Inhibition of ultraviolet-B-induced wrinkle formation by an elastase-inhibiting herbal extract: implication for the mechanism underlying elastase-associated wrinkles

BACKGROUND: Previously, we have demonstrated that fibroblast-derived elastase plays an essential role in the increased three-dimensional tortuosity of elastic fibers, contributing to the loss of skin elasticity in UV-B-exposed skin. This decrease in skin elasticity is closely associated with the formation of wrinkles induced by UV exposure. OBJECTIVE: To further clarify the role of elastase in the formation of wrinkles induced by UV exposure, we assessed the effects of an extract of Zingiber officinale (L.) Rose (which inhibits fibroblast-derived elastase) on the wrinkle formation induced by chronic UV-B irradiation. RESULTS: Topical application of an extract of Zingiber officinale (L.) Rose to rat or hairless mouse skin significantly inhibited the wrinkle formation induced by chronic UV-B irradiation at a suberythemal dose, which was accompanied by a significant prevention of the decrease in skin elasticity in both types of animal skin. In the rat hind limb skin, consistent with the inhibition of reduced skin elasticity, wrinkle prevention occurred concomitantly with a significant decrease in the curling and three-dimensional tortuosity of dermal elastic fibers. CONCLUSION: Our results indicate that herbal extracts with an ability to inhibit fibroblast-derived elastase may prove to be effective as anti-wrinkling agents, confirming the important role of elastase in UV-B-induced wrinkle formation.     

Selective inhibition of skin fibroblast elastase elicits a concentration-dependent prevention of ultraviolet B-induced wrinkle formation

We previously reported that wrinkle formation in the skin following long-term ultraviolet B irradiation is accompanied by decreases in skin elasticity and the curling of elastic fibers in the dermis. We further showed that wrinkles could be repaired by treatment with retinoic acid and that this was concomitant with the recovery of skin elasticity ascribed to the repair of damaged elastic fibers. Those studies suggested that decreasing the tortuosity of dermal elastic fibers is an important factor involved in inhibiting or repairing wrinkle formation. Therefore, it is of particular interest to determine whether the inhibition of elastase activity in vivo would prevent the damage of dermal elastic fibers and might abolish wrinkle formation associated with the loss of skin elasticity. Because the major elastase in the skin under noninflammatory conditions is skin fibroblast elastase, we used a specific inhibitor of that enzyme to assess its biologic role in wrinkle formation. The hind limb skins of Sprague-Dawley rats were irradiated with ultraviolet B at a suberythemal dose three times a week for 6 wk. During that period, 0.1-10.0 mM N-phenetylphosphonyl-leucyl-tryptophane, an inhibitor of skin fibroblast elastase, was applied topically five times a week. N-phenetylphosphonyl-leucyl-tryptophane application at concentrations of 0.1-1.0 mM abolished wrinkle formation in a concentration-dependent manner, with a peak for inhibition at 1.0 mM. This inhibition was accompanied by a continued low tortuosity of dermal elastic fibers and a maintenance of skin elasticity. Measurement of elastase activity after 6 wk of ultraviolet B irradiation demonstrated that whereas phosphoramidon-sensitive elastase activity was significantly enhanced in the ultraviolet B-exposed skin, there was no significant increase in that activity in the ultraviolet B-exposed, N-phenetylphosphonyl-leucyl-tryptophane-treated skin. These findings suggest that skin fibroblast elastase plays an essential part in the degeneration and/or tortuosity of elastic fibers induced by cumulative ultraviolet B irradiation.     

