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The development of a rat/human skin flap served by a defined and accessible vasculature on a congenitally athymic (Nude) rat
Abstract
Experience in microvascular surgery on rats and availability of athymic (nude) rats led us to believe that a long-term functional rat/human skin sandwich flap could be generated on a defined and experimentally accessible vasculature on nude rats. Such a system has been developed and validated. Microvasculature has been assessed. The volume of blood to the flap ranges from 1 to 2 ml/min, collateral circulation to the flap exists, but is negligible, and there is little change in the capillary blood flow as the flap ages. The flap can be utilized to study absorption of compounds from a half-cell diffusion chamber or from direct deposition on the skin, and can be utilized to study various parameters of percutaneous absorption, e.g., the effect of hydration on the stratum corneum. Transdermal flux can be determined. Altering the microcirculation directly affects the percutaneous absorption of compounds that are rapidly absorbed. The absorption of benzoic acid through an experimentally vasoconstricted area (iontophoresis of phenylephrine) significantly alters the time to peak absorption, with values being 14 times that of the control site. The system has been utilized to assess metabolic activity of skin in situ using [3H]adenine arabinoside and studying the appearance of its major metabolite, [3H]Ara-H, in flap blood, as well as the back diffusion of this compound into the donor chamber. Recently the human/rat skin sandwich flap component has been developed. With this system, it has been demonstrated that benzoic acid, when applied to the human skin component of the flap has an absorption profile which is quite different from that when benzoic acid is applied to rat skin, peak flux occurred 2 hr after application. This contrasts with 10 min to peak flux when the same experiment is carried out on the rat/rat skin sandwich flap. To our knowledge, the human/rat skin sandwich flap is the first example of a viable, functional human organ that is chronically maintained by a biologic support system which has the added distinction of being on an independent but accessible vasculature. The validation experiments strongly suggest that this system will be important in gaining insights into the more sophisticated in vivo components of skin, relative to toxicology and pharmacology.
The ultimate goal of many in vitro and in vivo drug and xenobiotic percutaneous absorption studies in animals is to predict penetration in humans. The optimal approach would be a quantitative one that not only would precisely predict chemical disposition in humans but also would allow one to determine the effect of different formulation, environmental, and dermatological variables on the rate and extent of dermal penetration. Knowledge of these effects and of the overall source of variability in dermal penetration is especially important when designing transdermal delivery systems for drugs with “narrow” therapeutic windows. In toxicological applications, if the chemicals to be tested are either very toxic or of unknown toxic potential in man, in vitro or animal testing is required. With the current societal emphasis on minimizing the use of animals in biological research, in vitro approaches are being stressed.
Several authors reviewed the experimental models for psoriasis (Krueger and Jorgensen 1990; Boehncke 1997; Nickoloff 1999; Rosenberg et al. 1999; Schön 1999; Mizutani et al. 2003). In addition to studies in animals, various in vitro investigations were carried out with autopsy material from human psoriatic lesions. Several studies were devoted to the T cell hypothesis of psoriasis (Bos and de Rie 1999). Lymphocytes may change epidermal growth homeostasis, leading to increased keratinocyte proliferation and abnormal differentiation of these apoptotic cells that end their life cycle as corneocytes.
Gene Defect And Knockout RodentsReconstituted ModelsTransgenic and Knock-In Alternative ModelsMulti-Gene Engineering and Conditional KnockoutsAdvantages and Limitations of Alternative ModelsSummaryReferences
Skin Sensitization TestingGuinea Pig Maximization AssayPurpose and RationaleA variety of methods are available for the prospective identification of skin sensitizing chemicals. The methods that have been used are the Draize rabbit irritancy test (Draize et al. 1944; Phillips et al. 1972), the occluded guinea pig patch test (Buehler 1965), and the guinea pig maximization test (Magnusson and Kligman 1969; Magnusson 1980). The guinea pig maximization test is an adjuvant sensitization test requiring intradermal injections of the test substance, in combination with Freund's complete adjuvant, which stimulates non‐specifically the immune system of treated animals, enhancing their ability to respond to sensitizing chemicals.ProcedureGroups of 15–20 guinea pigs (Hartley strain) are used. On day 0 an area of 4 × 6 cm over the shoulder region is clipped short with an electric clipper. Three pairs of intradermal injections are made simultan ...
