Evaluation of Aβ Deposits in the Hippocampus of a Rat Model of Alzheimer’s Disease After Intravenous Injection of Human Adipose Derived Stem Cells by Immuno- and Thioflavin S-Costaining

authors:

avatar Maryam Doshmanziari 1 , avatar Arash Sarveazad 2 , avatar Fatemeh Moradi 1 , avatar Marjan Shariatpanahi ORCID 3 , 4 , avatar Esfandiar Doshmanziari 5 , avatar Sara Simorgh 1 , avatar Mina Eftekharzadeh 1 , 4 , *

Department of Anatomy, Iran University of Medical Sciences, Tehran, Iran
Colorectal Research Center, Iran University of Medical Sciences, Tehran, Iran
Department of Toxicology and Pharmacology, School of Pharmacy, International Campus, Iran University of Medical Sciences, Tehran, Iran
Neuroscience Research Center (NRC), Iran University of Medical Sciences, Tehran, Iran
Faculty of Management and Accounting, Islamic Azad University, Islamshahr, Iran

how to cite: Doshmanziari M, Sarveazad A, Moradi F, Shariatpanahi M, Doshmanziari E , et al. Evaluation of Aβ Deposits in the Hippocampus of a Rat Model of Alzheimer’s Disease After Intravenous Injection of Human Adipose Derived Stem Cells by Immuno- and Thioflavin S-Costaining. Thrita J Neu. 2018;7(2):e88367. https://doi.org/10.5812/thrita.88367.

Abstract

Background:

Alzheimer's disease (AD) is a progressive neuropsychiatric disorder that gradually impairs memory and behavioral functions. Amyloid beta (Aβ) is considered as the most toxic substance in the brain of AD patients.

Objectives:

The present study was designed to evaluate Aβ deposits by Immuno- and Thioflavin S-costaining in the hippocampus of a rat model of AD after intravenous injection of human adipose-derived stem cells (hADSCs).

Methods:

Thirty-two male rats were included in the four groups of control, sham, AD and hADSCs. The hADSCs characterization was confirmed by the flow cytometry technique. Immuno- and Thioflavin S-costaining was utilized for detecting Aβ plaques in the hippocampus of a rat model of AD following injection of hADSCs.

Results:

Statistical analysis revealed that Aβ plaques increased significantly in the AD group compared to the control and sham groups. The administration of hADSCs significantly decreased immunoreactivity and Thio-S-positive plaques in the AD group. We also found that the plaques detected by anti-Aβ antibody (immunohistochemical staining) were significantly more than those distinguished by Thioflavin-S in all the groups.

Conclusions:

Results showed that hADSCs played an effective role in decreasing amyloids aggregation following migration to the hippocampus of the rat model of AD.

1. Background

Alzheimer's disease (AD) is a progressive neuropsychiatric disorder that gradually impairs memory and behavioral functions (1). The global prevalence of AD was estimated at 47 million people in 2015 and this rate is expected to approximately triple by 2050 (2, 3). AD features include amyloid plaques and neurofibrillary tangles (NFTs). Amyloid plaques (deposits) are produced by the accumulation of Aβ proteins and neurofibrillary tangles created by the aggregation of hyper-phosphorylated Tao proteins (4, 5). Aβ is considered as the most toxic substance in the brain of AD patients (6). Thioflavin staining is the most suitable and standardized method for screening Aβ (7). Thioflavin-S is used for detecting Aβ plaques that can bind to distinctive β-pleated sheet of amyloid fibrils (8-11). Thioflavin-S results from methylation of dehydrothiotoluidine with sulfonic acid (12). In addition, monoclonal mouse antibodies are increasingly employed to detect amyloid deposits. Different mechanisms are presented for Aβ accumulation such as elevated synthesis and high propensity for aggregation (13). Thus, approaches to prevent and reduce Aβ deposition are mainly examined as therapeutic strategies for AD treatment (14, 15).

Adipose-derived stem cells (ADSCs) are an appropriate source of stem cells in clinical studies as these cells can be achieved via liposuction or from subcutaneous adipose tissues (16-18). hADSCs are the most suitable stem cells due to their ability to cross the blood-brain barrier, migrating to the damaged sites of the brain without any ethical concerns, immune rejection or tumorigenesis (19). This is the first attempt to use Immuno- and Thioflavin S-costaining to evaluate Aβ plaques in a rat model of AD following intravenous injection of hADSCs. Regarding the previous investigations, there is a scarcity of reports on using this technique for evaluating Aβ plaques after stem cell injection.

