Modifications of gene expression detected in peripheral blood after brain ischemia treated with remote postconditioning.

BACKGROUND: A stroke is an acute damage to a certain area of a nerve tissue of the brain. In developed countries, it ranks second among the most often causes of death and is also the leading cause of disability. Recent findings emphasize the significant neuroprotective effect of conditioning on the course and rate of recovery after ischemic attack; however the molecular mechanism of ischemic tolerance induced by conditioning is still not completely explored. METHODS AND RESULTS: The purpose of this study is an identification of changes in gene expression induced by stimulation of reaction cascades after activation of the neuroprotective mechanism using an experimental rat model of global ischemia. The induction of neuroprotective cascades was stimulated by the application of early and delayed form of remote ischemic postconditioning. The quantitative qRT-PCR method was used to assess the rate of change in ADM, BDNF, CDKN1A, CREB, GADD45G, IL6, nNOS, and TM4SF1 gene expression levels 72 h after ischemic attack. The detected results confirm the neuroprotective effect of both forms of postconditioning. Participation of neuroprotection-related gene expression changes was observed once as an early one (CREB, GADD45G), once as a delayed one (ADM, IL6), or both (BDNF, CDKN1A, nNOS, TM4SF1) postconditioning forms, depending on the particular gene. CONCLUSIONS: Our results characterize impact of ischemic tolerance on the molecular level. We predict ischemic tolerance to be consisted of complex combination of early and delayed remote postconditioning.

Rapid remote conditioning mediates modulation of blood cell paracrine activity and leads to the production of a secretome with neuroprotective features.

The indirect use of the protective potential of stem cells in the form of cell secretomes has become an attractive strategy in regenerative medicine. In the present work, we studied the paracrine activity of blood cells that could be modulated towards a neuroprotective nature using in vivo remote conditioning (i.e. tolerant blood cells). The increased neuronal survival mediated by the tolerant secretome was clearly confirmed in vitro in a model of glutamate toxicity in a primary culture of rat cortical neurons and in vivo in a pre- and post-treatment of rats that were subjected to transient occlusion of the middle cerebral artery. Bioinformatic-based analysis of the protein profile revealed higher amounts of proteins released by the tolerant blood cells; 29 proteins were recognised as secreted. More than half of these secreted proteins were involved in the biological processes of the response to the stimulus (GO:0050896) and the response to chemicals (GO:0042221). The protective phenotype was most likely mediated by the synergistic effect of multiple identified proteins, including unique to the tolerant secretome (ceruloplasmin, D-3-phosphoglycerate dehydrogenase) and was promoted by the co-participation of several reaction pathways. The most probably of these pathways were post-translation protein modification, MAP2K and MAPK activation and platelet activation. Taken together, our results demonstrate that properly stimulated blood cells could serve as a source for cell-free-based therapies of regenerative medicine.

Neuroprotection mediated by remote preconditioning is associated with a decrease in systemic oxidative stress and changes in brain and blood glutamate concentration.

It has been shown that ischemia of remote organs can generate resistance to ischemic conditions within sensitive brain tissues. However, only limited information about its mechanism is available. In the present paper, we used hind-limb ischemia by tourniquet to generate early remote ischemic tolerance in rats. The main objective was to investigate the role of glutamate in the process of neuroprotection and discover parameters that are affected in the blood of ischemia-affected animals. Our results showed that pretreatment with a hind-limb tourniquet caused a decrease in neurodegeneration by about 30%. However, we did not observe neurological deficit recovery. When compared to ischemia, glutamate concentration decreased in all observed brain regions (cortex, CA1 and dentate gyrus of hippocampus), regardless of their sensitivity to blood restrictions. In contrast to this, the blood levels raised significantly from 26% to 29% during the first four days of postischemic reperfusion. Pretreatment of animals reduced systemic oxidative stress-as represented by lymphocytic DNA damage-by about 80%, while changes in blood antioxidant enzymes (catalase, superoxide dismutase) were not detected. With these data we can further hypothesize that hind-limb-tourniquet preconditioning could accelerate brain-to-blood efflux of glutamate which could positively impact neuronal survival of ischemia-affected brain regions. Moreover, remote preconditioning improved systemic oxidative stress and did not seem to be affected by enzymatic antioxidant defenses in the blood.

Temperature-dependent competition between adsorption and aggregation of a cellulose ether--simultaneous use of optical and acoustical techniques for investigating surface properties.

