Biotechnologie 2000. Teilprojekt 1: Lichtgetriebene Reparatur von UV-geschaedigter DNA Abschlussbericht

SIGLEAvailable from TIB Hannover: F00B893+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman

The human stratum corneum layer: An effective barrier against dermal uptake of different forms of topically applied micronised titanium dioxide

Electron microscopy visualisation and light microscopic investigations of three different application forms of titanium dioxide proved that neither surface characteristics, particle size nor shape of the micronised titanium dioxide result in any dermal absorption of this substance: Micronised titanium dioxide is solely deposited on the outermost surface of the stratum corneum and cannot be detected in deeper stratum corneum layers, the human epidermis and dermis

The outermost stratum corneum layer is an effective barrier against dermal uptake of topically applied micronized titanium dioxide

In order to help clarify the controversially discussed dermal uptake properties of micronized titanium dioxide (TiO), we conducted extensive in vitro dermal absorption studies with 'Franz-type' diffusion cells on excised porcine skin. After biopsies and chemical fixation, the overall localization of TiO in the skin was analyzed by means of transmission electron microscopy (TEM). The lateral and vertical distribution of TiO within the stratum corneum (SC) was investigated by tape stripping and subsequent scanning electron microscopy (SEM) in combination with energy dispersive X-ray analysis (EDXA). TiO was found exclusively on the outermost SC layer. The surface deposit, as displayed by TEM, featured clearly distinguishable agglomerates as well as single particles with a characteristic cubic shape and a primary particle size of about 20-50 nm. Concurrently, SEM/EDXA micrographs first showed an even distribution of TiO on the skin surface. After 10-fold stripping, however, TiO was found to be localized only in the furrows and not on the partially removed ridges of the skin surface. SEM/EDXA micrographs of the adhesive tape strips revealed a characteristic pattern of stripped material and free regions. This pattern was an imprint of the skin's topography. Hence, tape stripping initially removed TiO and SC layers only from the ridges and not from the deeper furrows. Continued stripping increasingly yielded material from the deeper contours of the SC surface. TiO was found only in traces in the upper part of the follicle without any evidence of uptake into the follicular epithelium. This indicates that there is not any relevant penetration via the follicular route. We conclude that due to the microtopography of the skin, the strip number normally does not reflect the SC layer number. Accordingly, tape stripping results should always be interpreted with care, especially in the case of topically applied particles, as even higher numbers of subsequent strips may still sample material from the outermost SC layer of the deeper furrows, which could be interpreted falsely as penetrated material. Our results clearly demonstrate that TiO homogeneously and completely covers the outermost SC layer. It is neither delivered to the SC nor to the underlying skin layers when applied topically to porcine skin in vitro in the cosmetic vehicle used here. These findings underscore the safety of this micronized inorganic UV filter

Optical investigations to avoid the disturbing influences of furrows and wrinkles quantifying penetration of drugs and cosmetics into the skin by tape stripping

Furrows and wrinkles, as typical structures of human skin, represent a reservoir for topically applied substances. This reservoir can influence penetration experiments of topically applied substances into the stratum corneum by tape stripping. Optical methods such as laser-scanning microscopy, optical coherent tomography, and the microscopical investigation of histological sections obtained by biopsies were used to check a special protocol, which avoids these potential disturbances. The use of a transparent adhesive film with high flexibility and the realization of an intense contact to the stretched skin by pressing the tape with a roll, moved laterally on the tape, are the prerequisites to obtain correct data. The application of this experimental technique and the performance of tape stripping allow the determination of the horny layer profile and the local distribution of topically applied substance, undisturbed by the characteristic structure of the natural skin. These results demonstrate that the presented tape stripping procedure is a valuable tool to determine, quantitatively, the penetration and the bioavailability of drugs and cosmetics inside the human stratum corneum, in relation to the horny layer profile. © 2005 Society of Photo-Optical Instrumentation Engineers