2. Methods

2.1. Chemicals

Aβ (1-42), DMEM, DiI, Thioflavin-S and DAPI stains were supplied from Sigma (St. Louis, MO, USA). Ketamine (Alfasan, Holland) and Xylazine (Pantex Holland B.V.) were utilized for anesthesia before surgery. Anti-Aβ antibody was purchased from Abcam (Cambridge, MA, USA). Other chemicals in this research were supplied from commercial sources.

2.2. Animals

In this study, we used 32 male Wistar rats weighing 250 - 320 gr. The rats were purchased from the animal house of Iran University of Medical Sciences. The animals were kept under standard conditions with a 12-hour light/dark cycle at 25°C. Normal rat chow and water were provided ad libitum. All the experiments were performed under the standard guidelines for the use and care of laboratory animals approved by the Ethics Committee of Iran University of Medical Sciences (90/11/5931).

2.3. Experimental Design

Animals were randomly divided into four groups of eight as follows: control group: The rats did not undergo any surgery or injection; sham group: The Hamilton syringe (without any medication) was entered to the hippocampus of the rats by stereotaxic surgery; Alzheimer's model (AD) group: The rats received Aβ injection in their hippocampus through stereotaxic surgery; and stem cell treatment group (AD + Sc): The rats received 3 × 106 hADSCs by intravenous administration three weeks after intra-hippocampal Aβ injection.

2.4. Aβ Preparation and Surgery

Lyophilized peptide powder of Aβ1-42 was dissolved in sterile water to reach the final concentration of 2 mmol/L. The final solution was incubated for 48 hours at 37°C. Anesthesia was induced by intraperitoneal (IP) injection of ketamine hydrochloride (80 mg/kg) and xylazine (8 mg/kg). Afterwards, the rats were located in the stereotaxic frame (Stoelting Co, USA). Each animal received 5 μL of Aβ injection in the right and left CA1 hippocampus (each side 5 μL) (AP: 3.8, ML: 2.4, DV: 2.9) according to the Paxinos and Watson Atlas (20).

2.5. Isolation and Culture of hADSCs

hADSCs were separated from adipose tissues obtained from the abdominal superficial layers of 25 to 45-year-old patients during liposuction in the operation room of Rasoul-e-Akram General Hospital, Tehran, Iran. hADSCs were isolated according to the protocol of Dubois et al. (21). For removing blood vessels, adipose tissue was washed in 1% P/S solution, and 0.1% collagenase and 1.0% bovine serum albumin (BSA) were added. The cell pellets were washed with PBS, centrifuged, suspended in DMEM /F12 culture medium (10% fetal bovine serum [FBS] and 1% P/S) and incubated (37°C, 5% CO2, 98% moisture) until the fifth passage.

2.6. hADSCs Characterization

For characterization of hADSCs, flow cytometry was used according to a previous study (22). Cells were incubated with anti-CD44, anti-CD90, anti-CD73 (conjugated to FITC), anti-CD34 and anti-CD45 (conjugated to PE) for 30 minutes. The cells in the 5th passage were labeled with DiI. 106 cells were suspended in 1 ml of phosphate buffered saline (PBS), and then 5 μL of DiI solution (50 μg DiI powder solved in 50 μL DMSO) was added.

2.7. Immuno- and Thioflavin S-costaining

Three months after hADSCs injection, the rats were anesthetized with ketamine and xylazine, perfused by 4% paraformaldehyde in 0.1 mol/L PBS (pH = 7.4). The brains were taken out and fixed for 24 hours in 10% formalin solution. Coronal sections were prepared at 5 μm thickness by microtome after routine paraffin processing.

