ORIGINAL INVESTIGATIONdownloads.lww.com/wolterskluwer_vitalstream_com/... · to develop a novel skin-assessment technique known as skin blotting, based on the leakage of secreted

ORIGINAL INVESTIGATION

Skin Blotting: A Noninvasive Technique for Evaluating Physiological Skin Status

Takeo Minematsu, PhD; Motoko Horii, MHS; Makoto Oe, PhD; Junko Sugama, PhD; Yuko Mugita, MHS; Lijuan Huang, PhD; Gojiro Nakagami, PhD; and Hiromi Sanada, PhD

ABSTRACT OBJECTIVE: The skin performs important structural and physiological functions, and skin assessment represents an important step in identifying skin problems. Although noninvasive techniques for assessing skin status exist, no such techniques for monitoring its physiological status are available. This study aimed to develop a novel skin-assessment technique known as skin blotting, based on the leakage of secreted proteins from inside the skin following overhydration in mice. The applicability of this technique was further investigated in a clinical setting. DESIGN: Skin blotting involves 2 steps: collecting proteins by attaching a damp nitrocellulose membrane to the surface of the skin, and immunostaining the collected proteins. The authors implanted fluorescein-conjugated dextran (F-DEX)Ycontaining agarose gels into mice and detected the tissue distribution of F-DEX under different blotting conditions. They also analyzed the correlations between inflammatory cytokine secretion and leakage following ultraviolet irradiation in mice and in relation to body mass index in humans. MAIN RESULTS: The F-DEX in mice was distributed in the deeper and shallower layers of skin and leaked through the transfollicular and transepidermal routes, respectively. Ultraviolet irradiation induced tumor necrosis factor secretion in the epidermis in mice, which was detected by skin blotting, whereas follicular tumor necrosis factor was associated with body mass index in obese human subjects. These results support the applicability of skin blotting for skin assessment. CONCLUSIONS: Skin blotting represents a noninvasive technique for assessing skin physiology and has potential as a predictive and diagnostic tool for skin disorders. KEYWORDS: skin blotting, skin assessment, physiological status, secreted protein

ADV SKIN WOUND CARE 2014;27:272Y9

INTRODUCTION Skin assessment can provide information to help prevent skin breakdown or damage, including pressure ulcers, skin tears, and dermatitis.1,2 Noninvasive skin-assessment techniques have been developed, including skin microscopy,3 transepidermal water loss,4

optical coherence tomography,5 and high-frequency ultrasound.6

Although these techniques are useful for analyzing structural and functional skin conditions, no methodology exists for evaluating the physiological status of the skin directly. The skin’s homeostatic equilibrium is maintained by several

secreted factors including cytokines, chemokines, growth factors, enzymes, and extracellular-matrix components. Signaling of secreted proteins, including the epidermal growth factor and transforming growth factor A families, regulates the proliferation and differen-tiation of keratinocytes.7 Turnover of the dermis depends on the secretion of extracellular matrix and its degradation by matrix met-

alloproteinases.8 Skin disorders, especially skin inflammation, are also associated with release of several cytokines from leukocytes and mast cells.9 Although the secreted-protein profiles reflect the skin condition, the existence of the skin barrier makes their ap-plication for noninvasive skin assessments difficult. The skin barrier consists of sebum, intercellular lipids in the cor-

neal layer of the epidermis, and keratinocyte tight junctions. This bar-rier regulates transepidermal water loss and restricts the invasion of foreign irritants and pathogens.10,11 Previous studies have demon-

strated the existence of structural and functional changes in the epi-dermis in macerated skin.12,13 Overhydration disrupts the lamellar structure of intercellular lipids and expands the interstitial spaces among keratinocytes in the epidermis. Thus, although only com-

pounds with a molecular weight of less than 500 daltons (d) are able to penetrate the dermis under normal conditions,10 the skin barrier is unable to restrict the transdermal permeation of large water-soluble molecules, such as dextran and bovine serum albumin, from the outside