Epidermal changes caused by chronic low-dose UV irradiation induce wrinkle formation in hairless mouse

To investigate the effects of chronic low-dose UV irradiation on the skin, hairless mice were irradiated with a 1/3 minimal erythemal dose (MED) of UV. We examined the relationship between visible changes and skin damage in the dermis and epidermis. Hairless mice were irradiated with UVB (20 mJ/cm2) and UVA (14 J/cm2) three times a week for 10 weeks, followed by a 24-week non-irradiation period. Visible fine wrinkling was present after 4 weeks of irradiation, and the wrinkling progressively worsened throughout the period of irradiation. The wrinkles remained after irradiation was discontinued. In dermal components, no significant histological changes in the collagen fibers and elastic fibers were found, and the amount of hydroxyproline was also not changed. Thus, in the epidermis, there was a significant increase in the number of stratum corneum layers and the terminal-differentiation marker, filaggrin, positive cells. The intensity of staining for the differentiation marker, keratin 1, was reduced. These changes were accompanied by wrinkle formation, and remained after discontinuance of irradiation. These findings suggested that chronic low-dose UV irradiation induces structural and quantitative changes in the epidermis as a result of keratinization impairment, and that this damage in the epidermis is an important causative factor in wrinkle formation.     

Relationships between changes in mechanical properties of the skin, wrinkling, and destruction of dermal collagen fiber bundles caused by photoaging

BACKGROUND/PURPOSE: Long-term exposure to ultraviolet (UV) radiation induces various cutaneous changes that differ from those because of physiological aging, including structural destruction of dermal collagen fiber bundles (DCFBs), which comprise the major component of the dermis. Wrinkling, a representative change in skin surface associated with photoaging, is often seen at the corners of the eyes and in the space between the eyebrows. These are locations where the skin contracts repeatedly and routinely. Lowered resiliency to skin contraction induced by marked structural changes in DCFBs may represent one cause of photo-induced wrinkles. Using animal models of photoaging, changes in mechanical properties of the skin caused by UV irradiation were measured, and relationships between UV-induced changes were analyzed. METHODS: Animal models of photoaging were prepared by irradiating hairless mice with UVB light. Dorsal skin surface replicas of animals were taken using silicon rubber, and volume of wrinkles was calculated using an image analyzer. Stress of the skin against horizontal contraction was measured using a new device called the Resiliometer. Three-dimensional organization of dermal collagen structures in skin samples collected from the back of each animal was observed under scanning electron microscopy, and compactness of DCFBs was assessed from electron micrography. RESULTS: With time and therefore increasing UV dose, deep wrinkles formed on the backs of mice. Volume of wrinkles peaked at 8 weeks. All parameters obtained from Resiliometer measurements were increased by irradiation. DCFB structure was degraded in a radiation dose-dependent manner. DCFB grading was significantly correlated with each Resiliometer parameter. Significant correlations were also observed between each Resiliometer parameter and volume of wrinkles. CONCLUSION: Stress of the skin against horizontal contraction obtained using the Resiliometer changes following UV irradiation, correlating with photo-induced wrinkling and destruction of DCFBs. These results support the hypothesis that changed force of restitution to skin contraction induced by marked structural changes in DCFBs represents one cause of photo-induced wrinkles. The resiliometric parameter may offer a good indicator for monitoring the condition of DCFB structure, as changes in these would induce failure in restitution to skin contraction, leading to wrinkling.     

A histological study of human wrinkle structures: comparison between sun-exposed areas of the face, with or without wrinkles, and sun-protected areas

Wrinkles are a major topic in dermocosmetology; the purpose of this work has been to go deeper into the knowledge of cutaneous damage underlying these modifications of skin surface. Up to now, the number of published works about the histological structure of wrinkles is not very large. Therefore to complete the findings, we studied 46 subjects of both sexes, between 57 and 98-year-old, enabling us to obtain 157 skin biopsies of wrinkles (face) and sun-protected areas (abdomen). We used different histological techniques involving histochemistry, immunohistochemistry, electron microscopy and quantification by image analysis in addition to classic standard techniques. This study has allowed us to confirm published structural modifications of wrinkles, but also to display many other original alterations. The increased thinning of the epidermis atrophied with age is confirmed by the study of desmoplakins outlining the cellular contours of keratinocytes. Such a thinning is accompanied by a decrease in several markers of epidermal differentiation at the bottom of the wrinkles: filaggrin, keratohyalin granules and transglutaminase I, disturbing desquamation and the capacity of the horny layer to retain water. The dermoepidermal junction is modified by a decrease of collagen IV and VII, which, combined with fewer and fewer oxytalan fibres under wrinkles, weakens this interface. The deposition of abnormal elastotic tissue in the dermis, with an interruption of these deposits under wrinkles and an atrophy of dermal collagen more pronounced under wrinkles, boosts the magnitude and depth of wrinkles. The composition of the other dermal constituents is also altered with, more particularly, a marked decrease of chondroitin sulphates in the papillary dermis under wrinkles, combined with an asymmetrical variation of glycosaminoglycans on both edges of wrinkles. The atrophy of the hypodermis, also more marked under wrinkles, with a thickening of fibrous lines, also makes the depth of wrinkles more pronounced. Wrinkle formation appears at the same time as numerous modifications in different cutaneous structures, which may be mutually amplified. Such a study by pointing out altered elements in skin physiology, makes the development of specific treatments possible in order to mitigate this unwelcome cutaneous deterioration.     