For immunofluorescent staining, brain sections were processed according to the regular protocols (23). Briefly, the sections were fixed in 4% formaldehyde for 20 minutes and immunostained with the application of 1:100 dilution of primary anti-Aβ rabbit polyclonal antibody followed by goat anti-rabbit FITC-conjugated secondary antibody at 1:200 dilution. Thereafter, the sections were put in 10% formalin for 10 minutes and rinsed in PBS. The sections were incubated in 0.25% potassium permanganate (10 minutes), and then washed in PBS, incubated in 2% potassium metabisulfite and 1% oxalic acid until they seemed white. Then, the sections were washed in water and stained for 10 minutes with a solution of 0.015% Thioflavin-S in 50% ethanol and water. After drying the sections, they were immersed into Histo-Clear (24). DAPI was used for counterstaining the nuclei. The sections were mounted on slides and evaluated under fluorescence microscope (Labomed microscope equipped with an Invenio 6EIII camera).

2.8. Statistical Analysis

The data were analyzed by using the Graph pad Prism program (GraphPad software, Inc. USA). We used One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post hoc test and two-way ANOVA with Bonferroni post hoc test. Data are presented as Mean ± SEM (standard error of mean). P-value less than 0.05 is considered statistically significant.

3. Results

3.1. hADSCs Characterization

hADSCs showed positive staining for the specific mesenchymal surface markers CD73, CD44 (Figure 1A) and CD90 (Figure 1B). hADSCs indicated high levels of CD73, CD44 and CD90, which were expressed in 92.28%, 93.65% and 83.5% of the total cell population, respectively. However, a small proportion of hADSCs represented hematopoietic stem cell surface markers, as well as CD34 and CD45, which were expressed in 18.67% and 16.45% of cells, respectively (Figure 1C).

Flow cytometry analysis of surface markers showed that hADSCs express high level of CD73 (92.28%), CD44 (93.65%) (A) and CD90 (83.5%) (B) express low level of CD34 (18.67%) and CD45 (16.45%) (C).
Flow cytometry analysis of surface markers showed that hADSCs express high level of CD73 (92.28%), CD44 (93.65%) (A) and CD90 (83.5%) (B) express low level of CD34 (18.67%) and CD45 (16.45%) (C).

3.2. Homing of hADSCs

As shown in Figure 2, hADSCs that were labeled DiI migrated from the site of delivery to the CA1 region of the hippocampus (DiI-labeled hADSCs = red and DAPI-stained nuclei = blue).

Homing of hADSCs in CA1 region of the hippocampus. Nuclei (DAPI - Blue) (A), hADSCs labeled with DiI (Red) (B).
Homing of hADSCs in CA1 region of the hippocampus. Nuclei (DAPI - Blue) (A), hADSCs labeled with DiI (Red) (B).

3.3. Immuno- and Thioflavin S-Costaining

Figure 3 shows immunofluorescent double staining of primary anti-Aβ rabbit polyclonal antibody followed by goat anti-rabbit FITC-conjugated secondary antibody (red), Thioflavin-S staining (green) and merge picture (yellow) in the CA1 region of the hippocampus in the AD and AD + Sc groups. Data showed more distinct Aβ immunoreactivity and Thioflavin-S stain in the AD group compared to the treatment group. Statistical analysis revealed that there was no significant difference in immunoreactive positive plaques between the control and sham groups, but these plaques increased significantly in the AD group relative to the control and sham groups (***P-value < 0.001). Nonetheless, the administration of hADSCs significantly decreased immunoreactivity positive plaques in the AD group (###P-value < 0.001; Figure 4A).

Immunofluorescent staining. Primary anti-amyloid beta rabbit polyclonal antibody followed by goat anti-rabbit FITC-conjugated secondary antibody (red), Thioflavin-S staining (green) and merge picture (yellow) in CA1 region of hippocampus in AD and AD + Sc groups (scale bar 20 µm).
Immunofluorescent staining. Primary anti-amyloid beta rabbit polyclonal antibody followed by goat anti-rabbit FITC-conjugated secondary antibody (red), Thioflavin-S staining (green) and merge picture (yellow) in CA1 region of hippocampus in AD and AD + Sc groups (scale bar 20 µm).
Immunoreactive positive plaques in different groups (values are expressed as mean ± SEM). N = 6, ***P < 0.001 different from control group, ###P < 0.001 different from Aβ group (A), N = 6, ***P < 0.001 different from control group, ###P < 0.001 different from Aβ group (B), *P < 0.05; **P < 0.01 show significant difference between Aβ immunostaining compared to Thioflavine-S staining (C).
Immunoreactive positive plaques in different groups (values are expressed as mean ± SEM). N = 6, ***P < 0.001 different from control group, ###P < 0.001 different from Aβ group (A), N = 6, ***P < 0.001 different from control group, ###P < 0.001 different from Aβ group (B), *P < 0.05; **P < 0.01 show significant difference between Aβ immunostaining compared to Thioflavine-S staining (C).