Takeo Minematsu, PhD, is a Project Lecturer, Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, The University of Tokyo, Japan. Motoko Horii, MHS, is an Assistant Professor, Faculty of Nursing, Josai International University, Chiba, Japan. Makoto Oe, PhD, is a Project Lecturer, Department of Advanced Nursing Technology, Graduate School of Medicine, The University of Tokyo. Junko Sugama, PhD, is a Professor, Department of Clinical Nursing, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan. Yuko Mugita, MHS, is a Doctoral Student; Lijuan Huang, PhD, is a Project Researcher; Gojiro Nakagami, PhD, is a Lecturer; and Hiromi Sanada, PhD, is a Professor, all at the Department of Gerontological Nursing/Wound Care Management, Graduate School of Medicine, The University of Tokyo, Japan. The authors have disclosed that they have no financial relationships related to this article. This study was supported by JSPS KAKENHI grant no. 23249088. Submitted May 31, 2013; accepted in revised form October 18, 2013.

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to the inside of the skin under overhydrated conditions. The authors hypothesized that large water-soluble molecules might similarly permeate from the inside to the outside under wet conditions. The amounts of protein leaking from inside the skin are likely to

be extremely small, and effective methods are therefore needed to detect and measure such leakage. The authors previously reported that nitrocellulose membranes, which are commonly used to fix proteins in Western blotting, could be used for the collection of extremely small volumes of cutaneous-wound exudate and for the immunological analysis of its protein components.14 In the current study, the authors collected soluble molecules leaking from inside the skin barrier by attaching a nitrocellulose membrane to the surface of the skin under wet conditions, using a technique referred to as ‘‘skin blotting.’’ The results of the study suggest that soluble molecules distri-

buted in the deeper dermis or subcutaneous tissue were leaked via the transfollicular route, whereas those in the shallow layer of the dermis or epidermis were leaked via the transepidermal route. Fur-thermore, clinical investigations performed in healthy volunteers indicated the applicability and practicality of this technique for as-sessing human skin.

METHODS Animal Experiments Sixty-five 7-week-old male C57BL/6J mice purchased from SLC Japan (Shizuoka, Japan) were maintained under controlled light (12-hour light and 12-hour dark) and temperature (25-C T 2-C) conditions, with free access to food and water, and were accli-matized for 1 week. Two separate experiments were conducted. In the first experiment, the dorsal hair was removed using a de-pilatory cream. After 4 days, 50 KL of agarose gel droplet (approx-imately 8-mm diameter) dissolved in sterilized phosphate-buffered saline plus fluorescein-conjugated dextran (F-DEX, 3 kd) at 0, 0.1, 1, or 10 Kg/mL was inserted into the subcutaneous space of the center of back via an approximately 1-cm-wide skin incision on the scapula. The agarose gel droplet was placed around the T12 level of the thoracic spine, with approximately 1.5 cm between the in-cision and the edge of the droplet. The incision was sutured and covered with a 1-cm-wide hydrocolloid dressing (DuoDERM Extra Thin, ConvaTec, Skillman, New Jersey) to prevent leakage of F-DEX from the incision. After 1, 3, 6, or 24 hours, skin blotting on the center of the back was performed under anesthesia for 1, 5, or 10 minutes. Five animals were assigned to each of the different conditions. Skin tissue was harvested immediately after blotting, fixed by 4% paraformaldehyde, and embedded in optimal cutting temperature compound. Frozen sections were counterstained with 4],6-diamidino-2-phenylindole. In the second experiment, skin blotting was used to detect acute

inflammation induced by ultraviolet B (UVB) irradiation. Ultra-

violet B exposure induces inflammation and upregulates inflam-

matory cytokines, including tumor necrosis factor (TNF), in the shallow layers of the skin.15 Mice were irradiated with UVB (290Y380 nm, 6 W) for 1 or 4 minutes at a distance of 10 cm under anesthesia 4 days after hair removal, with the left side of the body covered with aluminum foil (5 animals in each group). Skin blotting was performed the following day on the right (irradiated) and left (control) sides for 10 minutes, under anesthesia. Skin tissue was harvested immediately after blotting and divided into 2 pieces; 1 piece was fixed and embedded in paraffin for immunofluores-

cence analysis of TNF, and the other piece was processed for Western blotting of TNF. Experimental protocols were approved by the Animal Research