Correlation of skin phototype with facial wrinkle formation

Facial wrinkle formation is a representative sign of photoaging. Skin can be divided into different skin phototypes (SPTs) depending on its sensitivity to solar radiation. We have determined the relationship between SPTs and deep and fine wrinkle scores on the faces of 230 Japanese subjects. This group was chosen because it is relatively easy to quantify acute and chronic sun damage in Japanese individuals. SPT in the Japanese subjects was classified into one of four groups, SPT-I to -IV according to Fitzpatrick's classification method. The subjects were further classified into indoor or outdoor workers. After photographing the outer canthus, deep and fine wrinkles were discriminated visually and scored from 1–11 and from 1–9, respectively. UV light was used as a photographic light source for clear depiction of the fine wrinkles. Higher scores were recorded for deep wrinkles in individuals with SPT-I, who are more sensitive to sunlight than those with SPT-III or IV, who are more tolerant to sunlight. However, the same tendency did not seem to be apparent for fine wrinkle scores. A high incidence of SPT-IV was seen in outdoor workers. Early detection of SPT-I in individuals who are sensitive to sunlight, may help to predict and prevent photoaging of deep wrinkles. Sensitivity to sunlight may play a part in determining an individual's choice of working environment.     

Possible involvement of gelatinases in basement membrane damage and wrinkle formation in chronically ultraviolet B-exposed hairless mouse

A number of studies indicate that matrix metalloproteinase might be involved in photoaging, but little is known about their direct contribution to ultraviolet-induced histologic and morphologic changes in the skin in vivo. This study reports the relationship between changes of matrix metalloproteinase activities and ultraviolet B-induced skin changes in hairless mouse. The role of matrix metalloproteinase in the skin changes was studied by topical application of a specific matrix metalloproteinase inhibitor. The backs of mice were exposed to ultraviolet B three times a week for 10 wk. Histologic studies showed that the basement membrane structure was damaged, with epidermal hyperplasia, in the first 2 wk of ultraviolet B irradiation, followed by the appearance of wrinkles, which gradually extended in the latter half of the ultraviolet B irradiation period. We observed enhancement of type IV collagen degradation activity, but not collagenase or matrix metalloproteinase-3 activity, in extracts of ultraviolet B-irradiated, wrinkle-bearing skin. Gelatin zymographic analysis revealed that gelatinases, matrix metalloproteinase-9 and matrix metalloproteinase-2, were significantly increased in the extract. In situ zymographic study clarified that the activity was specifically localized in whole epidermis of ultraviolet B-irradiated, wrinkled skin in comparison with normal skin. The activity was induced around the basal layer of the epidermis by a single ultraviolet exposure of at least one minimal erythema dose. Furthermore, topical application of a specific matrix metalloproteinase inhibitor, CGS27023A, inhibited ultraviolet B-induced gelatinase activity in the epidermis, and its repeated application prevented ultraviolet B-induced damage to the basement membrane, as well as epidermal hyperplasia and dermal collagen degradation. Ultraviolet B-induced wrinkles were also prevented by administration of the inhibitor. These results, taken together, suggest that ultraviolet B-induced enhancement of gelatinase activity in the skin contributes to wrinkle formation through the destruction of basement membrane structure and dermal collagen in chronically ultraviolet B-exposed hairless mouse, and thus topical application of matrix metalloproteinase inhibitors may be an effective way to prevent ultraviolet B-induced wrinkle formation.     