The number of Thio-S-positive plaques was not significantly different between the control and sham groups; however, the number of Thio-S-positive plaques increased significantly in the AD group compared to the control and sham groups (***P-value < 0.001). Intravenous injection of hADSCs significantly decreased the number of Thio-S-positive plaques in the AD group (###P-value < 0.001; Figure 4B).

In this research, we also investigated Aβ plaques in the CA1 region of the hippocampus in different groups using Aβ immunostaining compared to Thioflavin-S staining.

As shown in Figure 4C, we found that the plaques detected by anti-Aβ antibody were significantly more than those distinguished by Thioflavin-S in all the groups. The results showed the significant effect of staining method (F (1, 40) = 42.48, P < 0.0001) and groups (F (3, 40) = 144.58, P < 0.0001) on the positive Aβ plaques in the CA1 region of the hippocampus. However, the interaction of these two factors was non-significant (F (3, 40) = 0.15, P = 0.928).

4. Discussion

The first neuropathological criterion for the diagnosis of AD is the aggregation of Aβ peptides that can form plaques. These plaques are toxic to neurons and lead to apoptosis and synaptic loss. Upregulated neuroinflammatory factors and loss of synaptic markers can lead to AD progression by causing cognitive disturbances (25-29). Immunofluorescent staining using anti- Aβ antibody can detect Aβ plaques in a rat model of AD. Additionally, Thioflavin-S staining technique is utilized for the detection of plaques (9). In our study, AD model was confirmed by histological analysis, while Thioflavin-S and immunoreactive positive plaques increased significantly in the AD group compared to the control group.

Our study showed that Aβ plaques as toxic elements increased in the AD group. These results are consistent with a previous report about staining of Aβ in AD. That report showed that Aβ immunohistochemical staining could detect both fibrillar and non-fibrillar Aβ, whereas Thioflavin-S identified β-pleated fibrillar amyloids (30). We also observed that the plaques detected by anti-Aβ antibody were significantly more than those distinguished by Thioflavin-S in all the groups (comparison of staining methods). It may be related to the detection of both fibrillar and non-fibrillar Aβ by immunohistochemical staining (anti-Aβ antibody) compared to the detection of fibrillar Aβ by Thioflavin-S staining. There are achievable therapeutic strategies for AD aiming to prevent and reduce Aβ deposits (14). Stem cell transplantation has been reported to prevent cell death by reducing Aβ deposits in neurodegenerative disorders.

Studies have shown that stem cells could increase synaptic density mainly through elevated secretion of neurotrophic and growth factors, which play an important role in the treatment of neurodegenerative diseases (16, 31, 32). This was the first attempt to measure the amount of Aβ deposits by Immuno- and Thioflavin S-costaining in a rat model of AD following hADSCs intravenous administration.

We found that after the migration of stem cells to the hippocampus of AD rats, a significant decrease in fluorescence intensity of Thioflavin-S and Aβ expression level was observed in the hADSCs treatment group, showing the effective role of hADSCs in decreasing amyloids aggregation. It seems that hADSCs as therapeutic targets apply neuroprotective effects related to Aβ clearance. A previous research showed that Aβ synthesis blockade is not effective in decreasing Aβ levels compared to Aβ clearance (33). Investigations showed that mesenchymal stem cells significantly enhanced neuronal survival against Aβ toxicity in AD models (34). hADSCs could induce endogenous microglial activation, which led to removing Aβ aggregates in AD animal models (35). Furthermore, bone marrow-derived cells could differentiate into functional microglia and cause Aβ clearance as a therapeutic effect (36). Further in-depth studies are still necessary to clarify the detailed mechanisms.

4.1. Conclusions

Our results indicated that hADSCs served an effective role in decreasing amyloid aggregation by using Immuno- and Thioflavin-S-costaining. As Aβ toxicity is the major reason for neuronal death in AD, hADSCs may be a promising candidate for AD therapy due to their high potential for clearance of Aβ deposits.

