Full-thickness Rat Skin Research Articles

Biological effects after percutaneous absorption of thyrotropin-releasing hormone and its analogue M-TRH

Besides its well known endocrinological effects, thyrotropin-releasing hormone (TRH) has potential clinical value in the treatment of neurotrauma and various neurologic and psychiatric disorders. The aim of this study was to assess if transdermal delivery of TRH and its analogue, M-TRH, in the presence of enhancers, is an effective means for administration of the peptides. Using the in vitro diffusion cell method, the effect of ethanol and a terpene on the transdermal penetration of the peptides across full-thickness rat skin were studied. Steady-state permeability values for TRH and M-TRH were 8.7 ± 2.2 and 6.7 ± 1.4 μg/cm 2 h, respectively. The addition of 3% terpene in combination with 47% ethanol increased the penetration of TRH and M-TRH to 16.2 ± 1.7 and 14.6 ± 2.1 μg/cm 2 h, respectively. Rats were studied in vivo for release of thyroid-stimulating hormone (TSH) as a biologic effect after transdermally delivered peptide. Topical application of TRH and M-TRH induced an increase in TSH serum concentration from 0.32 ± 0.09 ng/ml to 32.6 ± 5.0 and 22.9 ± 7.6 ng/ml, respectively, after 30 min. The addition of terpene and ethanol in combination with TRH or M-TRH, increased the TSH release to 43.0 ± 3.8 and 48.4 ± 4.0 ng/ml, respectively. It is concluded that, in the rat, peptides can be absorbed through the skin with retained biologic activity, and in amounts sufficient to elicit a physiological response.

Evidence for a Novel Glutamate-Mediated Signaling Pathway in Keratinocytes

Phenotypic alterations in keratinocyte behavior are essential for maintaining epidermal integrity during growth and wound repair and rely on co-ordinated cell signaling events. Numerous growth factors and cytokines have been shown to be instrumental in guiding such changes in keratinocyte activity; here we provide evidence which proposes a novel epidermal signaling pathway mediated by the excitatory amino acid glutamate. Glutamate is the major excitatory neurotransmitter at synaptic junctions within the central nervous system; however, we have identified expression in vivo of several regulatory molecules associated with glutamate signaling in keratinocytes. In resting rat skin epidermis, different classes of glutamate receptors, transporters, and a recently described clustering protein were shown to display distinct distribution patterns, supportive of a multifunctional cellular communication pathway. Immunoreactive N-methyl-D-aspartate-type, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate-type, and metabotropic-type glutamate receptors were colocalized with the specific glutamate transporter EAAC1 in basal layer keratinocytes, and GLT-1, a related transporter, was expressed suprabasally. In full-thickness rat skin wounds, marked modifications in the distribution of N-methyl-D-aspartate receptors and EAAC1 were observed during re-epithelialization, and alterations in N-methyl-D-aspartate receptor expression accompanied embryonic epidermal development, implicating glutamate signaling in these important biologic events. Furthermore, we provide evidence that these receptors are functional in vitro. These data provide strong evidence to support a role for glutamate in the control of epidermal renewal, and therefore suggest potentially novel therapeutic targets for the treatment of skin disease and enhancement of wound healing.

The Effect of Graft-Bed Irradiation on the Healing of Rat Skin Grafts

This study explores the possible side effects on healing skin grafts of irradiation, commonly used intraoperatively following surgical tumor removal. The experimental model involved the delivery of a single 10-Gy dose of electron radiation to the recipient bed of a skin wound, followed by attachment of a full thickness rat skin autograft. Skin graft repair was assessed by light microscopy, immunohistochemistry, and transmission electron microscopy over a 3-week period for grafted and grafted-irradiated groups. Graft-bed irradiation reduced fibrinogen, fibrin, and fibronectin deposition in the wound. It also produced brief changes in the extent of both re-epithelialization and granulation tissue formation, and reduced the diameter of collagen fibrils in the granulation tissue. Despite these changes, the results suggest that graft-bed irradiation only delays the healing process, producing no serious clinical complications at the time points studied.

