Evaluation of elastin fibres in young and aged eyelids and abdominal skin using computational 3D structural analysis.

Background: Aging-related degeneration of elastic fibres causes skin wrinkles and loss of elasticity. A correlation has been reported between dermal elastic fibre degradation and wrinkles. However, the mechanism of wrinkle formation is complex and unclear. To establish methods for treating wrinkles, it is necessary to understand the aging-related morphological alterations underlying elastin fibre degradation or disappearance. Objectives: To image and analyse aging-related three-dimensional (3D) morphological alterations of elastic fibres in the eyelid and abdominal skin. Methods: Excised human eyelid and abdominal skin tissues were examined. The structure of elastic fibres in the skin tissues was examined via nuclear, tropoelastin and fibrillin-1 immunostaining. Then, 3D imaging was performed using a confocal laser microscope and tissue decolourization technology. Images were analysed using a computational method. Results: The decolourization technology made it possible to image elastin fibres in 3D, and we devised a method for analyzing the elastin fibre structure using computational methods. It was quantitatively shown that the eyelid skin has a more complex fibrous structure than the abdomen, and the fibres became curved, shortened and thickened with age. Conclusions: We provide a novel 3D analysis method for elastin fibres and report age-related alterations in elastin fibre structure in the human eyelid and abdominal skin. This method contributes to the understanding of elastin fibre degeneration in more detail than conventional methods. Applying this 3D analysis method to skin tissues will contribute to a better understanding of age-related changes in fibres and to the development of novel wrinkle treatments.

Enolase-1 expression in the stratum corneum is elevated with parakeratosis of atopic dermatitis and disrupts the cellular tight junction barrier in keratinocytes.

OBJECTIVE: Previous studies have shown that enolase-1 (ENO1) in the stratum corneum (SC) is more highly expressed in patients with atopic dermatitis (AD) than in healthy individuals, suggesting that it is a novel biomarker for evaluating skin condition in patients with AD. However, the mechanism underlying high ENO1 expression in the SC and its pathological relevance in AD are unclear. In this study, the relationship between ENO1 expression and keratinization of epidermis was investigated, and the role of high ENO1 expression in keratinocytes was characterized. METHODS: ENO1 expression and morphological characteristics were examined in SC from the cheeks of 24 patients with AD. Additionally, the localization of ENO1 in the excised human epidermis was observed. Moreover, to analyse the role of ENO1 in cellular barrier function, tight junction proteins (TJs) and transepithelial electrical resistance (TEER) in keratinocytes with ENO1 overexpression were evaluated. Furthermore, the localization of ENO1 and plasminogen in keratinocytes was evaluated by immunostaining, and the cellular barrier function in keratinocytes was examined after treatment with tranexamic acid (TXA). RESULTS: ENO1 expression was substantially correlated with the rate of nucleated corneocytes in AD. In addition, ENO1 localized in the basal to spinous layers, but was its expression dramatically decreased in healthy human SC. ENO1 overexpression in human epidermal keratinocytes reduced the expression of TJs (claudin-4, E-cadherin, tricellulin, and occludin) and TEER, and treatment with anti-ENO1 IgG reversed these effects. ENO1 colocalized with plasminogen in keratinocytes. Treatment with TXA rescued the ENO1-induced reductions in TJ and TEER expression. CONCLUSION: We found a substantial correlation between ENO1 expression and the rate of nucleated corneocytes in AD and decreased ENO1 expression with nuclear disappearance. These results suggest that high ENO1 expression in the SC of AD is caused by deficient keratinization, which is an AD characteristic. Moreover, ENO1 overexpression in keratinocytes promoted dysfunction of TJ dynamics, leading to reduced integrity of the cellular barrier, and these effects might be mediated by plasmin activity. We propose that ENO1 is a useful indicator of parakeratosis and might have a potential role in cellular TJ barrier function in the epidermis.

Galectin-7 in the stratum corneum: a biomarker of the skin barrier function.

