Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) disrupt ultrastructure of brain endothelial cells.

<p>Cells were treated with no addition (control) (A,D,E), LPS (B,F,G) or UCB (C,H,I) and were analyzed by transmission electron microscopy. Black arrows, tight intercellular junctions; grey arrows, disruption of the plasma membrane; arrowheads, invaginations of the plasma membrane; a, apoptotic cells bodies; m, mitochondria; N, cell nuclei; RER, rough endoplasmic reticulum; v, vacuole. Representative results from one of four independent experiments are shown.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) modify the distribution of β-catenin in brain endothelial cells.

<p>Cells in mono-culture or co-cultured with astrocytes were fixed and immunostained with an antibody against β-catenin to evaluate its cellular localization (scale bars, 40 and 20 µm, respectively). Disruption of the monolayer with gaps between endothelial cells (*), alterations in protein patterns (arrowheads) with the presence of dot-like staining (yellow arrow), and perinuclear distribution (arrows) are indicated. Representative results from one of two independent experiments are shown.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) disrupt the endothelial monolayer.

<p>Permeability to sodium fluorescein (Na-F_Pe) (A) and transendothelial electrical resistance (TEER) (B) were determined after exposure. All values presented are means ± S.E.M. from at least four independent experiments performed in duplicate. *<i>P<</i>0.05, **<i>P<</i>0.01 and ***<i>P<</i>0.001 <i>vs.</i> respective control; ^§<i>P<</i>0.05 <i>vs.</i> LPS at the same time-point;^#<i>P</i><0.05 from 4 h.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) activate metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2) released by brain microvascular endothelial cells.

<p>A representative gel from one experiment is shown, where MMP-2 and MMP-9 were identified by their apparent molecular mass of 67 and 92 kDa, respectively (A). The intensity of the bands was quantified by scanning densitometry and results for MMP-9 (B) and MMP-2 (C) were standardized with respect to total protein content. Results are mean ± S.E.M. from at least four independent experiments performed in duplicate. *<i>P</i><0.05 and **<i>P</i><0.01 <i>vs.</i> respective control; ^##<i>P</i><0.01 from 4 h.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) alter <i>zonula occludens</i>-1 (ZO-1) expression in brain endothelial cells.

<p>Cells, either in mono-culture or co-cultured with astrocytes, were fixed and immunostained with an antibody against ZO-1 to evaluate its cellular localization and pattern of expression, as well as integrity of the monolayer. Disruption of the monolayer with gaps between endothelial cells (*), alterations in protein patterns (arrowheads) and perinuclear distribution (arrows) are indicated. Representative results from one of two independent experiments are shown. Scale bar, 20 µm.</p

Resting and electrical stimulation evoked[³H]5-HT and [³H]NA efflux in the hippocampus of P2rx7+/+ and P2rx7−/− mice, respectively.

<p>Tissue slices were loaded with [³H]5-HT or [³H]NA and then superfused with Krebs’ solution. After 60 min preperfusion, slices were stimulated electrically with the following parameters: 25 V, 1 msec, 2 Hz, 240 shocks. The release of radioactivity was expressed in Bq/g. For the calculation of the resting [³H]5-HT/[³H]NA efflux, the tritium content of the sample collected immediately before the first electrical stimulation period was taken into account. Electrical stimulation-induced [³H]5-HT/[³H]NA efflux (S1) was expressed by calculating the net release in response to electrical stimulation by the area-under-the-curve method. The number of experiments are in parentheses. *P<0.05, significantly different from P2rx7+/+ mice, calculated by the Student’s t-test.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) induce cell death in brain microvascular endothelial cells.

<p>Culture medium was collected for determination of lactate dehydrogenase (LDH) activity (A). Nuclei were stained with Hoechst 33258 dye and morphological features of apoptosis are pointed (arrows) (B). The number of apoptotic nuclei was counted and results were expressed as percentage of the total number of nuclei (C). Cell lysates were obtained for caspase-3 activity determination (D). Results are mean ± S.E.M. from at least five independent experiments performed in duplicate. Scale bar, 20 µm. *<i>P<</i>0.05, **<i>P<</i>0.01 and ***<i>P<</i>0.001 <i>vs.</i> respective control; ^§<i>P<</i>0.05, ^§§<i>P<</i>0.01 <i>vs.</i> LPS at the same time-point; ^#<i>P</i><0.05 and ^##<i>P</i><0.01 from 4 h.</p

Lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) alter expression of claudin-5 in brain endothelial cells.

