Major effects on blood-retina barrier passage by minor alterations in design of polybutylcyanoacrylate nanoparticles.

Because the blood-brain barrier (BBB) is an obstacle for drug-delivery, carrier systems such as polybutylcyanoacrylate (PBCA) nanoparticles (NPs) have been studied. Yet, little is known of how physiochemical features such as size, surfactants and surface charge influence BBB passage in vivo. We now used a rat model of in vivo imaging of the retina - which is brain tissue and can reflect the situation at the BBB - to study how size and surface charge determine NPs' ability to cross the blood-retina barrier (BRB). Interestingly, for poloxamer 188-modified, DEAE-dextran-stabilised, fluorescent PBCA NPs, decreasing the average zeta-size from 272 nm to 172 nm by centrifugation reduced the BRB passage of the NPs substantially. Varying the zeta potential within the narrow range of 0-15 mV by adding different amounts of stabiliser revealed that 0 mV and 15 mV were less desirable than 5 mV which facilitated the BRB passage. Moreover, whether the fluorescent marker was adsorbed or incorporated also influenced the transport into the retina tissue. Thus, minor changes in design of nano-carriers can alter physicochemical parameters such as size or zeta potential, thus substantially influencing NPs' biological distribution in vivo, possibly by interactions with blood constituents and peripheral organs.

Electrical brain stimulation induces dendritic stripping but improves survival of silent neurons after optic nerve damage.

Repetitive transorbital alternating current stimulation (rtACS) improves vision in patients with chronic visual impairments and an acute treatment increased survival of retinal neurons after optic nerve crush (ONC) in rodent models of visual system injury. However, despite this protection no functional recovery could be detected in rats, which was interpreted as evidence of "silent survivor" cells. We now analysed the mechanisms underlying this "silent survival" effect. Using in vivo microscopy of the retina we investigated the survival and morphology of fluorescent neurons before and after ONC in animals receiving rtACS or sham treatment. One week after the crush, more neurons survived in the rtACS-treated group compared to sham-treated controls. In vivo imaging further revealed that in the initial post-ONC period, rtACS induced dendritic pruning in surviving neurons. In contrast, dendrites in untreated retinae degenerated slowly after the axonal trauma and neurons died. The complete loss of visual evoked potentials supports the hypothesis that cell signalling is abolished in the surviving neurons. Despite this evidence of "silencing", intracellular free calcium imaging showed that the cells were still viable. We propose that early after trauma, complete dendritic stripping following rtACS protects neurons from excitotoxic cell death by silencing them.

The role of lipolysis in human orosensory fat perception.

Taste perception elicited by food constituents and facilitated by sensory cells in the oral cavity is important for the survival of organisms. In addition to the five basic taste modalities, sweet, umami, bitter, sour, and salty, orosensory perception of stimuli such as fat constituents is intensely investigated. Experiments in rodents and humans suggest that free fatty acids represent a major stimulus for the perception of fat-containing food. However, the lipid fraction of foods mainly consists of triglycerides in which fatty acids are esterified with glycerol. Whereas effective lipolysis by secreted lipases (LIPs) liberating fatty acids from triglycerides in the rodent oral cavity is well established, a similar mechanism in humans is disputed. By psychophysical analyses of humans, we demonstrate responses upon stimulation with triglycerides which are attenuated by concomitant LIP inhibitor administration. Moreover, lipolytic activities detected in minor salivary gland secretions directly supplying gustatory papillae were correlated to individual sensitivities for triglycerides, suggesting that differential LIP levels may contribute to variant fat perception. Intriguingly, we found that the LIPF gene coding for lingual/gastric LIP is not expressed in human lingual tissue. Instead, we identified the expression of other LIPs, which may compensate for the absence of LIPF.

Surfactants, not size or zeta-potential influence blood-brain barrier passage of polymeric nanoparticles.

Nanoparticles (NP) can deliver drugs across the blood-brain barrier (BBB), but little is known which of the factors surfactant, size and zeta-potential are essential for allowing BBB passage. To this end we designed purpose-built fluorescent polybutylcyanoacrylate (PBCA) NP and imaged the NP's passage over the blood-retina barrier - which is a model of the BBB - in live animals. Rats received intravenous injections of fluorescent PBCA-NP fabricated by mini-emulsion polymerisation to obtain various NP's compositions that varied in surfactants (non-ionic, anionic, cationic), size (67-464nm) and zeta-potential. Real-time imaging of retinal blood vessels and retinal tissue was carried out with in vivo confocal neuroimaging (ICON) before, during and after NP's injection. Successful BBB passage with subsequent cellular labelling was achieved if NP were fabricated with non-ionic surfactants or cationic stabilizers but not when anionic compounds were added. NP's size and charge had no influence on BBB passage and cell labelling. This transport was not caused by an unspecific opening of the BBB because control experiments with injections of unlabelled NP and fluorescent dye (to test a "door-opener" effect) did not lead to parenchymal labelling. Thus, neither NP's size nor chemo-electric charge, but particle surface is the key factor determining BBB passage. This result has important implications for NP engineering in medicine: depending on the surfactant, NP can serve one of two opposite functions: while non-ionic tensides enhance brain up-take, addition of anionic tensides prevents it. NP can now be designed to specifically enhance drug delivery to the brain or, alternatively, to prevent brain penetration so to reduce unwanted psychoactive effects of drugs or prevent environmental nanoparticles from entering tissue of the central nervous system.