Annette Bunge and her research group have had the central theme of mathematically modeling the dermal absorption process. Most of the research focus has been on estimating dermal absorption for the purpose of risk assessment, for exposure scenarios in the environment and in the occupational setting. Her work is the basis for the United States Environmental Protection Agency's estimations for dermal absorption from contaminated water. It is also the basis of the dermal absorption estimates used in determining if chemicals should be assigned a 'skin notation' for potential systemic toxicity following occupational skin exposure. The work is truly translational in that it started with mathematical theory, is validated with preclinical and human experiments, and then is used in guidelines to protect human health. Her valued research has also extended into the topical drug bioavailability and bioequivalence assessment field. © 2013 S. Karger AG, Basel.
A new in-situ experimental method was designed to evaluate the cutaneous disposition of to0ically-applied drugs. Flurbiprofen was used as a model drug.A disc-shaped agar gel (3.85 cm diam. and 0.5 cm thickness) was subcutaneously implanted in the abdominal region of hairless rats as a drug receptor, and a drug donor cell was placed above it. No significant pharmacokinetic modification of flurbiprofen was observed during the experimental procedure. A bolus injection and a constant intravenous infusion of flurbiprofen were applied, followed by analysis of flurbiprofen levels in the plasma and agar gels. The clearance rate of flurbiprofen from the systemic circulation to the gel could thus be calculated. Flurbiprofen (1% gel formulation) was then topically applied to the skin. The amount of flurbiprofen that migrated from the formulation to the systemic circulation and the amount that migrated directly to the agar gel across the skin, over 1Oh, were separately evaluated (±s.e.) to be 211 ±36.4`and 1.43 ±0.158 μg, respectively. Thus, most of the flurbiprofen was absorbed into systemic circulation.The technique has proved to be an effective tool for the quantitative evaluation of the cutaneous disposition of a topically-applied drug.
The possibility of predicting the behavior of in vivo systems based on physical and chemical parameters determined by in vitro experiments is examined using benzoic acid. The physical and chemical parameters governing percutaneous absorption of benzoic acid—permeability, partition coefficient, and skin thickness—were determined by in vitro experiments as described in Ref. 1. These parameters were used, in combination with benzoic acid elimination kinetics, to predict the results of in vivo experiments using a comprehensive mathematical model. The in vivo system consists of a congenitally athymic (nude) rat with a surgically constructed human skin sandwich (HSSF) flap on which a donor cell is placed. To apply the in vitro parameters to an in vivo system requires a suitable pharmacokinetic model describing distribution and elimination for benzoic acid in the nude rat. Blood concentrations of benzoic acid following a bolus intravenous injection are closely described by a two-compartment open pharmacokinetic model with elimination occurring from only one compartment. The mathematical model of the rat-donor cell system combines this two-compartment model of the rat with a percutaneous absorption model to provide useful estimates of the measured in vivo blood levels. Comparisons of predicted and measured results suggest that the parameters determined by in vitro experimentation can be used to predict the behavior of complex in vivo systems, if a suitable mathematical model is available.
Topical antiviral chemotherapy has a number of potential advantages over systemic drug delivery for the treatment of cutaneous herpes simplex virus infections, including convenience; higher target tissue drug levels and greater efficacy; and specific targeting of the drug to the site of infection, with reduced cost and reduced exposure of the remainder of the body to drug side effects. Realization of these possibilities has been slow in part because of the paucity of 'active' topical drug formulations with effective penetration-enhancing agents and a technical barrier-our failure as yet to measure drug levels in the epidermis, which could guide formulation development. Recent success with a topical treatment for herpes simplex labialis should stimulate continued laboratory and clinical research in this field.
Cutaneous blood flow has been directly quantitated in vivo for the first time without animal death utilizing the rat skin sandwich flap. This was accomplished by conducting experiments that made a direct correlation between two instruments: a laser Doppler velocimeter and an electromagnetic blood flow meter. Data demonstrate that the correlation between these two instruments is high and reproducible (r = 0.96) with a small (1.3%) coefficient of variation. Blood flow to skin in the unmanipulated state varies from 0.7 to 1.2 mls/min in an anesthetized rat. Application of the blood flow correlation to the determination of percutaneous absorption of caffeine across human skin and benzoic acid across rat skin demonstrates that assuming cutaneous blood flow is a particular value day to day in any skin type results in an apparent wide range of total compound absorbed across that skin on independent occasions. Utilizing actual blood flow measurements to calculate the amount of chemical absorbed reduces the range of variability in the total amount of chemical absorbed and provides a more accurate knowledge of events occurring during a particular time of the absorption process. Quantitation of cutaneous blood flow will be useful in physiologic and pharmacologic studies where actual cutaneous blood flow is likely to be important to the processes studied, e.g., delivery of drug to skin, metabolism within the skin, and disposition of drug to blood and skin following topical drug application.
