Identifying STEDable BF2-Azadipyrromethene Fluorophores.

BF2-azadipyrromethenes are highly versatile fluorophores used for cellular and in vivo imaging in the near-infrared and far-red regions of the spectrum. As of yet, their use in conjunction with super-resolution imaging methodologies has not been explored. In this report, a series of structurally related BF2-azadipyrromethenes has been examined for their suitability for use with stimulated emission depletion (STED) nanoscopy. The potential for STED imaging was initially evaluated using aqueous solutions of fluorophores as an effective predictor of fluorophore suitability. For live cell STED imaging in both 2D and 3D, several far-red emitting BF2-azadipyrromethenes were successfully employed. Image resolution below the diffraction limit of a confocal microscope was demonstrated through measurement of distinct intracellular features including the nuclear membrane, nuclear lamina invaginations, the endoplasmic reticulum, and vacuoles. As the STED ability of BF2-azadipyrromethene fluorophores has now been established, their use with this super-resolution method may be expected to increase in the future.

Exploiting directed self-assembly and disassembly for off-to-on fluorescence responsive live cell imaging.

A bio-responsive nanoparticle was formed by the directed self-assembly (DSA) of a hydrophobic NIR-fluorophore with poloxamer P188. Fluorophore emission was switched off when part of the nanoparticle, however upon stimulus induced nanoparticle dis-assembly the emission switched on. The emission quenching was shown to be due to fluorophore hydration and aggregation within the nanoparticle and the turn on response attributable to nanoparticle disassembly with embedding of the fluorophore within lipophilic environments. This was exploited for temporal and spatial live cell imaging with a measurable fluorescence response seen upon intracellular delivery of the fluorophore. The first dynamic response, seen within minutes, was from lipid droplets with other lipophilic regions such as the endoplasmic reticulum, nuclear membranes and secretory vacuoles imageable after hours. The high degree of fluorophore photostability facilitated continuous imaging for extended periods and the off to on switching facilitated the real-time observation of lipid droplet biogenesis as they emerged from the endoplasmic reticulum. With an in-depth understanding of the principles involved, further assembly controlling functional responses could be anticipated.

Dual Color Imaging from a Single BF2-Azadipyrromethene Fluorophore Demonstrated in vivo for Lymph Node Identification.

Dual emissions at ~700 and 800 nm have been achieved from a single NIR-AZA fluorophore 1 by establishing parameters in which it can exist in either its isolated molecular or aggregated states. Dual near infrared (NIR) fluorescence color lymph node (LN) mapping with 1 was achieved in a large-animal porcine model, with injection site, channels and nodes all detectable at both 700 and 800 nm using a preclinical open camera system. The fluorophore was also compatible with imaging using two clinical instruments for fluorescence guided surgery. Methods: An NIR-AZA fluorophore with hydrophilic and phobic features was synthesised in a straightforward manner and its aggregation properties characterised spectroscopically and by TEM imaging. Toxicity was assessed in a rodent model and dual color fluorescence imaging evaluated by lymph node mapping in a large animal porcine models and in ex-vivo human tissue specimen. Results: Dual color fluorescence imaging has been achieved in the highly complex biomedical scenario of lymph node mapping. Emissions at 700 and 800 nm can be achieved from a single fluorophore by establishing molecular and aggregate forms. Fluorophore was compatible with clinical systems for fluorescence guided surgery and no toxicity was observed in high dosage testing. Conclusion: A new, biomedical compatible form of NIR-dual emission wavelength imaging has been established using a readily accessible fluorophore with significant scope for clinical translation.

Psychological stress suppresses innate IFN-gamma production via glucocorticoid receptor activation: reversal by the anxiolytic chlordiazepoxide.

Studies in humans and in animals indicate that psychological stress can modulate immune responses. Here we demonstrate that exposure to psychological stress (restraint stress) suppresses innate interferon (IFN)-gamma production in mice following an in vivo lipopolysaccharide (LPS) challenge. IFN-gamma signaling was also impaired by stress, as indicated by reduced STAT1 phosphorylation and reduced expression of the IFN-gamma-inducible genes, inducible nitric oxide synthase (iNOS) and IFN-gamma-inducible protein 10 (IP-10/CXCL10). Furthermore, restraint stress suppressed production of the IFN-gamma inducing cytokine interleukin (IL)-12 and increased production of the anti-inflammatory cytokine IL-10, which can inhibit both IL-12 and IFN-gamma production. However, using IL-10 knockout mice, we demonstrate that IL-10 does not mediate the suppressive effect of restraint stress on innate IFN-gamma production. Restraint stress increased corticosterone concentrations in serum and spleen, and consistent with a role for glucocorticoids in the immunosuppressive actions of stress, pre-treatment with the glucocorticoid receptor antagonist mifepristone completely blocked the stress-related suppression of innate IFN-gamma production. Addition of exogenous IL-12 to LPS-stimulated spleen cells reversed the suppressive effect of both restraint stress and corticosterone on IFN-gamma production. These data suggest that reduced IL-12 production is a key event in stress-induced suppression of innate IFN-gamma production. Finally, we demonstrate that pre-treatment with the anxiolytic drug chlordiazepoxide prevents the suppressive effect of stress on innate IFN-gamma production, and also attenuates the stress-induced increase in circulating corticosterone concentrations.

