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Transit Phases and Beta Amyloid Aggregation/Clearance in Al-Induced Alzheimer’s Disease in Rat Brain Hippocampus: Synchrotron Fourier Transform Infrared Microspectroscopy (SFTIRM) Study - Volume 2 Issue 1 (Jan-June), - (6 months)

Pages: 30-43

Category: Original Research

Published Date: 21-06-2025

Safaa K. H. Khalil1, W. El hotaby1, Gehan. A-R Ahmed1*, Gihan Kamel2,3 , Hadeer H.A. Sherif1, Lamyaa Abbas1

Author Affiliation:

1   Spectroscopy Dept., Physics Research Institute, National Research Centre, 33 El Bohouth St. (former El Tahrir St.), Dokki, Giza, Egypt, P.O. 12622

2   SESAME (Synchrotron Light for Experimental Science and Applications in the Middle East), Jordan

3   Department of Physics, Faculty of Science, Helwan University, Cairo, Egypt

Keywords:

Alzheimer’s disease; Protein misfolding; Transit phase; Hippocampal tissue; SFTIRM; Multivariate analysis

Full Text:

Introduction

It is well known that the misfolding of proteins and the formation of beta-amyloids $(A\\beta)$ [Amyloidosis] and oxidative stress (OS) are considered the main reasons of many diseases, such as Alzheimer’s disease (AD, Parkinson’s disease, diabetes mellitus, etc.~[ref1,ref2,ref3,ref4,ref5]).

A living cell loses its function as a specific protein requires a distinct conformational structure to perform its role [ref6]. Alzheimer’s disease (AD), as a neurodegenerative disorder, is a typical characteristic form of amyloidosis [ref3,ref4,ref5,ref7]. The $\\beta$-secretase enzyme at the membrane surface cleaves the amyloid precursor protein (APP), resulting in the production of $A\\beta$ [ref8,ref9].

The pathological hallmarks of AD begin with unusual extracellular soluble $A\\beta$ deposition, followed by highly phosphorylated processes that lead to the transformation of $A\\beta$ through oligomeric intermediate phases into hard, insoluble neurofibrillary tangles (NFTs). These proteins form intracellularly and are known as tau proteins or plaques in hippocampal and cortical brain tissues [ref3,ref10,ref11,ref12,ref13]. Soluble $A\\beta$ (90% of $A\\beta$ is 40 amino acids long) and insoluble hydrophobic NFTs (10% of $A\\beta$ is 42 amino acids long) are neurotoxic and cause synaptic loss, neuronal death, and brain inflammation [ref1,ref8,ref14,ref15].

Aluminium (Al) is well known as a neurotoxic agent that promotes the production of soluble $A\\beta$ and insoluble NFTs associated with AD pathogenesis, due to its high affinity for membrane phospholipids and proteins [ref3,ref5,ref14,ref16].

AD has no cure; no efficient or promising therapy has yet reached the market [ref3,ref15]. Existing treatments aim to increase acetylcholine (ACh) concentration in the synaptic cleft through acetylcholinesterase inhibitors and anti–$A\\beta$ vaccines. However, these drugs demonstrate liver toxicity, modest therapeutic benefit, and do not prevent continuous misfolded protein formation, synaptic dysfunction, memory deterioration, and behavioural decline [ref17,ref18,ref19,ref20].

Despite advancements in synthetic drug development, medicinal plants remain an important source of therapeutics [ref21]. Herbal medications are preferred for their safety profiles and fewer side effects [ref22]. The pharmacological activity and medical uses of Lepidium sativum (LS) have been reported in both rat models and humans [ref21,ref22,ref23].

[ref23] investigated LS seeds and reported that they contain volatile essential oils, fatty oils, carbohydrates, proteins, fatty acids, amino acids, vitamins (including $\\beta$-carotene, riboflavin, niacin, and ascorbic acid), flavonoids, and glycosides, along with imidazole alkaloids and essential oils.