<p>Cells, either in mono-culture or co-cultured with astrocytes, were fixed and immunostained with an antibody against claudin-5 to evaluate its cellular localization, pattern of expression and integrity of the monolayer. Disruption of the monolayer with gaps between endothelial cells (*) and alterations in protein patterns (arrowheads) are indicated. Representative results from one of two independent experiments are shown. Scale bar, 20 µm.</p

Effects of lipopolysaccharide (LPS) and unconjugated bilirubin (UCB) on endothelial integrity in mono-cultures and co-cultures.

<p>Permeability to sodium fluorescein (Na-F_Pe) (A, B) and transendothelial electrical resistance (TEER) (C, D) were determined in mono-cultures (A,C) and co-cultures (B,D) after 24 h exposure. All values presented are means ± S.E.M. from at least two independent experiments performed in triplicate. **<i>P<</i>0.01 and ***<i>P<</i>0.001 <i>vs.</i> respective control; ^§§<i>P<</i>0.01 and ^§§§<i>P<</i>0.001 <i>vs.</i> LPS at the same time-point.</p

The effect of the genetic deletion of P2rx7 on ATP-evoked tritiated (A, C) and endogenous (B) glutamate efflux (D) mRNA expression and (E–I) immunofluorescence staining of the NR2B subunit of the NMDA receptors in acute hippocampal slices.

<p>A/10 mM ATP was used to induce [³H]Glu release from hippocampal slices of P2rx7+/+ and P2rx7−/− mice. After a 60-min preperfusion, the basal extracellular [³H]Glu efflux was lower in P2rx7−/− mice. 6-min perfusion of ATP (10 mM) resulted in a transient increase in the efflux of [³H]Glu in P2rx7+/+ mice, which peaked at 6 min after ATP administration and gradually decreased to baseline levels after 12 min. The ATP-evoked [³H]Glu efflux is substantially decreased in the hippocampus of P2rx7−/− mice, and the residual efflux is abolished by the selective P2X1 receptor antagonist NF449 (100 nM), which was applied at 15 min before ATP perfusion. [³H]Glu release is expressed as a percentage of the amount of radioactivity in the tissue at the sample collection time (fractional release). For the evaluation of the basal tritium outflow, the tritium content of the first four consecutive 3-min samples were taken into account. The curves represent the mean ± SEM of 8–12 identical experiments. B/HPLC analysis. Samples indicated in (A) as S1 and S2 were analyzed. ATP perfusion (10 mM) significantly increases the endogenous Glu level in the effluent. Results are expressed as pmol/3 min. N = 8, ***P<0.001. C/Concentration-response relationship of ATP-evoked [³H]Glu efflux in P2rx7+/+ and P2rx7−/− mice. Experiments were performed according to the protocol shown in (A), using different concentrations of ATP, as indicated in the abscissa. The net ATP-induced release was calculated and expressed as the fractional release (%). The curves represent the mean ± S.E.M. of 4–12 identical experiments. D/Changes in the mRNA expression levels of the NMDA-NR2B receptor in hippocampus obtained from P2rx7+/+ and P2rx7−/− mice. Immediately after the 60-min incubation, the brain slices were removed and total RNA was extracted from the hippocampus and reverse transcribed to cDNA. Quantitative SYBR Green real-time PCR was performed using specific primers, as described in Methods, and cDNA as a template. The experiments were repeated two times with similar results. The expression level of the NR2B receptor was normalized to that of the distinct housekeeping gene, 18S rRNA. The data are displayed as the mean ± SEM. Asterisk indicates significant difference from the P2rx7+/+ mice (*P<0.05, Student’s t-test). E-I/Immunofluorescence staining for NR2B on hippocampal sections of P2rx7+/+ and P2rx7−/− mice. Besides the typical dotted staining most likely presenting NR2B - immunolabelled terminals (arrows, F, H) some staining is observable around cell bodies especially in the pyramidal cell layer (three arrowheads G). The whole staining is more intense in the hippocampal section of P2rx7−/− mouse (G, I). The most intense staining is observed in the CA3 regions (CA3 on E, G and F, H), while stratum oriens (or) shows the least immunoreactivity either in P2rx7+/+ and P2rx7−/− sample. Contrary to P2rx7+/+ staining (E), intense immunofluorescence illustrates hilus region on P2rx7−/− (G) sample. Images acquired at higher magnification (F,H) also show blood vessels (arrowheads), stained at the same level in both sections (background staining). bars: 50 µm in F, H, 500 µm in E, G. I. Immunofluorescence staining intensity for NR2B in P2rx7+/+ and P2rx7−/− mice. Average intensity was quantified with NIH ImageJ program (U.S. National Institutes of Health, Bethesda, MD) and is expressed in arbitrary units. Asterisk indicates significant difference from the wild type mice (*P<0.05, Student’s t-test, n = 3).</p