Committee of The University of Tokyo, and all animals were treated according to guidelines established by the Japanese Association for Laboratory Animal Science (1987).

Clinical Study The authors investigated the relevance of evaluating inflammation in the deeper layers of the skin by focusing on the skin conditions in obesity, given that expanding tissue mass is known to result in hypoxia and inflammation of adipose tissue.16,17 The authors per-formed skin blotting for TNF in 59 adult volunteers. A cross-sectional study was conducted between June 2011 and November 2011. Healthy adult volunteers older than 20 years were recruited from 2 private companies. Exclusion criteria included atopic der-matitis, psoriasis, and other dermatopathies. Body weights and heights of the volunteers were measured, and the body mass index (BMI) was calculated (in kilograms per meter squared). Skin blotting was performed in the center of the rear surface of a thigh. Immuno-

staining was performed by 1 researcher, and membrane staining was evaluated by a second researcher blinded to BMI category. Approval was obtained from the Ethics Committee of the

Graduate School of Medicine and Faculty of Medicine, The Uni-versity of Tokyo, before performing this study.

Skin Blotting Circular (8-mm diameter) or square (1 � 1 cm) pieces of nitro-cellulose membrane (BioRad, Hercules, California) were used for skin blotting. Circular membrane pieces were used in human sub-jects to minimize the effects of tape removal. Square membranes were used in mice to ensure that the membrane was larger than the agarose gel drop. The pieces of membrane were prewetted with 2 and 5 KL of normal saline, respectively, and used for skin blot-ting. Pieces of membrane were attached to the skin surface with nonpermeable plastic tape. Collected membranes were stored at 4-C until use. Endogenous alkaline phosphatase and peroxidase activities were inactivated by incubation of the membrane with 500 Kg/mL levamisole (Sigma-Aldrich, St Louis, Missouri) and 0.3%

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hydrogen peroxide in 20% methanol at room temperature. Mem-

brane staining was performed using a standard protocol for in-direct methods with antifluorescein (Southern Biotech, Birmingham, Alabama) and anti-TNF antibodies (Santa Cruz Biotechnology, Santa Cruz, California).

Western blotting Ten milligrams of the frozen tissues were homogenized in 100 mL of the lysis buffer (2% SDS, 50 mM Tris, 5% 2-mercaptoethanol, and 2.5% sucrose) and boiled for 10 minutes. Protein was separated by SDS-PAGE (200 V, 40 minutes) using 15% acrylamide gel and transferred to a nitrocellulose membrane by semi-dry blotting (30 V, 2 hours). The separated TNF was immunologically detected by the same method used for skin blotting, as previously mentioned.

Histological analysis The paraffin sections were deparaffinized in a series of ethanol and xylene. Antigen retrieval by autoclaving (121-C, 15 minutes) in cit-rate buffer (pH 6) was performed. Following the reaction with anti-TNF antibody, immunoreaction was visualized with biotinylated anti-Goat IgG (Jackson ImmunoResearch, West Grove, Pennsylvania) and FITC-conjugated avidin (BioLegend, San Diego, California). Finally, sections were embedded with Dapi-Fluoromount-G (Southern Biotechnology, Birmingham, Alabama).

Statistical Analysis The significance of differences in TNF signal intensity between mice irradiated for 1 and 4 minutes was analyzed using Student t tests. The distributions of follicular TNF signals among BMI cat-egories were evaluated using Fisher exact tests. Associations be-tween epidermal TNF signal intensity and BMI were analyzed by Spearman rank correlation tests. P G .05 was considered significant.