OBJECTIVE: Skin barrier disruption often occurs in diseased and damaged skin conditions such as atopic dermatitis (AD). We focused the galectin-7 protein (Gal-7) as a biomarker of skin condition and assessed whether the content of Gal-7 in stratum corneum (scGal-7) could be used as an indicator of skin barrier disruption and as an index of local skin symptoms in AD patients. METHODS: Alteration of Gal-7 expression levels in keratinocyte and scGal-7 contents after barrier disruption by sodium dodecyl sulphate were evaluated in vitro and in vivo, respectively. Correlation between scGal-7 content and transepidermal water loss (TEWL) was examined in 126 healthy subjects. We performed single measurements of scGal-7 contents in 34 AD patients and serial measurements of 15 inpatients among them. SC samples were collected by the tape-stripping method, and scGal-7 content was determined using enzyme-linked immunosorbent assay. RESULTS: Gal-7 expression in keratinocytes increased after barrier disruption. The scGal-7 content reflected the disruption of the skin barrier. The scGal-7 contents and TEWL values correlated in healthy subjects. The scGal-7 level was higher in AD patients than in healthy subjects. The scGal-7 contents in the cheek and neck of AD patients significantly correlated with the total and local skin lesion severity scores. Serial measurements in the inpatients showed that the scGal-7 contents in the cheek and neck decreased in tandem with local severity scores in response to treatment. CONCLUSION: Measurement of scGal-7 content in tape-stripped samples was useful for the evaluation of the skin barrier function in dry skin conditions such as AD.

Effects of methyl paraben on skin keratinocytes.

Some ingredients of dermatological formulations result in skin irritation and allergy. In particular, preservatives have been reported extensively as a cause of allergic contact dermatitis. The study focused on parabens which have been used extensively as antimicrobial preservatives in foods, drugs and cosmetics. The aim of this study was to clarify the effects of the daily use of methyl paraben (MP) on human skin. The concentrations of MP in the stratum corneum (SC) of the human forearm were measured using the cup method and GC-MS after daily applications of MP containing formulations. The study also investigated the effects of long-term exposure to MP on keratinocytes in vitro. Normal human keratinocytes and the skin equivalents were cultured in the medium containing MP. The following changes were analysed: proliferating ability, apoptotic cells, morphological changes, mRNA and protein expressions. After 1 month of daily applications of MP containing formulations, MP remained unmetabolized and persisted slightly in the SC. MP decreased the proliferating ability of keratinocytes and changed the cell morphology. MP also decreased the expressions of hyaluronan synthase 1 and 2 mRNAs and type IV collagen. In contrast, it increased the expressions of involucrin and HSP27. Furthermore, MP influenced the epidermal differentiation of the skin equivalent. These results suggest that MP exposure through application of dermatological formulations results in MP persistence and accumulation in the SC, and that MP might influence the aging and differentiation of keratinocytes.

Influence of transferrin on manganese uptake in rat brain.

To evaluate the influence of transferrin (Tf) on manganese (Mn) uptake in the brain, pH 8.6 buffer-treated (54)MnCl(2), which has a higher affinity for Tf than untreated (54)MnCl(2), and Tf-bound (54)Mn were prepared. When pH 8.6 buffer-treated (54)MnCl(2) and untreated (54)MnCl(2) were incubated with apo-Tf in Tris (2-amino-2-hydroxymethylpropane-1,3-diol)-HCl buffer, the percentage of the total (54)MnCl(2) bound to Tf was approximately 85% and 10%, respectively. One hour after intravenous (iv) injection of pH 8.6 buffer-treated (54)MnCl(2) and untreated (54)MnCl(2), both tracers were concentrated similarly in the choroid plexus in the ventricles and distributed in other brain regions. Six days after iv injection, both pH 8.6 buffer-treated (54)MnCl(2) and untreated (54)MnCl(2) tracers were concentrated in the superior olivary complex, inferior colliculi, and red nuclei, although the former radioactivity was lower than the latter. Moreover, Tf-bound (54)Mn was prepared and injected iv into rats. The radioactivity from Tf-bound (54)Mn, which was also concentrated in the same regions, e.g., the superior olivary complex, was the lowest of all three traces. Tf-bound (54)Mn was stable during incubation with serum for 1 hr. It is likely that more Mn is transported into the brain when Mn is not bound to Tf. When Tf-bound (54)Mn and (54)MnCl(2) were unilaterally injected into the lateral ventricle, radioactivity was distributed only around the ipsilateral ventricle in the Tf-bound (54)Mn group 7 days after injection, whereas it was distributed more extensively in the (54)MnCl(2) group. It is likely that Tf-bound Mn in the CSF is less readily transported into the brain parenchymal cells than the non-Tf-bound form. These results suggest that Mn is transported into the brain efficiently via a Tf-independent uptake system.

Manganese uptake into rat brain during development and aging.

Manganese (Mn) is an essential metal and plays an important role in the brain. To evaluate Mn uptake into the brain during development and aging, 54Mn concentrations in the brain of rats aged from 5 days to 95 weeks were measured after injection of 54MnCl2. 54Mn concentration in the brain of 5-day-old rats was the highest of all age groups tested. The liver and blood of 5-day-old rats also showed the highest 54Mn concentrations among the age groups. These results suggest that Mn is required in a high amount during infancy and that a sufficient Mn supply is critical for normal brain development. The high uptake of Mn into the brain of neonatal rats may be due to high levels of Mn in the blood, which may be supplied from the liver. In the 5-day-old brain, 54Mn was relatively concentrated in the hippocampal CA3 and dentate gyrus and the pons. In the aging brain, 54Mn was relatively concentrated in the inferior colliculi, olivary nuclei and red nuclei.