To study the influence of cutaneous blood flow upon the transdermal penetration of drugs, an original in vivo model, the bipediculated dorsal flap (BDF) of the hairless rat is described. This model is constituted by a dorsal cutaneous muff on both sides of the spinal column. The blood circulation was maintained symmetrically on both skin insertion pedicles. Two radiotracers, 86Rb and 201Tl (respective half-lives: 18 and 3 days), known to measure nutritional blood flows, are compared in this model. In the back skin, over and under the flap, mean blood flow (expressed as percentage of injected dose per gram skin) was similar with Rb (9.5 +/- 3.1) x 10(-2) and Tl (10.2 +/- 2.1) x 10(-2). Equivalent values were obtained in BDF skin (non ligated pedicle): Rb (8.4 +/- 1.3) x 10(-2), Tl (9.5 +/- 2.7) x 10(-2). In BDF skin, cutaneous blood flow was lower by 70-91% in the ligated pedicle (compared to the nonligated) for both radioisotopes. Identical 86Rb and 201Tl distributions suggest the use of 201Tl for further experiments of cutaneous blood flow.
Using excised human skin and tissue grafted to athymic mice, the in vitro and in vivo delivery and metabolism of a salicylate diester were compared. Concentration profiles of this drug and its metabolites were obtained for the outer several hundred microns of the skin. These results show significant differences in the extent of enzymatic cleavage and distribution of metabolites between in vitro and in vivo studies. Furthermore, these data suggest that in vitro results may overestimate metabolism because of increased enzymatic activity and/or decreased capillary removal.
The assessment of cutaneous metabolism during in vitro percutaneous absorption studies requires maintenance of the viability of the skin section. With the use of flowthrough diffusion cells, Eagle's minimal essential medium (MEM), Hepes-buffered Hanks' balanced salt solution (HHBSS), or Dulbecco modified phosphate-buffered saline (DMPBS), acting as receptor fluids, were able to sustain aerobic and anaerobic glucose utilization, testosterone and estradiol metabolism, and histopathological appearance of perfused rat skin sections for 24 hr. Fetal bovine serum supplements were not required for survival and appeared to inhibit the extraction of the metabolite estrone from the receptor fluid fractions in estradiol absorption/metabolism experiments. The use of phosphate-buffered saline (PBS) resulted in elimination of aerobic and anaerobic glucose utilization in 12 hr and declining appearance of steroid metabolites in receptor fluid fractions during the 24-hr percutaneous absorption/metabolism studies. Histopathological examination of skin sections perfused with PBS for 24 hr showed autolysis of the viable epidermis and dermis. The results demonstrate that an appropriate receptor fluid, such as MEM, HHBSS, or DMPBS, is required for percutaneous absorption studies in which cutaneous metabolism of the penetrating compound is to be considered.
In the present study, human apolipoprotein E (apoE) was monitored in the circulation of athymic mice and rats bearing human epidermal grafts. Human apoE was detected in the systemic circulation of graft-bearing animals as long as the graft remained on the animal. Within 24 hr of graft removal, human apoE was not detectable in plasma, indicating that apoE resulted from continuous production of the protein by grafted keratinocytes. These results show that proteins as large as apoE (299 amino acids) traverse the epidermal-dermal barrier and achieve systemic distribution where they may produce effects on distal tissues. The feasibility of using grafts of genetically-altered keratinocytes for the delivery of secreted proteins is clearly reinforced by the demonstration that an epidermally derived protein exhibits systemic distribution. Finally, by virtue of its systemic distribution, apoE produced in a peripheral tissue such as skin, may function in the reverse transport of cholesterol from peripheral tissues to the liver.
