## 4 Discussion
On the other hand, when endothelial cells were treated with a lower concentration such as 10 uM A\(\beta_{1-42}\), only a few locations showed cell-A\(\beta\) matrix aggregates bound, and cells maintained their homeostasis (Figure 4A,C). It is interesting to note that A\(\beta\) mimicked CAA pathogenesis by depositing on the wall of BBB against the ECM rather than passing through the compromised BBB into the brain lane. We believe that this will be the initial stage of A\(\beta\) accumulation on the BBB. Whether the aggregate of the cell-A\(\beta\) matrix complex structure will experience a reduction over time to allow the transport of A\(\beta_{1-42}\) into the brain region has not been investigated. Investigating if A\(\beta\) transporter expression occurs on the ECs of our model will be necessary for future research. It has been reported that A\(\beta\) causes damage to TJs and directly impairs BBB integrity, confirming the relationship between A\(\beta\) and AD [35; 36]. Using a transwell assay technique, human brain endothelial cells (hCMEC/D3) have been used as a BBB model and treated to 0-40 uM of A\(\beta\)s. The concentration of A\(\beta\)s has been shown to increase the penetration ability of FITC-dextran: 40 uM of A\(\beta\)s has been shown to produce the highest permeability when compared to the control of 0 uM, while 5 uM has been found to produce a relatively small change [37]. We also confirmed that a low concentration of 10 uM and a higher concentration of 30 uM of A\(\beta_{1-42}\) for 48 h also induced structural damage in TJ (Figure 4A, \(p<0.001\)). The FITC-dextran perfusion assay with microfluidic technology demonstrated increased perfusion with A\(\beta_{1-42}\) in a concentration-dependent manner (Figure 3, \(p<0.001\)), suggesting that A\(\beta_{1-42}\) alters TJ architecture and digests BBB integrity [36; 37]. Significant differences in the perfusion of FITC-dextran were found between the engineered BBB treated with A\(\beta_{1-42}\) and the control (untreated), highlighting a possible neurotoxic effect of A\(\beta_{1-42}\). Figure 6: Schematic illustration of amyloid beta (A\(\beta\)) plaque aggregation with endothelial cells in the microfluidic device. Research rationale. (**A**): fully developed and functionally engineered BBB was challenged with different concentrations of A\(\beta_{1-42}\). (**B**): A\(\beta\) accumulates on the endothelial cells and forms cell–A\(\beta\) aggregates, eventually forming the thicker dysfunctional BBB. At this stage (**B**), the higher concentrations (20 uM and 30 uM) of A\(\beta\) produce a higher A\(\beta\)–cell aggregate that may diffuse into the brain region in a small amount. A low concentration such as 10 uM forms fewer aggregates with the cells, allowing individual oligomers to penetrate the barrier and reach the brain-like region. However, the A\(\beta\)–cells 3D complex structures at the barrier do not deter the permeability of FITC through the broken BBB and endothelial cells. Of note is the fact that the concentrations of A\(\beta_{1-42}\) used in this study are higher than the physiological concentration of A\(\beta_{40/42}\) in the blood [38]. However, we did not observe any defined mechanism of A\(\beta_{1-42}\) localization. There is published evidence that when the endothelial cells are impaired they release proinflammatory cytokines, increasing neuroinflammation and secondary alteration, ultimately resulting in the disruption of the TJ and BBB [39]. The cell stretching due to accumulation of A\(\beta_{1-42}\) may be due to impaired cell cytoskeleton, retard movement of the endothelial cells, and cause their death [40]. A\(\beta\) affects endothelial cells (ECs) in the brain microvasculature directly or indirectly by changing TJP distribution, encouraging EC mortality, increasing oxidative stress, and stimulating glial cell production of pro-inflammatory cytokines [38; 41]. In CAA mice models and human patients, A\(\beta\) buildup has a deleterious effect on brain artery walls, resulting in BBB disruption and possible bleeding [42; 43].