Age and skin structure and function, a quantitative approach (II): protein, glycosaminoglycan, water, and lipid content and structure

BACKGROUND/PURPOSE: The aging process has been studied with fervor recently, given our shifting demographics. As age's effects are so manifest in the skin's appearance, structure, mechanics, and barrier function, it is not surprising that much effort has been made in research to better understand them. Quantitative measurements permitted by bioengineering have allowed us to objectively and precisely study aging skin. These overviews piece together the immense amounts of information that have emerged from recent technological advances in dermatological research in order to develop a unified understanding of the quantitative effects of age on skin. METHODS: We performed a literature search on age-related changes in protein, glycosaminoglycan (GAG), water, and lipid content and structure, searching Pub-med, Em-Base, Science Citation Index, and the UCSF dermatological library's collection of books on the topic of aging skin. RESULTS: Collagen becomes sparser and less soluble in intrinsically aged skin, but is thickened and more soluble in extrinsically aged areas. Elastin is degraded slowly and accumulates damage with intrinsic aging; also, increased synthesis of abnormally structured elastin occurs in photoexposed areas. This leads to an age-related accumulation of aberrant elastoic material, clumped in the papillary dermis. Generally, age leads to increased folding and decreased interaction of proteins with water. Also, despite increased GAGs in aged skin, these are abnormally deposited on the elastoic material and cannot interact properly with water. Hence, in aged skin, water is found in the tetrahedron form, bound to itself rather than other molecules. Lipid content appears to decrease with age, although the proportion of different lipid classes seems to remain fairly constant. CONCLUSION: Much work remains to be carried out to reach a consensus on the effects of age on skin structure and function. Future studies would be benefited by increased standardization of skin sites tested, methodology, and increased sample sizes.     

Effects of ageing on dermal echogenicity

Background/aims: Changes in the dermis associated with ageing can be detected by high-frequency skin ultrasonography. In photoaged skin, this technique shows a subepidermal low echogenic band (SLEB) that is probably an ultrasound manifestation of elastosis and oedema in the papillary dermis. Since some authors found an association between age and SLEB thickness or its echogenicity on exposed sites, it has been proposed to use these parameters to quantify skin photoageing.
Methods: To determine whether SLEB can be used as a quantitative marker of ageing, its prevalence was determined on forearm skin in a group of 55 individuals (age 18–57 years). The size of SLEB has been measured by quantifying the number of low echogenic pixels in the subepidermal area, which is an accurate method for assessing SLEB severity.
Results: The prevalence of SLEB increased with age, but SLEB was also present in young subjects. The echogenicity of the subepidermal area did not show any age dependence. However, when a ratio of echogenicity between upper and lower dermis was calculated, a linear dependence on age was found.
Conclusions: This study indicates that skin echogenicity measured as a ratio between the upper and lower dermis may be used to objectively estimate photoageing.     

Magnetic resonance chemical shift microimaging of aging human skin in vivo: initial findings

Background/aims: In recent decades, interest has increased in the chronological and environmental factors governing the aging of skin. Various methods have been used for determining water and lipid content of human skin as a function of subject age. Magnetic resonance chemical shift imaging (CSI) offers a noninvasive technique for observing detailed distributions of water, lipids and other chemicals in the skin, and thus may be useful in dermatogerontology. Methods: Human skin was examined in vivo on nine healthy volunteers, both male and female. Localized ¹H spectra of the skin were obtained from voxels 78 μm thick and parallel to the skin surface. Unique water and lipid profiles were observed for different individuals, showing the composition and microstructure of epidermis, dermis and hypodermis in each subject. These allowed the quantification of skin thickness in vivo by the first appearance of triacylglyceride olefinic protons at the dermal-hy-podermal junction, or alternatively by the degree of lipid infiltration into dermis. Results: The relative concentration of free water in the skin, normalized to skin thickness, was observed to be slightly greater in older subjects and also in tanned subjects. More significantly, a microstructural feature common to every subject, i.e., the position of a possible capillary plexus in the dermis, showed migration toward the skin surface with advancing age. Conclusions: Such observations are consistent with previous studies of skin aging by other techniques and show promise for CSI in dermatogerontology as a non-invasive means for determination of skin water in vivo.     

