Ruthenium Tetroxide Staining Research Articles

Efficacy of Combined Peroxisome Proliferator-Activated Receptor-α Ligand and Glucocorticoid Therapy in a Murine Model of Atopic Dermatitis

Although topical glucocorticoids (GCs) display potent anti-inflammatory activity in inflamed skin, they also can exert numerous harmful effects on epidermal structure and function. In contrast, topical applications of ligands of peroxisome proliferator-activated receptor-α (PPARα) not only reduce inflammation, and also improve cutaneous barrier homeostasis. Therefore, we examined whether sequential topical GCs followed by topical Wy14643 (a ligand of PPARα) might be more effective than either alone for atopic dermatitis (AD) in a hapten (oxazolone)-induced, murine model with multiple features of AD (Ox-AD). Despite expected anti-inflammatory benefits, topical GC alone induced: i) epidermal thinning; ii) reduced expression of involucrin, loricrin and filaggrin; and iii) allowed outside-to-inside penetration of an epicutaneous tracer. While Wy14643 alone yielded significant therapeutic benefits in mice with mild or moderate Ox-AD, it was less effective in severe Ox-AD. Yet, topical applications of Wy14643 after GC was not only significantly effective comparable to GC alone, but it also prevented GC-induced structural and functional abnormalities in permeability barrier homeostasis. Moreover, rebound flares were largely absent after sequential treatment with GC and Wy14643. Together, these results show that GC and PPARα ligand therapy together is not only effective but also prevents development of GC-induced side effects, including rebound flares, in murine AD.

Human skin surface lipid film: an ultrastructural study and interaction with corneocytes and intercellular lipid lamellae of the stratum corneum.

Sebum is a complex mixture of lipids, which is secreted by mammalian sebaceous glands, and forms a fluid film over the skin surface. After sebum is secreted, it becomes mixed with lipid from the keratinizing epithelium and forms the skin surface lipid film (SSLF). Until now, direct fine structural observation of the SSLF has been lacking. In the present work, we viewed the detailed structures of the human SSLF by ruthenium tetroxide staining. The results showed that the SSLF formed an amorphous sheet of variable thickness on the skin surface instead of forming lipid droplets, as had been the usual assumption. In general, its thickness was < 0.5 microm or even negligible in sebum-poor extremities. However, in the sebum-rich face, its thickness was > 4 microm in focal areas. Consistent with the thickness of SSLF, the sebum quantity showed great regional variation. It varied from 1 microg/cm2 (leg) to 189 +/- 42.7 microg/cm2 (mean +/- SD: face). The SSLF was composed of numerous fine granules of about 4-5 nm in a random orientation. Within the SSLF, variable amounts of deranged lipid lamellae derived from corneocytes were mixed with sebum. As well as on the skin surface, a similar amount of sebum was also found between the desquamating corneocytes in the uppermost several layers of the stratum corneum (SC). We also observed the presence of intercellular lipid lamellae in the outer layers of the SC: their lipid envelope remained intact even in desquamated corneocytes. Our results provide some new insights concerning the structure of the SSLF and its relationship with the SC.

Permeability barrier abnormality of hairless mouse epidermis after topical corticosteroid: characterization of stratum corneum lipids by ruthenium tetroxide staining and high-performance thin-layer chromatography.

Topical corticosteroids (TCS) are among the most frequently used topical therapeutics. Recently, it has been shown that TCS not only has antiproliferative actions, but also inhibits the differentiation of the epidermis and finally perturbates stratum corneum (s.c.) barrier function. It is well established that epidermal barrier function resides within the intercellular lipids of the SC. However, to date, little is known about the effects of TCS on the structure and composition of s.c. lipids. We therefore used hairless mouse skin to study the sequential changes of the s.c. permeability barrier and their intercellular lipids by ruthenium tetroxide staining and high-performance thin-layer chromatography (HPTLC) during topical use of corticosteroids. The results demonstrated a progressive increase in transepidermal water loss accompanied by a diminution in the SC intercellular lipid lamellae, which showed a normal structure of individual lamella. Analysis of lipid composition by HPTLC after a 6-week application of TCS also showed an obvious decrease in all the main components of s.c. lipids, which are known to constitute the permeability barrier of the skin. In light of these results, our work provides direct morphological evidence that TCS deteriorates the permeability barrier of epidermis when applied to normal skin.

