Ventromedial medullary pathway mediating cardiac responses evoked from periaqueductal gray.

Periaqueductal gray (PAG) is a midbrain region that projects to areas controlling behavioral and autonomic outputs and is involved in the behavioral and physiological components of defense reactions. Since Raphe Pallidus (RPa) is a medial medullary region comprising sympathetic premotor neurons governing heart function, it is worth considering the PAG-RPa path. We assessed: i) whether PAG projects to RPa; ii) the amplitude of cardiac responses evoked from PAG; iii) whether cardiovascular responses evoked from PAG rely on RPa. Experiments conducted in Wistar rats (±300 g) were approved by Ethics Committee CEUA-UFG (092/18). Firstly, (n = 3), monosynaptic retrograde tracer Retrobeads was injected into RPa; PAG slices were analyzed. Other two groups (n = 6 each) were anesthetized with urethane (1.4 g/kg) and chloralose (120 mg/kg) and underwent craniotomy, tracheostomy, catheterization of femoral artery and vein and of cardiac left ventricle. In one group, we injected the GABAA receptor antagonist, bicuculline methiodide (BMI - 40 pmol/100 nL) into lateral/dorsolateral PAG. Another group was injected (100 nL) with the GABAA receptor agonist muscimol (20 mM) into RPa, 20 min before BMI into PAG. The results were: i) retrogradely labelled neurons were found in PAG; ii) PAG activation by BMI caused positive chronotropism and inotropism, which were accompanied by afterload increases; iii) RPa inhibition with Muscimol reduced heart rate, arterial and ventricular pressures; iv) the subsequent PAG activation still increased arterial pressure, cardiac chronotropy and inotropy, but these responses were significantly attenuated. In conclusion, PAG activation increases cardiac chronotropy and inotropy, and these responses seem to rely on a direct pathway reaching ventromedial medullary RPa neurons.

Lipid dynamics in LPS-induced neuroinflammation by DESI-MS imaging.

It is well-established that bacterial lipopolysaccharides (LPS) can promote neuroinflammation through receptor Toll-like 4 activation and induces sickness behavior in mice. This phenomenon triggers changes in membranes lipid dynamics to promote the intracellular cell signaling. Desorption electrospray ionization mass spectrometry (DESI-MS) is a powerful technique that can be used to image the distribution of lipids in the brain tissue directly. In this work, we characterize the LPS-induced neuroinflammation and the lipid dynamics in C57BL/6 mice at 3 and 24 h after LPS injection. We have observed that intraperitoneal administration of LPS (5 mg/kg body weight) induces sickness behavior and triggers a peripheral and cerebral increase of pro- and anti-inflammatory cytokine levels after 3 h, but only IL-10 was upregulated after 24 h. Morphological analysis of hypothalamus, cortex and hippocampus demonstrated that microglial activation was present after 24 h of LPS injection, but not at 3 h. DESI-MS revealed a total of 14 lipids significantly altered after 3 and 24 h and as well as their neuroanatomical distribution. Multivariate statistical analyzes have shown that ions associated with phosphatidylethanolamine [PE(38:4)] and docosatetraenoic acid [FA (22:4)] could be used as biomarkers to distinguish samples from the control or LPS treated groups. Finally, our data demonstrated that monitoring cerebral lipids dynamics and its neuroanatomical distribution can be helpful to understand sickness behavior and microglial activation after LPS administration.

Functionalized nanomaterials: are they effective to perform gene delivery to difficult-to-transfect cells with no cytotoxicity?

Nanodiamonds (NDs), multiwalled carbon nanotubes (MWCNTs) and gold nanorods (NRs) can be functionalized to promote gene delivery to hard-to-transfect cells with higher transfection efficiency than cationic lipids, and inducing less cell death.

Sarcosine preconditioning induces ischemic tolerance against global cerebral ischemia.

Brain ischemic tolerance is an endogenous protective mechanism activated by a preconditioning stimulus that is closely related to N-methyl-d-aspartate receptor (NMDAR). Glycine transporter type 1 (GlyT-1) inhibitors potentiate NMDAR and suggest an alternative strategy for brain preconditioning. The aim of this work was to evaluate the effects of brain preconditioning induced by sarcosine, a GlyT-1 inhibitor, against global cerebral ischemia and its relation to NMDAR. Sarcosine was administered over 7 days (300 or 500 mg/kg/day, ip) before the induction of a global cerebral ischemia model in Wistar rats (male, 8-week-old). It was observed that sarcosine preconditioning reduced cell death in rat hippocampi submitted to cerebral ischemia. Hippocampal levels of glycine were decreased in sarcosine-treated animals, which was associated with a reduction of [(3)H] glycine uptake and a decrease in glycine transporter expression (GlyT-1 and GlyT-2). The expression of glycine receptors and the NR1 and NR2A subunits of NMDAR were not affected by sarcosine preconditioning. However, sarcosine preconditioning reduced the expression of the NR2B subunits of NMDAR. In conclusion, these data demonstrate that sarcosine preconditioning induces ischemic tolerance against global cerebral ischemia and this neuroprotective state is associated with changes in glycine transport and reduction of NR2B-containing NMDAR expression.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Trypan blue exclusion assay by flow cytometry.

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.