Investigation into the dosage of dietary sphingomyelin concentrate in relation to the improvement of epidermal function in hairless mice.

We previously found that dietary sphingomyelin (SPM) concentrate from bovine milk improved epidermal function. In this study, we investigated the dosage of dietary SPM concentrate from bovine milk in relation to the improvement of epidermal function. Thirteen-week-old hairless male mice were separated into four experimental groups, each fed one of four types of experimental diet: the control group, the low SPM group, the medium SPM group and the high SPM group. The mice were each fed the experimental diet for 6 weeks. The stratum corneum hydration and the transepidermal water loss (TEWL) were measured using a Corneometer and a Tewameter at 3 weeks and 6 weeks. After the feeding period, ceramides in the stratum corneum were analyzed. We found that the stratum corneum hydration in all the SPM groups was significantly higher than that in the control group, whereas TEWL in all the SPM groups was significantly lower than that in the control group. Ceramides increased significantly in mice fed the medium SPM diet and statistically tended to increase in mice fed the high SPM diet. Our results indicate that a daily intake of 17 mg SPM concentrate is enough to improve epidermal function in hairless mice.

Functional barrel cortex in 'hairless', nude mice.

Although nude mice are not truly hairless, they demonstrate abnormal hair structure and growth patterns, which are related to their genetic state. Whereas wild-type mice are born with visible vibrissae, nude mice are distinguishable at birth by the lack of visible vibrissae, which do not appear until approximately postnatal day 6. Additionally, adult nude mice have abnormal whisker cycling patterns in which structurally normal whisker follicles produce fragile whiskers which break or fallout leaving follicles whiskerless for several days before a fine replacement whisker appears and develops. The current study shows that despite these abnormal periods of whisker deprivation, the barrel cortex of nude mice develops a normal structural appearance viewed with cytochrome oxidase staining. Additionally, intrinsic optical imaging studies of barrel cortex responses to single whisker stimulation do not appear altered from normal despite periodic loss of adjacent whiskers.

The thymus of the hairless rhino-j (hr/rh-j) mice.

The hairless (hr) gene is expressed in a large number of tissues, primarily the skin, and a mutation in the hr gene is responsible for the typical cutaneous phenotype of hairless mice. Mutant hr mouse strains show immune defects involving especially T cells and macrophages, as well as an age-related immunodeficiency and an accelerated atrophy of the thymus. These data suggest that the hr mutation causes a defect of this organ, although hr transcripts have not been detected in fetal or adult mice thymus. The present study analyses the thymus of young (3 mo) and adult (9 mo) homozygous hr-rh-j mice (a strain of hairless mice) by means of structural techniques and immunohistochemistry to selectively identify thymic epithelial cells, dendritic cells, and macrophages. There were structural alterations in the thymus of both young and adult rh-rh-j mice, which were more severe in older animals. These alterations consisted of relative cortical atrophy, enlargement of blood vessels, proliferation of perivascular connective tissue, and the appearance of cysts. hr-rh-j mice also showed a decrease in the number of epithelial and dendritic cells, and macrophages. Taken together, present results strongly suggest degeneration and accelerated age-dependent regression of the thymus in hr-rh-j mice, which could explain at least in part the immune defects reported in hairless mouse strains.

Delayed induction of pigmented spots on UVB-irradiated hairless mice.

Human skin exposed to solar radiation for a long time subsequently develops pigmented spots, which are named solar lentigines. Since no animal model of this process is currently available, we attempted to induce similar spots in pigmented hairless mice. The mice were irradiated at 38 or 94 mJ/cm(2) three times/week for various periods of time (1-8 weeks) under an ultraviolet light source (Toshiba FL-SE; UVB). Skin pigmentation of irradiated mice was visually observed and skin color was determined with a colorimeter for 78 weeks. Uniform pigmentation was induced, but persisted only during exposure, disappearing completely within 2 weeks after cessation of exposure. At about 28 weeks after the first exposure, pigmented spots suddenly began to appear. These pigmented spots were less than 2 mm in diameter and light brown in color. The length of the latent period until appearance and the extent of development of these spots were dependent on the exposure period. Histological examination revealed increased numbers of active melanocytes and melanin granules in the affected epidermis. These pigmented spots closely resemble solar lentigines in humans, and the mice should be useful as an animal model of solar lentigines.