RESULTS The authors used a mouse model to demonstrate the leakage and collection of the soluble molecule F-DEX from agarose gels im-

planted into the subcutaneous space on the back of the mice. At 24 hours after implantation of the agarose gel (10 Kg/mL

F-DEX), slightly wet nitrocellulose membranes were attached to the center of the back using nonpermeable plastic tape for 1, 5, and 10 minutes, respectively. Immunostaining of the membrane for F-DEX revealed a clear spot-type signal (representing skin fol-licles) and a relatively weak interspot signal (Figure 1A, upper panels). Both signals increased with increasing attachment dura-tion. The F-DEX distribution was revealed by observing fluorescein in skin sections harvested just after membrane attachment (Figure 1A, lower panels). Fluorescence intensity was increased in the corneal layer of mice with 1-minute membrane attachment compared with control mice without membrane attachment. Fluorescein condensed

at the surface of the corneal layer in mice with 5- and 10-minute attachment. These results indicate that soluble F-DEX was at-tracted from below the epidermal corneal layer and absorbed by the blotting membrane. When skin blotting of dorsal skin implanted with different

concentrations of F-DEX (0, 0.1, 1, and 10 Kg/mL) was performed for 10 minutes, the fluorescence intensity increased with increas-ing F-DEX concentration in the skin tissue harvested just after blotting (Figure 1B, upper panels). Skin blotting detected both spot-type and interspot immunoreactivities for 0.1, 1, and 10 Kg/mL F-DEX, whereas no signal was detected on membranes from 0 Kg/ mL F-DEX samples (Figure 1B, lower panels). The intensity of the interspot signal was significantly correlated with the F-DEX con-centration in the implanted gel (Figure 1C, r = 0.945, P G .001), whereas the intensity of the spot-type signal was not.

To clarify the origin of the molecules detected by skin blotting, the authors attached membranes 1, 3, and 6 hours after implan-

tation of 10 Kg/mL F-DEX gel. After 1 hour of implantation, skin blotting detected only spot signals, whereas interspot signals were remarkably higher at 6 hours compared with 3 hours after im-

plantation (Figure 1D, upper panels). Observation of fluorescein in skin tissue revealed that F-DEX was distributed in the sub-cutaneous tissue and deeper layer of the dermis after 1 hour of implantation, had diffused to the papillary layer of the dermis by 3 hours, and reached the surface of the corneal layer of the epi-dermis at 6 hours (Figure 1D, lower panels). These results indicate that F-DEX distributed in the deeper dermis, and subcutaneous fat tissue was detected through the transfollicular route and shown as spot-type signals, whereas F-DEX in the epidermis and papillary layer of the dermis was detected through the transepidermal route and shown as interspot signals. In mice subjected to UVB irradiation, no macroscopic signs of

inflammation, including redness or swelling, were observed imme-

diately after or 1 day after irradiation in either group. Severe skin damage appeared only in the mice irradiated for 4 minutes by 7 days after irradiation (Figure 2A). Western blotting, immunofluorescence, and skin blotting for TNF were performed 1 day after irradia-tion. Western blotting revealed increased TNF expression in the 4-minuteYexposed group compared with the 1-minuteYexposed group (Figure 2B). The TNF expression was upregulated mainly in the epidermis of irradiated skin (Figure 2C). Skin blotting not only detected TNF on the irradiated side, but also detected slight expres-sion on the opposite nonirradiated side in both groups (Figure 2D). Spot-type signals were not observed in any membranes, indicating that skin blotting detected subclinical inflammation in the shallow layers of the skin. The intensity of the TNF signal on the irradiated side was normalized by dividing it by the signal intensity for the control side and was significantly elevated in the 4-minute group compared with the 1-minute group (P G .001, Figure 2E).