Biopolymeric agents for skin wrinkle treatment

Skin aging is caused by several factors capable of deteriorating dermal matrix and is visibly noticed in skin color and skin contour deformities. In addition to the prevention of skin aging by application of antioxidants and sunscreens, treatment of skin wrinkles with those of dermal fillers is also recommended. Dermal filler products with enhanced injectability and longer duration are being developed continuously. Biodegradable polymers such as skin elastic fibers and dermal matrix mimetic used for treatment of skin wrinkle are summarized in this article. Additionally, the importance of amino acids, enzymes, and proteins in aesthetic of skin is addressed. Thus, elective agents are proposed for the dermatologists, cosmetic formulators, and the individuals facing skin aging problems. The candidate natural peptides from marine sources are additionally presented for widening the choice of actives application for treating aging.     

From skin microrelief to wrinkles. An area ripe for investigation

Skin microrelief alters progressively with age. Wrinkles do not result from these changes but are superimposed upon them. Wrinkles result from structural changes in the epidermis, dermis and hypodermis. Four types of wrinkles can be recognized. Type 1 wrinkles are atrophic. Type 2 wrinkles are elastotic. Type 3 wrinkles are expressional. Type 4 wrinkles are gravitational. Each type of wrinkle is characterized by distinct microanatomical changes and each type of wrinkle develops in specific skin regions. Each is likely to respond differently to treatment. Skin microrelief and skin folds can be identified on histological examination. By contrast, only minimal dermal changes are found beneath permanent or reducible wrinkles compared with immediately adjacent skin. A series of objective and non-invasive methods is available to quantify the severity of wrinkling.     

The microanatomical basis of facial frown lines

We studied facial frown lines on cadaver skin. These wrinkles persisting after death were kept unmodified during the collecting procedure; some included the underlying bone. Their microanatomical basis lies in the hypodermis where trabeculae of the retinacula cutis are broader and much shorter underneath the wrinkle than in the surrounding skin. These trabeculae contain striated muscle cells. The hypertrophy of the extracellular matrix of the hypodermal septae is probably related to repetitive mechanical stimuli generated by the muscle cells.     

Unusual wrinkle formation after temporary skin fixation followed by UVB irradiation in hairless mouse skin

Abstract Generally, many wrinkles form on the human face, and temporary wrinkles eventually become permanent. We evaluated the effects of temporary skin fixation on wrinkle formation after UVB irradiation using the back skin of hairless mice. In the group treated with UVB irradiation immediately after production using cyanoacrylate resin of an artificial groove parallel to the midline, wrinkles formed parallel to the midline, an uncommon direction for wrinkle formation in this mouse model. These wrinkles did not disappear even when the skin was stretched. No such changes were observed in the group in which only the temporary groove alone was produced without UVB irradiation. In 3-D surface parameter analysis, all roughness parameters in the group treated with UVB irradiation immediately after production of an artificial groove were significantly increased relative to the age-matched control group. In contrast, no differences were observed between the group in which only the temporary groove alone was produced without UVB irradiation and age-matched controls. The results of this study suggest that both a temporary groove in the skin and UVB irradiation are necessary for wrinkle formation in the back skin of hairless mice.     

Skin ageing: clinical and histopathologic study of permanent and reducible wrinkles