Epidermal change can alter mechanical properties of hairless mouse skin topically treated with 1alpha, 25-dihydroxyvitamin D(3).

Wrinkle formation caused by photoaging clearly involves changes of extracellular matrix components and mechanical properties of the skin. Recently, it was reported that the topical application of 1alpha, 25-dihydroxyvitamin D(3) to hairless mouse skin induced wrinkling. Here we have evaluated the effect of topical application of 1alpha, 25-dihydroxyvitamin D(3), which causes skin wrinkling, on the mechanical properties of the hairless mouse (HR/ICR) skin, using a commercially available non-invasive in vivo instrument. The elasticity element of the skin was unchanged, but the viscosity element significantly increased. Histologically, the epidermis became remarkably thick, but no conspicuous changes were observed in the dermis. Changes in the mechanical properties of the skin after 1alpha, 25-dihydroxyvitamin D(3) treatment take place through epidermal physical variation, especially changes of viscosity elements. It is suggested that the visco-elastic properties of the epidermis are also attributable to the morphology as well as the mechanical properties of the skin.

Skin microvascular adaptations during maturation and aging of hairless mice.

Using intravital fluorescence microscopy in the ears of hairless mice, we determined skin microvascular adaptations during the process of aging from juvenile to adult and senescent life (6-78 wk). Despite an increase of ear area within the first 36 wk, the number and branching pattern of both arteriolar and venular microvessels remained constant during the whole life period. Both arterioles and venules exhibited an increase in length, diameter, and intervascular distance up to the age of 36 wk. With the increase of the size of the ears, the observation that cutaneous capillary density remained unchanged implied new capillary formation. During aging to 78 wk, capillary density in the ears was reduced to approximately 40%. Functional analysis revealed an appropriate hyperemic response to a 2-min period of ischemia during late juvenile and adult life, which, however, was markedly reduced during senescence. Thus, except for capillaries, there is no indication for age-related new vessel formation. The process of aging from adult to senescent life does not cause any significant remodeling but is associated with a decrease of nutritive perfusion and a functional impairment to respond to stimuli such as ischemia.

Changes in the cerebellar cortex of hairless Rhino-J mice (hr-rh-j).

A mutation in the hr gene is responsible for typical epithelium phenotype in hairless mice. As this gene is expressed at high levels not only in the skin but also in the brain, the aim of the study was to clarify its role in the central nervous system. We have analyzed by morphological and immunocytochemical methods (calbindin D-28k, phosphorylated and 200 kDa neurofilament protein) the cerebellum of a mutated mouse strain, the hairless (hr-rh-j) type carrying the homozygous hr gene rhino mutation. The cerebellar cortex was studied in young (3 months) and adult (9 months) wild type and mutated mice. No major structural change was found in any of the groups and neuronal density or neuronal arrangement were similar in mutated animals to their age-matched controls. Nevertheless there were changes in shape and size of the Purkinje neurons in the old mutated animals respect to their normal littermates, while the molecular and the granule cell layers were apparently invariable. Calbindin (CB) immunohistochemistry revealed a significant decrease in the expression of this protein in the Purkinje cells of the aged mutated mice. Immunohistochemistry for a neurofilament protein (NFP) showed a reduction of staining in all the cerebellar cortex layers in the older animals, which was much more evident in the (hr-rh-j) mutated mice. These results suggest that hr gene is involved in the structural maintenance of the mature cerebellar cortex, rather than in the development. Our findings may also be consistent with an accelerated aging of the central nervous system in rh-rh-j mice.

Molecular and functional aspects of the hairless (hr) gene in laboratory rodents and humans.