Transdermal delivery of fentanyl by electroporation. II. Mechanisms involved in drug transport.

The aim of the present report was to systematically analyze the mechanisms involved in fentanyl transdermal transport by skin electroporation. The study was performed in vitro with full-thickness hairless rat skin, skin electroporation being carried out with five exponentially-decaying pulses of 100 V applied voltage and around 600 ms pulse duration. Transport during and after pulsing are both important in transdermal delivery of fentanyl by skin electroporation. Rapid transport occurred during pulsing due to electrophoresis and diffusion through highly permeabilized skin. No electroosmosis was observed. The slow post-pulse passive transport was explained by lasting changes in skin permeability. Measurements of fentanyl quantities in the skin demonstrated that pulses rapidly loaded the viable part of the skin with fentanyl and hence rapidly overcame skin barrier. The different contributions of the transport mechanisms appear to depend on the physicochemical parameters of the transported molecule as well as the solution, suggesting that mechanistic analysis and careful consideration of formulation variables are essential for the development and optimization of drug delivery by skin electroporation.

The percutaneous absorption of hydroquinone (HQ) through rat and human skin in vitro

Because of the potential for human contact with photographic developer solutions containing hydroquinone (HQ), the rates of percutaneous absorption of HQ through human stratum corneum and full-thickness rat skin have been measured in vitro using 5% aqueous solutions of HQ as the donor solutions. The studies were performed using infinite doses of aqueous solutions containing 14C-labeled HQ in Franz-type diffusion cells. The measured absorption rate (mean ± S.D.) of HQ through human stratum corneum was 0.52 ± 0.13 μg/cm 2/h, while that for full-thickness rat skin was 1.1 ± 0.65 μg/cm 2/h. The ratio (rat/human) of the permeability constants ( K p) was 2.4. Using the definitions suggested by Marzulli et al. (1969) Toxicol. Appl. Pharmacol. Suppl. 3, 76–83, HQ would be classified as ‘slow’ with respect to its absorption through human stratum corneum.

A freeze-injured skin graft model for the quantitative study of basic fibroblast growth factor and other promoters of angiogenesis in wound healing

A new in vivo model has been developed for the quantitative study of promoters and potential promoters of angiogenesis. Full thickness rat skin autografts received a reproducible and uniform freeze injury, before being applied to full thickness wounds, in order to delay revascularisation. Blood flow in the grafts was measured during the healing period using noninvasive (laser Doppler flowmetry) and invasive (133Xe clearance) techniques. The increase in blood flow over a period of 10-14 days was taken as an index of angiogenesis. These measurements were corroborated by histological assessment of the graft vasculature, using a laminin stain to highlight vascular basement membrane. Freeze injury delayed but did not ultimately prevent full graft revascularisation (p < 0.01 for laser Doppler flowmetry and 133Xe clearance). Application of the angiogenic agent basic fibroblast growth factor (bFGF), in slow release pellet form, stimulated angiogenesis in cryoinjured grafts in a dose-related fashion. Doses of 500 and 5000 ng bFGF produced significant stimulation (500 ng bFGF, p < 0.001, and 5000 ng bFGF, p < 0.01, for both laser Doppler flowmetry and 133Xe clearance; increased vessel profile counts, p < 0.05, at each time point tested for both doses) while 50 ng bFGF had no significant effect. By contrast, 500 ng bFGF had no measurable effect on uninjured grafts. In addition, bFGF-stimulated angiogenesis in cryoinjured grafts was antagonised by a neutralising antibody to bFGF, demonstrating the specificity of action of bFGF in this model.