Adsorption of the temperature-responsive polymer hydroxypropylmethylcellulose (HPMC) from an aqueous solution onto hydrophobized silica was followed well above the bulk instability temperature (T(2)) in temperature cycle experiments. Two complementary techniques, QCM-D and ellipsometry, were utilized simultaneously to probe the same substrate immersed in polymer solution. The interfacial processes were correlated with changes in polymer aggregation and viscosity of polymer solutions, as monitored by light scattering and rheological measurements. The simultaneous use of ellipsometry and QCM-D, and the possibility to follow layer properties up to 80 °C, well above the T(2) temperature, are both novel developments. A moderate increase in adsorbed amount with temperature was found below T(2), whereas a significant increase in the adsorbed mass and changes in layer properties were observed around the T(2) temperature where the bulk viscosity increases significantly. Thus, there is a clear correlation between transition temperatures in the adsorbed layer and in bulk solution, and we discuss this in relation to a newly proposed model that considers competition between aggregation and adsorption/deposition. A much larger temperature response above the T(2) temperature was found for adsorbed layers of HPMC than for layers of methyl cellulose. Possible reasons for this are discussed.

Layer-by-layer assemblies of chitosan and heparin: effect of solution ionic strength and pH.

The growth of polysaccharide multilayers consisting of positively charged chitosan (CH) and negatively charged heparin (HEP) was monitored in situ by employing a quartz crystal microbalance (QCM-D) and dual-polarization interferometry (DPI). The main focus was on how the physicochemical properties of the solution affect the growth and structure of the resulting multilayer film. These results showed that when increasing the ionic strength of the polysaccharide solutions at a fixed pH, both the "dry" (optical) (DPI) mass and wet (QCM) mass of the adsorbed multilayer film increased. The same effect was found when increasing the pH while keeping the ionic strength constant. Furthermore, the growth of multilayers showed an exponential-like behavior independent of the solution conditions that were used in this study. It was also established that chitosan was the predominant species present in the chitosan-heparin multilayer film. We discuss the viscoelastic properties of the adsorbed layers and their variation during the multilayer buildup. Interestingly and contrary to common interpretation of the QCM-D results, we found that under one particular solution condition (pH 4.2 and 30 mM NaCl) the increase in the dissipation of oscillation energy from the adsorbed layer was a consequence of layer stiffening rather than indicating a more hydrated and viscous film. On the basis of the widely used Voigt viscoelastic model for an adsorbed layer, we show that it is the film viscosity and shear that define the layer viscoelasticity (structure) of the film and not the absolute value of energy dissipation, which in fact can be very misleading.

Lubrication, adsorption, and rheology of aqueous polysaccharide solutions.

Aqueous lubrication is currently at the forefront of tribological research due to the desire to learn and potentially mimic how nature lubricates biotribological contacts. We focus here on understanding the lubrication properties of naturally occurring polysaccharides in aqueous solution using a combination of tribology, adsorption, and rheology. The polysaccharides include pectin, xanthan gum, gellan, and locus bean gum that are all widely used in food and nonfood applications. They form rheologically complex fluids in aqueous solution that are both shear thinning and elastic, and their normal stress differences at high shear rates are found to be characteristic of semiflexible/rigid molecules. Lubrication is studied using a ball-on-disk tribometer with hydrophobic elastomer surfaces, mimicking biotribological contacts, and the friction coefficient is measured as a function of speed across the boundary, mixed, and hydrodynamic lubrication regimes. The hydrodynamic regime, where the friction coefficient increases with increasing lubricant entrainment speed, is found to depend on the viscosity of the polysaccharide solutions at shear rates of around 10(4) s(-1). The boundary regime, which occurs at the lowest entrainment speeds, depends on the adsorption of polymer to the substrate. In this regime, the friction coefficient for a rough substrate (400 nm rms roughness) is dependent on the dry mass of polymer adsorbed to the surface (obtained from surface plasmon resonance), while for a smooth substrate (10 nm rms roughness) the friction coefficient is strongly dependent on the hydrated wet mass of adsorbed polymer (obtained from quartz crystal microbalance, QCM-D). The mixed regime is dependent on both the adsorbed film properties and lubricant's viscosity at high shear rates. In addition, the entrainment speed where the friction coefficient is a minimum, which corresponds to the transition between the hydrodynamic and mixed regime, correlates linearly with the ratio of the wet mass and viscosity at ∼10(4) s(-1) for the smooth surface. These findings are independent of the different polysaccharides used in the study and their different viscoelastic flow properties.