Figure 1. DETECTION OF F-DEX BY SKIN BLOTTING

Skin blotting was performed under different combinations of experimental conditions, including membrane attachment, F-DEX concentration, and postimplantation period. A, Membrane attachment: 1, 5, and 10 minutes; F-DEX concentration: 10 Kg/mL; postimplantation period: 24 hours. B, F-DEX concentration: 0, 0.1, 1, and 10 Kg/mL; membrane attachment: 10 minutes; postimplantation period: 24 hours. C, Postimplantation period: 1, 3, and 6 h; membrane attachment: 10 minutes; F-DEX concentration: 10 Kg/mL. Scale bar = 1 mm in skin-blotting membrane; 25 Km in Aand 50 Km in tissue sections in B and D. Each panel shows 1 representative experiment of 5 duplicates. C, Significant correlation between fluorescence intensity and F-DEX concentration analyzed by Spearman correlation.

The demographic characteristics of the subjects and the results no medication. Both follicular and epidermal signals were detected of skin blotting in the clinical study are shown in Tables 1 and 2, (Figure 3). Only follicular TNF values were significantly associated respectively. All participants had no comorbidities and were taking with BMI, and consistently elevated follicular TNF levels were





Figure 2. SKIN-BLOTTING DETECTION OF SUBCLINICAL INFLAMMATORY STATUS INDUCED BY UVB IRRADIATION

The left side of the body was covered with foil, and the animal was irradiated with UVB for 1 or 4 minutes, respectively. A, No macroscopic inflammatory signs were observed immediately after (day 0) or 1 day after irradiation, but severe skin damage appeared in the 4-minute-irradiation group on day 7. Subclinical inflammatory status on day 1 was revealed by Western blotting (B) and immunofluorescence analysis for TNF (C). Color chart in A: 1-cm square. Scale bar in C = 50 Km. D, Skin blotting detected only epidermal signals. E, The signal intensity (mean T SEM) was significantly higher in the 4-minute-irradiation group compared with the 1-minute group (t test, P G .001). Panels show 1 representative experiment of 5 duplicates.

observed only in subjects with BMI greater than 30 kg/m2. Corre- epidermis. This methodology is based on the principle of leakage lations with epidermal TNF were not significant. of soluble factors through the transfollicular and transepidermal

routes under slightly hydrated skin conditions. The passage of DISCUSSION soluble molecules by both these routes was previously dem-

This study demonstrated the feasibility of skin blotting for onstrated in studies of transdermal drug delivery18 and skin the detection of soluble factors distributed in the dermis and maceration.12,13





Table 1.

DEMOGRAPHIC CHARACTERISTICS OF SUBJECTS

Variable Value

Age, median (range), y 49 (33Y66) Female 49 (33Y66) Male 51 (40Y57)

Sex, n (%) Female 20 (33.9) Male 39 (66.1)

Body mass index, median (range), kg/m2

Female 22.1 (16.4Y38.8) Male 23.4 (18.6Y33.7)

The dorsal hair of mice was removed completely with depilatory cream 4 days before the experiment, because abundant dorsal hair could inhibit the application of skin blotting. Depilation-induced infiltration of inflammatory cells was quenched by 4 days after hair removal, as demonstrated in preliminary experiments (data not shown). Hair follicles are one of the major skin appendages. Transfol-

licular penetration of compounds is recognized as the most impor-

tant route for transdermal drug delivery,19 because the anagen follicle and its fibrous tract extend to the subcutis,20 and the lumen of the follicle can act as a long-term reservoir.18 The authors’ previous study on transdermal F-DEX penetration in overhydrated rat skin sug-gested that the barrier function of follicles was vulnerable.12 Similarly, skin blotting identified F-DEX in the deeper layers of the dermis and subcutaneous fat tissue as follicular signals. Follicular F-DEX signals were rapidly detected within 1 minute after membrane at-tachment with high sensitivity, possibly because of the histological and functional characteristics of the hair follicle. Furthermore, no