Effect of Penetration Enhancers on the in Vitro Transport of Ephedrine Through Rat Skin and Human Epidermis from Matrix Based Transdermal Formulations

The potential of skin as a site for administration of systemically active Ephedrine HCl (EH) has been recognised. The effect of penetration enhancers, i.e., Dimethyl sulfoxide (DMSO), Dimethyl acetamide (DMA), Dimethyl formamide (DMF) and Azone on the in-vitro transport of EH from matrix based transdermal formulations through full thickness rat skin and human epidermis was investigated. The highest flux with minimum time lag through rat skin and human epidermis was observed from the batch containing Azone as penetration enhancer.

A comparative study of the rates of in vitro percutaneous absorption of eight chemicals using rat and human skin

In vitro percutaneous absorption studies were carried out for eight chemicals using full thickness rat skin and human stratum corneum. The purpose of the studies was to compare the rates of absorption for the two species. For each of the chemicals, the observed rate using full thickness rat skin was greater than that observed for human stratum corneum. The ratios of the rates (rat/human) varied from 1.7 to 5.8 with a mean value of 3.1. The chemicals tested were tritiated water, 2-ethoxyethyl acetate, diethylene glycol monobutyl ether, urea, di(2-ethylhexyl) phthalate, 2-ethylhexanol, ethyl 3-ethoxypropionate, and 2-propoxyethanol. The chemicals were chosen to represent a wide range of physical properties and permeability constant values. It was concluded that rat skin was more permeable than human skin for each of these eight chemicals. This conclusion is supported by similar findings from studies in other laboratories and suggests that results from studies in the rat overestimate skin absorption in man.

In vitro penetration of the dopamine D2 agonist N-0923 with and without Azone

Abstract The penetration of the dopaminergic D2 agonist S (−)-2-( N -propyl- N -2-thienylethylamino)-5-hydroxytetralin (N-0923) was evaluated in vitro on full-thickness rat skin, using the Franz diffusion cell. The drug was tested as the N-0923 · HCl salt as well as the N-0923 base. The penetration-enhancing effect of Azone was studied in a vehicle concentration of 5%, or after pretreating shaven rat skin in vivo, at several time intervals before the in vitro experiment, with a solution containing 1 or 5% Azone. From the vehicle containing 60% ethanol, 20% propylene glycol and 20% water, the extent of penetration through rat skin was relatively low for the base and even lower for the salt. The introduction of Azone into the vehicle in a final concentration of 5% resulted in a 6-fold decrease in penetration for the free base. Nevertheless, penetration of the salt increased about 12-fold in extent during the first 12 h, after which it declined to almost zero. The lag time was reduced from 13 to 5 h. Pretreatment of rat skin in vivo with an ethanol-propylene glycol-water solution containing 5% Azone resulted in a 12-fold increase in flux for the salt, a 2-fold increase in flux for the base and a decrease in lag time to about the same values for both N-0923 · HCl and N-0923 base. In vivo pretreatment of rat skin with a solution containing 1% Azone had no significant effect on the flux and lag time vs controls. This study indicates that Azone may have potential in facilitating the transdermal application of N-0923, principally through pretreatment of the skin to enhance absorption. This may allow it to circumvent its considerable oral first-pass metabolism.

Percutaneous absorption of nicotinic acid, phenol, benzoic acid and triclopyr butoxyethyl ester through rat and human skin in vitro: Further validation of an in vitro model by comparison with in vivo data