Moreover, LS contains several active ingredients relevant to AD management:

  1. Natural antioxidants — high lipid content, low antioxidant concentration, and high oxygen turnover make the brain susceptible to ROS damage mediated by Al, leading to oxidative stress and AD-like pathology [ref24]. ROS can cause lipid peroxidation, ATPase inhibition, protein oxidation, and redox modulation of ion channels [ref25]. LS acts as a radical scavenger, reducing oxidative stress damage.
  2. Silicon (Si) — a metal chelator that reduces AD risk and protects cognitive function. It decreases Al absorption in the digestive tract, lowering its accumulation in the brain [ref26,ref27].
  3. Sinapic acid — exerts antioxidant, antimicrobial, anti-inflammatory, anticancer, and anxiolytic effects [ref28,ref29,ref30]. Its derivative, sinapine (sinapoyl choline), functions as an acetylcholinesterase inhibitor, offering therapeutic potential in AD.

Several studies report the therapeutic benefits of LS in AD-like rat models at hippocampal and cortical levels using conventional FTIR spectroscopy. LS reduces brain atrophy and hydrocephalus, observed via FTIR, MRI, histopathology, EPR, and behavioural assessments [ref3,ref5,ref16].

Synchrotron FTIR microscopy (SFTIRM) with chemical mapping enables highly accurate biochemical profiling of biological samples, including protein secondary structures such as $\\alpha$-helix, $\\beta$-sheet, and random coils within the amide I band (1600–1700 cm−1) [ref31,ref32,ref33]. SFTIRM is 100–1000 times brighter than conventional thermal sources [ref30]. The high flux density dramatically improves the S/N ratio over very small regions [ref34,ref35].

The aim of this study is to utilize SFTIRM to detail how misfolded proteins and $A\\beta$ formation occur during Al induction in rat hippocampal tissue, and to investigate the therapeutic action of LS in reversing misfolding through transitional phases.

A previous ATR-IR study by the author on AlCl3-induced AD demonstrated LS-mediated protective and curative effects, suggesting potential pathways of protein misfolding reversal. The current SFTIRM approach aims to provide high-resolution mechanistic details of Al toxicity and LS treatment effects in the rat brain hippocampus.

Materials and Methods

Animals & Experimental Design

Twenty-four Albino Wister male rats weighing between 120 to 140 g were used for this study. Animals were grouped randomly in cages, the humidity, the temperature, the exposure to light and dark were applied as described in full details in our previous work [ref16]. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. This study was approved by Experimental Animal Research and Ethics Committee, NRC, Cairo; NRC ethical approval (17 038- FWA 00014747). All methods are reported in accordance with ARRIVE guidelines. Rats were allowed to acclimatize to the laboratory environment for one week before the experiment [ref3,ref5,ref14,ref16]. All rats received the same basic diet during the experimental period (65 days). Diet and water supplied ad-libitum.

Animals' Groups

Four groups of rats (n=6 rats) were divided randomly as follows:

  1. Control group (Cont): these rats were injected with normal saline intra-peritoneal (i.p.) and given normal saline by oral gavage during the experimental period.
  2. AD-induced group (AD): rats were injected i.p with AlCl3 dissolved in distilled water (10 mg/kg of body weight) daily and were given normal saline by oral gavage.
  3. Curative group (Cur): rats were injected i.p with AlCl3 dissolved in distilled water for the whole experimental period (10 mg/kg of body weight) daily, and are given normal saline by oral gavage for the first 3 weeks and then switched to LS water extract (20 mg/kg) via oral gavage daily for the rest of the experiment [ref3].
  4. Sham group (LS): rats were i.p injected with normal saline daily and were given LS water extract (20 mg/kg) via oral gavage daily throughout the duration of the experiment.

Preparation of Lepidium sativum aqueous extract

LS seeds were purchased from a local market and the aqueous extract was prepared according to the Moroccan traditional phytotherapy [ref4,ref37]. The aqueous extract was filtered using a Millipore filter (Millipore 0.2 mm, St Quentin en Yvelines, France). The filtrate was then freeze-dried and the desired dose (20 mg of lyophilized aqueous extract of LS seeds per kg body weight) was then prepared and reconstituted in 0.5 ml of distilled water. The aqueous extracts were reconstituted daily, just before administration. This method is described in full details in our previous work (Ahmed et al. 2020b, a).

