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.

How Alpha Linolenic Acid May Sustain Blood–Brain Barrier Integrity and Boost Brain Resilience against Alzheimer’s Disease

“…DHA supplementation has shown inconclusive results of both positive and negative effects related to BBB integrity [ 59 ] and improvement in AD symptoms [ 60 ]. This may be due to the inhibitory effects of DHA on ALA metabolism [ 55 ] and the lower formation of intermediate n-3 FA like EPA, also needed for membrane phospholipids building.…”

Modulation of the Blood–Brain Barrier by Sigma-1R Activation

“…These morphological changes support the functional studies indicating a decrease in RBMVEC electrical resistance measured with ECIS. Similar changes were produced in brain microvascular endothelial cells by activation of GPR55 [35] and FFA1 receptors [49], or by agonists such as thrombin [33,47] and platelet-activating factor (PAF) [36].…”

“…This stateof-the-art technology was developed to assess neuronal function in vivo using fluorescence probes [50]. We optimized the use of a miniscope to assess the changes in BBB permeability in awake, freely moving rats [28,49,51]. Miniscope imaging of brain microcirculation allows the high-resolution, real-time, visualization of fluorescent tracer extravasation at selected regions of interest (ROIs) in awake rats while avoiding potential impairment of BBB by anesthetics [51].…”

“…Rat brain microvascular endothelial cells (RBMVEC) from Cell Applications, Inc. (San Diego, CA, USA) were cultured in rat brain endothelial basal medium enriched with endothelial growth supplement, according to the manufacturer's instructions at 37 • C and 5% CO 2 , as previously reported [35,49]. Cells were grown on T75 flasks coated with an attachment factor (Cell Applications, Inc.).…”

Two-pore channel-2 and inositol trisphosphate receptors coordinate Ca2+ signals between lysosomes and the endoplasmic reticulum

“…HeLa cells, U2OS cells, wild-type HEK-293 and HEK-293 cells engineered using CRISPR-Cas technology to lack all the three-native endogenous IP 3 receptors 36 were maintained in Dulbecco’s Modified Eagle Medium, supplemented with 10% (v/v) Fetal Bovine Serum, 100 μg/mL streptomycin and 100 units/mL penicillin (all from Invitrogen) at 37°C in a humidified atmosphere with 5% CO 2 . Rat brain microvascular endothelial cells (Cell Applications, Inc. (San Diego, CA, USA) were cultured in rat brain endothelial cell basal medium and rat brain endothelial cell growth supplement in flasks coated with attachment factor according to the manufacturer’s instructions (Cell Applications, Inc.) 51 , 52 …”