Toxicity of polymeric nanoparticles in vivo and in vitro.

Polybutylcyanoacrylate nanoparticles (PBCA NPs) are candidates for a drug delivery system, which can cross the blood-brain barrier (BBB). Because little is known about their toxicity, we exposed cells to PBCA NPs in vitro and in vivo and monitored their life and death assays. PBCA NPs were fabricated with different surfactants according to the mini-emulsion technique. Viabilities of HeLa and HEK293 cells after NP incubation were quantified by analysing cellular metabolic activity (MTT-test). We then repetitively injected i.v. rhodamine-labelled PBCA NP variations into rats and monitored the survival and morphology of retrogradely labelled neurons by in vivo confocal neuroimaging (ICON) for five weeks. To test for carrier-efficacy and safety, PBCA NPs loaded with Kyotorphin were injected in rats, and a hot plate test was used to quantify analgesic effects. In vitro, we found dose-dependent cell death which was, however, only detectable at very high doses and mainly seen in the cultures incubated with NPs fabricated with the tensids SDS and Tween. However, the in vivo experiments did not show any NP-induced neuronal death, even with particles which were toxic at high dose in vitro, i.e. NPs with Tween and SDS. The increased pain threshold at the hot plate test demonstrated that PBCA NPs are able to cross the BBB and thus comprise a useful tool for drug delivery into the central nervous system (CNS). Our findings showing that different nanoparticle formulations are non-toxic have important implications for the value of NP engineering approaches in medicine.

Local Li coordination and ionic transport in methacrylate-based gel polymer electrolytes.

The local Li cation coordination motifs and the interactions between the hosting methacrylate-based polymer membrane and the liquid electrolyte [1 M LiPF6 in ethylene carbonate (EC)/dimethyl carbonate (DMC)] are studied by employing liquid and solid-state NMR spectroscopy. At low temperatures, two different coordination modes for Li cations are identified with the help of dipolar-based solid-state NMR techniques, one of which is the exclusive coordination by DMC molecules, while the other is a co-coordination by the polymer and DMC molecules. At room temperature, Li cations are found to be extremely mobile, coordinated by EC and DMC molecules as well as the copolymer, as found by liquid-state NMR spectroscopy.

Transcorneal electrical stimulation alters morphology and survival of retinal ganglion cells after optic nerve damage.

Traumatic optic nerve injury leads to retrograde death of retinal ganglion cells (RGCs), but transcorneal electrical stimulation (TES) can increase the cell survival rate. To understand the mechanisms and to further define the TES-induced effects we monitored in living animals RGC morphology and survival after optic nerve crush (ONC) in real time by using in vivo confocal neuroimaging (ICON) of the retina. ONC was performed in rats and ICON was performed before crush and on post-lesion days 3, 7 and 15 which allowed us to repeatedly record RGC number and size. TES or sham-stimulation were performed immediately after the crush and on post-injury day 11. Three days after ONC we detected a higher percentage of surviving RGCs in the TES group as compared to sham-treated controls. However, the difference was below significance level on day 7 and disappeared completely by day 15. The death rate was more variable amongst the TES-treated rats than in the control group. Morphological analysis revealed that average cell size changed significantly in the control group but not in stimulated animals and the morphological alterations of surviving neurons were smaller in TES-treated compared to control cells. In conclusion, TES delays post-traumatic cell death significantly. Moreover, we found "responder animals" which also benefited in the long-term from the treatment. Our in vivo cellular imaging results provide evidence that TES reduces ONC-associated neuronal swelling and shrinkage especially in RGCs which survived long-term. Further studies are now needed to determine the differences of responders vs. non-responders.

Transcorneal alternating current stimulation after severe axon damage in rats results in "long-term silent survivor" neurons.

Transcorneal alternating current stimulation (tACS) was proposed to decrease acute death of retinal ganglion cells after optic nerve transection in rats, but it is not known if cell survival is long-term and associated with functional restoration. We therefore evaluated the effects of tACS in a rat model of optic nerve crush using anatomical, electrophysiological and behavioural measures. Rats were trained in a brightness discrimination visual task and the retinal ganglion cell number was quantified with in vivo confocal neuroimaging. Thereafter, severe optic nerve crush or sham crush was performed and rats were treated under anaesthesia either with tACS or sham stimulation immediately after the lesion and on day 3, 7, 11, 15, 19 and 23. Brightness discrimination was evaluated for 6 weeks and retinal ganglion cells were counted in vivo on post-crush days 7 and 28. In additional rats we studied the influence of tACS on bioelectrical activity. On post-lesion day 28, the tACS-treated group showed a neuronal survival of 28.2% which was significantly greater than in sham operates (8.6%). All animals with optic nerve crush were significantly impaired in brightness discrimination and did not recover performance, irrespective to which group they belonged. In accordance with this, there was no significant influence of the stimulation on EEG power spectra. In conclusion, tACS induced long-term neuronal protection from delayed retrograde cell death, but in this case of severe axonal damage tACS did not influence functional restoration and EEG signals recorded over the visual cortex.