For many years, hairless and rhino mouse mutants have provided a useful and extensively exploited model for studying different aspects of skin physiology, including skin aging, pharmacokinetic evaluation of drug activity and cutaneous absorption, skin carcinogenesis, and skin toxicology. Interestingly, however, hairless and rhino mice have rarely been studied for their primary cellular defect - hairlessness - and thus, the hairless gene itself and its physiological functions have been largely overlooked for decades. The recent identification of the human homolog of the hairless gene on human Chromosome 8p12 confirmed the clinical significance of the phenomenon of "hairlessness" in humans, which was predicted on the basis of similarities between hairless mice and a congenital hair disorder characterized by atrichia with papules. Mutations in the hairless gene of mice provide instructive models for further studies of hr gene function, and may facilitate insights into the pathophysiology of different human disorders associated with the disruption of hr gene activity. We provide an overview of current data on the structure and expression patterns of the hr gene, and of mutations at the hairless locus in mice and humans, including the genetic basis of different alleles, the pathology of hairlessness, reproductive and immunological defects, and susceptibility to dioxin toxicity. On the basis of our current understanding of hairlessness, we speculate on the putative functions of the hr gene product in skin physiology, and particularly, in hair follicle biology.

Chronic UVA (365-nm) irradiation induced scratching in hairless mice: dose-time dependency and the effect of ketanserin.

In a study on the dose-response relationship for longwave UVA (UVA1; 340-400 nm) carcinogenesis in hairless mice scratch marks appeared after months of daily exposure as an unwanted side effect. Tumor induction in the highest of the 4 tested dose groups (receiving a daily dose of 430 kJ/m2 of 365-nm radiation) could not be determined because extensive scarification occurred prior to the development of any tumors. The induction of scratch marks could be scored and quantified in all 4 dose groups tested. The UVA1 dose-dependencies for the induction of tumors and scratch marks were compared. We found that the induction of scratch marks depended mainly on the cumulative UVA1 exposure, whereas tumor induction showed a lesser dose-dependency. An attempt was made to prevent the apparent pruritogenic effect of UVA1 irradiation and to understand its mechanism. The influence of ketanserin, a serotonin/histamine antagonist, on the UVA1 induction of scratch marks was tested in groups of 8 mice daily irradiated with 430 kJ/m2. No difference was found between treated and untreated animals. Histological examination of skin biopsies from irradiated mice from the 430-kJ/m2 dose group from the UVA1 carcinogenic experiment, showed no changes in numbers of mast cells or other inflammatory features when compared to skin biopsies from unirradiated control mice. This indicated that UVA1-induced scratching is not mediated through mast cell release of serotonin and/or histamine. An adequate therapeutic treatment which can prevent UVA1-induced scratching would enable us to test tumor induction with UVA1 over a larger dose range, and may provide additional insight in how this radiation damages the skin. It remains conjectural whether there exists an analogous UVA-induced pruritus in human skin.

Cold-induced changes in brown adipose tissue thermogenic capacity of immunocompetent and immunodeficient hairless mice.

Mild cold acclimation (22 degrees C, 3 weeks) of hairless mice was shown to increase 5-fold the brown adipose tissue uncoupling protein content in immunodeficient BALB/c nu/nu mice, but by only 2.3-fold in immunocompetent BFU mice. The difference in activation of brown adipose tissue thermogenic capacity was due to a 2-fold increase in the content of brown adipose tissue in nu/nu mice only, which was paralleled by an increase in brown adipose tissue protein but not DNA content. Likewise, only in nu/nu mice the cold acclimation increased the reaction of natural killer cells in blood and peritoneal exudate with a shift from spleen to lymph nodes and increased the phagocytic index. The results indicate that the immune system may influence the defence against cold at the level of brown adipose tissue thermogenesis.

Effects of systemic indomethacin, meclizine, and BW755C on chronic ultraviolet B-induced effects in hairless mouse skin.