Psychological stress increases expression of IL-10 and its homolog IL-19 via beta-adrenoceptor activation: reversal by the anxiolytic chlordiazepoxide.

Recent studies from our laboratory indicate that psychological stress is a potent inducer of the anti-inflammatory cytokine interleukin (IL)-10, raising the possibility that the IL-10 family of cytokines may be key mediators of stress-induced immunosuppression. In this study we examined the impact of psychological stress (restraint stress) on expression of IL-10, and the novel IL-10 family members IL-19, IL-20 and IL-24 in mouse spleen following an in vivo challenge with lipopolysaccharide (LPS). We found that stressor exposure significantly augmented LPS-induced IL-10 expression. Similarly, IL-19 expression was induced by LPS, and this was significantly enhanced by restraint stress. In contrast, expression of IL-24 was not significantly altered by LPS or stress, and expression of IL-20 was largely not detectable in vivo in either saline or LPS-treated animals. Consistent with a role for sympathetic nervous system (SNS) activation in stress-induced immune regulation, the sympathetic neurotransmitter noradrenaline increased LPS-induced IL-10 and IL-19 expression in splenocytes and dendritic cells, and the ability of noradrenaline to induce expression of these cytokines was blocked by pre-treatment with the beta-adrenoceptor antagonist propranolol. Similarly, pre-treatment of mice with the peripherally acting beta-adrenoceptor antagonist nadolol completely blocked the stress-induced increase in IL-10 and IL-19 mRNA expression. Finally, pre-treatment with the benzodiazepine anxiolytic chlordiazepoxide prevented the stress-induced increase in IL-10 and IL-19 expression. Taken together, these data demonstrate that psychological stress induces expression of the IL-10 and its homolog IL-19 via activation of beta-adrenoceptors, and the ability of stress to induce these cytokines is prevented by treatment with the anxiolytic chlordiazepoxide. The findings suggest that stress enhances the production of immunosuppressive cytokines, which may impact on stress-related disease processes.

Noradrenaline reuptake inhibitors limit neuroinflammation in rat cortex following a systemic inflammatory challenge: implications for depression and neurodegeneration.

Evidence suggests that noradrenaline has a tonic anti-inflammatory action in the central nervous system (CNS) via its ability to suppress microglial and astrocytic activation, and inhibit production of inflammatory mediators. Consequently it is suggested that noradrenaline may play an endogenous neuroprotective role in CNS disorders where inflammatory events contribute to pathology. Here we demonstrate that acute treatment of rats with the noradrenaline reuptake inhibitors (NRIs) desipramine and atomoxetine elicited anti-inflammatory actions in rat cortex following a systemic challenge with bacterial lipopolysaccharide (LPS). This was characterized by a reduction in cortical gene expression of the pro-inflammatory cytokines interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha), the enzyme inducible nitric oxide synthase (iNOS), and the microglial activation markers CD11b and CD40. These anti-inflammatory actions of NRIs were associated with reduced activation of nuclear factor-kappa B (NF-kappaB); a transcription factor that is considered the major regulator of inflammation in the CNS. To determine whether NRI administration directly altered glial expression of these inflammatory markers, primary cortical glial cells were exposed in vitro to the NRIs desipramine or atomoxetine. In vitro treatment with NRIs largely failed to alter mRNA expression of IL-1beta, TNF-alpha, iNOS, CD11b and CD40, following stimulation with LPS. Similarly, LPS-induced TNF-alpha and IL-1beta protein production from glial cells was unaffected by NRI treatment. In contrast, in vitro exposure of cultured glial cells to noradrenaline suppressed IL-1beta, TNF-alpha, iNOS and CD40 expression. These results suggest that in vivo administration of NRIs limit inflammatory events in the brain, probably by increasing noradrenaline availability. Overall, this study has yielded significant insights into the ability of noradrenaline-augmentation strategies to limit neuroinflammation.