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. 2016 Dec;36(12):2108-2121.
doi: 10.1177/0271678X16642233. Epub 2016 Mar 31.

Blood-brain barrier breakdown promotes macrophage infiltration and cognitive impairment in leptin receptor-deficient mice

Alexis M Stranahan  1 , Shuai Hao  2 , Aditi Dey  2 , Xiaolin Yu  2 , Babak Baban  3   4
Affiliations

Blood-brain barrier breakdown promotes macrophage infiltration and cognitive impairment in leptin receptor-deficient mice

Alexis M Stranahan et al. J Cereb Blood Flow Metab. 2016 Dec.

Abstract

Accumulating evidence indicates that obesity accelerates the onset of cognitive decline. While mechanisms are still being identified, obesity promotes peripheral inflammation and increases blood-brain barrier (BBB) permeability. However, no studies have manipulated vascular permeability in obesity to determine whether BBB breakdown underlies memory deficits. Protein kinase Cβ (PKCβ) activation destabilizes the BBB, and we used a PKCβ inhibitor (Enzastaurin) to block BBB leakiness in leptin receptor-deficient (db/db) mice. Enzastaurin reversed BBB breakdown in db/db mice and normalized hippocampal function without affecting obesity or metabolism. Flow cytometric analysis of forebrain mononuclear cells (FMCs) from db/db mice revealed macrophage infiltration and induction of the activation marker MHCII in microglia and macrophages. Enzastaurin eliminated macrophage infiltration and MHCII induction, and protein array profiling revealed parallel reductions in IL1β, IL6, MCP1, and TNFα. To investigate whether these signals attract peripheral monocytes, FMCs from Wt and db/db mice were plated below migration inserts containing peritoneal macrophages. Peritoneal macrophages from db/db mice exhibit increases in transmigration that were blocked by recombinant IL1RA. These studies indicate that BBB breakdown impairs cognition in obesity and diabetes by allowing macrophage infiltration, with a potential role for IL1β in trafficking of peripheral monocytes into the brain.

Keywords: Diabetes; hippocampus; inflammation; learning and memory; microglia; obesity.

