Distinct patterns of cerebral extravasation by Evans blue and sodium fluorescein in rats.

The Evans blue dye (EBD; 961 Da) and the sodium fluorescein dye (NaF; 376 Da) are commonly used inert tracers in blood-brain barrier (BBB) research. They are both highly charged low molecular weight (LMW) tracers with similar lipophobic profiles. Nevertheless, the EBD binds to serum albumin (69,000 Da) to become a high molecular weight (HMW) protein tracer when injected into the circulation, whereas the NaF remains an unbound small molecule in the circulation. In this study, rats were injected with equal doses of either EBD or NaF to monitor their blood and tissue distribution. The EBD was largely confined to the circulation with little accumulation in the peripheral organ and even less accumulation in the central tissue, whereas the NaF distributed more evenly between the blood and the peripheral organ but was also largely excluded from the central tissue. Importantly, the EBD crossed the BBB most effectively at the prefrontal cortex and the cerebellum, and most poorly at the striatum. In marked contrast, the NaF was evenly distributed throughout the brain. Finally, the EBD exhibited this same peculiar tissue distribution profile when administered by either bolus injection or slow infusion. Our study suggests that different regions of the brain are equally permeable to LMW inert dyes like the NaF, but are markedly different in permeability to HMW proteins such as EBD-labelled serum albumin

The Acclimation Mechanisms of Chlamydomonas reinhardtii against Nitrosative Stress: A Role of NADPH Oxidase (RBOL2) in the Regulation of Nitric Oxide-Mediated ER Stress and Glutathione Redox State

Nitric oxide (NO) is a signal in the modulation of acclamatory responses to stress in plants. Here, the metabolic shift of Chlamydomonas reinhardtii to sub-lethal NO stress was approached by exposure to 0.1 mM S-nitroso-N-acetylpenicillamine (SNAP), a NO donor, in the presence or the absence of the NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO). NO did not cause growth impairment but induced a decrease in glutathione (GSH) levels and redox state. NO upregulated the expression of glutathione-associated genes, glutathione synthetase (GSH1), and glutathione reductase (GSHR1) genes while it decreased that of the proteins associated with ER stress-induced unfolded protein response (UPR). Furthermore, the expression of NADPH oxidase isoform, respiratory burst oxygenase-like 2 (RBOL2), instead of RBOL1 increased under NO stress. NO-induced upregulation of GSH1 and GSHR1 upregulation and the downregulation of most UPR genes were not found in rbol2 mutant. The presence of cPTIO suppressed the NO-induced changes in GSH availability, UPR, and RBOL expression. Overall, NADPH oxidase (RBOL2)-dependent and -independent signaling pathways involve in the inhibition of UPR and the enhancement of GSH availability by NO

Peripheral accumulation of Evans blue dye (EBD) and sodium fluorescein dye (NaF).

<p>(A) Representative images of rats before and after receiving EBD or NaF at equal doses (80 mg/kg i.v. bolus into the femoral vein). Post-injection images were taken at 2 h post-injection. (B) Blood concentration of EBD or NaF prior to and at 5, 30, 60, 90, and 120 min post-injection. Samples were collected from a cannulated femoral artery. (C) Peripheral EBD concentration in the blood and the liver at 120 min post-injection. Data are shown as mean±SEM. **p<0.01, comparison by paired t test. (D) Peripheral NaF concentration in the blood and the liver at 120 min post-injection. Data are shown as mean±SEM. *p<0.05, comparison by paired t test.</p

Central extravasation of sodium fluorescein dye (NaF) in a dose-dependent manner.

<p>Regional brain distribution of extravasated NaF in the prefrontal cortex, the motor cortex, the striatum, and the cerebellum following bolus injection (2 ml/kg i.v. into the femoral vein) of 1%, 2%, or 8% NaF. Data are shown as mean±SEM. **p<0.01 when compared by TWO-WAY repeated measures ANOVA followed by Tukey’s multiple comparisons test.</p

Illustration of the brain regions collected for analysis.

<p>Illustration of the brain regions collected for analysis.</p

Peripheral accumulation of Evans blue dye (EBD) following bolus injection and slow infusion.

<p>(A) Representative images of rats before and after receiving 4% EBD via bolus injection or slow infusion (1 ml/hr) at equal doses (2 ml/kg i.v. into the femoral vein). (B) Blood concentration of EBD prior to and at 5, 30, 60, 90, and 120 min post-bolus injection or slow infusion. Samples were collected from a cannulated femoral artery. Data are shown as mean±SEM. *p<0.05 and **p<0.01, comparison by TWO-WAY repeated measures ANOVA followed by Sidak’s multiple comparisons test. (C) Peripheral EBD concentration in the liver at 120 min post-bolus injection or slow infusion. Data are shown as mean±SEM. n.s., no significant difference when compared by unpaired t test.</p

Central extravasation of Evans blue dye (EBD) following bolus injection and slow infusion.

<p>Regional brain distribution of extravasated EBD in the prefrontal cortex, the motor cortex, the striatum, and the cerebellum following bolus injection (left panel) or slow infusion (right panel) (80 mg/kg i.v. into the femoral vein). Data are shown as mean±SEM. Statistics used are described in the figure.</p

Erratum to: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition) (Autophagy, 12, 1, 1-222, 10.1080/15548627.2015.1100356

non present