Influence of ionic strength changes on the structure of pre-adsorbed salivary films. A response of a natural multi-component layer.

Salivary films coating oral surfaces are critically important for oral health. This study focuses on determining the underlying nature of this adsorbed film and how it responds to departures from physiological conditions due to changes in ionic strength. Under physiological conditions, it is found that pre-adsorbed in vitro salivary film on hydrophobic surfaces is present as a highly hydrated viscoelastic layer. We follow the evolution of this film in terms of its effective thickness, hydration and viscoelastic properties, as well as adsorbed mass of proteins, using complementary surface characterisation methods: a Surface Plasmon Resonance (SPR) and a Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D). Our results support a heterogeneous model for the structure of the salivary film with an inner dense anchoring layer and an outer highly extended hydrated layer. Further swelling of the film was observed upon decreasing the salt concentration down to 1mM NaCl. However, upon exposure to deionised water, a collapse of the film occurs that was associated with the loss of water contained within the adsorbed layer. We suggest that the collapse in deionised water is driven by an onset of electrostatic attraction between different parts of the multi-component salivary film. It is anticipated that such changes could also occur when the oral cavity is exposed to food, beverage, oral care and pharmaceutical formulations where drastic changes to the structural integrity of the film is likely to have implications on oral health, sensory perception and product performance.

Sequential adsorption of bovine mucin and lactoperoxidase to various substrates studied with quartz crystal microbalance with dissipation.

Mucin and lactoperoxidase are both natively present in the human saliva. Mucin provides lubricating and antiadhesive function, while lactoperoxidase has antimicrobial activity. We propose that combined films of the two proteins can be used as a strategy for surface modification in biomedical applications such as implants or biosensors. In order to design and ultilize mixed protein films, it is necessary to understand the variation in adsorption behavior of the proteins onto different surfaces and how it affects their interaction. The quartz crystal microbalance with dissipation (QCM-D) technique has been used to extract information of the adsorption properties of bovine mucin (BSM) and lactoperoxidase (LPO) to gold, silica, and hydrophobized silica surfaces. The information has further been used to retrieve information of the viscoelastic properties of the adsorbed film. The adsorption and compaction of BSM were found to vary depending on the nature of the underlying bare surface, adsorbing as a thick highly hydrated film with loops and tails extending out in the bulk on gold and as a thinner film with much lower adsorbed amount on silica; and on hydrophobic surfaces, BSM adsorbs as a flat and much more compact layer. On gold and silica, the highly hydrated BSM film is cross-linked and compacted by the addition of LPO, whereas the compaction is not as pronounced on the already more compact film formed on hydrophobic surfaces. The adsorption of LPO to bare surfaces also varied depending on the type of surface. The adsorption profile of BSM onto LPO-coated surfaces mimicked the adsorption to the underlying surface, implying little interaction between the LPO and BSM. The interaction between the protein layers was interpreted as a combination of electrostatic and hydrophobic interactions, which was in turn influenced by the interaction of the proteins with the different substrates.

Interactions between chitosan and SDS at a low-charged silica substrate compared to interactions in the bulk--the effect of ionic strength.

The effect of ionic strength on association between the cationic polysaccharide chitosan and the anionic surfactant sodium dodecyl sulfate, SDS, has been studied in bulk solution and at the solid/liquid interface. Bulk association was probed by turbidity, electrophoretic mobility, and surface tension measurements. The critical aggregation concentration, cac, and the saturation binding of surfactants were estimated from surface tension data. The number of associated SDS molecules per chitosan segment exceeded one at both salt concentrations. As a result, a net charge reversal of the polymer-surfactant complexes was observed, between 1.0 and 1.5 mM SDS, independent of ionic strength. Phase separation occurs in the SDS concentration region where low charge density complexes form, whereas at high surfactant concentrations (up to several multiples of cmc SDS) soluble aggregates are formed. Ellipsometry and QCM-D were employed to follow adsorption of chitosan onto low-charged silica substrates, and the interactions between SDS and preadsorbed chitosan layers. A thin (0.5 nm) and rigid chitosan layer was formed when adsorbed from a 0.1 mM NaNO3 solution, whereas thicker (2 nm) chitosan layers with higher dissipation/unit mass were formed from solutions at and above 30 mM NaNO3. The fraction of solvent in the chitosan layers was high independent of the layer thickness and rigidity and ionic strength. In 30 mM NaNO3 solution, addition of SDS induced a collapse at low concentrations, while at higher SDS concentrations the viscoelastic character of the layer was recovered. Maximum adsorbed mass (chitosan + SDS) was reached at 0.8 times the cmc of SDS, after which surfactant-induced polymer desorption occurred. In 0.1 mM NaNO3, the initial collapse was negligible and further addition of surfactant lead to the formation of a nonrigid, viscoelastic polymer layer until desorption began above a surfactant concentration of 0.4 times the cmc of SDS.

