter release at presynaptic end, making its clearance difficult [83]. Previous studies also showed that mRNA expression of PSD95 and CamkIV were reduced in both cortex and hippocampus after amyloid induction [47] that were upregulated after curcuminoid treatment. Lead exposure decreases Homer1 expression in rat offspring’s in hippocampal cells [84]. Another study suggested that protein expression levels of Kalrn were downregulated in hippocampus of developing rat brain after treatment with Sevofurane anesthesia, whereas no effect was noted in adult rat brain, which is relevant to our studies, however we have only measured the mRNA expression level and studies were not validated at the protein level in this study [85]. Similarly, exposure to carbofuran also resulted in significant downregulation of mRNA expression of Nrg in female rats [86]. Increased levels of copper does not modify the mRNA expression levels of member of shank family genes in mice [87], whereas in vitro studies have resulted in alterations of biometal profile of ASD by decreasing the expression of shank gene [88]. Our literature search has shown that no studies have been performed in post-exposure recovery of these genes on Al exposure.
The gene expression results can be correlated with proteins expression; however, caution is needed in this respect as expression analysis depends on various factors. Earlier studies have suggested that gene expression can be correlated with respective protein expression. Study results on data set of 2044 proteins showed positive correlation of 0.66 between mRNA expression and protein [89], which indicates that mRNA expression changes can be correlated with proteins [90] in our study.
The main role of the presynapse is to ensure neurotransmitter release, which is maintained by Ca2+ influx by voltage dependent channels. This triggers the neurotransmitter fusion with synaptic vesicles for their release [91]. Binding of neurotransmitters causes ion channels in the postsynaptic membrane to open or close and changes the membrane potential of the postsynaptic membrane molecules, thus mediating information transfer across synapses [92]. Al interaction with cholinergic system has been well reported, that disrupts the Ca2+ regulation by reducing the nicotinic acetylcholine gene expression [29]. It also downregulates the expression of PKC by inhibiting expression of NMDAR through CamkII [93]. Therefore, any modifications induced by Al may alter Ca2+ release or its binding at target site, which may disrupt the release of neurotransmitters through presynaptic vesicles in the synapse (Fig. 14). In the FTIR studies, no difference was noted in the wavenumber shift of the deoxyribose band (990 cm−1), which indicates that change in phosphate may be due to alterations in lipids [30]. This suggests that changes in synaptic vesicles induced by Al may be due to alterations in lipid content and altered genes expression, as synaptic vesicle lipids are crucial for recruiting components of the exo- and endocytic machinery [94]. Furthermore, alterations in membrane fluidity lead to change in membrane-bound enzymes, ion channels, and receptors [95]. The decrease in mRNA expression of Nrg1/2 may be due to changes in membrane fluidity, which may further disrupt the function of associated receptors and ion channels. In many cases, lipid peroxidation may serve as an initial step in events that cause oxidative modification of DNA and proteins [30, 96]. This contributes to excitotoxicity and neuronal injury, by decreasing the activity of Ca2+ pump [95], which may have altered the expression of CamkIV and other studied genes.
To understand Al induced toxicity and post-exposure recovery effect on cellular morphology of cortex and hippocampal tissues, Congo red and silver stain confers best information. Congo red staining is used to study in vitro formation of amyloid fibrils by forming lipophilic derivatives that penetrates the blood brain barrier [97]. Similarly, silver staining is used for identification and detection of pathological deposits in brain as amyloid beta and neurofibrillary tangles [98]. In our study, increased number of Congo red positive cells in cortical layers in 45DAl group with no recovery in its relative group suggested an irreversible damage. Meanwhile in the hippocampal subfields DG, CA1, CA2 and CA3 Congo red deposited in both 30DAl and 45DAl groups with only recovery in 30DR group. Results of study are in accordance with the earlier studies where deposition occurred in DG of mouse hippocampus after Al exposure [99]. Similarly, Congo red cells are formed in Al-induced Alzheimer’s disease model in cerebellum of rat brain [100]. In terms of recovery, studies conducted by Verma and colleagues that showed no difference between Al exposed and recovery group [33]. Results from silver staining showed black colored deposition in cells in both cortical layers and hippocampal regions in both 30DAl and 45DAl groups with recovery only in 30DR groups, while only DG showed recovery in 45DR group. Reports of earlier research have also suggested that Al exposure can lead to shrinkage of pyknotic Purkinje cells with formation of dark irregular amyloid plaques in cerebellum [100], hippocampus and cortex [33] of rat brain.