New method for the characterization of domain morphology of polymer blends using ruthenium tetroxide staining and low voltage scanning electron microscopy (LVSEM)

A new method has been developed for the analysis of domain morphology of stained polyolefin blends by low voltage scanning electron microscopy (LVSEM). The component polymers of the blend are differentiated by heavy staining with RuO 4. LVSEM at low accelerating voltages provides high resolution imaging and minimal beam damage to the sample. The method is routinely applied to the analysis of domain morphology in moulded samples, fibres and films, to failure analysis and to the analysis of layer morphology of co-extruded films.

Transmission electron microscopy of 3F/PMDA-polypropylene oxide triblock copolymer based nanofoams

Transmission electron microscopy was performed on a polymeric nanofoam material, derived from a triblock copolymer composed of a fluorinated polyimide center block, 3F/PMDA (derived from pyromelletic dianhydride (PMDA) and 1,1-bis(4-aminophenyl)-1-phenyl-2,2,2-trifluoroethane (3F)) and polypropylene oxide (PO) end blocks. The cast and imidized polymer exhibits a microphase-separated morphology consisting of PO microdomains within a polyimide matrix. The final nanofoam material is obtained by decomposing PO microdomains into low molecular weight products, which diffuse out of the polyimide matrix leaving nanometer length scale voids. Ruthenium tetroxide staining prior to microscopy was used to enhance the contrast between the 3F/PMDA matrix and the PO microdomains or voids, which permitted a more detailed view of the microstructure of both the foamed and unfoamed materials. From the power spectra of the micrographs, spatial correlation between the PO microdomains in the unfoamed material and between the voids in the foam were found. An interdomain separation distance of ca. 37 nm was observed. Analysis of the image yielded an average area of 411 nm2 for the PO domains. The analysis indicated that the PO domains were oblong, having average major and minor dimensions of 35 and 12.5 nm, respectively. An autocorrelation of the image showed that the domain center of masses were positioned 41 nm apart, in close agreement with the domain spacing (ca. 37 nm) found as described above. © 1997 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 35: 1067–1076, 1997

Characterization of thin Polymeric Nanofoam films by Transmission Electron Microscopy and Small Angle Neutron Scattering

ABSTRACTThin film polymer nanofoams are produced from triblock copolymers of a fluorinated polyimide, 3F/PMDA (derived from pyromelletic dianhydride (PMDA) and 1,1-bis(4-aminophenyl)-1-phenyl-2,2,2-trifluoroethane (3F)) as the center block and polypropylene oxide (PO) as the end blocks. The nanofoam is produced using a three step process: 1.) spin casting the triblock copolymer onto a silicon substrate, 2.) thermal treatment in an Argon atmosphere to imidize the center block and 3.) thermal treatment in air to degrade the PO domains and form nanoscale voids. This process was characterized using both transmission electron microscopy (TEM) and small angle neutron scattering (SANS). For the TEM studies, Ruthenium tetroxide staining was used to enhance the contrast between the polyimide (PI) matrix and the PO microdomains or voids, which permitted a more detailed view of the microstructure of both the foamed and unfoamed materials. From the two dimensional Fourier transform of the micrographs the spatial correlation between the PO microdomains in the unfoamed material and between the voids in the foam were found. An interdomain separation distance of -37 nm was observed. SANS was performed to follow the imidization and foaming processes both in-situ and on a Si substrate. The SANS results indicated that the films are homogeneous when spun from solution and that the microphase separation of the PO domains occurs during the imidization step. The subsequent foaming step leaves the morphology generally intact with the PO domains converting to voids. A peak was found in the SANS curve with a spacing of about 26 nm, which is qualitative agreement with the TEM data.