Wrinkles are modifications of the skin associated with cutaneous ageing and develop preferentially on sun-exposed skin. The aim of the study was to analyse the clinicopathological features of wrinkles, among the different types of skin relief modifications. Despite its importance in dermato-cosmetology and skin ageing, few studies have been specifically devoted to wrinkles. In the present study, we analyzed the histological features of the pre-auricular wrinkle compared to retro-auricular skin, obtained from sixteen patients undergoing facial surgery; skin samples were immediately processed for routine histology and histochemical staining. Four types of skin depressions could be defined according to their depth: folds, permanent wrinkles, reducible wrinkles and skin micro-relief. Two different types of pre-auricular wrinkles were observed: (i) permanent wrinkles which were conserved after sampling and, (ii) reducible wrinkles which required in vivo staining to be visible at histology. Histological analysis of the epidermis and dermis of the skin forming the pre-auricular wrinkle revealed a normal skin morphology, identical to that of the skin immediately adjacent to the wrinkle. This was particularly striking for the reducible wrinkles which could not be individualized in the absence of in vivo staining. Both types of wrinkles comprised skin modifications observed in sun-exposed skin, however, in the upper dermis, permanent wrinkles showed a more pronounced accumulation of basophilic fibers, i.e. actinic elastosis, than reducible wrinkles did. These data suggest that the development of wrinkles could be secondary to actinic elastosis and to the disappearance of microfibrils and collagen fibers at the dermal-epidermal junction.     

Carbon dioxide laser treatment promotes repair of the three-dimensional network of elastic fibres in rat skin

BACKGROUND: We have previously reported that ultraviolet (UV) B irradiation induces a loss of linearity in the three-dimensional structure of dermal elastic fibres, which results in the reduction of elastic properties of the skin and leads to wrinkle formation. We further reported that repair of wrinkles by all-trans retinoic acid is accompanied by recovery of the linearity of elastic fibres. Carbon dioxide (CO2) lasers are widely used for treating wrinkles in cosmetic surgery. OBJECTIVES: To perform CO2 laser treatment of wrinkles induced in rat skin by UVB irradiation and to evaluate changes in the three-dimensional structure of dermal elastic fibres during wrinkle repair. METHODS: Wrinkles were induced in the hind limb skin of Sprague-Dawley rats by UVB irradiation (130 mJ cm-2 three times weekly for 6 weeks), followed by CO2 laser treatment (11.3 J cm-2). The surface appearance of the skin was evaluated by replica observation 6 and 10 weeks after CO2 laser treatment followed by measurement of mechanical properties using a Cutometer. Subsequently, perfusion fixation and digestion with formic acid were performed and elastic fibres were observed by scanning electron microscopy (SEM). Image analysis of SEM micrographs was carried out to evaluate the linearity in the three-dimensional structure of elastic fibres. RESULTS: Six weeks after CO2 laser treatment, all parameters of skin mechanical properties in the UVB-irradiated group recovered to levels of the control non-irradiated group, accompanied by repair of wrinkles and a significant increase in linearity of the three-dimensional structure of elastic fibres. CONCLUSIONS: These findings indicate that CO2 laser treatment has a therapeutic potential to repair wrinkles to non-irradiated levels through recovery of the three-dimensional structure of elastic fibres.     

Morphological study of the relationship between solar elastosis and the development of wrinkles on the forehead and lateral canthus

OBJECTIVE To identify whether there is a relationship between solar elastosis and the development of wrinkles in human skin. DESIGN Wrinkle depth was measured on the forehead and lateral canthus of human cadavers using image analysis. The thickness of the dermis was measured in skin sections obtained around wrinkles and stained with Elastica-van Gieson. SETTING Gross Anatomy Section, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan. SUBJECTS Fifty-eight male and female cadavers (age range at death, 29-93 years). MAIN OUTCOME MEASURES The ratio of solar elastosis dermal thickness to full dermal thickness (elastosis ratio) was calculated and compared between the deepest point of a wrinkle (wrinkle point) and a point within 1 mm where no wrinkle existed (nonwrinkle point). The relationship between elastosis ratios and wrinkle depths was investigated. RESULTS Advanced solar elastosis was present at nonwrinkle points but was present a little bit at wrinkle points. On the forehead, a positive correlation between elastosis ratios and wrinkle depths was observed at nonwrinkle points but not at wrinkle points. On the lateral canthus, a positive correlation between elastosis ratios and wrinkle depths was observed at nonwrinkle points, as well as at wrinkle points until the wrinkle became deeper than one-half of the original dermal thickness (0.6 mm). Solar elastosis on the lateral canthus ceased developing at this point, but the wrinkle developed further. CONCLUSIONS Solar elastosis tends to commence with the development of a wrinkle until the wrinkle becomes deeper than 0.6 mm. This tendency is less evident at wrinkle points than at nonwrinkle points.     