In vivo stimulation-induced release of manganese in rat amygdala.

The role of manganese ions in neural functions is poorly understood because of the low level of manganese in the brain. An excess of this ion is associated with neurological disorders such as extrapyramidal symptoms. We demonstrated that manganese may be taken up by piriform neurons (tertiary olfactory neurons) after release from the terminals of secondary olfactory neurons, in which 54Mn taken up by the soma may be anterogradely transported [A. Takeda, Y. Kodama, S. Ishiwatari, S. Okada, Manganese transport in the neural circuit of Rat CNS, Brain Res. Bull. (45) (1998) 149-152]. Here we demonstrate for the first time that 54Mn previously taken up into the amygdala is released with neurotransmitters into the extracellular space during stimulation with high K+. The results suggest that the role of manganese ions in the amygdala, and probably in the olfactory system, is dynamically linked to neural signalling processes.

Manganese transport in the neural circuit of rat CNS.

To study manganese (Mn) transport in the neural circuit of rat CNS, brain isotope distribution after 54Mn injection into the brain was analyzed by autoradiography. One day after 54MnCl2 injection into the striatum, 54Mn was highly distributed in the ipsilateral thalamus, hypothalamus, and substantia nigra. When 54MnCl2 was bilaterally injected into the striata after unilateral treatment with colchicine or vehicle into the medial forebrain bundle, 54Mn was distributed in both sides of the substantia nigra of vehicle-treated rats. On the other hand, unilateral colchicine treatment caused a decrease of 54Mn distribution in the ipsilateral substantia nigra, suggesting that Mn is subjected to axonal transport in the striatonigra and/or nigrostriatal pathways. In the case of unilateral injection of 54MnCl2 into the olfactory bulb, 54Mn was distributed in the ipsilateral piriform, amygdaloid areas (the primary olfactory cortex), and entorhinal area (the secondary olfactory cortex). These results suggest that Mn is subject to widespread axonal transport in the neural circuits. Moreover, Mn may be taken up by the piriform neurons (the third olfactory neuron) after release from the secondary olfactory neuron terminals and transported to the entorhinal area.

A possible role of "steroidogenic factor" in the corticoidogenic response to ACTH; effect of ACTH, cycloheximide and aminoglutethimide on the content of cholesterol in the outer and inner mitochondrial membrane of rat adrenal cortex.

The role of a "steriodogenic factor" in the corticoidogenic response to adrenocorticotropic hormone (ACTH) was studied in this experiment. ACTH caused an accumulation of cholesterol within the adrenocortical mitochondria in rat after pretreatment with either cycloheximide (CH) or aminoglutethimide (AG). The cholesterol distribution in these cholesterol-rich mitochondria was examined by measuring the cholesterol concentrations in the outer and inner mitochondrial membranes. In the case of pretreatment with CH, cholesterol was accumulated in the outer membrane and not in the inner membrane. In contrast, in the case of pretreatment with AG, cholesterol was accumulated in the inner membrane and not in the outer membrane. It could be concluded from the presented data that the function of a "steroidogenic factor" in the corticoidogenic response to ACTH might take place at the step of cholesterol translocation from the outer mitochondrial membrane to the inner membrane.

A possible regulatory role of Ca++-calmodulin system in cellular cholesterol ester hydrolysis in the steroidogenic response to ACTH in bovine adrenocortical cells.

In order to corroborate the regulatory role of Ca++-calmodulin system in the steroidogenic response to adrenocorticotropic hormone (ACTH), the effects of calmodulin inhibitors (chlorpromazine, trifluoperazine, and W-7) on cortisol production and cellular cholesterol ester hydrolysis induced by ACTH in bovine adrenocortical cells were examined. Three calmodulin inhibitors diminished not only the cholesterol ester hydrolysis and cortisol production induced by ACTH in the presence of Ca++, but also inhibited the Ca++-induced hydrolysis and cortisol production in the absence of ACTH. Neither cortisol production in crude mitochondrial fraction nor the ACTH-induced Ca++-influx was affected by chlorpromazine. These results indicate that Ca++f-calmodulin system plays a significant regulatory role in the supply of free cholesterol to the adrenal mitochondria in the steroidogenic response to ACTH.