Blood-Brain Barrier Disruption Mediated by FFA1 Receptor-Evidence Using Miniscope

Abstract

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), obtained from diet and dietary supplements, have been tested in clinical trials for the prevention or treatment of several diseases. n-3 PUFAs exert their effects by activation of free fatty acid (FFA) receptors. FFA1 receptor, expressed in the pancreas and brain, is activated by medium- to long-chain fatty acids. Despite some beneficial effects on cognition, the effects of n-3 PUFAs on the blood-brain barrier (BBB) are not clearly understood. We examined the effects of FFA1 activation on BBB permeability in vitro, using rat brain microvascular endothelial cells (RBMVEC), and in vivo, by assessing Evans Blue extravasation and by performing live imaging of brain microcirculation in adult rats. AMG837, a synthetic FFA1 agonist, produced a dose-dependent decrease in RBMVEC monolayer resistance assessed with Electric Cell-Substrate Impedance Sensing (ECIS); the effect was attenuated by the FFA1 antagonist, GW1100. Immunofluorescence studies revealed that AMG837 produced a disruption in tight and adherens junction proteins. AMG837 increased Evans Blue content in the rat brain in a dose-dependent manner. Live imaging studies of rat brain microcirculation with miniaturized fluorescence microscopy (miniscope) showed that AMG837 increased extravasation of sodium fluorescein. Taken together, our results demonstrate that FFA1 receptor activation reduced RBMVEC barrier function and produced a transient increase in BBB permeability.

Keywords: BBB; ECIS; brain microvascular endothelial cells; n-3 PUFAs; omega-3 fatty acids.

Figure 1

FFA1 mediated an acute decrease in the normalized resistance of RBMVEC monolayer assessed with Electric Cell–Substrate Impedance Sensing (ECIS). (A) Representative example of a decrease in normalized resistance produced by AMG837 (10 μM); pretreatment with the FFA1 antagonist GW1100 (10 μM) reduced the effect of AMG837. (B) Comparison of the effect of AMG (1–20 μM) on RBMVEC resistance in the absence and presence of GW1100 (10 μM); AMG837 (2.5, 5, 10, 20 μM) produced a dose-dependent decrease in the normalized resistance of RMBVEC monolayer. p < 0.05 as compared to the effect of AMG837 (1 μM) (*) or to the effect of the same concentration of AMG837 in the presence of GW1100 (10 μM) (**). (C) Representative example of a decrease in normalized resistance produced by DHA (10 μM); pretreatment with GW1100 (10 μM) markedly decreased the effect of DHA. (D) Comparison of the reduction in normalized resistance produced by DHA (10 μM) in the absence and presence of GW1100 (10 μM). * p < 0.05.

Figure 2

FFA1-mediated effect on normalized RBMVEC resistance returned to basal levels within 24 h after treatment with AMG837. (A) Representative example of normalized resistance recorded with ECIS during treatment with AMG837 (10 μM) in the first 2 and after 24 h. (B) Comparison of the effect of AMG837 on normalized resistance at 2 and 24 h. * p < 0.05.

Figure 3

AMG37 produced a disruption in tight and adherens junction proteins and F-actin in RBMVEC. Distribution of tight junction accessory protein ZO-1, adherens junction protein VE-cadherin, and cytoskeleton component F-actin in control RBMVEC and RBMVEC treated with AMG837 (10 µM, 30 min). Nuclei are stained with DAPI. Treatment with AMG837 produced a disruption in ZO-1 and VE-cadherin, reorganization of F-actin and the formation of intercellular gaps (arrows).

Figure 4

AMG837 increased Evans Blue content in the brain. (A) Systemic administration of AMG837 (0.1–3 mg/kg, i.v.) increased Evans Blue content in the brain, determined at 2 h after treatment, in a dose-dependent manner, indicating an increase in BBB permeability. GW1100 (2 mg/kg, i.v.) also increased Evans Blue. P < 0.05 as compared to control or vehicle-injected rats (*) or compared with AMG 837 (1–3 mg/kg) (**). (B) Comparison of Evans Blue concentration in the rat brain in control rats, and 2 and 72 h after AMG (2 mg/kg, i.v.). p < 0.05 as compared to control (*) or to effect of AMG at 72 h (**).

Figure 5

AMG837 increases sodium fluorescein (Na-F) extravasation in brain microvessels visualized with miniscope. (A) Pseudocolor images of Na-F fluorescence in rat prefrontal cortex before (control), 30 min and 72 h after injection of AMG837. (B) Comparison of Na-F extravasation (%) in brain microvessels in control and AMG (2 mg/kg)-treated rats, at 30 min and 72 h, determined by averaging the fluorescence intensity for 10 ROIs in the vicinity of microvessels 15 min after i.v. administration of Na-F. AMG837 increased Na-F extravasation at 30 min after injection. p < 0.05 as compared to control (*) or to effect of AMG at 72 h (**).