The effect of metabolites of the epidermal barrier on the development of phototoxic dermatitis

Mechanical removal of the horny layer immediately after UV irradiation of the skin did not result in development of erythema when irradiated in the minimal erythemal dose (MED). When a patient was irradiated in a dose of 2 MED, the intensity of erythema was drastically reduced. This leads to a conclusion that the metabolites accumulating on the surface of the skin and in the horny layer during their oxidation with UV light are capable of return diffusion into the skin where they act as erythema mediators. Simultaneous recording of lipid peroxidation and oxygen consumption in the removed horny layer has demonstrated the relationship between the intensity of oxidation processes in the horny layer and the UV irradiation dose.     

Dietary compound ellagic acid alleviates skin wrinkle and inflammation induced by UV‐B irradiation

Please cite this paper as: Dietary compound ellagic acid alleviates skin wrinkle and inflammation induced by UV‐B irradiation. Experimental Dermatology 2010; 19 : e182–e190. Abstract: Ellagic acid, a polyphenol compound present in berries and pomegranate, has received attention as an agent that may have potential bioactivities preventing chronic diseases. This study examined photoprotective effects of ellagic acid on collagen breakdown and inflammatory responses in UV (ultraviolet)‐B irradiated human skin cells and hairless mice. Ellagic acid attenuated the UV‐B‐induced toxicity of HaCaT keratinocytes and human dermal fibroblasts. Non‐toxic ellagic acid markedly prevented collagen degradation by blocking matrix metalloproteinase production in UV‐B‐exposed fibroblasts. Anti‐wrinkle activity of ellagic acid was further investigated in hairless mice exposed to UV‐B, in which it attenuated UV‐B‐triggered skin wrinkle formation and epidermal thickening. Topical application of 10 μmol/l ellagic acid diminished production of pro‐inflammatory cytokines IL‐1β and IL‐6, and blocked infiltration of inflammatory macrophages in the integuments of SKH‐1 hairless mice exposed to UV‐B for 8 weeks. In addition, this compound mitigated inflammatory intracellular cell adhesion molecule‐1 expression in UV‐B‐irradiated keratinocytes and photoaged mouse epidermis. These results demonstrate that ellagic acid prevented collagen destruction and inflammatory responses caused by UV‐B. Therefore, dietary and pharmacological interventions with berries rich in ellagic acid may be promising treatment strategies interrupting skin wrinkle and inflammation associated with chronic UV exposure leading to photoageing.     

Light and scanning electron microscopic studies on wrinkles in aged persons' skin

Wrinkles in six aged persons (67–82 years of age) have been investigated by light microscopy (LM) and scanning electron microscopy (SEM). There are two types of wrinkles. One is a deep wrinkle which develops on the sun-exposed skin and does not disappear on stretching (permanent wrinkle). The LM and SEM showed less elastotic change in the upper dermis in the area of wrinkle than in that of the surroundings. The other type is a shallow wrinkle which develops on sun-protected skin and disappears on stretching (temporary wrinkle). The LM and SEM showed the decrease or loss of the elastic fibres in the papillary dermis as seen in ageing skin.     

Loss of skin elasticity precedes to rapid increase of wrinkle levels

BACKGROUND: Decreased skin elasticity is considered as a factor that promotes wrinkle formation. However, the relationship between decreased skin elasticity and the formation of wrinkles is not fully understood. OBJECTIVE: The purpose of this study is to characterize the relationship between skin elasticity and wrinkle formation using quantitative methods. METHODS: Skin elasticity at the corner of the eye was measured using a Cutometer SEM575. Wrinkle levels at the corner of the eye were determined from three-dimensional analysis of surface replicas. Ninety healthy female volunteers living in Tokyo, Japan (aged 18-76 years) were examined in this study. RESULTS: In each scatter plot examined, women with lower U(r)/U(f) values or with higher U(v)/U(e) values showed a tendency for increased R(a) and R(max) values. However, this study determined widely scattered values for both types of parameters. These relationships were then reanalyzed in relation to age. Results were compared within a young (YNG) group of 27 women (aged 18-29), a middle-aged (MED) group of 26 women (aged 30-48) and an elderly (OLD) group of 37 women (aged 50-76). For elasticity parameters, U(r)/U(f) and U(v)/U(e) values, significant differences were found between the YNG and MED groups and also between the MED and OLD groups. For roughness parameters, a significant difference was found only between the MED and OLD groups. These results suggest that changes in R(a) or R(max) values lag about 20 years behind changes in skin elasticity values. CONCLUSIONS: Despite a strong relationship, elasticity parameters are unlikely to be an independent predictor of wrinkle levels. The amount of UV exposure to skin with decreased elasticity seems to be the variable that determines wrinkle levels. This finding represents another complexity in the mechanism of wrinkle formation.     

