Langerhans cell granules in porcine epidermis.
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Animals↗
Biomedical subjects
N A Monteiro-Riviere
Publications and source records attributed to N A Monteiro-Riviere.
Effects of organic solvent vehicles on the viability and morphology of isolated perfused porcine skin.
Although many organic solvents are known to be cutaneous irritants, they are commonly utilized as vehicles in percutaneous absorption and toxicity studies. The isolated perfused porcine skin flap (IPPSF) is an alternative animal model that has been used to study percutaneous absorption and cutaneous toxicity. The purpose of this study was to evaluate the effect of five organic solvents (ethanol, acetone, dimethyl sulfoxide (DMSO), toluene, and cyclohexane) on biochemical viability parameters, vascular response, and epidermal morphology of the IPPSF. Cumulative glucose utilization (CGU), the ratio of lactate production/glucose utilization (L/CGU ratio), and the leakage of lactate dehydrogenase (LDH) were used as biochemical indicators of alterations in glucose metabolism and flap viability. Only ethanol resulted in a statistically significant decrease in the average rate of CGU over the perfusion period. All of the solvent treatments resulted in slight increases in LDH release versus the controls. Vascular resistance (VR) was measured to examine the response of the cutaneous vasculature to these solvents, and most treatments resulted in a decreased VR in the terminal phases of perfusion. Ethanol was the only solvent to cause an apparent increase in terminal VR. Light microscopy demonstrated a moderate increase in intracellular edema in the DMSO, toluene, and acetone flaps. Ultrastructural evaluation showed focal blebbing of the nuclear envelope and vesiculation of the rough endoplasmic reticulum in cells of the stratum basale and stratum spinosum layers with DMSO treatment. The IPPSF allowed the evaluation of subtle biochemical, vascular, and morphological changes associated with non-occlusive topical exposure to these organic solvents. These findings support the necessity of documenting vehicle effects which might mask or otherwise alter subtle, but potentially important, compound-specific responses.
Acetone↗
Laser Doppler measurements of cutaneous blood flow in ageing mice and rats.
Laser Doppler velocimetry (LDV) is a non-invasive technique that measures capillary blood perfusion parameters (blood flow, volume and velocity) and can be used non-invasively to evaluate the effects of ageing on cutaneous blood perfusion. Male Fischer-344 rats (R) of 1, 2, 3, 8, 12, 16, 20 and 24 months and C57BL/6N mice (M) 1, 2, 3, 9, 15, 19 and 22 months (n = 6/mo) were used. Animals were anesthetized with ketamine/xylazine and LDV-measured blood flow was made on the backs of all animals. Five readings for each parameter were recorded in order to obtain a mean value of the individual. Skin biopsies (4 mm) were removed after LDV for assessing epidermal and dermal thickness and number of epidermal cell layers. The number of viable epidermal layers in M remained constant while in R it decreased with age. Epidermal thickness in both M and R decreased from 2-3 months and then remained constant. Dermal thickness decreased from 3 to 22 months in M, and increased in R from 1 to 2 months and remained constant. Blood flow of M increased between 1 and 2 months, remained constant to 19 months, then increased at 22 months; flow in R was constant except at 2 months. Thus age differences in epidermal and dermal thickness, and blood flow of M and R occur and should be considered when evaluating cutaneous toxicity studies in different-aged animals. These changes may potentially alter dermal absorption and/or distribution of xenobiotics.
Aging↗
The isolated perfused porcine skin flap as an in vitro model for percutaneous absorption and cutaneous toxicology.
The isolated perfused porcine skin flap (IPPSF) is a new perfused skin model which allows in vitro cutaneous pharmacology and toxicology studies to be conducted in a viable skin preparation which has a normal anatomical structure and a functional microcirculation. The purpose of this review is to (1) outline the background of this field which indicated the need for this type of model; (2) outline the surgical procedures needed to create and harvest viable preparations; (3) overview the criteria (biochemical, physiological, and histological) used to assess viability during an experiment; (4) present results of percutaneous absorption, cutaneous metabolism, transdermal delivery (passive and active), and skin distribution experiments conducted to date; (5) present the strategy developed to quantitate percutaneous absorption and cutaneous drug distribution using compartmental and physiological-based pharmacokinetic models; (6) assess the correlation of IPPSF data to in vivo results; (7) define the biochemical, physiological and histological (LM, TEM, enzyme histochemistry) response of the IPPSF to topically applied cutaneous vesicants; (8) overview where this type of in vitro model fits into the overall framework of cutaneous toxicology and pharmacology research; and (9) outline possible paths for future development. This review should provide the reader with an appreciation of some unique problems in this field which require an in vitro model that is closely integrated in structure and function to the in vivo setting.
