The pivotal role of astrocytes in an in vitro stroke model of the blood-brain barrier

Abstract

Stabilization of the blood-brain barrier during and after stroke can lead to less adverse outcome. For elucidation of underlying mechanisms and development of novel therapeutic strategies validated in vitro disease models of the blood-brain barrier could be very helpful. To mimic in vitro stroke conditions we have established a blood-brain barrier in vitro model based on mouse cell line cerebEND and applied oxygen/glucose deprivation (OGD). The role of astrocytes in this disease model was investigated by using cell line C6. Transwell studies pointed out that addition of astrocytes during OGD increased the barrier damage significantly in comparison to the endothelial monoculture shown by changes of transendothelial electrical resistance as well as fluorescein permeability data. Analysis on mRNA and protein levels by qPCR, western blotting and immunofluorescence microscopy of tight junction molecules claudin-3,-5,-12, occludin and ZO-1 revealed that their regulation and localisation is associated with the functional barrier breakdown. Furthermore, soluble factors of astrocytes, OGD and their combination were able to induce changes of functionality and expression of ABC-transporters Abcb1a (P-gp), Abcg2 (bcrp), and Abcc4 (mrp4). Moreover, the expression of proteases (matrixmetalloproteinases MMP-2, MMP-3, MMP-9, and t-PA) as well as of their endogenous inhibitors (TIMP-1, TIMP-3, PAI-1) was altered by astrocyte factors and OGD which resulted in significant changes of total MMP and t-PA activity. Morphological rearrangements induced by OGD and treatment with astrocyte factors were confirmed at a nanometer scale using atomic force microscopy. In conclusion, astrocytes play a major role in blood-brain barrier breakdown during OGD in vitro.

Omega-3 polyunsaturated fatty acids mitigate blood–brain barrier disruption after hypoxic–ischemic brain injury

“…In addition, activated MMPs were also deposited in and around blood vessels. Notably, negligible MMP activation was observed in astrocytes (Figure 6C), suggesting proteolytic activation of MMP-9 following release from astrocytes (Maddahi et al, 2009; Neuhaus et al, 2014). As expected, n-3 PUFA treatment dramatically suppressed the extensive activation of MMPs in the brain parenchyma at the same time point (Figure 6B and 6C).…”

Penetration of blood–brain barrier and antitumor activity and nerve repair in glioma by doxorubicin-loaded monosialoganglioside micelles system

“…36 As shown in Figure 2, HUVECs and C6 cells treatment with free DOX displayed DOX fluorescence in cytoplasm after 0.5 h, and the fluorescence was distributed in nucleus within a short period of 2-4 h. In comparison, the red fluorescence was observed after 0.5 h and enhanced in nuclei after incubation with DOX/GM1. There was no significant difference in fluorescence intensity between 2-h and 4-h incubation.…”

Characterisation of a mouse cerebral microvascular endothelial cell line (bEnd.3) after oxygen glucose deprivation and reoxygenation

“…These findings are consistent with the kinetics of BBB disruption in rodent models of stroke. Indeed, a number of studies have shown that BBB hyperpermeability occurs as early (2–4 hours) after ischaemia and is sustained up to 72 hours later . Moreover, previous OGD studies using bEnd.3 cells have shown paracellular hyperpermeability to occur in response to 2–3 hours of OGD exposure …”