The in vitro percutaneous absorption of three model compounds, nicotinic acid, phenol and benzoic acid, and the herbicide triclopyr butoxyethyl ester (triclopyr BEE) has been investigated in flow-through diffusion cells using skin from male Fischer 344 rats and humans. After the application of the four chemicals to the epidermal surface of unoccluded full-thickness rat skin, the absorption of each compound across the skin and into the receptor fluid at 72 hr reached 3.7 ± 0.3, 5.7 ± 0.6, 26.7 ± 3.7 and 48.3 ± 1.2% (mean ± SD, n = 2–7) of the applied dose for triclopyr BEE, nicotinic acid, phenol and benzoic acid, respectively. After the application of the four chemicals to the epidermal surface of unoccluded full-thickness human skin, the absorption of each compound across the skin and into the receptor fluid at 72 hr was significantly ( P < 0.05) less than through rat skin, reaching 0.7 ± 0.1, 0.7 ± 0.2, 18.8 ± 1.3 and 37.8 ± 6.9% (mean ± SD, n = 2–7) of the applied dose for triclopyr BEE, nicotinic acid, phenol and benzoic acid, respectively. Occlusion of the skin surface with teflon caps often significantly ( P < 0.05) enhanced the percutaneous absorption of the model compounds, although this effect was not uniform, varying with the compound under study and the skin (rat or human) used. When rat skin was occluded with teflon caps, the extent of absorption at 72 hr reached 8.6 ± 0.8, 36.2 ± 1.7 and 51.8 ± 3.3% (mean ± SD, n = 3–4) for nicotinic acid, phenol and benzoic acid, respectively. Corresponding values for human skin occluded with teflon caps were 3.3 ± 1.6, 47.1 ± 0.5 and 65.5 ± 7.1% (mean ± SD, n = 3–4). The experiments on the absorption of each model compound through rat and human skin were repeated and there was generally good agreement between the results from the two sets of experiments. The in vitro data reported compare favourably with data obtained by other workers using both in vitro and in vivo methodologies. The in vitro : in vivo correlation supports the use of the flow-through diffusion cell system as a model for the prediction of percutaneous absorption in vivo in the rat and in humans.

A Comparative Study of the Rates of in Vitro Percutaneous Absorption of Eight Chemicals Using Rat and Human Skin

In vitro percutaneous absorption studies were carried out for eight chemicals using full thickness rat skin and human stratum corneum. The purpose of the studies was to compare the rates of absorption for the two species. For each of the chemicals, the observed rate using full thickness rat skin was greater than that observed for human stratum corneum. The ratios of the rates (rat/human) varied from 1.7 to 5.8 with a mean value of 3.1. The chemicals tested were tritiated water, 2-ethoxyethylacetate, diethylene glycol monobutyl ether, urea, di(2-ethylhexyl)phthalate, 2-ethylhexanol, ethyl 3-ethoxypropionate, and 2-propoxyethanol. The chemicals were chosen to represent a wide range of physical properties and permeability constant values. It was concluded that rat skin was more permeable than human skin for each of these eight chemicals. This conclusion is supported by similar findings from studies in other laboratories and suggests that results from studies in the rat overestimate skin absorption in man.

Destabilization of whole skin lipid bio-liposomes induced by skin penetration enhancers and FT-IR/ATR (Fourier transform infrared/attenuated total reflection) analysis of stratum corneum lipids.

Whole skin lipid bio-liposomes (skin bio-liposomes), in size ranging from 2 to 8 microns, were prepared by a reverse phase evaporation technique using rat full thickness skin. Leakage of an encapsulated fluorescence probe, ANTS (delta-amino-1,3,6-naphthalene-trisulfonate), was measured by adding transdermal penetration enhancers (penetrants) into the medium where the skin bio-liposomes were present. Oleylamine induced a fast release of ANTS from the liposomes compared to lauryl-amine which showed a weak action. With these penetrants, the degree of ANTS release from the prepared bio-liposomes was found to correlate well with the results of frequency changes in the CH-asymmetric stretching band near 2920 cm-1 in the rat stratum corneum. The penetrant which caused relatively strong leakage of ANTS induced the significantly large shift of the peak toward the higher wave-numbers due to the perturbation in the structure of lipids of the stratum corneum. The skin bio-liposomes prepared from the rat full thickness skin could be useful in evaluating the penetrants.