Animals↗
Cutaneous laser-Doppler velocimetry in nine animal species.
The assessment of cutaneous microcirculation by laser-Doppler velocimetry (LDV) has been primarily limited to human studies. The purpose of this investigation was to establish normal values in various species and anatomic sites for blood flow, velocity, and volume as determined by LDV. Microcirculation was measured with a laser-Doppler velocimeter in 54 animals, 6 healthy animals from each of 9 species. The standard sites used were the buttocks, convex surface of the ear, metacarpal pad, humeroscapular junction, thoracolumbar junction, ventral portion of the abdomen, dorsal metacarpus (hooved animals), and ventral surface of the tail (horse). Significant differences in blood flow, velocity, and volume were measured between species and sites within species. The ventral portion of the abdomen consistently had the highest relative blood flow across all species except the monkey. Measurements in the canine metacarpal pad had a high SD, possibly indicating the stratum corneum and epidermis to be too thick for LDV. Our findings provide baseline data in several species, with application of LDV in comparative dermatologic research.
Animals↗
Development of surgical techniques for preparation of in vitro-isolated perfused porcine skin flaps for percutaneous absorption studies.
We developed a single-pedicle, axial pattern tubed skin flap that could be transferred to an in vitro perfusion apparatus. On the basis of results of prosections, angiography, contact radiography, and surviving-length studies, it was concluded that a single-pedicle, axial pattern skin flap measuring 4 cm x 12 cm incorporating the caudal superficial epigastric artery would survive to its entire length. Subsequently, a surgical (stage 1) procedure was developed for the routine preparation of single-pedicle, axial pattern tubed skin flaps. Healing after the stage-1 procedure was evaluated by visual inspection and fluorescein angiography. Stage-1 procedures were performed successfully 149 of 160 (93%) times. A second surgical (stage 2) procedure was developed for routine cannulation of the caudal superficial epigastric artery and harvest of the tubed skin flap. Stage-2 procedures were performed successfully 136 of 144 (94%) times.
Animals↗
Differential down-regulation of epidermal protein kinase C by 12-O-tetradecanoylphorbol-13-acetate and diacylglycerol: association with epidermal hyperplasia and tumor promotion.
A single topical application of 2 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) to CD-1 mouse skin resulted in a rapid decrease in cytosolic, particulate, and total epidermal protein kinase C (PKC) activity at 6 h, which remained decreased by 70% at 96 h. This dose of TPA produced epidermal hyperplasia as determined by an increase in the number of nucleated epidermal cell layers. A single application of 10 mumol sn-1,2-didecanoylglycerol, a model sn-1,2-diacylglycerol and complete tumor promoter, induced ornithine decarboxylase to an extent similar to that of 2 nmol TPA. However, sn-1,2-didecanoylglycerol produced an 80% increase in particulate PKC activity that was accompanied by a 45% decrease in cytosolic PKC activity, resulting in no net change in total PKC activity. Unlike TPA, this dose of sn-1,2-didecanoylglycerol did not produce a hyperplastic response. Additional dosing regimens were examined to determine whether the down-regulation of particulate PKC activity was associated with hyperplasia and tumor promotion. A tumor-promoting dosing regimen consisting of multiple applications of 5 or 10 mumol sn-1,2-didecanoylglycerol twice daily for 1 week resulted in more than a 60% decrease in cytosolic and particulate PKC activity and a marked epidermal hyperplasia. Twice-weekly application of 10 mumol sn-1,2-didecanoylglycerol, a nonpromoting dosing rate, for 1 week decreased cytosolic PKC activity but increased particulate PKC activity and did not produce hyperplasia. Dosing regimens utilizing multiple applications of TPA decreased both particulate and cytosolic PKC activity and were also hyperplastic. PKC activity was also measured in epidermal papillomas from mice initiated with 7,12-dimethylbenz[a]- anthracene and promoted with either sn-1,2-didecanoylglycerol or TPA. Cytosolic- and particulate-associated PKC activity in these papillomas was decreased by at least 70% and 40%, respectively, when compared with epidermis and whole skin. After 2 months without promoter treatment, both cytosolic and particulate PKC activity remained decreased in the papillomas, whereas epidermal PKC activity returned to control values by 2 to 3 weeks following cessation of several weeks of TPA treatment. Collectively, these data demonstrate that the down-regulation of epidermal PKC is associated with and may be a permissive event for epidermal hyperplasia and tumor promotion.
Acetone↗
Cutaneous toxicity of 2-chloroethyl methyl sulfide in isolated perfused porcine skin.