Acknowledgements

References

  • 1.

    Castellani RJ, Rolston RK, Smith MA. Alzheimer disease. Dis Mon. 2010;56(9):484-546. [PubMed ID: 20831921]. [PubMed Central ID: PMC2941917]. https://doi.org/10.1016/j.disamonth.2010.06.001.

  • 2.

    Baumgart M, Snyder HM, Carrillo MC, Fazio S, Kim H, Johns H. Summary of the evidence on modifiable risk factors for cognitive decline and dementia: A population-based perspective. Alzheimers Dement. 2015;11(6):718-26. [PubMed ID: 26045020]. https://doi.org/10.1016/j.jalz.2015.05.016.

  • 3.

    Hebert LE, Weuve J, Scherr PA, Evans DA. Alzheimer disease in the United States (2010-2050) estimated using the 2010 census. Neurology. 2013;80(19):1778-83. [PubMed ID: 23390181]. [PubMed Central ID: PMC3719424]. https://doi.org/10.1212/WNL.0b013e31828726f5.

  • 4.

    Chiarini A, Armato U, Gardenal E, Gui L, Dal Pra I. Amyloid beta-exposed human astrocytes overproduce phospho-tau and overrelease it within exosomes, effects suppressed by calcilytic NPS 2143-further implications for Alzheimer's therapy. Front Neurosci. 2017;11:217. [PubMed ID: 28473749]. [PubMed Central ID: PMC5397492]. https://doi.org/10.3389/fnins.2017.00217.

  • 5.

    Bloom GS. Amyloid-beta and tau: The trigger and bullet in Alzheimer disease pathogenesis. JAMA Neurol. 2014;71(4):505-8. [PubMed ID: 24493463]. https://doi.org/10.1001/jamaneurol.2013.5847.

  • 6.

    Prasansuklab A, Tencomnao T. Amyloidosis in alzheimer's disease: The toxicity of amyloid beta (A beta ), mechanisms of its accumulation and implications of medicinal plants for therapy. Evid Based Complement Alternat Med. 2013;2013:413808. [PubMed ID: 23762130]. [PubMed Central ID: PMC3671299]. https://doi.org/10.1155/2013/413808.

  • 7.

    Li M, Zhao A, Ren J, Qu X. N-methyl mesoporphyrin IX as an effective probe for monitoring Alzheimer's disease beta-amyloid aggregation in living cells. ACS Chem Neurosci. 2017;8(6):1299-304. [PubMed ID: 28281745]. https://doi.org/10.1021/acschemneuro.6b00436.

  • 8.

    Rajamohamedsait HB, Sigurdsson EM. Histological staining of amyloid and pre-amyloid peptides and proteins in mouse tissue. Methods Mol Biol. 2012;849:411-24. [PubMed ID: 22528106]. [PubMed Central ID: PMC3859432]. https://doi.org/10.1007/978-1-61779-551-0_28.

  • 9.

    Urbanc B, Cruz L, Le R, Sanders J, Ashe KH, Duff K, et al. Neurotoxic effects of thioflavin S-positive amyloid deposits in transgenic mice and Alzheimer's disease. Proc Natl Acad Sci U S A. 2002;99(22):13990-5. [PubMed ID: 12374847]. [PubMed Central ID: PMC137824]. https://doi.org/10.1073/pnas.222433299.

  • 10.

    Watanabe H, Ono M, Matsumura K, Yoshimura M, Kimura H, Saji H. Molecular imaging of beta-amyloid plaques with near-infrared boron dipyrromethane (BODIPY)-based fluorescent probes. Mol Imaging. 2013;12(5):338-47. [PubMed ID: 23759374]. https://doi.org/10.2310/7290.2013.00049.

  • 11.

    Darvesh S, Cash MK, Reid GA, Martin E, Mitnitski A, Geula C. Butyrylcholinesterase is associated with beta-amyloid plaques in the transgenic APPSWE/PSEN1dE9 mouse model of Alzheimer disease. J Neuropathol Exp Neurol. 2012;71(1):2-14. [PubMed ID: 22157615]. [PubMed Central ID: PMC3246090]. https://doi.org/10.1097/NEN.0b013e31823cc7a6.

  • 12.