Findings of our studies indicate that, although Al is given while keeping the total exposure same for two different time points, it has differential effect on rat brain tissues. This implies that exposure to Al has not just dose-dependent but also has time-dependent effect. Consequently, it also provides answer to our next question that why similar recovery period has different effect. The possible reason for this could be the fact that when Al was given in time-dependent manner has different effect, then clearance or removal of metal would also be dependent on time, therefore, short-term exposure lead to better recovery compared to the prolonged exposure. This could be a major application of our study in terms of studying the toxic effects of Al metal on the human population.
Limitations of our study are that we have used only one time point of recovery. To better understand the phenomenon of recovery, more time points are needed in future studies. The accumulation and elimination of Al can be studied in a better way when levels of Al are measured at multiple time points, not only in brain tissues but also in plasma, which will provide detailed insight into the accumulation pattern as well as the removal of Al from the body. Moreover, along with the mRNA expression, proteins level and their functional studies can further elucidate the exact mechanisms underlying Al-induced neurotoxicity.
Conclusion
In conclusion, Al exposure for prolonged period resulted in irreversible damage to lipid and protein component of membrane, mRNA expression levels of synaptic genes and accumulation of red and black colored deposits in cortical and hippocampal tissues. However, recovery after 30 days of Al resulted in improvement in lipid and protein profile of brain tissues and inhibition of plaques aggregation occurred due to Al exposure. Self-recovery of 20 days in Al exposed rats for 45 days did not cause any notable improvement that suggested period of 20 days is too short for self-recovery after prolonged exposure even when using the same dose which was being used for the 30 days. It could be due to the reason that in the rat brain Al has half-life of more than 100 days and Al elimination from brain is slow process when given at lower doses. It appears that when given high dose for shorter duration of 30 days, there might be some saturation of the system, that did not cause Al accumulation, or somehow the elimination may be faster, this should be further studied with Al detection and measuring its kinetics. In conclusion, further studies need to be done with post-exposure recovery at varying time-points to better understand the phenomenon of self-recovery after Al exposure.
Data availability
The data will be made available upon reasonable request.
Abbreviations
- Al:
-
Aluminum
- FTIR:
-
Fourier transform infrared spectroscopy
- HCA:
-
Hierarchical cluster analysis
- PSD:
-
Post synaptic density
- Syp:
-
Synaptophysin
- SNAP25:
-
Synaptosomal-associated protein 25
- Nrxn1, 2:
-
Neurexin 1, 2
- CamkIV:
-
Calcium/calmodulin-dependent protein kinase IV
- Kalrn:
-
Kalirin
- Nrg1, 2:
-
Neuregulin 1, 2
- Actb:
-
Beta actin
- ATR:
-
Attenuated total reflection
- qRT-PCR:
-
Real-time PCR
- FWHM:
-
Full width at half maximum
- DG:
-
Dentate gyrus
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Acknowledgements
We like to acknowledge Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Pakistan, and Higher Education Commission (HEC) of Pakistan for providing support and facilities for this study. First author is recipient of Indigenous 5000 Ph.D. Scholarship program, HEC. HEC is not involved in study design, data interpretation, writing of manuscript, and decision to submit article for publication.
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H.A.: Methodology, Formal analysis, Investigation, Visualization, Writing - original draft; T.A.: Conceptualization, Methodology, Investigation, Visualization, Resources, Writing - original draft, Writing - review & editing, Supervision. All authors reviewed the manuscript.
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Asghar, H., Ahmed, T. Comparative Study of Time-Dependent Aluminum Exposure and Post-Exposure Recovery Shows Better Improvement in Synaptic Changes and Neuronal Pathology in Rat Brain After Short-Term Exposure. Neurochem Res 48, 2731–2753 (2023). https://doi.org/10.1007/s11064-023-03936-6
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DOI: https://doi.org/10.1007/s11064-023-03936-6