Chronic exposure of hairless mice to ultraviolet B (UVB) radiation is associated with inflammation as well as an altered macromolecular composition of the dermis. This study was designed to determine whether or not various systemic anti-inflammatory agents inhibit chronic UVB-induced changes in the macromolecular content of the dermis and, if so, whether each agent had the same or different effects. The agents and doses were chosen for their ability to inhibit the changes induced by a single exposure to UVB radiation (increased vasopermeability, neutrophil accumulation, and skin-fold thickness). Indomethacin, a cyclooxygenase inhibitor, and meclizine, an H1 histamine receptor antagonist, were administered from slow-release pellets. BW755C, a combined cyclooxygenase and lipoxygenase inhibitor, was administered intraperitoneally 30 min prior to UVB exposure. Animals were exposed to UVB three times per week for 20-26 weeks or were unirradiated. The elastin, glycosaminoglycan and collagen content of the skin were determined by measuring the desmosine, uronic acid, and hydroxyproline levels, respectively. The amount of each macromolecule per area of skin increased after chronic UVB exposure. The increase in desmosine was inhibited by indomethacin; the increase in hydroxyproline was inhibited by meclizine and BW755C. None of the agents inhibited the uronic acid increase. These results suggest that chronic inflammation contributes to the dermal changes seen in chronically UVB-exposed skin and that different inflammatory mediators are involved in the increases observed in elastin, glycosaminoglycans, and collagen.

Age and site variability of skin blood perfusion in the hairless mouse ear determined by laser Doppler flowmetry.

Laser Doppler Flowmetry (LDF) was used to characterize skin blood perfusion variations present within the Homozygous Hairless mouse ear microvasculature as a function of 1) arteriolar level within the microvasculature, 2) animal age and 3) LDF probe distance above the tissue being studied. To this end blood perfusion measurements (arbitrary perfusion units) were made in the ear at vascular sites in the vicinity of primary vessels (site A), secondary vessels (site B), tertiary vessels (site C), and peripheral zones distant from larger vessels. These were done without any invasive procedures save anesthesia administration and were carried out at three different tissue-probe distances (0.5, 0.75 and 1.0 mm) on three groups of eight mice each with ages of three, five and eight weeks. Results show a significant gradient in perfusion from sites A to D in each age group and a significant increase in perfusion with increasing age. The effect of probe-tissue distance variations on perfusion values was found to be significant between 0.5 mm and 1.0 mm but overall differences were small. In addition to these specific findings the present results demonstrate the potential of LDF as a means of detecting, evaluating and correlating perfusion changes, either by itself or as a complementary method to detailed microvascular measurements using conventional in vivo microscopy.

Effects of chronic exposure ultraviolet-A including 2% ultraviolet-B on free radical reduction systems in hairless mice.

Chronic ultraviolet (UV) irradiation is known to cause a variety of changes in the skin, including wrinkles, pigmented spots and carcinogenesis. To explore time dependent changes in several parameters with chronic UV irradiation, we examined the molecular changes in connective tissue, intracellular defence enzymes and free radical antioxidant substances in hairless mice skin caused by chronic exposure to UV-A including 2% UV-B. Connective tissue changes were estimated using hydroxyproline and isodesmosine assays as a measure of collagen and elastin concentrations, respectively. After 6 weeks irradiation, the insoluble collagen and elastin were both substantially elevated, as were the activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Continued UV irradiation resulted in a steady decline in SOD and lipid soluble antioxidants, while the GSH-Px remained elevated, suggesting that SOD and lipid soluble antioxidants in the skin may be involved in protecting it from UV damage and deteriorate with chronic irradiation.

The hairless mouse ear for in vivo studies of skin microcirculation.

The homozygous (hr/hr) hairless mouse ear was introduced in 1980 by Eriksson and coworkers as a model for in vivo studies of the skin microcirculation. Herein we expand on this work, presenting results of in vivo microvascular parameter measurements and morphologic studies in the intact ear. The in vivo measurements include microvascular diameter, RBC velocity, capillary density, and the frequency and amplitude of arteriolar vasomotion. In connection with the in vivo studies, a detailed anatomic description of the overall and vascular anatomy is given. Additionally, the preparation techniques for carrying out these in vivo and morphologic studies in the mouse ear are presented in detail.

Pretreatment with long-wave ultraviolet light inhibits ultraviolet-induced skin tumor development in hairless mice.