Previous research has shown the isolated perfused porcine skin flap (IPPSF) to be a novel in vitro experimental model for investigating xenobiotic percutaneous absorption. In this study, the IPPSF was used to biochemically and morphologically assess the dermatotoxicity of 2-chloroethyl methyl sulfide (CEMS), a monofunctional analog of the vesicant, sulfur mustard. IPPSFs were perfused in a recirculating perfusion system and were treated with 97% CEMS (n = 4) or served as controls (n = 4). Additional IPPSFs were perfused in a nonrecirculating perfusion system and were treated with CEMS (n = 4) or were controls (n = 4). After dosing, each IPPSF was perfused for 8 hr. Cumulative glucose utilization (GU) and lactate production/glucose utilization ratio (L/GU ratio) were used as viability parameters. The average rate of GU for CEMS was significantly lower than control (p less than 0.05) in the recirculating and nonrecirculating IPPSFs. The L/GU ratio for CEMS was not significantly different (p greater than 0.05) from control for either perfusion system. CEMS resulted in a marked increase in vascular resistance versus control in both perfusion systems. Gross vesicles and bullae formation occurred in six of the CEMS-treated IPPSFs. Light microscopy revealed subepidermal vesicle formation above the basement membrane and extensive basal cell pyknosis in all IPPSFs treated with CEMS. No macroscopic or microscopic lesions were noted in the control flaps. Transmission electron microscopy revealed separation between the lamina lucida and the lamina densa of the basal lamina, with intracellular vacuolization and mitochondrial swelling occurring in the stratum basale and stratum spinosum cells of IPPSFs treated with CEMS. These lesions are similar to those described after human exposure to sulfur mustard. Full characterization of the morphological and biochemical changes seen after topical exposure of the IPPSF to vesicants may shed light on the pathogenesis of cutaneous toxicity of these compounds in vivo and serve as a relevant model to assess protective strategies against vesicant exposure.
Animals↗
Interspecies and interregional analysis of the comparative histologic thickness and laser Doppler blood flow measurements at five cutaneous sites in nine species.
Studies in dermatology, cutaneous pharmacology, and toxicology utilize skin from different animal species and body sites. However, regional differences exist in topical chemical percutaneous absorption studies in man and in animal. The objective of this study was to compare epidermal thickness and number of cell layers across species and body sites using both formalin-fixed paraffin and frozen sections. Cutaneous blood flow determined by laser Doppler velocimetry (LDV) was compared to histologic data. Six animals of each of the following species were used: monkeys, pigs, dogs, cats, cows, horses, rabbits, rats, and mice. Cutaneous blood flow was determined and 6-mm skin biopsies were taken directly from the following sites: buttocks, ear, humeroscapular joint, thoracolumbar junction, and abdominal area. When the two histologic methods were compared across all species and body sites, the thickness of the epidermis was significantly greater, and the thickness of the stratum corneum significantly less, in paraffin sections versus frozen sections (p less than 0.05). There were no differences in the number of viable cell layers determined by both methods. The values for LDV-determined blood flow did not significantly correlate (p greater than 0.05) to epidermal or stratum corneum thickness. However, regional and species differences were noted in all these parameters. In conclusion, these data indicate that thickness and LDV blood flow are independent and must be evaluated separately when comparisons are made between species and body sites. This work provides a data base for future comparative studies in which a knowledge of skin thickness or blood flow might be important variables.
Animals↗
Altered epidermal morphology secondary to lidocaine iontophoresis: in vivo and in vitro studies in porcine skin.
Iontophoresis is the process of delivering ionic drugs across the skin using electric current. Iontophoresis of lidocaine hydrochloride in 30 pigs in vivo and in 112 in vitro isolated perfused porcine skin flap (IPPSF) preparations produced a drug-specific alteration in the epidermis greater than or equal to 10 min after dosing. By light microscopy, this change was characterized by the appearance of flattened dark basophilic staining nuclei oriented parallel to the stratum corneum in the stratum granulosum and spinosum layers. In severe cases, this alteration extended into the deeper usually vacuolated stratum basale. The stratum corneum appeared normal. This unique morphological alteration showed an abrupt change from the stratum basale to stratum granulosum. An immune-mediated etiology can be ruled out since this alteration is observed both in vivo and in vitro. The severity of this change, graded on a scale of 0-3 (no change to severe), was best correlated to total transcutaneous lidocaine flux as estimated in IPPSF studies and to flux as estimated by current (mA-hr) in vivo. Electron microscopic changes following iontophoresis showed specific alterations in the tonofilaments of the epidermal cells. The tonofilaments appeared unrecognizable and resembled an amorphous matrix. In pigs followed through 10 days to study the resolution of this alteration, the epidermis reverted to normal within 6 days with no additional manifestations. In conclusion, lidocaine iontophoresis can induce in swine a unique dose-dependent non-immune-mediated epidermal alteration which is expected to have minimal toxicological significance.