Assessment of aging of the human skin by in vivo ultrasonic imaging

The ultrasonic imaging technique that we have developed provides cross-sectional images of human skin in vivo with a resolution of about 80 microns axially (i.e., deep into the skin) and 250 microns lateral (parallel to the surface). In order to study aging skin, we obtained ultrasonic images from the mid-forearm (volar and dorsal sides) of 142 women. Ultrasonically, on the images, the dermis appears composed of two bands: a dark superficial one where the ultrasonic waves are propagated in a relatively homogeneous or non-echogenic medium, and a deeper one, which is lighter in color, suggesting a heterogeneous medium. Our results show that skin is thicker on the dorsal than on the volar forearm. In contrast to previously published results, skin thickness remains constant until the seventh decade of life, diminishing thereafter. The relative thickness of the two bands show marked variations with age: a progressive thickening of the dark band, from zero in infants to approximately 75% of total skin thickness in aged subjects, while the light band shows the inverse trend. Comparing the amplitude of the bands on the volar and dorsal forearm, the relative thickness of the dark band is larger on the dorsal (exposed) side and increases with age. These findings and the analysis of variously stained biopsies taken in some of our patients lead us to assign this dark band to a zone in the upper dermis where the collagen network is delicate, dense, and well organized. This is supported by some data in the literature. The thickness of this subepidermal non-echo-genic band appears to be a far more sensitive marker of skin aging at the dermal level than is the measurement of skin thickness.     

Bioestimulación cutánea con plasma rico en plaquetas autólogo. Estudio controlado con ecografía

Introduction

It is well known that, due to the presence of growth factors, platelet-rich plasma (PRP), is able to produce histological changes in the dermis that reproduce the process of biological tissue repair without previous damage. Biostimulation with PRP has been used clinically for skin rejuvenation of the face and neck. Skin ultrasound can be helpful to evaluate several aspects of skin aging. This study was designed to perform an objective evaluation of the beneficial effects of this treatment on skin aging.

Material and methods

Autologus PRP was injected by mesotherapy in 10 women, aged between 45 and 60 years, in the face, neck and cleavage. Three-monthly sessions were carried out and the final results were measured 1 month after the final treatment. Pictures were taken and skin ultrasound was performed of the same area of the face before and after the treatment.

Results

All patients noted an improvement in their skin quality: the skin was brighter, more hydrated, more compact and plumper. An increase of dermal thickness and a decrease in subepidermic low echogenic band (SLEB) was observed on skin ultrasound. However, this improvement was not statistically significant, probably due to the small number of patients.

Conclusions

Biotestimulation with autologous PRP improved skin quality and reversed ultrasound signs of skin aging. More studies are required to confirm these results.     

Skin from various ethnic origins and aging: an in vivo cross-sectional multimodality imaging study

Background: Ethnic differences in skin structural features have not been thoroughly investigated, and the few reported studies are contradictory. Thus, we have carried out a set of in vivo measurements on the skin of about 400 volunteers from various ethnic origins living in the same environment.
Methods: Female subjects were distributed into four ethnic groups: African Americans, Mexicans, Caucasians, and Chinese. Inter- and intra-ethnic skin structural differences, according to age and anatomic site, were investigated using three non-invasive skin-imaging methods: ultrasound (US) at 25 and 150 MHz, and optical coherence tomography (OCT).
Results: The thickness of the skin is higher on the cheek compared with the dorsal and ventral forearm, with no ethnic or age-related specificity. We confirm that the sub-epidermal non-echogenic band is a sensitive marker of skin aging, and reveal for the first time that it is less pronounced in African Americans. From OCT images, we bring out evidence that the thickness of the dermal–epidermal junction (DEJ) decreased with age, and was higher in African Americans than in Caucasians. Finally, by comparing US images at 150 MHz with OCT images, we show that papillary dermis thickness can be measured and appears to be quite constant irrespective of age or ethnic group.
Conclusion: Our study confirms that skin imaging is very attractive to further our knowledge of the morphology of skin from various ethnic origins. Regarding age effects, quantitative parameters have shown that they would be delayed in African Americans compared with all other ethnic populations.     