Determination of penetration profiles of topically applied substances by means of tape stripping and optical spectroscopy: UV filter substance in sunscreens

Penetration profiles of topically applied drugs and cosmetic products provide important information on their efficacy. The application of tape stripping in combination with UV/VIS spectroscopy is checked to determine the local position of topically applied substances inside the stratum corneum, the penetration profile. The amount of corneocytes removed with each tape strip is quantified via the particle-dependent absorption, the pseudoabsorption, in the visible spectral range. The concentration of a typical UV filter substance, 4-methylbenzylidene camphor, is determined by optical spectroscopy using the tape strips removed originally. In this case, a time-dependent increase in the absorbance must be taken into account. Laser scanning microscopic investigations confirm that the nonhomogeneous distribution of the filter substance, on the strips, can explain this spectroscopic behavior. When reaching a homogeneous distribution, the UV spectroscopic signal reflects the correct concentration. These spectroscopic values are compared with high performance liquid chromatography (HPLC) data. The values obtained with both methods for the concentrations of 4-methylbenzylidene camphor are in good agreement. The data obtained are used to illustrate the determination of a penetration profile of a UV filter substance. The results demonstrate that the described protocol is well suited to characterize, in a simple manner, topically applied substances that have a characteristic UV/VIS absorption band. © 2005 Society of Photo-Optical Instrumentation Engineers

Distribution of sunscreens on skin

The effectiveness of sunscreens was originally achieved by incorporation of soluble organic UV absorbers such as cinnamates and others into cosmetic formulations. Determinations of the sun protection factor (SPF) of emulsions containing different organic UV absorbers clearly indicate that the efficacy depends on the absorption characteristics of each single UV filter substance. Nowadays, micronised pigments such as titanium dioxide or zinc oxide have also been found to be protective against harmful UV rays. Our investigations using optical and electron microscopy proved that neither surface characteristics, particle size nor shape of the micronised pigments result in any dermal absorption of this substance. Micronised titanium dioxide is solely deposited on the outermost surface of the stratum corneum and cannot be detected in deeper stratum corneum layers, the human epidermis and dermis

Overexpression of the Fibroblast Growth Factor Receptor 1 (FGFR1) in a Model of Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a severe condition that affects many people and results in high health care costs. Therefore, it is essential to find new targets for treatment. The fibroblast growth factor receptor 1 (FGFR1) signalling pathway has a history of being explored for SCI treatment. Several groups have examined the effect of high availability of different FGFR1 ligands at the injury site and reported corticospinal tract (CST) regeneration as well as improved motor functions. In this study, we investigated overexpression of the FGFR1 in rat corticospinal neurons in vivo after injury (unilateral pyramidotomy) and in cerebellar granule neurons (CGNs) in vitro. We show that overexpression of FGFR1 using AAV1 intracortical injections did not increase sprouting of the treated corticospinal tract and did not improve dexterity or walking in a rat model of SCI. Furthermore, we show that overexpression of FGFR1 in vitro resulted in decreased neurite outgrowth compared to control. Thus, our results suggest that the FGFR1 is not a suitable therapeutic target after SCI

Timeline and scheme of in vivo experiment.

<p>A) Rats were pre-trained on the Montoya staircase test and the horizontal ladder for three weeks. One week before the SCI (pyramidotomy) animals were injected with AAVs overexpressing FGFR1 or mCherry into the sensorimotor cortex innervating the less preferred paw. During spinal cord surgeries the medullary pyramids were cut unilaterally in the brainstem innervating the preferred paw of all animals. Three days post-surgery the animals were assessed behaviourally and thereafter every week for ten weeks. Four weeks prior to the end of the study all animals were injected with BDA on the same side as to the vector injection. B) Scheme of the different surgical procedures. 1) Injection of AAVs expressing either CMV-FGFR1-2A-EGFP or CMV-mCherry-2A-EGFP, 2) unilateral pyramidotomy, 3) BDA injection for axon tracing, and 4) counting fibres that sprouted over the cervical midline.</p