Administration, Cutaneous↗
In vitro penetration of pesticides through human newborn foreskin.
The in vitro dermal penetration of 14C-labelled parathion, fenvalerate, carbofuran, and lindane through fresh full-thickness human newborn foreskin was determined at 1, 6, 24, and 48 h. The pesticides were applied to a constant dosing area (0.031 cm2), and a fixed dose (1.18 microgram), for each of the compounds studied. 90%, or greater, of the labelled pesticides were recovered in all cases. Carbofuran showed the greatest mean penetration of 82% followed by parathion and lindane with mean penetrations of 79 and 66%, respectively. Fenvalerate exhibited a mean penetration of 9% which is significantly lower than that of the other three compounds. No difference was noted in the penetration of pesticides through human skin from blacks and whites.
Epidermis↗
Comparison of the effect of sn-1,2-didecanoylglycerol and 12-O-tetradecanoylphorbol-13-acetate on cutaneous morphology, inflammation and tumor promotion in CD-1 mice.
Since sn-1,2-didecanoylglycerol mimics 12-O-tetradecanoylphorbol-13-acetate (TPA) by inducing ornithine decarboxylase activity and stimulating DNA synthesis in mouse epidermis [Smart, R.C., Huang, M.-T. and Conney, A.H. Carcinogenesis, 7, 1865 (1986)], we have investigated morphological changes induced by TPA and sn-1,2-didecanoylglycerol in the epidermis and we have also examined sn-1,2-didecanoylglycerol as a possible complete tumor promoter. It was determined that topical application of 2.5 or 10 mumol of sn-1,2-didecanoylglycerol induced epidermal ornithine decarboxylase activity to about the same extent as the application of 1 or 2 nmol of TPA respectively. Therefore, these doses of TPA and sn-1,2-didecanoylglycerol were used in most of our studies. Single or multiple application (2 X/week for 4 weeks) of 1, 2 or 5 nmol of TPA to the skin of CD-1 mice produced a dose-dependent increase in the number of epidermal non-cornified cell layers, epidermal thickness, leukocyte infiltration and intracellular edema. In contrast, neither single nor multiple application (2 X/week for 4 weeks) of 2.5 or 10 mumol sn-1,2-didecanoylglycerol produced any of these responses. However, when 5 mumol sn-1,2-didecanoylglycerol was applied topically twice a day (10 mumol/day) for 5 days there was a significant increase in the number of epidermal non-cornified cell layers and epidermal thickness. The effects of TPA and sn-1,2-didecanoylglycerol were compared using the mouse ear inflammation model. Application of TPA caused edema, but sn-1,2-didecanoylglycerol had little or no effect. sn-1,2-Didecanoylglycerol was then evaluated as a complete tumor promoter utilizing the mouse skin two-stage model. CD-1 mice were initiated with 200 nmol 7,12-dimethylbenz[a]anthracene and then treated with 1 nmol TPA or 2.5 mumol sn-1,2-didecanoylglycerol twice a week for 28 weeks. A 28 weeks, 28% of the mice treated with TPA had developed tumors, while none of the mice treated with 2.5 mumol sn-1,2-didecanoylglycerol developed tumors. The data indicate that topical application of 2.5 mumol sn-1,2-didecanoylglycerol induced ornithine decarboxylase activity to the same extent as a tumor-promoting dose of 1 nmol TPA, but it did not cause morphological changes in the epidermis when applied once or when applied twice a week for 4 weeks and did not function as a complete tumor promoter when applied twice a week for 28 weeks.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
A method for preparing mouse skin for assessing in vitro dermal penetration of xenobiotics.
A method is described for preparing mouse skin for assessment of in vitro penetration of dermally applied compounds. Separation of the epidermis from the dermis was attempted using a dermatome, heat, trypsin and collagenase. When mouse skin was incubated in a collagenase solution and separated using water, the hypodermis and part of the dermis were separated from the epidermis while leaving the hair follicles and hair shafts intact. Morphologically, the skins prepared for in vitro use appeared to offer similar barriers to topically applied compounds as those found in vivo.
Administration, Topical↗
On the definition of viability in isolated perfused skin preparations.
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Animals↗
The isolated perfused porcine skin flap (IPPSF). I. A novel in vitro model for percutaneous absorption and cutaneous toxicology studies.
