Nanoparticles: A Hope for the Treatment of

Inflammation in the Alzheimer`s diseases

Submitted by
Raghad Alharbi, Rawan Almalki, Atheer Alahmadi
Department of Biochemistry, Faculty of Science, King Abdulaziz University

Introduction Results and discussion

Alzheimer disease (AD) typically affects behavior, memory and thinking. In this study, acquisition and retention of
spatial learning and memory are studied in a rat animal model of AD after intrahippocampal (IH) injections of model
nanoparticles (1).
Nanoparticles (NPs) can be used to modulate Aβ aggregation. They have been proved to be attractive and efficient
materials for detection, bioimaging, diagnosis, drug delivery, drug therapy, repair and regeneration (2).
Over/underestimation of cell viability by the MTT assay may be due to both adaptive metabolic and mitochondrial
reprogramming of cells subjected to drug treatment-mediated stress and inhibitor off-target effects (3). In the present
study, we evaluated the effect of the brain penetration and lack of selectivity for the soluble Aβ oligomers, which are
implicated as upstream drivers of neurodegeneration. Development of NPs that can effectively inhibit Aβ oligomer
formation or block their toxicity; we evaluated the effect of TPP- MoS2 QDs (NPs) in a rat model of AD using
Aluminum Chloride (AlCl3).
Methodology
I - Cellular Experiments
i. Cell cultures
BV2microglial cells were maintained in minimum essential medium Dulbecco's modified Eagle's medium
supplemented with10% fetal bovine serum and 1% penicillin-streptomycin solution. Cells were cultured in a
humidified atmosphere with 5% CO at 37 °C.
ii.Dose determination for TPP- MoS2 QDs (NPs) treatment
The cytotoxic dose of TPP- MoS2 QDs (NPs) was examined in BV-2 microglial cells using MTT assay.
The most interesting finding is that only the concomitant presence of Aβ, and microglial activation abnormalities could
elicit the cognitive impairment. The experimental results showed that the neuroinflammation involving microglia and
cytokines, especially the neuritic plaques composed of aggregates of β-amyloid protein, also play a major risk in
AD. Upon investigation, we found that microglial processes in Ab, their total number of inclusions were significantly
reduced in numbers in Ab. Comparing sub regions, fibrillar materials were observed only in processes near plaques
(Figure 1). Cell survival is measured by re-incubation in MTT for 48 h. MTT was converted to blue formazan crystals
by the dehydrogenases in active mitochondria in live cells. After incubation, medium was discarded, and absorbance of
the solution read at 490 nm. These results indicate that Ab-induced microglial proliferation is mediated by microglial
release of TNF-alpha (4).
In this study male albino wistar rats were administered with D-gal 60 mg/kg.bwt intra peritoneally (I.P) injected
and AlCl3 (10 mg/kg.bwt.) was orally administered once daily for 21 days (Figure 2). Performance of the rats were
evaluated through behavioral assessments; Y maze test. The results of this experiment on rats treated with D-gal
60 + AlCl3 10 mg/kg.bwt showed near ideal cognitive impairments (3). The results of the Y-maze test confirmed the
spatial memory deficit and the decreased general activity in the AlCl3-treated rats, as evidenced by total arm entries by
38 and 53%, respectively, as compared to the control group (Figure 3).
The results showed that Aluminum has been shown to induce brain oxidative damage, neuronal death and a significant
increase in expression of TNFα. Compared with saline controls, AlCl3 significantly increased brain TNFα (4). It also
increased amyloid Aβ concentrations. AlCl3 impaired motor strength and memory performance and caused brain
neurodegeneration. AlCl3-injected rats provoked oxidative stress in the hippocampus as accentuated by the elevated
content of TNFα. TNF alpha on the hippocampus were examined. The presence of PCR products was visualized by
3.5% agarose gel electrophoresis. TNFα is predicted to have MW 17.3 (Figure 4) (1).

iii. MTT Cell viability assay

BV2microglial Cells viability was measured using3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) at 490 nm.

II - Evaluation of 21-day repeated oral dose toxicity of aluminum chloride in rats

All animal experiments were approved by the Animal Ethics Committee of king abdulaziz University and were
conducted in compliance with the ARRIVE guidelines.

i-Aluminum chloride toxicity model induction

Rats induced with AlCl3 10 mg/kg b. wt. daily for 21 days by oral gavage (1). Blood samples were collected in
ethylene diamine tetra acetic acid (EDTA) anticoagulant tubes. Then the rats were sacrificed by decapitation under
thiopental anesthesia and brain homogenate (intrahippocampal (IH)) was collected for further analysis.
Figure 1: Light microscopy of Immortalized microglial Figure 2: Aluminum chloride toxicity
Blood samples were collected. After euthanasia via CO2 inhalation, and brain homogenate was collected for further cell line (IMG). A- Ramified Microglia morphology. B- model induction (Samaila et.,al. 2018).
analysis. Amoeboid microglial activated by Aβ stimulation.

ii- Behavioral test - Y-maze test

Working memory was evaluated using the Y-maze test. The movement trajectory of each mouse and time spent
searching for food were recorded with a camera and analyzed using Any-maze software under the condition of
researchers blinded to treatment groups.

III - DNA Extraction and Determination of Inflammatory Marker

DNA was isolated from blood, using salting out method. to precipitate protein and isopropanol to precipitate DNA.
The sample was submitted to a DNA extraction method in which two different concentrations of ammonium acetate (2
and 4M) were compared with a phenol-chloroform extraction method and with a commercial DNA isolation kit. DNA
was qualified and quantified by spectrophotometer analysis, electrophoresis, and amplification by PCR.
Rat TNF a
Cytokine typing was performed by polymerase chain reaction with sequence-specific primers (PCR-SSP) assay.
The forward and reverse primer pairs of TNF-α used for the PCR assay were 5’- AGG CAA TAG GTT TTG AGG Is predicted to have MW 17.3

GCC AT -3’ and 5’- TCC TCC CTG CTC CGA TTC -3’, respectively. Briefly, amplification was carried out using a
thermal cycler Techne Flexigene apparatus. The presence or absence of PCR products was visualized by 3.5% agarose Figure 4: Rat TNF a
gel electrophoresis.

References Conclusion
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Acknowledgements
4. N.A.P. Atchan, S.T. Shivashankara, S. Piazza, D.A. Tchamgoue, G. Beretta, M. Dell’Agli, P. gni, G. Agbor Authors cordially acknowledge our principle supervisor Prof. Safaa Yousef Qusti for suggesting the point, the
Agbor, J.R. Kuiaté, U.V. Manjappara Polyphenol-rich extracts of Xylopia and Aframomum species show metabolic
Department of Biochemistry. Ohoud Alomari, Lubna Alshikh and Sara Ameen who assisted us practically,
benefits by lowering hepatic lipid accumulation in die induced obese Mice ACS Omega, 7 (2022), pp. 11914-11928.
great thanks. Laboratory of regenerative medicine unit, King Fahad Center, King Abdulaziz University for
5. Samaila Musa Chiroma , Mohamad Aris Mohd Moklas , Che Norma Mat Taib , Mohamad Baharuldin, Zulkhairi
Amon (2018). D-galactose and aluminium chloride induced rat model with cognitive impairments. Biomed providing laboratory facilities with equipment and chemicals.
Pharmacother. 2018 Jul;103:1602-1608.