Immunohistochemical detection of TAS2R38 protein in human taste cells.

The sense of taste plays an important role in the evaluation of the nutrient composition of consumed food. Bitter taste in particular is believed to serve a warning function against the ingestion of poisonous substances. In the past years enormous progress was made in the characterization of bitter taste receptors, including their gene expression patterns, pharmacological features and presumed physiological roles in gustatory as well as in non-gustatory tissues. However, due to a lack in TAS2R-specifc antibodies the localization of receptor proteins within gustatory tissues has never been analyzed. In the present study we have screened a panel of commercially available antisera raised against human bitter taste receptors by immunocytochemical experiments. One of these antisera was found to be highly specific for the human bitter taste receptor TAS2R38. We further demonstrate that this antibody is able to detect heterologously expressed TAS2R38 protein on Western blots. The antiserum is, however, not able to interfere significantly with TAS2R38 function in cell based calcium imaging analyses. Most importantly, we were able to demonstrate the presence of TAS2R38 protein in human gustatory papillae. Using double immunofluorescence we show that TAS2R38-positive cells form a subpopulation of PLCbeta2 expressing cells. On a subcellular level the localization of this bitter taste receptor is neither restricted to the cell surface nor particularly enriched at the level of the microvilli protruding into the pore region of the taste buds, but rather evenly distributed over the entire cell body.

In vivo visualisation of nanoparticle entry into central nervous system tissue.

Because the potential neurotoxicity of nanoparticles is a significant issue, characterisation of nanoparticle entry into the brain is essential. Here, we describe an in vivo confocal neuroimaging method (ICON) of visualising the entry of fluorescent particles into the parenchyma of the central nervous system (CNS) in live animals using the retina as a model. Rats received intravenous injections of fluorescence-labelled polybutyl cyanoacrylate nanoparticles that had been synthesised by a standard miniemulsion polymerisation process. We performed live recording with ICON from before and up to 9 days after particle injection and took photomicrographs of the retina. In addition, selective retrograde labelling of the retinal ganglion cells was achieved by stereotaxic injection of a fluorescent dye into the superior colliculus. Using ICON, we observed vascular kinetics of nanoparticles (wash-in within seconds), their passage to the retina parenchyma (within minutes) and their distribution (mainly cellular) under in vivo conditions. For the detection of cell loss--which is important for the evaluation of toxic effects--in another experiment, we semi-quantitatively analysed the selectively labelled retinal neurons. Our results suggest that the dye per se does not lead to neuronal death. With ICON, it is possible to study nanoparticle kinetics in the retina as a model of the blood-brain barrier. Imaging data can be acquired within seconds after the injection, and the long-term fate of cellular uptake can be followed for many days to study the cellular/extracellular distribution of the nanoparticles. ICON is thus an effective and meaningful tool to investigate nanoparticle/CNS interactions.

G protein-coupled receptors in human fat taste perception.

In contrast to carbohydrates and proteins, which are detected by specialized taste receptors in the forms of their respective building blocks, sugars, and L-amino acids, the third macronutrient, lipids, has until now not been associated with gustatory receptors. Instead, the recognition of fat stimuli was believed to rely mostly on textural, olfactory, and postingestive cues. During the recent years, however, research done mainly in rodent models revealed an additional gustatory component for the detection of long-chain fatty acids (LCFAs), the main taste-activating component of lipids. Concomitantly, a number of candidate fat taste receptors were proposed to be involved in rodent's gustatory fatty acid perception. Compared with rodent models, much less is known about human fat taste. In order to investigate the ability of the human gustatory system to respond to fat components, we performed sensory experiments with fatty acids of different chain lengths and derivatives thereof. We found that our panelists discriminated a "fatty" and an irritant "scratchy" taste component, with the "fatty" percept restricted to LCFAs. Using functional calcium-imaging experiments with the human orthologs of mouse candidate fat receptors belonging to the G protein-coupled receptor family, we correlated human sensory data with receptor properties characterized in vitro. We demonstrated that the pharmacological activation profile of human GPR40 and GPR120, 2 LCFA-specific receptors associated with gustatory fat perception in rodents, is inconsistent with the "scratchy" sensation of human subjects and more consistent with the percept described as "fatty." Expression analysis of GPR40 and GPR120 in human gustatory tissues revealed that, while the GPR40 gene is not expressed, GPR120 is detected in gustatory and nongustatory epithelia. On a cellular level, we found GPR120 mRNA and protein in taste buds as well as in the surrounding epithelial cells. We conclude that GPR120 may indeed participate in human gustatory fatty acid perception.