follicular TNF signal was detected in the dorsal skin of UVB-irradiated mice in which inflammation was induced in the epidermis and papillary layer of the dermis, suggesting that the follicular signal specifically reflects the physiological status of the deeper layers of the skin. In the authors’ previous study on skin maceration, 30-minute

hydration of the skin surface with an agarose gel containing nor-mal saline was insufficient to induce the transdermal penetration of 460-d fluorescein from the outside to the inside.12 However, leak-age of 3-kd F-DEX from the inside to the outside in the current study was achieved by as little as 10-minute hydration with a wet nitrocellulose membrane. These results suggest that the inhibitory effect of the skin barrier on the passage of soluble molecules differs between leakage and invasion. In addition, the electrostatic pro-perties of the nitrocellulose membrane may attract molecules from the layer below the corneal layer of epidermis, as indicated by the F-DEX distribution 1 minute after attachment of the membrane. The authors used 3-kd F-DEX and 17-kd TNF as markers for skin blotting in this study. Tan et al13 showed transdermal penetration of bovine serum albumin with a molecular weight of approximately 70 kd. Further studies are needed to identify the range of molecular weights detectable by skin blotting.

The epidermal signal intensity correlated significantly with the concentration of F-DEX in the implanted agarose, whereas no sig-nificant correlation was observed for the follicular signals. The intensity of the epidermal signal in UVB-irradiated mice also re-flected the irradiation period and the fluorescence intensity of TNF immunostaining in skin tissue. These results indicate that skin blot-ting qualitatively measured soluble tissue factors. No macroscopic skin damage was apparent 1 day after irradiation, but severe dam-

age was observed by 1 week after irradiation. Skin blotting for TNF

Table 2.

SKIN-BLOTTING ASSESMENT OF ASSOCIATION BETWEEN BMI AND TNF SECRETION IN SKIN TISSUE BMI (kg/m2)

G20 20 to G25 25 to G30 Q30

Subjects Total 6 29 15 9 Male 3 20 9 7 Female 3 9 6 2

Follicular signal,a n (%) P Total 3 (50.0) 5 (17.2) 7 (46.7) 9 (100.0) G.001 Male 2 (66.7) 4 (20.0) 7 (77.8) 7 (100.0) G.001 Female 1 (33.3) 1 (11.1) 0 (0.0) 2 (100.0) .016

Epidermal signal (AU), median (Q1, Q3)b R Total 1.345 (1.176, 1.399) 1.186 (0.015, 1.529) 0.962 (0.030, 1.402) 0.030 (0.016, 1.174) .396 Male 1.415 (1.143, 1.462) 1.134 (0.017, 1.477) 0.999 (0.033, 1.472) 0.030 (0.021, 1.216) .312 Female 1.338 (1.230, 1.345) 1.363 (0.014, 1.592) 0.856 (0.229, 1.219) 0.473 (0.236, 0.709) .411

Q1: 25th percentile, Q3: 75th percentile. aFisher exact test. bSpearman rank correlation.




https://inside.12

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Figure 3. TNF SIGNAL DETECTED IN CLINICAL SUBJECTS BY SKIN BLOTTING

Fifty-nine subjects were recruited and categorized as lean (BMI G20 kg/m2), normal (BMI 20 to G25 kg/m2), overweight (BMI 25 to G30 kg/m2), and obese (BMI Q30 kg/m2). Skin blotting was performed to detect follicular and epidermal TNF signals. Panels include samples stained at the same time.

may therefore be useful for detecting subclinical skin inflammation. It is possible, however, that the permeability of larger molecules was lower than that of smaller molecules. In addition, individual differences in skin-barrier function and epidermal thickness may also affect epidermal detection. The effects of molecular weight, skin-barrier function, and epidermal thickness on epidermal signal intensity need to be clarified to establish skin blotting as a quan-titative skin-assessment technique.