The isolated perfused porcine skin flap (IPPSF) has been developed as an alternative in vitro tool for examining the pharmacokinetics and mechanisms of percutaneous absorption. In this study, dosing solutions of seven 14C-radiolabeled compounds representing three chemical classes--organic acid/base [benzoic acid (B), caffeine (C)], organophosphate (OP) pesticides (diisopropylfluorophosphidate, malathion, parathion), and steroid hormones (progesterone, testosterone)--were prepared in ethanol and applied topically at a surface concentration of 40 micrograms cm-2 to the IPPSF. A three-compartment pharmacokinetic model used to stimulate mass transfer from the surface (C1), diffusion through epidermis and dermis (C2), and transfer into the capillary perfusate (C3), was developed based on flux through the IPPSF from 0 to 8 hr. This basic model accurately stimulated measured IPPSF fluxes for five of seven compounds, including the OPs and steroids. The model was modified to simulate the shunting of drug to fast and slow release pathways, which occurred for B 3-4 hr postapplication, and to account for flow-dependent flux increases seen for C at 6 hr postapplication. The latter may be due to a direct pharmacologic effect, since C is a known vasodilator. Extrapolated (to 6 days) areas under the curve from the model simulations were compared with in vivo percutaneous absorption estimates, obtained from 6-day excretion studies in pigs. The in vivo-in vitro correlation, based on simple linear regression across compounds, was excellent (R2 = 0.88, R = 0.94, p less than 0.002). These results suggest that xenobiotic penetration in the 8-hr IPPSF experiments is highly predictive of in vivo absorption totals (6-day studies). In addition, since pig and human skin are similar physiologically and pharmacologically, the IPPSF may eventually have applications in formulating human dermal risk assessment models.
Cultures of human epidermal cells may be used to generate epidermis on athymic recipients. We describe two novel techniques for grafting such cultures. Both techniques permit the generation of typical human epidermis within 7 d. Both techniques result in less graft contraction than conventional grafting, and there is no difficulty in distinguishing the human epidermis generated by the graft from the epidermis of the recipient animal. Starting with a single human biopsy, epidermis may be generated on a great many experimental animals; such grafts should therefore provide uniform material for investigation of the properties of human epidermis.
The percutaneous absorption and distribution profile of hexamethylene lauramide (hexahydro-1-lauroyl-1H-azepine) were examined using a rat skin-flap model. After a topical dose to the skin flap, the drug concentrations in the vasculature at the site of drug application and in the systemic blood were monitored simultaneously. Hexamethylene lauramide penetrated the skin and reached a steady state in stratum corneum, viable epidermis, dermis, and cutaneous blood in 3 hr. Its concentration in the skin was much higher than that in the blood. Its apparent concentration in the epidermis was 19 times that in the dermis and about 3000 times that in the cutaneous blood. The percutaneous absorption of 14C-hexamethylene lauramide resulted in ascending systemic blood concentrations throughout the experimental period, whereas the cutaneous blood levels remained steady. The topically absorbed hexamethylene lauramide was quantitatively recovered in urine (85%) and feces (13%). The half-lives of urinary and fecal excretion of 14C-hexamethylene lauramide were 17 and 30 hr, respectively. Hexamethylene lauramide, when topically coadministered in an experimental formulation, enhanced the skin penetration of hydrocortisone with increased drug contents in the stratum corneum (2-fold) and with increased hydrocortisone concentrations in the cutaneous blood (3.4-fold) and the systemic blood (3.5-fold). The results indicated that the high concentration and retention of hexamethylene lauramide in stratum corneum and viable epidermis may contribute to its penetration enhancement effect in the skin. A steady state in percutaneous tissues was observed before the drug reached distribution equilibrium systemically. The systemic blood concentration of a topically applied agent therefore may not reflect its percutaneous kinetic processes before a systemic distribution equilibrium is reached.(ABSTRACT TRUNCATED AT 250 WORDS)
Gradual rejection of topically engrafted human split-thickness skin grafts (HSTSG) occurred in greater than 90% of congenitally athymic (nude) rats between 21 and 42 days of grafting. Engraftment and rejection of HSTSG is accompanied by a partial restoration of some cell-mediated immune components, the mixed lymphocyte response and lysis of human target cells. Histologic features of the rejection process were those seen in a host-versus-graft reaction. Immunofluorescent analysis of skin undergoing rejection demonstrated IgG at the basement membrane zone in most grafts. Nude rats rejecting HSTSG had circulating IgG which bound to the basement membrane zone and blood vessels of human skin. Nude rats treated with cyclosporine injections for 21 days had an enhanced survival of HSTSG, 120 or more days.