Brain tissue sections for SFTIRM measurement

At the desired time interval and overnight fasting, the rats were sacrificed by cervical dislocation at the end of 42d and 65d. The whole brain of each rat (3 from each group) rapidly and carefully removed from the skull. The isolated hippocampal brain tissues were quickly fixed in 4% buffered formaldehyde and embedded in paraffin according to routine tissue processing for pathological examination [ref3,ref5, ref14,ref16]. For each sample, 5-μm-thick sections were cut and deposited on Mirr-IR slides for SFTIRM mapping studies. Tissue slides were de-waxed by immersing them in xylene for 5min and repeating this step twice with fresh xylene. The slides then were immersed in 100% ethanol for another 5min and allowed to dry in air at room temperature [ref38].

Synchrotron-FTIRM Measurements

SFTIRM data were collected at the IR beamline of SESAME (Synchrotron light for Experimental Science and Applications in the Middle East) light source, Jordan. The beam was directed to a Nicolet Continuum IR microscope (Thermo Fisher Scientific©, USA) equipped with a liquid-nitrogen-cooled mercurycadmium-telluride (MCT) detector [ref3,ref5,ref14,ref16]. Mapping was performed with Atlus mapping software (Thermo Fisher Scientific, USA). Samples’ sections were fixed on MirrIR slides (Kevley Technologies, UK) and mounted on Prior scan motorized stage for data collection. Background spectra of tissue free region were collected before each map to assess its contribution. 128 spectra were recorded in reflection mode in the region 4000–650 cm−1 with a spectral resolution of 4 cm−1 using an aperture of 20×20 μm2. Area maps were collected with 10 μm step size increment (~42 spectra per map). Each spectrum was baseline corrected and normalized to the absorbance of amide I (~1654 cm−1) using OMNIC 8.3 software. Gaussian decomposition was used for overlapping bands and second derivative analysis applied using Savitzky-Golay filter (9-point smoothing).

Materials and Methods

Animals & Experimental Design:

Twenty-four Albino Wister male rats weighing between 120 to 140 g were used for this study. Animals were grouped randomly in cages, the humidity, the temperature, the exposure to light and dark were applied as described in full details in our previous work ~[16]. All applicable international, national and/or institutional guidelines for the care and use of animals were followed. This study was approved by Experimental Animal Research and Ethics Committee, NRC, Cairo; NRC ethical approval (17 038- FWA 00014747). All methods are reported in accordance with ARRIVE guidelines. Rats were allowed to acclimatize to the laboratory environment for one week before the experiment ~[3], [5], [14], [16]. All rats received the same basic diet during the experimental period (65 days). Diet and water supplied ad-libitum.

Animals' Groups:

  1. Control group (Cont): these rats were injected with normal saline intra-peritoneal (i.p.) and given normal saline by oral gavage during the experimental period.
  2. AD-induced group (AD): rats were injected i.p with AlCl3 dissolved in distilled water (10 mg/kg of body weight) daily and were given normal saline by oral gavage (Li et al., 2012).
  3. Curative group (Cur): rats were injected i.p with AlCl3 dissolved in distilled water for the whole experimental period (10 mg/kg of body weight) daily, and are given normal saline by oral gavage for the first 3 weeks (to ensure the development of AD-like pathogenesis) and then switched to LS water extract (20 mg/kg) via oral gavage daily for the rest of the experiment ~[3].
  4. Sham group (LS): rats were i.p injected with normal saline daily and were given LS water extract (20 mg/kg) via oral gavage daily throughout the duration of the experiment.

Preparation of Lepidium sativum aqueous extract:

LS seeds were purchased from a local market and the aqueous extract was prepared according to the Moroccan traditional phytotherapy ~[4], [37]. The aqueous extract was filtered using a Millipore filter (Millipore 0.2 mm, St Quentin en Yvelines, France). The filtrate was then freeze-dried and the desired dose (20 mg of lyophilized aqueous extract of LS seeds per kg body weight) was then prepared and reconstituted in 0.5 ml of distilled water. The aqueous extracts were reconstituted daily, just before administration. This method is described in full details in our previous work (Ahmed et al. 2020b, a).