Effect of adsorbed layer surface roughness on the QCM-D response: focus on trapped water.

The effect of surface roughness on the quartz crystal microbalance with dissipation monitoring (QCM-D) response was investigated with emphasis on determining the amount of trapped water. Surfaces with different nanoroughnesses were prepared on silica by self-assembly of cationic surfactants with different packing parameters. We used surfactants with quaternary ammonium bromide headgroups: the double-chained didodecyltrimethylammonium bromide (C12)2DAB (DDAB), the single-chained hexadecyltrimethylammonium bromide C16TAB (CTAB), and dodecyltrimethyl-ammonium bromide C12TAB (DTAB). The amount of trapped water was obtained from the difference between the mass sensed by QCM-D and the adsorbed amount detected by optical reflectometry. The amount of water, which is sensed by QCM-D, was found to increase with the nanoroughness of the adsorbed layer. The water sensed by QCM-D cannot be assigned primarily to hydration water, because it differs substantially for adsorbed surfactant layers with similar headgroups but with different nanoscale topographies.

Lack of association between a cationic protein and a cationic fluorosurfactant.

Surface tension, 19F and 1H NMR spectroscopy, and cryotransmission electron microscopy are used to characterize the state of association in aqueous solutions of a fluorosurfactant CF3(CF2)nSO2NH(CH2)3-4N(CH3)3+ I- (n = 8, 6) with and without lysozyme added. In the absence of lysozyme, we find monomers, small aggregates, and large vesicles to coexist, with the individual fluorosurfactant molecules exchanging slowly (>1 ms) among those states. When both lysozyme and fluorosurfactant are present in the solution, they have no measurable influence on the physical state of the other. In contrast, a hydrogenated cationic surfactant with the same headgroup, hexadecyltrimethylammonium bromide, is shown to associate to lysozyme.

Mucin-chitosan complexes at the solid-liquid interface: multilayer formation and stability in surfactant solutions.

The adsorption of a biologically important glycoprotein, mucin, and mucin-chitosan complex layer formation on negatively charged surfaces, silica and mica, have been investigated employing ellipsometry, the interferometric surface apparatus, and atomic force microscopy techniques. Particular attention has been paid to the effect of an anionic surfactant sodium, dodecyl sulfate (SDS), with respect to the stability of the adsorption layers. It has been shown that mucin adsorbs on negatively charged surfaces to form highly hydrated layers. Such mucin layers readily associate with surfactants and are easily removed from the surfaces by rinsing with solutions of SDS at concentrations > or =0.2 cmc (1 cmc SDS in 30 mM NaCl is equal to 3.3 mM). The mucin adsorption layer is negatively charged, and we show how a positively charged polyelectrolyte, chitosan, associates with the preadsorbed mucin to form mucin-chitosan complexes that resist desorption by SDS even at SDS concentrations as high as 1 cmc. Thus, a method of mucin layer protection against removal by surfactants is offered. Further, we show how mucin-chitosan multilayers can be formed.

Dendritic structures based on bis(hydroxymethyl)propionic acid as platforms for surface reactions.

In this paper we present results related to the self-assembly of different generations of disulfide-cored 2,2-bis(hydroxymethyl)propionic acid-based dendritic structures onto gold surfaces. These molecular architectures, ranging from generation 1 to generation 3, contain removable acetonide protecting groups at their periphery that are accessible for hydrolysis with subsequent formation of OH-terminated surface-attached dendrons. The deprotection has been investigated in detail as a versatile approach to accomplish reactive surface platforms. A special focus has been devoted to the comparison of the properties of the layers formed by hydrolysis of the acetonide moieties directly on the surface and in solution, prior to the layer formation.