To date, invasive sampling, such as by biopsy, has been used to analyze protein synthesis to evaluate the physiological status of the skin.21,22 Compared with these techniques, skin blotting has the advantages of noninvasive sampling and quantitative anal-ysis of secreted proteins. Skin functions are regulated by several secreted proteins.7Y9 Protein secretion is a complex process involv-ing translocation across the endoplasmic reticulum membrane, N-glycosylation and folding in the endoplasmic reticulum lumen, modification in the Golgi apparatus, and release from the secre-tory granules to the extracellular space following translation.23 Direct measurement of secretion is essential for evaluation of the phys-iological status of the skin, given that each step of the protein-secretion process is regulated independently. Skin blotting is the first method to realize the quantitative analysis of protein secretion within the tissue, although immunohistochemistry can be used to analyze secretion quantitatively in tissue sections.

Tape stripping using adhesive tape is a widely accepted tech-nique for examining skin conditions.24 In this study, F-DEX dispersed from the subcutaneous space to the corneal layer of the epidermis within 6 hours after implantation, supporting the idea that analysis of the corneum can reflect the present status of the skin. However, adhesion and removal of relatively large pieces of adhesive tape need to be repeated to provide sufficiently large samples for biochemical analysis.25 Therefore, this method may not be feasible in patients with vulnerable skin, such as older adults. Skin blotting is more suitable in these cases because molecules can be collected from the deeper layers of the skin with no invasive procedures. The current cross-sectional study of skin blotting for TNF in

obese subjects indicated the applicability and practicality of this method in the clinical setting. In addition, the results demonstrated an interesting aspect of obese skin; follicular signals were detected in all obese subjects (BMI Q30 kg/m2). Excessive maturation induces inflammatory responses and alters the secretion of several cyto-kines.26,27 The detection of follicular TNF signals probably indicated the subclinical inflammatory status of subcutaneous adipose tissue. The authors recently reported that obesity-induced oxidative stress promoted the expression of collagen-degrading enzymes in sub-cutaneous adipose tissue, resulting in decreased density of dermal collagen fibers and increased skin fragility.16 Skin blotting for TNF represents a possible tool for assessing the mechanical vulnerability of skin in obese patients. The composition of sweat metabolites was previously shown

to reflect the ischemic condition of the skin due to pressure load-ing.28,29 Nitrocellulose membranes are able to absorb not only pro-teins, but also polar molecules. Metabolites with polarity, such as lactate and urine, are also possible targets for skin blotting; how-

ever, biochemical staining methods need to be developed for them. This study had some limitations. The animal experiments were

performed under invasive conditions, including implantation of agarose gel and 4-minute UVB irradiation, which induced severe skin damage 7 days after irradiation. Although the authors’ clinical study supported the applicability of this technique in humans, they were unable to analyze the distribution of TNF in skin tissues, and further studies are needed to confirm the validity of this meth-

odology in humans. Moreover, it is difficult to demonstrate the re-liability of this technique; because skin blotting alters the distribution of proteins in the skin tissue, the authors were unable to compare the results among subsequent samples. An artificial skin model will be required to confirm the reliability of this technique. Protein collection by skin blotting can be affected by skin properties such as barrier function and thickness. Thus, collection and evaluation methods may need to be adapted to account for specific skin disorders. In conclusion, skin blotting can be used to collect soluble mol-

ecules penetrating via transfollicular and transepidermal routes.




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This simple and noninvasive method represents a novel tool for assessing physiological skin status. Identification of effective and specific biomarkers for certain skin conditions will allow the es-tablishment of novel predictive and diagnostic tools for a range of skin disorders.& REFERENCES

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ORIGINAL INVESTIGATIONdownloads.lww.com/wolterskluwer_vitalstream_com/... · to develop a novel skin-assessment technique known as skin blotting, based on the leakage of secreted