The carcinogenic effects of long-wave ultraviolet radiation (UV-A) (320 to 400 nm) irradiation followed by exposure to broad-spectrum ultraviolet (UV) irradiation were studied in 200 lightly pigmented, hairless, hr/hr C3H/Tif mice. No skin tumors were observed in the group irradiated with UV-A for four weeks (total dose, 4050 kJ/m2, observed for 57 weeks). Ultraviolet exposure induced skin tumors in a dose-dependent manner. In a group exposed to UV irradiation for 13 weeks, 35% of the mice had developed tumors after 57 weeks. Twenty-six weeks of exposure resulted in 88% of the animals being affected. In contrast it was found that treatment with UV-A irradiation (four weeks, total dose up to 4200 kJ/m2) preceding exposure to UV irradiation (13 or 26 weeks) resulted in a significantly delayed tumor development. Exposure with UV-A induced no visible changes of the skin, and subsequent microscopic examination revealed no measurable changes in epidermal thickness or melanin content. Our results suggest that, depending on the exposure schedule, UV-A in addition to previously reported carcinogenic properties also may act as an antitumor agent.

Effect of essential fatty acid deficiency on cutaneous sterol synthesis.

The fact that the skin is a major site of total body sterologenesis, coupled both with the apparent absence of low density lipoprotein receptors on keratinocytes and with the lack of influence of serum cholesterol on epidermal sterologenesis, has created the impression that epidermal lipid synthesis might be autonomous, i.e., nonregulatable. Recent studies have shown, however, that disruption of cutaneous barrier function with acetone or detergents stimulates epidermal sterologenesis (J Lipid Res 26:418-427, 1985). To correlate further sterologenesis with barrier function, we measured de novo synthesis of cholesterol and total nonsaponifiable lipids in essential fatty acid-deficient (EFAD) hairless mice. Animals with defective barrier function, manifested by abnormal transepidermal water loss, demonstrated a 2-fold increase in epidermal cholesterol and total nonsaponifiable lipid synthesis over controls while synthesis in the dermis was unchanged. Epidermal sterologenesis in EFAD animals, repleted with linoleic acid either systematically or topically, returned toward normal as barrier function improved. Moreover, plastic occlusion of EFAD mouse skin normalized epidermal sterologenesis at 1 and 3 days. These results provide further evidence that epidermal sterologenesis is not entirely autonomous, and can be regulated by water barrier requirements.

[The effects of fur on the thermal regulation of mice (Mus musculus)].

In order to ascertain the thermal insulation function of fur in mice, food and water intakes and rectal and skin temperatures were observed in Jcl: ICR mice and a group of the same species with their fur clipped, as well as BALB/C nu/+ and nu/nu mice. Food and water intakes increased as the ambient temperature dropped. The rectal temperature remained almost unchanged at ambient temperatures of from 18 to 30 degrees C. Skin temperatures were highest at side of the abdomen, lower at the neck, and lowest at the head. There was a tendency for skin temperatures to increase as the ambient temperature rose. The skin temperatures of mice with fur were higher than those of mice without fur. The thermal insulation of fur was 0.25, 0.14 and -0.06 clo at 18, 22, 26 and 30 degrees C in ICR mice, and 0.36, 0.01, 0.01 and -0.03 clo at 18, 22, 26 and 30 degrees C in BALB/C mice, respectively. These results confirm that heat loss from the skin at low temperatures could be prevented by the presence of fur. It also appeared that the hairless mice mutant had a lower metabolic rate than the animals with their fur clipped.

Oxygen consumption, activity and body fat in normal and hairless mice.

The oxygen consumption spontaneous activity of A2G (hr/+), A2G (hr/hr) and NMRI mice in groups of 2, 3, or 5 were measured, and body fat content was also determined. Average rates of oxygen consumption were found to be lowest in the A2G (hr/+) and highest in the A2G (hr/hr) mice, and conversely for the proportion of total body fat. There was no difference in activity of A2G (hr/+) and A2G (hr/hr), but the NMRI mice were more active.