Percutaneous absorption occurs after passive diffusion through the different layers of the skin and its appendages. Thereafter, a resorption process into the cutaneous microcirculation brings the compounds into the systemic circulation. The objective of this in vivo study in the hairless rat was to compare the percutaneous absorption of two steroids on normal and appendage-free (scar) skin and to show if differences in absorption result only from the lack of hair follicles and sebaceous glands and/or from a modification of local blood flow. Percutaneous absorption was evaluated with estradiol and progesterone after 30 min, 2 and 6 h. Except after 30 min, the reservoir function of the stratum corneum of scar skin was approximately twice as high as in normal skin. Eighty to ninety percent of the estradiol and progesterone found in the stratum corneum were located in its superficial layers. Inversely, whatever the application time was, the concentrations of both steroids in the epidermis and dermis were significantly higher in normal skin than in scar skin with maximal difference between about 40 and 400 microns, the area of sebaceous gland localization. Cutaneous blood flow in full-thickness skin, assessed by the thallium-201 method, was globally identical in normal and in scar skin. In scar skin, at the level of papillary dermis, a decrease in blood flow due to the thicker viable epidermis and the flat dermoepidermal junction has been shown without implying an accumulation of drug in the epidermis and superficial dermis. Under these conditions, our results clearly demonstrate that the nutritional blood flow does not interfere with the percutaneous absorption of estradiol and progesterone in normal and scar skin. Thus, they confirm the significant contribution of hair follicles and sebaceous glands to drug penetration into the skin and subsequently the systemic circulation.
Disorders in which there is toxic buildup of circulating substrate may be treated by furnishing an enzyme reservoir capable of metabolically processing the excess substrate. The epidermal keratinocyte is a potential site for such a reservoir. In this study, we explore the capacity of genetically modified keratinocytes to metabolize extracellular substrate in a culture model that resembles in vivo epidermal architecture. Keratinocytes from adenosine deaminase (ADA)-deficient patients were transduced with a retroviral vector encoding the human ADA gene and the capacity of this tissue to deaminate deoxyadenosine (dAdo) in vitro was measured. The results show that at a substrate concentration of 10 microM, ADA-corrected keratinocytes deaminate dAdo at a rate of 0.38 nmol/min.10(6) cells. These results indicate that keratinocytes process extracellular substrate at rates that suggest complete substrate conversion in a single pass. This study provides a strong indication that the epidermis, the largest and most accessible tissue of the body, is a valuable site for designing clinically relevant gene therapies.
Many aspects related to the biology and the effective therapy of proliferative scars have remained undefined, in part due to a lack of an accurate and reproducible animal model with which to systematically study them. This report describes a new model for investigating the pathophysiology and manipulation of human proliferative scars.
Human proliferative scars (n = 86) were explanted into flaps based on isolated vascular pedicles in congenitally athymic rats. Serial analysis of the structural and functional integrity of the explanted scars was performed by microscopy and by measurement of human procollagen type III peptide (PIIIP) production, human factor VIII immunostaining, and in vitro cellular proliferation.
By these methods, both fibroblastic and epithelial components of explanted scar specimens retained the histologic characteristics of original human scar specimens, for up to 12 months. Over the same duration, scar explants continued to have high levels of human PIIIP, comparable to those found in original surgical specimens. The microvasculature of scar explants demonstrated a double basement membrane, with no staining of human factor VIII in the inner capillary endothelial layer, suggesting that host vessels were growing into ghost vessels of the human donor scar. Human factor VIII staining decreased over time. Fibroblasts cultured from explanted scar demonstrated less aggressive growth characteristics than those from original surgical specimens.
This new model is the first to allow such long-term maintenance and serial evaluation of human proliferative scar on an accessible, isolated vasculature. It may prove useful in further defining the biology and therapy of this widespread pathologic process.