Ultrasound biomicroscopy in the diagnosis of skin diseases

Ultrasound scanning is becoming an important diagnostic tool in dermatology. The major advantages of this technique are its non invasive non-ionizing nature and its relatively low cost. We aimed to evaluate the accuracy of ultrasound biomicroscopy (UBM) in the diagnosis of eight skin disorders namely, morphea, keloid, lichen planus, chronic eczema, psoriasis, port wine stain, seborrheic keratosis, and photo-aged skin, through correlation of its findings with clinical and pathological assessment. Fifty seven patients with the above diseases were examined by ultrasound biomicroscopy (UBM). Two areas, one of normal skin and the other from lesional skin, were examined for each patient. Skin biopsies were taken from the same lesion examined by UBM. In morphea, the dermal echogenicity was increased and the thickness of morphea plaques correlated significantly with disease severity. Keloids appeared as low echogenic images. In lichen planus and chronic eczema the dermis appeared as sound shadow. In psoriasis, an intermediate zone between the epidermis and dermis (B zone) was detected. Its thickness correlated significantly with the PASI score. Port wine stain lesions appeared hypoechoic. Seborrheic keratosis appeared as a sound shadow. In photo-aged skin a subepidermal low echogenic band (SLEB) was detected. We conclude that UBM is a non-invasive diagnostic tool in dermatology which can be used to give valuable information about disease progress and the effectiveness of therapy.     

Clinical,histologic, and ultrastructural changes after use of human growth factor and cytokine skin cream for the treatment of skin rejuvenation

The present study reports the clinical, histologic, and ultrastructural changes observed after the 6‐month application of a novel skin cream containing a mixture of human growth factors and cytokines. Participants were asked to apply the human growth factor and cytokine skin cream twice daily to their entire face over a period of 6 months. A 3‐mm punch biopsy was taken from the preauricular skin area before and after the treatment period. Further evaluations included photographic and clinical assessment of facial skin for wrinkles. After the treatment period, improved clinical appearance of periorbital and perioral wrinkles by 33% and 25% on average, respectively, was demonstrated. Histologic evaluation indicated moderate changes in the epidermal thickness as well as an increased fibroblast density in the superficial dermis at the end of the 6‐month treatment period. Ultrastructural changes consistent with new collagen formation were shown by electron microscopy. This study, together with earlier studies, corroborates that topical application of growth factors and cytokines are beneficial in reducing signs of facial skin aging.     

The Kollias legacy: Skin autofluorescence and beyond

In a paper published at the J Invest Dermatol in 1998 Nik Kollias and coworkers described distinct changes in skin native fluorescence associated with skin aging and photoaging, using in vivo fluorescence excitation spectroscopy. The assignment of the 295 nm band to tryptophan fluorescence had a profound significance influencing many later studies from multiple groups. The reproducible changes in skin native fluorescence suggested that aging causes predictable alterations in both the epidermis and the dermis, whereas chronic UV exposure induces the appearance of new fluorophores. This seminal, but insufficiently widely appreciated work deserves re‐examination as it points to important horizons in future experimental dermatology, such as cancer diagnostics, diabetes, wound healing, and understanding skin aging and photoaging mechanisms.     

An ultrasonographic monitoring of skin condition in patients receiving radiotherapy for head and neck cancers

Radiodermatitis is one of the commonest side effects of radiotherapy. They are usually assessed by semi‐quantitative clinical scores, which are not validated and may be subject to inter‐observer variability. A few previous studies suggested that high‐frequency ultrasonography (HF‐USG) is useful in the assessment of the acute phase of radiation dermatitis in breast cancer patients. (a) To monitor skin changes by HF‐USG during the course of radiotherapy due to head and neck cancers, and (b) to determine whether there is any connection between skin sonograms and the skin scoring criteria. This prospective, observational study includes patients diagnosed with head and neck cancers, treated with radiotherapy or concomitant chemoradiation. The final analysis includes six patients. In every patient, the HF‐USG as well as dermatological assessment (target lesion score—TLS and CACE v. 4.0) were performed 4×: before, in the middle, day after, and 3 months after radiotherapy. There were significant differences between non‐irradiated skin thickness and thickness of skin with clinically obvious radiodermatitis (TLS grade 1‐4; P < .0001), as well as between irradiated, unchanged skin thickness (TLS grade 0) and thickness of skin with clinically obvious radiodermatitis (TLS grade 1‐4; P = .0002). There was no significant difference between non‐irradiated and irradiated, unchanged skin thickness (TLS grade 0; P = .9318). In four patients, we demonstrated subepidermal low echogenic band (SLEB). HF‐USG can be useful tool to noninvasive and objective assessment of skin changes during radiotherapy.     