Effects of petrolatum on stratum corneum structure and function

Ointments (e.g., petrolatum) are thought to be occlusive, thereby blocking transcutaneous water loss and trapping water under the skin's surface. If this premise is correct, then petrolatum should delay barrier recovery after barrier perturbation, as shown previously in occluded murine skin. We reexamined the assumption that Vaseline Petroleum Jelly (VPJ) is occlusive, ascertaining both its site and mechanism of action. Barrier recovery was measured in VPJ-treated versus untreated sites after acetone-induced barrier disruption in human volunteers. Moreover, VPJ was localized within the stratum corneum (SC) with tracers and ruthenium tetroxide staining, which allowed visualization of the depth of VPJ penetration and its relation to intercellular membrane structures. VPJ accelerated, rather than impeded, barrier recovery. Moreover, VPJ was present within the interstices at all levels of the SC, where it replaced intercellular bilayers. VPJ neither forms nor acts like an epicutaneous impermeable membrane; instead, it permeates throughout the SC interstices, allowing normal barrier recovery despite its occlusive properties.

Ruthenium tetroxide staining of polybutylene terephthalate (PBT) and polyisobutylene- b-PBT segmented block copolymers

A ruthenium tetroxide (RuO 4) staining method has been evaluated for segmented polyisobutylene- b-polybutylene terephthalate (PIB- b-PBT). Solution cast films and melt pressed samples have been studied. For comparison PBT has also been studied. PBT and PIB- b-PBT could be stained with RuO 4 at room temperature. The observed structures on the PBT and PIB- b-PBT films were spherulitic with lamellae. In some regions in the PIB- b-PBT films large scale phase separation was observed. In the melt pressed PIB- b-PBT samples the spherulitic and lamellar structures were less well developed. The PBT segments appeared to have crystallized out in bundles and the PIB phase was present as microspheres with diameters of 3–6 nm.

Membrane Structures in Normal and Essential Fatty Acid-Deficient Stratum Corneum: Characterization by Ruthenium Tetroxide Staining and X-Ray Diffraction

Despite the importance of intercellular lamellar bilayers for stratum corneum (SC) barrier function, knowledge about the structure of these bilayers is limited due to their poor visualization and/or retention. Whereas substitution of ruthenium tetroxide (RuO4) for osmium tetroxide fixation provides clear images of these bilayers, the usefulness of RuO4 has been limited by its slow penetration and cytotoxicity. Utilizing a new fixation protocol for RuO4, we obtained clear images of lamellar domains at all levels of murine SC. Computer-aided image reconstructions demonstrated a lamellar spacing of 129 +/- 2 A, which agreed with x-ray diffraction data from parallel, unfixed samples (131 +/- 2 A), a spacing not affected by hydration. Furthermore, novel structures were seen in the intercellular spaces of normal SC. Finally, in murine essential fatty acid deficiency (EFAD), the overall lamellar spacing is comparable to normal [127 +/- 7 A by computer transform vs. 131.9 +/- 2 A (hydrated) and 129.6 +/- 2.2 A (dry) by x-ray diffraction]. Yet, these domains are structurally abnormal, displaying regions with either an excess or absence of lamellae. The new RuO4 protocol provides quantitative information about SC lamellar dimensions and morphologic abnormalities in bilayer distribution and substructure in EFAD stratum corneum that are not detected by either x-ray diffraction or computer-aided image reconstruction. Thus, the barrier abnormality in EFAD stratum corneum can be ascribed either to focal depletion of lamellae or abnormalities in lamellar substructure.

Quantitative Ruthenium Method for Analysis of Nitrogen Ion-Implanted Titanium Alloy (Ti-6AI-4V) and the Effect on Bacterial Adherence

ABSTRACTThe effects of nitrogen ion implantation of Ti-6AI-4V alloy on growth ofPseudomonas aeruginosabacteria on surfaces of the alloy have been investigated. Results for ion implanted samples were compared with controls with similarly smoothly polished surfaces and with controls that had intentionally roughened surfaces. The test consisted of exposing sterile alloy samples to a microbiological broth, to which 24 hour-old cultures ofPseudomonas aeruginosahad been added. After bioassociation at normal temperature 37°C, bacteria adhering to the surface were fixed and treated with a new ruthenium tetroxide staining method, and quantified by use of scanning electron microscopy (SEM), back-scattered electron imaging and EDAX energy dispersive microanalysis. For smooth samples of the alloy, after a 12 hour growth period, the retained bacteria (revealed by the biologically incorporated ruthenium), decreased monotonically with nitrogen dose out to a total fluence of approximately 7 × 1017/cm2in an affected depth of approximately 0.1500μm. The SEM confirmed that thePseudomonas aeruginosaadhered equally to control materials. The ruthenium studies revealed that the amount of bacterial adhesion is indirectly proportional to the nitrogen ion implantation of the titanium. The greater the percentage of nitrogen ion implantation in the titanium alloy, the less bacteria colonized the disk.