Brain tissue sections for SFTIRM measurement:

At the desired time interval and overnight fasting, the rats were sacrificed by cervical dislocation at the end of 42d and 65d. The whole brain of each rat (3 from each group) was rapidly and carefully removed from the skull. The isolated hippocampal brain tissues were quickly fixed in 4% buffered formaldehyde and embedded in paraffin according to routine tissue processing for pathological examination ~[3], [5], [14], [16]. For each sample, 5-μm-thick sections were cut and deposited on Mirr-IR slides for SFTIRM mapping studies. Tissue slides were de-waxed by immersing them in xylene for 5 min and repeating this step twice with fresh xylene. The slides then were immersed in 100% ethanol for another 5 min and allowed to dry in air at room temperature according to ~[38].

Synchrotron-FTIRM Measurements:

SFTIRM data were collected at the IR beamline of SESAME (Synchrotron light for Experimental Science and Applications in the Middle East) light source, Jordan. For the microspectroscopy beamline the beam is directed to a Nicolet Continuum IR microscope (Thermo Fisher Scientific©, USA) equipped with a liquid-nitrogen-cooled mercury-cadmium-telluride (MCT) detector ~[3], [5], [14], [16]. The Continuum microscope is coupled to 8700 Thermo Spectrometer and the data collection was carried out using Thermo OMNIC software. Mapping was performed with Atlus mapping software (Thermo Fisher Scientific, USA). The microscope and the spectrometer are purged with dry air to minimize water vapour and carbon dioxide interference with the spectra. Samples’ sections were fixed on MirrIR slides (Kevley Technologies, UK) and consequently mounted on Prior scan motorized stage for data collection. Background spectra of tissue free region were collected before each map to assess its contribution. 128 Spectra were recorded in reflection mode in the region 4000–650 cm−1 with a spectral resolution of 4 cm−1 using an aperture of 20×20 µm². Area maps were collected with 10 µm step size increment (approximately 42 spectra collected per map and the degree of freedom was between 3327). Each spectrum was baseline corrected in the 4000–650 cm−1, normalized to the absorbance of amide I (~1654 cm−1) using OMNIC 8.3 software program (Thermo Fisher Scientific Inc., Massachusetts, USA). To increase the resolution of the overlapping bands before any measurements, a Gaussian decomposition was used to localize the position of the bands in the spectra over the amide I and amide II bands over the range 1700–1500 cm−1 using the same software (baseline taken 1718–1477 cm−1). Second derivative analysis was carried out using Savitzky-Golay filter with 9 points of smoothing with the same software.

Statistical analysis:

The spectral differences among the groups under investigation were determined using multivariate exploratory techniques: Principal component analysis (PCA) and Hierarchical cluster analysis (HCA), exactly as described before in (Ahmed et al. 2020b, a). The multivariate classification (data recognition) was performed on the previously processed SFTIRM collected spectra between the intensities of the IR bands over the amide I and amide II bands.

Results & Discussion

In this study, we applied SFTIRM to screen the protein misfolding in rat hippocampal tissue in response to both Al toxicity and the influence of LS in reversing these protein misfolding, as proved earlier ~[3], [5], [14], [16] over the amide I and amide II range (1700–1500 cm−1). This section provides a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions.

  • SFTIRM spectra
  • Second derivative analysis

Very slight changes are observed from the raw spectra obtained from Cont, AD, Cur, and LS groups; the spectra are nearly superimposed (data not shown). For deeper spectral analysis, spectral second derivative and curve fitting were carried over the selected amide I & II regions from the computed averaged, normalized, smoothed, and baseline corrected spectra obtained from each group. Figure 1 illustrates the second derivatives of Cont, AD, Cur, and LS groups at the two tested time intervals [42d, 65d], respectively. The amide I band arises mainly from carbonyl C=O stretching vibrational modes of proteins, while the amide II band is due to N–H bending and C–N stretching of proteins ~[39]–[44]. Only the marked changes in the amide II second derivative bands are noticed in the early stage of the experiment at 42d (Figure 1).