Enhancing Effect of Pyrrolidone Derivatives on Transdermal Penetration of 5-Fluorouracil, Triamcinolone Acetonide, Indomethacin, and Flurbiprofen

The enhancing effects of pyrrolidone derivatives on the transdermal penetration of 5-fluorouracil, triamcinolone acetonide, indomethacin, and flurbiprofen were studied by using an in vitro technique and full-thickness rat skin. The enhancers included 1-methyl (1), 1-hexyl (2), and 1 -lauryl-2- pyrrolidone (3). Penetrants with various physicochemical properties were used. Flurbiprofen penetrated through skin rapidly after application alone. 5-Fluorouracil, triamcinolone acetonide, and indomethacin showed little penetration. Pyrrolidone derivatives enhanced the penetration of penetrants, especially the lipophilic compounds 2 and 3, which showed a great enhancing effect on the penetration of 5-fluorouracil and indomethacin. Pyrrolidone derivatives also enhanced the solubility of these penetrants in isopropyl myristate. Compounds 2 and 3 showed greater enhancing effects on the solubility and penetration of hydrophilic penetrants than those of lipophilic penetrants. These results suggest that the pyrrolidone derivatives enhance the flux of penetrants in skin by increasing the solubility of penetrants in the stratum corneum. Compounds 1 and 2 were detected in the receptor phase. All enhancers accumulated to a great extent in the skin. These derivatives also enhanced the skin retention of drug. It is concluded that these pyrrolidone derivatives are useful for transdermal drug delivery, although further studies are necessary before they could be used clinically.

3‐D model of vascular network in rat skin obtained by stereo vision techniques

The quantitative analysis of the depth of injury, penetration of therapeutic agents in tissues, and the regeneration of vascular patency after a graded degree of thermal injury requires a knowledge of the shape and spatial configuration of the vascular networks in the tissue. We have applied computational stereo vision techniques to describe the 3-D configuration of microvessels in full thickness rat skin vascular casts produced by perfusion of Yellow Microfil latex solution through the aorta. The principal concern is to describe the 3-D structure of vascular networks using a set of 3-D space curves. This representation is computed by integrating monocular and binocular processing; the 2-D curve representation of blood vessels computed through monocular analysis is integrated with disparity data to yield a space curve representation for each vessel. A connection diagram is also computed to indicate the connections existing among the computed space curve representations.

Transdermal delivery of 5-fluorouracil and its alkylcarbamoyl derivatives

The transdermal absorption of 5-fluorouracil and its alkylcarbamoyl derivatives containing various lipophilic pro-moieties, including butylcarbamoyl, hexylcarbamoyl and octylcarbamoyl groups, was investigated in the rat. All compounds showed various degrees of lipophilicity and instability at physiological pH. Transdermal permeation of these drugs was examined by employing an in vitro technique using a diffusion device which was mounted with a full-thickness rat skin as the diffusion membrane. On application of the alkylcarbamoyl derivatives to the rat skin, 5-fluorouracil appeared in the receptor phase. 1-Butylcarbamoyl and 1-hexylcar-bamoyl derivatives showed 3–4-fold greater penetration than that of 5-fluorouracil. A lag time was observed before steady-state diffusion of the drug was established which was prolonged with increasing alkyl chain length. The increase in lipophilicity of the derivatives was found to enhance their binding with keratin, a component of the skin. The accumulation of both derivatives and 5-fluorouracil in the skin was noted. Drugs accumulated rapidly in the skin within 2 h after topical application and reached a constant level thereafter. In particular, the 1-butylcarbamoyl derivative, which permeated rapidly through the skin, showed a high level of accumulation in the skin. Co-application of 5-fluorouracil and the 1-octylcarbamoyl derivative resulted in an additive effect of skin absorption of 5-fluorouracil.