The Elastic Tissue of the Skin

In order to separate the changes of actinic damage from those of simple aging, we studied the elastic fibers in low and high sun-exposed skins of normal subjects at different ages. Low sun-exposed skin shows chronologic aging lesions only. These begin at age 30 with a disappearance of oxytalan fibers and with some abnormalities in the reticular and deep dermis; at age 40, aging changes are established: no oxytalan fibers, marked abnormalities, and lysis of elaunic and elastic fibers. In high sun-exposed skin, age-related lesions also occur but are associated with more or less precocious elastotic degeneration in reticular and deep dermis. Both types of aging fibers are revealed by the antielastin antibody HB 8, disappear with elastase, but resist collagenase. Actinic elastosis clearly originates from elastin. The two types of change differ in electron microscopic appearance: with spontaneous aging, elastic fibers are disintegrated (loose and porous fibers); in actinic damage, elastotic fibers are thicker and have accentuated microfibril dense masses. The age-associated lesions could be due to the activity of protease of fibroblastic origin whereas the elastotic degeneration is probably due to the actinic stimulation of fibroblasts.     

Cellulite and skin ageing: is there any interaction?

Objective  This study aimed to identify the characteristics of cellulite in women of different age and to appreciate whether cellulite could interfere with skin ageing or not.
Methods  94 healthy females, divided into three age groups (21–30yrs; 31–40yrs; 51–60yrs) and two grade groups of cellulite (grade 2; grade 0 or control group), were investigated using non invasive techniques. The "orange peel appearance" was quantified by measuring the shadowed surfaces under low angle light. The biomechanichal properties were measured (extensibility-retractability-elasticity). The thicknesses of the skin structures were also evaluated using ultrasound. Echogenicity of the dermis was recorded and dermis density determined in two bands (superficial and low dermis).
Results  In grade 2, the shadowed surfaces are significantly different according to age; i.e. smaller and more numerous after age of 30; the total skin thickness including hypodermis is increased of about 30% irrespective to age, compared to control group.
The biomechanical properties of the skin are significantly modified as age increases without any grade effect. In grade 2, retractability and elasticity parameters are altered from age 30 whilst only from age 50 in the control group. Echogenicities of the superficial and deep dermis also decrease from age 30 and become significantly lower than the ones of grade 0.
Conclusion  Population with cellulite presents earlier skin ageing characteristics than the control population. Two sub-populations may exist: the under 30 age with large dimpled surfaces, normal biomechanical and density properties; and the over 30 age with smaller and numerous dimpled surfaces and already altered dermis properties. This premature skin ageing should be prevented accordingly.     

Quantitative evaluation of chronological ageing and photoageing in vivo: studies on skin echogenicity and thickness

Skin ageing is divided into chronological ageing and photoageing due to the cumulative effects of solar ultraviolet radiation. It is, however, difficult to measure the degree of photoageing and chronological ageing in humans in vivo . Here, we have evaluated the usefulness of ultrasonography for measurement of chronological ageing and photoageing in vivo . Twenty megahertz ultrasonography was performed in 90 individuals (29 men, 61 women, age 18–94) to describe age-related changes in sun-exposed regions with different levels of sun exposure (dorsal and ventral forearm, forehead, ankle) and non-exposed buttock skin. Skin thickness and skin echogenicity in different layers of the dermis were measured in ultrasound images. Additionally, cutaneous photodamage was scored clinically. Age-related changes were dependent on body site as well as layer of the dermis. A progressive, age-related decrease in echogenicity of the upper dermis was found in sun-exposed regions (dorsal forearm, forehead), but not in moderately exposed regions (ventral forearm, ankle). In the buttock an increase in echogenicity was observed. The echogenicity of the lower dermis increased in all examined sites. Skin thickness increased with age in the forehead and buttock, but decreased in the extremity skin. Our findings show that photoageing causes a decrease in echogenicity in the upper dermis. In contrast, chronological ageing is associated with an increase in echogenicity in the lower dermis. Although both increases and decreases in skin thickness were observed in different anatomical regions, there was no general relationship between skin thickness and age. Dermal echogenicity was deemed valuable for in vivo study of chronological ageing and photoageing.     

