Lipopolysaccharide-induced blood-brain barrier disruption: roles of cyclooxygenase, oxidative stress, neuroinflammation, and elements of the neurovascular unit

Abstract

Background: Disruption of the blood-brain barrier (BBB) occurs in many diseases and is often mediated by inflammatory and neuroimmune mechanisms. Inflammation is well established as a cause of BBB disruption, but many mechanistic questions remain.

Methods: We used lipopolysaccharide (LPS) to induce inflammation and BBB disruption in mice. BBB disruption was measured using (14)C-sucrose and radioactively labeled albumin. Brain cytokine responses were measured using multiplex technology and dependence on cyclooxygenase (COX) and oxidative stress determined by treatments with indomethacin and N-acetylcysteine. Astrocyte and microglia/macrophage responses were measured using brain immunohistochemistry. In vitro studies used Transwell cultures of primary brain endothelial cells co- or tri-cultured with astrocytes and pericytes to measure effects of LPS on transendothelial electrical resistance (TEER), cellular distribution of tight junction proteins, and permeability to (14)C-sucrose and radioactive albumin.

Results: In comparison to LPS-induced weight loss, the BBB was relatively resistant to LPS-induced disruption. Disruption occurred only with the highest dose of LPS and was most evident in the frontal cortex, thalamus, pons-medulla, and cerebellum with no disruption in the hypothalamus. The in vitro and in vivo patterns of LPS-induced disruption as measured with (14)C-sucrose, radioactive albumin, and TEER suggested involvement of both paracellular and transcytotic pathways. Disruption as measured with albumin and (14)C-sucrose, but not TEER, was blocked by indomethacin. N-acetylcysteine did not affect disruption. In vivo, the measures of neuroinflammation induced by LPS were mainly not reversed by indomethacin. In vitro, the effects on LPS and indomethacin were not altered when brain endothelial cells (BECs) were cultured with astrocytes or pericytes.

Conclusions: The BBB is relatively resistant to LPS-induced disruption with some brain regions more vulnerable than others. LPS-induced disruption appears is to be dependent on COX but not on oxidative stress. Based on in vivo and in vitro measures of neuroinflammation, it appears that astrocytes, microglia/macrophages, and pericytes play little role in the LPS-mediated disruption of the BBB.

Fig. 1

Dose-dependence of LPS on BBB permeability to ¹⁴C-Sucrose. Only the 3 mg/kg dose increased BBB permeability. **p < 0.01, n = 9/group

Fig. 2

Effects of LPS on body weight, brain weight, and BBB permeability. Panel a shows that all doses of LPS had a significant effect on body weight (n = 6/group). Panel b shows no dose of LPS had an effect on brain weight (n = 6/group). Panel c shows that BBB permeability was increased to ¹⁴C-sucrose 24 but not 4 h after 3 mg/kg of LPS (n = 3–4/group). Panel d shows that 3 mg/kg LPS increased BBB permeability to albumin (n = 3/group). *p < 0.05; ***p < 0.001

Fig. 3

Effects of indomethacin and N-acetylcysteine on LPS-induced BBB disruption. Panel a shows that pre-treatment with indomethacin prevented LPS-induced BBB disruption (n = 7–8/group), whereas panel b shows that N-acetylcysteine was without effect (9–10/group). *p < 0.05; **p < 0.01; ****p < 0.001

Fig. 4

Effects of LPS and indomethacin on microglial/macrophage and astrocytic activation. Microglial/macrophage activation was increased by LPS but not blocked by indomethacin as assessed by Iba1 (left-hand panels) and by F4/80 (middle panels) immunohistochemistry. Astrocytic activation was increased by LPS and blocked by indomethacin as assessed by GFAP staining (right-hand panels). The bottom three panels show quantification of Iba1 (n = 5–6/statistical cell), F4/80 (n = 5–6), and GFAP (n = 4–5) immunohistochemical staining. *p < 0.05, **p < 0.01, ***p < 0.001, Mag = ×20

Fig. 5

Effects of LPS plasma TREM2. Plasma TREM2 levels are increased by LPS, an effect blocked by treatment with indomethacin (n = 8/group). *p < 0.05

Fig. 6

Effects of LPS on TEER of BMECs and cytoarchitecture of ZO-1, claudin-5, and occludin. Panels a and b show that LPS added either 24 or 48 h earlier to monocultures of BMECs decreased their TEER in a dose-dependent manner, consistent with disruption of the BBB. Immunostaining for ZO-1 shows increasing dose-dependent cytoarchitectural disorganization for ZO-1 (panels c–f). Immunostaining for claudin-5 (panels g and h) does not clearly show such disorganization, whereas immunostaining for occludin does (panels i and j). *p < 0.05; ***p < 0.001

Fig. 7

Effect of LPS and indomethacin (Indo) on TEER and ¹⁴C-sucrose permeability in monolayers of BEC monocultures (EOO, n = 10), BEC + astrocyte co-cultures (EOA, n = 5), BEC + pericyte co-cultures (EPO, n = 5), and BEC + pericyte + astrocyte tri-cultures (EPA, n = 10). Panel a shows that in monocultures of BECs, LPS increased permeability to sucrose and that indomethacin partially blocked this effect. Panel b shows that LPS increased permeability of monolayer monocultures of BEC as measured by TEER and that indomethacin had no effect on LPS-induced permeability. Co-cultures of BECs with astrocytes (panels c and d) or pericytes (panels e and f) or tri-cultures of BECs with pericytes and astrocytes (panels g and h) did not produce results substantially different from those obtained with monocultures of BECs. Y-axis is %Control of Pe in units of microliters per minute per square centimeter. *p < 0.05, ***p < 0.001; without bar compares to respective 0 value; with bar compares LPS 10 μg/ml without indomethacin to LPS 10 μg/ml with indomethacin

Fig. 8

Effect of LPS and indomethacin on ¹⁴C-sucrose (upper panel) and Tc-Alb permeabilities (lower panel) in monocultures of BECs. Results show that indomethacin partially blocked the LPS-induced permeability for both sucrose and albumin. *p <0.05, ***p <0.001; without bar compares to respective 0 value; with bar compares indicatedgroups