The severity of wrinkling at the forehead is related to the degree of ptosis of the upper eyelid

Background: Most studies on wrinkle formation have focused on changes in the dermal condition that promote the fixation of transiently formed wrinkles. Little is known about the age‐dependent changes in transient wrinkle formation in response to altered facial expression or the mechanism leading to fixed wrinkle formation. Objective: To clarify the mechanism of wrinkle formation at the forehead, we investigated the factors that influence the severity of transient wrinkling and the relationship of transient with fixed wrinkles, using a newly established method to evaluate transient wrinkle formation. Methods: Transient wrinkles were generated by requesting subjects to gaze in an upward direction. Foreheads of the subjects with or without an upward gaze at a fixed angle were photographed and the severity of wrinkles at the forehead was graded from 0 to 5 in 50 healthy Japanese female volunteers in their 20s, 40s, or 60s. Skin elasticity was measured using a Cutometer^®. Frontalis muscle activity and ptosis of the upper eyelid were estimated by measuring movement of the eyebrow during upward gazing and the position of the upper eyelid of the open eye, respectively. Results: Wrinkles formed transiently at the forehead by upward gazing were highly reproducible in each subject. Their severity increased with aging and was highly correlated to that of fixed wrinkles (R=0.81, P<0.001). Therefore, this method appears to be suitable for studying the mechanism of transient wrinkle formation and the relationship between transient and fixed wrinkles at the forehead. The severity of transient wrinkles was correlated with elevation of the eyebrow during upward gazing (R=0.69, P<0.001), but not with dermal elasticity. This suggests that transient wrinkles are induced by increased frontalis muscle activity during upward gazing. Frontalis muscle activation was negatively correlated with upper eyelid position (R=−0.37, P<0.05), which descended with aging, meaning ptosis of the upper eyelid, and negatively correlated with the severity of transient wrinkles induced by upward gazing (R=−0.43, P<0.05). Furthermore, the upper eyelid position was also negatively correlated with the severity of fixed wrinkles (R=−0.44, P<0.05). Conclusion: These results suggest that ptosis of the upper eyelid is associated with increased activation of the frontalis muscle during upward gazing and increased severity of transient and fixed wrinkling at the forehead.     

Botulinum toxin A: a 9-month clinical and 3D in vivo profilometric crow's feet wrinkle formation study.

OBJECTIVE: The objective of this study was to determine the duration of the effects of a defined dose of BTX-A on crow's feet formation wrinkles. MATERIALS AND METHODS: Twenty-five patients each received one injection of 12 units of BTX-A in the orbicularis oculi muscle on both sides of the face. The results were evaluated at 3, 6 and 9 months. Standardized photographs were taken of the wrinkle formations at rest at each step of the 9-month period following the treatment, and the wrinkles were then rated on photographs by blinded observers. Three-dimensional (3D) in vivo profilometry was also used to quantify the degree of improvement. RESULTS: Both the self-assessments by the patients themselves and the objective assessments of the 3D profilometry study provided similar results, but the independent observer assessment results were slightly more optimistic. There was a statistically significant difference observed between baseline and the evaluations at both 3 and 6 months. CONCLUSION: BTX-A is a safe and effective method for treating crow's feet wrinkles. In both the observer assessment and the 3D skin profilometric assessment, a clear improvement was shown at 6 months.