Ruthenium tetroxide staining of semicrystalline polyester polyurethane films

Thin films of segmented polyurethanes (PU) based on poly(ethylene adipate)glycol, 4,4′-diphenyl-methanediisocyanate and 1,4-butanediol were investigated. Film-covered slides with a very clear spherulitic structure, visible by means of a polarized microscope, were vapour-stained with ruthenium tetroxide (RuO 4). Exposure to the RuO 4 vapours for 30–60 min revealed lamellar morphology of the spherulites. Thus, the staining procedure using RuO 4 is a valuable technique for study of semicrystalline polyester PU films.

Avian permeability barrier function reflects mode of sequestration and organization of stratum corneum lipids: Reevaluation utilizing ruthenium tetroxide staining and lipase cytochemistry

The epidermis of avians and terrestrial mammals has evolved distinct, but related mechanisms to survive in a terrestrial environment. In both phyla, stratum corneum lipids form the basis of the cutaneous permeability barrier, but barrier function is less efficient in avians. Whereas in mammals the epidermal lamellar body (LB) secretes its contents into the intercellular spaces, in the feathered epidermis of avians, its distinctive secretory organelle, the multigranular body (MGB), does not secrete its contents into the stratum corneum intercellular spaces. Yet, neutral lipid-enriched droplets, derived from the cytosolic breakdown of MGB. ultimately are squeezed through membrane pores into the stratum corneum interstices. In this study we determined: a) using ruthenium tetroxide (RuO 4) fixation, whether these droplets form membrane structures after deposition in the stratum corneum interstices; and b) the similarities and differences between avian MGB and mammalian LB, using enzyme cytochemistry as a marker for secretion, and optical diffraction computer-aided image analysis and reconstruction to compare the internal structure of MGB vs. LB. MGB were shown to possess a similar lamellar substructure to LB in RuO 4-fixed specimens, exhibiting comparable dimensions on optical diffraction and computer transform analysts. Moreover, the intercellular lipids of avian stratum corneum lacked membrane-substructure, as was present in parallel samples of mammalian stratum corneum. Thus, both the absence of MGB secretion, and the failure of intercellular lipids to form membrane bilayers may explain the inherent differences in barrier function in these two taxa.

Some examples of ruthenium tetroxide staining technique for TEM of polymers

Staining is essential for examination of polymers under the electron microscope. Organic polymers consist exclusively of light atoms, e.g. carbon, oxygen, hydrogen and nitrogen, with the consequence that natural contrast is faint and completely inadequate. The aim of staining is to treat the polymer molecules with chemical substances, which must contain heavy atoms, and thus to modify them so that the electron density is drastically increased on the molecular site. Frequently, a hardening and structure-stabilizing effect is also achieved.In the last few years, ruthenium tetroxide staining has developed into a very useful method. Ruthenium tetroxide (RuO4) is a very strong oxidizing agent that can react not only with unsaturated, but also with many saturated polymers. For this reason, the method is less selective than others, yet it attracts interest for some polymer systems, because it allows differences in the rate of reaction and reaction intensity between various polymers to be exploited. This will be explained in the light of a few examples.

Synthesis and structural study of an ABA block copolymer consisting of poly(γ‐benzyl L‐glutamate) as the a block and poly(ethylene oxide) as the B block

AbstractABA‐type block copolymers composed of poly(γ‐benzyl L‐glutamate) (PBLG) as the A component and poly(ethylene oxide) (PEO) as the B component were synthesized by polymerization of γ‐benzyl L‐glutamate N‐carboxyanhydride initiated by primary amines located at both ends of the PEO chain. The morphology examined by transmission electron microscopy using ruthenium tetroxide staining revealed a lamellar type of microphase separation of the copolymers. From circular dichroism measurements in ethylene dichloride solution, as well as from infrared spectra measurements in the solid state, it was found that the polypeptide block exists in the α‐helical conformation, as in PBLG homopolymer. Wide‐angle X‐ray diffraction patterns for the block copolymers depend on the casting solvent and show basically similar reflections as the PBLG homopolymer.