Drug fraction transported from a topical formulation on skin to subsutaneous tissues or muscles is dependent on the physicochemical properties of the entrapped drug. Cutaneous disposition of model drugs, antipyrine(ANP), lidocaine (LC) and piroxicam (PXC) as well as flurbiprofen (FP) was thus evaluated in hairless rats in which an agar gel disc was subcutaneously inserted into abdominal region as a drug receptor and a drug donor cell was placed above it. Time courses of plasma level and agar gel amount were measured after topical application of 50% ANP, 3% LC, 1% PXC and 1% FP in hydroxypropylcellulose gel. Percutaneous absorption clearance of unionized from, CL*ab was proportional to true octanol/water distribution coefficient and the order of FP>PXC>LC>ANP, suggesting that permeation of the drug was determined mainly by its distribution from the formulation to the skin barrier. PXC, however, had a relatively low flux compared to the other three drugs, probably due to its high molecular weight and meltiong point. Migration clearance of unionized form from systemic cirsulation to the subcutaneous agar gel, CL*g was also influenced by the lipophilicity of drugs. On the other hand, fraction from the formulation to the systemic circulation was in the order of PXC>FP>ANP>LC.This fraction was much higher than the direct migration fraction from the formulation to the subcutaneous agar gel. Factors determining for these fractions are still unclear. A drug having a low lipophilicity and a low prorein binding, however, had a tendency to have a great targeting ability to the subcutaneous agar gel. In addition, most of the drug in the agar gel was contributed by the direct flow from formulation, not from the systemic circulation. The present in situ experimental method is a useful tool to evaluate skin disposition of drugs. Detailed understanding of the skin disposition of drugs from several formulations will enable the finding of a good drug and formulation candidates.
Cutaneous disposition of topically applied flurbiprofen (FP) was evaluated using a new in situ experimental model in hairless rats. A disk-shaped agar gel (3.85 cm in diameter and 0.5 cm in thickness) was subcutaneously implanted in the abdominal region of rats as a drug receptor, and a drug donor cell was subsequently placed above this agar gel. No significant pharmacokinetic modification of FP was observed as a result of this experimental procedure. A bolus injection and a constant intravenous infusion of FP were applied to the rats, followed by an analysis of FP levels in the plasma and agar gels. Using these results, the clearance rate of FP from the systemic circulation to the gel could be calculated. FP (1% gel formulation, 1.0 g/3.14 cm(2)) was then topically applied to the skin of these rats. From these experiments, the amount of FP that migrated from the formulation to the systemic circulation and the amount of FP that migrated directly to the agar gel across the skin, over 10 h, were separately evaluated to be 235.4 and 2.0 microg, respectively. Thus, most of the FP was absorbed into the systemic circulation. The effect of endogeneous vasoactive compounds and penetration enhancers on the FP disposition within skin was also determined. Epinephrine and bradykinin were used as vasoactive compounds that were entrapped in agar gel, and an isopropyl myristate system (IPM system) and a l-menthol-ethanol-glycerin-water system (MEGW system) were used as enhancers in the formulation. Epinephrine enhanced the direct delivery of FP into the agar gel to more than ten times its former level, in spite of the fact that it had no effect on systemic delivery. Bradykinin strengthened systemic delivery slightly, without changing the quantity of FP in the gel. IPM increased only the systemic delivery of FP, as was the case with bradykinin, whereas the MEGW system markedly increased both the blood concentration and the quantity of FP in the gel (13 and 200 times, respectively). This technique has proven to be an effective tool for the quantitative evaluation of cutaneous disposition of a topically applied drug.
Most of the drug fraction penetrating the skin after topical application is taken up by the cutaneous blood flow, although the rest directly migrates into deeper tissues such as the subcutis and muscle. A new in situ experimental hairless rat model was designed to distinguish these fractions of topically applied drugs. Flurbiprofen, a non-steroidal anti-inflammatory drug, was selected as the model drug. In this model, two agar gel discs were subcutaneously inserted into the abdominal region of hairless rats as a drug receptor, and a topical formulation containing the drug was placed above either side of the gel disc. Plasma and agar levels of flurbiprofen were followed every 2 h over 10 h. The migration fraction of the drug into the systemic circulation and that directly to subcutaneous tissues were calculated to be 99.8% and 0.2% against the total amount which penetrated the skin, and the drug ratios into agar gel from the systemic circulation and not from the systemic circulation (i.e. directly migrated from the formulation) were 16.0% and 84.0%, respectively, at 10 h. This in situ drug disposition profile in skin was similar to the in vivo profile calculated from the in vivo muscle amount of flurbiprofen using muscle clearance. These results clearly suggest that the present in situ experimental model is a valuable tool for easy analysis of the skin disposition of topically applied drugs.