Differences in Human Skin Between the Epidermal Growth Factor Receptor Distribution Detected by EGF Binding and Monoclonal Antibody Recognition

Two methods have been used to examine epidermal growth factor (EGF) receptor distribution in human scalp and foreskin. The first employed [125I]EGF viable explants and autoradiography to determine the EGF binding pattern while the second used a monoclonal antibody to the human EGF receptor to map the distribution on frozen skin sections of an extracellular epitope on the EGF receptor. The [125I]EGF binding experiments showed accessible, unoccupied EGF receptors to be present on the epidermal basal cells (with reduced binding to spinous cells), the basal cells of the hair shaft and sebaceous gland, the eccrine sweat glands, capillary system, and the hair follicle outer root sheath, generally similar in pattern to that previously reported for full-thickness rat skin and human epidermis. The same areas also bound EGF-R1 but in addition the monoclonal antibody recognized a cone of melanin containing presumptive cortex cells, excluding the medulla, lying around and above the upper dermal papilla of anagen hair follicles, epithelial cells around the lower dermal papilla region, and in some tissue samples the cell margins of the viable differentiating layers of the epidermis. In a control study, to clarify whether EGF-R1 could recognize molecules unrelated to the EGF receptor, the EGF binding and EGF-R1 recognition profiles were compared on cultures of SVK14 cells, a SV40 transformed human keratinocyte cell line. EGF binding and EGF-R1 monoclonal antibody distribution on these cells was found to be similar, indicating that, at least for SVK14 cells, EGF-R1 binding provides a reliable marker for EGF binding. Explanations for the discrepancies between these two methods for determining EGF receptor distribution in human skin are discussed, including the possibility that latent EGF receptors, unable to bind [125I]EGF, may be present in some differentiating epithelial compartments.

Effect of protein depletion on skin graft "take" in rats.

Protein malnutrition, with reduction of serum albumin from a normal level of 3.3 gm/dl to a 6-week depleted value of 2.1 gm/dl, does not appear to adversely affect the "take" of rat full-thickness skin grafts.

Skin graft survival on avascular defects.

Full-thickness skin grafts placed on bare rabbit ear cartilage were revascularized, whereas split-skin grafts on the same kinds of areas failed. The contrast is most likely due to differences in the skin graft thickness and the vascular patterns in the grafts. Full-thickness rat skin grafts placed over the same sized underlying silicone sheet implants did not survive. When the graft was made 3 mm larger on all sides than the implant, all or a portion of the graft over the implant survived. Quantitation of the area of graft survival is presented. Prepared grafts (replaced on their donor areas for 48 hours) on prepared beds and fresh grafts on prepared beds had slightly larger areas of graft surviving over an avascular defect. On the basis of this investigation, we suggest that a full-thickness skin graft placed on a prepared peripheral bed may make possible the greatest area of bridging over an avascular defect.

Characterization of Aging Skin via Thermal Analysis

Abstract Samples of full-thickness rat skin from birth to 3 years were characterized by thermal analysis. The techniques involve differential scanning calorimetry, thermomechanical analysis, and thermogravimetry. In the dry state the skin samples exhibit thermally induced transverse dimensional changes at 33, 130, 192, and 250[ddot] C. The 130 and 192[ddot]C expansions are complimented by enthalpy changes. As the animal ages, the degree of the individual expansion decreases as the energy associated with the transition increases. This observation is consistent with the theory of increased cross-linking with age. The relationship observed in the dry state is also present in hydrated samples. Thermogravimetric results indicate an initial weight loss up to 100[ddot]C, which is due to dehydration, followed by a major weight loss between 250 and 450[ddot]C. The decomposition products in the higher temperature range also varied with age. Work with recast collagen films and elastin aid in the interpretation of th...

Histidine decarboxylase activity of autografted and allografted rat skin.

SummaryIntracellular histamine formation from histidine decarboxylase enzyme activity was measured in autografts and allografts of full thickness rat skin from 1-21 days after grafting by a sensitive and specific isotope dilution method. Histidine decarboxylase activity of autografted skin was eleva