This article describes the development of a novel in vitro alternative animal model for dermatology and cutaneous toxicology. A single-pedicle, axial-pattern, island-tubed skin flap was created in crossbred Yorkshire weanling pigs in one surgical procedure, then transferred 2 or 6 days later to a computer-controlled temperature-regulated perfusion chamber for 10-to 12-hr studies. Perfusate consisted of Krebs-Ringer bicarbonate buffer (pH 7.4) containing albumin and glucose. Viability was assessed by glucose utilization, lactate production, an absence of significant concentrations of the intracellular enzyme lactate dehydrogenase in the perfusate, and light and electron microscopy. A mean lactate to glucose ratio of 1.6 for flaps harvested 2 days after surgery and 1.8 for flaps taken 6 days after surgery suggested primarily anaerobic glycolysis. This preparation would be a humane alternative animal model for studies in cutaneous toxicology, physiology, oncology, and percutaneous drug absorption and metabolism.
Animals↗
Ultrastructural evaluation of acute nasal toxicity in the rat respiratory epithelium in response to formaldehyde gas.
Ultrastructural lesions were induced by formaldehyde (HCHO) gas in the rat nasal respiratory epithelium. Male F-344 rats, 7-9 weeks old, were exposed to 0.5 or 2 ppm (6 hr/day) for 1 or 4 days and to 6 ppm (6 hr/day) of HCHO for 1 day and sacrificed immediately or 18 hr after 1, 2, or 4 days of exposure. Other groups were exposed to 15 ppm (6 hr/day) of HCHO for 1 and 2 days. Ultrastructural changes to 0.5 or 2 ppm were limited to altered cilia with wing-like projections occasionally seen on the tips of the ciliary shafts. Autophagic vacuoles were present in some of the basal cells while neutrophils were seen in the basal and suprabasal layers after 1 day exposure to 6 ppm of formaldehyde. Hypertrophy of goblet and ciliated cells were noted in animals exposed to 6 ppm of formaldehyde and sacrificed 18 hours after 1, 2, or 4 days of exposure. Some nonciliated cells formed apical blebs containing an abundance of SER. Ciliated-mucous cells were observed after 2 and 4 days of exposure to 6 ppm of formaldehyde. Nonkeratinized squamous cells containing microfilaments were seen as early as 4 days after exposure to 6 ppm and at 1 and 2 days after exposure to 15 ppm. Loss of microvilli in ciliated cells occurred in all exposure levels. At 15 ppm for 1 and 2 days, nucleolar segregation was observed in basal and cuboidal cells and internalized cilia were noted. These results demonstrate that short-term exposure to 6 or 15 ppm of HCHO caused respiratory epithelial injury which was not cell specific, but was dose related in severity.
Animals↗
Dose-response studies of gentamicin nephrotoxicity in rats with experimental renal dysfunction. II. Polyvinyl alcohol glomerulopathy.
Animal studies involving concurrent pathophysiologic states, including experimental renal dysfunction, are useful for a proper understanding of the mechanisms of gentamicin nephrotoxicity and acute renal failure. This study examined gentamicin nephrotoxicity in a model of glomerular dysfunction in rats. Administration of medium molecular weight polyvinyl alcohol (PVA) produced a glomerulopathy, with characteristic accumulation of macromolecular PVA in the glomerular mesangium without altering glomerular filtration or causing proteinuria. Subsequent daily doses of gentamicin ranging from 0 to 120 mg/kg elicited a dose-response nephrotoxicity in both control and PVA-pretreated rats after 6 or 12 days of drug. Based on statistical analysis of renal clearances of creatinine, urea, sodium, and potassium; serum creatinine and urea nitrogen; urinary N-acetyl-beta-D-glucosaminidase excretion (6 days only); in vitro renal cortical transport of tetraethylammonium (TEA) (6 days); and quantified light-microscopic data (12 days), PVA induced an early (6 days) sensitivity to gentamicin nephrotoxicity. By 12 days, there were no differences in the responses of control and PVA rats to gentamicin. Single-dose gentamicin clearance, volume of distribution, and half-life were not altered by PVA and renal concentrations at 6 days were generally lower in these rats. Results of TEA transport studies tend to rule out PVA-induced metabolic lesions in the proximal tubular epithelium as the mechanism for the early sensitivity. This investigation demonstrates altered gentamicin nephrotoxicity in rats with an otherwise benign glomerulopathy and, combined with similar conclusions from a related study in subtotally nephrectomized rats, presents further evidence that the underlying pathophysiologic state of the kidney is an important factor in the renal response to nephrotoxins.
Animals↗
Ultrastructure of the integument of the domestic pig (Sus scrofa) from one through fourteen weeks of age.
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Aging↗