This study determined and compared the percutaneous penetration and absorption of an organophosphorus (OP) pesticide, parathion (PA), using three experimental skin models: namely the human abdominal- and pig-ear skin in vitro models and the Human Skin grafted onto a nude mouse (HuSki) in vivo model. The percentage of topically applied dose absorbed and the doses present in the stratum corneum and skin were systematically determined at 24 h under similar experimental conditions. The three experimental skin models were first compared. Then, the advantages of the HuSki model for in vivo PA skin absorption studies were evaluated compared with the pig in vivo model previously used by others. Lastly, the relevance of each skin model to predict the permeability of human skin to PA in vivo was assessed by comparing our results with previously published in vivo human volunteer values. It was demonstrated that (a) pig-ear skin is relevant for predicting the in vitro human abdominal skin absorption taking into account a 2-3 times higher skin permeability to PA, (b) using ethanol as the vehicle, the absorption of PA was 4-5 times higher in the HuSki model than in the pig model but supports the usefulness of the HuSki model to easy mass balance studies, (c) both human in vitro and HuSki models closely predict the in vivo human volunteer absorption at 24 h when acetone is used as a vehicle but the HuSki model overcomes the known limitations of in vitro models for studying the fate of PA in the different skin layers after topical application.
IN 1953 an athymic hairless mutation was observed in a colony of outbred hooded rats maintained at the Rowett Research Institute. A breeding colony of mutant animals was maintained with difficulty until the early 1960s, when it died out. However, the recessive mutant gene (to which the symbol rnu for Rowett nude has been assigned) was apparently maintained at low gene frequency within the random bred colony. Homozygous mutants were recovered in 1975, and a breeding colony was again established, first in conventional, and later in germ-free conditions, where good breeding performance and survival were obtained. In February 1977 a breeding nucleus of these rats was established at the MRC Laboratory Animals Centre in conventional, germ-free and specific pathogen-free conditions. The observations and data reported here were obtained with small numbers of animals bred in clean, conventional conditions, and suggest the potential usefulness of nude rats in biomedical research.
This work describes an instrument for the noninvasive measurement of cutaneous blood flow velocity. The system utilizes the Doppler shift of laser light backscattered from moving red blood cells in the cutaneous microcirculation, the shift being obtained by an optical heterodyning technique. Comparison is made between this technique and the 133xenon clearance technique in measuring cutaneous flow in the forearms of normal volunteers. Variations in flow were obtained by inducing different degrees of solar erythema with an ultraviolet sunlamp. A Y on X linear regression yielded a regression coefficient = 0.89 (p less than 0.001, n = 16) between the two methods. The laser Doppler method appear to represent a practical technique for clinical evaluation of cutaneous blood flow in any skin surface.
A study of human skin grafts on congenitally athymic (nude) rats Description and inhibition of rejection of human skin split-thickness grafts by congenitally athymic (nude) rats. Submitted for publication Laser Doppler measurement of cutaneous blood flow
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Per-cutaneous absorption Skin on an isolated accessible vasculature as a model system to study skin pathophysiology Role of microcirculation in percutaneous absorption An experimental skin sandwich flap on an independent vascular supply for the study of percutaneous absorption and metabo-lism
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~CHEUPLEIN, R. J., AND BRONAUGH, R. L. (1983). Per-cutaneous absorption. In Biochemistry and Physiology oftheSkin (L. A. Goldsmith, Ed.), p. 1255-1295. Oxford Univ. Press, New York. WOJCIECHOWSKI, 2. J., HUETHER, S. E., LEONARD, L. G., AND KRUEGER, G. G. (1984). Skin on an isolated accessible vasculature as a model system to study skin pathophysiology. Ciin. Res. 32(2), 482A. WOJCIECHOWSKI, Z. J., BURTON, S. A., BTELENZ, T. J., AND KRUU;ER, G. G. (1985a). Role of microcirculation in percutaneous absorption. Clin. Res. 33(l), 2 1 A. WOJCIECHOWSKI, Z. J., PERSHING, L. K., HUETHER, S. E., LEONARD, L. G., BURTON, S. A., HIGUCHI, W. I., AND KRUEGER, G. G. (1985b). An experimental skin sandwich flap on an independent vascular supply for the study of percutaneous absorption and metabo-lism. Submitted for publication.
The nude mouse in the biology and pathology of skin
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