    Ly PT, Cai F, Song W. Detection of neuritic plaques in Alzheimer's disease mouse model. J Vis Exp. 2011;(53). [PubMed ID: 21841757]. [PubMed Central ID: PMC3197440]. https://doi.org/10.3791/2831.

  • 13.

    Wyss-Coray T, Yan F, Lin AH, Lambris JD, Alexander JJ, Quigg RJ, et al. Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice. Proc Natl Acad Sci U S A. 2002;99(16):10837-42. [PubMed ID: 12119423]. [PubMed Central ID: PMC125059]. https://doi.org/10.1073/pnas.162350199.

  • 14.

    Yang H, Xie Z, Wei L, Yang H, Yang S, Zhu Z, et al. Human umbilical cord mesenchymal stem cell-derived neuron-like cells rescue memory deficits and reduce amyloid-beta deposition in an AbetaPP/PS1 transgenic mouse model. Stem Cell Res Ther. 2013;4(4):76. [PubMed ID: 23826983]. [PubMed Central ID: PMC3854736]. https://doi.org/10.1186/scrt227.

  • 15.

    Kim S, Chang KA, Kim J, Park HG, Ra JC, Kim HS, et al. The preventive and therapeutic effects of intravenous human adipose-derived stem cells in Alzheimer's disease mice. PLoS One. 2012;7(9). e45757. [PubMed ID: 23049854]. [PubMed Central ID: PMC3458942]. https://doi.org/10.1371/journal.pone.0045757.

  • 16.

    Gimble JM, Katz AJ, Bunnell BA. Adipose-derived stem cells for regenerative medicine. Circ Res. 2007;100(9):1249-60. [PubMed ID: 17495232]. [PubMed Central ID: PMC5679280]. https://doi.org/10.1161/01.RES.0000265074.83288.09.

  • 17.

    Lee ST, Chu K, Jung KH, Im WS, Park JE, Lim HC, et al. Slowed progression in models of Huntington disease by adipose stem cell transplantation. Ann Neurol. 2009;66(5):671-81. [PubMed ID: 19938161]. https://doi.org/10.1002/ana.21788.

  • 18.

    Puissant B, Barreau C, Bourin P, Clavel C, Corre J, Bousquet C, et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells: Comparison with bone marrow mesenchymal stem cells. Br J Haematol. 2005;129(1):118-29. [PubMed ID: 15801964]. https://doi.org/10.1111/j.1365-2141.2005.05409.x.

  • 19.

    Chan TM, Chen JY, Ho LI, Lin HP, Hsueh KW, Liu DD, et al. ADSC therapy in neurodegenerative disorders. Cell Transplant. 2014;23(4-5):549-57. [PubMed ID: 24816450]. https://doi.org/10.3727/096368914X678445.

  • 20.

    Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 6th ed. Oxford: Elsevier Academic; 2007.

  • 21.

    Dubois SG, Floyd EZ, Zvonic S, Kilroy G, Wu X, Carling S, et al. Isolation of human adipose-derived stem cells from biopsies and liposuction specimens. Methods Mol Biol. 2008;449:69-79. [PubMed ID: 18370084]. https://doi.org/10.1007/978-1-60327-169-1_5.

  • 22.

    Sarveazad A, Babahajian A, Bakhtiari M, Soleimani M, Behnam B, Yari A, et al. The combined application of human adipose derived stem cells and Chondroitinase ABC in treatment of a spinal cord injury model. Neuropeptides. 2017;61:39-47. [PubMed ID: 27484347]. https://doi.org/10.1016/j.npep.2016.07.004.

  • 23.

    Donaldson JG. Immunofluorescence staining. Curr Protoc Cell Biol. 2015;69:4 3 1-7. [PubMed ID: 26621373]. https://doi.org/10.1002/0471143030.cb0403s69.

  • 24.

    Bussiere T, Bard F, Barbour R, Grajeda H, Guido T, Khan K, et al. Morphological characterization of Thioflavin-S-positive amyloid plaques in transgenic Alzheimer mice and effect of passive Abeta immunotherapy on their clearance. Am J Pathol. 2004;165(3):987-95. [PubMed ID: 15331422]. [PubMed Central ID: PMC1618604]. https://doi.org/10.1016/S0002-9440(10)63360-3.

  • 25.