It seems that, at early stage of treatment [42d], the noticed spectral changes in the hippocampal proteins secondary structure begin mainly in the NH bending of proteins, i.e., in the amide II bands (Figure 1a). This may be due to the formation of peptide bonds between NH2 and C=O of amino acids during protein aggregation. The most significant spectral changes in band intensities, HBW, band positions, disappearance and appearance of new bands are clearly shown in AD group at 65d rather than the Cont and the other LS treated groups [Cur & LS] for both amide I and amide II bands (Figure 1b). Figure 1 revealed a broad amide I band in AD second derivative spectrum centered at ~1643 cm−1 with a doublet in the amide II band at ~1533 & 1546 cm−1, shifted to lower frequency, and appearance of a new band at 1585 cm−1 compared to the Cont second derivative spectrum.

Cur spectrum showed several small bands in the amide II region compared to the AD second derivative spectrum [Figure 1b black stars]. The C=O stretching carbonyl bands in the AD group are depleted compared to Cont and the other LS tested groups [black arrow]. This observation agrees with Catherine et al., ~[45], who reported disappearance of the lipid carbonyl band at 1738 cm−1 in the SFTIR spectrum of Aβ42 aggregated plaque.

(A) SFTIRM normalized, mean second derivative spectra from Cont, AD, Curative, and LS groups over the amide I and amide II region after 42 days, with marked alterations in AD 2nd derivative spectra (black arrows). (B) Normalized, mean second derivative spectra after 65 days showing substantial changes in the AD 2nd derivative spectrum for both amide I and II compared to control and LS-treated groups. The intensity, HBW, and position of the amide modes are affected by Al and LS treatment.

Curve Fitting Analysis

To quantify the secondary structure of hippocampal proteins, Gaussian curve fitting was performed on the amide I and amide II regions (1700–1500 cm−1) from the baseline corrected spectra. The contribution of α-helix, β-sheet, β-turn, and random coil structures was calculated as a percentage of total amide absorbance. AD-induced rats exhibited a significant increase in β-sheet content with a concurrent decrease in α-helix and random coil structures compared to Cont. Curative treatment with LS partially restored α-helix content and reduced β-sheet aggregation, suggesting a protective effect against Al-induced protein misfolding. LS alone had no significant effect compared to Cont.

Multivariate Analysis

Principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed on the processed SFTIRM spectra to evaluate the discrimination among groups. PCA scores plot revealed clear separation of AD from Cont and LS-treated groups along the first two principal components, explaining more than 85% of the spectral variance. Curative group spectra clustered closer to Cont, indicating partial reversal of AD-like alterations. HCA dendrogram confirmed these groupings, with AD forming a distinct cluster while Cur and LS spectra were more similar to Cont, supporting the potential of LS in mitigating Al-induced structural protein changes.

Discussion

The present study demonstrates that Al exposure induces protein misfolding and aggregation in rat hippocampus, as evidenced by SFTIRM analysis. The observed spectral shifts and increased β-sheet content are consistent with previous reports linking Al toxicity to neurodegenerative-like protein alterations. Importantly, LS treatment attenuated these changes, restoring protein secondary structure closer to control levels. The second derivative analysis highlighted early alterations in amide II bands, reflecting N–H bending disruptions, which may serve as sensitive biomarkers for early neurotoxicity.

These results suggest that LS exerts a neuroprotective effect, potentially via antioxidant and anti-aggregatory mechanisms, consistent with prior studies on its bioactive constituents. Multivariate analysis confirmed that LS treatment partially reverses AD-like protein misfolding patterns induced by Al, supporting its therapeutic potential.

Conclusion

This study highlights the utility of SFTIRM in monitoring hippocampal protein misfolding and demonstrates that AlCl3 induces significant alterations in protein secondary structures in rats. LS aqueous extract exhibits protective and curative effects, mitigating Al-induced protein aggregation and preserving α-helix content while reducing β-sheet accumulation. These findings underscore the potential of LS as a natural therapeutic agent for early intervention in Al-related neurodegeneration.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by [Author Names]. The first draft of the manuscript was written by [Author Names] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Declarations

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflicts of Interest

The authors declare that they have no conflict of interest.

Data Availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. Additional data are available from the corresponding author on reasonable request.

Ethical Approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This study was approved by Experimental Animal Research and Ethics Committee, NRC, Cairo; NRC ethical approval (17 038- FWA 00014747).

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