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Figures

Figure 1.
Figure 1.
Oral administration of the blood–brain barrier sealing compound Enzastaurin does not impact food intake, obesity, or adiposity in leptin receptor-deficient mice. (a) Treatment with Enzastaurin, a PKCβ inhibitor previously shown to prevent BBB leakiness in models of infectious disease, does not cause weight loss in db/db mice when administered daily during the final two weeks of the study (db/Enz; 50 mg/kg/d, PO). Vehicle-treated db/db and Wt mice received daily oral administration of 0.5% methyl cellulose (db/Veh, Wt/Veh). (b) Enzastaurin administration had no effect on food intake during the two-week treatment period. (c) db/db mice reliably exhibit greater adiposity, based on increased weights of individual fat depots relative to body weight. The magnitude of this effect was unchanged by Enzastaurin treatment. (d) The severity of insulin resistance in db/db mice was unaffected by systemic Enzastaurin administration. For all graphs, bars or symbols depict the mean of (n = 10) mice in each group. Error bars represent the SEM and asterisks (*) denote statistical significance at p < 0.05 following one-way ANOVA (a, b, c) or repeated measures one-way ANOVA (d) with Bonferonni’s post hoc.
Figure 2.
Figure 2.
Genetic obesity promotes blood–brain barrier breakdown. (a) Penetration of sodium fluorescein (NaFl), a low molecular weight fluorophore that does not pass the intact BBB, is more evident on hippocampal sections from vehicle-treated db/db mice (db/Veh). Increased NaFl penetrance was observed relative to vehicle-treated Wt mice (Wt/Veh) and db/db mice administered the PKCβ inhibitor Enzastaurin (db/Enz). (b) Quantitative fluorometry in hippocampal extracts reveals that hippocampal fluorescein penetrance is significantly increased in db/Veh mice relative to db/Enz mice and Wt/Veh controls. This pattern was not evident in cortical extracts, suggestive of regional differences in vulnerability of the BBB. (c) Western blotting for the tight junction proteins claudin 5 and occludin revealed PKCβ-dependent reductions in tight junction protein expression in db/Veh mice. (d) Representative images of western blot membranes for claudin 5 and occludin. For all graphs, bars depict the mean of (n = 6–8) mice per group, error bars represent the sem, and asterisks (*) denote statistical significance at p < 0.05 following one-way ANOVA with Bonferonni’s post hoc.
Figure 3.
Figure 3.
Pharmacological reinstatement of BBB integrity restores hippocampus-dependent memory and long-term potentiation. (a) Spatial recognition memory in the Y-maze is impaired in vehicle-treated db/db mice (db/Veh), but is normalized with Enzastaurin treatment (db/Enz). (b) Novel object recognition memory (left panel) is reduced in db/Veh mice and restored in db/Enz mice. There were no effects of genotype or Enzastaurin treatment on the total time spent exploring in each trial (right panel). (c) Reinstatement of BBB integrity in db/Enz mice rescues long-term potentiation at medial perforant path synapses on dentate gyrus granule neurons in hippocampal slices (left panel). db/Veh mice exhibit smaller increases in the slope of the field excitatory postsynaptic potential (fEPSP) during minutes 50–60 of recording (right panel), but db/Enz mice are indistinguishable from Wt mice. (d) The ratio of the presynaptic fiber volley (FV) and the postsynaptic fEPSP slope across a range of stimulation intensities was not influenced by genotype or Enzastaurin treatment. (e) Quantification of reductions in fEPSP slope after sequential stimuli (S1, S2) revealed no differences in presynaptic paired-pulse depression. (f) Direct application of the PKCβ inhibitor Enzastaurin had no effect on LTP deficits in hippocampal slices from db/db mice (left panel). PKCβ antagonism also did not influence plasticity in Wt slices (right panel). For all graphs, the height of bars or symbols depicts the average of (n = 9–11) mice per condition (a, b), or (n = 12–16) slices from mice in each condition (c–f). Error bars represent the SEM and asterisks (*) denote statistical significance at p < 0.05 following one-way ANOVA (a-e) or two-way ANOVA (f) with Bonferonni's post hoc.
Figure 4.
Figure 4.
Blood–brain barrier breakdown promotes macrophage infiltration and classical activation in resident microglia from db/db mice. (a) Experimental timeline for Enzastaurin treatment and isolation of forebrain mononuclear cells (FMCs). (b) Cells were gated on forward and side scatter (FSC, SSC) and nonviable cells were excluded using the dead cell marker Sytox Orange. (c) Analysis of CD45hi macrophages and CD45low microglia within the population of CD11b+ cells reveals macrophage infiltration in db/Veh mice. Increases in CD45hi/CD11b+ cells were not detected in db/db mice treated with Enzastaurin (db/Enz). (d) Fluorescence intensity for the monocyte marker Ly6C also distinguishes between brain-penetrant macrophages (Ly6Chi) and resident microglia (Ly6Clow). Accumulation of Ly6Chi/CD11b + macrophages in db/Veh mice was eliminated by Enzastaurin treatment. For all histograms, filled regions represent labeling with fluorescence-conjugated antibodies and empty regions show fluorescence with isotype control. (e) Scatterplots show representative distributions of CD45hi macrophages and CD45low microglia that express the classical activation marker MHCII. Graph (far right) shows coordinated induction of MHCII in microglia and macrophages from db/Veh mice that is reversed by Enzastaurin. For all graphs, bar height shows the average of (n = 8) mice per group, error bars represent the SEM, and asterisks (*) denote statistical significance at p < 0.05 following one-way ANOVA with Bonferonni’s post hoc.
Figure 5.
Figure 5.
Induction of pro-inflammatory cytokines in brain-penetrant macrophages and resident microglia is eliminated by reinstating BBB integrity. (a) Protein array analysis of conditioned media from forebrain mononuclear cells (FMCs) isolated from vehicle-treated db/db and Wt mice (db/Veh, Wt/Veh) and from db/db mice administered the PKCβ inhibitor Enzastaurin (db/Enz). Graphs represent data from db/Veh and db/Enz cells, with each target normalized to the corresponding mean intensity in samples from Wt/Veh cells. FMCs from db/Veh mice released significantly more interleukin 1β (IL1β), interleukin 6 (IL6), monocyte chemoattractant protein 1 (MCP1) and tumor necrosis factor-α (TNFα), but induction of these pro-inflammatory cytokines was not detected in cells from db/Enz mice. (b) Quantification of interleukin 1β (IL1β) by western blotting in cell lysates revealed increased IL1β in db/Veh cells, but not in db/Enz cells. (c) Reinstatement of BBB integrity in db/Enz mice reduced levels of IL6 in cell lysates. (d) FMCs from db/Veh mice, but not db/Enz mice, exhibit increases in MCP1 protein. (e) PKCβ antagonism prevents increases in TNFα in cell lysates from db/db mice. For all graphs, bar height depicts the mean fold change from Wt/Veh (A) or the group mean (B-E) from (n = 6) mice per condition. Error bars represent the SEM and asterisks (*) denote statistical significance at p < 0.05 following one-way ANOVA with Bonferonni’s post hoc.
Figure 6.
Figure 6.
Interleukin 1 mediates attraction between peripheral macrophages and forebrain mononuclear cells in genetic obesity. (a) Schematic diagram represents experimental design for analysis of migration in peritoneal macrophages (Mac) and forebrain mononuclear cells (FMCs). Micrographs to the right of each schematic show representative images of macrophage transmigration in each condition visualized with the nuclear marker DAPI. Scalebar = 100 microns. (b) Transmigration is selectively enhanced when peritoneal macrophages from db/db mice are plated above FMCs from another db/db mouse. (c) Incubation with recombinant interleukin 1 receptor antagonist (rIL1RA) prevents increases in transmigration. (d) Incubation with a neutralizing antibody against interleukin 6 (IL6 nAb) does not influence transmigration. (e) Antibody-based inhibition of tumor necrosis factor alpha (anti-TNFa) does not affect transmigration. (f) The monocyte chemoattractant protein 1 (MCP1) receptor antagonist RS504393 does not prevent increases in transmigration in db/db macrophages plated above db/db FMCs. For all graphs, bar height shows the average of (n = 8–11) mice per condition, error bars represent the SEM, and asterisks (*) denote statistical significance at p < 0.05 following 2 × 2 ANOVA.
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