Tobacco smoke causes premature skin aging

Smoking tobacco is the most preventable cause of morbidity and is responsible for more than three million deaths a year worldwide. In addition to a strong association with a number of systemic diseases, smoking is also associated with many dermatological conditions, including poor wound healing, premature skin aging, squamous cell carcinoma, melanoma, oral cancer, acne, psoriasis, and hair loss. This review focuses on the effects of smoking on premature skin aging. It has been long established that smoking has deleterious effects on skin. Epidemiological studies indicate that smoking is an important environmental factor in premature skin aging. In vitro studies indicate that tobacco smoke extract impairs the production of collagen and increases the production of tropoelastin and matrix metalloproteinases (MMP), which degrade matrix proteins, and also causes an abnormal production of elastosis material. Smoking increases MMP levels, which leads to the degradation of collagen, elastic fibers, and proteoglycans, suggesting an imbalance between biosynthesis and degradation in dermal connective tissue metabolism. Reactive oxygen species are also involved in tobacco smoke-induced premature skin aging. Scavengers of reactive oxygen species ameliorate the induction of MMP. Tobacco smoke extract also impacts dermal connective tissue in nude mice. Thus, in vitro and in vivo evidence indicates that smoking tobacco leads to accelerated aging of the skin. These findings might be useful to motivate those patients who are more concerned about their appearance than the potential internal damage associated with smoking to stop smoking.     

Histopathologic characteristics and ultrastructure of aging skin

Histologic and ultrastructural examination of skin biopsy specimens from younger compared to older persons documents age-related structural alteration in the epidermis, dermal-epidermal junction, dermis, and the epidermal appendages including hair follicles, sebaceous glands, and sweat ducts and glands. The fine, regular epidermal surface patterns change to coarser and less regular ridges with aging. Epidermal projections into the dermis are retracted and the dermal-epidermal junction is flattened. The dermis becomes thinner; there is less fibrous collagen and less elastic fiber in older skin, but the elastic component may appear increased compared to collagen. Elastic fibers may become frayed, porous, and matted together. The density of blood vessels is reduced and, in particular, there are fewer capillary loops in the papillary dermis. There are fewer as well as structurally altered hair follicles, sebaceous glands, and sweat glands with increasing age.     

Mapping the human face: biophysical properties

Background: Facial skin exhibits unique biophysical properties that are distinct from skin belonging to other areas of the body. Small to large regional differences in biophysical properties between facial sites are observed. Technological advances in dermatological research allow a quantitative study of the biophysical qualities of the face and its relation to skin elsewhere. However, comprehensive studies examining inter‐regional variations using each of the six standard biophysical parameters have been few. We summarize findings on the biophysical parameters used to explore the human face as well as regional differences in skin reactivity to chemical irritants. Methods: We performed a literature search using Pubmed, Embase, Science Citations Index, and the UCSF's dermatological library on biophysical parameters and skin physiology pertaining to the human face. Results: Distinct regional differences in transepidermal water loss (TEWL), capacitance, blood flow, sebum, pH, and temperature were demonstrated in facial skin. However, studies cannot be compared with each other because each uses different anatomical sites, skin conditions, and measurement techniques. Intraregional differences in TEWL, sebum, and temperatures were observed on the cheeks and appeared to follow characteristic distribution patterns. Higher blood flow levels and skin temperatures were generally observed in areas with dense networks of blood vessels such as the nose and perioral region. Areas such as the forehead, nose, and chin consistently showed higher sebum casual levels, but variability in sebum levels between sites was also observed. The susceptibility of the face to hexyl nicotinate, sodium lauryl sulfate, and benzoic acid differed depending on location and age. Conclusion: Establishing a standardized biophysical profile of the human face will help to improve therapeutics, and further our understanding of differences in chemical reactivity and disease distribution. Future research necessitates standardization of the anatomical sites studied, sample size, and experimental protocols.     