    Reiss AB, Arain HA, Stecker MM, Siegart NM, Kasselman LJ. Amyloid toxicity in Alzheimer's disease. Rev Neurosci. 2018;29(6):613-27. [PubMed ID: 29447116]. https://doi.org/10.1515/revneuro-2017-0063.

  • 26.

    Duncan T, Valenzuela M. Alzheimer's disease, dementia, and stem cell therapy. Stem Cell Res Ther. 2017;8(1):111. [PubMed ID: 28494803]. [PubMed Central ID: PMC5427593]. https://doi.org/10.1186/s13287-017-0567-5.

  • 27.

    Serrano-Pozo A, Frosch MP, Masliah E, Hyman BT. Neuropathological alterations in Alzheimer disease. Cold Spring Harb Perspect Med. 2011;1(1). a006189. [PubMed ID: 22229116]. [PubMed Central ID: PMC3234452]. https://doi.org/10.1101/cshperspect.a006189.

  • 28.

    Morales-Zavala F, Casanova-Morales N, Gonzalez RB, Chandia-Cristi A, Estrada LD, Alvizu I, et al. Functionalization of stable fluorescent nanodiamonds towards reliable detection of biomarkers for Alzheimer's disease. J Nanobiotechnology. 2018;16(1):60. [PubMed ID: 30097010]. [PubMed Central ID: PMC6085760]. https://doi.org/10.1186/s12951-018-0385-7.

  • 29.

    Rao JS, Kellom M, Kim HW, Rapoport SI, Reese EA. Neuroinflammation and synaptic loss. Neurochem Res. 2012;37(5):903-10. [PubMed ID: 22311128]. [PubMed Central ID: PMC3478877]. https://doi.org/10.1007/s11064-012-0708-2.

  • 30.

    Wisniewski HM, Wen GY, Kim KS. Comparison of four staining methods on the detection of neuritic plaques. Acta Neuropathol. 1989;78(1):22-7. [PubMed ID: 2472039]. https://doi.org/10.1007/BF00687398.

  • 31.

    Park J, Lee N, Lee J, Choe EK, Kim MK, Lee J, et al. Small molecule-based lineage switch of human adipose-derived stem cells into neural stem cells and functional GABAergic neurons. Sci Rep. 2017;7(1):10166. [PubMed ID: 28860504]. [PubMed Central ID: PMC5579051]. https://doi.org/10.1038/s41598-017-10394-y.

  • 32.

    Radhakrishnan S, Anna Trentz O, Parthasarathy VK, Sellathamby S. Human adipose tissue-derived stem cells differentiate to neuronal-like lineage cells without specific induction. Cell B Res Ther. 2017;6(1). https://doi.org/10.4172/2324-9293.1000131.

  • 33.

    Yoon SS, Jo SA. Mechanisms of amyloid-beta peptide clearance: Potential therapeutic targets for Alzheimer's disease. Biomol Ther (Seoul). 2012;20(3):245-55. [PubMed ID: 24130920]. [PubMed Central ID: PMC3794520]. https://doi.org/10.4062/biomolther.2012.20.3.245.

  • 34.

    Shin JY, Park HJ, Kim HN, Oh SH, Bae JS, Ha HJ, et al. Mesenchymal stem cells enhance autophagy and increase beta-amyloid clearance in Alzheimer disease models. Autophagy. 2014;10(1):32-44. [PubMed ID: 24149893]. [PubMed Central ID: PMC4389879]. https://doi.org/10.4161/auto.26508.

  • 35.

    Ma T, Gong K, Ao Q, Yan Y, Song B, Huang H, et al. Intracerebral transplantation of adipose-derived mesenchymal stem cells alternatively activates microglia and ameliorates neuropathological deficits in Alzheimer's disease mice. Cell Transplant. 2013;22 Suppl 1:S113-26. [PubMed ID: 24070198]. https://doi.org/10.3727/096368913X672181.

  • 36.

    Takamatsu K, Ikeda T, Haruta M, Matsumura K, Ogi Y, Nakagata N, et al. Degradation of amyloid beta by human induced pluripotent stem cell-derived macrophages expressing Neprilysin-2. Stem Cell Res. 2014;13(3 Pt A):442-53. [PubMed ID: 25460605]. https://doi.org/10.1016/j.scr.2014.10.001.