Heat shocks enhance procollagen type I and III expression in fibroblasts in ex vivo human skin

Background: The well‐known characteristics of aging skin are the development of fine lines and wrinkles, but changes in skin tone, skin texture, thickness and moisture content are also aspects of aging. Rejuvenation of the skin aims at reversing the signs of aging and can be established in the epidermis as well as in the dermis. Aged dermis, in fact, has a degenerated collagen matrix. To regenerate this matrix, fibroblasts need to be stimulated into synthesizing new collagen. Aims: In this study, the effects of heat shocks of different temperatures on human dermal fibroblasts in ex vivo skin on the expression of procollagen 1, procollagen 3, heat shock protein (hsp)27, hsp47, and hsp70 are investigated. Materials and methods: The heat shocks were applied on ex vivo skin samples by immersing the samples in heated phosphate‐buffered saline of 45 °C or 60 °C. Metabolic activity was measured and at similar time points propidium–iodide–calceine staining was performed to establish cell viability. Quantitative polymerase chain reaction (qPCR) was performed after the heat shock to determine gene expression levels relative to the reference temperature. Furthermore, PicroSirius Red and hematoxylin stainings were performed to visualize the collagen network and the cells. Results: The skin samples were shown to be viable and metabolically active. Histology indicated that the heat shocks did not influence the structure of the collagen network or cell appearance. qPCR results showed that in contrast to the 45 °C heat shock the 60 °C heat shock resulted in significant upregulations of procollagen type I and III, hsp70 and hsp47. Conclusion: A 60 °C, heat shock stimulates the human dermal fibroblasts in ex vivo skin to upregulate their procollagen type I and type III expression.     

Sonography of the skin at 100 MHz enables in vivo visualization of stratum corneum and viable epidermis in palmar skin and psoriatic plaques

A main drawback of 20-25 MHz ultrasound units for skin imaging is their limited resolution. We used a transducer with a center frequency of 95 MHz and a resolution of 8.5 microm axially and 27 microm laterally - an almost 10-fold increase compared with 20 MHz. By means of a new scanning technology we reached a depth of field of 3.2 mm. We examined normal palmar skin, normal glabrous skin on the abdomen, the upper back, the calf and the dorsal forearm, and 35 lesions of psoriasis vulgaris. From 11 psoriatic plaques biopsies were taken for correlation with the sonograms. In normal palmar skin, the horny layer is represented as an echopoor band below the skin entry echo, traversed by echorich coils, which correspond to eccrine sweat gland ducts. The thickness of this band significantly increases after occlusive application of petrolatum. Its lower border is defined by an echorich line, representing the stratum corneum/stratum Malpighii-interface. Underneath, a second echopoor band is visible, which corresponds to the viable epidermis plus the papillary dermis, bordered by the scattered echo reflexes of the reticular dermis. This band is also visible in glabrous skin; however, the stratum corneum cannot be detected. In psoriatic lesions, the thickened horny layer appears echorich; after application of petrolatum, its echodensity decreases. Below, the acanthotic epidermis plus the dermis with the inflammatory infiltrate are represented as an echopoor band. There is an excellent correlation between the sonometric thickness of this band and the histometric thickness of the acanthosis plus the infiltrated dermis. Our results show that 100 MHz sonography is a valuable tool for in vivo examination of the upper skin layers.     

Control of androgen receptor expression in human keratinocytes and in a reconstituted human epidermis model with selective antisense oligonucleotides

Clinical evidence of correlations between menopause and endogenous skin aging gave input to various studies investigating the relevance of estrogens for skin functions that are associated with skin aging and their possible therapeutic effects. Skin thickness and bone density are significantly decreased already six months after menopause and are increased after the same period of hormone replacement (HRT). Fibroblast and keratinocyte function is stimulated by estrogen application, and among other effects significant increases of collagen fibres have been demonstrated six months after the onset of HRT ( 1 ). Topical use of estrogen compounds was found to diminish skin aging symptoms. The effects of conjugated estrogens (0,625% Premarin) were studied in 60 postmenopausal women ( 2 ). In another study the effects of estradiol 0,01% and estriol 0,3% were compared ( 3 ). Both studies documented significant reductions of wrinkles without any systemic side effects of the treatments. Recently significant increases of epidermal thickness during estradiol have been described ( 4 ). For cosmetic purposes phytoestrogens seem a promising alternative to the medical treatment. Isoflavone containing cosmetical creams were shown to improve skin dryness and wrinkles ( 5 ). In various studies mainly beneficial effects of systemic HRT on skin aging parameters have been documented. Although skin aging is certainly no indication for systemic hormone supplementation the beneficial action of such treatment on aging symptoms of the skin are a positive side aspect of such treatment.